CN113398242B - Antibody coupling medicine and application thereof - Google Patents

Abstract

The invention discloses an antibody coupling medicament and application thereof. The antibody-coupled drug is an antibody coupled with drug dolastatin, the antibody comprises Fc of a 9G8 nano antibody, a 7D12 nano antibody and an IgG1 antibody, two Fc chains of a double-chain structure are respectively fused with the 9G8 nano antibody and the 7D12 nano antibody of one molecule, the amino acid sequence of the 9G8 nano antibody is used for coupling the dolastatin after serine at the 7 th position and/or the 71 th position is mutated into cysteine by a sequence shown as SEQ ID No.1, and the DAR value of the antibody-coupled drug is 1-2. The invention makes the fusion protein easily connect with small molecular toxin through the site-specific cysteine mutation of the conserved site of the nano antibody, the uniformity of the coupling product is good, and the invention shows good tumor killing ability in vitro and in vivo experiments. Compared with the currently marketed cetuximab, the fusion protein conjugate has stronger CDC effect and can overcome a series of drug-resistant mutations of EGFR extracellular regions.

Description

Antibody coupling medicine and application thereof

Technical Field

The invention relates to the technical field of biological pharmacy, in particular to an antibody coupling medicament and application thereof.

Background

Antibody-conjugated drugs are antibody-modified drugs that combine an antibody with a cytotoxic small molecule drug, and are of interest to people due to the combination of targeting of the antibody and the killing of chemotherapeutic drugs. If the antibody part is regarded as a targeted guidance system, the high-efficiency cytotoxic micromolecule drug connected to the antibody part is a drug loading responsible for killing, and the effect of killing the target at a fixed point can be realized theoretically. By coupling the chemical small molecules to the antibody, the therapeutic window of the small molecule drug can be widened, the side effects can be reduced, and the long half-life period of the antibody drug and various antibody-mediated immune effects can be exerted.

Epidermal Growth Factor Receptor (EGFR) is a type of transmembrane glycoprotein of the cell surface tyrosine kinase receptor family that functions to regulate cell proliferation, growth, differentiation, and migration. Under normal conditions, the signaling pathway of EGFR is tightly regulated, and in many cancers, due to abnormal overexpression of EGFR, the downstream signaling pathway is dysregulated, and the pathogenesis of some tumors related to epidermal growth is closely related. The common EGFR overexpression cancers at present comprise lung cancer, colorectal cancer, squamous cell carcinoma and the like, and the abnormal overexpression of the EGFR is closely related to the poor recovery and survival of a plurality of cancer patients.

After being combined with EGFR on the surface of tumor cells, the EGFR-targeting ADC drug can enter the tumor cells through endocytosis to release cytotoxic small molecules to exert a killing effect, and can effectively overcome partial mutations of intracellular downstream signals of the EGFR, such as KRAS, BRAF mutation and the like. However, it has been clinically found that some patients treated with cetuximab for a period of time produce EGFR extracellular point mutations such as S492R and G465R, which directly affect the affinity of EGFR antibodies. In the face of such drug-resistant point mutations, the original effective ADC drugs also affect the efficacy of the mutated tumor tissues because they cannot effectively bind to the mutated tumor tissues.

Traditional ADC drugs typically select either the lysine or cysteine of the antibody itself as the conjugation site for the small molecule drug. The antibody has more lysine, and the coupling product is a mixture of coupling multiple lysine sites. Cysteine coupling sites are relatively few, the cysteine sites which are exposed on the surface of the antibody and can be coupled can only be generated after reducing interchain disulfide bonds of the antibody, and the antibody with a normal structure has 4 pairs of interchain disulfide bonds in total, so that the antibody with the normal structure has 8 cysteine sites at most for coupling. Conventional non-site-directed coupling methods all produce coupling products that are highly heterogeneous, and the final reaction product is a mixture of components containing different DAR ratio values. Because the coupling sites of the components are different, the pharmacokinetics, the efficacy and the safety of the components in vivo are different to different degrees.

Complement was originally identified as a class of proteins involved in innate immunity, which primarily serves to protect against bacterial invasion, activate the release of inflammatory factors, and mediate lysis in this first line of defense, thereby gaining time for subsequent acquired immunity. In the last 70 th century, the relationship between complement and malignant tumors was investigated, and it was found that in specific in vivo and in vitro environments, tumor cells can activate complement, and it was later found that the process of killing tumor cells by complement is closely related to antibody-mediated immune effects. After the antibody binds to the surface of the tumor cell, the complement protein can be bound to the surface of the target cell at the same time, and the tumor cell can be killed by complement-dependent cytotoxicity (CDC). Currently marketed anti-EGFR antibody drugs such as cetuximab and panitumumab, panitumumab itself is an IgG2 type antibody, the immune effect itself is weak, and almost no CDC effect, while cetuximab of IgG1 type is also weak in vitro and in vivo CDC effect.

Disclosure of Invention

The invention provides an antibody conjugate formed by coupling nano antibody Fc fusion protein aiming at EGFR and dolastatin (MMAE) and a preparation method and application thereof aiming at the defects in the prior art.

An antibody coupling medicine is prepared by coupling medicine dolastatin on an antibody,

the antibody comprises Fc of 9G8 nano antibody, 7D12 nano antibody and IgG1 type antibody, wherein the Fc of the IgG1 type antibody is a double-chain structure,

the 9G8 nano antibody and the 7D12 nano antibody are fused and then fused with the Fc of the IgG1 type antibody, and two Fc chains of a double-chain structure are respectively fused with a molecule of the 9G8 nano antibody and the 7D12 nano antibody,

the amino acid sequence of the 9G8 nano antibody is shown in SEQ ID No.1,

the amino acid sequence of the 7D12 nanometer antibody is used for coupling dolastatin after serine at the 7 th site and/or the 71 th site is mutated into cysteine by the sequence shown in SEQ ID No.3,

the amino acid sequence of the Fc of the IgG1 type antibody is shown in SEQ ID No.7,

the DAR value of the antibody-coupled drug is 1-2.

One molecule of antibody may have a molecule of drug coupled to both chains, in which case the DAR value is 2, or only one of the chains may have a molecule of drug coupled to it, in which case the DAR value is 1, and both are effective. In most cases, the two forms are prepared as a mixture, and can be purified to obtain a homogeneous antibody-coupled drug, or a mixture of both DAR forms.

Preferably, the C-terminus of the 9G8 nanobody is fused to the N-terminus of the 7D12 nanobody, and the C-terminus of the 7D12 nanobody is fused to the N-terminus of the Fc. More preferably, the 9G8 nano antibody and the 7D12 nano antibody are connected through flexible connecting peptide, the flexible connecting peptide is (GGGGS) n, and n is a natural number of 1-3. Further preferably, the amino acid sequence of the single chain of the two chains of the antibody, in which serine at position 7 and/or 71 of the 7D12 nanobody is not mutated to cysteine, is shown in SEQ ID No. 9. Further preferably, the antibody is conjugated to a drug and the Fc amino acid sequence is mutated from E to G at position 215 (residues numbered according to the EU index of Kabat (Edelman et al, proc. natl. acad. sci. usa,63(1969)78-85), position 215 on the Fc corresponding to position 430 in the full anti-heavy chain).

The invention also provides a preparation method of the antibody coupled drug, which comprises the following steps:

(1) the antibody is obtained through recombinant expression and purification,

(2) the reducing agent treatment is used to open the inter-chain disulfide bonds of the antibody, the oxidizing agent is used to reconnect the inter-chain disulfide bonds,

(3) and (3) reacting mc-vc-MMAE with the antibody to enable the maleimide group and the sulfhydryl group to generate Michael addition reaction so as to obtain the antibody coupled drug coupled with the drug.

The MC-VC-MMAE is formed by connecting an MMAE and a VC joint and is called MC-VC-PAB-MMAE in full. MMAE is firstly connected with a vc joint, and after purification preparation, the maleimide group of the MMAE and the interchain disulfide bond of the antibody generate addition reaction to prepare the antibody coupling drug.

In step (2), the reducing agent is used to open the interchain disulfide bonds of the antibody, and then the oxidizing agent is used to reconnect the interchain disulfide bonds, so that the mutated cysteines are exposed and the cysteines of the interchain disulfide bonds are not exposed. TCEP can be generally used as the reducing agent, and DHAA can be generally used as the oxidizing agent.

The invention also provides application of the antibody coupled drug in preparation of antitumor drugs. Preferably, the tumor type against which the anti-tumor drug is directed is an EGFR-positive solid tumor.

The invention makes the fusion protein easily connect with small molecular toxin through the site-specific cysteine mutation of the conserved site of the nano antibody, the uniformity of the coupling product is good, and the invention shows good tumor killing ability in vitro and in vivo experiments, and can be easily expanded to other antibody coupling drugs based on the nano antibody. Compared with the currently marketed cetuximab, the fusion protein conjugate has stronger CDC effect and can overcome a series of drug-resistant mutations of EGFR extracellular region, such as S492R and G465R. The drug-resistant mutation sites of the EGFR extracellular region can cause that the currently marketed cetuximab or panitumumab cannot effectively have affinity with EGFR, thereby influencing the drug effect. The fusion protein conjugate proves that CDC effect and small molecular toxin killing effect in ADC medicine can mutually act synergistically, and shows better anti-tumor effect in experimental model, thereby widening the research idea of ADC medicine.

Drawings

FIG. 1 is a schematic diagram of a fusion protein (A) and a chemical structural formula of mc-vc-MMAE (B).

FIG. 2 shows the results of SDS-PAGE analysis of the coupled products, from left to right in 4 lanes, lanes 1 and 2: the results in the non-reduced state, the 3 rd and 4 th lanes are the results in the reduced state, the 1 st and 3 rd lanes are the purified unconjugated products, and the 2 nd and 4 th lanes are the purified conjugated products.

FIG. 3 is a graph showing the results of HPLC analysis of the coupled product.

FIG. 4 shows the results of flow cytometry analysis of the affinity of fusion proteins for A431 of target cells, using the mutation of the S7C cysteine site followed by the attachment of MMAE.

FIG. 5 is a graph showing the killing effect of the fusion protein and its conjugate on A431, MDA-MB-468 and BxPC-3 cells.

FIG. 6 is a graph showing the killing effect of the fusion protein and its conjugate on 293T cells expressing the EGFR extracellular domain S492R and the G465R mutation.

FIG. 7 is a graph of the induction of apoptosis in A431 and MDA-MB-468 cells by fusion proteins and conjugates thereof.

FIG. 8 is a graph showing the CDC killing effect of the fusion protein and its conjugate with cetuximab on EGFR-transfected NIH-3T3 cells and MDA-MB-468 cells.

Fig. 9 is a graph of the endocytosis efficiency of the fusion protein and its conjugate after binding to EGFR on the a431 cell surface with cetuximab.

FIG. 10 is a graph of tumor growth in BALB/c Nude mice for different doses of the fusion protein conjugate group, the fusion protein group, and the control ADC group.

FIG. 11 is a graph showing the change in body weight of BALB/c Nude mice administered with different doses of the fusion protein conjugate group, the fusion protein group and the control ADC group.

FIG. 12 is a graph of tumor growth in BALB/cNude mice for the 5mg/kg dose fusion protein conjugate group, the fusion protein group, and the control ADC group.

FIG. 13 is a graph showing the change in body weight of BALB/cNude mice in the 5mg/kg dose fusion protein conjugate group, the fusion protein group and the control ADC group after administration.

Detailed Description

Example 1

Preparation of 97 m-vcMAE conjugate.

The nano antibody Fc fusion protein taking EGFR as a target is formed by fusing 7D12 and 9G8 nano antibodies, wherein the sequences of the nano antibodies are from the literature (Karl R.Schmitz et al, Structure.2013; 21(7): 1214-1224), and are abbreviated as 97m below. The sequence of the Fc segment of the fusion protein was stored in the laboratory. In this example, 7D12 and 9G8 were linked by a flexible linker peptide (GGGGS) by PCR₃And connecting the two, connecting the C terminal with IgG1 heavy chain constant region, and finally obtaining the DNA sequence.

The sequence is as follows:

9G 8: the amino acid sequence is shown as SEQ ID No.1, and the gene sequence is shown as SEQ ID No. 2.

7D 12: the amino acid sequence is shown as SEQ ID No.3, and the gene sequence is shown as SEQ ID No. 4.

Flexible connecting peptide: the amino acid sequence is shown as SEQ ID No.5, and the gene sequence is shown as SEQ ID No. 6.

Heavy chain constant region (Fc) of IgG1 type: the amino acid sequence is shown as SEQ ID No.7, and the gene sequence is shown as SEQ ID No. 8.

97 m: the amino acid sequence is shown as SEQ ID No.9, and the gene sequence is shown as SEQ ID No. 10.

The constructed DNA sequence of 97m is used as a template, the 7 th or 71 th conservative serine site in the nano antibody sequence of 97m is mutated into cysteine by a PCR method, the constructed gene sequence is connected to a pMH3 expression vector, the constructed expression vector is transfected to human embryonic kidney cell HEK293, culture solution supernatant is collected, and the fusion protein is obtained after the purification of a protein A affinity column. Fig. 1A is a schematic diagram of a fusion protein, two kinds of nanobodies are connected in series, each nanobody can be mutated by 1-2 mutation sites, and the fusion protein in this embodiment is selected to perform cysteine site-directed mutation at S7 position of the nanobody.

The fusion protein solution after expression and purification was replaced with PBS buffer, 50-fold molar equivalent of TCEP reducing agent was added, and reduction was carried out at 37 ℃ for 3 hours. Then, the solution was replaced with a PBS solution containing 2mM EDTA at 4 ℃ by an ultrafiltration tube to remove the residual TCEP in the solution, thereby obtaining an antibody having an interchain disulfide bond opened. Then, 5 times molar amount of DHAA was added to the antibody solution as an oxidizing agent, and the solution was re-oxidized at room temperature in the dark for 2h to re-link the interchain disulfide bonds of the antibody. And finally, adding mc-vc-MMAE (hereinafter referred to as vcMMAE) into the reaction solution, reacting for 2h at room temperature to enable a maleimide group and a sulfhydryl group to generate Michael addition reaction, wherein FIG. 1B shows the chemical structure of the vcMMAE, and the specific synthetic process refers to the patent US2018/0036423A 1. After the reaction is finished, the reaction solution is replaced by PBS at 4 ℃ by a centrifugal ultrafiltration tube, residual micromolecules in the reaction solution are removed, or the product is directly purified by a protein A affinity chromatography column again to obtain an antibody conjugate 97m (S7C) -vcMMAE, which is hereinafter referred to as 97m-vcMMAE, and the obtained conjugate product is sterilized by a filter membrane with the aperture of 0.22 mu m and then is stored at-80 ℃ for a long time.

The products before and after conjugation were analyzed by SDS-PAGE, and as shown in FIG. 2, the bands after conjugation shifted upward compared to the bands before conjugation both under non-reducing and reducing conditions, indicating that the overall molecular weight was indeed increased compared to that before conjugation. Under reducing conditions, the band was slightly above 55kD, which is consistent with the theoretical molecular weight in view of the glycosylation effect of the Fc region.

Example 2

And (3) determining the drug antibody coupling ratio of the conjugate.

In order to quantitatively detect the DAR value after coupling of toxic small molecules, the coupling product was analyzed by hydrophobic interaction chromatography. The ADC concentration after coupling is diluted to 1mg/mL and then injected. Because the vcMMAE micromolecule has stronger hydrophobicity, the components with different drug micromolecule coupling ratios in the final coupling product can be distinguished by utilizing the change of hydrophobicity after coupling the micromolecules, and the specific analysis conditions are as follows:

a chromatographic column: TSKgel Butyl-NPR hydrophobic chromatography column (2.5 μm,4.6 mm. times.3.5 cm); mobile phase A: 2M (NH)₄)₂SO₄，150mM Na₃PO₄pH 7.0; mobile phase B: 150mM Na₃PO₄pH 7.0; mobile phase C: isopropyl alcohol; flow rate: 0.8 mL/min; column temperature: 25 ℃; sample introduction amount: 10 mu L of the solution; the gradient elution conditions are shown in table 1.

TABLE 1

Time (min)	A％	B％	C％
					0	75	25	0
15	0	75	25
				16	75	25	0
21	75	25	0

FIG. 3A is a hydrophobic chromatogram of a conjugated vc-MMAE after mutation of serine at position 71 to cysteine of the nanobody 7D12 sequence in a fusion protein. As can be seen from the figure, the unconjugated fraction in the conjugated product was 3.303%, DAR1 fraction was 20.59%, and the drug-antibody conjugation ratio in the final conjugated product was 1.73.

FIG. 3B is a hydrophobic chromatogram of a conjugated vc-MMAE after mutation of serine at position 7 of the Nanobody 7D12 sequence to cysteine in a fusion protein. As can be seen from the figure, the unconjugated fraction in the conjugated product was 6.579%, the DAR1 fraction was 3.845%, and the drug-antibody conjugation ratio in the final conjugated product was 1.83.

Example 3

And (3) flow affinity detection.

Culturing EGFR-expressing cells such as A431 in DMEM medium containing double antibody, and culturing the cells at 5 × 10 after logarithmic growth phase⁵The cell number of individuals was incubated with a series of serially diluted antibodies or ADCs on ice in 1% BSA in PBS for 30 min.

After incubation, cells were washed twice with pre-chilled PBS to remove proteins non-specifically adsorbed to the cell surface. And finally, adding a FITC-labeled goat anti-human IgG secondary antibody diluted at a ratio of 1:200 in a dark environment, and continuously placing on ice for incubation for 30min in the dark. After the incubation is finished, the cells are washed by precooled PBS, and the cell suspension after the PBS is resuspended is subjected to an up-flow cytometry analysis to detect the average fluorescence intensity (MFI) of the cells.

And (3) detecting the affinity of the fusion protein with EGFR positive cells before and after coupling by using a flow cytometry, wherein the strength of the MFI signal represents the change of the strength of the binding force.

The detection results are shown in fig. 4, the difference of the affinity of the conjugate coupled with vcMMAE after the site-directed mutagenesis of 97m and S7C without cysteine mutation is not large, and the conjugate basically maintains the affinity of the fusion protein before the conjugation.

Example 4

And (5) detecting cytotoxicity.

Three tumor cells MDA-MB-468, A431 and BxPC-3 with the surface expressing the EGFR are selected as target cells, the in-vitro killing activity of the ADC on the tumor cells is detected, and the quantity of the EGFR expressed by the three tumor cells is reduced in sequence. Culturing the tumor cells, centrifuging to collect the cells after the cells grow to logarithmic growth phase, and collecting the cells according to 5 × 10⁴Cell density of one/mL was added to a 96 well cell culture plate, and 100 μ L of cell suspension was added per well. Placing 96-well culture plate at 37 deg.C and containing 5% CO₂The cell culture box is continuously incubated overnight until the cells are attached to the wall and the growth state is good. After the antibody or ADC to be tested is filtered and sterilized by a 0.22 mu m filter membrane, the antibody or ADC is diluted into a series of antibody solutions by a gradient of a corresponding complete culture medium for culturing tumor cells, the antibody solutions are added into a 96-well cell culture plate according to the dosage of 100 mu L/well, three multiple wells are arranged in parallel in each concentration, wherein the complete culture medium without the medicine is used as a negative control well, the cells and the medicine are not added, and the wells with the complete culture medium are used as blank control wells. Continuously placing at 37 deg.C with 5% CO₂After incubation for 72 hours, the 96-well plate was removed, the medium in the plate was discarded, and a medium containing 10% of CCK8 reagent (Dong ren chemical Co., Ltd.) was added to the plate in a volume of 100. mu.L per well, and the plate was returned to the 37 ℃ cell incubator to wait for color development. And after the required time for color development, putting the 96-hole enzyme label plate into an enzyme label instrument to read the OD450 value, recording the reading and calculating the cell killing rate.

The results are shown in FIG. 5, where OFA ADCs were control ADCs conjugated to vcMAE based on the anti-CD 20 antibody Aframumab, preparation method reference (Zhaohui Li et al, Investigational New drugs.2014; 32, 75-86). No significant cell killing effect was observed for OFA ADCs as they failed to bind EGFR and three tumor cells did not express CD 20. After coupling vcMAE, 97 m-vcMAE has obvious improvement on killing of EGFR positive A431, MDA-MB-468 and BxPC-3 tumor cells, and the IC50 values are 0.055nM, 0.15nM and 1.24nM respectively.

Example 5

And (3) overcoming the cytotoxicity detection of EGFR extracellular region point mutation.

Transfecting 293T cells with full-length plasmids with S492R or G465R point mutation EGFR, selecting monoclonals with high transfection efficiency, and finally obtaining 293T cells with surface high expression bands with S492R or G465R point mutation EGFR, which are named as 293T-S492R and 293T-G465R respectively. The in vitro killing activity of the ADC on tumor cells in the presence of point mutations in the extracellular region was tested. Culturing the cells to be used, collecting the cells by centrifugation after the cells have grown to logarithmic growth phase, suspending the cells with fresh complete medium and following a 1X 10 basis⁴Cell density per well was added to 96 well cell culture plates, 100 μ L of cell suspension was added per well. Placing 96-well culture plate at 37 deg.C and containing 5% CO₂The cell culture box is continuously incubated overnight until the cells are attached to the wall and the growth state is good. The ADC to be tested was sterilized by filtration through a 0.22 μm filter and the drug was diluted to a series of concentration gradients in the corresponding complete medium of the cultured cells. The cells were added to the plate at a dose of 100. mu.L/well, and three wells were arranged in parallel. Continuously placing at 37 deg.C with 5% CO₂After incubation for 72 hours, the 96-well plate was removed, the medium in the plate was discarded, and a medium containing 10% of CCK8 reagent (Dong ren chemical Co., Ltd.) was added to the plate in a volume of 100. mu.L per well, and the plate was returned to the 37 ℃ cell incubator to wait for color development. And after the required time for color development, putting the 96-hole enzyme label plate into an enzyme label instrument to read the OD450 value, recording the reading and calculating the cell killing rate.

The results are shown in FIG. 6, where cetuximab ADC is a control ADC coupled to vcMMAE based on the marketed anti-EGFR antibody drug cetuximab, a preparation method reference (Carolin Sellmann et al, Journal of Biological chemistry.2016; 291, 25106-. Because the S492R and G465R point mutations in the extracellular region of EGFR affect the affinity of cetuximab, cetuximab ADC does not kill target cells efficiently. And the S492R and G465R point mutation do not affect the affinity of 97m, so that 97 m-vcMAE can overcome the point mutation of the extracellular region and still play a role in killing.

Example 6

And (4) analyzing apoptosis.

The apoptotic effect of ADC on a431 tumor cells was examined. Collecting A431 cells at 2.5X 10⁵Cell density per mL was plated in 6 well cell culture plates. Respectively adding the diluted antibody or antibody conjugated drug in complete culture medium to ensure that the final concentration in the well is 5 μ L/mL, 0.7 μ L/mL and 0.1 μ L/mL, and continuously returning to 37 deg.C containing 5% CO₂In a cell culture incubator. After the drug and the cells are incubated for 48h, tumor cells in a 6-well plate are collected, the cells are washed twice by precooled PBS, 100 mu L of 1 × Annexin V (Annexin V) binding solution, 5 mu L of Propidium Iodide (PI) and 5 mu L of Annexin are added, the cell suspension is gently blown, and the incubation is continued for 20min in the dark at room temperature. Adding 300 mu L PBS into a centrifuge tube, detecting on a flow machine, and detecting the percentage of apoptotic cells by a flow cytometry analyzer.

As shown in FIG. 7, in A431 cells, 97 m-vcMAE and 97m at the administration concentration of 5 μ g/mL both showed obvious effects of inducing apoptosis of target cells, wherein the apoptosis promoting effect of 97 m-vcMAE is obviously enhanced. 97m were early-withered 5.66% and late-withered 5.37%, whereas 97m-vcMMAE were early-withered 43.32% and late-withered 29.41%. The result shows that after the fusion protein 97m is coupled with MMAE in A431 cells, the apoptosis inducing effect reaches the maximum killing already at the administration concentration of 0.7 mu g/mL, and the apoptosis inducing effect and the concentration of 5 mu g/mL have little difference.

Example 7

Complement dependent killing activity.

Transfecting NIH-3T3 cells with plasmids with wild EGFR, selecting monoclonals with high transfection efficiency, and finally obtaining the NIH-3T3 cells with high surface expression of EGFR, which are named as 3T 3-EGFR. Selecting constructed 3T3-EGFR or MDA-MB-468 tumor cells from target cells, resuspending the cells, and adjusting cell concentration to 1 × 10⁵Perml, plated in 96-well plates at 0.1 ml/well. The fusion protein and its conjugate after gradient dilution were added to the sample wells at an addition of 50. mu.l/well. Wherein 97m-E430G-vcMMAE is the introduction of E430G mutation in the Fc region of 97m-vcMMAE to enhance its CDC effect (mutation site is position 215 as calculated from the amino acid sequence of Fc as shown in SEQ ID No.7, whereas residues are numbered according to the EU index of Kabat (Edelman et al, Proc. Natl. Acad. Sci. USA,63 (1969)) corresponds to position 430 in the full-length antibody heavy chain), the specific engineering method comes from the invention patent: 201880010285.3. the negative control wells were filled with the same volume of complete medium. In addition, a blank well without cells was set as a background, and 3 duplicate wells were set. And finally, adding 50 mu l/hole of human serum into the sample hole, the negative control hole and the blank hole to serve as a complement source, and uniformly mixing to ensure that the final proportion of the human serum is 20%. And (3) placing the 96-well plate in a cell culture box containing 5% carbon dioxide at 37 ℃ for culturing for 1-4 h, and adding 100 mu l of RPMI-1640 culture medium containing 10% CCK-8 reagent into each well to be detected. And (3) placing the cell culture plate in an incubator containing 5% carbon dioxide at 37 ℃ for culture and waiting for color development, placing the 96-hole ELISA plate in an ELISA reader for reading the OD450 value after the required time of color development, and recording the reading.

The results are shown in FIG. 8, and 97m and its conjugate both showed better CDC activity than cetuximab in the constructed NIH-3T3 model cells. And the conjugate 97 m-E430G-vcMAE with enhanced CDC effect can exert better CDC activity. In natural tumor cells MDA-MB-468, 97 m-E430G-vcMAE has more obvious killing effect.

Example 8

And (4) detecting the endocytosis efficiency.

A431 cells in a petri dish were carefully digested with pancreatin, collected and divided into two equal numbers of samples. The test antibody diluted in PBS with 1% BSA was used to resuspend the cells in the centrifuge tube and incubated on ice for 30 min. After removing the antibody not bound to the cell surface by washing twice with pre-cooled PBS, the cells were resuspended. One portion was placed at 4 ℃ and the other at 37 ℃. And after the standing time reaches the time required by endocytosis detection, transferring all cells to be detected to ice, adding FITC labeled goat anti-human IgG secondary antibody diluted by 1:200 in a dark environment, and continuously placing on the ice for incubation for 30min in the dark. And (3) after the incubation is finished, washing the cells by using precooled PBS, and mechanically detecting the average fluorescence intensity value of the cell surface on the cell suspension after the PBS is resuspended. The endocytosis efficiency of the cell surface bound antibody is calculated by calculating the percentage reduction of the average fluorescence intensity of the antibody to be detected incubated at 37 ℃ compared with the average fluorescence intensity of the antibody to be detected incubated at 4 ℃.

The results are shown in fig. 9, the fusion protein 97m and its conjugate have better endocytosis efficiency than cetuximab, and the endocytosis efficiency is not greatly affected before and after the conjugation.

Example 9

And (4) detecting the in vivo biological activity.

A Balb/c Nude female Nude mouse is adopted to construct a transplantation tumor model. A431 tumor cells in log phase growth were collected and resuspended in precooled PBS. A431 cell suspension at 4X 10⁶The density of each was inoculated into the right underarm of 6-8 week female nude mice. The tumor volume of the nude mice is 100mm³To 150mm³When the mice are randomly divided into 6 groups, each group comprises 6 mice, and the mice are respectively PBS blank control group, OFA ADC 6mg/kg, 97m 6mg/kg, 97 m-vcMAE 2mg/kg and 97 m-vcMAE 0.6mg/kg, wherein the OFA ADC is a negative control ADC prepared on the basis of anti-CD 20 antibody Aframumab. The drug concentration was adjusted and 100. mu.L of the drug was administered to the tail vein of each group of nude mice at the above administration dose. The experimental time was calculated from the first administration, once every four days for four times, and the body weight and tumor size of nude mice were measured every two to three days. The specific formula for tumor volume is: v ═ L × W²)/2. Wherein V is the tumor volume (mm)³) L is the measured tumor length (mm) and W is the measured tumor width (mm).

The results are shown in fig. 10, and both the 97m and 97 m-vcMMAE-administered groups significantly inhibited tumor growth compared to the OFA ADC group and the PBS group of the two negative controls. The tumor inhibiting effect of 97 m-vcMAE showed a dose-dependent trend, and the tumor inhibiting effect of the high, medium and low concentration groups was reduced in turn, wherein the high and medium dose groups could observe a significant reduction in tumor volume, while the low dose group could still observe a slower growth trend of tumor volume compared to the PBS group due to less drug reaching the tumor site.

FIG. 11 is the body weight data of the mice in each group during the experiment, and it can be observed that the body weight of the tumor-bearing mice in each group does not show obvious reduction in the whole experiment period, and the whole body weight shows a slight increase trend, which indicates that no obvious side effect is generated on the mice in the experiment period no matter the 97 m-vcMAE or 97m groups with different dosages.

Example 10

And (4) detecting the in vivo biological activity after strengthening the CDC activity.

In example 7, CDC activity was observed upon co-incubation of MDA-MB-468 with the conjugate. To compare the in vivo biological activities of CDC-potentiated, double-epitope antibody-conjugated drugs 97 m-vcMAE and 97 m-E430G-vcMAE, a model of MDA-MB-468 transplantable tumor was constructed using Balb/c Nude female Nude mice. MDA-MB-468 tumor cells were collected in logarithmic growth phase, resuspended in precooled PBS, and the cell suspension was inoculated into the right underarm of 6-8 week female nude mice. The cell inoculation amount of MDA-MB-468 is 5.5X 10⁶Each. The tumor volume of the nude mice is increased to 100mm³To 150mm³In size, nude mice were randomly divided into 4 groups of 5 mice each, PBS blank control group, 97m 5mg/kg, 97 m-vcMAE 5mg/kg and 97 m-E430G-vcMAE ADC 5 mg/kg. The drug concentration of each group is adjusted, and 100 mu L of the drug is administered to each nude mouse by tail vein injection according to the administration dose. The experimental time was calculated starting with the first day of administration, while the body weight and the size of tumor of nude mice were measured every two to three days.

As a result, as shown in FIG. 12, after a single dose of 5mg/kg, the 97m group exhibited a significant tumor suppression effect as compared with the PBS group, and the tumor remained at the size of the administration time within 11 days of administration. In the groups administered with a single dose of 5mg/kg of 97 m-vcMAE and 97 m-E430G-vcMAE, the group administered with 97 m-E430G-vcMAE showed a better antitumor effect, and within one week after administration, tumors on the body surface of all mice in the group administered disappeared, and no recurrence of tumors was observed until the end of the experiment. In the 97 m-vcMAE-administered group, two of 5 mice eventually showed tumor disappearance, but 3 mice showed no tumor disappearance and an increase in the later stage. Combined with the in vitro killing result, this shows that in the tumor cells with effective complement, 97 m-E430G-vcMAE can exert the killing effect of self-micromolecule MMAE and further enhance the anti-tumor effect through CDC reinforcement.

In addition, as can be seen from the body weight data of the mice in fig. 13, the body weight of the tumor-bearing mice in each group showed a tendency to increase in the whole experiment period, indicating that neither the fusion protein nor the conjugate thereof has serious toxic and side effects on the mice in the experiment period.

Sequence listing

<110> Zhejiang university

<120> antibody coupling medicine and application thereof

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 127

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Val Ala Ile Asn Trp Ser Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Gly Tyr Gln Ile Asn Ser Gly Asn Tyr Asn Phe Lys Asp Tyr

100 105 110

Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 2

<211> 381

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gaggtgcagc tggtggagtc cggcggcgga ctggtgcagg ctggaggatc cctgcggctg 60

tcctgtgctg ccagcggcag aaccttctcc agctatgcca tgggctggtt taggcaggcc 120

cctggcaagg agcgggagtt tgtggtggct atcaactggt cctccggcag cacctactac 180

gctgactccg tgaagggccg gttcaccatc agcagggaca acgccaagaa caccatgtac 240

ctgcagatga actccctgaa gcctgaggat accgctgtgt actactgcgc tgccggctat 300

cagatcaata gcggcaacta taacttcaag gactacgagt acgattattg gggccagggc 360

acccaggtga ccgtgtcctc c 381

<210> 3

<211> 124

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly

1 5 10 15

Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr

20 25 30

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ser Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Asp

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Ala Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 4

<211> 372

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

caggtgaagc tggaggagtc tggcggcggc tctgtgcaga ccggcggatc tctgagactg 60

acatgcgctg cttctggaag aacatctaga tcctatggca tgggctggtt tagacaggct 120

cctggaaagg agagagagtt tgtgtctgga atttcttgga gaggagattc tactggctac 180

gctgattctg tgaaaggaag attcacaatt tctagagata atgccaagaa taccgtggac 240

ctgcagatga actctttgaa gcctgaagat acagctatct actactgtgc tgctgctgcc 300

ggctctgctt ggtatggaac cctgtatgaa tatgattatt ggggacaggg cacacaggtg 360

accgtgtctt ct 372

<210> 5

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 6

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggtggtggcg gttcaggcgg aggtggctct ggcggtggcg gatcg 45

<210> 7

<211> 232

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

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

115 120 125

Arg Arg Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asn Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 8

<211> 699

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 60

gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120

acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240

tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300

ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 360

atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420

gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 480

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540

cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 600

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660

tacacgcaga agagcctctc cctgtctccg ggtaaatga 699

<210> 9

<211> 498

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Val Ala Ile Asn Trp Ser Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Gly Tyr Gln Ile Asn Ser Gly Asn Tyr Asn Phe Lys Asp Tyr

100 105 110

Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

130 135 140

Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly Ser Leu

145 150 155 160

Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr Gly Met

165 170 175

Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Gly

180 185 190

Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp Ser Val Lys Gly

195 200 205

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Asp Leu Gln

210 215 220

Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala

225 230 235 240

Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp Tyr Trp

245 250 255

Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp

260 265 270

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

275 280 285

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

290 295 300

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

305 310 315 320

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

325 330 335

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

340 345 350

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

355 360 365

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

370 375 380

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

385 390 395 400

Thr Leu Pro Pro Ser Arg Asn Glu Leu Thr Lys Asn Gln Val Ser Leu

405 410 415

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

420 425 430

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

435 440 445

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

450 455 460

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

465 470 475 480

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

485 490 495

Gly Lys

<210> 10

<211> 1497

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gaggtgcagc tggtggagtc cggcggcgga ctggtgcagg ctggaggatc cctgcggctg 60

tcctgtgctg ccagcggcag aaccttctcc agctatgcca tgggctggtt taggcaggcc 120

cctggcaagg agcgggagtt tgtggtggct atcaactggt cctccggcag cacctactac 180

gctgactccg tgaagggccg gttcaccatc agcagggaca acgccaagaa caccatgtac 240

ctgcagatga actccctgaa gcctgaggat accgctgtgt actactgcgc tgccggctat 300

cagatcaata gcggcaacta taacttcaag gactacgagt acgattattg gggccagggc 360

acccaggtga ccgtgtcctc cggtggtggc ggttcaggcg gaggtggctc tggcggtggc 420

ggatcgcagg tgaagctgga ggagtctggc ggcggctctg tgcagaccgg cggatctctg 480

agactgacat gcgctgcttc tggaagaaca tctagatcct atggcatggg ctggtttaga 540

caggctcctg gaaaggagag agagtttgtg tctggaattt cttggagagg agattctact 600

ggctacgctg attctgtgaa aggaagattc acaatttcta gagataatgc caagaatacc 660

gtggacctgc agatgaactc tttgaagcct gaagatacag ctatctacta ctgtgctgct 720

gctgccggct ctgcttggta tggaaccctg tatgaatatg attattgggg acagggcaca 780

caggtgaccg tgtcttctga gcccaaatct tgtgacaaaa ctcacacatg cccaccgtgc 840

ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac 900

accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 960

gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 1020

aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 1080

caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 1140

gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 1200

accctgcccc catcccggga ggagatgacc aagaaccagg tcagcctgac ctgcctggtc 1260

aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 1320

aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag 1380

ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 1440

gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaatga 1497

Claims (7)

1. An antibody coupling medicine is an antibody coupled with medicine dolastatin and is characterized in that,

the antibody comprises Fc of 9G8 nano antibody, 7D12 nano antibody and IgG1 type antibody, wherein the Fc of the IgG1 type antibody is a double-chain structure,

the 9G8 nano antibody and the 7D12 nano antibody are fused and then fused with the Fc of the IgG1 type antibody, and two Fc chains of a double-chain structure are respectively fused with a molecule of the 9G8 nano antibody and the 7D12 nano antibody,

the amino acid sequence of the 9G8 nano antibody is shown in SEQ ID No.1,

the amino acid sequence of the 7D12 nanometer antibody is used for coupling dolastatin after serine at the 7 th position or the 71 th position is mutated into cysteine by the sequence shown as SEQ ID No.3,

the amino acid sequence of the Fc of the IgG1 type antibody is shown in SEQ ID No.7,

the DAR value of the antibody-coupled drug is 1-2.

2. The antibody-coupled drug of claim 1, wherein the C-terminus of the 9G8 nanobody is fused to the N-terminus of the 7D12 nanobody, and the C-terminus of the 7D12 nanobody is fused to the N-terminus of the Fc.

3. The antibody-coupled drug of claim 2, wherein the 9G8 nm antibody is connected with the 7D12 nm antibody through a flexible connecting peptide, wherein the flexible connecting peptide is (GGGGS) n, and n is a natural number of 1-3.

4. The antibody-coupled pharmaceutical of claim 3, wherein the amino acid sequence of the single chain of the two chains of the antibody in which serine at position 7 or 71 of the 7D12 nanobody is not mutated to cysteine is shown in SEQ ID No. 9.

5. The antibody-coupled pharmaceutical of claim 4, wherein the amino acid sequence of Fc is further mutated from E to G at position 215.

6. A method of preparing an antibody-coupled drug according to any one of claims 1 to 5, comprising the steps of:

(1) the antibody is obtained through recombinant expression and purification,

(2) the reducing agent treatment is used to open the inter-chain disulfide bonds of the antibody, the oxidizing agent is used to reconnect the inter-chain disulfide bonds,

(3) and (3) reacting mc-vc-MMAE with the antibody to enable the maleimide group and the sulfhydryl group to generate Michael addition reaction so as to obtain the antibody coupled drug coupled with the drug.

7. The use of an antibody-coupled drug as claimed in any one of claims 1 to 5 in the preparation of an anti-tumor drug,

the anti-tumor drug is directed against solid tumors which are EGFR positive in tumor type.

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