Anti-b7-h3 nanobodies and fusion proteins comprising the same and multispecific antibodies

Abstract

The application discloses an anti-B7-H3 nanobody and a fusion protein and a multispecific antibody containing the same, and belongs to the technical field of biological medicines. ‑8 The nanobody is preferably BH-C6, and the affinity thereof reaches 6.47*10 ‑9 M. The fusion protein C6-Fc (containing C220A mutation) constructed has an expression amount of 573.1 mg / L, a purity of >98.9%, and an affinity of 3.62*10 ‑9 M. The "2+1" asymmetric trispecific antibody C6-A1-X1 constructed further simultaneously targets B7-H3, PD-L1 and LAG3, has an expression amount of 670.47 mg / L, a purity of >94%, and an affinity of 3.40 nM, 9.83 nM and 75 pM respectively. The antibody of the application can be used for tumor treatment, diagnosis and molecular imaging.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an anti-B7-H3 nanobody and a fusion protein and multispecific antibody containing it, as well as its preparation method and application. Background Technology

[0002] B7-H3 is an important immune checkpoint molecule in the B7 family, belonging to the immunoglobulin superfamily. It is widely and highly expressed on the surface of various solid tumor cells, including lung cancer, breast cancer, brain tumors, and pancreatic cancer, and is also present in small amounts in immune cells such as dendritic cells and macrophages, as well as in normal tissues such as the placenta and kidneys. Its core function is to inhibit T cell activation, proliferation, and cytokine secretion by binding to an unidentified receptor, thereby constructing a tumor immunosuppressive microenvironment and helping tumors evade immune system surveillance.

[0003] In clinical practice, high expression levels of B7-H3 can serve as a diagnostic marker and prognostic indicator for various tumors. Meanwhile, traditional B7-H3-targeting antibodies have been used in cancer treatment. These are mostly IgG monoclonal antibodies with a molecular weight of approximately 150 kDa. They work by blocking the B7-H3-mediated immunosuppressive pathway, reactivating the killing effect of T cells on tumor cells. However, these traditional antibodies have significant limitations: their large molecular weight makes it difficult to penetrate the stromal barrier of dense solid tumors such as brain tumors and pancreatic cancer, allowing them to act only on tumor surface cells; they easily bind to B7-H3 expressed at low levels in normal tissues, causing adverse reactions such as liver damage and immune inflammation; and their production process is complex and costly, requiring intravenous administration, which leads to poor compliance for patients who cannot tolerate frequent intravenous infusions (such as brain tumor patients).

[0004] Nanobodies, derived from the variable region of heavy chain antibodies from camelids, have a molecular weight of only about 15 kDa, which is 1 / 10 of that of traditional antibodies. They can precisely overcome the aforementioned bottlenecks: their small molecular structure can efficiently penetrate deep into solid tumors and act on tumor cells that traditional antibodies cannot reach; their antigen binding specificity is stronger, which can reduce off-target binding with normal tissues and reduce the risk of adverse reactions; at the same time, they have the advantages of simple production process, low cost, and high stability, and can also be developed into flexible drug delivery formulations such as intratumoral injection and local sustained release, which can meet the treatment needs of tumors in special locations such as brain tumors.

[0005] Despite reports of B7-H3-targeting antibodies, existing technologies still have the following shortcomings: single-target nanobodies cannot completely overcome the complexity of tumor immune escape; the small molecular weight of nanobodies results in a short half-life in vivo, which limits their clinical application; how to construct structurally stable and functionally synergistic multispecific antibodies, especially multi-target combinations with B7-H3 as the core target, remains a technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, this invention aims to provide a novel anti-B7-H3 nanobody, as well as a fusion protein and multispecific antibody constructed based on the nanobody, in order to solve the technical problems of poor penetration of traditional antibodies, short half-life of nanobodies, and limited efficacy of single-target therapy.

[0007] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One objective of this invention is to provide a nanobody that can specifically bind to B7-H3, which 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 selected from the group consisting of CDR1, CDR2 and CDR3 and their homologous sequences, and a backbone region selected from the group consisting of FR1, FR2, FR3 and FR4 and their homologous sequences. The amino acid sequence of CDR1 is shown in SEQ ID NO.3 or SEQ ID NO.10; The amino acid sequence of CDR2 is shown in SEQ ID NO.4 or SEQ ID NO.11; The amino acid sequence of CDR3 is shown in SEQ ID NO.5 or SEQ ID NO.13; The amino acid sequence of FR1 is shown in SEQ ID NO.6 or SEQ ID NO.13; The amino acid sequence of FR2 is shown in SEQ ID NO.7 or SEQ ID NO.14; The amino acid sequence of FR3 is shown in SEQ ID NO.8 or SEQ ID NO.15; The amino acid sequence of FR4 is shown in SEQ ID NO.9 or SEQ ID NO.16.

[0008] Preferably, the nanobody includes nanobody BH-A1 and nanobody BH-C6, whose amino acid sequences are SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0009] A second objective of this invention is to provide a polynucleotide for encoding the nanobody, wherein the nucleotide sequence for encoding nanobody BH-A1 is shown in SEQ ID NO.17 and the nucleotide sequence for encoding nanobody BH-C6 is shown in SEQ ID NO.18.

[0010] A third objective of this invention is to provide an expression vector containing the polynucleotide, or a nucleotide sequence containing a codon that omits 1-5 amino acid residues in the nucleotide sequence and / or a missense mutation of 1-5 base pairs.

[0011] The fourth objective of this invention is to provide a host cell containing the expression vector.

[0012] The fifth objective of this invention is to provide a method for preparing the nanobody, characterized in that it includes culturing the host cell under conditions expressing the nanobody and isolating the nanobody from the culture.

[0013] The sixth objective of this invention is to provide a fusion protein comprising the nanobody and a heterologous protein domain.

[0014] The seventh objective of this invention is to provide a multispecific antibody comprising the nanobody and one or more additional antigen-binding domains.

[0015] The eighth objective of this invention is to provide the application of the nanobody, the nucleotide, the fusion protein, and / or the multispecific antibody in the preparation of tumor therapeutic drugs.

[0016] The ninth objective of this invention is to provide the application of the nanobody, the nucleotide, the fusion protein, and / or the multispecific antibody in the preparation of a tumor detection kit.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. Excellent nanobody performance The nanobody BH-C6 obtained by screening in this invention has high affinity (KD reaches 6.47 × 10⁻⁶). -8 It exhibits excellent thermal stability, maintaining its binding activity with B7-H3 even after treatment at 80°C for 2 hours, significantly superior to traditional monoclonal antibodies. This high thermal stability is beneficial for antibody production, storage, and transportation.

[0018] 2. Highly efficient target cell binding ability Flow cytometry showed that BH-C6 bound to B7-H3 positive tumor cells at a rate of 87.6%, demonstrating its good target recognition ability and its potential for in vivo diagnosis and targeted therapy.

[0019] 3. Excellent fusion protein production performance The nanobody-Fc fusion protein C6-Fc constructed in this invention exhibits an expression level as high as 573.1 mg / L in the HEK293 expression system, with a purity >98.9%, far exceeding the expression level of traditional antibodies. This high expression level, combined with the simplified purification process of nanobodies, can significantly reduce production costs and improve drug accessibility.

[0020] 4. The affinity remains unchanged after fusion. The affinity of the fusion protein C6-Fc for B7-H3 is 3.62 × 10⁻⁶. -9M is comparable to or even better than monomeric nanobodies, proving that Fc fusion does not affect the binding activity of nanobodies, providing a theoretical and technical reference for the large-scale production of nanobody fusion proteins and their application in in vivo diagnostics and targeted therapy.

[0021] 5. Multi-target synergistic design with B7-H3 as the core This invention successfully constructed an IgG-like trispecific antibody, C6-A1-X1, with a "2+1" asymmetric structure. The "2" represents two different targets (B7-H3 and PD-L1) at the N-terminus of the two polypeptide chains, while the "1" represents the same target (LAG3) carried by both polypeptide chains at their C-terminus, existing in a bivalent form. This antibody simultaneously targets three immune checkpoint molecules: B7-H3, PD-L1, and LAG3. In the HEK293 system, its expression level reached 670.47 mg / L with a purity >94%, and its affinity for all three targets reached therapeutic levels (B7-H3: 3.40 nM, PD-L1: 9.83 nM, LAG3: 75 pM). By placing BH-C6 as the "tumor-targeting core" at the N-terminus of the first polypeptide chain, the molecule is preferentially enriched in the tumor microenvironment where B7-H3 is highly expressed, while simultaneously exerting a blocking effect on other immune checkpoint molecules, achieving a dual function of "tumor targeting + immune activation." This design is particularly suitable for dense solid tumors such as liver cancer, brain tumors, and pancreatic cancer, which are difficult for traditional antibodies to penetrate.

[0022] 6. Compatibility with multiple heterodimerization technologies The multispecific antibodies 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.

[0023] 7. Scalability of platform technology This invention successfully verified the feasibility of constructing multispecific antibodies based on BH-C6. This technology platform can be rapidly expanded to other target combinations, providing a technical foundation for the development of next-generation multispecific tumor immunotherapy drugs.

[0024] 8. Broad clinical application prospects The nanobodies, fusion proteins, and multispecific 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. Simultaneously, they can be used for in vitro diagnostics and in vivo molecular imaging of B7-H3 expression, achieving integrated diagnosis and treatment. Attached Figure Description

[0025] Figure 1This is a graph showing the titer of alpaca (Vicugna pacos) immune serum in this invention. The horizontal axis represents the serum dilution factor, and the vertical axis represents the corresponding absorbance OD450 value.

[0026] Figure 2 The results of agarose gel electrophoresis of total RNA from alpaca PBMCs in this invention are shown.

[0027] Figure 3 This is an agarose gel electrophoresis image of the VHH gene amplified using different amounts of cDNA template in this invention.

[0028] Figure 4 The results of colony PCR agarose gel electrophoresis in this invention are shown.

[0029] Figure 5 This is the result of the library sequence diversity alignment in this invention.

[0030] Figure 6 The results of identifying positive clones using the sandwich phage ELISA method in this invention.

[0031] Figure 7 This is an SDS-PAGE protein electrophoresis image of the B7-H3 nanobodies BH-A1 and BH-C6 in this invention. In the image, lane M: molecular weight marker; lane 1: nanobodies BH-C6; lane 2: nanobodies BH-A1.

[0032] Figure 8 The figure shows the experimental results of the thermal stability of the B7-H3 nanobodies BH-A1 and BH-C6 in this invention.

[0033] Figure 9 This invention demonstrates the flow cytometry detection of the binding of the B7-H3 nanobody BH-C6 to target cells. A. Negative control group (Blank blank control); B. Tumor target cell positive group (BH-C6 nanobody). The binding rate of the BH-C6 nanobody to target cells was 87.6%, while the binding rate of the blank control group was 1.16%.

[0034] Figure 10 This is an SDS-PAGE protein electrophoresis image of the nanobody-Fc fusion protein (C6-Fc) of this invention. In the image, lane M: molecular weight marker; lane R: molecular weight of the fusion protein in the reduced state (39.703 kDa); lane NR: molecular weight of the fusion protein in the non-reduced state (79.406 kDa).

[0035] Figure 11This is the SEC-HPLC chromatogram of the nanobody-Fc fusion protein (C6-Fc) of this invention, showing the peak values ​​at 214 nm and 280 nm. The main peak accounts for over 98.9%, indicating extremely high purity of the fusion protein, which mainly exists in the form of correctly folded antibody monomers.

[0036] Figure 12 The results show the SPR affinity of the nanobody-Fc fusion protein (C6-Fc) in this invention. It can be seen that the fusion protein C6-Fc has koff(1 / s) = 1.12E-03, kon(1 / Ms) = 3.10E+05, and KD(M) = 3.62E-09.

[0037] Figure 13 This is an SDS-PAGE protein electrophoresis image of the asymmetric trispecific nanobody C6-A1-X1 in this invention. 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 antibody structure is characterized by: the first polypeptide chain having a B7-H3 nanobody BH-C6 at the N-terminus and a LAG3 nanobody X1 at the C-terminus; the second polypeptide chain having a PD-L1 nanobody PL-A1 at the N-terminus and a LAG3 nanobody X1 at the C-terminus. X1 is fused to the C-terminus of both chains, achieving bivalent binding to LAG3.

[0038] Figure 14 This is the SEC-HPLC chromatogram of the asymmetric trispecific nanobody C6-A1-X1 in this invention, showing the peak values ​​of the protein 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

[0039] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0040] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0041] Example 1: Preparation of B7-H3 nanobody This embodiment provides a novel B7-H3 nanobody, the preparation method of which includes the following steps: (1) Immunize alpacas: One mg of B7-H3 protein (Acro, B7B-H52E7) 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 B7-H3 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 B7-H3 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 titers. 100 mL of peripheral blood was collected after serum titer testing. The results showed that this immunization protocol stimulated the alpaca to produce high-titer antibodies, ensuring the diversity of the gene library.

[0042] Figure 1 This is a graph showing the titer of immunized alpaca (Vicugna pacos) serum. A positive result is defined as a positive-to-negative serum ratio ≥2.1. Therefore, the serum titer of the immunized alpaca reached 1:512000, indicating a very good immunization effect and the production of a large number of antibodies in the alpaca's body.

[0043] (2) RNA extraction from peripheral blood lymphocytes: Total RNA was extracted using LeukoLOCK™ (ThermoFisher) following the instructions. 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.

[0044] Table 1 Total RNA volume and detection results

[0045] (3) Construction of nanobody gene library: The construction of a nanobody gene library includes the following steps: a. Synthesize cDNA strand: Following the instructions of the SuperScript® III First-Strand Synthesis System for RT-PCR, 123µg of RNA was reverse transcribed to synthesize a cDNA strand; b. Amplification of the alpaca VHH gene: Using the cDNA strand from step a as a template, PCR was performed using primers HS and Hanti1 (Table 5) to amplify the VHH gene of alpaca heavy chain antibody IgG3. PCR was also performed using primers HS and Hanti2 (Table 5) to amplify the VHH gene of alpaca heavy chain antibody IgG2. Figure 3 The results showed that the VHH gene originated from heavy chain antibodies IgG2 and IgG3. Therefore, the VHH gene amplified using these two primer pairs included both the VHH gene of IgG2 and the VHH gene of IgG3, resulting in a more diverse gene library compared to the VHH-P1 and VHH-P2 (which only amplified the VHH gene of IgG2) used by other research institutions.

[0046] The reaction mixture was as follows: 2 μL cDNA; 1.3 μL HS primers; 1.3 μL Hanti1 or Hanti2 primers; 44.4 μL TaqEnzyme Mix; reaction program: 94℃, 3 min; 94℃, 30 s, 55℃, 30 s, 72℃, 1 min, 32 cycles; 72℃, 5 min. The PCR products were analyzed by electrophoresis. Electrophoresis results. Figure 3 The results showed that using 0.1 μl as template amplified a clear and non-spam band (approximately 500 bp single band). 0.1 μl was selected as the optimal template amount for amplification, and the PCR product was recovered by gel excision.

[0047] c. Linearization treatment of pComb3X phage vector by SfiⅠ restriction enzyme digestion: The inventors discovered that the SfiⅠ enzyme is sensitive to methylated DNA. Therefore, *E. coli* C2925 was selected as the host bacterium for amplification of this vector. *E. coli* C2925 lacks nonspecific endonuclease I (endA1) activity and methyltransferase, so the NNA sequence will not be methylated, making it suitable for amplifying high-quality pComb3X plasmids without affecting the SfiⅠ restriction enzyme digestion effect. Therefore, *E. coli* C2925 competent cells were purchased, and the pComb3X phage vector was transformed into *E. coli* C2925 competent cells. 1 μL of pComb3X phage vector was added to thawed *E. coli* C2925 competent cells, incubated in ice water for 10-20 min, heat-shocked at 42℃ for 60-90 s, incubated on ice for 2 min, and then added to SOC medium at 25-37℃. The cells were incubated at 37℃ and 250 rpm for 1 h. Spread 200 μL of bacterial culture onto an ampicillin-resistant plate and incubate overnight at 37°C. The next day, select single clones and incubate overnight at 37°C and 250 rpm on ampicillin-resistant LB medium. Extract the plasmid using a plasmid extraction kit. Perform enzyme digestion in a 0.2 μL PCR tube: add 1 μg of plasmid pComb3X, 4 μL of SfiⅠ enzyme, 4 μL of CutSmart Buffer, and bring the volume to 50 μL with ultrapure deionized water. Incubate the tube at 50°C for 12 h. The next day, electrophoresis the digestion product, excise the gel, recover the large fragment, and dissolve it in ultrapure deionized water.

[0048] d. Based on the principle of homologous recombination, the VHH gene was ligated into the pComb3X vector: Using the ClonExpress UltraOne Step Cloning Kit, 200 ng of the pComb3X vector fragment (digested with SfiI and then gel-recovered), 60 ng of the VHH gene, 5 μL of 2×ClonExpress Mix, and ultrapure deionized water were added to a final volume of 10 μL. The mixture was incubated at 50°C for 5 minutes, and then immediately cooled on ice. The ligation product was purified and recovered using a PCR product purification kit. The results showed that the use of homologous recombination enzymes increased the ligation efficiency between the vector and the VHH gene, and the absence of DNA ligase in the entire system reduced the efficiency of vector self-ligation. Figure 4 This increased storage capacity to 8.56 × 10⁻⁶. 9 pfu.

[0049] e. Electroporation of the recombinant vector: Add 3 μL of the purified ligation product from step d to 50 μL of *E. coli* ER2738 electroporation competent cells. Gently stir 1-2 times with a tip to avoid air bubbles. Add the electroporation system to a cooled 1.0 mm electroporation cuvette. Gently shake the cuvette with your wrist to allow the cells to sink to the bottom. Immediately place it in an electroporator. Electroporation conditions: 1400-1600 V, 200-400 Ω, 10 μF, 3.5-4.5 ms. Immediately add 975 μL of preheated recovery medium. Mix the cells by blowing up and down three times. Transfer to a bacterial culture tube and incubate at 37°C and 250 rpm for 1 h. Dilute 1 μL of the bacterial culture with 10... -1 10 -3 10 -5 10 -7 The library capacity was determined by plating after fold expansion (LB for ampicillin resistance). Forty-eight clones were randomly selected from the plate for colony PCR to verify ligation efficiency. Figure 4 Of the 48 single clones selected, 46 were positive, with a positive rate of 96%. The primers for colony PCR were primer HS and primer Hback (Table 5). After sequencing, 48 randomly selected single clones were transcribed and translated into protein sequences using GENtle software. Sequence diversity alignment showed that all 48 sequences were independent sequences, with 100% diversity, and the bacterial library diversity met the requirements. Figure 5 ).

[0050] f. Gene bank rescue: Transfer the transformed bacteria revived in step e into 200 mL of SB medium containing ampicillin and tetracycline, and culture at 37°C and 250 rpm until the bacterial logarithmic phase. Add 10 12 PFU M13KO7 helper phage was incubated at 37°C for 30 min, followed by shaking for 1 h. Kanamycin (final concentration 70 μg / mL) was then added and the culture was incubated overnight. The next day, the overnight culture was centrifuged (13000 rpm, 4°C) for 10 min. The supernatant was collected, and 5×PEG / NaCl was added. After incubating on ice for 2 h, the culture was centrifuged again. The precipitate was resuspended in a protective buffer (PBS solution containing 1× protease inhibitor, 0.02% sodium azide, and 0.5% BSA). The solution was filtered through a 0.22 μm filter and aliquoted, then stored at -80°C to obtain 3 mL of nanobody phage library with a titer of 3.6 × 10⁻⁶. 13 pfu / ml.

[0051] (4) Selecting and identifying B7-H3 nanobodies: The phage library obtained in step f was subjected to affinity panning using magnetic beads coupled with streptavidin to obtain the first output product. 10 μL of this product was used for titer determination. The remaining products were amplified and subjected to second and third rounds of screening. Ninety-six clones were then picked from the culture plates obtained in the third round of screening and incubated overnight at 37°C. Positive clones were identified by phage ELISA. The results showed that, compared with solid-phase screening methods using coated antigen proteins, magnetic bead liquid-phase screening exposed more epitopes and reduced the probability of some epitopes being blocked due to antigen embedding during solid-phase screening, thus achieving comprehensive screening of more epitope antibodies.

[0052] The specific method for affinity screening is as follows: a. Take 200 μL of streptomycin affinity magnetic beads, wash twice with 1 mL TBST, and then add 1 mL of blocking solution (3% BSA for the first round of screening, 3% skim milk for the second round of screening, and alternate between 3% BSA and 3% skim milk), and block at 4℃ and 150-160 rpm for 1 h. b. Add the phage library to the same blocking solution and block and remove impurities by shaking at 4°C and 150-160 rpm. c. After sealing, centrifuge the magnetic beads at low speed (2000-3000 rpm) for 30 seconds, discard the supernatant, and wash 3 times with 1 mL TBST. d. After washing, the magnetic beads were added to 200 μL of magnetic bead binding buffer (20 mM pH 7.5 Tris-HCl, 0.5 MNAcl, 1 mM EDTA) and 30 μL of biotinylated B7-H3 protein. The mixture was vortexed at 4 °C and 130-150 rpm for 30 min. Then, the purified phage library was added and the mixture was vortexed at 4 °C and 150-160 rpm for 1 h. e. Centrifuge the reaction solution at low speed (2000-3000 rpm) for 30 seconds, discard the supernatant, add 1 mL of TBST and wash 10 times. Add 200 μL of glycine-hydrochloric acid at pH 2.2 to the magnetic beads, and shake at 150-200 rpm for 15 minutes to bind. Centrifuge at low speed (2000-3000 rpm) for 30 seconds, discard the supernatant and immediately add 1.6 μL of Tris-HCl at pH 9.1 to neutralize the elution product, thus obtaining the first product (output). f. Take 10 μL of the product obtained in step e to determine the titer. Amplify the remaining products as follows: Add 3-5 mL of ER2738 bacteria in the logarithmic growth phase to the eluted product. Incubate at 37°C for 30-45 minutes. Then add 5 mL of SB medium preheated at 37°C containing 200 μg ampicillin and 60 μg tetracycline resistance. Incubate at 37°C and 220-250 rpm for 1 hour. Add 500 μg ampicillin and continue incubating at 37°C and 220-250 rpm for 1 hour. Add 10¹² pfu of M13KO7 helper phage and incubate at 37°C for 30 minutes. Transfer to 91 mL of preheated SB medium containing 9.4 mg ampicillin and 920 μg tetracycline. Incubate at 220-250 rpm for 1 hour. Add kanamycin to a final concentration of 70 μg / mL and incubate overnight. The following day, the overnight bacteria were centrifuged at 13,000 rpm at 4°C for 10 min. The supernatant was collected and 5×PEG / NaCl was added. After incubating on ice for 2-4 h, the mixture was centrifuged again. The resulting precipitate was resuspended in 0.01M PBS buffer for the next round of panning.

[0053] g. The amplification buffer from the first round of product was used in the second round of screening; similarly, the amplification buffer from the second round of product was used in the third round of screening. The volume of biotinylated B7-H3 protein used in the second and third rounds was reduced to 6 μL and 1.5 μL, respectively.

[0054] h. Select 58 clones from the plates after three rounds of screening for sandwich Phage Elisa identification of positive clones.

[0055] (5) Sandwich Phage ELISA for identifying positive clones: Alpaca immunoglobulin G, purified and isolated using a protein A agarose gel chromatography column, was embedded in ELISA plates, and positive clones were identified by sandwich phage ELISA. Figure 6 The absorbance values ​​of the sandwich Phage ELISA were obtained. Of the 58 clones selected, 54 were positive, a positive rate of 93.10%. The steps for identifying positive clones using the sandwich Phage ELISA are as follows: a. Protein A agarose gel affinity chromatography was used to separate and purify anti-B7-H3 immunoglobulin G (IgG). 50 mL of peripheral blood was collected from alpacas on day 7 after the last immunization. The serum was centrifuged at 2000-3000 rpm for 10 min to obtain anti-B7-H3 serum. The serum was serially diluted, and the anti-B7-H3 serum titer was measured. Binding buffer (0.02 mol / L sodium phosphate, pH 7.0), elution buffer (0.1 mol / L glycine-hydrochloric acid, pH 2.7), and neutralization buffer (1 mol / L Tris-HCl, pH 9.0) were prepared. All buffers were sterilized before use by filtering through a 0.45 μm filter. The bottom tip of the chromatography column was cut off, and the cap was removed. The column was placed on a rack, and equilibrated with 10-20 mL of binding buffer. The serum sample was slowly added, followed by 15-20 mL of binding buffer. 5-8 mL of elution buffer was added, and the eluted fraction was collected. The eluent was subjected to SDS-PAGE gel electrophoresis to determine its purity and concentration. Simultaneously, B7-H3 protein was plated, and the biological activity of the collected IgG was detected using ELISA.

[0056] b. Dilute the anti-B7-H3 immunoglobulin isolated in the previous step to 5-10 μg / mL with coating buffer, add 100 μL to each well of a 96-well plate, and coat overnight at 4°C. The next day, after washing the plate, add B7-H3 protein and incubate at 37°C for 1-2 hours; after washing again, add 300 μL of 5% skim milk blocking solution to each well and block for 1 hour. After washing and drying, store at 4°C for later use. Use a sterile toothpick to pick 46 single colonies and inoculate them into a 96-well plate with 800 μL of ampicillin-resistant medium per well. Incubate at 37°C with shaking at 250 rpm for approximately 5-6 hours until the bacterial OD600 is approximately 0.6-0.8. Add 10 μL of 5% skim milk blocking solution to each well. 11 PFU M13KO7 infection was performed, incubated at 37°C for 30 min, followed by incubation at 37°C with shaking at 250 rpm for 1 h. Kanamycin was added to a final concentration of 70 μg / mL, and incubated overnight at 37°C with shaking at 250 rpm. The next day, the plates were centrifuged at 6000 rpm for 15 min, and the supernatant was collected. After removing impurities with 3% skim milk, the supernatant was added to a 96-well ELISA plate stored at 4°C and incubated at room temperature with shaking at 150-160 rpm for 1 h. The plates were washed three times, and anti-M13 secondary antibody was added. Incubation was performed at room temperature with shaking at 150-160 rpm for 30 min. Goat anti-rabbit HRP antibody was added and incubated at 150-160 rpm for 30 min. TMB colorimetric analysis was then performed.

[0057] (6) Expression and purification of B7-H3 nanobody: The ELISA strain with the strongest positive signal obtained in step (5) was used to extract plasmids using a plasmid kit (provided by Qiagen) to transform Escherichia coli Top10 F'. Single clones were picked and cultured overnight at 37°C. The plasmids were added to 100 mL of SB medium at a ratio of 1:100 and cultured at 37°C for 3-4 h until the OD600 reached 0.6-0.8. IPTG (final concentration 0.5 mmol / L) was added and expression was induced overnight at 26°C. The next morning, the cells were collected by centrifugation (10000 rpm, 10 min) and lysed at room temperature for 30 min using bacterial protein extraction reagent (B-PER) to release the protein. The cells were centrifuged again (12000 rpm, 15 min) and the supernatant was collected and added to a nickel column. The cells were bound at 4°C for 3-6 h and washed with 20 mmol / M imidazole for 4 column volumes. 5 mL of 50 mmol / L and 100 mmol / L imidazole washing buffer were collected to obtain nanobodies.

[0058] The PAGE electrophoresis results of the nanobody are as follows: Figure 7 As shown in the figure, the molecular weight of the nanobodies BH-C6 and BH-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 BH-A1 are shown in SEQ ID NO.3, CDR2 in SEQ ID NO.4, and CDR3 in SEQ ID NO.5; the amino acid sequence of FR1 is shown in SEQ ID NO.6; the amino acid sequence of FR2 is shown in SEQ ID NO.7; the amino acid sequence of FR3 is shown in SEQ ID NO.8; and the amino acid sequence of FR4 is shown in SEQ ID NO.9.

[0062] The amino acid sequences of CDR1, CDR2, and CDR3 of nanobody BH-C6 are shown in SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively; the amino acid sequence of FR1 is shown in SEQ ID NO.13; the amino acid sequence of FR2 is shown in SEQ ID NO.14; the amino acid sequence of FR3 is shown in SEQ ID NO.15; and the amino acid sequence of FR4 is shown in SEQ ID NO.16.

[0063] The details of each sequence are as follows: ① Nanobody BH-A1 (SEQ ID NO.1): DVQLQESGGGLVQPGGSLRLSCAASGFTFSTSAIHWARQAPGKGTDWVSRIYAPGSAGSAYYADSVKGRFTASRDNAKNTVYLQMNSLKPEDTAVYYCGTGTIVDGTRIGSWGLGTQVTVSS CDR1 of nanobody BH-A1 (SEQ ID NO.3): GFTFSTS CDR2 of nanobody BH-A1 (SEQ ID NO.4): YAPGSAGS CDR3 (SEQ ID NO.5) of nanobody BH-A1: GTIVDGTRIGS FR1 of nanobody BH-A1 (SEQ ID NO.6): DVQLQESGGGLVQPGGSLRLSCAAS FR2 of nanobody BH-A1 (SEQ ID NO.7): AIHWARQAPGKGTDWVSRI FR3 of nanobody BH-A1 (SEQ ID NO.8): YYADSVKGRFTASRDNAKNTVYLQMNSLKPEDTAVYYCGT FR4 of nanobody BH-A1 (SEQ ID NO.9): WGLGTQVTVSS Nucleotide sequence of nanobody BH-A1 (SEQ ID NO. 17): GATGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTACTTCTGCCATACACTGGGCCCGCCAGGCTCCAGGGAAGGGGACCGACTGGGTGTCCAGAATTTACGCGCCAGGTAGCGCAGGTAGCGCATACTACGCAGACTCCGTGAAGGGCCGATTCACCGCCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTATTGTGGGACTGGGACGATAGTGGATGGTACACGTATTGGTTCCTGGGGCCTGGGGACCCAGGTCACCGTCTCCAGC ② Nanobody BH-C6 (SEQ ID NO.2): DVQLQESGGGLVQPGGSLRLSCAASGFTLDVYGIGWFRQAPGKEREGISCFVKDANVPYYADSVKGRFTVSTDNAKNTVYLQMNSLKPEDTAVYYCARTSACVLLDGQPSHYGMDYWGKGTQVTVSS CDR1 of nanobody BH-C6 (SEQ ID NO.10): GFTLDVY CDR2 of nanobody BH-C6 (SEQ ID NO.11): VKDANV CDR3 of nanobody BH-C6 (SEQ ID NO.12): TSACVLLDGQPSHYGMDY FR1 of nanobody BH-C6 (SEQ ID NO.13): DVQLQESGGGLVQPGGSLRLSCAAS FR2 of nanobody BH-C6 (SEQ ID NO.14): GIGWFRQAPGKEREGISCF FR3 of nanobody BH-C6 (SEQ ID NO.15): PYYADSVKGRFTVSTDNAKNTVYLQMNSLKPEDTAVYYCAR FR4 of nanobody BH-C6 (SEQ ID NO.16): WGKGTQVTVS The nucleotide sequence of nanobody BH-C6 (SEQ ID NO.18): GATGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTAGATGTTTATGGCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGATCTCATGTTTTGTTAAAGATGCAAATGTCCCATACTATGCAGACTCCG TGAAGGGCCGATTCACCGTCTCCACGGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTTTATTACTGTGCACGCACTTCGGCTTGTGTCCTTTTGGACGGGCAGCCCTCCACTACGGCATGGACTACTGGGGCAAAGGGACCCAGGTCACCGTCTCCAGC Example 2: Affinity Detection of B7-H3 Nanobodies (1) 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, and 500 nM. Prepare sample protein B7-H3 (10 μg / mL). Check the integrity of the Anti-His probe (GatorBio, catalog number 160009) to avoid air bubbles. Label the 96-well plate according to the experimental design layout.

[0064] (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.

[0065] (3) The start-up experiment is shown in Table 2 below.

[0066] Table 2 Experiment Start-up Procedure

[0067] The results are shown in Table 3 below.

[0068] Table 3. Affinity details of B7-H3 nanobodies

[0069] Results analysis: The values ​​of koff, kon, and KD at different concentrations were highly consistent, 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 BH-C6 is higher than that of nanobody BH-A1.

[0070] Example 3: Thermostability Experiment of Nanobody B7-H3 protein was coated onto ELISA plates. 100 μL of 1 μg / mL B7-H3 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. The nanobody of this invention and a commercially available B7-H3 monoclonal antibody (B7H3-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. 100 μL of each nanobody was added to each well, and the plate was incubated at room temperature for 1 hour, followed by washing three times with PBST.

[0071] For the nanobody groups BH-A1 and BH-C6, because the nanobodies have His tags, HRP-labeled His-mAb (Cell Signaling) was added to each well, incubated at room temperature for 40 minutes, washed three times with PBST, and then TMB was added for color development for 10 minutes. After terminating the reaction with 2M sulfuric acid, the UV absorbance (OD450 value) at 450 nm was measured using a microplate reader.

[0072] In the B7-H3 mAb group, HRP-labeled anti-rabbit IgG secondary antibody (Cell Signaling) 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.

[0073] The results are as follows Figure 8 As shown in the figure. The results show that the nanobody structure is stable, and nanobodies BH-A1 and BH-C6 still exhibit B7-H3 antigen binding activity after treatment at 80℃ for 2 hours. This indicates that compared with B7-H3-mAb, the nanobody structure of the present invention is more stable and has better heat resistance.

[0074] Example 4: Flow cytometry detection of the binding of nanobodies to target cells Tumor cells A549 (B7-H3) with high expression of B7-H3 were selected. +Using 1×10⁻⁶ cells as target cells, the binding efficiency of BH-C6 to target cells was detected. 5 Tumor cells were resuspended in 500 μL PBS (2% BSA), and BH-C6 nanobody was added. Incubation was performed at 4°C for 30-40 min with gentle shaking at 150-160 rpm to prevent non-specific binding. After washing the cells with PBS, His-Tag (D3I1O) Rabbit mAb (Alexa Fluor® 647 Conjugate, Cell Signaling Technology, #14931) was added to the cell culture and incubated at 4°C for 30 min. Cells were then washed and analyzed by flow cytometry. Figure 9 Flow cytometry analysis showed that the binding rate of nanobody BH-C6 to target cells was 87.6%, indicating that nanobody BH-C6 binds to target cells with high affinity for B7-H3 molecules.

[0075] Example 5: Expression and purification of nanobody-Fc fusion protein (C6-Fc) (1) Synthesize the fusion gene of nanobody BH-C6 and Fc nucleotide sequences. The fusion gene sequence is designed as follows: signal peptide sequence - C6 sequence - Fc (Human IgG1 C220A) sequence, and E is designed at both ends. coR I and H ind The restriction enzyme site III. The C6-Fc fusion gene was cloned into the pcDNA3.1 eukaryotic expression vector (Thermo Fisher Scientific, catalog number V79020), transformed into E. coli DH5α competent cells, and positive clones were selected for amplification. The plasmid was then extracted and sequenced for DNA identification. The plasmid was named pcDNA3.1-C6-Fc.

[0076] Signal peptide amino acid (SEQ ID NO.23): MGWSCIILFLVATATGVHS Signal peptide nucleotide (SEQ ID NO.24): GGCTGGTCTTGTATCATCCTGTTCCTGGTGGCTACAGCTACTGGAGTGCACAGC Fc amino acid sequence (human IgG1 Fc, containing the C220A mutation, SEQ ID NO.25): EPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Fc nucleotide sequence (SEQ ID NO.26): GAGCCCAAATCCGCCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGCTGGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCCGCCCCCATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGCAAG (2) Cell culture. HEK293T cells were revived and passaged 3 times. Culture conditions: 120 rpm, 8% CO2, 37℃. Cell passage density was controlled at 0.3 × 10⁻⁶ cells / year. 6 pcs / ml

[0077] (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 100 μg of plasmid pcDNA3.1-C6-Fc and 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 (15,000g, 20-30 minutes) to remove cell debris and particulate matter to prevent clogging of the chromatography column.

[0078] (4) Protein A affinity chromatography purification of protein. 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 the column with 1×PBS at a flow rate of 1 mL / min for at least 10 column volumes of 1×PBS 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 the 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.

[0079] (5) Detection of the fusion protein C6-Fc. The protein concentration reached 3.88 mg / mL using NanoDrop, and the protein expression level of this HEK293 expression system reached 573.1 mg / L. SDS-PAGE was used to detect (…). Figure 10 The molecular weight of the protein in its reduced state is 39.703 kDa, and that in its non-reduced state is 79.406 kDa. The purity of the fusion protein was determined using SEC-HPLC with an LC-20AT high-performance liquid chromatography (HPLC) instrument and a gel permeation column. The water was replaced with the mobile phase (100 mM sodium phosphate + 100 mM sodium sulfate, pH 6.6), and the flow rate was slowly increased to 1.000 ml / min until the baseline stabilized. 25 μL of antibody was injected into the corresponding numbered vial, and the vial was placed in the appropriate position on the instrument. The injection time was 15 min. The data was analyzed, processed, and saved. The mobile phase was then replaced with deionized water, and the sample was rinsed for 1.5 h. The detection results (…) Figure 11The study found that the main peak accounted for over 98.9% of the total protein, indicating extremely high purity of the C6-Fc fusion protein, which primarily existed as correctly folded antibody monomers. The binding performance of the C6-Fc fusion protein to the target antigen B7-H3 was assessed using SPR. Figure 12 It is evident that koff(1 / s) = 1.12E-03, kon(1 / Ms) = 3.10E+05, and KD(M) = 3.62E-09. The fusion protein C6-Fc expressed by the HEK293 expression system exhibits the same high affinity as the standalone nanobody BH-C6, indicating that the fusion of Fc does not affect the binding activity of the nanobody. Furthermore, the C220A mutation introduced into the Fc region effectively eliminates disulfide bond mismatches that may be caused by cysteine ​​residues, improving the uniformity of the fusion protein. This provides a theoretical and technical reference for the large-scale production of nanobody fusion proteins and their application in in vivo diagnostics and targeted therapy.

[0080] Example 6: Construction of IgG-like asymmetric trispecific nanobody C6-A1-X1 containing BH-C6 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.

[0081] A trispecific nanobody was constructed using B7-H3 nanobody BH-C6, PD-L1 nanobody PL-A1, and LAG3 nanobody X1. The design employed an asymmetric structure of "N-terminal dual targets + C-terminal common target": BH-C6 was placed at the N-terminus of the first polypeptide chain as the "core targeting arm," enabling it to efficiently recognize B7-H3-positive tumor cells and achieve tumor-targeted enrichment; simultaneously, PL-A1 at the N-terminus of the second polypeptide chain blocked the PD-L1 immunosuppressive pathway, and X1, carried by both chains at the C-terminus, achieved bivalent binding to LAG3, enhancing the blocking effect on the LAG3 pathway. The specific structure is as follows: The first polypeptide chain comprises, from the N-terminus to the C-terminus, BH-C6 nanobody (SEQ ID NO.2), the first Fc region (containing heterodimerization modification complementary to the second Fc region, including a protrusion mutation), (G4S)3 linker peptide (SEQ ID NO.31), and anti-LAG3 nanobody X1 (SEQ ID NO.29). The second polypeptide chain comprises, from the N-terminus to the C-terminus, the anti-PD-L1 nanobody PL-A1 (SEQ ID NO.27), the second Fc region (containing modifications that promote heterodimerization, including cavity mutations and C220A mutations), the (G4S)3 linker peptide (SEQ ID NO.31), and the anti-LAG3 nanobody X1 (SEQ ID NO.29).

[0082] (1) Sequence Design The amino acid sequence of the anti-PD-L1 nanobody PL-A1 (SEQ ID NO.27): DVQLQESGGGLVQAGGSLRLSCAASGHDFSNYAMGWFRQAPGKEREFVAVITWIGGSTYYADSVKGRFTISRDNAKNTLYLQMNNLKPEDTAVYYCAARVLGWGVQVLFRTNPADFGSWGQGTQVTVSS Nucleotide sequence of anti-PD-L1 nanobody PL-A1 (SEQ ID NO.28): GATGTGCAGCTGCAGGAGAGCGGAGGAGGACTGGTTCAGGCTGGAGGAAGCCTGAGACTGAGCTGTGCCGCTAGCGGACACGACTTTAGCAACTACGCCATGGGATGGTTTAGGCAGGCCCCCGGAAAGGAGAGAGTTTGTGGCAGTGATCACCTGGATAGGAGGAAGCACCTACTACGCCGACTCCGTGA AAGGCAGATTCACCATCAGCAGAGACAACGCTAAGAACACACTGTACCTGCGATGAACAACCTGAAGCCCGAGGACACCGCTGTGTACTACTGCGCAGCCAGAGTGCTGGGCTGGGGAGTTCAAGTCCTCTTTAGAACCAACCCAGCTGACTTCGGCTCCTGGGGACAAGGAACACAAGTGACCGTCAGCAGC The amino acid sequence of the anti-LAG3 nanobody X1 (SEQ ID NO.29): QVQLQESGGGLVQPGGSLRLSCAASGFTLENYAIGWFRQAPGKEREGVSCISRSGGSTKYADSVKGRFTISRDHAKNTVFLQMNSLKPEDTAIYYCGKARDCTGPWGGSDYWGKGTQVTVSS Anti-LAG3 nanobody X1 nucleotide sequence (SEQ ID NO.30): CAAGTCCAACTCCAAGAGAGCGGCGGCGGACTGGTTCAACCAGGAGGATCACTCAGACTCTCTTGCGCCGCCAGCGGATTCACCCTGGAAAATTACGCCATCGGCTGGTTCAGACAGGCCCCTGGAAAAGAGAGAAGGAGTGAGCTGTATTAGCAGAAGCGGGGGGAGCACCAAATACGCT GACAGTGTGAAGGGCAGATTTACCATCAGCAGAGACCATGCCAAGAACACAGTGTTCCTGCAGATGAACAGCCTGAAGCCCGAGGACACCGCAATCTACTACTGCGGCAAGGCCCGGGACTGCACAGGACCTTGGGGAGGAAGTGACTACTGGGGCAAGGGGACACAAGTGACCGTGAGCAGC (G4S)3 linker peptide amino acid sequence (SEQ ID NO.31): GGGGSGGGGSGGGGS (G4S)3 linker peptide nucleotide sequence (SEQ ID NO.32): GGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCG First Fc region (including protrusion mutation, SEQ ID NO.33): DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK The nucleotide sequence of the first Fc region (SEQ ID NO.34): GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA Second Fc region (including cavity mutation and C220A mutation, SEQ ID NO.35): EPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Second Fc region nucleotide sequence (with cavity mutation and C220A mutation, SEQ ID NO.36): GAGCCCAAATCTGCCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGAGCTGCGCCGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (2) Polypeptide chain design First polypeptide chain: Signal peptide (SEQ ID NO.23) - BH-C6 (SEQ ID NO.2) - First Fc region (SEQ ID NO.35) - (G4S)3 (SEQ ID NO.31) - X1 (SEQ ID NO.29) Second polypeptide chain: Signal peptide (SEQ ID NO.23) - PL-A1 (SEQ ID NO.27) - Second Fc region (SEQ ID NO.33) - (G4S)3 (SEQ ID NO.31) - X1 (SEQ ID NO.29) (3) Construction of expression vector The coding gene sequences of the two polypeptide chains were synthesized, and NotI and XbaI restriction sites were designed at both ends, respectively. The synthesized genes were cloned into the pcDNA3.4 eukaryotic expression vector, transformed into E. coli DH5α competent cells, and positive clones were selected, amplified, and then the plasmids were extracted for DNA sequencing identification. The recombinant plasmids were named pcDNA3.4-C6-Fc-X1 and pcDNA3.4-A1-Fc-X1, respectively.

[0083] (4) Cell culture and transfection HEK293T cells were revived and passaged three times. Culture conditions: 120 rpm, 8% CO2, 37°C; cell density was maintained at 0.3 × 10⁻⁶ cells / year. 6 When the cell density reaches 70%, plasmid transfection is performed. Add 150 μL of Lipofectamine™ 3000 to 2.5 ml of Opti-MEM medium, labeling this solution 1. Add 100 μg of plasmids pcDNA3.4-C6-Fc-X1 and 100 μg of plasmid pcDNA3.4-A1-Fc-X1, along with 200 μL of P3000, to 2.5 ml of Opti-MEM medium, labeling this solution 2. Add solution 2 to solution 1, mix well, and incubate at 37°C for 15 minutes. Then, add the mixed transfection solution dropwise to the cell culture medium while shaking. Incubate on a shaker at 37°C, 120 rpm, and 8% CO2 for 5-7 days. Collect the cell culture supernatant and centrifuge (15,000 g, 20-30 minutes) to remove cell debris and particulate matter.

[0084] (5) Protein purification 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 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. Then elute contaminating proteins with 1×PBS at a flow rate of 1 mL / min for at least 10 column volumes. Elute the column with sodium acetate buffer (pH 3.4) at a flow rate of 1 mL / min for 5 min. Immediately after elution, collect small aliquots of the collected solution using centrifuge tubes. Measure the absorbance of each aliquot at 280 nm using NanoDrop. Combine the high-concentration protein solutions and place them into pretreated dialysis bags, then seal. Immerse the dialysis bags in a large volume of dialysis buffer (120 mM NaAc-HAc + 70 mM Arginine, pH 5.5). Dialyze at 4°C, changing the buffer solution every 3-4 hours for a total of 3-4 times. After dialysis, remove the protein solution, aliquot it, and store it at -80°C.

[0085] (6) Protein detection The protein concentration reached 8.21 mg / mL and the protein expression level reached 670.47 mg / L, as detected by NanoDrop. SDS-PAGE was used for further analysis. Figure 13 It can be seen that the molecular weight of the protein in the reduced state is 68.503 kDa, and the molecular weight of the protein in the non-reduced state is 137.44 kDa, which is consistent with the expected dimer structure.

[0086] The purity of the fusion protein was determined by SEC-HPLC using a high-performance liquid chromatography (HPLC) system and a gel chromatography column. Experimental conditions: TSKgel G3000SWxl column, 100 mM PB (pH 7.0) mobile phase, 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, and acquisition time 15 min. Detection results (…) Figure 14 The study found that the main peak accounted for 94.732%, and the trispecific nanobody C6-A1-X1 had high purity and mainly existed in the form of correctly folded antibody.

[0087] The binding performance of the trispecific nanobody C6-A1-X1 to the target antigen was detected using SPR, and the results are shown in Table 4. The trispecific antibody achieved therapeutic binding to all three targets, and all binding domains maintained their original affinity. This demonstrates that the "2+1" asymmetric structural design and the introduction of mutations in each Fc region of this invention do not affect the functional activity of each antigen-binding domain, while simultaneously improving the uniformity of the fusion protein.

[0088] Table 4. Binding of the trispecific nanobody C6-A1-X1 to various targets

[0089] The above results demonstrate that this invention successfully constructed an asymmetric trispecific antibody with the B7-H3 nanobody BH-C6 as its core. This antibody not only maintains the affinity of each binding domain but also exhibits high expression levels and high purity, providing theoretical and technical references for the large-scale production of nanobody fusion proteins and their application in in vivo diagnostics and targeted therapy.

[0090] Table 5. Sequences of the amplified products or primers used in the examples.

[0091] [Sequence List Free Text] The amino acid sequences of the nanobody described in SEQ ID NO.1-2.

[0092] The CDR sequences described in SEQ ID NO.3-5 and 10-12 are artificially designed nanobody complementarity-determining region sequences.

[0093] The FR sequences described in SEQ ID NO. 6-9 and 13-16 are artificially designed nanobody backbone region sequences.

[0094] The nucleotide sequences described in SEQ ID NO.17-18.

[0095] The amino acid sequence of the signal peptide described in SEQ ID NO.23.

[0096] The Fc amino acid sequence described in SEQ ID NO.25 is an artificially designed sequence of human IgG1 Fc containing the C220A mutation.

[0097] The Fc nucleotide sequence described in SEQ ID NO.26 is an artificially designed sequence of human IgG1 Fc containing the C220A mutation.

[0098] The amino acid sequence of the anti-PD-L1 nanobody PL-A1 described in SEQ ID NO.27 is an artificially designed sequence.

[0099] The nucleotide sequence of the anti-PD-L1 nanobody PL-A1 described in SEQ ID NO.28 is an artificially designed sequence.

[0100] The amino acid sequence of the anti-LAG3 nanobody X1 described in SEQ ID NO.29 is an artificially designed sequence.

[0101] The anti-LAG3 nanobody X1 nucleotide sequence described in SEQ ID NO.30 is an artificially designed sequence.

[0102] The amino acid sequence of the (G4S)3 linker peptide described in SEQ ID NO.31 is an artificially designed sequence.

[0103] The (G4S)3 linker nucleotide sequence described in SEQ ID NO.32 is an artificially designed sequence.

[0104] The amino acid sequence of the first Fc region described in SEQ ID NO.33 is an artificially designed sequence containing a protrusion mutation.

[0105] The nucleotide sequence of the first Fc region described in SEQ ID NO.34 is an artificially designed sequence containing a protrusion mutation.

[0106] The amino acid sequence of the second Fc region described in SEQ ID NO.35 is an artificially designed sequence containing cavity mutations and C220A mutations.

[0107] The second Fc region nucleotide sequence described in SEQ ID NO.36 is an artificially designed sequence containing cavity mutations and C220A mutations.

[0108] 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. A nanobody, characterized in that, It can specifically bind to B7-H3 and is composed of the variable region of the heavy chain antibody; the variable region of the heavy chain antibody includes a complementary region of the antigen determinant selected from the group consisting of CDR1, CDR2 and CDR3 and their homologous sequences, and a backbone region selected from the group consisting of FR1, FR2, FR3 and FR4 and their homologous sequences. The amino acid sequence of CDR1 is shown in SEQ ID NO.3 or SEQ ID NO.10; The amino acid sequence of CDR2 is shown in SEQ ID NO.4 or SEQ ID NO.11; The amino acid sequence of CDR3 is shown in SEQ ID NO.5 or SEQ ID NO.13; The amino acid sequence of FR1 is shown in SEQ ID NO.6 or SEQ ID NO.13; The amino acid sequence of FR2 is shown in SEQ ID NO.7 or SEQ ID NO.14; The amino acid sequence of FR3 is shown in SEQ ID NO.8 or SEQ ID NO.15; The amino acid sequence of FR4 is shown in SEQ ID NO.9 or SEQ ID NO.

16.

2. The Nanobody according to claim 1, characterized in that, The nanobodies include nanobodies BH-A1 and BH-C6, whose amino acid sequences are SEQ ID NO.1 and SEQ ID NO.2, respectively.

3. A polynucleotide, comprising, It is used to encode the nanobody of claim 2, wherein the nucleotide sequence for encoding nanobody BH-A1 is shown in SEQ ID NO.17, and the nucleotide sequence for encoding nanobody BH-C6 is shown in SEQ ID NO.

18.

4. An expression vector, characterized by, It includes the polynucleotide of claim 3, or a nucleotide sequence obtained by deleting 1-5 amino acid residues from the codons of the nucleotide sequence of claim 3 and / or by performing missense mutations of 1-5 base pairs.

5. A host cell, characterized in that, It includes the expression vector as described in claim 4.

6. A method for preparing nanobodies as described in claim 2, characterized in that, The process includes culturing the host cells of claim 5 under conditions expressing the nanobody, and isolating the nanobody from the culture.

7. A fusion protein, characterized in that, It includes the nanobody and heterologous protein domain as described in any one of claims 1-2.

8. A multispecific antibody, characterized in that, It includes the nanobody as described in any one of claims 1-2, and one or more additional antigen-binding domains.

9. The use of the nanobody of claim 2, the nucleotide of claim 3, the fusion protein of claim 7, and / or the multispecific antibody of claim 8 in the preparation of tumor therapeutic drugs.

10. The use of the nanobody of claim 2, or the nucleotide of claim 3, the fusion protein of claim 7, and / or the multispecific antibody of claim 8 in the preparation of a tumor detection kit.

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