CN107875398B - Preparation method of antibody conjugate drug, antibody conjugate drug and application - Google Patents

Abstract

The invention discloses a preparation method of an antibody conjugate drug, the antibody conjugate drug and application. In the preparation method, under the catalysis of Sortase A enzyme, an LPXTG sequence and an oligoglycine linker generate transpeptidation reaction, so that the anti-CD20 monoclonal antibody is connected with a first connecting arm; and then the azide linker and the alkynyl linker undergo a cycloaddition reaction, so that the first connecting arm is connected with the second connecting arm. The antibody coupling drug is prepared by an enzyme-chemical method, so that the dosage of expensive drugs is reduced, and the cost is saved. The bifunctional micromolecules are adopted, so that the steric hindrance of the Sortase A during the transpeptidation reaction is reduced, and the flexibility is good. Effectively controlling the coupling sites and the number of the drugs on the antibody, enhancing the uniformity and improving the in vivo and in vitro anti-tumor activity of the antibody on the basis of keeping the original antibody affinity.

Description

Preparation method of antibody conjugate drug, antibody conjugate drug and application

Technical Field

The invention relates to the technical field of biology, in particular to a preparation method of an antibody conjugate drug, the antibody conjugate drug and application.

Background

Cancer, one of the most serious diseases, threatens the life health of human beings, and has great toxic and side effects due to poor target selectivity of the traditional chemotherapy; however, the conventional monoclonal antibody therapy is often poor in curative effect after being used by tumor patients due to weak tumor killing capability. Antibody-conjugated drugs (ADCs) are used as an innovative treatment option, and are formed by connecting antibodies or fragments thereof and cytotoxic drugs, so that the antibody-conjugated drugs have the advantages of both biological drugs and chemical drugs, namely, the antibody-conjugated drugs simultaneously utilize the unique targeting property of the antibodies and the extremely strong cytotoxicity of small-molecule chemical drugs, and show unique advantages in cancer treatment.

As early as a century ago, the German immunologist Paul Ehrlich proposed the concept of "magic bullets" (Perez HL, Cardarelli PM, Desmopander S, et al]Drug Discov Today,2014,19, 869-881). ADCs comprise antibody, cytotoxic drug and linker, and selectively deliver the "warhead" drug to target cells with antibody as carrier, and combine with antigen, and perform endocytosisEnters lysosome and is hydrolyzed by hydrolase such as cathepsin in the lysosome to release cytotoxic drugs, and tumor cells are killed specifically. Compared with chemotherapy and combination therapy, the selectivity of the ADCs is good, off-target toxicity can be obviously reduced, and the therapeutic window is widened. In recent years, Brentuximab vegetin (

SGN-35) and Trastuzumab (

T-DM1), etc., have achieved significant clinical success, again demonstrating that ADCs are a powerful weapon against cancer.

ADCs currently on the market achieve drug-antibody conjugation by covalently linking cytotoxic drugs through lysine residues or cysteine residues produced by reduction of interchain disulfide bonds in antibodies. Since antibodies contain multiple lysine or cysteine residues, drug to antibody ratio (DAR) of ADCs obtained in this manner varies from 0 to 8, and the conjugation sites have large variability and large heterogeneity, which poses a serious challenge to batch-to-batch consistency of ADCs. In addition, these mixtures often have different pharmacodynamic, Pharmacokinetic (PK) and safety issues, which present great difficulties for the quality control and clinical use of ADCs.

In recent years, various site-directed coupling techniques have been developed to achieve uniformity of ADCs. One of the methods is to generate uniform ADCs by mutating an amino acid on an antibody to cysteine through genetic engineering and then forming a thioether bond with a toxin drug modified by maleic amide for site-specific coupling on the premise of not influencing the affinity of the antibody. However, clinical trials have found that the linker is unstable in the circulation and can exchange with the reactive thiol group of free cysteines in plasma, leading to premature release of the cytotoxic drug. Therefore, in the site-directed conjugation of ADCs, not only is the uniformity of ADC achieved, but also the stability of the linker in vitro and in the circulatory system is ensured to avoid the systemic toxicity brought by ADCs, and only after the ADCs are endocytosed, the linker can be effectively disconnected and active cytotoxic drugs can be released to kill tumor cells. In addition, suitable mutation sites are crucial to the activity and tolerance of ADCs, and these sites vary from antibody to antibody, and need to be rescreened by PHESELECTOR technology, thus greatly reducing the universality.

Based on the disadvantages of the above chemical coupling methods, the enzymatic coupling techniques have been further developed with the continuing efforts of researchers. Sortase a is an enzyme which catalyzes a transpeptidation reaction and is isolated from staphylococcus aureus, cysteine at position 184 of the Sortase nucleophilically attacks a peptide bond between threonine (T) and glycine (G) in a LPXTG (X represents any one of natural amino acids) tag to form a covalent thio intermediate, and simultaneously releases glycine and a carboxyl-terminal peptide fragment 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, the Sortase A is widely used for protein engineering and protein site-directed modification. Thus, ADCs catalyze transpeptidation as an efficient method for generating uniform ADCs by controlled site-directed modification. Since Sortase a is less efficient in catalysis and the reaction is reversible, a large excess of oligoglycine-modified toxin drug (100-fold molar mass) is required to react with the antibody in order to shift the equilibrium towards the production of ADCs. The toxic drugs are expensive and easily cause dangerous toxic waste pollution in the production process, so that the original attraction of the toxic drugs in industrial mass production is lost. In addition, oligoglycine-modified toxin drugs are difficult to access site coupling due to steric hindrance, resulting in low coupling efficiency compared to isotype control substrates of smaller molecular weight. While these existing methods are effective to some extent, new methods are urgently needed in view of the simplicity, flexibility and versatility of the coupling techniques. As an alternative, enzyme-chemistry methods have great potential in the field of site-directed conjugation.

Disclosure of Invention

The invention aims at the problem of heterogeneity caused by the fact that the traditional antibody coupling drug adopts the cysteine residue generated by reducing the lysine residue or the interchain disulfide bond on the antibody to couple the drug; the thiol-maleimide linker is unstable in the circulation system and is not universal for screening mutation sites in chemical conjugation; the anti-CD20 antibody-dolastatin conjugate obtained by the preparation method has the advantages of greatly improving the uniformity and the coupling efficiency, targeting CD20 positive tumor cells, having good in-vivo and in-vitro anti-tumor effects and being more suitable for large-scale production.

A preparation method of an antibody conjugated drug comprises the following steps:

(1) providing an anti-CD20 monoclonal antibody with an LPXTG sequence at the C terminal, a first connecting arm, and dolastatin or dolastatin derivatives coupled with a second connecting arm, wherein the two ends of the first connecting arm are respectively provided with an oligoglycine linker and an azido linker, and the two ends of the second connecting arm are provided with an alkynyl linker and a self-elimination linker;

(2) under the catalysis of Sortase A enzyme, the LPXTG sequence and the oligoglycine linker generate transpeptidation reaction, so that the anti-CD20 monoclonal antibody is connected with the first connecting arm;

(3) and the azide joint and the alkynyl joint generate cycloaddition reaction, so that the first connecting arm is connected with the second connecting arm.

The oligoglycine linker is typically 1-5 glycine residues.

The heavy chain amino acid sequence of the anti-CD20 monoclonal antibody is shown as SEQ ID No.1, and the light chain amino acid sequence is shown as SEQ ID No. 2.

The heavy chain and the light chain of the anti-CD20 monoclonal antibody are connected with LPXTG sequences at the C ends. Such a complete antibody has 4 LPXTG sequences and carries a high proportion of the drug.

The LPXTG sequence is LPETG, and a flexible joint is arranged between the anti-CD20 monoclonal antibody and the LPXTG sequence and consists of a plurality of glycine residues and/or serine residues. 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 flexible joint is a GGGGS sequence or a GGGGSGGGGS sequence. Most preferably, the flexible linker is a GGGGSGGGGS sequence.

The first connecting arm is a difunctional small molecule GGG-PEG-N₃(GPN for short). One terminal three glycine residues were used for coupling to the LPXTG sequence under the enzyme Sortase a. N is a radical of₃Is azido and is used for carrying out azide-alkyne cycloaddition reaction with alkynyl.

The self-eliminating linker is p-aminobenzoic acid, and the alkynyl linker is dibenzylcyclooctyne.

Be equipped with valine-citrulline dipeptide joint between self-elimination joint and the alkynyl joint, connect through the polyethylene glycol joint between alkynyl joint and the valine-citrulline dipeptide joint. The valine-citrulline dipeptide linker can be cleaved by the lysosomal enzyme cathepsin B.

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

The invention also provides the application of the antibody conjugate drug in preparing anti-tumor drugs. The anti-tumor drug can be used for inducing apoptosis of tumor cells with CD20 protein molecules expressed on the cell surfaces. Specifically, the anti-tumor drug is an anti-colon cancer drug, an anti-leukemia drug, an anti-ovarian cancer drug, an anti-gastric cancer drug, an anti-lung cancer drug, an anti-breast cancer drug or an anti-liver cancer drug. Furthermore, the anti-tumor drug is an anti-lymphoma or leukemia drug.

The preparation method of the antibody coupling drug comprises the steps of firstly enabling an anti-CD20 antibody containing an LPXTG sequence to perform a transpeptidation reaction with bifunctional small molecule GPN under the catalysis of a Sortase A enzyme to introduce azide groups on the antibody, and then enabling the anti-CD20 antibody and the bifunctional small molecule GPN to perform a second coupling reaction with a drug connected with a linker containing alkynyl through an azide-alkynyl cycloaddition reaction (SPAAC) initiated by high-efficiency and high-specificity force, wherein the SPAAC reaction belongs to the most classical reaction in click chemistry. The reaction can be carried out in aqueous solution, and has the advantages of simple operation, no need of exogenous catalyst, mild reaction condition and high reaction rate. The most outstanding advantage is that the reaction yield is high, almost approaches to one hundred percent, the dosage of the medicine MMAE can be greatly reduced, the cost investment is saved, and meanwhile, the toxic wastes which are easy to generate danger are reduced in the production process, thereby creating the possibility for realizing industrial large-scale production.

The invention has the advantages that:

(1) the invention uses enzyme-chemical method to prepare antibody coupling drug, which can reduce the usage of the expensive dolastatin, save the cost and provide possibility for realizing large-scale production.

(2) The invention uses enzyme-chemical method to prepare antibody coupling drug, and uses double-function small molecule with oligoglycine and azide group to reduce steric hindrance when the Sortase A enzyme is converted into peptide. Compared with the direct enzymatic coupling of oligoglycine-bearing dolastatin, the enzyme-chemical method has better flexibility.

(3) The invention utilizes an enzyme-chemical method to prepare the antibody coupling drug, effectively controls the coupling sites and the number of the drugs on the antibody, and compared with the traditional non-fixed-point coupling method, the uniformity of the prepared ADC is greatly improved. And the conjugate can improve the in vivo and in vitro anti-tumor activity of the antibody on the basis of keeping the original affinity of the antibody.

Drawings

FIG. 1 is a schematic diagram of the structure of various variants of anti-CD20 antibody engineered.

FIG. 2 shows a double-functional small molecule GGG-PEG-N₃The chemical structural formula of (1).

FIG. 3 is the chemical structural formula of cyclooctyne-containing dolastatin derivative DBCO-PEG-vc-PAB-MMAE.

FIG. 4 is a schematic diagram of the preparation of an antibody-conjugated drug by an enzyme-chemical method.

Figure 5 is a comparison of the efficiency of coupling of 6 variants of anti-CD20 antibody to MMAE, where lane 1: n is 0, m is 0; lane 2: n is 1, m is 0; lane 3: n is 2, m is 0; lane 4: n is 0, m is 1; lane 5: n is 1, m is 1; lane 6: n is 2 and m is 1.

FIG. 6 is a graph showing the results of reverse phase high performance liquid chromatography (RP-HPLC) analysis of the conjugation of an anti-CD20 antibody to a light-heavy chain of a drug, wherein the antibody or conjugate sample is reduced to a light-heavy chain by DTT before being loaded on a column; l0 is an unconjugated light chain, L1 is a light chain conjugated to a dolastatin; h0 is an unconjugated heavy chain, H1 is a heavy chain conjugated to a dolastatin; OFA is a light and heavy chain C-terminal LPETG-tagged anti-CD20 antibody (n-2, m-1); OFA-GPN-vcMAE is an anti-CD20 antibody conjugate drug prepared by an enzyme-chemical method.

FIG. 7 is a graph showing the results of Hydrophobic Interaction Chromatography (HIC) analysis of the conjugation of an anti-CD20 antibody conjugated drug, wherein E0 indicates that the drug-antibody conjugation ratio (DAR) is 0; e1 indicates a DAR of 1; e2 indicates DAR is 2; e3 indicates DAR is 3; e4 indicates a DAR of 4.

Fig. 8 is a graph showing the results of molecular weight determination of the anti-CD20 antibody and its antibody-conjugated drug, wherein a is the molecular weight of the light chain and the heavy chain of the anti-CD20 antibody variant (n-2, m-1); panel B shows the light and heavy chain molecular weights of the anti-CD20 antibody conjugate drug OFA-GPN-vcMMAE.

FIG. 9 is a graph showing the results of Q-TOF mass spectrometry analysis of the coupling site of the anti-CD20 antibody-conjugated drug OFA-GPN-vcMMAE.

FIG. 10 is a graph showing the results of comparing the affinities of the anti-CD20 monoclonal antibody and its dolastatin conjugate to Daudi cells.

FIG. 11 is a diagram showing the results of detecting the endocytosis rates of anti-CD20 antibody and its conjugate by flow cytometry, wherein OFA-vcMMAE is ADC prepared by direct Sortase A enzyme catalysis; OFA-GPN-vcMMAE are enzyme-chemically prepared ADCs, p <0.05, p <0.01 and p <0.001 were considered statistically significant.

FIG. 12 is a diagram of the results of confocal laser microscopy analysis of intracellular localization of the anti-CD20 antibody-conjugated drug OFA-GPN-vcMMAE after endocytosis, wherein, the diagram A is the position of ADC, the diagram B is the position of LAMP-1, and the diagram C is the relative position of the two overlapping.

FIG. 13 is a graph showing the results of an analysis of anti-CD20 antibody and its conjugate for inducing Ramos apoptosis, wherein A is OFA; panel B is OFA-vcMMAE; panel C is OFA-GPN-vcMMAE.

FIG. 14 is a graph showing the results of in vitro cytotoxicity assays of anti-CD20 antibody-conjugated drugs, wherein A is Daudi cells; panel B shows Ramos cells; panel C is K562 cells.

FIG. 15 is a graph showing the in vivo antitumor activity of the anti-CD20 antibody-conjugated drug and the change in body weight of each group of mice, wherein Panel A is a graph showing the results of the change in tumor volume; and the graph B is a graph of the change result of the body weight of the mouse.

Detailed Description

Example 1

Preparation of anti-CD20 antibody variants.

1. Constructing an expression vector of the anti-CD20 antibody with light and heavy chains having LPETG labels.

Ofatumumab (ofatumumab, OFA), is a fully humanized targeting anti-CD20 monoclonal antibody, and the gene sequences encoding the heavy chain and the light chain thereof are shown as SEQ ID No.3 and SEQ ID No.4, respectively.

Respectively amplifying sequences of a heavy chain encoding gene and a light chain encoding gene from plasmids (a heavy chain expression vector H and a light chain expression vector L, obtained by previous experiments of the inventor and described in Chinese invention patent with the application number of ZL 201310046396.9) containing the heavy chain encoding gene and the light chain encoding gene of the monoclonal antibody by a PCR technology, and respectively adding a signal peptide sequence (the nucleotide sequence is shown as SEQ ID No. 5) and a code sequence (GGGGS) at the N end and the C end of the heavy chain encoding gene and the light chain encoding gene by a PCR method_nLPETG(GHHHHHH)_m(n-0, 1, 2; m-0, 1, 6 combinations) amino acid sequences, cloning the sequences to T vectors respectively, and after the sequences are verified to be correct by sequencing, cloning the sequences to eukaryotic expression vectors pMH3 respectively, namely pMH3-SP-H-C (containing a heavy chain) and pMH3-SP-L-C (containing a light chain), wherein the C-terminal sequences are selected according to 6 combinations of different n and m values. Primer design is shown in table 1.

TABLE 1

The underlined parts GAATTC and GCGGCCGC indicate the cleavage sites for EcoR I and Not I, respectively.

2. Expression of recombinant anti-CD20 antibody variants was purified.

(1) Transient HEK293F cell

Coli DH 5. alpha. transformed with pMH3-SP-H-C and pMH3-SP-L-C plasmids, respectively, was inoculated at 1: 100 to 100mL of Amp + -resistant LB medium and shake-cultured overnight at 37 ℃. Collecting thallus by centrifugation at 8000rpm, extracting the target expression plasmid according to the operation method of the endotoxin-free plasmid large-scale extraction kit, measuring the plasmid concentration by using a NanoDrop instrument, filtering and sterilizing by using a 0.22 mu m filter membrane, and storing at-20 ℃.

A tube of cryopreserved HEK293F cells (approximately 10) was removed from the liquid nitrogen tank⁶Individual cells/tube), put into a 37 ℃ constant temperature water bath to be quickly melted, and centrifuged at 800rpm for 5 min. Discarding frozen stock solution on a superclean bench; taking an SMM 293-TI serum-free culture medium (Sinobiological Inc., production batch No. RZ11MA0303 of manufacturer), gently suspending the cells, transferring the cells into a 100mL cell shaking flask, supplementing the SMM 293-TI serum-free culture medium to 30mL, adding 0.5% serum, and adding 5% CO₂Shaker, 37 ℃ culture, 120 rpm. Passages were allowed by shaking for 2 days, and the HEK293F cell suspension was transferred to 500mL cell shake flasks, supplemented with 0.5% serum in SMM 293-TI serum-free medium to 120mL, and shaking was continued for 2 days. And then expanding and culturing to 1L of cell shake flask, supplementing SMM 293-TI serum-free culture medium to 250mL, simultaneously adding 0.5% serum, and continuing shake flask culture.

When the cell density reaches 2.0-3.0X 10⁶At one/mL, transfection may be performed. During transfection, 2 centrifugal tubes were added with 10mL of medium, one centrifugal tube was added with 2mL of PEI (1mg/mL), the other centrifugal tube was added with 300. mu.g of heavy chain plasmid (pMH3-SP-H-C) and 300. mu.g of light chain plasmid (pMH3-SP-L-C), and the C-terminal sequences of the heavy chain plasmid and the light chain plasmid were in agreement. And (3) after fully mixing, adding the plasmid diluent into the PEI diluent, fully mixing again, and standing for 15 min. After adding 50mL of medium to the 1L cell shake flask, the transfection solution was poured into the flask. After 4 days, the whole cell suspension was collected, centrifuged at 4000rpm for 30min and the supernatant was filtered through a 0.45 μm filter.

(2) ProteinA column purification

The Protein was purified using an AKTA purifier apparatus, and the antibody without His tag was a Hitrap Protein A HP affinity column (1mL), and the antibody with His tag was a Hitrap Protein A HP affinity column (1mL) or a Hitrap Ni-NTA affinity column (5 mL). The Hitrap Protein A HP affinity column takes 50mM Tris-HCl, 150mM NaCl (pH7.4) as a binding buffer solution and 1M sodium acetate (pH3.0) as an elution buffer solution; HiTrap Ni-NTA affinity column with 50mM NaH₂PO₄300mM NaCl (pH7.4) as binding buffer, 50mM NaH₂PO₄300mM NaCl and 500mM imidazole (pH7.4) are used as elution buffer, firstly 5% of elution buffer is used for removing the foreign protein, and then 50% of elution buffer is used for eluting the target protein and collecting the target protein.

At 4 deg.C, with Millipore

The solution of the purified antibody was replaced with 50mM Tris-HCl, 150mM NaCl (pH7.4) by an Ultra centrifugation ultrafiltration tube (membrane cut-off molecular weight: 30kDa), and the concentration was measured with NanoDrop Protein A280 and stored at-80 ℃.

A schematic of the structure of the anti-CD20 antibody variants is shown in figure 1.

Example 2

The anti-CD20 antibody conjugate drug is prepared by an enzyme-chemical method.

1. Expression and purification of Sortase A enzyme

Specific procedures reference is made to the document Pan, L.Q., et al (2017) Sortase A-generated high level patent anti-CD20-MMAE conjugates for efficacy evaluation of B-linkage lymphoma. Small.13(6).

2. Sortase A enzyme catalyzed transpeptidation reaction

Double-functional small molecule GGG-PEG-N₃(GPN for short) is purchased from Nanjing Lining biopharmaceutical Co., Ltd., and the chemical structure is shown in FIG. 2. In a reaction system of 50mM Tris and 150mM NaCl (pH 7.4): 2 μ M recombinant anti-CD20 antibody variant (prepared as described in example 1), 50 μ M Sortase A enzyme, 200 μ M bifunctional small molecule, 5 μ M CaCl₂. The presence of a calcium ion binding site in Sortase A increases the catalysis of the enzymeEfficiency. Reacting at 37 ℃ for 12-24 h. Coupling a Sortase A catalytic antibody with bifunctional small molecule GPN, and purifying by a Protein A affinity column to obtain an antibody OFA-GPN carrying azide groups.

3. Click chemistry

Cyclooctyne-containing dolastatin derivative DBCO-PEG-vc-PAB-MMAE is purchased from Nanjing Binning biopharmaceutical Co., Ltd, and has a chemical structure shown in FIG. 3. DBCO-PEG-vc-PAB-MMAE is a abbreviation of dibenzylcyclooctyne-polyethylene glycol-valve-cltruline-p-aminobenzoyloxycarbenyl-monomenthyl auristatin E, and is a dolastatin derivative, comprising a dibenzylcyclooctyne linker (DBCO) for connecting azide, a polyethylene glycol linker (PEG), a dipeptide linker (valine-citrulline, vc) which can be broken by a lysosomal enzyme cathepsin B, a self-elimination linker (p-aminobenzoic acid, PAB) and a high-toxicity small-molecule drug dolastatin (MMAE).

The antibody OFA-GPN carrying the azide group and the cyclooctyne-containing dolastatin derivative DBCO-PEG-vc-PAB-MMAE react in PBS buffer solution according to the proportion of 1: 8 (the proportion of the azide and the alkynyl functional groups is 1: 2) at room temperature for overnight reaction. The reaction solution was ultrafiltered overnight with PBS until the amount of dolastatin derivative in the system was less than 0.1 nM.

4. Screening of antibody formats with the highest coupling efficiency

The enzyme-chemical method (the preparation process is shown in figure 4) is adopted to compare the coupling efficiency of 6 anti-CD20 antibody variants (different m and n values), a reaction sample is detected by a western blot (the western blot is operated as follows: 12% separation gel and 5% stacking gel are prepared, 6 protein samples with the same quantity are added into a sample loading hole, the electrophoresis conditions are 90V and 25min, 160V and 40min, the membrane transferring and sealing are carried out after the electrophoresis is finished, the MMAE resistant primary antibody is incubated for 1h, after the membrane is washed for 3 times, an HRP marked secondary antibody is incubated for 1h, and after the membrane is washed for 3 times, the DAB method is used for color development), the qualitative conditions of the light and heavy chains of the antibody are connected with the MMAE are obtained, the result is shown in figure 5, so that the antibody form with the highest coupling efficiency (n is 2, m is 1) is selected as the optimal selection of the invention, the subsequent experiments are all selected, and the structure of the obtained product is OFA- (GGS)₂LPET-GGG-PEG-N₃-DBCO-PEG-vc-PAB-MMAE, hereinafter referred to as OFA-GPN-vcMMAE.

Example 3

Analysis of the properties of the anti-CD20 antibody conjugate drug prepared by enzyme-chemical method.

1. RP-HPLC analysis

A chromatographic column: varian PLRP-S

column (8 μm, 150X 25 mm); column temperature: 80 ℃; mobile phase A: 0.1% aqueous trifluoroacetic acid; mobile phase B: and (3) acetonitrile. Gradient elution conditions: 25% B (3 min); 25-50% B (25 min); 50-95% B (2 min); 95-25% B (1 min); 25% B (2 min). Flow rate: 0.6 mL/min; the sample was reduced with DTT at 37 ℃ for 30 min.

The result is shown in fig. 6, the anti-CD20 antibody OFA still has an unreduced peak under the DTT reduction condition, and the light and heavy chain peak-off time of the anti-CD20 antibody coupling drug OFA-GPN-vcMMAE and the light and heavy chain peak-off time of the Shanghai rabbit toxin are similar, so that a combined peak (L1+ H1) appears, and the drug antibody coupling ratio (DAR) cannot be accurately judged.

2. HIC analysis

The anti-CD20 antibody conjugate drug OFA-GPN-vcMAE was further analyzed for conjugation with HIC.

A chromatographic column: TOSOH Butyl-NPR (4.6 mm. times.3.5 cm, 2.5 μm); column temperature: room temperature; mobile phase A: 1.5M (NH)₄)₂SO₄，25mM NaH₂PO₄(pH 7.0); mobile phase B: 75% 25mM Na₃PO₄(pH7.0), 25% isopropyl alcohol (IPA). Gradient conditions: a 15min linear gradient from mobile phase a to mobile phase B; flow rate: 0.8 mL/min.

As a result, as shown in FIG. 7, the DAR of the anti-CD20 antibody conjugate drug was determined to be about 3.3 based on the peak area size of each component.

3. LC-MS analysis

Further, molecular weight determination and coupling site determination were performed using a Waters acquisition UPLC ultra high performance liquid chromatograph in combination with a Waters Xevo-G2S Q-TOF quadrupole-time-of-flight mass spectrometer.

(1) Light and heavy chain molecular weight determination

The sample was diluted to 2mg/mL, reduced by DTT (37 ℃ C., 40min), and then subjected to molecular weight measurement using a LC-MS. A chromatographic column: waters UPLC MassREPTM desalting column; column temperature: 80 ℃; mobile phase A: 0.1% aqueous formic acid; mobile phase B: 0.1% formic acid acetonitrile solution; the elution conditions were a linear gradient from 10% B to 90% B over 12 minutes. Mass spectrum conditions: capillary voltage 2.5 kV; the taper hole voltage is 60V; the mass analysis range is 2000-; the ion source temperature is 120 ℃; the atomization temperature is 500 ℃; the atomization flow rate is 800L/Hr.

In the process of synthesizing the conjugate OFA-GPN-vcCMMAE by the anti-CD20 antibody through an enzyme-chemical two-step method, after a GGHHHHHHHH sequence is removed from a LPETGGHHHHHH sequence at the tail ends of a light chain and a heavy chain, a bifunctional small molecule GPN (the molecular weight is reduced to 609.72Da) is connected, and then, a dolastatin is connected through click chemistry, and after two steps, the molecular weight of the light chain and the molecular weight of the heavy chain are theoretically increased to 1047.22 Da. The molecular weight was determined by the LC-MS method, and the results are shown in FIG. 8, where the molecular weight of the light chain was increased by 1048.6875Da and the molecular weight of the heavy chain was increased by 1049.461Da, both of which were consistent with the calculated theoretical molecular weights, indicating that the light and heavy chains of the antibody were both linked to dolastatin by the enzyme-chemical method.

(2) Determination of conjugation sites

Diluting a sample to 4mg/mL by using guanidine hydrochloride-Tris solution (pH8.0), adding DTT (final concentration of 0.02mol/L), reacting for 2.5h, adding iodoacetamide (final concentration of 0.06M), reacting for 40min at room temperature in a dark place, performing centrifugal ultrafiltration at 12000rpm and 4 ℃ for 2h to obtain a 0.01M Tris solution (pH7.4) as a displacement buffer solution, adding trypsin, performing enzyme digestion at 37 ℃ for 24h, and terminating the enzyme digestion reaction by using formic acid (final concentration of 1%) to be detected.

A chromatographic column: waters UPLC BEH 300C 18 chromatography column; column temperature: 40 ℃; mobile phase A: 0.1% aqueous formic acid; mobile phase B: 0.1% formic acid acetonitrile solution; the elution conditions were a linear gradient from 5% B to 95% B over 12 minutes. Mass spectrum conditions: capillary voltage 2.0 kV; the taper hole voltage is 150V; the mass analysis range is 50-8000 Da; the ion source temperature is 90 ℃; the atomization temperature is 400 ℃; the atomization flow rate was 700L/Hr.

The results are shown in FIG. 9, and the secondary mass spectrum results of the polypeptide fragments are arranged to confirm that the fragment LPETGGG-PEG-N₃Presence of DBCO-PEG-vc-PAB-MMAE (LPET-GPN-vcMMAE), arrangement of y and b ions, determined by molecular weight, in Table 2The LPET sequence was modified to GPN-vcMMAE, further indicating that dolastatin is conjugated to the target site.

TABLE 2

Arrangement of	Theoretical molecular weight (Da)	Actual molecular weight (Da)	Molecular weight error (Da)	Decorative article
					b11	744.328	744.3276	-0.0004	-
b13	970.4229	970.423	0.0001	-
					y13*	3016.5522	3016.5647	0.0125	GPN-vcMMAE
y13-H2O*	2998.5396	2998.554	0.0144	GPN-vcMMAE
					y14-H2O*	3055.5459	3055.5754	0.0295	GPN-vcMMAE
y14*	3073.5767	3073.5862	0.0095	GPN-vcMMAE

Example 4

Affinity of anti-CD20 antibody conjugate drugs for CD20+ tumor cells.

The effect of conjugated dolastatin on antibody affinity was assessed by flow cytometry on Daudi cells.

1×10⁶Each Daudi cell was incubated in a 1% BSA-PBS solution (pH7.4) containing 5. mu.g/mL of antibody or ADC for 30min at 4 ℃ with PBS as a control. Washed 3 times with pre-cooled PBS, and added FITC-labeled goat anti-human IgG (H + L) polyclonal antibody (diluted 1: 250 with 1% BSA-PBS) and incubated at 4 ℃ in the dark for 30 min. After 3 washes with pre-chilled PBS, the Mean Fluorescence Intensity (MFI) on the cell surface was measured by flow cytometry.

As a result, as shown in fig. 10, the affinity of the anti-CD20 antibody conjugate drug prepared by an enzyme-chemistry method for Daudi cells was decreased compared to the anti-CD20 antibody (OFA), but the decrease was small in magnitude because the conjugation site was far from the antigen binding site.

Example 5

Endocytosis of anti-CD20 antibody-conjugated drug.

ADCs need to be first endocytosed before they can become active in the tumor cell. Therefore, for ADCs, endocytic capacity is particularly important.

Daudi cells were incubated at 4 ℃ for 30min in 1% BSA-PBS solutions (pH7.4) containing different concentrations of ADCs, with PBS as a control. Washed 3 times with pre-chilled PBS and incubated for 30min at 4 ℃ with addition of FITC-labeled goat anti-human IgG (H + L) polyclonal antibody (diluted 1: 250 with 1% BSA-PBS). After washing with pre-cooled PBS 3 times, the Mean Fluorescence Intensity (MFI) on the cell surface was measured by flow cytometry. And drawing an MFI-c curve by taking the concentration as an abscissa and the MFI as an ordinate, and selecting a range in a linear relation as a standard curve. The corresponding concentration is calculated through the MFI on the cell surface, the volume is all fixed to be 200 mu L, and the endocytosis amount of the ADCs can be further calculated. In parallel, a linear range of concentrations (2. mu.g/mL) was selected, and in five replicates, Daudi cells were incubated in 2. mu.g/mL of ADCs in 1% BSA-PBS (pH7.4) at 4 ℃ for 30min, washed 3 times with pre-cooled PBS, incubated at 37 ℃ for 2H, centrifuged, and the supernatant (containing the dissociated ADCs from the cell surface) was incubated at 4 ℃ for 30min, after washing, cells incubated at 37 ℃ for 2H and cells incubated with the supernatant were incubated with FITC-labeled goat anti-human IgG (H + L) polyclonal antibody (diluted 1: 250 with 1% BSA-PBS) at 4 ℃ for 30 min. After washing with precooled PBS for 3 times, detecting the MFI on the cell surface by using a flow cytometer, and calculating the corresponding concentration according to an MFI-c standard curve. Endocytosis rate ═ amount of surface bound at 4 ℃ -amount of surface remaining after 2h at 37 ℃ + amount of dissociation from surface)/amount of surface bound at 4 ℃ x 100%.

As a result, as shown in FIG. 11, OFA was very weakly endocytosed by Daudi cells, which is consistent with the literature report that the anti-CD20 monoclonal antibody is not easily endocytosed. After coupling vcMAE, the OFA-vcMAE prepared by direct Sortase A enzyme catalysis (prepared by previous experiments of the inventor, and the specific preparation method is shown in Chinese invention patent with application number CN 201610555042.0) and the OFA-GPN-vcMAE prepared by an enzyme-chemical method have obviously increased endocytosis, and the 2h endocytosis rate is respectively more than 30% and 40%, which shows that the coupling of vcMAE can promote the endocytosis of anti-CD20 antibody.

Example 6

anti-CD20 antibodies are conjugated to the intracellular localization of the drug after endocytosis.

The intracellular localization of the enzyme-chemically prepared anti-CD20 antibody conjugate drug OFA-GPN-vcMMAE after endocytosis by Daudi cells was further determined by confocal.

1×10⁵One/well of Daudi cells were incubated with 5. mu.g/mL of OFA-GPN-vcMAE at 37 ℃ and 5% CO₂Incubate for 6h under conditions. Washing with PBS for 3 times, washing away unbound ADC, centrifuging at 1000rpm for 5min to fix suspended Daudi cells on the glass slide, wherein the cell density is determined by that the cells are not adhered to each other; fixing the cells with 4% paraformaldehyde at room temperature for 15 min; after 3 times of PBS washing, treating the cells for 10min at room temperature by PBS containing 0.1 percent TritonX-100 and 0.2 percent BSA to ensure that cell membranes become permeable, and washing 3 times by PBS; blocking with 2% BSA in PBS at room temperature for 30 min; dropping anti-LAMP-1 rabbit monoclonal antibody diluted by 1: 500 with PBS (1% BSA) on the cells, incubating for 45min, and washing for 3 times with PBS; dropping FITC-labeled goat anti-human IgG (H + L) diluted 1: 250 with 1% BSA PBS and Cy 5-labeled goat anti-human IgG (H + L) on the cells, incubating for 45min in the dark, and washing for 3 times with PBS; dropping DAPI diluted 1: 1000 with 1% BSA PBS on the cells, staining cell nuclei for 3min in dark, and washing with PBS for 3 times; dropping an anti-quenching agent on the cells, covering a cover glass, carefully removing bubbles, and sealing; finally, the intracellular localization of the anti-CD20 antibody conjugated drug is observed by a laser confocal microscope.

DAPI is a nuclear stain (blue); FITC is a fluorescent label (green) carried on the secondary antibody and indicates the position of the ADC; LAMP-1 is a lysosomal associated membrane protein-1, indicating the location of lysosomes (red). As a result, as shown in FIG. 12, OFA-GPN-vcMMAE was endocytosed by Daudi cells and co-localized with LAMP-1, confirming that the conjugate reached lysosome, since the dipeptide linker (vc) was highly stable in plasma, ADC reached lysosome after endocytosis, and the lysosomal protease cleaved the dipeptide linker, releasing the active drug, killing the cells.

Example 7

The anti-CD20 antibody conjugated drug induces apoptotic activity.

In order to evaluate the mechanism of toxicity of the anti-CD20 antibody conjugated drug, the anti-CD20 antibody conjugated drug was tested for the activity of inducing CD20+ apoptosis by the annexin V/PI double staining method.

Ramos cells, 5X 10, were cultured in six-well plates⁴One well, each well was spiked with antibody or ADCs at a final concentration of 5. mu.g/mL, at 37 ℃ and 5% CO₂Incubation was continued for 72h under these conditions. After incubation, cells are centrifuged, supernatant is removed, the cells are washed for 3 times by PBS, then an annexin V/PI double staining kit is used for immunostaining the cells, 100 mu L of binding solution, 5 mu L of annexin V-FITC and 5 mu L of PI are added, the mixture is shaded for 15min at room temperature, 400 mu L of binding solution is added, and the percentages of early apoptotic cells (annexin V +/PI-) and late apoptotic cells (annexin V +/PI +) are analyzed by a flow cytometer, so that the apoptosis activity of the antibodies and the conjugates thereof induced by the antibodies is analyzed.

The results are shown in fig. 13, OFA-vcMMAE and OFA-GPN-vcMMAE caused extremely high apoptosis rates of Ramos cells with rates of inducing apoptosis of 97.6% (90.2% early + 7.4% late + 91.7% early + 6.8% late), respectively, and significantly higher than antibody OFA not conjugated with MMAE, indicating that the ability of ADCs to induce apoptosis is mainly dependent on highly toxic dolastatin.

Example 8

In vitro cytotoxicity of anti-CD20 antibody conjugated drugs.

2 tumor cells positive for CD20 (Ramos and Daudi) and negative for CD20 (K562) were selected to test the in vitro specific anti-tumor activity of anti-CD20 antibodies and their conjugates.

5000 cells/well were plated on 96-well cell plates (100. mu.L of RPMI-1640 medium containing 10% calf serum), 100. mu.L/well of different concentrations of antibody or ADCs (diluted with 10% calf serum medium) were added, mixed, and incubated at 37 ℃ and 5% CO₂The saturated water vapor carbon dioxide incubator is cultured for 4 days, so that the ADCs can fully exert the anti-tumor activity. Blank wells were replaced with an equal volume of RPMI-1640 medium containing 10% calf serum, and negative control wells were supplemented with the same number of tumor cells in RPMI-1640 medium containing 10% calf serum. After 4 days of incubation, 10% by volume of CCK-8 reagent was added to each well, incubation was continued for 4h, and OD at 450nm was read with a microplate reader. The relative fineness of each well was then calculatedCell survival rate: relative cell viability (%) ═ experimental-blank)/(negative-blank x 100%.

The results are shown in fig. 14, 2 conjugates OFA-vcMMAE and OFA-GPN-vcMMAE can effectively kill 2 tumor cells positive to CD20, both show higher activity than OFA, and the activity of OFA-GPN-vcMMAE prepared by an enzyme-chemical method is higher than that of OFA-vcMMAE prepared by direct Sortase a enzyme catalysis. Significantly, the IC50 of 2 conjugates was over 1000 times lower than that of unconjugated antibody, demonstrating that ADC conjugated to dolastatin significantly improved cytotoxicity. Moreover, both showed high toxicity only to CD20 positive cells, while low toxicity to CD20 negative K562 cells, confirming that ADCs specifically kill only CD20 positive target cells.

Example 9

anti-CD20 antibody coupled with the in vivo anti-tumor activity of the drug.

In a Ramos lymphoma PDX model, the in vivo anti-tumor activity of an anti-CD20 antibody conjugate drug prepared by an enzyme-chemical method is explored.

15 nude mice were randomly divided into the following 3 groups: physiological saline group, OFA-vcMAE (5mg/kg) positive control group and OFA-GPN-vcMAE (5mg/kg) experimental group. Each nude mouse had 100. mu.L of 8X 10 subcutaneous injections in the right underarm of the forelimb⁶Individual Ramos cells, with average tumor size up to 450mm³When (the tumor volume of the conventional initial administration is 50-150mm³) The drug is administered according to the above groups. The administration mode is tail vein injection, and the administration period is 4 times and 4 times. The body weight and tumor size of nude mice were measured periodically, the length and width were expressed as L and W, respectively, the tumor volume was V, and the calculation formula was V ═ L × W²)/2。

As a result, as shown in FIG. 15A, the tumors grew rapidly in the saline group, and the mean tumor volume had exceeded 2000mm at the last administration³. In contrast, OFA-GPN-vcMMAE and OFA-vcMMAE were able to significantly inhibit tumor growth compared to saline group<0.001 and x p<0.01. Tumors were effectively reduced in the OFA-GPN-vcMMAE group and the OFA-vcMMAE group in the first week of dosing. After one week, the OFA-vcMMAE group tumors gradually increased, while OFA-GPN-vcMMAE still can effectively control the growth of tumors, indicating that the anti-tumor effect of OFA-GPN-vcMMAE isThe tumor activity was stronger than OFA-vcMMAE, both of which were consistent with in vivo effects. Remarkably, 3 of 5 tumor-bearing nude mice in the OFA-GPN-vcMMAE group had lost their tumor after drug treatment and did not recur.

At the same time, body weights of each group of mice were measured periodically to characterize potential systemic toxicity. If the anti-CD20 antibody conjugate drug is unstable in vivo, the virulent dolastatin conjugated to the antibody can be liberated to kill normal cells, thereby affecting the malfunction of certain organs of the mouse and leading to the irreversible and rapid reduction of body weight.

The body weight changes of the mice in each group were seen in FIG. 15B, and the body weights of the mice in the OFA-GPN-vcMMAE group and the OFA-vcMMAE group were slightly decreased compared with the saline group. However, the weight of the mice in the OFA-GPN-vcMAE group gradually recovered to be normal, which indicates that the OFA-GPN-vcMAE does not cause serious irreversible damage to the mice and is relatively safe. The body weight of the mice in the OFA-vcMAE group is continuously reduced, which shows that compared with the OFA-vcMAE prepared by direct Sortase A enzyme catalysis, the OFA-GPN-vcMAE prepared by the enzyme-chemical method not only improves the curative effect, but also reduces the treatment coefficient.

Sequence listing

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Claims (4)

1. A preparation method of an antibody conjugated drug is characterized by comprising the following steps:

(1) providing a C terminal with (GGGGS)₂LPETGGHHHHHH, a CD20 monoclonal antibody, a first connecting arm and dolastatin coupled with a second connecting arm, wherein the two ends of the first connecting arm are respectively provided with an oligoglycine joint and an azido joint, and the two ends of the second connecting arm are provided with an alkynyl joint and a self-elimination joint;

(2) under the catalysis of Sortase A enzyme, an LPETG sequence and an oligoglycine linker generate transpeptidation reaction, so that the anti-CD20 monoclonal antibody is connected with a first connecting arm;

(3) the azide linker and the alkynyl linker generate cycloaddition reaction to ensure that the first connecting arm is connected with the second connecting arm,

the heavy chain and the light chain of the anti-CD20 monoclonal antibody are connected with (GGGGS) at the C ends₂LPETGGHHHHHH, the sequence of the sequence is,

the first connecting arm is a difunctional small molecule GGG-PEG-N₃，

The self-elimination joint is p-aminobenzoic acid, the alkynyl joint is dibenzylcyclooctyne,

the heavy chain amino acid sequence of the anti-CD20 monoclonal antibody is shown as SEQ ID No.1, and the light chain amino acid sequence is shown as SEQ ID No. 2.

2. The method according to claim 1, wherein a valine-citrulline dipeptide linker is provided between the self-eliminating linker and the alkynyl linker, and the alkynyl linker and the valine-citrulline dipeptide linker are connected by a polyethylene glycol linker.

3. An antibody-conjugated drug produced by the production method according to any one of claims 1 to 2.

4. The use of the antibody conjugate of claim 3 in the preparation of an anti-tumor medicament.

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