WO2021083409A1

WO2021083409A1 - Anti-cd97 monoclonal antibody, variable region and constant region sequences thereof, and anti-brain tumor application

Info

Publication number: WO2021083409A1
Application number: PCT/CN2020/137694
Authority: WO
Inventors: 尹金龙; 师冰洋; 吴海刚
Original assignee: 河南大学
Priority date: 2019-10-29
Filing date: 2020-12-18
Publication date: 2021-05-06
Also published as: CN110627903B; CN110627903A

Abstract

An anti-CD97 monoclonal antibody, variable region and constant region sequences thereof, and application thereof. The variable region sequences of the anti-CD97 monoclonal antibody comprise nucleotide sequences such as a heavy chain sequence as represented by SEQ ID NO: 1 and a light chain sequence as represented by SEQ ID NO: 2. The anti-CD97 monoclonal antibody prepared with the variable region sequences is able to bind specifically to CD97, and may be used in the preparation of medication for treating diseases (such as brain tumor) related to excessive or uncontrolled expression of CD97.

Description

Anti-CD97 monoclonal antibody, its variable region and constant region sequence and its anti-brain tumor application

Cross-references to related applications

This application claims the priority of the Chinese patent application filed with the Chinese Patent Office on October 29, 2019, with the application number 201911038259.4, titled "anti-CD97 monoclonal antibody, its variable and constant region sequences and applications", all of which The content is incorporated in this application by reference.

Technical field

It relates to the field of biotechnology, in particular to an anti-CD97 monoclonal antibody with specific binding to CD97 biological activity, its variable region and constant region sequences, and its application against brain tumors.

Background technique

Malignant glioma is the most common type of brain tumors in clinic, and its incidence rate is as high as 60% in brain tumors. Among them, glioblastoma multiforme (Glioblastoma, GBM) is the most malignant type of malignant glioma. The incidence of malignant glioma in my country is 1-4/100. Glioma stem cells (GSC) have the characteristics of general stem cells, that is, self-renewal ability and differentiability. They play a decisive role in the invasion, drug resistance, and radiation resistance of glioma.

CD97 is a dimeric glycoprotein containing seven transmembrane structures. Its molecular weight is between 75 and 85kDa. It is mainly expressed continuously on the surface of macrophages/monocytes and granulocytes, while high levels have not been found in other blood cells. expression. The biological function of CD97 is mainly concentrated in the rheumatoid synovium causing inflammation (Arthritis and Rheumatism, 1999, 42(4): 650-658.), as an indicator of thyroid cancer dedifferentiation (Cancer Research, 1997, 57(9) ): 1798-1806.), regulating the adhesion and invasiveness of epidermal cells (Blood, 2005, 105(7): 2836-2844.) and so on. According to the analysis of clinical samples, high expression of CD97 in glioma can significantly enhance its aggressiveness and reduce the survival rate of patients (PloS One, 2013, 8(4): e62765.), showing that it is a glioma Characteristics of potential biological targets of stem cells.

Antibody is a protein molecule secreted by lymphatic B cells, which is composed of two identical heavy and light chains. The light and heavy chains contain variable and constant regions. Through the specific binding of the antibody and the antigen, the activity of the antigen molecule can be neutralized, and the purpose of disease treatment can be finally realized. However, there is currently no anti-CD97 monoclonal antibody with therapeutic properties.

Summary of the invention

The purpose of this application is to provide anti-CD97 monoclonal antibodies, their variable region and constant region sequences and applications. The anti-CD97 monoclonal antibody prepared from the variable region sequence described in this application can specifically bind to CD97, and can be used for the preparation of drugs for treatment of diseases (such as brain tumors) related to excessive and out-of-control expression of CD97.

This application provides the variable region sequence of the anti-CD97 monoclonal antibody, the variable region sequence includes the nucleotide sequence of the heavy chain shown in SEQ ID NO: 1 and the nucleotide sequence of the heavy chain shown in SEQ ID NO: 2 The sequence of the light chain.

The application also provides the constant region sequence of the anti-CD97 monoclonal antibody, the constant region sequence includes a heavy chain with a nucleotide sequence as shown in SEQ ID NO: 3 and a nucleotide sequence as shown in SEQ ID NO: 4 Light chain sequence.

The present application also provides an anti-CD97 chimeric antigen receptor CD97-scfv based on the variable region sequence of the above technical solution. The nucleotide sequence of the CD97-scfv is shown in SEQ ID NO: 5.

Optionally, the amino acid sequence of the CD97-scfv is the sequence shown in SEQ ID NO: 6.

The application also provides T lymphocytes containing the anti-CD97 chimeric antigen receptor CD97-scfv described in the above technical solution.

This application also provides an anti-CD97 monoclonal antibody prepared based on the variable region sequence described in the above technical solution, the constant region sequence described in the above technical solution, or the anti-CD97 chimeric antigen receptor CD97-scfv described in the above technical solution.

This application also provides the aforementioned variable region sequences, the aforementioned constant region sequences, the aforementioned anti-CD97 chimeric antigen receptor CD97-scfv, the aforementioned T lymphocytes or the aforementioned anti-CD97 monoclonal antibodies in the preparation of prevention or treatment. The application of drugs for diseases caused by overexpression of CD97 and diseases caused by uncontrolled expression of CD97.

Optionally, the aforementioned diseases include brain tumors.

Optionally, the above-mentioned disease is malignant glioma.

Optionally, the above disease is astroblastoma, astrocytoma, glioblastoma multiforme, oligodendroglioma, neuroblastoma, ependymoblastoma or ependymal cell tumor.

The above variable region sequence, the above constant region sequence, the above anti-CD97 chimeric antigen receptor CD97-scfv, the above T lymphocytes or the above anti-CD97 monoclonal antibody inhibit the proliferation or decrease of glioma stem cells Application at the level of glioma stem cells.

Optionally, the above-mentioned glioma stem cells are at least one of the following glioma stem cells: glioma stem cell line 83, glioma stem cell line CSC2, and glioma stem cell line 528.

The above-mentioned variable region sequence, the above-mentioned constant region sequence, the above-mentioned anti-CD97 chimeric antigen receptor CD97-scfv, the above-mentioned T lymphocytes or the above-mentioned anti-CD97 monoclonal antibody are used in the preparation of inhibitors of glioma stem cells Applications.

The above-mentioned variable region sequence, the above-mentioned constant region sequence, the above-mentioned anti-CD97 chimeric antigen receptor CD97-scfv, the above-mentioned T lymphocytes or the above-mentioned anti-CD97 monoclonal antibody can prevent or treat diseases caused by overexpression of CD97 The application of diseases caused by out-of-control CD97 expression in at least one of the following diseases:.

Diseases caused by overexpression of CD97 and uncontrolled expression of CD97. Uncontrolled expression also includes protein misfolding, abnormal mutations and so on. Such as: liver cancer, pancreatic cancer, thyroid cancer, kidney cancer, ovarian cancer, bowel cancer, breast cancer, etc. The application also provides a medicine for treating diseases caused by overexpression of CD97 or uncontrolled expression of CD97, which includes the above-mentioned variable region sequence, the above-mentioned constant region sequence, the above-mentioned anti-CD97 chimeric antigen receptor CD97-scfv, The above-mentioned T lymphocytes or the above-mentioned anti-CD97 monoclonal antibody.

Optionally, the above-mentioned medicine also includes pharmaceutically acceptable additives or excipients.

Optionally, the dosage form of the aforementioned pharmaceutical composition is selected from the group consisting of tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections, sprays or injections.

The application also provides a pharmaceutical composition, which includes the above-mentioned variable region sequence, the above-mentioned constant region sequence, the above-mentioned anti-CD97 chimeric antigen receptor CD97-scfv, the above-mentioned T lymphocyte or the above-mentioned anti-CD97 single Cloned antibodies.

Optionally, the aforementioned pharmaceutical composition further includes a combination drug/therapy, and the combination drug/therapy is at least one of the following drugs:

Chemotherapeutics, radiotherapy, photosensitizers, photothermal agents, immunotherapy, thyroxine receptor agonists, antagonists and function modifiers, sodium-glucose cotransporter 2 inhibitors.

Application of the above-mentioned medicine or pharmaceutical composition in the prevention or treatment of diseases caused by overexpression of CD97 or uncontrolled expression of CD97.

This application provides the constant region and variable region sequences of the anti-CD97 monoclonal antibody. Compared with other traditional biological targets, CD97 is a specific biological target closely related to the invasiveness of glioma. By inhibiting its activity, it can reduce the invasiveness of glioma stem cells, inhibit tumor proliferation and increase Survival rate of patients. The anti-CD97 monoclonal antibody prepared from the variable region sequence provided in this application can specifically bind to CD97, and can be used for the preparation of drugs for treatment of diseases (such as brain tumors) related to excessive and out-of-control expression of CD97. The test results show that interference or silencing of CD97 protein can significantly inhibit the proliferation of glioma stem cells.

Description of the drawings

Figure 1 is a schematic diagram of the CD97 monoclonal antibody light chain and heavy chain plasmid provided by this application, wherein Figure 1-1 is a schematic diagram of the CD97 monoclonal antibody light chain, and Figure 1-2 is a schematic diagram of the CD97 monoclonal antibody heavy chain plasmid;

Figure 2 is a graph of the results of the CD97 antibody and CD97 protein binding capacity test provided by this application, in which Figure 2-1 is the antigen-antibody binding capacity test result, and Figure 2-2 is the antigen-antibody binding capacity test fitting result;

Figure 3 shows that the proliferation of glioma stem cell lines is significantly inhibited after silencing CD97. Figure 3A is a flow cytometry chart showing the efficiency of CD97 interfering RNA transfection into glioma stem cell line 83; Figure 3B shows the protein Western blot (WB) results; Figure 3C shows the Western blot (WB) results; Figure 3D shows the inhibition of proliferation of glioma stem cell line 83 when CD97 is silenced; Figure 3E shows the effect of CD97 silence on the glioma stem cell line CSC2 Proliferation inhibition results; Figure 3F shows the proliferation inhibition results of glioma stem cell line 528 by CD97 being silenced.

Detailed ways

This application preferably uses the pUC plasmid for the expression of the light chain and the heavy chain. As shown in Figure 1, the pUC plasmid JK4-PL (Figure 1-1) expresses antibody light chain fragments, and the expression site ranges from KnpI (1486) to Xba I ( 2193). The pUC plasmid JK4-P-H (Figure 1-2) expresses antibody heavy chain fragments, and the expression site ranges from KnpI (1486) to Xba I (2880).

The application also provides an anti-CD97 chimeric antigen receptor CD97-scfv based on the variable region sequence of the above technical solution, and the nucleotide sequence of the CD97-scfv is shown in SEQ ID NO: 5:

Among them, the underlined front part (positions 1 to 321) is the light chain variable region, the non-tilted and underlined (positions 322 to 375) are linker, and the underlined and inclined (positions 376 to 735) ) Is the variable region of the heavy chain.

In this application, the amino acid sequence of the CD97-scfv is as shown in SEQ ID NO:6.

Among them, the underlined front part (position 1 to 107) is the light chain variable region, the non-tilted and underlined (position 108 to 125) is the linker, and the underlined and inclined (position 126 to 245) ) Is the variable region of the heavy chain.

This application also provides an anti-CD97 monoclonal antibody prepared based on the aforementioned variable region sequence, the aforementioned constant region sequence, or the aforementioned anti-CD97 chimeric antigen receptor CD97-scfv.

Optionally, an anti-CD97 monoclonal antibody prepared based on the aforementioned variable region sequence or the aforementioned anti-CD97 chimeric antigen receptor CD97-scfv.

The variable region sequences of the above technical solutions, the constant region sequences of the above technical solutions, the anti-CD97 chimeric antigen receptor CD97-scfv of the above technical solutions, the above T lymphocytes or the above anti-CD97 monoclonal antibodies are used in the preparation of prevention or treatment Application in medicine for diseases caused by overexpression of CD97 and diseases caused by out-of-control.

In this application, the diseases include brain tumors.

Optionally, the aforementioned brain tumor may be a primary or secondary brain-like tumor, and the primary brain-like tumor may be an intramedullary or extramedullary tumor. Intramedullary tumors can be gliomas, neurocytomas, and germ-like stromal tumors, and extramedullary tumors can be stromal tumors, epithelial tumors, teratomas, and pineal tumors.

Optionally, the above-mentioned disease is malignant glioma.

Optionally, the above-mentioned malignant glioma may be astroblastoma, astrocytoma, glioblastoma multiforme, oligodendroglioma, neuroblastoma, ependymoma , Ependymoma, etc.

Optionally, the aforementioned disease is glioblastoma multiforme.

Optionally, the above-mentioned inhibiting the proliferation of glioma stem cells includes, but is not limited to, slowing, stagnating or stopping the proliferation of glioma stem cells.

It should be noted that in other embodiments, the glioma stem cells are not limited to the above-mentioned glioma stem cells.

The dosage form of the above inhibitor is selected from the group consisting of tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections, sprays or injections.

The aforementioned inhibitors include, but are not limited to, competitive inhibitors, non-competitive inhibitors, anti-competitive inhibitors, and irreversible inhibitors of glioma stem cells.

The above-mentioned variable region sequence, the above-mentioned constant region sequence, the above-mentioned anti-CD97 chimeric antigen receptor CD97-scfv, the above-mentioned T lymphocytes or the above-mentioned anti-CD97 monoclonal antibody can prevent or treat diseases caused by overexpression of CD97 , Application in diseases caused by out-of-control expression of CD97.

The application also provides a medicine for treating diseases caused by overexpression of CD97 or uncontrolled expression of CD97, which includes the above-mentioned variable region sequence, the above-mentioned constant region sequence, the above-mentioned anti-CD97 chimeric antigen receptor CD97-scfv, and the above-mentioned anti-CD97 chimeric antigen receptor CD97-scfv. T lymphocytes or the aforementioned anti-CD97 monoclonal antibody.

The chemotherapeutic drugs can be selected from alkylating agents, antimetabolites, anti-cancer antibiotics, plant-based chemotherapeutics, hormones, immune preparations, etc.

The photosensitizer can be an ionic photoinitiator or a free radical photoinitiator.

Application of the above-mentioned medicine or pharmaceutical composition in the prevention or treatment of diseases caused by overexpression of CD97 and diseases caused by uncontrolled expression of CD97.

Thyroxine receptor agonists include, but are not limited to, thyroxine hormone beta receptor agonists.

In the following, the anti-CD97 monoclonal antibody, its variable region and constant region sequences and applications described in this application will be further described in detail with reference to specific examples. The technical solutions of this application include but are not limited to the following examples.

Example 1

The binding constant of CD97-His (11280-H08H) and CD97 antibody (Mouse Mab) is measured using the ProteOn XPR36 biosensor, the chip uses GLC (Bio-Rad, Hercules, CA), and the temperature is 25°C. CD97 (170RU) is coupled to the ligand channel using a coupling agent (400mM EDC and 100mM SNHS mixed 1:1, diluted 1/500), time is 3 minutes, the concentration is 15μg/mL, the buffer solution is 10mM acetic acid Sodium solution (pH=4.5), and finally 1M ethanolamine hydrochloric acid solution was used to seal the remaining activated sites. The surface of the chip is washed with IgG diluted buffer solution and reprocessed. The experimental mobile phase uses PBST (pH=7.4).

The purified CD97 mouse antibody was injected within 3 minutes through the "one-shot kinetic" mode, and tested at four concentrations of 0.8333nM, 1.67nM, 3.33nM, and 6.67nM. The results of the CD97 antibody and CD97 protein binding capacity test are shown in Figure 2 and Table 1. Figure 2-1 is the antigen-antibody binding capacity test result, and Figure 2-2 is the antigen-antibody binding capacity test fitting result (Figure 2) The curve from bottom to top corresponds to the four concentrations of 0.8333nM, 1.67nM, 3.33nM, and 6.67nM). As can be seen from Figure 2, the monoclonal CD97 antibody obtained from this sequence has excellent binding ability to the CD97 antigen molecule.

Interfering with or silencing CD97 protein can significantly inhibit the proliferation of glioma stem cells, and the results are shown in Figure 3, where Figure 3A is flow cytometry to characterize the efficiency of CD97 interfering RNA transfection into glioma stem cell line 83; Figure 3B shows the result of Western blot (WB), which shows that CD97 was silenced after transfection of CD97 interfering RNA with the glioma stem cell line CSC2; Figure 3C shows the result of Western blot (WB), which shows that the glioma stem cell line 528 is transfected After CD97 interfering RNA, CD97 was silenced; Figure 3D shows that silencing CD97 can significantly inhibit the proliferation of glioma stem cell line 83; Figure 3E shows that CD97 silence can significantly inhibit the proliferation of glioma stem cell line CSC2; Figure 3F It shows that silencing CD97 can significantly inhibit the proliferation of glioma stem cell line 528.

Table 1. Test results of binding ability of CD97 antibody and CD97 protein

The above are only the preferred embodiments of this application. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of this application, several improvements and modifications can be made, and these improvements and modifications are also Should be regarded as the scope of protection of this application

Industrial applicability

The anti-CD97 monoclonal antibody prepared by the variable region sequence of the application can specifically bind to CD97, and can be used for the preparation of drugs for the treatment of diseases related to excessive expression of CD97 and out of control. T lymphocytes containing the anti-CD97 chimeric antigen receptor CD97-scfv can be used to prevent or treat diseases caused by excessive CD97 expression and out-of-control CD97 expression, including a series of diseases such as brain tumors. This application also provides a pharmaceutical composition comprising anti-CD97 chimeric antigen receptor CD97-scfv or anti-CD97 monoclonal antibody. The pharmaceutical composition also includes a combination drug/therapy for the prevention or treatment of CD97 overexpression and CD97 Diseases caused by uncontrolled expression.

Claims

The variable region sequence of an anti-CD97 monoclonal antibody, characterized in that the variable region sequence includes a nucleotide sequence as shown in SEQ ID NO: 1 and a heavy chain as shown in SEQ ID NO: 2 The sequence of the light chain.
The constant region sequence of the anti-CD97 monoclonal antibody is characterized in that the constant region sequence includes the heavy chain with the nucleotide sequence shown in SEQ ID NO: 3 and the light chain with the nucleotide sequence shown in SEQ ID NO: 4 Chain sequence.
An anti-CD97 chimeric antigen receptor CD97-scfv based on the variable region sequence of claim 1, wherein the nucleotide sequence of the CD97-scfv is shown in SEQ ID NO: 5.
The anti-CD97 chimeric antigen receptor CD97-scfv according to claim 3, wherein the amino acid sequence of the CD97-scfv is as shown in SEQ ID NO: 6.
T lymphocytes comprising the anti-CD97 chimeric antigen receptor CD97-scfv of claim 3 or 4.
An anti-CD97 monoclonal prepared based on the variable region sequence of claim 1, the constant region sequence of claim 2, or the anti-CD97 chimeric antigen receptor CD97-scfv of any one of claims 3-4 Antibody.
The variable region sequence of claim 1, the constant region sequence of claim 2, the anti-CD97 chimeric antigen receptor CD97-scfv of any one of claims 3-4, and the T lymphocyte of claim 5 Use of the cell or the anti-CD97 monoclonal antibody of claim 6 in the preparation of drugs for preventing or treating diseases caused by overexpression of CD97 and diseases caused by uncontrolled expression of CD97.
The use according to claim 7, wherein the disease includes brain tumors.
The use according to claim 7 or 8, wherein the disease is malignant glioma.
The application according to any one of claims 7-9, wherein the disease is astroblastoma, astrocytoma, glioblastoma multiforme, oligodendroglioma, neuroblastoma Blastoma, ependymoma, or ependymoma.
The variable region sequence of claim 1, the constant region sequence of claim 2, the anti-CD97 chimeric antigen receptor CD97-scfv of any one of claims 3-4, the T of claim 5 The use of lymphocytes or the anti-CD97 monoclonal antibody of claim 6 in inhibiting the proliferation of glioma stem cells or reducing the level of glioma stem cells.
The use according to claim 11, wherein the glioma stem cells are at least one of the following glioma stem cells: glioma stem cell line 83, glioma stem cell line CSC2, and glioma stem cells Department 528.
The variable region sequence of claim 1, the constant region sequence of claim 2, the anti-CD97 chimeric antigen receptor CD97-scfv of any one of claims 3-4, the T of claim 5 Use of lymphocytes or the anti-CD97 monoclonal antibody of claim 6 in the preparation of inhibitors of glioma stem cells.
The variable region sequence of claim 1, the constant region sequence of claim 2, the anti-CD97 chimeric antigen receptor CD97-scfv of any one of claims 3-4, the T of claim 5 The use of lymphocytes or the anti-CD97 monoclonal antibody of claim 6 in the prevention or treatment of diseases caused by overexpression of CD97 and diseases caused by uncontrolled expression of CD97.
A medicine for treating diseases caused by overexpression of CD97 or uncontrolled expression of CD97, characterized in that it comprises the variable region sequence of claim 1, the constant region sequence of claim 2, and any of claims 3-4 One of the anti-CD97 chimeric antigen receptor CD97-scfv, the T lymphocyte of claim 5, or the anti-CD97 monoclonal antibody of claim 6.
The drug for treating diseases caused by overexpression of CD97 or uncontrolled expression of CD97 according to claim 15, wherein the drug further comprises pharmaceutically acceptable additives or excipients.
The drug for treating diseases caused by overexpression of CD97 or uncontrolled expression of CD97 according to claim 15 or 16, characterized in that the dosage form of the pharmaceutical composition is selected from the group consisting of tablets, pills, powders, suspensions, gels, Emulsion, cream, granules, nanoparticles, capsules, suppositories, injections, sprays or injections.
A pharmaceutical composition, characterized in that it comprises the variable region sequence of claim 1, the constant region sequence of claim 2, and the anti-CD97 chimeric antigen receptor of any one of claims 3-4. CD97-scfv, the T lymphocyte of claim 5, or the anti-CD97 monoclonal antibody of claim 6.
The pharmaceutical composition according to claim 18, wherein the pharmaceutical composition further comprises a combination drug/therapy, and the combination drug/therapy is at least one of the following drugs:

Chemotherapeutics, radiotherapy, photosensitizers, photothermal agents, immunotherapy, thyroxine receptor agonists, antagonists and function modifiers, sodium-glucose cotransporter 2 inhibitors.
A drug according to any one of claims 15-17 or a pharmaceutical composition according to any one of claims 18-19 in the prevention or treatment of diseases caused by overexpression of CD97 and diseases caused by uncontrolled expression of CD97 application.