Anti-HER2 single-chain antibody and application thereof

Abstract

The invention discloses an anti-HER2 single-chain antibody and application thereof, belongs to the technical field of molecular biology and protein engineering, and aims to solve the technical problems that hydrophobic amino acid residues of existing scFv easily cause protein aggregation and low solubility, and the stability and functional activity of the protein are affected. The structure of the single-chain antibody is X-m-Y, X is a light chain variable region or a heavy chain variable region, m is a connecting peptide, and Y is a heavy chain variable region or a light chain variable region corresponding to X; and hydrophobic amino acids in the amino acid sequence of the single-chain antibody are selectively replaced by hydrophilic amino acids. The protein coding technology is applied to hydrophilic optimization of HER2 targeting scFv, potential interference of a traditional modification strategy on CDR conformation is overcome, the hydrophilicity of the HER2 targeting scFv is remarkably improved, and the HER2 targeting scFv can stably exist in an aqueous solution; the compound can be used as a new target spot in preparation of HER2 positive non-small cell lung cancer, breast cancer or gastric cancer treatment drugs and in screening of drugs for treating or preventing lung diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and protein engineering, and relates to an anti-HER2 single-chain antibody, in particular to an anti-human epidermal growth factor receptor-2 (HER2) single-chain antibody with protein coding processing and its application. Background Art

[0002] Non-small cell lung cancer (NSCLC) holds a prominent position among the world's leading causes of malignant tumor mortality, with epidemiological data indicating that this pathological type accounts for approximately 85% of new lung cancer cases. In recent years, breakthroughs in the clinical application of molecularly targeted therapies and immune checkpoint inhibitors have significantly improved progression-free survival in patients with specific molecular subtypes. However, tumor microenvironment heterogeneity, acquired resistance mechanisms, and limitations in preclinical translational research models remain key bottlenecks in achieving both therapeutic response and clinical benefit. Of particular note, aberrant activation of the human epidermal growth factor receptor 2 (HER2) signaling system, specifically manifested by hyperfunction of the receptor tyrosine kinase domain, has been shown to be significantly associated with the pathological mechanisms of NSCLC invasion and metastasis. HER2 belongs to the ERBB receptor tyrosine kinase family. Its overexpression or gene amplification can promote tumor cell proliferation, invasion, and chemoresistance by activating signaling pathways such as PI3K / AKT and MAPK. Breakthrough data on HER2-targeted therapies are frequently presented at top conferences such as ASCO, WCLC, ESMO, and AACR, and are included in prestigious journals such as NEJM and JCO, highlighting the high level of attention within the field. While HER2-targeted therapies have achieved remarkable results in breast cancer, they still face challenges in NSCLC, such as low response rates and inconsistent efficacy. Therefore, developing efficient and specific HER2-targeted strategies and establishing drug evaluation platforms that can mimic the in vivo tumor microenvironment are key areas of current translational medicine research.

[0003] Pertuzumab is a recombinant humanized monoclonal antibody targeting HER2. Its originator is Roche's Perjeta. This drug is primarily used to treat HER2-positive breast cancer, with applications in other indications, such as colorectal cancer. Numerous domestic companies have innovated based on this approach and developed several new products. In the targeted treatment of HER2-positive tumors, monoclonal antibodies, such as trastuzumab and pertuzumab, along with tyrosine kinase inhibitors, such as afatinib, have established a standard clinical treatment regimen.

[0004] However, current clinical data indicate that these treatment options still face multiple challenges, including acquired drug resistance, drug-related toxicity, and heterogeneous treatment responses. Traditional monoclonal antibodies, such as trastuzumab and pertuzumab, have large molecular weights and poor tissue penetration, making them difficult to effectively penetrate the dense tumor stroma and prone to triggering immunogenic reactions, limiting their application in solid tumors. The development of a new generation of antibodies is imperative.

[0005] Single-chain antibodies (scFv) have attracted much attention in the biomedical field due to their significant advantages, such as low molecular weight, strong tissue permeability, and ease of genetic engineering modification. They have become a key research direction in the exploration of new targeted therapeutic molecules. scFv is composed of the variable region of the light chain (VL) and the variable region of the heavy chain (VH) of an antibody, covalently linked by a class of connecting short peptides [such as flexible peptide (G4S)3]. It retains the antigen binding ability of the complete antibody and effectively avoids non-targeted immune activation mediated by the crystallizable fragment (Fc). For example, the invention patent with application number 202410996016.6 discloses a mouse-derived single-chain antibody against porcine IgA and its application. The single-chain antibody includes a light chain variable region, a connecting peptide, and a heavy chain variable region, or includes a heavy chain variable region, a connecting peptide, and a light chain variable region. However, the clinical application of scFv still faces two major bottlenecks: First, the hydrophobic amino acid residues of scFv easily lead to protein aggregation and low solubility, affecting its stability and functional activity; second, the small molecular weight of scFv also shortens its half-life, making it impossible to avoid glomerular filtration clearance. Therefore, how to improve the properties of scFv through protein engineering strategies while ensuring its efficient recognition of antigen conformational epitopes constitutes a key scientific issue that needs to be addressed in this field. Summary of the Invention

[0006] The purpose of the present invention is to provide an anti-HER2 single-chain antibody and its application in order to solve the technical problem that the hydrophobic amino acid residues of existing scFvs easily lead to protein aggregation and low solubility, affecting their stability and functional activity. By improving the hydrophilicity and biological activity of the antibody, the defects of existing antibodies such as weak tissue penetration caused by excessive molecular weight, difficulty in penetrating dense tumor matrix, and easy induction of immunogenic reactions are solved.

[0007] In order to achieve the above-mentioned purpose, the present invention specifically adopts the following technical solutions: An anti-HER2 single-chain antibody having a structure of XmY, wherein X is a light chain variable region or a heavy chain variable region, m is a connecting peptide, and Y is a heavy chain variable region or a light chain variable region corresponding to X; characterized in that hydrophobic amino acids in the amino acid sequence of the single-chain antibody are selectively replaced with hydrophilic amino acids.

[0008] This anti-HER2 single-chain antibody adopts the XmY structural strategy, including the following: Antibodies can be classified from multiple dimensions. Table 1 provides a detailed division based on different classification criteria, and Pertuzumab is selected as a model for demonstration.

[0009] Table 1 Overview of monoclonal antibody classification

[0010] In the XmY structural strategy, the linker peptide m is a powerful molecular engineering tool, far more than a simple "connector." The "(G4S)3 flexible linker peptide connecting VL and VH" is selected as a representative linker between the m protein sequences in "XmY." These linkers can be self-assembling peptides, chiral self-assembling peptides, or amino acid polymers. Their main types and structural characteristics include but are not limited to: 1. Flexible Linkers: Structural Characteristics: Primarily composed of small, hydrophilic amino acids, particularly glycine (G) and serine (S). Typical sequence patterns include repeating units such as (GGGGS)n, (G)n, (GGGS)n`, and `(EAAAK)n (although EAAAK is often considered rigid, it also exhibits some flexibility in short bursts). They are typically 5-25 amino acids in length. Their structural role: They form a random coil or highly flexible polypeptide chain between the linked domains. They impose no steric constraints, allowing the linked domains to move, rotate, and fold independently with relative freedom. 2. Rigid Linkers: Structural Characteristics: They are rich in proline (P) and glutamic acid (E), or are designed to form specific secondary structures (such as α-helices). Typical sequences include (EAAAK)n (α-helical tendency), (PAPAP)n, and (AEAAAKEAAAKA)n. Their length is generally more precisely controlled than that of flexible linkers. Their structural role: They form relatively rigid rod-like or helical structures. Fixed spatial distance and relative orientation between linked domains, restricting their relative motion and reducing nonspecific interactions between domains. 3. Cleavable Linkers: Structural features: Contains sequences recognized by specific conditions or enzymes. Enzymatic Cleavage Sites: Specific protease recognition sequences. Common examples: Thrombin: LVPRGS; Enterokinase: DDDDK; Factor Xa: IEGR; TEV protease: ENLYFQG / S; Furin: RX(K / R)R (cleavage occurs after R); Cathepsins: GFLG, FRRG; Chemical Cleavage Sites; Acid-sensitive Linkers: Contain chemical bonds sensitive to low pH (such as hydrazone bonds and cis-aconamide bonds), often introduced as modifications alongside dipeptides such as Val-Cit and Phe-Lys. The sequence itself may be designed to contain these dipeptides or analogs (such as Cit-Val for ADCs). Reduction-sensitive linkers: These contain a disulfide bond (-Cys-Cys-) or are designed to attach to a Cys residue, cleaved by reduction in high intracellular glutathione concentrations. Photocleavable linkers: These contain groups sensitive to specific wavelengths of light (such as o-nitrobenzyl) and may include modified amino acids or specialized designs. Inteins: Special "self-splicing" protein elements. Although relatively long (>100 aa), their splicing reaction occurs at the junction of specific short sequences (exteins) at either end, ultimately achieving seamless fusion (no linker residue) or controlled release. Their structural role: Under specific conditions (enzymes, low pH, reducing environment, light), the linker disconnects, releasing the fused protein or functional domain.The structure of the linker itself maintains the connection before cleavage. 4. Subcellular Localization Signal Peptides: Structural Characteristics: Their primary function is to guide localization, but they also act as linkers when connecting proteins. Nuclear localization signals: rich in basic amino acids (e.g., PKKKRKV); mitochondrial localization signals: amphipathic α-helices (e.g., MLSLRQSIRFFKPATRTLCSSRYLL); endoplasmic reticulum retention signals: KDEL (mammalian) / HDEL (yeast). Structural Role: After directing the fusion protein to a specific organelle, these sequences may be cleaved by signal peptidases (e.g., secretory signal peptides) or retained (e.g., KDEL). When acting as linkers, their structural characteristics (e.g., the positive charge of the NLS) may influence the interactions of the linker domains. 5. Self-Assembling Peptides / Domain Linkers: Structural Characteristics: Short peptides designed to form specific higher-order structures (e.g., coiled-coil, β-pleated sheet) are used to connect protein monomers and enable self-assembly into multimeric structures. Examples include leucine zippers (`L / EXXXLXXLXXXL), isoleucine zippers (IAAL E3, IAAL K3). Structural role: By forming specific oligomerization structures (dimers, trimers, tetramers, etc.), multiple fusion protein monomers are stably connected and assembled into complexes with higher-order functions. Its core functions include: 1) Spatial separation: Preventing steric hindrance between linked protein domains, ensuring that each can independently and correctly fold into its native conformation, and reducing unnecessary non-functional interactions between domains. 2) Conformation and orientation control: Rigid linkers precisely fix the distance and relative angle between linked domains, which is crucial for functions requiring specific spatial arrangements (such as bispecific antibody binding). 3) Enhanced stability: Suitable linkers can reduce the exposure of hydrophobic patches at domain interfaces, improving the solubility of fusion proteins. 4. Functional Domain Release: Cleavable linkers allow the linked functional domain (e.g., toxin, activator) to be cleaved and released at a specific time point (e.g., after drug delivery to target cells) or at a specific location (e.g., within a specific organelle), allowing it to exert its activity. 5. Facilitation of Purification and Detection: Attaching affinity tags (e.g., His-tag, GST, MBP) or epitope tags (e.g., FLAG, HA, Myc) allows for purification, detection, or immobilization of fusion proteins.These tags themselves are linked to the target protein via linkers. Linkers with enzyme cleavage sites are often used to remove the tag after purification. 6. Subcellular Targeting: Linkers containing targeting signals can direct fusion proteins to specific organelles (such as the nucleus, mitochondria, and endoplasmic reticulum). 7. Mediating Multimerization: Self-assembling peptide linkers can promote the self-assembly of fusion protein monomers into multimers (such as dimers and trimers) with specific functions and higher affinity. Key application areas include antibody engineering, recombinant protein expression and purification, protein structure and function research, drug delivery systems, synthetic biology and biomaterials, and cell and gene therapy. These linkers come in a variety of types (flexible, rigid, cleavable, with targeting signals, and self-assembly) and distinct structural features (amino acid composition, secondary structure propensity, and length). Their core function is to regulate the folding, conformation, spacing, orientation, stability, active release, localization, and assembly of the linker domains. They possess indispensable application value in cutting-edge fields such as antibody-based therapeutics (ADCs, BsAbs), recombinant protein production, basic research, drug delivery, synthetic biology, and cell therapy. Linker selection and optimization are crucial for successfully constructing functional fusion proteins and require rational design and experimental validation based on specific application requirements. A flexible linker peptide (G4S)3 with 15 amino acid residues was used to connect VL and VH, demonstrating the potential for such connections, yielding amino acid sequences such as aHs.

[0011] Using the XmY structural strategy, an antibody, such as pertuzumab, was selected as a model to construct a HER2 single-chain antibody. The highly hydrophilic anti-HER2 single-chain antibody sequence and its application were successfully expressed. The m linker, modeled by a flexible peptide (G4S)3, can connect the X light chain variable region to the Y heavy chain variable region [or vice versa] to construct an anti-HER2 single-chain antibody. Based on the similarity between the chemical structure and electron density maps of the amino acids, the hydrophobic amino acids in the scFv were selectively replaced with hydrophilic amino acids to produce the protein-encoded anti-HER2 single-chain antibody.

[0012] Furthermore, the hydrophobic amino acids include one or more of tryptophan Trp, phenylalanine Phe, valine Val, leucine Leu, isoleucine Ile, alanine Ala, proline Pro and methionine Met; and / or The hydrophilic amino acids include one or more of glycine Gly, serine Ser, threonine Thr, cysteine ​​Cys, tyrosine Tyr, aspartic acid Asp and glutamic acid Glu.

[0013] Furthermore, the ratio of selective amino acid replacement in the transmembrane region sequence is 10%-100%, for example, it can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.

[0014] Furthermore, the amino acid sequence of the anti-HER2 single-chain antibody is selected from: (a) the amino acid sequence shown in SEQ ID NO: 1; or (b) the amino acid sequence shown in SEQ ID NO: 2; or (c) an amino acid sequence derived from the amino acid sequence defined in (a) by substitution and / or deletion and / or addition of one or more amino acid residues, and having the same function as the amino acid sequence defined in (a); or (d) An amino acid sequence derived from the amino acid sequence defined in (b) by substitution and / or deletion and / or addition of one or more amino acid residues, and having the same function as the amino acid sequence defined in (b).

[0015] This anti-HER2 single-chain antibody is composed of 214 amino acid residues, with an aHs molecular weight of 25.8kDa, a QaHs of 26.1kDa, an isoelectric point of 8.29, a QaHs of 8.22, a GRAVY of -0.308, and a QaHs of -1.461. Physicochemical property prediction analysis showed that the scFv before and after protein encoding modification showed similar physicochemical properties such as molecular weight and isoelectric point. The GRAVY decreased from -0.308 to -1.461, significantly reducing the protein's hydrophobicity.

[0016] This anti-HER2 single-chain antibody did not significantly alter the molecular weight (difference <2%) and isoelectric point (shift <0.1 pH unit) of the scFv, suggesting that its core structural framework was preserved. Furthermore, sequence similarity in the CDR regions (62.5%-100% similarity in CDR1-3 for VL and 66.7%-90% similarity in CDR1-3 for VH) further demonstrated that the conformational stability of the key antigen-binding site was not significantly affected.

[0017] The optimized QaHs amino acid sequence was used to construct an expression vector. Restriction sites were selected, and the QaHs gene sequence was synthesized through gene synthesis. Enzyme digestion, ligation, and transformation into host cells were performed. Colony-directed PCR was used to assess transformation status, and colonies that met the requirements were preserved and sequenced. DNA sequencing confirmed the correct results, indicating successful construction of the engineered expression strain.

[0018] A nucleotide encoding the above-mentioned anti-HER2 single-chain antibody, wherein the nucleotide sequence of the nucleotide is selected from: (e) the nucleotide sequence shown in SEQ ID NO: 3; or (f) the nucleotide sequence shown in SEQ ID NO: 4; or (g) a nucleotide sequence that has at least 69% similarity to the nucleotide sequence specified in (e) and encodes a protein with the same function; or (h) A nucleotide sequence that has at least 69% similarity to the nucleotide sequence defined in (f) and encodes a protein with the same function.

[0019] It is understood that (g) is a nucleotide sequence that has at least 69% similarity to the nucleotide sequence defined in (e), and encodes a protein with the same function, wherein (g) is a nucleotide sequence that has at least 69%, 70%, 73%, 75%, 76%, 78%, 80%, 81%, 83%, 85%, 87%, 88%, 89%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or 99.9% similarity to the nucleotide sequence defined in (e), and encodes a protein with the same function; (h) has at least 69% similarity to the nucleotide sequence defined in (f), and encodes a nucleotide sequence of the same functional protein, wherein (h) has at least 69%, 70%, 73%, 75%, 76%, 78%, 80%, 81%, 83%, 85%, 87%, 88%, 89%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or 99.9% similarity to the nucleotide sequence defined in (f), and encodes a nucleotide sequence of the same functional protein.

[0020] An expression vector comprising the above-mentioned nucleotides; the restriction enzyme cleavage site of the expression vector is selected from one or more combinations of NdeI, EcoRI, and XhoI; the expression vector is selected from one or more combinations of DNA, RNA, viral vectors, plasmids, transposons, and other gene transfer systems.

[0021] Furthermore, the vector is a pET-30a(+) vector.

[0022] A host cell containing the above-mentioned nucleotide or the above-mentioned expression vector.

[0023] Furthermore, the host cells include Escherichia coli, yeast cells, and mammalian cells.

[0024] Furthermore, the host cell is a BL21 (DE3) Escherichia coli competent cell or a BL21 (DE3) Escherichia coli competent cell.

[0025] The constructed expression vector was transformed into host cells for expression. Protein expression was verified by SDS-PAGE and Western blot. The correct bands appeared after induced expression, while no distinct bands were observed in the empty control group. The expressed QaHs protein was purified using a protein purifier and assessed by SDS-PAGE combined with Coomassie Brilliant Blue staining. Purity analysis confirmed that the optimized QaHs protein had a purity exceeding 90%.

[0026] A biomaterial, wherein the biomaterial is selected from one of the following (A) to (E), (A) the aforementioned nucleotide; (B) an expression cassette, a recombinant vector, a recombinant primary cell, or a recombinant cell line containing the nucleotide described in (A); (C) the aforementioned anti-HER2 single-chain antibody; (D) the aforementioned expression vector; (E) the aforementioned host cell.

[0027] A pharmaceutical composition comprising the above-mentioned anti-HER2 single-chain antibody and one or more pharmaceutically acceptable carriers.

[0028] Use of the above-mentioned anti-HER2 single-chain antibody; and / or the above-mentioned expression vector; and / or the above-mentioned host cell; and / or the above-mentioned biological material; and / or the above-mentioned pharmaceutical composition in the preparation of products for screening, treating or preventing HER2-positive non-small cell lung cancer, breast cancer or gastric cancer.

[0029] Use of the above-mentioned anti-HER2 single-chain antibody; and / or the above-mentioned expression vector; and / or the above-mentioned host cell; and / or the above-mentioned biological material; and / or the above-mentioned pharmaceutical composition in the preparation of products for screening, treating or preventing lung diseases.

[0030] The beneficial effects of the present invention are as follows: 1. This invention, for the first time, applies protein encoding technology to optimize the hydrophilicity of a HER2-targeting scFv, overcoming the potential interference of traditional modification strategies on the conformation of the CDR region, significantly improving its hydrophilicity and enabling its stability in aqueous solution. Through prokaryotic expression, QaHs was successfully expressed and purified, resulting in innovative drug candidate compounds, providing an important tool and platform for studying their structure, function, and drug screening.

[0031] 2. In the present invention, the hydrophobic amino acids in the amino acid sequence of the single-chain antibody are selectively replaced with hydrophilic amino acids, which significantly improves its solubility without changing the structure and function of the antibody, allowing it to exist stably in aqueous solution. The selectively replaced single-chain antibody is successfully expressed and purified through prokaryotic expression. The selectively replaced single-chain antibody can enhance the hydrophilicity and biological activity of the antibody, solving the defects of existing antibodies such as weak tissue penetration due to excessive molecular weight, difficulty in penetrating dense tumor matrix, and easy induction of immunogenic reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Figure 3 shows the design of the single-chain antibody of the present invention under the XmY structural strategy and the amino acid sequences of the single-chain antibody before and after protein encoding technology processing. The results show that the substitution rate of hydrophobic amino acid residues in QaHs reached 20.75%, indicating that protein encoding technology may significantly reduce the hydrophobicity of the antibody, but may also reduce the antibody's antigen-binding ability. Figure 2 Using the XmY structural strategy, we predicted the hydrophobicity, secondary structure, and physicochemical properties of single-chain antibodies before and after protein encoding. The results preliminarily demonstrate that protein encoding technology can effectively enhance the hydrophilicity of scFv while maintaining its binding capacity, laying the foundation for subsequent recombinant expression and functional verification. Figure 3 The three-dimensional structure prediction and three-dimensional structure Pull-type diagram of a single-chain antibody before and after processing using the protein encoding technology of the present invention under the XmY structure strategy are shown. The results show that the prediction model meets the reliable standards in terms of spatial conformation and stereochemical parameters, meeting the structural accuracy requirements for molecular docking and functional studies. Figure 4 The docking of single-chain antibodies (scFvs) with the HER2 receptor molecule before and after protein encoding using the XmY structural strategy was performed. The results showed that the binding sites of the two scFvs to the HER2 receptor and the hydrogen bonds formed were similar to those previously studied for the HER2 receptor binding epitope, indicating that the designed scFv models have biological plausibility for binding to the HER2 receptor. Figure 5 The nucleotide sequence of the single-chain antibody designed and synthesized using the protein encoding technology of the present invention before and after treatment using the XmY structural strategy was shown. The results showed that its base composition was 100% identical to the designed sequence, and its open reading frame remained intact, with no frameshift mutations. Figure 6The single-chain antibody gene sequences obtained using the protein encoding technology of the present invention before and after processing under the XmY structural strategy were obtained. The results showed that the amplification and purification of the aHs and QaHs genes met expectations, laying the foundation for subsequent cloning into expression vectors and protein expression experiments. Figure 7 This is the construction of a single-chain antibody recombinant plasmid vector before and after protein encoding technology treatment using the XmY structural strategy. The results show that the scFv recombinant plasmid vector construction process is stable and reliable, laying the foundation for subsequent transformation engineering bacteria experiments; Figure 8 The results show the construction of single-chain antibody engineering bacteria before and after protein encoding technology treatment using the XmY structural strategy. The results show that the transformation, identification, and storage process of scFv recombinant plasmid vectors is stable and reliable, providing a standardized experimental system for subsequent protein expression research. Figure 9 The results were based on the XmY structural strategy and were used to characterize the expression of single-chain antibodies before and after protein encoding. The results showed that both aHs and QaHs scFvs were successfully expressed in prokaryotic cells, and the expression products possessed the expected molecular characteristics. Figure 10 The optimized expression of single-chain antibodies before and after protein encoding technology treatment was performed under the XmY structural strategy. The optimal expression conditions for aHs and QaHs were determined to be: induction temperature 37°C, induction time 9 hours, and inducer IPTG concentration 0.5 mM. Figure 11 Single-chain antibody affinity chromatography purification; ABaHs inclusion body treatment and affinity chromatography purification; CDQaHs inclusion body treatment and affinity chromatography purification; where M: protein molecular weight standard; 1: before IPTG induction; 2: after IPTG induction; 3: supernatant after bacterial disruption; 4: precipitate after bacterial disruption; 5-7: inclusion body washing; 8: inclusion body denaturation; 9: before purification; 10: flow-through; 11: wash solution; 12: after purification; Figure 12 The results show the refolding and concentration of single-chain antibodies before and after protein encoding using the XmY structural strategy. These experimental data confirm that the purification process established in this study is highly efficient and reproducible, making it suitable for large-scale production of scFvs. Figure 13 The biological activity of single-chain antibodies (scFvs) before and after treatment with the protein encoding technology of the present invention, using the XmY structural strategy, was tested. The results showed that the scFv had selective inhibitory effects on normal lung cells and tumor organoids, which may be related to the expression level of its target. Figure 14This is the construction and preliminary testing of organoid chips using the XmY structural strategy, before and after protein encoding technology treatment. The results show that scFv has an inhibitory effect on tumor organoids. DETAILED DESCRIPTION

[0033] The meaning of tumor in the present invention generally refers to: malignant tumors, which are characterized by rapid growth, invasive growth, interlaced with normal tissues, and unclear boundaries. The degree of tissue differentiation is low, the atypia is significant, and the cell morphology is abnormal. It has the ability to metastasize and can spread to distant organs through lymphatic channels, blood channels or implantation, and has a high recurrence rate after treatment. If classified based on tissue origin: including epithelial tissue tumors, squamous cell carcinomas, adenocarcinomas such as lung adenocarcinoma, gastric cancer, pancreatic ductal adenocarcinoma, easy early hematogenous metastasis, undifferentiated cancers, mesenchymal tissue tumors such as fibrosarcoma, liposarcoma, osteosarcoma, neural tissue tumors such as gliomas (such as glioblastoma) and schwannomas, blood and lymphatic system tumors such as leukemia, lymphoma, etc.

[0034] The present invention establishes an XmY structural strategy, selecting an antibody (pertuzumab) as a model, where X represents the light chain variable region or the heavy chain variable region, m represents a linker, and Y represents the heavy chain variable region or the light chain variable region. Using this method, a monoclonal antibody was selected as a model and a model was established, resulting in a novel structure and function. The present invention will be further described below with reference to the following examples. The examples described are only some of the examples of the present invention and are not intended to be exhaustive. Based on the examples of the present invention, any other examples obtained by persons of ordinary skill in the art without inventive effort are also within the scope of protection of the present invention.

[0035] Example 1 This example is used for the design and optimization of the amino acid sequence of an anti-HER2 single-chain antibody, specifically: (1) Query databases such as NCBI, PDB, and UniProt. In the XmY structure strategy, select a cloned antibody. Here, the monoclonal antibody Pertuzumab is selected as a model to obtain the amino acid sequence of the monoclonal antibody.

[0036] (2) Using the VL and VH of the monoclonal antibody as the basic framework, the X and Y in "XmY" are formed respectively, and a connection model is selected as a representative and a flexible connecting peptide (G4S)3 of 15 amino acid residues is used to connect VL and VH to obtain the aHs amino acid sequence.

[0037] (3) Protein encoding technology was used to modify and transform aHs. In the structure of XmY, the hydrophobic amino acids contained in the protein were systematically and logically replaced with hydrophilic amino acids to obtain the QaHs amino acid sequence, which is shown in SEQ ID NO: 1.

[0038] The results showed that the substitution rate of hydrophobic amino acid residues in QaHs reached 20.75%, indicating that protein encoding technology can significantly improve the hydrophilicity of antibodies. This further demonstrates that the structural strategy in XmY is reasonable, the method is reliable, and has the potential for widespread application in the future.

[0039] Example 2 This example is used for bioinformatics prediction of anti-HER2 single-chain antibodies, specifically: (1) Prediction of scFv physicochemical properties: Use the ProtParam tool in the Expasy database to input the scFv amino acid sequence (sequence shown in SEQ ID NO: 1) to predict and analyze its physicochemical properties.

[0040] (2) Prediction of scFv hydrophobic properties: Use the protein hydrophobic property analysis tool in the NovoPro database to input the scFv amino acid sequence (sequence as shown in SEQ ID NO: 1) to predict and analyze its hydrophobic properties.

[0041] (3) Prediction of scFv secondary structure: Use the protein secondary structure prediction tool in the NovoPro database to input the scFv amino acid sequence (sequence shown in SEQ ID NO: 1) to predict and analyze its secondary structure.

[0042] The results showed that protein encoding technology can effectively improve the hydrophilicity of scFv while maintaining its binding ability, laying the foundation for subsequent recombinant expression and functional verification. Furthermore, bioinformatics predictions showed that the structural strategy of XmY is reasonable, the method is reliable, and has the potential for widespread application in the future.

[0043] Example 3 This example is used to predict the three-dimensional structure of an anti-HER2 single-chain antibody, specifically: (1) Use the 3D structure prediction tool in AlphaFold 3, input the scFv amino acid sequence (sequence as shown in SEQ ID NO: 1) for 3D structure modeling, and download the model file.

[0044] (2) Use the PROCHECK tool in SAVES to evaluate the rationality of the predicted 3D structure and select the most reasonable scFv 3D structure.

[0045] The results show that the prediction model meets the reliable standards in terms of spatial conformation and stereochemical parameters, and meets the structural accuracy requirements of molecular docking.

[0046] Example 4 This example is used for docking of anti-HER2 single-chain antibody with HER2 receptor molecules, specifically: (1) Query databases such as NCBI, PDB, and UniProt to obtain high-resolution 3D structure files of the HER2 receptor.

[0047] (2) Use PyMOL software to remove other molecules such as ligand molecules and solvent molecules other than the Pertuzumab-HER2 complex and save the model file.

[0048] (3) Upload the model files of scFv and HER2 receptor to the following websites: AlphaFold 3, ClusPro, GRAMM, and ZDOCK, perform molecular docking of scFv (i.e., anti-HER2 single-chain antibody) with HER2 receptor, and download the docking model files.

[0049] (4) Use the PDBePISA tool to evaluate the rationality of molecular docking based on the interaction free energy ΔG < 0, and select the most reasonable model file.

[0050] (5) PyMOL software was used to display the amino acid residues that interact with the HER2 receptor and the hydrogen bonds formed by scFv (i.e., anti-HER2 single-chain antibody).

[0051] The results showed that the sites where the two scFvs bind to the HER2 receptor and the hydrogen bonds formed are similar to the previous binding epitopes of the Pertuzumab-HER2 complex, indicating that the designed scFv model has biological rationality in binding to the HER2 receptor.

[0052] Example 5 This example is used to design and synthesize the nucleotide sequence encoding the anti-HER2 single-chain antibody, specifically: (1) The codon preference of E. coli coding sequences was systematically analyzed using the GenSmart™ codon optimization tool, and the amino acid sequence was reverse translated into the corresponding nucleotide sequence.

[0053] (2) The scFv nucleotide sequence synthesis work will be completed by the company.

[0054] The results showed that its base composition was 100% consistent with the designed sequence, and its open reading frame remained intact without any frameshift mutation.

[0055] Example 6 This example is used to obtain the gene sequence of an anti-HER2 single-chain antibody, specifically: (1) PCR amplification reaction of gene sequence 1) Analyze the gene sequences of aHs and QaHs and the restriction enzyme cleavage sites of the plasmid vector pET-30a(+) using SnapGene software. PCR primers for the two scFv gene sequences were designed and synthesized by the company. The primer sequences are shown in Table 2. The underlined portions indicate the designed restriction enzyme cleavage sites. The amplified product is expected to be approximately 750 bp.

[0056] Table 2 Single-chain antibody gene sequence primer names and sequences

[0057] 2) Prepare a reaction mixture by adding the ingredients in the order listed in Table 3 to a centrifuge tube in an ice bath.

[0058] Table 3 PCR reaction system for single-chain antibody gene sequence

[0059] 3) Set up the PCR reaction program according to the conditions shown in Table 4.

[0060] Table 4 PCR reaction procedures for single-chain antibody gene sequences

[0061] 4) Vortex the mixture from step 2) and centrifuge briefly. Immediately transfer the mixture to the PCR instrument set in step 3) for amplification.

[0062] 5) After the reaction environment temperature drops to room temperature, remove the PCR product and purify the PCR product.

[0063] (2) Purification of gene sequence PCR products 1) Transfer the PCR reaction product to a 1.5 mL centrifuge tube, add Buffer B3 at a 1:5 volume ratio, and gently vortex to mix until the system is homogenized.

[0064] 2) Transfer the mixture to the adsorption column and centrifuge at 8000 × g for 30 seconds. Discard the waste liquid and return the purification column to the original collection tube.

[0065] 3) Inject 500 μL of rinse solution into the column and centrifuge at 9000 × g for 30 seconds. Discard the waste solution and return the column to the collection tube.

[0066] 4) Repeat step 3) once.

[0067] 5) Place the adsorption column and collection tube in a centrifuge and centrifuge at 9000×g for 1 min.

[0068] 6) Place the adsorption column in a clean centrifuge tube. Apply 20 μL of elution buffer to the center of the adsorption membrane. Let stand at room temperature for 2 minutes. Place the adsorption column and collection tube in a centrifuge and centrifuge at 9000 × g for 1 minute to collect the DNA solution.

[0069] 7) Take a certain amount of DNA solution and test it using agarose gel electrophoresis. Store the remaining DNA solution at -20°C for use in subsequent experiments.

[0070] (3) Agarose gel electrophoresis detection 1) Mix an appropriate amount of DNA solution with DNA loading buffer at a ratio of 5:1.

[0071] 2) Inject 5 μL of DNA Ladder and the mixed solution from step 1) into the agarose gel wells according to the preset loading order.

[0072] 3) Set the voltage to 100 V during electrophoresis. Stop electrophoresis when the dye reaches the edge of the agarose gel. Remove the gel and place it in the instrument to photograph and analyze the nucleic acid properties.

[0073] The results showed that the amplification and purification of aHs and QaHs genes were in line with expectations, laying the foundation for subsequent cloning into expression vectors and protein expression experiments.

[0074] Example 7 This example is used to construct a recombinant plasmid vector for an anti-HER2 single-chain antibody, specifically: (1) Enzyme digestion reaction between gene sequence and plasmid vector 1) Prepare a reaction mixture by adding the ingredients to a centrifuge tube in the order listed in Table 5.

[0075] Table 5 Reaction system of gene sequence and plasmid vector restriction enzyme digestion

[0076] 2) Incubate at 37°C for 15 minutes.

[0077] 3) The experimental steps for purification of gene sequence enzyme digestion products are the same as before.

[0078] 4) Agarose gel recovery of plasmid vector digestion products: a. Mix the digested plasmid vector with DNA loading buffer in a ratio of 5:1.

[0079] b. Inject 5 μL of DNA Ladder and the mixed solution from step a. into the agarose gel wells according to the preset loading order.

[0080] c. Run electrophoresis at a constant voltage of 100 V until the dye migrates to the edge of the gel. Cut the gel containing the single target DNA band, accurately weigh it, and transfer it to a new centrifuge tube.

[0081] d. Add 3 times the volume of sol solution and incubate at 50℃ for 10 min until the gel is completely dissociated.

[0082] e. Transfer the mixture to the adsorption column, let it stand at room temperature for 2 minutes, then centrifuge at 12000 rpm for 60 seconds. After removing the waste liquid, return the adsorption column to the collection tube.

[0083] f. Inject 600 μL of rinse solution into the adsorption column, centrifuge at 12,000 rpm for 60 seconds, remove the waste liquid, and return the adsorption column to the collection tube.

[0084] g. Repeat step f.

[0085] h. Place the adsorption column back into the collection tube and centrifuge at 12,000 rpm for 2 minutes to thoroughly remove the remaining rinse solution. Dry the adsorption column at room temperature.

[0086] i. Place the adsorption column in a clean centrifuge tube, add 30 μL of elution buffer to soak the center of the adsorption membrane, let it stand for 2 minutes, and then centrifuge at 12,000 rpm for 2 minutes to collect the DNA solution.

[0087] j. Take a certain amount of DNA solution and test it using agarose gel electrophoresis. Store the remaining DNA solution at -20°C for use in subsequent experiments.

[0088] 5) The agarose gel electrophoresis test procedure is the same as before.

[0089] (2) Ligation reaction between gene sequence and plasmid vector 1) Prepare the reaction mixture by adding the ingredients into a centrifuge tube in the order shown in Table 6.

[0090] Table 6 Gene sequence and plasmid vector ligation reaction system

[0091] 2) Incubate at 22°C for 10 min.

[0092] The results showed that the construction process of scFv recombinant plasmid vector was stable and reliable, laying the foundation for subsequent transformation engineering bacteria experiments.

[0093] Example 8 This example is used to construct an engineered bacterium for producing an anti-HER2 single-chain antibody, specifically: (1) Transformation of recombinant plasmid vector into Escherichia coli 1) Remove three tubes of BL21(DE3) competent cells from the -80°C cryostat and quickly transfer to an ice-water bath for thawing.

[0094] 2) Take three tubes of BL21(DE3) competent cells and add aHs-pET-30a(+), QaHs-pET-30a(+), and empty-pET-30a(+) plasmids, respectively. Gently tap the bottom of the tube 2-3 times to mix the contents, then quickly place the tube in an ice-water bath and let it stand for 30 minutes.

[0095] 3) Quickly transfer the tube from the ice bath to a 42°C water bath. Maintain the heat shock for 45 seconds. Immediately terminate the reaction and return the tube to the ice bath for another 120 seconds. Avoid shaking the tube during this step.

[0096] 4) Add 900 μL of antibiotic-free LB medium to the centrifuge tube and gently invert several times to mix. Incubate the tube in a shaker at 37°C, 200 rpm for 1 hour.

[0097] 5) Gently vortex to resuspend the cells. Spread an appropriate amount of the suspension evenly onto LB solid medium containing 50 μg / mL kanamycin. Allow the plate to stand upright until the suspension is completely absorbed. Invert the plate and incubate in a 37°C incubator for 12 hours.

[0098] (2) Identification of positive monoclonal colonies by direct colony PCR 1) Gene sequence analysis of the pET-30a(+) vector plasmid was performed using SnapGene software, and universal primers were constructed. These primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. The primer sequences are shown in Table 7.

[0099] Table 7 Names and sequences of universal primers for plasmid vectors

[0100] 2) When milky white monoclonal colonies with a diameter of 1-2 mm form on the surface of LB solid medium, pick several monoclonal colonies and transfer them to a sterile centrifuge tube pre-filled with 10 µL of ultrapure water. Gently pipette the solution to mix thoroughly to prepare a dilution suspension of the colonies.

[0101] 3) Thaw 2× E. coli Colony Direct PCR Mix at room temperature, gently mix by inverting, and centrifuge at low speed for a few seconds.

[0102] 4) Prepare a reaction mixture by adding the ingredients to a centrifuge tube in the order shown in Table 8 in an ice bath.

[0103] Table 8 Colony direct PCR reaction system

[0104] 5) Set up the PCR reaction program according to the conditions shown in Table 9.

[0105] Table 9 Colony direct PCR reaction procedure

[0106] 6) Vortex the mixture from step 4) and centrifuge briefly. Immediately transfer the mixture to the PCR instrument set in step 5) for amplification.

[0107] 7) After the reaction system has reached room temperature, remove the PCR product.

[0108] 8) The agarose gel electrophoresis test procedure is the same as before.

[0109] (3) DNA sequencing to identify positive monoclonal colonies 1) Inoculate the bacteria that tested positive in the initial colony PCR screening into LB liquid medium containing 50 μg / mL kanamycin. Incubate the culture at 200 rpm in a 37°C constant temperature shaking incubator. Terminate the culture when the OD600 of the culture solution is approximately 0.6.

[0110] 2) Take an appropriate amount of amplified bacterial sample and send it to Beijing Qingke Biotechnology Co., Ltd. for sequencing analysis.

[0111] (4) Preservation of positive strains 1) Measure the bacterial suspension with accurate sequencing results and add an equal volume of 50% glycerol suspension to mix thoroughly.

[0112] 2) Aliquot into cryovials (1 mL per tube), label them as aHs-pET-30a(+)-BL21(DE3), QaHs-pET-30a(+)-BL21(DE3), and Empty-pET-30a(+)-BL21(DE3), and store them in a -80°C cryogenic freezer.

[0113] (5) Preservation of recombinant plasmid 1) Take 15 mL of each aHs-pET-30a(+)-BL21(DE3) and QaHs-pET-30a(+)-BL21(DE3) bacterial suspension, centrifuge at 13,000 rpm for 1 min, collect the bacterial pellet, and completely remove the supernatant.

[0114] 2) Add 500 μL of buffer P1 to the resulting bacterial pellet and resuspend by vortexing.

[0115] 3) Add 500 μL of buffer P2, gently invert and mix 10 times, and lyse at room temperature for 5 minutes.

[0116] 4) Inject 500 μL of Buffer E3, invert and mix 10 times, and let stand at room temperature for 5 minutes. Centrifuge at 13,000 rpm for 5 minutes, remove the supernatant, pass it through the adsorption column, and transfer the filtrate to a clean centrifuge tube.

[0117] 5) Add 450 μL of isopropanol to the filtrate system and mix thoroughly.

[0118] 6) Equilibrate the adsorption column with 200 μL of Buffer PS, centrifuge at 13,000 rpm for 2 minutes, discard the waste liquid, and return the adsorption column to the collection tube.

[0119] 7) Transfer the mixed solution prepared in step 5) to the adsorption column that has completed the equilibrium treatment.

[0120] 8) After centrifugation at 13,000 rpm for 1 minute, discard the waste liquid and return the adsorption column to the collection tube.

[0121] 9) Inject 750 μL of buffer PW into the adsorption column, centrifuge at 13,000 rpm for 1 min, and discard the waste liquid.

[0122] 10) Place the adsorption column back into the collection tube and centrifuge at 13,000 rpm for 1 minute.

[0123] 11) Transfer the adsorption column to a new centrifuge tube, add 100 μL of Buffer EB, and let it stand at room temperature for 5 minutes. Centrifuge at 13,000 rpm for 2 minutes to collect the DNA solution. Label the samples as aHs-pET-30a(+) and QaHs-pET-30a(+) and store at -20°C.

[0124] The results showed that the transformation, identification and storage process of scFv recombinant plasmid vector was stable and reliable, providing a standardized experimental system for subsequent protein expression research.

[0125] Example 9 This example is used to identify the induced expression of an anti-HER2 single-chain antibody, specifically: (1) scFv-induced expression 1) Use a sterile inoculating loop to pick the aHs-pET-30a(+)-BL21(DE3), QaHs-pET-30a(+)-BL21(DE3), and empty-pET-30a(+)-BL21(DE3) strains stored at -80°C and inoculate them into LB liquid medium containing 50 μg / mL kanamycin. Incubate the culture at 37°C with constant shaking at 200 rpm for 12 h to allow bacterial recovery.

[0126] 2) The next day, transfer the activated bacterial suspension to LB liquid medium containing 50 μg / mL kanamycin at a 1:100 inoculum ratio and expand the culture under the same temperature control parameters.

[0127] 3) When the OD600 of the culture system reaches 0.6, add IPTG inducer at a final concentration of 1 mM, maintain a constant temperature of 37°C, and continue shaking to induce expression for 12 hours to complete the induced expression of the target protein.

[0128] (2) scFv protein preparation 1) After terminating the induced expression, transfer the bacterial suspension to a centrifuge tube and centrifuge at 6000 rpm for 10 min at 4°C to obtain the bacterial pellet. After the centrifugation is complete, completely aspirate and discard the remaining supernatant.

[0129] 2) Add an appropriate volume of bacterial protein to prepare lysis buffer and resuspend the bacterial pellet at the bottom of the centrifuge tube.

[0130] 3) Rinse the ultrasonic cell disruptor probe three times with ultrapure water. Place the mixed bacterial solution in an ice-water bath for ultrasonic disruption. Set the ultrasonic equipment power to 400 W and use an intermittent mode of sonication with a 3-second pause followed by a 6-second sonication cycle for a total of 30 minutes.

[0131] 4) After sonication, transfer the sample to a centrifuge and centrifuge at 6000 rpm for 30 minutes at 4°C. Collect the supernatant (soluble protein) and the precipitated inclusion body protein.

[0132] (3) SDS-PAGE 1) Pipette the supernatant into a new pre-chilled centrifuge tube and resuspend the inclusion body pellet in an appropriate volume of bacterial protein preparation lysis buffer.

[0133] 2) Take an appropriate amount of protein sample and mix it with protein loading buffer in a 4:1 ratio, and place it in a 100℃ metal bath for 5 minutes for heat denaturation.

[0134] 3) Add 5 μL of Protein Ladder and the mixed solution from step 2) to the SDS-PAGE gel loading wells according to the predetermined order.

[0135] 4) Set the voltage to 100 V during electrophoresis and stop when bromophenol blue reaches the edge of the SDS-PAGE gel.

[0136] (4) Coomassie Brilliant Blue Staining 1) After electrophoresis, rinse the gel gently with ultrapure water, slowly discarding the liquid to avoid structural damage. Add Coomassie Brilliant Blue stain and place on a shaker at a constant speed for 60 minutes to ensure adequate and uniform staining.

[0137] 2) After staining, carefully remove the staining solution, add an appropriate amount of ultrapure water, and destain on a shaker for 30 minutes. Repeat this step twice. Then, destain overnight with an appropriate amount of ultrapure water. The next day, place the gel in an instrument to photograph and analyze protein expression.

[0138] (5) Western Blot 1) After electrophoresis, remove the gel and immediately immerse it in transfer buffer.

[0139] 2) After activating the PVDF membrane with methanol for 10 seconds, rinse it with ultrapure water for 60 seconds and then immerse it in transfer buffer for later use.

[0140] 3) Assemble the transfer cassette using the wet transfer method and place it in the electrophoresis tank. Place the tank in an ice bath and transfer the membrane at a constant current of 200 mA for 40 minutes.

[0141] 4) After transfer, remove the PVDF membrane and wash it three times with ultrapure water, each time for 10 minutes, on a shaker.

[0142] 5) Submerge the PVDF membrane in blocking solution and block on a shaker at room temperature for 60 minutes. Wash the membrane three times with TBST buffer for 10 minutes each.

[0143] 6) Immerse the PVDF membrane with diluted Anti-6×His tag recombinant antibody and incubate overnight at 4°C on a shaker. Wash the PVDF membrane three times with TBST buffer for 10 minutes each.

[0144] 7) Immerse the PVDF membrane in diluted goat anti-rabbit secondary antibody and incubate on a shaker at room temperature for 60 minutes. Wash the membrane three times with TBST buffer for 10 minutes each.

[0145] 8) Prepare chromogenic solution A and chromogenic solution B in a 1:1 volume ratio. Place the PVDF membrane on a clean wax plate and evenly drip the chromogenic solution onto the membrane. Place the membrane in a luminescence instrument, take a photo, and save the image.

[0146] The results showed that both aHs and QaHs scFv were successfully expressed in the prokaryotic system, and the expression products had the expected molecular characteristics.

[0147] Example 10 This example is used to optimize the induced expression of an anti-HER2 single-chain antibody, specifically: (1) Induction temperature optimization 1) Resuscitate and expand the aHs-pET-30a(+)-BL21(DE3) and QaHs-pET-30a(+)-BL21(DE3) strains using the aforementioned method.

[0148] 2) When the OD600 of the bacterial solution reaches 0.6, add IPTG to a final concentration of 0.5 mM for induction.

[0149] 3) Aliquot the bacterial solution into shake tubes and place them in incubators at 16°C, 23°C, 30°C, and 37°C, respectively, with shaking at 200 rpm for 6 hours to induce expression of the target protein.

[0150] 4) The rest of the experimental steps are the same as before.

[0151] (2) Optimization of induction time 1) The experimental steps for strain recovery and expansion culture are the same as before.

[0152] 2) When the OD600 of the bacterial solution reaches 0.6, add IPTG to a final concentration of 0.5 mM for induction.

[0153] 3) Aliquot the bacterial solution into shake tubes, place them in an incubator, and culture at the optimal induction temperature with shaking at 200 rpm for 3, 6, 9, and 12 hours to induce the expression of the target protein.

[0154] 4) The rest of the experimental steps are the same as before.

[0155] (3) Optimization of inducer concentration 1) The experimental steps for strain recovery and expansion culture are the same as before.

[0156] 2) When the OD600 of the bacterial solution reaches ≈ 0.6, divide the bacterial solution into shake tubes and add IPTG at final concentrations of 0.25mM, 0.5mM, 0.75mM, and 1mM, respectively.

[0157] 3) Place the cells in an incubator and culture at the optimal induction temperature and 200 rpm for the optimal induction time to induce expression of the target protein.

[0158] 4) The rest of the experimental steps are the same as before.

[0159] The results showed that the optimal induction expression conditions for aHs and QaHs were: induction temperature 37°C, induction time 9h, and inducer IPTG concentration 0.5mM.

[0160] Example 11 This example is used for affinity chromatography purification of anti-HER2 single-chain antibodies, specifically: (1) Acquisition, washing and denaturation of inclusion bodies 1) Resuscitate and expand the aHs-pET-30a(+)-BL21(DE3) and QaHs-pET-30a(+)-BL21(DE3) strains using the aforementioned method.

[0161] 2) When the OD600 of the bacterial solution reaches 0.6, add IPTG to a final concentration of 0.5 mM for induction. Transfer the culture system to a 37°C constant temperature shaking incubator and continue induction culture at 200 rpm for 9 h.

[0162] 3) Lyse the bacteria using the aforementioned method to obtain inclusion bodies.

[0163] 4) Add an appropriate amount of inclusion body wash buffer to thoroughly resuspend the pellet. Centrifuge in a refrigerated centrifuge at 6000 rpm for 30 minutes at 4°C to collect the pellet. Repeat this step twice.

[0164] 5) Take an appropriate amount of inclusion body denaturation buffer to fully resuspend the precipitate. Place the solution in a refrigerated centrifuge and centrifuge at 6000 rpm at 4°C for 30 minutes. Collect the supernatant.

[0165] (2) Affinity chromatography purification 1) Flush the protein purifier tubing with ultrapure water at a flow rate of 5 mL / min and at least 5 times the volume of the chromatography column.

[0166] 2) Unscrew the stoppers at both ends of the chromatography column and connect the two ends of the chromatography column to the protein purification instrument pipeline.

[0167] 3) Equilibrate the column at a flow rate of 5 mL / min using at least 5 column volumes of ultrapure water to replace the storage buffer in the chromatography system.

[0168] 4) Equilibrate the column with at least 5 column volumes of inclusion body denaturing buffer at a flow rate of 5 mL / min until the UV absorbance shows a stable baseline.

[0169] 5) Slowly inject the denatured inclusion body sample into the protein purification instrument pipeline at a flow rate of 1 mL / min.

[0170] 6) Connect pump A of the protein purifier to the inclusion body denaturation buffer and pump B to the inclusion body elution buffer. Adjust the imidazole concentration in the buffer by controlling the ratio of pumps A to B, adjusting the imidazole concentration to 0 mM, 25 mM, 50 mM, and 75 mM. For each imidazole concentration, flush the column at a flow rate of 5 mL / min, using a flush volume of at least five times the column volume. This will elute nonspecifically bound proteins until the UV absorbance shows a stable baseline.

[0171] 7) Elute the target protein at a flow rate of 5 mL / min using inclusion body elution buffer with a volume of at least 5 times the column volume.

[0172] 8) Rinse the column at a flow rate of 5 mL / min with five column volumes of inclusion body denaturation buffer, ultrapure water, and 20% ethanol. Store the column in 20% ethanol at 4°C to prevent bacterial contamination.

[0173] The results showed that the purification process established in this study is efficient and reproducible, and is suitable for large-scale preparation of scFv.

[0174] Example 12 This example is used for the renaturation and concentration of anti-HER2 single-chain antibodies, specifically: (1) Non-interfering protein concentration determination 1) Remove 12 1.5 mL centrifuge tubes and label them in duplicate. Aliquot the standard protein solution into 0, 4, 8, 12, 20, and 25 μL portions according to the gradient principle, ensuring equal volumes between tubes with the same number.

[0175] 2) Take two other 1.5 mL centrifuge tubes and add 50 μL of the protein sample solution to each tube.

[0176] 3) Add 0.5 mL of Precipitation Reagent I to each tube, vortex mix for 30 seconds, and equilibrate at room temperature for 3 minutes.

[0177] 4) Add 0.5 mL of Precipitation Reagent II and vortex for 30 seconds.

[0178] 5) Centrifuge at 13,000 rpm for 15 min at 4°C to allow the protein to form a visible or invisible precipitate at the bottom of the tube.

[0179] 6) Remove the supernatant completely and place the tube upside down on filter paper to drain any remaining droplets.

[0180] 7) Inject 100 μL of copper ion solution and 400 μL of ultrapure water and continue shaking for 30 seconds to ensure complete dissolution of the precipitate.

[0181] 8) Add 1 mL of freshly prepared chromogenic reagent and invert repeatedly to mix evenly.

[0182] 9) Transfer 200 μL of the mixture to a 96-well microtiter plate and measure the absorbance at wavelength A480 within 10 minutes.

[0183] 10) Establish a linear regression model between the standard protein amount and the corresponding OD mean value.

[0184] 11) Based on the standard curve equation, calculate the concentration of the protein solution to be tested using the mean OD value of the samples.

[0185] (2) Gradient dialysis renaturation of inclusion bodies 1) Cut the dialysis bag into appropriate lengths, immerse it in dialysis bag cleaning solution I, and boil it for 10 minutes.

[0186] 2) After thoroughly washing the dialysis bag with ultrapure water, immerse the dialysis bag in dialysis bag cleaning solution II and boil for 10 minutes.

[0187] 3) After the solution has cooled, ensure that the dialysis bag is always immersed in the solution and clean the dialysis device with ultrapure water before use.

[0188] 4) Dilute the protein sample to 0.1 mg / mL using inclusion body denaturation buffer and slowly inject the protein sample into the dialysis device.

[0189] 5) Transfer the dialysis apparatus to refolding buffer solutions with decreasing urea concentrations. Perform dialysis in stages under continuous stirring at 4°C, replacing the refolding buffer solution every 12 hours.

[0190] 6) After urea gradient refolding is complete, transfer the dialysis apparatus to PBS-glycerol refolding buffer and dialyze at 4°C for 12 hours under continuous stirring.

[0191] (3) Protein concentration 1) Transfer the protein solution in the dialysis bag to a centrifuge tube and centrifuge at 6000 rpm at 4°C for 30 min.

[0192] 2) Take the supernatant protein solution and add it to an ultrafiltration centrifuge tube. Centrifuge at 3000×g at 4°C for 45 minutes.

[0193] 3) After centrifugation, collect the protein solution contained in the upper layer of the ultrafiltration tube.

[0194] (4) Removal of endotoxins and filtration sterilization 1) Take 500 μL of the protein sample to be processed and place it in a 1.5 mL endotoxin-free centrifuge tube.

[0195] 2) Measure 100 μL of Endotoxin Removal Solution A and add it to the centrifuge tube. Vortex the tube to mix it evenly. The solution will become turbid.

[0196] 3) Add 120 μL of Endotoxin Removal Solution B and mix thoroughly. The solution should still be turbid.

[0197] 4) Add 1 μL of Separation Indicator and mix thoroughly.

[0198] 5) Incubate at 4°C for 5 min, at which point the solution becomes transparent.

[0199] 6) Incubate at 37°C for 1 min. The solution will become milky white and turbid.

[0200] 7) Centrifuge at 14,000 × g for 5 minutes at room temperature to separate the two phases. The endotoxin-removed protein is located in the upper aqueous phase, and the endotoxin is located in the organic phase at the bottom of the centrifuge tube.

[0201] 8) Immediately transfer the upper aqueous phase to a 1.5 mL centrifuge tube and discard the lower organic phase to prevent further mixing of the aqueous and organic phases after prolonged storage, which would affect the endotoxin removal effect.

[0202] 9) After removing endotoxin, the solution was sterilized by filtration through a 0.22 μm filter. The resulting scFv solution was aliquoted and stored in a -80°C ultra-low temperature storage box for subsequent experiments.

[0203] The results showed that the target protein bands were clear and the scFv met the standards for biopharmaceutical applications.

[0204] Example 13 This example is used to test the biological activity of anti-HER2 single-chain antibodies, specifically: (1) PDOs and BEAS-2B cells were seeded into 96-well cell culture plates suitable for chemiluminescence detection according to the aforementioned method, with approximately 10,000 cells seeded per well. The effects of scFv on PDOs and normal lung cells were tested, respectively. At the same time, wells containing complete medium without cells or drug were set up as blank controls, and wells containing complete medium without drug were set up as negative controls.

[0205] (2) The next day, scFv was added to the complete culture medium of the experimental group to a final concentration of 1×10 -4 ~1×10 - 9 M, Place the 96-well cell culture plate in a cell culture incubator at 37°C with 5% CO2 and incubate for 48 h.

[0206] (3) After the drug incubation, cell viability was tested using the same method as before.

[0207] The results showed that the IC50 of aHs in the BEAS-2B model was 718.8 μM, while that of QaHs was 907.5 μM. In the PDOs model, the IC50 of aHs was 11.9 μM, while that of QaHs was 13.5 μM, with statistically significant differences (P < 0.05). These results demonstrate that the scFv has selective inhibitory effects on normal lung cells and tumor organoids, potentially related to their HER2 expression levels. This study provides a preliminary pharmacodynamic theoretical foundation for subsequent in vivo experiments and clinical translation.

[0208] Example 14 This example is used for the construction and preliminary testing of organoid chips, specifically: (1) An organoid chip was designed with an upper vascular chamber and a lower cell chamber arranged in parallel. The two chambers were separated by a polycarbonate porous membrane, which was synthesized by the Mifluidic Microfluidic Chip Laboratory.

[0209] (2) PDOs were inoculated into the lower cell chamber at a density of 1000 cells / μL according to the aforementioned method. The upper vascular chamber was continuously perfused with complete culture medium (5 μL / min) using a microfluidic pump. The organoid chip was placed in a cell culture incubator at 37°C with 5% CO2 and cultured for 24 h.

[0210] (3) QaHs was added to the complete culture medium of the experimental group to a final concentration of 13.5 μM, while the control group continued to perfuse the complete culture medium. The organoid chip was placed in a cell culture incubator at 37°C with 5% CO2 and incubated for 48 h.

[0211] (4) After drug incubation, Live-Dead staining and fluorescence microscopy were performed, using the same methods as before.

[0212] The results showed that the PDOs in the control group exhibited a large green fluorescent area, with only a limited red fluorescent area, indicating that the cells maintained good activity, demonstrating that the organoid chip and microperfusion system can stably maintain the organoid culture environment. In the experimental group, after 48 hours of QaHs treatment, the PDOs showed significant morphological changes and a clear red fluorescent area was observed, indicating low cell survival rate, confirming the inhibitory effect of the scFv on tumor organoids.

[0213] The above-described embodiments merely represent specific implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of the present application. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the technical concept of the present application, and all such variations and improvements fall within the scope of protection of the present application.

Claims

1. An anti-HER2 single-chain antibody having the structure: XmY, wherein X is a light chain variable region or a heavy chain variable region, m is a connecting peptide, and Y is a heavy chain variable region or a light chain variable region corresponding to X; characterized in that: The hydrophobic amino acids in the amino acid sequence of the single-chain antibody are selectively replaced with hydrophilic amino acids.

2. The anti-HER2 single-chain antibody according to claim 1, wherein: The hydrophobic amino acids include one or more of tryptophan Trp, phenylalanine Phe, valine Val, leucine Leu, isoleucine Ile, alanine Ala, proline Pro and methionine Met; and / or The hydrophilic amino acids include one or more of glycine Gly, serine Ser, threonine Thr, cysteine ​​Cys, tyrosine Tyr, aspartic acid Asp and glutamic acid Glu.

3. The anti-HER2 single-chain antibody according to claim 1, wherein: The ratio of selective amino acid replacement is 10%-100%.

4. The anti-HER2 single-chain antibody according to claim 1, wherein: The amino acid sequence of the anti-HER2 single-chain antibody is selected from: (a) the amino acid sequence shown in SEQ ID NO: 1; or (b) the amino acid sequence shown in SEQ ID NO: 2; or (c) an amino acid sequence derived from the amino acid sequence defined in (a) by substitution and / or deletion and / or addition of one or more amino acid residues, and having the same function as the amino acid sequence defined in (a); or (d) An amino acid sequence derived from the amino acid sequence defined in (b) by substitution and / or deletion and / or addition of one or more amino acid residues, and having the same function as the amino acid sequence defined in (b).

5. A nucleotide encoding an anti-HER2 single-chain antibody according to any one of claims 1 to 4, characterized in that: The nucleotide sequence of nucleotides is selected from the group consisting of: (e) the nucleotide sequence shown in SEQ ID NO: 3; or (f) the nucleotide sequence shown in SEQ ID NO: 4; or (g) a nucleotide sequence that has at least 69% similarity to the nucleotide sequence specified in (e) and encodes a protein with the same function; or (h) A nucleotide sequence that has at least 69% similarity to the nucleotide sequence defined in (f) and encodes a protein with the same function.

6. An expression vector, characterized in that: The expression vector comprises the nucleotide according to claim 5; the restriction enzyme site of the expression vector is selected from a combination of one or more of NdeI, EcoRI, and XhoI; the expression vector is selected from a combination of one or more of DNA, RNA, viral vector, plasmid, and transposon.

7. A host cell, characterized in that: The host cell contains the nucleotide according to claim 5 or the expression vector according to claim 6.

8. A biomaterial, characterized in that: The biological material is selected from one of the following (A) to (E): (A) the nucleotide according to claim 5; (B) an expression cassette, a recombinant vector, a recombinant primary cell, or a recombinant cell line containing the nucleotide described in (A); (C) the anti-HER2 single-chain antibody according to any one of claims 1 to 4; (D) the expression vector according to claim 6; (E) the host cell according to claim 7.

9. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the anti-HER2 single-chain antibody according to any one of claims 1 to 4 and one or more pharmaceutically acceptable carriers.

10. Use of the anti-HER2 single-chain antibody according to any one of claims 1 to 4; and / or the nucleotide according to claim 5; and / or the expression vector according to claim 6; and / or the host cell according to claim 7; and / or the biomaterial according to claim 8; and / or the pharmaceutical composition according to claim 9 in the preparation of products for screening, treating or preventing HER2-positive non-small cell lung cancer, breast cancer or gastric cancer.

11. Use of the anti-HER2 single-chain antibody according to any one of claims 1 to 4; and / or the nucleotide according to claim 5; and / or the expression vector according to claim 6; and / or the host cell according to claim 7; and / or the biomaterial according to claim 8; and / or the pharmaceutical composition according to claim 9 in the preparation of products for screening treatment or prevention of lung diseases.

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