Nanobodies against sialoadhesin and related biomaterials and uses thereof

Abstract

The application discloses a nanobody against sialic acid-binding immunoglobulin-like lectin 15 and a related biomaterial and application thereof, and belongs to the technical field of immunotherapy biological medicine. The nanobody against sialic acid-binding immunoglobulin-like lectin 15 or an antigen-binding fragment thereof provided by the application comprises three complementarity determining regions CDR1, CDR2 and CDR3, the nanobody is S1, the amino acid sequence of CDR1 of the S1 is SEQ ID No. 6, the amino acid sequence of CDR2 of the S1 is SEQ ID No. 7, and the amino acid sequence of CDR3 of the S1 is SEQ ID No. 8. The nanobody against sialic acid-binding immunoglobulin-like lectin 15 provided by the application can play a role in the development of a therapeutic drug for a cancer patient with high expression of Siglec-15.

Description

Technical Field

[0001] The present invention belongs to the field of immunotherapy biomedicine technology, and specifically relates to nano antibodies against sialic acid-binding immunoglobulin-like lectin 15 and related biomaterials and applications. Background Art

[0002] Cancer has become a major threat to human health and a leading cause of death. Cancer is a generic term for a diverse group of diseases that can affect any part of the body. It is characterized by abnormal cell differentiation and proliferation, uncontrolled growth, invasiveness, and metastasis. Cancer is a neoplastic entity formed when cells in local tissues, under the influence of various carcinogenic factors, lose normal genetic regulation of their growth, leading to abnormal proliferation and differentiation.

[0003] Currently, cancer treatment primarily relies on a multidisciplinary approach tailored to the patient's pathological type, stage, and underlying health status. Treatment options include surgery, radiotherapy, and chemotherapy. These treatments significantly reduce patients' quality of life and are limited in effectiveness for advanced tumors, presenting significant limitations. In recent years, immunotherapy based on immune checkpoints, such as programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1), has been shown to prolong overall survival in some cancer patients. Clinical trials have demonstrated that the five-year survival rate for lung cancer patients receiving pembrolizumab as a first-time treatment can reach 23.3%. Among these patients, those with a PD-L1 tumor proportion score (TPS) ≥50% have a median survival of 35.4 months and a five-year survival rate of 29.6%. Immunotherapy, alone or in combination with chemotherapy, has become an important and widely used approach for cancer treatment. Although PD-L1 expression levels and tumor mutation burden (TMB) testing have predictive effects on the efficacy of ICIs, only approximately 20% of cancer patients benefit from them, and patients may develop drug resistance after several months of treatment. Therefore, the search for more new immune-related targets and drugs remains an important research direction in cancer immunotherapy today.

[0004] Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) is a novel immunosuppressive factor discovered using genome-scale T cell activity arrays combined with bioinformatics analysis of human cancer databases. As a member of the immunoglobulin superfamily, Siglec-15 is a highly conserved type I transmembrane protein with a structure consisting of two immunoglobulin-like domains and a lysine-rich transmembrane domain. These lysine residues play a crucial role in the interaction between Siglec-15 and its adaptor protein, DNAX activation protein 12 (DAP12). When the extracellular end of DAP12 binds to Siglec-15, a complex is formed that triggers phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) domain within DAP12. This process activates multiple signaling pathways, including phospholipase Cγ and tyrosine kinase Syk, affecting the activity of immune cells in the tumor microenvironment, particularly by inhibiting T cell function and regulating the function of antigen-presenting cells, thereby promoting tumor immune escape. Studies have shown that inhibiting Siglec-15 function may promote normalization of the immune system in patients with malignant tumors. Similar to PD-1 expression, Siglec-15 is widely upregulated in human tumor tissues, including lung, colon, thyroid, uterine, bladder, kidney, and liver cancers, while expression is very low or absent in normal tissues. Siglec-15 shares high homology with PD-L1, with similar domain structures and modes of action. However, unlike the PD-1 / PD-L1 signaling pathway, Siglec-15 primarily influences tumor immunity by regulating the activity of effector T cells. Furthermore, Siglec-15 and PD-L1 expression are mutually exclusive, suggesting that Siglec-15 may offer a new treatment option for cancer patients refractory to PD-1 / PD-L1 therapy, making it an emerging target for tumor immunotherapy.

[0005] Antibody drugs play a vital role in current cancer treatment and are widely considered to be one of the first choices for biological drugs for cancer treatment. Chen Lieping's team developed a monoclonal antibody against Siglec-15. The results showed that anti-Siglec-15 antibodies can block the inhibitory effect of Siglec-15 on effector T cells and confirmed that Siglec-15 exerts an immunosuppressive effect independently of B7-H1 / PD-1. Xiao Xueyun's team prepared anti-Siglec-15 antibodies targeting Siglec-15. The results showed that anti-Siglec-15 antibodies have a certain therapeutic effect on lung adenocarcinoma and can kill tumor cells. They may act by intervening in macrophage polarization and affecting the tumor microenvironment of lung adenocarcinoma. In 1993, Belgian scientists first reported the discovery of heavy chain antibodies that naturally lack light chains in the peripheral blood of camelids. Cloning their variable regions can obtain single-domain antibodies composed only of heavy chain variable regions, also known as nanobodies (variable domain of heavy chain of heavy-chain antibody, VHH). Compared to traditional antibodies, nanobodies have the advantage of being easily optimized and modified in tumor immunotherapy. In addition, nanobodies are highly expressed in prokaryotic and eukaryotic hosts and have low immunogenicity, and have been applied to the treatment of solid tumors. However, due to their small molecular weight and easy clearance by the kidneys, many studies have fused nanobodies with the Fc segment (hFc) of human IgG to enhance the activity of nanobodies in vivo. Currently, there is still an unmet need for tumor therapy in this field. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to develop a nanobody that can effectively bind to Siglec-15. The technical problem to be solved is not limited to the technical subject matter described, and those skilled in the art can clearly understand other technical subjects not mentioned herein through the following description.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] The present invention provides a nanobody targeting Siglec-15 or an antigen-binding fragment containing the nanobody.

[0009] In a first aspect, the present invention provides a nanobody or an antigen-binding fragment thereof against sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15), wherein the nanobody or the antigen-binding fragment thereof comprises three complementary determining regions CDR1, CDR2 and CDR3, and the nanobody is S1;

[0010] The amino acid sequence of the CDR1 of S1 is SEQ ID No. 6, the amino acid sequence of the CDR2 of S1 is SEQ ID No. 7, and the amino acid sequence of the CDR3 of S1 is SEQ ID No. 8.

[0011] In addition to the complementary determining regions, the above-mentioned Nanobody also contains four framework regions FR1, FR2, FR3 and FR4;

[0012] The amino acid sequence of FR1 of S1 is SEQ ID No. 2, the amino acid sequence of FR2 of S1 is SEQ ID No. 3, the amino acid sequence of FR3 of S1 is SEQ ID No. 4, and the amino acid sequence of FR4 of S1 is SEQ ID No. 5.

[0013] The amino acid sequence of S1 in the above-mentioned Nanobody is SEQ ID No.1.

[0014] The above term "antigen binding fragment" refers to an antigen binding fragment of an antibody and antibody analogs, which generally include at least a portion of the antigen binding region or variable region (e.g., one or more CDRs) of a parental antibody. The antigen binding fragment retains at least some of the binding specificity of the parental antibody. Typically, when activity is expressed on a molar basis, the antigen binding fragment retains at least 10% of the parental binding activity. Specifically, the antigen binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parental antibody to the target.

[0015] In a second aspect, the present invention provides a heavy chain antibody against sialic acid-binding immunoglobulin-like lectin 15, wherein the heavy chain antibody comprises the aforementioned Nanobody.

[0016] The above-mentioned heavy chain antibody includes a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is SEQ ID No.1 or has an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with SEQ ID No.1.

[0017] The heavy chain antibody is S1, and the amino acid sequence of S1 is SEQ ID No.11.

[0018] In a third aspect, the present invention provides a biomaterial, wherein the biomaterial is any one of the following:

[0019] D1) a monoclonal antibody comprising the aforementioned Nanobody or the aforementioned heavy chain antibody;

[0020] D2) Small molecule antibodies containing the above-mentioned nanobodies.

[0021] The small molecule antibody may be any of the following:

[0022] F1, Fab antibody;

[0023] F2, Fv antibody;

[0024] F3, single-chain antibody;

[0025] F4, Fab′ fragment.

[0026] The term "Fab' fragment" contains one antibody light chain and a portion of one antibody heavy chain including the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, whereby an interchain disulfide bond can form between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule.

[0027] The term "F(ab')2 fragment" contains two light chains and two heavy chains comprising a portion of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0028] The term "nanobody (single-domain antibody)" (VHH) refers to a polypeptide consisting of the variable region of an antibody heavy chain. Single-domain antibodies can be prepared by genetically engineering the variable region of an antibody heavy chain (VH) to produce an antibody containing only the VH fragment. The antigen-binding ability and stability of single-domain antibodies are essentially the same as those of full-length antibodies.

[0029] The term "minimum recognition unit (MRU)" refers to a structure containing only a single CDR in the variable region, with a molecular mass of only about 1% of that of a complete antibody, which can bind to the corresponding antigen.

[0030] The term "Fab antibody" refers to a heterodimer formed by the heavy chain (Fd) and an intact light chain bound by disulfide bonds, containing only a single antigen-binding site. Fab antibodies can be prepared by ligating the genes encoding the heavy chain (Fd) and the complete light chain, and fusing them with a bacterial protein signal peptide gene. This allows for secretory expression of Fab antibodies (Fab fragments) in Escherichia coli, with a complete three-dimensional fold and intra- and inter-chain disulfide bonds. The heavy chain (Fd) refers to approximately half of the H chain portion of a Fab (comprising approximately 225 amino acid residues, including the VH, CH1, and part of the hinge region).

[0031] The term "Fv antibody" refers to a compound composed solely of the heavy and light chain variable regions of an antibody. The heavy and light chain variable regions are linked by non-covalent bonds. Fv antibodies can be prepared by constructing separate vectors containing the VH and VL genes, co-transfecting cells to express them, and then assembling them into a functional Fv antibody. Alternatively, a stop codon can be inserted between the VH and VL genes in the vector to express the two small protein fragments separately, which are then non-covalently bound to form an Fv antibody (Fv fragment).

[0032] The term "single-chain antibody" (ScFv) refers to a polypeptide composed of the heavy and light chain variable regions of an antibody linked by a short peptide. ScFv can be prepared by linking the light and heavy chain variable region genes with an oligonucleotide linker to express a single polypeptide chain, known as a single-chain antibody (ScFv). The polypeptide chain spontaneously folds into its native conformation, maintaining the specificity and affinity of the Fv.

[0033] In a fourth aspect, the present invention provides genetic material, wherein the genetic material is any one of the following:

[0034] g1) a nucleic acid molecule encoding the aforementioned Nanobody or antigen-binding fragment thereof, or the aforementioned Nanobody-hFc fusion protein;

[0035] g2) An expression cassette, recombinant vector, recombinant cell or recombinant bacterium containing the nucleic acid molecule described in g1).

[0036] In a fifth aspect, the present invention claims protection for a drug against sialic acid-binding immunoglobulin-like lectin 15, said drug comprising the aforementioned nanobody or antigen-binding fragment thereof or the aforementioned heavy chain antibody.

[0037] The medicament further includes a physiologically or pharmaceutically acceptable excipient, diluent or carrier.

[0038] Herein, the above-mentioned "physiologically or pharmaceutically acceptable carriers or diluents" refer to those carriers and diluents that have no significant irritation to organisms and will not impair the biological activity and performance of the agent in the pharmaceutical composition.

[0039] As used herein, a "physiologically or pharmaceutically acceptable excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of the agent. Carrier materials herein include, but are not limited to, water-soluble carrier materials (e.g., polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethyl cellulose, cholesterol stearate, etc.), and enteric-soluble carrier materials (e.g., cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Water-soluble carrier materials are preferred.

[0040] In a sixth aspect, the present invention claims protection for any of the following applications:

[0041] M1) Use of the aforementioned genetic material in the preparation of the aforementioned Nanobody or antigen-binding fragment thereof or the aforementioned heavy chain antibody;

[0042] M2) Use of the aforementioned Nanobody or antigen-binding fragment thereof or the aforementioned heavy chain antibody in the preparation of the aforementioned medicament;

[0043] M3) Use of the aforementioned Nanobody or antigen-binding fragment thereof or the aforementioned heavy chain antibody or the aforementioned genetic material or the aforementioned drug in the preparation of a product for treating tumors that positively express sialic acid-binding immunoglobulin-like lectin 15;

[0044] M4) Use of the aforementioned Nanobody or antigen-binding fragment thereof or the aforementioned Nanobody-hFc fusion protein or the aforementioned nucleic acid molecule or an expression cassette, recombinant vector, recombinant cell or recombinant bacteria or pharmaceutical composition containing the aforementioned nucleic acid molecule in the preparation of a product for detecting the content of sialic acid-binding immunoglobulin-like lectin 15 in a tumor.

[0045] The nanobody against sialic acid-binding immunoglobulin-like lectin 15 provided by the present invention can play a role in the development of therapeutic drugs for cancer patients with high Siglec-15 expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 For the isolation of peripheral blood lymphocytes from camels.

[0047] Figure 2 This is the binding (part) of phage clones to the target antigen after Phage-ELISA screening. The odd-numbered columns are the target antigen; the even-numbered columns are the control antigen.

[0048] Figure 3 SDS-PAGE electrophoresis detection of the purified anti-Siglec-15 nanoantibody fusion protein.

[0049] Figure 4 Figure 1 shows the binding activity of anti-Siglec-15 nanobody-hFc fusion protein. (a) shows the binding curve of S1 to recombinant mouse Siglec-15 protein. (b) shows the binding curve of S1 to recombinant human Siglec-15 protein.

[0050] Figure 5 Specific detection of anti-Siglec-15 nanobody-hFc fusion protein.

[0051] Figure 6 This is a test of the binding activity of anti-Siglec-15 nanobody-hFc fusion protein to NCI-H157-S15.

[0052] Figure 7 This is the kinetic curve of the interaction between anti-Siglec-15 nanobody-hFc fusion protein and Siglec-15.

[0053] Figure 8 This is the time-quantity curve of the metabolism of anti-Siglec-15 nanobody-hFc fusion protein in nude mice.

[0054] Figure 9 This is the fluorescence distribution in normal mice after tail vein injection of fluorescently labeled antibodies.

[0055] Figure 10 This is the fluorescence distribution in tumor-bearing mice after tail vein injection of fluorescently labeled antibodies.

[0056] Figure 11 It is the blocking effect of anti-Siglec-15 nanobody-hFc fusion protein and CD44.

[0057] Figure 12 It is the blocking effect of anti-Siglec-15 nanobody-hFc fusion protein and MAG.

[0058] Figure 13 It is the blocking effect of anti-Siglec-15 nanobody-hFc fusion protein and Sialyl-Tn.

[0059] Figure 14 Evaluation of the anti-tumor activity of the anti-Siglec-15 nanobody-hFc fusion protein. (a) Schematic diagram of subcutaneous tumor model construction and subsequent antibody administration. Nude mice (n=6) were subcutaneously inoculated with NCI-NCI-H157-S15 cells on their backs. Seven days later, tumor-bearing mice were treated with 10 mg / kg S1 or PBS (control group). (b) Curves showing changes in mouse body weight over time in different groups. (c) Curves showing growth of tumor volume over time in each group. (d) Tumor weight in each group at the end of treatment. (e) Images of tumors in different groups. Statistical analysis of tumor volume, mouse body weight, and mouse tumor weight at the same time point was performed using one-way analysis of variance (ANOVA) and Dunnett's multiple comparison test. P < 0.05 indicates statistically significant differences. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0060] Figure 15Figure 3. Changes in cytokine levels in mouse peripheral blood serum. (a) Changes in TGF-β levels; (b) Changes in IL-6 levels; (c) Changes in IL-10 levels. One-way analysis of variance (ANOVA) and Dunnett's multiple comparison test were used to statistically analyze the differences between the antibody-treated and PBS groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The Blank group represents healthy female BALB / c-Nude mice not inoculated with tumor cells. The PBS group represents tumor-bearing mice injected with PBS via the tail vein. The S1 group represents tumor-bearing mice injected with antibodies via the tail vein.

[0061] Figure 16 Figure 4 shows the distribution of TAMs in mouse tumor tissue. (a) Representative results of flow cytometric analysis of the proportion of TAMs in tumor tissue; (b) Quantitative analysis of the proportion of TAMs in tumor tissue. Statistical analysis of the treatment groups versus the PBS control group was performed using one-way analysis of variance (ANOVA) and Dunnett's multiple comparison test. *P < 0.05.

[0062] Figure 17 Flow cytometric analysis workflow for TAMs cells. DETAILED DESCRIPTION

[0063] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0064] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0065] The pTSE-hFc in the following examples is obtained by connecting the gene for the Fc domain of human immunoglobulin G to the pCMV vector. pTSE-hFc has been described in: "Xie Qing, Li Zhiying, Zhang Wei, et al. Screening and identification of antibodies against the protective antigen V of Yersinia pestis [J]. Chinese Journal of Pathogenic Biology, 2022, 17(03): 266-271." The public can obtain this biomaterial from the Military Medical Research Institute of the Chinese Academy of Military Sciences. This biomaterial is only used to repeat the experiments of the present invention and cannot be used for other purposes.

[0066] The NEN-SCFV described in the following examples was generated by ligating the genes for the bacteriophage surface protein pill and the arabinose operon into a pET vector. NEN-SCFV has been described in "Chen L, Lu J, Yue J, Wang R, Du P, Yu Y, Guo J, Wang X, Jiang Y, Cheng K, Yang Z and Zheng T (2023) A humanized antihuman adenovirus 55 monoclonal antibody with good neutralizationability. Front. Immunol. 14: 1132822. doi: 10.3389 / fimmu.2023.1132822. Epub 2023 Mar 16. PMID: 37006289; PMCID: 10060833." The public can obtain this biological material from the Military Medical Research Institute of the Chinese People's Liberation Army Academy of Military Sciences. This biological material is only used to repeat the experiments of the present invention and cannot be used for other purposes.

[0067] Example 1. Construction of anti-Siglec-15 nanobody library

[0068] 1. Camel Immunity

[0069] Recombinant human Siglec-15 protein (Beijing Sino Biological Technology Co., Ltd., 13976-H08H) was mixed with Freund's complete adjuvant (Sigma, F5881) in a 1:1 ratio, emulsified by oscillation, and injected subcutaneously into healthy adult Bactrian camels at multiple points. Booster immunizations were performed every two weeks. Except for the first immunization with Freund's complete adjuvant, subsequent immunizations used Freund's incomplete adjuvant (Sigma, F5506).

[0070] 2. Isolation of peripheral blood lymphocytes from camel blood

[0071] Recombinant human Siglec-15 protein without adjuvant was used as the immunogen for pulse immunization. Within one week of the pulse immunization, 100-150 mL of peripheral blood was collected from camels. Peripheral blood lymphocytes were separated from camel blood using lymphocyte separation medium (stemcell, 07851). After centrifugation of the mixture of whole blood and lymphocyte separation medium, the results were as follows: Figure 1 As shown: The liquid in the centrifuge tube is divided into four layers from top to bottom: plasma layer, PBMC layer, lymphocyte separation fluid layer and red blood cell layer.

[0072] 3. Nested PCR amplification of VHH gene fragments

[0073] Total RNA was extracted from isolated peripheral blood mononuclear cells (PBMCs) using the OMEGA EZNA Total RNA Kit I (OMEGA, R6834), and then reverse transcribed to cDNA using the Invitrogen Superscript III First-strand Synthesis System for RT-PCR Kit (Invitrogen, 18080-051). In the first round of PCR, the synthesized cDNA was used as a template to amplify the antibody leader peptide to the CH2 region using the designed IgG-specific upstream primers CALL001 and CALL002. In the second round of PCR, the VHH fragment was amplified using the recovered first-round PCR product as a template and the designed VHH-F and VHH-R fragments.

[0074] Table 1 Primer sequences used in two rounds of PCR

[0075] Primer name Primer sequence (5'-3') CALL001 GTCCTGGCTGCTCTTCTACAAGG CALL002 GGTACGTGCTGTTGAACTGTTCC VHH-F CGGCCATGGCGGTCTCTAGCTGCTCTCACTG VHH-R TCCCGCGGCCCGCTGACWGGGTGAGAYGGGTGAC

[0076] 4. Electroporation products

[0077] The amplified VHH gene was digested with restriction endonucleases Nco I-HF (purchased from Beijing New England Biotechnology Co., Ltd., Catalog No. R3193V) and Not I-HF (purchased from Beijing New England Biotechnology Co., Ltd., Catalog No. R3189V). The NEN-SCFV vector was digested with Nco I-HF, Not I-HF, and EcoRI-HF (purchased from Beijing New England Biotechnology Co., Ltd., Catalog No. R3101V). The digested products were ligated using T4 DNA ligase (NEN-SCFV vector:VHH gene fragment molar ratio of 1:3) to obtain the recombinant vector NEN-SCFV-VHH. The recombinant plasmid NEN-SCFV-VHH was transformed into competent E. coli TG1 cells (Solarbio, C1170) by electroporation. An anti-Siglec-15-specific phage antibody library was constructed, and the phage antibody library was diluted in series to determine its capacity and transformation efficiency. After identification, the storage capacity of this antibody library reached 4.1×10 9 cfu.

[0078] Example 2: Screening of anti-Siglec-15 specific phage nanobody library

[0079] The constructed immune library was screened using the solid phase screening method to obtain anti-Siglec-15 nanoantibodies.

[0080] Take the constructed anti-Siglec-15 nanoantibody library bacterial solution and transfer it to 2YT medium containing ampicillin, culture it to the logarithmic growth phase, and add helper phage M13K07 (NEB, N0315S) for display. The next day, collect the culture supernatant and concentrate the phage with 20% PEG / 2.5M NaCl solution (1L solution contains 200g PEG6000, 146.25g NaCl) to obtain high-titer antibody library display products for subsequent screening. Screening is carried out using the solid phase method (experimental protocol refers to Phage Display: General Experimental Guide / (US) Clarkson (Clackson, T), (US) Lowman (Lowman, HB); (US) Translated by Ma Lan et al. Chemical Industry Press, 2008.5). The specific steps are as follows:

[0081] The recombinant human Siglec-15 protein was coated on the immunotube with 0.05M NaHCO3 coating solution (pH=9.6) and incubated overnight at 4°C. The coating concentrations for each round of selection were 20, 10, and 5 μg / mL, respectively. The immunotube was washed 3 times with PBS the next day for 3 min each time. After blocking with blocking solution (2% bovine serum albumin) at room temperature for 2 h, the phage library solution was added and incubated at room temperature for 2 h. The tube was shaken at a low speed of 200 rpm for 20 min. The tube was washed 10 times with PBST (1×PBS+0.1% Tween-20) and then washed 5 times with PBS. After washing, 1 mL of elution solution (glycine-hydrochloric acid, pH=2.2) was added and shaken at 400 rpm for 20 min. The eluate in the target antigen well was taken out and neutralization solution (1M The cells were neutralized with Tris-HCl (pH=8.0); Escherichia coli TG1 in logarithmic phase growth was infected, the cells were allowed to stand at room temperature for 30 min, and then cultured at 37°C for 1 h to produce and purify phages for the next round of screening. The same screening process was repeated for three rounds. The enrichment results are shown in Table 2:

[0082] Table 2 Analysis of enrichment degree of anti-Siglec-15 phage nanobody library screening

[0083] Number of screenings Input amount (cfu) Output (cfu) Output / Input 1 <![CDATA[5.00×10 11 ]]> <![CDATA[2.80×10 5 ]]> <![CDATA[5.60×10 -5 ]]> 2 <![CDATA[3.00×10 11 ]]> <![CDATA[2.70×10 6 ]]> <![CDATA[9.00×10 -4 ]]> 3 <![CDATA[1.00×10 11 ]]> <![CDATA[1.60×10 8 ]]> <![CDATA[1.60×10 -3 ]]>

[0084] Well-separated single clones were selected from the culture dishes containing phage after the three rounds of screening described above and inoculated into 96-well plates (250 μL / well) containing 2YT-GA medium (1 L of 2YT medium contains 16 g Typtone, 10 g Yeast extract, 5 g NaCl, 100 μg / mL ampicillin, and 20% glucose). Two negative control wells (no clone or clone inoculated with other antigens) were left, and the plates were cultured at 37°C until the logarithmic growth phase. M13KO7 helper phage was added at an MOI of ≈50 and 100 μL / well was added to the deep-well plates cultured with single phage clones. The plates were inoculated at room temperature for 30 minutes and then incubated at 150 rpm at 37°C for 1 hour. The deep-well plates were centrifuged at room temperature (2000 rpm for 10 minutes), the supernatant discarded, and expression was induced with arabinose at a final concentration of 1 mM. The plates were cultured overnight to obtain phage particles displaying the antibody variable regions.

[0085] Example 3: Identification of specific nanobody-positive clones using phage-ELISA

[0086] The recombinant human Siglec-15 protein was used as the antigen for coating, and the adjacent column was coated with an irrelevant antigen as a control, and the coating was carried out overnight at 4°C. The ELISA plate coated overnight was washed 6 times with PBST on a plate washer, blocked with 200 μL / well of blocking solution (3 g skim milk powder added to 100 mL of PBS), and placed in an incubator at 37°C for 2 h. Then, 100 μL / well of the phage supernatant induced and expressed overnight was added to the 96-well plate and placed in an incubator at 37°C for incubation. The plate was incubated for 1.5 h; the plate was washed 6 times with shaking using a plate washer, and HRP-labeled M13 mouse monoclonal antibody (Beijing Sino Biological Technology Co., Ltd., 1973-MM05T-H) diluted 10,000-fold with blocking solution was added to the ELISA plate at 100 μL / well, and the plate was incubated at 37°C in an incubator for 30-45 min; the plate was washed 6 times with shaking using a plate washer, and the color development solution (9 mL of color development solution, 1 mL of 10× OPD, 10 μL of 30% H2O2 per ELISA plate) was added for color development, and the reaction was allowed to proceed for 10-20 min; the reaction was terminated by adding 2 M H2SO4 stop solution (50 μL / well); the plate was read using a microplate reader, and the optical density was measured at a wavelength of 492 / 630 nm; the color development reaction was observed, and a positive clone was determined when the ratio of the absorbance value to the control well was greater than 10.

[0087] Some Phage-ELISA test results are shown in Figure 2 The odd-numbered columns are the target antigens, and the even-numbered columns are the control antigens. The bacterial culture corresponding to the positive clones was sent to a biotechnology service company for sequencing, and the DNA sequence of the insert was obtained. Ultimately, the sequence of a phage clone S1 that specifically binds to Siglec-15 was obtained.

[0088] The amino acid sequence of S1 is shown in SEQ ID No. 1, including framework regions (FR: FR1, FR2, FR3, FR4) and complementarity determining regions (CDR: CDR1, CDR2, CDR3). The four parts of the framework region are sequentially recorded as SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5; the three parts of the complementarity determining region are sequentially recorded as SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8.

[0089] Wherein SEQ ID No.1:

[0090] EVQLVESGGGVVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSGISRSGRTTSYADSVKGRFTISRDNAK NTLYLQLNNLKTEDTATYYCAKDVGHDCYQGSWCLPYSSTRGQGTQVTVSS.

[0091] SEQ ID No. 2: EVQLVESGGGVVQPGGSLRLSCAAS.

[0092] SEQ ID No. 3: MNWVRQAPGKGLEWVSG.

[0093] SEQ ID No. 4: SYADSVKGRFTISRDNAKNTLYLQLNNLKTEDTATYYC.

[0094] SEQ ID No. 5: RGQGTQVTVSS.

[0095] SEQ ID No. 6: GFTFSTYA.

[0096] SEQ ID No. 7: ISRSGRTT.

[0097] SEQ ID No. 8: AKDVGHDCYQGSWCLPYSST.

[0098] The gene encoding the above-mentioned anti-Siglec-15 nanobody S1 has a nucleotide sequence as shown in SEQ ID No. 9, wherein SEQ ID No. 9:

[0099] 5'-Gaggtgcagctggtggagtctgggggaggcgtggtgcagcctggggggtctctgagactctcctgtgcagcct ctggattcaccttcagtacttatgccatgaactgggtccgccaggctccagggaaggggctcgagtgggtctcaggtattagtagaagtggtcgtacaacatcctatgcagactccgtgaagggccgattcaccatctccagagacaacgccaaga acacgctgtatttgcaattgaacaacctgaaaactgaggacacggccacgtattactgtgcaaaagatgtcgggcatgctgctatcaaggctcttggtgtctgccgtattcgtcaactcggggccaggggacccaggtcaccgtctcctca-3'.

[0100] The sequences of the complementarity determining regions are defined according to the IMGT numbering system.

[0101] Example 4. Preparation of anti-Siglec-15 nanobody-hFc fusion protein

[0102] 1. Construction of anti-Siglec-15 nanobody-hFc fusion protein eukaryotic expression plasmid

[0103] (1) Construction of pTSE-VHH-hFc

[0104] According to the gene sequence of the anti-Siglec-15 nanobody S1 (nucleotide sequence is SEQ ID No.9, amino acid sequence is SEQ ID No.1), its carboxyl terminus is connected to the Fc segment of human immunoglobulin to form the anti-Siglec-15 nanobody-hFc fusion protein S1. The amino acid sequence of S1 is as SEQ ID No.11, and the base sequence is as SEQ ID No.10.

[0105] Using conventional molecular biology techniques, the small fragment between the Sal I and Nhe I restriction sites of the pTSE-hFc expression vector (pTSE-hFc was modified by the inventors by connecting the gene of the Fc domain of human immunoglobulin to the pCMV vector) was replaced with a DNA molecule with a nucleotide sequence of SEQ ID No. 9 (coding gene of S1), while keeping the other nucleotides of the pTSE-hFc vector unchanged, to obtain a recombinant expression plasmid pTSE-VHH-hFc containing the gene encoding the nanobody-hFc fusion protein S1 (i.e., the fusion protein expression plasmid pTSE-VHH-hFc).

[0106] SEQ ID No.10:

[0107]

[0108] SEQ ID No.11:

[0109] EVQLVESGGGVVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSGISRSGRTTSYADSVKGRFTISRDNAKNTLYLQLNNLKTEDTATYYCAKDVGHDCYQGSWCLPYSSTRGQGTQVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK*.

[0110] The sequences of the complementarity determining regions of the above-mentioned Nanobody-hFc fusion protein are defined according to the IMGT numbering system.

[0111] 2. Expression and purification of anti-Siglec-15 nanobody-hFc fusion protein

[0112] The constructed S1 fusion protein expression plasmid was transfected into FreeStyle TM HEK293-F cells (Invitrogen, R79007) were used to monitor cell activity daily. When the cell activity dropped from 95-100% to 80-85%, the cell supernatant was collected and purified to obtain the nanobody-hFc fusion protein S1. The purified antibody was analyzed by SDS-PAGE electrophoresis. Figure 3 As shown: The molecular weight of the antibody is consistent with expectations. The band size under S1 reducing conditions is approximately 40 kDa, and the band size under non-reducing conditions is approximately 80 kDa.

[0113] Example 5. Evaluation of the properties of Nanobody-hFc fusion protein S1

[0114] 1. ELISA assay to detect the binding activity between nanobody-hFc fusion protein S1 and recombinant human and mouse Siglec-15 proteins

[0115] The experimental method is as follows: recombinant human Siglec-15 protein (Beijing Yiqiao Shenzhou Science and Technology Co., Ltd., 13976-H08H) and recombinant mouse Siglec-15 protein (Beijing Yiqiao Shenzhou Science and Technology Co., Ltd., 5A4074-M31H) were coated on an enzyme-linked plate at 200 ng / well using carbonate coating buffer (pH = 9.6) and incubated at 4°C overnight; the next day, the coating solution in the enzyme-linked plate was discarded, and the plate was washed 6 times with PBST (0.1% Tween-20). The residual liquid in the well was drained, and 200 μL / well of blocking solution (3% skim milk powder) was added, and the plate was blocked at 37°C for 2 h; the blocking solution was discarded, the plate was washed 6 times with PBST, the residual liquid was patted dry, and the plate prepared in Example 4 was coated with blocking solution. The nanobody-hFc fusion protein S1 was diluted 3-fold (initial concentration 80 μg / mL), and 100 μL / well of the diluted antibody was added to the plate and incubated at 37°C for 1.5 h; the primary antibody was discarded, and the cells were washed 6 times with PBST and patted dry; goat anti-human IgG (HRP, 1:4000) was diluted with blocking solution, and 100 μL / well was added to a 96-well plate and incubated at 37°C for 45 min; the secondary antibody was discarded, and the cells were washed 6 times with PBST (0.1% Tween-20), and the residual liquid was patted dry. 50 μL / well of peroxidase substrate colorimetric solution was added for color development, and the cells were protected from light for 15 min. The color development effect was observed, and 50 μL / well of 2M sulfuric acid was added to terminate the reaction after complete color development; the readings were read on a microplate reader, and the detection wavelengths were OD 492 / OD 630 , record the test value, then take the logarithm of 10 of the antibody concentration as the horizontal axis and the OD value as the vertical axis, calculate the antibody concentration when the OD value is 50% of the highest platform value, and record it as EC 50 value.

[0116] The results are as follows Figure 4 As shown in the figure, the horizontal axis is the logarithm of the protein molar concentration (mAb concentration lognM), and the vertical axis is the optical density value. The analysis showed that the binding half effective concentration (EC 50 ) is 0.04 nM, and the half effective concentration (EC 50 ) is 0.04nM.

[0117] 2. Identification of the specificity of nanobody-hFc fusion protein S1 by ELISA

[0118] The experimental method is as follows: recombinant human Siglec-15 protein (Beijing Sino Biological Science and Technology Co., Ltd., 13976-H08H), recombinant human Siglec-2 protein (Beijing Sino Biological Science and Technology Co., Ltd., 11958-H08H), recombinant human Siglec-3 protein (Beijing Sino Biological Science and Technology Co., Ltd., 12238-H08H), recombinant human Siglec-4a protein (MedChemExpress, 538-MG), recombinant human Siglec-6 protein (Beijing Sino Biological Science and Technology Co., Ltd., 13976-H08H), recombinant human Siglec-15 protein (Beijing Sino Biological Science and Technology Co., Ltd., 13976-H08H), recombinant human Siglec-2 protein (Beijing Sino Biological Science and Technology Co., Ltd., 11958-H08H), recombinant human Siglec-3 protein (Beijing Sino Biological Science and Technology Co., Ltd., 12238-H08H), recombinant human Siglec-4a protein (MedChemExpress, 538-MG), recombinant human Siglec-6 protein (Beijing Sino Biological Science and Technology Co., Ltd., 13976-H08H) were coated with carbonate coating buffer (pH = 9.6). Co., Ltd., 12241-H08H), recombinant human Siglec-8 protein (MedChemExpress, HY-P71313), recombinant human Siglec-9 protein (MedChemExpress, HY-P70739), and recombinant mouse Siglec-15 protein (Beijing Sino Biological Technology Co., Ltd., 5A4074-M31H) were coated on enzyme-linked plates at a protein concentration of 200 ng / well and incubated at 4°C overnight; the coating solution was discarded, and the 96-well plate coated overnight was washed 6 times with PBST (0.1% Tween-20), each time for 1 min. The residual liquid in the wells was drained, 200 μl / well of ELISA blocking solution was added, and the wells were blocked at 37°C for 2 h; the blocking solution was discarded, the wells were washed 6 times with PBST (0.1% Tween-20), patted dry, and 100 μL / well of the solution of Nanobody-hFc fusion protein S1 prepared in Example 4 (diluted with blocking solution to a concentration of 15 μg / mL) was added, and incubated at 37°C for 1.5 h; the primary antibody was discarded, the wells were washed 6 times with PBST (0.1% Tween-20), and patted dry. Goat anti-human IgG (HRP, 1:4000) was diluted with blocking solution, and 100 μL / well was added to a 96-well plate and incubated at 37°C for 45 min. The secondary antibody was discarded, and the plate was washed 6 times with PBST (0.1% Tween-20). The residual liquid was patted dry, and 50 μL / well of peroxidase substrate colorimetric solution was added for color development. The plate was protected from light for 15 min, and the color development effect was observed. After complete color development, 50 μL / well of 2M sulfuric acid was added to terminate the reaction. The plate was read with a microplate reader, and the detection wavelengths were OD 492 / OD 630 , record the detection values ​​and analyze using GraphPadPrism 8 software.

[0119] The results are as follows Figure 5 As shown, S1 binds to Siglec-15 protein with good specificity and only binds to recombinant human Siglec-15 and recombinant mouse Siglec-15 antigens.

[0120] 3. Detection of the binding activity between nanobody-hFc fusion protein S1 and NCI-H157-S15 by cell ELISA

[0121] The experimental method is as follows: NCI-H157-S15 cells (human non-small cell lung adenocarcinoma cells in logarithmic growth phase and in good growth condition, kindly provided by Professor Feng Jiannan of the Institute of Toxicology and Pharmacology, Academy of Military Medical Sciences, and described in the non-patent literature "Wu J, Peng J, Zhou Y, Zhang R, Wang Z, Hu N, Zhang D, Quan G, Wu Y, Feng J, Shen B, Zhao J, Zhang Y, Yang K, Luo L. Screening and Identification of a Novel Anti-Siglec-15 Human Antibody 3F1 and Relevant Antitumor Activity. Mol Pharmacol. 2022 Sep; 102(3): 161-171. doi: 10.1124 / molpharm.121.000470 IF: 3.2Q2 B3. Epub 2022 Jun 28. PMID: 35764384. ", which is named "NCI-H157 (S15+)" in this document. The public can obtain this biological material from the Military Medical Research Institute of the Chinese People's Liberation Army Academy of Military Sciences. This biological material is only used to repeat the experiments of this invention and cannot be used for other purposes). Digestion and counting were performed, and the cell concentration was adjusted to 2×10 5 / mL, 100 μL / well was inoculated into a 96-well culture plate, and allowed to adhere overnight at 37°C and 5% CO2; the liquid in the 96-well culture plate was discarded, 200 μL / well 4% paraformaldehyde was added, and the plate was fixed at 4°C for 30 min; the liquid in the 96-well plate was discarded, 200 μL / well RPMI1640 medium was added, and the plate was blocked on ice for 2 h; the liquid in the 96-well culture plate was discarded, and 100 μL of a solution of the nanobody-hFc fusion protein S1 prepared in Example 4 (initial concentration 100 μg / mL) diluted 5-fold was added to each well, and a total of 1 5 gradients, incubated at 4°C for 1.5h; washed the 96-well plate with RPMI1640 medium, added HRP-labeled goat anti-human IgG antibody (1:4000) diluted with RPMI1640 medium, and incubated on ice for 1h; washed the 96-well plate 3 to 5 times with RPMI1640 medium, washed twice with pre-cooled PBS, patted dry the residual liquid, added peroxidase substrate colorimetric solution 50μL / well for color development, protected from light for 15min, observed the color development effect, and added 2M sulfuric acid 50μL / well to terminate the reaction after complete color development; read the plate with a microplate reader, and the detection wavelengths were OD 492 / OD 630, record the test value, then take the logarithm of 10 of the antibody concentration as the horizontal axis and the OD value as the vertical axis, calculate the antibody concentration when the OD value is 50% of the highest platform value, and record it as EC 50 value.

[0122] The results are as follows Figure 6 As shown in the figure, the horizontal axis is the logarithm of the protein molar concentration (mAb concentration lognM), and the vertical axis is the optical density value. S1 can bind to Siglec-15 on the surface of NCI-H157-S15 tumor cells in a concentration-dependent manner. EC 50 The value is 0.40nM.

[0123] 4. Through ForteBIO TM Octet QK e Systematic detection of nanobody-hFc fusion protein S1 affinity

[0124] The experimental method is as follows:

[0125] 1. Probe activation: Add 200 μL HBS-EP to each well of the probe fixing box + Buffer, select 7 AHC (Anti-human IgG Fc) probes and place them in the fixing box to soak for 30 minutes;

[0126] 2. Solution preparation: use HBS-EP + Nanobody-hFc fusion protein S1 was diluted to 200 nM in the buffer (purchased from Sitofan Biotechnology Co., Ltd., cat. no. BR100188), and antigen Siglec-15 (Beijing Sino Biological Technology Co., Ltd., 5A4074-M31H) was diluted to 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, and 15.6 nM, respectively;

[0127] 3. Set the reaction program in the order of Baseline-Loading-Baseline-Association-Dissociation. See Table 3 for details:

[0128] Table 3 ForteBIO antibody affinity determination procedure

[0129]

[0130]

[0131] 4. Data acquisition: Add the sample to be tested (200 μL / well) to the 96-well test plate, place the probe fixing box and the test plate into the test instrument, check that everything is correct, and run the test program; Data analysis: Import the experimental data into Data Analysis 7.0 software for processing and analysis, and calculate parameters such as affinity.

[0132] The results are shown in Table 4 and Figure 7 As shown, S1 and recombinant human Siglec-15 protein exhibited typical binding kinetics, and the binding between S1 and Siglec-15 was very strong and difficult to dissociate after binding. The KD values ​​of the three antibodies were calculated using a 1:1 binding model in Analysis Software 7.0. The KD value of S1 binding to recombinant human Siglec-15 protein was 0.13nM, indicating good affinity between the antigen and antibody and suitable for subsequent development.

[0133] Table 4 Equilibrium dissociation constants of antibodies

[0134]

[0135] Kon: association rate constant, indicating the rate of product formation per unit time; Kdis: dissociation rate constant, indicating the percentage of product degradation per unit time; KD: equilibrium dissociation constant, indicating the strength of affinity interaction; KD = Kon / Kdis. All data were calculated using a 1:1 binding model in Analysis Software 7.0.

[0136] 5. Detection of the metabolism of nanobody-hFc fusion protein S1 in nude mice by ELISA

[0137] Nanobody-hFc fusion protein S1 solution: The Nanobody-hFc fusion protein S1 prepared in Example 4 was diluted to 0.8 μg / μL with PBS to obtain a Nanobody-hFc fusion protein S1 solution.

[0138] The experimental method is as follows:

[0139] Sixteen 6-8 week old female nude mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were randomly divided into two groups, each with 8 mice. One group of nude mice was injected with a solution of nanobody-hFc fusion protein S1 (100 μg / mouse) via the tail vein, and the other group of nude mice was injected with sterile PBS buffer via the tail vein as a control. Blood was collected by tail cutting at 1 h, 1.5 h, 3 h, 6 h, 12 h, 24 h, 48 h, 96 h, and 7 d after administration. After the blood samples were allowed to stand at 4 ° C overnight, the upper serum was collected by centrifugation at 3000 × g for 30 min. , and stored at -20°C after aliquoting; the ELISA method was used to detect the concentration of the administered antibody in the blood. The difference between the ELISA steps and "III. Detecting the binding activity between nanobody-hFc fusion protein S1 and NCI-H157-S15 by cell ELISA experiment" is that the nanobody-hFc fusion protein S1 was replaced by the mouse serum collected above, and the rest of the operations were the same as "III. Detecting the binding activity between nanobody-hFc fusion protein S1 and NCI-H157-S15 by cell ELISA experiment".

[0140] The results are as follows Figure 8 As shown in the figure, with the increase of administration time, the antibody concentration in the blood gradually decreased, and the antibody could still be detected in the blood 7 days after injection. S1 showed typical pharmacokinetic characteristics.

[0141] VI. Detection of the metabolism of nanobody-hFc fusion protein S1 in nude mice using in vivo imaging

[0142] The experimental method is as follows:

[0143] Antibodies were labeled using Alexa Fluor 750 dye according to the instructions. Two 6-8 week old female nude mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were injected with the nanobody-hFc fusion protein S1 (200 μg / mouse) prepared in Example 4, and one 6-8 week old female nude mouse was injected with sterile PBS buffer. Images were taken using a small animal in vivo imaging system at 1 h, 12 h, 24 h, and 72 h after administration. The experimental data were imported into AniView Phoenix software for processing and analysis.

[0144] To fluorescently label the test antibody: Prepare a 1 M sodium bicarbonate solution. Adjust the antibody concentration to 1.0 mg / mL, then add 1 / 10 the volume of 1 M sodium bicarbonate solution. Add 100 μL of antibody to the Alexa Fluor dye vial. Invert to mix and incubate at room temperature for 1 hour. Centrifuge at 1100 × g for 3 minutes. Add the reaction mixture and centrifuge at 1100 × g for 5 minutes to collect the labeled antibody.

[0145] The results are as follows Figure 9As shown, the three mice at 1h, 12h, 24h and 72h were PBS group mice and nanobody-hFc fusion protein group mice, respectively. As the administration time increased, the fluorescence signal gradually weakened. At 1h, the antibody was widely distributed in most tissues and organs throughout the body, and the fluorescence signal was strongest in the bladder of nude mice, indicating that some antibodies had been metabolized by multiple hydrolases in the body and entered the bladder through the kidneys. At 72h, there was no fluorescence signal, which was different from the experimental results of the above-mentioned ELISA detection of antibody metabolism in nude mice. The possible reason is that the fluorescence used for antibody labeling will fall off from the upper body during metabolism in the body, resulting in the difference in experimental results.

[0146] VII. The experimental method for detecting the targeting of nanobody-hFc fusion protein S1 in a xenograft mouse model using in vivo imaging technology is as follows:

[0147] The basic method is the same as the experimental method for detecting S1 metabolism in nude mice using in vivo imaging technology. The difference is that the healthy BALB / c female nude mice were replaced with BALB / c nude mice subcutaneously inoculated with NCI-H157-S15 cells. Construction of the xenograft model: NCI-H157-S15 cells in the logarithmic growth phase and in good growth condition were obtained, digested with 0.25% trypsin, and prepared into a cell suspension. The cell concentration of the above cell suspension was adjusted to 3×10 6 / mL, and each mouse was injected subcutaneously with 100μL of cell suspension in the right axilla. After inoculation, the mice were kept under SPF conditions for one week. The model was considered successful when a round mass could be felt subcutaneously. Then, a vernier caliper was used to accurately measure and record the long and short diameters of the tumor until the experiment was completed. The tumor volume was calculated using the following formula: Tumor volume = [L × W 2 ] / 2, where W represents the width of the tumor and L represents the length of the tumor. When the tumor volume is about 100 mm 3 , injection of fluorescently labeled antibodies.

[0148] The results are as follows Figure 10 As shown, after administration, the fluorescence intensity of each tissue is at the peak concentration level, and the fluorescence intensity gradually decreases with time. At 1 hour, the antibody is widely distributed in most tissues and organs throughout the body, and the fluorescence signal at the tumor site is stronger. At 12-72 hours, the fluorescence at the tumor site is strongest, indicating that the antibody targets the tumor tissue site.

[0149] Example 6. Analysis of the blocking effect of nanobody-hFc fusion protein

[0150] 1. Determination of S1 blocking the binding of Siglec-15 to CD44 by indirect ELISA

[0151] The experimental method is as follows:

[0152] CD44 protein (Suzhou Nearshore Protein Technology Co., Ltd., C579) was diluted to 1 μg / mL in carbonate coating buffer (pH 9.6) and coated onto a 96-well ELISA plate. The plate was then incubated overnight at 4°C or for 2 hours at 37°C. The coating buffer was discarded from the 96-well plate, and the plate was rinsed six times with PBST. 3% skim milk powder was added and the plate was incubated at 37°C for 2 hours to prevent nonspecific binding. Discard the blocking solution from the 96-well plate, rinse six times with PBST, add 50 μL / well of Siglec-15-Fc biotin (100 ng / μL, Suzhou Nearshore Protein Technology Co., Ltd., CY14) and 50 μL / well of the test antibody (100 ng / μL) to the corresponding ELISA plate, and incubate at 37°C for 1.5 hours. Discard the primary antibody solution, rinse the wells six times with PBST, add 100 μL / well of streptavidin-HRP diluted in 3% skim milk (1:6000), and incubate at 37°C for 45 minutes. Discard the solution from the 96-well plate, rinse six times with PBST, add 100 μL / well of peroxidase substrate colorimetric solution, and incubate at 37°C for 15-20 minutes. The reaction was stopped with 2M H2SO4 (50 μL / well), and the absorbance was measured at 492 / 630 nm using a microplate reader (Molecular Devices, USA).

[0153] The results are as follows Figure 11 As shown, T7 is an irrelevant control antibody (anti-tetanus toxin THc domain neutralizing antibody) and S1 can block the interaction between Siglec-15 and CD44.

[0154] 2. Determination of S1 blocking the binding of Siglec-15 to MAG by indirect ELISA

[0155] The experimental method is as follows:

[0156] MAG protein (Suzhou Nearshore Protein Technology Co., Ltd., C897) was diluted to 1 μg / mL in carbonate coating buffer (pH 9.6) and coated onto a 96-well ELISA plate. The plate was then incubated overnight at 4°C or for 2 hours at 37°C. The coating buffer was discarded from the 96-well plate, and the plate was rinsed six times with PBST. 3% skim milk powder was added and the plate was incubated at 37°C for 2 hours to prevent nonspecific binding. The blocking buffer in the 96-well plate was discarded, and the plates were rinsed six times with PBST. 50 μL / well of Siglec-15-Fc biotin (100 ng / μL, Suzhou Nearshore Protein Technology Co., Ltd., CY14) and 50 μL / well of the test antibody (100 ng / μL) were added to the corresponding ELISA plates and incubated at 37°C for 1.5 h. The primary antibody solution was discarded, the wells were rinsed six times with PBST, and 100 μL / well of streptavidin-HRP diluted in 3% skim milk (1:6000) was added and incubated at 37°C for 45 min. The 96-well plate was discarded, rinsed six times with PBST, and 100 μL / well of peroxidase substrate development solution was added and incubated at 37°C for 15-20 min. The reaction was stopped with 2 M H2SO4 (50 μL / well), and the absorbance was measured at 492 / 630 nm using a microplate reader (Molecular Devices, USA).

[0157] The results are as follows Figure 12 As shown, T7 is an irrelevant control antibody (anti-tetanus toxin THc domain neutralizing antibody) and S1 can block the interaction between Siglec-15 and MAG.

[0158] 3. Flow cytometry detection of the blocking effect of S1 and NCI-H157 (Sialyl-Tn antigen)

[0159] NCI-H157 cells in the logarithmic growth phase were taken, digested and counted, and the cell concentration was adjusted to 5×10 5 Cells were washed with PBS solution, and 50 μL of Siglec-15-Fc-biotin (Suzhou Jinan Protein Technology Co., Ltd., CY14) (final concentration 20 μg / mL) and 50 μL of antibody (final concentration 100 μg / mL) were added to the corresponding test tubes. Siglec-15-Fc-biotin without antibody (final concentration 20 μg / mL) was used as a positive control (Positive), and Streptavidin-APC (Invitrogen, S911) was used as a negative control. All reactions were incubated at 4°C for 30 minutes. All reactions were incubated at 4°C in the dark for 30 minutes. 100 μL of secondary antibody (Streptavidin-APC) diluted 1:3000 was added to each well and incubated at 4°C for 30 minutes. After washing twice, the cells were detected at the next level.

[0160] The results are as follows Figure 13 As shown, S1 can block the interaction between Siglec-15 and Sialyl-Tn.

[0161] Example 7. Evaluation of anti-tumor activity of nanobody-hFc fusion protein

[0162] 1. Nanobody-hFc fusion protein S1 inhibits tumor growth

[0163] Successfully established tumor-bearing mice (see "VII. Detection of the targeting activity of Nanobody-hFc fusion protein S1 in a xenograft mouse model by in vivo imaging" in Example 5 for modeling methods) were marked with pins in descending order of tumor size and randomly assigned to two groups of six mice each using a random number table. The specific procedures for each group were as follows:

[0164] Treatment group (i.e., S1 in the figure): S1 solution was administered once every two days via the tail vein. The S1 solution was prepared from physiological saline and the Nanobody-hFc fusion protein S1 prepared in Example 4. Each mouse was dosed at 10 mg / kg. Tumor size and mouse body weight were monitored during administration. All data were collated in Excel, and statistical analysis and data charting were performed using GraphPad Prism 8.0 software. Tumor volume, mouse body weight, and mouse tumor weight in each group at the same time point were statistically analyzed using one-way analysis of variance (ANOVA) and Dunnett's multiple comparison test. P < 0.05 indicated statistical significance.

[0165] Negative control group (i.e., PBS in the figure): S1 solution was replaced with an equal amount of normal saline, and the rest of the procedures were the same as those of the treatment group.

[0166] The results are as follows Figure 14 As shown in the results, the S1 treatment group had a significant inhibitory effect on tumor growth compared with the PBS group (P < 0.001). The tumor morphology showed that the tumors in the antibody group were significantly smaller than those in the isotype control group. During the first 21 days of treatment, there was no significant difference in the weight of mice between the treatment groups and the control group, and all of them increased steadily. From the 21st day to the end of treatment, the weight of the control group mice decreased significantly compared with the S1 treatment group mice (P < 0.05, Figure 15 At the end of the experiment, the average tumor weight of the S1 treatment group was 0.3185 g, and the average tumor weight of the PBS group was 2.0795 g. The tumor weight of the antibody treatment group was significantly lower than that of the isotype control group, and the difference was statistically significant (P < 0.05, Figure 15 These results indicate that the S1 antibody has significant anti-tumor activity and can be used as a candidate drug targeting Siglec-15 for anti-tumor therapy.

[0167] 2. Detection of cellular molecular changes in mouse serum using ELISA TGF-β cytokine detection kit and Luminex multifactor detection technology

[0168] 1. TGF-β cytokine detection experimental method:

[0169] (1) Preparation before the experiment:

[0170] a) Take the test kit out of the refrigerator 20 minutes in advance and equilibrate to room temperature;

[0171] b) Preparation of washing solution: dilute the concentrated washing solution with double distilled water (1:24);

[0172] c) Preparation of standard working solution: Centrifuge the standard at 10,000 × g for 1 min, then add 0.9 mL of universal diluent and let stand for 15 minutes to fully dissolve. Then gently mix (concentration is 2,000 pg / mL). Subsequently, serially dilute the standard to 2,000, 1,000, 500, 250, 125, 62.5, 31.25, and 0 pg / mL.

[0173] d) Preparation of biotinylated antibody working solution: 15 minutes before use, centrifuge the concentrated biotinylated antibody at 800 × g for 1 minute. Dilute the 100× concentrated biotinylated antibody to the working concentration using biotinylated antibody diluent.

[0174] e) Preparation of enzyme conjugate working solution: 15 minutes before use, centrifuge the concentrated HRP enzyme conjugate at 800 × g for 1 minute. Dilute the 100× concentrated HRP enzyme conjugate to the working concentration with enzyme conjugate diluent.

[0175] f) Specimen pretreatment: Add 5 μL of serum and 10 μL of activation solution to 225 μL of universal diluent, mix thoroughly, incubate at 4°C for 2 min, and then add 10 μL of stop solution.

[0176] (2) Set up standard wells, blank wells, and sample wells. Add 100 μL of serially diluted standard to the standard wells, 100 μL of universal diluent to the blank wells, and 100 μL of the sample to be tested (the serum from the mice in the treatment group and the negative control group in the "I. Nanobody-hFc fusion protein S1 inhibits tumor growth" of this example) to the remaining wells. Cover the ELISA plate with a film and incubate at 37°C for 90 min.

[0177] (3) Shake off all liquid in the wells without washing. Add 100 μL of biotinylated antibody working solution to each well, cover the plate with film, and incubate at 37°C for 60 min.

[0178] (4) Shake off all liquid in the wells and pat dry on clean absorbent paper. Add 350 μL of washing solution to each well and soak for 1 min. Shake off all liquid in the ELISA plate and pat dry. Repeat this step 3 times.

[0179] (5) Add 100 μL of enzyme conjugate working solution to each well, cover the plate with film, and incubate at 37°C for 30 min.

[0180] (6) Shake off all liquid in the wells and wash the plate 5 times using the same method as step (4).

[0181] (7) Add 90 μL of substrate solution (TMB) to each well, cover the ELISA plate, and incubate at 37°C in the dark for about 15 minutes.

[0182] (8) Add 50 μL of stop solution to each well to terminate the reaction.

[0183] (9) Immediately measure the optical density (OD450 value) of each well at a wavelength of 450 nm using a microplate reader.

[0184] 2. Luminex multi-factor detection experimental method:

[0185] (1) Sample preparation: The blood of the mice in the treatment group of the "1. Nanobody-hFc fusion protein S1 inhibits tumor growth" of this example and the blood of the mice in the negative control group were placed in a centrifuge tube and allowed to stand at 4°C for 45 minutes. Subsequently, the tubes were centrifuged at 4°C (3000×g, 30 minutes) to separate the serum to obtain the serum of the mice in the treatment group and the serum of the mice in the negative control group.

[0186] (2) Preparation before the experiment:

[0187] a) Preparation of washing solution: dilute the concentrated washing solution with double distilled water at a ratio of 1:24;

[0188] b) Preparation of standard working solution: Reconstitute the LXSAMSM-25 standard according to the dilution method provided in the COA, then thoroughly mix with Diluent RD6-52. Let stand at room temperature for 15 minutes. Then, take 100 μL from each standard vial and add it to a new EP tube. Then, make up to 1 mL with Diluent RD6-52. This EP tube will serve as the highest concentration for the standard curve. Prepare the standard and blank control using a 3-fold dilution method.

[0189] c) Sample processing: After mixing, the samples were centrifuged at 12,000 × g for 5 min. Then, the samples were diluted 2-fold with Diluent RD6-52, i.e., 75 μL sample (serum from treated mice or serum from negative control mice) + 75 μL Diluent RD6-52;

[0190] d) Beads preparation: Before removing the bottle cap, centrifuge briefly, taking care not to invert the bottle, to mix the pre-mixed beads mixture;

[0191] e) Biotin-antibody mixture preparation: Before removing the cap, briefly centrifuge the bottle, taking care not to invert the bottle, to gently mix the pre-mixed detection antibody mixture;

[0192] f) Preparation of PE-Streptavidin: Before removing the cap, centrifuge briefly, taking care not to invert the bottle, to gently mix the premixed PE-Streptavidin.

[0193] (3) According to the experimental design, add 50 μL of standard or sample to the corresponding well.

[0194] (4) Add 50 μL of resuspended beads to each well, cover securely with a foil cover, and incubate on a shaker at 800 rpm at room temperature for 2 h.

[0195] (5) Place the microplate on a magnetic stand, wait for 1 minute, remove the liquid, and add 200 μL of wash buffer to each well. Wait for another 1 minute, then remove the liquid. Repeat the above steps for 3 washes.

[0196] (6) Add 50 μL of the diluted biotin-antibody mixture to each well, cover securely with a foil cover, and incubate on a shaker at 800 rpm at room temperature for 1 h.

[0197] (7) Repeat step (4).

[0198] (8) Add 50 μL of diluted streptavidin-PE to each well, cover securely with foil, and incubate on a shaker at 800 rpm for 30 min at room temperature.

[0199] (9) Repeat step 4.

[0200] (10) Add 100 μL of wash buffer to each well, resuspend the beads, and incubate on a shaker at 800 rpm for 2 min at room temperature. Finally, analyze the beads.

[0201] The results are as follows Figure 15 As shown in the figure, the concentrations of TGF-β, IL-6, and IL-10 in the peripheral blood serum of mice in the PBS group were significantly higher than those in the S1 group. High levels of TGF-β, IL-6, and IL-10 may affect tumor progression by weakening the immune response, promoting inflammatory response, and regulating tumor cell growth in the tumor environment.

[0202] 3. Detection of the distribution of immune cells in mouse tumor tissues by flow cytometry

[0203] The experimental method is as follows:

[0204] (1) Sample preparation: After euthanizing the mice in the treatment group and the negative control group, spray them with 75% alcohol for disinfection. Use a surgical scalpel and tweezers to carefully separate the tumor from the surrounding tissue, and quickly place the tumor tissue in a six-well plate containing DMEM high-glucose medium. After all the tumor tissue is peeled off, aspirate the DMEM medium reserved in the six-well plate, use surgical scissors to cut the tumor tissue into a slurry, add 2mL of digestion solution (DMEM high-glucose medium containing 5% FBS, add collagenase I and deoxyribonuclease I to make the final concentrations of 1mg / mL and 200μg / mL respectively), and place in a 37℃ incubator for digestion for 30min. After digestion, remove the six-well plate and place it on ice, and add DMEM high-glucose medium (containing 5% FBS) to terminate digestion. The tumor tissue suspension was passed through a cell strainer, and the tissue blocks on the strainer were rinsed with DMEM high-glucose medium. The suspension containing tumor cells was collected and centrifuged at 850 × g for 5 min at 4°C. The supernatant was discarded, and 1 mL of PBS was added and mixed to obtain a tumor cell suspension. 100 μL of cell suspension was taken and counted, and the cell density was adjusted to 2 × 10 7 Fixable Viability Stain 780 (purchased from Biolegend, USA, Catalog No. 565388), Anti-mouse F4 / 80 (purchased from Biolegend, USA, Catalog No. 565411), Anti-mouse CD11b (purchased from Biolegend, USA, Catalog No. 552850), Anti-mouse Ly-6C (purchased from Biolegend, USA, Catalog No. 563011), Anti-mouse Ly-6G (purchased from Biolegend, USA, Catalog No. 127614), Anti-mouse CD45.2 (purchased from Biolegend, USA, Catalog No. 563686).

[0205] (2) Blocking before staining: Take 100 μL of cell suspension into a 96-well plate, add anti-Fc receptor antibody at a ratio of 100:3, and incubate at 4°C in the dark for 30 min.

[0206] (3) Set up a blank control group tube, a fluorescence subtraction control group, and an experimental group, prepare the antibody mixture according to Table 5, calculate the required volume of each flow cytometry antibody, and prepare 70 μL of the corresponding flow cytometry antibody mixture in each well.

[0207] Table 5 Flow cytometry antibody staining color matching

[0208] Flow cytometry antibodies Fluorescence channels Dilution ratio FixableViabilityStain780 APC-Cy7 1000× Anti-mouseF4 / 80 BV421 200× Anti-mouseCD11b PE-Cy7 200× Anti-mouseLy-6C BV605 200× Anti-mouseLy-6G APC 200× Anti-mouseCD45.2 BV786 200×

[0209] (4) After blocking, centrifuge the sample at 850 × g for 5 min at 4°C. Discard the supernatant and add 70 μL of flow cytometry antibody mixture to each well. Stain in the dark at 4°C for 30 min.

[0210] (5) After staining, centrifuge at 850 × g for 5 min at 4°C, discard the supernatant, and add 100 μL PBS buffer to wash the cells. Repeat this operation twice.

[0211] (6) Add 200 μL of PBS buffer to each well to resuspend the cells, store at 4°C in the dark, and filter with a 70 μm cell sieve before detection.

[0212] (7) Analysis steps: First, use FSC-H and SSC-H to circle single cells, and then use FVS 780 + Circle the live cells and finally circle the tumor-associated macrophages (CD45 + Ly-6G - CD11b + f / 4 / 80 + ).

[0213] The results are as follows Figure 16 As shown in the figure, compared with the PBS control group, the proportion of TAMs in tumor tissue in the S1 group was significantly increased (P<0.05). Figure 17 Flow cytometric analysis workflow for TAMs cells.

[0214] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A nanobody against sialic acid-binding immunoglobulin-like lectin 15, characterized in that The nanobody comprises three complementary determining regions, CDR1, CDR2 and CDR3, and the nanobody is S1; The amino acid sequence of the CDR1 of S1 is SEQ ID No. 6, the amino acid sequence of the CDR2 of S1 is SEQ ID No. 7, and the amino acid sequence of the CDR3 of S1 is SEQ ID No.

8.

2. The Nanobody according to claim 1, characterized in that The Nanobody comprises, in addition to the complementarity determining regions, four framework regions FR1, FR2, FR3 and FR4; The amino acid sequence of FR1 of S1 is SEQ ID No. 2, the amino acid sequence of FR2 of S1 is SEQ ID No. 3, the amino acid sequence of FR3 of S1 is SEQ ID No. 4, and the amino acid sequence of FR4 of S1 is SEQ ID No.

5.

3. The Nanobody according to claim 1 or 2, characterized in that The amino acid sequence of S1 is SEQ ID No.

1.

4. An anti-sialic acid-binding immunoglobulin-like lectin 15 heavy chain antibody, characterized in that: The heavy chain antibody comprises the Nanobody according to claim 1.

5. The heavy chain antibody according to claim 4, characterized in that The heavy chain antibody includes a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is SEQ ID No.

1.

6. The heavy chain antibody according to claim 4 or 5, characterized in that The heavy chain antibody is S1-Fc, and the amino acid sequence of S1-Fc is SEQ ID No.

11.

7. Genetic material, characterized in that The genetic material is any of the following: g1) a nucleic acid molecule encoding the Nanobody according to any one of claims 1 to 3, or the heavy chain antibody according to any one of claims 4 to 6; g2) An expression cassette, recombinant vector, recombinant cell or recombinant bacterium containing the nucleic acid molecule described in g1).

8. An antibody drug against sialic acid-binding immunoglobulin-like lectin 15, characterized in that: The drug comprises the Nanobody according to any one of claims 1 to 3 or the heavy chain antibody according to any one of claims 4 to 6.

9. Application, which can be any of the following: M1) Use of the genetic material of claim 7 in the preparation of a Nanobody according to any one of claims 1 to 3 or a heavy chain antibody according to any one of claims 4 to 6; M2) Use of the Nanobody of any one of claims 1 to 3, or the heavy chain antibody of any one of claims 4 to 6, or the genetic material of claim 7, or the antibody drug of claim 8 in the preparation of a product for treating a tumor that positively expresses sialic acid-binding immunoglobulin-like lectin 15, wherein the positive-expressing tumor is a human non-small cell lung adenocarcinoma; M3) Use of the Nanobody according to any one of claims 1 to 3, the heavy chain antibody according to any one of claims 4 to 6, the nucleic acid molecule according to claim 7, or an expression cassette, recombinant vector, recombinant cell or recombinant bacteria or pharmaceutical composition containing the nucleic acid molecule according to claim 7 in the preparation of a product for detecting the content of sialic acid-binding immunoglobulin-like lectin 15 in a tumor.

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