CN107936118B

CN107936118B - Antibody-dolastatin conjugate as well as preparation method and application thereof

Info

Publication number: CN107936118B
Application number: CN201711052440.1A
Authority: CN
Inventors: 来骏; 吴姗姗; 王赟; 陈枢青
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2017-10-30
Filing date: 2017-10-30
Publication date: 2021-03-30
Anticipated expiration: 2037-10-30
Also published as: CN107936118A

Abstract

The invention discloses a preparation method of an antibody-dolastatin conjugate, which comprises the following steps: (1) providing a monoclonal antibody of an anti-MART-1 protein presentation peptide EAAGIGILTV/HLA-A2 complex with an LPXTG sequence at the C terminal, dolastatin with an oligoglycine linker or a dolastatin derivative; (2) under the catalysis of Sortase A enzyme, the LPXTG sequence and the oligoglycine linker generate transpeptidation reaction, so that the monoclonal antibody is coupled with the dolastatin or dolastatin derivative with the oligoglycine linker; (3) after the reaction is finished, separating and purifying to obtain the antibody-dolastatin conjugate. The invention leads each molecule of antibody to carry 4 molecules of MMAE toxin through site-specific coupling, has good uniformity and shows good tumor killing capability in vivo experiments; compared with the existing ADCs target point, the CLA 12-vcMAE ADCs expand the selection area of the current ADCs target point, and demonstrate the view that intracellular proteins can also be used as the ADCs drug target point through the presentation of MHC I molecules.

Description

Antibody-dolastatin conjugate as well as preparation method and application thereof

Technical Field

The invention relates to the technical field of biological pharmacy, in particular to an antibody-dolastatin conjugate and a preparation method and application thereof.

Background

MART-1 protein is a melanoma-associated antigen which is expressed only in melanocytes and most of melanoma cells, and is highly expressed in melanoma cells, and according to incomplete statistics, 100% of melanoma samples have MART-1 protein expression (Barrow C et al, Clin Cancer Res.2006 Feb 1; 12(3 Pt 1): 764-. Therefore, the drug designed by taking the MART-1 protein as a target point is selected to have wider application in melanoma.

In the last 20 years, the rapid development of the monoclonal antibody field makes it have a significant role in the clinical tumor treatment field, and for example, a series of monoclonal antibodies such as cetuximab, trastuzumab and rituximab all show good clinical treatment effects on respective tumor indications. They usually achieve the goal of inhibiting tumor cell growth and killing tumor cells by inhibiting the key signaling pathways of tumor cells and inducing ADCC, CDC effects through the Fc region (Weiner LM et al, Nat Rev immunol.2010 May; 10(5): 317-.

Antibody-conjugated drugs (ADCs) are novel biological drugs combining monoclonal drugs and chemical modification/conjugation technology, which link monoclonal antibodies and toxic small molecules together through a linker technology, have not only excellent targeting property of the monoclonal drugs but also high cytotoxicity of small molecule poisons, so that the monoclonal drugs can quickly and accurately reach tumor sites, and release the toxic small molecules in cells after being endocytosed by tumor cells, thereby achieving the purpose of quickly and strongly killing the tumor cells. However, we have noticed that the target to which they are directed, whether monoclonal antibodies or antibody-conjugated drugs are used clinically, is still the extracellular region of the membrane protein on the surface of the cell membrane, and there is still no way to express or mutate tumor-associated proteins in the cell.

Major Histocompatibility Complex (MHC) for short is specifically classified into two types. The MHC class I molecule compound consists of MHC class I molecules, beta 2-microglobulin and presentation peptide with the length of 8-11 amino acids, wherein the MHC class I molecules can be mainly classified into three categories of HLA-A, B, C. Cells present polypeptides produced by proteasome hydrolysis of self proteins to the cell surface via their MHC class I molecules (York IA et al, Annu Rev Immunol. 1996; 14: 369-. Based on this theory, amino acids 26-35 of the melanoma associated antigen MART-1 protein: EAAGIGILTV is being presented to the cell surface by HLA-A2 molecules. It is this theory that intracellular proteins can also be targets for antibodies and even antibody-conjugated drugs.

The monoclonal antibody has a stable structure and good targeting property, but the stability or targeting property of the antibody may be reduced by common coupling modes such as amino and sulfhydryl coupling modes.

Sortase A is an enzyme which is separated from staphylococcus aureus and catalyzes a transpeptidation reaction, cysteine at the 184 position of the Sortase A nucleophilically attacks a peptide bond between threonine (T) and glycine (G) in a label of LPXTG (X represents any one of natural amino acids) to form a covalent thio intermediate, and simultaneously releases glycine and a carboxyl C terminal peptide segment thereof. This intermediate forms a new peptide bond between threonine and the captured glycine substrate by capturing the glycine substrate in the solvent, releasing the sortase a enzyme for further catalysis. Because the reaction has high specificity, simple operation and mild reaction conditions, sortase A is widely used for protein engineering and protein site-directed modification.

Disclosure of Invention

The invention provides an antibody-dolastatin conjugate formed by coupling an antibody of EAAGIGILTV/HLA-A2 and dolastatin (MMAE), a preparation method and application thereof, aiming at the defects in the prior art.

A method for preparing an antibody-dolastatin conjugate, comprising the steps of:

(1) providing a monoclonal antibody of an anti-MART-1 protein presentation peptide EAAGIGILTV/HLA-A2 complex with an LPXTG sequence at the C terminal, dolastatin with an oligoglycine linker or a dolastatin derivative;

(2) under the catalysis of Sortase A enzyme, the LPXTG sequence and the oligoglycine linker generate transpeptidation reaction, so that the monoclonal antibody is coupled with the dolastatin or dolastatin derivative with the oligoglycine linker;

(3) after the reaction is finished, separating and purifying to obtain the antibody-dolastatin conjugate.

The heavy chain amino acid sequence of the monoclonal antibody of the anti-MART-1 protein presentation peptide EAAGIGILTV/HLA-A2 compound is shown as SEQ ID No.1, and the light chain amino acid sequence is shown as SEQ ID No. 3.

The C ends of the heavy chain and the light chain of the monoclonal antibody are connected with LPXTG sequences. The C-terminal is located in the constant region and far away from the variable region of the antibody, so that the coupling has small influence on the activity of the antibody. The C end of the heavy chain light chain is connected with LPXTG sequence for coupling the dolastatin, the proportion of the dolastatin coupled on a single antibody can be increased as much as possible, one molecule of the antibody comprises two heavy chains and two light chains, thus theoretically one molecule of the antibody can be coupled with four molecules of dolastatin, and the proportion of the drugs carried by the antibody is high.

The heavy and light chains of the monoclonal antibody are linked to GWSHPQFEK sequences after the LPXTG sequence. The sequence can increase the stability of a complex between the sortase A enzyme and the tail end of an antibody, thereby increasing the coupling efficiency, and the glycine residue at the tail end of the recognition sequence of the sortase A enzyme can be cut off together after the coupling, so that the activity of the final antibody-dolastatin conjugate is not influenced.

The light chain LPXTG sequence of the monoclonal antibody is connected with a flexible joint in front, and the flexible joint is GGGGS pentapeptide. The flexible joint is introduced, the problem that the prior toxic drug with oligoglycine cannot be coupled on the light chain of the antibody is solved, the coupling efficiency is greatly improved, and the antibody coupling drug with higher drug-antibody coupling ratio (DAR) is obtained.

The oligoglycine joint is a GGG sequence, and a valine-citrulline dipeptide joint is arranged between the oligoglycine joint and the dolastatin or the dolastatin derivative. The valine-citrulline dipeptide linker can be cleaved by the lysosomal enzyme cathepsin B.

The invention also provides the antibody-dolastatin conjugate prepared by the preparation method.

The invention also provides application of the antibody-dolastatin conjugate in preparing antitumor drugs.

The invention also provides an anti-tumor drug which comprises the antibody-dolastatin conjugate.

The tumor is melanoma with double positive of HLA-A2 and MART-1.

The invention leads each molecule of antibody to carry 4 molecules of MMAE toxin through site-specific coupling, has good uniformity and shows good tumor killing capability in vivo experiments; compared with the existing ADCs target point, the CLA 12-vcMAE ADCs expand the selection area of the current ADCs target point, and demonstrate the view that intracellular proteins can also be used as the ADCs drug target point through the presentation of MHC I molecules.

Drawings

FIG. 1 shows that CLA12 ADCs and CLA12 monoclonal antibody are subjected to high performance liquid chromatography PLRP-S in a reduced state

Analytical results of the reverse column.

FIG. 2 is a graph showing the results of analysis of CLA12 ADCs and CLA12 monoclonal antibody in a non-reduced state on a high performance liquid HIC column.

FIG. 3 is the secondary fragment pattern of GGG-vcMMAE after mass spectrometry.

Fig. 4 is a graph showing the results of mass spectrometry analysis of the pancreatin-digested CLA12 ADCs polypeptides, and 3 GGG-vcMMAE characteristic ions were extracted, wherein fig. A, B, C is a graph showing the analysis of three characteristic ions, and fig. D is a graph showing the total mass spectrometry analysis.

FIG. 5 is a characteristic peak diagram of extracting target peptide with LPETGGG-vcMMAE from a primary diagram of a ultraviolet absorption diagram and a primary diagram of a mass spectrum after the CLA12 ADCs polypeptide which is digested by pancreatin is analyzed by the mass spectrum, wherein, a diagram A is the ultraviolet absorption diagram, and a diagram B is the primary diagram of the mass spectrum.

FIG. 6 is a diagram of the secondary fragment of CLA12 ADCs polypeptide after trypsinization with LPETGGG-vcMMAE target peptide at the C-terminal of the antibody heavy chain after mass spectrometry.

FIG. 7 is a diagram of the secondary fragment of CLA12 ADCs polypeptide after trypsinization with LPETGGG-vcMMAE target peptide at the C-terminal of the antibody light chain after mass spectrometry.

FIG. 8 is a graph showing the affinity of CLA12 ADCs and CLA12 monoclonal antibody for MEL624.38, a target cell, at a concentration of 5. mu.g/mL, using flow cytometry.

FIG. 9 is a graph of the killing effect of CLA12 ADCs and CLA12 monoclonal antibodies on MEL624.38, MCF7, SK-MEL 28 and K562 cell lines.

Fig. 10 is a graph of the induction of MEL624.38 apoptosis by CLA12 ADCs and CLA12 monoclonal antibody, wherein, graph a is negative control group RPIM-1640, graph B is an experimental group, graph C is an indication graph, and graph D is CLA12 control group.

FIG. 11 is a graph showing the in vivo tumor growth in mice treated with different doses of CLA12 ADCs, CLA12 monoclonal antibody and control ADCs.

FIG. 12 is a graph showing the change in body weight of SCID-beige mice dosed with different doses of CLA12 ADCs, CLA12 monoclonal antibody and control ADCs.

Detailed Description

Example 1

Preparation of CLA12HL-vcMMAE conjugate.

The monoclonal antibody targeting the EAAGIGILTV/HLA-A2 complex was CLA12, the sequence of which was derived from U.S. Pat. No.: US20100158927a 1. The gene sequence of the antibody light and heavy chain variable region (the gene sequence of the heavy chain variable region is shown as SEQ ID No.5, and the gene sequence of the light chain variable region is shown as SEQ ID No. 6) is synthesized by the generation of bioengineering biological engineering (Shanghai), the constant region sequence of the antibody light and heavy chain is stored in a laboratory, the light and heavy chain variable region is respectively connected with a human Lamda light chain constant region and a human IgG1 type heavy chain constant region by a PCR method, the end of the light and heavy chain C is connected with an LPETG sequence, and the recombinant antibody obtained by expression is named as CLA12 HL.

(1) CLA12 monoclonal antibody light heavy chain C terminal transformation, through PCR method add coding LPETG polypeptide nucleic acid sequence separately in the light heavy chain C terminal of the antibody, wherein Lamda light chain constant region and LPETG sequence are connected through GGGGS pentapeptide linker, and add GWSHPQFEK sequence after light heavy chain LPETG sequence to increase the stability of the complex between sortase A enzyme and antibody C terminal, thus increase the coupling efficiency, after the light heavy chain sequence is constructed, connect them separately with pMH3 expression vector and transfect to Chinese hamster ovary Cell (CHO) steadily, choose the high monoclonal antibody of expression level to express, and purify the antibody with protein A affinity column. The final gene sequence of the heavy chain is shown as SEQ ID No.2, the gene sequence of the light chain is shown as SEQ ID No.4, the amino acid sequence of the heavy chain is shown as SEQ ID No.1, and the amino acid sequence of the light chain is shown as SEQ ID No. 3.

(2) Expressing Sortase A enzyme, obtaining base sequence corresponding to 60-210 amino acid by PCR method, adding His6 label at N end, constructing in expression vector PET28a, expressing by Escherichia coli Rosetta strains 2(DE3), adding IPTG to make final concentration 1mM when bacterial liquid OD600 is about 0.5. Finally, the product was purified by an affinity column (HiTrap Ni-NTAcolumn, GE).

(3) The coupling reaction system is as follows: 50mM Tris-HCl, 150mM NaCl, 5mM CaCl₂The CLA12 HL-vcMAE conjugate is prepared by reacting at room temperature for 8h, wherein the pH is 7.5, the CLA12HL antibody is 2 mu M, the sortase A enzyme is 10 mu M, the GGG-vcMAE (Nanjing Linning biopharmaceutical Co., Ltd., GGG-vcMAE, GGG represents three glycine residues, vc represents a dipeptide linker (valine-citrulline) which can be broken by the lysosome enzyme cathepsin B, and MMAE is high-toxicity small-molecule drug sea rabbit toxin).

(4) The CLA12HL-vcMMAE conjugate is purified by the previous high performance liquid chromatography and mass spectrometry, and the result shows that the antibody is basically connected with dolastatin in terms of weight and the reaction is basically complete, so the product is purified by directly using an affinity chromatography column (HiTrap Protein A HP column, GE) to obtain uniform CLA12 ADCs, and the obtained product is sterilized by a filter membrane with the aperture of 0.22 mu m and stored at-20 ℃ for later use.

Example 2

Identification of CLA12HL-vcMMAE conjugates.

(1) Performing high performance liquid chromatography identification on CLA12HL-vcMMAE conjugate drug, respectively adding small amount of TCEP powder into CLA12HL-vcMMAE conjugate drug and CLA12HL monoclonal antibody 20 μ g, mixing, incubating at 37 deg.C for 10min, and performing liquid chromatography with PLRP-S

The analysis was carried out on a reversed phase column, and the detection wavelength was 280 nm.

As shown in FIG. 1, the CLA12HL monoclonal antibody has two peaks of a light chain (L0) and a heavy chain (H0), and the CLA12 HL-vcMAE also has two peaks of a light chain (L0) and a heavy chain (H0), but the positions of the two peaks of the light chain and the heavy chain are obviously shifted backwards compared with the CLA12HL monoclonal antibody, and the positions of the two peaks of the light chain and the heavy chain are not overlapped with the positions of the two peaks of the CLA12HL monoclonal antibody, which indicates that the CLA12 HL-vcMAE light chain and the heavy chain are respectively connected with GGG-vcMAE.

(2) Performing high performance liquid phase identification on the CLA12 HL-vcMAE coupled drug, taking 20 mu g of each CLA12 HL-vcMAE coupled drug and CLA12HL monoclonal antibody, analyzing by using an HIC hydrophobic column, and detecting the wavelength to be 280 nm.

The detection result is shown in fig. 2, the CLA12HL monoclonal antibody has a single peak at about 6min, the CLA12HL-vcMMAE has a main single peak at about 11min, and 3 very small peaks at about 9min and 10min, so the CLA12HL-vcMMAE conjugate is judged to have good homogeneity as a main product, and the results shown in fig. 1 show that basically 1 molecule of CLA12HL monoclonal antibody is basically connected with 4 molecules of GGG-vcMMAE.

(3) The CLA12 HL-vcMAE conjugate drug mass spectrum identification comprises diluting a test sample to be tested with CLA12 HL-vcMAE conjugate drug in guanidine hydrochloride-Tris solution (pH 8.0), adding dimercaptothreitol (final concentration 20mM) for reaction for 1.5h, adding iodoacetamide (final concentration 60mM) for reaction at room temperature in a dark place for 40min to block free cysteine residues, centrifuging at 12000rpm/min4 ℃ to replace buffer solution with 100mM phosphate (pH7.4), adding trypsin for digestion at 37 ℃ for 24h, and terminating the digestion reaction with formic acid (final concentration 1%) to be tested.

The columns used in this assay were: acquity UPLC BEH 300C 18, sample size 10 μ L. The liquid phase separation conditions are shown in table 1, and the mass spectrum parameter settings are shown in table 2.

TABLE 1 ultra-high performance liquid phase separation of polypeptides after enzyme digestion with CLA12HL-vcMMAE conjugate drugs.

Table 2 sets the mass spectrum parameters of the polypeptide after the enzyme digestion of CLA12HL-vcMMAE coupled drug.

Capillary voltage	2.5kV	Voltage of taper hole	80V
				Mass analysis scope	50 to 2000Da	MS^ECollision energy	20 to 45eV
Ion source temperature	90℃	Temperature of atomization	400℃
				Velocity of atomization	700L/Hr	Internal standard	Leucine enkephalin

And (3) detection results: firstly, a secondary map of GGG-vcMMAE micromolecules is shown in figure 3, wherein ions with molecular weights of 506.4110, 686.5637 and 718.5949 are characteristic fragments of GGG-vcMMAE; secondly, as shown in fig. 4, the total ion flow diagram of CLA12HL-vcMMAE is subjected to extraction of the feature fragments of GGG-vcMMAE, and all three feature fragments have single peaks with higher intensity at 38.3min and 39.7 min; thirdly, as shown in fig. 5, combining the total ion flow diagram and the ultraviolet absorption diagram of CLA12HL-vcMMAE, and we observed that the molecular weights of the two peaks at 38.3min and 39.7min are 2895.476Da and 1734.998Da, respectively, which are consistent with the theoretical values of polypeptide fragments with LPETGGG-vcMMAE (2895.477Da and 1735Da), and it was preliminarily judged that the polypeptides with LPETGGG-vcMMAE connected to the C-terminal of the light chain and the C-terminal of the heavy chain correspond to the two peaks at 38.3min and 39.7min, respectively; fourthly, as shown in fig. 6 and table 3(38.3min), fig. 7 and table 4(39.7min), the two peaks at 38.3min and 39.7min were further analyzed for secondary fragments, and it was found that the corresponding peptide fragment actually contained the LPET sequence, and that there were modifications found to GGG-vcMMAE on a part of y ions, and characteristic fragments of GGG-vcMMAE were also found on the secondary map of the whole peptide fragment. Thus, it was determined from mass spectrometry that the coupling site of GGG-vcMMAE indeed follows the LPET sequence.

The peaks in Table 338.3 min were subjected to further secondary fragmentation analysis data.

The peaks in Table 439.7 min were subjected to further secondary fragmentation analysis data.

Example 3

The biological activity of the CLA12HL-vcMMAE conjugate was tested.

1. Flow affinity assay

(1) Take 5X 10⁵Each MEL624.38 cell was incubated with 5 μ g/mL CLA12 monoclonal antibody and CLA12HL-vcMMAE in 1% BSA in PBS at 4 ℃ for 30 min;

(2) after two PBS washes, goat anti-human IgG (H + L) polyclonal antibody (diluted 1: 200) was added and incubated at 4 ℃ for 30 min; after two washes with PBS, the Mean Fluorescence Intensity (MFI) of the cells was measured with a loss cell analyzer.

The flow cytometry analyzer detects the binding of CLA12 and CLA12 HL-vcCMMAE to MART-1, HLA-A2 double positive cells MEL624.38, and the strength of the binding force is shown by the average fluorescence intensity of FITC after secondary antibody labeling.

As shown in FIG. 8, the affinity of CLA12 monoclonal antibody and CLA12 HL-vcMAE was almost the same for MEL624.38 of the same number of MART-1, HLA-A2 double positive cells at the same concentration.

2. Cytotoxicity assays

(1) MEL624.38, MCF7, SK-MEL 28, K562, four cell lines were plated in 96-well plates with 2500 cells per well in separate media, CLA12 monoclonal antibody and CLA12HL-vcMMAE were serially diluted in gradient and added to each well in order to give final concentrations of drugs: 10. mu.g/mL, 5. mu.g/mL, 1. mu.g/mL, 0.5. mu.g/mL, 0.1. mu.g/mL, 0.05. mu.g/mL, 0.01. mu.g/mL, 0.005. mu.g/mL, 0.001. mu.g/mL, 0.0005. mu.g/mL, 3 attachment wells per drug concentration. After 120h, 10uL of CCK-8 (Dongren chemical technology, Shanghai, Ltd.) was added to each well, and after 1h, the OD value at 450nm was measured, and the cell viability was calculated.

The results are shown in FIG. 9, and the IC of CLA12HL-vcMMAE for MART-1+, HLA-A2+ cell MEL624.38₅₀The value is about: 1.01 μ g/mL IC for MART-1-, HLA-A2+ cells MCF7₅₀Values of about 72.24. mu.g/mL, IC for MART-1+, HLA-A2-cell SK-MEL 28₅₀The value is about: 16.66 μ g/mL IC for MART-1-, HLA-A2-cell K562₅₀The value is about: 26.14. mu.g/mL, wherein the IC of CLA12 monoclonal against double positive cells MEL624.38₅₀The value is about: 64.25 mu g/mL, the ADCs obtained by the invention have obviously improved killing capability on positive cells compared with the original monoclonal antibody, but have a cross reaction phenomenon at a cell level, particularly on MART-1+, HLA-A2-cell SK-MEL 28.

3. Apoptosis assay

(1) Respectively at 5X 10⁴Plating MEL624.38 cells, adding 1 μ g/mL CLA12 monoclonal antibody and CLA12 HL-vcCMMAE, culturing for 72 hr, centrifuging, removing culture medium, washing with PBS, and collecting 2 × 10⁵MEL624.38 cells were incubated with 500. mu.L of binding buffer, 5. mu.L of annexin V, 10. mu.L of PI (propidium iodide) at room temperature in the absence of light for 5 min. The flow cytometer detected the percentage of apoptotic cells. The detection results are shown in fig. 10.

As can be seen from fig. 10, the apoptosis rate of CLA12 monoclonal antibody treatment was very low, which is almost not different from that of blank administration group, while CLA12HL-vcMMAE induced apoptosis phenomenon is much, and the premature/wann withering rate is 16.6% and 8.2%, respectively, showing that CLA12HL-vcMMAE has stronger killing ability to positive cell MEL624.38 than CLA12 monoclonal antibody.

4. SCID-beige mouse in vivo antitumor assay

Culturing MEL624.38 cells in vitro to logarithmic phase, digesting with pancreatin, washing twice with RPMI-1640, adding appropriate amount of sterile PBS, mixing, adding into high concentration matrigel (Corning Corp.), mixing, and temporarily cooling in ice;

the skin of the right underarm of the anterior limb of SCID-beige mice is sterilized with 75% alcohol, and each injection is 0.2mL, namely 10⁷Tumor cells with an average tumor size of 180mm in vivo³At the time, 25 mice were randomly divided into 5 groups of 5 mice each;

for 5 groups of SCID-beige rat tail veins: no drug administration group, CLA12 monoclonal antibody group (15mg/kg), OFA-vcMAE group (DAR ≈ 4), CLA12 HL-vcMAE low dose group (5mg/kg) and CLA12 HL-vcMAE high dose group (15 mg/kg). The dosing interval was 3 days, for a total of 4 doses.

As can be seen from fig. 11, the CLA12 monoclonal antibody group (15mg/kg), the CLA12HL-vcMMAE low dose group (5mg/kg) and the CLA12HL-vcMMAE high dose group (15mg/kg) all inhibited tumor growth, wherein the CLA12HL-vcMMAE high dose group had the most significant inhibitory effect, and it was found that the ADCs drug had a slow onset, the drug concentration had to be maintained at a high level at day 4 after the administration, the high dose group had two mice with completely eliminated tumors, and at day 7 after the administration, all the mice with high dose had completely eliminated tumors, and no recurrence was observed until day 60. The CLA12 monoclonal antibody group (15mg/kg) and the CLA12 HL-vcMAE low-dose group (5mg/kg) have slightly different tumor inhibition effects, a slight difference can be seen on the 4 th day after the administration, the longer the time is, the more obvious the difference between the tumor inhibition effects of the two groups is, and the CLA12 HL-vcMAE low-dose group has slightly better tumor inhibition capability than the CLA12 monoclonal antibody group. Statistical analysis is carried out on data after the last administration, and compared with the OFA-vcMAE group, the CLA12 HL-vcMAE low dose group and the CLA12 HL-vcMAE high dose group, the tumor inhibition capability is obviously different.

As can be seen from FIG. 12, the effect of MEL-624.38 cells on SCID-beige mice was greater, reaching a tumor volume of 800mm for the non-drug administered group³When the mice grow, the body weight of the mice tends to decrease, and the mice except the CLA12HL-vcMMAE high-dose group grow to 800-1000mm in the tumor body³After, make upThe body weight of mice also decreased, but the CLA12HL-vcMMAE high dose group showed a tendency of complete elimination of tumor bodies in the body of the mice after administration, and the body weight of the mice increased all the time, indicating that the CLA12HL-vcMMAE at high dose did not significantly poison the mice while killing tumor cells.

The experimental results show that the Sortase A enzyme can be used for carrying out fixed-point coupling on the monoclonal antibody and the dolastatin to obtain uniform ADCs (adenosine receptor ligands) with DAR being approximately equal to 4, and the obtained CLA12HL-vcMMAE conjugate has good stability and targeting property, can effectively kill tumor cells in vitro and in a mouse body, but has slow effect, and needs to maintain the drug concentration at a higher level, and the CLA12HL-vcMMAE has good state all the time in the process of killing the tumor, and has no obvious toxic or side effect on other tissues of the mouse.

Sequence listing

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ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac 840

accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 900

gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 960

aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 1020

caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 1080

gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 1140

accctgcccc catcccggga ggagatgacc aagaaccagg tcagcctgac ctgcctggtc 1200

aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 1260

aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag 1320

ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 1380

gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaactacct 1440

gaaactggag gttggtcaca tcctcaattt gaaaagtaa 1479

<210> 3

<211> 265

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Lys Leu Arg Val Gly Gln Thr Leu Gly Thr Ile Pro Arg Gln Cys

1 5 10 15

Glu Val Leu Leu Leu Leu Leu Leu Leu Gly Leu Val Asp Gly Gln Ser

20 25 30

Val Val Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Arg Lys Val

35 40 45

Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val

50 55 60

Ser Trp Tyr Gln Gln Val Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr

65 70 75 80

Glu Asp Asp Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

85 90 95

Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln Thr Gly

100 105 110

Asp Glu Ala Asp Tyr Phe Cys Ala Thr Trp Asp Arg Thr Val Asn Val

115 120 125

Val Arg Phe Gly Gly Gly Thr Arg Leu Thr Val His Ser Gln Pro Lys

130 135 140

Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln

145 150 155 160

Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly

165 170 175

Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly

180 185 190

Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala

195 200 205

Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser

210 215 220

Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val

225 230 235 240

Ala Pro Thr Glu Cys Ser Gly Gly Gly Gly Ser Leu Pro Glu Thr Gly

245 250 255

Gly Trp Ser His Pro Gln Phe Glu Lys

260 265

<210> 4

<211> 798

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgaagctca gagtaggaca gactctgggc actatcccca ggcagtgtga agttctcctc 60

ctgctgctgc tgttgggtct agtggatggt cagtctgtcg tgacgcagcc gccctcagtg 120

tctgcggccc caggacggaa ggtcaccatc tcctgctctg gaagcagctc caacattggg 180

agtaattatg tgtcctggta ccagcaagtc ccagggacag cccccaaact cctcatctat 240

gaagatgata agcgaccctc agggattcct gaccgattct ctggctccaa gtctgggacg 300

tccgccaccc tgggcatcac cggactacag actggggacg aggccgatta tttctgcgcc 360

acgtgggata ggactgtcaa tgttgtacgt ttcggcggag ggaccaggct gaccgtccac 420

agtcagccca aggctgcccc ctcggtcact ctgttcccgc cctcctctga ggagcttcaa 480

gccaacaagg ccacactggt gtgtctcata agtgacttct acccgggagc cgtgacagtg 540

gcttggaaag cagatagcag ccccgtcaag gcgggagtgg agaccaccac accctccaaa 600

caaagcaaca acaagtacgc ggccagcagc tatctgagcc tgacgcctga gcagtggaag 660

tcccacagaa gctacagctg ccaggtcacg catgaaggga gcaccgtgga gaagacagtg 720

gcccctacag aatgttcagg cggcggagga tccctacctg aaactggagg ttggtcacat 780

cctcaatttg aaaagtga 798

<210> 5

<211> 354

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cagctgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtagtagtt actactgggg ctggatccgc 120

cagcccccag ggaaggggct ggagtggatt gggagtatct attatagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagtcacc atatccgtag acacgtccaa gaaccagttc 240

tccctgaagc tgagctctgt gaccgccgca gacacggctg tgtattactg tgcgagacat 300

gtgggccacg aacttgacta ctggggccag ggaaccctgg tcaccgtctc aagc 354

<210> 6

<211> 333

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cagtctgtcg tgacgcagcc gccctcagtg tctgcggccc caggacggaa ggtcaccatc 60

tcctgctctg gaagcagctc caacattggg agtaattatg tgtcctggta ccagcaagtc 120

ccagggacag cccccaaact cctcatctat gaagatgata agcgaccctc agggattcct 180

gaccgattct ctggctccaa gtctgggacg tccgccaccc tgggcatcac cggactacag 240

actggggacg aggccgatta tttctgcgcc acgtgggata ggactgtcaa tgttgtacgt 300

ttcggcggag ggaccaggct gaccgtccac agt 333

<210> 7

<211> 10

<212> PRT

<213> Homo sapiens

<400> 7

Glu Ala Ala Gly Ile Gly Ile Leu Thr Val

1 5 10

<210> 8

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Leu Pro Glu Thr Gly

1 5

<210> 9

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Gly Gly Gly Gly Ser

1 5

<210> 10

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Gly Gly Gly

1

<210> 11

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Gly Trp Ser His Pro Gln Phe Glu Lys

1 5

Claims

1. A preparation method of an antibody-dolastatin conjugate is characterized by comprising the following steps:

(1) providing a monoclonal antibody of an anti-MART-1 protein presentation peptide EAAGIGILTV/HLA-A2 complex with an LPXTG sequence at the C terminal, dolastatin with an oligoglycine linker;

(2) under the catalysis of Sortase A enzyme, the LPXTG sequence and the oligoglycine linker generate transpeptidation reaction, so that the monoclonal antibody is coupled with the dolastatin with the oligoglycine linker;

(3) after the reaction is finished, separating and purifying to obtain the antibody-dolastatin conjugate,

2. The method of claim 1, wherein the oligoglycine linker is a GGG sequence and a valine-citrulline dipeptide linker is provided between the oligoglycine linker and the dolastatin.

3. An antibody-dolastatin conjugate prepared by the method of claim 1 or 2.

4. The use of the antibody-dolastatin conjugate of claim 3 for the preparation of an anti-tumor agent, wherein the tumor is melanoma that is double positive for HLA-A2 and MART-1.

5. An antitumor drug comprising the antibody-dolastatin conjugate according to claim 3, wherein the tumor is melanoma with double positive of HLA-A2 and MART-1.