Human anti-TREM2 antibodies for treatment of neurodegenerative diseases

Abstract

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

Description

Technical Field

[0001] The present invention relates to a protein comprising or consisting of an immunoglobulin (Ig) heavy chain variable region or a light chain variable region. The present invention also relates to a protein comprising an antibody light chain variable region and an antibody heavy chain variable region, and an antibody comprising an antibody light chain variable region and an antibody heavy chain variable region. The present invention also relates to a pharmaceutical composition comprising the protein or antibody. The protein or antibody is capable of binding to human TREM2, preferably to the stem region of hTREM2. The protein or antibody is typically an agonist of hTREM2, preferably an agonist antibody against hTREM2. Therefore, the protein, antibody and pharmaceutical composition can be used for treatment, in particular for treating or preventing neurodegenerative diseases, such as Alzheimer's disease. The present invention also relates to a method for treating or preventing neurodegenerative diseases, such as Alzheimer's disease. Background of the Invention

[0003] Neurodegenerative diseases such as Alzheimer's disease (AD) result in age-related, progressive deterioration of neuronal structure, 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 therapies have been approved for the treatment of AD. Aducanumab (marketed as Aduhelm), an antibody against amyloid-β protein (Aβ), has been controversial in its use and approval. This antibody targets amyloid plaques, a key sign of Alzheimer's disease, thereby reducing plaque burden in the brain. Lecanemab (marketed as Leqembi) is another recently approved FDA-approved antibody. Leqembi also targets Aβ, more specifically protofibrils. Both therapies carry a high risk of infusion-related reactions, cerebral edema, and microbleeds (van Dyck et al., 2022). Of the many Aβ-based therapies, Aduhelm and Leqembi are the only ones confirmed to date for the treatment of AD. Therefore, new strategies to intervene in plaque deposition are needed.

[0004] Triggering receptor expressed on myeloid cells 2 (TREM2) is a transmembrane receptor expressed on myeloid cells that is essential for microglial activation. TREM2 mutations have been identified in neurodegenerative diseases such as AD, leading to loss of TREM2 function through diverse mechanisms (Gernot Kleinberger et al., 2017; Schlepckow et al., 2017; Song et al., 2017; Ulland et al., 2017). TREM2-mediated signaling in microglia induces a transition from homeostatic microglia to disease-associated microglia (DAMs) (Keren-Shaul et al., 2017). This transition is phenotypicly characterized by enhanced phagocytosis, migration, and cell survival. Activation of TREM2 signaling is mediated by 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. α-Secretase-mediated shedding of the TREM2 extracellular domain leads to the release of soluble TREM2 (sTREM2), thereby terminating signaling (G Kleinberger et al., 2014; Wunderlich et al., 2013). The function of sTREM2 is still not fully understood. Altered sTREM2 levels have been reported in the CSF of AD patients and have been proposed as a potential disease biomarker (Zhong & Chen, 2019). sTREM2 levels exhibit dynamic changes during AD progression, reaching their highest levels in the early symptomatic stages of the disease (Suárez-Calvet et al., 2016). Furthermore, sTREM2 has been observed to positively correlate with phosphorylated tau and total tau (Suárez-Calvet et al., 2016). Furthermore, studies have demonstrated that sTREM2 has a signaling function (Zhong et al., 2017, 2019).

[0005] Unlike the prior art, the object of the present invention is to provide a remedial method for treating or preventing neurodegenerative diseases / disorders such as AD. SUMMARY OF THE INVENTION

[0007] In order to solve this problem, the present invention provides:

[0008] 1) A protein comprising or consisting of a heavy chain variable region or comprising or consisting of a light chain variable region, wherein

[0009] 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 1 or 2 amino acid residues substituted compared to SEQ ID NO: 2, and

[0010] 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 1 or 2 amino acid residues substituted compared to SEQ ID NO: 1; preferably, the protein is an agonist of human TREM2.

[0011] 2) The protein according to 1), which comprises an antibody heavy chain comprising the heavy chain variable region.

[0012] 3) The protein according to 1) or 2), comprising two identical antibody heavy chains, each heavy chain comprising the heavy chain variable region and at least one, preferably at least two, Ig heavy chain constant regions.

[0013] 4) A protein, preferably a protein according to any one of 1) to 3), comprising a light chain variable region and a heavy chain variable region, wherein

[0014] 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 1 or 2 amino acid residues substituted compared to SEQ ID NO: 1, and

[0015] 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 1 or 2 amino acid residues substituted compared to SEQ ID NO: 2.

[0016] 5) A protein, preferably a protein according to 4), comprising an antibody light chain and an antibody heavy chain, wherein

[0017] The light chain comprises a light 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 1 or 2 amino acid residues substituted compared to SEQ ID NO: 1, and

[0018] 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 1 or 2 amino acid residues substituted compared with SEQ ID NO: 2.

[0019] 6) A protein, preferably a protein according to 4) or 5), comprising a light chain variable region and a heavy chain variable region, wherein

[0020] The light chain (LC) variable region comprises, preferably in CDR-L3, an amino acid sequence having SEQ ID NO: 5 or a fragment of an amino acid sequence having 1 amino acid residue substitution compared to the amino acid sequence of SEQ ID NO: 5; and

[0021] The heavy chain (HC) variable region preferably comprises a fragment comprising the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having 1 amino acid residue substitution compared to the amino acid sequence of SEQ ID NO: 8 in CDR-H3.

[0022] 7) The protein according to any one of 4) to 6), wherein

[0023] In the N-terminal to C-terminal direction, the light chain variable region comprises, preferably in CDRs L1 and L3, the following amino acid stretch:

[0024] a fragment (CDR-L1) having the amino acid sequence of SEQ ID NO: 3, and

[0025] a fragment (CDR-L3) having the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having 1 amino acid residue substituted compared to the amino acid sequence of SEQ ID NO: 5; and

[0026] In the N-terminal to C-terminal direction, the heavy chain (HC) variable region comprises, preferably in CDRs H2 and H3, the following amino acid stretch:

[0027] a fragment (CDR-H2) having the amino acid sequence of SEQ ID NO: 7, and

[0028] A fragment (CDR-H3) having the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having 1 amino acid residue substituted compared to the amino acid sequence of SEQ ID NO: 8.

[0029] 8) The protein according to 6) or 7), wherein the light chain (LC) variable region comprises, in the N-terminal to C-terminal direction, the following amino acid stretch, preferably in CDRs L1 to L3:

[0030] a fragment (CDR-L1) having the amino acid sequence of SEQ ID NO: 3,

[0031] a fragment (CDR-L2) having the amino acid sequence of SEQ ID NO: 4, and

[0032] a fragment (CDR-L3) having the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having 1 amino acid residue substituted compared to the amino acid sequence of SEQ ID NO: 5; and

[0033] In the N-terminal to C-terminal direction, the heavy chain (HC) variable region comprises, preferably comprises the following amino acid sequence segments in CDRH1 to H3:

[0034] a fragment (CDR-H1) having the amino acid sequence of SEQ ID NO: 6,

[0035] a fragment (CDR-H2) having the amino acid sequence of SEQ ID NO: 7, and

[0036] A fragment (CDR-H3) having the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having 1 amino acid residue substituted compared to the amino acid sequence of SEQ ID NO: 8.

[0037] 9) The protein according to any one of 4) to 8), wherein the protein is a single-chain antibody (scFv), a Fab fragment, a F(ab)2 fragment or an immunoglobulin (Ig); and / or

[0038] 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 a first fusion protein fragment and a second fusion protein fragment.

[0039] 10) The protein according to any one of 4) to 9), comprising an antibody light chain (subunit) and an antibody heavy chain (subunit),

[0040] The light chain comprises the light chain variable region and the light chain constant region,

[0041] The heavy chain comprises the heavy chain variable region and at least one heavy chain constant region, preferably at least constant region CH1.

[0042] 11) The protein according to 10), wherein the heavy chain constant region comprises 1, 2 or 3 Ig heavy chain constant domains, preferably 3 heavy chain constant domains.

[0043] 12) The protein according to any one of 1) to 8), wherein the protein is an immunoglobulin selected from IgG, IgA, IgD, IgE and IgM, or the protein is a fusion protein comprising the Ig and an additional fusion protein fragment.

[0044] 13) The protein according to 12), wherein the Ig is IgG or a fusion protein comprising IgG and an additional fusion protein fragment.

[0045] 14) The protein according to any one of 1) to 13), comprising the following light chain:

[0046] (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

[0047] (b) The amino acid sequence of the light chain is or comprises an amino acid sequence having 1 or 2 amino acid residues substituted compared to the amino acid sequence of SEQ ID NO: 9, 10 or 11.

[0048] 15) The protein according to any one of 4) to 14), wherein the light chain is a kappa light chain or a lambda light chain.

[0049] 16) The protein according to any one of 4) to 15), comprising the following heavy chain:

[0050] (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

[0051] (d) the amino acid sequence of the heavy chain is or comprises an amino acid sequence having 1 or 2 amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 12, 13, 14 or 15.

[0052] 17) The protein according to any one of 4) to 16), wherein the protein is a fully human Ig.

[0053] 18) The protein according to any one of 4) to 17), which comprises an antibody heavy chain or Fc part that is unable to bind to an Fc receptor, or has reduced binding to an Fc receptor, or has a mutated constant region that has reduced binding to an Fc receptor.

[0054] 19) The protein according to 18), wherein the amino acid sequence of the heavy chain has a substitution from P to G at the position corresponding to position 334 of SEQ ID NO: 12 or 13, so as to prevent or reduce binding of the protein to an Fc receptor.

[0055] 20) A protein according to any one of 1) to 3) and 4) to 19), which comprises a binding domain capable of binding to human transferrin receptor 1 (hTfR1) to allow the protein to cross the blood-brain barrier, preferably, the protein comprises a heavy chain with a modified CH3 domain or comprises a C-terminal extension of the CH3 domain allowing binding to hTfR1.

[0056] 21) A protein according to any one of 1) to 20), wherein the protein is capable of binding to human TREM2 via its variable region, preferably to the stem region of hTREM2; typically, the protein or antibody is an hTREM2 agonist.

[0057] 22) An antibody comprising an Ig light chain variable region as defined in 4) and an Ig heavy chain variable region as defined in 4).

[0058] 23) The antibody according to 22), comprising:

[0059] Light chain, of which

[0060] (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

[0061] (b) the amino acid sequence of the light chain is or comprises an amino acid sequence having 1 or 2 amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 9, 10 or 11; and

[0062] Heavy chain, of which:

[0063] (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

[0064] (d) the amino acid sequence of the heavy chain is or comprises an amino acid sequence having 1 or 2 amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 12, 13, 14 or 15.

[0065] 24) The antibody according to 22) or 23), comprising

[0066] The two light chains are as follows:

[0067] (a) the amino acid sequence of the light chain is or comprises the amino acid sequence of SEQ ID NO: 10 or 11, or

[0068] (b) the amino acid sequence of the light chain is or comprises an amino acid sequence having 1 or 2 amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 10 or 11; and

[0069] The two heavy chains are as follows:

[0070] (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

[0071] (d) the amino acid sequence of the heavy chain is or comprises an amino acid sequence having 1 or 2 amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 12, 13, 14 or 15.

[0072] 25) A pharmaceutical composition comprising the protein or antibody according to any one of 1) to 24) and a pharmaceutically acceptable carrier.

[0073] 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.

[0074] 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 an early stage patient or in an early stage of the disease in said patient.

[0075] 28) The protein or antibody or pharmaceutical composition for the use according to 26) or 27), which comprises administering the protein or antibody parenterally to a mammal, preferably intravenously, subcutaneously or intraperitoneally.

[0076] 29) A nucleic acid molecule encoding the protein, polypeptide, light chain and / or heavy chain defined in any one of 1) to 24).

[0077] 30) A nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 25 or 26.

[0078] 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).

[0079] 32) A method for treating or preventing a neurodegenerative disease such as Alzheimer's disease, comprising administering to a mammal in need thereof a protein or antibody as defined in any one of 1) to 24) or a pharmaceutical composition according to 25).

[0080] The inventors surprisingly identified antibodies that can strongly induce human TREM2 / DAP-triggered SYK phosphorylation, which is a key TREM2-dependent effector pathway in AD. Antibody M07 increased SYK phosphorylation by up to 60-fold, while other antibody clones showed no or little activation, even though M07 binds to the same epitope on the TREM2 extracellular domain as H08 and M03.

[0081] Using phage display technology, the inventors obtained fully human anti-TREM2 antibodies, which were initially used for antigen binding screening. The selected fully human IgG1-LALA modified antibodies were used to determine binding affinity to the human TREM2 extracellular domain, activation of human TREM2 signaling (human TREM2 / DAP-dependent SYK phosphorylation), and most importantly, efficacy in a complex and relevant AD model using human brain cells differentiated from human induced pluripotent stem cells (hiPSCs).

[0082] LALA modification significantly reduces the effector function of Ig antibodies (especially IgG, and especially IgG1 antibodies), which is crucial for the study of human immunity and neuronal cells. The fully human backbone of the antibodies we generate has significant advantages over existing humanized antibodies based on clones identified in non-human animal immune systems (such as US2017240631A1 (Alector AL-002) and WO2020172450 A1 (Denali)), because the use of fully human antibodies is expected to reduce immune complications when repeatedly used for prophylaxis or treatment in humans.

[0083] Initially, the inventors identified a number of structurally similar and related antibodies (with heavy chain amino acid sequence homology of 90% or more) that all bind with high affinity (less than 10 -9 M) binds to the extracellular domain (ec) of human TREM2. To the inventors' surprise, they subsequently found that only one of these antibodies (antibody M07) could strongly induce SYK phosphorylation triggered by human TREM2 / DAP, which is a key TREM2-dependent effector pathway in AD. M07 increased SYK phosphorylation by up to 60-fold, while no or almost no activation was observed with the other antibody clones, even though M07 binds to the same epitope on the extracellular domain of TREM2 as H08 and M03.

[0084] The increase in human TREM2 / DAP-dependent pSYK levels induced by M07 is much greater than that observed with published and patented anti-human TREM2 agonist antibodies (especially any human anti-human TREM2 antibody). The hT2AB antibody disclosed by AMGEN (WO2022120373 A1) was used in a pSYK assay similar to ours, and the result was a 12-fold increase over baseline (Ellwanger et al, 2021). Alector introduced a variety of antibodies against human anti-TREM2 in its patent application (US2017240631 A1). Phosphorylation of SYK is shown at the protein level, and antibodies #22, #45, and #65 are reported to have an approximately 3-4-fold increase in human dendritic cells. In human macrophages, a 6-fold increase in SYK phosphorylation was observed. Denali presented several anti-hTREM2 antibodies in its patent application (WO2020172450A1), one of which is CL0020188. Compared to a control antibody, this antibody increased pSYK levels by 4-fold in HEK293 cells expressing TREM2.

[0085] In addition, the inventors have used an innovative, complex and relevant AD model that uses human brain cells differentiated from human induced pluripotent stem cells (hiPSCs). No anti-TREM2 antibodies known in the prior art have been analyzed in equally complex AD models using hiPSC-derived neurons and microglia. For example, WO2020172450 A1 (Denali) discloses a phagocytosis assay using hiPSC-derived microglia and amyloid-beta protein. However, the analysis was not performed in co-culture with neurons. Therefore, it is impossible to determine the benefit of amyloid-beta protein phagocytosis on neurons. AD can also be studied in other disease models, but these models have inherent limitations. Most researchers still conduct research in mouse models, but this is generally not predictive of the clinical efficacy of anti-AD drug candidates.

[0086] The present invention solves the above-mentioned problems and surprisingly provides fully human anti-human TREM2 antibodies that are able to strongly activate human TREM2-dependent human pSYK signaling, so that beneficial effects can be observed in relevant AD models in human brain cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] 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 under reducing buffer conditions. After reduction / boiling, the antibody is separated into light and heavy chains detected at 25 kDa and 50 kDa.

[0089] Figure 2 : ELISA-based EC50 values ​​for antibody 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.

[0090] Figure 3 : Cartoon of human TREM2, highlighting the epitope peptide within the stem region. Adapted from (Reifschneider et al, 2022). The amino acid sequence shown is that of SEQ ID NO: 27.

[0091] Figure 4 ELISA-based EC50 values ​​for binding of rat H01 and various human antibodies to epitope peptides derived from the human TREM2 stalk domain: H01 and M05 did not bind to peptide sequences present in the human TREM2 stalk region, while H08, M03, and M07 did bind with measured EC50 values ​​ranging from 329 to 832 pM. Mean of N=2 technical replicates.

[0092] Figure 5 : p-SYK signaling in HEK293-Flp-In hTREM2 / hDAP12 after antibody treatment (40 μg / ml): AlphaLISA assay for pSYK showed significant activation of the signaling pathway after addition of human anti-TREM2 antibodies H08, M03, and M07 and rat anti-TREM2 antibody H01. Antibody M07 had a much stronger activation effect than any other antibody tested (an average increase of 36-fold over baseline). H05 and isotype controls for rat or human antibodies did not result in activation of the signaling pathway. Shown are means + / - SEM; one-way Anova and Brown-Forsythe post hoc test were used; 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.

[0093] Figure 6: Titration of p-SYK signaling in HEK293-Flp-In hTREM2 / hDAP12 cells after antibody treatment: A. Titration curves of anti-TREM2 antibodies H01 and M07 show that M07 has a much stronger activation effect on pSYK signaling at different antibody concentrations. B. Titration curves of anti-TREM2 antibody M07 show that M07 has a strong activation effect on pSYK signaling at different antibody concentrations. Shown are mean + / - SEM; n = 9; EC50 = 4.387 nM; KD = 2.19 nM.

[0094] Figure 7 : An iPSC-derived microglia-neuron co-culture model of Alzheimer's disease detected neurite degeneration and dead nuclei using amyloid beta protein (Aβ). A. Timeline of co-culture of neurons and microglia. Amyloid beta protein was added to simulate Alzheimer's disease, and TREM2 antibodies were added in parallel to test neuroprotection. B. Representative images of co-cultures containing microglia (Iba1) and neurons (βIII tubulin) under control conditions (culture medium) and 5μM amyloid beta protein conditions. C. As the dose of amyloid beta protein increased, neurite degeneration and an increase in the number of dead nuclei were detected. Shown are mean values ​​+ / - SD; n = 6 technical replicates. D. Representative images of ICC staining of MAP2 and DAPI, and Cellprofiler analysis of the number of neurites and dead nuclei.

[0095] Figure 8: Anti-hTREM2 M07 antibody reduces amyloid β-dependent neurite degeneration and cell death in a microglia neuron co-culture system. A. Addition of amyloid β with anti-hTREM2 antibody M07 (0.6 μM Aβ+M07 antibody) significantly reduced neurite degeneration and the number of dead nuclei compared to addition of Aβ alone (0.6 μM Aβ) or addition of Aβ with isotype control antibody (0.6 μM Aβ+isotype antibody). B. Representative images of ICC staining of MAP2 and DAPI, and Cellprofiler analysis of the number of neurites and dead nuclei. Shown are mean values ​​+ / - SD; n = 6 technical replicates.

[0096] Figure 9 Titration of p-SYK signaling in iPSC-derived microglia after antibody treatment: Titration curves of the anti-TREM2 antibody M07 show the activation of pSYK signaling by M07 at different antibody concentrations. n = 1. Detailed Description of the Invention

[0098] The proteins and antibodies of the present invention are capable of binding to the extracellular (ec) domain of human TREM2, in particular to its stem region. In addition, the proteins and antibodies of the present invention have excellent ability to activate human TREM2, in particular 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 promising active drugs for treating neurodegenerative diseases (such as AD).

[0099] definition

[0100] As used herein, a protein refers to a monomeric protein, i.e., a protein comprising one subunit or polypeptide molecule; or a multimeric protein, i.e., a protein comprising two or more subunits or polypeptide molecules. An example of a monomeric protein is a single-chain antibody (scFv) or a single-domain antibody. Examples of multimeric proteins include an Ig Fab fragment, an Ig F(ab)2 fragment, or an immunoglobulin that can be a tetramer. Proteins may have modifications on the amino acid residue side chains, such as those described below for polypeptides.

[0101] A polypeptide refers to a polypeptide molecule, not a sequence stretch or portion of a molecule. The amino acid residue side chains of a polypeptide may contain chemical modifications on 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 include glycosylation (e.g., glycosylation of heavy chain asparagine residues), side chain oxidation, and the addition (linkage) of markers, tags, labels, or other proteins or polypeptides. Modifications of amino acid residue side chains are not limited to small molecule components, but may also be other polypeptides or protein domains.

[0102] The term "region" of a protein or polypeptide refers to a domain of the protein or polypeptide, i.e., a stretch (or fragment) of the amino acid sequence of the polypeptide or protein. The polypeptide or protein comprises the region as a stretch or fragment of the amino acid sequence of the polypeptide or protein. The protein or polypeptide comprises at least one more amino acid residue than the region, stretch or fragment thereof. The terms "region" and "domain" are used interchangeably herein.

[0103] The term "amino acid sequence" refers to the primary structure of a polypeptide, region, domain, fragment, or stretch. Amino acid sequences are often defined by reference to a reference sequence identified by a SEQ ID NO. Unless a subrange of a reference sequence is specified, a reference sequence refers to the entire amino acid sequence of that reference sequence.

[0104] A (polypeptide) extension or fragment refers to a plurality of (contiguous) amino acid residues within a polypeptide molecule, wherein the polypeptide molecule comprises more amino acid residues than the extension or fragment.

[0105] The term "antibody" refers to a protein with an immunoglobulin fold that specifically binds to an antigen through its variable region, here to the extracellular (ec) domain of human TREM2, in particular to the epitope of SEQ ID NO: 16. The term covers polyclonal and monoclonal antibodies, single domain antibodies, heavy chain antibodies and single chain antibodies. The term "antibody" as used herein also includes Ig fragments that retain binding specificity through their variable regions, including but not limited to Fab, F(ab')2, scFv and bivalent scFv. Antibodies may comprise light chains, which may be divided into kappa chains or lambda chains. Antibodies may comprise heavy chains, which may be divided into gamma chains, μ chains, alpha chains, delta chains or epsilon chains, which in turn define the immunoglobulin classes: IgG, IgM, IgA, IgD and IgE, respectively. Preferred antibodies herein are immunoglobulins (Ig). The antibodies and Ig used in the present invention are preferably monoclonal and further fully human.

[0106] Immunoglobulin (Ig) is a globulin type protein (naturally present in serum or other body fluids) that has antibody activity, that is, it specifically binds to antigens through its variable region. The Ig molecule contains two light chains (L) and two heavy chains (H) (or polypeptide chains or subunits), which are linked together by disulfide bonds. Ig can form oligomeric structures, for example, IgM is a pentameric Ig. Based on the antigenicity and structural differences of the H chain, Ig can be divided into five categories: IgG, IgM, IgA, IgD and IgE. The Ig region is a structural domain of Ig. The structural domains of Ig are variable domains and constant domains. The Ig heavy chain has a variable domain (or region) and three different constant domains, called C H 1. C H 2 and C H 3. Ig light chains have two domains: a variable domain and a constant domain.

[0107] 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).

[0108] The term "heavy chain" or "antibody heavy chain" refers to an antibody comprising an Ig heavy chain (HC) variable region and at least one Ig constant region (usually comprising at least C H Preferably, a heavy chain or antibody heavy chain refers to a polypeptide comprising an Ig heavy chain variable region (or domain) and three Ig constant regions (or domains). H 1. C H 2 and C H 3's (complete) Ig heavy chain.

[0109] Proteins and antibodies of the present invention

[0110] The protein of the present invention comprises or consists of an (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 1 or 2 amino acid residues substituted compared to SEQ ID NO: 2. Alternatively, the protein of the present invention comprises or consists of an (Ig) light 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 1 or 2 amino acid residues substituted compared to SEQ ID NO: 1. An example of such a protein is a single domain antibody or a nanobody. A single domain antibody is composed of the variable domains of a heavy chain or a light chain, preferably of the variable domains of a heavy chain.

[0111] In another embodiment, the protein of the present invention can be a heavy chain antibody. In this embodiment, the protein generally comprises (one or preferably two) heavy chains or consists of (one or preferably two) heavy chains, each heavy chain 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 1 or 2 amino acid residue substitutions compared to SEQ ID NO:2. In addition to the variable region, the heavy chain preferably also comprises at least one constant region (domain), such as two constant regions or five constant regions. The protein may comprise two (preferably identical) subunits or polypeptides 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. An example of such a heavy chain antibody is V H H antibody (camelid). Alternatively, the protein may comprise or consist of two (preferably identical) subunits or polypeptides, each comprising the variable region and five heavy chain constant regions. An example is V NAR Antibodies (Cartilaginous Fish Type). In all of these embodiments, the CDRs of the heavy chain variable domain are as follows.

[0112] In the preferred embodiment described below, the protein of the present invention comprises a light chain variable region and a heavy chain variable region.

[0113] Therefore, 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 1 or 2 amino acid residues substituted compared to SEQ ID NO: 1, and

[0114] 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 1 or 2 amino acid residues substituted compared to SEQ ID NO: 2.

[0115] Preferably, the protein comprises an antibody light chain and an antibody heavy chain. The antibody light chain and heavy chain are polypeptides comprising a light chain variable region as defined above and a heavy chain variable region as defined above, respectively. The variable region (light chain and heavy chain) comprises three hypervariable regions, commonly referred to as complementarity determining regions (CDRs), numbered CDR1 to CDR3 from the N-terminus to the C-terminus. The CDRs are preceded and separated by regions of less variability, commonly referred to as framework regions (FRs), numbered FR1 to FR4 from the N-terminus to the C-terminus of the chain.

[0116] The light chain (LC) variable region may comprise, preferably in CDR-L3, a fragment of the amino acid sequence of SEQ ID NO: 5 or a fragment 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 may comprise, preferably in CDR-H3, a fragment of the amino acid sequence of SEQ ID NO: 8 or a fragment of an amino acid sequence having one amino acid residue substitution compared to the amino acid sequence of SEQ ID NO: 8. In this embodiment and the following embodiments, these CDRs and chains preferably do not have such amino acid substitutions.

[0117] Preferably, in the N-terminal to C-terminal direction, the light chain variable region comprises, preferably in CDRs L1 and L3, the following amino acid stretch:

[0118] a fragment of the amino acid sequence of SEQ ID NO: 3 (CDR-L1), and

[0119] the amino acid sequence of SEQ ID NO: 5 or a fragment of an amino acid sequence having one amino acid residue substituted compared to the amino acid sequence of SEQ ID NO: 5 (CDR-L3); and

[0120] In the N-terminal to C-terminal direction, the heavy chain (HC) variable region comprises, preferably in CDRs H2 and H3, the following amino acid stretch:

[0121] a fragment of the amino acid sequence of SEQ ID NO: 7 (CDR-H2), and

[0122] The amino acid sequence of SEQ ID NO: 8 or a fragment of the amino acid sequence having one amino acid residue substitution compared to the amino acid sequence of SEQ ID NO: 8 (CDR-H3).

[0123] More preferably, in N-terminal to C-terminal direction, the light chain (LC) variable region comprises, preferably in CDRs L1 to L3, the following amino acid stretch:

[0124] a fragment of the amino acid sequence of SEQ ID NO: 3 (CDR-L1),

[0125] a fragment of the amino acid sequence of SEQ ID NO: 4 (CDR-L2), and

[0126] the amino acid sequence of SEQ ID NO: 5 or a fragment of an amino acid sequence having one amino acid residue substituted compared to the amino acid sequence of SEQ ID NO: 5 (CDR-L3); and

[0127] In the N-terminal to C-terminal direction, the heavy chain (HC) variable region comprises, preferably in CDRs H1 to H3, the following amino acid sequence segment:

[0128] a fragment of the amino acid sequence of SEQ ID NO:6 (CDR-H1),

[0129] a fragment of the amino acid sequence of SEQ ID NO:7 (CDR-H2),

[0130] and the amino acid sequence of SEQ ID NO: 8 or a fragment (CDR-H3) of the amino acid sequence having one amino acid residue substituted compared to the amino acid sequence of SEQ ID NO: 8.

[0131] Preferably, the CDRs do not have amino acid residue substitutions compared to a given reference sequence. As mentioned above, these segments (CDRs) are usually not present continuously in the chain or region, but are separated by framework regions, as shown in SEQ ID NOs: 1 and 2.

[0132] 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 reverse order from N-terminus to C-terminus. The variable region is as defined above, and the CDRs are preferably also as defined above. scFv generally does not contain a constant region.

[0133] However, in addition to the variable region, the light chain and heavy chain of the protein of the present invention generally also contain one or more Ig constant domains, or all the domains of the respective constant regions of (complete) Ig light chain and heavy chain. Thus, the protein may comprise an antibody light chain and an antibody heavy chain, wherein:

[0134] The light chain comprises a light 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 1 or 2 amino acid residues substituted compared to SEQ ID NO: 1, and

[0135] 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 in which there is no one or no (i.e., no) amino acid residue substitution are preferred. The CDRs are preferably as described above.

[0136] Since the light chain of a natural immunoglobulin contains one Ig constant domain or region, the light chain of the protein of the present invention preferably contains a constant region (especially one). The light chain can be a kappa light chain or a lambda light chain, with the former being preferred. Since the heavy chain of a natural immunoglobulin contains three constant domains (usually referred to as C H1. C H 2 and C H 3), therefore the heavy chain of the protein of the present invention generally comprises at least one constant domain, preferably C H 1 domain. 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 present invention comprises three (Ig) constant domains, such as C H 1. C H 2 and C H 3 domains (from N-terminus to C-terminus).

[0137] Therefore, the protein of the present invention can be an Ig Fab fragment, that is, it can contain a light chain and a heavy chain; the former contains a polypeptide containing a light chain (κ or λ) or consists of the polypeptide, and the latter contains a heavy chain variable region and an (Ig) heavy chain constant region or one (Ig) heavy chain constant region (usually CH1) or consists of the foregoing. The light chain and heavy chain are usually covalently linked by a disulfide bond.

[0138] Alternatively, the protein of the present invention may be a F(ab)2 fragment of Ig, which comprises two Fab fragments linked by one or more disulfide bonds.

[0139] The protein of the present invention may comprise an Ig light chain and a (i.e., complete or full) Ig heavy chain. A complete heavy chain means that the heavy chain comprises three Ig constant domains in addition to the variable domains. Thus, the heavy chain preferably comprises three constant domains C in addition to the variable domains. 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 (usually identical) Ig light chains and two usually (but not necessarily identical) Ig heavy chains. In IgG, the protein can be IgG1, IgG2, IgG3, or IgG4, depending on the heavy chain. It is preferably IgG1, IgG2, or IgG3, more preferably IgG1, such as clone M07 described and used in the Examples.

[0140] Preferred proteins or antibodies of the present invention comprise a light chain, wherein

[0141] (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 11; or

[0142] (b) the amino acid sequence of the light chain is or comprises an amino acid sequence based on a kappa-1 light chain backbone having 1 or 2 amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 9 or 10 (preferably SEQ ID NO: 10); or

[0143] (c) The amino acid sequence of the light chain may also be or may include an amino acid sequence based on a lambda light chain backbone having 1 or 2 amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 11.

[0144] In the above embodiments, in particular embodiments (a), (b) and (c), the protein or antibody preferably further comprises a heavy chain, wherein:

[0145] (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 the amino acid sequence of SEQ ID NO: 13 or 15, or

[0146] (e) The amino acid sequence of the heavy chain is or comprises an amino acid sequence having 1 or 2 amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 12, 13, 14 or 15 (preferably the amino acid sequence of SEQ ID NO: 13 or 15).

[0147] In the above embodiment, further preferably, 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. Therefore, the protein or antibody may comprise or consist of:

[0148] Two light chains,

[0149] (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 the amino acid sequence of SEQ ID NO: 10; or

[0150] (b) the amino acid sequence of the light chain is or comprises an amino acid sequence having 1 or 2 amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 9, 10 or 11 (preferably the amino acid sequence of SEQ ID NO: 10),

[0151] and two heavy chains, of which:

[0152] (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 the amino acid sequence of SEQ ID NO: 13 or 15, or

[0153] (d) The amino acid sequence of the heavy chain is or comprises an amino acid sequence having 1 or 2 amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 12, 13, 14 or 15 (preferably the amino acid sequence of SEQ ID NO: 13 or 15).

[0154] The two light chains and the two heavy chains preferably have the same amino acid sequence. Embodiments with one or no amino acid residue substitutions are preferred. The CDRs are as defined above.

[0155] A further embodiment of the protein or antibody of the present invention is 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 (preferably the latter) or 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).

[0156] 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 chain may have 1 or 2 amino acid residue substitutions in the sequence portion outside of the heavy chain CDRs defined above.

[0157] 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 chain may have 1 or 2 amino acid residue substitutions in the sequence portion outside of the heavy chain CDRs defined above.

[0158] As defined above, the protein of the present invention is preferably an antibody, more preferably an Ig, comprising a light chain variable region (Ig) as defined above and a heavy chain variable region (Ig) as defined above. The antibody preferably comprises a light chain, wherein:

[0159] (a) the amino acid sequence of the light chain is or comprises the amino acid sequence of SEQ ID NO: 9 or 10, or

[0160] (b) the amino acid sequence of the light chain is or comprises an amino acid sequence having 1 or 2 amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 9 or 10 (preferably SEQ ID NO: 10), and

[0161] Heavy chain, of which:

[0162] (c) the amino acid sequence of the heavy chain is or comprises SEQ ID NO: 12, 13, 14 or 15, preferably SEQ ID NO: 13 or 15, or

[0163] (d) the amino acid sequence of the heavy chain is or comprises an amino acid sequence having 1 or 2 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). Preferred embodiments are similar to those given above for the protein of the present invention.

[0164] Alternatively, the protein of the present invention may be an antibody, preferably an Ig, comprising a light chain variable region (Ig) as defined above and a heavy chain variable region (Ig) as defined above. The antibody preferably comprises a light chain, wherein:

[0165] (a) the amino acid sequence of the light chain is or comprises the amino acid sequence of SEQ ID NO: 11, or

[0166] (b) the amino acid sequence of the light chain is or comprises an amino acid sequence having 1 or 2 amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 11, and

[0167] Heavy chain, of which:

[0168] (c) the amino acid sequence of the heavy chain is or comprises SEQ ID NO: 12, 13, 14 or 15, preferably SEQ ID NO: 13 or 15, or

[0169] (d) the amino acid sequence of the heavy chain is or comprises an amino acid sequence having 1 or 2 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). Preferred embodiments are similar to those given above for the protein of the present invention.

[0170] The protein of the present invention or the polypeptide of the protein may be a fusion protein. The fusion protein comprises a polypeptide comprising any of the above-mentioned chains, regions or domains as a first fragment, and a second fragment (preferably located at the C-terminus of the first fragment). The second fusion protein fragment can provide additional functions for the protein or antibody, such as binding to a receptor (not 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 fragment, wherein a fusion protein comprising IgG is preferred. Preferably, the fusion protein is a fusion protein of any such Ig, wherein the fusion protein comprises two Ig light chains and two heavy chains, wherein the polypeptide of at least one (preferably two) heavy chain is a fusion protein comprising a second fusion protein fragment.

[0171] The protein or antibody of the present invention may have further modifications to promote protein or antibody transport to the subject's brain. For parenteral administration, such as intravenous administration, the protein or antibody may have modifications that promote its passage through the blood-brain barrier (BBB). It is known in the art that proteins or antibodies pass through the BBB and are delivered to the brain. For review, see Pardridge (2015), Expert Opinion on Drug Delivery, 12: 2, 207-222 (DOI: 10.1517 / 17425247.2014.952627). Established solutions utilize the binding of the protein to be delivered to the human transferrin receptor (hTfR). Denali Therapeutics' WO2018152285 and WO2018152326 and Kariolis et al. (Science Translational Medicine, Vol. 12, No. 545; DOI: 10.1126 / scitranslmed.aay1359) describe amino acid residue substitutions in the antibody heavy chain CH3 region to achieve specific binding to TfR. WO2014033074 and WO2015101588 (Roche) describe BBB shuttling modules comprising a brain effector entity, a linker, and a monovalent binding entity that binds to a BBB receptor such as TfR.

[0172] Therefore, the protein or antibody of the present invention may comprise a binding domain capable of binding to hTfR1 to allow the protein to cross the BBB. Preferably, the protein or antibody comprises a heavy chain with a modified CH3 domain that can bind to hTfR1. The heavy chain CH3 domain can be modified as described in WO2018152285, WO2018152326, or Kariolis et al. Alternatively, for similar purposes, the protein of the present invention may comprise a fusion protein as described above.

[0173] In order to treat or prevent neurological disorders or diseases, the protein or antibody of the present invention may have reduced Fc effector function to prevent adverse or unnecessary effects on the immune system. To this end, the protein of the present invention may lack the CH2 and CH3 domains of the antibody heavy chain. However, preferably, the protein comprises a heavy chain containing CH2 and CH3 domains, but the CH2 domain has one or more amino acid residue substitutions that reduce the Fc effector function. An example of such a mutation is the well-known L234A and L235A double mutation, particularly described in the review by Wang et al (Protein Cell 2018, 9 (1), 63-73; doi.org / 10.1007 / s13238-017-0473-8), which is used to reduce the binding of the protein to Fc receptors, thereby reducing effector function. Corresponding mutations are generally preferred for all embodiments of the present invention in which the protein comprises a heavy chain with a CH2 domain. Each of the sequences in SEQ ID NOs: 12 to 15 contains this double mutation at 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 also have a P to G substitution at a position corresponding to position 334 of SEQ ID NOs: 13 or 15 (resulting in a LALA-PG triple mutation) to prevent or reduce binding of the protein to Fc receptors. This mutation is described in detail in WO2012130831 A1.

[0174] Nucleic acid molecules

[0175] The present invention provides a nucleic acid molecule encoding a protein, polypeptide, light chain and / or heavy chain as defined above. The nucleic acid molecule can be a plasmid or vector comprising one or more constructs or cistrons encoding the protein, polypeptide, light chain and / or heavy chain, and gene regulatory elements for expressing them in suitable cells. In embodiments where the protein comprises two or more different polypeptide molecules, the plasmid or vector can comprise two or more constructs or cistrons, one for each polypeptide to be expressed. Alternatively, the present invention provides a kit for two nucleic acid molecules, one nucleic acid molecule encoding the first polypeptide of the protein of the present invention, and another nucleic acid molecule encoding the second polypeptide. The nucleic acid molecule can comprise the nucleotide sequence of SEQ ID NO:25 or 26.

[0176] cell

[0177] The present invention provides a cell, preferably a eukaryotic cell, comprising a protein of the present invention or a nucleic acid molecule of the present invention, such as those described above. The cell is preferably used to produce and express the protein of the present invention. For use in humans, the cell is preferably a human cell so as to impart human-like glycosylation to the protein or antibody. However, the cell may have genetically engineered glycosylation machinery to impart the desired glycosylation to the protein.

[0178] Protein or antibody production

[0179] Protein or antibody of the present invention can be expressed by the nucleic acid molecule encoding it in a suitable expression system, as is generally known in the art. For heterologous oligomeric proteins (e.g., immunoglobulins), light and heavy chains can be expressed by bicistronic plasmids in the same cell (preferably eukaryotic cells), as described in the Examples. Light and heavy chains can be expressed in the form of containing an N-terminal leader sequence, thereby guiding the secretion of the leader sequence. The leader sequence should be able to be excised after secretion or in the cell secretory pathway, and then the light and heavy chains can be assembled to form an oligomeric protein, preferably without a leader sequence. For human applications, the cell system used for expression is preferably human, so as to give the protein or antibody with human-like glycosylation as needed.

[0180] Pharmaceutical compositions and preparations

[0181] The present invention also provides pharmaceutical compositions comprising the proteins or antibodies of the present invention. Such 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. The preferred solvent is water, which may additionally contain excipients.

[0182] Examples of excipients include 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 by mixing proteins or antibodies in a manner known to those skilled in the art, such as by conventional mixing, dissolution, or lyophilization processes.

[0183] For desired parenteral administration, the pharmaceutical composition can be administered as a solution, typically by injection or infusion. For injection, a preparation can be formulated by dissolving, suspending or emulsifying the protein or antibody in an aqueous solvent, which may contain conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives. In some embodiments, the compound can be formulated in an aqueous solution, such as in a physiologically compatible buffer, such as a physiological saline buffer. Injectable preparations can exist in unit dosage form, such as in an ampoule or multidose container, with or without the addition of a preservative. The composition can be in the form of a suspension, solution or emulsion in an oily or aqueous vehicle.

[0184] However, the pharmaceutical composition may alternatively be a solid composition, for example in lyophilized form. The solid form may be reconstituted with a suitable solution or vehicle before use, as described above.

[0185] Typically, pharmaceutical compositions for in vivo administration are sterile. Sterilization can be performed according to methods known in the art, such as sterile filtration of solutions or by irradiation.

[0186] Treatment or prevention

[0187] The protein or antibody of the present invention is used for treatment or prevention. The disease or condition to be prevented or treated is a neurodegenerative disease, such as Alzheimer's disease. In order to treat or prevent the patient's disease or condition, 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.

[0188] In the treatment or prevention of neurodegenerative diseases, the patient to whom the protein or antibody is administered is preferably a patient in the early stages of a neurodegenerative disease (e.g., Alzheimer's disease), because the therapeutic effect in the early stages of the disease is expected to be higher than in the late stages. Therefore, treatment in the early stages prevents or inhibits the progression of the disease to a more advanced or more severe stage.

[0189] The stage of a neurodegenerative disease, such as Alzheimer's disease, can be determined using established methods. One such method is the Mini-Mental State Examination (MMSE) or Folstein test, which is based on a 30-point questionnaire and is widely used in clinical and research settings to measure cognitive impairment. The MMSE can be a version described in 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 screening for dementia. It is also used to assess the severity and progression of cognitive impairment and to track an individual's cognitive changes over time. Therefore, the MMSE is an effective method for documenting an individual's response to treatment. A score of 24 or above (out of 30) indicates normal cognition. Scores below this value may indicate severe (≤9 points), moderate (10-18 points), or mild (19-23 points) cognitive impairment. Raw scores may also need to be adjusted for education and age. Low to very low scores are strongly associated with the presence of dementia, although other psychiatric conditions can also cause abnormal results on the MMSE test. The presence of purely physical problems may interfere with comprehension without proper attention; for example, a patient may be physically unable to hear or read instructions correctly, or may have motor impairments that affect writing and drawing skills.

[0190] Another method used to determine the stage of neurodegenerative diseases such as Alzheimer's disease is the CDR global score (Clinical Dementia Rating). The CDR is a global rating scale used to stage patients diagnosed with dementia. The CDR assesses cognitive, behavioral and functional aspects of Alzheimer's disease and other dementias. It is not a mental status examination or checklist, but rather the assessor makes a judgment on six categories based on all available information. The CDR's scoring system relies heavily on memory scores, but the CDR has good inter-rater reliability when staging dementia. The CDR is a scale widely used in Alzheimer's disease centers and dementia research. The assessment of the CDR is based on semi-structured interviews with the subject and caregiver (informant) and the clinical judgment of the clinician. The calculation of the CDR is based on tests in six different cognitive and behavioral areas, such as memory, orientation, judgment and problem solving, community affairs, family and hobby performance, and personal care. CDR is based on a scale of 0-3: no dementia (CDR=0), suspected dementia (CDR=0.5), MCI (CDR=1), moderate cognitive impairment (CDR=2), and severe cognitive impairment (CDR=3). Two groups of questions were asked, one to the information provider and the other to the subject. The information provider's group of questions included questions about the subject's memory, the subject's judgment and problem-solving ability, the subject's community affairs, the subject's family life and hobbies, and personal issues related to the subject. The subject's group included questions related to memory, questions related to orientation, and questions about judgment and problem-solving ability. This method is described in Handbook of Clinical Neurology, Volume 167, 2019, pages 89-104, Chapter 6 - Cognitive and neuropsychological examination of the elderly.

[0191] Therefore, the present invention provides a protein or antibody for treating or preventing an early stage neurodegenerative disease such as Alzheimer's disease in a patient, preferably as follows:

[0192] - the patient has a cognitive impairment score of 23 or less, preferably a score of 10 to 23, more preferably a score of 19 to 23 on the Mini-Mental State Examination (MMSE) test, or

[0193] - The patient has a cognitive impairment score of 0.5 or higher and 2 or lower on the Clinical Dementia Rating Scale (CDR Global Score).

[0194] The protein or antibody is preferably administered parenterally. Preferred administration routes include intravenous, subcutaneous, and intraperitoneal administration.

[0195] The protein or antibody can be administered to a subject in a therapeutically effective amount or dosage. The dosage range for each administration is from about 0.01 mg / kg to about 500 mg / kg, or from about 0.1 mg / kg to about 200 mg / kg, or from about 1 mg / kg to about 100 mg / kg, or from about 10 mg / kg to about 50 mg / kg. However, dosage can vary according to various factors, including the frequency of administration, the selected route of administration, the formulation of the composition, patient response, severity of disease, and the judgment of the prescribing physician. Dosage can increase or decrease over time according to the needs of individual patients. The patient can initially receive a low dose, which is then increased to an effective dose that the patient can tolerate. The determination of the effective dose is within the capabilities of those skilled in the art.

[0196] The protein or antibody may be administered once every 1 to 6 weeks, preferably once every 2 to 4 weeks. Example

[0197] The present invention is not limited to the examples described below.

[0198] I. Materials and Methods

[0199] Identification of antibody clones

[0200] A portion of the human TREM-2 extracellular domain was used as a coating antigen for phage display screening of a phage library containing a complete repertoire of human antibody sequences (with a diversity of at least 5 × 10 10The researchers used a phage array to generate a complete human IgG1 heavy chain, comprising a sequence of 100 heavy chain IgG1s, each containing 100 heavy chain IgG1s, each containing 100 heavy chain IgG1s. The sequence was then assembled into a 100 light chain IgG1 sequence, which was sequenced using a 100 light chain IgG1 sequence. The sequence was then assembled into a 100 light chain IgG1 sequence, each containing 100 heavy chain IgG1s ... A restriction site for AvrII was added to the beginning of the heavy chain gene, and a restriction site for BstZ171 was added to the end. Similarly, a restriction site for EcoRV was added to the beginning of the light chain gene, and a restriction site for PacI was added to the end. These DNA fragments were then used to insert the heavy and light chain genes into the corresponding multiple cloning sites (AvrII-BstZ171 and EcoRV-PacI) of the bicistronic vector pCHOv1, which is a Freedom TS This plasmid is part of the CHO-S kit (Thermo Fisher catalog number: A13696-01). The resulting vector is generated by GeneArt in Regensburg, undergoes comprehensive quality control, and is column-purified. This plasmid allows the expression of two different proteins in the same cell under the control of two hybrid modified cytomegalovirus (CMV) promoters. In particular, it can be used to express both heavy and light antibody chains in a single cell, such as Chinese hamster ovary (CHO) cells.

[0201] An IgG1-LALA isotype control was created using the heavy and light chain sequences of an anti-green fluorescent protein (GFP) antibody.

[0202] Transform E. coli DH5α with 1 ng of plasmid DNA. Streak the transformed bacteria onto agar plates containing 50 μg / mL kanamycin and incubate overnight at 37°C. The next day, pick colonies and culture them in LB medium containing 50 μg / mL kanamycin and incubate overnight at 37°C. Perform Maxi Prep using NucleoBond Xtra Maxi EF, a Maxi kit for obtaining endotoxin-free plasmid DNA (Machery Nagel, Cat. No. 740424.50).

[0203] The coding sequences for the light and heavy chains of clone M07 are given in SEQ ID NOs: 25 and 26, respectively.

[0204] The term "clone M07" refers to an IgG1 antibody of the present invention that is expressed from the coding sequences encoding the kappa-1 light chain of SEQ ID NO: 9 and the heavy chain of SEQ ID NO: 12. During secretion, the N-terminal leader sequence 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 were performed using antibody M07 comprising two of the mature light chains and two of the heavy chains. The heavy chain polypeptides of SEQ ID NOs: 12 and 13 were expressed in C H The 3 domains contain a binding fragment (TFN) for binding to human transferrin receptor (hTfR). SEQ ID NOs: 14 and 15 are heavy chains corresponding to SEQ ID NOs: 12 and 13, but in C H The 3 heavy chains lack the binding fragment (TFN) and have the corresponding wild-type sequence instead of TFN. All heavy chains contain the LALA double mutation.

[0205] HEK293-Flp-In cell culture

[0206] HEK293-Flp-In cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing GlutaMAX I and supplemented with 10% (v / v) fetal calf serum (FCS), 1% (v / v) penicillin / streptomycin, and 0.4% (v / v) hygromycin B.

[0207] CHO-S kits and gene transfer, selection, and purification

[0208] CHO-S cells (as Freedom TSCHO-S test kit (part of Thermo Fisher catalog number A13696-01) is commercially available) thawed and grown in cell culture using recommended culture medium according to the manufacturer's instructions. According to the manufacturer's instructions, the pCHOv1 plasmid containing the respective heavy and light chain sequences under the control of two different hybrid CMV promoters is subjected to gene transfer. After the successfully transfected CHO-S cells are positively selected, the cells are cultured under selective pressure according to the recommendation of the manufacturer's instructions. Supernatants are collected from time to time and prepared for antibody purification. In brief, the supernatant (~250 ml) is diluted with binding buffer (20 mM sodium phosphate, pH=7.0) in a ratio of 1:4 and then loaded onto 1 ml Protein A column (Cytiva catalog number 17040201) with the help of a peristaltic pump. Next, the column was 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). The protein-containing fractions (detected by Nano-Drop) were combined and dialyzed overnight with 1x PBS. The dialyzed sample was concentrated and the concentration was determined by Nano-Drop.

[0209] Differentiation of hiPSC-derived microglia (hiMGL)

[0210] hiMGLs were differentiated from iPSCs as described by 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 to obtain approximately 30 small colonies per well. Cells were fed with 2 ml of HemA medium (HPC Differentiation Kit, StemCell Technologies) on day 0 and half-fed with 1 ml on day 2. The medium was changed to 2 ml of HemB on day 3, half-fed on days 5 and 7, and topped with 1 ml on day 10. Non-adherent HPCs were harvested on day 12 and frozen or continued for microglial differentiation. HPCs were frozen in BamBanker (Wako) at 1 million cells per ml. They were then thawed directly onto GelTrex-coated 6-well plates, and 1 million cells were evenly distributed across 6 wells containing 2 ml of iMGL medium freshly supplemented with 25 ng / ml M-CSF, 100 ng / ml IL-34, and 50 ng / ml TGF-β. 1 ml of medium was added every other day. During microglial differentiation, cells were passaged 1:2 every 6–8 days, depending on confluence. A very similar differentiation protocol has been recently published (McQuade et al, 2018). We did not use CD200 and CX3CL1, as they appear to have no effect on hiMGL gene expression, as determined by Nanostring analysis (data not shown). Experiments using hiMGL were performed on day 16 of differentiation.

[0211] Differentiation of hiPSC-derived cortical neurons

[0212] For the differentiation of cortical neurons, neural progenitor cells (NPCs) were first differentiated from hiPSCs, and then NPCs were further differentiated into cortical neurons according to the method described by Gregg et al., 2016 and improved. Specifically, hiPSCs were grown on GelTrex-coated plates to 100% confluence. Neural induction (NI) medium containing 10 μM SB431542 and 250 nM LDN193189 was added to the cells (day 0 in vitro (DIV0)) and maintained for 12 days. At ~DIV2 and ~DIV8, Accutase was used and ROCK inhibitor was used to separate the cells into single cells. At DIV2, 260,000 cells in each 12 wells were passaged in a GelTrex-coated 12-well plate. At DIV8, 200 μl of 30 million cells / ml cell suspension was inoculated into 1.1 cm 26-well plates coated with poly-L-ornithine and laminin (PLO / lam). During neural induction, rosettes should become visible. Starting from DIV12, NI medium was replaced with neural maintenance (NM) medium and 20ng / ml bFGF was added during the first four days. At DIV22, rosettes were isolated using STEMdiff neural rosette selection reagent (STEMCELL Techn.) and seeded into NM medium containing bFGF in 6-well plates coated with PLO / lam. At DIV29, rosettes were passaged using accutase. At DIV39, NPCs were frozen in neural progenitor cell freezing medium (STEMCELL Techn.).

[0213] For cortical neuronal differentiation, NPCs were thawed in NM medium containing bFGF in PLO / lam-coated 6-well plates. 1.5 million NPCs were differentiated in maturation medium consisting of NB / B27 medium (Neurobasal medium, penicillin-streptomycin, 1x B27 supplement) and 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) in PLO / lam-coated 6-well plates. After one week, 50,000 cells were passaged into 96-well plates using Accutase and ROCK inhibitor. Half of the medium was replaced every two to three days. After two weeks of neuronal differentiation, microglia were co-cultured. An IntegraASSIST PLUS pipetting robot was used for coating, cell seeding, and medium changes.

[0214] Neuron-microglia co-culture

[0215] Two days before adding microglia to neurons, PD0332991 was removed from the maturation medium. HiMGLs on 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. After co-culturing neurons and microglia for two weeks, amyloid-β was added.

[0216] Amyloid β processing

[0217] Human amyloid β (1-42) (rPeptide, A-1170-02) or scrambled human amyloid β (1-42) (rPeptide, A-1004-1) were incubated overnight at 37°C for aggregation. Every 3 to 4 days, the specified concentration of amyloid β was added to the co-culture system by replacing half of the culture medium. Anti-TREM2 antibodies were added simultaneously with amyloid β for 1 to 2 weeks.

[0218] p-SykAlphaLISA

[0219] 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 culture medium in a 96-well plate at a density of 50,000 cells / well and incubated overnight in a cell culture incubator at 37 ° C. The next day, the culture medium was removed and 50 μl of antibody diluted in culture medium was added to the cells. After incubation at 37 ° C for 5 minutes, the treatment solution was removed and the cells were lysed on a plate shaker (about 350 rpm) with 50 μl of lysis buffer supplemented with phosphatase inhibitors (VWR) for 10 minutes. Then, CLARIOstar Plus After analysis by Plate Reader (BMG Labtech), 30 μl of lysate was used for further incubation steps with acceptor and donor beads (1 hour each).

[0220] For microglia, 96-well plates were pre-coated with antibodies overnight at 4°C and microglia were added at a density of 60,000 cells / well the next day. After incubation at 37°C for 10 minutes, the treatment solution was removed and the cells were lysed with 50 μl of lysis buffer supplemented with phosphatase inhibitors (VWR) on a shaker (approximately 350 rpm) for 10 minutes. Plus After analysis by Plate Reader (BMG Labtech), 30 μl of lysate was used for further incubation steps with acceptor and donor beads (1 hour each).

[0221] ELISA-based binding assay for anti-TREM2 antibodies

[0222] Anti-TREM2 antibodies binding to the human or mouse peptide fragments of the TREM2 extracellular domain (ecTREM2) or the TREM2 stem region were quantified in ELISA assays. All operations were performed at room temperature and incubated on a microtiter plate shaker. ELISA plates were incubated with 60 μl / well (final concentration 0.5 μg / ml) containing the corresponding ligand (ecTREM2 ( Diagnostika, 11084-H08H) or human or mouse TREM2 peptide fragment) was coated with coating buffer (NaHCO3) for 1 hour. The coated plate was washed three times with PBS-T (PBS, 0.1% Tween-20), blocked with 100 μl / well blocking solution (PBS-T, 3% milk powder) for 1 hour, and then washed again. The antibody was pre-diluted in PBS at a ratio of 1:10 and set to a concentration of 1 μg / ml. A dilution step was used to perform a serial dilution of 1:3 and 1:10. 50 μl / well of the diluted AB was transferred to a blocked ELISA plate and incubated for 1 hour. The plate was washed three times with PBS-T and then incubated with anti-human-Strep-POD (Jackson Immunoresearch, #109-035-098) diluted in PBS-T at 1:10000 for 1 hour. After washing three times, bound POD was detected by incubation with 100 μl / well of TMB substrate (Thermo Scientific, #34029) until a maximum optical density (OD) of approximately 1 to 2 was reached. Finally, the colorimetric reaction was stopped with 100 μl / well stop solution (1 M H2SO4), and the OD was measured at 450 nm with a reference wavelength of 595 nm in a plate reader (SpectraMax i3xl).

[0223] Immunocytochemistry (ICC) and imaging

[0224] Cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature and washed three times with PBS. For permeabilization and blocking, cells were incubated with PBS containing 3% normal goat serum (Abcam) and 0.3% Triton X-100 for 1 hour at room temperature and then washed three times. The following primary antibodies were incubated overnight at 4°C: rabbit anti-synaptophysin 1 (1:500, Synptic Systems, #106103) and mouse anti-MAP2 (1:1500, Sigma Aldrich, #M9942). After washing three times, 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 washing three times with PBS, cells were stained with 10 μM DAPI. After washing three times with PBS, cells were imaged using an EVOSM7000 imaging system with a 20x objective. Thirteen pictures were taken per well, with six wells per condition.

[0225] CellProfiler analysis

[0226] Image analysis was performed using CellProfiler software (Carpenter et al., 2006). A custom CellProfiler pipeline was generated for automated analysis of synapse number, total and dead nuclei, and neurite area. Binary images were used to ensure correct image segmentation and identification of targets. In rare cases, cell culture issues (e.g., pipetting errors) may result in outliers in specific wells. Individual wells were visually inspected to ensure significant differences from the mean, and outliers were statistically identified using the Grubbs method (α = 0.05). Identified outliers were then removed.

[0227] Neurite Analysis: Neurites were detected using the MAP2 channel. To enhance neurite structure, a tubular enhancement method was used. Neurites were detected using the minimum cross-entropy thresholding method. To account for cell density, neurite area was normalized to the total number of nuclei per image.

[0228] Nuclei analysis: Nuclei were detected using the DAPI channel. DAPI images were corrected for illumination, and nuclei were detected using the Otsu thresholding method. Dead nuclei were defined as the sum of apoptotic nuclei and small bright nuclei. Apoptotic nuclei were defined as at least two small apoptotic bodies in close proximity. To enhance the structure of apoptotic bodies, speckle enhancement was used. Apoptotic bodies were detected using the Otsu thresholding method. Small bright nuclei were filtered from the nuclei based on intensity and area.

[0229] Statistical analysis

[0230] For comparisons of more than two groups, one-way ANOVA was used. Statistical significance was set as: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0231] II. Results

[0232] Example 1: Cloning and analysis of antibodies against the hTREM2 stem region

[0233] As described in the Methods, we identified several antibody clones that bind with high affinity to the extracellular domain of TREM2 and to epitope peptides derived from the stem region of human TREM2. These clones are highly related to each other in structure but are not identical.

[0234] Based on BLAST amino acid sequence alignment, the heavy chain gene of clone H08 (including a 19-aa leader peptide) is 95% homologous to the heavy chain gene of clone M07 (22 of 471 amino acids differ, 87% homologous when only the variable regions of the two antibody clones are considered). The light chain gene of clone H08 (including a 22-aa leader peptide) is 88% homologous to the light chain gene of clone M07 (27 of 243 amino acids differ).

[0235] The heavy chain gene of clone M07 (including a 19-aa leader peptide) is 96% homologous to M05 (18 of 471 amino acids differ, 90% homologous when only the variable regions of the two antibody clones are considered). The light chain gene of clone M07 (including a 22-aa leader peptide) is 85% homologous to M05 (40 of 239 amino acids differ).

[0236] Similarly, the heavy chain gene of clone M07 (containing a 19-aa leader peptide) is 96% homologous to M03 (18 of 471 amino acids differ, 90% homologous when only the variable regions of the two antibody clones are considered). The light chain gene of clone M07 (containing a 22-aa leader peptide) is 82% homologous to M03 (41 of 239 amino acids differ).

[0237] Example 2: Analysis of antibody M07 targeting the hTREM2 stem region

[0238] All antibody clones, including M07, were compared to each other and to an established rat agonist antibody against hTREM2 (H01) and a fully human control antibody (IgG1-LALA isotype). Rat anti-human TREM2 antibody H01 was identified and purified after immunization in rats. This antibody binds to the extracellular domain of TREM2—for which a specific DNA sequence is currently unavailable.

[0239] Figure 1 Shown is an SDS gel (silver stain) of M07 under reducing and non-reducing conditions. Under non-reducing non-boiling (NRNB) conditions, the antibody can be detected at approximately 150 kDa. Under reducing / boiling (RB) conditions, we detected the antibody light chain at 25 kDa and the heavy chain at 50 kDa ( Figure 1 ).

[0240] We determined the binding affinity of all antibody clones (including M07) as well as control antibody H01 and a negative control (not shown here) to ecTREM2 using an ELISA-based binding assay ( Figure 2 Among them, H08, M03, and M07, as well as the rat control antibody H01, showed an affinity for human ecTREM2 of less than 350 pM (190 pM, 350 pM, 204 pM, and 274 pM, respectively), while antibody M05 did not bind to the ecTREM2 domain ( Figure 2 ).

[0241] Next, we wanted to determine the exact antibody binding epitope of TREM2. We investigated a peptide fragment (DAGDLWFPG SEQ ID NO: 16) that represents a peptide from the human TREM2 stem region ( Figure 3 : oval) and the ELISA binding assay was performed again.

[0242] 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 ).

[0243] Example 3: Activation of TREM2 signaling using p-SYK assay

[0244] To detect the activation of TREM2 signaling by anti-TREM2 antibodies, we used the p-SYKAlphaLISA assay, which detects phosphorylation of human pSYK following ligand binding to human TREM2. To analyze the activation of the TREM2 pathway, HEK293 cells stably expressing human TREM2 and human DAP12 were incubated with culture medium or with culture medium containing various antibodies at a concentration of 40 μg / ml at 37°C for 5 minutes, and then the level of SYK phosphorylation was measured. Appropriate controls (human IgG1-LALA antibody and rat IgG isotype antibody) were also used in this assay. Treatment of HEK293-Flp-In hTREM2 / hDAP12 cells with M07 resulted in a significant increase in p-SYK levels compared to baseline controls (isotype or culture medium only). The activation caused by M07 was on average 36-fold higher than baseline, making it much more potent in activating pSYK than either H01 or M03 ( Figure 5 ), while H08 induced very little activation and M05 did not induce any activation at all. The following table shows the increase relative to baseline.

[0245]

[0246] We also performed serial titrations of the best activating antibody clones (H01 and M07) ( FIG6 ).

[0247] To test activation of the TREM2 signaling pathway in cell types that endogenously express the TREM2 receptor, we differentiated microglia from hiPSCs. pSYK phosphorylation in hiPSC-derived microglia was detected by the previously described pSYK AlphaLISA assay using increasing concentrations of the M07 antibody. Treatment of microglia with M07 resulted in a significant concentration-dependent increase in p-SYK levels ( Figure 9 ).

[0248] Example 4: Induction of Neurodegeneration in Alzheimer's Disease Model

[0249] Amyloid-β was added to a microglia-neuron co-culture system to induce neurodegeneration as a model of Alzheimer's disease. Under control conditions containing only culture medium, microglia exhibited the expected branched morphology, while the addition of amyloid-β resulted in an amoeboid morphology ( Figure 7 B) A dose-dependent effect of amyloid-β on neurodegeneration was observed. Neurite degeneration is a hallmark of neurodegeneration and can be mediated by MAP2 + Neuronal death is another hallmark of neurodegeneration and can be quantified by measuring the number of dead nuclei / total nuclei ( Figure 7C , 7D). This neurodegeneration model was used to determine the neuroprotective effect of the anti-hTREM2 antibody M07 on neurons.

[0250] Figure 8 shows that the addition of anti-hTREM2 antibody M07 significantly reduced amyloid beta-dependent neurodegeneration compared to the isotype antibody. Specifically, M07 antibody reduced neurite degeneration at 1 and 2 weeks of antibody addition. In terms of the number of dead cells per nucleus, M07 antibody reduced the number of dead nuclei at 2 weeks of antibody addition.

[0251] Example 5: hTREM2 Antibody Reduces Neurodegeneration in a hiPSC-Derived Microglia-Neuron Co-culture Model of Alzheimer's Disease

[0252] Amyloid-β was added to a microglia-neuron co-culture system to induce neurodegeneration as a model of Alzheimer's disease ( Figure 7 A). Microglia displayed the expected branched morphology in control conditions with culture medium alone, whereas the addition of amyloid-β resulted in an amoeboid morphology ( Figure 7 B). The dose-dependent effect of amyloid beta on neurodegeneration can be measured. Neurite degeneration is a hallmark of neurodegeneration and can be measured by the ratio of MAP2+ neurite area to live nuclei. Neuronal death is another hallmark of neurodegeneration and can be quantified by measuring 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.

[0253] Figure 8 shows that the addition of the anti-hTREM2 antibody M07 significantly reduced amyloid beta-dependent neurodegeneration compared to the isotype antibody. Specifically, the M07 antibody reduced neurite degeneration at 1 and 2 weeks of antibody addition. In terms of the number of dead cells per nucleus, the M07 antibody reduced the number of dead cell nuclei at 2 weeks of antibody addition.

[0254] III. Discussion and Conclusion

[0255] We used phage display technology to generate fully human anti-TREM2 antibodies, which were initially screened for antigen binding. The 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).

[0256] The LALA modification significantly reduced the effector function of IgG1 antibodies, which is crucial for the study of human immune cells and neuronal cells. Compared with existing humanized antibodies based on clones identified in the immune system of non-human animals (for example, US2017240631A1-Alector AL-002 and WO2020172450 A1-Denali), the fully human backbone of our generated antibodies has an advantage because the use of fully human antibodies can reduce immune complications when repeatedly used for prophylaxis or treatment in humans.

[0257] Initially, we identified several structurally similar and related antibodies (with heavy chain amino acid sequence identity of 90% or more) that could bind to the cytokine with high affinity (less than 10 -9 M) bind to the extracellular domain of human TREM2. To our surprise, we found that only one of these antibodies strongly induced human TREM2 / DAP-triggered SYK phosphorylation, a key TREM2-dependent effector pathway in AD. M07 increased SYK phosphorylation by up to 60-fold, while little or no activation was observed with the other antibody clones, despite binding to the same epitope on the TREM2 extracellular domain as H08 and M03.

[0258] The increase in human TREM2 / DAP-dependent pSYK levels induced by M07 is much stronger than that observed with published and patented anti-human TREM2 agonist antibodies (especially any human anti-human TREM2 antibody). The hT2AB antibody disclosed by AMGEN (WO2022120373 A1) was used in a pSYK assay comparable to ours, and the result was a 12-fold increase over baseline (Ellwanger et al, 2021). Alector disclosed a variety of antibodies against human anti-TREM2 in its patent application (US2017240631 A1). Phosphorylation of SYK was shown at the protein level, with antibodies #22, #45, and #65 reported to have an approximately 3-4 fold increase in human dendritic cells. In human macrophages, a 6-fold increase in SYK phosphorylation was observed. Denali presented several anti-hTREM2 antibodies in its patent application (WO2020172450 A1), one of which is CL0020188. In HEK293 cells expressing TREM2, this antibody increased pSYK levels by 4-fold compared to a control antibody. Data from Fassler et al. showed anti-hTREM2 antibody-mediated pSYK activation at the protein level, but this increase was not quantified (Fassler et al., 2021).

[0259] In addition, the inventors also used an innovative, complex and relevant AD model using human brain cells differentiated from human induced pluripotent stem cells (hiPSCs). No anti-TREM2 antibodies known in the art have been analyzed in similarly complex AD models using hiPSC-derived neurons and microglia. For example, WO2020172450 A1 (Denali) discloses a phagocytic assay using hiPSC-derived microglia and amyloid beta. However, the analysis was not performed in co-culture with neurons. Therefore, it is impossible to determine the benefits of amyloid beta phagocytosis on neurons.

[0260] AD can also be studied in other disease models that have inherent limitations. Most researchers still conduct studies in mouse models, which often fail to predict the clinical efficacy of anti-AD drug candidates.

[0261] The present invention addresses the above problems and surprisingly provides fully human anti-human TREM2 antibodies that are able to potently activate human TREM2-dependent pSYK signaling, such that beneficial effects can be observed in relevant AD models in human brain cells. Previously, potent activators have only been described for mouse TREM2 in mouse cells.

[0262] Cited references

[0263] Abud,EM,Ramirez,RN,Martinez,ES,Healy,LM,Nguyen,CHH,Newman,SA,Yeromin,AV,Scarfone,VM,Marsh,SE,Fimbres,C.,Caraway,CA, Fote,GM,Madany,AM,Agrawal,A.,Kayed,R.,Gylys,KH,Cahalan,MD,Cummings,BJ,Antel,JP,…Blurton-Jones,M.(2017).iPSC-Derived Human Microglia-like Cells toStudy Neurological Diseases.Neuron,94(2),278-293.e9.https: / / doi.org / 10.1016 / j.neuron.2017.03.042

[0264] Carpenter,A.E.,Jones,T.R.,Lamprecht,M.R.,Clarke,C.,Kang,I.H.,Friman,O.,Guertin,D.A.,Chang,J.H.,Lindquist,R.A.,Moffat,J.,Golland,P.,&Sabatini,D.M.(2006).CellProfiler:Image analysis software for identifying and quantifyingcell phenotypes.Genome Biology,7(10).https: / / doi.org / 10.1186 / gb-2006-7-10-r100

[0265] Ellwanger,D.C.,Wang,S.,Brioschi,S.,Shao,Z.,Green,L.,Case,R.,Yoo,D.,Weishuhn,D.,Rathanaswami,P.,Bradley,J.,Rao,S.,Cha,D.,Luan,P.,Sambashivan,S.,Gilfillan,S.,Hasson,S.A.,Foltz,I.N.,van Lookeren Campagne,M.,&Colonna,M.(2021).Prior activation state shapes the microglia response to antihumanTREM2 in a mouse model of Alzheimer’s disease.Proceedings oftheNationalAcademy ofSciences ofthe UnitedStates ofAmerica,118(3),1–12.https: / / doi.org / 10.1073 / PNAS.2017742118

[0266] English,H.,Hong,J.,&Ho,M.(2020).Ancient species offers contemporarytherapeutics:An update on shark VNAR singledomain antibody sequences,phagelibraries and potential clinical applications.Antibody Therapeutics,3(1),1–9.https: / / doi.org / 10.1093 / ABT / TBAA001

[0267] Fassler,M.,Rappaport,M.S., C.B.,&George,J.(2021).Engagement ofTREM2 by a novel monoclonal antibody induces activation of microglia andimproves cognitive function in Alzheimer’s disease models.Journal ofNeuroinflammation,18(1),1–18.https: / / doi.org / 10.1186 / s12974-020-01980-5

[0268] Gregg,A.,Teo,S.,Tessier-Lavigne,M.,Jacob,S.,Olsen,K.M.,Noggle,S.,Paquet,D.,Kwart,D.,Chen,A.,&Sproul,A.(2016).Efficient introduction ofspecific homozygous and heterozygous mutations using CRISPR / Cas9.Nature,533(7601),125–129.https: / / doi.org / 10.1038 / nature17664

[0269] Harmsen,M.M.,&De Haard,H.J.(2007).Properties,production,andapplications of camelid single-domain antibody fragments.Applied MicrobiologyandBiotechnology,77(1),13–22.https: / / doi.org / 10.1007 / s00253-007-1142-2

[0270] Keren-Shaul,H.,Spinrad,A.,Weiner,A.,Matcovitch-Natan,O.,Dvir-Szternfeld,R.,Ulland,T.K.,David,E.,Baruch,K.,Lara-Astaiso,D.,Toth,B.,Itzkovitz,S.,Colonna,M.,Schwartz,M.,&Amit,I.(2017).A Unique Microglia TypeAssociated with Restricting Development of Alzheimer’s Disease.Cell,169(7),1276-1290.e17.https: / / doi.org / 10.1016 / j.cell.2017.05.018

[0271] Kleinberger,G,Yamanishi,Y.,Suárez-Calvet,M.,Czirr,E.,Lohmann,E.,Juyvers,E.,Struyfs,H.,Pettkus,N.,Wenninger-Weinzierl,A.,Mazaheri,F.,T ahirovic,S.,Lleó,A.,Alcolea,D.,Fortea,J.,Willem,M.,Lammich,S.,Molinuevo,J.,Sánchez-Valle,R.,Antonell,A.,…Haass,C.(2014).TREM2mutations implicated inneurodegeneration impair cell surface transport and phagocytosis.ScienceTranslational Medicine,6(243).https: / / doi.org / 10.1126 / scitranslmed.3009093

[0272] Kleinberger,Gernot,Brendel,M.,Mracsko,E.,Wefers,B.,Groeneweg,L.,Xiang,X.,Focke,C.,Deuβing,M.,Suárez-Calvet,M.,Mazaheri,F.,Pa rhizkar,S.,Pettkus,N.,Wurst,W.,Federle,R.,Bartenstein,P.,Mueggler,T.,Arzberger,T.,Knuesel,I.,Rominger,A.,&Haass,J.(2017).The FTD-like syndrome causing TREM2T66M mutation impairs microglia function,brain perfusion,and glucosemetabolism.The EMBO Journal,36(13),1837–1853.https: / / doi.org / 10.15252 / embj.201796516

[0273] McQuade,A.,Coburn,M.,Tu,C.H.,Hasselmann,J.,Davtyan,H.,&Blurton-Jones,M.(2018).Development and validation ofa simplified method to generate humanmicroglia from pluripotent stem cells.Molecular Neurodegeneration,13(1),67.https: / / doi.org / 10.1186 / s13024-018-0297-x

[0274] Reifschneider,A.,Robinson,S.,van Lengerich,B., J.,Logan,T.,Heindl,S.,Vogt,M.A.,Weidinger,E.,Riedl,L.,Wind,K.,Zatcepin,A., I.,Haberl,S.,Nuscher,B.,Kleinberger,G.,Klimmt,J., J.K.,Liesz,A.,Bürger,K.,…Haass,C.(2022).Loss of TREM2 rescues hyperactivation of microglia,butnot lysosomal deficits and neurotoxicity in models of progranulindeficiency.The EMBO Journal,41(4),1–25.https: / / doi.org / 10.15252 / embj.2021109108

[0275] Schlepckow,K.,Kleinberger,G.,Fukumori,A.,Feederle,R.,Lichtenthaler,S.F.,Steiner,H.,&Haass,C.(2017).An Alzheimer-associated TREM2 variant occursat the ADAM cleavage site and affects shedding and phagocytic function.EMBOMolecular Medicine,9(10),1356–1365.https: / / doi.org / 10.15252 / emmm.201707672

[0276] Song,W.,Hooli,B.,Mullin,K.,Jin,S.C.,Cella,M.,Ulland,T.K.,Wang,Y.,Tanzi,R.E.,&Colonna,M.(2017).Alzheimer’s disease-associated TREM2variantsexhibit either decreased or increased ligand-dependent activation.Alzheimer’sand Dementia,13(4),381–387.https: / / doi.org / 10.1016 / j.jalz.2016.07.004

[0277] Suárez-Calvet,M.,Caballero,M. A.,Kleinberger,G.,Bateman,R.J.,Fagan,A.M.,Morris,J.C.,Levin,J.,Danek,A.,Ewers,M.,&Haass,C.(2016).Early changes inCSF sTREM2 in dominantly inherited Alzheimer’s disease occur after amyloiddeposition and neuronal injury.Science Translational Medicine,8(369),34–38.https: / / doi.org / 10.1126 / scitranslmed.aag1767

[0278] Suárez-Calvet,M.,Kleinberger,G.,Araque Caballero,M. Brendel,M.,Rominger,A.,Alcolea,D.,Fortea,J.,Lleó,A.,Blesa,R.,Gispert,JD,Sánchez-Valle,R.,Antonell,A.,Ra mi,L.,Molinuevo,JL,Brosseron,F.,Traschütz,A.,Heneka,MT,Struyfs,H.,Engelborghs,S.,…Haass,J.(2016).sTREM2 cerebrospinal fluid levelsare a potential biomarker for microglia activity in early-stage Alzheimer'sdisease and associate with neuronal injury markers.EMBO Molecular Medicine,8(5),466–476.https: / / doi.org / 10.15252 / emmm.2015306

[0279] Ulland,TK,Song,WM,Huang,SCC,Ulrich,JD,Sergushichev,A.,Beatty,WL,Loboda,AA,Zhou,Y.,Cairns,NJ,Kambal,A.,Login icheva,E.,Gilfillan,S.,Cella,M.,Virgin,HW,Unanue,ER,Wang,Y.,Artyomov,MN,Holtzman,DM,&Colonna,M.(2017).TREM2 Maintains Microglial Metabolic Fitness inAlzheimer's Disease.Cell,170(4),649-663.e13.https: / / doi.org / 10.1016 / j.cell.2017.07.023

[0280] Wunderlich,P.,Glebov,K.,Kemmerling,N.,Tien,N.T.,Neumann,H.,&Walter,J.(2013).Sequential proteolytic processing of the triggering receptor expressedon myeloid cells-2(TREM2)protein by ectodomain shedding andγ-secretase-dependent intramembranous cleavage.Journal of Biological Chemistry,288(46),33027–33036.https: / / doi.org / 10.1074 / jbc.M113.517540

[0281] Zhong,L.,Chen,X.-F.,Wang,T.,Wang,Z.,Liao,C.,Wang,Z.,Huang,R.,Wang,D.,Li,X.,Wu,L.,Jia,L.,Zheng,H.,Painter,M.,Atagi,Y.,Liu,C.-C.,Zhang,Y.-W.,Fryer,J.D.,Xu,H.,&Bu,G.(2017).Soluble TREM2 induces inflammatory responses andenhances microglial survival.The Journal of ExperimentalMedicine,jem.20160844.https: / / doi.org / 10.1084 / jem.20160844

[0282] Zhong,L.,&Chen,X.F.(2019).The Emerging Roles and TherapeuticPotential of Soluble TREM2 in Alzheimer’s Disease.Frontiers in AgingNeuroscience,11(November),1–9.https: / / doi.org / 10.3389 / fnagi.2019.00328

[0283] Zhong,L.,Wang,T.,&Zhuo,R.(2019).Soluble TREM2 amelioratespathological phenotypes by modulating microglial functions in an Alzheimer'sdisease model.Nature Communications,904,1–16. https: / / doi.org / 10.1038 / s41467- 019-09118-9

[0284] Nucleotide and amino acid sequences

[0285] SEQ ID NO: 1 Variable region or domain of clone M07 light chain (107aa):

[0286] DIQLTQSPLSLSASAGDRVTITCRASQSIRDYLGWYQQKPGKAPKLLIYAASKLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYHTPPFTFGQGTKVEI

[0287] SEQ ID NO:2 Variable region or domain of clone M07 heavy chain (122aa):

[0288] EVQLLESGGGLVQPGGSLRLTCAASGFTFSSYAMSWVRQAPGKGLEWVSVINGRGSNTYYADSVKGRFTITRDNSKNTLYLEMNSLRAEDTAVYYCARVRAYSGPSYGFDYWGQGTLVTVSS

[0289] CDR-L1 of SEQ ID NO:3M07:QSIRDY

[0290] CDR-L2 of SEQ ID NO:4M07:AAS

[0291] CDR-L3 of SEQ ID NO:5M07:QQSYHTPPFT

[0292] CDR-H1 of SEQ ID NO: 6M07: GFTFSSYA

[0293] CDR-H2 of SEQ ID NO:7M07:NGRGSNT

[0294] CDR-H3 of SEQ ID NO:8M07:ARVRAYSGPSYGFDY

[0295] SEQ ID NO: 9 Kappa-1 light chain of M07 (237 aa) with an N-terminal leader sequence of 22 amino acid residues, the sequences in the proposed CDR 1 to CDR 3 are underlined:

[0296] MDMRVPAQLLGLLLLWLSGARCDIQLTQSPLSLSASAGDRVTITCRAS QSIRDY LGWYQQKPGKAPKLLIY AAS KLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSYHTPPFT FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0297] Kappa-1 light chain (215 aa) of SEQ ID NO: 10M07, the sequence / sequences in the putative CDR 1 to CDR 3 are underlined:

[0298] DIQLTQSPLSLSASAGDRVTITCRAS QSIRDY LGWYQQKPGKAPKLLIY AAS KLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSYHTPPFT FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0299] Lambda light chain (213 aa) of SEQ ID NO: 11M07, the sequence / sequences in the proposed CDR 1 to CDR 3 are underlined:

[0300] DIQLTQSPLSLSASAGDRVTITCRAS QSIRDY LGWYQQKPGKAPKLLIY AA

[0301] S KLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSYHTPPFT FQG

[0302] KVEIKGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKAD

[0303] SSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGST

[0304] VEKTVAPTEC

[0305] SEQ ID NO: 12 Heavy chain complete IgG1-LALA-TFN of clone M07 with N-terminal leader sequence (containing C H The sequence of the proposed CDR 1 to CDR 3 is underlined:

[0306] MELGLSWIFLLAILKGVQCEVQLLESGGGLVQPGGSLRLTCAAS GFTFSSY

[0307] A MSWVRQAPGKGLEWVSVI NGRGSNT YYADSVKGRFTITRDNSKNTLYL

[0308] EMNSLRAEDTAVYYC ARVRAYSGPSYGFDY WGQGTLVTVSSASTKGPSV

[0309] FPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ

[0310] SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT

[0311] CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN

[0312] WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV

[0313] SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP

[0314] SDIAVEWESYGTEWSSYKTTPPVLDSDGSFFLYSKLTVTKSEWQQGFVFS

[0315] CSVMHEALHNHYTQKSLSLSPGK

[0316] SEQ ID NO:13 clone of the heavy chain of fully IgG1-LALA-TFN (452aa) of M07, the sequences in the so-called CDR 1 to CDR3 are underlined as follows:

[0317] EVQLLESGGGLVQPGGSLRLTCAAS GFTFSSYA MSWVRQAPGKGLEWVS

[0318] VI NGRGSNT YYADSVKGRFTITRDNSKNTLYLEMNSLRAEDTAVYYC AR

[0319] VRAYSGPSYGFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG

[0320] CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG

[0321] TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFP

[0322] PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE

[0323] EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ

[0324] PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESYGTEWSSYK <00​​​​​​SEQ ID NO: 14 Heavy chain complete IgG1-LALA (471 aa) with N-terminal leader sequence, the sequences in the proposed CDR 1 to CDR 3 are underlined:

[0328] MELGLSWIFLLAILKGVQCEVQLLESGGGLVQPGGSLRLTCAAS GFTFSSY

[0329] A MSWVRQAPGKGLEWVSVI NGRGSNT YYADSVKGRFTITRDNSKNTLYL

[0330] EMNSLRAEDTAVYYC ARVRAYSGPSYGFDY WGQGTLVTVSSASTKGPSV

[0331] FPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ

[0332] SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT

[0333] CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN

[0334] WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV

[0335] SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP

[0336] SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

[0337] CSVMHEALHNHYTQKSLSLSPGK

[0338] SEQ ID NO: 15 Heavy chain complete IgG1-LALA (452 ​​aa), the sequence in the proposed CDR 1 to CDR 3 is underlined:

[0339] EVQLLESGGGLVQPGGSLRLTCAAS GFTFSSYA MSWVRQAPGKGLEWVS

[0340] VI NGRGSNT YYADSVKGRFTITRDNSKNTLYLEMNSLRAEDTAVYYC AR

[0341] VRAYSGPSYGFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG

[0342] CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG

[0343] TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFP

[0344] PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE

[0345] EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ

[0346] PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK

[0347] TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL

[0348] SLSPGK

[0349] SEQ ID NO: 16: Peptide fragment representing an epitope from the hTREM2 stem region:

[0350] DAGDLWFPG

[0351] Kappa-1 light chain of SEQ ID NO: 17H08, the sequence / sequences in the putative CDR 1 to CDR 3 are underlined:

[0352] AIRMTQSPDSLPVSLGERATINCKSS QSVLYGSNNKNY LAWYQQKPGQPP

[0353] KLLIY WAS TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYYSTP

[0354] LT FGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV

[0355] QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE

[0356] VTHQGLSSPVTKSFNRGEC

[0357] SEQ ID NO: 18 Clone H08's heavy chain complete IgG1-LALA, the sequences in the so-called CDR 1 to CDR 3 are underlined as follows:

[0358] EVQLVQSGGGLVQPGGSLRLSCAAS GFTFSSYA MSWVRQAPGKGLEWVS

[0359] A ISGSGGST YYADSVKGRFTISRDNAKRSLYLQMNDLRVEDTAVYYC AR

[0360] TRTNVFDF WGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD

[0361] YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI

[0362] CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKD

[0363] TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS

[0364] TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ

[0365] VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV

[0366] LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0367] K

[0368] The complete IgG1 Kappa-1 light chain of SEQ ID NO: 19M03, the sequences in the proposed CDR 1 to CDR 3 are underlined:

[0369] DIRLTQPPSVSGAPGQRVTISC SGSSSNIGS LFVSWYQQLPGTAPKLLIY SN

[0370] SQ HPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYC SAYDQFSNSVV FG

[0371] GGTKLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK

[0372] VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH

[0373] QGLSSPVTKSFNRGEC

[0374] The complete IgG1 lambda light chain of SEQ ID NO: 20M03, the sequences in the proposed CDR 1 to CDR 3 are underlined:

[0375] DIRLTQPPSVSGAPGQRVTISC SGSSSNIGS LFVSWYQQLPGTAPKLLIY SN

[0376] SQ HPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYC SAYDQFSNSVV FG

[0377] GGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW

[0378] KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE

[0379] GSTVEKTVAPTEA

[0380] SEQ ID NO: 21 Heavy chain complete IgG1-LALA of clone M03; the sequence / sequences in the proposed CDR 1 to CDR 3 are underlined:

[0381] EVQLVQSGGGLVQPGGSLRLSCAAS GFTFSSYG ITWVRQAPGKGLEWVSF

[0382] I SGGGSYT YYADSVKGRFTISRDNAKRTLYLQMNSLRAEDTAVYYC ARS

[0383] GRAYFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD

[0384] YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI

[0385] CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKD

[0386] TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS

[0387] TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ

[0388] VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV

[0389] LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0390] KYTEWSS

[0391] SEQ ID NO: 22M05 complete IgG1 Kappa-1 light chain, the sequence of the proposed CDR 1 to CDR 3 is underlined:

[0392] DIVMTQPPSVSVTPGQRVTISC RSSSSNIGS LFVSWYQQLPGTAPKLLIY SN

[0393] SQ HPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYC SAYDQFSNSVV FG

[0394] GGTKLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK

[0395] VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH

[0396] QGLSSPVTKSFNRGEA

[0397] The complete IgG1 lambda light chain of SEQ ID NO: 23M05, the sequences in the proposed CDR 1 to CDR 3 are underlined:

[0398] DIVMTQPPSVSVTPGQRVTISC RSSSSNIGS LFVSWYQQLPGTAPKLLIY SN

[0399] SQ HPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYC SAYDQFSNSVV FG

[0400] GGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW

[0401] KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE

[0402] GSTVEKTVAPTEA

[0403] SEQ ID NO: 24 Heavy chain complete IgG1-LALA of clone M05, the sequence in the proposed CDR 1 to CDR 3 is underlined:

[0404] EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSNA MTWVRQAPGKGLEWVS

[0405] VI GSSGSYT YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARS

[0406] GTTGFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY

[0407] FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC

[0408] NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDT

[0409] LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST

[0410] YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

[0411] YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL

[0412] DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0413] YTEWSS

[0414] Coding sequence (723bp) of the Kappa-1 light chain of antibody M07 with an N-terminal leader sequence: SEQ ID NO:25

[0415] CATCATGGACATGAGAGTGCCCGCTCAGCTGCTGGGACTGCTGTTGTT

[0416] GTGGCTGTCTGGCGCTAGATGCGACATCCAGCTGACCCAGTCTCCACT

[0417] GTCTCTGTCTGCCTCTGCTGGCGACAGAGTGACCATCACCTGTCGGGC

[0418] CTCTCAGTCTATCAGAGACTACCTCGGCTGGTATCAGCAGAAGCCTGG

[0419] CAAGGCTCCCAAGCTGCTGATCTACGCTGCCTCTAAACTGCAGTCCGG

[0420] CGTGCCCTCTAGATTCTCTGGCTCTGGATCTGGCACCGACTTCACCCTG

[0421] ACCATCAGTTCTCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAG

[0422] CAGTCCTATCACACCCCTCCATTCACCTTTGGCCAGGGCACCAAGGTG

[0423] GAAATCAAGAGAACCGTGGCCGCTCCTTCCGTGTTCATCTTCCCACCA

[0424] TCTGACGAGCAGCTGAAGTCCGGCACAGCTTCTGTCGTGTGCCTGCTG

[0425] AACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAGGTGGACAA

[0426] TGCCCTGCAGTCTGGCAACTCCCAAGAGTCTGTGACCGAGCAGGACTC

[0427] CAAGGACTCTACCTACAGCCTGTCCTCCACACTGACCCTGTCTAAGGC

[0428] CGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTGACCCACCAGG

[0429] GACTGTCTAGCCCCGTGACCAAGTCTTTCAACAGAGGCGAGTGCTGAT

[0430] TAAT

[0431] Coding sequence (1424bp) of the heavy chain of antibody M07 with an N-terminal leader sequence:

[0432]

[0433] SEQ ID NO: 27 The amino acid sequence of hTREM2 is as follows Figure 3 shown.

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 1 or 2 amino acid residue substitutions compared to SEQ ID NO: 2, wherein the protein is capable of activating human TREM2-dependent pSYK signaling.

2. A protein, preferably a protein according to claim 1, 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 1 or 2 amino acid residues substituted 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 1 or 2 amino acid residues substituted 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, 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 1 or 2 amino acid residues substituted 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 residues substituted compared to SEQ ID NO: 1; The protein is capable of activating human TREM2-dependent pSYK signaling.

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

5. The protein according to any one of claims 2 to 4, wherein In the N-terminal to C-terminal direction, the light chain variable region comprises, and preferably comprises within CDRs L1 and L3, the following amino acid stretch: a fragment of the amino acid sequence of SEQ ID NO: 3 (CDR-L1), and the amino acid sequence of SEQ ID NO: 5 or a fragment of an amino acid sequence having one amino acid residue substituted compared to the amino acid sequence of SEQ ID NO: 5 (CDR-L3); and In the N-terminal to C-terminal direction, the heavy chain (HC) variable region comprises, and preferably comprises within CDRs H2 and H3, the following amino acid stretch: a fragment of the amino acid sequence of SEQ ID NO: 7 (CDR-H2), and The amino acid sequence of SEQ ID NO: 8 or a fragment of the amino acid sequence having one amino acid residue substitution compared to the amino acid sequence of SEQ ID NO: 8 (CDR-H3).

6. The protein according to claim 4 or 5, wherein in the N-terminal to C-terminal direction, the light chain (LC) variable region comprises, preferably in CDRs L1 to L3, the following amino acid stretch: a fragment of the amino acid sequence of SEQ ID NO: 3 (CDR-L1), a fragment of the amino acid sequence of SEQ ID NO: 4 (CDR-L2), and the amino acid sequence of SEQ ID NO: 5 or a fragment of an amino acid sequence having one amino acid residue substituted compared to the amino acid sequence of SEQ ID NO: 5 (CDR-L3); and In the N-terminal to C-terminal direction, the heavy chain (HC) variable region comprises, preferably comprises the following amino acid sequence segments in CDRH1 to H3: a fragment of the amino acid sequence of SEQ ID NO:6 (CDR-H1), a fragment of the amino acid sequence of SEQ ID NO: 7 (CDR-H2), and The amino acid sequence of SEQ ID NO: 8 or a fragment of the amino acid sequence having one amino acid residue substitution compared to the amino acid sequence of SEQ ID NO: 8 (CDR-H3).

7. The protein according to any one of claims 1 to 6, wherein the protein is a single-chain antibody (scFv), a Fab fragment, a F(ab)2 fragment or an immunoglobulin (Ig); and / or The protein is a fusion protein, which comprises a single-chain antibody (scFv), a Fab fragment, a F(ab)2 fragment or an immunoglobulin (Ig) as a first fusion protein fragment and a second fusion protein fragment.

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

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

10. The protein according to any one of claims 2 to 9, comprising the following 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) the amino acid sequence of the light chain is or comprises an amino acid sequence having 1 or 2 amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 9, 10 or 11; and / or The protein comprises the following 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, or (d) the amino acid sequence of the heavy chain is or comprises an amino acid sequence having 1 or 2 amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 12, 13, 14 or 15.

11. The protein according to any one of claims 1 to 10, comprising a binding domain capable of binding to human transferrin receptor 1 (hTfR1) to allow the protein to cross the blood-brain barrier, preferably, the protein comprises a C having a modified H The heavy chain of the 3-domain or contains a C that allows binding to hTfR1 H C-terminal extension of the 3-domain.

12. The protein according to any one of claims 1 to 11, wherein the protein is capable of binding to human TREM2 via its variable region, preferably to the stem region of hTREM2; and / or the protein activates human TREM2-dependent human pSYK signaling; and / or the protein 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. The antibody according to claim 13, comprising: Light chains, of which: (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 amino acid sequence of the light chain is or comprises an amino acid sequence having 1 or 2 amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 9, 10 or 11; and Heavy chain, of which: (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 amino acid sequence of the heavy chain is or comprises an amino acid sequence having 1 or 2 amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 12, 13, 14 or 15.

15. The antibody according to claim 13 or 14, comprising The following two light chains: (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) the amino acid sequence of the light chain is or comprises an amino acid sequence having 1 or 2 amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 10 or 11; and The following 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, or (d) the amino acid sequence of the heavy chain is or comprises an amino acid sequence having 1 or 2 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 treatment 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. The protein or antibody for use according to claim 17 or 18, wherein the use is in a method for treating or preventing a neurodegenerative disease such as Alzheimer's disease in a patient at an early stage of the disease.

20. The protein or antibody for use according to any one of claims 17 to 19, wherein the use is in a method for treating or preventing a neurodegenerative disease such as Alzheimer's disease at the stage of the disease in a patient who: - the patient has a cognitive impairment score of 23 or less, preferably a score of 10 to 23, more preferably a score of 19 to 23 on the Mini-Mental State Examination (MMSE) test, or - The patient has a cognitive impairment score of 0.5 or higher and 2 or lower on the Clinical Dementia Rating Scale (CDR Global Score).

21. The protein or antibody or pharmaceutical composition for use according to any one of claims 17 to 20, which comprises administering the protein or antibody parenterally to a mammal, preferably intravenously, subcutaneously or intraperitoneally.

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 the protein of any one of claims 1 to 15 or the nucleic acid molecule of claim 22.

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

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 the disease.

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

Images