An anti-pd-l1 nanobody and an asymmetric trispecific antibody comprising the same, and a preparation method and application thereof

Abstract

The application discloses an anti-PD-L1 nanobody, an asymmetric trispecific antibody containing the same, and a preparation method and application thereof, and belongs to the technical field of biological medicines. The nanobody contains a CDR region as shown in SEQ ID NO. 1-3 or 8-10, the affinity of which is 7.91 nM and 44.2 nM respectively, and the activity is still maintained after being treated at 80 DEG C for 2 hours. The application further constructs an asymmetric trispecific antibody C6-A1-X1, the structure of which is that the N terminals of two chains are respectively targeted to B7-H3 and PD-L1, and the C terminals are commonly targeted to LAG3, the expression amount of the antibody reaches 670.47 mg / L, the purity is greater than 94%, the affinity of the antibody with B7-H3, PD-L1 and LAG3 is 3.40 nM, 9.83 nM and 75 pM respectively, and the half-life is as long as 6.2 hours due to the bivalent combination of LAG3. The antibody can be used for tumor treatment, diagnosis and molecular imaging.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and particularly relates to an anti-PD-L1 nanobody and an asymmetric trispecific antibody containing it, as well as its preparation method and application. Background Technology

[0002] PD-L1 is a key immune checkpoint molecule in tumor immunotherapy, widely expressed on the surface of various cells, including tumor cells and antigen-presenting cells and macrophages in the tumor microenvironment. It binds to the PD-1 receptor on the surface of T cells, transmitting an inhibitory signal that weakens the immune response of T cells, thereby helping tumors evade recognition and clearance by the immune system. Clinically, the expression level of PD-L1 has become an important biomarker for determining whether patients with various solid tumors, such as non-small cell lung cancer and melanoma, are suitable for PD-1 / PD-L1 inhibitor therapy. Currently, the mainstream treatment is traditional monoclonal antibodies, which restore the immune activity of T cells by blocking the binding of PD-1 / PD-L1, and have achieved significant clinical efficacy.

[0003] However, traditional IgG antibodies still face several clinical challenges due to their molecular structure: First, their large molecular weight (approximately 150 kDa) limits their penetration into solid tumors, making it difficult to cross the dense matrix barrier and resulting in uneven drug distribution within the tumor, affecting the immune activation effect on deep tumor cells; second, antibodies may bind off-target to PD-L1, which is expressed at low levels in non-tumor tissues, triggering immune-related adverse events such as interstitial pneumonia and colitis, thus limiting treatment safety; in addition, traditional antibody preparation processes are complex and costly, and they require intravenous infusion, which restricts patient compliance and ease of use.

[0004] In recent years, nanobodies, as a novel form of antibody, have shown great potential to overcome the aforementioned bottlenecks. Derived from the variable region of heavy chain antibodies naturally lacking the light chain in camel-like animals, their molecular weight is only about one-tenth that of traditional antibodies (approximately 15 kDa), possessing unique structural and functional advantages: their smaller molecular size endows them with stronger tissue penetration capabilities, enabling more uniform distribution deep within solid tumors and achieving more effective immune pathway blocking; simultaneously, although their antigen-binding domain has a simple structure, it exhibits high affinity and high specificity, helping to reduce off-target risks and improve treatment safety; furthermore, nanobodies also have advantages such as high stability, good solubility, ease of genetic engineering modification and production, lower production costs, and suitability for development into various dosage forms such as subcutaneous injection and intratumoral local administration, which helps improve patient convenience and compliance.

[0005] Despite reports of anti-PD-L1 nanobodies, existing technologies still have the following shortcomings: single-target nanobodies are difficult to overcome the complexity of tumor immune escape; the construction methods of multispecific antibodies are complex, with low heterodimerization efficiency and low expression levels; in particular, how to design structurally stable and functionally synergistic multispecific antibodies remains a technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0006] To address the above technical problems, this invention provides a novel anti-PD-L1 nanobody and an asymmetric trispecific antibody constructed based on this nanobody, thereby solving the technical problems of poor penetration, limited efficacy of single-target antibodies, and low construction efficiency of multispecific antibodies in traditional antibodies. The specific solution is as follows: This invention provides a nanobody that specifically binds to PD-L1 and includes a complementarity-determining region (CDR) selected from the group consisting of: (a) CDR1 as shown in SEQ ID NO.1, CDR2 as shown in SEQ ID NO.2, and CDR3 as shown in SEQ ID NO.3; or (b) CDR1 as shown in SEQ ID NO.8, CDR2 as shown in SEQ ID NO.9, and CDR3 as shown in SEQ ID NO.10; Or a variant thereof, wherein the variant has at least 90% sequence identity with the above-described CDR sequence and retains binding activity with PD-L1.

[0007] Preferably, the backbone region (FR) sequence of the nanobody is selected from: (a) FR1, FR2, FR3, and FR4 as shown in SEQ ID NO. 4, 5, 6, and 7; or (b) FR1, FR2, FR3, and FR4 as shown in SEQ ID NO. 11, 12, 13, and 14; Or a variant thereof, wherein the variant has at least 90% sequence identity with the above-described FR sequence.

[0008] In a preferred embodiment, the nanobody is: (a) The amino acid sequence PL-A1 as shown in SEQ ID NO.15; or (b) The amino acid sequence PL-A2 as shown in SEQ ID NO.16; Or its functionally active fragments or derivatives.

[0009] The present invention also provides a polynucleotide encoding the above-mentioned nanobody, preferably comprising the nucleotide sequence shown in SEQ ID NO.17 or SEQ ID NO.18.

[0010] The present invention also provides an expression vector comprising the above-mentioned polynucleotides, and a host cell comprising the expression vector.

[0011] The present invention also provides a method for preparing the above-mentioned nanobody, comprising the steps of culturing the host cells and isolating and purifying the nanobody from the culture.

[0012] The present invention also provides a multispecific antibody comprising at least one of the above-described nanobody and one or more additional antigen-binding domains.

[0013] The present invention also provides an asymmetric trispecific antibody comprising: (a) A first polypeptide chain, which comprises, from the N-terminus to the C-terminus, a first antigen-binding domain that specifically binds to a first target, a first dimerizing domain, a first linker peptide, and a third antigen-binding domain that specifically binds to a third target. (b) A second polypeptide chain, which, from the N-terminus to the C-terminus, comprises, in sequence: a second antigen-binding domain that specifically binds to a second target, a second dimerizing domain, a second linker peptide, and a third antigen-binding domain that specifically binds to a third target; Wherein, at least one of the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain is the aforementioned nanobody, the first dimerization domain and the second dimerization domain combine with each other to form a dimer, and the third antigen-binding domain exists simultaneously at the C-terminus of the first polypeptide chain and the second polypeptide chain, thereby achieving bivalent binding to the third target.

[0014] Preferably, the first antigen-binding domain is a nanobody that specifically binds to B7-H3, the second antigen-binding domain is the aforementioned nanobody that specifically binds to PD-L1, and the third antigen-binding domain is a nanobody that specifically binds to LAG3. This asymmetric design of "N-terminal dual targets + C-terminal common target" enables the antibody molecule to simultaneously target tumor cells (B7-H3), the immunosuppressive pathway (PD-L1), and effector cell regulation (LAG3), and the blocking effect on the LAG3 pathway is enhanced by the C-terminal bivalent LAG3 nanobody.

[0015] In one specific embodiment, the first antigen-binding domain comprises the C6 nanobody shown in SEQ ID NO.21, the second antigen-binding domain comprises the PD-L1 nanobody shown in SEQ ID NO.15 or SEQ ID NO.16, and the third antigen-binding domain comprises the X1 nanobody shown in SEQ ID NO.25.

[0016] Preferably, the first and second dimerizing domains are immunoglobulin Fc regions or functionally active fragments thereof. More preferably, the first and second Fc regions contain modifications that promote heterodimer formation, said modifications being selected from the group consisting of: spatial complementarity modifications, charge complementarity modifications, chain exchange modifications, or combinations thereof.

[0017] Preferably, the first linker peptide and the second linker peptide are independently selected from (G4S)n, where n is an integer from 1 to 5, preferably 3.

[0018] The present invention also provides a pharmaceutical composition comprising the above-described nanobody or the above-described asymmetric trispecific antibody, and a pharmaceutically acceptable carrier.

[0019] The present invention also provides the application of the above-mentioned nanobodies, asymmetric trispecific antibodies or polynucleotides in the preparation of drugs for the treatment of tumors or the detection of PD-L1 expression.

[0020] The present invention also provides a detection kit comprising the above-mentioned nanobody or the above-mentioned asymmetric trispecific antibody.

[0021] The present invention also provides a fusion protein comprising the above-mentioned nanobody and a heterologous protein domain, preferably an Fc domain, a toxin protein, a cytokine, or a fluorescent protein.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) Excellent nanobody performance The nanobodies PL-A1 and PL-A2 obtained by screening in this invention have high affinity (KD of 7.91 nM and 44.2 nM, respectively) and exhibit excellent thermal stability, maintaining their binding activity with PD-L1 even after treatment at 80°C for 2 hours, which is significantly superior to traditional monoclonal antibodies. This high thermal stability is beneficial for antibody production, storage, and transportation, reducing cold chain costs.

[0023] (2) Unique “2+1” asymmetric trispecific antibody structure design This invention is the first to construct an IgG-like trispecific antibody with a "2+1" asymmetric structure. The N-terminus of each of the two polypeptide chains carries an antigen-binding domain targeting different targets (the first and second targets), while the C-terminus of both chains shares an antigen-binding domain targeting the same target (the third target), forming an asymmetric distribution of "two different targets at the N-terminus + one common target at the C-terminus." This design allows a single antibody molecule to simultaneously recognize three different targets, and the C-terminal target achieves bivalent binding, enhancing the blocking effect against key immune checkpoints.

[0024] The C6-A1-X1 antibody of this invention comprises, at its N-terminus, B7-H3 nanobody C6 (targeting tumor cells) and PD-L1 nanobody A1 (blocking the PD-1 / PD-L1 immunosuppressive pathway), respectively, and at its C-terminus, bivalently fused with LAG3 nanobody X1 (enhancing the blocking of the LAG3 immune checkpoint). This design achieves a triple function of "tumor targeting + immune activation + effector cell regulation." SPR assays show that the affinity for all three targets reaches therapeutic levels (B7-H3: 3.40 nM, PD-L1: 9.83 nM, LAG3: 75 pM), and the bivalent binding to LAG3 results in a dissociation half-life of up to 6.2 hours, demonstrating sustained target occupancy and potentially overcoming primary or acquired resistance to single PD-1 / PD-L1 blockade therapies.

[0025] (3) Excellent production performance The asymmetric trispecific antibody of this invention exhibits an expression level as high as 670.47 mg / L in the HEK293 expression system, with a purity >94%, far exceeding the expression levels of traditional multispecific antibodies (typically 50-200 mg / L). This high expression level, combined with the simplified purification process of nanobodies, can significantly reduce production costs and improve drug accessibility.

[0026] (4) Compatibility of multiple heterodimerization technologies The asymmetric trispecific antibody of this invention can be constructed using various heterodimerization techniques, including spatial complementarity modification (such as knots-into-holes), charge complementarity modification (such as charge pairs), chain exchange modification (such as SEED bodies), and combinations thereof. Those skilled in the art can select appropriate modification methods according to actual needs, demonstrating the flexibility and universality of the technical solution of this invention.

[0027] (5) Excellent target binding properties The asymmetric trispecific antibody C6-A1-X1 of this invention maintains high affinity for all three targets, particularly with LAG3, achieving a binding affinity of 75 pM and a dissociation half-life of 6.2 hours, demonstrating sustained target occupancy and potentially achieving longer-lasting efficacy. Simultaneously, all binding domains retain their original affinity, proving that the asymmetric structural design of "N-terminal dual targets + C-terminal common target" does not affect the functional activity of each antigen-binding domain.

[0028] (6) Broad clinical application prospects The nanobodies and asymmetric trispecific antibodies of this invention can not only be used for systemic drug delivery to treat various solid tumors, but can also be developed into various drug delivery forms such as intratumoral injection formulations and local sustained-release formulations, which are particularly suitable for dense solid tumors such as brain tumors and pancreatic cancers that are difficult for traditional antibodies to penetrate. At the same time, they can be used for in vitro diagnosis of PD-L1 expression and in vivo molecular imaging, realizing integrated diagnosis and treatment.

[0029] (7) Scalability of platform technology The nanobody screening and asymmetric trispecific antibody construction platform established in this invention can be rapidly extended to other target combinations, providing a technical foundation for the development of next-generation multispecific tumor immunotherapy drugs. Attached Figure Description

[0030] Figure 1 This is a graph showing the titer of alpaca (Vicugna pacos) immune serum in Example 1. The horizontal axis represents the serum dilution factor, and the vertical axis represents the corresponding absorbance OD450 value.

[0031] Figure 2 The results of agarose gel electrophoresis of total RNA from alpaca PBMCs in Example 1 are shown.

[0032] Figure 3 The results of colony PCR agarose gel electrophoresis in Example 1 are shown.

[0033] Figure 4 This is the result of sequence diversity alignment in the Chinese library in Example 1.

[0034] Figure 5 The results of positive clone identification by sandwich phage ELISA in Example 1 are shown.

[0035] Figure 6 This is an SDS-PAGE protein electrophoresis image of PD-L1 nanobodies PL-A1 and PL-A2 from Example 1. In the image, lane M: molecular weight marker; lane 1: nanobodies PL-A2; lane 2: nanobodies PL-A1.

[0036] Figure 7 The figures show the binding and dissociation kinetics curves of nanobodies PL-A1, PL-A2, and PD-L1 protein in Example 2. A. Binding and dissociation kinetics curve of nanobodies PL-A1 and PD-L1 protein; B. Binding and dissociation kinetics curve of nanobodies PL-A2 and PD-L1 protein.

[0037] Figure 8 The figure shows the thermal stability test results of PD-L1 nanobodies PL-A1 and PL-A2 in Example 3.

[0038] Figure 9This is an SDS-PAGE protein electrophoresis image of the asymmetric trispecific nanobody C6-A1-X1 from Example 4. Lane M: molecular weight marker; Lane R: molecular weight of the fusion protein in the reduced state (68.503 kDa); Lane NR: molecular weight of the fusion protein in the non-reduced state (137.44 kDa). The structural characteristics of this antibody are: the first polypeptide chain (Knob chain) has a B7-H3 nanobody C6 at its N-terminus and a LAG3 nanobody X1 at its C-terminus; the second polypeptide chain (Hole chain) has a PD-L1 nanobody A1 at its N-terminus and a LAG3 nanobody X1 at its C-terminus. X1 is fused to the C-terminus of both chains, achieving bivalent binding to LAG3.

[0039] Figure 10 The image shows the SEC-HPLC chromatogram of the trispecific nanobody C6-A1-X1 in Example 4, along with the peak values ​​at 214 nm and 280 nm. The main peak accounts for 94.732%, indicating high purity of the fusion protein, which mainly exists in the form of correctly folded antibody monomers. Detailed Implementation

[0040] Example 1: Preparation of PD-L1 nanobodies This embodiment provides a novel PD-L1 nanobody, the preparation method of which includes the following steps: 1. Construction of Immunized Alpaca and Phage Display Libraries (1) Immunize alpacas: 1 mg of PD-L1 protein (Acro, PD1-H5223) expressed in HEK293 eukaryotic cells was emulsified with Freund's complete adjuvant, totaling 2 mL, and administered as the first immunization to a healthy adult alpaca via multiple subcutaneous injections. On day 15, 0.5 mg of PD-L1 protein was emulsified with Freund's complete adjuvant, totaling 2 mL, and administered as the second immunization via multiple subcutaneous injections. Subsequent immunizations were performed every 15 days using 0.5 mg of PD-L1 protein emulsified with Freund's incomplete adjuvant, totaling 2 mL. A total of four immunizations were administered. Peripheral blood was collected on the day of each immunization and on day 7 after the last immunization to determine the titer.

[0041] Figure 1 This is a graph showing the titer of immunized alpaca (Vicugna pacos) serum. A positive / negative serum ratio ≥2.1 is considered positive. The resulting alpaca serum titer reached 1:1024000, confirming the production of high-titer specific antibodies in the alpaca and providing a high-quality source of B cells for subsequent library construction.

[0042] (2) Total RNA extraction and VHH gene amplification: Peripheral blood was collected from immunized alpacas, and lymphocytes were isolated. Total RNA was extracted from the lymphocytes using the TRIzol method or a commercial RNA extraction kit. Electrophoresis results are shown below. Figure 2 As shown in Table 1, the total RNA volume and detection results are as follows. Figure 2 Two clear major ribosomal RNA bands, 28S rRNA and 18S rRNA, were observed in the lane. The 28S band was approximately twice as bright as the 18S band (28S:18S ≈ 2:1), indicating that the total RNA was of good integrity and high purity, meeting the requirements for subsequent experiments.

[0043] Table 1 Total RNA volume and detection results

[0044] Using extracted total RNA as a template, cDNA first strand was synthesized via reverse transcription using Oligo(dT) primers. To obtain diverse heavy chain antibody variable region (VHH) genes, specific primer pairs (such as HS / Hanti2 and HS / Hanti1, sequences shown in Table 7) targeting alpaca heavy chain antibody IgG2 and IgG3 subtypes were designed for PCR amplification. The PCR amplification system was as follows: 2 μL cDNA; 1.3 μL HS primer; 1.3 μL Hanti1 or Hanti2 primer; 44.4 μL Taq Enzyme Mix; reaction program: 94℃, 3 min; 94℃, 30 s, 55℃, 30 s, 72℃, 1 min, 32 cycles; 72℃, 5 min. The optimized PCR amplification program effectively enriched the target band of approximately 500 bp. By expanding the PCR reaction system (a total of 288 reactions), sufficient VHH gene fragments were obtained. The DNA in the PCR reaction solution was purified and recovered using a universal DNA purification and recovery kit (TIANGEN, model DP214-03). The recovered DNA volume was 1200 μL, the concentration was 82 ng / μl, and the total amount was 98.4 μg.

[0045] (3) Phage vector processing and VHH gene library construction: To prepare a compatible linearized vector, the SfiI restriction endonuclease, sensitive to DNA methylation, was selected. Therefore, during vector amplification, a methylation-deficient *E. coli* strain (e.g., C2925) was used to extract pComb3X phage plasmids to ensure SfiI digestion efficiency. The extracted pComb3X plasmid was fully digested with SfiI, and the large vector backbone fragment was recovered by agarose gel electrophoresis. The digestion reaction system consisted of 1 μg pComb3X plasmid, 4 μL SfiI enzyme, 4 μL CutSmart Buffer, and ultrapure water / deionized water to a final volume of 50 μL. Digestion was carried out at 50°C for 12 h. The next day, the digestion product was electrophoresed, and the large fragment was recovered by gel extraction. A total of 11 μg of vector was digested, and 7.5 μg of the large vector fragment was recovered at a concentration of 50 ng / μl.

[0046] The recovered VHH gene fragment was ligated to the linearized pComb3X vector backbone using homologous recombination (instead of the traditional T4 ligase method). The homologous recombination system consisted of 200 ng of pComb3X vector backbone, 60 ng of VHH gene fragment, 5 μL of 2×ClonExpress Mix, and ultrapure deionized water to a final volume of 10 μL. The reaction was carried out at 50°C for 5 minutes, followed by immediate cooling on ice. This strategy leverages the high efficiency of homologous recombination enzymes, significantly improving ligation efficiency and reducing the background of vector self-ligation. After purification, the ligation product was transformed into *E. coli* ER2738 competent cells via electroporation. The calculated original library size (Table 2) reached 1.4 × 10⁻⁶. 9 With a pfu level of above, randomly selected clones were subjected to colony PCR verification, and the positive rate was as high as 96%. Figure 3 Furthermore, sequencing results showed that the inserted fragment exhibited good sequence diversity. Figure 4 ).

[0047] Table 2. Detection Indicators for Bacterial Library

[0048] (4) Packaging and rescue of phage libraries The constructed bacterial culture was expanded to the logarithmic growth phase, and 10... 12 PFU M13KO7 helper phage was used for infection. After static infection for 30 min, the medium was changed and kanamycin (final concentration 70 μg / mL) was added for resistance selection, followed by overnight culture. The next day, the culture supernatant was collected by centrifugation (13000 rpm, 4℃, 10 min), and phage particles were precipitated using PEG / NaCl solution. The precipitate was resuspended in a protective buffer containing 1× protease inhibitor, 0.02% sodium azide, and 0.5% BSA. After sterilization by filtration through a 0.22 μm filter, a high-titer phage display library was obtained, with a titer of 6.8 × 10⁻⁶. 13pfu / ml can be used for subsequent affinity screening.

[0049] 2. Affinity screening and identification of PD-L1-targeting nanobodies (1) Liquid phase panning strategy: To fully expose the antigenic epitopes, a biotin-streptavidin-mediated liquid-phase panning method was employed. First, the biotinylated PD-L1 antigen was co-incubated with streptavidin magnetic beads to form an "antigen-magnetic bead" complex. Simultaneously, the prepared phage library was mixed with blocking buffer (3% BSA for the first round of screening, 3% skim milk for the second round, with alternating use of 3% BSA and 3% skim milk) to remove non-specific phages that might bind to the magnetic beads or proteins.

[0050] The blocked phage library was incubated with the antigen-magnetic bead complex at 4°C with gentle shaking (150-160 rpm) to allow specific phages to bind to the antigen. The magnetic beads were separated using a magnetic rack and washed repeatedly with TBST to remove non-specifically bound phages. Finally, the bound phages were eluted using an acidic elution buffer (e.g., glycine-hydrochloric acid, pH 2.2). The eluent was centrifuged at low speed (2000-3000 rpm) for 30 seconds, and the supernatant was immediately neutralized with Tris-HCl (pH 9.1) to obtain the first round of screening products.

[0051] (2) Multi-round screening and enrichment: The phages eluted in the first round were used to infect ER2738 bacteria in the logarithmic growth phase, and then amplified and purified to serve as the input library for the next round of screening. In the subsequent two rounds of screening, the amount of biotinylated antigen was gradually reduced (from 30 μL to 1.5 μL) and the washing intensity was increased to enrich high-affinity specific clones. After three rounds of panning, the phage yield / input ratio significantly improved, indicating that specific phages were effectively enriched.

[0052] The results of the three rounds of screening are shown in Table 3 below. The enrichment level is calculated by dividing the screening titer by the input titer; a higher value indicates a higher enrichment level of the antibody in the input library. The fold difference is calculated by dividing the screening titer by the control titer; a higher value indicates a higher concentration of positive antibodies in the screening library. The third screening result shows that after three rounds of screening, the enrichment level was 1.0 × 10⁻⁶. -5 The difference is 13.3 times, so monoclonal ELISA verification can be performed first.

[0053] Table 3 Results of the three rounds of screening

[0054] h. Select 51 clones from the plates after three rounds of screening for sandwich Phage Elisa identification of positive clones.

[0055] (3) Identification of positive clones: Single clones were randomly selected from the plates after the third round of screening and inoculated into 96-well deep-well plates for culture. Phages in the culture supernatant were directly used for Phage ELISA detection. The specific steps were: coating the ELISA plate with purified alpaca IgG, then adding PD-L1 protein, and finally detecting with HRP-labeled anti-M13 antibody. After screening, the positive clone rate exceeded 80%. Figure 5 ).

[0056] 3. Expression, purification, and performance validation of nanobodies (1) Construction and transformation of expression carriers: Positive monoclonal plasmids identified by ELISA were extracted, and their coding sequences were obtained by sequencing. The VHH gene with the correct sequence was subcloned into the pPICZαA vector containing a histidine tag. The recombinant plasmid was transformed into the suitable expression host strain, Pichia pastoris X33.

[0057] (2) Induced expression and affinity purification: Positive transformants were selected and cultured. They were added to 30 ml of BMGY medium and cultured at 30°C with shaking (250 rpm) until the OD600 value was approximately 2-4. The cells were centrifuged at 3000 rpm for 5 min, collected, and resuspended in 5 ml of autoclaved water. The pellet was centrifuged at 3000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in 3 mL of BMMY medium, transferred to BMMY medium, and inducing expression at 30°C and 250 rpm. The supernatant was collected, added to a nickel column, and incubated at 4°C for 3-6 h. Four column volumes were washed with 20 mmol / L imidazole, and 5 mL of 50 mmol / L and 100 mmol / L imidazole wash buffer were collected respectively to obtain the nanobody.

[0058] The PAGE electrophoresis results of the nanobody are as follows: Figure 6 As shown in the figure, the molecular weight of the nanobodies PL-A2 and PL-A1 prepared in this invention is approximately 15 kDa.

[0059] The positive clones obtained in step (4) are selected for sequencing to obtain the nucleotide sequence of the nanobody, and then the amino acid sequence of the nanobody is obtained according to the codon table.

[0060] The nanobody is composed of a variable region of a heavy chain antibody; the variable region of the heavy chain antibody includes an antigenic determinant complementary region and a backbone region; the backbone region is selected from FR1, FR2, FR3 and FR4 and the group consisting of amino acid sequences having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% identity with them; the antigenic determinant complementary region is selected from CDR1, CDR2 and CDR3 and the group consisting of amino acid sequences having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% identity with them.

[0061] Specifically, in this invention: The amino acid sequences of CDR1 of nanobody PL-A1 are shown in SEQ ID NO.1, CDR2 in SEQ ID NO.2, and CDR3 in SEQ ID NO.3; the amino acid sequence of FR1 is shown in SEQ ID NO.4; the amino acid sequence of FR2 is shown in SEQ ID NO.5; the amino acid sequence of FR3 is shown in SEQ ID NO.6; and the amino acid sequence of FR4 is shown in SEQ ID NO.7.

[0062] The amino acid sequences of CDR1 of nanobody PL-A2 are shown in SEQ ID NO.8, CDR2 in SEQ ID NO.9, and CDR3 in SEQ ID NO.10; the amino acid sequence of FR1 is shown in SEQ ID NO.11; the amino acid sequence of FR2 is shown in SEQ ID NO.12; the amino acid sequence of FR3 is shown in SEQ ID NO.13; and the amino acid sequence of FR4 is shown in SEQ ID NO.14.

[0063] The amino acid sequences of PL-A1 and PL-A2 are shown in SEQ ID NO.15 and SEQ ID NO.16.

[0064] The details of each sequence are as follows: ① CDR1 (SEQ ID NO.1) of nanobody PL-A1: GHDFSNY CDR2 of nanobody PL-A1 (SEQ ID NO.2): TWIGGS CDR3 (SEQ ID NO.3) of nanobody PL-A1: ARVLGWGVQVLFRTNPADFGS FR1 of nanobody PL-A1 (SEQ ID NO.4): DVQLQESGGGLVQAGGSLRLS CAAS FR2 of nanobody PL-A1 (SEQ ID NO.5): AMGWFRQAPGKEREFVAVI FR3 of nanobody PL-A1 (SEQ ID NO.6): TYYADSVKGRFTISRDNAKNT LYLQMNNLKPEDT AV YYCAA FR4 of nanobody PL-A1 (SEQ ID NO.7): WGQGTQVTVS Nanobody PL-A1 (SEQ ID NO.15): DVQLQESGGGLVQAGGSLRLSCAASGHDFSNYAMGWFRQAPGKEREFVAVITWIGGSTYYADSVKGRFTISRDNAKNTLYLQMNNLKPEDTAVYYCAARVLGWGVQVLFRTNPADFGSWGQGTQVTVSS Nucleotide sequence of nanobody PL-A1 (SEQ ID NO. 17): GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACACGACTTCAGTAACTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGTTATTACCTGGATTGGTGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAACCTGAAACCTGAGGACACGGCCGTGTATTACTGTGCGGCACGGGTCCTCGGCTGGGGTGTTCAGGTTCTCTTCCGCACGAACCCCGCTGACTTTGGTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGC ② CDR1 of nanobody PL-A2 (SEQ ID NO.8): G F T F H Y Y CDR2 of nanobody PL-A2 (SEQ ID NO.9): S S S D G S CDR3 of nanobody PL-A2 (SEQ ID NO.10): D G W R L C R A D Y Y D G M D Y FR1 of nanobody PL-A2 (SEQ ID NO.11): D V Q L Q E S G G G L V Q P G G S L R L S C A A S FR2 of nanobody PL-A2 (SEQ ID NO.12): A I G W F R Q A P G K E R E G V S C I FR3 of nanobody PL-A2 (SEQ ID NO.13): T Y Y A D S V K G R F T V S R D N A K N T V Y L Q M N S L K P E D T AV Y Y C A A FR4 of nanobody PL-A2 (SEQ ID NO.14): WGKGTQVTVSS Nanobody PL-A2 (SEQ ID NO.16): DVQLQESGGGLVQPGGSLRLSCAASGFTFHYYAIGWFRQAPGKEREGVSCISSDGSTYYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCAADGWRLCRADYYDGMDYWGKGTQVTVSS Nucleotide sequence of nanobody PL-A2 (SEQ ID NO.18): GATGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCCATTATTATGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGGTCTCATGTATTAGTAGTAGTGATGGTAGCACATACTATGCAGACT CCGTGAAGGGCCGATTCACCGTCTCCAGAGACAACGCCAAGAACACGGTCTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTTTATTACTGTGCAGCGGACGGGTGGCGACTATGCCGAGCCGATTATTACGACGGCATGGACTACTGGGGCAAAGGGACCCAGGTCACCGTCTCCAGC Example 2: Affinity Detection of PD-L1 Nanobodies (1) Procedure: Preparation of reagents and consumables: Prepare Q Buffer (PBS + 0.02% Tween 20 + 0.2% BSA). Dilute the nanobody to be detected with Q Buffer to different concentrations: 7.813 nM, 15.625 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM. Prepare sample protein PD-L1 (10 μg / mL). Check the integrity of the Anti-His probe (Gator Bio, catalog number 160009) to avoid air bubbles. Label the 96-well plate (Gator Bio, catalog number 130150) according to the experimental design layout.

[0065] (2) Probe installation and identification: Take the probe out of the packaging and hold the white base part; insert the probe vertically into the probe slot of the instrument and hear a "click" sound. Select the probe type as "Anti-His" in the software.

[0066] (3) The start-up experiment is shown in Table 4 below.

[0067] Table 4 Experiment Start-up Procedure

[0068] The dynamic data are shown in Table 5 below.

[0069] Table 5 Affinity of PD-L1 nanobodies

[0070] Results analysis: The values ​​of koff, kon, and KD were consistent across all concentrations, indicating good experimental stability. R 2 The values ​​are generally high (>0.95), indicating that the fitting curves agree well with the experimental data. Relatively speaking, the affinity of nanobody PL-A1 is higher than that of nanobody PL-A2. The binding and dissociation kinetics curves of the two nanobodies with PD-L1 are shown below. Figure 7 As shown.

[0071] Example 3: Thermal stability experiment of nanobody PD-L1 protein was coated onto ELISA plates. 100 μL of 1 μg / mL PD-L1 protein was added to each well, and the plate was incubated overnight at 4°C. After washing three times with PBST, 300 μL of 5% skim milk was added to each well, and the plate was blocked at 37°C for 1 hour. 100 μL of the nanobody of this invention and a commercially available PD-L1 monoclonal antibody (PD-L1-mAb, Cell Signaling) were added to each well after being treated at different temperatures (4°C, 37°C, 60°C, 70°C, 80°C, 90°C) for 2 hours. After incubation at room temperature for 1 hour, the plate was washed three times with PBST.

[0072] For the nanobody PL-A1 and PL-A2 groups, because the nanobodies have His tags, HRP-labeled His-mAb (Cell Signaling, catalog number 12698) was added to each well, incubated at room temperature for 40 minutes, washed 3 times with PBST, added TMB for color development for 10 minutes, and the reaction was terminated with 2M sulfuric acid. The UV absorbance (OD450 value) at 450 nm was then measured using a microplate reader.

[0073] For the PD-L1 mAb group, HRP-labeled anti-rabbit IgG secondary antibody (Cell Signaling, catalog number 7074) was added, and the mixture was incubated at room temperature for 30 minutes. After washing the plate three times with PBST, TMB was added for color development for 10 minutes. The reaction was terminated with 2M sulfuric acid, and the UV absorbance (OD450 value) at 450 nm was measured using an ELISA reader.

[0074] The results are as follows Figure 8 As shown in the figure. The results show that the nanobody structure is stable, and nanobodies PL-A1 and PL-A2 still exhibit PD-L1 antigen binding activity after treatment at 80℃ for 2 hours. This indicates that compared with PD-L1-mAb, the nanobody structure of the present invention is stable and has good heat resistance.

[0075] Example 4: Construction of IgG-like asymmetric trispecific nanobody C6-A1-X1 containing nanobody PL-A1 This embodiment constructs a trispecific antibody with a “2+1” asymmetric structure, where “2” represents two different targets (B7-H3 and PD-L1) at the N-terminus of the two polypeptide chains, and “1” represents the same target (LAG3) carried by the C-terminus of the two polypeptide chains, which exists in a bivalent form.

[0076] A trispecific nanobody was constructed using nanobodies PL-A1 (PD-L1), C6 (B7-H3), and X1 (LAG3). The nanobodies with two N-terminal targets, C6 (B7-H3) and A1 (PD-L1), are located on two different polypeptide chains. The nanobodies with one C-terminal target, X1 (LAG3), are fused to the C-terminus of both Fc regions, achieving bivalent binding to LAG3. This asymmetric distribution of "dual N-terminal targets + common C-terminal target" allows a single molecule to recognize three different targets simultaneously, and the bivalent C-terminal target enhances the blocking effect on the LAG3 pathway, which is a unique feature of the inventor's design.

[0077] (1) Two heavy chain antibody gene sequences were synthesized. The first polypeptide chain sequence was designed as: signal peptide-C6-Fc(HumanIgG1Mutation1)-(G4S)3-X1. The second polypeptide chain sequence was designed as: signal peptide-A1-Fc(HumanIgG1Mutation2)-(G4S)3-X1. These two heavy chain antibody sequences were inserted into the NotI and XbaI multiple cloning sites of the eukaryotic vector pcDNA3.4 (Thermo Fisher Scientific, catalog number A14697), respectively. The vector was identified by enzyme digestion and gene sequencing, and the recombinant plasmids were named pcDNA3.4-C6-Fc-X1 and pcDNA3.4-A1-Fc-X1, respectively.

[0078] (SEQ ID NO.19):MGWSCIILFLVATATGVHS Report Ad (SEQ ID NO.20): GGCTGGTCTTGTATCATCCTGTTCCTGGTGGCTACAGCTACTGGAGTGCACAGC C60000000000000000000(SEQ ID NO.21): DVQLQESGGGLVQPGGSLRLSCAASGFTLDVYGIGWFRQAPGKEREGISCFVKDANVPYYADSVKGRFTVSTDNAKNTVYLQMNSLKPEDTAVYCARTSACVLLDGQPSHYGMDYWGKGGTQVTVSS C60000000000000000(SEQ ID NO.22): GATGTGCAGCTGCAGGAGAGCGGAGGAGGACTGGTTCAGCCTGGAGGAAGCCTGAGACTGAGCTGTGCCGCTAGCGGATTTACCCTGGACGTGTACGGGATTGGATGGTTTAGACAGGCCCCTGGCAAAGAAAGGGAGGGAATTTCTTGCTTTGTCAAGGATGCTAACGTGCCCTACTACGCCGATTCCG TGAAGGGCAGATTCCAGTGTCCACAGACAGCCAAGAACACAGTGTACCTGCAGATGAACAGCCCTGAAGCCCGAGGATACCGCCGTGTATTACTGCCCCCGGACCTCTGCATGCGTGCTGCTGGATGGTCAGCCCTCTCACTACGGAATGGACTACTGGGGGAAGGGAGCACCAGGAGGAGGAGCCTC Fc(Human IgG1 Mutation1)(SEQ ID NO.23): DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Fc (Human IgG1 Mutation1) Nucleotide (SEQ ID NO.24): GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA Amino acid sequence of Nanobody X1 (SEQ ID NO.25): QVQLQESGGGLVQPGGSLRLSCAASGFTLENYAIGWFRQAPGKEREGVSCISRSGGSTKYADSVKGRFTISRDHAKNTVFLQMNSLKPEDTAIYYCGKARDCTGPWGGSDYWGKGTQVTVSS Nucleotide sequence of Nanobody X1 (SEQ ID NO.26): CAAGTCCAACTCCAAGAGAGCGGCGGCGGACTGGTTCAACCAGGAGGATCACTCAGACTCTCTTGCGCCGCCAGCGGATTCACCCTGGAAAATTACGCCATCGGCTGGTTCAGACAGGCCCCTGGAAAAGAGAGAGAAGGAGTGAGCTGTATTAGCAGAAGCGGGGGGAGCACCAAATACGCTGACAGTGTGAAGGGCAGATTTACCATCAGCAGAGACCATGCCAAGAACACAGTGTTCCTGCAGATGAACAGCCTGAAGCCCGAGGACACCGCAATCTACTACTGCGGCAAGGCCCGGGACTGCACAGGACCTTGGGGAGGAAGTGACTACTGGGGCAAGGGGACACAAGTGACCGTGAGCAGC Amino acid sequence of Fc (Human IgG1 Mutation2) (SEQ ID NO.27): EPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Nucleotide sequence of Fc (Human IgG1 Mutation2) (SEQ ID NO.28): GAGCCCAAATCTGCCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTC AAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCC ATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCCAAGAACCAGGTCAGCCTGAGCTGCGCCGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGTCCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (G4S)3 (SEQ ID NO.29): GGGGSGGGGSGGGGS (2) Cell culture. HEK293T cells were revived and passaged 3 times. Culture conditions: 120 rpm, 8% CO2, 37℃, cell density controlled at 0.3 × 10⁻⁶. 6 Plasmid transfection was performed when the cell density reached 70% at a density of 1 cell / ml.

[0079] (3) Transfection and expression. Take 150 μL of Lipofectamine™ 3000 (Thermo Fisher Scientific, catalog number L3000075) and add it to 2.5 ml of Opti-MEM medium, which is named Solution 1; take 50 μg of plasmids pcDNA3.4-C6-Fc-X1 and pcDNA3.4-A1-Fc-X1, 200 μL of P3000 (Thermo Fisher Scientific, catalog number L3000075), and add them to 2.5 ml of Opti-MEM medium, which is named Solution 2. Add Solution 2 to Solution 1, mix well, incubate at 37°C for 15 minutes, and then add the mixed transfection solution dropwise to the cell culture medium while shaking. Place the mixture on a shaker at 37°C, 120 rpm, and 8% CO2 for 5-7 days. Collect the cell culture medium supernatant and centrifuge (15,000 g, 20-30 minutes) to remove cell debris and particulate matter to prevent clogging of the chromatography column.

[0080] (4) Protein A affinity chromatography purification of the trispecific nanobody C6-A1-X1. Equilibrate the Protein A column with 1×PBS phosphate buffer (pH 7.2-7.4). Wash the column with at least 20 column volumes of 1×PBS buffer at a flow rate of 1 mL / min until the UV baseline (280 nm absorbance) is completely stable. Replace the PBS with the sample to be purified and load the sample at a flow rate of 1 mL / min for 4 min to ensure that the antibody binds to the column. Then elute with 1×PBS to remove contaminating proteins at a flow rate of 1 mL / min for at least 10 column volumes of 1×PBS. Continue washing until the UV absorbance returns to the baseline level, indicating that non-specifically bound impurities have been washed away. At this point, only the target antibody that specifically binds to Protein A remains in the column. Elute the column with sodium acetate buffer at pH 3.4 at a flow rate of 1 mL / min for 5 min to ensure that the acidic buffer reacts fully with the column and that all antibodies are effectively eluted. Immediately after elution, collect small volumes (e.g., 0.5-1 mL / tube) of protein solution using centrifuge tubes. Rapidly measure the absorbance of each collected solution at 280 nm using a NanoDrop instrument. Transfer the high-concentration protein solution (elution peak merging tubes) into a pre-treated dialysis bag and seal it. Immerse the dialysis bag in a large volume (typically 200-1000 times the sample volume) of dialysis buffer (120 mM NaAc-HAc + 70 mM Arginine, pH 5.5). Stir slowly on a magnetic stirrer at 4°C. The external buffer needs to be changed several times (every 3-4 hours, for a total of 3-4 times) to ensure complete replacement. After dialysis, remove the protein solution, aliquot it, and store at -80°C.

[0081] (5) Detection of the trispecific nanobody C6-A1-X1. The protein concentration reached 8.21 mg / mL and the protein expression level reached 670.47 mg / L using NanoDrop. SDS-PAGE was used to detect... Figure 9 The molecular weight of the protein in the reduced state was 68.503 kDa, and the molecular weight of the protein in the non-reduced state was 137.44 kDa, consistent with the expected dimer structure. The purity of the fusion protein was determined by SEC-HPLC using an LC-20AT high-performance liquid chromatograph and a gel permeation column. Experimental conditions: column: TSKgel G3000SWxl; mobile phase: 100 mM PB (pH 7.0); flow rate: 1 ml / min; injection volume: 20 μl (concentration ≤ 4 mg / ml) or 50 μg (concentration > 4 mg / ml); column temperature: 35℃; detection wavelengths: 214 nm and 280 nm; acquisition time: 15 min. Detection results ( Figure 10 The main peak was found to account for 94.732%, indicating high purity of the trispecific nanobody C6-A1-X1, which mainly existed in the form of correctly folded antibodies. The binding performance of the trispecific nanobody C6-A1-X1 to the target antigens was detected using SPR (Table 6), following the method described in Example 2. It was observed that the trispecific nanobody C6-A1-X1 bound to all three targets to a therapeutic level. The expression level of this trispecific nanobody was high in the HEK293 expression system (670.47 mg / L), and SPR confirmed that all binding domains maintained their original affinity. This complex molecular design not only resulted in successful expression but also ensured excellent functional activity of all three binding domains, providing theoretical and technical reference for the large-scale production of nanobody fusion proteins and their application in in vivo diagnostics and targeted therapy.

[0082] Table 6. Binding of the trispecific nanobody C6-A1-X1 to various targets.

[0083] The sequence text mentioned above: The CDR sequences described in SEQ ID NO.1-3 and 8-10 are artificially designed nanobody complementarity-determining region sequences.

[0084] The FR sequences described in SEQ ID NO.4-7 and 11-14 are artificially designed nanobody backbone sequences.

[0085] SEQ ID NO.15-16 describes the amino acid sequences of nanobodies.

[0086] SEQ ID NO.17-18 describes the nucleotide sequences of nanobodies.

[0087] The amino acid sequence of the signal peptide described in SEQ ID NO.19.

[0088] The amino acid sequence of the C6 nanobody described in SEQ ID NO.21.

[0089] The amino acid sequences of the Fc region described in SEQ ID NO. 23 and 27.

[0090] The amino acid sequence of the X1 nanobody described in SEQ ID NO.25.

[0091] SEQ ID NO.29 describes a linker peptide sequence.

[0092] The sequences of the amplified products or primers used in the above embodiments are shown in Table 7 below.

[0093] Table 7. Sequences of the amplified products or primers used in the examples.

[0094] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An anti-PD-L1 nanobody, characterized in that, The nanobody is composed of a heavy chain, which includes three complementarity-determining regions, namely CDR1, CDR2, and CDR3; the nanobody is any one of the following: (1) Nanobody 1: CDR1 with amino acid sequence as shown in SEQ ID NO.1, CDR2 with amino acid sequence as shown in SEQ ID NO.2 and CDR3 with amino acid sequence as shown in SEQ ID NO.3; (2) Nanobody 2: CDR1 with amino acid sequence as shown in SEQ ID NO.8, CDR2 with amino acid sequence as shown in SEQ ID NO.9 and CDR3 with amino acid sequence as shown in SEQ ID NO.

10.

2. The anti-PD-L1 nanobody according to claim 1, characterized in that, The backbone region of the nanobody 1 includes amino acid sequences FR1, FR2, FR3, and FR4 as shown in SEQ ID NO. 4, 5, 6, and 7, respectively.

3. The anti-PD-L1 nanobody according to claim 2, characterized in that, The backbone region of the nanobody 2 includes amino acid sequences FR1, FR2, FR3, and FR4 as shown in SEQ ID NO. 11, 12, 13, and 14, respectively.

4. The nucleic acid encoding the anti-PD-L1 nanobody according to any one of claims 1-3.

5. The nucleic acid according to claim 4, characterized in that, The nucleic acid sequence encoding nanobody 1 is shown in SEQ ID NO. 17; the nucleic acid sequence encoding nanobody 2 is shown in SEQ ID NO.

18.

6. A mammalian system expression vector containing the nucleic acid of claim 4.

7. A method for constructing an asymmetric trispecific antibody, characterized in that, Includes the following steps: (1) Construct a eukaryotic expression vector containing the coding sequences of the first and second polypeptide chains; (2) Transfect the expression vector into eukaryotic host cells; (3) Culture the host cells to express the asymmetric trispecific antibody; (4) Separate and purify the asymmetric trispecific antibody; The first polypeptide chain, from its N-terminus to its C-terminus, sequentially comprises: a first antigen-binding domain as shown in SEQ ID NO. 21, an Fc region 1 as shown in SEQ ID NO. 23, and a third antigen-binding domain as shown in SEQ ID NO. 25, wherein the Fc region 1 and the third antigen-binding domain are connected by a linker peptide; the second polypeptide chain, from its N-terminus to its C-terminus, sequentially comprises: a second antigen-binding domain as shown in SEQ ID NO. 15, an Fc region 2 as shown in SEQ ID NO. 27, and a third antigen-binding domain as shown in SEQ ID NO. 25, wherein the Fc region 2 and the third antigen-binding domain are connected by a linker peptide.

8. The construction method according to claim 7, characterized in that, The linker peptides in both the first and second polypeptide chains are (G4S)n, where n is an integer from 1 to 5.

9. An asymmetric trispecific antibody constructed by the method of claim 8.

10. The use of the nanobody according to any one of claims 1-3, the nucleic acid according to claim 4, or the asymmetric trispecific antibody according to claim 9 in the preparation of a tumor-treating drug or a PD-L1 detection reagent.

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