Coupling drug for fusing albumin binding peptide with single-domain antibody and preparation method and application of coupling drug

Abstract

The invention discloses a coupling drug of albumin binding peptide fused with a single-domain antibody and a preparation method and application thereof, and relates to the technical field of biological pharmacy, the coupling drug is obtained by fusing albumin binding peptide and the single-domain antibody to obtain fusion protein and coupling drug molecules with the fusion protein; the fusion form of the albumin binding peptide and the single-domain antibody comprises a single-target monoclonal antibody mode: the C terminal or the N terminal of the albumin binding peptide is fused with one single-domain antibody; in the single-target double-antibody mode, the C end and the N end of the albumin binding peptide are simultaneously fused with one single-domain antibody or are respectively fused with two same single-domain antibodies; and a double-target double-antibody mode: the C end and the N end of the albumin binding peptide are respectively fused with two different single-domain antibodies. By means of the characteristic of high-affinity binding of the albumin binding peptide and the serum albumin, the long-acting effect of the coupling medicine in vivo can be achieved, the effect that the coupling medicine is enriched to a tumor part in vivo in a targeted mode can be achieved, and the anti-tumor purpose is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and in particular to a conjugated drug of an albumin-binding peptide-fused single-domain antibody, and a preparation method and application thereof. Background Art

[0002] Cancer and tumor diseases seriously threaten human life and health, and their clinical treatment, control and prevention have always been the hot spots and difficulties of world research. Small molecule toxic chemical drug therapy (chemotherapy) is one of the commonly used treatments for inoperable cancer and tumor diseases. There are many small molecule toxic chemotherapy drugs for anti-tumor in clinical practice, such as paclitaxel, docetaxel, doxorubicin, irinotecan, maytansine, auristatin, etc. This type of drug is characterized by having a strong tumor cell killing activity, but it also has a normal killing effect on human cells. In addition, this type of drug also has defects such as weak cell penetration and fast metabolic half-life in the body, which limits its clinical application.

[0003] Biological targeted drugs are a type of drug development model that has emerged in the past 10 to 20 years, such as antibody-drug conjugates (ADC) or protein / peptide conjugates (PDC). Thanks to the high specificity and high targeting of antibody / protein / peptide ligand molecules to specific receptors on the surface of tumor cells, biological targeted drugs have the advantages of being safer and having better efficacy than traditional chemical toxic drugs. In particular, antibody-drug conjugates, which act as carriers to give small molecule drugs long-term efficacy and tumor targeting in vivo, have become a popular direction and mainstream trend in the development of biological targeted drugs in the current biopharmaceutical industry. However, the only drawback is that the biological manufacturing process of biological targeted drugs is complex and costly, and the price of the drugs is extremely expensive, making it difficult to popularize them to a large number of patients.

[0004] Single-domain antibodies, also known as nanoantibodies, are a type of single-chain heavy chain variable region miniaturized antibodies (VHH) derived from alpaca or shark animal screening systems. While having the high targeting characteristics of traditional antibodies, they also have the properties of small molecular weight (about 13kDa) and greater stability. Another major advantage of single-domain antibodies is that they can be efficiently expressed and correctly folded in eukaryotic microbial expression systems and prokaryotic microbial expression systems, such as Escherichia coli (E. coli) or yeast (Yeast), and are easy to clone and produce on a large scale, with great cost advantages. In contrast, traditional complete antibodies (IgG) can only be expressed in mammalian cell expression systems (such as CHO cells), and the expression level is low and the expression cycle is long (the culture cycle is up to 14 days); in addition, traditional complete antibodies have high requirements for culture media, which leads to extremely high costs in their biomanufacturing process, which is also one of the main reasons why ADCs are extremely expensive and out of reach for the general public. Therefore, reducing the biopharmaceutical cost of biological targeted drugs is conducive to the further promotion of biological targeted drugs in clinical anti-tumor treatment and their popular use in patient groups.

[0005] Serum albumin is the largest protein component in the blood, and its metabolic half-life in the human body is as long as about 19 days. This is because the molecular weight of human serum albumin is difficult to be cleared by glomerular filtration. In addition, human serum albumin can bind to the FcRn receptor on human cells and can be recycled by the body system. Therefore, drug delivery based on albumin binding has always been the focus of drug research. Among them, the long-acting strategy of fusing with a polypeptide domain that binds to albumin with high affinity is widely used in the design of macromolecular drugs, such as long-acting interferon, granulocyte colony stimulating factor, etc. Protein domains that can bind to serum albumin include domains screened from natural microbial proteins, such as the albumin binding domain (albumin binding domain, ABD) in streptococcal G protein, and anti-albumin antibody domains (anti-HSAantibody / VHH) obtained by artificial screening, design and modification. In addition, a variety of albumin-binding peptides obtained through molecular display technology screening all have the ability to bind to albumin and have extremely high albumin affinity. They specifically bind to plasma albumin at picomolar concentrations. With the help of the long circulation of albumin in the body, they can indirectly prolong the metabolic time of the conjugated drug in the body and exert a long-lasting effect. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a conjugated drug of an albumin-binding peptide fused single-domain antibody and a preparation method and application thereof. The conjugated drug can achieve a long-lasting effect in vivo by virtue of the high-affinity binding of the albumin-binding peptide domain to serum albumin. At the same time, by utilizing the binding ability of the fused single-domain antibody to specific targets on the surface of tumor cells, toxic small molecule drugs can be delivered to tumor cells with high specificity and high targeting, thereby achieving drug enrichment in tumor cells and achieving the goal of killing tumor cells or inhibiting the growth of tumor cells.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] An albumin-binding peptide-fused single-domain antibody conjugated drug is obtained by fusing the albumin-binding peptide with the single-domain antibody to obtain a fusion protein, and then conjugating the drug molecule with the fusion protein;

[0009] Fusions of albumin binding peptides and single domain antibodies include:

[0010] a1, single-target mAb model: the C-terminus or N-terminus of the albumin binding peptide is fused to a single domain antibody;

[0011] a2, single-target dual antibody mode: the C-terminus and N-terminus of the albumin-binding peptide are simultaneously fused with one single-domain antibody or respectively with two identical single-domain antibodies;

[0012] a3, Dual-target dual-antibody mode: the C-terminus and N-terminus of the albumin-binding peptide are fused with two different single-domain antibodies respectively.

[0013] Furthermore, the albumin binding peptide is fused to the C-terminus and / or N-terminus of the single-domain antibody via a flexible connecting peptide, and then expressed via a yeast expression system or an Escherichia coli expression system to obtain a fusion protein; the flexible connecting peptide contains 1-4 cysteines, and the drug molecule is coupled to the cysteine ​​residues in the flexible connecting peptide region of the fusion protein via a covalent coupling chemical bond.

[0014] Furthermore, the albumin-binding peptide is derived from a domain in streptococcal G protein that binds to human serum albumin or a domain in an anti-human serum albumin antibody that binds to human serum albumin, as well as modifications based thereon; the homology of amino acids in the albumin-binding peptide is higher than 80%, and the molecular weight of the albumin-binding peptide is 5-15 kDa; the single-domain antibody is an antibody heavy chain variable region protein derived from alpaca or shark and composed of a single domain.

[0015] Furthermore, the albumin binding peptide is derived from the domain in Streptococcus G protein that binds to human serum albumin, and its amino acid sequence is shown in SEQ ID NO.1.

[0016] Furthermore, the general formula of the flexible connecting peptide is (Gm S)n i C or G i C-(G m S) n; wherein G is glycine, S is serine, and C is cysteine; m, i, and n are selected from natural integers in the range of 0-10.

[0017] Furthermore, the amino acid sequence of the flexible connecting peptide is shown as SEQ ID NO.2 or SEQ ID NO.3.

[0018] Furthermore, the molecular weight of the drug molecule does not exceed 2000Da; the drug molecule is selected from one or a combination of at least two of monomethyl auristatin D or its derivatives, monomethyl auristatin E or its derivatives, monomethyl auristatin F or its derivatives, and maytansine or its derivatives.

[0019] The present invention also provides a method for preparing the above-mentioned albumin binding peptide fused single domain antibody conjugate drug, comprising:

[0020] S1, by means of genetic engineering, the albumin binding peptide and the single domain antibody are fused through a flexible connecting peptide, and the fusion amino acid sequence is converted into a gene sequence, and the codon preference is optimized by codon preference software, and the engineered bacteria are transformed, positive bacteria are selected, and then cultured and induced for expression, and purified by affinity chromatography, hydrophobic chromatography and ion exchange chromatography to obtain a fusion protein of the albumin binding peptide and the single domain antibody;

[0021] S2, dissolving the fusion protein in a slightly alkaline buffer solution and pre-treating it with 0.2-1.0 mM tris(2-carboxyethyl)phosphine to obtain solution A; dissolving the drug molecule in an organic solvent to obtain solution B, then adding solution B dropwise to solution A, stirring gently, reacting at room temperature in the dark for 1-24 hours, and removing the precipitate by centrifugation to obtain a crude coupling product; the reaction molar ratio of drug molecule to fusion protein is 1-10:1;

[0022] S3, purifying the crude conjugated product by desalting to obtain the conjugated drug of the albumin-binding peptide fused single-domain antibody.

[0023] Furthermore, in step S2, the pH of the alkaline buffer solution is 6.5-8.0, the pretreatment time is 30 minutes, and the organic solvent is a dimethyl sulfoxide solution with a volume percentage of 10-50%; and during desalting and purification in step S3, the chromatography column filler is Sephadex-G25.

[0024] The invention provides an application of a conjugated drug of an albumin-binding peptide-fused single-domain antibody in the preparation of an anti-tumor drug.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The albumin-binding peptide-fused single-domain antibody drug conjugate of the present invention can achieve a long-term effect of the drug conjugate in vivo by virtue of the high affinity binding property of the albumin-binding peptide to serum albumin. At the same time, by utilizing the high specific binding characteristics of the single-domain antibody to the surface receptors of tumor cells, the drug conjugate can achieve the effect of targeted enrichment to the tumor part in vivo, achieving the purpose of anti-tumor, while the drug toxicity is greatly reduced.

[0027] (2) The biopreparation process of the albumin-binding peptide-fused single-domain antibody-drug conjugates in the present invention is simple, easy to operate, has simpler quality control, a shorter production cycle, and lower costs. The preparation of the coupling carriers in traditional antibody-drug conjugates, namely antibody molecules, requires the use of advanced mammalian cell expression systems, which have the disadvantages of long cycles and high prices. The albumin-binding peptide-fused single-domain antibodies in the present invention can be recombinantly expressed in high yields in yeast expression systems or Escherichia coli expression systems, and have the great advantages of extremely short cycles and lower costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the structure of the albumin binding peptide fused single domain antibody-drug conjugate in the present invention;

[0029] Figure 2 This is the nucleic acid electrophoresis identification diagram of the target gene sequence in Example 1;

[0030] Figure 3 This is the expression identification diagram of the target gene in Escherichia coli in Example 1;

[0031] Figure 4-6 This is a diagram showing the results of recombinant protein purification in Example 1;

[0032] Figure 7-9 The fluorescence emission spectrum characterization result of the albumin binding peptide fused single domain antibody fusion protein in Experiment 1;

[0033] Figure 10-12 The circular dichroism characterization results of the albumin binding peptide fused single domain antibody fusion protein in Experiment 1;

[0034] Figure 13-15 The MALDI-TOF characterization results of the albumin binding peptide fused single domain antibody fusion protein in Experiment 1;

[0035] Figure 16-19 This is the MALDI-TOF spectrum of the albumin binding peptide fusion single domain antibody coupled to the MMAE drug in Experiment 1;

[0036] Figure 20-23 This is the HSA binding validation result of the albumin binding peptide fusion single domain antibody coupled to the MMAE drug in Experiment 1;

[0037] Figure 24-27 The results of in vitro binding validation of albumin-binding peptide-fused single-domain antibody-drug conjugates with human serum albumin;

[0038] Figure 28-31 The albumin binding peptide fused single domain antibody conjugate drug and its growth inhibition curve on A549 cells;

[0039] Figure 32-35 Schematic diagram of the uptake of albumin binding peptide fused single domain antibody coupled to Cy5 in A549 cells;

[0040] Figure 36-40 Schematic diagram of the in vivo activity assay of albumin binding peptide fusion single domain antibody coupled to MMAE drug, free MMAE and blank control PBS on transplanted tumor nude mice in Experiment 5;

[0041] Fig.41 The pharmacokinetic results (drug-time curve) of the albumin-binding peptide-fused single-domain antibody-coupled MMAE drug and free MMAE in SD rats in Experiment 6;

[0042] Figure 42-44 These are the in vivo imaging images and fluorescence quantum yield graphs of each group of mice in Experiment 7. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with the accompanying drawings and examples, and the methods of the present invention include but are not limited to the following examples. The experimental methods used in the examples are conventional methods unless otherwise specified, and the instruments, reagents, consumables, experimental animals, etc. used are all commercially available unless otherwise specified.

[0044] This embodiment provides a drug conjugate of an albumin binding peptide fused single domain antibody, such as Figure 1 As shown, the C-terminus or N-terminus of the albumin binding peptide is fused with a single domain antibody (single-target monoclonal antibody mode) alone or simultaneously with a single domain antibody (single-target dual antibody mode) or two identical or different single domain antibodies (dual-target dual antibody mode) through a flexible connecting peptide, and the albumin binding peptide fusion single domain antibody protein is expressed by a low-cost recombinant protein expression system such as a yeast expression system or an Escherichia coli expression system, and then the drug molecule (toxic small molecule drug derivative) is coupled to the free thiol group on the mutated cysteine ​​(Cys) residue in the flexible connecting peptide region of the fusion protein by covalent chemical bonds to achieve fixed-point and quantitative coupling, and finally the albumin binding peptide fusion single domain antibody conjugated drug is obtained; the covalent chemical bond is selected from but not limited to one or a combination of at least two of a carbon-sulfur single bond (drug-CS-polypeptide) and a sulfur-sulfur single bond (drug-SS-polypeptide).

[0045] The albumin binding peptide is derived from the human serum albumin binding domain in Streptococcus G protein or the human serum albumin binding domain in anti-human serum albumin antibody, as well as the modified product based thereon, with an amino acid homology of more than 80% and a molecular weight of 5-15 kDa. The human serum albumin binding domain in Streptococcus G protein is particularly preferred. The amino acid sequence of a typical albumin binding peptide is shown in SEQ ID NO.1:

[0046] SEQ ID NO.1 (albumin binding peptide amino acid sequence):

[0047] LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP

[0048] Single-domain antibodies are a type of antibody heavy chain variable region protein composed of a single domain and originating from species such as alpacas or sharks. They are prepared through animal immunization and molecular display and screening techniques, and can bind to specific biological target molecules.

[0049] A flexible connecting peptide between the albumin binding peptide and the single domain antibody; its general sequence formula is (G m S)n i C or G i C-(G m S)n; wherein G is glycine, S is serine, and C is cysteine; m, i, and n are selected from natural integers in the range of 0-10; the amino acid sequence of a typical flexible connecting peptide is shown in SEQ ID NO.2 or SEQ ID NO.3. The flexible connecting peptide region between the albumin binding peptide and the single domain antibody contains cysteine ​​mutation points for site-specific and quantitative coupling of small molecule drugs, the number of which is 1-4, preferably 1-2.

[0050] SEQ ID NO.2: GGGGSGGGGCGGGGS

[0051] SEQ ID NO.3: GGGGCGGGGSGGGGS

[0052] The drug molecule is a small molecule compound that has the effect of inhibiting the growth of tumor cells and / or killing the tumor cells, and its molecular weight does not exceed 2000Da. The drug molecule is selected from, but not limited to, any one of monomethyl auristatin D or its derivatives (Mc-MMAD or Vc-MMAD), monomethyl auristatin E or its derivatives (Mc-MMAE or Vc-MMAE), monomethyl auristatin F or its derivatives (Mc-MMAF) or maytansine (DM1) or its derivatives, or a combination of at least two thereof.

[0053] At present, there are also targeted polypeptides used to prepare fusion proteins. The molecular weight of the targeted polypeptide is relatively small, generally 3-10 amino acids in length. The single-domain antibody used in this embodiment is a variable region single-domain structure of a heavy chain antibody obtained by immunizing alpacas or sharks that participates in the binding of target antigens, with a molecular weight of about 13kDa and about 120 amino acids involved. Compared with targeted polypeptides, single-domain antibodies have better affinity and higher binding specificity for target antigens. Albumin binding peptides are a form of long-acting peptides, and their mechanism is to achieve long-term effect by binding to serum albumin and relying on the long-term effect of albumin in the body. Its main function is to be able to bind to albumin, and its advantage is that the molecular weight is relatively small, about 5-6kDa, and it is easy to express efficiently in a simple prokaryotic expression system.

[0054] In this embodiment, an albumin-binding peptide with long-acting properties in vivo is fused with a single-domain antibody with tumor-targeted penetration properties to obtain a fusion protein with both long-acting properties in vivo and tumor-targeted properties. The fusion protein is used as a coupling carrier for toxic small molecule drugs, and the small molecule drugs or their derivatives are site-specifically chemically coupled with the albumin-binding peptide-fused single-domain antibody to prepare an albumin-binding peptide-fused single-domain antibody-coupled drug with long-acting properties in vivo and tumor-targeted properties.

[0055] Example 1

[0056] Construction of recombinant fusion protein of albumin-binding peptide fused to single-domain antibody

[0057] (1) Construction of amino acid sequence:

[0058] VHH EGFR -The amino acid sequence of ABD (single-target monoclonal antibody mode) is shown in SEQ ID NO.4:

[0059] SEQ ID NO.4:

[0060] Question

[0061] ABD-VHH HER2 The amino acid sequence of (single-target monoclonal antibody mode) is shown in SEQ ID NO.5:

[0062] SEQ ID NO.5:

[0063] LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALPGGGGCGGGGSGGGGSQVQLVQSGGGLVQAGGSLRLSCAASGRTFSSYAMAWFRQAPGKEREFVAAISWSGANIYVADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAVKLGFAPVEERQYDYWGQGTQVTVSSHHHHHH

[0064] VHH EGFR -ABD-Cys-VHH HER2 The amino acid sequence of (dual-target dual-antibody mode, containing a cysteine ​​drug coupling site) is shown in SEQ ID NO.6:

[0065] SEQ ID NO.6:

[0066] QVKLEESGGGLVQAGDSLRVSCAASGRDFSDYVMGWFRQAPGKEREFVAAISRNGLTTRYADSVKGRFTISRDNDKNMVYLQMNSLKPEDTAVYYCAVNSAGTYVSPRSREYDYWGQGTQVTVSSGGGGSGGGGSGGGGSLAEAKVLANRELDKYGVSDFYKRLI NKAKTVEGVEALKLHILAALPGGGGCGGGGSGGGGSQVQLVQSGGGLVQAGGSLRLSCAASGRTFSSYAMAWFRQAPGKEREFVAAISWSGANIYVADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAVKLGFAPVEERQYDYWGQGTQVTVSSHHHHHH

[0067] VHH EGFR -ABD-VHH HER2 The amino acid sequence of (dual-target dual-antibody mode, containing two cysteine ​​drug coupling sites) is shown in SEQ ID NO.7:

[0068] SEQ ID NO.7:

[0069] QVKLEESGGGLVQAGDSLRVSCAASGRDFSDYVMGWFRQAPGKEREFVAAISRNGLTTRYADSVKGRFTISRDNDKNMVYLQMNSLKPEDTAVYYCAVNSAGTYVSPRSREYDYWGQGTQVTVSSGGGGSGGGGCGGGGSLAEAKVLANRELDKYGVSDFYKRLI NKAKTVEGVEALKLHILAALPGGGGCGGGGSGGGGSQVQLVQSGGGLVQAGGSLRLSCAASGRTFSSYAMAWFRQAPGKEREFVAAISWSGANIYVADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCAVKLGFAPVEERQYDYWGQGTQVTVSSHHHHHH

[0070] (2) Nucleotide sequence:

[0071] VHH EGFR -The nucleotide sequence of ABD (single-target monoclonal antibody mode) is shown in SEQ ID NO.8:

[0072] SEQ ID NO.8:

[0073] CAGGTGAAACTGGAAGAAAGCGGTGGTGGTCTGGTTCAGGCAGGCGATAGCCTGCGTGTTAGCTGTGCAGCAAGCGGTCGTGATTTTAGCGATTATGTTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAACGTGAATTTGTTGCAGCAATTAGCCGTAATGGTCTGACCACACGTTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATAACGACAAAAACATGGTTTACCTGCAGATGAATAGCCTGAAACCGGAAGATACCGCAGTGTATTATTGTGCAGTTAATAGCGCAGGCACCTATGTTAGTCCGCGTAGCCGTGAATATGATTATTGGGGTCAGGGCACCCAGGTTACCGTTAGCTCAGGTGGTGGTGGTAGCGGTGGCGGTGGCTGTGGTGGTGGCGGTTCACTGGCAGAAGCAAAAGTTCTGGCAAATCGTGAACTGGATAAATATGGTGTGAGCGATTTCTATAAACGCCTGATCAATAAAGCCAAAACCGTTGAAGGTGTTGAAGCACTGAAACTGCATATTCTGGCAGCACTGCCTCATCACCATCATCATCATTAA

[0074] ABD-VHH HER2 (Single-target monoclonal antibody mode) The nucleotide sequence is as shown in SEQ ID NO.9:

[0075] SEQ ID NO.9:

[0076] CTGGCAGAAGCAAAAGTTCTGGCAAATCGTGAACTGGATAAATATGGTGTGAGCGATTTCTATAAACGCCTGATCAATAAAGCCAAAACCGTTGAAGGTGTTGAAGCACTGAAACTGCATATTCTGGCAGCACTGCCTGGTGGCGGAGGTTGTGGAGGTGGCGGTAGTGGTGGTGGTGGAAGCCAGGTTCAGCTGGTTCAGTCCGGTGGCGGACTGGTGCAAGCCGGTGGTAGTCTGCGTCTGAGCTGTGCCGCATCAGGTCGTACCTTTAGCAGCTATGCAATGGCATGGTTCCGCCAGGCTCCAGGCAAAGAACGCGAGTTCGTGGCAGCCATTAGTTGGAGCGGTGCAAACATTTATGTTGCCGATTCAGTGAAAGGTCGCTTCACAATTTCACGTGATAATGCCAAAGATACCGTGTATCTGCAAATGAATTCACTGAAACCTGAGGATACAGCCGTTTACTACTGTGCCGTTAAACTGGGTTTTGCACCGGTGGAAGAACGTCAGTATGACTACTGGGGACAGGGTACACAGGTGACAGTGAGCAGCCATCACCATCATCATCATTAA

[0077] VHH EGFR -ABD-Cys-VHH HER2 (Double-target bispecific antibody mode, containing a cysteine drug conjugate site) The nucleotide sequence is shown in SEQ ID NO.10:

[0078] SEQ ID NO.10:

[0079] CAGGTGAAACTGGAAGAAAGCGGTGGTGGTCTGGTTCAGGCAGGCGATAGCCTGCGTGTTAGCTGTGCAGCAAGCGGTCGTGATTTTAGCGATTATGTTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAACGTGAATTTGTTGCAGCAATTAGCCGTAATGGTCTGACCACACGTTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATAACGACAAAAACATGGTTTACCTGCAGATGAATAGCCTGAAACCGGAAGATACCGCAGTGTATTATTGTGCAGTTAATAGCGCAGGCACCTATGTTAGTCCGCGTAGCCGTGAATATGATTATTGGGGTCAGGGCACCCAGGTTACCGTTAGCTCAGGTGGTGGTGGTAGCGGTGGCGGTGGCTCTGGTGGTGGCGGTTCACTGGCAGAAGCAAAAGTTCTGGCAAATCGTGAACTGGATAAATATGGTGTG AGCGATTTCTATAAACGCCTGATCAATAAAGCCAAAACCGTTGAAGGTGTTGAAGCACTGAAACTGCATATTCTGGCAGCACTGCCTGGTGGCGGAGGTTGTGGAGGTGGCGGTAGTGGTGGTGGTGGAAGCCAGGTTCAGCTGGTTCAGTCCGGTGGCGGACTGGTGCAAGCCGGTGGTAGTCTGCGTCTGAGCTGTGCCGCATCAGGTCGTACCTTTAGCAGCTATGCAATGGCATGGTTCCGCCAGGCTCCAGGCAAAGAACGCGAGTTCGTGGCAGCCATTAGTTGGAGCGGTGCAAACATTTATGTTGCCGATTCAGTGAAAGGTCGCTTCACAATTTCACGTGATAATGCCAAAGATACCGTGTATCTGCAAATGAATTCACTGAAACCTGAGGATACAGCCGTTTACTACTGTGCCGTTAAACTGGGTTTTGCACCGGTGGAAGAACGTCAGTATGACTACTGGGGACAGGGTACACAGGTGACAGTGAGCAGCCATCACCATCATCATCATTAA

[0080] VHH EGFR -ABD-VHH HER2 The nucleotide sequence of (dual-target dual-antibody mode, containing two cysteine ​​drug coupling sites) is shown in SEQ ID NO.11:

[0081] SEQ ID NO.11:

[0082] CAGGTGAAACTGGAAGAAAGCGGTGGTGGTCTGGTTCAGGCAGGCGATAGCCTGCGTGTTAGCTGTGCAGCAAGCGGTCGTGATTTTAGCGATTATGTTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAACGTGAATTTGTTGCAGCAATTAGCCGTAATGGTCTGACCACACGTTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATAACGACAAAAACATGGTTTACCTGCAGATGAATAGCCTGAAACCGGAAGATACCGCAGTGTATTATTGTGCAGTTAATAGCGCAGGCACCTATGTTAGTCCGCGTAGCCGTGAATATGATTATTGGGGTCAGGGCACCCAGGTTACCGTTAGCTCAGGTGGTGGTGGTAGCGGTGGCGGTGGCTGTGGTGGTGGCGGTTCACTGGCAGAAGCAAAAGTTCTGGCAAATCGTGAACTGGATAAATATGGTGTGAGCGATTTCTATAAACGCCTGATCAATAAAGCCAAAACCGTTGAAGGTGTTGAAGCACTGAAACTGCATATTCTGGCAGCACTGCCTGGTGGCGGAGGTTGTGGAGGTGGCGGTAGTGGTGGTGGTGGAAGCCAGGTTCAGCTGGTTCAGTCCGGTGGCGGACTGGTGCAAGCCGGTGGTAGTCTGCGTCTGAGCTGTGCCGCATCAGGTCGTACCTTTAGCAGCTATGCAATGGCATGGTTCCGCCAGGCTCCAGGCAAAGAACGCGAGTTCGTGGCAGCCATTAGTTGGAGCGGTGCAAACATTTATGTTGCCGATTCAGTGAAAGGTCGCTTCACAATTTCACGTGATAATGCCAAAGATACCGTGTATCTGCAAATGAATTCACTGAAACCTGAGGATACAGCCGTTTACTACTGTGCCGTTAAACTGGGTTTTGCACCGGTGGAAGAACGTCAGTATGACTACTGGGGACAGGGTACACAGGTGACAGTGAGCAGCCATCACCATCATCATCATTAA

[0083] (3) Specific steps for obtaining recombinant plasmid:

[0084] VHH EGFR -ABD-VHH HER2 The sequence was optimized for E. coli preferred codons and then commissioned for synthesis and inserted into the pET32a plasmid vector. EGFR -ABD-VHH HER2 -pET32a plasmid was used as a template to design upstream and downstream primers, and VHH was obtained by PCR amplification. EGFR -ABD-pET32a and ABD-VHH HER2 -pET32a gene sequence, in which VHH was amplified EGFR -ABD-pET32a sequence PCR reaction conditions are 98℃ pre-denaturation for 2min, 98℃ for 10s, 57℃ for 15s, 72℃ for 90s, and 72℃ extension for 5min after 35 cycles; ABD-VHH was amplified HER2 -pET32a sequence PCR reaction conditions are 98℃ pre-denaturation for 2min, 98℃ for 10s, 47℃ for 15s, 72℃ for 90s, and 72℃ extension for 5min after 35 cycles. EGFR -ABD-Cys-VHH HER2 The sequence can be VHH EGFR -ABD-VHH HER2 Template, obtained by PCR mutation of a single amino acid codon. The nucleic acid electrophoresis diagram of the target gene is as follows Figure 2 As shown, the expression and identification of the target gene in E. coli is shown in Figure 3 As shown, where:

[0085] Figure 2 ,Lane1:VHH EGFR -ABD-VHH HER2 -pET32a; Lane2:1kb DNAMarker; Lane3:VHH EGFR -ABD-pET32a; Lane4:ABD-VHH HER2 -pET32a; Lane5:1kb DNAMarker.

[0086] Figure 3 , Lane1:ABD-VHH HER2 -pET32a before induction; Lane2:ABD-VHH HER2 -After pET32a induction; Lane3:ABD-VHH HER2 -pET32a lysis supernatant; Lane4: ABD-VHH HER2-pET32a lysis and precipitation; Lane5: VHH EGFR -ABD-pET32a before induction; Lane6:VHH EGFR -ABD-pET32a after induction; Lane7:VHH EGFR -ABD-pET32a lysis supernatant; Lane8: VHH EGFR -ABD-pET32a bacterial lysis precipitation; Lane9: VHH EGFR -ABD-VHH HER2 -pET32a before induction; Lane10:VHH EGFR -ABD-VHH HER2 -After pET32a induction; Lane11:VHH EGFR -ABD-VHH HER2 -pET32a lysis supernatant; Lane12: VHH EGFR -ABD-VHH HER2 -pET32a bacterial lysis and precipitation; Lane 13: Marker.

[0087] VHH EGFR -ABD-pET32a sequence amplification upstream primer sequence is shown in SEQ ID NO.12, amplification downstream primer sequence is shown in SEQ ID NO.13; ABD-VHH HER2 -The upstream primer sequence for amplifying the pET32a sequence is shown in SEQ ID NO.14, and the downstream primer sequence for amplifying the pET32a sequence is shown in SEQ ID NO.15.

[0088] SEQ ID NO.12:

[0089] 5'-ATTCTGGCAGCACTGCCTCATCACCATCATCATCATTAAGATATCATCGG-3'

[0090] SEQ ID NO.13: 5'-AGGCAGTGCTGCCAGAATATG-3'

[0091] SEQ ID NO.14: 5'-CTGGCAGAAGCAAAAG-3'

[0092] SEQ ID NO.15: 5'-AACTTTTGCTTCTGCCAGCATATGTATATCTCCTTCTTAAAG-3'

[0093] (4) The correctly sequenced recombinant plasmid was transformed into E. coli Origami B strain and screened using ampicillin-resistant plates.

[0094] (5) Microbial culture: Use LB medium to culture until OD 600 When the cell density was about 3.0, IPTG with a final concentration of 50 μM was added to induce the expression of the target gene protein. After induction at 20°C for 20 hours, the bacteria were obtained by centrifugation, and 50 mmol / L Tris-HCl, 300 mM NaCl, pH 7.5 lysis solution was added at a ratio of 1:10. The cells were broken using an ultrasonic cell disruptor and the supernatant was obtained by centrifugation.

[0095] (6) Purification of target protein: The albumin-binding peptide-fused single-domain antibody recombinant fusion protein in the supernatant of the bacterium was purified by nickel affinity chromatography. Before loading, the column was pre-washed with buffer B (50mmol / L Tris-HCl, 300mMNaCl, 500mM imidazole, pH 7.5) for 3 to 5 column volumes, and then equilibrated with buffer A (50mmol / L Tris-HCl, 300mMNaCl, pH 7.5) for 3 to 5 column volumes. After loading, equilibrate with buffer A for 10 column volumes, and finally elute the impurities with 15% buffer B for 5 column volumes, then elute the protein with 50% and 100% buffer B, collect the eluted product, and identify the purification result by 15% SDS-PAGE. The target protein (fusion protein) is as follows: Figure 4 , 5 , 6, where:

[0096] Figure 4 , Lane1:ABD-VHH HER2 -pET32a lysis supernatant; Lane 2: flow-through; Lane 3: 15% B wash; Lane 4: 50% B-①; Lane 5: 50% B-②; Lane 6: 50% B-③; Lane 7: 100% B-①; Lane 8: 100% B-②; Lane 9: Marker;

[0097] Figure 5 ,Lane1:VHH EGFR -ABD-pET32a lysis supernatant; Lane 2: flow-through; Lane 3: 15% B elution; Lane 4: 50% B-①; Lane 5: 50% B-②; Lane 6: 100% B-①; Lane 7: 100% B-②; Lane 8: Marker;

[0098] Figure 6 ,Lane1:VHH EGFR -ABD-VHH HER2-pET32a lysis supernatant; Lane 2: flow-through; Lane 3: 15% B elution; Lane 4: 50% B-①; Lane 5: 50% B-②; Lane 6: 100% B; Lane 7: Marker.

[0099] Example 2

[0100] Preparation of albumin-binding peptide-fused single-domain antibody coupled to MMAE drug.

[0101] (1) Synthetic conjugated drug VHH EGFR -ABD-MMAE, ABD-VHH HER2 -MMAE, VHH EGFR -ABD-Cys-VHH HER2 -MMAE, VHH EGFR -ABD-VHH HER2 -2MMAE:

[0102] The VHH purified by nickel affinity chromatography in Example 1 EGFR -ABD, ABD-VHH HER2 、VHH EGFR -ABD-Cys-VHH HER2 and VHH EGFR -ABD-VHH HER2 Replace with 50mM Tris-HCl, 100mM NaCl, pH 7.5 buffer and perform protein quantification (BCA method), add 0.2mM-1.0mM TCEP and incubate at room temperature for 30 minutes, then add 10mM MMAE solution at a molar ratio of 1:2-1:10, mix well and incubate at 4°C overnight in the dark.

[0103] (2) Isolation and purification of conjugated drug VHH EGFR -ABD-MMAE, ABD-VHH HER2 -MMAE, VHH EGFR -ABD-Cys-VHH HER2 -MMAE and VHH EGFR -ABD-VHH HER2 -MMAE:

[0104] After adding MMAE and coupling at 4°C overnight, the uncoupled MMAE small molecules were removed by desalting, and VHH EGFR -ABD-MMAE, ABD-VHH HER2 -MMAE, VHH EGFR -ABD-Cys-VHH HER2 -MMAE and VHH EGFR -ABD-VHHHER2 -MMAE was replaced with 50 mM Tris-HCl, 100 mM NaCl, pH 7.5 buffer and stored frozen at -20°C.

[0105] The following tests were performed on the albumin-binding peptide fusion single-domain antibody recombinant fusion protein and the albumin-binding peptide fusion single-domain antibody coupled to the MMAE drug prepared in Examples 1 and 2 above:

[0106] Test 1

[0107] Results of recombinant fusion proteins fused to single domain antibodies with albumin binding peptides.

[0108] (1) Fluorescence emission spectrum identification: The secondary structure of the albumin binding peptide fusion single domain antibody recombinant fusion protein was identified by fluorescence spectrophotometer. First, the protein sample buffer was replaced with 50mM Tris-HCl, 100mM NaCl, pH 7.5, concentrated and adjusted to a protein concentration of about 0.1mg / mL, and the sample was placed in a quartz cuvette sample pool with a light path of 1.0cm. The excitation wavelength was set to 280nm, the emission wavelength was set to 300-400nm, the scanning wavelength interval was 1.0nm, and the sample scanning speed was 600nm / min. The fluorescence emission spectrum characterization results are shown in Figure 2. Figure 7 , 8 , as shown in 9, Figure 7 ABD-VHH HER2 Fluorescence emission spectra; Figure 8 For VHH EGFR -ABD fluorescence emission spectrum; Fig. 9 For VHH EGFR -ABD-VHH HER2 Fluorescence emission spectra.

[0109] (2) Circular dichroism spectroscopy identification: The secondary structures of the albumin-binding peptide-fused single-domain antibody recombinant fusion protein and their respective MMAE-conjugated drugs were identified by circular dichroism analysis. First, the protein sample buffer was replaced with 50mM Tris-HCl, 100mM NaCl, pH 7.5, and the protein concentration was concentrated and adjusted to 0.2-0.3mg / mL. A sample cell with a thickness of 0.1cm was used, the scanning wavelength interval was 1.0nm, the scanning was repeated 3 times, the scanning wavelength range was 190-260nm, and the sample scanning speed was 1200nm / min. The circular dichroism characterization results are shown in Figure 2. Fig.10 , 11 , as shown in 12, Fig.10 ABD-VHH HER2 Circular dichroism spectra; Fig.11 For VHH EGFR -ABD circular dichroism spectrum; Fig.12 For VHH EGFR -ABD-VHH HER2 Circular dichroism spectrum; the results show that the albumin binding peptide fused single domain antibody recombinant fusion protein is folded in an orderly manner and contains α-helix and β-fold structure.

[0110] (3) MALDI-TOF-MS: The albumin binding peptide fusion single domain antibody recombinant fusion protein was determined by MALDI mass spectrometry. The target method was: first, 1 μl of the sample was naturally air-dried, then 1 μl of matrix (acetonitrile / water (7:3), containing 0.1% TFA) was added and naturally air-dried, and then tested on the machine. The MALDI-TOF characterization results are shown in Fig.13 , 14 , 15, the MALDI-TOF spectrum of the coupled MMAE drug is as shown in Figure 16-19 As shown, Fig.13 ABD-VHH HER2 Mass spectra; Fig.14 For VHH EGFR -ABD mass spectrum; Fig.15 For VHH EGFR -ABD-VHH HER2 Mass spectra; Fig.16 ABD-VHH HER2 -MMAE mass spectrum; Fig.17 For VHH EGFR -ABD-MMAE mass spectrum; Fig.18 For VHH EGFR -ABD-Cys-VHH HER2 -MMAE mass spectrum; Fig.19 For VHH EGFR -ABD-VHH HER2 -2MMAE mass spectrum. According to the results of each sample, VHH EGFR -ABD theoretical molecular weight 20.6kDa, actual molecular weight 20.8kDa, molecular weight after coupling with MMAE 22.0kDa; ABD-VHH HER2 Theoretical molecular weight 20.2kDa, actual molecular weight 20.3kDa, molecular weight after coupling with MMAE 21.6kDa; VHH EGFR -ABD-VHH HER2 Theoretical molecular weight 35.0kDa, actual molecular weight 35.2kDa, VHH EGFR -ABD-Cys-VHH HER2 -MMAE (coupled with one MMAE molecule) molecular weight 36.4 kDa, VHH EGFR -ABD-VHH HER2-2MMAE (coupled with two MMAE molecules) has a molecular weight of 37.7 kDa. It can be considered that the molecular weights of the albumin-binding peptide-fused single-domain antibody recombinant fusion protein and their respective MMAE-coupled drugs are in line with expectations.

[0111] HSA binding verification: The ability of the albumin binding peptide fusion single domain antibody recombinant fusion protein to bind to HSA was verified by size exclusion chromatography SEC. The mobile phase was 50mM PB, 100mM Na2SO4, pH 7.4, and the flow rate was 0.5mL / min. The results of HSA in vitro verification are as follows Figure 20-23 As shown, Fig. 20 ABD-VHH HER2 In vitro binding HSA results; Fig.21 For VHH EGFR - Results of ABD binding to HSA in vitro; Fig. 22 For VHH EGFR -ABD-Cys-VHH HER2 In vitro binding HSA results; Fig.23 For VHH EGFR -ABD-VHH HER2 In vitro binding to HSA results. The results showed that VHH EGFR -ABD, ABD-VHH HER2 、VHH EGFR -ABD-Cys-VHH HER2 and VHH EGFR -ABD-VHH HER2 Both can bind to HSA.

[0112] Test 2

[0113] Verification of the binding ability of albumin binding peptide fusion single domain antibody coupled to MMAE and human serum albumin. The ability of albumin binding peptide fusion single domain antibody coupled to MMAE and HSA was verified by size exclusion chromatography SEC. The mobile phase was 50mM PB, 100mM Na2SO4, pH 7.4, and the flow rate was 0.5mL / min. The results are shown in Figure 24-27 Display, where Fig.24 ABD-VHH HER2 -MMAE binding to HSA in vitro results; Fig.25 For VHH EGFR - Results of ABD-MMAE binding to HSA in vitro; Fig.26 For VHH EGFR -ABD-Cys-VHH HER2 -MMAE binding to HSA in vitro results; Fig. 27 For VHH EGFR -ABD-VHH HER2-2MMAE in vitro binding to HSA. The results showed that ABD-VHH HER2 -MMAE, VHH EGFR -ABD-MMAE, VHH EGFR -ABD-Cys-VHH HER2 -MMAE and VHH EGFR -ABD-VHH HER2 -2MMAE can bind to HSA.

[0114] Test 3

[0115] The cytotoxicity test of albumin binding peptide fused single domain antibody coupled with MMAE drug on A549 cells. 4 / well were inoculated into a 96-well plate and incubated at 37°C, 5% CO2 for 24h. After discarding the old culture medium, the albumin-binding peptide-fused single-domain antibody-coupled MMAE drug (100μl / well) diluted with complete culture medium was added. In addition, a negative control (containing cells + complete culture medium) and a blank control (containing no cells + complete culture medium) were set up and incubated at 37°C, 5% CO2 for 48h. After 48h of culture, the liquid in the wells was aspirated. 100μl of complete culture medium containing 10% CCK8 was added to each well and incubated at 37°C in the dark for 30min. The absorbance at 450nm was measured, the cell survival rate was calculated, and the curve was drawn. Graphpad Prism was used for analysis, and the results are shown in the figure. Figure 28-31 As shown, Fig.28 ABD-VHH HER2 -MMAE inhibits the growth of A549 cells; Fig.29 For VHH EGFR -ABD-MMAE inhibits the growth of A549 cells; Fig.30 For VHH EGFR -ABD-Cys-VHH HER2 -MMAE inhibits the growth of A549 cells; Fig.31 For VHH EGFR -ABD-VHH HER2 -2MMAE inhibits the growth of A549 cells. The results show that ABD-VHH HER2 -MMAE, VHH EGFR -ABD-MMAE, VHH EGFR -ABD-Cys-VHH HER2 -MMAE, and VHH EGFR -ABD-VHH HER2-MMAE IC50 values ​​for A549 cells were 0.5891nM, 0.7327nM, 0.5282nM and 0.2913nM, respectively.

[0116] Test 4

[0117] Verification of A549 cell uptake of albumin-binding peptide-fused single-domain antibody coupled to Cy5. TCEP was added to the albumin-binding peptide-fused single-domain antibody and reacted at room temperature for 30 minutes in the dark. Sulfo Cy5 (1 mg of SulfoCy5 was weighed and 100 ul of sterile water was added to fully dissolve it to prepare a 10 mg / mL stock solution) was added at a molar ratio of 1:2 for coupling and reacted at room temperature for 4 hours. Desalt the mixture into PBS buffer using a desalting column. A Sulfo Cy5 standard curve was established and the concentration of albumin-binding peptide-fused single-domain antibody coupled to Cy5 was measured at a wavelength of 649 nm. A549 cells were cultured at 2.5×10 5 / mL density was plated into 12-well plates at a volume of 1mL / well, and incubated at 37°C, 5% CO2 for 36h. The old culture medium was discarded, and new complete culture medium was added at 1mL / well. 1mL of 5μM albumin-binding peptide-fused single-domain antibody coupled to Cy5 was added to each well of the experimental group, an equal volume of Sulfo Cy5 was added to the control group, and an equal volume of PBS was added to the blank group. The plates were incubated in a 37°C, 5% CO2 incubator in the dark for 1, 2, and 6 hours. The culture medium was discarded, the cells were collected, and the cells were washed 1-2 times with pre-cooled PBS and then resuspended. The cell uptake was detected by flow cytometry. The schematic diagram of the uptake of albumin-binding peptide-fused single-domain antibody coupled to Cy5 in A549 cells is shown in the figure. Figure 32-35 As shown, Fig.32 ABD-VHH HER2 - Cy5 uptake in A549 cells; Fig.33 For VHH EGFR - Uptake of ABD-Cy5 in A549 cells; Fig.34 For VHH EGFR -ABD-Cys-VHH HER2 - Cy5 uptake in A549 cells; Fig.35 For VHH EGFR -ABD-VHH HER2 -2Cy5 uptake in A549 cells. The results showed that the albumin binding peptide fusion single domain antibody coupled to Cy5 can be taken up by A549 cells, and as the incubation time increases, the drug accumulates in the cells.

[0118] Test 5

[0119] Verification of the anti-tumor efficacy of albumin-binding peptide-fused single-domain antibody-coupled MMAE drug in tumor-bearing mice (nude mice, A549 cell subcutaneous tumor model).

[0120] A549 cells (2×10 6 A549 cell subcutaneous transplant tumor model was established. When the tumor volume grew to 100 mm 3 Around, randomly divided into VHH EGFR -ABD-MMAE, ABD-VHH HER2 -MMAE, VHH EGFR -ABD-Cys-VHH HER2 -MMAE, VHH EGFR -ABD-VHH HER2 -2MMAE, MMAE and PBS groups (n=8) were given drug treatment. The whole course of treatment required 6 injections, which were injected subcutaneously. The single dose was VHH EGFR -ABD-MMAE 2mg / kg, ABD-VHH HER2 -MMAE 2mg / kg, VHH EGFR -ABD-Cys-VHH HER2 -MMAE 3.5mg / kg, VHH EGFR -ABD-VHH HER2 -2MMAE 3.5mg / kg, MMAE 0.13mg / kg (except VHH EGFR -ABD-VHH HER2 -2MMAE group, each group contained the same amount of MMAE). The tumor volume and mouse weight were recorded every 2-3 days. Figure 36-40 As shown, Fig.36 Figure 2 is the change diagram of tumor volume in each group; Fig.37 Statistical graph of tumor weight in each group; Fig.38 Actual pictures of tumors in each group; Fig.39 , 40 is the weight change diagram of each group, where Fig.39 The weight change graphs for each group include error bars. Fig.40 The weight change chart of each group is shown with the error bars removed. The results show that VHH EGFR -ABD-MMAE, ABD-VHH HER2 -MMAE, VHH EGFR -ABD-Cys-VHH HER2 -MMAE, VHH EGFR -ABD-VHH HER2 -2MMAE showed significantly better antitumor activity than MMAE.

[0121] Test 6

[0122] Pharmacokinetic validation of albumin-binding peptide-fused single-domain antibody-coupled MMAE drug in SD rats.

[0123] Thirty SD rats were randomly divided into five groups, with six rats in each group. VHH was injected once through the tail vein. EGFR -ABD-MMAE 2mg / kg, ABD-VHH HER2 -MMAE 2mg / kg, VHH EGFR -ABD-Cys-VHH HER2 -MMAE 3.5mg / kg, VHH EGFR -ABD-VHH HER2 -2MMAE 3.5mg / kg, MMAE 0.13mg / kg (except VHH EGFR -ABD-VHH HER2 -2MMAE group, each group contained the same amount of MMAE, and each group contained the same amount of albumin binding peptide fusion single domain antibody). Blood was collected from the eye sockets at 0.5, 1, 2, 6, 12, 24, 50, 74, 122, and 170 hours after administration. Blood was collected with a sodium heparin anticoagulant tube and then centrifuged to separate plasma. The content of albumin binding peptide fusion single domain antibody coupled to MMAE drug in each experimental group was determined by ELISA method. The specific operation steps included:

[0124] ① Use coating buffer to dilute EGFR (HER2) protein to 1 μg / mL, plate it on a 96-well ELISA plate at a volume of 100 ul / well, place the ELISA plate in a wet box, and incubate at 4°C for 12 hours;

[0125] ② Washing: After coating, discard the coating solution, pat dry on absorbent paper, add PBST at 200 μl / well, discard, pat dry, and repeat 5 times;

[0126] ③ Blocking: Add 5% BSA at 200 μl / well and block at 37°C for 2 h;

[0127] ④ Washing: After blocking, discard the blocking solution, pat dry on absorbent paper, add PBST at 200 μl / well, discard, pat dry, and repeat 5 times;

[0128] ⑤ Sample addition: Take out the required plasma samples from the -80℃ refrigerator in advance, thaw at 4℃, mix the serum of each drug group at the same time point in equal volumes under light-proof conditions, dilute the samples by a certain multiple with PBST, add protein at 100μl / well, set 3 replicate wells for the samples at each time point, and incubate at 37℃ for 2h after sample addition;

[0129] ⑥ Washing: After incubation, discard the sample solution, pat dry on absorbent paper, add PBST at 200 μl / well, discard, pat dry, and repeat 5 times;

[0130] ⑦ Incubate with secondary antibody: HRP-anti-MMAE (diluted 1:4000 with PBST) at 100 μl / well in a 96-well plate and incubate at 37°C for 2 h;

[0131] ⑧ Washing: After incubation, discard the secondary antibody, pat dry on absorbent paper, add PBST at 200μl / well and discard, pat dry, repeat 6 times to fully wash away the secondary antibody. During the washing period, take appropriate amounts of H2O2 colorimetric solution and TMB colorimetric solution according to your own required amount, mix the two in a volume ratio of 1:1, and gently mix to make a reaction solution;

[0132] ⑨ Add reaction solution: 100 μl / well, incubate at 37°C in the dark for 5-30 min;

[0133] ⑩ Color development: Add 50 μl of 2M sulfuric acid to each well to terminate the reaction, and measure the OD value of each well at a wavelength of 450 nm within 15 minutes;

[0134] LC-MS was used to measure the MMAE content and changes in plasma samples at each time point in the MMAE group. The specific operation requirements include:

[0135] ① Sample processing: Plasma samples were thawed at 4°C, and blood samples of 6 mice in the same group at the same time point were mixed in equal volumes. 100ul of sample was taken, and 300ul of HPLC-grade acetonitrile was added. The samples were vortexed for 30s, centrifuged at 12000rpm for 15min, and the supernatant was taken for sample preparation. The samples were repeated three times.

[0136] ②Chromatographic conditions: mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 100% acetonitrile, injection volume is 5ul, column temperature is 35℃, flow rate is 1mL / min;

[0137] Elution procedure:

[0138] Time (min) A(％) B(％) 0 90 10 1 90 10 2 5 95 4 5 95 4.5 90 10 5.5 90 10

[0139] ③Mass spectrometry conditions: Agilent 1290 Infinity II and ULTIVO mass spectrometer were used in series. The ULTIVO mass spectrometer was used in MRM mode to analyze the MMAE sample. The mass-to-charge ratio (m / z) of the MMAE ion pair was 718.5→686.5. The CE was 30V and the Fragmentor was 150V.

[0140] The pharmacokinetic results of albumin binding peptide fusion single domain antibody coupled to MMAE drug and free MMAE in SD rats (drug-time curve) are shown in Fig.41 The half-life of free MMAE in rats is about 8.242h, while ABD-VHH HER2 -MMAE, VHHEGFR -ABD-MMAE, VHH EGFR -ABD-Cys-VHH HER2 -MMAE and VHH EGFR -ABD-VHH HER2 The half-lives of -2MMAE in rats were approximately 28.799h, 27.541h, 69.315h and 35.710h, respectively. In comparison, the in vivo half-life of the albumin-binding peptide-fused single-domain antibody-coupled MMAE drug was significantly prolonged.

[0141] Test 7

[0142] The targeting of albumin binding peptide fused single domain antibody coupled to Cy5 was verified in vivo in tumor-bearing mice (nude mice, A549 cell subcutaneous tumor model). A549 cells (2×10 6 A549 cell subcutaneous transplant tumor model was established. When the tumor volume grew to 100 mm 3 Around, randomly divided into VHH EGFR -ABD-Cy5, ABD-VHH HER2 -Cy5, VHH EGFR -ABD-Cys-VHH HER2 -Cy5, VHH EGFR -ABD-VHH HER2 -2Cy5 and Cy5 control groups were given 5 mg / kg (Cy5 equivalent) of the drug by tail vein injection, and the images were taken using a live imaging system at 5, 13, 26, 50, 74, and 98 hours. Figure 42-44 The results showed that the tumor targeting effect of the drug appeared in each experimental group 5 hours after administration ( Fig.42 ), VHH EGFR -ABD-Cy5 and ABD-VHH HE The fluorescence quantum yield of the R2-Cy5 group decreased gradually after 13 h, while that of the VHH EGFR -ABD-Cys-VHH HER2 -Cy5 and VHH EGFR -ABD-VHH HER2 -2Cy5 still had an increase in fluorescence quantum yield from 13h to 26h, proving that the drug continued to accumulate in the tumor site after 13h.

[0143] The above embodiment is only one of the preferred implementation modes of the present invention and should not be used to limit the protection scope of the present invention. Any changes or modifications that are made to the main design concept and spirit of the present invention and have no substantive significance, and the technical problems they solve are still consistent with the present invention, should be included in the protection scope of the present invention.

Claims

1. A conjugate drug of an albumin-binding peptide fused single-domain antibody, characterized in that: It is obtained by fusing albumin-binding peptide with single-domain antibody to obtain fusion protein, and then coupling drug molecules with fusion protein; Fusions of albumin binding peptides and single domain antibodies include: a1, single-target mAb model: the C-terminus or N-terminus of the albumin binding peptide is fused to a single domain antibody; a2, single-target dual antibody mode: the C-terminus and N-terminus of the albumin-binding peptide are simultaneously fused with one single-domain antibody or respectively with two identical single-domain antibodies; a3, Dual-target dual-antibody mode: the C-terminus and N-terminus of the albumin-binding peptide are fused with two different single-domain antibodies respectively.

2. The albumin-binding peptide-fused single-domain antibody conjugate drug according to claim 1, characterized in that: The albumin binding peptide is fused to the C-terminus and / or N-terminus of the single-domain antibody via a flexible connecting peptide, and then expressed via a yeast expression system or an Escherichia coli expression system to obtain a fusion protein; the flexible connecting peptide contains 1-4 cysteines, and the drug molecule is coupled to the cysteine ​​residues in the flexible connecting peptide region of the fusion protein via a covalent coupling chemical bond.

3. The albumin-binding peptide-fused single-domain antibody conjugate drug according to claim 2, characterized in that: The albumin-binding peptide is derived from a domain in streptococcal G protein that binds to human serum albumin or a domain in an anti-human serum albumin antibody that binds to human serum albumin, as well as a modified form thereof; the homology of amino acids in the albumin-binding peptide is higher than 80%, and the molecular weight of the albumin-binding peptide is 5-15 kDa; the single-domain antibody is an antibody heavy chain variable region protein derived from alpaca or shark and composed of a single domain.

4. The albumin-binding peptide-fused single-domain antibody conjugate drug according to claim 3, characterized in that: The albumin binding peptide is derived from the structural domain in Streptococcus G protein that binds to human serum albumin, and its amino acid sequence is shown in SEQ ID NO.

1.

5. The albumin binding peptide fused single domain antibody conjugate drug according to claim 4, characterized in that: The general formula of the flexible connecting peptide is (G m S)n i C or G i C-(G m S)n; wherein G is glycine, S is serine, and C is cysteine; m, i, and n are selected from natural integers in the range of 0-10.

6. The albumin-binding peptide-fused single-domain antibody conjugate drug according to claim 5, characterized in that: The amino acid sequence of the flexible connecting peptide is shown as SEQ ID NO.2 or SEQ ID NO.

3.

7. The albumin-binding peptide-fused single-domain antibody conjugate drug according to claim 6, characterized in that: The molecular weight of the drug molecule does not exceed 2000Da; the drug molecule is selected from one or a combination of at least two of monomethyl auristatin D or its derivatives, monomethyl auristatin E or its derivatives, monomethyl auristatin F or its derivatives, maytansine or its derivatives.

8. The method for preparing the albumin binding peptide fused single domain antibody conjugate drug according to any one of claims 1 to 7, characterized in that: include: S1, by means of genetic engineering, the albumin binding peptide and the single domain antibody are fused through a flexible connecting peptide, and the fusion amino acid sequence is converted into a gene sequence, and the codon preference is optimized by codon preference software, and the engineered bacteria are transformed, positive bacteria are selected, and then cultured and induced for expression, and purified by affinity chromatography, hydrophobic chromatography and ion exchange chromatography to obtain a fusion protein of the albumin binding peptide and the single domain antibody; S2, dissolving the fusion protein in a slightly alkaline buffer solution and pre-treating it with 0.2-1.0 mM tris(2-carboxyethyl)phosphine to obtain a solution A; dissolving the drug molecule in an organic solvent to obtain a solution B, then adding the solution B dropwise to the solution A, stirring gently, reacting at room temperature in the dark for 1-24 hours, and removing the precipitate by centrifugation to obtain a crude coupling product; The reaction molar ratio of drug molecule to fusion protein is 1-10:1; S3, purifying the crude conjugated product by desalting to obtain the conjugated drug of the albumin-binding peptide fused single-domain antibody.

9. The method for preparing the albumin binding peptide fused single domain antibody conjugate drug according to claim 8, characterized in that: In step S2, the pH of the alkaline buffer solution is 6.5-8.0, the pretreatment time is 30 minutes, and the organic solvent is a dimethyl sulfoxide solution with a volume percentage of 10-50%; in step S3, the chromatography column filler during desalting and purification is Sephadex-G25.

10. Use of the albumin binding peptide fused single domain antibody conjugate drug according to any one of claims 1 to 9 in the preparation of anti-tumor drugs.

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