Anti-ILT2 antibody and application thereof

Abstract

The invention relates to the technical field of antibody drugs, in particular to an anti-ILT2 antibody or an antigen binding fragment thereof, a pharmaceutical composition containing the anti-ILT2 antibody or the antigen binding fragment thereof and application of the anti-ILT2 antibody or the antigen binding fragment thereof. The anti-ILT2 antibody disclosed by the invention has remarkable anti-tumor activity and can be applied to preparation of anti-tumor medicines.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody drugs, and particularly relates to an anti-ILT2 antibody or an antigen-binding fragment thereof, a pharmaceutical composition comprising the anti-ILT2 antibody or the antigen-binding fragment thereof, and their applications. Background Art

[0002] Immunoglobulin-like transcript 2 (ILT2, also known as LILRB1, CD85j, LIR1, MIR7) is a member of the ILT family, and there are currently four members in this family: ILT1, ILT2, ILT3, and ILT4. ILT2 is most similar to ILT4 and has approximately 80% homology. The ligands of ILT2 include classical (HLA-A, HLA-B, and HLA-C) and non-classical (HLA-E, HLA-F, and HLA-G) major histocompatibility complex class I molecules (MHCIs), and the strongest known interacting partner of ILT2 in the human genome is HLA-G. ILT2 is mainly expressed on immune cells such as T cells, NK cells, B cells, monocytes, macrophages, and dendritic cells, and is the most widely expressed member of the ILT family. ILT2 belongs to a typical type I transmembrane protein. The extracellular part contains 4 immunoglobulin domains. The transmembrane region consists of 20 amino acids, and the cytoplasmic region is a long cytoplasmic tail containing 4 immunoreceptor tyrosine-based inhibitory motifs (ITIMs). When the tyrosine residues of the ITIMs of ILT2 are activated by its ligands, it is phosphorylated itself and recruits the tyrosine phosphatase SHP-1 to play a role in transmitting extracellular signals into cells, and an immunosuppressive effect occurs in NK cells, monocytes, macrophages, DC cells, T cells, and B cells.

[0003] Physiological functions related to the occurrence and development of cancer have been found on both ILTs expressed on immune cells and tumor cells themselves. More and more evidence indicates that ILT2 may be a molecular target for immunotherapy. ILT2 is upregulated in NK cells of some cancer patients and is also upregulated on the surface of tumor-associated macrophages (TAMs). ILT2 can negatively regulate the normal physiological functions of various immune cells such as NK cells, DC cells, T cells, and B cells by interacting with ligands such as HLA-G on tumor cells, such as inhibiting the proliferation, migration, and cytokine release of immune cells.

[0004] Blocking ILT2 can enhance the killing effect of immune cells on solid tumors and hematological malignancies. In tumor immunotherapy, enhancing T cell activity can significantly inhibit and kill tumors. After ILT2 binds to its ligand HLA-G, it significantly inhibits the proliferation and cytokine release of T cells, and the antibody of ILT2 can relieve this inhibitory effect. The antibody of ILT2 also has a strong positive stimulating effect on another type of tumor-killing cell, NK cells. Specific antibodies can restore the inhibitory effect of NK cell killing toxicity. ILT2 blockade enhances the in vitro immune response of NK cells to solid tumor cells (breast cancer and melanoma) and hematological cancer cells (such as acute myeloid leukemia (AML)), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and multiple myeloma (MM). In addition to lymphocytes, blocking ILT2 can also enhance the phagocytosis of cancer cells by macrophages. In addition to immune cells, ILT2 is also directly expressed on certain tumors and pre-tumor cells, such as acute myeloid leukemia (AML) cells, T cell lymphoma cells, and tumor B cells.

[0005] The high expression of ILT2 and its main ligand HLA-G is negatively correlated with the prognosis of many cancer patients. Antibodies against ILT2 have potential cancer treatment capabilities, indicating that the ILT2 target has broad application prospects. Therefore, the research and development of specific antibody drugs that can act on ILT2 have important clinical significance. Summary of the Invention

[0006] On the one hand, the present invention provides an anti-ILT2 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises complementarity-determining region 1 (HCDR1) of the heavy chain variable region, complementarity-determining region 2 (HCDR2) of the heavy chain variable region, and / or complementarity-determining region 3 (HCDR3) of the heavy chain variable region, and the light chain variable region comprises complementarity-determining region 1 (LCDR1) of the light chain variable region, complementarity-determining region 2 (LCDR2) of the light chain variable region, and / or complementarity-determining region 3 (LCDR3) of the light chain variable region.

[0007] In some embodiments, the present invention provides an anti-ILT2 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and / or a light chain variable region, wherein:

[0008] (1) The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 selected from the following groups:

[0009] (a1) The amino acid sequences shown in SEQ ID NO: 1, 2, and 3;

[0010] (a2) The amino acid sequences shown in SEQ ID NO: 7, 8, and 9;

[0011] (a3) The amino acid sequences shown in SEQ ID NO: 13, 14, and 15;

[0012] (a4) The amino acid sequences shown in SEQ ID NO: 1, 19, and 20;

[0013] (a5) The amino acid sequences shown in SEQ ID NO: 7, 21, and 9; and

[0014] (a6) An amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in (a1), (a2), (a3), (a4), or (a5); and

[0015] (2) The light chain variable region comprises LCDR1, LCDR2, and LCDR3 selected from the following group:

[0016] (b1) The amino acid sequences shown in SEQ ID NO: 4, 5, and 6;

[0017] (b2) The amino acid sequences shown in SEQ ID NO: 10, 11, and 12;

[0018] (b3) The amino acid sequences shown in SEQ ID NO: 16, 17, and 18;

[0019] (b4) The amino acid sequences shown in SEQ ID NO: 22, 11, and 12; and

[0020] (b5) An amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in (b1), (b2), (b3), or (b4).

[0021] In some specific embodiments, the present invention provides an anti-ILT2 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are SEQ ID NO: 1, 2, and 3, SEQ ID NO: 1, 19, and 20, respectively, or an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, 2, and 3 or SEQ ID NO: 1, 19, and 20, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are SEQ ID NO: 4, 5, and 6, respectively, or an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, 5, and 6; or

[0022] The HCDR1, HCDR2, and HCDR3 are SEQ ID NO:7, 8, and 9, SEQ ID NO:7, 21, and 9, respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:7, 8, and 9 or SEQ ID NO:7, 21, and 9, and the LCDR1, LCDR2, and LCDR3 are SEQ ID NO:10, 11, and 12, SEQ ID NO:22, 11, and 12, respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:10, 11, and 12 or SEQ ID NO:22, 11, and 12; or

[0023] The HCDR1, HCDR2, and HCDR3 are SEQ ID NO:13, 14, and 15, respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:13, 14, and 15, and the LCDR1, LCDR2, and LCDR3 are SEQ ID NO:16, 17, and 18, respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:16, 17, and 18.

[0024] In some embodiments, the anti-ILT2 antibody or antigen-binding fragment thereof according to the present invention, wherein the HCDR1, HCDR2, and HCDR3 are SEQ ID NO:1, 2, and 3, respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:1, 2, and 3, and the LCDR1, LCDR2, and LCDR3 are SEQ ID NO:4, 5, and 6, respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:4, 5, and 6; or

[0025] The HCDR1, HCDR2, and HCDR3 are SEQ ID NO:1, 19, and 20, respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:1, 19, and 20, and the LCDR1, LCDR2, and LCDR3 are SEQ ID NO:4, 5, and 6, respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:4, 5, and 6; or

[0026] The HCDR1, HCDR2, and HCDR3 are SEQ ID NO:7, 8, and 9 respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:7, 8, and 9, and the LCDR1, LCDR2, and LCDR3 are SEQ ID NO:10, 11, and 12 respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:10, 11, and 12; or

[0027] The HCDR1, HCDR2, and HCDR3 are SEQ ID NO:7, 21, and 9 respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:7, 21, and 9, and the LCDR1, LCDR2, and LCDR3 are SEQ ID NO:22, 11, and 12 respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:22, 11, and 12.

[0028] In some embodiments, the anti-ILT2 antibody or antigen-binding fragment thereof according to the present invention, wherein the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:33, SEQ ID NO:35, and SEQ ID NO:37, amino acid sequences obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37 and having the same function as SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, and amino acid sequences having at least 85% sequence identity with SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO:34, SEQ ID NO:36, and SEQ ID NO:38, amino acid sequences obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38 and having the same function as SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38, and amino acid sequences having at least 85% sequence identity with SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38.

[0029] In some specific embodiments, the anti-ILT2 antibody or antigen-binding fragment thereof according to the present invention, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 33, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 33, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 33, and the amino acid sequence of the light chain variable region is SEQ ID NO: 34, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 34, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 34.

[0030] In some specific embodiments, the anti-ILT2 antibody or antigen-binding fragment thereof according to the present invention, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 35, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 35, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 35, and the amino acid sequence of the light chain variable region is SEQ ID NO: 36, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 36, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 36.

[0031] In some specific embodiments, the anti-ILT2 antibody or antigen-binding fragment thereof according to the present invention, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 37, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 37, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 37, and the amino acid sequence of the light chain variable region is SEQ ID NO: 38, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 38, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 38.

[0032] In some embodiments, an anti-ILT2 antibody or antigen-binding fragment thereof according to the present invention, wherein the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 42 and having the same function as SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 42, and an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 42, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, or SEQ ID NO: 47 and having the same function as SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, or SEQ ID NO: 47, and an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, or SEQ ID NO: 47.

[0033] In some embodiments, an anti-ILT2 antibody or an antigen-binding fragment thereof according to the present invention, wherein the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, or SEQ ID NO:51 and having the same function as SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, or SEQ ID NO:51, and an amino acid sequence having at least 85% sequence identity with SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, or SEQ ID NO:51, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, or SEQ ID NO:56 and having the same function as SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, or SEQ ID NO:56, and an amino acid sequence having at least 85% sequence identity with SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, or SEQ ID NO:56.

[0034] In some specific embodiments, an anti-ILT2 antibody or an antigen-binding fragment thereof according to the present invention, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO:42, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:42 and having the same function as SEQ ID NO:42 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:42, and the amino acid sequence of the light chain variable region is SEQ ID NO:47, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ IDNO:47 and having the same function as SEQ ID NO:47 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:47.

[0035] In some specific embodiments, the anti-ILT2 antibody or antigen-binding fragment thereof according to the present invention, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 48, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO: 48 and having the same function as SEQ ID NO: 48, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 48, and the amino acid sequence of the light chain variable region is SEQ ID NO: 52, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO: 52 and having the same function as SEQ ID NO: 52, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 52.

[0036] In some specific embodiments, the present invention provides an anti-ILT2 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are SEQ ID NO: 1, 2 and 3 respectively, LCDR1, LCDR2 and LCDR3 of the light chain variable region are SEQ ID NO: 4, 5 and 6 respectively, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 33, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO: 33 and having the same function as SEQ ID NO: 33, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 33 and the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO: 1, 2 and 3, and the amino acid sequence of the light chain variable region is SEQ ID NO: 34, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO: 34 and having the same function as SEQ ID NO: 34, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 34 and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO: 4, 5 and 6.

[0037] In some specific embodiments, the present invention provides an anti-ILT2 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are SEQ ID NO:7, 8, and 9 respectively, the LCDR1, LCDR2, and LCDR3 of the light chain variable region are SEQ ID NO:10, 11, and 12 respectively, the amino acid sequence of the heavy chain variable region is SEQ ID NO:35, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:35 and having the same function as SEQ ID NO:35, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:35 and the HCDR1, HCDR2, and HCDR3 are as shown in SEQ ID NO:7, 8, and 9, and the amino acid sequence of the light chain variable region is SEQ ID NO:36, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:36 and having the same function as SEQ ID NO:36, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:36 and the LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NO:10, 11, and 12.

[0038] In some specific embodiments, the present invention provides an anti-ILT2 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are SEQ ID NO:13, 14, and 15 respectively, the LCDR1, LCDR2, and LCDR3 of the light chain variable region are SEQ ID NO:16, 17, and 18 respectively, the amino acid sequence of the heavy chain variable region is SEQ ID NO:37, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:37 and having the same function as SEQ ID NO:37, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:37 and the HCDR1, HCDR2, and HCDR3 are as shown in SEQ ID NO:13, 14, and 15, and the amino acid sequence of the light chain variable region is SEQ ID NO:38, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:38 and having the same function as SEQ ID NO:38, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:38 and the LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NO:16, 17, and 18.

[0039] In some specific embodiments, the present invention provides an anti-ILT2 antibody or an antigen-binding fragment thereof, which comprises a heavy-chain variable region and a light-chain variable region, wherein HCDR1, HCDR2, and HCDR3 of the heavy-chain variable region are SEQ ID NO: 1, 2, and 3 respectively, LCDR1, LCDR2, and LCDR3 of the light-chain variable region are SEQ ID NO: 4, 5, and 6 respectively, the amino acid sequence of the heavy-chain variable region is selected from SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 and having the same function as SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, and an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41 and wherein HCDR1, HCDR2, and HCDR3 are as shown in SEQ ID NO: 1, 2, and 3, and the amino acid sequence of the light-chain variable region is selected from SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, or SEQ ID NO: 47 and having the same function as SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, or SEQ ID NO: 47, and an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, or SEQ ID NO: 47 and wherein LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NO: 4, 5, and 6.

[0040] In some specific embodiments, the present invention provides an anti-ILT2 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are SEQ ID NO: 1, 19 and 20 respectively, the LCDR1, LCDR2 and LCDR3 of the light chain variable region are SEQ ID NO: 4, 5 and 6 respectively, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 42, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO: 42 and having the same function as SEQ ID NO: 42, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 42 and the HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO: 1, 19 and 20, and the amino acid sequence of the light chain variable region is SEQ ID NO: 47, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO: 47 and having the same function as SEQ ID NO: 47, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 47 and the LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO: 4, 5 and 6.

[0041] In some specific embodiments, the present invention provides an anti-ILT2 antibody or an antigen-binding fragment thereof, which comprises a heavy-chain variable region and a light-chain variable region, wherein the HCDR1, HCDR2 and HCDR3 of the heavy-chain variable region are SEQ ID NO:7, 8 and 9 respectively, the LCDR1, LCDR2 and LCDR3 of the light-chain variable region are SEQ ID NO:10, 11 and 12 respectively, the amino acid sequence of the heavy-chain variable region is selected from SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:49, SEQ ID NO:50 or SEQ ID NO:51 and having the same function as SEQ ID NO:49, SEQ ID NO:50 or SEQ ID NO:51, and an amino acid sequence having at least 85% sequence identity with SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51 and with the HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:7, 8 and 9, and the amino acid sequence of the light-chain variable region is selected from SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 and SEQ ID NO:56, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 or SEQ ID NO:56 and having the same function as SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 or SEQ ID NO:56, and an amino acid sequence having at least 85% sequence identity with SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 or SEQ ID NO:56 and with the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:10, 11 and 12.

[0042] In some specific embodiments, the present invention provides an anti-ILT2 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are SEQ ID NO:7, 21 and 9 respectively, the LCDR1, LCDR2 and LCDR3 of the light chain variable region are SEQ ID NO:22, 11 and 12 respectively, the amino acid sequence of the heavy chain variable region is SEQ ID NO:48, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:48 and having the same function as SEQ ID NO:48, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:48 and the HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO:7, 21 and 9, and the amino acid sequence of the light chain variable region is SEQ ID NO:52, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:52 and having the same function as SEQ ID NO:52, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:52 and the LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO:22, 11 and 12.

[0043] In some specific embodiments, the present invention provides an anti-ILT2 antibody or an antigen-binding fragment thereof, which comprises a heavy chain and a light chain, wherein the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are SEQ ID NO:1, 19 and 20 respectively, the LCDR1, LCDR2 and LCDR3 of the light chain variable region are SEQ ID NO:4, 5 and 6 respectively, the amino acid sequence of the heavy chain is SEQ ID NO:61, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:61 and having the same function as SEQ ID NO:61, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:61 and the HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO:1, 19 and 20, and the amino acid sequence of the light chain is SEQ ID NO:62, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:62 and having the same function as SEQ ID NO:62, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:62 and the LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO:4, 5 and 6.

[0044] In some specific embodiments, the present invention provides an anti-ILT2 antibody or an antigen-binding fragment thereof, which comprises a heavy chain and a light chain, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are SEQ ID NO:7, 21 and 9 respectively, and LCDR1, LCDR2 and LCDR3 of the light chain variable region are SEQ ID NO:22, 11 and 12 respectively. The amino acid sequence of the heavy chain is SEQ ID NO:63, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:63 and having the same function as SEQ ID NO:63, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:63 and wherein HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO:7, 21 and 9. And the amino acid sequence of the light chain is SEQ ID NO:64, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:64 and having the same function as SEQ ID NO:64, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:64 and wherein LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO:22, 11 and 12.

[0045] In some embodiments, the present invention provides an anti-ILT2 antibody or an antigen-binding fragment thereof, wherein the antibody is a monoclonal antibody. In some embodiments, the present invention provides an anti-ILT2 antibody or an antigen-binding fragment thereof, wherein the antibody is a murine monoclonal antibody, a chimeric antibody, a humanized antibody, a bispecific antibody or a fully human antibody.

[0046] In some specific embodiments, the anti-ILT2 antibody according to the present invention is a murine antibody, which further contains a heavy chain constant region of murine IgG1, IgG2a, IgG2b, IgG2c, IgG3 or a variant thereof, and a light chain constant region of murine κ, λ chain or a variant thereof.

[0047] In some preferred embodiments, the heavy chain of the anti-ILT2 chimeric antibody or an antigen-binding fragment thereof further comprises a heavy chain constant region of murine IgG1, IgG2a, IgG2b, IgG2c, IgG3 or a mutant sequence thereof, preferably comprises a human IgG1, IgG2 or IgG4 heavy chain constant region.

[0048] In some embodiments, the present invention provides a humanized anti-ILT2 antibody or an antigen-binding fragment thereof, wherein the heavy chain comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, and the light chain comprises a light chain constant region of human κ, λ chain or a variant thereof.

[0049] In some preferred embodiments, the anti-ILT2 humanized antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region of human IgG4 or a variant thereof, and a light chain constant region of human kappa chain or a variant thereof.

[0050] In some embodiments, the anti-ILT2 antibody or antigen-binding fragment thereof of the present invention binds to ILT2 and inhibits the direct interaction between said ILT2 and beta-2 microglobulin (B2M). In some specific embodiments, the anti-ILT2 antibody or antigen-binding fragment thereof of the present invention inhibits the interaction between said ILT2 and HLA protein or MHC-I protein by inhibiting the direct interaction between ILT2 and B2M.

[0051] In some embodiments, the anti-ILT2 antibody or antigen-binding fragment is a bispecific or multispecific antibody or antigen-binding fragment that binds to the ILT2 protein and binds to one or more additional binding targets, preferably said additional binding targets are one or more tumor antigens. In some specific embodiments, the one or more additional binding targets are immunomodulatory molecules.

[0052] In some embodiments, the present invention provides an anti-ILT2 antibody or antigen-binding fragment thereof, wherein the antigen-binding fragment is Fab, Fab', Fv, scFv, F(ab')2, F(ab)2, dAb or a single domain antibody.

[0053] Another aspect of the present invention provides an isolated nucleic acid encoding the anti-ILT2 antibody or antigen-binding fragment thereof according to the present invention.

[0054] In some specific embodiments, the isolated nucleic acid according to the present invention comprises:

[0055] A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO:33, SEQ ID NO:35 or SEQ ID NO:37; and a nucleotide sequence encoding a light chain variable region such as SEQ ID NO:34, SEQ ID NO:36 or SEQ ID NO:38.

[0056] In some preferred embodiments, the isolated nucleic acid according to the present invention comprises:

[0057] A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO:33; and a nucleotide sequence encoding a light chain variable region such as SEQ ID NO:34; or

[0058] A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO:35; and a nucleotide sequence encoding a light chain variable region such as SEQ ID NO:36; or

[0059] A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO:36; and a nucleotide sequence encoding a light chain variable region such as SEQ ID NO:37.

[0060] In some specific embodiments, the isolated nucleic acid according to the present invention comprises:

[0061] A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:42; and a nucleotide sequence encoding a light chain variable region such as SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 or SEQ ID NO:47.

[0062] In some preferred embodiments, the isolated nucleic acid according to the present invention comprises:

[0063] A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO:42; and a nucleotide sequence encoding a light chain variable region such as SEQ ID NO:47.

[0064] In some preferred embodiments, the isolated nucleic acid according to the present invention comprises:

[0065] A nucleotide sequence encoding a heavy chain variable region amino acid sequence as shown in SEQ ID NO:57; and a nucleotide sequence encoding a light chain variable region amino acid sequence as shown in SEQ ID NO:58.

[0066] In some specific embodiments, the isolated nucleic acid according to the present invention comprises:

[0067] A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 or SEQ ID NO:51; and a nucleotide sequence encoding a light chain variable region such as SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 or SEQ ID NO:56.

[0068] In some preferred embodiments, the isolated nucleic acid according to the present invention comprises:

[0069] A nucleotide sequence encoding a heavy chain variable region amino acid sequence as shown in SEQ ID NO:59; and a nucleotide sequence encoding a light chain variable region amino acid sequence as shown in SEQ ID NO:60.

[0070] Another aspect of the present invention provides an expression vector that expresses the anti-ILT2 antibody or antigen-binding fragment thereof of the present invention. The expression vector according to the present invention comprises the isolated nucleic acid molecule of the present invention.

[0071] Another aspect of the present invention provides a chimeric antigen receptor (CAR) fusion protein that comprises the anti-ILT2 antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the chimeric antigen receptor fusion protein comprises the anti-ILT2 antibody or antigen-binding fragment thereof of the present invention, which is a single-chain variable fragment (scFv) of VH and VL against the ILT2 antigen. The scFv of VH and VL against the ILT2 antigen has HCDR1, HCDR2, and HCDR3 of the heavy-chain variable region and LCDR1, LCDR2, and LCDR3 of the light-chain variable region described in the above embodiments.

[0072] Another aspect of the present invention provides a host cell transformed with the expression vector as described above.

[0073] In some embodiments, the host cell according to the present invention is selected from prokaryotic cells and eukaryotic cells. In some embodiments, the host cell is a bacterium, preferably Escherichia coli. In another preferred embodiment, the host cell is a mammalian cell.

[0074] Another aspect of the present invention provides a method for preparing the anti-ILT2 antibody or antigen-binding fragment thereof of the present invention, comprising the steps of expressing the antibody in the host cell and isolating the antibody from the host cell.

[0075] Another aspect of the present invention provides a pharmaceutical composition that comprises the anti-ILT2 antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the present invention provides a pharmaceutical composition that comprises the anti-ILT2 antibody or antigen-binding fragment thereof of the present invention and further comprises other active components, such as other antibodies, targeting drugs, etc. In some embodiments, the pharmaceutically acceptable carrier is selected from antioxidants, polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, sugar alcohols, ions, and surfactants. In a specific embodiment, the pharmaceutically acceptable carrier is a buffered aqueous solution. In another specific embodiment, the pharmaceutically acceptable carrier is in the form of liposomes.

[0076] The anti-ILT2 humanized antibody or its antigen-binding fragment of the present invention can be mixed with a pharmaceutically acceptable carrier, diluent or excipient to prepare a pharmaceutical preparation suitable for oral or parenteral administration. The administration methods include, but are not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous, intracerebral, intraocular, intratracheal, subcutaneous, intranasal routes. The preparation can be administered by any route, such as by infusion or bolus injection, and by the route of absorption through the epithelium or skin mucosa (such as oral mucosa or rectum, etc.). The administration can be systemic or local. The preparation can be prepared by methods known in the art and contains carriers, diluents or excipients commonly used in the field of pharmaceutical preparations.

[0077] Another aspect of the present invention provides a method for treating cancer, which includes administering the anti-ILT2 antibody or its antigen-binding fragment of the present invention or the pharmaceutical composition of the present invention to an individual in need thereof.

[0078] Another aspect of the present invention provides the use of the anti-ILT2 antibody or its antigen-binding fragment of the present invention or the pharmaceutical composition of the present invention in the preparation of a drug for preventing and / or treating tumors. In some embodiments, the tumor is a tumor expressing HLA-G or MHC-I. In some embodiments, the anti-ILT2 antibody or its antigen-binding fragment of the present invention is combined with an anti-PD-L1 / PD-1 antibody for treating tumors expressing HLA-G or MHC-I. In some embodiments, the tumors are selected from cholangiocarcinoma, cervical cancer, urogenital cancer, testicular cancer, prostate cancer, thyroid cancer, ovarian cancer, nervous system cancer, eye cancer, lung cancer, soft tissue cancer, bone cancer, pancreatic cancer, bladder cancer, skin cancer, intestinal cancer, liver cancer, rectal cancer, colorectal cancer, esophageal cancer, gastric cancer, gastroesophageal cancer, breast cancer, kidney cancer, head and neck cancer, leukemia and lymphoma. In some embodiments, the tumors are selected from breast cancer, cholangiocarcinoma, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, head and neck cancer, liver cancer, lung cancer, kidney cancer, skin cancer, urogenital cancer and pancreatic cancer. In some embodiments, the tumors are selected from breast cancer, cholangiocarcinoma, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, head and neck cancer, liver cancer, lung cancer, kidney cancer, skin cancer, urogenital cancer, pancreatic cancer and leukemia. In some embodiments, the tumors are selected from urothelial carcinoma, triple-negative breast cancer, non-small cell lung cancer, melanoma, squamous cell carcinoma, hepatocellular carcinoma, gallbladder cancer, cholangiocarcinoma and KRAS wild-type colorectal cancer. In some embodiments, the tumors are selected from acute myeloid leukemia (AML), T cell lymphoma and B cell lymphoma. In some specific embodiments, the tumor is monocytic acute myeloid leukemia. In some embodiments, the tumors are selected from breast cancer, cervical cancer, colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, thyroid cancer, ovarian cancer cells, glioblastoma multiforme, melanoma.

[0079] The anti-ILT2 antibody or its antigen-binding fragment provided by the present invention has a significant anti-tumor effect, does not affect the binding of ILT2 to its ligand, does not affect its normal function, and at the same time, the immunogenicity of the humanized antibody is greatly reduced, effectively eliminating the rejection reaction of the human immune system to exogenous monoclonal antibodies. It can be used in the preparation of drugs for treating various tumor diseases and has broad market prospects.

[0080] Definitions

[0081] Unless otherwise defined, the meanings of scientific and technical terms used herein are those commonly understood by those skilled in the art. The nomenclature and techniques used in cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (such as electroporation, lipofection). Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions or methods commonly used in the art or described herein. The foregoing techniques and methods are generally used as described in many comprehensive and more specific references well known in the art and cited and discussed in this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual) (2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)). The nomenclature and laboratory methods and techniques used in analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are well known and commonly used in the art.

[0082] In the present invention, the term "at least 80% sequence identity" means at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity. In the present invention, the term "at least 85% sequence identity" means at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity. In some preferred embodiments, the sequence identity described in the present invention can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. The sequence comparison and determination of the percentage of identity between two sequences can be carried out by the BLASTN / BLASTP algorithm on the website of the National Center For Biotechnology Institute.

[0083] In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged in a three-dimensional space in positions relative to each other to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy chain and light chain are referred to as "complementary determining regions" or "CDRs". The assignment of amino acids to each domain is defined according to Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Maryland (1987 and 1991)) or Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987), Chothia et al., Nature 342:878-883 (1989).

[0084] "Antibody" as used in the present invention refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. The antibody can be a whole antibody and any antigen-binding fragment or single chain thereof. "Antibody" as used in the present invention includes any protein or peptide containing at least a part of the Ig molecule having the biological activity of binding to an antigen. Examples of "antibody" of the present invention include, but are not limited to, the CDR of the heavy chain or light chain or its ligand-binding portion, the variable region of the heavy chain or light chain, the constant region of the heavy chain or light chain, the framework region or any part thereof.

[0085] "Antigen-binding fragment" as described in the present invention refers to a Fab fragment, a Fab' fragment, an F(ab')2 fragment having antigen-binding activity, and an Fv fragment, an scFv fragment that binds to human ILT2. The Fv fragment contains the variable region of the antibody heavy chain and the variable region of the light chain, but no constant region, and is the smallest antibody fragment having all the antigen-binding sites. Generally, the Fv antibody also contains a polypeptide linker between the VH and VL domains and is capable of forming the structure required for antigen binding. Two antibody variable regions can also be linked into a single polypeptide chain with different linkers, called a single-chain antibody or single-chain Fv (scFv). The anti-ILT2 antibody of the present invention can be a single-chain variable region fragment (scFv), which is a single-chain polypeptide derived from an antibody and retains the ability to bind to an antigen. Examples of scFv include antibody polypeptides formed by recombinant DNA technology, in which the Fv regions of the immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked via a spacer sequence. Various methods for preparing scFv are well known to those skilled in the art.

[0086] The antibody described in the present invention refers to an immunoglobulin molecule or its immunologically active part, that is, a molecule containing an antigen-binding site that specifically binds to an antigen (reacting immunologically therewith). "Specific binding" means that the antibody reacts with one or more antigenic determinants of the antigen and does not react with other polypeptides or binds to other polypeptides with very low affinity (Kd>10-6). Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single-chain, Fab, Fab', and F(ab')2 fragments, Fv, scFv, and Fab expression libraries. Monoclonal antibodies (mAbs) are antibodies obtained from a single clone of cell lines, and the cell lines are not limited to eukaryotic, prokaryotic, or phage clone cell lines. Monoclonal antibodies or antigen-binding fragments can be recombinantly obtained using techniques such as hybridoma technology, recombinant technology, phage display technology, and synthetic techniques such as CDR grafting or other existing techniques.

[0087] The "murine antibody" described in the present invention is a monoclonal antibody against human ILT2 prepared according to the knowledge and skills in the art. When preparing, the test subject is injected with hILT2 antigen, and then hybridomas expressing antibodies with the required sequence or functional characteristics are isolated.

[0088] The "chimeric antibody" described in the present invention is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To establish a chimeric antibody, first establish a hybridoma secreting murine-specific monoclonal antibody, then clone the variable region gene from the murine hybridoma cells, and then clone the constant region gene of the human antibody as needed. Connect the murine variable region gene with the human constant region gene to form a chimeric gene and insert it into a human vector. Finally, express the chimeric antibody molecule in a eukaryotic industrial system or a prokaryotic industrial system.

[0089] The "humanized antibody" described in the present invention is also called a CDR-grafted antibody, which is an antibody produced by transplanting the CDR sequence of a mouse into the framework (FR) of the variable region of a human antibody. Such variable region framework sequences can be obtained from public DNA databases or published references, for example, obtained from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr or from the Journal of Immunoglobulin, 2001 ISBN012441351. Description of the Drawings

[0090] Figure 1 It is the experimental result (ELISA) of the binding activity of the anti-ILT2 humanized antibody to human ILT2, where the abscissa is the antibody concentration (nM) and the ordinate is the absorbance value at OD450.

[0091] Figure 2These are the experimental results (ELISA) of the anti-ILT2 humanized antibody blocking the binding of hHLA-G to human ILT2. The abscissa is the antibody concentration (nM), and the ordinate is the absorbance value at OD450.

[0092] Figure 3 These are the experimental results (FACS) of the binding activity of the anti-ILT2 humanized antibody to human ILT2. The abscissa is the antibody concentration (nM), and the ordinate is the mean fluorescence intensity.

[0093] Figure 4 These are the experimental results (FACS) of the anti-ILT2 humanized antibody blocking the binding of hHLA-G to human ILT2. The abscissa is the antibody concentration (nM), and the ordinate is the mean fluorescence intensity. Detailed implementation manners

[0094] The following representative examples are for better illustrating the present invention rather than limiting the protection scope of the present invention. In the following examples, the experimental methods without specified conditions are usually carried out according to conventional conditions, such as the antibody technology experimental manual of Cold Spring Harbor, the molecular cloning manual, etc., or according to the conditions recommended by the raw material or commodity manufacturers. The materials and reagents used in the examples are commercially available unless otherwise specified.

[0095] Example 1: Preparation of ILT2 antigen protein and anti-ILT2 positive control antibody

[0096] 1. Construction of expression vectors for antigen protein and positive control antibody

[0097] 1.1 Construction of expression vector for antigen protein

[0098] Synthesize the gene fragment encoding the complete human ILT2 protein, the amino acid sequence of which is shown in SEQ ID NO:23, and then clone it into the eukaryotic expression plasmid pTargeT to obtain its expression plasmid hILT2-pTarget. Synthesize the gene fragment encoding HLA-G, the amino acid sequence of which is designed as shown in SEQ ID NO:24, and then clone it into the eukaryotic expression plasmids pTargeT and pCDNA3.1 to obtain its expression plasmids hHLA-G-pTargeT and hHLA-G-pCDNA3.1.

[0099] Fuse the amino acid sequence of the extracellular region of human ILT2 protein with the hIgG1 Fc, mIgG1 Fc or His-tag amino acid sequence, and the amino acid sequences are designed as shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27 respectively. After codon optimization of the above amino acid sequences, the tagged ILT2-ECD gene fragments hILT2-ECD-hFc, hILT2-ECD-mFc, and hILT2-ECD-His are synthesized. After homologous recombination with the linear vector pSHiG3.0, the plasmids hILT2-ECD-hFc-pSHiG3.0, hILT2-ECD-mFc-pSHiG3.0, and hILT2-ECD-his-pSHiG3.0 are obtained.

[0100] Fuse the amino acid sequence of the D1D2 region in the extracellular region of human ILT2 protein with the hIgG1 Fc, mIgG1 Fc or His-tag amino acid sequence, and the amino acid sequences are designed as shown in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30 respectively. After codon optimization of the above amino acid sequences, the tagged ILT2-D1D2 gene fragments hILT2-D1D2-hFc, hILT2-D1D2-mFc, and hILT2-D1D2-His are synthesized. After homologous recombination with the linear vector pSHiG3.0, the plasmids hILT2-D1D2-hFc-pSHiG3.0, hILT2-D1D2-mFc-pSHiG3.0, and hILT2-D1D2-his-pSHiG3.0 are obtained.

[0101] 1.2 Construction of the expression vector of the positive control antibody 15G8

[0102] Use the humanized antibody 15G8 disclosed in the patent application WO2021 / 028921A1 as the positive control antibody, and the amino acid sequence of 15G8 is as follows:

[0103] Amino acid sequence of the 15G8 heavy chain: SEQ ID NO:31;

[0104] Amino acid sequence of the 15G8 light chain: SEQ ID NO:32.

[0105] 15G8 was prepared according to the method disclosed in WO2021 / 028921A1. After codon optimization of the amino acids of the 15G8 humanized antibody (the heavy chain subtype is human IgG4-PE (S228P, L235E mutants), and the light chain subtype is human κ), the full-length heavy chain and full-length light chain DNA sequences of the positive control antibody 15G8 were obtained. After homologous recombination of the full-length heavy chain DNA fragment of 15G8 with the linear vector pSHiG3.0, the eukaryotic expression plasmid 15G8-IgG4-PE-pSHiG3.0 was obtained. After homologous recombination of the full-length light chain DNA fragment of 15G8 with the linear vector pSHiG3.0, the eukaryotic expression plasmid 15G8-hK-pSHiG3.0 was obtained.

[0106] 2. Expression and purification of antigen proteins and positive control antibodies

[0107] 2.1 Expression of tagged antigen proteins and positive control antibodies

[0108] One day before transfection, count the seed cells, and the seeding density is 1×10 6 cells / mL. Dilute the cells with pre-warmed fresh Freestyle293 medium (Gibco) to 250 mL. The shaking culture conditions are 37 °C, 8% CO2, humidity > 80%, 95 ± 5 rpm. On the day of transfection, count the cells inoculated for 24 hours, calculate the amounts of plasmid and PEI, and the ratio of PEI transfection reagent: plasmid = 2:1. Take two centrifuge tubes, add 8 mL of pre-warmed medium to each, add the calculated amounts of plasmid and PEI transfection reagent respectively, invert and mix well, let stand at room temperature for 5 min, then slowly add the PEI dilution to the plasmid dilution, invert and mix well, let stand at room temperature for 20 min, and then slowly add the plasmid and PEI mixture to the cell shaking flask and place it on the shaker for culture. Among them, the tagged antigen protein plasmids hILT2-ECD-hFc-pSHiG3.0, hILT2-ECD-mFc-pSHiG3.0, hILT2-ECD-his-pSHiG3.0, hILT2-D1D2-hFc-pSHiG3.0, hILT2-D1D2-mFc-pSHiG3.0, hILT2-D1D2-his-pSHiG3.0 are transfected respectively; the positive control antibody 15G8 heavy chain plasmid 15G8-IgG4-PE-pSHiG3.0 and the light chain plasmid 15G8-hK-pSHiG3.0 are co-transfected. Feed supplements are carried out according to different protein tags, samples are collected on the 6th day, count before collecting the bottles, and record the cell density and viability.

[0109] 2.2 Purification by affinity chromatography column

[0110] Purification was carried out using AKTA (GE, AKTA pure-150) with an affinity chromatography column according to the protein properties. The affinity chromatography columns suitable for different proteins are shown in Table 1.

[0111] Table 1 Affinity chromatography columns suitable for different proteins

[0112]

[0113]

[0114] 3. Construction of stable cell lines

[0115] The eukaryotic expression plasmids hILT2-pTargeT and hHLA-G-pTargeT were transfected into CHO-K1 cells and JEG-3 cells by Lip3000 transfection method respectively, and the eukaryotic expression plasmid HLA-G-pcDNA3.1 was transfected into NUGC4 cells by exponential wave electroporation method. They were cultured in an incubator at 37 °C and 5% CO2. After 1-2 days, they were cultured under pressure with media containing 1000 μg / ml G418, 500 μg / ml G418, and 500 μg / ml G418 respectively. 14 days after transfection, the positive rate of the pool was detected by FACS. The transfected cells were plated (0.8 - 1.1 cells per well, 200 μl / well). The CHO-K1 stable hILT2 cells were incubated with PE anti-human CD85j (ILT2) Antibody; the JEG-3 and NUGC4 stable hHLA-G cells were incubated with PE anti-human HLA-G Antibod antibody. The mean value at a wavelength of 585 nm was read using a flow cytometer (ACEA, Novocyte 2060R), and data analysis was performed using GraphPad. The positive cell lines were subcloned, and the CHO-K1 cell line with high-level expression of hILT2 molecule, the JEG-3 cell line with high-level expression of hHLA-G molecule, and the NUGC4 cell line with high-level expression of hHLA-G molecule were selected respectively, and named CHO-K1-hILT2, JEG-3-hHLA-G, and NUGC4-hHLA-G.

[0116] Example 2: Preparation of anti-ILT2 murine monoclonal antibody

[0117] 1. Preparation of hybridoma monoclonal

[0118] 1.1 Animal immunization

[0119] BALB / c experimental mice were immunized by the method of co-immunizing with hILT2-ECD-mFc, hILT2-ECD-His, hILT2-D1D2-mFc, and hILT2-D1D2-His antigen proteins and Freund's complete adjuvant. The initial immunization antigen dosage was 50 μg, and 25 μg of antigen was used for later immunization. The initial immunization method was intraperitoneal injection, and the later immunization method was subcutaneous injection.

[0120] 1.2 Hybridoma fusion

[0121] 1.2.1 Preparation stage

[0122] Prepare B cells. Sacrifice the mice that were boost-immunized 3 - 4 days in advance; soak them in 75% alcohol for 5 min, transfer them to the operating table, and place them on a tray; cut open the skin of the lower abdomen of the mice, hold it with straight forceps, pull up the abdominal skin of the mice, aseptically cut open the peritoneum, take out the spleen and put it into a six-well plate containing 5 mL of PBS, transfer it to the laminar flow hood, use forceps and straight forceps to decompose the spleen capsule, and release the spleen cells; collect the spleen cell suspension, filter the cell suspension through a cell sieve into a 50 mL centrifuge tube, and add PBS to 20 mL. Centrifuge at 1570 rpm for 7 min, discard the supernatant; add 2 mL of red blood cell lysate (Sigma) to each mouse, lyse the red blood cells at room temperature for 2 min, add PBS to 20 mL, centrifuge at 1570 rpm for 7 min, and discard the supernatant. Generally, the spleen of 1 mouse is resuspended with 10 - 15 mL of PBS and counted. Prepare 1×10 8 Sp2 / 0 cells. Place the Sp2 / 0 cells in a 50 mL centrifuge tube, centrifuge at 1570 rpm at room temperature for 7 min. Discard the supernatant, resuspend the cells with PBS, and count. Take Sp2 / 0 cells and fuse them with mouse spleen cells (fusion ratio 1:1.5 - 1:2), centrifuge at 1570 rpm for 7 min. Resuspend the cells with 20 mL of electroporation buffer, centrifuge at 1570 rpm for 7 min, repeat once and then discard the supernatant, and resuspend the cells with (the total cell density during electroporation is 1 - 2×10 7 cells / mL) electroporation buffer (BTX), and add the cell suspension to a 9 mL electroporation fusion chamber for fusion.

[0123] 1.2.2 Electro-fusion

[0124] After adding the cell suspension to a 9 mL electroporation fusion chamber and completing the electroporation, let it stand for 1 min, aspirate the cell suspension and transfer it to a 50 mL centrifuge tube containing 20 mL of H-SFM medium (Gibco) (18 ml of incubation medium: 2 ml of 10% FBS (Gibco), 200 μl of 1·BIOMYC-3 (Bioind)), and incubate at room temperature for 20 min.

[0125] 1.2.3 Plating

[0126] The fused cell suspension was added to H-SFM medium (2% FBS, 1% 1·BIOMYC-3, 2% 1·HAT (Gibco)), and the cells were adjusted to a density of 2 - 2.7×10 5 cells / mL for plating, 150 μL / well, plating a 96-well plate, and placed in an incubator at 37°C and 5% CO2 for culture.

[0127] 1.3 Hybridoma screening

[0128] After 7 - 10 days of fusion culture, hybridoma supernatants that could bind to hILT2-ECD-hFc or hILT2-ECD-His protein were screened out by combined ELISA. Positive mother clones were selected and detected by blocking ELISA to screen out hybridoma clones that blocked the binding of HLA-G to hILT2-ECD.

[0129] The positive cell lines were subcloned by the limiting dilution method. After 7 - 10 days of culture, the binding activity of the subcloned supernatants to hILT2-his protein was detected by combined ELISA. The selected subclones were expanded in a 24-well plate. Five days later, pre-cell line detection was performed, and the binding activity of the subclones was detected by ELISA and FACS to obtain positive monoclonal cell lines. Multiple cell lines with good activity were selected and named L08, L16, and L43. The detection results are shown in Table 2 below.

[0130] Table 2 Detection results of positive cell lines

[0131] <![CDATA[Combined with ELISA OD 450 value]]> <![CDATA[Blocking ELISA OD 450 value]]> Combined with FACS M2% Parent L08 3.2313 0.1255 99.55％ L16 2.4510 0.1226 99.8％ L43 2.3636 0.9915 98.17％

[0132] The experimental results showed that the murine anti-L08, L16, and L43 of the present invention had good ability to bind to ILT2 and block the binding of HLA-G to ILT2.

[0133] 1.4 Preparation and sequencing of murine monoclonal antibodies

[0134] 1.4.1 Preparation of murine monoclonal antibodies

[0135] The positive cell lines L08, L16, and L43 were expanded in culture. The culture conditions were H-SFM medium containing 10% FBS and 1·BIOMYC-3. When the cell confluence > 80%, the cells were passaged and expanded in culture. When the culture reached 50 mL, the supernatant was collected and the antibody was purified. An antibody with good purity by SDS-PAGE gel electrophoresis was obtained. 1.4.2 Sequencing of murine monoclonal antibodies

[0136] Two vials of cells in a 10-cm culture dish were cryopreserved. The cells in a 15-cm culture dish were rinsed and collected into a 1.5-mL EP tube. After extracting mRNA (Qiagen) and reverse transcribing it to obtain cDNA (Thermo), specific primers were designed based on the variable regions of different subtypes of mouse antibodies. Using the cDNA as a template, PCR amplification of the antibody variable region genes was performed to obtain the gene fragments of the variable regions of the mouse antibody light and heavy chains, respectively. The sequences are shown in Tables 3 and 4 below.

[0137] Table 3 Amino acid sequence of anti-ILT2 murine monoclonal antibody

[0138] Antibody Amino acid sequence of the heavy chain variable region Amino acid sequence of the light chain variable region L08 SEQ ID NO:33 SEQ ID NO:34 L16 SEQ ID NO:35 SEQ ID NO:36 L43 SEQ ID NO:37 SEQ ID NO:38

[0139] Table 4 Amino acid sequence of the HCDR region of anti-ILT2 murine monoclonal antibody

[0140]

[0141]

[0142] Example 3: Construction of anti-ILT2 chimeric antibody

[0143] The gene fragments of the mouse antibody light and heavy chain variable regions were respectively homologously recombined with a linearized eukaryotic expression plasmid containing the human antibody light chain (κ) or heavy chain (IgG4) constant region, and then transformed into competent Escherichia coli DH5α cells. The mixture was evenly spread on the surface of an agar plate containing the corresponding antibiotic and incubated overnight in a 37°C constant temperature incubator. Then, several single colonies were picked for DNA sequencing. The correctly sequenced chimeric antibodies were respectively labeled as L08CHI, L16CHI, and L43CHI. The amino acid sequences of the heavy chain variable regions and light chain variable regions of antibodies L08CHI, L16CHI, and L43CHI are the same as those of the corresponding mouse antibodies L08, L16, and L43.

[0144] The correctly sequenced positive clones were inoculated into 2·YT liquid medium containing the corresponding antibiotic and cultured with shaking at 37°C for more than 12 hours. Then, the bacterial cells were collected for plasmid extraction to obtain the chimeric antibody light and heavy chain expression plasmids, and a nucleic acid quantifier was used to detect the concentration and purity of the plasmids.

[0145] The chimeric antibody plasmids were transfected into HEK293E cells to express and purify a large amount of antibody, and purity detection, activity analysis, and affinity detection were performed. The detection results are shown in Table 5 below.

[0146] Table 5 Activity determination of chimeric antibody

[0147]

[0148] The experimental results show that the chimeric antibodies L08CHI, L16CHI, and L43CHI of the present invention have good abilities to bind to ILT2, block the binding of HLA-G to ILT2, and have high affinity for ILT2. Example 4: Construction and production of anti-ILT2 humanized antibodies

[0149] Based on the activity analysis of the chimeric antibodies, chimeric antibodies such as L08CHI and L16CHI were selected for humanized antibody modification.

[0150] First, by comparing with the mouse antibody sequences in the Immunoglobulin Gene Database (IMGT), the murine germlines of the variable regions of the L16CHI and L08CHI antibodies were confirmed. After homologous alignment, the FR region of the heavy chain variable region sequence of the L08CHI antibody was most similar to the murine antibody germline gene IGHV2-3*01, and the FR region sequence of the light chain variable region sequence was most similar to the murine antibody gene IGKV10-96*01; the FR region of the heavy chain variable region sequence of the L16CHI antibody was most similar to the murine antibody germline gene IGHV3-6*01, and the FR region sequence of the light chain variable region sequence was most similar to the murine antibody gene IGKV14-111*01.

[0151] The sequences to be humanized were numbered according to Kabat to determine the CDR regions and FR regions, and the CDR3 sequence was input into IGBLAST after incomplete processing https: / / www.ncbi.nlm.nih.gov / igblast / ) Select templates with high homology scores, replace the amino acids that are different between human and mouse in the FR region with human amino acids, perform back mutations on the key amino acids in the FR region, and perform point mutations on the PTM, glycosylation, and oxidation-risk amino acids in the CDR region. For the heavy chain of L08CHI, the gemline template IGHV4-4*08 was selected, and 4 humanized heavy chain variable regions were obtained. The sequences are shown in SEQ ID NO:39, 40, and 41 (HCDR1, HCDR2, and HCDR3 are SEQ ID NO:1, 2, and 3) and SEQ ID NO:42 (HCDR1, HCDR2, and HCDR3 are SEQ ID NO:1, 19, and 20); for the light chain, the template IGKV1D-33*01 was selected, and 5 humanized light chain variable regions were obtained. The sequences are shown in SEQ ID NO:43, 44, 45, 46, and 47 (LCDR1, LCDR2, and LCDR3 are SEQ ID NO:4, 5, and 6). For the heavy chain of L16CHI, the gemline template IGHV4-31*02 was selected, and 4 humanized heavy chain variable regions were obtained. The sequences are shown in SEQ ID NO:48 (HCDR1, HCDR2, and HCDR3 are SEQ ID NO:7, 21, and 9) and SEQ ID NO:49, 50, 51 (HCDR1, HCDR2, and HCDR3 are SEQ ID NO:7, 8, and 9); for the light chain, the template IGKV1-16*01 was selected, and 5 humanized light chain variable regions were obtained. The sequences are shown in SEQ ID NO:52 (LCDR1, LCDR2, and LCDR3 are SEQ ID NO:22, 11, and 12) and SEQ ID NO:53, 54, 55, 56 (LCDR1, LCDR2, and LCDR3 are SEQ ID NO:10, 11, and 12).

[0152] Reverse transcribe the amino acid sequences of the humanized antibody light and heavy chain variable regions designed above into the corresponding nucleotide sequences, generate oligonucleotide fragments with complementary sequences between adjacent fragments, anneal and ligate the oligonucleotide fragments by Overlap PCR, and then amplify the complete light and heavy chain variable region nucleotide fragments using specific primers; co-transform the purified light chain variable region nucleotide fragments with a eukaryotic expression plasmid containing the human κ light chain constant region into competent Escherichia coli DH5α cells, and co-transform the purified heavy chain variable region nucleotide fragments with a eukaryotic expression plasmid of the IgG4-PE heavy chain constant region into competent Escherichia coli DH5α cells. Spread the competent cells of the transformed plasmids evenly on the surface of agar plates containing the corresponding antibiotics, and after overnight culture in a 37°C constant temperature incubator, pick several colonies for DNA sequencing respectively.

[0153] Inoculate the correctly sequenced positive clones into 2×YT liquid medium containing the corresponding antibiotics, shake culture at 37°C for more than 12 hours, then collect the bacterial cells for plasmid extraction to obtain the expression plasmids of humanized antibody light chain and heavy chain, and use a nucleic acid quantitative analyzer to detect the concentration and purity of the plasmids.

[0154] Transfect the plasmid into HEK293E cells, express and purify to obtain a large amount of antibody, and perform purity detection, activity analysis and affinity detection.

[0155] Select humanized antibodies with good purity, activity and affinity, and label them as HuL08 and HuL16. The sequences are shown in Table 6 and Table 7.

[0156] Table 6 Sequences of anti-ILT2 humanized antibodies

[0157]

[0158] The amino acid sequences of HCDR1, HCDR2 and HCDR3 of antibody HuL08 are SEQ ID NO:1, 19 and 20 respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are SEQ ID NO:4, 5 and 6 respectively. The amino acid sequences of HCDR1, HCDR2 and HCDR3 of antibody HuL16 are SEQ ID NO:7, 21 and 9 respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are SEQ ID NO:22, 11 and 12 respectively.

[0159] Table 7 Complete sequences of anti-ILT2 humanized antibodies

[0160] Humanized antibody Amino acid sequence of the heavy chain Amino acid sequence of the light chain HuL08 SEQ ID NO:61 SEQ ID NO:62 HuL16 SEQ ID NO:63 SEQ ID NO:64

[0161] Example 5: Determination of the binding activity of anti-ILT2 antibody to ILT2 (ELISA)

[0162] Analyze the binding activity of the antibody by ELISA. Coat the protein hILT2-ECD-His (0.2 μg / ml, 100 μL per well) onto a 96-well ELISA plate. The anti-ILT2 antibody of the present invention is used as the primary antibody and is added to the ELISA plate starting from 0.5 μg / mL and diluted 4-fold serially. Incubate at 37°C for 2 h. The positive control antibody is 15G8. HRP-Goat anti-Human IgG (Jackson) diluted 1:5000 is used as the secondary antibody, and after adding the secondary antibody, incubate at 37°C for 1 h. Add the chromogenic solution TMB (3,3',5,5'-tetramethylbenzidine) (Beijing Solarbio Science & Technology Co., Ltd.), and after termination, read the OD450 value using an ELISA reader (Thermo, Multiskan FC). Use GraphPad to generate the EC50, and the results are shown in Table 8. Figure 1 as shown.

[0163] Table 8 Binding activity of anti-ILT2 humanized antibody to human ILT2

[0164] Humanized antibody EC50 value (nM) HuL08 0.01733 HuL16 0.01148 15G8 0.01288

[0165] The experimental results show that the humanized anti-ILT2 antibodies HuL08 and HuL16 of the present invention have good ability to bind to human ILT2.

[0166] Example 6: Blocking the binding of anti-ILT2 antibody to hHLA-G and ILT2 (ELISA)

[0167] The blocking activity of the anti-ILT2 antibody of the present invention to block the binding of HLA-G to ILT2 was detected by ELISA. hHLA-G-his (2 μg / mL, 100 μL per well) was coated on a 96-well ELISA plate. The anti-ILT2 antibody of the present invention was used as the primary antibody and was added to the ELISA plate starting from 6 μg / mL and diluted 2.5-fold in gradient, with a total of 8 concentrations. The positive control antibody was 15G8; the ligand solution was a 2 μg / mL hILT2-ECD-mFc solution. The two were mixed at a 1:1 volume ratio, incubated at 37 °C for 0.5 h, then added to the ELISA plate, and incubated at 37 °C for 2 h. HRP-Goat anti-Mouse IgG (Jackson) was diluted 1:5000 and used as the secondary antibody. After adding the secondary antibody, it was incubated at 37 °C for 1 h. The chromogenic solution TMB was added, and after termination, the OD450 value was read using an ELISA reader (Thermo, Multiskan FC). IC50 was generated using GraphPad, and the results are shown in Table 9. Figure 2 as shown.

[0168] The experimental results show that the humanized anti-ILT2 antibodies HuL08 and HuL16 of the present invention have the ability to block the binding of HLA-G to ILT2.

[0169] Table 9 Ability of anti-ILT2 humanized antibody to block the binding of hHLA-G to ILT2

[0170] Humanized antibody IC50 value (nM) HuL08 3.249 HuL16 3.933 15G8 3.654

[0171] The experimental results show that the humanized anti-ILT2 antibodies HuL08 and HuL16 of the present invention have good ability to block the binding of hHLA-G to human ILT2.

[0172] Example 7: Determination of the binding of anti-ILT2 antibody to cell surface ILT2 (FACS)

[0173] The binding activity of the antibody to cell surface ILT2 was analyzed by FACS. The CHO K1-ILT2 cells stably expressing ILT2 were seeded at 1×10 per well 5Seed 96-well U-shaped plates with cells; Starting from 20 μg / mL, the anti-ILT2 antibody of the present invention was used as the primary antibody and serially diluted 4-fold and added to the U-shaped plates, with a total of 8 concentrations. Incubate at 4 °C for 1.5 h. The positive control was 15G8; The secondary antibody was PE Goat anti-Human IgG (abcam), added to the U-shaped plates at 0.8 μL per well, and incubated at 4 °C for 45 min. Read the mean value at a wavelength of 585 nm using a flow cytometer (ACEA, Novocyte2060R), and perform data analysis using GraphPad to generate the EC50 value. The results are shown in Table 10, Figure 3 as shown.

[0174] Table 10 Binding activity of anti-ILT2 humanized antibody and ILT2

[0175] Humanized anti EC50 value (nM) HuL08 0.2978 HuL16 0.5072 15G8 0.2917

[0176] The experimental results showed that the humanized anti-ILT2 antibodies HuL08 and HuL16 of the present invention had good ability to bind to cell surface ILT2.

[0177] Example 8: Anti-ILT2 antibody blocks the binding of cell surface hHLA-G to ILT2 (FACS)

[0178] The blocking activity of the anti-ILT2 antibody of the present invention in blocking the binding of cell surface hHLA-G to ILT2 was detected by FACS. Seed JEG-3-hHLA-G cells stably expressing hHLA-G at 1×10 5 cells per well in 96-well U-shaped plates; Starting from 40 μg / mL, the anti-ILT2 antibody of the present invention was used as the primary antibody and serially diluted 2-fold and added to the enzyme-linked immunosorbent assay (ELISA) plates, with a total of 8 concentrations. The positive control antibody was 15G8; The ligand solution was 8 μg / mL hILT2-ECD-mFc solution. The two were mixed at a volume ratio of 1:1, incubated at 37 °C for 0.5 h and then added to the U-shaped plates, and incubated at 4 °C for 1.5 h. The secondary antibody was PE Goat anti-Mouse IgG (biolegend), added to the U-shaped plates at 0.8 μL per well, and incubated at 4 °C for 45 min. Read the mean value at a wavelength of 585 nm using a flow cytometer (ACEA, Novocyte 2060R), and perform data analysis using GraphPad to generate the IC50 value. The results are shown in Table 11, Figure 4 as shown.

[0179] Table 11 Ability of anti-ILT2 humanized antibody to block the binding of hHLA-G to ILT2

[0180] Humanized antibody IC50 value (nM) HuL08 24.04 HuL16 29.04 15G8 20.05

[0181] The experimental results show that the humanized anti-ILT2 antibodies HuL08 and HuL16 of the present invention have the ability to block the binding of cell surface hHLA-G to ILT2.

[0182] Example 9: Determination of the Affinity of Anti-ILT2 Antibodies for Human ILT2 (Fortebio)

[0183] Dilute ILT2-His to a concentration of 5 μg / ml with SD buffer (0.02% Tween20 + 0.1% BSA solution). Dilute the humanized anti-ILT2 antibody with SD buffer at a 4-fold concentration gradient. Select an AHC sensor to immobilize the antigen and perform the affinity determination according to the operating procedures of fortebio Octet RED96. The specific parameters and experimental results are shown in Table 12.

[0184] Table 12 Determination of the Affinity of Anti-ILT2 Antibodies for Human ILT2 Protein

[0185] Antibody KD (M) Kon (1 / Ms) Kdis (1 / s) 15G8 <![CDATA[2.02×10 -9 > <![CDATA[4.35×10 5 > <![CDATA[1.76×10 -3 > HuL08 <![CDATA[3.40×10 -9 > <![CDATA[8.73×10 5 > <![CDATA[1.48×10 -3 <!-- 18 -->]]> HuL16 <![CDATA[7.76×10 -10 > <![CDATA[4.40×10 5 > <![CDATA[3.42×10 -4 >

[0186] The experimental results show that the ILT2 humanized antibodies HuL08 and HuL16 of the present invention have good affinity for binding to human ILT2 protein.

[0187] Example 10: ILT2 Antibodies Enhance the Killing Function of Human NK Cells

[0188] 1. Experimental Materials

[0189] (1) Experimental Cells and Reagents

[0190] Human NK cells are derived from PBMC amplification; human gastric cancer cell line NUGC4-HLA-G cells (prepared according to Example 1); To-Pro-3 is purchased from Biolegend.

[0191] (2) Test Articles and Controls

[0192] Negative control IgG4, positive control 15G8, HuL08;

[0193] 2. Experimental Method

[0194] 2.1 Antibody Preparation

[0195] Dilute the antibody with RPMI-1640 medium containing 5% FBS to different concentration working solutions according to the preparation scheme.

[0196] 2.2 Effector Cell Treatment & Sample Addition

[0197] ① Resuscitate human NK cells from different sources with RPMI-1640 medium containing 5% FBS, count and adjust the viable cell concentration to 1×10 6cells / mL, inoculated into a non-TC-treated 96-well round bottom plate (100 μL / well);

[0198] ② Add the corresponding antibodies to the wells of different drug groups and incubate in an incubator for about 0.5 h;

[0199] 2.3 Target cell treatment (CFSE staining incubation)

[0200] ① Digest and collect target cells, count, and stain with CFSE;

[0201] ② Resuspend CFSE(+) target cells at 1×10 6 cells / mL in RPMI-1640 medium containing 5% FBS and inoculate. The effector-to-target ratio is 2:1. For the no effector cell and no antibody group, add the corresponding volume of RPMI-1640 medium containing 5% FBS to make up the volume to 200 μL;

[0202] ③ Centrifuge the well plate at 200 g for 1 min and incubate in a cell incubator for 24 h;

[0203] 2.4 Sample collection and detection

[0204] After 24 h, collect all cells, stain with To-Pro-3, and detect the mortality rate of CFSE+ target cells by flow cytometry. The negative control is when human NK cells and target cells exist alone or in the presence of isotype control IgG4. In the control group, the NUGC4-HLA-G group means there are NUGC4-HLA-G target cells, no effector cells, and no antibodies; the No drug group means there are NUGC4-HLA-G cells and effector cells, no antibodies; the IgG4 group means there are NUGC4-HLA-G cells and effector cells, and IgG4 is added. The experimental results are shown in Table 13. One-way ANOVA model was used to calculate the statistical significance. Compared with the IgG4 group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0205] Table 13 Effects of anti-ILT2 antibody on the killing function of human NK cells against NUGC4-HLA-G cells

[0206] Antibody Dose (μg / ml) Target cell death (%) NUGC4 - HLA - G - 17.68 No drug - 22.16 IgG4 50 26.31 HuL08 0.1 35.37** HuL08 1 42.84**** HuL08 10 47.23**** 15G8 50 41.08****

[0207] The experimental results show that the anti-ILT2 antibody of the present invention can significantly enhance the killing function of human NK cells against the tumor target cells of human gastric cancer cell line NUGC4-HLA-G in a concentration-dependent manner.

[0208] Example 11: Antitumor test of human tumor cell mouse xenograft model

[0209] 1. Experimental materials

[0210] (1) Experimental cells and animals

[0211] Human gastric cancer cell line NUGC4-HLA-G cells;

[0212] huHSG-NCG-hIL15 mice, female, 6 - 8 weeks old, weighing 18 - 20 grams, purchased from Jiangsu Genscript Biotech Co., Ltd.;

[0213] (2) Test articles and reference substances

[0214] Negative control vehicle PBS, positive control substances 15G8, HuL08, HuL16;

[0215] 2. Experimental methods

[0216] NUGC4-HLA-G xenograft tumor model

[0217] Inoculate human tumor cells NUGC4-HLA-G cells on the right rear side of mice, and the number of inoculated cells is 5×10 6 / mouse. Wait until the tumor grows to an average volume of 80 - 120 mm 3 and then start grouping. Administer the drug via the tail vein twice a week. Measure the tumor diameter with vernier calipers twice a week, and calculate the tumor volume. The formula for calculating the tumor volume is: V = 0.5a×b 2 , where a and b represent the long diameter and short diameter of the tumor respectively. The tumor growth inhibition rate is calculated using the following formula: TGI(%) = [1 - (Ti - T0) / (Vi - V0)]×100, where Ti is the average tumor volume of a certain dosing group on a certain day, T0 is the average tumor volume of this dosing group at the start of dosing; Vi is the average tumor volume of the vehicle control group on a certain day (the same day as Ti), and V0 is the average tumor volume of the vehicle control group at the start of dosing. Record the tumor volumes measured continuously for 34 days of drug administration, and plot the tumor volume growth curve using GraphPad Prism. After the experiment, detect the tumor weight and calculate the tumor inhibition rate (TGI)% of each group. The results are shown in Table 14.

[0218] Table 14 Anti-ILT2 antibody's antitumor effect on NUGC4-HLA-G xenograft tumor model of human gastric cancer cell line

[0219] Antibody Dose (mg / kg) <![CDATA[TGI TW (%)]]> PBS 0 0 15G8 20 48.27 HuL08 5 41.95 HuL16 5 37.10

[0220] The experimental results show that the humanized anti-ILT2 antibodies HuL08 and HuL16 of the present invention have good in vivo proliferation inhibitory effects on human gastric cancer cell line NUGC4-HLA-G.

[0221] Although the present invention has been described in detail above, those skilled in the art understand that various modifications and changes can be made to the present invention without departing from the spirit and scope of the present invention. The scope of the rights of the present invention is not limited to the detailed description above, but shall be attributed to the claims.

Claims

1. An anti-ILT2 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein: (1) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 selected from the following groups: (a1) the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3; (a2) the amino acid sequences shown in SEQ ID NOs: 7, 8 and 9; (a3) the amino acid sequences shown in SEQ ID NOs: 13, 14 and 15; (a4) the amino acid sequences shown in SEQ ID NOs: 1, 19 and 20; (a5) the amino acid sequences shown in SEQ ID NOs: 7, 21 and 9; and (a6) an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in (a1), (a2), (a3), (a4) or (a5); and (2) The light chain variable region comprises LCDR1, LCDR2 and LCDR3 selected from the following group: (b1) the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6; (b2) the amino acid sequences shown in SEQ ID NOs: 10, 11 and 12; (b3) the amino acid sequences shown in SEQ ID NOs: 16, 17 and 18; (b4) the amino acid sequences shown in SEQ ID NOs: 22, 11 and 12; and (b5) An amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in (b1), (b2), (b3) or (b4).

2. The anti-ILT2 antibody or antigen-binding fragment thereof according to claim 1, which has: The HCDR1, HCDR2 and HCDR3 are SEQ ID NOs: 1, 2 and 3, SEQ ID NOs: 1, 19 and 20, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3 or SEQ ID NOs: 1, 19 and 20, respectively, and the LCDR1, LCDR2 and LCDR3 are SEQ ID NOs: 4, 5 and 6, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively; or The HCDR1, HCDR2 and HCDR3 are respectively SEQ ID NOs: 7, 8 and 9, SEQ ID NOs: 7, 21 and 9, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 7, 8 and 9 or SEQ ID NOs: 7, 21 and 9, and the LCDR1, LCDR2 and LCDR3 are respectively SEQ ID NOs: 10, 11 and 12, SEQ ID NOs: 22, 11 and 12, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 10, 11 and 12 or SEQ ID NOs: 22, 11 and 12; or The HCDR1, HCDR2 and HCDR3 are SEQ ID NOs: 13, 14 and 15, respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 13, 14 and 15, and the LCDR1, LCDR2 and LCDR3 are SEQ ID NOs: 16, 17 and 18, respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 16, 17 and 18.

3. The anti-ILT2 antibody or antigen-binding fragment thereof according to claim 2, wherein The HCDR1, HCDR2 and HCDR3 are SEQ ID NOs: 1, 2 and 3, respectively, or amino acid sequences having at least 85% sequence identity thereto, and the LCDR1, LCDR2 and LCDR3 are SEQ ID NOs: 4, 5 and 6, respectively, or amino acid sequences having at least 85% sequence identity thereto; or The HCDR1, HCDR2 and HCDR3 are SEQ ID NOs: 1, 19 and 20, respectively, or amino acid sequences having at least 85% sequence identity thereto, and the LCDR1, LCDR2 and LCDR3 are SEQ ID NOs: 4, 5 and 6, respectively, or amino acid sequences having at least 85% sequence identity thereto; or The HCDR1, HCDR2 and HCDR3 are SEQ ID NOs: 7, 8 and 9, respectively, or amino acid sequences having at least 85% sequence identity thereto, and the LCDR1, LCDR2 and LCDR3 are SEQ ID NOs: 10, 11 and 12, respectively, or amino acid sequences having at least 85% sequence identity thereto; or The HCDR1, HCDR2 and HCDR3 are SEQ ID NOs: 7, 21 and 9, respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 7, 21 and 9, and the LCDR1, LCDR2 and LCDR3 are SEQ ID NOs: 22, 11 and 12, respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 22, 11 and 12.

4. The anti-ILT2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein: The amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:33, SEQ ID NO:35 and SEQ ID NO:37, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:33, SEQ ID NO:35 or SEQ ID NO:37 and having at least 85% sequence identity with SEQ ID NO:33, SEQ ID NO:35 or SEQ ID NO:37, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO:34, SEQ ID NO:36 and SEQ ID NO:38, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:34, SEQ ID NO:36 or SEQ ID NO:38 and having at least 85% sequence identity with SEQ ID NO:34, SEQ ID NO:36 or SEQ ID NO:38; or The amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:42 and having the same function as SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:42, and an amino acid sequence having at least 85% sequence identity with SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:42, and the amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:47, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 or SEQ ID NO:47 and having the same function as SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45 NO:44, SEQ ID NO:45, SEQ ID NO:46 or SEQ ID NO:47, and an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 or SEQ ID NO:47; or The amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 or SEQ ID NO:51 and an amino acid sequence having at least 85% sequence identity with SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 or SEQ ID NO:51, and the amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 and SEQ ID NO:56, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 or SEQ ID NO:56 and NO:53, SEQ ID NO:54, SEQ ID NO:55 or SEQ ID NO:56, and amino acid sequences that have at least 85% sequence identity to SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 or SEQ ID NO:

56.

5. The anti-ILT2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein The amino acid sequence of the heavy chain variable region is SEQ ID NO:33, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:33 and having at least 85% sequence identity with SEQ ID NO:33, and the amino acid sequence of the light chain variable region is SEQ ID NO:34, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:34 and having at least 85% sequence identity with SEQ ID NO:34; The amino acid sequence of the heavy chain variable region is SEQ ID NO:35, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:35 and having at least 85% sequence identity with SEQ ID NO:35, and the amino acid sequence of the light chain variable region is SEQ ID NO:36, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:36 and having at least 85% sequence identity with SEQ ID NO:36; or The amino acid sequence of the heavy chain variable region is SEQ ID NO:37, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:37 and having at least 85% sequence identity with SEQ ID NO:37, and the amino acid sequence of the light chain variable region is SEQ ID NO:38, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:38 and having at least 85% sequence identity with SEQ ID NO:38; or The amino acid sequence of the heavy chain variable region is SEQ ID NO:42, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:42 and having at least 85% sequence identity with SEQ ID NO:42, and the amino acid sequence of the light chain variable region is SEQ ID NO:47, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:47 and having at least 85% sequence identity with SEQ ID NO:47; or The amino acid sequence of the heavy chain variable region is SEQ ID NO:48, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:48 and having the same function as SEQ ID NO:48, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:48, and the amino acid sequence of the light chain variable region is SEQ ID NO:52, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:52 and having the same function as SEQ ID NO:52, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:

52.

6. The anti-ILT2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody is a murine monoclonal antibody, a chimeric antibody, a humanized antibody, a bispecific antibody or a fully human antibody.

7. An isolated nucleic acid encoding the anti-ILT2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. The nucleic acid of claim 7, comprising: A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO: 33, SEQ ID NO: 35 or SEQ ID NO: 37; and a nucleotide sequence encoding a light chain variable region such as SEQ ID NO: 34, SEQ ID NO: 36 or SEQ ID NO: 38; or A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:42; and a nucleotide sequence encoding a light chain variable region such as SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 or SEQ ID NO:47; or A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 or SEQ ID NO:51; and a nucleotide sequence encoding a light chain variable region such as SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 or SEQ ID NO:

56.

9. An expression vector comprising the nucleic acid according to claim 7 or 8.

10. A host cell transformed with the expression vector according to claim 9, wherein the host cell is selected from prokaryotic cells and eukaryotic cells, preferably mammalian cells.

11. A method for preparing the anti-ILT2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, comprising the steps of expressing the antibody or antigen-binding fragment thereof in the host cell according to claim 10, and isolating the antibody or antigen-binding fragment thereof from the host cell. 12 . A pharmaceutical composition comprising the anti-ILT2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

13. Use of the anti-ILT2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 12 in the preparation of a drug for preventing and / or treating tumors.

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