阅读说明：本技术 抗tspan8单克隆抗体及其用途 (anti-TSPAN 8 monoclonal antibody and application thereof ) 是由 王红霞 葛伟玉 杨光 朱容萱 马培祥 周天浩 李军建 许静轩 于 2019-10-30 设计创作，主要内容包括：本发明公开了一种抗TSPAN8单克隆抗体及其用途,该抗体能与TSPAN8蛋白特异性结合；所述抗体的重链可变区由如SEQ ID NO：1所示的核酸序列或其同源性序列编码；所述抗体的轻链可变区由如SEQ ID NO：2所示的核酸序列或其同源性序列编码,可用于制备治疗和/或预防由TSPAN8介导促进肿瘤细胞上皮间质转化及干性增强的分子调控机制及关键分子靶点的疾病的药物组合物。本发明提供的抗TSPAN8单克隆抗体具有高亲和力,特异性强,稳定性好,且具有抑制乳腺癌细胞系的增殖和迁移能力,且可以抑制肿瘤的生长。(The invention discloses anti-TSPAN 8 monoclonal antibodies and application thereof, wherein the antibodies can be specifically combined with TSPAN8 protein, the heavy chain variable region of the antibodies is coded by a nucleic acid sequence shown as SEQ ID NO: 1 or a homologous sequence thereof, and the light chain variable region of the antibodies is coded by a nucleic acid sequence shown as SEQ ID NO: 2 or a homologous sequence thereof, and can be used for preparing a pharmaceutical composition for treating and/or preventing diseases of a key molecular target spot and a molecular regulation mechanism for promoting epithelial-mesenchymal transition and dryness enhancement of tumor cells mediated by TSPAN 8.)

antibodies, wherein said antibodies specifically bind to TSPAN8 protein;

the heavy chain variable region of the antibody consists of the amino acid sequence shown as SEQ ID NO: 1 or a homologous sequence thereof;

the variable region of the light chain of the antibody consists of the amino acid sequence shown as SEQ ID NO: 2 or a homologous sequence thereof.

2. The antibody of claim 1, wherein the antibody is a single chain antibody or a diabody.

3. The antibody of claim 1, wherein the antibody is a monoclonal antibody.

4. The antibody of claim 1, wherein the antibody is a fully human antibody.

5. The antibody of claim 1, further comprising a heavy chain constant region selected from the group consisting of an IgG1 or IgG4 subtype and a light chain constant region comprising kappa.

Use of the antibody of any of claims 1-5 for the preparation of a pharmaceutical composition for the treatment and/or prevention of a disease associated with TSPAN8 mediation.

7, polynucleotides encoding the heavy chain variable region and/or the light chain variable region of claim 1.

8, , wherein the vector comprises the polynucleotide of claim 7.

genetically engineered host cell, characterized in that it comprises the vector of claim 8.

Technical Field

The invention relates to the field of biological medicines, in particular to a fully human anti-TSPAN 8 monoclonal antibody and application thereof.

Background

Tetraspanins, also known as the tetraspanin superfamily (TM4SF), is composed of 33 family members, including TSPAN8, differentiation (CD) -associated CD63, CD37, CD53, CD81, and CD9, among others. The Tetraspanins family is commonly characterized by four highly conserved membrane spans, which play important biological regulation in a variety of cellular functions.

Invasion and metastasis of tumors and drug resistance are the main causes of clinical treatment failure and death of patients. In a Tumor Microenvironment (TME), a tetraspanin protein TSPAN8 promotes a molecular regulatory mechanism and key molecular targets for Tumor cell Epithelial Mesenchymal Transition (EMT) and dryness (Stemness) enhancement. There is increasing evidence that TSPAN8 promotes tumor cell migration, invasion and metastasis in various types of human cancers, including ovarian and gastric cancers colorectal, liver, pancreatic, glioma, and others.

The monoclonal antibody medicine has the features of high targeting property, high specificity, low toxic side effect, etc. Monoclonal antibodies targeting tetraspanin TSPAN8 specifically bind TSPAN8, mediating antitumor therapeutic effects.

The antibody library technology bypasses the hybridoma process required in the conventional monoclonal antibody development process, and can obtain various antibody genes and antibody molecule fragments even without an immunization process, the phage antibody library is also the antibody library which is most -fold in the earliest application, the phage antibody library is divided into an immune library and a non-immune library according to the source of the antibody genes, and the non-immune library is a natural library, a semi-synthetic library and a fully-synthetic library.

At present, a fully human antibody against TSPAN8 having a high affinity anti-tumor effect is still lacking in China.

Disclosure of Invention

The invention aims to screen a monoclonal antibody or a fragment thereof for resisting TSPAN8 from a full-human combined natural antibody library by taking an extracellular segment sequence of TSPAN8 as an antigen, wherein the antibody has high affinity, can inhibit proliferation and migration of a breast cancer cell line and has an anti-tumor effect.

In order to achieve the aim, the invention provides monoclonal antibodies against TSPAN8 and application thereof.

According to of the present invention, there are provided kinds of antibody heavy chain variable regions, wherein the nucleic acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 1 or the homologous sequence thereof.

In the second aspect of the present invention, kinds of antibody light chain variable regions are provided, and the nucleic acid sequence encoding the antibody light chain variable regions is shown in SEQ ID No. 2 or its homologous sequence.

Preferably, the antibody comprises: single chain antibodies, diabodies.

Preferably, the antibody is a monoclonal antibody.

Preferably, the antibody is a monoclonal antibody specific for the TSPAN8 protein.

Preferably, the antibody further comprises a heavy chain constant region selected from the group consisting of IgG1 or IgG4 subtype and a light chain constant region selected from the group consisting of kappa.

In a third aspect of the present invention, polynucleotides encoding the heavy chain variable region and/or the light chain variable region described above are provided.

In a fourth aspect of the invention, there are provided vectors comprising the polynucleotide of the third aspect.

In a fifth aspect of the invention, there are provided genetically engineered host cells comprising the vector of the fourth aspect.

The invention has the following beneficial effects:

the invention screens specific inhibitory monoclonal antibodies (TS04-IgG) aiming at the TSPAN8 protein functional region by taking an extracellular segment sequence of the TSPAN8 protein functional region as an antigen, and the monoclonal antibody (TS04-IgG) resisting the TSPAN8, which is provided by the invention, has the advantages of high affinity, strong specificity and good stability, has the capacity of inhibiting the proliferation and migration of a breast cancer cell line, and can inhibit the growth of tumors.

Drawings

FIG. 1 is an SDS-PAGE analysis of TSPAN8-LEL-FC fusion proteins in the examples.

FIG. 2 is the results of the combinatorial antibody library screening based on differential enrichment in the examples.

FIG. 3 shows the result of the phage monoclonal ELISA assay in the examples.

FIG. 4 is an SDS-PAGE analysis of antibody expression purification in the examples.

FIG. 5 is an analysis of the interaction forces of TS01-IgG to TS08-IgG and TSPAN8-LEL-FC in the examples.

FIG. 6 shows the cell membrane co-localization studies of TS03-IgG to TS05-IgG in the examples.

FIG. 7 shows the affinity analysis of TSPAN8-LEL-FC at the concentration of TS04-IgG gradient in example.

FIG. 8A is an analysis of the apparent binding affinity of TS 04-IgG.

FIG. 8B is a flow cytometry method to detect the binding capacity of TS04-IgG to cells.

FIG. 9 shows the melting point profile of TS 04-IgG.

FIG. 10A is a graph showing the results of TS04-IgG migration inhibition in vitro.

FIG. 10B is a statistical graph of the ability of TS04-IgG to inhibit migration of breast cancer cells in vitro.

FIG. 11A is a graph showing the results of the ability of monoclonal antibodies to inhibit proliferation of MCF-10A cells in this example.

FIG. 11B is a statistical plot of the ability of monoclonal antibodies to inhibit MDA-MB-231 cell proliferation in this example.

FIG. 12A is an in vivo imaging of TS04-IgG in a mouse breast cancer xenograft model.

FIG. 12B is a TS04-IgG ability to inhibit tumor growth.

Detailed Description

The invention provides monoclonal antibodies against TSPAN8 and application thereof.

As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which form the binding and specificity of each particular antibody for its particular antigen.however, the variability is not evenly distributed throughout the antibody variable region.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of class , i.e., the individual antibodies contained in the population are identical except for a few naturally occurring mutations that may be present.

As used herein, the term "variable region" is used interchangeably with "Complementary Determining Region (CDR)".

the antigen binding properties of an antibody can be described by 3 specific regions in the heavy and light chain variable regions, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction, the CDRs forming a loop structure, the β fold formed by the FRs in between being folded in spatial proximity to each other, the CDRs on the heavy chain and the CDRs on the corresponding light chain constituting the antigen binding site of the antibody.

The antibody of the invention refers to polypeptides having the binding activity of the TSPAN8 protein, comprising the CDR regions described above, which term also includes variants of polypeptides comprising the CDR regions described above, which variants include, but are not limited to, deletions, insertions and/or substitutions of or more (typically 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, and the addition of or more (typically within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and/or N-terminus.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes under high or low stringency conditions with DNA encoding an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.

In the present invention, "conservative variant" refers to the amino acid sequence of the antibody of the present invention, wherein at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids having similar or similar properties to form a polypeptide.

The invention also provides polynucleotide molecules encoding the above antibodies or fragments or fusion proteins thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The coding region sequence can be identical to SEQ ID No.: 1. 2, or a degenerate variant thereof. As used herein, "degenerate variant" means in the present invention a variant that encodes a polypeptide having the same amino acid sequence as the polypeptide of the present invention, but which has an amino acid sequence identical to SEQ ID No.: 1. 2, or a variant thereof.

The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be obtained by PCR amplification, recombination or artificial synthesis methods methods are available in which the sequence is synthesized, especially when the length of the fragment is short, usually, a large-length fragment is obtained by synthesizing a plurality of small fragments and then ligating them together, and furthermore, the coding sequence of the heavy chain and the expression tag can be fused at to form a fusion protein.

Once the sequence has been obtained, it can be obtained in large quantities by recombinant methods, usually by cloning it into a vector, transferring it into a cell, and isolating the sequence from the expanded host cell by conventional methods.

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.

The host cell may be a prokaryotic cell such as a bacterial cell, or a lower eukaryotic cell such as a yeast cell, or a higher eukaryotic cell such as a mammalian cell, representative examples are bacterial cells of Escherichia coli, Streptomyces, Salmonella typhimurium , fungal cells such as yeast, insect cells of Drosophila S2 or Sf9, animal cells of CHO, COS7, 293 cells, and the like.

In the invention, the principle of flow cytometry is that under the pressure of , sheath fluid carries cells to enter a flow irradiation chamber through the center of a nozzle, laser irradiates the cells to scatter and refract at an analysis point of the flow irradiation chamber, scattered light (including Forward scattered light and side scattered light) is emitted, fluorescein carried by the cells is excited by the laser and emits fluorescence, Forward scattered light (FSC) and side scattered light (SSC) detectors convert the scattered light into electric signals, the fluorescence is collected by a condenser, the fluorescence of different colors is turned to different photomultiplier detectors by a double-color reflector, the fluorescence signals are converted into electric signals, and the electric signals are subjected to data processing and then input into a computer for storage, thus analyzing or sorting the cells.

In the present invention, "Nc" represents negative, and "Nc-IgG" represents negative control.