Development and application of TIM-3 targeting single-domain antibody

Abstract

The invention discloses development and application of a single-domain antibody targeting TIM-3, and belongs to the technical field of molecular biology. Aiming at the unique advantages of a single-domain antibody in the prior art, the invention provides a TIM-3-targeting single-domain antibody, the amino acid sequence of the TIM-3-targeting single-domain antibody is shown as SEQ ID NO.1, and the single-domain antibody has relatively strong affinity with TIM-3 positive cells 293TTIM-3 and A549TIM-3, and can be combined with TIM-3 protein expressed on the surfaces of the cells; through NOTA coupling modification and positron radionuclide Ga68 labeling, a nuclide probe can be prepared and applied to PET / CT imaging of a mouse tumor model. The TIM-3-targeting single-domain antibody provided by the invention can be prepared by mass expression of in-vitro engineering bacteria, can be applied to preparation of protein detection antibodies or therapeutic antibodies, and has important commercial value in clinical disease diagnosis and treatment.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology and relates to a single-domain antibody and its applications. Background Technology

[0002] TIM-3 (T-cell immunoglobulin and mucin domain-containing protein 3) is an important immune checkpoint molecule, belonging to the TIM gene family and encoding a type I transmembrane protein. TIM-3 is primarily expressed on the surface of immune cells, including CD4+. + Helper T cells, CD8 + Cytotoxic T cells, regulatory T cells, natural killer cells, and dendritic cells, among others, participate in the negative regulation of the immune response through interactions with their ligands, playing a crucial role in maintaining immune tolerance and preventing excessive inflammation. The TIM-3 structure comprises an extracellular immunoglobulin variable region (IgV) domain, a mucin-like domain, a transmembrane region, and a cytoplasmic tail. The IgV domain is responsible for binding to ligands (such as galactolectin-9, HMGB1, and CEACAM-1). The mucin domain is rich in threonine and serine residues and can undergo O-glycosylation modification, affecting protein stability and function. The cytoplasmic tail contains conserved tyrosine phosphorylation sites, participating in the transduction of downstream signaling pathways.

[0003] The human TIM-3 gene (HAVCR2) is located on chromosome 5q33.2, is approximately 15 kb long, and consists of 10 exons and 9 introns. Multiple isoforms can be generated through alternative splicing, the most common being the full-length membrane-bound form (TIM-3FL) and the soluble form (sTIM-3). TIM-3FL is highly expressed on the surface of activated T cells, while sTIM-3 may regulate signal transduction through competitive ligand binding. Under normal physiological conditions, TIM-3, by binding to its ligand galactolectin-9, induces Th1 cell apoptosis or functional exhaustion, thereby limiting the inflammatory response. However, in the tumor microenvironment, TIM-3 is persistently highly expressed on tumor-infiltrating lymphocytes, leading to T cell dysfunction and promoting immune escape. Studies have found that TIM-3 is significantly upregulated in T cells of various malignant tumors (such as melanoma, lung cancer, liver cancer, and hematologic malignancies), and its expression level is closely related to disease progression and poor prognosis. Monoclonal antibody drugs targeting TIM-3 can restore the anti-tumor activity of T cells by blocking the TIM-3 signaling pathway. They have shown synergistic therapeutic potential when used in combination with PD-1 / PD-L1 inhibitors in multiple clinical trials.

[0004] Currently, the immune checkpoint molecule TIM-3, as a promising emerging target for tumor immunotherapy, has demonstrated enormous clinical application potential. Numerous pharmaceutical companies and research institutions worldwide have begun developing TIM-3-targeted drugs; these are primarily monoclonal antibody drugs, but also include various innovative drug forms such as bispecific antibodies (e.g., simultaneously targeting PD-1 / TIM-3 or LAG-3 / TIM-3). Sabatolimab is a humanized monoclonal antibody with high affinity and specificity that specifically binds to TIM-3, thereby blocking its interaction with ligands (such as galactochetin-9 and CEACAM-1). Through this blocking effect, Sabatolimab can reverse the exhaustion state of T cells, restore their proliferative capacity and cytokine secretion function, and reactivate the anti-tumor immune response. Although no TIM-3 targeted drugs have yet been officially approved for marketing worldwide, Sabatolimab is one of the most advanced representative drugs in clinical research and development. It has shown encouraging efficacy and safety in multiple clinical trials for the treatment of myelodysplastic syndromes and acute myeloid leukemia, and is actively being explored for combination with existing standard therapies or other immunotherapies.

[0005] Since the first discovery of heavy chain antibodies in camels, single-domain antibodies (VHHs), also known as nanobodies, derived from them, have seen rapid development in multiple fields. With a molecular weight of only 12-15 kDa, significantly smaller than traditional monoclonal antibodies (approximately 150 kDa), VHHs are the smallest intact antigen-binding fragments, giving them stronger tissue penetration and faster tumor uptake efficiency. This allows them to more effectively penetrate solid tumors, thereby improving therapeutic efficacy. Single-domain antibodies also exhibit high solubility and stability and can be mass-produced in bacteria. Furthermore, the longer CDR3 of VHHs enables them to recognize and bind to hidden epitopes that are difficult for traditional antibodies to reach, such as enzyme active sites, thus expanding their potential applications. In recent years, drug development based on single-domain antibodies has progressed rapidly, demonstrating great potential in areas such as tumor targeted therapy, immunomodulation, diagnostic imaging, and the treatment of infectious diseases. Their unique physicochemical properties and functional advantages have made single-domain antibodies a hot topic in the development of next-generation antibody drugs, laying a solid foundation for their clinical translation and industrial application. Summary of the Invention

[0006] Taking advantage of the unique advantages of single-domain antibodies in the prior art, this invention provides a method for developing and applying a single-domain antibody targeting TIM-3.

[0007] One of the objectives of this invention is to provide a single-domain antibody targeting TIM-3, wherein the amino acid sequence of the single-domain antibody targeting TIM-3 is shown in SEQ ID NO.1.

[0008] In a preferred embodiment of the present invention, the single-domain antibody targeting TIM-3 includes complementarity-determining regions CDR1, CDR2 and CDR3.

[0009] In a preferred embodiment of the present invention, the amino acid sequence of CDR1 is shown in SEQ ID NO.2, the amino acid sequence of CDR2 is shown in SEQ ID NO.3, and the amino acid sequence of CDR3 is shown in SEQ ID NO.4.

[0010] In a preferred embodiment of the present invention, the single-domain antibody targeting TIM-3 further includes the framework regions FR1, FR2, FR3 and FR4.

[0011] In a preferred embodiment of the present invention, the amino acid sequence of FR1 is shown in SEQ ID NO.5, the amino acid sequence of FR2 is shown in SEQ ID NO.6, the amino acid sequence of FR3 is shown in SEQ ID NO.7, and the amino acid sequence of FR4 is shown in SEQ ID NO.8.

[0012] A second objective of this invention is to provide a nucleic acid comprising a nucleic acid sequence encoding the aforementioned single-domain antibody targeting TIM-3.

[0013] A third objective of this invention is to provide an expression vector containing the aforementioned nucleic acid.

[0014] A fourth objective of this invention is to provide a host cell containing the aforementioned expression vector.

[0015] The fifth objective of this invention is to provide a detection nuclide probe containing the aforementioned single-domain antibody.

[0016] The sixth objective of this invention is to provide the application of the above-mentioned single-domain antibody targeting TIM-3 in the preparation of in vivo imaging probes, in vitro detection probes or therapeutic antibodies, wherein the application refers to the detection of tissues or cells expressing TIM-3.

[0017] Beneficial effects of the present invention: The present invention provides a single-domain antibody targeting TIM-3, wherein the single-domain antibody targeting TIM-3 reacts with TIM-3 positive cells 293T. TIM-3 and A549 TIM-3It exhibits strong affinity; mouse PET / CT imaging results show that the TIM-3-targeting single-domain antibody-nucleoside probe can detect TIM-3-expressing tumor tissue in vivo. The TIM-3-targeting single-domain antibody provided by this invention can be prepared through large-scale expression in vitro using engineered bacteria. It can be used to prepare protein detection antibodies or therapeutic antibodies, enabling in vitro and in vivo detection of TIM-3-expressing tumor tissue, and has significant commercial value in clinical disease diagnosis and treatment. Attached Figure Description

[0018] Figure 1 The image shows the identification results of the TIM-3 expression plasmid in Example 1; M: DL1000 DNA molecular weight standard; Figure 2 This is a graph showing the identification results of TIM-3 ECD recombinant protein expression in Example 1; M represents the protein molecular weight standard. Figure 3 The TIM-3 recombinant protein Ni in Example 1 2+ Affinity purification results (image); Figure 4 This is a graph showing the serum antibody titer determination results after alpaca immunization with TIM-3 protein in Example 2; Figure 5 This is a graph showing the results of cDNA library and phage library capacity detection in Example 3; Figure 6 This is a seroconversion detection image of TIM-3 alpaca immunization in Example 4; Figure 7 This is a graph showing the recovery rate of the phage panning library based on the membrane protein display system in Example 4; Figure 8 This is a graph showing the indirect ELISA identification results of the induced expression of single-domain antibodies after three rounds of screening in Example 4; Figure 9 This is a flow cytometry identification result of the induced expression of single-domain antibodies after three rounds of panning in Example 4; Figure 10 This is a diagram showing the prokaryotic expression and purification of the TIM-3-targeting single-domain antibody in Example 5; Figure 11 This is a flow cytometry diagram showing the binding of the TIM-3 single-domain antibody to the TIM-3 membrane protein in Example 5. Figure 12 This is a graph showing the titer results of the TIM-3-targeting single-domain antibody in Example 5; Figure 13 This is a fluorescence detection result of TIM-3 overexpression in A549 cells in Example 6; Figure 14 This is a Western blot result of TIM-3 overexpression in A549 cells in Example 6; Figure 15 A549 in Example 6 TIM-3 Overexpression cell lines 68 Ga-NOTA-VHH TIM-3 A schematic diagram illustrating the endocytosis effect of the probe; Figure 16 In Example 6 68 Ga-NOTA-VHH TIM-3 PET imaging results showing the distribution of probes in a mouse tumor model; Figure 17 Injecting different groups of mice in Example 6 68 Ga-NOTA-VHH TIM-3 Schematic diagram of tumor uptake after probe insertion; Figure 18 Injecting different groups of mice in Example 6 68 Ga-NOTA-VHH TIM-3 Schematic diagram of tumor / muscle ratio after probe examination; Figure 19 For example, A549 / A549 in Example 6 TIM-3 Subcutaneous tumor model mice injected 68 Ga-NOTA-VHH TIM-3 Schematic diagram of biodistribution results after probe insertion. Detailed Implementation

[0019] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0021] The Western blot used in this embodiment includes the following steps: S1: Collect protein samples for SDS-PAGE polyacrylamide gel electrophoresis, and perform constant voltage electrophoresis at 120V. S2: After electrophoresis in S1, the protein sample in the gel was transferred to a nitrocellulose membrane and transferred at a constant current of 230 mA for 2 h in an ice box. S3: After the transfer is completed, place the membrane from S2 into the blocking solution (5% skim milk powder) and seal it at room temperature for 2 hours; S4: Wash the membrane after blocking in S3 three times with 1×TBST solution for 10 min each time, then add primary antibody solution and incubate at room temperature for 2 h. S5: Wash the membrane after primary antibody incubation in S4 three times with 1×TBST solution for 10 min each time, then add secondary antibody solution and incubate at room temperature for 1 h; S6: The membrane after incubation with the secondary antibody in S5 was washed three times with 1×TBST solution for 10 min each time. Then, a developing solution was prepared for development, followed by development, photography, and storage in a gel imaging system.

[0022] Example 1: Preparation of TIM-3 Immunogen (1) The extracellular segment of the target gene TIM-3 was amplified using pUC19-TIM-3 plasmid as a template; (2) The eukaryotic expression plasmid pCAGGS-IL2-TIM-3-His was constructed using overlap extension PCR technology. The recombinant plasmid obtained above was verified by PCR, and the results are as follows: Figure 1 As shown, the positive plasmids were sequenced, and after successful sequencing alignment, a large number of plasmids were extracted for later use. (3) The recombinant plasmid pCAGGS-TIM-3-His obtained in (2) was transiently transfected into HEK293 suspension cells for protein expression, and the protein supernatant was collected for Ni 2+ After affinity purification and protein concentration determination, the protein was stored at -80℃ for later use.

[0023] In this embodiment, the TIM-3 protein obtained above was subjected to Western spectroscopy, and the results are as follows: Figure 2 As shown, the target band appears at the 43kDa position, indicating that TIM-3 protein expression was successful and can be used for subsequent immunization.

[0024] The example describes the Ni reaction of the TIM-3 protein obtained above. 2+ Affinity purification identification, results as follows Figure 3 As shown, the target protein was obtained at 43 kDa by elution with an imidazole gradient, and the TIM-3 protein was successfully purified.

[0025] Example 2: Animal Immunization Procedure (1) 0.5 mg of the TIM-3 recombinant protein obtained in Example 1 was mixed with 1 mL of Freund's adjuvant and emulsified to obtain a TIM-3 immunogen mixture, which was stored at 4°C for later use. (2) Select alpacas and record their ear numbers. Inject 0.4 mL of the TIM-3 immunogen mixture obtained in (1) into the subcutaneous tissue of the alpaca's buttocks on both sides, with two injection points on each side. Observe for 30 min after the immunization injection to confirm that the alpaca is in good condition and has no discomfort symptoms. Inject immunization once every 2 weeks for a total of 4 immunizations. Before each immunization injection, collect blood from the alpaca's jugular vein, taking 10 mL of blood each time. Separate the serum and store it at -80℃ for later use. On the 5th day after the last immunization injection, draw 100 mL of peripheral blood from the alpaca's neck to obtain the immunized alpaca peripheral anticoagulated blood sample, i.e., the immune blood sample. (3) Add 3 mL of cell separation solution to a 15 mL centrifuge tube, then slowly add 3 mL of the serially diluted immune blood sample obtained in (2). Pre-cool the centrifuge and centrifuge at 400 g for 30 min. Observe the blood separation in the centrifuge tube. Use a 200 μL pipette to aspirate the upper layer of immune cells in the middle cotton-like layer to a new 15 mL centrifuge tube and save the upper plasma in a new centrifuge tube. Store at -80℃. Add 10 mL of room temperature PBS buffer to the above centrifuge tube and centrifuge at 400 g for 20 min. Discard the supernatant. Then add 5 mL of room temperature PBS buffer and centrifuge at 400 g for 20 min. Count the number of cells using a hemocytometer, centrifuge and discard the supernatant. Use Trizol (Sigma) to dissolve and separate lymphocytes according to the number of cells and the instructions. Store at -80℃.

[0026] This embodiment detects seroconversion in serum from alpacas immunized with TIM-3, and the results are as follows: Figure 4 As shown, the ELISA plate was coated with TIM-3 protein. The primary antibody was serially diluted alpaca immune serum, and the secondary antibody was HRP-labeled goat anti-alpaca antibody. The ELISA results showed that the alpaca immune serum titer reached 1:256k, indicating that the serum seroconversion was successful after antigen immunization.

[0027] Example 3: Preparation of a single-domain antibody library targeting TIM-3 Total RNA was extracted from the lymphocytes obtained in Example 2 using an RNA extraction kit (Invitrogen), and a cDNA library was generated using a reverse transcription kit (Invitrogen). Nested PCR was performed using alpaca heavy chain antibody and primers specific to the variable region of the heavy chain antibody to amplify the VHH gene fragment. The primer sequences were the upstream primer FR1-RSCF as shown in SEQ ID NO.9 and the downstream primer VHH-RSCB as shown in SEQ ID NO.10. The VHH gene fragment was ligated to the phage expression vector pComb3 by enzyme digestion to construct a recombinant phage vector. The recombinant phage vector was then transformed into E. coli TG1 competent cells by electroporation for amplification. With the help of helper phages, VHH was displayed on the surface of the phages, forming a VHH phage library, which is a single-domain antibody library targeting TIM-3.

[0028] This embodiment tests the capacity of the obtained cDNA library and phage library as follows: Figure 5 As shown, the cDNA library size is 5.3 × 10⁻⁶. 7 pfu / mL, phage library capacity 2.1×10 13 cfu / mL.

[0029] Example 4: Panning and Identification of Single-Domain Antibodies Targeting TIM-3 1. Construction of a 293T cell line overexpressing TIM-3 (1) The TIM-3 target gene was amplified using pUC19-TIM-3 plasmid as a template; (2) The lentiviral plasmid pLVSIN-TIM-3 was constructed by overlapping extension PCR technology. The recombinant plasmid obtained above was sequenced. After successful sequencing alignment, a large number of plasmids were extracted for later use. (3) Using the recombinant plasmid pLVSIN-TIM-3 / pLVSIN-vector obtained in (2) together with the packaging plasmids gag-pol and VSV-G, 293T mammalian cells were transiently transfected to package the virus. The packaged lentivirus was then used to infect 293T cells to construct a TIM-3 overexpressing cell line, 293T. TIM-3 and 293T vector .

[0030] With 293T vector As a control, the primary antibody used was alpaca immune serum, and the secondary antibody was FITC-labeled goat anti-alpaca secondary antibody (Thermo) against 293T. TIM-3 Cells were identified by flow cytometry, and the results were as follows: Figure 6 As shown, the serum of non-immunized alpacas was effective against 293T. TIM-3 and 293T vectorCell lines did not bind, and only 293T cells bound to alpaca serum after immunization. TIM-3 The presence of a cell line indicates that the cell line has been successfully constructed.

[0031] 2. Screening of single-domain antibodies based on phage display technology (1) Negative washing S1: Prepare 293T negative wash cells vector Cell lines, cell count, using 3.5 × 10⁻⁶ cells per cycle. 8 One cell; S2: Wash the cells from S1 twice with PBS, resuspending them each time with 30 mL of PBS. Centrifuge at 4℃ and 500 g for 5 min, discard the supernatant, and resuspend them with 2 mL of PBS for the last time before placing them in cryovials. S3: Centrifuge the resuspended cells obtained in S2 at 4℃ and 500 g for 5 min, discard the supernatant, then add 500 μL of phage-milk / PBS to resuspend, and incubate at room temperature for 30 min; then centrifuge at 13000 rpm for 2 min, and collect the phage supernatant, i.e., phage elution buffer.

[0032] (2) Positive washing S1: Prepare 293T positive wash cells TIM-3 Cell lines, count cell numbers, use 5 × 10⁻⁶ cells each time. 8 One cell; S2: Wash the cells from S1 twice with PBS, resuspending them each time with 30 mL of PBS. Centrifuge at 4℃ and 500 g for 5 min, discard the supernatant, and resuspend them with 2 mL of PBS for the last time before placing them in cryovials. S3: Centrifuge the resuspended cells obtained in S2 at 4°C and 500 g for 5 min, discard the supernatant, then add 500 μL of negative phage elution buffer obtained in (1), resuspend, and incubate at room temperature for 30 min; wash five times with PBS, centrifuge at 500 g at room temperature for 2 min, and remove the PBS; add 150 μL of Elution Buffer (glycine solution, pH=2.2), react at room temperature for 10 min; add 10 μL of 2MTrisBase solution (glycine solution, pH=9.0) to neutralize; then centrifuge at 13000 rpm for 2 min and collect the phage elution buffer; S4: Take 10 μL of the phage elution buffer obtained in S3 and dilute it 5 times (103). 1-5 (Dilution), add 90 μL OD to each EP tube. 600The TG1 bacterial suspension was prepared at 0.5-0.6 and incubated in a water bath at 37°C for 15 min. Then, the bacterial suspensions of five different gradients were spread onto 2×YT culture plates containing ampicillin resistance and incubated upside down overnight at 37°C. The number of single colonies on the culture plates was counted to calculate the titer. S5: Take 10 mL of the remaining phage elution buffer from S3 and add it to the OD200. 600 Add 0.5-0.6 μL of TG1 bacterial culture and let stand at room temperature for 30 min; add 0.1% ampicillin and continue culturing at 37℃ and 250 rpm for 30 min; then add 100 μL of helper phage, let stand at room temperature for 30 min, centrifuge at 8000 rpm for 10 min, transfer the precipitate after centrifugation to 100 mL of 2×YT culture plate containing 0.1% ampicillin and kanamycin resistance, and incubate overnight at 37℃ and 220 rpm. The next day, concentrate the phage for later use.

[0033] (3) The VHH phage library obtained in Example 3 was compared with the 293T phage library obtained in Part 1 of this Example. vector / 293T TIM-3 The cell lines were incubated and subjected to three rounds of negative and positive washes to obtain VHH phages that specifically bind to and are highly enriched with the TIM-3 membrane antigen, which constitutes the positive phage screening library.

[0034] This embodiment detects the phage recovery rate of the obtained positive phage screening library, and the results are as follows: Figure 7 As shown, the amount of phage recovered increased progressively with each round, ultimately reaching a phage recovery rate of 1.0 × 10⁻⁶. -4 .

[0035] 3. Indirect ELISA identification Take the phage elution buffer obtained from the third round of positive wash in this example, spread it on a plate, randomly select 96 bacterial clones, and culture them statically overnight at 37°C. Take 10 μL of each of the above bacterial solutions and transfer them to 2xYT medium in a 96-well plate (1 mL / well). Culture until the logarithmic phase. Add 50% sterile glycerol to the remaining bacterial solution, shake well, and store at -80°C. Add IPTG to a final concentration of 0.2 mM and induce expression for 12 h at 37°C and 220 rpm. Centrifuge at 4000 rpm and 4°C for 15 min, then place the bacterial cells at -20°C and freeze for 30 min. After returning to room temperature, resuspend the bacterial cells in PBS (100 μL / well) and shake at 300 rpm and 4°C for 30 min. Centrifuge at 4000 rpm and 4°C for 10 min to obtain the supernatant, which is the crude extract of single-domain antibodies.

[0036] The recombinant TIM-3 ECD protein obtained in Example 1 was diluted with coating buffer and added to wells at 400 ng / well and 100 μL / well, respectively, and incubated overnight at 4°C. On the second day, the plates were washed three times with PBST for 2 min each time. 300 μL of 5% skim milk was added to each well to block the ELISA plate for 2 h. The plate was then washed three times with PBST for 2 min each time. 100 μL of the crude nanobody extract obtained above was added to each well as the primary antibody and incubated at 37°C for 2 h. The plate was washed three times with PBST for 2 min each time. 100 μL of Anti-HA-HRP antibody was added to each well as the secondary antibody and incubated at 37°C for 1 h. The plate was washed three times with PBST for 2 min each time. 100 μL of TMB chromogenic solution was added to each well and the reaction was carried out at 37°C. 50 μL of 2M sulfuric acid was added to terminate the reaction, and the OD was read. 450 nm value.

[0037] In this embodiment, the crude extract of the induced single-domain antibody was detected by indirect ELISA, and the identification results are as follows: Figure 8 As shown, OD 450 >1.0 was identified as a positive colony.

[0038] 4. Flow cytometry identification This embodiment utilizes the constructed 293T TIM-3 Cell lines were identified by flow cytometry of the phages induced to express single-domain antibodies after the three rounds of panning. The primary antibody was phage C6 and a homotype control phage, and the secondary antibody was APC-anti-HA1.1 flow cytometry antibody (Biolegend). Results are as follows: Figure 9 As shown, compared with the negative control group and the isotype control group, the C6 phage obtained by screening was deflected by 99.7%, indicating that the C6 phage obtained by screening can specifically bind to TIM-3 on the membrane surface. Subsequently, the phage was infected with TG1 and its sequence was determined.

[0039] Example 5: Prokaryotic expression, purification, and identification of TIM-3-targeting single-domain antibody The positive sequence identified in Part 2 of Example 4 was cloned, and the prokaryotic expression vector pET22b-TIM-3-VHH-6his was constructed using overlap extension technology. The positive plasmid was transformed into Rosetta (DE3) competent cells for induced expression. The cells were picked and placed in 6 mL of LB liquid medium containing ampicillin, and cultured at 37°C and 220 rpm / min for 12-15 h to obtain a bacterial culture. The above 2 mL of bacterial culture was activated and placed in LB liquid medium containing 100 μg / mL ampicillin, and cultured at 37°C and 220 rpm / min for 3-4 h. When the OD value of the bacterial culture reached 0.4, the above bacterial culture was induced to express for 12 h at 16°C using 0.2 mM IPTG. After centrifugation, the supernatant was collected, which is the single-domain antibody solution containing TIM-3.

[0040] Through Ni 2+ The single-domain antibody obtained in this example was purified using a Sepharose Excel affinity chromatography column and the AKATA protein purification system, followed by SDS-PAGE purification and identification. The results are as follows: Figure 10 As shown, this embodiment successfully obtained purified single-domain antibody protein targeting TIM-3.

[0041] The purified single-domain antibody protein was further identified using 293T. vector and 293T TIM-3 The cell line was used as the research object and identified by flow cytometry. The primary antibody was a purified single-domain antibody, and the secondary antibody was an APC-anti-His flow cytometry antibody (Biolegend). Results are as follows: Figure 11 As shown, 293T TIM-3 The cell line incubated with the single-domain antibody group showed a 99.1% deviation compared to the control group, indicating that the purified single-domain antibody can specifically bind to the TIM-3 protein on the membrane surface.

[0042] TIM-3 protein was coated onto ELISA plates. The primary antibody consisted of serially diluted single-domain antibody protein targeting TIM-3 / isotype control, and the secondary antibody consisted of HRP-labeled goat anti-alpaca antibody. Results were as follows: Figure 12 As shown, the ELISA results indicate that the equilibrium dissociation constant (KD) of the single-domain antibody is 1.96 nM.

[0043] Example 6: Application of single-domain antibodies targeting TIM-3 1. 68 Ga-NOTA-VHH TIM-3 Probe preparation (1) The N-terminus of the single-domain antibody that was identified as positive in Example 5 was linked to a cysteine ​​residue so that it could be coupled with NOA. The purification method of the modified single-domain antibody was the same as that in Example 5. Take 50 μg of purified single-domain antibody and dilute it to a total volume of 90 μL with reaction buffer. Add 10 μL of 100 mM TCEP solution to the above solution, gently pipette to mix, and incubate at 37°C for 1 hour or at room temperature (25°C) for 1.5 hours to completely reduce any disulfide bonds that may have formed, ensuring that the thiol groups of Cys are completely free and active. Add the reaction solution at a molar ratio of 5:1 (NOTA: single-domain antibody). Add the calculated Nota-maleimide DMSO solution (2 μL) directly to the unpurified reduced single-domain antibody solution (100 μL), gently vortex or flick the tube wall to mix thoroughly, seal the reaction tube tightly, wrap it with aluminum foil to protect it from light, and react at room temperature (25°C) on a shaker or rotary mixer for 2 hours. Take a PD-10 column, open the cap, let the stock solution flow out, and add 25 Rinse the column with 102 mL of purification / storage buffer (PBS, pH=7.4) to equilibrate it. Carefully add the entire mixture (approximately 102 μL) to the column packing material using a pipette. After the sample has completely entered the column bed, add 0.4 mL of PBS and discard the eluent. Then add another 1.5 mL of PBS and collect this eluent in a new 1.5 mL centrifuge tube. The resulting 1.5 mL liquid is the purified NOA-single-domain antibody conjugate. Perform LC-MS analysis on a small amount of the sample to confirm successful NOA conjugation (approximately 700 Da). Further identify the purified NOA-single-domain antibody conjugate by flow cytometry using the same method as in Example 5.

[0044] (2) Add 0.5M-1.0M HEPES buffer to the NOTA-single-domain antibody conjugate solution to precisely adjust the pH of the system to 3.8-4.5. Then, add the purified and pre-concentrated... 68 GaCl3 solution (dissolved in 0.1M HCl) was added to the reaction mixture. The reaction tube was sealed, and the mixture was vortexed until homogeneous. The mixture was then heated at 95°C for 10 minutes. After the reaction was complete, the mixture was cooled to room temperature to obtain the desired product. 68 Ga-NOTA-VHH TIM-3 Probe.

[0045] 2. Construction of A549 cell line overexpressing TIM-3 The recombinant plasmid pLVSIN-TIM-3-gcGFP was constructed and lentivirus was packaged, following the same steps and methods as in Example 4. The packaged virus solution was then used to infect the A549 cell line to construct the A549-TIM-3-gcGFP cell line. After construction, the results were detected using fluorescence microscopy and confocal microscopy; the results are as follows. Figure 13As shown, TIM-3 is expressed in almost all cells; subsequently, A549 cells were extracted. TIM-3 Cell line proteins and A549 cell line proteins were compared, with A549 protein as a control. The primary antibody used was a TIM-3 specific antibody (Proteintech), and the secondary antibody was an HRP-labeled goat anti-mouse secondary antibody (Thermo) against A549. TIM-3 Western spectroscopy was performed on the cell lines, and the results were as follows: Figure 14 As shown, the presence of the target band at 40kDa indicates that the cell line was successfully constructed.

[0046] 3.A549 TIM-3 cell lines 68 Ga-NOTA-VHH TIM-3 In vitro binding assay of probe A549 and A549 TIM-3 Cell lines were seeded at the same density in 24-well plates and incubated at 37°C with 5% CO2 for 24-48 hours. Experiments were performed when the cell density reached 80%-90% confluence. The experiments included A549 (wild-type) as a negative control group (TIM-3 non-expressing) and A549 as a negative control group. TIM-3 : TIM-3 overexpression experimental group; A549 TIM-3 +αTIM-3: TIM-3 antibody blocking group, used to confirm the specificity of binding.

[0047] A549 TIM-3 In the +αTIM-3 group, an excess of anti-TIM-3 antibody was added before probe incubation, and the cells were incubated at 37°C for 60 minutes to block the TIM-3 receptors on the cell surface; the count for each group was 2 × 10⁻⁶. 5 The experiment was conducted using individual cells, with each group receiving an injection containing the same level of radioactivity. 68 Ga-NOTA-VHH TIM-3 Fresh culture medium for the probe; cells were returned to the incubator and incubated for 30 min, 60 min, and 120 min, respectively, with radioactivity measured at different time points. Results are as follows: Figure 15 As shown, it indicates 68 Ga-NOTA-VHH TIM-3 The probe can be used with A549 TIM-3 Cell line specific binding.

[0048] 4. 68 Ga-NOTA-VHH TIM-3 PET / CT imaging of probes in mouse tumor models A549 and A549 were subcutaneously injected into the right back of nude mice (purchased from Liaoning Changsheng Biotechnology Co., Ltd.) respectively. TIM-3 Cells (5×10) 6(1 mouse per tumor), until the tumor grows to approximately 100-300 mm. 3 The experiment was conducted at different sizes. An experimental group was established: inoculated with A549. TIM-3 Cells (n=4); Negative control group: inoculated with A549 cells (n=3); TIM-3 antibody blocking group: mice in the blocking group were intraperitoneally injected with a certain dose of unlabeled anti-TIM-3 protein (n=4) 6 hours before the injection of the radioactive probe. All mice were injected with the same radioactive activity via the tail vein. 68 Ga-NOTA-VHH TIM-3 Probe; at predetermined time points (0.5 hours, 1 hour, 2 hours) after probe injection, mice were anesthetized and placed in a small animal PET scanner for scanning.

[0049] The results are as follows Figure 16-19 As shown, the tumor sites are circled in red ellipses. The results indicate that the tumors in the experimental group appear as clear "hot spots" on PET images, and the quantitative analysis (%ID / g) is significantly higher than that in the A549 group. This suggests that the probe can efficiently accumulate in tumors with high TIM-3 expression. The radioactive uptake of the tumors in the TIM-3 antibody blocking group is significantly lower than that in the unblocked experimental group. This indicates that the accumulation of the probe in the tumor is a specific process mediated by the TIM-3 receptor. 68 Ga-NOTA-VHH TIM-3 Probe uptake peaked at 1 hour, then slightly decreased or plateaued at 2 hours, indicating rapid and stable probe binding to the target. (Proof) 68 Ga-NOTA-VHH TIM-3 The probe can efficiently and specifically target tumors with high TIM-3 expression, and can also be rapidly cleared by the kidneys with a good target / background ratio.

[0050] Therefore, the single-domain antibody targeting TIM-3 provided by the present invention can specifically bind to the TIM-3 protein on the cell membrane surface and can be used as a detection and therapeutic antibody.

[0051] The contents not described in detail in this specification are well-known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A single-domain antibody targeting TIM-3, characterized in that, The amino acid sequence of the single-domain antibody targeting TIM-3 is shown in SEQ ID NO.

1.

2. The single-domain antibody targeting TIM-3 according to claim 1, characterized in that, The single-domain antibody targeting TIM-3 includes complementarity-determining regions CDR1, CDR2, and CDR3.

3. The single-domain antibody targeting TIM-3 according to claim 2, characterized in that, The amino acid sequence of CDR1 is shown in SEQ ID NO.2, the amino acid sequence of CDR2 is shown in SEQ ID NO.3, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

4.

4. The single-domain antibody targeting TIM-3 according to claim 1, characterized in that, The single-domain antibody targeting TIM-3 also includes the framework regions FR1, FR2, FR3, and FR4.

5. The single-domain antibody targeting TIM-3 according to claim 4, characterized in that, The amino acid sequence of FR1 is shown in SEQ ID NO.5, the amino acid sequence of FR2 is shown in SEQ ID NO.6, the amino acid sequence of FR3 is shown in SEQ ID NO.7, and the amino acid sequence of FR4 is shown in SEQ ID NO.

8.

6. A nucleic acid, characterized in that, The nucleic acid includes a nucleic acid sequence encoding a single-domain antibody targeting TIM-3 as described in any one of claims 1 to 5.

7. An expression carrier, characterized in that, The expression vector contains the nucleic acid as described in claim 6.

8. A host cell, characterized in that, The host cell contains the expression vector as described in claim 7.

9. A radionuclide probe for detection, characterized in that, The radionuclide probe contains a single-domain antibody targeting TIM-3 as described in any one of claims 1 to 5.

10. The use of the single-domain antibody targeting TIM-3 according to any one of claims 1 to 5 in the preparation of in vivo imaging probes, in vitro detection probes, or therapeutic antibodies, characterized in that, The application refers to the detection of tissues or cells expressing TIM-3.

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