Anti-TREM2 nanobody or antigen-binding fragment thereof, and use

Abstract

An anti-TREM2 nanobody or antigen-binding fragment thereof, and a use thereof. The anti-TREM2 nanobody or antigen-binding fragment thereof contains a VHH chain, and the VHH chain comprises complementarity-determining regions CDR1, CDR2, and CDR3. The anti-TREM2 nanobody or antigen-binding fragment thereof has strong ADCC effect induction activity, and can be used for enhancing immune responses and / or treating immune-related diseases, such as cancer, of individuals. The anti-TREM2 nanobody or antigen-binding fragment thereof, or a TREM2-binding molecule containing the anti-TREM2 nanobody or antigen-binding fragment thereof can be used to eliminate, incapacitate, or reduce the number of non-stimulatory myeloid cells.

Description

Anti-TREM2 nanoantibodies or antigen-binding fragments thereof and applications Technical Field

[0001] The present application relates to the field of biomedicine technology, and specifically to an anti-TREM2 nanobody or its antigen-binding fragment and its application. Background Art

[0002] Triggering receptor 2 on myeloid cells (TREM2) belongs to the immunoglobulin superfamily and is a transmembrane receptor. The physiological functions of TREM2 include cell maturation, cell proliferation, cell survival, phagocytosis, and inflammation regulation. TREM2 is considered an important pathology-induced immune signaling hub and plays a crucial role in myeloid cell activation and survival. TREM2 has multiple ligands, primarily free negatively charged molecules bound to the plasma membrane, including bacterial products, DNA, lipoproteins, and phospholipids. After binding to ligands, TREM2 transmits signals into the cell via DNAX activating protein 10 (DAP10) and DNAX activating protein 12 (DAP12), subsequently activating multiple downstream signaling pathways and producing diverse effects. TREM2 is a marker of macrophage infiltration in tumors. TREM2 expression on the surface of tumor-associated macrophages (TAMs) has been found in various tumors and is negatively correlated with tumor survival. Binding of TREM2 to its ligands promotes high expression of CD163 by TAMs, amplifying inflammatory responses in the local tumor microenvironment and promoting tumor development and progression.

[0003] An increasing number of studies have found that TREM2-expressing TAMs accumulate in a variety of tumors and are associated with T cell exhaustion and resistance to immune checkpoint inhibitor therapy.

[0004] Therefore, TREM2 may be an ideal target for targeting tumor myeloid infiltration and enhancing the efficacy of immune checkpoint inhibitors.

[0005] Summary of the Invention

[0006] In response to the prior art, the present application provides an anti-TREM2 nanobody or an antigen-binding fragment thereof and applications thereof, wherein the anti-TREM2 nanobody or the antigen-binding fragment thereof contains a VHH chain, and the VHH chain comprises complementary determining regions CDR1, CDR2 and CDR3; the anti-TREM2 nanobody or the antigen-binding fragment thereof, and the TREM2 binding molecule comprising the anti-TREM2 nanobody or the antigen-binding fragment thereof have a strong activity of inducing ADCC effect, and can be used to enhance the immune response and / or for treating an individual's immune-related diseases such as cancer. The use of the anti-TREM2 nanobody or the antigen-binding fragment thereof, or the TREM2 binding molecule comprising the anti-TREM2 nanobody or the antigen-binding fragment thereof can kill non-stimulatory myeloid cells, causing the non-stimulatory myeloid cells to lose their ability or reduce their number.

[0007] In a first aspect, the present application provides an anti-TREM2 nanobody or an antigen-binding fragment thereof, comprising a VHH chain, wherein the VHH chain comprises complementary determining regions CDR1, CDR2, and CDR3 with the amino acid sequences shown below:

[0008] The CDR1 amino acid sequence is one of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 36, 39, 41, 44, 47, 49, 52, 54, 57, 59, 63, 66 or 68;

[0009] The CDR2 amino acid sequence is one of SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 37, 40, 42, 45, 48, 50, 53, 55, 58, 60, 61, 64, 67 or 69;

[0010] The CDR3 amino acid sequence is one of the sequences shown in SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 38, 43, 46, 51, 56, 62 or 65.

[0011] In some embodiments of the present application, the VHH chain comprises:

[0012] (1) CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO: 3;

[0013] (2) CDR1 shown in SEQ ID NO: 4, CDR2 shown in SEQ ID NO: 5, and CDR3 shown in SEQ ID NO: 6;

[0014] (3) CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 8, and CDR3 shown in SEQ ID NO: 9;

[0015] (4) CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 11, and CDR3 shown in SEQ ID NO: 12;

[0016] (5) CDR1 shown in SEQ ID NO: 13, CDR2 shown in SEQ ID NO: 14, and CDR3 shown in SEQ ID NO: 15;

[0017] (6) CDR1 shown in SEQ ID NO: 16, CDR2 shown in SEQ ID NO: 17, and CDR3 shown in SEQ ID NO: 18;

[0018] (7) CDR1 shown in SEQ ID NO: 19, CDR2 shown in SEQ ID NO: 20, and CDR3 shown in SEQ ID NO: 21;

[0019] (8) CDR1 shown in SEQ ID NO: 22, CDR2 shown in SEQ ID NO: 23, and CDR3 shown in SEQ ID NO: 24;

[0020] (9) CDR1 shown in SEQ ID NO: 25, CDR2 shown in SEQ ID NO: 26, and CDR3 shown in SEQ ID NO: 27;

[0021] (10) CDR1 shown in SEQ ID NO: 28, CDR2 shown in SEQ ID NO: 29, and CDR3 shown in SEQ ID NO: 30;

[0022] (11) CDR1 shown in SEQ ID NO: 31, CDR2 shown in SEQ ID NO: 32, and CDR3 shown in SEQ ID NO: 33;

[0023] (12) CDR1 shown in SEQ ID NO: 34, CDR2 shown in SEQ ID NO: 35, and CDR3 shown in SEQ ID NO: 12;

[0024] (13) CDR1 shown in SEQ ID NO: 36, CDR2 shown in SEQ ID NO: 37, and CDR3 shown in SEQ ID NO: 38;

[0025] (14) CDR1 shown in SEQ ID NO:39, CDR2 shown in SEQ ID NO:40, and CDR3 shown in SEQ ID NO:18;

[0026] (15) CDR1 shown in SEQ ID NO:41, CDR2 shown in SEQ ID NO:42, and CDR3 shown in SEQ ID NO:43;

[0027] (16) CDR1 shown in SEQ ID NO:44, CDR2 shown in SEQ ID NO:45, and CDR3 shown in SEQ ID NO:46;

[0028] (17) CDR1 shown in SEQ ID NO:47, CDR2 shown in SEQ ID NO:48, and CDR3 shown in SEQ ID NO:38;

[0029] (18) CDR1 shown in SEQ ID NO:49, CDR2 shown in SEQ ID NO:50, and CDR3 shown in SEQ ID NO:51;

[0030] (19) CDR1 shown in SEQ ID NO: 52, CDR2 shown in SEQ ID NO: 53, and CDR3 shown in SEQ ID NO: 18;

[0031] (20) CDR1 shown in SEQ ID NO: 54, CDR2 shown in SEQ ID NO: 55, and CDR3 shown in SEQ ID NO: 56;

[0032] (21) CDR1 shown in SEQ ID NO: 57, CDR2 shown in SEQ ID NO: 58, and CDR3 shown in SEQ ID NO: 9;

[0033] (22) CDR1 shown in SEQ ID NO: 59, CDR2 shown in SEQ ID NO: 60, and CDR3 shown in SEQ ID NO: 12;

[0034] (23) CDR1 shown in SEQ ID NO: 19, CDR2 shown in SEQ ID NO: 61, and CDR3 shown in SEQ ID NO: 62;

[0035] (24) CDR1 shown in SEQ ID NO: 63, CDR2 shown in SEQ ID NO: 64, and CDR3 shown in SEQ ID NO: 65;

[0036] (25) CDR1 shown in SEQ ID NO: 66, CDR2 shown in SEQ ID NO: 67, and CDR3 shown in SEQ ID NO: 12; or

[0037] (26) CDR1 shown in SEQ ID NO:68, CDR2 shown in SEQ ID NO:69, and CDR3 shown in SEQ ID NO:18.

[0038] In a preferred embodiment, the VHH chain comprises:

[0039] (1) CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO: 3;

[0040] (2) CDR1 shown in SEQ ID NO: 4, CDR2 shown in SEQ ID NO: 5, and CDR3 shown in SEQ ID NO: 6; or

[0041] (3) CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 11, and CDR3 shown in SEQ ID NO: 12.

[0042] In some embodiments of the present application, the VHH chain further comprises a framework region FR, which comprises FR1, FR2, FR3, and FR4;

[0043] The amino acid sequence of FR1 is selected from the amino acid sequence shown in any one of SEQ ID NOs: 80 to 83, or a sequence having one or more amino acid substitutions, deletions, additions, or any combination thereof (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the amino acid sequence shown in SEQ ID NOs: 80 to 83;

[0044] The amino acid sequence of FR2 is the amino acid sequence shown in SEQ ID NO: 84, 85 or 106, or a sequence having one or more amino acid substitutions, deletions, additions, or any combination thereof (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the amino acid sequence shown in SEQ ID NO: 84, 85 or 106;

[0045] The amino acid sequence of FR3 is selected from the amino acid sequence shown in any one of SEQ ID NOs: 86 to 89 and 107, or a sequence having one or more amino acid substitutions, deletions, additions, or any combination thereof (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to any one of the amino acid sequences shown in SEQ ID NOs: 86 to 89 and 107;

[0046] The amino acid sequence of FR4 is the amino acid sequence shown in SEQ ID NO: 90, or a sequence having one or more amino acid substitutions, deletions, additions, or any combination thereof (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to the amino acid sequence shown in SEQ ID NO: 90;

[0047] Preferably, the substitution is a conservative substitution.

[0048] In some embodiments, the framework region FR comprises:

[0049] (1) FR1 shown in SEQ ID NO: 80, FR2 shown in SEQ ID NO: 84, FR3 shown in SEQ ID NO: 86, and FR4 shown in SEQ ID NO: 90;

[0050] (2) FR1 shown in SEQ ID NO: 80, FR2 shown in SEQ ID NO: 84, FR3 shown in SEQ ID NO: 87, and FR4 shown in SEQ ID NO: 90;

[0051] (3) FR1 shown in SEQ ID NO:81, FR2 shown in SEQ ID NO:84, FR3 shown in SEQ ID NO:86, and FR4 shown in SEQ ID NO:90;

[0052] (4) FR1 shown in SEQ ID NO:82, FR2 shown in SEQ ID NO:84, FR3 shown in SEQ ID NO:88, and FR4 shown in SEQ ID NO:90;

[0053] (5) FR1 shown in SEQ ID NO:83, FR2 shown in SEQ ID NO:84, FR3 shown in SEQ ID NO:86, and FR4 shown in SEQ ID NO:90;

[0054] (6) FR1 shown in SEQ ID NO:81, FR2 shown in SEQ ID NO:84, FR3 shown in SEQ ID NO:89, and FR4 shown in SEQ ID NO:90;

[0055] (7) FR1 shown in SEQ ID NO:83, FR2 shown in SEQ ID NO:84, FR3 shown in SEQ ID NO:88, and FR4 shown in SEQ ID NO:90;

[0056] (8) FR1 shown in SEQ ID NO:81, FR2 shown in SEQ ID NO:84, FR3 shown in SEQ ID NO:87, and FR4 shown in SEQ ID NO:90;

[0057] (9) FR1 shown in SEQ ID NO:83, FR2 shown in SEQ ID NO:84, FR3 shown in SEQ ID NO:87, and FR4 shown in SEQ ID NO:90;

[0058] (10) FR1 shown in SEQ ID NO: 83, FR2 shown in SEQ ID NO: 85, FR3 shown in SEQ ID NO: 87, or FR4 shown in SEQ ID NO: 90; or

[0059] (11) FR1 shown in SEQ ID NO: 83, FR2 shown in SEQ ID NO: 106, FR3 shown in SEQ ID NO: 107, and FR4 shown in SEQ ID NO: 90.

[0060] In some embodiments, the VHH chain has the structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0061] In some embodiments, the VHH chain comprises any one of the amino acid sequences shown in SEQ ID NOs: 70-79 or 108-109, or a variant thereof, which has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the amino acid sequences shown in SEQ ID NOs: 70-79 or 108-109, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions) compared to SEQ ID NOs: 70-79 or 108-109.

[0062] In some embodiments, compared to the amino acid sequences shown in SEQ ID NOs: 70-79 or 108-109, the different amino acids in the amino acid sequence having at least 80% identity or the amino acid sequence having one or more amino acid substitutions, deletions or additions are mainly or entirely located in the FR region of the VHH chain.

[0063] Preferably, the variant sequence retains the activity of specifically binding to human TREM2, murine TREM2 or cynomolgus monkey TREM2.

[0064] Preferably, the substitution is a conservative substitution. In this article, a conservative substitution means that another amino acid of the same type (having similar chemical properties or functions) can be used for substitution. As an example, amino acids can be divided into the following categories based on their side chain properties: (1) non-polar amino acids: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar amino acids: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic amino acids: Asp (D), Glu (E); (4) basic amino acids: Lys (K), Arg (R), His (H). Alternatively, amino acids can be divided into the following categories based on common side chain properties: (1) hydrophobic amino acids: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic amino acids: Cys, Ser, Thr, Asn, Gln; (3) acidic amino acids: Asp, Glu; (4) basic amino acids: His, Lys, Arg; (5) amino acids that affect chain orientation: Gly, Pro; (6) aromatic amino acids: Trp, Tyr, Phe.

[0065] In some specific embodiments, the VHH chain comprises any one of the amino acid sequences shown in SEQ ID NOs: 70 to 79 and 108 to 109. In a preferred embodiment, the VHH chain has the amino acid sequence shown in SEQ ID NO: 73, 75 or 77.

[0066] In some embodiments, the anti-TREM2 nanobody is selected from at least one of a camel-derived nanobody, a humanized nanobody, and a fully human nanobody, and the anti-TREM2 nanobody is preferably a humanized nanobody.

[0067] Optionally, the TREM2 nanobody or antigen-binding fragment thereof contains or does not contain an antibody heavy chain constant region. Preferably, the TREM2 nanobody or antigen-binding fragment thereof contains an antibody heavy chain constant region. In some embodiments, the antibody heavy chain constant region is preferably an antibody heavy chain constant region sequence in the absence of a CH1 fragment. In some embodiments, the TREM2 nanobody or antigen-binding fragment thereof contains an antibody constant region sequence having CH2 and CH3 fragments; or the antibody heavy chain constant region is an antibody Fc region (in this case, the nanobody is also referred to as a "heavy chain antibody").

[0068] In some embodiments, the antibody heavy chain constant region is connected to the VHH chain with or without a linker peptide.

[0069] In some embodiments, the antibody heavy chain constant region is from human, camel, alpaca, mouse, rat, rabbit, or sheep. In some specific embodiments, the antibody heavy chain constant region is from human. In some embodiments, the antibody heavy chain constant region is selected from the heavy chain constant region of IgG, IgM, IgA, IgE, or IgD, or a variant thereof. The heavy chain constant region is preferably an IgG heavy chain constant region or a variant thereof, such as an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region or a variant thereof; the heavy chain constant region is more preferably an IgG1 heavy chain constant region or a variant thereof. In some specific embodiments, the heavy chain constant region is of the human IgG1 subtype.

[0070] In some embodiments, the heavy chain constant region or variant thereof comprises one or more mutations in the IgG1 Fc region that enhance antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the heavy chain constant region or variant thereof comprises mutations at positions 234, 235, and 243, or mutations at positions 239, 330, and 332 of the IgG1 Fc region; in some embodiments, the heavy chain constant region or variant thereof comprises at least one mutation at positions 234, 235, 243, 239, 330, or 332 of the IgG1 Fc region. In some specific embodiments of the present application, the IgG1 Fc region comprises mutations of S239D, A330L, and I332E. In some embodiments, the Fc region binds to an Fcγ receptor, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, or FcγRIIIb.

[0071] In some embodiments, the amino acid sequence of the antibody heavy chain constant region is shown in SEQ ID NO: 104, and the amino acid sequence of the heavy chain constant region comprising a mutation that enhances ADCC is shown in SEQ ID NO: 105.

[0072] In some embodiments, the full-length amino acid sequence of the anti-TREM2 Nanobody is any one of the amino acid sequences shown in SEQ ID NOs: 91 to 101, or a variant sequence thereof, and the variant sequence has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the amino acid sequences shown in SEQ ID NOs: 91 to 101, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions) compared to SEQ ID NOs: 91 to 101.

[0073] Preferably, the variant sequence retains the activity of specifically binding to human TREM2, murine TREM2 or cynomolgus monkey TREM2.

[0074] Preferably, the substitution is a conservative substitution. In some embodiments, the full-length amino acid sequence of the anti-TREM2 nanobody is any one of the amino acid sequences shown in SEQ ID NOs: 91 to 101. In a preferred embodiment, the anti-TREM2 nanobody has the amino acid sequence shown in SEQ ID NO: 94, 96, 98 or 101.

[0075] As a non-limiting example, the anti-TREM2 Nanobody or antigen-binding fragment thereof has one or more of the following properties:

[0076] (1) Capable of binding to human TREM2 or cells expressing human TREM2;

[0077] (2) able to bind to murine TREM2 or cells expressing murine TREM2;

[0078] (3) able to bind to cynomolgus monkey TREM2 or cells expressing cynomolgus monkey TREM2;

[0079] (4) competing with one or more TREM2 ligands for binding to TREM2;

[0080] (5) inhibiting and / or blocking the binding of TREM2 to ligands;

[0081] (6) inhibiting and / or blocking intracellular signal transduction mediated by TREM2 binding ligands;

[0082] (7) Induce cytokine or chemokine secretion;

[0083] (8) when containing an Fc region, having at least one of antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and antibody-mediated phagocytosis (ADCP) activity;

[0084] (9) Enhance immune response;

[0085] (10) Enhanced ADCC activity when the Fc region is included.

[0086] In some embodiments, the anti-TREM2 nanobody or antigen-binding fragment thereof has ADCC activity. ADCC can occur when the antibody binds to an antigen on the surface of a pathogenic or tumorigenic target cell.

[0087] The second aspect of the present application provides a TREM2 binding molecule, which comprises the nanobody or antigen-binding fragment thereof described in the first aspect. In some embodiments, the TREM2 binding molecule is selected from at least one of a monoclonal antibody, a polyclonal antibody, a neutralizing antibody, an antagonistic antibody, a fucosylated antibody, a heavy chain antibody (HcAb), a chimeric antibody, a monovalent antibody, a multivalent antibody, a monospecific antibody, a multispecific antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a dAb fragment, an isolated CDR region, and a scFv.

[0088] In some embodiments, the TREM2-binding molecule is a multivalent antibody comprising at least two VHH chains; preferably, the multivalent antibody is a bivalent antibody, a trivalent antibody, or a tetravalent antibody. More preferably, the multivalent antibody is a bivalent antibody.

[0089] In some embodiments, the TREM2-binding molecule is a multispecific antibody that specifically binds to TREM2 and additionally specifically binds to one or more other targets; preferably, the multispecific antibody, such as a bispecific antibody, further comprises at least one second antibody having a second binding specificity for a second target. Preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody. More preferably, the multispecific antibody is a bispecific antibody.

[0090] In some embodiments, the one or more TREM2 ligands are selected from Escherichia coli cells, apoptotic cells, nucleic acids, anionic lipids, zwitterionic lipids, negatively charged phospholipids, phosphatidylserine, sulfatides, phosphatidylcholine, sphingomyelin, membrane phospholipids, lipidated proteins, proteolipids, lipidated peptides, and lipidated amyloid-β peptide.

[0091] In some embodiments, the cytokine or chemokine is selected from IFN-γ, TNF-α, CXCL1, or CXCL10.

[0092] In some embodiments, the anti-TREM2 nanobody or antigen-binding fragment thereof is capable of specifically killing, depleting, or disabling TREM2+ myeloid cells. Optionally, the TREM2+ myeloid cells are non-stimulatory myeloid cells, for example, CD45+, HLA-DR+, CD11c+, CD14+, and BDCA3- non-stimulatory myeloid cells; optionally, the TREM2+ myeloid cells are intratumoral myeloid cells. Optionally, the TREM2+ cells are selected from dendritic cells, macrophages, monocytes, and neutrophils; the macrophages are preferably tumor-associated macrophages (TAMs).

[0093] In some embodiments, the TREM2 binding molecule has ADCC activity. ADCC can occur when an antibody binds to an antigen on the surface of a pathogenic or neoplastic target cell.

[0094] The third aspect of the present application provides a nucleic acid molecule, which encodes the anti-TREM2 nanobody or its antigen-binding fragment described in the first aspect of the present application or encodes the TREM2 binding molecule described in the second aspect.

[0095] In some embodiments, the nucleic acid molecule includes a nucleic acid molecule encoding the heavy chain variable region and / or light chain variable region of the anti-TREM2 nanobody or its antigen-binding fragment described herein, respectively.

[0096] The fourth aspect of the present application provides a recombinant vector comprising the nucleic acid molecule described in the third aspect of the present application.

[0097] The fifth aspect of the present application provides a host cell, which comprises the nucleic acid molecule described in the third aspect of the present application or the recombinant vector described in the fourth aspect of the present application.

[0098] In some embodiments, the vector is a cloning vector; in other embodiments, the vector is an expression vector. The expression vector can optionally be any expression vector capable of expressing the anti-TREM2 Nanobody or antigen-binding fragment thereof, or the TREM2 binding molecule described herein.

[0099] In a sixth aspect, the present application provides a method for preparing an anti-TREM2 nanobody or an antigen-binding fragment thereof, or the TREM2 binding molecule, comprising culturing the host cell described in the fifth aspect of the present application. In some embodiments, the method further comprises isolating and purifying the expression product of the host cell, for example, using a Protein A affinity column for purification.

[0100] In some embodiments, the host cell is a prokaryotic cell. In other embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is selected from yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments thereof. Mammalian cells are, for example, Chinese hamster ovary cells (CHO), human kidney epithelial cell line 293 cells or 293T cells. In some embodiments, the host cell is a recombinant CHO cell.

[0101] In the seventh aspect of the present application, there is provided the use of the anti-TREM2 nanobody or its antigen-binding fragment described in the first aspect of the present application, the TREM2 binding molecule described in the second aspect of the present application, the nucleic acid molecule described in the third aspect of the present application, the recombinant vector described in the fourth aspect of the present application, or the host cell described in the fifth aspect of the present application in the preparation of a drug.

[0102] In some embodiments, the drug is a drug for preventing and / or treating a disease or condition associated with TREM2. In some embodiments, the disease or condition associated with TREM2 is cancer; in some embodiments, the disease or condition associated with TREM2 is a lipid-related disease or condition; in some embodiments, the disease or condition associated with TREM2 is a fibrotic disease or condition; in some embodiments, the drug is a drug for modulating an immune response in a subject; in some specific embodiments, the drug is a drug for increasing an anti-tumor immune response in a subject and / or a drug for inducing a memory immune response in a subject.

[0103] In some embodiments, the present application relates to the anti-TREM2 nanobody or antigen-binding fragment thereof described in the first aspect of the present application, the TREM2 binding molecule described in the second aspect of the present application, the nucleic acid molecule described in the third aspect of the present application, the recombinant vector described in the fourth aspect of the present application, or the host cell described in the fifth aspect of the present application for preventing and / or treating a disease or condition related to TREM2, or regulating an immune response in a subject. Alternatively, the present application relates to a method for preventing and / or treating a disease or condition related to TREM2, or regulating an immune response in a subject, comprising administering to a subject in need thereof the anti-TREM2 nanobody or antigen-binding fragment thereof described in the first aspect of the present application, the TREM2 binding molecule described in the second aspect of the present application, the nucleic acid molecule described in the third aspect of the present application, the recombinant vector described in the fourth aspect of the present application, or the host cell described in the fifth aspect of the present application.

[0104] In the eighth aspect of the present application, there is provided the use of the anti-TREM2 nanobody or antigen-binding fragment thereof described in the first aspect, the TREM2 binding molecule described in the second aspect of the present application, the nucleic acid molecule described in the third aspect of the present application, the recombinant vector described in the fourth aspect of the present application, or the host cell described in the fifth aspect of the present application in the preparation of a reagent for diagnosing TREM2-related diseases or conditions.

[0105] In some embodiments, the TREM2-related disease or condition is cancer. In some embodiments, the TREM2-related disease or condition is a lipid-related disease or condition. In some embodiments, the TREM2-related disease or condition is a fibrotic disease or condition. In some embodiments, the agent is capable of modulating an immune response in a subject. In some specific embodiments, the agent is capable of increasing an anti-tumor immune response in a subject and / or inducing a memory immune response in a subject.

[0106] In some embodiments, the present application relates to the anti-TREM2 nanoantibody or its antigen-binding fragment described in the first aspect of the present application, the TREM2 binding molecule described in the second aspect of the present application, the nucleic acid molecule described in the third aspect of the present application, the recombinant vector described in the fourth aspect of the present application, or the host cell described in the fifth aspect of the present application for diagnosing TREM2-related diseases or conditions.

[0107] The ninth aspect of the present application provides a kit for detecting TREM2, which comprises the anti-TREM2 nanobody or its antigen-binding fragment described in the first aspect of the present application, the TREM2 binding molecule described in the second aspect of the present application, or the nucleic acid molecule described in the third aspect of the present application.

[0108] The tenth aspect of the present application provides a method for treating or preventing a disease or condition in a subject in need thereof, comprising administering to the subject the anti-TREM2 nanobody or antigen-binding fragment thereof described in the first aspect of the present application, or the TREM2 binding molecule described in the second aspect of the present application.

[0109] In some embodiments, the disease or condition is a TREM2-related disease or condition. In some embodiments, the TREM2-related disease or condition is cancer. In some embodiments, the TREM2-related disease or condition is a lipid-related disease or condition. In some embodiments, the TREM2-related disease or condition is a fibrotic disease or condition.

[0110] The eleventh aspect of the present application provides a method for enhancing the immune response of a subject, comprising administering to the subject the anti-TREM2 nanobody or its antigen-binding fragment described in the first aspect of the present application, or the TREM2 binding molecule described in the second aspect of the present application; preferably, the enhancing the immune response of the subject is to increase the subject's anti-tumor immune response and / or induce the subject's memory immune response.

[0111] In some specific embodiments, the subject is a mammal, such as a human. In some embodiments, the immune response is an adaptive immune response. In some embodiments, the immune response is an innate immune response.

[0112] In some embodiments, the cancer is selected from melanoma, renal cancer, hepatobiliary cancer, head and neck squamous cell carcinoma (HNSC), pancreatic cancer, colorectal cancer, bladder cancer, glioblastoma, prostate cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, esophageal cancer, kidney cancer, leukemia, lymphoma or mesothelioma. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is breast cancer.

[0113] In some embodiments, the lipid-related disease or disorder is selected from obesity, fatty liver disease, heart disease, stroke, atherosclerosis, diabetes, osteoarthritis, gout, sleep apnea, or hypertension.

[0114] In some embodiments, the fibrotic disease or disorder is a liver disease. Optionally, the liver disease is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

[0115] In some embodiments, the present application provides a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen binding domain comprises the anti-TREM2 nanobody or its antigen-binding fragment described herein or the TREM2 binding molecule described herein.

[0116] Beneficial effects of this application:

[0117] The anti-TREM2 nanoantibodies or antigen-binding fragments thereof of the present application have binding affinity and, when containing an Fc region, have antibody-dependent cellular cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and antibody-mediated phagocytosis (ADCP) activity, and in particular have a strong activity of inducing ADCC effects, and can be used to enhance the immune response and / or for treating individual immune-related diseases such as cancer, including using anti-TREM2 nanoantibodies or antigen-binding fragments thereof to kill non-stimulatory myeloid cells, thereby disabling the ability of non-stimulatory myeloid cells or reducing their number.

[0118] The nanoantibodies targeting TREM2 in the present application block TREM2-mediated signal transduction or eliminate tumor-associated macrophages (TAMs) expressing TREM2 in the tumor microenvironment through at least one of the ADCC effect, CDC effect, and ADCP effect (especially through the ADCC effect), thereby helping to enhance the anti-tumor immune response.

[0119] The anti-TREM2 nanobody or its antigen-binding fragment of the present application can cross-bind with human, mouse, or monkey TREM2 overexpressing cell lines or cross-bind with human or monkey TREM2 overexpressing cell lines. The anti-TREM2 nanobody or its antigen-binding fragment of the present application contains a VHH chain and has good biological activity.

[0120] The TREM2 binding molecules of the present application comprise the anti-TREM2 nanoantibodies or antigen-binding fragments thereof, and also have cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, antibody-mediated phagocytosis (ADCP) activity, binding affinity, and in particular have a strong activity of inducing ADCC effects, and can be used to enhance immune responses and / or for treating individual immune-related diseases such as cancer, including using anti-TREM2 nanoantibodies or antigen-binding fragments thereof to kill non-stimulatory myeloid cells, thereby disabling the ability of non-stimulatory myeloid cells or reducing their number.

[0121] The TREM2 binding molecules of the present application block TREM2-mediated signal transduction or eliminate tumor-associated macrophages (TAMs) expressing TREM2 in the tumor microenvironment through at least one of the ADCC effect, CDC effect, and ADCP effect (especially through the ADCC effect), thereby helping to enhance the anti-tumor immune response. The TREM2 binding molecules of the present application can also cross-bind with human, mouse, and monkey TREM2-overexpressing cell lines or cross-bind with human and monkey TREM2-overexpressing cell lines. The TREM2 binding molecules of the present application also have good biological activity.

[0122] In the present application, the inventors have devoted a lot of creative work to conduct in-depth research and transformation of camel-derived antibodies, thereby developing humanized nanobodies. The humanized nanobodies of the present application not only retain (or even improve) the functions and properties of the parent camel-derived nanobodies, such as cross-binding with human, mouse, and monkey TREM2 overexpressing cell lines or cross-binding with human and monkey TREM2 overexpressing cell lines with very high affinity, but also have a very high degree of humanization and can be safely administered to human subjects without inducing or less immunogenic response. The present application also includes nanobodies screened from the humanized nanoantibody library, which have a very high degree of humanization without modification and can also be safely administered to human subjects without inducing or less immunogenic response. BRIEF DESCRIPTION OF THE DRAWINGS

[0123] Figure 1-1 is a diagram showing the binding test results of the alpaca immune antibody library and humanized nanobody library molecules of this application with human TREM2 protein;

[0124] Figure 1-2 is a diagram showing the binding test results of the alpaca immune antibody library and humanized nanobody library molecules of this application with human TREM2 protein;

[0125] Figures 1-3 are graphs showing the binding test results of the alpaca immune antibody library and humanized nanobody library molecules of this application with human TREM2 protein;

[0126] Figures 1-4 are graphs showing the binding test results of the alpaca immune antibody library and humanized nanobody library molecules of this application with human TREM2 protein;

[0127] Figures 1-5 are diagrams showing the binding test results of the alpaca immune antibody library molecules of this application and the mouse TREM2 protein;

[0128] Figures 1-6 are graphs showing the binding test results of the alpaca immune antibody library and humanized nanobody library molecules of this application with the cynomolgus monkey TREM2 protein;

[0129] Figures 1-7 are graphs showing the binding test results of the alpaca immune antibody library and humanized nanobody library molecules of this application with the cynomolgus monkey TREM2 protein;

[0130] Figures 1-8 are graphs showing the binding test results of the alpaca immune antibody library and humanized nanobody library molecules of this application with the cynomolgus monkey TREM2 protein;

[0131] Figures 1-9 are graphs showing the binding test results of the alpaca immune antibody library and humanized nanobody library molecules of this application with the cynomolgus monkey TREM2 protein;

[0132] Figure 2-1 is a diagram showing the binding test results of the alpaca immune antibody library and humanized nanobody library molecules of this application with human TREM2 overexpressing cell lines;

[0133] Figure 2-2 is a diagram showing the binding test results of the alpaca immune antibody library and humanized nanobody library molecules of this application with human TREM2 overexpressing cell lines;

[0134] Figure 2-3 shows the binding test results of the alpaca immune antibody library and humanized nanobody library molecules of this application with human TREM2 overexpressing cell lines;

[0135] Figures 2-4 are graphs showing the binding test results of the alpaca immune antibody library and humanized nanobody library molecules of this application with human TREM2 overexpressing cell lines;

[0136] Figures 2-5 are graphs showing the binding test results of the alpaca immune antibody library and humanized nanobody library molecules of this application with mouse TREM2 overexpressing cell lines;

[0137] Figure 2-6 is a diagram showing the binding test results of the alpaca immune antibody library and humanized nanobody library molecules of this application with mouse TREM2 overexpressing cell lines;

[0138] Figures 2-7 are graphs showing the binding test results of the alpaca immune antibody library and humanized nanobody library molecules of this application with mouse TREM2 overexpressing cell lines;

[0139] Figure 2-8 is a diagram showing the binding test results of the alpaca immune antibody library and humanized nanobody library molecules of this application with the cynomolgus monkey TREM2 overexpressing cell line;

[0140] Figure 2-9 is a diagram showing the binding test results of the alpaca immune antibody library and humanized nanobody library molecules of this application with the cynomolgus monkey TREM2 overexpressing cell line;

[0141] Figure 2-10 is a diagram showing the binding test results of the alpaca immune antibody library and humanized nanobody library molecules of this application with the cynomolgus monkey TREM2 overexpressing cell line;

[0142] Figure 3-1 is a graph showing the binding test results of the humanized nanobody of the present invention and the human TREM2 overexpressing cell line;

[0143] Figure 3-2 is a graph showing the binding test results of the humanized nanobody of the present invention and the human TREM2 overexpressing cell line;

[0144] Figure 3-3 is a graph showing the binding test results of the humanized nanobody of the present invention and the human TREM2 overexpressing cell line;

[0145] Figure 3-4 is a graph showing the binding test results of the humanized nanobody of the present invention and the mouse TREM2 overexpressing cell line;

[0146] Figure 3-5 is a graph showing the binding test results of the humanized nanobody of the present invention and the mouse TREM2 overexpressing cell line;

[0147] Figure 3-6 is a graph showing the binding test results of the humanized nanobody of the present invention and the cynomolgus monkey TREM2 overexpressing cell line;

[0148] Figures 3-7 are the results of the binding test between the humanized nanobody of the present invention and the cynomolgus monkey TREM2 overexpressing cell line;

[0149] Figure 3-8 is a graph showing the binding test results of the humanized nanobody of the present invention and the cynomolgus monkey TREM2 overexpressing cell line;

[0150] Figure 4-1 is a graph showing the ADCC effect of the humanized Nanobody and humanized Nanobody library molecules of the present invention;

[0151] Figure 4-2 is a graph showing the ADCC effect of the humanized Nanobody and humanized Nanobody library molecules of the present invention;

[0152] Figure 5-1 is a graph showing the in vivo efficacy of the nanobody of the present application for tumor growth;

[0153] Figure 5-2 is a graph showing the in vivo efficacy and body weight change trends of the Nanobody of the present application;

[0154] Figure 6 is a graph showing the ADCC effect of the nanobody enhanced by mutation modification in this application. DETAILED DESCRIPTION

[0155] In order to make the present application easier to understand, the present application will be described in detail below with reference to embodiments. These embodiments are merely illustrative and do not limit the scope of application of the present application.

[0156] Unless otherwise indicated, the terms "comprise, comprise, and comprising" or their equivalents (contain, contains, containing, include, includes, including) used herein are open-ended expressions and mean that in addition to the listed elements, components, and steps, other unspecified elements, components, and steps may also be included.

[0157] Unless otherwise indicated, all numbers used herein expressing amounts of ingredients, measurements, or reaction conditions are to be understood as modified in all instances by the term "about." When used in conjunction with a percentage, the term "about" can mean, for example, ±1%, preferably ±0.5%, and more preferably ±0.1%.

[0158] Unless the context clearly indicates otherwise, singular terms herein include plural referents and vice versa. Similarly, the word "or" herein is intended to include "and" unless the context clearly indicates otherwise.

[0159] Unless otherwise indicated, the terms "optional" or "optionally" as used herein mean that the object or event it modifies exists or does not exist, or occurs or does not occur.

[0160] Unless otherwise indicated, the term "treat" as used herein means that after administration, the disease or its related symptoms can be inhibited, contained, alleviated, improved, slowed down, relieved or eliminated, the progression of the disease or its related symptoms can be delayed, postponed, slowed down, stopped or terminated, or the recurrence of the disease or its related symptoms can be prevented, controlled or reduced.

[0161] Unless otherwise indicated, the term "subject" as used herein encompasses any vertebrate, e.g., mammals and non-mammals, such as humans, non-human primates, sheep, dogs, cats, horses, cows, chickens, pigs, mice, etc. Preferably, the subject in this application is a human. The term "subject" as used herein can be used interchangeably with the term "patient."

[0162] The term "antibody" refers to a polypeptide encoded by an immunoglobulin gene and capable of binding to an antigen. When referring to an antibody, unless otherwise indicated, it is an isolated antibody.

[0163] "Conventional" antibodies are tetrameric (such as the case of G-type immunoglobulins (IgG) in humans), consisting of two identical polypeptide chains called light chains and two identical polypeptide chains called heavy chains. The variable domain is located at the N-terminus of each chain ("VH" for the heavy chain and "VL" for the light chain), and the two variable domains VH and VL are capable of recognizing antigens. Each variable domain typically contains four "framework regions" (called FR1, FR2, FR3, and FR4) and three regions directly responsible for antigen binding, called "CDRs" (called CDR1, CDR2, and CDR3). Typically, each variable domain has the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Typically, binding to the antigen is achieved through a pair of variable domains (i.e., the light chain variable domain VL and the heavy chain variable domain VH) (such as the case of human IgG), that is, six CDRs participate in the formation of one antigen binding site.

[0164] The term "heavy-chain antibody" or "HcAb" refers to a functional antibody that contains heavy chains but lacks the light chains typically found in four-chain antibodies. HcAbs are known to be produced by camelids (e.g., camels, llamas, or alpacas). Heavy-chain antibodies lack light chains and the heavy chain constant region 1 (CH1), consisting of only two heavy chains consisting of a variable region (VHH) and another constant region, connected to the constant region by a hinge-like structure. Each heavy chain of a camelid heavy-chain antibody contains one variable region (VHH) and two constant regions (CH2 and CH3).

[0165] The antigen-binding fragment of a heavy chain antibody includes an isolated CDR region, a VHH, and a single-chain heavy chain antibody. By fusing the VHH with the constant region of human IgG Fc, a heavy chain antibody can have the CH2 and CH3 of human IgG Fc. "Heavy chain variable region domain of a heavy chain antibody," "VHH," and "nanobody" are used interchangeably.

[0166] The term "chimeric antibody" broadly refers to an engineered antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. For example, a chimeric antibody comprises a variable region derived from a non-human animal antibody and a constant region from an antibody of human origin.

[0167] The framework region FR refers to a part of the variable domain that serves as a scaffold for the CDRs.

[0168] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined herein using a number of numbering schemes well known in the art, including: Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 Jan;27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309(3):657-70 (“Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm,” PNAS, 1989, 86(23):9268-9272 (“AbM” numbering scheme).

[0169] In some embodiments, the boundary of CDR or FR is different depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, and the Chothia scheme is based on structural information. The numbering of Kabat and Chothia schemes is all based on the most common antibody region sequence length, wherein insertion (for example " 30a ") is provided by inserting letters and disappearance occurs in some antibodies. These two schemes are placed in different positions with certain insertions and deletions (" indels (indels) "), thereby producing different numberings. The Contact scheme is based on the analysis of complex crystal structure, and is similar to the Chothia numbering scheme in many aspects. The AbM scheme is a compromise between Kabat and Chothia definitions, and it is based on the scheme used by the AbM antibody modeling software of Oxford Molecular. In this article, the identity percentage (homology degree) between the sequences can be determined by, for example, using the free available computer program (for example BLASTp or BLASTn with default settings) that is generally used for this purpose on the World Wide Web (for example www.ncbi.nlm.nih.gov) to compare two sequences.

[0170] In a first aspect, the present application provides an anti-TREM2 nanobody or an antigen-binding fragment thereof, comprising a VHH chain, wherein the VHH chain comprises complementary determining regions CDR1, CDR2, and CDR3 with the amino acid sequences shown below:

[0171] The CDR1 amino acid sequence is one of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 36, 39, 41, 44, 47, 49, 52, 54, 57, 59, 63, 66 or 68;

[0172] The CDR2 amino acid sequence is one of SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 37, 40, 42, 45, 48, 50, 53, 55, 58, 60, 61, 64, 67 or 69;

[0173] The CDR3 amino acid sequence is one of the sequences shown in SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 38, 43, 46, 51, 56, 62 or 65.

[0174] In some embodiments of the present application, the VHH chain comprises:

[0175] (1) CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO: 3;

[0176] (2) CDR1 shown in SEQ ID NO: 4, CDR2 shown in SEQ ID NO: 5, and CDR3 shown in SEQ ID NO: 6;

[0177] (3) CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 8, and CDR3 shown in SEQ ID NO: 9;

[0178] (4) CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 11, and CDR3 shown in SEQ ID NO: 12;

[0179] (5) CDR1 shown in SEQ ID NO: 13, CDR2 shown in SEQ ID NO: 14, and CDR3 shown in SEQ ID NO: 15;

[0180] (6) CDR1 shown in SEQ ID NO: 16, CDR2 shown in SEQ ID NO: 17, and CDR3 shown in SEQ ID NO: 18;

[0181] (7) CDR1 shown in SEQ ID NO: 19, CDR2 shown in SEQ ID NO: 20, and CDR3 shown in SEQ ID NO: 21;

[0182] (8) CDR1 shown in SEQ ID NO: 22, CDR2 shown in SEQ ID NO: 23, and CDR3 shown in SEQ ID NO: 24;

[0183] (9) CDR1 shown in SEQ ID NO: 25, CDR2 shown in SEQ ID NO: 26, and CDR3 shown in SEQ ID NO: 27;

[0184] (10) CDR1 shown in SEQ ID NO: 28, CDR2 shown in SEQ ID NO: 29, and CDR3 shown in SEQ ID NO: 30;

[0185] (11) CDR1 shown in SEQ ID NO: 31, CDR2 shown in SEQ ID NO: 32, and CDR3 shown in SEQ ID NO: 33;

[0186] (12) CDR1 shown in SEQ ID NO: 34, CDR2 shown in SEQ ID NO: 35, and CDR3 shown in SEQ ID NO: 12;

[0187] (13) CDR1 shown in SEQ ID NO: 36, CDR2 shown in SEQ ID NO: 37, and CDR3 shown in SEQ ID NO: 38;

[0188] (14) CDR1 shown in SEQ ID NO:39, CDR2 shown in SEQ ID NO:40, and CDR3 shown in SEQ ID NO:18;

[0189] (15) CDR1 shown in SEQ ID NO:41, CDR2 shown in SEQ ID NO:42, and CDR3 shown in SEQ ID NO:43;

[0190] (16) CDR1 shown in SEQ ID NO:44, CDR2 shown in SEQ ID NO:45, and CDR3 shown in SEQ ID NO:46;

[0191] (17) CDR1 shown in SEQ ID NO:47, CDR2 shown in SEQ ID NO:48, and CDR3 shown in SEQ ID NO:38;

[0192] (18) CDR1 shown in SEQ ID NO:49, CDR2 shown in SEQ ID NO:50, and CDR3 shown in SEQ ID NO:51;

[0193] (19) CDR1 shown in SEQ ID NO: 52, CDR2 shown in SEQ ID NO: 53, and CDR3 shown in SEQ ID NO: 18;

[0194] (20) CDR1 shown in SEQ ID NO: 54, CDR2 shown in SEQ ID NO: 55, and CDR3 shown in SEQ ID NO: 56;

[0195] (21) CDR1 shown in SEQ ID NO: 57, CDR2 shown in SEQ ID NO: 58, and CDR3 shown in SEQ ID NO: 9;

[0196] (22) CDR1 shown in SEQ ID NO: 59, CDR2 shown in SEQ ID NO: 60, and CDR3 shown in SEQ ID NO: 12;

[0197] (23) CDR1 shown in SEQ ID NO: 19, CDR2 shown in SEQ ID NO: 61, and CDR3 shown in SEQ ID NO: 62;

[0198] (24) CDR1 shown in SEQ ID NO: 63, CDR2 shown in SEQ ID NO: 64, and CDR3 shown in SEQ ID NO: 65;

[0199] (25) CDR1 shown in SEQ ID NO: 66, CDR2 shown in SEQ ID NO: 67, and CDR3 shown in SEQ ID NO: 12; or

[0200] (26) CDR1 shown in SEQ ID NO:68, CDR2 shown in SEQ ID NO:69, and CDR3 shown in SEQ ID NO:18.

[0201] In a preferred embodiment, the VHH chain comprises:

[0202] (1) CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO: 3;

[0203] (2) CDR1 shown in SEQ ID NO: 4, CDR2 shown in SEQ ID NO: 5, and CDR3 shown in SEQ ID NO: 6; or

[0204] (3) CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 11, and CDR3 shown in SEQ ID NO: 12.

[0205] The scope of the above-mentioned CDR has been defined according to the AbM numbering rules, but those skilled in the art will understand that CDR sequences numbered according to any one or more of the ImMunoGenTics (IMGT) numbering rules, the Kabat numbering rules, the Chothia numbering rules and the Contact numbering rules (based on analysis of available complex crystal structures) also fall within the scope of protection of this application.

[0206] In the present application, anti-TREM2 nanobodies or antigen-binding fragments thereof can also be conservatively modified. Conservative modifications refer to amino acid modifications that do not significantly affect or change the binding properties of the antibody. For example, conservative amino acid substitutions can be substituted with another amino acid of the same type (having similar chemical properties or functions). As an example, amino acids can be divided into the following categories based on their side chain properties: (1) non-polar amino acids: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar amino acids: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic amino acids: Asp (D), Glu (E); (4) basic amino acids: Lys (K), Arg (R), His (H). Alternatively, amino acids can be divided into the following categories based on common side chain properties: (1) hydrophobic amino acids: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic amino acids: Cys, Ser, Thr, Asn, Gln; (3) acidic amino acids: Asp, Glu; (4) basic amino acids: His, Lys, Arg; (5) amino acids that affect chain orientation: Gly, Pro; (6) aromatic amino acids: Trp, Tyr, Phe.

[0207] The anti-TREM2 nanobodies or antigen-binding fragments thereof and their variants described in the present application are all capable of specifically binding to TREM2.

[0208] The anti-TREM2 nanoantibodies or antigen-binding fragments thereof of the present application are isolated anti-TREM2 nanoantibodies or antigen-binding fragments thereof.

[0209] In some embodiments of the present application, the VHH chain further comprises a framework region FR, wherein the framework region comprises FR1, FR2, FR3, and FR4;

[0210] The amino acid sequence of FR1 is selected from the amino acid sequence shown in any one of SEQ ID NOs: 80 to 83, or a sequence having one or more amino acid substitutions, deletions, additions, or any combination thereof (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the amino acid sequence shown in SEQ ID NOs: 80 to 83;

[0211] The amino acid sequence of FR2 is the amino acid sequence shown in SEQ ID NO: 84, 85 or 106, or a sequence having one or more amino acid substitutions, deletions, additions, or any combination thereof (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the amino acid sequence shown in SEQ ID NO: 84, 85 or 106;

[0212] The amino acid sequence of FR3 is selected from the amino acid sequence shown in any one of SEQ ID NOs: 86 to 89 and 107, or a sequence having one or more amino acid substitutions, deletions, additions, or any combination thereof (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to any one of the amino acid sequences shown in SEQ ID NOs: 86 to 89 and 107;

[0213] The amino acid sequence of FR4 is the amino acid sequence shown in SEQ ID NO: 90, or a sequence having one or more amino acid substitutions, deletions, additions, or any combination thereof (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to the amino acid sequence shown in SEQ ID NO: 90;

[0214] Preferably, the substitution is a conservative substitution.

[0215] In some embodiments, the framework region FR comprises:

[0216] (1) FR1 shown in SEQ ID NO: 80, FR2 shown in SEQ ID NO: 84, FR3 shown in SEQ ID NO: 86, and FR4 shown in SEQ ID NO: 90;

[0217] (2) FR1 shown in SEQ ID NO: 80, FR2 shown in SEQ ID NO: 84, FR3 shown in SEQ ID NO: 87, and FR4 shown in SEQ ID NO: 90;

[0218] (3) FR1 shown in SEQ ID NO:81, FR2 shown in SEQ ID NO:84, FR3 shown in SEQ ID NO:86, and FR4 shown in SEQ ID NO:90;

[0219] (4) FR1 shown in SEQ ID NO: 82, FR2 shown in SEQ ID NO: 84, FR3 shown in SEQ ID NO: 88, and FR4 shown in SEQ ID NO: 90;

[0220] (5) FR1 shown in SEQ ID NO:83, FR2 shown in SEQ ID NO:84, FR3 shown in SEQ ID NO:86, and FR4 shown in SEQ ID NO:90;

[0221] (6) FR1 shown in SEQ ID NO:81, FR2 shown in SEQ ID NO:84, FR3 shown in SEQ ID NO:89, and FR4 shown in SEQ ID NO:90;

[0222] (7) FR1 shown in SEQ ID NO:83, FR2 shown in SEQ ID NO:84, FR3 shown in SEQ ID NO:88, and FR4 shown in SEQ ID NO:90;

[0223] (8) FR1 shown in SEQ ID NO:81, FR2 shown in SEQ ID NO:84, FR3 shown in SEQ ID NO:87, and FR4 shown in SEQ ID NO:90;

[0224] (9) FR1 shown in SEQ ID NO:83, FR2 shown in SEQ ID NO:84, FR3 shown in SEQ ID NO:87, and FR4 shown in SEQ ID NO:90;

[0225] (10) FR1 shown in SEQ ID NO: 83, FR2 shown in SEQ ID NO: 85, FR3 shown in SEQ ID NO: 87, or FR4 shown in SEQ ID NO: 90; or

[0226] (11) FR1 shown in SEQ ID NO: 83, FR2 shown in SEQ ID NO: 106, FR3 shown in SEQ ID NO: 107, and FR4 shown in SEQ ID NO: 90.

[0227] Human FR germline sequences can be obtained, for example, from the ImMunoGeneTics (IMGT) website.

[0228] In some embodiments, the VHH chain has the structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0229] In some embodiments, the VHH chain comprises any one of the amino acid sequences shown in SEQ ID NOs: 70-79 or 108-109, or a variant thereof, which has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the amino acid sequences shown in SEQ ID NOs: 70-79 or 108-109, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions) compared to SEQ ID NOs: 70-79 or 108-109.

[0230] In some embodiments, compared to the amino acid sequences shown in SEQ ID NOs: 70-79 or 108-109, the different amino acids in the amino acid sequence having at least 80% identity or the amino acid sequence having one or more amino acid substitutions, deletions or additions are mainly or entirely located in the FR region of the VHH chain.

[0231] Preferably, the variant sequence retains the activity of specifically binding to human TREM2, mouse TREM2 or cynomolgus monkey TREM2. Preferably, the substitution is a conservative substitution.

[0232] In some specific embodiments, the VHH chain comprises any one of the amino acid sequences shown in SEQ ID NOs: 70 to 79 or 108 to 109. In a preferred embodiment, the VHH chain has the amino acid sequence shown in SEQ ID NO: 73, 75 or 77.

[0233] In some embodiments, the anti-TREM2 nanobody is selected from at least one of a camelid nanobody, a humanized nanobody, and a fully human nanobody. In some specific embodiments, the anti-TREM2 nanobody is preferably a humanized nanobody.

[0234] Optionally, the TREM2 Nanobody or antigen-binding fragment thereof contains or does not contain an antibody heavy chain constant region. Preferably, the TREM2 Nanobody or antigen-binding fragment thereof contains an antibody heavy chain constant region. In some embodiments, the VHH chain of the humanized Nanobody may contain one or more fully human framework region sequences. In some embodiments, the antibody heavy chain constant region is preferably an antibody heavy chain constant region sequence without the CH1 fragment. In some embodiments, the TREM2 Nanobody or antigen-binding fragment thereof contains an antibody constant region sequence having CH2 and CH3 fragments; or the antibody heavy chain constant region is an antibody Fc region.

[0235] In some embodiments, the antibody heavy chain constant region is connected to the VHH chain with or without a linker peptide.

[0236] In some embodiments, the antibody heavy chain constant region is from human, camel, alpaca, mouse, rat, rabbit, or sheep. In some specific embodiments, the antibody heavy chain constant region is from human. In some embodiments, the antibody heavy chain constant region is selected from the heavy chain constant region of IgG, IgM, IgA, IgE, or IgD, or a variant thereof. The heavy chain constant region is preferably an IgG heavy chain constant region or a variant thereof, such as an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region or a variant thereof; the heavy chain constant region is more preferably an IgG1 heavy chain constant region or a variant thereof. In some specific embodiments, the heavy chain constant region is of the human IgG1 subtype.

[0237] In some embodiments, the heavy chain constant region or variant thereof comprises one or more mutations in the IgG1 Fc region that enhance antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the heavy chain constant region or variant thereof comprises mutations at positions 234, 235, and 243 of the IgG1 Fc region, or mutations at positions 239, 330, and 332. In some specific embodiments of the present application, the IgG1 Fc region comprises mutations of S239D, A330L, and I332E. In some embodiments, the Fc region binds to an Fcγ receptor, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, or FcγRIIIb.

[0238] In some embodiments, the full-length amino acid sequence of the anti-TREM2 Nanobody is any one of the amino acid sequences shown in SEQ ID NOs: 91 to 101, or a variant sequence thereof, and the variant sequence has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the amino acid sequences shown in SEQ ID NOs: 91 to 101, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions) compared to SEQ ID NOs: 91 to 101.

[0239] Preferably, the variant sequence retains the activity of specifically binding to human TREM2, murine TREM2 or cynomolgus monkey TREM2.

[0240] Preferably, the substitutions are conservative substitutions.

[0241] In some specific embodiments, the full-length amino acid sequence of the anti-TREM2 Nanobody is any one of the amino acid sequences shown in SEQ ID NOs: 91 to 101. In a preferred embodiment, the anti-TREM2 Nanobody has the amino acid sequence shown in SEQ ID NO: 94, 96, 98 or 101.

[0242] As a non-limiting example, the anti-TREM2 Nanobody or antigen-binding fragment thereof has one or more of the following properties:

[0243] (1) Capable of binding to human TREM2 or cells expressing human TREM2;

[0244] (2) able to bind to murine TREM2 or cells expressing murine TREM2;

[0245] (3) able to bind to cynomolgus monkey TREM2 or cells expressing cynomolgus monkey TREM2;

[0246] (4) competing with one or more TREM2 ligands for binding to TREM2;

[0247] (5) inhibiting and / or blocking the binding of TREM2 to ligands;

[0248] (6) inhibiting and / or blocking intracellular signal transduction mediated by TREM2 binding ligands;

[0249] (7) Induce cytokine or chemokine secretion;

[0250] (8) when containing an Fc region, having at least one of antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and antibody-mediated phagocytosis (ADCP) activity;

[0251] (9) Enhance immune response;

[0252] (10) Enhanced ADCC activity when the Fc region is included.

[0253] In some embodiments, the anti-TREM2 nanobody or antigen-binding fragment thereof has ADCC activity. ADCC can occur when the antibody binds to an antigen on the surface of a pathogenic or tumorigenic target cell.

[0254] The second aspect of the present application provides a TREM2 binding molecule, which comprises the nanobody or antigen-binding fragment thereof described in the first aspect. In some embodiments, the TREM2 binding molecule is selected from at least one of a monoclonal antibody, a polyclonal antibody, a neutralizing antibody, an antagonistic antibody, a fucosylated antibody, a heavy chain antibody (HcAb), a chimeric antibody, a monovalent antibody, a multivalent antibody, a monospecific antibody, a multispecific antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a dAb fragment, an isolated CDR region, and a scFv.

[0255] In some embodiments, the TREM2-binding molecule is a multivalent antibody comprising at least two VHH chains; preferably, the multivalent antibody is a bivalent antibody, a trivalent antibody, or a tetravalent antibody. More preferably, the multivalent antibody is a bivalent antibody.

[0256] In some embodiments, the TREM2-binding molecule is a multispecific antibody that specifically binds to TREM2 and additionally specifically binds to one or more other targets. Preferably, the multispecific antibody, such as a bispecific antibody, further comprises at least one second antibody having a second binding specificity for a second target. Preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody. More preferably, the multispecific antibody is a bispecific antibody.

[0257] In some embodiments, the one or more TREM2 ligands are selected from Escherichia coli cells, apoptotic cells, nucleic acids, anionic lipids, zwitterionic lipids, negatively charged phospholipids, phosphatidylserine, sulfatides, phosphatidylcholine, sphingomyelin, membrane phospholipids, lipidated proteins, proteolipids, lipidated peptides, and lipidated amyloid-β peptide.

[0258] In some embodiments, the cytokine or chemokine is selected from IFN-γ, TNF-α, CXCL1, or CXCL10.

[0259] In some embodiments, the anti-TREM2 nanobody or antigen-binding fragment thereof is capable of specifically killing, depleting or disabling TREM2+ myeloid cells. Optionally, the TREM2+ myeloid cells are non-stimulatory myeloid cells, for example: CD45+, HLA-DR+, CD11c+, CD14+ and BDCA3- non-stimulatory myeloid cells. Optionally, the TREM2+ myeloid cells are intratumoral myeloid cells. Optionally, the TREM2+ cells are selected from dendritic cells, macrophages, monocytes and neutrophils; the macrophages are preferably tumor-associated macrophages (TAMs).

[0260] In some embodiments, the TREM2 binding molecule has ADCC activity. ADCC can occur when an antibody binds to an antigen on the surface of a pathogenic or neoplastic target cell.

[0261] To assess the activity of the ADCC effect of the target molecule, an in vitro ADCC assay such as that described in U.S. Patent No. 5,500,362 or 5,821,337 can be performed. Effector cells that can be used for the assay include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the activity of the ADCC effect of the target molecule can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 95: 652-656 (1998).

[0262] The third aspect of the present application provides a nucleic acid molecule, which encodes the anti-TREM2 nanobody or its antigen-binding fragment described in the first aspect of the present application or encodes the TREM2 binding molecule described in the second aspect.

[0263] In some embodiments, the nucleic acid molecule includes a nucleic acid molecule encoding the heavy chain variable region and / or light chain variable region of the anti-TREM2 nanobody or its antigen-binding fragment described in the present application, or a nucleic acid molecule encoding the TREM2 binding molecule described in the second aspect.

[0264] The fourth aspect of the present application provides a recombinant vector comprising the nucleic acid molecule described in the third aspect of the present application.

[0265] The term "vector" refers to a tool for expressing a target gene in a host cell, such as a plasmid vector, a cosmid vector, and a viral vector such as a phage vector, a lentiviral vector, an adenoviral vector, a retroviral vector, and an adeno-associated viral vector. A recombinant vector can be constructed by or by manipulating a plasmid (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, etc.), a phage (e.g., λgt4λB, λ 8 Charon, λΔz1, M13, etc.) or viral vectors (e.g., SV40, etc.), which are commonly used in the art.

[0266] In a recombinant vector, a nucleic acid molecule can be operably linked to a promoter. The term "operably linked" is intended to refer to a functional connection between a target nucleotide sequence and an expression control sequence (e.g., a promoter sequence). When "operably linked," a regulatory element can control the transcription and / or translation of the target polynucleotide.

[0267] In some embodiments, the vector is a cloning vector; in other embodiments, the vector is an expression vector. The expression vector can optionally be any expression vector capable of expressing the anti-TREM2 Nanobody or antigen-binding fragment thereof described herein.

[0268] In order to use in a host such as a prokaryotic or eukaryotic cell, a recombinant vector can be constructed accordingly. For example, when constructing a vector as an expression vector for a prokaryotic host, the vector generally includes a strong promoter for transcription (e.g., pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. On the other hand, expression vectors for eukaryotic hosts include replication origins that can be operated in eukaryotic cells, such as f1 replication origin, SV40 replication origin, pMB1 replication origin, adenovirus replication origin, AAV replication origin, and BBV replication origin, but are not limited thereto. In addition, expression vectors generally include promoters derived from mammalian cell genomes (e.g., metallothionein promoters) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV, etc.), and a polyadenylation sequence as a transcription termination sequence. Recombinant cells can be prepared by introducing the recombinant vector into a suitable host cell. Any host cell known in the art can be used in the present disclosure as long as it allows for continuous cloning and expression of recombinant vectors in a stable manner. Examples of prokaryotic host cells that can be used in the present disclosure can be selected from Escherichia coli (E. coli) such as E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus species such as Bacillus subtilis and Bacillus thuringiensis, and Enterobacteriaceae strains such as Salmonella typhimurium, Serratia marcescens and various Pseudomonas species. Eukaryotic host cells that can be used for transformation can be selected from, but are not limited to, Saccharomyces cerevisiae, insect cells, and animal cells, such as Sp2 / 0, CHO (Chinese Hamster Ovary) K1, CHO DG44, CHO S, CHO DXB11, CHO GS 8 KO, PER.C6, W138, BHK, COS.7, 293, HepG2, Huh7, 3T3, RIN, MDCK etc. Can use the well-known method in the related art that nucleic acid molecule or the recombinant vector carrying nucleic acid molecule is introduced (transfected) into host cell.For example, when host cell is prokaryotic cell, CaCl or electroporation method can be used to carry out this transfection.For eukaryotic host cell, can use but not limited to microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection or particle bombardment to realize gene introduction.

[0269] In order to select transformed host cells, the phenotype associated with the selection marker can be utilized according to methods well known in the art. For example, when the selection marker is a gene that confers resistance to certain antibiotics, the host cells can be grown in culture medium in the presence of antibiotics to select target transformants.

[0270] The fifth aspect of the present application provides a host cell, which comprises the nucleic acid molecule described in the third aspect of the present application or the recombinant vector described in the fourth aspect of the present application.

[0271] The host cell in this application can be any suitable host cell for expressing anti-TREM2 nanobodies or antigen-binding fragments thereof. In some embodiments, the host cell is a prokaryotic cell. In other embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies or antigen-binding fragments thereof. Mammalian cells are, for example, Chinese hamster ovary cells (CHO), human kidney epithelial cell line 293 cells or 293T cells. In some embodiments, the host cell is a recombinant CHO cell.

[0272] In a fifth aspect, the present application provides a method for preparing an anti-TREM2 nanobody or an antigen-binding fragment thereof, comprising culturing the host cell described in the fourth aspect of the present application. Preferably, the method further comprises isolating and purifying the expression product of the host cell.

[0273] Use of the anti-TREM2 nanobody or its antigen-binding fragment described in the first aspect of this application, the TREM2 binding molecule described in the second aspect of this application, the nucleic acid molecule described in the third aspect of this application, the recombinant vector described in the fourth aspect of this application, or the host cell described in the fifth aspect of this application in the preparation of drugs.

[0274] In some embodiments, the drug is a drug for preventing and / or treating a disease or condition associated with TREM2. In some embodiments, the disease or condition associated with TREM2 is cancer. In some embodiments, the disease or condition associated with TREM2 is a lipid-related disease or condition. In some embodiments, the disease or condition associated with TREM2 is a fibrotic disease or condition.

[0275] In some embodiments, the drug is a drug that modulates the subject's immune response. In some specific embodiments, the drug is a drug that increases the subject's anti-tumor immune response and / or induces a subject's memory immune response.

[0276] In the eighth aspect of the present application, there is provided the use of the anti-TREM2 nanobody or antigen-binding fragment thereof described in the first aspect, the TREM2 binding molecule described in the second aspect of the present application, the nucleic acid molecule described in the third aspect of the present application, the recombinant vector described in the fourth aspect of the present application, or the host cell described in the fifth aspect of the present application in the preparation of a reagent for diagnosing TREM2-related diseases or conditions.

[0277] In some embodiments, the TREM2-related disease or condition is cancer. In some embodiments, the TREM2-related disease or condition is a lipid-related disease or condition. In some embodiments, the TREM2-related disease or condition is a fibrotic disease or condition. In some embodiments, the agent is capable of modulating an immune response in a subject. In some specific embodiments, the agent is capable of increasing an anti-tumor immune response in a subject and / or inducing a memory immune response in a subject.

[0278] The ninth aspect of the present application provides a kit for detecting TREM2, which comprises the anti-TREM2 nanobody or its antigen-binding fragment described in the first aspect of the present application, the TREM2 binding molecule described in the second aspect of the present application, or the nucleic acid molecule described in the third aspect of the present application.

[0279] The tenth aspect of the present application provides a method for treating or preventing a disease or condition in a subject in need thereof, comprising administering to the subject the anti-TREM2 nanobody or antigen-binding fragment thereof described in the first aspect of the present application, or the TREM2 binding molecule described in the second aspect of the present application.

[0280] In some embodiments, the disease or condition is a TREM2-related disease or condition. In some embodiments, the TREM2-related disease or condition is cancer. In some embodiments, the TREM2-related disease or condition is a lipid-related disease or condition. In some embodiments, the TREM2-related disease or condition is a fibrotic disease or condition.

[0281] In an eleventh aspect of the present application, there is provided a method for enhancing an immune response in a subject, comprising administering to the subject the anti-TREM2 nanobody or antigen-binding fragment thereof described in the first aspect of the present application, or the TREM2 binding molecule described in the second aspect of the present application. Preferably, the enhancing the immune response in the subject is to increase the subject's anti-tumor immune response and / or induce a memory immune response in the subject.

[0282] In some specific embodiments, the subject is a mammal, such as a human. In some embodiments, the immune response is an adaptive immune response. In some embodiments, the immune response is an innate immune response.

[0283] Cancer cells that can be killed by the anti-TREM2 nanobodies or antigen-binding fragments thereof, and TREM2 binding molecules of the present application include, but are not limited to, cancer cells from the following: bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, pancreas, testicles, tongue, cervix or uterus.

[0284] Additionally, cancer may specifically be of the following histological types, although not limited thereto: tumor, malignant neoplastic disease.

[0285] In some embodiments, the cancer is selected from melanoma, renal cancer, hepatobiliary cancer, head and neck squamous cell carcinoma (HNSC), pancreatic cancer, colorectal cancer, bladder cancer, glioblastoma, prostate cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, esophageal cancer, kidney cancer, leukemia, lymphoma or mesothelioma. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is breast cancer.

[0286] In some embodiments, the lipid-related disease or disorder is selected from obesity, fatty liver disease, heart disease, stroke, atherosclerosis, diabetes, osteoarthritis, gout, sleep apnea, or hypertension.

[0287] In some embodiments, the fibrotic disease or disorder is a liver disease. Optionally, the liver disease is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

[0288] The appropriate dosage of the anti-TREM2 nanobodies or antigen-binding fragments thereof, TREM2 binding molecules, and pharmaceutical compositions described herein can be determined by a clinician based on factors such as the age, weight, sex, general condition, and severity of the disease of the subject; they can be administered by any conventional route known in the art, such as parenteral, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal administration.

[0289] Those skilled in the art can adjust the dosage regimen to provide the optimal desired response (e.g., clinical response) based on common knowledge in the art. For example, a single large dose can be administered, multiple divided doses can be administered over time, or the single dose can be reduced or increased in proportion to the severity of the therapeutic situation. For example, when the antibody is administered as a sustained-release formulation, it can be administered at a reduced frequency.

[0290] Example

[0291] The solution of the present application will be further described below with reference to embodiments. However, those skilled in the art will appreciate that the scope of protection of the present application is not limited to these embodiments.

[0292] Unless otherwise specified, the reagents, materials, or instruments involved in the following examples are commercially available.

[0293] Example 1. Expression of positive control antibody

[0294] Pionyr Immunotherapeutics' TREM2 antibody PY314 was used as a positive control antibody. The variable region amino acid sequence of PY314 is described in WO2019118513. It uses the IgG1 subtype and enhances its ADCC effect through the heavy chain constant region S239D / I332E / A330L mutations. Its sequence is shown below:

[0295] PY314 heavy chain sequence, SEQ NO ID: 102:

[0296] PY314 light chain sequence, SEQ NO ID: 103:

[0297] The light chain and heavy chain genes of PY314 were synthesized and subcloned into the pcDNA3.4 expression vector, respectively, and transfected into Expi CHO cells. After culture at 37°C and 5% CO2, the cell culture was collected, centrifuged, and purified using a Protein A affinity column to obtain the PY314 antibody.

[0298] Example 2: Construction and screening of alpaca immune antibody library

[0299] Alpacas were immunized with human TREM2 protein (purchased from Kaika) and cross-immunization with human TREM2 protein and mouse TREM2 protein (purchased from Kaika) for 5 times, every other week. The TREM2 protein for the first immunization was emulsified with Freund's complete adjuvant, with an antigen amount of 500 μg / mouse. The subsequent immunization adjuvant was Freund's incomplete adjuvant, with an antigen amount of 500 μg / mouse. The injection method was subcutaneous multi-point injection. Two weeks after the end of the immunization, blood was collected for serum titer detection. After the titer test was qualified, 100 mL of blood was collected to isolate PBMC cells. RNA was extracted according to the instructions of the RNA extraction kit (purchased from Omega) and cDNA was obtained by reverse transcription according to the instructions of the reverse transcription kit (purchased from TaKaRa). The gene fragment encoding the VHH fragment was amplified by PCR using universal degenerate primers (synthesized by Jin Weizhi). Finally, the VHH gene fragment was inserted into the phage display vector by double enzyme digestion and ligation. The ligation product was recovered using a recovery kit (purchased from Omega), and finally transformed into competent Escherichia coli SS320 (purchased from Lucigen) using an electroporator (purchased from Bio-Rad), and coated on a 2-YT solid plate containing ampicillin resistance. Thus, an alpaca immune antibody library was constructed, and the antibody library was packaged using helper phage M13KO7 (purchased from NEB).

[0300] The alpaca immune antibody library was screened by solid phase and liquid phase cross-screening, and specific VHH antibodies against human, mouse, and crab-eating macaque TREM2 were enriched by trypsin elution.

[0301] Alpaca immune antibody library selection (Kingfisher method):

[0302] (1) Background subtraction: The phage suspension of the alpaca immune antibody library was diluted with 5% BSA and blocked, and incubated with Dynabeads (purchased from Invitrogen), and the phages after negative screening incubation were collected.

[0303] (2) Dynabeads blocking and coating: The magnetic beads were bound and washed using the Kingfisher magnetic bead screening system (purchased from Thermo scientific), and 5% BSA was incubated with the magnetic beads.

[0304] (3) Positive screening: The phage suspension collected after negative screening was incubated with Dynabeads coated and blocked with biotin-labeled TREM2 protein (purchased from Kaijia), and the binding and washing were performed using the Kingfisher magnetic bead screening system.

[0305] (4) Elution: Use Trypsin (purchased from Shanghai Biotechnology) to elute the phage.

[0306] (5) Infection of library plates: After elution, the phage solution was thoroughly mixed with logarithmic phase SS320 cells and incubated at 37°C for 30 min. The cells were then spread onto 2YT-Car+-Tet+ plates and cultured in a 37°C incubator overnight.

[0307] (6) Titer: The eluted phage solution was diluted 10-fold with logarithmic phase SS320 cells, incubated at 37°C for 30 min, mixed and dropped 2 μL onto a plate, and cultured in a 37°C incubator overnight.

[0308] (7) Statistics: Calculate phage input, output, etc., prepare the input phage by scraping and perform two rounds of screening. Each round of screening uses a 3-fold gradient antigen concentration reduction to obtain antibody clones with higher affinity.

[0309] Alpaca immune antibody library selection (immunotube method):

[0310] (1) Background subtraction: The phage suspension of the alpaca immune antibody library was blocked with 5% PBSM.

[0311] (2) Immunotube coating and blocking: TREM2 protein (purchased from KaiXia) was coated with 5% PBSM for blocking.

[0312] (3) Positive screening: incubate the phage suspension with the protein-coated and sealed immunotubes, and perform binding and washing according to the immunotube screening system method.

[0313] (4) Elution: Use Trypsin (purchased from Shanghai Biotechnology) to elute the phage.

[0314] (5) Infection of library plates: After elution, the phage solution was thoroughly mixed with logarithmic phase SS320 cells and incubated at 37°C for 30 min. The cells were then spread onto 2YT-Car+-Tet+ plates and cultured in a 37°C incubator overnight.

[0315] (6) Titer: The eluted phage solution was diluted 10-fold with logarithmic phase SS320 cells, incubated at 37°C for 30 min, mixed and dropped 2 μL onto a plate, and cultured in a 37°C incubator overnight.

[0316] (7) Statistics: Calculate phage input, output, etc., prepare the input phage by scraping and perform three rounds of screening. Each round of screening uses a 3-fold gradient antigen concentration reduction to obtain antibody clones with higher affinity.

[0317] The enrichment of the above different output pools was tested by ELISA using the following steps:

[0318] (1) Coating: 2 μg / mL human, mouse, or cynomolgus monkey TREM2 (all purchased from Kejia) was added to the ELISA plate at a rate of 30 μL per well, incubated at 4°C overnight, and washed three times with PBST.

[0319] (2) Blocking: Block with 5% PBSM at room temperature for 1 hour, and wash the plate three times with PBST.

[0320] (3) Primary antibody incubation: Dilute the VHH or phage expression supernatant of the above different output sets with 5% PBSM in a 5-fold gradient, add 30 μL / well of the diluted supernatant, incubate at room temperature for 1 hour, and wash the plate 3 times with PBST.

[0321] (4) Secondary antibody incubation: Add the secondary antibody Anti-mouse-VHH-HRP (purchased from Sino biological) diluted 1:5000 in PBS to the VHH supernatant, and add the secondary antibody Anti-M13-HRP (purchased from Sino biological) diluted 1:20000 in 5% PBSM to the phage, 30 μL / well, incubate at room temperature for 1 hour, and wash the plate 6 times with PBST.

[0322] (5) Termination: Add 30 μL / well of TMB (purchased from SurModics) to develop color at room temperature for 5-10 minutes, then add 30 μL / well of stop solution (2 M sulfuric acid) to terminate the reaction, and read the data using a microplate reader at OD 450.

[0323] Select output sets that can cross-enrich with human, mouse, and cynomolgus monkey TREM2 (all purchased from Kaijia) at the VHH level or phage level, or can cross-enrich with human and cynomolgus monkey TREM2. Pick monoclonal clones from the output sets of the first and second rounds of screening for phage ELISA screening against human, mouse, and cynomolgus monkey TREM2. A total of 1596 clones were picked in the primary screening, and 185 positive clones with human, mouse, and monkey cross-binding activity at the protein ELISA level were obtained, and 518 positive clones with human and monkey cross-binding activity were obtained, of which 13 were sequence-unique molecules. Heavy chain antibodies were constructed for 11 of these molecules, using the human IgG1 subtype to obtain heavy chain antibodies VHH-Fc (hIgG1).

[0324] The heavy chain antibody construction process is as follows: the genes of the 11 VHH molecules mentioned above and the genes of the heavy chain constant region CH2 and CH3 fragments of human IgG1 (Fc region of human IgG1) are cloned into the pcDNA3.4 expression vector respectively, and the heavy chain antibody expression plasmid with the correct sequence is obtained by sequencing analysis. TMThe purified heavy chain antibody expression plasmid was transfected into Expi CHO cells according to the instructions of CHO transfection reagent (purchased from Thermo). After incubation at 37°C and 5% CO2, the supernatant was collected by centrifugation and purified by Protein A affinity chromatography to obtain the heavy chain antibody.

[0325] Example 3: Screening of humanized nanobody library

[0326] The humanized nanoantibody library (constructed by Sanyou Bio) was screened by solid-phase and liquid-phase cross-screening, and specific VHH antibodies against human, mouse, and crab-eating macaque TREM2 were enriched by trypsin and glycine-hydrochloric acid elution.

[0327] Humanized Nanobody Library Panning (Kingfisher Method)

[0328] (1) Background subtraction: The humanized nanobody library phage suspension was diluted with a final concentration of 2.5% BSA and blocked, incubated with Dynabeads, and the phages after negative screening incubation were collected.

[0329] (2) Dynabeads blocking and coating: The magnetic beads were bound and washed using the Kingfisher magnetic bead screening system (purchased from Thermo scientific), and 5% BSA was incubated with the magnetic beads.

[0330] (3) Positive screening: The phage suspension collected after negative screening was incubated with Dynabeads coated and blocked with biotin-labeled TREM2 protein (purchased from Kaijia), and the binding and washing were performed using the Kingfisher magnetic bead screening system.

[0331] (4) Elution: Use Trypsin (purchased from Shanghai Biotechnology) to elute the phage.

[0332] (5) Infection of library plates: After elution, the phage solution was thoroughly mixed with logarithmic phase SS320 cells and incubated at 37°C for 30 min. The cells were then spread onto 2YT-Car+-Tet+ plates and cultured in a 37°C incubator overnight.

[0333] (6) Titer: The eluted phage solution was diluted 10-fold with logarithmic phase SS320 cells, incubated at 37°C for 30 min, mixed and dropped 2 μL onto a plate, and cultured in a 37°C incubator overnight.

[0334] (7) Statistics: Calculate phage input, output, etc., prepare the input phage by scraping and perform three rounds of screening. Each round of screening uses a 3-fold gradient antigen concentration reduction to obtain antibody clones with higher affinity.

[0335] Humanized Nanobody Library Panning (Immunotube Method)

[0336] (1) Background subtraction: The humanized nanobody library phage suspension was blocked with 5% PBSM.

[0337] (2) Immunotube coating and blocking: TREM2 protein (purchased from KaiXia) was coated with 5% PBSM for blocking.

[0338] (3) Positive screening: incubate the phage suspension with the protein-coated and sealed immunotubes, and perform binding and washing according to the immunotube screening system method.

[0339] (4) Elution: Use Trypsin (purchased from Shanghai Biotechnology) to elute the phage.

[0340] (5) Infection of library plates: After elution, the phage solution was thoroughly mixed with logarithmic phase SS320 cells and incubated at 37°C for 30 min. The cells were then spread onto 2YT-Car+-Tet+ plates and cultured in a 37°C incubator overnight.

[0341] (6) Titer: The eluted phage solution was diluted 10-fold with logarithmic phase SS320 cells, incubated at 37°C for 30 min, mixed and dropped 2 μL onto a plate, and cultured in a 37°C incubator overnight.

[0342] (7) Statistics: Calculate phage input, output, etc., prepare the input phage by scraping and perform three rounds of screening. Each round of screening uses a 3-fold gradient antigen concentration reduction to obtain antibody clones with higher affinity.

[0343] The enrichment of different output sets was detected by ELISA using the steps described in Example 2, and output sets that could cross-enrich with human, mouse, and cynomolgus monkey TREM2 at the VHH level or phage level, or that could cross-enrich with human and cynomolgus monkey TREM2, were selected. Monoclonal clones of the screened output sets were selected for primary screening against human, mouse, and cynomolgus monkey TREM2 by phage ELISA. A total of 1,140 clones were selected in the primary screening, and 33 positive clones with human, mouse, and monkey cross-binding activity at the protein ELISA level were obtained, and 177 positive clones with human and monkey cross-binding activity were obtained, of which 55 were sequence-unique molecules. Heavy chain antibodies of 42 of these molecules were constructed using the human IgG1 subtype, and the heavy chain antibody construction process was as described in Example 2. The representative nanobody CDR sequences obtained by screening the alpaca immune antibody library and the humanized nanobody library in Example 2 and Example 3 are shown in Table 1, among which nanobody C76 was screened from the alpaca immune antibody library and is a camel-derived antibody, requiring further antibody humanization (see Example 6 for details). The remaining nanobodies were screened from the humanized nanoantibody library and have a high degree of humanization, so no further antibody humanization is required.

[0344] Table 1. CDR sequences of alpaca immune antibody library and humanized nanobody library molecules

[0345] Example 4. ELISA binding activity of alpaca immune antibody library and humanized nanobody library molecules

[0346] The binding activity of the alpaca immune antibody library and the humanized nanoantibody library molecules to human, mouse, and crab-eating macaque TREM2 was detected by ELISA according to the following steps.

[0347] (1) Coating: Add 2 μg / mL human, mouse, or cynomolgus monkey TREM2 (all purchased from Kaijia) to the ELISA plate at a rate of 30 μL per well, incubate at 4°C overnight, and wash the plate three times with PBST.

[0348] (2) Blocking: Block with 5% PBSM at room temperature for 2 hours, and wash the plate three times with PBST.

[0349] (3) Primary antibody incubation: dilute the antibody to be tested in 1% PBSM in a 3-fold gradient to a final concentration of 3, 0.333, 0.111, 0.037, 0.0123, 0.00412, 0.00137, and 0.00045 μg / mL, respectively. Add 30 μL / well of the diluted antibody and incubate at room temperature for 1 hour. Wash the plate three times with PBST.

[0350] (4) Secondary antibody incubation: Add secondary antibody Anti-human-Fc-IgG-HRP (purchased from abcam) diluted 1:8000 with 1% milk, incubate at room temperature for 1 hour, and wash the plate 6 times with PBST.

[0351] (5) Color development and termination: 30 μL / well of TMB (purchased from SurModics) was added to develop color at room temperature for 5-10 minutes, and then 30 μL / well of stop solution (2 M sulfuric acid) was added to terminate the reaction. The OD 450 data was read using a microplate reader.

[0352] Figures 1-1 to 1-4 show the binding of alpaca immune antibody library and humanized nanoantibody library molecules to human TREM2 protein, Figure 1-5 shows the binding of alpaca immune antibody library molecules to mouse TREM2 protein, and Figures 1-6 to 1-9 show the binding of alpaca immune antibody library and humanized nanoantibody library molecules to crab-eating macaque TREM2 protein. It can be seen that some alpaca immune antibody library and humanized nanoantibody library molecules can cross-bind to human, mouse, and monkey TREM2 proteins or cross-bind to human and monkey TREM2 proteins.

[0353] Example 5: FACS binding activity of alpaca immune antibody library and humanized nanobody library molecules

[0354] Flow cytometry was used to detect the binding activity of the alpaca immune antibody library and the humanized nanoantibody library molecules with CHO-K1 cells overexpressing human TREM2, mouse TREM2, and crab-eating macaque TREM2 (all purchased from Yoshiman Gene), respectively, as follows.

[0355] (1) According to 1×10 5 Cells were seeded at 100 μg / well, centrifuged and washed with FACS buffer (DPBS + 1% BSA).

[0356] (2) Add 100 μL of the alpaca immune antibody library or humanized nanobody library molecules after concentration gradient dilution (initial concentration 50 μg / mL, 4-fold dilution with FACS buffer) to the cells, incubate at 4°C for 60 minutes, and wash the cells twice by centrifugation with FACS buffer.

[0357] (3) Add 100 μL of PE-labeled anti-Human Fc (purchased from Abcam) to the cells and incubate at 4°C for 30 min.

[0358] (4) Resuspend the cells in FACS buffer and detect using flow cytometry.

[0359] Figures 2-1 to 2-4 show the binding of the alpaca immune antibody library and the humanized nanobody library molecules to human TREM2-overexpressing cell lines, Figures 2-5 to 2-7 show the binding of the alpaca immune antibody library and the humanized nanobody library molecules to mouse TREM2-overexpressing cell lines, and Figures 2-8 to 2-10 show the binding of the alpaca immune antibody library and the humanized nanobody library molecules to cynomolgus monkey TREM2-overexpressing cell lines, where PY314 antibody was used as a positive control. It can be seen that the alpaca immune antibody library and the humanized nanobody library molecules can cross-bind to human, mouse, and monkey TREM2-overexpressing cell lines or to human and monkey TREM2-overexpressing cell lines.

[0360] Example 6: Humanization of Alpaca Immune Antibody Library Molecules

[0361] Nanobody C76, obtained from the alpaca immune antibody library, was humanized. Humanization design was performed based on the amino acid sequence of the alpaca-derived nanobody VHH chain, retaining the CDR region sequence of the original antibody. Based on the results of germline alignment and antibody structure simulation, a humanized antibody template was selected. After humanization, the framework region was backmutated to design candidate humanized antibody sequences. Ten humanized antibody molecules were designed based on the nanobody C76 antibody sequence. The VHH chains of the designed humanized antibodies were gene-synthesized and subcloned into the pcDNA3.4-hIgG1 expression vector. After sequencing verification, the constructed vector was transiently expressed in Expi CHO cells and purified using Protein A affinity chromatography to obtain the humanized nanobody. The VHH chain sequence and full-length sequence (i.e., the heavy chain sequence) of the C76 humanized nanobody are shown in Table 2.

[0362] Table 2. C76 humanized nanobody VHH chain sequence and full-length sequence

[0363] Example 7: ELISA binding activity of humanized Nanobodies

[0364] The binding activity of the C76 humanized Nanobody to human, mouse, and cynomolgus monkey TREM2 was detected by ELISA according to the steps described in Example 4, and compared with the parent Nanobody. Table 3 shows the binding of the C76 humanized Nanobody to human, mouse, and cynomolgus monkey TREM2 proteins. It can be seen that the C76 humanized Nanobody retains good binding activity compared to the parent Nanobody.

[0365] Table 3. EC binding of C76 humanized nanobody ELISA 50 Results Summary Table

[0366] Example 8: FACS binding activity of humanized Nanobodies

[0367] According to the steps described in Example 5, flow cytometry was used to detect the binding activity of C76 humanized nanoantibodies to CHO-K1 cells overexpressing human TREM2, mouse TREM2, and crab-eating monkey TREM2.

[0368] Figures 3-1 to 3-3 show the binding of humanized nanobodies to human TREM2-overexpressing cell lines, Figures 3-4 to 3-5 show the binding of humanized nanobodies to mouse TREM2-overexpressing cell lines, and Figures 3-6 to 3-8 show the binding of humanized nanobodies to crab-eating macaque TREM2-overexpressing cell lines. It can be seen that the C76 humanized nanobody retains good FACS binding activity compared to the parent nanobody.

[0369] Example 9: ADCC effect of nanobodies

[0370] The following steps were used to detect the ability of nanoantibodies to mediate ADCC effects, using human PBMC cells as effector cells and CHO-K1 cells overexpressing human TREM2 (purchased from Yoshimitsu Gene) as target cells.

[0371] (1) According to 1×10 4 hu-TREM2-CHO-K1 cells were inoculated at 50 μL / well and incubated at 37°C for 30 min. At the same time, target cell plus effector cell wells (only target cells and effector cells were added), target cell non-lysis wells (only target cells were added), and target cell full lysis wells (only target cells were added) were set.

[0372] (2) The nanobody was serially diluted with 1840 medium containing 1% FBS and added to the cells at 50 μL / well to achieve final concentrations of 2, 0.2, 0.02, and 0.002 μg / mL, respectively. The cells were incubated at 37°C for 30 min.

[0373] (3) According to 5×10 5 cells / well, 100 μL / well PBMC cells (prepared by Sanyou Biotechnology) were added and incubated at 37°C for 4 h.

[0374] (4) Add 10 μL of 10% Triton X-100 to the target cell lysis well and incubate at 37°C for 30 min.

[0375] (5) After centrifugation, 60 μL of supernatant was taken from each well and mixed with LDH reagent in equal proportions. The mixture was incubated at room temperature for 30 min and the OD value was measured at 490 nm.

[0376] (6) Calculate the lysis rate according to the following formula: lysis rate = (OD value of sample well - OD value of target cell plus effector cell well) / (OD value of target cell fully lysed well - OD value of target cell non-lysed well)

[0377] Figures 4-1 and 4-2 show the ADCC effect results of nanobodies. It can be seen that humanized nanobodies C76-VHH4, C76-VHH6, C76-VHH8 and humanized nanobody library molecules E028 and D29 all have strong activity in inducing ADCC effect.

[0378] Example 10: In vivo efficacy study of nanobodies

[0379] The efficacy of nanoantibodies C76-VHH4 and E028 in a mouse tumor model was studied in vivo. To better simulate the efficacy in humans, TREM2 humanized C57BL / 6 mice (purchased from Shanghai Model Organisms) with the human TREM2 gene were used. Female TREM2 humanized mice were subcutaneously inoculated with MC38 mouse colon cancer cells at a dose of 2.5E6 per mouse to establish a subcutaneous tumor-bearing model. The tumors were grown to 100 mm. 3 Around 3 d, the mice were divided and dosed according to the schedule shown in Table 5. During the study, the survival and general condition of the mice were observed every day, and the tumor volume and body weight were measured twice a week.

[0380] The tumor growth curve and body weight change trend graph at the end of the study are shown in Figure 5-1 and Figure 5-2, respectively. The tumor growth inhibition rate (TGI) is shown in Table 4. The research results show that nanoantibodies C76-VHH4 and E028 both have significant tumor inhibitory effects, and the tumor inhibitory effects of C76-VHH4 and E028 are comparable to those of PY314. There was no significant decrease in the body weight of animals in each group during the experiment.

[0381] Table 4. In vivo efficacy study plan for nanobodies

[0382] Example 12: ADCC-enhancing modification of nanobodies linked to Fc regions

[0383] In order to further enhance the ADCC effect of the nanobody C76-VHH4 linked to the Fc region of human IgG1, the constant region of the antibody was subjected to S239D / I332E / A330L mutation modification and represented by HC. C76-VHH4-HC represents the C76-VHH4 molecule after mutation modification, and the sequence is shown in Table 5.

[0384] Table 5. ADCC-enhanced Nanobody sequences

[0385] Example 13: ADCC effect of nanobodies after mutation modification

[0386] The ability of the mutated Nanobody C76-VHH4-HC to mediate ADCC effects was detected using the steps described in Example 9, and the Nanobody C76-VHH4 before mutation was used as a control. The final concentrations of C76-VHH4-HC added to the cells were 5, 1.667, 0.556, 0.185, 0.0617, 0.0206, 0.0069, and 0.0023 nM, respectively, and the final concentrations of C76-VHH4 added to the cells were 100, 20, 4, 0.8, 0.16, 0.032, 0.0064, and 0.0013 nM, respectively.

[0387] Figure 6 shows the ADCC effect results of the Nanobody after mutation modification. It can be seen that the activity of the ADCC effect induced by the mutation-modified C76-VHH4-HC is significantly enhanced compared with that before mutation modification.

[0388] The sequences of the present application are listed in Table 6 below.

[0389] Table 6

[0390] For the purposes of description and disclosure, all patents, patent applications, and other publications are expressly incorporated herein by reference. These publications are provided solely because their disclosure predates the filing date of the present application. All statements regarding the dates of these documents or the representations of their contents are based on the information available to the applicant and do not constitute any admission as to the correctness of the dates of these documents or the contents of these documents. Furthermore, any citation of these publications herein does not constitute an admission that such publications become part of the common general knowledge in the art in any country.

[0391] Those skilled in the art will recognize that the scope of the present application is not limited to the various specific implementation modes and examples described above, but that various modifications, replacements, or recombinations can be made without departing from the spirit of the present application, which all fall within the scope of protection of the present application.

Claims

1. An anti-TREM2 nanobody or an antigen-binding fragment thereof, comprising a VHH chain, wherein the VHH chain comprises complementary determining regions CDR1, CDR2 and CDR3 whose amino acid sequences are as follows: The CDR1 amino acid sequence is one of the sequences shown in SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 36, 39, 41, 44, 47, 49, 52, 54, 57, 59, 63, 66 or 68; The CDR2 amino acid sequence is one of the sequences shown in SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 37, 40, 42, 45, 48, 50, 53, 55, 58, 60, 61, 64, 67 or 69; The CDR3 amino acid sequence is one of the sequences shown in SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 38, 43, 46, 51, 56, 62 or 65.

2. The anti-TREM2 nanobody or antigen-binding fragment thereof according to claim 1, wherein The VHH chain comprises: (1) CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO: 3; (2) CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6; (3) CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8, and CDR3 shown in SEQ ID NO:9; (4) CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 11, and CDR3 shown in SEQ ID NO: 12; (5) CDR1 shown in SEQ ID NO: 13, CDR2 shown in SEQ ID NO: 14, and CDR3 shown in SEQ ID NO: 15; (6) CDR1 shown in SEQ ID NO: 16, CDR2 shown in SEQ ID NO: 17, and CDR3 shown in SEQ ID NO: 18; (7) CDR1 shown in SEQ ID NO: 19, CDR2 shown in SEQ ID NO: 20, and CDR3 shown in SEQ ID NO: 21; (8) CDR1 shown in SEQ ID NO:22, CDR2 shown in SEQ ID NO:23, and CDR3 shown in SEQ ID NO:24; (9) CDR1 shown in SEQ ID NO:25, CDR2 shown in SEQ ID NO:26, and CDR3 shown in SEQ ID NO:27; (10) CDR1 shown in SEQ ID NO:28, CDR2 shown in SEQ ID NO:29, and CDR3 shown in SEQ ID NO:30; (11) CDR1 shown in SEQ ID NO:31, CDR2 shown in SEQ ID NO:32, and CDR3 shown in SEQ ID NO:33; (12) CDR1 shown in SEQ ID NO:34, CDR2 shown in SEQ ID NO:35, and CDR3 shown in SEQ ID NO:12; (13) CDR1 shown in SEQ ID NO:36, CDR2 shown in SEQ ID NO:37, and CDR3 shown in SEQ ID NO:38; (14) CDR1 shown in SEQ ID NO:39, CDR2 shown in SEQ ID NO:40, and CDR3 shown in SEQ ID NO:18; (15) CDR1 shown in SEQ ID NO:41, CDR2 shown in SEQ ID NO:42, and CDR3 shown in SEQ ID NO:43; (16) CDR1 shown in SEQ ID NO:44, CDR2 shown in SEQ ID NO:45, and CDR3 shown in SEQ ID NO:46; (17) CDR1 shown in SEQ ID NO:47, CDR2 shown in SEQ ID NO:48, and CDR3 shown in SEQ ID NO:38; (18) CDR1 shown in SEQ ID NO:49, CDR2 shown in SEQ ID NO:50, and CDR3 shown in SEQ ID NO:51; (19) CDR1 shown in SEQ ID NO:52, CDR2 shown in SEQ ID NO:53, and CDR3 shown in SEQ ID NO:18; (20) CDR1 shown in SEQ ID NO:54, CDR2 shown in SEQ ID NO:55, and CDR3 shown in SEQ ID NO:56; (21) CDR1 shown in SEQ ID NO:57, CDR2 shown in SEQ ID NO:58, and CDR3 shown in SEQ ID NO:9; (22) CDR1 shown in SEQ ID NO:59, CDR2 shown in SEQ ID NO:60, and CDR3 shown in SEQ ID NO:12; (23) CDR1 shown in SEQ ID NO: 19, CDR2 shown in SEQ ID NO: 61, and CDR3 shown in SEQ ID NO: 62; (24) CDR1 shown in SEQ ID NO:63, CDR2 shown in SEQ ID NO:64, and CDR3 shown in SEQ ID NO:65; (25) CDR1 shown in SEQ ID NO:66, CDR2 shown in SEQ ID NO:67, and CDR3 shown in SEQ ID NO:12; or (26) CDR1 shown in SEQ ID NO:68, CDR2 shown in SEQ ID NO:69, and CDR3 shown in SEQ ID NO:18; Preferably, the VHH chain comprises: (1) CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO: 3; (2) CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6; or (3) CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:

12.

3. The anti-TREM2 nanobody or antigen-binding fragment thereof according to claim 1 or 2, wherein: The VHH chain also comprises FR1, FR2, FR3, FR4: The FR1 has an amino acid sequence shown in any one of SEQ ID NOs: 80 to 83, or a sequence having one or more amino acid substitutions, deletions, additions, or any combination thereof compared to the amino acid sequences shown in SEQ ID NOs: 80 to 83; The FR2 has an amino acid sequence as shown in SEQ ID NO: 84, 85 or 106, or a sequence having one or more amino acid substitutions, deletions, additions or any combination thereof compared to the amino acid sequence as shown in SEQ ID NO: 84, 85 or 106; The FR3 has an amino acid sequence shown in any one of SEQ ID NOs: 86 to 89 and 107, or a sequence having one or more amino acid substitutions, deletions, additions or any combination thereof compared to any one of the amino acid sequences shown in SEQ ID NOs: 86 to 89 and 107; The FR4 has the amino acid sequence shown in SEQ ID NO: 90, or a sequence having one or more amino acid substitutions, deletions, additions, or any combination thereof compared to the amino acid sequence shown in SEQ ID NO:

90.

4. The anti-TREM2 nanobody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein The VHH chain comprises any one of the amino acid sequences shown in SEQ ID NOs: 70 to 79 or a variant sequence thereof, wherein the variant sequence has an amino acid sequence that is at least 80% identical to any one of the amino acid sequences shown in SEQ ID NOs: 70 to 79 or 108 to 109 or has one or more amino acid substitutions, deletions or additions compared to SEQ ID NOs: 70 to 79 or 108 to 109; Preferably, the variant sequence retains the activity of specifically binding to human TREM2, mouse TREM2 or cynomolgus monkey TREM2; Preferably, the substitution is a conservative substitution; Preferably, compared with the amino acid sequences shown in SEQ ID NOs: 70 to 79 or 108 to 109, the amino acids with at least 80% identity or the amino acids with substitution, deletion or addition of one or more amino acids are mainly or entirely located in the FR region of the VHH chain.

5. The anti-TREM2 nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein The VHH chain has the amino acid sequence shown in SEQ ID NO: 73, 75 or 77.

6. The anti-TREM2 nanobody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein The anti-TREM2 nanobody is selected from at least one of a camel-derived nanobody, a humanized nanobody, and a fully human nanobody, and the anti-TREM2 nanobody is preferably a humanized nanobody; Optionally, the TREM2 Nanobody or antigen-binding fragment thereof contains or does not contain an antibody heavy chain constant region; Preferably, the TREM2 nanobody or its antigen-binding fragment comprises an antibody heavy chain constant region, and the antibody heavy chain constant region is preferably an antibody heavy chain constant region sequence without a CH1 fragment; more preferably, the TREM2 nanobody or its antigen-binding fragment comprises an antibody constant region sequence having CH2 and CH3 fragments; or the antibody heavy chain constant region is an antibody Fc region; Preferably, the antibody heavy chain constant region is connected to the VHH chain with or without a connecting peptide; Preferably, the antibody heavy chain constant region is from human, camel, alpaca, mouse, rat, rabbit or sheep; Preferably, the antibody heavy chain constant region is selected from the heavy chain constant region of IgG, IgM, IgA, IgE or IgD or a variant thereof, and the heavy chain constant region is preferably the heavy chain constant region of IgG or a variant thereof; the heavy chain constant region is more preferably the heavy chain constant region of IgG1 or a variant thereof. Preferably, the anti-TREM2 nanobody also comprises a heavy chain constant region having an amino acid sequence as shown in SEQ ID NO: 104, SEQ ID NO:

105.

7. The anti-TREM2 nanobody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein The full-length amino acid sequence of the anti-TREM2 Nanobody is any one of the amino acid sequences shown in SEQ ID NOs: 91 to 101 or a variant sequence thereof, wherein the variant sequence has an amino acid sequence that is at least 80% identical to any one of the amino acid sequences shown in SEQ ID NOs: 91 to 101 or has one or more amino acid substitutions, deletions or additions compared to SEQ ID NOs: 91 to 101; Preferably, the anti-TREM2 nanobody has the amino acid sequence shown in SEQ ID NO: 94, 96, 98 or 101.

8. A TREM2 binding molecule, wherein: It comprises a Nanobody or an antigen-binding fragment thereof as described in any one of claims 1 to 7; Preferably, the TREM2 binding molecule is selected from at least one of a monoclonal antibody, a polyclonal antibody, a neutralizing antibody, an antagonistic antibody, a fucosylated antibody, a heavy chain antibody, a chimeric antibody, a monovalent antibody, a multivalent antibody, a monospecific antibody, a multispecific antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a dAb fragment, an isolated CDR region, and a scFv.

9. The TREM2 binding molecule of claim 8, wherein The TREM2 binding molecule is a multivalent antibody, comprising at least two VHH chains; Preferably, the multivalent antibody is a bivalent antibody, a trivalent antibody or a tetravalent antibody. More preferably, the multivalent antibody is a bivalent antibody.

10. The TREM2 binding molecule according to claim 8 or 9, wherein The TREM2 binding molecule is a multispecific antibody that specifically binds to TREM2 and additionally specifically binds to one or more other targets; Preferably, the multispecific antibody further comprises at least one second antibody having a second binding specificity for a second target. Preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody or a tetraspecific antibody. More preferably, the multispecific antibody is a bispecific antibody.

11. A nucleic acid molecule encoding the anti-TREM2 nanobody or antigen-binding fragment thereof as described in any one of claims 1-7 or the TREM2 binding molecule as described in any one of claims 8-10.

12. A recombinant vector comprising the nucleic acid molecule according to claim 11.

13. A host cell comprising the nucleic acid molecule of claim 11 or the recombinant vector of claim 12.

14. An anti-TREM2 Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 7, a TREM2 binding molecule according to any one of claims 8 to 10, a nucleic acid molecule according to claim 11, a recombinant vector according to claim 12, or a host cell according to claim 13 for diagnosing, preventing and / or treating a TREM2-related disease or disorder, or modulating an immune response in a subject; Preferably, the disease or disorder is cancer, a lipid-related disease or disorder, or a fibrotic disease or disorder; Preferably, regulating the subject's immune response is increasing the subject's anti-tumor immune response and / or inducing the subject's memory immune response.

15. A kit for detecting TREM2, comprising the anti-TREM2 nanobody or its antigen-binding fragment as described in any one of claims 1-7, the TREM2 binding molecule as described in any one of claims 8-10, or the nucleic acid molecule as described in claim 11.

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