Anti-ILT4 single-domain antibody and application thereof

Abstract

The invention provides an anti-ILT4 single domain antibody and an antigen binding fragment thereof. Also provided are methods of using the same for treating or preventing a disease in a subject; and methods of making the antibodies and antigen binding fragments thereof.

Description

An anti-ILT4 single-domain antibody and its application Technical Field

[0001] The present invention belongs to the fields of tumor immunotherapy and molecular immunology, and specifically relates to an anti-ILT4 single-domain antibody and applications thereof. Background Art

[0002] ILT4 (Immunoglobulin-like transcript 4), also known as CD85D, LILRB2, LIR2, and MIR10, is encoded by the LILRB2 gene and belongs to the family of activating and inhibitory immunoglobulin-like transcripts (ILTs) that regulate immune cell activation. ILT4 is a classic type I transmembrane protein with four extracellular tandem Ig-like domains, a 23-amino acid transmembrane domain, and a cytoplasmic tail with three immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Physiologically, ILT4 is mainly expressed in innate immune cells such as monocytes, macrophages, dendritic cells (DCs), and granulocytes. Ligand binding to ILT4 recruits SH-2 containing SHP-1 or SHP-2 phosphatases in immune cells, thereby inhibiting calcium mobilization of monocytes and dendritic cells and suppressing their activation signals.

[0003] Studies have shown that ILT4 is also highly expressed in various solid tumors, including NSCLC, breast cancer, esophageal cancer, and pancreatic cancer. In a pancreatic endothelial cell malignant transformation model, ILT4 expression is significantly induced during the multi-step carcinogenesis process. Clinically, ILT4 expression levels in tumor cells from patients with non-small cell lung cancer and breast cancer are positively correlated with poor cell differentiation, increased regional lymph node metastasis, advanced cancer stages, and poor patient survival. Other studies have shown that ILT4 directly controls the behavior of malignant tumor cells through the following pathways: 1. Promoting tumor proliferation and growth; 2. Increasing tumor invasion and metastasis; 3. Maintaining an immunosuppressive tumor microenvironment; 4. Producing tolerogenic dendritic cells; 5. Inhibiting the development and function of T effector cells; and 6. Inducing various regulatory T cell (Treg) subsets. In addition to signaling pathway-mediated regulation of tumor biology, ILT4 can also activate the PI3K / AKT / mTOR signaling and NF-κB pathways to directly and / or indirectly modulate adaptive anti-tumor immune responses.

[0004] These studies suggest that ILT4 has the potential to serve as a new immune checkpoint target in tumor immunotherapy. Given that the current success rate of immune checkpoint therapy is still not as expected, there is an urgent need to develop drugs targeting new and alternative checkpoint molecules / signals to improve anti-tumor immunotherapy.

[0005] Summary of the Invention

[0006] To achieve the above objectives, the present invention provides an anti-ILT4 single-domain antibody or an antigen-binding fragment thereof.

[0007] In some embodiments, the single-domain antibody or antigen-binding fragment thereof comprises CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 respectively comprise an amino acid sequence represented by SEQ ID NO: 1-3, or a sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 1-3, or an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence of SEQ ID NO: 1-3.

[0008] In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences represented by SEQ ID NOs: 1-3, respectively.

[0009] In some embodiments, the single-domain antibody or antigen-binding fragment thereof comprises an amino acid sequence represented by SEQ ID NO: 4, 6, or a sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 4, 6, or an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence of SEQ ID NO: 4, 6.

[0010] In some embodiments, the single-domain antibody or antigen-binding fragment thereof further comprises an immunoglobulin Fc region.

[0011] In some embodiments, the dissociation constant KD between the single domain antibody or antigen binding fragment thereof and ILT4 is less than 10 nM. In some embodiments, the dissociation constant KD between the single domain antibody or antigen binding fragment thereof and ILT4 is less than 1 nM.

[0012] In some embodiments, the single domain antibody or antigen-binding fragment thereof is camelid, chimeric, humanized, or fully human.

[0013] The present invention provides a polynucleotide encoding the single-domain antibody or antigen-binding fragment thereof according to any one of the above schemes. In some embodiments, the polynucleotide is selected from the polynucleotide sequences corresponding to SEQ ID NOs: 5 and 8.

[0014] The present invention provides a recombinant vector, a transgenic cell line, a bacteriophage, a recombinant bacterium or a viral vector, wherein the recombinant vector, the transgenic cell line, the bacteriophage, the recombinant bacterium or the viral vector contains any of the polynucleotides described above.

[0015] The present invention provides an isolated host cell containing the above-mentioned recombinant vector, transgenic cell line, phage, recombinant bacteria or viral vector.

[0016] In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a CHO cell.

[0017] The present invention provides an antibody expression method, which uses the above-mentioned recombinant vector, transgenic cell line, phage, recombinant bacteria or viral vector to express antibody protein in any of the above-mentioned host cells.

[0018] The present invention provides a use of the above-mentioned single-domain antibody or antigen-binding fragment thereof in the preparation of a medicament, wherein the medicament contains the single-domain antibody or antigen-binding fragment thereof and chemotherapy, for treating tumors in human patients, wherein the antibody and the chemotherapy are formulated to provide a therapeutic effect greater than the sum of the effects of the individual agents.

[0019] The present invention provides a construct comprising any of the above-mentioned single-domain antibodies or antigen-binding fragments thereof, and a second part selected from a second antibody or antigen-binding fragment thereof, a detectable marker, a drug, gold nanoparticles / nanorods, nanomagnetic particles, viral coat proteins or viral particles, radionuclides, liposomes, chemotherapeutic agents, or a combination thereof.

[0020] In some embodiments, the second antibody or antigen-binding portion thereof has a different binding specificity than any of the above-mentioned single domain antibodies or antigen-binding fragments thereof. In some embodiments, the antigen of the second antibody or antigen-binding portion thereof is selected from a tumor-associated antigen (TAA) or an immune checkpoint.

[0021] In some embodiments, the detectable label is a radionuclide.

[0022] In some embodiments, the drug is selected from the group consisting of a toxin, a cytokine, or an enzyme.

[0023] The present invention provides a pharmaceutical composition comprising any of the above-mentioned single-domain antibodies or antigen-binding fragments thereof.

[0024] In some embodiments, the composition further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an immunotherapeutic agent.

[0025] The present invention provides a use of the above-mentioned pharmaceutical composition in preparing a drug for treating diseases associated with ILT4, wherein the disease includes tumors or autoimmune diseases.

[0026] The present invention provides a use of the above-mentioned single-domain antibody or antigen-binding fragment thereof in the preparation of a medicament for treating and / or preventing and / or diagnosing a disease.

[0027] In some embodiments, the disease is selected from the group consisting of human brain astroglioblastoma, human pharyngeal cancer, adrenal tumor, AIDS-related cancer, alveolar soft tissue sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, renal chromophobe cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymoma, Ewing tumor, extraskeletal myxoid chondrosarcoma, fibrous dysplasia, fibrous dysplasia, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, pancreatic islet cell tumor, Kaposi sarcoma, kidney cancer, leukemia, liposarcoma / malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid cancer, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, metastatic kidney cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymic cancer, thymoma, metastatic thyroid cancer, or uterine cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG1 is a graph showing the binding curve of the antibody of the present invention to the ILT4 protein.

[0029] FIG2 is a diagram showing the single-point binding effect of the antibody of the present invention on CHO-ILT4 cells.

[0030] FIG3 is a multi-point binding curve diagram of the antibody of the present invention and CHO-ILT4 cells.

[0031] FIG4 is a diagram showing the single-point binding effect of the antibody of the present invention (chimeric antibody) on CHO-ILT4 cells.

[0032] FIG5 is a multi-point binding curve diagram of the antibody of the present invention (chimeric antibody) and CHO-ILT4 cells.

[0033] FIG6 is a diagram showing the effect of the antibody of the present invention (chimeric antibody) in blocking the binding of ILT4 to HLA-G.

[0034] FIG7 is a graph showing an activation experiment of the antibody (chimeric antibody) M1 of the present invention.

[0035] FIG8 is a multi-point binding curve diagram of the antibody of the present invention (humanized antibody) and CHO-ILT4 cells.

[0036] FIG9 is a graph showing the blocking effect of the antibody of the present invention (humanized antibody) on the binding of ILT4 to HLA-G.

[0037] FIG10 is a graph showing an M1 activation experiment of the antibody (humanized antibody) of the present invention. DETAILED DESCRIPTION

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this application, suitable methods and materials are described below. In the event of a conflict, the patent specification will control.

[0039] There are a variety of methods / systems in the art to define and describe CDRs, and these systems and / or definitions have been developed and refined over the years, including Kabat, Chothia, IMGT, AbM, and Contact. Kabat is the most commonly used and defines CDRs based on sequence variability; Chothia defines CDRs based on sequence variability based on the position of the structural loop region; the IMGT system defines CDRs based on sequence variability and position within the variable domain structure; AbM is based on the AbM antibody modeling software from Oxford Molecular and is a compromise between Kabat and Chothia; Contact defines CDRs based on the analysis of complex crystal structures and is similar to Chothia in many aspects. The numbering of amino acid positions (e.g., amino acid residues in the Fc region) and target regions (e.g., CDRs) in the present invention uses the Kabat system.

[0040] The term "single-domain antibody" as used herein refers to a fragment comprising a single variable domain of an antibody, also known as a nanobody, which, like a complete antibody, can selectively bind to a specific antigen. Single-domain antibodies are much smaller, at approximately 11-15 kDa, compared to the 150-160 kDa of a complete antibody.

[0041] The term "chimeric antibody" as used herein refers to an immunoglobulin or antibody whose variable region is derived from a first species and whose constant region is derived from a second species. A chimeric immunoglobulin or antibody can be constructed, for example, by genetic engineering from immunoglobulin gene segments belonging to different species.

[0042] The term "humanized antibody" as used herein refers to an antibody comprising at least one humanized antibody chain. The term "humanized antibody chain" refers to an antibody chain having a variable region comprising a variable framework region and complementarity determination substantially derived from a human antibody. Regions (CDRs) substantially derived from non-human antibodies (e.g., at least one CDR, two CDRs, or three CDRs). In some embodiments, the humanized antibody chain further comprises a constant region.

[0043] As used herein, the term "identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to obtain maximum percent sequence identity. Comparisons for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST software or the FASTA program package. The term "at least 80% identity" means that the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence is greater than 80%, including 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0044] The term "vector" as used herein generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers an inserted nucleic acid molecule into a host cell and / or between host cells. The term can include vectors primarily used to insert DNA or RNA into cells, vectors primarily used for replication of DNA or RNA, and expression vectors used for transcription and / or translation of DNA or RNA. Also included are vectors that provide more than one of the above functions. An "expression vector" is a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell.

[0045] The term "macrophage M1" used in this article can be used interchangeably with "M1 macrophages" and "classically activated macrophages". Macrophages are divided into two subpopulations, M1 macrophages and M2 macrophages, based on their function and the level of secretion of inflammatory factors. M1 macrophages (classically activated macrophages) are mainly activated by LPS and IFNγ, secreting high levels of IL2 and lower levels of IL10, mainly promoting the occurrence and development of inflammation, sterilization and phagocytosis, while M2 macrophages (alternatively activated macrophages) are mainly activated by IL4 inflammatory factors, mainly suppressing M1 macrophages by secreting anti-inflammatory cytokines such as IL10, and playing a role in wound healing and tissue repair.

[0046] As used herein, "TNFα" is a pro-inflammatory cytokine produced primarily by macrophages, monocytes, certain T lymphocytes and NK cells, as well as brain and liver cells, and is involved in normal inflammatory and immune responses. In this study, whether macrophages secrete TNFα was used to determine whether they were M1 macrophages.

[0047] The term "KD" as used herein refers to the dissociation equilibrium constant for a particular antibody-antigen interaction. Typically, an antibody binds to an antigen with a dissociation equilibrium constant (KD) of less than about 1E-8 M, such as less than about 1E-9 M, 1E-10 M, or 1E-11 M, or less, as determined, for example, by biofilm interferometry. The smaller the KD value, the greater the affinity.

[0048] As used herein, the term "pharmaceutical composition" comprises a single-domain antibody or antigen-binding fragment thereof of the present invention. Typically, the single-domain antibody or antigen-binding fragment thereof of the present invention can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous medium. The pharmaceutical composition can be administered via conventional routes, including but not limited to intratumoral, intraperitoneal, intravenous, or topical administration. The pharmaceutical composition can be used directly to bind to ILT4 protein molecules and thus be used to treat tumors. Furthermore, other therapeutic agents may be administered concurrently.

[0049] The present invention will be further described below with reference to the accompanying drawings and specific examples, and the protection content of the present invention is not limited to the following examples. It should also be understood that the terms used in the examples of the present invention are for the purpose of describing specific embodiments, rather than for the purpose of limiting the scope of protection of the present invention. Without departing from the spirit and scope of the inventive concept, variations and advantages that can be imagined by those skilled in the art are all included in the present invention, and the scope of protection of the present invention is defined by the appended claims and any equivalents thereof.

[0050] Example 1: Immune library construction

[0051] An alpaca was immunized with Human LILRB2 (manufacturer: KACTUS, Cat: LIL-HM4B2) for a total of four immunizations, biweekly, with each injection of 0.5 mg of protein in complete Freund's adjuvant. Two weeks after immunization, 100 mL of blood was collected for library construction, and 1 mL of blood was used for immune titer testing. PBMCs were isolated, RNA was extracted, and reverse-transcribed into cDNA. VHHs were amplified for library construction.

[0052] Example 2: Antibody Screening

[0053] The constructed alpaca immune library was screened, and specific VHH antibodies against the Human LILRB2 (manufacturer: KACTUS, Cat: LIL-HM4B2) protein were enriched by trypsin elution; the enrichment of different output sets was detected by ELISA screening. After initial ELISA screening, all pools were well enriched at the phage level, and a total of 51 molecules with unique sequences were obtained. Among them, the antibody with better activity (sequence: SEQ ID NO: 4) was screened as the antibody of the present invention.

[0054] Example 3: Binding of the Antibody of the Present Invention to ILT4 Protein

[0055] Human LILRB2 (manufacturer: KACTUS, Cat: LIL-HM4B2) was diluted to 1 μg / mL and coated in a 96-well ELISA plate at 100 μL / well at 4°C overnight. Pour off the coating solution, wash the plate with 300 μL per well of 1×PBST, wash 3 times with a plate washer, and pat dry on a dust-free paper. Prepare 3% skim milk powder, 300 μL / well, incubate at 37°C for 1 hour, pour off the blocking solution, wash the plate with 300 μL per well of 1×PBST, wash 3 times with a plate washer, pat dry on a dust-free paper to block the protein. The antibody of the present invention was diluted to 1 μg / mL with 3% skim milk powder, and diluted 3 times with this as the initial concentration, with a total of 7 dilution gradients. Set up 1 blank well and add only the diluent. 100 μL / well, incubate at 37°C for 1 hour. Discard the liquid in the wells, wash each well with 300 μL of 1×PBST, wash 3 times with a plate washer, and pat dry on a dust-free paper. TM Rabbit Anti-Camelid VHH Cocktail [HRP] (Manufacturer: Genscript, Cat: A06016-200) was diluted 1:10,000, 100 μL / well, and incubated at 37°C for 45 minutes. Discard the secondary antibody solution, wash the plate six times with 1×PBST (300 μL per well) using a plate washer, and pat dry on lint-free paper. Add 100 μL / well of a single-component TMB colorimetric mixture (Manufacturer: Solebol, Cat: PR1200), and develop at 37°C in the dark for 7 minutes. Terminate the colorimetric reaction by adding 1M HCl (83 mL 37% concentrated hydrochloric acid + 917 mL pure water) as a stop solution, 100 μL / well. Read the plate at 450 nm on a microplate reader to obtain a binding curve for the antibody of the present invention and the ILT4 protein, as shown in Figure 1. The results indicate that the antibody of the present invention has good binding ability to the ILT4 protein.

[0056] Example 4: Single-site binding of the antibody of the present invention to CHO-ILT4 cells

[0057] Dilute the antibody of the present invention to 5 μg / mL (final concentration 2.5 μg / mL) with diluent (PBS + 2% FBS). Wash CHO-ILT4 cells (manufacturer: Kangyuan Bochuang, Cat: KC-1473) twice, group them into 1E5 / well, wash them twice, and centrifuge to remove the supernatant. Add protein and antibody at 100 μL / well, with a total volume of 200 μL, and incubate at 4°C for 1 hour. After washing the cells, add the secondary antibody MonoRab at a dilution of 1:500. TMRabbit Anti-Camelid VHH Cocktail [PE] (Manufacturer: Genscript, Cat: A02018-200) was incubated at 4°C in the dark for 0.5 h. PE fluorescence was then measured using flow cytometry. The results showed that the antibody of the present invention clearly and specifically binds to CHO-ILT4 cells (see Figure 2).

[0058] Example 5: Multi-point binding of the antibody of the present invention to CHO-ILT4 cells

[0059] The antibody of the present invention was diluted to 5 μg / mL (final concentration 2.5 μg / mL) with diluent (PBS + 2% FBS), and then diluted 5-fold, with a total of 7 dilution gradients. CHO-ILT4 cells (manufacturer: Kangyuan Bochuang,

[0060] The cells were washed twice, divided into groups (1E5 / well), washed twice, and centrifuged to remove the supernatant. Protein and antibody were added at 100 μL / well, with a total volume of 200 μL, and incubated at 4°C for 1 hour. After washing the cells, the secondary antibody MonoRab was added at a dilution of 1:500. TM Rabbit Anti-Camelid VHH Cocktail[PE](Manufacturer: Genscript, Cat:

[0061] A02018-200), incubated at 4°C in the dark for 0.5 h. PE fluorescence readings were then detected by flow cytometry. The results showed that the antibody of the present invention can clearly bind specifically to CHO-ILT4 cells, and its bound EC 50 The value was 0.36 μg / mL, see Figure 3.

[0062] Example 6: Single-site binding of the antibody of the present invention (chimeric antibody) to CHO-ILT4 cells

[0063] The antibody (chimeric antibody) of the present invention (VHH and Fc sequences are SEQ ID NO: 4 and SEQ ID NO: 7, respectively) and an unrelated antibody, IgG4, were diluted to 5 μg / mL (final concentration 2.5 μg / mL) using a diluent (PBS + 2% FBS). CHO-ILT4 cells (Manufacturer: Kangyuan Bochuang, Cat: KC-1473) were washed twice, grouped at 1E5 per well, washed twice, and centrifuged to remove the supernatant. Protein and antibody were added at 100 μL / well for a total volume of 200 μL, and incubated at 4°C for 1 hour. After washing the cells, a secondary antibody, F(ab')2 Goat anti-human IgG Fcγ Antibody, was added at a 1:500 dilution and incubated at 4°C in the dark for 0.5 hour. PE fluorescence readings were then measured using a flow cytometer. The results showed that the antibody (chimeric antibody) of the present invention clearly and specifically bound to CHO-ILT4 cells, as shown in Figure 4.

[0064] Example 7: Multi-point binding of the antibody of the present invention (chimeric antibody) to CHO-ILT4 cells

[0065] The antibody of the present invention (chimeric antibody) and the irrelevant antibody IgG4 were diluted to 5 μg / mL (final concentration 2.5 μg / mL) with diluent (PBS + 2% FBS), and then diluted 5 times, with a total of 7 dilution gradients. The CHO-ILT4 cells (manufacturer: Kangyuan Bochuang, Cat: KC-1473) were washed twice, grouped at 1E5 / well, washed twice, and centrifuged to remove the supernatant. Protein and antibody were added at 100 μL / well, with a total volume of 200 μL, and incubated at 4°C for 1 hour. After washing the cells, the secondary antibody F(ab')2Goat anti-human IgG FcγAntibody was added at a ratio of 1:500 and incubated at 4°C in the dark for 0.5 hours. The PE fluorescence reading was then detected by flow cytometry. The results showed that the antibody of the present invention (chimeric antibody) can obviously bind specifically to CHO-ILT4 cells, and its bound EC 50 The value was 0.057 μg / mL, see Figure 5.

[0066] Example 8: Antibodies of the present invention (chimeric antibodies) block the binding of ILT4 to HLA-G

[0067] Human HLA-G biotin (manufacturer: KATUS, Cat: HLG-HM41CTB) was diluted to 300 nM (final concentration 100 nM) in diluent (PBS + 2% FBS). The antibody of the present invention (chimeric antibody) and an unrelated antibody, IgG4, were diluted to 60 μg / mL (final concentration 20 μg / mL). CHO-ILT4 cells (manufacturer: Kangyuan Bochuang, Cat: KC-1473) were washed twice, grouped at 1E5 per well, washed twice, and centrifuged to remove the supernatant. Protein and antibody were added at 50 μL / well for a total volume of 100 μL, and incubated at 4°C for 1 hour. After washing the cells, secondary antibody SA FITC was added at a 1:100 dilution and incubated at 4°C in the dark for 0.5 hour. FITC fluorescence was then measured using a flow cytometer. The results showed that the antibody of the present invention (chimeric antibody) completely inhibited the binding of the ILT4 cell line to HLA-G (see Figure 6).

[0068] Example 9: M1 activation experiment of the antibody of the present invention (chimeric antibody)

[0069] Resuscitated PBMC (ID#: PCH20201200031, CAT#: FPB004-C) were used to isolate monocytes using a monocyte isolation kit. Monocytes were resuspended in 1640 medium (with 10% inactivated FBS) and M-CSF was added to a final concentration of 100 ng / mL. The cells were plated into Danish 6-well plates at a density of 2E6 / mL and stimulated for 7 days. Adherent macrophages were obtained. After trypsin digestion and centrifugation, the cells were resuspended in 1640 medium (with 10% inactivated FBS) and added to untreated Corning 96-well plates at 1E5 / 100 μL / well. The antibody of the present invention (chimeric antibody) and the unrelated antibody IgG4 were diluted to 20 μg / mL (final concentration 10 μg / mL), and then diluted 5-fold, with a total of 8 gradients, and 50 μL / well were added to the well plate. Lipopolysaccharide (LPS) was diluted to 400 ng / mL (final concentration of 100 ng / mL) and added to the culture plate at 50 μL / well. The final volume was 200 μL and the cells were incubated in an incubator for 24 hours. After 24 hours, the supernatant was collected and assayed for TNFα cytokine levels. The results showed that the antibody (chimeric antibody) of the present invention effectively activated M1 macrophages (see Figure 7).

[0070] Example 10: Affinity of the Antibodies of the Present Invention and the Antibodies of the Present Invention (Humanized Antibodies) to ILT4

[0071] AHC Biosensors (Manufacturer: ForteBio. Inc., Cat: 18-5060) were pre-wetted with PBST buffer for 20 minutes and added to Human LILRB2 / CD85d / ILT4 Protein, His Tag (Manufacturer: KATUS, Cat: LIL-HM4B2) diluted to 20 μg / mL in PBST, resulting in a target protein loading thickness of 1 nm. The antibody of the present invention and the antibody (humanized antibody) of the present invention (VHH and Fc sequences of SEQ ID NO: 6 and SEQ ID NO: 7, respectively) were diluted to 100 nM in PBST and added to the above-mentioned sensor. Binding was performed for 180 seconds and dissociation for 200 seconds. The binding and dissociation curves of the antibody of the present invention and the antibody (humanized antibody) of the present invention with ILT4 were fitted, and their KD values ​​were calculated to be 1.07E-10M and <1.0E-12M, respectively.

[0072] Example 11: Multi-point binding of the antibody of the present invention (humanized antibody) to CHO-ILT4 cells

[0073] The antibody of the present invention (humanized antibody) and the irrelevant antibody IgG4 were diluted to 10 μg / mL (final concentration 5 μg / mL) with diluent (PBS + 2% FBS), and then diluted 5 times, with a total of 7 dilution gradients. The CHO-ILT4 cells (manufacturer: Kangyuan Bochuang, Cat: KC-1473) were washed twice, grouped at 1E5 / well, washed twice, and centrifuged to remove the supernatant. Protein and antibody were added at 100 μL / well, with a total volume of 200 μL, and incubated at 4°C for 1 hour. After washing the cells, the secondary antibody FITC F(ab')2Goat anti-human IgG FcγAntibody was added at a ratio of 1:500 and incubated at 4°C in the dark for 0.5 hours. The TITC fluorescence reading was then detected by flow cytometry. The antibody of the present invention (humanized antibody) can obviously bind specifically to CHO-ILT4 cells, and its bound EC 50 The value was 0.057 μg / mL, see Figure 8.

[0074] Example 12: Antibodies of the present invention (humanized antibodies) block the binding of ILT4 to HLA-G

[0075] Human HLA-G biotin (manufacturer: KATUS, Cat: HLG-HM41CTB) was diluted to 300 nM (final concentration 100 nM) in diluent (PBS + 2% FBS). The humanized antibody of the present invention and an unrelated IgG4 antibody were diluted to 40 μg / mL (final concentration 20 μg / mL). The humanized antibody of the present invention was further diluted seven times, five-fold, for a total of eight dilutions. CHO-ILT4 cells (manufacturer: Kangyuan Bochuang, Cat: KC-1473) were washed twice, grouped at 1E5 cells / well, washed twice, and centrifuged to remove the supernatant. Protein and antibody were added at 50 μL / well for a total volume of 100 μL and incubated at 4°C for 1 hour. After washing the cells, secondary antibody SA FITC was added at a 1:500 dilution and incubated at 4°C in the dark for 0.5 hour. FITC fluorescence was then measured using a flow cytometer. The results showed that the antibody of the present invention (humanized antibody) can completely inhibit the binding of ILT4 cell line to HLA-G, as shown in FIG9 .

[0076] Example 13: Activation experiment of the antibody (humanized antibody) M1 of the present invention

[0077] Resuscitated PBMC (ID#: PCH20201200031, CAT#: FPB004-C) were used to isolate monocytes using a monocyte isolation kit. Monocytes were resuspended in 1640 medium (with 10% inactivated FBS) and M-CSF was added to a final concentration of 100 ng / mL. The cells were plated into Danish 6-well plates at a density of 2E6 / mL and stimulated for 7 days. Adherent macrophages were obtained. After trypsin digestion and centrifugation, the cells were resuspended in 1640 medium (with 10% inactivated FBS) and added to untreated Corning 96-well plates at 1E5 / 100 μL / well. The antibody of the present invention (humanized antibody) and the unrelated antibody IgG4 were diluted to 3.2 μg / mL (final concentration 1.67 μg / mL), and then the antibody of the present invention (humanized antibody) was diluted 6-fold, with a total of 5 gradients, and 50 μL / well was added to the well plate. Lipopolysaccharide (LPS) was diluted to 400 ng / mL (final concentration: 100 ng / mL) and added to the culture plate at 50 μL / well. The final volume was 200 μL and the cells were incubated in an incubator. After 24 hours, the supernatant was collected and assayed for TNFα cytokine levels. The results showed that the humanized antibody of the present invention effectively activated M1 macrophages (see Figure 10).

[0078] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. An anti-ILT4 single-domain antibody or an antigen-binding fragment thereof, characterized in that: The single-domain antibody or antigen-binding fragment thereof comprises CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 respectively comprise an amino acid sequence represented by SEQ ID NO: 1-3, or a sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 1-3, or an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence of SEQ ID NO: 1-3.

2. The single-domain antibody or antigen-binding fragment thereof according to claim 1, characterized in that The CDR1, CDR2 and CDR3 respectively comprise the amino acid sequences represented by SEQ ID NOs: 1-3.

3. The single-domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, characterized in that The single-domain antibody or antigen-binding fragment thereof comprises an amino acid sequence represented by SEQ ID NO: 4, 6, or a sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 4, 6, or an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence of SEQ ID NO: 4, 6.

4. The single-domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that The single-domain antibody or antigen-binding fragment thereof further comprises an immunoglobulin Fc region.

5. The single-domain antibody or antigen-binding fragment thereof according to claim 4, characterized in that The dissociation constant KD between it and ILT4 is less than 10 nM, preferably less than 1 nM.

6. The single-domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that The single domain antibodies or antigen-binding fragments thereof include chimeric, humanized or fully human.

7. A polynucleotide, characterized in that It encodes the single domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. The polynucleotide according to claim 7, wherein The polynucleotide is selected from the polynucleotide sequences corresponding to SEQ ID NO: 5 and 8.

9. A recombinant vector, transgenic cell line, bacteriophage, recombinant bacteria or viral vector, characterized in that: The recombinant vector, transgenic cell line, phage, recombinant bacteria or viral vector contains the polynucleotide according to any one of claims 7-8.

10. An isolated host cell, characterized in that It contains the recombinant vector, transgenic cell line, phage, recombinant bacteria or viral vector according to claim 9.

11. The host cell according to claim 10, characterized in that The host cell is a prokaryotic cell.

12. The host cell according to claim 10, characterized in that The host cell is a eukaryotic cell.

13. The host cell according to claim 12, characterized in that The eukaryotic cell is a mammalian cell.

14. The host cell according to claim 13, characterized in that The mammalian cells are CHO cells.

15. A method for expressing an antibody, characterized in that: The antibody protein is expressed in a host cell according to any one of claims 10 to 14 using the recombinant vector, transgenic cell line, phage, recombinant bacteria or viral vector according to claim 9.

16. Use of the single-domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 in the preparation of a medicament, characterized in that: The medicament contains the single domain antibody or antigen-binding fragment thereof and chemotherapy for treating tumors in human patients, wherein the single domain antibody or antigen-binding fragment thereof and chemotherapy are formulated to provide a therapeutic effect greater than the sum of their respective effects.

17. A construct, characterized in that The construct contains a single domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, and a second part selected from a second antibody or antigen-binding fragment thereof, a detectable label, a drug, gold nanoparticles / nanorods, nanomagnetic particles, viral coat proteins or viral particles, radionuclides, liposomes, chemotherapeutic agents or combinations thereof.

18. A pharmaceutical composition, characterized in that It comprises the single domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

19. The pharmaceutical composition according to claim 18, characterized in that The composition may also comprise an additional therapeutic agent.

20. The pharmaceutical composition according to claim 19, characterized in that The additional therapeutic agent is an immunotherapeutic agent.

21. Use of the pharmaceutical composition according to any one of claims 18 to 20 in the preparation of a medicament for treating a disease associated with ILT4, wherein the disease comprises a tumor or an autoimmune disease.

22. Use of the single-domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 in the preparation of a medicament for treating and / or preventing and / or diagnosing a disease.

23. The use according to claim 22, characterized in that The disease is selected from the group consisting of human brain astroblastoma, human pharyngeal cancer, adrenal tumor, AIDS-related cancer, alveolar soft tissue sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, renal chromophobe cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymoma, Ewing tumor, extraskeletal myxoid chondrosarcoma, fibrous dysplasia, fibrous dysplasia, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, pancreatic islet cell tumor, Kaposi's muscle tumor, kidney cancer, leukemia, liposarcoma / malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid cancer, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, metastatic kidney cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymic cancer, thymoma, metastatic thyroid cancer, or uterine cancer.

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