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WO2021083248A1 - Anticorps monoclonal anti-tspan8 et son utilisation - Google Patents

Anticorps monoclonal anti-tspan8 et son utilisation Download PDF

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Publication number
WO2021083248A1
WO2021083248A1 PCT/CN2020/124627 CN2020124627W WO2021083248A1 WO 2021083248 A1 WO2021083248 A1 WO 2021083248A1 CN 2020124627 W CN2020124627 W CN 2020124627W WO 2021083248 A1 WO2021083248 A1 WO 2021083248A1
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antibody
igg
tspan8
cells
protein
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Chinese (zh)
Inventor
王红霞
曹源
许静轩
葛伟玉
朱容萱
周天浩
李军建
杜少倩
虞越
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Shanghai First Peoples Hospital
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the field of biomedicine, in particular to a fully human anti-TSPAN8 monoclonal antibody and its use.
  • Tetraspanins also known as the four-pass transmembrane protein superfamily (TM4SF) are composed of 33 family members, including TSPAN8, CD63, CD37, CD53, CD81, and CD9 related to differentiation (CD).
  • TSPAN8 transmembrane protein superfamily
  • CD63 CD63
  • CD37 CD37
  • CD53 CD53
  • CD81 CD9 related to differentiation
  • CD9 CD9 related to differentiation
  • TSPAN8 tumor cell epithelial-mesenchymal transition
  • EMT tumor cell epithelial-mesenchymal transition
  • stemness enhancement More and more evidence shows that TSPAN8 promotes tumor cell migration, invasion and metastasis in many types of human cancers, including ovarian cancer and gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, glioma, etc.
  • Monoclonal antibody drugs have the characteristics of strong targeting, high specificity and low side effects.
  • Monoclonal antibodies targeting the four-pass transmembrane protein TSPAN8 can specifically bind to TSPAN8, thereby mediating the effect of anti-tumor therapy.
  • Fully human antibodies are the main direction for the development of therapeutic antibodies.
  • the emergence of antibody library technology provides a good technical platform for the preparation and screening of human antibodies.
  • the antibody library technology bypasses the hybridoma process necessary in the previous monoclonal antibody development process, and can obtain various antibody genes and antibody molecular fragments without even going through the immunization process.
  • the phage antibody library is the earliest and most widely used antibody library.
  • the phage antibody library is divided into immune library and non-immune library according to the source of antibody gene. Non-immune library is also natural library, semi-synthetic library and fully synthetic library.
  • the screening of the phage antibody library simulates the process of antibody affinity maturation.
  • the antigen is wrapped on a solid-phase medium and added to the phage antibody library to be screened.
  • the affinity is panned through several rounds of "adsorption-washing-elution-amplification". Specific antibodies.
  • the purpose of the present invention is to target the extracellular sequence of TSPAN8 as an antigen, and to screen out anti-TSPAN8 monoclonal antibodies or fragments thereof from a fully human combined natural antibody library.
  • the antibodies have high affinity and can inhibit breast cancer cell lines. Proliferation and migration, with anti-tumor effect.
  • the present invention provides an anti-TSPAN8 monoclonal antibody and its application.
  • the first aspect of the present invention provides a heavy chain variable region of an antibody, and the nucleic acid sequence encoding the heavy chain variable region of the antibody is shown in SEQ ID NO: 1 or its homologous sequence.
  • the second aspect of the present invention provides a light chain variable region of an antibody.
  • the nucleic acid sequence encoding the light chain variable region of the antibody is shown in SEQ ID NO: 2 or its homologous sequence.
  • the antibodies include: single-chain antibodies and double-chain antibodies.
  • the antibody is a monoclonal antibody.
  • the antibody is a monoclonal antibody specific to TSPAN8 protein.
  • the antibody further comprises a heavy chain constant region selected from IgG1 or IgG4 subtypes and a light chain constant region selected from kappa.
  • the third aspect of the present invention provides a polynucleotide encoding the aforementioned heavy chain variable region and/or light chain variable region.
  • the fourth aspect of the present invention provides a vector comprising the polynucleotide as described in the third aspect.
  • the fifth aspect of the present invention provides a genetically engineered host cell, which comprises the vector as described in the fourth aspect.
  • the sequence of the extracellular segment of the TSPAN8 protein functional region of the present invention is screened as an antigen to obtain an inhibitory monoclonal antibody (TS04-IgG) specific to the TSPAN8 protein functional region.
  • the anti-TSPAN8 monoclonal antibody (TS04-IgG) provided by the present invention Has high affinity, strong specificity, good stability, and has the ability to inhibit the proliferation and migration of breast cancer cell lines, and can inhibit the growth of tumors.
  • Figure 1 shows the SDS-PAGE analysis of the TSPAN8-LEL-FC fusion protein in the example.
  • Figure 2 shows the results of screening combinatorial antibody libraries based on differential enrichment in the Examples.
  • Figure 3 shows the results of phage monoclonal ELISA identification in the examples.
  • Figure 4 is an SDS-PAGE analysis of antibody expression and purification in the example.
  • Figure 5 shows the analysis of the interaction forces between TS01-IgG to TS08-IgG and TSPAN8-LEL-FC in the Examples.
  • Figure 6 shows the study of co-localization of TS03-IgG to TS05-IgG on cell membrane in the Examples.
  • Figure 7 shows the affinity analysis with TSPAN8-LEL-FC at TS04-IgG gradient concentration in the example.
  • Figure 8A shows the apparent binding affinity analysis of TS04-IgG.
  • Figure 8B shows the binding ability of TS04-IgG to cells detected by flow cytometry.
  • Figure 9 shows the melting point curve of TS04-IgG.
  • Figure 10A is a graph showing the results of TS04-IgG inhibiting the migration ability of breast cancer cells in vitro.
  • Figure 10B is a statistical diagram of the ability of TS04-IgG to inhibit the migration of breast cancer cells in vitro.
  • Figure 11A is a graph showing the results of monoclonal antibodies inhibiting the proliferation ability of MCF-10A cells in this example.
  • Fig. 11B is a statistical diagram of the ability of monoclonal antibodies to inhibit the proliferation of MDA-MB-231 cells in this example.
  • Figure 12A shows the in vivo imaging of TS04-IgG in a mouse breast cancer xenograft model.
  • Figure 12B is an analysis of the ability of TS04-IgG to inhibit tumor growth.
  • the invention provides an anti-TSPAN8 monoclonal antibody and its application.
  • variable means that certain parts of the variable region of the antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the variable regions of antibodies. It is concentrated in three fragments called complementarity determining regions (CDR) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved part of the variable region is called the framework region (FR).
  • CDR complementarity determining regions
  • FR framework region
  • the variable regions of the natural heavy chain and light chain each contain four FR regions, which are roughly in a ⁇ -sheet configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial ⁇ -sheet structure.
  • the CDRs in each chain are closely held together by the FR region and form the antigen binding site of the antibody together with the CDRs of the other chain. Constant regions do not directly participate in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.
  • the term "monoclonal antibody (monoclonal antibody)” refers to an antibody obtained from a substantially homogeneous population, that is, the single antibodies contained in the population are the same, except for a few naturally occurring mutations that may exist. Monoclonal antibodies are highly specific to a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (usually with different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized through hybridoma culture and will not be contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristics of the antibody, which is obtained from a substantially uniform antibody population, which should not be interpreted as requiring any special method to produce the antibody.
  • variable region and “complementarity determining region (CDR)” are used interchangeably.
  • variable regions which are divided into 4 framework regions (FR), 4
  • FR framework regions
  • the amino acid sequence of FR is relatively conservative and does not directly participate in the binding reaction. These CDRs form a circular structure, and the ⁇ sheets formed by the FRs between them are close to each other in space structure, and the CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen binding site of the antibody.
  • the amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.
  • variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding to antigen. Therefore, the present invention includes those molecules with CDR-bearing monoclonal antibody light chain and heavy chain variable regions, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably 98%) with the CDRs identified here.
  • the above) homology that is, the sequence of homology.
  • the antibody of the present invention refers to a polypeptide having TSPAN8 protein binding activity and including the above-mentioned CDR regions.
  • the term also includes variant forms of polypeptides containing the above-mentioned CDR regions that have the same functions as the antibodies of the present invention. These variants include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid deletion , Insertion and/or substitution, and adding one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal.
  • amino acids with similar or similar properties are substituted, the function of the protein is usually not changed.
  • adding one or several amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • the variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and DNA that can hybridize with the coding DNA of the antibody of the present invention under high or low stringency conditions.
  • the encoded protein, and the polypeptide or protein obtained by using the antiserum against the antibody of the present invention.
  • “conservative variants” refer to at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids compared with the amino acid sequence of the antibody of the present invention. Similar or similar amino acids are replaced to form polypeptides.
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the coding region sequence may be the same as the coding region sequence shown in SEQ ID NO.: 1 and 2 or a degenerate variant.
  • degenerate variant in the present invention refers to a nucleic acid sequence that encodes a nucleic acid sequence that has the same amino acid sequence as the polypeptide of the present invention, but differs from the coding region sequence shown in SEQ ID NO.: 1 and 2 .
  • the full-length nucleotide sequence or fragments of the antibody of the present invention can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • a feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences.
  • the coding sequence of the heavy chain and the expression tag can be fused together to form a fusion protein.
  • the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • the biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules that exist in an isolated form.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
  • the present invention also relates to a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • the host cell can 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.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, and 293 cells.
  • the principle of flow cytometry under a certain pressure, the sheath liquid takes the cells through the center of the nozzle into the flow-type irradiation chamber, and at the analysis point of the flow-type irradiation chamber, the cells are scattered and refracted by laser irradiation. Scattered light (including forward scattered light and side scattered light) is emitted; at the same time, the fluorescein carried by the cell is excited by the laser and emits fluorescence. Forward scatter (FSC) and side scatter (SSC) detectors convert scattered light into electrical signals.
  • FSC forward scatter
  • SSC side scatter
  • the fluorescence is collected by the condenser, and the fluorescence of different colors is turned to different photomultiplier detectors by the two-color mirror, and the fluorescence signal is also converted into an electrical signal. These electrical signals are then digitally processed and then input into a computer and stored, and then the cells can be analyzed or sorted.
  • Nc represents negative
  • Nc-IgG represents negative control group
  • a method for obtaining an anti-TSPAN8 monoclonal antibody includes the following steps:
  • TSPAN8-LEL Use gene recombination technology to clone the gene sequence (TSPAN8-LEL) encoding the extracellular segment of TSPAN8 (TSPAN8-LEL), C-terminal FC-tag into pFUSE expression vector, construct human TSPAN8-LEL-FC fragment, and express it in HEK293F cells Obtain the TSPAN8-LEL-FC fusion protein;
  • TSPAN8-LEL-FC fusion protein As the antigen, through three rounds of enrichment and screening of the antibody library, eight ScFv sequences with higher affinity were obtained from the fully human ScFv phage library, which were named TsC01-TsC08.
  • S3 The above eight positive ScFv sequences, namely TsC01-TsC08, were cloned into a eukaryotic expression vector, transfected into HEK293F cells, expressed and purified to obtain a full anti-TSPAN8 monoclonal antibody, and named TS01-IgG to TS08- IgG.
  • S4 Screening of high stability and high affinity anti-TSPAN8 monoclonal antibody TS04-IgG through ELISA, SPR, flow cytometry and melting point test.
  • TS04-IgG The ability of TS04-IgG to inhibit the proliferation and migration of breast cancer cell line (MDA-MB-231) was verified by transwell migration test and CCK8 proliferation test. Through a mouse xenograft model, it was found that TS04-IgG can inhibit tumor growth in mice.
  • the preparation method of a method for obtaining an anti-TSPAN8 monoclonal antibody provided by the present invention is as follows:
  • TSPAN8-LEL sequence The gene sequence encoding the extracellular segment of TSPAN8 (amino acids 110-205; called TSPAN8-LEL sequence) and the C-terminal FC-tag were cloned into the pFUSE expression vector (pfuse fc2; Invivogen) using gene recombination technology to construct human TSPAN8 -LEL-FC fragment.
  • HEK293F cells were transfected and cultured for 4 days to express heterologous recombinant protein.
  • the TSPAN8-LEL-FC recombinant protein secreted into the culture medium was collected, and on the KTA protein purifier (GE Healthcare), the protein A HP column (#17'3-03; GE Healthcare) was used with citric acid The salt buffer (pH 3.4) is eluted to obtain the purified fusion protein.
  • the purified fusion protein was concentrated to 7.82 mg/mL and stored in pH 7.4 PBS buffer.
  • lanes 1 and 2 are the undenatured TSPAN8-LEL-FC fusion protein
  • lane 3 is the protein maker
  • lanes 4 and 5 are the denatured TSPAN8-LEL-FC fusion protein.
  • TSPAN8-LEL-FC fusion protein Dilute the TSPAN8-LEL-FC fusion protein to 10ug/mL, coat a 96-well microtiter plate with 100 ⁇ L/well and keep at 4°C overnight, then add 50g/L skimmed milk powder blocking solution and block overnight at 4°C to 50 ⁇ L/well
  • Add the combined human monoclonal scFv antibody natural phage library (10 11 diversity) carry out multiple rounds of affinity enrichment and fix it on protein A-coated magnetic beads, incubate at 37°C for 2 hours, wash off unbound phage antibodies, and use glycine -Hydrochloric acid (pH 2.2) to elute the bound phage, the eluate was infected with XL1-Blue in the logarithmic growth phase, and super-infected with VCSM13 helper phage, cultured overnight at 30°C to amplify positive phage clones, and proceed to the next step Round screening.
  • the positive phage clones obtained after the third round of screening were infected with Xl1-Blue competent, and directly spread on a fixed medium containing 100ug/mL ampicillin, cultured overnight at 37°C, and the next day picking monoclonal colonies were placed in LB culture.
  • VCSM13 helper phage was added to super-infect the preparation of monoclonal phage antibodies. After centrifugation, the supernatant containing the phage antibody was subjected to monoclonal ELISA identification.
  • a 96-well ELISA plate was coated with 50 ⁇ L/well plus 2ng/m concentration of TSPAN8-LEL-FC antigen, each monoclonal was equipped with 3 multiple wells, incubated at room temperature for 2h, and added after washing HRP-labeled anti-M13 secondary antibody was incubated for 1h at room temperature, then washed with PBST for 8 times and then added 50 ⁇ L HRP substrate 2,2'-azinobis (3-ethylbenzothiazolin-6-sulfonic acid)-diammonium salt solution (#11684302001 ; Roche) is added to each hole. After incubating at room temperature for 20 minutes in the dark, add 50mol/L H 2 S0 4 to each well to stop the reaction, and measure the absorbance at 405 nm.
  • TsC01-TsC08 wherein, the heavy chain of TsC04 was obtained by gene sequencing.
  • the variable region is the nucleic acid sequence shown in SEQ ID NO: 1
  • the light chain variable region of TsC04 is the nucleic acid sequence shown in SEQ ID NO: 2.
  • the light chain region and the heavy chain region of the three positive scFv (TsC03-TsC05) gene sequences with the best specificity were cloned into a plasmid with a complete constant heavy chain domain and a complete constant light chain domain to construct Heavy chain carrier and light chain carrier. Then the equimolar heavy chain vector and light chain vector were co-transfected into HEK293F and then expressed and cultured for 4 days to obtain fully human IgG. After centrifuging the cell culture medium, HiTrap was used to purify the full-length IgG antibody secreted into the culture medium.
  • the protein A HP column (#17'3-03; GE Healthcare) was eluted with a citrate buffer (pH 3.4) and purified to obtain an anti-TSPAN8 monoclonal
  • the whole antibody is named TS01-IgG to TS08-IgG.
  • the eight anti-TSPAN8 monoclonal antibodies were all concentrated to 1 mg/mL and stored in pH 7.4 PBS buffer.
  • lanes 1, 2, and 3 are undenatured TS04-IgG, TS05-IgG, and TS06-IgG, respectively, lane 4 is the protein marker, lanes 5, 6, and 7 They are denatured TS04-IgG, TS05-IgG, TS06-IgG, and the purity of TS04-IgG, TS05-IgG, and TS06-IgG are all greater than 90%, and stored at -20°C.
  • the breast cancer cell line (MDA-MB-231) with high expression of TSPAN8 was collected and resuspended in ice-cold FACS buffer (PBS, 0.05% BSA, 2mM EDTA). Then an equal amount of MDA-MB-231 cells (50,000 cells per tube) and 50nm TS01-IgG to TS08-IgG were incubated at 4°C for 20 minutes, washed with 1 mL of ice-cold FACS buffer, rotated, and resuspended in 100 ⁇ L in ice. Contains a cold FACS buffer that recognizes human Fc at a volume of 1:800 diluted Alexa555-coupled secondary antibody.
  • the cells were washed twice and resuspended in FACS buffer, then they were sorted and analyzed on a flow cytometer (LSRFortessaTM X-20; BD) to determine the relative binding level of the antibody , Record the average fluorescence intensity of Alexa555 and analyze it. As shown in Figure 5, compared with the control group, the average fluorescence intensity after treatment with TS03-IgG, TS04-IgG, and TS05-IgG was increased, indicating that TS03-IgG, TS04-IgG, and TS05-IgG can all interact with TSPAN8 on the cell membrane surface. Combine.
  • the 293T cells expressing TSPAN8-GFP were seeded on a 24-well plate with cover slips at a concentration of 2 ⁇ 10 5 cells/mL for cell climbing. After culturing for 24 hours, the cells were fixed in 4% paraformaldehyde and incubated in a blocking solution (PBS solution containing 5% calf serum) for 30 minutes. The obtained cells were incubated with 2 ⁇ g/mL TS03-IgG, TS04-IgG, and TS05-IgG at 4°C overnight, and then washed three times with PBS for 5 minutes each, and combined with Alexa555-conjugated goat anti-human two Antibiotics (Life Technology) are incubated together.
  • a blocking solution PBS solution containing 5% calf serum
  • the SPR-combined study was conducted on Biacore T200 (GE Healthcare).
  • the antigen can be coupled with the amine on the surface of the biosensor chip CM5 series S (GE Healthcare). Fix the recombinant protein TSPAN8-LEL-FC on the surface of the chip.
  • the antibody TS04-IgG was diluted with a running buffer (pH 7.4) containing 25 mM Hepes, 500 mM NaCl, 0.05% EDTA and 0.05% DDM at a concentration of 0, 3.125, 6.25, 12.5, 25, 50, and 100 nM.
  • Measure the binding/dissociation kinetics as the change in the resonance unit of the SPR signal, and use the Biacore T200 evaluation software with a single-cycle kinetic model to calculate the apparent binding constant km.
  • the flow cytometry method detects the binding ability of TS04-IgG to cells.
  • the 50nM TS04-IgG is filled into the capillary and installed in the precise position of the thermometer, and then fixed in the center of the b-shaped tube. The temperature is heated and measured, and the melting point curve is finally obtained, as shown in Figure 9, which is the melting point curve of TS04-IgG.
  • the measured Tm 78.2°C, and the melting point is greater than 37°C (normal body temperature of the human body), indicating that the antibody has high stability.
  • Breast cancer epithelial cells MDA-MB-231 were starved in serum-free DMEM/F12 (3:1) medium for 12 hours, trypsinized and then resuspended in DMEM/F12 medium containing 0.1% FBS, press 1.0 ⁇ 10 5 /100 ⁇ L was added to the upper chamber, each group contained 50nM TS03-IgG, 50nM TS04-IgG, 50nM TS05-IgG. Add 600L of 10% FBS DMEM/F12 medium in the lower chamber, use PBS as a TS-IgG blank control, and culture for 24 hours.
  • FIG. 10A it is the result of TS04-IgG inhibiting the migration ability of breast cancer cells in vitro.
  • MDA-MB-231, MCF-10A, 4175 cells in good condition and in the proliferation stage for experiment.
  • the cells are routinely digested, centrifuged, and counted. The concentration of each cell was adjusted to 1000 cells per 100 ⁇ L of culture medium, and 4 96-well flat bottom plates were taken to make time gradients of 0h, 24h, 48h, 72h, respectively. Inoculate the cells into a 96-well plate, make 5-6 replicate wells for each cell, and add 100 ⁇ L of culture medium to each well. After adding the cells, place them in a 37°C incubator and culture them. The cells will be attached 24 hours after inoculation.
  • TS03-IgG, TS04-IgG, and TS05-IgG have no significant effect on the growth of breast cystic epithelial cells MCF-10A cells with low expression of TSPAN8; TS03-IgG, TS04-IgG and TS05-IgG can inhibit the growth of breast cancer epithelial cells MDA-MB-231 with high TSPAN8 expression, and TS04-IgG has the most obvious inhibitory effect on tumor cell growth.
  • DMEM/F12 Medium Gibco
  • Fetal Bovine Serum Gibco
  • Double Antibody Gibco
  • 35mm Petri Dish Nest
  • 25mm Cell Slide Nest
  • Phosphate Buffer/PBS Boster
  • Fluorescence Co- Focusing microscope Leica
  • 35mm cell culture dish Corning
  • inverted microscope Leica
  • flow analysis instrument LSRFortessaTM X-20; BD
  • antibody purification instrument 100 GE Healthcare
  • FuGENE transfection reagent Promega
  • Protein A coated magnetic beads 21925; Pierce
  • Anti-M13 HRP-conjugated secondary antibody #27–9421-01; GE/Amersham/Whatman
  • Goat anti-human Fc conjugated HRP secondary antibody #A0170; Sigma
  • microplate reader Enspire; PerkinElmer
  • Biacore T200 GE Healthcare
  • Transwell chamber Invitrogen
  • Cell culture medium DMEM/F12 medium + 10% fetal bovine serum + 1% double antibody (volume fraction);
  • SPR running buffer 25mM Hepes, 500mM NaCl, 0.05% EDTA, 0.05% DDM.
  • the gene recombination technology is used to screen the extracellular sequence of the TSPAN8 protein functional region as an antigen to obtain an inhibitory type specific to the TSPAN8 protein functional region.
  • the monoclonal antibody TS04-IgG has been verified by ELISA, SPR, flow cytometry and melting point test to verify the affinity and stability of TS04-IgG.
  • the transwell migration test and CCK8 proliferation test verified the ability of TS04-IgG to inhibit the proliferation and migration of breast cancer cell line (MDA-MB-231). Further through the mouse xenograft model, it was found that TS04-IgG can inhibit tumor growth in vivo.
  • the inhibitory monoclonal antibody TS04-IgG against TSPAN8 protein functional region provided by the present invention has high affinity, strong specificity, good stability, and has the ability to inhibit the proliferation and migration of breast cancer cell lines.
  • Tumor has obvious inhibitory effect and can be used to treat or prevent diseases with TSPAN8-mediated promotion of tumor cell Epithelial Mesenchymal Transition (EMT) and Stemness (Stemness) enhanced molecular regulation mechanisms and key molecular targets.
  • EMT Epithelial Mesenchymal Transition
  • Stemness Stemness

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Abstract

La présente invention concerne un anticorps monoclonal anti-TSPAN8 et son utilisation, l'anticorps pouvant inhiber la prolifération et la migration de lignées cellulaires du cancer du sein, et étant capable d'inhiber la croissance tumorale. L'anticorps peut être utilisé pour traiter et/ou prévenir des maladies à médiation par TSPAN8.
PCT/CN2020/124627 2019-10-30 2020-10-29 Anticorps monoclonal anti-tspan8 et son utilisation Ceased WO2021083248A1 (fr)

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