WO2018000324A1

WO2018000324A1 - Method for introducing antibody into cell

Info

Publication number: WO2018000324A1
Application number: PCT/CN2016/087889
Authority: WO
Inventors: 张军方
Original assignee: 张军方
Priority date: 2016-06-30
Filing date: 2016-06-30
Publication date: 2018-01-04
Also published as: CN107223136B; CN107223136A

Abstract

A method for introducing an antibody into a cell, comprising a fusion expression of a transmembrane peptide and antibody. The antibody can pass through the cell membrane and enter the cell to bind with a target molecule. The antibody also retains a specific antigen-binding property. The method successfully introduced the antibody into the cell using the fusion expressed transmembrane peptide and exerted an intracellular antibody effect. The invention successfully realized a method for performing an intracellular targeted antibody therapy and significantly increased a curative effect.

Description

Method for introducing an antibody into a cell

Technical field

The present invention relates to the field of antibody drug technology, and more particularly to a method for introducing an antibody into a cell by means of a penetrating peptide.

Background technique

In molecular biology, an intrabody refers to an antibody that recognizes a protein in a cell and functions in the cell. Due to the lack of effective means for introducing antibodies into cells, intracellular antibodies are mainly achieved by means of expressing antibody molecules in target cells, and genetic modification techniques are used to appropriately modify antibody molecules to have different subcellular localizations, such as Mitochondria, Golgi, lysosomes or nuclei, which can block certain important processes or interfere with the processing and secretion processes of target molecules, thereby exerting their biological functions. The early interaction between proteins in cells by antibodies is achieved by microinjection, which studies the stability and function of mature antibodies in cells. However, there are few reports on this aspect, and it may be that this technology requires complicated operations. There is therefore an urgent need to develop more widely used techniques for introducing antibody molecules into living cells.

Naturally produced antibodies are derived from B cells and function outside the cell. Currently, antibody drugs used clinically are generally targeted to cell surface targets, which are transported to the target site through the circulatory system, while intracellular antibodies are accessible. An antibody that exerts its action inside a target cell. Naturally produced antibodies are highly complex ordered structures, and the structure of antibodies plays an important role in maintaining antibodies to recognize antigens and causing immune effects. Although antibody molecules can be expressed in different cells, due to the different environments of different types of cells, the resulting antibody structure will eventually differ greatly, or even the antigen will not be recognized at all. Moreover, in clinical applications, the production of intracellular antibodies by means of delivery of antibody genes to target cell expression is limited by the vector. Other methods for introducing intracellular antibodies into cells for gene therapy, including viral transport systems and non-viral transport systems, also face various problems. The viral transport system includes adenoviruses, lentiviruses, and adeno-associated viruses, and has problems such as safety (such as causing oncogene expression) and immune rejection. The use of non-viral transport systems, such as naked DNA, various DNA wrapping techniques, although the safety is improved, but there are other problems, such as poor targeting, turn Low dyeing efficiency and low transient expression of gene expression.

Cell-penetrating peptides (CPPs) are short peptides, usually less than 30 amino acids, which not only have the ability to cross cell membranes, but also serve as vectors to transfer biological macromolecules together into cells. At present, the most recent study is the HIV-I transcriptional activator TAT, which was first discovered by Green et al in 1988. Subsequent studies have shown that the transduction function exists in amino acids 49-57, which is called protein transduction. Domain, PTD), after which many polypeptide molecules with similar transmembrane functions were discovered.

The introduction of hydrophilic substances into living cells by transmembrane peptides has strong application potential in both basic research and therapeutics. Moreover, the goods that are transduced in practice can be unrestricted by size, thereby greatly increasing the range of goods that can be transferred. Transmembrane peptides have many advantages in mediating the entry of biomolecules into cells, as follows: First, the transmembrane peptide transduced cargo is not affected by the molecular weight and its properties, and has a wide range of applications. Second, studies have shown that cell penetrating peptide toxicity It is relatively small and has good clinical application potential; again, cell penetrating peptide can cross various types of tissue cells without significant specificity and can be widely applied to the treatment of different diseases; finally, cell penetrating peptide transmembrane It has strong ability to enter the tissue cells through the mucosa, not limited to intravenous injection, and can be used for oral or nasal mucosal administration. In theory, cell penetrating peptides can carry a wide variety of goods, including: small molecule proteins, peptides, plasmids, nucleic acids, oligonucleotides, liposomes and nanoparticles.

In the technique of introducing an intrabody into a cell, a transmembrane peptide has not been successfully used to introduce an antibody into a cell while maintaining the biological function of the antibody. Therefore, the study of transmembrane peptide-mediated intracellular antibody introduction into cell technology is of great significance and application value, and will also provide new ideas for the study of therapeutic antibodies against intracellular targets.

Summary of the invention

The present invention provides a method for introducing an antibody into a cell, which can successfully introduce a fusion-expressed antibody into a cell using a penetrating peptide, and maintain the biological activity of the antibody unaffected, and the antibody can be used in the preparation of an antibody drug. .

According to a first aspect of the invention, the invention provides a method of introducing an antibody into a cell, by The transmembrane peptide is fused to an antibody, and the antibody can penetrate the cell membrane and enter the cell to bind to the target molecule, and the antibody retains a specific binding function to the antigen.

Further, the above antibodies include intact antibodies as well as antibody fragments.

Further, the transmembrane peptide is linked to the C-terminus of the heavy chain of the above antibody.

Further, the transmembrane peptide is directly linked to the above antibody or mediated by a linker sequence.

Further, the sequence of the above transmembrane peptide is selected from any one of SEQ ID NOS: 5-17.

Further, the transmembrane peptide is a transcriptional activator TAT _(48-60) derived from HIV-I, and the antibody is a monoclonal antibody against the HBx protein, and the transmembrane peptide is linked to the heavy chain of the antibody. The amino acid sequence is SEQ ID NO: 1, and the amino acid sequence of the light chain of the above antibody is SEQ ID NO: 2.

According to a second aspect of the present invention, the present invention provides the use of the transmembrane peptide and antibody fusion expression product obtained in the method of the first aspect for the preparation of an antibody drug.

Further, the above antibody drug is an antitumor drug for treating and/or preventing a tumor.

According to a third aspect of the present invention, the present invention provides a method for introducing an antibody drug conjugate into a cell, wherein the antibody can penetrate the cell membrane and enter the cell to bind to the target molecule by fusion expression of the transmembrane peptide and the antibody. The antibody retains a specific binding function to the antigen, and a drug molecule is coupled to the above antibody.

Further, the above-conjugated drug is a cytotoxic drug or a radionuclide.

Further, the above-conjugated drug is an antitumor drug.

Further, the transmembrane peptide is a transcriptional activator TAT _(48-60) derived from HIV-I, and the antibody is a monoclonal antibody against HBx protein, and the transmembrane peptide is formed by being linked to the heavy chain of the antibody. The amino acid sequence is SEQ ID NO: 1, and the amino acid sequence of the light chain of the above antibody is SEQ ID NO: 2.

Further, the above-conjugated drug molecule is Monomethyl auristatin E (MMAE).

According to a fourth aspect of the present invention, the present invention provides the use of a product obtained by fusion of a transmembrane peptide obtained by a method of the third aspect with an antibody and conjugated with a drug molecule for the preparation of an antibody drug.

The method for introducing an antibody into a cell of the present invention can successfully introduce an antibody into a cell by expressing the transmembrane peptide and the antibody, and can maintain the biological activity of the antibody without being affected, and the antibody can be used for preparation of an antibody drug. in.

DRAWINGS

1 is a reaction kinetics curve of HBx after fusion expression of TAT at different ends of a 6D12 antibody according to an embodiment of the present invention; wherein, the control indicates a 6D12 antibody that does not express TAT, and C:H-TAT indicates fusion to a C-terminus of the heavy chain. C: L-TAT indicates fusion to the C-terminus of the light chain, N:H-TAT indicates fusion to the N-terminus of the heavy chain, and N:L-TAT indicates fusion to the N-terminus of the light chain;

2 is a schematic diagram showing the expression of TAT fusion at the Fc end of an antibody in one embodiment of the present invention;

3 is a diagram showing experimental results of recombinantly expressed TAT-6D12 entering cells in an embodiment of the present invention, using a confocal microscope for immunofluorescence;

4 is a diagram showing the results of an experiment in which several endocytosis inhibitors can block the entry of TAT-6D12 into cells according to an embodiment of the present invention, using a confocal microscope for immunofluorescence;

Figure 5 is a graph showing the results of an experiment of the activity of intracellular TAT-6D12 on HBx in one embodiment of the present invention;

6 is a graph showing experimental results of intracellular colocalization of TAT-6D12 and HBx in an embodiment of the present invention, using a laser confocal microscope;

Figure 7 is a diagram showing the results of an experiment in which TAT-6D12 which enters a cell can degrade its target HBx according to an embodiment of the present invention;

Figure 8 is a diagram showing the results of intracellular colocalization of TAT-6D12 and Trim21 in an embodiment of the present invention, using a laser confocal microscope;

Figure 9 is a graph showing the results of an experiment in which the mutated TAT-6D12 cannot degrade intracellular HBx in one embodiment of the present invention;

Figure 10 is a graph showing the results of a comparison between the transmembrane peptide and the ADC-expressing TAT-3G11-MMAE treatment group and the control group 3G11-MMAE in an embodiment of the present invention, and the treatment group significantly reduces the tumor volume and enhances the antitumor effect. .

detailed description

The present invention will be further described in detail below with reference to the accompanying drawings.

For the first time, the present invention successfully combines a transmembrane peptide with an antibody to form a fusion antibody capable of penetrating the cell membrane and exerting an antibody activity in the cell. The present invention confirmed by specific experiments that a transmembrane peptide is capable of introducing an antibody fused thereto into a cell. Based on the general commonality of various transmembrane peptides and the general commonality of various antibodies, it is known that the transmembrane peptide of the present invention forms a fusion antibody with an antibody and has a transmembrane function and an antibody activity, and is widely applicable to various kinds of wearing. Membrane peptides and antibodies.

Suitable transmembrane peptides according to their source characteristics include, but are not limited to, transmembrane peptides derived from natural proteins, Such as TAT, pVEC and penetratin; model transmembrane peptides, which mimic the structure of transmembrane peptides through artificially synthesized amphipathic alpha-helical structures, such as MAP and (Arg)7; fully artificially designed synthetically Membrane peptides, such as Transportan and MPG, are usually chimeric polypeptides, amphiphilic molecules. Some typical transmembrane peptides and their source and functional peptide sequences useful in the present invention are shown in Table 1.

Table 1 Types of cell penetrating peptides

Suitable antibodies useful in the present invention refer to intact antibodies as well as antibody fragments such as Fab fragments, single-chain Fv (ScFv), bispecific monoclonal antibodies (BsAb), disulfide-stabilized antibodies ( Disulfied-stablized Fv, dsFv), single domain antibody (SDAb). The antibody may be a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody, and the antibody of the present invention is a recombinantly expressed antibody, preferably a recombinantly expressed antibody as a monoclonal antibody. The recombinantly expressed antibody is capable of directly forming a fusion antibody by fusion with a transmembrane peptide by recombinant expression.

The present inventors have found that when the transmembrane peptide is expressed in fusion with an antibody, the transmembrane peptide is linked to the heavy chain C-terminus of the antibody and is linked to other parts of the antibody (for example, the heavy chain N-terminus, the light-chain C-terminus, and the light-chain N-terminus, etc.). Better ability to keep antibody activity unaffected. Therefore, it is preferred that the transmembrane peptide is linked to the heavy chain C-terminus of the antibody.

The link between the transmembrane peptide and the antibody may be a direct linkage or a linkage mediated by a linker. A typical but non-limiting sequence of linkages such as (GGGS)4. Directly linking the transmembrane peptide to the antibody is preferred without affecting the activity of the antibody. In some cases, direct attachment between the transmembrane peptide and the antibody affects the activity of the antibody, and this effect can be abolished by ligation of the linker. In such cases, it is preferred that the transmembrane peptide and the antibody are linked by a sequence. . Specifically, the manner of attachment can be selected depending on the specific antibody characteristics and the type and characteristics of the transmembrane peptide employed.

In one embodiment of the invention, the transmembrane peptide is a transcriptional activator TAT _(48-60) derived from HIV-I, and the antibody is a monoclonal antibody against the HBx protein, which is an antibody obtained by recombinant expression. TAT _(48-60) and the monoclonal antibody against the HBx protein form a fusion antibody by recombinant expression, the two are directly linked together, and preferably the TAT _{(48-60) is} ligated to the weight of the monoclonal antibody against the HBx protein. Chain C end. In the preferred embodiment, the amino acid sequence formed by the transmembrane peptide linked to the C-terminus of the heavy chain of the antibody is SEQ ID NO: 1 (see Sequence Listing), and the amino acid sequence of the light chain of the antibody is SEQ ID NO: 2 (see Sequence table).

The fusion antibody of the present invention can be directly used as an antibody drug, and can also be used in the preparation of a drug. The antibody drug carries a penetrating peptide, so it can penetrate the cell membrane into the cell, and the antibody can target the intracellular target, thereby improving the targeting and therapeutic effect of the treatment. Such an antibody drug is preferably an antitumor drug for treating and/or preventing a tumor. Due to the lack of targeting in tumor therapy and the negative killing problem to normal tissues, the fusion antibody of the present invention can penetrate the cell membrane into the tumor cells, thereby overcoming the difficulty that the antibody drug is difficult to enter the cell, thereby targeting the intracellular target. Molecular tumors have obvious curative effects.

In further studies, the fusion antibodies of the invention can be combined with current antibody drug conjugates (ADCs) to form antibody drug conjugates with a penetrating peptide, the antibody drug conjugates comprising A fusion antibody and a conjugated drug, wherein the fusion antibody comprises a transmembrane peptide and an antibody fused together, and the fusion antibody is capable of penetrating the cell membrane into the cell to bind to the target molecule. On the one hand, the antibody can exert specific binding to the target molecule, thereby eliminating the target molecule or eliminating its influence; on the other hand, the coupled drug can also exert its therapeutic effect targeted, and improve the targeting and therapeutic effect. The conjugated drug may be a cytotoxic drug or a radionuclide or the like, which may be an antitumor drug for treating and/or preventing a tumor.

In one embodiment of the invention, the antibody drug conjugate with a penetrating peptide is formed, comprising a transcriptional activator TAT _(48-60) derived from HIV-I as a penetrating peptide, a single against the HBx protein. The antibody was cloned as an antibody, and Monomethyl auristatin E (MMAE) was used as a coupled drug molecule. Wherein, the transmembrane peptide and the antibody form a fusion antibody in a directly linked form by recombinant expression, and the coupled drug is coupled to the fusion antibody by chemical coupling, preferably by EDC after activation (1-Ethyl- 3-(3A-dimethylaminopropyl)-carbodiimide hydrochloride) is coupled together.

The fusion antibody of the present invention comprises a transmembrane peptide and an antibody fused together, can be successfully introduced into a cell, and the biological activity of the antibody is not affected, and the fusion antibody can be used in the preparation of an antibody drug.

The present invention will be further described in conjunction with the specific embodiments. It is to be understood that the embodiments are merely illustrative and not restrictive.

Example 1: Transmembrane peptide TAT _(48-60) and anti-HBx protein antibody form fusion antibody TAT-6D12

In this example, TAT _(48-60) (specific amino acid sequence: GRKKRRQRRRPPQ) was added to the heavy chain C-terminus (C:H-TAT) of the recombinantly expressed 6D12 antibody, which was lightly expressed by fusion expression. There are four ways of chain C (C: L-TAT), heavy chain N (N: H-TAT) or light chain N (N: L-TAT).

First, it was evaluated whether the reactivity of the modified antibody to HBx changed. The kinetic curves of each fusion antibody were evaluated by indirect chemiluminescence immunoassay using recombinant HEBx protein-coated plates at different concentrations. The results shown in Figure 1 showed that TAT _{(48-60) was} added to the heavy or light chain N. The end causes the complete disappearance of the antibody to the HBx protein reactivity, while the C-terminus of the light chain, while retaining the reactivity with the HBx protein, the reactivity decreased by nearly two orders of magnitude compared to the unmodified antibody, only Fusion expression of TAT _(48-60) to the heavy chain C-terminus of the antibody (C:H-TAT), the Fc end of the antibody, did not affect the reactivity of 6D12 to HBx. A schematic diagram of the expression of TAT fusion at the Fc end of the antibody is shown in Figure 2.

Example 2: Transmembrane effect of fusion-expressed TAT-6D12 antibody

The recombinant plasmid constructed by the 6D12Fc-end fusion TAT _(48-60) was transfected into CHO cells for expression, and the antibody secreted into the culture supernatant was purified by Protein A affinity chromatography to carry out biological function analysis. Recombinantly expressed TAT-6D12 and unmodified 6D12 were separately added to the HuH7 cell culture medium at a final concentration of 0.3 mg/ml, incubated for 12 h, and then immunofluorescence was used to analyze whether TAT-6D12 could enter the cell by confocal microscopy. . The results are shown in Figure 3. The recombinantly expressed TAT-6D12 can enter the HuH7 cells significantly (note: the fluorescence is green, the gray color after transformation), the entry efficiency is close to 100%, very efficient, and it is diffusely distributed. Antibodies are distributed in the cytoplasm. Furthermore, in cell lines of different species and tissues, such as human hepatoma cell line HepG2, murine hepatoma cell line Hepa1-6, human embryonic kidney cell line HEK-293, human lung cancer cell line A549, etc., it was found that TAT-6D12 can be used. Effectively traversing the cell membrane into the cell (results not shown). It is suggested that recombinant expression of TAT-6D12 can enter the cell non-specifically and efficiently.

Example 3: Transmembrane mechanism of fusion-expressed antibody TAT-6D12

The mechanism of TAT-mediated transmembrane has not been thoroughly studied. The latest research results show that endocytosis plays an important role in the internalization process. It is generally believed that the macromolecular transport of TAT is dependent on endocytosis. . Therefore, several commonly used endocytosis inhibitors were used to investigate whether TAT-6D12 can be blocked from entering the cell. The mechanism of action of these commonly used endocytosis inhibitors is shown in Table 2. These inhibitors were first added to HuH7 cell culture medium, then the final concentration of 0.3mg/ml TAT-6D12 was added to the cell culture medium, incubated for 12h, and then immunofluorescence was used to analyze TAT-6D12 into the cells by confocal microscopy. Whether the process is blocked. As shown in Fig. 4, three inhibitors, Filipin, Cytochalasin D (Cyto D) and Chloroquine (Chlo), can significantly block the entry of TAT-6D12 into cells, methyl -β-cyclodextrin (Mβ-β-cyclodextrin, MβCD) also inhibited the internalization of TAT-6D12 to a certain extent. In summary, TAT-6D12 crosses the cell membrane into the cell and is a process dependent on endocytosis, and clathrin-mediated endocytosis (CME), clathrin-independent endocytosis (CIE) Four modes of clathrin-independent endocytosis, macropocycytosis and Lipid raft may be involved in the TAT-mediated antibody entry process.

Table 2 Several commonly used endocytosis inhibitors and mode of action

(CCVs, clathrin-coated vesicles, clathrin-coated vesicles)

Example 4: TAT-6D12 entering cells still has reactivity against HBx

To investigate whether TAT-6D12 internalized into cells also has reactivity against HBx, TAT-6D12 at a final concentration of 0.3 mg/ml was incubated with HuH7 cells for 0.5 h, 2 h, 4 h, 6 h, 8 h, 10 h, respectively. For 12 h, it was washed 5 times with PBS, and the cells were lysed with non-denaturing lysate DDM, and the cell lysate was added to an HBx-coated ELISA plate to examine its affinity for HBx. The results shown in Figure 5 show that TAT-6D12 entering the cell still has good reactivity against HBx (note: the fluorescence is green and the gray color after transformation), suggesting that TAT-6D12 can maintain its activity in the cell.

Example 5: TAT-6D12 entering cells can recognize its target HBx

In order to investigate whether TAT-6D12 internalized into cells can recognize HBx protein in cells, it is investigated by immunofluorescence whether they are co-localized in cells. As shown in Figure 6, green fluorescence represents HBx protein (note: upper right image, fluorescence is green, converted to grayish white), red fluorescence represents TAT-6D12 (note: lower left, fluorescence is red, converted to grayish white) By taking a laser confocal microscope and then merging the pictures together, the result shows that the green fluorescence overlaps with the red fluorescence (note: the lower right picture, the fluorescence is green and red, and turned into grayish white), indicating intracellular TAT. -6D12 forms a complex with HBx protein, and TAT-6D12 entering the cell can recognize its target HBx.

Example 6: TAT-6D12 entering cells can degrade its target HBx

Western blot was used to detect whether TAT-6D12 entering the cell degraded its target HBx, and the internal reference protein was tubulin (TUBLIN), which was used to indicate that the total amount of protein used was consistent. The results shown in Figure 7 show that the amount of HBx in the TAT-6D12 treated group was significantly less than that in the 6D12 treated group and the control antibody treated group, indicating that TAT-6D12 entering the cell could degrade its target HBx.

Example 7: Interaction of TAT-6D12 into TRIM21 into cells

Recent studies have shown that when an adenovirus infects a cell, antibodies adsorbed on the surface of the adenovirus are brought into the cell together and can bind to the Fc receptor 21, the antibody Fc receptor in the cytoplasm, to stimulate intracellular immunity. React to destroy the virus. As an Fc receptor present inside the cell, TRIM21 binds to IgG more strongly than all Fc receptors known to date. TRIM21 recognizes antibody molecules adsorbed on the surface of the virus and then catalyzes the formation of K48 and KK63 ubiquitinated chains by the function of its E3 ubiquitination ligand. The catalytically formed K48 is able to rapidly eliminate viral particles within a few hours through the AAA ATPase VCP and proteasome degradation systems. At the same time, the K63 chain catalyzed by TRIM21 activates downstream innate immune signaling pathways, such as NFκB, AP-1 and IRF3/5/7, which results in the up-regulation of antiviral molecules such as cytokines and chemokines, upregulation of NKG2D ligands. And MHC class II molecules, leaving the cells in an antiviral state. It is hypothesized that the mechanism of action of the genetically engineered TAT-6D12 antibody, which acts in the cell, is likely to pass a similar pathway, and therefore this hypothesis is validated. TRIM21 is currently the only reported antibody Fc receptor in the cytoplasm, and it is likely that the engineered antibody that can enter the cell will function through this Fc receptor. In order to study whether there is an interaction between TRIM21 and TAT-6D12 entering the cell, by immunofluorescence detection method, Confocal microscopy was used to see if they were co-localized within the cell. The results shown in Figure 8 indicate that the green signal representing TAT-6D12 (note: the upper left image, the fluorescent signal is green, the gray color after conversion) and the red signal representing TRIM21 (note: the upper right image, the fluorescent signal is originally red, The grayish whites after the transformation overlap, showing an interaction between TAT-6D12 and TRIM21 to form a complex.

Example 8: The mutated TAT-6D12 antibody cannot degrade intracellular HBx protein

In order to further confirm that the antibody plays a role in the cell, it is dependent on the pattern of TRIM21 molecule. First, the TAD-6D12 clone (TAT-6D12-CH3 ^- ) with CH3 deletion was constructed and then expressed and purified. The results showed that TAT-6D12-CH3 ^- and HBx There was no significant change in binding activity, indicating that the Fc-terminal CH3 region of the antibody plays an important role in mediating intracellular immunity.

To further evaluate the role of TAT-6D12 in cells, depending on the interaction with TRIM21, H433A, N434A, H435A, and TRIM21 in the antibody by point mutations in hot-spots that bind to antibodies. Mediates the role of intracellular immunity. The results shown in Fig. 9 show that the mutated TAT-6D12 cannot form a complex with TRIM21 and thus cannot degrade the HBx protein in the cell.

Example 9: Transmembrane peptides improve the therapeutic effect of ADC

In recent years, antibody drug conjugates (ADCs) have been developed, in which cytotoxic drugs or radionuclides are coupled to antibodies and brought into target cells, thereby achieving efficient and specific killing of tumor cells. The development direction of clinical application of a new generation of antibody technology. Although ADC has achieved great success and attracted wide attention, ADC development technology requires high complexity and complex process. The low efficiency of ADC phagocytosis by target cells is the bottleneck of clinical application. The data shows that there are only a few ADC molecules. It can enter tumor cells, and the efficiency of phagocytosis is crucial for the improvement of anti-tumor efficacy.

This study found that fusion of transmembrane peptides with antibodies can significantly improve the internalization efficiency of antibodies. This technology can be applied to the development of ADC drugs, improve internalization efficiency and killing ability to tumors. The data in this study show that the penetrating peptide is not targeted. However, after the fusion of the antibody against the HBs (3G11) and the transmembrane peptide (TAT), the antibody can be introduced into the cell on the one hand, and the transmembrane can be improved on the other hand. The targeting of the peptide, TAT-3G11, was more efficient in HBV-positive liver cancer cells (Hepa 1-6-N10). Since the antibody does not have significant cytotoxicity after entering the cell, in order to improve the lethality against the liver cancer cells, the previously studied small molecule toxin MMAE (Monomethyl Auristain E) is coupled with TAT-3G11, namely TAT-3G11-MMAE. Wherein, the amino acid sequence formed by fusion of TAT with the heavy chain C-terminus of the 3G11 antibody is SEQ ID NO: 3 (see the Sequence Listing), and the amino acid sequence of the light chain of the 3G11 antibody is SEQ ID NO: 4 (see Sequence Listing).

In vitro cell assays indicate that TAT-3G11-MMAE is positive for HBV-positive liver cancer cells (Hepa 1-6-N10) has more significant cytotoxicity, and subsequent animal experiments (Fig. 10) also confirmed that the conjugate has a significant therapeutic effect on tumors formed by Hepa 1-6-N10. The above experimental results show that the fusion expression of transmembrane peptide and ADC can improve the internalization efficiency of target cells and improve its anti-tumor effect. It has important guiding significance for the development strategy of ADC drugs.

The above is a further detailed description of the present invention in connection with the specific embodiments, and the specific embodiments of the present invention are not limited to the description. It will be apparent to those skilled in the art that the present invention may be made without departing from the spirit and scope of the invention.

Claims

A method for introducing an antibody into a cell, characterized in that, by fusion expression of a transmembrane peptide and an antibody, the antibody is capable of penetrating a cell membrane and entering a cell to bind to a target molecule, and the antibody retains a specific binding function to the antigen. .
The method of claim 1 wherein said antibody comprises an intact antibody and an antibody fragment.
The method of claim 1 wherein said transmembrane peptide is linked to the heavy chain C-terminus of said antibody.
The method according to claim 3, wherein the transmembrane peptide is directly linked to the antibody or mediated by a linker sequence.
The method according to claim 1, wherein the sequence of the transmembrane peptide is selected from any one of SEQ ID NOS: 5-17.
The method according to claim 5, wherein said transmembrane peptide is a transcriptional activator TAT (48-60) derived from HIV-I, said antibody is a monoclonal antibody against HBx protein, and said The amino acid sequence formed by the transmembrane peptide linked to the heavy chain of the antibody is SEQ ID NO: 1, and the amino acid sequence of the light chain of the antibody is SEQ ID NO: 2.
Use of the transmembrane peptide and antibody fusion expression product obtained in the method of claim 1 for the preparation of an antibody drug.
The use according to claim 7, wherein the antibody drug is an antitumor drug for treating and/or preventing a tumor.
A method for introducing an antibody drug conjugate into a cell, characterized in that, by fusion expression of a transmembrane peptide and an antibody, the antibody is capable of penetrating a cell membrane and entering a cell to bind to a target molecule, the antibody remaining to the antigen The binding function is specific, and a drug molecule is coupled to the antibody.
The method of claim 9 wherein said conjugated drug is a cytotoxic drug or a radionuclide.
The method of claim 9 wherein said conjugated drug is an anti-tumor drug.
The method according to claim 9, wherein said transmembrane peptide is a transcriptional activator TAT (48-60) derived from HIV-I, said antibody is a monoclonal antibody against HBx protein, and said The amino acid sequence formed by the transmembrane peptide linked to the heavy chain of the antibody is SEQ ID NO: 1, and the amino acid sequence of the light chain of the antibody is SEQ ID NO: 2.
The method of claim 9 wherein said coupled drug molecule is methyl Auristatin E.
Use of a transmembrane peptide obtained by the method of claim 9 in fusion with an antibody and conjugated with a drug molecule for the preparation of an antibody drug.
The use according to claim 14, wherein the antibody drug is an antitumor drug for treating and/or preventing a tumor.