WO2023177232A1 - Anticorps monoclonal anti b7-h3 et son utilisation - Google Patents

Anticorps monoclonal anti b7-h3 et son utilisation Download PDF

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WO2023177232A1
WO2023177232A1 PCT/KR2023/003515 KR2023003515W WO2023177232A1 WO 2023177232 A1 WO2023177232 A1 WO 2023177232A1 KR 2023003515 W KR2023003515 W KR 2023003515W WO 2023177232 A1 WO2023177232 A1 WO 2023177232A1
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cancer
antibody
cells
npb40
cell
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Korean (ko)
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류춘제
서효선
한산하
최문주
최홍서
이현민
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세종대학교산학협력단
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to a monoclonal antibody that specifically binds to human B7-H3, and specifically, to a monoclonal antibody that recognizes B7-H3 expressed on the surface of various cancer cells, including human pluripotent stem cells and liver cancer, and to the monoclonal antibody that specifically binds to human B7-H3. It relates to a hybridoma producing a clonal antibody, a diagnostic kit containing the monoclonal antibody, and a composition for anticancer treatment containing the monoclonal antibody.
  • Human pluripotent stem cells are cells that have the ability to proliferate indefinitely and differentiate into all cells that make up the body.
  • the first human pluripotent stem cells human embryonic stem cells (hESC) were established from human frozen embryos by Dr. James Thomson's team in the United States in 1998, and by gene introduction in 2007 by Dr. Shinya Yamanaka's team in Japan.
  • Human Induced Pluripotent Stem Cell hiPSC
  • human embryonic stem cells were created through egg nuclear transfer by Dr. Shoukhrat Mitalipov's team at the University of Oregon, and currently, three types of hPSC exist.
  • pluripotent stem cells can be separated, and pluripotent stem cells before implantation are called naive pluripotent, and those after implantation are called primed pluripotent. Pure state and quasi-state pluripotent stem cells have many differences in cell shape, growth rate, differentiation ability, degree of methylation, germline transmission ability, etc.
  • the quasi-state grows in a largely flat state and has a slow growth rate, while the pure state It grows in a small dome shape, grows well even when separated into single cells with trypsin, has a fast growth rate, is not yet concentrated in the developmental stage, is expected to have the ability to differentiate into more cells, has a lower degree of methylation, and has germline transmission. It is known as an early pluripotent stem cell with superior capabilities, and can be induced from quasi-human pluripotent stem cells to pure human pluripotent stem cells under various culture conditions.
  • Oncofetal antigens are important target molecules currently used in the diagnosis or treatment of cancer, such as CA-125, CEA, AFP, cancer /testes antigen, POA, etc.
  • Cripto-1, Glypican-3, 5T4, and M2A are known as oncofetal antigens, and antibodies targeting these are being developed as antibodies for cancer treatment.
  • oncofetal antigens was not easy due to ethical issues regarding human embryos, and as human embryonic stem cells were established and cultured in 1998, oncofetal antigens on the surface of human embryonic stem cells such as 5T4, Cripto, and EpCAM were discovered, and these The molecules are being applied as target molecules for antibodies for cancer treatment.
  • hPSC human embryonic stem cells
  • hiPSC human induced pluripotent stem cells
  • B7-H3 (CD276, PD-L3) is an immune checkpoint member of the B7 and CD28 families and consists of two identical pairs of human immunoglobulin variable domains and immunoglobulin constant domains.
  • B7-H3 was identified by a database search of cDNA libraries of human dendritic cells. Although B7-H3 is known to be a T cell immunostimulatory molecule, accumulating evidence shows that B7-H3 also has an immunosuppressive function, reducing the cytotoxic activity of interferons and natural killers released from T cells.
  • B7-H3 The transcript of B7-H3 is co-expressed in most normal tissues and solid tumors, but the protein is highly expressed only in tumors such as lung, liver, leukemia, colon, rectum, stomach, breast, kidney, stomach, glioma, ovary, and pancreas. do. B7-H3 expression is also found in tumor-related vasculature and stroma. Overexpression of B7-H3 is closely correlated with advanced tumors and shorter overall survival in many cancers, including breast, ovarian, lung, liver, and gastric cancer.
  • the survival rate is significantly reduced when B7-H3 is overexpressed in liver cancer, breast cancer, ovarian cancer, lung cancer, and stomach cancer. Even though it is not expressed in the early stage of cancer, it is overexpressed in the late stage of cancer, making it a target for late-stage cancer treatment. It has high value, and has recently been suggested as a cancer stem cell marker for squamous cell carcinoma and pancreatic cancer stem cell. Additionally, many studies suggest that beyond its role in immune evasion, B7-H3 promotes tumorigenesis through mediation of anti-apoptosis, pro-proliferation, angiogenesis, and tumor microenvironment.
  • B7-H3 is considered an attractive target for immunotherapy therapy in various carcinomas.
  • antibody-based treatments and CAR-T cell therapies targeting B7-H3 are being developed for many advanced cancers, including TNBC, ovarian cancer, pancreatic cancer, NSCLC, glioblastoma, and melanoma.
  • TNBC TNBC
  • ovarian cancer pancreatic cancer
  • NSCLC NSCLC
  • glioblastoma melanoma
  • melanoma melanoma.
  • the receptor or ligand for B7-H3 has not yet been identified, it is still difficult to draw a clear mechanistic picture of cancer progression caused by B7-H3 in the cancer immune evasion mechanism.
  • NPB40 an antibody that simultaneously binds to pure human pluripotent stem cells and quasi-human pluripotent stem cells from a group of monoclonal antibodies prepared by injecting pure human pluripotent stem cells into mice.
  • the NPB40 antigen was rarely expressed in normal cells, such as peripheral blood monocytes or hepatocytes, but was highly expressed in liver cancer cells, embryonic cancer, pancreatic cancer, colon cancer, osteosarcoma, melanoma, lung cancer, and neuroblastoma cells.
  • NPB40 antigen revealed that it was B7-H3, and it was assumed that the NPB40 antibody that recognizes B7-H3 could be developed as a cancer treatment antibody as a new antibody that recognizes the oncofetal epitope co-expressed in human pluripotent stem cells and cancer cells.
  • injection of NPB40 showed an effective anticancer effect in a liver cancer mouse model transplanted with human liver cancer cells. Therefore, this invention was completed by confirming that NPB40 is an antibody that can be developed as an antibody for treating cancer, including liver cancer.
  • the purpose of the present invention is to provide a monoclonal antibody that recognizes the B7-H3 protein expressed on the surface of human pure pluripotent stem cells, human quasi-pluripotent stem cells, and cancer cells.
  • the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, a composition for diagnosing cancer, and a method for providing information for diagnosing cancer using the function of the monoclonal antibody.
  • the purpose of the present invention is to provide a hybridoma that produces the monoclonal antibody.
  • a heavy chain comprising heavy chain complementary determine region 1 (HCDR1) containing SEQ ID NO: 1, HCDR2 containing SEQ ID NO: 2, and HCDR3 containing SEQ ID NO: 3; And an antibody comprising a light chain comprising light chain complementary determine region 1 (LCDR1) comprising SEQ ID NO: 4, LCDR2 comprising SEQ ID NO: 5, and LCDR3 comprising SEQ ID NO: 6.
  • the antibody according to 1 above comprising a heavy chain containing SEQ ID NO: 7 and a light chain containing SEQ ID NO: 8.
  • the antibody is an antibody that binds to B7-H3 on the surface of human naive pluripotent stem cells, human prime pluripotent stem cells, and human cancer cells.
  • a pharmaceutical composition for preventing or treating cancer containing the antibody of any one of 1 to 3 above.
  • composition for preventing or treating cancer according to item 4 above further comprising an anti-cancer substance conjugated to the antibody.
  • composition for preventing or treating cancer according to item 4 above further comprising at least one from the group consisting of NK-cell (natural killer cell), macrophage, and neutrophil.
  • a composition for diagnosing cancer containing the antibody of any one of items 1 to 3 above.
  • composition for diagnosing cancer according to item 11 above wherein the cancer is a cancer derived from cancer cells expressing B7-H3 protein on the cell membrane.
  • a method of providing information for cancer diagnosis comprising the step of treating a separated sample with the composition of 11 above and detecting a protein to which the antibody is bound.
  • cancer is selected from the group consisting of liver cancer, pancreatic cancer, osteosarcoma, skin cancer, lung cancer, neuroblastoma, uterine cancer, prostate cancer, and colon cancer.
  • the monoclonal antibody of the present invention can recognize B7-H3 protein expressed on the surface of human pure pluripotent stem cells, human quasi-pluripotent stem cells, and cancer cells. It can be preferably used in pharmaceutical compositions for preventing or treating cancer, compositions for diagnosing cancer, etc.
  • FIG 1A shows that H9 cells, which are human pluripotent stem cells (hPSC), were cultured in primed human pluripotent stem cell (primed H9) medium and medium induced by pure human pluripotent stem cells (naive H9). In the pure human pluripotent stem cell medium, small cells appear. The dome-shaped shape of pure H9 cells can be seen.
  • Figures 1B and 1C show semi-human pluripotent stem cell markers SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, CD24, and CD90 and pure human pluripotent stem cell markers CD7, This picture shows the expression of CD77 and CD130.
  • Figure 2 is a diagram showing the analysis of the expression of 31 naive hPSC-related genes in naive hPSC and primed hPSC using RNA-seq method, showing that the expression of 15 naive hPSC-related genes increased in naive hPSC.
  • Figure 3 shows that through FACS analysis, the monoclonal antibody NPB40 of the present invention binds to primed human pluripotent stem cells (primed H9) and pure human pluripotent stem cells (naive H9), but does not bind to human embryonic carcinoma cells 2102EP and NT-2. It shows that it does not bind to mouse embryonic fibroblasts (MEF) and human peripheral blood mononuclear cells (PBMC). At this time, the solid line is the monoclonal antibody NPB40, and the red background is negative control data containing only the secondary antibody.
  • primed human pluripotent stem cells primed human pluripotent stem cells
  • naive H9 pure human pluripotent stem cells
  • PBMC peripheral blood mononuclear cells
  • Figure 4A is a diagram showing the extent to which the monoclonal antibody NPB40 of the present invention binds to various cell lines or primary cells through flow cytometry analysis.
  • the solid line is the monoclonal antibody and the red background is 2. This is negative control data containing only primary antibodies.
  • the monoclonal antibody NPB40 binds weakly to normal human hepatocytes, but binds strongly to four types of human liver cancer cells, Huh7, HepG2, SNU-387, and SNU-449.
  • NPB40 was used in three types of human pancreatic cancer cells, BxPC-3, PANC-1, and SNU-213, two types of human colon cancer cells, Colo-205 and HCT-116, and two types of human osteosarcoma cells, U2-OS and Saos-2. It shows that it binds well to many cancer cells, including human skin cancer cells A375, human lung cancer cells A549, and human neuroblastoma SH-SY5Y.
  • Figure 4B shows the cell surface of 2102Ep human embryonic cancer cells, Huh7, and SNU-449 human liver cancer cells stained with monoclonal antibody NPB40 through cell immunochemical staining, showing that monoclonal antibody NPB40 recognizes cell surface proteins of cancer cells. .
  • Figure 5A shows that proteins immunoprecipitated using the monoclonal antibody NPB40 from the eluate of NT2/D1 human embryonic cancer stem cells whose cell surface was labeled with biotin were separated through 10% SDS-PAGE and analyzed by Nitrogen by Western blotting. After transferring to a cellulose membrane, the sample was analyzed with streptavidin-HRP (SA-HRP). The same experiment was performed without adding antibodies, which was used as a negative control (No Ab). Arrows indicate proteins immunoprecipitated by NPB40.
  • Figure 5B shows immunoprecipitation performed in the same manner as Figure 5A, separated through 10% SDS-PAGE, and then the polyacrylamide gel was stained with PageBlue.
  • Figure 5C shows the results of LC-MS/MS analysis of the protein recovered after immunoprecipitation with the monoclonal antibody NPB40 in Figure 5B.
  • the amino acid sequence of the NPB40 antigen is identical to the amino acid sequence of the B7-H3 protein, and the matching portion is This picture is marked in red.
  • Figure 6A shows the monoclonal antibody NPB40 and the known rabbit anti-B7-H3 polyclonal in the cell lysate after biotin-labeling the surface of NT2/D1 human embryonic cancer cells to confirm that the antigen recognized by the monoclonal antibody NPB40 is B7-H3.
  • This picture shows the protein immunoprecipitated with antibody ( ⁇ -B7-H3) detected through Western blotting and streptavidin-HRP (SA-HRP) reaction.
  • Figure 6B shows the protein immunoprecipitated from the cell lysate of NT2/D1 human embryonic cancer cells with the monoclonal antibody NPB40 and a known rabbit anti-B7-H3 polyclonal antibody, and the cell lysate (whole lysate) was analyzed by Western blotting. It was detected by reacting with anti-B7-H3 polyclonal antibody.
  • Figures 6C and 6D show that to reconfirm that the antigen of the monoclonal antibody NPB40 is B7-H3, cell lysates of human embryonic kidney cells HEK293T overexpressing B7-H3 were tested with known mouse anti-His monoclonal antibodies and rabbit anti-B7- After immunoprecipitation with H3 polyclonal antibody and monoclonal antibody NPB40, it was detected by Western blotting and reaction with mouse anti-His monoclonal antibody or rabbit anti-B7-H3 polyclonal antibody.
  • Figure 7A shows a B7-H3 knockdown experiment performed on liver cancer cell lines Huh7, HepG2, SNU387, and SNU449, which were recovered after transfection of negative control siRNA (siCon) or siRNA against B7-H3 (siB7-H3) into liver cancer cells.
  • Western blotting was performed to analyze the expression of B7-H3 protein in one cell lysate, using GAPDH protein expression as a control.
  • Figures 7B and 7C show that liver cancer cells recovered after performing B7-H3 knockdown as in Figure 7A were analyzed through flow cytometry analysis, and the known rabbit anti-B7-H3 polyclonal antibody ( ⁇ -B7-H3) was used. It shows the degree of bonding.
  • FIG. 7B The solid and dotted lines in Figure 7B are monoclonal antibodies, the red background is negative control data containing only secondary antibodies, and Figure 7C is a graph statistically showing the average fluorescence intensity by repeating the experiment in Figure 7B three or more times.
  • Figures 7D and 7E are diagrams showing the degree of binding of the monoclonal antibody NPB40 of the present invention by analyzing liver cancer cells recovered after B7-H3 knockdown as shown in Figure 7A through flow cytometry analysis.
  • the solid and dotted lines in Figure 7D are monoclonal antibodies, the red background is negative control data containing only secondary antibodies, and Figure 7E is a graph statistically showing the average fluorescence intensity by repeating the experiment in Figure 7D three or more times.
  • *** indicates p value ⁇ 0.005.
  • Figure 8A shows hepatoma cells treated with negative control siRNA (siCon) or siRNA against B7-H3 (siB7-H3) recovered after performing B7-H3 knockdown as in Figure 7A, in 6-well cells coated with gelatin. This is a picture showing the degree of adhesion analyzed by reacting to a culture plate. This is a picture of attached liver cancer cells stained with crystal violet.
  • Figure 8B shows the same experiment as Figure 8A in Huh7 liver cancer cell line, cells were eluted with 0.1% SDS, absorbance was measured three times repeatedly at OD 570 nm , and statistical processing was performed.
  • Figure 8C is a diagram showing the same experiment as Figure 8A in HepG2 liver cancer cell line, cells were eluted with 0.1% SDS, absorbance was measured three times at OD 570nm , and statistical processing was performed.
  • Figures 8D and 8E show flow cytometry in liver cancer cells Huh7 in which B7-H3 knockdown was performed by treating negative control siRNA (siCon) or siRNA against B7-H3 (siB7-H3), as in Figure 8A.
  • Figure 8E is a picture analyzing the expression of the cell adhesion proteins E-Cadherin, Integrin- ⁇ 2, and integrin- ⁇ V.
  • Figure 8E is a statistical result of the experiment in Figure 8D repeated three times.
  • Figures 8F and 8G show HepG2 liver cancer cells subjected to B7-H3 knockdown by treating negative control siRNA (siCon) or siRNA against B7-H3 (siB7-H3), as shown in Figure 8A, through flow cytometry.
  • Figure 8G is a picture analyzing the expression of the cell adhesion proteins E-Cadherin, Integrin- ⁇ 2, and integrin- ⁇ V.
  • Figure 8G is a statistical result of the experiment in Figure 8F repeated three times.
  • *** represents p value ⁇ 0.005, ** represents p value ⁇ 0.01, and * represents p value ⁇ 0.05.
  • Figure 9A shows single cells of liver cancer cells Huh7 cells in which B7-H3 knockdown was performed by treating negative control siRNA (siCon) or siRNA against B7-H3 (siB7-H3), as shown in Figure 8A, in a 6-well cell.
  • siCon negative control siRNA
  • siB7-H3 siRNA against B7-H3
  • FIG 8A This is a photograph of colonies formed by inoculating a plate and culturing for two weeks stained with a crystal violet solution.
  • the graph is a graph that statistically compares colony forming ability by repeating the same experiment three times and analyzing the number of colonies.
  • Figure 9B shows single cells of HepG2 cells in which B7-H3 knockdown was performed by treating negative control siRNA (siCon) or siRNA against B7-H3 (siB7-H3), as shown in Figure 8A, in a 6-well cell.
  • siCon negative control siRNA
  • siB7-H3 siB7-H3
  • This is a photograph of colonies formed by inoculating a plate and culturing for 2 weeks stained with a crystal violet solution.
  • the graph is a graph that statistically compares the colony forming ability by repeating the same experiment three times and analyzing the number of colonies.
  • *** indicates p value ⁇ 0.005.
  • Figures 10A and 10B show the results of analysis of HepG2 cells separated into B7-H3-high and B7-H3-low using biotinylated NPB40 antibody using PE-conjugated SA and FACSCalibur.
  • Figure 10B shows the results of the same experiment three times. This is a graph that was statistically compared repeatedly.
  • Figures 10C and 10D are photographs of colonies formed by culturing HepG2 cells isolated using the NPB40 antibody in Figure 10A in a 6-well plate for 2 weeks, stained with crystal violet solution, and Figure 10D shows colony forming ability by analyzing the number of colonies. This is a statistically compared graph.
  • *** represents p value ⁇ 0.005, and ** represents p value ⁇ 0.01.
  • Figures 11A and 11B analyze the internalization of monoclonal antibody NPB40 into human embryonic cancer cells NT2/D1, liver cancer cells Huh7, HepG2, and SNU449, and human pancreatic cancer cells BxPC-3, PANC-1, and SNU-213 through flow cytometry. It was done.
  • the solid line is the monoclonal antibody NPB40 reacted at 4°C
  • the dotted line is the monoclonal antibody NPB40 reacted at 37°C after reaction at 4°C
  • the red background includes only the secondary antibody.
  • Figure 11B is a diagram comparing the degree of attachment of NPB40 on the cell surface of each cancer cell by repeating the same experiment in Figure 11A three times and statistically processing the relative average fluorescence intensity. ** indicates p value ⁇ 0.01, *** indicates p value ⁇ 0.005.
  • Figures 12A and 12B are illustrations showing DNA amplified from DNA fragments corresponding to the variable regions of the monoclonal antibody NPB40 heavy chain and light chain genes, respectively, through polymerase chain reaction.
  • Figures 12C and 12D show the monoclonal antibody NPB40 heavy chain and light chain gene variable regions cloned into the pBluescript-KS(+) vector, respectively.
  • the heavy chain DNA was cloned into EcoRI and SalI, respectively
  • Figure 12D the light chain DNA was cloned into HindIII and SalI. The heavy chain and light chain gene variable regions cloned into the pBluescript-KS(+) vector were confirmed.
  • Figure 13 is a diagram showing the base sequence and amino acid sequence of the variable region of the monoclonal antibody NPB40 heavy chain gene, with the CDR (Complementarity Determining Region) that binds to the antigen indicated in bold.
  • Figure 14 is a diagram showing the base sequence and amino acid sequence of the variable region of the monoclonal antibody NPB40 light chain gene, with the CDR (Complementarity Determining Region) that binds to the antigen indicated in bold.
  • Figure 15 shows an agarose gel electrophoresis photograph of the gene fusion process to produce the NPB40 antibody into a chimeric antibody containing human IgG1.
  • Figure 15A is a diagram showing the fusion of the heavy chain signal peptide and the NPB40 heavy chain variable region by recombinant PCR, and
  • Figure 15B shows the cloning process by cutting with EcoRI and ApaI into the pdCMV-dhfr vector containing the human IgG1 constant region.
  • Figure 15C is a diagram showing the fusion of the light chain signal peptide and the NPB40 light chain variable region by recombinant PCR
  • Figure 15D shows the cloning process by cutting with HindIII and BsiWI into the pdCMV-dhfr vector containing the human kappa constant region.
  • Figure 16 shows the amino acid sequence (blue) and constant region (red) of the heavy chain variable region of the mouse-human chimeric NPB40 antibody (Chi-NPB40) containing the signal sequence, the signal peptide sequence (black), the antigen and The residue positions of the binding CDR amino acids are underlined.
  • Figure 17 shows the amino acid sequence (blue) and constant region (red) of the light chain variable region of the mouse-human chimeric NPB40 antibody (Chi-NPB40) containing the signal sequence, the signal peptide sequence (black), the antigen and The residue positions of the binding CDR amino acids are underlined.
  • Figure 18A shows the results of SDS-PAGE and Coomassie blue staining for the mouse-derived NPB40 antibody and chimeric antibody of the present invention.
  • Figure 18B shows the results of Western blotting on the above antibody, and the secondary antibody used was one that binds to the IgG gamma and kappa chains of human antibodies.
  • Figure 19 is a diagram analyzing the internalization of Chi-NPB40 antibody into human liver cancer cells Huh7 and SNU-449 through flow cytometry.
  • the red background represents the chimeric antibody Chi-NPB40 reacted at 4°C
  • the white background represents the chimeric antibody Chi-NPB40 reacted at 37°C after the reaction at 4°C
  • the gray background represents only the secondary antibody.
  • Figures 20A and 20B measure the cell survival rate of Huh7 cells treated with NPB40 or Chi-NPB40.
  • Huh7 cells were treated with 0 nM to 100 nM NPB40 or Chi-NPB40 and treated with 12.7 nM ⁇ -HFc-CL-DMDM for 48 hours. After that, it was analyzed.
  • Figure 20A is a photo of cells and Figure 20B shows cell viability measured using CCK-8. The scale bar is 100 ⁇ m.
  • Figures 20C and 20D measure the cell survival rate of Huh7 cells treated with NPB40 or Chi-NPB40.
  • Huh7 cells were treated with 0 nM to 100 nM NPB40 or Chi-NPB40 and treated with 12.7 nM ⁇ -HFc-CL-MMAF for 48 hours. After that, it was analyzed.
  • Figure 20C is a photo of cells and Figure 20D shows cell viability measured using CCK-8. The scale bar is 100 ⁇ m.
  • * represents p value ⁇ 0.05, ** represents p value ⁇ 0.01, and *** represents p value ⁇ 0.005.
  • Figures 21A and 21B measure the cell survival rate of HepG2 cells treated with NPB40 or Chi-NPB40. HepG2 cells were treated with 0 nM to 250 nM NPB40 or Chi-NPB40 and treated with 12.7 nM ⁇ -HFc-CL-DMDM for 48 hours. After that, it was analyzed.
  • Figure 21A is a photo of cells and Figure 21B shows cell viability measured using CCK-8. The scale bar is 100 ⁇ m.
  • Figures 21C and 21D measure the cell survival rate of HepG2 cells treated with NPB40 or Chi-NPB40.
  • HepG2 cells were treated with 0 nM to 250 nM NPB40 or Chi-NPB40 and treated with 12.7 nM ⁇ -HFc-CL-MMAF for 48 hours. After that, it was analyzed.
  • Figure 21C is a photo of cells and Figure 21D shows cell viability measured using CCK-8. The scale bar is 100 ⁇ m.
  • * represents p value ⁇ 0.05, ** represents p value ⁇ 0.01, and *** represents p value ⁇ 0.005.
  • Figures 22A and 22B measure the cell viability of SNU-449 cells treated with NPB40 or Chi-NPB40.
  • SNU-449 cells were treated with 0 nM to 250 nM NPB40 or Chi-NPB40 and treated with 12.7 nM ⁇ -HFc-CL-MMAF. It was analyzed after processing for 48 hours.
  • Figure 22A is a photo of cells and Figure 22B shows cell viability measured using CCK-8. The scale bar is 100 ⁇ m.
  • * indicates p value ⁇ 0.05
  • *** indicates p value ⁇ 0.005
  • FIG 23 confirms the ADCC function of Chi-NPB40.
  • Huh7 cells which are liver cancer cells, were treated with a human IgG isotype antibody corresponding to Chi-NPB40 at a concentration of 0.01 ⁇ g/ml to 100 ⁇ g/ml, then ADCC core kit (Promega). The ADCC response occurring in the included Jurkat effector cells was measured by observing luminescence.
  • * indicates p value ⁇ 0.05, and *** indicates p value ⁇ 0.005.
  • Figure 24A shows cancer growth observed in nude mice xenografted with Huh7 cells, a liver cancer cell, by administering mouse IgG isotype antibodies and NPB40, respectively. Each antibody was administered intravenously once every two days at a dose of 10 mg/kg. Statistical processing was performed using three cancer xenograft models in each of the experimental and control groups. In Figure 24A, * indicates p value ⁇ 0.05 and *** indicates p value ⁇ 0.005.
  • Figure 24B compares the sizes of cancer masses obtained from mice sacrificed 20 days after the start of drug administration, which is the final time point in Figure 24A.
  • the present invention relates to a heavy chain comprising a heavy chain complementary determine region 1 (HCDR1) comprising SEQ ID NO: 1, a HCDR2 comprising SEQ ID NO: 2, and a heavy chain comprising HCDR3 comprising SEQ ID NO: 3; and light chain complementary determine region 1 (LCDR1) comprising SEQ ID NO: 4, LCDR2 comprising SEQ ID NO: 5, and LCDR3 comprising SEQ ID NO: 6. .
  • HCDR1 heavy chain complementary determine region 1
  • HCDR2 comprising SEQ ID NO: 2
  • HCDR3 comprising SEQ ID NO: 3
  • LCDR1 light chain complementary determine region 1
  • LCDR2 comprising SEQ ID NO: 5
  • LCDR3 comprising SEQ ID NO: 6.
  • the monoclonal antibody of the present invention can bind to the B7-H3 protein of pure human pluripotent stem cells, quasi-human pluripotent stem cells, and cancer cells.
  • the na ⁇ ve human pluripotent stem cell refers to a human pluripotent stem cell before implantation, and is said to have pluripotency in a naive or ground state.
  • Human pluripotent stem cells after implantation refer to primed human pluripotent stem cells that have pluripotency in a quasi-primed state.
  • pure human pluripotent stem cells are distinguished from semi-pluripotent stem cells that grow in a colony-like form in that individual cells grow separately in a small dome shape.
  • pure human pluripotent stem cells are further divided, they can be divided into stem cells in the ground state, and stem cells with pure pluripotency in the intermediate, formative, or naive-like states.
  • the cancer cells correspond to the cancer cells of the present invention without limitation as long as they express B7-H3 on the cell surface.
  • the heavy and light chain sequences of the antibody of the present invention are not limited as long as it has the HCDR sequences of SEQ ID NOs: 1 to 3 and the LCDR sequences of SEQ ID NOs: 4 to 6.
  • it may have a heavy chain sequence including SEQ ID NO: 7 and a light chain sequence including SEQ ID NO: 8, but is not limited thereto.
  • SEQ ID NO: 7 refers to the heavy chain variable region sequence shown in FIG. 16 and SEQ ID NO: 8 refers to the light chain variable region sequence shown in FIG. 17.
  • SEQ ID Nos: 1 to 8 are as shown in Table 1.
  • an antibody may specifically refer to a monoclonal antibody
  • a monoclonal antibody refers to a protein molecule that is directed to a single antigenic site (single epitope) and binds specifically to it.
  • the monoclonal antibody of the present invention binds specifically to B7-H3, a cell surface molecule of pure human pluripotent stem cells, quasi-human pluripotent stem cells, and cancer cells, and thus, pure human pluripotent stem cells, quasi-human pluripotent stem cells. It is a protein molecule that recognizes cell surface molecules of cells and cancer cells.
  • the main region of an antibody that recognizes a specific epitope of an antigen and forms an antigen-antibody complex is the variable region of the heavy and light chains, especially the complementarity determining region (CDR).
  • CDR complementarity determining region
  • the present invention includes the variable region of the above-mentioned monoclonal antibody of the present invention, especially its chimeric antibody, humanized antibody, etc. including the CDR.
  • variable regions of the monoclonal antibodies of the present invention are also included in the scope of the present invention.
  • the invention also includes functional fragments of the antibody molecule as well as intact forms having two full-length light chains and two full-length heavy chains, as long as they have the binding properties described above.
  • Functional fragments of antibody molecules refer to fragments that possess at least an antigen-binding function and include Fab, F(ab'), F(ab')2, and Fv.
  • the present invention relates to a pharmaceutical composition for preventing or treating cancer containing the above antibody.
  • the composition can be used for prevention or treatment regardless of whether the cancer is caused by cancer cells expressing B7-H3 on the cell surface.
  • cancer cells expressing B7-H3 on the cell surface For example, liver cancer, pancreatic cancer, osteosarcoma, skin cancer, lung cancer, human embryonic cancer, neuroblastoma, uterine cancer, prostate cancer, colon cancer, stomach cancer, biliary tract cancer, kidney cancer, bladder cancer, ovarian cancer, brain tumor, etc.
  • the composition of the present invention when the composition of the present invention is treated with the human embryonic cancer cell NT2/D1, liver cancer cell lines Huh7, HepG2, and SNU449 cell lines, and pancreatic cancer cell lines BxPC-3, PANC-1, and SNU-213 cell lines, anticancer activity occurs. It was confirmed that it was effective. However, it is not limited to this.
  • ADCC antibody-dependent cellular cytotoxicity
  • F c ⁇ R F c gamma receptor
  • F c ⁇ R F c gamma receptor
  • NK cells natural killer cells
  • the pharmaceutical composition of the present invention can specifically bind to cancer cells expressing B7-H3, and when treated by including an antibody prepared in the form of IgG1 in the composition, B7 bound to the antibody by the ADCC mechanism -It may be effective in preventing and treating cancer by killing H3-positive cancer cells.
  • ICI therapy immune checkpoint inhibitor therapy
  • Cancer cells suppress immune function or avoid the cancer cell removal mechanism by immune cells through mechanisms such as T cell immunotolerance or immune-editing.
  • ICI expresses proteins on the cell surface that interfere with the destruction of cancer cells and activates inhibitory immune checkpoints to avoid attacks by immune cells.
  • ICI therapy is a treatment method based on the principle that antibodies that bind to proteins that activate these inhibitory immune checkpoints inhibit the function of those proteins, thereby activating the cancer cell death mechanism by immune cells.
  • the pharmaceutical composition of the present invention can specifically bind to B7-H3, which performs the function of an inhibitory immune checkpoint, and therefore, when treated with IgG4 containing the CDR of the antibody, the antibody NK cell, It can also induce cancer cell death by phagocytes and neutrophils.
  • the pharmaceutical composition for preventing or treating cancer containing the antibody may be a conjugation of an antibody and an anti-cancer substance, and the conjugation may be achieved by a linker sequence or a secondary antibody that binds to the antibody. It may be possible.
  • the secondary antibody refers to an antibody that specifically binds to the amino acid sequence of another antibody (primary antibody) that binds to an antigen, and differs from the primary antibody that binds directly to the target antigen in the object to which it binds. It corresponds to a term with .
  • the linker sequence is used to connect an anticancer substance and an antibody.
  • the linker sequence is attached to the Fc sequence of the antibody, and the linker sequence and the anticancer substance are connected to form an antibody-linker-anticancer substance (antibody-linker-anticancer substance). It can be manufactured and used in the form of a linker-drug.
  • the linker or the secondary antibody are all used to bind the antibody of the present invention to an anti-cancer substance, and if it is used to conjugate an antibody and a drug, the linker or the secondary antibody can be used by those skilled in the art regardless of the secondary antibody sequence or linker sequence. It can be used depending on the method.
  • ⁇ -HFc-CL-DMDM AH-102DD, Moradec, USA
  • ⁇ -HFc-CL-MMAF (AH-102AF, Moradec) antibody was used.
  • the anticancer substance may be used without limitation as long as it is an anticancer substance known in the art, for example, erlotinib (TARCEVA(TM), Genentech/OSI Pharm.), bortezomib (VELCADE(TM), Millenium Pharm.
  • erlotinib TARCEVA(TM), Genentech/OSI Pharm.
  • bortezomib VELCADE(TM)
  • Millenium Pharm for example, erlotinib (TARCEVA(TM), Genentech/OSI Pharm.), bortezomib (VELCADE(TM), Millenium Pharm.
  • dynemycin A dynemycin including; bisphosphonates such as clodronate; esperamicin; and the neocarzinostatin chromophore and the related chromoprotein enedine antibiotic chromophore), aclasinomycin, actinomycin, anthramycin, azaserine, and bleo Mycin, cactinomycin, carabicin, carminomycin, carzinophylline, Chlomomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN (TM) Doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin
  • the prevention or treatment of cancer may be induced by an antibody-drug conjugate (hereinafter referred to as ADC) mechanism of action.
  • ADC is a method of injecting a drug by conjugating it to an antibody, and delivers the drug into the cell by inducing internalization of the antibody in cells expressing an antigen that specifically binds to the antibody, thereby delivering the drug in a cell-specific manner. It corresponds to the principle treatment method.
  • chi-NPB40 a mouse-human chimeric antibody of the present invention, is conjugated with the previously mentioned ⁇ -HFc-CL-DMDM (AH-102DD, Moradec, USA) and auristatin.
  • the pharmaceutical composition of the present invention is formulated into a unit dosage form suitable for administration into the patient's body, preferably a preparation useful for the administration of protein drugs, according to a method commonly used in the pharmaceutical field, and is formulated as a preparation commonly used in the art.
  • Methods of administration include intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastric, topical, sublingual, intravaginal, or rectal routes. It may be administered via parenteral administration routes, but is not limited to these.
  • Dosage forms suitable for this purpose include various preparations for oral administration such as tablets, pills, dragees, powders, capsules, syrups, solutions, gels, suspensions, emulsions, microemulsions, and injections such as ampoules for injection.
  • Preparations for parenteral administration such as injections and sprays such as hypospray, are preferred.
  • preparations for injection or infusion they may take the form of a suspension, solution, or emulsion, and may contain formulation agents such as suspending agents, preservatives, stabilizers, and/or dispersants.
  • the antibody molecules may be formulated in a dried form that can be reconstituted in an appropriate sterile liquid prior to use.
  • the antibody can be administered to mammals, including humans, at an amount of 0.01 to 50 mg/kg of body weight per day, preferably 0.1 to 20 mg/kg of body weight, once or divided into several times.
  • the actual dosage of the active ingredient must be determined in light of various related factors such as the disease to be prevented or treated, the severity of the disease, the route of administration, the patient's weight, age and gender, drug combination, reaction sensitivity, and resistance/response to treatment. It should be understood that the above dosages are to be determined and therefore do not limit the scope of the present invention in any way.
  • the present invention relates to hybridomas producing the above antibodies.
  • hybridoma refers to hybridoma cells or hybridoma cell lines, which are cells created by artificially fusing two different types of cells, using polyethylene glycol (PEG) to cause cell fusion. It refers to a cell or cell line in which two or more homogeneous cells or heterogeneous cells are fused using a substance or a certain type of virus, and the different functions of each cell are integrated into one cell.
  • the present invention provides a hybridoma producing the monoclonal antibody NPB40. Specifically, the hybridoma of the present invention is obtained by injecting 2 Lymphocytes were isolated and prepared by fusing them with myeloma cancer cells. Hybridomas that secrete monoclonal antibodies can be cultured in large quantities in vitro or in vivo.
  • the monoclonal antibody produced by the hybridoma may be used without purification, but to obtain the best results, it should be purified to high purity (e.g., 95% or more) according to a method well known in the technical field to which the present invention pertains. It is desirable. These purification techniques include, for example, gel electrophoresis, dialysis, salt precipitation, chromatography, etc., for separation from the culture medium or ascites fluid. In one example of the present invention, for mass production of monoclonal antibodies, hybridoma culture medium was purified using protein G-Sepharose column chromatography. However, any method for cultivating and purifying the hybridoma can be any method known in the art, and is not limited thereto.
  • the present invention relates to a composition for diagnosing cancer containing the above antibody.
  • the antibody can specifically bind to the B7-H3 protein of cancer cells.
  • the B7-H3 protein is overexpressed in various cancer cells, so if the B7-H3 antigen is confirmed in the target cell, it can be said that there is a high probability that the cell is a cancer cell. Therefore, when a composition containing the antibody is treated with a cancer cell expressing B7-H3, the antibody binds to the B7-H3 antigen of the cancer cell to form an antigen-antibody complex, so it does not bind to the cancer cell that binds to the antibody. Can be classified into cells.
  • Cancers that can be diagnosed using the cancer diagnostic composition are preferably cancers known to overexpress B7-H3, such as liver cancer, pancreatic cancer, osteosarcoma, skin cancer, lung cancer, human embryonic cancer, neuroblastoma, uterine cancer, and prostate cancer. , colon cancer, stomach cancer, biliary tract cancer, kidney cancer, bladder cancer, ovarian cancer, brain tumor, etc. However, it is not limited to this.
  • the present invention relates to a method of providing information for cancer diagnosis, including the step of treating a separated sample with the cancer diagnosis composition and detecting a protein to which the antibody is bound. If an antigen-antibody complex in which the antibody binds to a protein is detected, it can be determined that there is a high possibility of being diagnosed with cancer.
  • the sample may be body fluid, tissue, cell, whole blood, plasma, serum, etc., preferably serum, but is not limited thereto.
  • the formation of the antigen-antibody complex can be determined by tissue immunostaining, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation assay, immunodiffusion assay, Complement fixation assay, FACS, protein chip, etc. can be used.
  • tissue immunostaining radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation assay, immunodiffusion assay, Complement fixation assay, FACS, protein chip, etc.
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • Western blotting Western blotting
  • immunoprecipitation assay immunodiffusion assay
  • Complement fixation assay FACS, protein chip, etc.
  • FACS protein chip, etc.
  • Example 1 Culture of human pluripotent stem cells and establishment of pure human pluripotent stem cells
  • Human quasi-pluripotent stem cells H9 were cultured according to a previously described method according to the protocol provided by Wicell Research Institute (Korean Patent No. 2235935).
  • mouse embryonic fibroblasts (MEFs) from pregnant CF1 mice were extracted and cultured, treated with ⁇ -irradiation or mitomycin (10 ⁇ g/ml), and then used as feeder cells.
  • 20% (v/v) KOSR (knockout serum replacement), 1% (v/v) NEAA, 0.1mM ⁇ -mercaptoethanol, 100U/ml penicillin-G, 100 ⁇ g/ml streptomycin, and 8 ⁇ 12ng/ml bFGF were added.
  • nonaive human pluripotent stem cell refers to stem cells that express markers of pure human pluripotent stem cells, unlike primed hPSCs, in the ground state, or intermediate, or formative. It refers to stem cells that express markers of stem cells in the same intermediate state.
  • Quasi-human pluripotent stem cells H9 (primed hPSC) were used to induce conversion into pure human pluripotent stem cells (naive hPSC) and cultured, and the specific protocol followed a previously known method (Korean Patent No. 2235935).
  • semi-human pluripotent stem cells H9 were treated with collagenase IV (1 mg/ml) for 5 minutes, cut to an appropriate size using a yellow tip, and then transferred onto the previously laid MEF.
  • 2iL/X/F/P medium a pure hPSC cell induction medium (1 ⁇ M PD0325901, 3 ⁇ M CHIR99021, hLIF (20 ng/ml), 4 ⁇ M XAV939, 10 ⁇ M Forskolin, 2 ⁇ M Purmorphamine, 20% (v/v) KOSR , DMEM/F12, 1% (v/v) L-glutamine, 1% (v/v) NEAA, 0.1mM ⁇ -mercaptoethanol, 1x penicillin-G, streptomycin) (Zimmerlin et al., 2016, Development 143:4368 ) cells are similar to pure human pluripotent stem cells in a formal/intermediate state (Taei et al., 2020,
  • Human embryonic cancer cells NT-2 and 2102EP were cultured in DMEM medium supplemented with 10% FBS, 1% (v/v) NEAA, 100 U/ml penicillin-G, and 100 ⁇ g/ml streptomycin.
  • Human peripheral blood mononuclear cells (PBMC) were isolated using the method suggested by Ficoll-Paque Plus method (GE Healthcare, Seoul, Korea).
  • CD7, CD77, and CD130 known as background pure human pluripotent stem cell markers
  • SSEA3, SSEA4, TRA-1-60, TRA-1-81, and CD24, excluding CD90 was observed to decrease in pure human pluripotent stem cells
  • pure human pluripotent stem cell markers It was confirmed that pure human pluripotent stem cells were induced, with only the known expression of CD7 and CD77 increased in H9-2iL/X/F/P and CD130 not increased ( Figures 1B and 1C).
  • RNA from primed H9 and naive H9 cells was extracted using RNAiso PLUS (TAKARA) reagent, and the expression of 31 naive hPSC-related genes was compared through RNA-seq transcriptome analysis (3BIGS, Gyeonggi-do).
  • TAKARA RNAiso PLUS
  • 15 naive hPSC-related genes DPPA3/5, KLF2/4/5 were expressed in naive hPSC cells induced by the 2iL/X/F/P method (Zimmerlin et al., 2016, Development 143:4368).
  • pluripotent stem cells were immunized into the footpads of mice, and the specific protocol followed a previously well-described method (Korean Patent No. 2235935).
  • 2 ⁇ 10 6 quasi-human pluripotent stem cell H9 cells were injected into the right footpad of 11 6-week-old female Balb/c mice at -3, 0, 3, 6, 13, 17, 20, and 21 days, and the same Pure human pluripotent stem cell H9 cells were injected into the left sole on days 0, 3, 6, 13, 17, 20, and 21, respectively.
  • the immunized mouse was dislocated at the cervical spine and then injected from the left hind foot.
  • the popliteal lymph node was extracted with fat and muscle tissue removed, and the popliteal lymph node was ground and single-celled using a frosted slide glass with a bumpy surface.
  • 3 ml of DMEM medium for 1 minute, 17 ml for 1 minute, and 20 ml for 1 minute were sequentially added, left to stand for 5 minutes, and then centrifuged to collect the cells.
  • 2 Only hybridomas producing antibodies were selected using .
  • the Sandwich Enzyme Linked Immunosorbent Assay (ELISA) method was used to select clones expressing antibodies. Add 100 ⁇ l of hybridoma culture medium to a plate coated with 2 ⁇ g/ml of anti-mouse IgG or IgM antibody, which is a capture antibody, and react at 37°C for 1 hour, then add anti-mouse IgG-HRP or anti- It was reacted with a 1/5,000 dilution of mouse IgM-HRP (horseradish peroxidase, Sigma-Aldrich) for another hour.
  • ELISA Sandwich Enzyme Linked Immunosorbent Assay
  • Clones producing antibodies were selected first.
  • the culture medium of each clone was first selected through FACS analysis to select hybridomas that bind to pure human pluripotent stem cells.
  • 33 types of hybridomas were selected that simultaneously bind to quasi-human pluripotent stem cells and pure human pluripotent stem cells. were additionally selected and subcloned to secure stable clones.
  • the double antibody NPB40 was classified as a monoclonal antibody with IgG1 and ⁇ chains that simultaneously binds to pure and sub-human pluripotent stem cells (Figure 3), and its recognition antigen and characteristics were analyzed in this patent.
  • Flow cytometry was performed to observe the degree of binding of the monoclonal antibody NPB40 to various cells.
  • Various cancer cells were removed with 0.05% Trypsin-EDTA (Welgene, Gyeongsan, Korea), neutralized with cell culture medium containing 10% fetal bovine serum (VWR), and then passed through a 40 ⁇ m strainer (SPL, Pocheon, Korea).
  • SPL 40 ⁇ m strainer
  • Each single cell was mixed with PBA (1% bovine serum albumin, 0.02% NaN 3 in PBS ) at about 1 After washing twice with PBA, the primary antibody and the corresponding anti-mouse IgG-FITC (Invitrogen) were further reacted at 4°C for 20 minutes.
  • PBA 1% bovine serum albumin, 0.02% NaN 3 in PBS
  • PI sodium iodide-negative cells were analyzed for antibody reaction using FACSCalibur and Cell Quest software (BD sciences).
  • the monoclonal antibody NPB40 did not bind to normal human peripheral blood mononuclear cells (PBMC), but strongly bound to four types of human liver cancer cells, Huh7, HepG2, SNU-387, and SNU-449 ( Figure 4A, Table 2) Additionally, three types of human pancreatic cancer cells, BxPC-3, PANC-1, and SNU-213, two types of human colon cancer cells, Colo-205 and HCT-116, and two types of human osteosarcoma cells, U2OS and Saos2.
  • PBMC peripheral blood mononuclear cells
  • DAPI 4,6-diamidino-2-phenylindole
  • 0.1% PBST PBS containing 0.1% Triton-X-100
  • the monoclonal antibody NPB40 can be seen to bind well to the surface of 2102Ep cells, a human embryonic cancer cell line, and Huh7 and SNU-449 cells, a liver cancer cell line ( Figure 4B).
  • Biotinylation of the surface of the human embryonic cancer cell line NT2/D1 was performed by slightly modifying the protocol presented by EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific). Human embryonic cancer cells NT2/D1 cultured on a 100 mm cell culture plate were washed twice with PBS (pH 7.4), then 5 ml of cold PBS (pH 8.0) containing 0.5 mg of biotin dissolved in them was added, and reacted at 4°C for 30 minutes. .
  • biotin-labeled cells were lysed in lysis buffer (25mM Tris-HCl, pH 7.5, 250mM NaCl, 5mM EDTA, 1% Nonidet P-40, 2 ⁇ g/ml Aprotinin, 100 ⁇ g/ml PMSF). (phenylmethylsulphonyl fluoride), 5 ⁇ g/ml Leupeptin, 1mM NaF, 1mM Na 3 VO 4 ), reacted at 4°C for 30 minutes, centrifuged at 12,000 rpm for 40 minutes to remove nuclei, and stored at -70°C before use. It was stored in .
  • lysis buffer 25mM Tris-HCl, pH 7.5, 250mM NaCl, 5mM EDTA, 1% Nonidet P-40, 2 ⁇ g/ml Aprotinin, 100 ⁇ g/ml PMSF. (phenylmethylsulphonyl fluoride), 5 ⁇ g/ml Leupeptin, 1mM NaF, 1mM
  • Protein G agarose was added to the cell lysate of about 2.0 The Protein G agarose was recovered through centrifugation, and a sample washed five times in lysis buffer was used as a negative control.
  • 4 ⁇ g of antibody was added to the cell lysate from which proteins that non-specifically bind to Protein G agarose were removed, reacted at 4°C for 12 hours, and then added to 20 ⁇ l Protein G agarose. was added and reacted at 4°C for further 4 hours.
  • the immunoprecipitated immune mixture was washed five times in lysis buffer, 5X sample buffer was added to elute the antigen bound to the antibody, and boiled at 100°C for 10 minutes.
  • the negative control protein and the eluted protein were separated through 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and then subjected to Western blotting using a nitrocellulose membrane. This membrane was blocked using 5% skim milk powder for 1 hour at room temperature. After washing three times with 0.1% TBST (Tris-Buffered Saline, 0.1% Tween), streptavidin-HRP (SA-HRP, GE healthcare) was reacted at room temperature for 1 hour.
  • TBST Tris-Buffered Saline, 0.1% Tween
  • Polyacrylamide gels containing proteins immunoprecipitated by the monoclonal antibody NPB40 were stained with PageBlue Protein Staining Solution (Thermo Scientific) according to the supplier's protocol ( Fig. 5B ).
  • the stained protein band at the 110 kDa position presumed to be the protein immunoprecipitated by the monoclonal antibody NPB40, was cut from the gel and submitted for LC-MS/MS (Liquid Chromatography with Tandem Mass Spectrometry) analysis (ProteomeTech, Seoul, Korea).
  • the UniProt database https://www.uniprot.org/ was used to identify proteins from the analyzed mass spectrum.
  • Biotin-labeled cell eluate was prepared by the method described in Example 4-1 above, and this eluate was immunoprecipitated with monoclonal antibody NPB40 and rabbit anti-B7-H3 polyclonal antibody in the same manner as described above. Afterwards, the negative control protein without antibody (No Ab) and the eluted protein were separated through 10% SDS-PAGE and transferred to a nitrocellulose membrane for Western blotting. This membrane was blocked for 1 hour at room temperature using 5% skim milk powder. After washing three times with 0.1% TBST, streptavidin-HRP (SA-HRP) was added and reacted at room temperature for 1 hour.
  • SA-HRP streptavidin-HRP
  • the B7-H3 gene expression vector pCMV3-B7H3-His (Sino Biological) was purchased and His was expressed in human embryonic kidney cells HEK293T. Tagged recombinant B7-H3 was overexpressed, and the lysate of these cells was subjected to immunoprecipitation and Western immunoprecipitation using mouse anti-His monoclonal antibody (Invitrogen) and rabbit anti-B7-H3 polyclonal antibody ( ⁇ -B7-H3). Blots were performed.
  • This membrane was blocked for 1 hour at room temperature using 5% skim milk powder.
  • a known mouse anti-His monoclonal antibody or rabbit anti-B7-H3 polyclonal antibody ( ⁇ -B7-H3) was reacted at 4°C for 12 hours.
  • anti-mouse IgG-HRP (1:10,000; Millipore) or anti-rabbit IgG-HRP (1:15,000) was further reacted at room temperature for 1 hour. After washing three times with 0.1% TBST, it was confirmed with an ECL detection kit.
  • siRNA targeting the B7-H3 gene was purchased and transfected into liver cancer cell lines Huh7, HepG2, SNU-387, and SNU-449.
  • siRNAsiCon Genolution, Seoul, Korea
  • siRNA against B7-H3 siB7-H3
  • Lipofectamine RNAiMAX Invitrogen
  • the cells were removed with 0.05% Trypsin-EDTA (Welgene, Gyeongsan, Korea) and neutralized with cell culture medium containing 10% fetal bovine serum (VWR). After lysis with cell lysis buffer, 5X sample buffer was added and boiled at 100°C for 10 minutes. The siCon protein and siB7-H3 protein were separated through 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and then subjected to Western blotting using a nitrocellulose membrane.
  • Trypsin-EDTA Welgene, Gyeongsan, Korea
  • VWR fetal bovine serum
  • B7-H3 knockdown liver cancer cells Huh7 and HepG2 bind well to the cell culture plate.
  • B7-H3 knockdown liver cancer cell lines Huh7 and HepG2 were prepared , 3.0 It was placed in a well plate and cultured for 2-3 hours in an incubator at 37°C supplied with 5% CO 2 and 95% air. After washing twice with PBS (pH 7.4), the attached cells were fixed with 2% Paraformaldehyde at room temperature for 15 minutes and stained with 0.5% crystal violet (Sigma-aldrich) dissolved in 2% ethanol for 30 minutes at room temperature.
  • B7-H3 knockdown Huh7 cells prepared through the above experiment were removed with 0.05% Trypsin-EDTA (Welgene, Gyeongsan, Korea), neutralized with cell culture medium containing 10% fetal bovine serum (VWR), and the cells were seeded at 40 ⁇ m. Single cells were prepared by passing them through a strainer (SPL, Pocheon, Korea). 1.0 _ Analysis was performed using the Image J program, and the same experiment was repeated three times to statistically indicate the colony-forming ability of the cells ( Figure 9A).
  • B7-H3 deficiency reduced the number and size of Huh7 colonies by approximately 3.6-fold.
  • B7-H3 deficiency reduced the number and size of HepG2 colonies by approximately 1.4-fold (Figure 9B).
  • human hepatocytes HepG2 were treated with the monoclonal antibody NPB40 to determine whether cells expressing B7-H3 highly (B7-H3-high) or cells expressing B7-H3 at low levels (B7-H3) were used. After separation by sorting with -H3-low), clonogenic survival assay of each cell group was performed and compared.
  • NPB40 antibody was first biotinylated using the DSB-X biotin labeling kit (Thermo Fischer Scientific) according to the manufacturer's protocol.
  • B7-H3-high The group with high B7-H3 expression (B7-H3-high), which strongly bound to biotinylated NPB40, was separated using magnetic beads and then reacted with releasing buffer at room temperature for 10 minutes to remove the cells from the beads.
  • the group with low B7-H3 expression (B7-H3-low) did not bind to the magnetic beads, so the cell group was collected and centrifuged.
  • Each cell group was analyzed with PE conjugated SA (Phycoerythrin-conjugated streptavidin) and FACSCalibur, and it was confirmed that the B7-H3high cell group isolated by NPB40 had approximately twice as high B7-H3 expression as the low group ( Figure 10A, 10B).
  • Example 7 Analysis of B7-H3 cell membrane internalization after NPB40 treatment in liver cancer and pancreatic cancer cell lines
  • RNAiso plus reagent TaKaRa, Otsu, Japan
  • the amount of RNA was quantified by measuring A 260 of the obtained total RNA. Add 1 unit of DNase° per 1 ⁇ g of total RNA and react at 37°C for 30 minutes to remove remaining DNA, then add 1 ⁇ l of 50mM EDTA and react at 85°C for 5 minutes to inactivate DNase° and denature the total RNA. I ordered it.
  • a reverse transcription polymerase chain reaction mixture was prepared using total RNA and the Prime Script RT reagent Kit (TaKaRa), and cDNA was synthesized according to the supplier's protocol.
  • TaKaRa Prime Script RT reagent Kit
  • cDNA was synthesized according to the supplier's protocol.
  • TGG TGC AGC ATC-3' (SEQ ID NO: 15) oligonucleotide and 5'MK 5'-cgg aag ctt GAY ATT GTG MTS ACM CAR WCT MCA-3' (SEQ ID NO: 16), a primer corresponding to the N terminus of the kappa chain variable region. ) and were used, respectively.
  • EcoRI and SalI restriction enzyme sites were assigned to the 5'-primer end for the heavy chain and SalI restriction enzyme sites to the 3'-primer end.
  • 5'- HindIII at the end of the primer and SalI restriction enzyme site at the end of the 3'-primer were mixed with each heavy or light chain polymerase reaction primer and 1 unit of i-pfu DNA Polymerase (iNtRON Biotechnology, Seoul, Korea), first at 95°C. It was reacted once for 5 minutes and then 30 times for 1 minute at 95°C, 1 minute at 35°C, and 1 minute at 72°C.
  • the DNA fragment corresponding to the heavy chain variable region was estimated to be about 400bp in length, and the light chain Amplified DNA was obtained at a position corresponding to approximately 390 bp, which is the estimated length of the DNA fragment corresponding to the variable region ( Figures 12A and 12B).
  • the polymerase chain reaction product was first treated with EcoRI and SalI for the heavy chain and HindIII and SalI for the light chain, and then spread on a 1.0% (w/v) agarose gel using FavorPrep. DNA corresponding to approximately 400bp and 390bp was isolated using the GEL PCR Purification Kit (Favorgen, Pingtung, Taiwan).
  • pBluescript KS+ which was used as a vector for cloning the heavy chain gene, was treated with EcoRI and SalI
  • pBluescript KS+ which was used as a cloning vector for the light chain gene, was treated with HindIII and SalI and then isolated using the FavorPrep GEL PCR Purification Kit. These two DNAs were linked with T4 DNA ligase (New England Biolab, Massachusetts, USA) and transformed into E. coli DH5 ⁇ using the CaCl 2 method.
  • the above-mentioned clones were cultured overnight in 3 ml of LB medium containing 100 ⁇ g/ml ampicillin and then cultured using the DNA-Spin plasmid mini prep kit (Intron, Korea) according to the supplier's protocol. Plasmid DNA was isolated and E. coli clones with a DNA insert of about 400 bp for the heavy chain ( Figure 12C) and E. coli clones with a DNA insert of about 390 bp for the light chain were selected ( Figure 12D). The base sequence of each DNA insert was confirmed through Nucleotide sequencing (Bionics, Seoul, Korea).
  • the antigen recognition decision sites according to the antibody structure were organized through Kabat numbering.
  • the heavy chain corresponded to subgroup II and the light chain corresponded to subgroup V.
  • the antigen binding sites, CDR1, 2, and 3, for each sequence are indicated in dark color ( Figures 13 and 14).
  • mouse-derived monoclonal antibodies When mouse-derived monoclonal antibodies are used in humans, there is a possibility of causing immune side effects. To overcome this, mouse-derived monoclonal antibodies can be produced as mouse-human chimeric antibodies with fewer immune side effects and used as therapeutic antibodies.
  • To develop a mouse-human chimeric antibody (Chi-NB40) of the mouse-derived monoclonal antibody NPB40 the NPB40 antibody gene and the constant region of the human antibody were combined, and this was cloned into an expression vector to produce the Chi-NPB40 antibody. manufactured.
  • DNA corresponding to about 400bp and 390bp of the heavy and light chain genes of NPB40 obtained from mice was isolated through PCR (polymerase chain reaction) using a Gel extraction kit (FAVORGEN, Taiwan).
  • h2B7-H.C-SLIC-5 ⁇ (5 ⁇ -GCC AGT GTG CTG GAA TTC ACT) was prepared using the pdCMV-dhfr vector (Korean Patent No. 2120223) as a template.
  • PCR was performed using primers CTA ACC-3 ⁇ , SEQ ID NO. 17) and NPB40-Chi-HC-SP-3 ⁇ (5 ⁇ -CTG CAC CTG GGA GTG GAC ACC TGT AGT TA-3 ⁇ , SEQ ID NO. 18) primer. carried out.
  • NPB40-Chi-HC-SP-5 ⁇ (5 ⁇ -GTC CAC TCC CAG GTG CAG CTG CAG-3 ⁇ , SEQ ID NO: 19) primer was used using the DNA of the heavy chain variable region as a template. PCR was performed using primers and NPB40-chi-HC-3 ⁇ (5 ⁇ -CCT TGG TGG AGG CTG AGG AGA CTG TGA G-3 ⁇ , SEQ ID NO: 20).
  • the pdCMV-dhfr vector was used as a template, h2B7-L.C-SLIC-5 ⁇ (5 ⁇ -ATA GGG AGA CCC AAG CTT CGG CAC GAG CAG A-3 ⁇ ) , SEQ ID NO: 22) primer and NPB40-Chi-LC-SP-3 ⁇ (5 ⁇ -CAT CAC AAT ATC TCC TTC AAC ACC AGA CA-3 ⁇ , SEQ ID NO: 23) primer pair PCR was performed.
  • NPB40-Chi-LC-SP-5 ⁇ (5 ⁇ -GTT GAA GGA GAT ATT GTG ATG ACA CAG TC-3 ⁇ , SEQ ID NO: 24 )
  • PCR was performed using primers Chi63-B6-LC-SLIC-3 ⁇ (5 ⁇ -TGG TGC AGC CAC CGT ACG TTT GAT TTC CA-3 ⁇ , SEQ ID NO: 25).
  • the DNA prepared by the above PCR was mixed as a template, and primers h2B7-L.C-SLIC-5 ⁇ and Chi63-B6-LC-SLIC-3 ⁇ were used as a template. Recombinant PCR was performed using . As a result of running the PCR result on a 1% agarose gel, DNA corresponding to about 430 bp, in which the light chain variable region and light chain signal peptide sequence were linked, was confirmed (Figure 15C).
  • coli clones with a light chain gene insert of about 430 bp were selected.
  • DNA sequencing of antibody genes the above-mentioned clones were cultured overnight in 5 ml of LB medium containing 50 ⁇ g/ml ampicillin, and then plasmid DNA was isolated using the DNA-spinTM Plasmid DNA purification kit (INTRON, Korea). and confirmed the base sequence of each DNA insert (Bionics, Korea). It was confirmed that the DNA base sequences of the heavy and light chain variable regions of pdCMV-dhfr-chi-NPB40 were identical to the gene sequences of the existing NPB40 variable regions, and were correctly linked to the signal peptide sequence and the human heavy and light chain constant region genes ( Figure 16, Fig. 17).
  • the chimeric antibody linked to the NPB40 mouse variable region and the human constant region was named Chi-NPB40.
  • Chi-NPB40 The chimeric antibody linked to the NPB40 mouse variable region and the human constant region was named Chi-NPB40.
  • DMEM Biowest, France
  • 50 ⁇ g of pdCMV-dhfr-Chi-NPB40 expression vector and 75 ⁇ l of Polyethyleneimine (1 mg/ml) were mixed, mixed with 500 ⁇ l of transfection optimization medium, and sprinkled on the cell culture medium of the cultured cells. After culturing for 24 hours, the cell culture medium was collected and the antibodies were purified.
  • the collected cell culture medium was placed in a column filled with Protein G agarose beads (Amicogen, Korea) to allow the antibody to bind to the Protein G agarose beads. After washing the beads with PBS (pH 8.0), the beads were washed with 0.1M glycine. Antibodies were eluted from Protein G agarose beads by adding 10ml (pH2.8) and 1M Tris-HCl (pH9.0). Afterwards, the purity of the purified Chi-NPB40 chimeric antibody was confirmed through SDS-PAGE and Coomassie blue staining (Figure 18A).
  • Flow cytometry was performed to observe cell membrane internalization of the chimeric antibody Chi-NPB40 in liver cancer cell lines Huh7 and SNU449.
  • Cells were removed with 0.05% Trypsin-EDTA (Welgene, Gyeongsan, Korea), neutralized with cell culture medium containing 10% fetal bovine serum (FBS; VWR, PA, USA), and then passed through a 40 ⁇ m strainer to form single cells. It was prepared with Each single cell, approximately 1x10 5 per ml, was mixed with PBA (1% bovine serum albumin, 0.02% NaN 3 in PBS) and then reacted with the chimeric antibody Chi-NPB40 at 4°C for 30 minutes.
  • PBA 1% bovine serum albumin, 0.02% NaN 3 in PBS
  • the cells were washed with PBA to remove antibodies that did not bind to the cells, and the cells were suspended in 100 ⁇ l of culture medium and incubated at 37°C for 30 minutes so that the bound antibodies could be internalized into the cell membrane.
  • the primary antibody and the corresponding anti-human IgG-FITC Invitrogen were further reacted at 4°C for 20 minutes.
  • PI propidium iodide-negative cells were analyzed for antibody reaction using FACSCalibur and Cell Quest software (BD sciences).
  • the fluorescence intensity of the chimeric antibody decreased in human liver cancer cells Huh7 and SNU-449 when additional reaction was performed at 37°C ( Figure 19).
  • the chimeric antibody Chi-NPB40 which binds to B7-H3 on the surface of liver cancer cells at 4°C, can induce the internalization of B7-H3 into the cell when the cell is active at 37°C, which is similar to the mouse monoclonal antibody NPB40 and It shows that it performs the same function.
  • the NPB40 antibody with a mouse IgG1 constant region and the chimeric Chi-NPB40 antibody with a human IgG1 constant region bind to the surface of liver cancer cells and are internalized by the cells at a temperature of 37°C. Based on these characteristics, it was expected that when an anticancer drug was conjugated to the Chi-NPB40 antibody, the Chi-NPB40 antibody would deliver the drug inside the cancer cells and exhibit cytotoxicity.
  • drug-conjugated human IgG Fc region-specific secondary antibodies [anti-human IgG (Fc Specific) antibody-drug conjugates (ADCs)] were used.
  • ⁇ -HFc-CL-DMDM (AH-102DD, Moradec, USA) antibody conjugated with duocamycin, a DNA alkylation agent, and ⁇ -HFc-CL-MMAF (AH-102AF, conjugated with monomethyl auristatin, a tubulin polymerization inhibitor). Moradec) antibody was used.
  • 5,000 Huh7 cells were seeded into each well of a 96 well plate (SPL, Korea) using RPMI-1640 (Biowest, France) medium containing 10% FBS and cultured for 24 hours.
  • the existing culture medium was removed, and then the cells were treated with the chimeric Chi-NPB40 antibody diluted in the culture medium at various concentrations or the control mouse-derived NPB40 antibody at concentrations of 0, 0.01, 0.1, 1, 10, and 100 nM. did.
  • the medium containing the antibody was replaced with medium in which the secondary antibody was diluted to a concentration of 12.7 nM.
  • Cell Counting Kit-8 was used to determine the survival rate of the cells; CCK-8 (K1018, APExBIO, USA) was used.
  • liver cancer cell survival rate increased to 18% at a Chi-NPB40 antibody concentration of 100 nM and 38% at a Chi-NPB40 antibody concentration of 250 nM. It was confirmed that this decreased ( Figures 22A, 22B).
  • ADCC antibody-dependent cellular cytotoxicity
  • mice administered NPB40 showed lower cancer growth compared to the control group.
  • NPB40 reduced cancer growth in vivo. This confirms that NPB40 can be used for the prevention and treatment of cancer in vivo.

Abstract

La présente invention concerne : un anticorps monoclonal NPB40 se liant de manière spécifique à B7-H3 sur la surface de cellules souches pluripotentes naïves humaines, des cellules souches pluripotentes amorcées humaines et des cellules cancéreuses humaines ; un hybridome le produisant ; une composition pharmaceutique pour prévenir/traiter le cancer à l'aide de celui-ci ; une composition pour le diagnostic du cancer à l'aide de celui-ci ; et un procédé pour fournir des informations pour le diagnostic du cancer. La présente invention concerne en outre une composition ciblant B7-H3 et pouvant être utilisée pour la prévention, le traitement et le diagnostic du cancer.
PCT/KR2023/003515 2022-03-18 2023-03-16 Anticorps monoclonal anti b7-h3 et son utilisation WO2023177232A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015181267A1 (fr) * 2014-05-29 2015-12-03 Spring Bioscience Corporation Anticorps anti-b7-h3 et leurs utilisations diagnostiques
WO2019225787A1 (fr) * 2018-05-24 2019-11-28 에이비엘바이오 주식회사 Anticorps anti-b7-h3 et son utilisation
US10604582B2 (en) * 2014-09-17 2020-03-31 The United States Of America, As Represented By The Secretary, Department Of Health Anti-CD276 antibodies (B7H3)
KR20210094589A (ko) * 2018-11-22 2021-07-29 쑤저우 카노바 바이오파마슈티컬 컴퍼니 리미티드 항-b7-h3 항체

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Publication number Priority date Publication date Assignee Title
WO2015181267A1 (fr) * 2014-05-29 2015-12-03 Spring Bioscience Corporation Anticorps anti-b7-h3 et leurs utilisations diagnostiques
US10604582B2 (en) * 2014-09-17 2020-03-31 The United States Of America, As Represented By The Secretary, Department Of Health Anti-CD276 antibodies (B7H3)
WO2019225787A1 (fr) * 2018-05-24 2019-11-28 에이비엘바이오 주식회사 Anticorps anti-b7-h3 et son utilisation
KR20210094589A (ko) * 2018-11-22 2021-07-29 쑤저우 카노바 바이오파마슈티컬 컴퍼니 리미티드 항-b7-h3 항체

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