CN114796519A

CN114796519A - Preparation and application of protein coupled small molecule drug

Info

Publication number: CN114796519A
Application number: CN202110109069.8A
Authority: CN
Inventors: 洪章勇; 李文静; 李启育
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2021-01-27
Filing date: 2021-01-27
Publication date: 2022-07-29

Abstract

The invention discloses a design strategy, synthesis and application of a miniaturized Antibody Drug Conjugate (ADC), wherein the designed miniaturized ADC is shown as a general formula (I), and a miniaturized antibody Z is involved in the miniaturized ADC _HER2 A preparation method of a conjugate of a targeting group and a Drug molecule (Drug) through a polyethylene glycol (PEG) chain with a certain length and application in tumor treatment; researches show that the insertion of the PEG chain obviously prolongs the circulating half-life period of the miniaturized ADC drug, obviously reduces the cytotoxicity of the miniaturized ADC drug, and in an animal model with the same dosage, a drug molecule with the PEG chain length of 10kDa has the optimal tumor treatment capacity, and the half-life period is prolonged to help to improve the treatment capacity of the miniaturized ADC drug; z _HER2 ‑PEG‑Drug(I)。

Description

Preparation and application of protein coupled small molecule drug

Technical Field

The application relates to a design strategy of a miniaturized Antibody Drug Conjugate (ADC), a preparation method thereof and application thereof in preparing a drug for treating tumor. In the miniaturized ADC, a miniaturized antibody Z as a targeting group _HER2 The drug molecules are linked to each other by polyethylene glycol (PEG), optionally with a specific linker, as a linking moiety.

Background

Compared with monoclonal antibody drugs, the Antibody Drug Conjugate (ADC) can remarkably improve the killing capacity of the antibody by coupling the monoclonal antibody with high-activity small molecular toxin. However, the traditional ADC drugs use large monoclonal antibodies (-150 kDa), such as trastuzumab, as tumor targeting ligands, whose large molecular structure reduces the efficiency of penetration of drug molecules into solid tumor tissues, limiting the efficacy of this strategy in solid tumor therapy. In addition, because the monoclonal antibody has a large molecular weight (150 kDa), each antibody molecule needs to be coupled with a plurality of small-molecule drugs (4-8 small molecules) to achieve a satisfactory tumor killing capability, which makes the preparation and site-specific connection of the drugs very difficult. Furthermore, the synthesized ADC product molecules are often complex mixtures of multiple positional isomers with different numbers of small molecules, which makes purification and quality control difficult. The present invention, which we use miniaturized antibody analogues (affibodies) instead of monoclonal antibodies for ADC drug preparation, is expected to help remedy the above drawbacks. The affibody molecule is a very small protein with only 58 amino acids, and has excellent properties of high stability, easy preparation and the like. Furthermore, various high throughput screening techniques are currently available for the modification and affinity maturation of affibody structures. Furthermore, affibody molecules do not contain cysteine residues, so site-specific coupling to small molecule drugs can be achieved by introducing additional cysteine residues.

However, ADC conjugates based on affibody molecules may suffer from a short circulating half-life in vivo compared to traditional ADC drug molecules based on full-length monoclonal antibodies, which may limit the efficiency of drug accumulation at the tumor site, thereby affecting the tumor therapeutic capacity of the drug. The invention relates to a small-sized ADC based on affibody molecules, which is prepared by taking a bifunctional PEG chain with maleimide groups and activated ester as a connector, so as to realize site-specific coupling of small-molecule drugs and affibody molecules and prolong the circulating half-life of the drugs.

The invention selects the affibody molecule Z with good binding affinity with the HER2 receptor _HER2：2891 (abbreviation Z) _HER2 ) As a target molecule, a cysteine residue is introduced into the C end of the target molecule, and the fixed-point coupling of the affibody molecule, the PEG chain and the small molecule drug MMAE is realized through the reaction of the sulfydryl of the cysteine and the maleimide group and the reaction of the activated ester and the amino group. MMAE is a tubulin inhibitor used as a small molecule part of a toxin, N-terminal to oneThe specific valine citrulline PAB (Val-Cit-PAB) sequence is linked and can be selectively cleaved by cathepsin B in lysosomes, thereby releasing small MMAE in tumor cells. In the invention, MMAE is used as a small molecule drug to synthesize three PEG modified conjugates with different molecular weights, namely Z _HER2 -SMCC-MMAE(HM)、Z _HER2 PEG4K-MMAE (HP4KM) and Z _HER2 PEG10K-MMAE (HP10KM), and the circulating half-life and in vitro and in vivo antitumor effects of these conjugates were evaluated in detail.

Disclosure of Invention

The invention aims to provide a design strategy for miniaturizing an ADC.

The invention also aims to provide a preparation method and application of the miniaturized ADC.

The technical scheme of the invention is as follows:

a miniaturized ADC of the general formula (I):

Z _HER2 -PEG-Drug (I)

the miniaturized ADC is characterized in that Z is _HER2 Has the following structure: the C-terminal contains cysteine with sulfhydryl

The N-and C-termini contain and/or do not contain a tag of 6 histidines, respectively.

The miniaturized ADC is characterized in that the Drug moiety Drug is independently selected from Gly3-VC-PAB-MMAF group and Gly3-VC-PAB-MMAE group, and has the following structure:

gly3-VC-PAB-MMAE group is preferred.

The miniaturized ADC is characterized in that the repeating unit of the PEG is-CH ₂ CH ₂ O-; the molecular weight is independently selected from 3kDa, 4kDa, 5kDa, 6kDa, 7kDa, 8kDa, 9kDa, 10kDa, 11kDa or 12kDa, preferably 10 kDa.

The miniaturized ADC is characterized in that the PEG is bifunctional polyethylene glycol, wherein one end of the PEG has a structure of

A linker attached to the amine group; the other end has a structure of

A linker attached to a thiol group.

The preparation method of the miniaturized ADC comprises the following steps: wherein the PEG is linked to Gly3-VC-PAB-MMAE or Gly3-VC-PAB-MMAF to obtain PEG-Drug; then PEG-Drug is mixed with the Z _HER2 Coupling to obtain miniaturized ADCs of general formula (I).

In the present invention, we designed and practiced to prepare three PEG-mediated anti-HER 2 molecules of different molecular weights: 2891 (abbreviated as Z) _HER2 ) Conjugates with drug molecules were named HM, HP4KM and HP10KM (as shown in FIG. 1). In these molecules, Z _HER2 And drug molecules are positioned at the terminal and are connected through succinimidyl-trans-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) or a bifunctional PEG chain, namely maleimide and an activated ester linker, which can specifically and effectively react with a sulfhydryl group and an amine group respectively. According to the reaction of a thiol group with a maleimide group, in Z _HER2：2891 A cysteine residue is introduced into the C terminal of the (D) for site-specific coupling. The amino group in the Gly3-VC-PAB-MMAE molecule can perform specific reaction with the activated ester group at the other end of the difunctional SMCC or PEG chain to realize the connection of the three parts.

The product was analyzed for purity by HPLC and the purity of HM, HP4KM and HP10KM was greater than 95% (fig. 2A). Molecular weight characterization was performed using MALDI-TOF-MS, HM: 10092.69Da, theoretical molecular mass: 10101.1 Da; HP4 KM: 13765.90Da, theoretical molecular mass: 13782.97 Da; HP10 KM: 19721.93Da, theoretical molecular mass: 19782.97 Da.

The invention provides a preparation method of the miniaturized ADC, and a synthetic route of the miniaturized ADC is shown in a figure 1.

The invention also provides application of the miniaturized ADC in preparation of a medicine for treating tumors.

The invention evaluates the in vivo and in vitro activity of the conjugate through cell experiments and animal experiments. The results show that the introduction of PEG chains significantly extends the circulating half-life of miniaturized ADC drugs. With Z not incorporating PEG _HER2 -SMCC-MMAE (HM) compared to Z incorporating 4kDa or 10kDa PEG _HER2 PEG4K-MMAE (HP4KM) and Z _HER2 The half-life of PEG10K-MMAE (HP10KM) was extended 2.5-fold and 11.2-fold, respectively. Meanwhile, the introduction of PEG chain obviously reduces the cytotoxicity of the miniaturized ADC medicine. Compared to the HM, the cytotoxicity of HP4KM and HP10KM was reduced by 4.5-fold and 22-fold, respectively. In the same dose animal model, HP10KM has the most ideal tumor treatment capacity, and the target toxicity is reduced by more than 4 times compared with HM, and the above results show that the half-life period is prolonged to help improve the treatment capacity of the miniaturized ADCs. This strategy can be employed to extend half-life without affecting cytotoxicity in drug design, potentially contributing to further increase the therapeutic potential of miniaturized ADCs.

Drawings

FIG. 1 shows that PEG chains with different molecular weights and MMAE jointly modify vector Z _HER2：2891 Synthesis and drug release of cathepsin B;

FIG. 2 is a purity characterization and affinity and half-life determination of HM, HP4KM, and HP10KM conjugates (A) Z _HER2 And HPLC analysis of the conjugate, (B) enzyme linked immunosorbent assay of the conjugate binding to HER2 receptor, (C) ELISA assay of the circulating clearance of the conjugate in vivo, AUC: area from zero to infinity under the curve, (D) flow cytometry detection of binding specificity of the conjugate;

FIG. 3 is a graph showing the MTT assay for determining the cell killing ability of the conjugates against NCI-N87 cells (A), BT-474 cells (B), MCF-7 cells (C) and PC-3 cells (D), (E) EC HM, HP4KM, HP10KM and MMAE against different tumor cell lines ₅₀ Data;

FIG. 4 is a competitive inhibition assay to detect cytotoxicity and live/dead staining imaging experiment (C) of conjugate on NCI-N87 cells (A) and BT-474 cells (B), scale bar: 20 μm;

FIG. 5 is a flow cytometer detecting the apoptotic mechanism of binders on NCI-N87 cells (A) and PC-3 cells (B);

FIG. 6 is a graph showing that flow cytometry analysis of the conjugate selectively induced growth arrest in the G2 phase of NCI-N87 cells,% G2/M indicating the percentage of cells arrested in the G2/M phase;

FIG. 7 is the in vivo antitumor activity of the conjugate, tumor growth curve (A) and body weight change (B) of nude mice bearing tumor BALB/c, NCI-N87 bearing tumor BALB/H after intravenous injection of 1.5mg/kg conjugate in NCI-N87 bearing tumor _C Tumor growth curves (C) and body weight changes (D) after intravenous injection of 0.6mg/kg conjugate in nude mice, changes in serum ALT (E) and AST (F) after 4 administrations of BALB/C nude mice at a dose of 1.5mg/kg were determined by ELISA, (G) histological analysis of major tissues after conjugate treatment (200X).

Detailed Description

It will be appreciated by those skilled in the art that the following examples are intended to further illustrate the invention but are not intended to limit the invention. The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are not indicated by manufacturers, and are conventional products available on the market.

Acronyms

Cys: (ii) cysteine; his: (ii) histidine; PEG: polyethylene glycol; SMCC: 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester; IPTG: isopropyl-beta-D-thiogalactopyranoside; TCEP: tricarboxyethyl phosphine; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; HPLC: high performance liquid chromatography; MALDI-TOF-MS: analyzing a tandem time-of-flight mass spectrometer by matrix-assisted laser; PBS: a phosphate buffer; MTT: thiazole blue; EC (EC) ₅₀ The value: a semi-effective concentration; SPF: no specific pathogen.

Sources of Experimental materials

MMAE and Gly3-VC-PAB-MMAE are from Nanjing Linning biopharmaceutical Co., Ltd (Nanjing, China). Mal-PEG-NHS (4000Da and 10000Da) was purchased from Kyork technology, Inc. of Beijing, China. TCEP comes from TCI (Shanghai, China). The activity/cytotoxicity assay kit was purchased from Suzhou space (Suzhou) Inc. The APC-Annexin V/PI apoptosis assay kit was purchased from Biolegend (san Diego, Calif., USA). Human breast cancer cell lines BT-474 and MCF-7 were purchased from ATCC in the United states. The human gastric cancer cell line NCI-N87 and the prostate cancer cell line PC-3 were purchased from Chinese academy of sciences (Shanghai). BT-474 and NCI-N87 cells were cultured in RPMI1640 medium, MCF-7 cells and PC-3 cells in DMEM medium. All media were supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin streptomycin double antibody solution. BALB/c mice (female, 7 weeks old) and BALB/c nude mice (female, 6-8 weeks old) were purchased from Bekininowa laboratory animal science and technology Co., Ltd, Beijing, China, and the experiments were conducted according to the guidelines of the animal Committee of the university of south Keysterning (Tianjin, China).

EXAMPLE 1 preparation and purification of conjugates

1、Z _HER2 Cloning, expression and purification of

Z _HER2：2891 The coding sequence of (a) is based on the amino acid sequence described by Feldwisch et al, with a hexahistidine (6 × His) tag added at the N-terminus and the sequence GGGGC containing a cysteine residue for drug binding added at the C-terminus. This sequence was then inserted between the NcoI and XhoI restriction sites of the pET28a vector. The resulting plasmid, pET28a-Z, was subsequently used _HER2 BL21(DE3) pLysS E.coli was transformed and cultured in LB medium supplemented with 30. mu.g/mL kanamycin until OD600 reached 0.6. Subsequently, the bacterial solution was induced with beta-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.7mM and cultured with shaking on a bacterial shaker at 20 ℃ for 4-5 h. Collecting thallus, lysing cells, purifying cell lysate by Ni-NTA agarose column, dialyzing eluent with buffer solution (10mM disodium hydrogen phosphate, 2.7mM potassium chloride, 1.76mM potassium dihydrogen phosphate and 0.5mM NaCl, pH 7.4) for three times, and collecting the final Z _HER2 And the identification is carried out by SDS-PAGE and mass spectrum.

2. Preparation and purification of conjugates (FIG. 1)

Reduction of Z with TCEP prior to conjugation is hindered because the thiol groups in the molecule are readily oxidized to form disulfide bonds, preventing conjugation during purification and storage _HER2 . For reduction, Z is reacted at room temperature _HER2 (0.5 mM in reaction buffer) with TCEP (1: (N-methyl-L-alanine)Relative to Z _HER2：2891 1.1 eq) was shaken for 1h and excess TCEP was removed by passing through a PD-10 desalting column. Reduced Z _HER2 Directly used for the next coupling reaction.

In the preparation of HP4KM and HP10KM, bifunctional PEG4K or PEG10K was used. First 40mM bifunctional PEG4K or PEG10K was mixed with 80mM Gly3-VC-PAB-MMAE (1.2 equiv.) and N, N-diisopropylethylamine (DIPEA, 2 equiv.) and shaken at room temperature for 2 h. Then, the mixture is reacted with reduced Z _HER2 The reaction was carried out at room temperature for 6 hours in an equivalent ratio of 1.1: 1. The conjugate HM replaces the bifunctional PEG chain with a bifunctional linker SMCC, and the coupling strategy is the same as HP4KM and HP10 KM. All coupling products were purified by Ni-NTA resin to obtain HM, HP4KM and HP10KM conjugates (FIG. 2A) with a purity of greater than 95%, stored in PBS buffer at-80 ℃.

3. High performance liquid chromatography (FIG. 2A)

The conjugate was analyzed for properties by High Performance Liquid Chromatography (HPLC) using a C-4 column (250X 4.6mm) and an ultraviolet detector. The mobile phase consisted of a (0.14% TFA in H2O) and B (0.14% TFA in CH3 CN). The column was first kept at 90/10(V/V) a/B, then B concentration was kept at 10% over 5 minutes and 80% over 30 minutes. Subsequently, the concentration was restored to 20% in another 10 minutes. Prior to each injection, the column was equilibrated at a/B-90/10 (V/V) for 30 minutes. Detection was then carried out at 220 nm.

Example 2 ELISA-based receptor binding assay (FIG. 2B)

Human HER2 protein (ACRO Biosystems, Cat # HE2-H521y, Beijing, China) was seeded at 0.2. mu.g/well in 96-well plates and left overnight at 4 ℃. The plates were blocked with 3% Bovine Serum Albumin (BSA) solution at room temperature for 2h, washed and then the conjugate was added in a gradient dilution, incubated for another 2h, incubated with HRP-conjugated mouse anti-His tag antibody at room temperature for 1h, then TMB developing solution was added at 100. mu.L/well, left to stand at room temperature for 10min, then 2.0N H2SO4 (100. mu.L/well) was added to terminate the development and the absorbance was measured at 450 nm. The data were analyzed using GraphPad prism 6.0.

As a result: we used ELISA method to measureThe affinity of the binder to the HER2 receptor is determined. The results are shown in FIG. 2B, where the absorbance gradually increased with increasing conjugate concentration over a range of concentrations. HER2 protein and Z _HER2 The calculated KD values for HM, HP4KM and HP10KM were 135.6pM, 98.5pM, 161.7pM and 250.7pM, respectively (fig. 2B).

These results indicate that these binders have a very high affinity for their receptor HER 2. When MMAE-PEG chain with larger molecular weight is linked with Z _HER2 After protein coupling, the KD value and Z of the conjugate and HER2 receptor _HER2 There was no significant difference in comparison. Since the C-terminus is distant from its receptor binding region, the drug or PEG chain is placed at Z _HER2 Such a design strategy may help to reduce the effect of the affinity of the binder molecule. The high binding affinity of these molecules to the HER2 receptor lays the foundation for their specificity and high efficiency in killing HER2 over-expressed tumors.

Example 3 in vivo pharmacokinetics (FIG. 2C)

We used ELISA to determine the half-life of the conjugate in the blood circulation. BALB/c mice (female, 7 weeks old) were injected via tail vein with 5.0mg/kg doses of conjugate and blood samples were taken from the ocular veins at various time points (3, 60, 120, 240, 360, 600 and 1440 min). The plasma was allowed to stand at room temperature for 30 minutes, and then centrifuged at 4 ℃ to separate the serum. Human HER2 protein was coated on microtiter plates (5.0mg/mL) and allowed to stand at 4 ℃ for 18 h. The plates were washed with 0.1% PBS-T (PBS buffer containing 0.1% Tween-20) and blocked with 3% BSA solution for 1h, then serum samples diluted 1: 400 with 100. mu.L/well of PBS buffer were added. After 1h of incubation, the plates were again washed with PBS-T, incubated with mouse anti-His-tagged antibody (1: 2000 dilution, Cat. No. 37-2900; Life Technologies, Camarillo, CA, USA) at room temperature for 1h, and incubated with HRP-tagged goat anti-mouse IgG (1: 2000 dilution, Cat. No. CW 0102; CWBio, Beijing, China) at room temperature for an additional hour. After washing with PBS-T, TMB developing solution was added and incubated for 15 minutes in the absence of light, followed by terminating the developing reaction with 100. mu.L of 2.0N sulfuric acid and measuring the absorbance at 450 nm. A standard curve was drawn with the solution containing the quantified protein. The half-life of the conjugate in mice was calculated using GraphPad-Prism6.0 software.

As a result: the in vivo blood circulation half-life of the conjugate was determined by measuring the amount of residual protein in plasma using enzyme-linked immunosorbent assay (ELISA), calculating the clearance of the conjugate using GraphPad-prism6.0 software. As shown in FIG. 2C, the half-life of HM is only 19.6min, in combination with Z _HER2 The half-lives (18.5min) are similarly short, with limited residence time in tumor tissue and rapid clearance from the body. When long-chain PEG with the molecular weight of 4kDa or 10kDa is introduced, the half lives of HP4KM and HP10KM are respectively prolonged to 49.2min and 219.0min, and are prolonged by 2.5 times and 11.2 times.

Example 4 evaluation of Binders at cellular level

1. Cell recovery and culture:

removing human gastric cancer NCI-N87 cell line (HER2 high expression), human ductal carcinoma of mammary gland BT-474 cell line (HER2 high expression), human breast cancer MCF-7 cell line (HER2 low expression) and human prostate cancer PC-3 cell line (HER2 low expression) from liquid nitrogen, rapidly thawing in a 37 deg.C water bath, centrifuging at 1000rpm/min for 5min, discarding supernatant, adding NCI-N87 cells and BT-474 cells into preheated RPMI complete medium (10% serum and 1% antibiotic are added into base medium 1640), adding MCF-7 cells and PC-3 cells into DMEM complete medium, and placing into 5% CO ₂ And cultured overnight in an incubator at 37 ℃. The medium was changed the next day. And continuously culturing until the cells are fully paved at the bottom of the dish. Subculturing for 2-3 times.

2. Flow cytometry detection binding Selectivity (FIG. 2D)

The binding affinity and selectivity of these binders to HER2 receptor were analyzed by flow cytometry. We plated NCI-N87 and BT474 cells positively expressing the HER2 receptor and MCF-7 and PC-3 cells poorly expressing the HER2 receptor at a cell density of 1X 105 cells/well in 12-well plates and incubated at 37 ℃ for 24 hours in a cell incubator, then incubated with 50nM conjugate or free Z _HER2 Cells were incubated at 4 ℃ for 1 hour, then incubated with FITC-labeled mouse anti-His-Tag IgG for an additional 1 hour, and after final incubation with PBS buffer and washing, cell concentration was adjusted to 1.0X 106 cells/mL using a FACS-Calibur instrument (BD Biosciences, San Jose, Calif., USA)) Detection was performed and then data analysis was performed using FlowJo.

As a result: we used flow cytometry to determine the binding affinity and selectivity of these binders to the HER2 receptor. Human gastric cancer NCI-N87 cells and human breast cancer BT-474 cells with high expression of HER2, human breast cancer MCF-7 cells and human prostate cancer PC-3 cells with low expression of HER2 are taken as research objects. As shown in fig. 2D, all binders had very strong interactions with HER2 positive cell lines NCI-N87 and BT-474, with much stronger fluorescence intensities than the control. And the fluorescence intensity of the experimental group and the control group has almost no difference for the negative cell lines MCF-7 and PC-3. These results indicate that these conjugates have good binding affinity and targeting selectivity for HER2 receptor, while indicating that the coupling of MMAE to long PEG chains does not affect Z _HER2 Binding affinity to the cell surface.

MTT assay for in vitro cytotoxicity (FIGS. 3, 4A-B)

Human gastric cancer NCI-N87 cells and human breast cancer BT-474 cells with high expression of HER2, human breast cancer MCF-7 cells with low expression of HER2, and human prostate cancer PC-3 cells with little expression of HER2 were selected for the study. Cancer cells were seeded at a density of 5000 cells/well in 96-well plates and cultured in standard humidified cell culture chambers (37 ℃, 5% CO 2). After 24 hours, different concentrations of conjugate (2.56pM to 1.0. mu.M) were added to the wells and co-incubated for 72 hours at 37 ℃. Then, MTT solution (10. mu.L, 5.0mg/mL, final concentration 0.5mg/mL) was added thereto for further co-incubation for 4 hours. After removing the supernatant, formazan crystals were dissolved in 100. mu.L of dimethyl sulfoxide (DMSO), and the absorbance was measured at 490 nm. Statistical data analysis was performed using GraphPad Prism software (GraphPad software corporation, san diego, ca, usa).

The result of the MTT method for determining the cytotoxic activity is as follows: as shown in FIG. 3, the conjugates HM, HP4KM and HP10KM all showed very strong cytotoxicity to HER2 high expression cells, with their EC ₅₀ The values (concentration at which cell viability is reduced by 50%) are between 4.0 and 100 nM. Among them, HM activity was the strongest, and EC was high in HER2 expression in NCI-N87 and BT-474 cells ₅₀ Values were 4.94nM and 2.48nM, respectively. 4kDa PEG chain modified HP4KM pairEC of both cell lines ₅₀ The values were 31.9nM and 26.2nM, respectively, and the EC of 10kDa PEG chain-modified HP10KM on these two cell lines ₅₀ Values were 111.3nM and 83.5nM, respectively. In contrast, the introduction of PEG chains negatively affects the cytotoxicity of the conjugate. For MCF-7 or PC-3 cells with low expression of HER2, the cytotoxicity of all the conjugates is greatly reduced, and the EC ₅₀ The values were all above 1000nM, almost 50 times higher than those of HER2 high expressing cells. These data indicate that these conjugates can kill HER2 high expressing cancer cells with high selectivity by relying on the HER2 receptor. The free MMAE molecule has strong killing ability to cells with high expression of HER2 or low expression of HER2, has no target selectivity to the cells, and has EC ₅₀ The value is between 0.229 and 3.81 nM. Lack of the necessary cytotoxic selectivity results in strong toxic side effects, which is also the main reason why MMAE cannot be used clinically.

To further validate the role of the HER2 receptor in cytotoxicity of these conjugates, we used a competitive inhibition assay. Here, HER2 high expressing NCI-N87 and BT-474 cells (5000 cells/well) were seeded in 96 well plates first with 50. mu.M free Z _HER2 The proteins were preincubated for 2 hours to block HER2 receptor, and then incubated with different concentrations of conjugate for 72 hours. Cell viability was measured in a similar manner with MTT reagent.

Competitive inhibition assay results: with an excess of Z _HER2 Cells were preincubated (50 μ M, 2 hours) to block HER2 receptor, and the cytotoxicity of the conjugate was significantly reduced on NCI-N87 and BT-474 cells highly expressed in HER2 receptor. Under these conditions, the viability of the cells was above 90% even when the concentration of the conjugate was increased to 1.0. mu.M (FIGS. 4A-B). These results indicate that the cytotoxicity of these conjugates on cells is highly dependent on the expression of HER2 receptor on the cell surface.

The results show that the molecules have strong cytotoxic activity and good HER2 receptor dependence and selectivity. However, long-chain PEG modifications negatively impact the cytotoxicity of the conjugate. Modification with a 4kDa or 10kDa PEG chain reduced the cytotoxicity of the conjugate by about 6.5-fold and 22.5-fold, respectively. However, HP4KM and HP10KM still had good cytotoxicity. In addition, long PEG chain modifications did not alter the selectivity and receptor dependence of the conjugate.

4. In vitro cytotoxicity by live/dead staining (FIG. 4C)

NCI-N87 cells with high expression of HER2 and PC-3 cells with low expression of HER2 were seeded in 96-well plates at a cell density of 5000 cells/well, incubated at 37 ℃ for 24 hours, then 200nM of HM, HP4KM, HP10KM and MMAE were added, co-incubated at 37 ℃ for 72 hours, washed with PBS buffer, stained with calcein-AM, mixed with EthD-1 and PBS at a ratio of 1: 4: 1000, incubated for 15 minutes, and then imaged under an inverted fluorescence microscope (DMI 4000B, Leica USA).

As a result: as shown in figure 4C, when HER2 high expressing NCI-N87 cells were co-cultured with these compounds, the 200nM HM killed almost all cells completely with little green signal to live cells; HP4KM and HP10KM killed most of the cells, leaving only a small green signal of viable cells. HP4KM and HP10KM reduced cytotoxicity compared to HM due to conjugation with long PEG chains. For PC-3 cells with low expression of HER2, co-culture with HM, HP4KM and HP10KM did not result in PC-3 cell death, and almost all cells showed green signals from live cells. In contrast, free MMAE killed almost all NCI-N87 cells with high expression of HER2 and PC-3 cells with low expression of HER2 without selectivity. The results show that HM, HP4KM and HP10KM have strong cytotoxicity on HER2 positive cells and have good selectivity on HER2 receptors. With the modification of the PEG chain, the cytotoxic activity of the conjugate decreases. However, free MMAE was not cytotoxic selective.

Detection of apoptosis by APC-annexinv/PI apoptosis assay kit (FIG. 5)

HER2 high expression NCI-N87 cells and HER2 low expression PC-3 cells were seeded on 12-well plates at a density of 1.0X 105 cells/well, cultured in a cell incubator at 37 ℃ for 24h, then co-cultured with 200nM HM, HP4KM, HP10KM and MMAE for 24h, 48h and 72h, digested and washed with AV binding buffer, cell concentration was adjusted, APC-annexin V and PI were added, stained at room temperature for 20min, and stained cells were detected by flow cytometry.

As a result: as shown in figure 5, HM, HP4KM and HP10KM induced significant apoptotic signals when high HER2 expressing NCI-N87 cells were incubated with these molecules (figure 5A). However, little potent apoptosis was induced when PC-3 cells with low expression of HER2 were incubated with these molecules (fig. 5B). The free MMAE has stronger apoptosis induction effect on NCI-N87 cells with high expression of HER2 and PC-3 cells with low expression of HER2, the killing rates are 73.3 percent and 65.3 percent respectively, and no obvious selectivity exists. In addition, we also determined the effect of incubation time on apoptosis. In NCI-N87 cells, the experimental group HM, HP4KM, HP10KM incubated for 24h had almost no apoptosis signal, and when the incubation time was extended to 48h, the cells gradually entered early apoptosis (AV + PI +). After 72h incubation, many early apoptotic cell populations entered late apoptosis (AV + PI +). At this time, HM, HP4KM and HP10KM induced apoptosis rates of 65.6%, 41.3% and 32.8%, respectively. The apoptosis rate of the PBS group was less than 10% at all incubation times.

The above results indicate that HM, HP4KM and HP10KM conjugate significantly induced HER2 receptor-dependent apoptosis. As the culture time is prolonged, the cells gradually transition from early stages of apoptosis to late stages of apoptosis. Meanwhile, the long PEG chain modification introduced into the conjugate has certain negative effects on the apoptosis inducing activity, and the longer the PEG chain is, the more obvious the effect is.

6. Flow cytometry analysis of the Selective Induction of growth retardation G2 (FIG. 6)

NCI-N87 cells were cultured in 12-well plates at a density of 2.0X 105 cells per well. After 24 hours of culture, cells were co-cultured with 1.0 μ M conjugate or free MMAE for 16 hours at 37 ℃. Then, cells were harvested, fixed with pre-chilled ethanol solution (75% v/v) for 16h, washed twice with PBS, and stained with 50 μ g/mL Propidium Iodide (PI) solution in PBS buffer containing 0.1% Triton X-100, along with DNase-free RNaseA at 37 ℃ for 30min in the dark. The cells were harvested using a flow cytometer (BD-FACS-Calibur) and the data analyzed using ModFit software.

As a result: since MMAE inhibits cell division by inhibiting tubulin polymerization, we examined the effect of these conjugates and free MMAE on the NCI-N87 cell cycle (attachment)Fig. 6). Compared with the PBS control group, MMAE strongly inhibited NCI-N87 cell division, and the percentages of cells in the G2/M phase in the PBS and MMAE-treated groups were 3.7% and 72.0%, respectively. Similar to MMAE, HM, HP4KM and HP10KM conjugates also strongly inhibited cell division, and the percentage G2/M phase of cells treated with these conjugates was 71.2%, 67.7% and 57.2%, respectively. However, the ability of these conjugates to inhibit cell division also decreases with increasing molecular weight of the PEG chains. Free Z _HER2 There was little effect on the cell cycle, with only 10.9% of cells in the G2/M phase, with no significant difference compared to the PBS control group. These experimental results show that these conjugates have the function of inhibiting NCI-N87 cells to the G2/M phase, while PEG chain modification reduces the killing ability of the conjugates.

Example 5 evaluation of conjugates at animal level

1. In vivo antitumor Activity (FIG. 7)

The invention adopts a HER2 positive NCI-N87 xenograft mouse model to evaluate the in vivo anti-tumor activity. The nude mouse model was established by subcutaneously inoculating 5X 106NCI-N87 cells on the right side of female BALB/c nude mice. When tumor volume reached about 200mm3, mice were randomized into 4 groups (n-5) and injected with different doses of conjugate every 3 days for a total of 4 times, with PBS group as control. Tumor size and mouse body weight were monitored every 3 days until day 31. Tumor size was measured with a vernier caliper and tumor volume was calculated using the length x width 2/2 formula.

On day 31 post-treatment, blood samples (200. mu.L) were collected from mice receiving 1.5mg/kg dose injection treatment and serum was separated by centrifugation at 3000rpm for 10 min. ALT and AST levels in serum were determined using ALT and AST activity assay kits (Nanjing Jiancheng bioengineering research institute), respectively. The mice were then sacrificed and tissues and organs were collected and fixed with 4% paraformaldehyde. Specimens were embedded in paraffin, stained with hematoxylin-eosin (H & E), and histopathological changes were observed with a Nikon eclipse E100 microscope.

As a result: to further validate the therapeutic effect of these conjugates in vivo, we evaluated the in vivo tumor growth inhibition of these conjugates in the NCI-N87 gastric cancer xenograft model. Mice were randomized and treated with two different doses (1.5mg/kg and 0.6mg/kg) of conjugate. The results are shown in FIG. 7, where the tumors grew rapidly in the control mice receiving PBS injection, with an average tumor volume of 1000mm3 at day 25 and 1300mm3 at day 31 after treatment. The HM, HP4KM or HP10KM injected into the treatment group at the dosage of 1.5mg/kg or 0.6mg/kg can obviously inhibit the tumor growth, but the curative effect of the dosage of 1.5mg/kg is more obvious. At 31 days post-treatment, the tumor volumes of HM, HP4KM and HP10KM groups at this dose were 593mm3, 403mm3 and 245mm3, respectively, and the tumor inhibition rates were 54.38%, 69.00% and 81.15, respectively. At a dose of 0.6mg/kg, the efficacy is slightly reduced, but still very effective. At day 31 post-treatment, the tumor volumes of the HM, HP4KM, and HP10KM groups at this dose were 762mm3, 556mm3, and 454mm3, respectively, with tumor inhibition rates of 41.38%, 57.23%, and 65.07%, respectively.

In contrast, HP10KM has higher in vivo anti-tumor activity. Compared with HM and HP4KM, HP10KM has the longest PEG chain modification and the longest circulating half-life, which are 11.2-fold and 4.5-fold higher than HM and HP4KM, respectively. But its cytotoxicity was 22.5 times and 3.5 times lower than that of HM and HP4KM, respectively. This combined effect gave HP10KM a relatively optimal in vivo tumor suppression potential, which might indicate the importance of the circulating half-life of the conjugate to improve its tumor therapeutic potential, even at the expense of its cytotoxicity. Our results indicate that the introduction of PEG chains can improve the therapeutic effect of miniaturized ligand based ADC drugs; however, we need to use a suitable length of PEG chain for this modification and consider the effect of PEG chain on conjugate half-life and cytotoxic activity. The strategy has good application prospect in the development of anti-tumor drugs based on conjugates.

Toxicity of the conjugate was assessed by liver function assessment and immunohistochemical analysis of vital tissues and organs of the treated mice. The liver function test adopts a kit to test the contents of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) in the serum of the mice. As shown in FIGS. 7E-F, all of the conjugates showed low toxicity compared to the control group, and the ALT and AST levels were slightly elevated in these groups. AST levels were significantly lower in the HP10KM group than in the HM and HP4KM groups. Histological analysis of heart, liver, spleen, lung, kidney and tumor tissues showed no significant damage to major organs after treatment with these conjugates (fig. 7G). These results indicate that these conjugates have good safety in therapy.

2. Off-target toxicity assay

BALB/c mice (n ═ 5) were injected intravenously with the indicated doses of conjugate or free MMAE (2.0mg/kg, 5.0mg/kg, 10.0mg/kg, 15.0mg/kg and 20.0 mg/kg). Mice were tested and reported for mortality and death events within 2 weeks after injection.

Results of off-target toxicity test of conjugate on BALB/c mice: as shown in table 1, after injection of 5.0mg/kg free MMAE, all mice died soon after injection; when 2.0mg/kg free MMAE (not shown in table 1) was injected, 1 of 5 mice died, and the remaining mice were blinded, unable to open the eyes, and had severe ocular toxicity. These data indicate that free MMAE molecules can cause serious side effects and even death at doses of 2.0 or 5.0 mg/kg. The conjugate significantly increased the tolerated dose, and 100% survival was achieved in mice injected at 5.0mg/kg (HM), 10.0mg/kg (HP4KM), and 20.0mg/kg (HP10 KM). The Maximum Tolerated Dose (MTD) of HP10KM was at least 4-fold higher than the free MMAE or HM. The use of PEG modification, especially 10kDa modification, significantly reduced off-target toxicity of the conjugate to mice.

TABLE 1 off-target toxicity test results in mice

In summary, in the present invention, we propose a new method for designing and synthesizing miniaturized ADC molecules. Modification of the PEG chain and prolonging of the circulating half-life can significantly improve the tumor treatment capacity, however, the influence of the PEG chain on the half-life and cytotoxicity of the conjugate needs to be considered comprehensively, so that modification with a PEG chain of an appropriate length is important.

The general description and the specific embodiments of the present invention described above should not be construed as limiting the technical solution of the present invention. Those skilled in the art will appreciate that other technical solutions pertaining to the present invention can be formed by adding, subtracting or combining technical features disclosed in the above general description or/and the detailed description (including the examples) without departing from the structural elements of the present invention, and the scope of the present invention is also within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A miniaturized ADC of the general formula (I):

Z _HER2 -PEG-Drug

(I) 。

2. the miniaturized ADC of claim 1, wherein said Z is _HER2 Has the following structure: the C-terminal contains cysteine with sulfhydryl

3. The miniaturized ADC of claim 1, wherein the Drug moieties Drug are independently selected from the group consisting of Gly3-VC-PAB-MMAF group and Gly3-VC-PAB-MMAE group having the following structure:

gly3-VC-PAB-MMAE group is preferred.

4. The miniaturized ADC of claim 1, wherein the repeating unit in the PEG is-CH ₂ CH ₂ O-; the PEG has a molecular weight independently selected from 3kDa, 4kDa, 5kDa, 6kDa, 7kDa, 8kDa, 9kDa, 10kDa, 11kDa or 12kDa, preferably 10 kDa.

5. The miniaturized ADC of claim 1, wherein the PEG is a bifunctional polyethylene glycolWherein one end has a structure of

A linker attached to the amine group; the other end has a structure of

A linker attached to a thiol group.

6. A method of making the miniaturized ADC of any one of claims 1-5, comprising:

1) linking the PEG of claims 4-5 to Gly3-VC-PAB-MMAE or Gly3-VC-PAB-MMAF to obtain PEG-Drug;

2) combining PEG-Drug with Z as described in claim 2 _HER2 Coupling to obtain miniaturized ADCs of general formula (I).

7. Use of a miniaturized ADC according to any one of claims 1-5 in the manufacture of a medicament for the treatment of a tumor.