CN117964768A

CN117964768A - Anti-TREM 2 antibodies and uses thereof

Info

Publication number: CN117964768A
Application number: CN202410389594.3A
Authority: CN
Inventors: 王永辉; 闵皓; 尹巧汕
Original assignee: Shanghai Hongcheng Pharmaceutical Co ltd
Current assignee: Shanghai Hongcheng Pharmaceutical Co ltd
Priority date: 2024-04-02
Filing date: 2024-04-02
Publication date: 2024-05-03

Abstract

The invention discloses an anti-TREM 2 antibody and application thereof. The invention belongs to the technical field of biology, and aims to provide an isolated antibody or an antigen binding fragment thereof which binds to human myeloid cell trigger receptor 2. The antibody or antigen-binding fragment thereof provided by the present invention binds to human TREM2 and exhibits more excellent properties.

Description

Anti-TREM 2 antibodies and uses thereof

Technical Field

The invention belongs to the technical field of biology, and relates to an anti-TREM 2 antibody and application thereof.

Background

Immunotherapy brings great hope for all human to overcome cancer, but the current immunotherapy taking PD-1/PD-L1 as the main benefit group is smaller and still does not meet clinical needs, so finding the next immunotherapy target is extremely important so as to start a new immunotherapy era.

Myeloid cell trigger receptor 2 (trigger receptor expressed on myeloid cells-2, TREM 2) is expressed on the surface of M2 type tumor-associated macrophages in tumor immune microenvironment, and plays a role in tumor immunosuppression by regulating the functions of tumor-associated macrophages (TAMs); however, TAM in the microenvironment is often one of the reasons for poor efficacy of immune checkpoints such as PD-1/PD-L1, so TREM2 and immune checkpoints have combined value from a biological perspective. Preclinical gene knockout and neutralizing antibody studies also indicate that TREM2 mab and PD-1 mab have a synergistic effect in a sarcoma and colon carcinoma syngeneic model. The development of a new generation of anti-TREM 2 antibodies has important scientific and application values. Screening for TREM2 antibodies with a stronger affinity would enhance the therapeutic effect.

Disclosure of Invention

The technical problem to be solved by the present invention is to provide an isolated antibody or antigen binding fragment thereof that binds to human myeloid cell trigger receptor 2.

In a first aspect, the present invention provides an antibody or antigen binding fragment thereof having binding specificity to myeloid cell trigger receptor 2 (TREM 2), wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, and wherein:

(a) The HCDR1 is an amino acid sequence shown as SEQ ID NO. 2, the HCDR2 is an amino acid sequence shown as SEQ ID NO. 3, the HCDR3 is an amino acid sequence shown as SEQ ID NO. 4, the LCDR1 is an amino acid sequence shown as SEQ ID NO. 6, the LCDR2 is an amino acid sequence shown as SEQ ID NO. 7, and the LCDR3 is an amino acid sequence shown as SEQ ID NO. 8;

(b) The HCDR1 is an amino acid sequence shown as SEQ ID NO. 10, the HCDR2 is an amino acid sequence shown as SEQ ID NO. 11, the HCDR3 is an amino acid sequence shown as SEQ ID NO. 12, the LCDR1 is an amino acid sequence shown as SEQ ID NO. 14, the LCDR2 is an amino acid sequence shown as SEQ ID NO. 15, and the LCDR3 is an amino acid sequence shown as SEQ ID NO. 16; or (b)

(C) The HCDR1 is the amino acid sequence shown in SEQ ID NO. 18, the HCDR2 is the amino acid sequence shown in SEQ ID NO. 19, the HCDR3 is the amino acid sequence shown in SEQ ID NO. 20, the LCDR1 is the amino acid sequence shown in SEQ ID NO. 22, the LCDR2 is the amino acid sequence shown in SEQ ID NO. 23, and the LCDR3 is the amino acid sequence shown in SEQ ID NO. 24.

In some embodiments, the antibody or antigen binding fragment thereof comprises the CDRs shown in (a), and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No. 1, or an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID No. 1, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, as compared to the amino acid sequence shown in SEQ ID No. 1; and/or the light chain variable region comprises the amino acid sequence shown in SEQ ID No. 5, or an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID No. 5, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, as compared to the amino acid sequence shown in SEQ ID No. 5;

The antibody or antigen binding fragment thereof comprises the CDRs shown in (b), and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No. 9, or an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID No. 9, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, as compared to the amino acid sequence shown in SEQ ID No. 9; and/or the light chain variable region comprises the amino acid sequence set forth in SEQ ID No. 13, or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID No. 13, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, as compared to the amino acid sequence set forth in SEQ ID No. 13; or (b)

The antibody or antigen binding fragment thereof comprises the CDRs shown in (c), and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No. 17, or an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID No. 17, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, as compared to the amino acid sequence shown in SEQ ID No. 17; and/or the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 21, or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO. 21, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, as compared to the amino acid sequence set forth in SEQ ID NO. 21.

In some embodiments, the antibody or antigen-binding fragment thereof of any one of the above, further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

In some embodiments, the antibody or antigen binding fragment thereof of any one of the above, the light chain constant region is a kappa chain or lambda chain constant region.

In some embodiments, the antibody or antigen binding fragment thereof of any one of the above, the heavy chain constant region is selected from IgG, igM, igA, igE or IgD classes.

In some embodiments, the antibody or antigen binding fragment thereof of any one of the above, the heavy chain constant region is a heavy chain constant region selected from the subclasses IgG1, igG2, igG3, or IgG 4.

In some embodiments, the antibody or antigen-binding fragment thereof of any one of the above, further comprises a human IgG1 heavy chain constant region or variant thereof, and/or a human kappa light chain constant region or variant thereof.

In some embodiments, the antibody or antigen binding fragment thereof of any one of the above is a chimeric, humanized antibody.

In some embodiments, the antibody or antigen binding fragment thereof of any one of the above is a Fab, fv, or scFv fragment.

In some embodiments, the antibody or antigen-binding fragment thereof of any one of the above is a monoclonal antibody (including full length monoclonal antibodies), a polyclonal antibody, or a multispecific antibody (e.g., bispecific antibody).

In some embodiments, the antibody or antigen-binding fragment thereof of any one of the above is a chimeric antibody, comprising a light chain and a heavy chain;

The heavy chain comprises an amino acid sequence shown as SEQ ID NO. 27, or an amino acid sequence having at least 90% identity to the amino acid sequence shown as SEQ ID NO. 27, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, as compared to the amino acid sequence shown as SEQ ID NO. 27; the light chain comprises the amino acid sequence shown in SEQ ID NO. 28, or an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO. 28, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, as compared to the amino acid sequence shown in SEQ ID NO. 28;

The heavy chain comprises an amino acid sequence shown as SEQ ID NO. 29, or an amino acid sequence having at least 90% identity to the amino acid sequence shown as SEQ ID NO. 29, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, as compared to the amino acid sequence shown as SEQ ID NO. 29; the light chain comprises an amino acid sequence shown as SEQ ID NO. 30, or an amino acid sequence having at least 90% identity to the amino acid sequence shown as SEQ ID NO. 30, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, as compared to the amino acid sequence shown as SEQ ID NO. 30;

The heavy chain comprises the amino acid sequence shown in SEQ ID NO. 31, or an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO. 31, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, as compared to the amino acid sequence shown in SEQ ID NO. 31; the light chain comprises the amino acid sequence shown in SEQ ID NO. 32, or an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO. 32, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, as compared to the amino acid sequence shown in SEQ ID NO. 32;

the heavy chain comprises an amino acid sequence shown as SEQ ID NO. 35, or an amino acid sequence having at least 90% identity to the amino acid sequence shown as SEQ ID NO. 35, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, as compared to the amino acid sequence shown as SEQ ID NO. 35; the light chain comprises the amino acid sequence shown in SEQ ID NO. 36, or an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO. 36, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, as compared to the amino acid sequence shown in SEQ ID NO. 36;

The heavy chain comprises an amino acid sequence shown in SEQ ID NO. 37, or an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO. 37, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, as compared to the amino acid sequence shown in SEQ ID NO. 37; the light chain comprises the amino acid sequence shown in SEQ ID NO. 38, or an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO. 38, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, as compared to the amino acid sequence shown in SEQ ID NO. 38; or (b)

The heavy chain comprises an amino acid sequence shown as SEQ ID NO. 39, or an amino acid sequence having at least 90% identity to the amino acid sequence shown as SEQ ID NO. 39, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, as compared to the amino acid sequence shown as SEQ ID NO. 39; the light chain comprises the amino acid sequence shown in SEQ ID NO. 40, or an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO. 40, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions, or any combination thereof, as compared to the amino acid sequence shown in SEQ ID NO. 40.

In some embodiments, the antibody or antigen binding fragment thereof of any of the above has antibody-dependent cell-mediated cytotoxicity (ADCC).

In a second aspect, the invention provides a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as described in any one of the above;

The nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

In a third aspect, the invention provides a recombinant vector comprising the nucleic acid molecule described above.

In a fourth aspect, the present invention provides a recombinant cell comprising the above nucleic acid molecule and/or the above recombinant vector;

The recombinant cells are not human, nor animal and plant species at the various stages of formation and development.

The term "identity" as used herein may be assessed with the naked eye or in computer software such as the software program described in Ausubel et al eds. (2007) at Current Protocols in Molecular Biology. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position. The identity between two or more sequences may be expressed in percent (%), which may be used to evaluate the identity between related sequences. A polynucleotide sequence or amino acid sequence has a percentage (e.g., 90%, 95%, 98%, or 99%) of "sequence identity" with another sequence, meaning that when the sequences are aligned, the percentage of bases or amino acids in the two sequences that are compared are identical.

In a fifth aspect, the invention provides a method of making an antibody or antigen-binding fragment thereof of the invention, comprising culturing a recombinant cell comprising a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of the invention, under conditions suitable for expression of the antibody.

In some embodiments, the method further comprises: recovering the antibody or antigen binding fragment thereof from the recombinant cells or culture medium.

In a sixth aspect, the invention provides a composition comprising an antibody or antigen-binding fragment thereof of the invention or a nucleic acid molecule of the invention, a recombinant vector, a recombinant cell, and a pharmaceutically acceptable carrier.

In some embodiments, the above composition is a pharmaceutical composition.

In some embodiments, the composition of any of the above further comprises a checkpoint inhibitor;

preferably, the checkpoint inhibitor is selected from the group consisting of: checkpoint inhibitors of T cells, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-LAG-3 antibodies.

In a seventh aspect, the invention provides the use of an antibody or antigen binding fragment thereof according to any one of the preceding claims, a nucleic acid molecule according to any one of the preceding claims, a recombinant vector according to any one of the preceding claims, a recombinant cell according to any one of the preceding claims and/or a composition according to any one of the preceding claims for the preparation of a product as shown in any one of the following:

(1) Detecting a TREM2 product;

(2) A product that stimulates or enhances an immune response;

(3) A product for preventing and/or treating cancer;

Preferably, the cancer is selected from the group consisting of: melanoma, renal cancer, hepatobiliary cancer, squamous carcinoma of head and neck, pancreatic cancer, colon cancer, bladder cancer, glioblastoma, prostate cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, esophageal cancer, and mesothelioma.

The antibodies or antigen binding fragments thereof provided herein bind to human TREM2 and exhibit a number of superior properties, including the following:

1. The antibody or the antigen binding fragment thereof provided by the invention is combined with human TREM2 with high affinity, so that the immune response can be regulated and controlled;

2. The mutation of the Fc of the antibody or antigen binding fragment thereof of the present invention to S239D/A330L/I332E enhances the ADCC effect induced by the wild-type hIgG1 Fc.

Drawings

FIG. 1 shows the binding activity of a murine monoclonal antibody against human TREM2 to the 293T cell line 293T-hTREM2-NFAT-RE-EGFP expressing human TREM 2.

FIG. 2 shows the binding activity of a murine monoclonal antibody against Human TREM2 to Human TREM2 Protein (His Tag).

FIG. 3 shows the binding activity of a murine monoclonal antibody against human TREM2 to Cynomolgus TREM2 Protein (His Tag).

FIG. 4 shows the binding activity of a murine monoclonal antibody against human TREM2 to a Mouse TREM2 Protein (His Tag).

FIG. 5 is the binding activity of anti-human TREM2 chimeric antibodies to 293T-hTREM2-NFAT-RE-EGFP of a 293T cell line expressing human TREM 2.

FIG. 6 shows the binding activity of an anti-Human TREM2 chimeric antibody to Human TREM2 Protein (His Tag).

FIG. 7 shows the binding activity of the anti-human TREM2 chimeric antibody to Cynomolgus TREM2 Protein (His Tag).

FIG. 8 shows the binding activity of the anti-human TREM2 chimeric antibody to Mouse TREM2 Protein (His Tag).

Detailed Description

The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Unless otherwise indicated, all technical means used in the examples are routine in the art or according to the experimental methods suggested by the manufacturers of the kits and instruments. Reagents and biological materials used in the examples were obtained commercially unless otherwise specified.

Abbreviations and definitions

Unless otherwise indicated, the following terms shall have the meanings set forth below. Other terms or abbreviations have the meaning well known in the art.

An "antibody" refers to any form of antibody that exhibits a desired biological activity (e.g., inhibits ligand binding to its receptor or receptor signaling induced by inhibition of ligand). Thus, "antibody" is used in its broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), fully humanized, primatized, chimeric antibodies, single chain antibodies, and the like.

An "antigen binding fragment" refers to a portion of an antibody, such as F (ab ') 2, F (ab) 2, fab', fab, fv, scFv, and the like. Regardless of its structure, the antibody fragment binds to the same antigen that is recognized by the intact antibody. The term "antigen binding fragment" includes aptamers, stereoisomers, and diabodies. The term "antigen binding fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.

"Fab fragment" consists of a light chain and a heavy chain CH1 and variable domains. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

The "Fc region" contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic effect of the CH3 domain.

The "Fv region" comprises variable regions from both the heavy and light chains, but lacks constant regions.

"Single chain Fv antibody" (or "scFv antibody") refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, fv polypeptides additionally comprise a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding. For an overview of scFv, see U.S. Pat. No. 6423538.

Those skilled in the art will appreciate that classes of antibody heavy chains include gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε), some of which are also subclasses (e.g., γ1- γ4). The nature of this chain determines the "class" of antibody as IgG, igM, igA, igD or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., igG1, igG2, igG3, igG4, igG5, etc., have been well characterized and the functional specificities conferred are also known. All immunoglobulin classes are within the scope of the present disclosure. In some embodiments, the immunoglobulin molecule is an IgG class. IgG typically comprises two identical light chain polypeptides having a molecular weight of about 23,000 daltons and two identical heavy chain polypeptides having a molecular weight of about 53,000-70,000. The four chains are linked by disulfide bonds in a "Y" configuration, wherein the light chain starts at the "Y" mouth and continues through the variable region surrounding the heavy chain. Antibodies in the form of IgG1 are a subclass of IgG, with the heavy chain being the gamma 1 subclass. In some embodiments, the presently disclosed antibodies are IgG1.

As used herein, the term "heavy chain constant region" includes amino acid sequences derived from an immunoglobulin heavy chain. The polypeptide comprising a heavy chain constant region comprises at least one of: a CH1 domain, a hinge (e.g., upper hinge region, middle hinge region, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen binding polypeptide for use in the present disclosure may comprise: a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of the present disclosure comprises a polypeptide chain comprising a CH3 domain. Furthermore, antibodies for use in the present disclosure may lack at least a portion of a CH2 domain (e.g., all or a portion of a CH2 domain). As described above, one of ordinary skill in the art will appreciate that the heavy chain constant region can be modified such that it differs in amino acid sequence from a naturally occurring immunoglobulin molecule.

"Hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region comprises the following amino acid residues: amino acid residues from a "complementarity determining region" or "CDR" defined by the sequence alignment. "framework" residues or "FR" residues are variable domain residues other than the hypervariable region residues defined herein.

An "isolated antibody" is an antibody that is separated from all or part of a natural environmental component. The contaminating components of its natural environment are substances that interfere with the diagnostic or therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody is purified to the following extent: (1) More than 95% by weight of antibody, such as more than 99% by weight, as determined by the Lowry method; (2) A degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by use of a cup sequencer; or (3) homogeneity by SDS-PAGE stained with Coomassie blue or silver under reducing or non-reducing conditions. The isolated antibody includes an antibody in situ within the recombinant cell because at least one component of the antibody's natural environment will not be present. The isolated antibodies are typically prepared by at least one purification step. In some embodiments, the purity of the isolated antibody is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range between any two of these values (inclusive) or any value therein.

"Nucleic acid" or "polynucleotide" refers to a nucleic acid consisting of a single nucleotide: a polymer molecule consisting of adenine (a), cytosine (c), guanine (g), thymine (t) (or uracil (u) in RNA), such as DNA, RNA or modifications thereof. The nucleic acid molecule may be a natural nucleic acid molecule or a synthetic nucleic acid molecule or a combination of one or more natural nucleic acid molecules and one or more synthetic nucleic acid molecules. Examples of nucleic acids include, but are not limited to: genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.

An "isolated nucleic acid molecule" is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule. The isolated nucleic acid molecule differs from its naturally occurring form or environment. Thus, an isolated nucleic acid molecule is distinguished from a nucleic acid molecule that is present in its natural cell. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in a cell, such as a cell that normally expresses an antibody, e.g., the nucleic acid molecule is located at a chromosomal location different from that of a native cell.

"Monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, each antibody comprising the population being identical. Monoclonal antibodies are highly specific and can be directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include a plurality of different antibodies directed against a plurality of different determinants (epitopes), each monoclonal antibody is directed against only a single determinant on the antigen.

The term "chimeric antibody" refers to an antibody in which a portion of the heavy and/or light chains are derived from one source or species, while the remainder of the heavy and/or light chains are derived from a different source or species.

"Immune cells" include cells of hematopoietic origin and that play a role in the immune response. The immune cells include: b lymphocytes, T lymphocytes, natural killer cells, monocytes, macrophages, eosinophils, mast cells, basophils and granulocytes.

As used herein, a sequence "variant" refers to a sequence that differs from the sequence shown at one or more amino acid residues but retains the biological activity of the resulting molecule.

"Amino acid" refers to an organic compound containing both amino and carboxyl groups, such as an alpha-amino acid, which may be encoded by a nucleic acid directly or in precursor form. A single amino acid is encoded by a nucleic acid consisting of three nucleotides, a so-called codon or base triplet. The same amino acid may be encoded by different codons called "degeneracy of the genetic code". Amino acids include natural amino acids and unnatural amino acids. Natural amino acids include alanine (three-letter code: ala, one-letter code: a), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

"Conservatively substituted variant" or "conservative amino acid substitution" refers to an amino acid substitution known to those of skill in the art that is made without normally altering the biological activity of the resulting molecule. In general, it is well recognized by those skilled in the art that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity. Conservative substitutions may be made by amino acid substitutions that contain chemically similar side chains, such as: 1) Aliphatic side chain: glycine, alanine, valine, leucine and isoleucine; 2) Aliphatic hydroxyl side chains: serine and threonine; 3) Amide-containing side chains: asparagine and glutamine; 4) Aromatic side chain: phenylalanine, tyrosine, and tryptophan; 5) Basic side chain: lysine, arginine, and histidine; 6) Acidic side chain: aspartic acid and glutamic acid.

The term "about" as used herein means that the value is within an acceptable error range for the particular value being determined by one of ordinary skill in the art, which value depends in part on how the measurement or determination is made (i.e., the limits of the measurement system). Or "about" may mean a range of up to ±20%, such as ±10%, 5% or ±1% range. Unless otherwise indicated, when a particular value is found in the present disclosure and claims, the meaning of "about" should be assumed to be within an acceptable error range for that particular value.

When referring to ligand/receptor, antibody/antigen or other binding pair, "specific" binding refers to determining the presence or absence of a binding reaction for a protein, such as TREM2, in a heterogeneous population of proteins and/or other biological agents. Thus, under the specified conditions, a particular ligand/antigen binds to a particular receptor/antibody and does not bind in significant amounts to other proteins present in the sample.

The term "encoding" when applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide if it can be transcribed and/or translated to produce an mRNA of the polypeptide and/or fragment thereof in its native state or when manipulated by methods well known to those of skill in the art. The antisense strand is the complement of such a nucleic acid and from which the coding sequence can be deduced.

When referring to an animal, human, subject, cell, tissue, organ or biological fluid with "administration" and "treatment" it is meant that the exogenous drug, therapeutic, diagnostic agent or composition is contacted with the animal, human, subject, cell, tissue, organ or biological fluid. "administration" and "treatment" may refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating the cell includes contacting the agent with the cell and contacting the agent with a fluid, wherein the fluid is in contact with the cell. "administration" and "treatment" also mean in vitro and ex vivo treatment of cells, e.g., by agents, diagnostic agents, binding compositions, or by other cells.

The term "treatment" includes amelioration or cessation of a disorder or symptoms thereof. Treatment includes inhibition, e.g., reducing the overall frequency of onset of the disorder or symptoms thereof.

The term "preventing" includes avoiding the onset of a disorder or symptoms thereof.

The term "therapeutically effective amount" or "effective amount" as used herein refers to an amount effective to prevent or slow down a disease or disorder to be treated when an anti-TREM 2 antibody or antigen-binding fragment thereof is administered alone or in combination with another therapeutic agent to a cell, tissue or subject. A therapeutically effective dose further refers to an amount of the antibody or antigen binding fragment thereof sufficient to cause a alleviation of symptoms, such as treatment, cure, prevention, or alleviation of a relevant medical condition, or an increase in the rate of treatment, cure, prevention, or alleviation of the condition. The effective amount for a particular subject can vary depending upon a variety of factors, such as the disease to be treated, the overall health of the patient, the route of administration, and the dosage and severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects. When administered to an individual an active ingredient administered alone, a therapeutically effective amount refers to the individual ingredient. When a combination is administered, a therapeutically effective amount refers to the amount of the combination of active ingredients that produces a therapeutic effect, whether administered in combination, serially or simultaneously.

Pharmaceutical composition

The invention also provides a pharmaceutical composition. Such compositions comprise an effective amount of an antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

In some embodiments, the term "pharmaceutically acceptable carrier" refers to a substance listed in the pharmacopoeia approved by a regulatory agency of the government or otherwise generally recognized for use in animals, and particularly in humans. Furthermore, a "pharmaceutically acceptable carrier" will generally be any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid.

The term "carrier" refers to a diluent, adjuvant, excipient, or carrier used with the active ingredient for treatment. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal or vegetable origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. In some embodiments, when the pharmaceutical composition is administered intravenously, the carrier may be water. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences of e.w. Martin, incorporated herein by reference. Such compositions will contain a clinically effective dose of the antibody or antibody fragment, along with a suitable carrier, to provide a form of administration suitable for the patient. The formulation should be suitable for the mode of administration. The formulations may be packaged in ampules, disposable syringes or multiple dose vials made of glass or plastic.

In some embodiments, the pharmaceutical compositions of the present invention may be administered by any suitable route known in the art, including, but not limited to: oral, nasal, intradermal, subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic and/or cerebrospinal.

In some embodiments, the composition is formulated according to conventional procedures into a pharmaceutical composition suitable for intravenous injection into the human body. Compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffers. The pharmaceutical composition may also contain a solubilizing agent and a local anesthetic such as lidocaine, thereby alleviating pain at the injection site. In general, the active ingredients are supplied individually or in admixture in unit dosage form, such as in the form of a dry lyophilized powder or dry concentrate, in a sealed container (e.g., ampoule or pouch) that is indicative of the amount of active agent. In the case of administration of the composition by infusion, the composition may be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. In the case of administering the composition by injection, an ampoule of sterile water for injection or saline may be used so that the active ingredients may be mixed prior to administration.

The antibodies or antigen-binding fragments thereof of the invention include salt forms thereof. Pharmaceutically acceptable salts include those derived from anions such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and those derived from cations such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

Combination therapy

The antibodies or antibody fragments of the invention may be used in combination (e.g., contained in the same pharmaceutical composition) with at least one additional therapeutic agent, e.g., in combination with a checkpoint inhibitor;

in some embodiments, the checkpoint inhibitor is a checkpoint inhibitor of T cells, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody;

The anti-PD-1 antibody may be pembrolizumab Pembrolizumab, nivolumab, cimipu Li Shan anti Cemiplimab, s Lu Lishan anti Serplulimab, terlipp Li Shan anti Toripalimab;

The anti-PD-L1 antibody can be Ab Atezolizumab, ab Avelumab or Dewaruzumab Durvalumab;

the anti-CTLA-4 antibody can be ipilimumab Ipilimumab.

Example 1: production of murine monoclonal antibodies against human TREM2

1. Acquisition of hybridomas

The rats are immunized by TREM2 protein, SP2/0 cells are fused, positive clones are screened by ELISA, and hybridomas are obtained. The specific method comprises the following steps:

Animal immunization: the Protein immunization (first 100. Mu.g/rat, second 50. Mu.g/rat) was performed by intraperitoneal injection on SD female rats (8 weeks old, beijing Veitz laboratory animal technologies Co., ltd.) using the Human TREM2 Protein, his Tag (ACROBiosystems, cat# TR2-H52H 5) Protein as immunogen, diluted to 1:1 mg/mL with physiological saline, and then mixed with the equal volume of Quick adjuvant Quick Antibody Q5W (Beijing Boolong immune technologies Co., cat# KX 0210041). Protein immunization was repeated 5-6 times at intervals of 2 weeks, and 50. Mu.g of immunogen was injected intraperitoneally for impact immunization 14 days after the last immunization, and rat spleens were taken for cell fusion 3 days later.

Cell fusion: rat spleen cells were electrofused with Sp2/0-Ag14 cells (ATCC No. CRL-1581) at a cell number ratio of 2:1 (BTX electrofusion apparatus: ECM2001 ⁺), cultured in HAT medium (Sigma, cat# H0262) in 96-well plates, and hybridoma cell supernatant antibody screening was performed after 10 days.

ELISA screening of positive clones: the hybridoma supernatants were added at 50. Mu.L per well to 96-well flat bottom assay plates (Corning, cat# 9018) coated with Human TREM2 Protein (i.e., human TREM2 Protein, his Tag, described above), cynomolgus TREM2 Protein (ACROBiosystems, cat# TR2-C52H 3), or Mouse TREM2 Protein (Mouse TREM2 Protein, his Tag (ACROBiosystems, cat# TR2-M52H 3), respectively, and incubated at 37℃for 60 minutes. PBST was washed three times, and secondary antibodies (HRP Rabbit Anti-Rat IgG (whole molecular) (Sigma, cat# A5795), diluted 1:5000) were added to each well and incubated at 37℃for 30 minutes. PBST was washed three times, 100. Mu.L of TMB (Innovative, cat# EL 0009) was added to each well, and after 15 minutes of development, 50. Mu.L of sulfuric acid was added to each well to terminate the reaction. Reading an OD value on an enzyme labeling instrument, uniformly mixing cells in a positive clone culture hole, sucking the cells into a centrifuge tube, adding a proper amount of culture medium, uniformly mixing, sucking out a small amount of cell count, diluting hybridoma cells to contain 5 cells per mL according to a counting result, adding 0.2 mL diluted cell suspension into each hole of a 96-well plate, culturing for one week, selecting a culture supernatant containing single cell colonies, detecting according to the method, and performing corresponding cloning shown in table 1.

TABLE 1 binding Capacity of positive clone hybridoma supernatants to human TREM2, cynomolgus monkey TREM2 and mouse TREM2 proteins

Clone number	Human TREM2 (OD 450)	Cynomolgus monkey TREM2 (OD 450)	Mouse TREM2 (OD 450)
				342F9-2E6	3.66	0.70	3.19
334E9-2C11	4.00	3.43	4.00
				342H1-2B8	3.81	1.16	4.00

2. Preparation of murine monoclonal antibody against human TREM2

After the hybridoma cells in Table 1 were cultured in a serum-free medium for 10 days, the supernatant was collected, and a murine monoclonal antibody was purified by using a Protein A column (Bogurone (Shanghai) Biotechnology Co., ltd., cat# AA 0272) to obtain a purified monoclonal antibody (named after hybridoma). The activity and function of the purified murine monoclonal antibody against human TREM2 were then examined.

3. Functional identification of murine monoclonal antibody against human TREM2

Binding activity of murine monoclonal antibody against human TREM2 to human TREM2, cynomolgus TREM2, mouse TREM 2:

(1) Binding experiments of murine monoclonal antibodies against human TREM2 with cell lines expressing human TREM 2:

The 293T cell line 293T-hTREM-NFAT-RE-EGFP (product number: CS002, chongqing Ind. Biotechnology Co., ltd.) expressing human TREM2 was adjusted to a concentration of 1X10 ⁶ cells/mL with FACS buffer (PBS solution containing 1% FBS), placed in 96-well U-bottom plates at 100. Mu.L/well, and the supernatant was discarded after centrifugation. FACS buffer (PBS solution containing 1% FBS) was used to dilute the Anti-human TREM2 murine monoclonal antibody, the positive control antibody Anti-TREM2 Rat IgG1 (R & D Systems, cat# MAB 17291) to the initial working concentration, and then the FACS buffer was used for gradient dilution. The antibody diluted in gradient was added to the well plate at 100. Mu.L/well to resuspend the cells, and the mixture was stirred and incubated at 4℃for 1 hour. After incubation, the cells were centrifuged and washed three times with FACS buffer. AlexaFlour-647 labeled anti-rat secondary antibody (Invitrogen, cat# A21247) was diluted 1:1000 with FACS buffer (PBS solution with 1% FBS), 100 μl of secondary antibody diluent was added to each well, the cell pellet was resuspended, air-blown, mixed well, and incubated at 4deg.C for about 45 minutes. After incubation, the cells were centrifuged and washed three times with FACS buffer, and the cells were resuspended by adding FACS buffer to the well plate at 100 μl/well. Flow cytometer (BECKMAN COULTER cytoFLEX) reads the average fluorescence intensity (Median Fluorescence Intensity, MFI), analyzes the test data by using GRAPHPAD PRISM 8.0.0 software, takes the logarithm of the antibody concentration as the x axis and the corresponding MFI value as the y axis, selects a four-parameter equation regression model, fits the antibody dose-response curve, and calculates EC ₅₀.

(2) Binding experiments of murine monoclonal antibodies against human TREM2 with human TREM2 protein, cynomolgus TREM2 protein, mouse TREM2 protein:

The plate coating Protein is Human TREM2 Protein (i.e. the Human TREM2 Protein, his Tag), cynomolgus monkey TREM2 Protein (i.e. the Cynomolgus TREM2 Protein, his Tag) or Mouse TREM2 Protein (i.e. the Mouse TREM2 Protein, his Tag) with the concentration of 1 mug/mL, and the 96-well plate is coated at 50 mu L/well and 4 ℃ for overnight; adding 2% BSA blocking solution into the mixture according to 150 mu L/hole, and incubating the mixture for 1h at room temperature; PBST (containing 0.05% Tween 20) was added at 200. Mu.L/well and washed three times; using a blocking solution to prepare 300 mu L of mother solution with concentration of 10 mu g/mL, wherein the sample or positive control antibody is Anti-TREM2 Rat IgG1 (R & D Systems, product number: MAB 17291); diluting the mother solution with a sealing solution, wherein the dilution multiple is 3.16, and the total gradient is 11; adding a sample or a positive control antibody according to 50 mu L/hole, and incubating for 1h at room temperature; PBST (containing 0.05% Tween 20) was added at 200. Mu.L/well and washed three times; secondary antibodies (HRP Rabbit Anti-ray IgG (whole molecular) (Sigma, cat# a 5795)) were added, diluted 1:5000, added to each well at 50 μl/well, and incubated for 1h at room temperature; PBST (containing 0.05% Tween 20) was added at 200. Mu.L/well and washed three times; after drying, 50. Mu.L of TMB developing solution was added, the color developed was about 15 min, and the reaction was stopped with sulfuric acid. Microplate reader reading OD450. Analyzing the data by GRAPHPAD PRISM 8.0.0 software, taking the logarithm of the antibody concentration as the x axis, the corresponding OD450 value as the y axis, selecting a four-parameter equation regression model, fitting an antibody dose effect curve, calculating EC ₅₀,

The results are shown in FIGS. 1-4 and Table 2.

TABLE 2 binding Activity of murine monoclonal antibodies against human TREM2 with human TREM2, cynomolgus monkey TREM2 and mouse TREM2

The results showed that 342F9-2E6, 334E9-2C11 and 342H1-2B8 had strong binding activity to human TREM2 protein, of which 342H1-2B8 was better than the control. 342H1-2B8 also showed better binding activity to 293T cell line expressing human TREM2 than the control, comparable to the mouse TREM2 binding activity and control, and also binding activity to cynomolgus TREM 2.

Example 2: sequencing of murine monoclonal antibody against human TREM2 and functional identification of chimeric antibody

1. Sequencing of murine monoclonal antibody against human TREM2 and preparation of chimeric antibody

342F9-2E6, 334E9-2C11, 342H1-2B8 were sequenced and the heavy chain variable region, light chain variable region and CDR sequences are shown in Table 3.

TABLE 3 CDR sequences and variable region sequences of murine monoclonal antibodies against human TREM2 (determined according to Kabat protocol)

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Light and heavy chain variable regions were constructed onto human constant regions (IgG 1/K, table 4), corresponding chimeric antibodies were constructed (Table 5), and the sequences were verified by sequencing. Chimeric antibodies were named by adding a prefix of ch to the corresponding hybridoma clone numbers. For example, the chimeric antibody obtained by this example using hybridoma clone 342F9-2E6 was designated ch342F9-2E6 for in vitro functional identification.

The corresponding nucleic acid encoding the chimeric antibody was expressed using an Expi293 cell and purified using a Protein a column as follows:

The Expi293 cells express chimeric antibodies: the day before transfection, the density of the Expi293 cells (purchased from Gibco, cat# A14527) was diluted to 1.5X10. 10 ⁶ cells/mL and incubated in a 37℃8% CO ₂ shaker at 120 rpm. The next day, viable cell density and viability were measured, cell transfection density should be 3×10 ⁶ cells/mL, cell viability was greater than 95%. Preparation of PEI/plasmid complex: PEI (1 mg/mL, polysciences, cat# 24765-1) was mixed upside down. The plasmid was diluted with OPM-293 CD05 medium (Shanghai Aomo Pu Mai Biotechnology Co., ltd., product number: 81075-001) to a total plasmid volume of 1. Mu.g/mL, the volume of the medium for dilution of the plasmid was 1/20 of the transfection volume, and the mixture was gently mixed to a light-to-heavy chain ratio of 1:1.5. PEI reagent was diluted with OPM-293 CD05 medium, the volume of the diluted PEI medium was 1/20 of the transfection volume, mixed gently upside down, and incubated at room temperature for 5 minutes. Diluted PEI reagent was added to the diluted plasmid and mixed gently upside down. PEI/plasmid complex was incubated at room temperature for 15 minutes, then the solution was slowly added dropwise to the transfer flask, which was gently swirled during the addition. After transfection, shake flasks were incubated at 37℃in a shaker with 8% CO ₂ at 120: 120 rpm. On the next day after transfection (24 hours after transfection), 10% OPM-293 ProFeed (Shanghai Ao Pu Mai Biotech Co., ltd., cat# F081918) was added to the shake flask, the shake flask was gently rotated during the addition, and then returned to the shaker for further cultivation for 5-7 days, and the supernatant was harvested.

Protein a column purification antibody: preparing a gravity chromatographic column, opening an upper cover of the gravity chromatographic column, placing a gasket at the bottom of the gravity column, and compacting. Filler Protein a (Cytiva, cat No. 17549801) was prepared, and the required filler suspension volume was precisely calculated from the target filler volume and the filler suspension ratio, and the required filler suspension volume=target filler volume/filler suspension ratio. And (5) fully vortex oscillating the filler to ensure that the filler is completely suspended. And adding the filler suspension into the bottom of the gravity chromatographic column. At least 10 CV of equilibration buffer PBS was added to the gravity chromatography column and after equilibration, the exit pH was measured. If the target pH is not reached, the addition of equilibration buffer is continued until equilibration to the target pH. A volume of sample was slowly added to the gravity column. At least 10 CV of the elution buffer is added to the gravity chromatography column. The eluate was collected by slowly adding 5CV of elution buffer (10-50 mM NaAc, pH 3.0-pH 3.5) to the gravity column and incubating for 3-5 minutes. The elution step was repeated as necessary. And (3) neutralization: the pH was adjusted to the target pH with a neutralization buffer (1M Tris). Protein concentration was determined using Nanodrop. The buffer holding the antibody was replaced with PBS by ultrafiltration.

TABLE 4 constant region sequences

TABLE 5 chimeric anti-human TREM2 antibodies

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2. Functional identification of anti-human TREM2 chimeric antibodies

Binding activity of anti-human TREM2 chimeric antibody to human TREM2, cynomolgus monkey TREM2, mouse TREM 2:

(1) Binding experiments of anti-human TREM2 chimeric antibodies and human TREM2 expressing cell lines:

The 293T cell line 293T-hTREM-NFAT-RE-EGFP (product number: CS002, chongqing Ind. Biotechnology Co., ltd.) expressing human TREM2 was adjusted to a concentration of 1X 10 ⁶ cells/mL with FACS buffer (PBS solution containing 1% FBS), 100. Mu.L/well was placed in a 96-well U-bottom plate, and the supernatant was discarded after centrifugation. The anti-human TREM2 chimeric antibody, positive control antibody PI37012 (table 6) were diluted to the initial working concentration by FACS buffer (PBS solution containing 1% fbs), and then subjected to gradient dilution by FACS buffer. The cells were resuspended by adding 100. Mu.L/well of the gradient diluted antibody to the well plate, and incubated at 4℃for 1 hour. After incubation, the cells were centrifuged and washed three times with FACS buffer. The AlexaFlour-647 labeled anti-Human secondary antibody (Alexa Fluor 647 AffiniPure ™ Goat Anti-Human IgG (H+L) (Jackson Immuno, cat. No.: 109-605-003) was diluted 1:1000 with FACS buffer (PBS solution containing 1% FBS), 100. Mu.L of secondary antibody diluent was added to each well, the cell pellet was resuspended, air-blown, mixed well, incubated at 4℃for about 45 minutes, after incubation the cells were centrifuged, washed three times with FACS buffer, and the FACS buffer was added to the well plate to resuspend cells at 100. Mu.L/well, flow cytometer (BECKMAN COULTER cytoFLEX) read the average fluorescence intensity (Median Fluorescence Intensity, MFI), assay data were analyzed using GRAPHPAD PRISM 8.0.0 software, log antibody concentration was x-axis, corresponding MFI value was y-axis, a four parameter equation regression model was selected, and antibody dose response curve was fitted to calculate EC ₅₀.

(2) Binding experiments of anti-human TREM2 chimeric antibody with human TREM2 protein, cynomolgus TREM2 protein, mouse TREM2 protein:

The experiment was performed as in (2) in step 3 of example 1, with the positive control antibody replaced with PI37012 and the secondary antibody replaced with Anti-Human IgG HRP (Sigma, cat# A8867).

The results are shown in FIGS. 5-8 and Table 7.

TABLE 6 PI37012 antibodies

TABLE 7 binding Activity of anti-human TREM2 chimeric antibodies to human TREM2, cynomolgus monkey TREM2 and mouse TREM2

The results show that the anti-human TREM2 chimeric antibody is consistent with the positive control antibody PI37012, has strong binding activity to human TREM2 protein and 293T cell line expressing human TREM2, and also has binding activity to cynomolgus monkey TREM2 and mouse TREM 2.

EXAMPLE 3 Fc mutant anti-human TREM2 chimeric antibody-mediated ADCC Effect

The S239D/A330L/I332E mutation enhances activation of Jurkat-human FcgammaRIIIa (158V) -NFAT by anti-human TREM2 chimeric antibodies with THP-1, and the S239D/A330L/I332E mutation of the antibody constant region (Fc) can increase affinity with FcgammaRIIIa, increasing antibody-mediated ADCC effect (Greg A Lazar et al. Proc NATL ACAD SCI U S A. 2006 and Jeffrey L Nordstrom et al. Breast Cancer Res. 2011). This mutation has been applied clinically to enhance ADCC effect of antibodies (Rena Liu et al Antibodies (Basel).2020). The S239D/A330L/I332E (DLE) mutation was performed against the Fc of the human TREM2 chimeric antibody, respectively, to enhance the induced ADCC effect.

The Fc mutant anti-human TREM2 chimeric antibodies are shown in table 8.

Table 8 Fc mutant anti-human TREM2 chimeric antibodies

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It was found in clinical studies that anti-TREM 2 antibodies can clear Tumor Associated Macrophages (TAMs) in the tumor microenvironment by ADCC. anti-TREM 2 antibody dependent FcgammaIII activation was assayed using Jurkat-human FcgammaIIIa (158V) -NFAT incubated with TREM 2-expressed THP-1 cells. The Fab end of the TREM2 antibody is combined with a target TREM2 on a target cell THP-1, the Fc end of the TREM2 antibody is combined with an Fc gamma RIIIa receptor on an effector cell, so that an NFAT signal path in the effector cell is activated, and luciferase generated by activating the NFAT path is quantified to react with ADCC activity of the antibody, wherein the specific method is as follows:

THP-1 cells (available from Gibby Biotechnology (Shanghai) Inc. under the trade designation GM-C09479) were centrifuged at 300: 300 g for 5 minutes and the cell concentration was adjusted to 1.2X106 cells/mL using RMPI1640 medium. mu.L of each well was inoculated into a white opaque plate as target cells for use. RMPI1640 medium the anti-human TREM2 chimeric antibody, positive control antibody PI37012 and mutant positive control antibody PI37012_dle of the present application were diluted to a concentration of 40 μg/mL, and then subjected to 4-fold gradient dilution with medium. The collected Jurkat-human FcgammaRIIIa (158V) -NFAT cells (Jiman Biotechnology (Shanghai) Co., ltd.; cat. No. GM-C05619) were centrifuged at 300 g min, the supernatant was discarded, and the cell concentration was adjusted to 1.5X10 ⁶ cells/mL by adding RMPI1640 medium. Then 100. Mu.L of cells were added to each culture well containing target cells, while 50. Mu.L of gradient diluted antibody was added to each well. Induced overnight at 37℃under 5% CO ₂. Equilibrated at room temperature for at least 15 min, 100. Mu.L of luciferase substrate solution (Vazyme, cat# DD 1203) was added to each well, mixed and reacted at room temperature for 5 min in the absence of light, and the relative light unit values were read on the cell culture plates with an enzyme-labeled instrument. The experimental data are analyzed by GRAPHPAD PRISM 8.0.0 software, the logarithm of the concentration of the anti-TREM 2 antibody is taken as an x axis, the corresponding RLU value is taken as a y axis, a four-parameter equation regression model is selected, and a dose response curve of the anti-TREM 2 antibody is fitted.

The ADCC effect of the anti-TREM 2 antibody is shown in table 9.

TABLE 9 ADCC Effect of Fc mutant anti-human TREM2 chimeric antibodies

Remarks: N/A indicates inapplicability and no effective EC ₅₀ was obtained.

The results showed that the ch342F9-2E6, the ch342F9-2E6_DLE and the ch334E9-2C11, the ch334E9-2C11_DLE could dose-dependently activate Jurkat-human FγRIIIa (158V) -NFAT reporter cell, the EC ₅₀ of the activation activities of the ch342F9-2E6 and the ch342F9-2E6_DLE chimeric antibodies were 0.01075 μg/mL and 0.01969 μg/mL, respectively, and the EC ₅₀ of the activation activities of the ch334E9-2C11 and the ch334E9-2C11_DLE chimeric antibodies were 0.005415 μg/mL and 0.07286 μg/mL, respectively. From Span (Span) results, it was shown that the Fc mutant antibodies enhanced activation of Jurkat-human fcγriiia (158V) -NFAT compared to wild-type human IgG1 Fc.

Claims

1. An antibody or antigen-binding fragment thereof having binding specificity to myeloid cell trigger receptor 2 (TREM 2), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, and wherein:

The HCDR1 is the amino acid sequence shown in SEQ ID NO. 10, the HCDR2 is the amino acid sequence shown in SEQ ID NO. 11, the HCDR3 is the amino acid sequence shown in SEQ ID NO. 12, the LCDR1 is the amino acid sequence shown in SEQ ID NO. 14, the LCDR2 is the amino acid sequence shown in SEQ ID NO. 15, and the LCDR3 is the amino acid sequence shown in SEQ ID NO. 16.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein:

the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 9, or an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO. 9, or a sequence having one or more amino acid substitutions, deletions or insertions, or any combination thereof, as compared to the amino acid sequence shown in SEQ ID NO. 9; and/or the number of the groups of groups,

The light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 13, or an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO. 13, or a sequence having one or more amino acid substitutions, deletions or insertions, or any combination thereof, as compared to the amino acid sequence shown in SEQ ID NO. 13.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, further comprising a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

4. The antibody or antigen-binding fragment thereof of claim 3, wherein the antibody or antigen-binding fragment thereof is a chimeric antibody comprising a light chain and a heavy chain;

The heavy chain comprises an amino acid sequence as set forth in SEQ ID NO. 29, or an amino acid sequence having at least 90% identity to the amino acid sequence as set forth in SEQ ID NO. 29, or a sequence having one or more amino acid substitutions, deletions or insertions, or any combination thereof, as compared to the amino acid sequence as set forth in SEQ ID NO. 29; the light chain comprises an amino acid sequence shown as SEQ ID NO. 30, or an amino acid sequence having at least 90% identity to the amino acid sequence shown as SEQ ID NO. 30, or a sequence having one or more amino acid substitutions, deletions or insertions, or any combination thereof, as compared to the amino acid sequence shown as SEQ ID NO. 30; or (b)

The heavy chain comprises an amino acid sequence shown in SEQ ID NO. 37, or an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO. 37, or a sequence having one or more amino acid substitutions, deletions or insertions, or any combination thereof, as compared to the amino acid sequence shown in SEQ ID NO. 37; the light chain comprises the amino acid sequence shown in SEQ ID NO. 38, or an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO. 38, or a sequence having one or more amino acid substitutions, deletions or insertions, or any combination thereof, as compared to the amino acid sequence shown in SEQ ID NO. 38.

5. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-4.

6. A recombinant vector comprising the nucleic acid molecule of claim 5.

7. A recombinant cell comprising the nucleic acid molecule of claim 5 and/or the recombinant vector of claim 6;

8. A method of making an antibody or antigen-binding fragment thereof that binds TREM2, the method comprising culturing a recombinant cell comprising a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-4 under conditions suitable for expression of the antibody, optionally the method further comprising recovering the antibody or antigen-binding fragment thereof from the recombinant cell or culture medium.

9. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-4 or the nucleic acid molecule of claim 5 or the recombinant vector of claim 6 or the recombinant cell of claim 7, and a pharmaceutically acceptable carrier.

10. Use of the antibody or antigen binding fragment thereof of any one of claims 1-4, the nucleic acid molecule of claim 5, the recombinant vector of claim 6, the recombinant cell of claim 7 and/or the composition of claim 9 in the preparation of a product as shown in any one of the following:

(1) Detecting a TREM2 product;

(2) A product that stimulates or enhances an immune response;

(3) Products for preventing and/or treating cancer.