WO2016155420A1 - Utilisation d'anticorps ciblant spécifiquement wnt16b dans la préparation d'un médicament anti-tumoral - Google Patents

Utilisation d'anticorps ciblant spécifiquement wnt16b dans la préparation d'un médicament anti-tumoral Download PDF

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WO2016155420A1
WO2016155420A1 PCT/CN2016/073709 CN2016073709W WO2016155420A1 WO 2016155420 A1 WO2016155420 A1 WO 2016155420A1 CN 2016073709 W CN2016073709 W CN 2016073709W WO 2016155420 A1 WO2016155420 A1 WO 2016155420A1
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tumor
wnt16b
cells
psc27
sfrp2
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孙宇
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中国科学院上海生命科学研究院
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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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/136—Amines having aromatic rings, e.g. ketamine, nortriptyline having the amino group directly attached to the aromatic ring, e.g. benzeneamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin

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  • the present invention belongs to the field of biomedicine, and more particularly, the present invention relates to the use of an antibody that specifically targets WNT16B for the preparation of an antitumor drug.
  • P-glycoprotein which is an ATP-dependent drug efflux pump
  • MDR multidrug resistance-associated protein
  • GSTs glutathione S transferase
  • TOPO II topoisomerase II
  • protein kinase C The intracellular content and activity of PKC
  • tumor cells are not isolated, and their evolutionary path is affected by the surrounding microenvironment.
  • the biological effects imposed are strongly dependent on the latter.
  • the Tumor mass of the tumor is mostly stromal tissue.
  • the interaction of tumor cells with normal cells in their microenvironment is essentially through secreted proteins and cell surface proteins, which are critical for tumor growth and evolution.
  • tumor-matrix interaction has profound effects on many steps in the development of malignant tumors, including neovascularization, immune surveillance and escape, and through blood circulation and lymphatic metastasis.
  • the non-cancer component (Nonneoplastic) in the microenvironment can secrete a variety of factors, promoting the survival, growth and evolution of cancer cells at different stages of the tumor.
  • an antibody which specifically inhibits WNT16B for the preparation of a medicament for inhibiting tumors, including inhibiting tumor growth, or reversing tumor resistance.
  • the antibody that specifically inhibits WNT16B is an anti-WNT16B monoclonal antibody.
  • it is a monoclonal antibody (clone F4-1582) of WNT16B of BD Pharmingen.
  • the tumor is a tumor that is therapeutically damaged or a tumor that is resistant.
  • the tumor of the therapeutic injury comprises: a tumor treated with a chemotherapeutic drug, or a tumor treated by radiotherapy (such as irradiation); preferably, the chemotherapeutic drug comprises: mitoxantrone , doxorubicin, epirubicin, 5-fluorouracil, platinum compounds, bleomycin, mitomycin, etoposide and the like.
  • the inhibiting the tumor comprises: promoting tumor cell apoptosis, preventing the tumor from obtaining drug resistance from the microenvironment of the damage-activated or increasing the sensitivity of the tumor to the chemotherapeutic drug.
  • the tumor is a solid tumor.
  • the solid tumor comprises: prostate cancer, breast cancer.
  • a pharmaceutical composition for inhibiting tumors comprising: an antibody that specifically inhibits WNT16B; and a chemotherapeutic drug are provided.
  • kits for inhibiting tumors comprising: an antibody that specifically inhibits WNT16B; and a chemotherapeutic drug are provided.
  • the chemotherapeutic drug comprises: mitoxantrone, doxorubicin, epirubicin, 5-fluorouracil, platinum compound, bleomycin , mitomycin, etoposide and the like.
  • the tumor is a solid tumor; preferably, the tumor comprises: prostate cancer, breast cancer.
  • FIG. 1 Genome-wide expression profiling revealed that a soluble factor SFRP2 involved in the regulation of Wnt signaling pathway is highly up-regulated in prostate stromal cells (PSC27) treated with genotoxic agents. Heatmap compares cells with RNA that includes hydrogen peroxide (H 2 O 2 ), bleomycin, and radiation treatment in parallel with undamaged RNA signals. WNT16B and SFRP2 are indicated in blue and pink, respectively. These two extracellular factors are the core of this work and the target of related technologies.
  • PSC27 prostate stromal cells
  • FIG. 2A-2B molecular biology to identify the expression status of SFRP2 in prostate stromal cells.
  • Figure 2A Fluorescence quantitative qRT-PCR analysis of SFRP2 expression under several conditions. The other two are commonly used in the clinic. The chemotherapeutic agents mitoxantrone (MIT) and satraplatin (SAT) administered to patients with adenocarcinoma were involved in parallel experiments.
  • Figure 2B Western blot analysis of SFRP2 protein expression in PSC27 cells, including intracellular (IC) and conditioned media (CM) extracts.
  • IC intracellular
  • CM conditioned media
  • Figures 3A-3B Expression status of SFRP2 in breast stromal cells.
  • Figure 3A Fluorescence quantitative qRT-PCR analysis of SFRP2 expression in breast stromal cells (HBF1203). The chemotherapeutic agents cisplatin (CDDP), carboplatin (CBDCA), doxorubicin (DXR), and epirubicin (EPIR), which are commonly used in breast cancer patients, are involved in parallel experiments.
  • Figure 3B Western blot analysis of SFRP2 protein expression in HBF1203 cells, including intracellular (IC) and conditioned media (CM) extracts.
  • IC intracellular
  • CM conditioned media
  • FIG. 5 Fluorescence quantitative qRT-PCR analysis of the expression of a group of DDSP factors in a stromal cell (PSC27) after a DNA damage in a certain period of time.
  • the subjects listed in the figure are representative proteins, including MMP1, WNT16B, SFRP2, SPINK1, MMP3, IL8, EREG, ANGPTL4, CCL26, AREG.
  • Figures 6A-6B show differential expression characteristics of SFRP2 between different cell types.
  • Figure 6A Prostate stromal cell line (PSC27) and several epithelial cancer cell lines (BPH1, M12, PC3, DU145, LNCaP, VCaP) were simultaneously treated with genotoxic agents and qRT-PCR analysis compared their expression of SFRP2. The value is relative to the internal reference signal RPL13A.
  • Figure 6B Western blot analysis of the induction of SFRP2 expression at the protein level after radiation injury, using the same cell line as Figure 6A.
  • FIG. 7 Induction of SFRP2 expression in several types of cells in colorectal tissues after chemotherapy and radical surgery.
  • Laser capture microdissection (LCM) was used to obtain the above types of cells, followed by gene expression analysis.
  • Benign prostate epithelium, neoplastic prostate epithelium and prostate stroma represent benign colorectal epithelial cells, cancerous colorectal epithelial cells and cells in colorectal stromal tissue, respectively.
  • Each group of samples consisted of 10 patients, each representing one patient. The value is relative to the sample internal reference signal RPL13A.
  • the three cell samples after chemotherapy are labeled in green, blue, and red, respectively.
  • Figures 8A-8C systematic studies and determination of the inducible expression of SFRP2 are regulated by the NF- ⁇ B complex.
  • Figure 8A shows the presence of the NF- ⁇ B binding site in the 5' proximal upstream promoter region of the transcription start site (TSS) by fragment cloning and site-directed mutagenesis analysis of the proximal promoter region of SFRP2.
  • TSS transcription start site
  • a series of reporter vectors constructed for 11 potential NF- ⁇ B binding sites in the range of +198 bp to -4000 bp upstream of TSS 5'. Black box, wild sequence; white box, mutant sequence.
  • PSC27 undergoes radiation treatment after transfection of the reporter vector, and the signal is compared.
  • Figure 8B compares the reported signal intensity of NF- ⁇ B in PSC27 cells under conditions of genotoxic conditions (MIT, SAT) and biochemical reagents (TNF- ⁇ ).
  • NAT11-Luc2C is a positive control vector.
  • Figure 8C chromatin immunoprecipitation (ChIP) analysis.
  • Several sets of conditions include blank control, NF- ⁇ B sedimentation, total input RNA and no antibody (Ab) before and after treatment (pre) (RAD) for comparison.
  • the p1, p2, p3 and p4 used in the PCR reaction were 4 sets of primers for amplifying SFRP2, and WNT16B-p1 and IL8-p1 were two sets of control primers with known sequences.
  • Figures 9A-9D the biological importance of the transcriptional complex NF- ⁇ B for the inducible expression of DDSP secreting factors.
  • Figure 9A shows the nuclear transfer of NF- ⁇ B subunits p50/p105 and p65 after DNA damage by a stromal cell line expressing IKB ⁇ variant (PSC27 IKB ⁇ ). Cytosol and Nucleus are protein expression in the cytoplasm and nucleus, respectively.
  • Figure 9B in the case of NF- ⁇ B nuclear translocation hindered, PSC27 was analyzed for expression of WNT16B and IL8 after genotoxic BLEO, SAT and RAD injury, respectively.
  • FIG 9C NF- ⁇ B activity after stromal cell DNA damage was analyzed on the premise that NF- ⁇ B activity was inhibited by the drug Bay11-7082. The value is the luciferase signal produced by the reporter vector.
  • Figure 9D in the case where NF- ⁇ B intracellular activity was inhibited, PSC27 was analyzed for expression of WNT16B and IL8 after genotoxic BLEO, SAT and RAD lesions, respectively.
  • FIG. 10 Fluorescence quantitative qRT-PCR analysis of the expression changes of a group of DDSP factors under NF- ⁇ B specific inhibition of stromal cells before and after DNA damage.
  • the subjects listed in the figure are typical DDSP secreted proteins, including MMP1, WNT16B, SFRP2, SPINK1, MMP3, IL8, EREG, ANGPTL4, CCL26, AREG.
  • FIG 11A-11B shows changes in SFRP2 expression levels after treatment with SFRP2-specific small hairpin RNA (shRNA) in the PSC27 stromal cell line.
  • Figure 11A under undamaged conditions. SCR, scramble RNA; SFRP2#1 and SFRP2#2 are two shRNAs directed against SFRP2.
  • Figure 11A shows the expression of stromal cells WNT16B and SFRP2 under intact conditions, compared to two PSC27 stable lines that express WNT16B and SFRP2, respectively.
  • shRNA small hairpin RNA
  • FIG. 12 Biological relationship between activation of the canonical Wnt/ ⁇ -catenin signaling pathway and WNT16B and SFRP2.
  • the prostate cancer epithelial cell lines M12 and PC3 were cultured in vitro under various conditions of CM derived from PSC27 substrain, and the reporter gene firefly enzyme protein activity was detected.
  • PSC27-Pre original PSC27 line; PSC27-RAD, damaged PSC27 line; PSC27-RAD/WNT16B KD, first WNT16B knockout and then damaged PSC27 line; PSC27-RAD/SFRP2KD, first SFRP2 knockout and then damaged PSC27 line; PSC27-RAD/WNT16B-SFRP2KD, first knockdown of WNT16B and SFRP2 and then damaged PSC27 line; PSC27-SFRP2, PSC27 stable line with high expression of SFRP2; PSC27-WNT16B, PSC27 stable system with high expression of WNT16B PSC27-WNT16B-SFRP2, PSC27 stably transfecting WNT16B and SFRP2; constitutive ⁇ -catenin, a subline transfected with ⁇ -catenin expression vector.
  • TOPFlash/FOPFlash is a pair of reporting vectors for detecting canonical Wnt signaling pathways.
  • Figure 13 The canonical Wnt signaling pathway activated by WNT16B is assisted by SFRP2 and can be abolished by DKK1.
  • Figure 13A after treatment with PDC27CM containing WNT16B, ⁇ -catenin was stabilized in PC3 and the amount of protein was increased, while LRP6 was phosphorylated.
  • Figure 13B Immunoprecipitation analysis mediated by WNT16B antibody.
  • the membrane receptor FZD3/4/6 can be bound by WNT16B, a process that is promoted by SFRP2, but is completely inhibited once DKK1 is added.
  • FIG. 14A-14C Proliferation (A), migration (B) and invasion (C) activity of PC3 cells can be produced by PSC27-RAD
  • the CM has increased dramatically. Once SFRP2 was eliminated from PSC27, the above activity decreased significantly; in contrast, WNT16B knocked out from PSC27, the activity decreased more significantly; the effect of WNT16B and SFRP2 double knockout, the effect of WNT16B single elimination similar.
  • WNT16B significantly upregulates the tolerance of prostate cancer cells to chemotherapeutic agents.
  • Several prostate cancer cell lines showed significant apoptosis after being poisoned by mitoxantrone, but were all attenuated by CM produced by PSC27-RAD.
  • SFRP2 and WNT16B shRNA SFRP2 or shRNA WNT16B were knocked out from PSC27, the resistance of cancer cells decreased, but the latter caused more obvious effects.
  • Figure 16 The ability of WNT16B monoclonal antibody purified by affinity chromatography, refolded and renatured to recognize WNT16B antigen in PSC27 after DNA damage is compared with the antibody in its original state. Lanes 1-4, purified antibodies, lanes 5-8, unpurified antibodies.
  • Figure 17 Picture of PC3 cells under a bright field microscope. After the Mitoxantrone reached IC50 concentration in the culture medium, the cancer cells under several conditions showed different survival states. Among them, the addition of WNT16B-specific antibodies significantly reduced the resistance of cells to genotoxicity.
  • Figures 18A-18B show the resistance of PC3 cells to chemotherapeutic agents in vitro.
  • Figure 18A CM produced by PSC27, once used in PC3 cell culture, can significantly reduce the extent of apoptosis.
  • the WNT16B antibody is used to treat extracellular fluids in stromal cells, the resistance of cancer cells is significantly reduced.
  • XAV939 a canonical Wnt pathway specific inhibitor, served as a positive control for this experiment.
  • Figure 18B Survival curves of PC3 under the above conditions, demonstrate that the effect of WNT16B antibody is similar to that produced by direct use of XAV939.
  • FIG. 19 Intra-tissue response of immunodeficient mouse SCID via renal subcapsular transplantation (PC3/PSC27) to chemotherapeutic drugs.
  • Placebo (P) and WNT16B monoclonal antibodies did not significantly affect the transplanted tumor tissues of mice that were not administered with chemotherapy.
  • WNT16B antibody can cause significant blockade of ⁇ -catenin nuclear translocation in cancer cells, ie most ⁇ -catenin remains in the cytoplasm.
  • WNT16B antibody caused an increase in the degree of apoptosis in prostate cancer (PC3) tissues under chemotherapy conditions.
  • Figure 20A, placebo and WNT16B antibodies have little effect on tissue.
  • the use of the WNT16B antibody resulted in a significant increase in the proportion of apoptotic cells throughout the tissue.
  • FIG. 21 The effect of WNT16B antibody was determined by analysis of another prostate cancer cell line, VCaP, under similar conditions, further revealing the role of WNT16B in cancer cell acquired resistance.
  • FIG 22 Treatment of WNT16B antibody with immunodeficient mouse SCID trans-subcapsular transplantation (cancer cell/stromal cells). It was found that DNA-damaged stromal cells PSC27-RAD can be compared to intact stromal cells PSC27. Significantly increase the tumor volume. However, the use of the WNT16B antibody reduced the terminal volume by approximately 20%.
  • Figures 23A-23B show the physiological reality under clinical treatment conditions for chemotherapy and/or antibody administration to immunodeficient mice bearing a transplanted tumor.
  • Figure 23A is a schematic representation showing the sequence and time node of transplantation of recombinant tissue, chemotherapy administration, and antibody administration. Note that within two weeks after tumor implantation, the mice were not treated with drugs in order to allow the graft to be successfully absorbed by the tissue; after the transplantation was stabilized, the animals were dosed from the third week onwards.
  • Figure 23B at the end of the 8th week pre-clinical trial, the tumor volume under the various treatment conditions above was measured and analyzed. It was found that chemotherapy itself reduced tumors by nearly 40%, and the use of WNT16B antibodies further reduced tumor volume by about 35%.
  • FIG. 24 Mice bearing the VCaP/PSC27 xenografts showed a growth profile similar to that of PC3/PSC27 xenografts after treatment with Figure 23A.
  • the use of the WNT16B antibody can further reduce the tumor volume by about 30%.
  • Figure 25 Photograph of fresh tumors under pre-clinical conditions. Comparative analysis of the growth of transplanted tumors in mice that had not been treated with drugs, treated with chemotherapy alone, and treated with chemotherapy and antibody treatment.
  • FIG. 26 Immunofluorescence staining analysis of expression changes of epithelial-mesenchymal transition (EMT)-associated marker molecule E-cad (green) and canonical Wnt pathway marker protein ⁇ -catenin (red) in tissues.
  • EMT epithelial-mesenchymal transition
  • MIT chemotherapy causes E-cad to decline and ⁇ -catenin to rise, but combination therapy including WNT16B antibodies can reverse this trend, resulting in an increase in E-cad and a decrease in ⁇ -catenin.
  • FIG. 27 Western blot analysis of WNT16B expression in different organs of mice in the case of genotoxic chemotherapy and WNT16B antibody alone or in combination.
  • --actin is a solid tissue internal reference and Albumin is a serum internal reference.
  • an antibody that specifically targets WNT16B can effectively inhibit the biological activity of WNT16B, thereby promoting tumor cell apoptosis and preventing tumor from being acquired from the microenvironment of damage activation.
  • the role of the drug which ultimately reduces the resistance of the tumor to chemotherapy drugs.
  • DDSP DNA damage secretion program
  • the present inventors have found that human somatic cells exhibit a strong and long-lasting secretory phenotype during senescence, and this result is concentrated in the stromal cell population after DNA damage.
  • the inventors have named it "DNA damage secretion program” (DDSP), provided that the cells are subjected to a certain degree of DNA damage stress, that is, gene poison treatment.
  • DDSP is distinctive and unique in several ways. First, the procedure is relatively complex, with hundreds of proteins being induced to different degrees simultaneously; secondly, the response is dramatic, many transcripts and their encoded proteins are up-regulated by a factor of 10; and third, depending on the cell type The difference is that this program has obvious consistency and difference.
  • MMPs proteases
  • HGF growth factors
  • VEGF pro-angiogenic factors
  • IL6, IL8 pro-inflammatory cytokines
  • DDSP broad-spectrum secretory group of DDSP
  • protein factors that can cause epithelial cancer cells to undergo EMT transformation, including IL6, IL8, MMP3, MMP8, MMP10, MMP12, WNT16B.
  • IL6, IL8, MMP3, MMP8, MMP10, MMP12, WNT16B Preliminary results showed that prostate cancer cells showed a high proportion of stem cell populations and related expressions of CD133-positive, CD44 High /CD24 Low (with LSC High , Epcam Low /CD24 Low ) after cytosolic cell secretion culture after DNA damage.
  • Sex markers Snail, Twist, Zeb upregulation
  • stem cells are more tumorigenic both in vitro and in vivo.
  • the stromal cell-based DDSP secretory phenotype caused by the chemotherapy process itself can ultimately confer the biological characteristics of a cancer stem cell to the surrounding tumor cells, which will exhibit a stronger local invasion activity and a distant metastatic tendency.
  • the present invention provides a use of specifically targeting a WNT16B antibody for the preparation of a medicament for inhibiting tumors.
  • the tumor of the present invention is a solid tumor such as prostate cancer or breast cancer.
  • the tumor may be a tumor that is therapeutically damaged, or a tumor that acquires resistance under chemotherapeutic conditions.
  • therapeutic damage refers to the destruction of tumor cells after treatment with anti-tumor, primarily DNA damage (including single and double strand breaks).
  • the anti-tumor treatment includes, for example, chemotherapy (chemotherapy) or radiotherapy (such as radiation therapy).
  • the antibody that specifically inhibits WNT16B may be a monoclonal antibody or a polyclonal antibody.
  • the WNT16B protein can be used to immunize animals, such as rabbit mice, rats and the like, to produce polyclonal antibodies; various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.
  • cells expressing WNT16B or its antigenic fragments can be used to immunize animals to produce antibodies.
  • the antibodies may also be monoclonal antibodies, which may be prepared using hybridoma technology (see Kohler et al, Nature 256; 495, 1975; Kohler et al, Eur. J. Immunol. 6: 511, 1976; Kohler et al, Eur. J. Immunol. 6: 292, 1976; Hammerling et al, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981).
  • the present invention also provides a composition
  • a composition comprising an effective amount (e.g., 0.000001 to 50% by weight; preferably 0.00001 to 20% by weight; more preferably 0.0001-10% by weight) of the antibody specifically inhibiting WNT16B, and A pharmaceutically acceptable carrier.
  • the composition further comprises an effective amount (e.g., 0.000001 to 50% by weight; preferably 0.00001 to 20% by weight; more preferably, 0.0001 to 10% by weight) of a chemotherapeutic drug.
  • the composition can be used to inhibit tumors.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • pharmaceutical carriers which are not themselves essential active ingredients and which are not excessively toxic after administration.
  • the pharmaceutically acceptable carrier in the composition may contain a liquid such as water, saline, or a buffer.
  • auxiliary substances such as fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
  • the vector may also contain a cell transfection reagent.
  • the antibody or its pharmaceutical composition that specifically targets WNT16B can be administered to a mammal using a variety of methods well known in the art. These include, but are not limited to, subcutaneous injection, intramuscular injection, transdermal administration, topical administration, implantation, sustained release administration, and the like; preferably, the administration mode is parenterally administered.
  • the effective amount of the antibody or the pharmaceutical composition containing the same for inhibiting WNT16B according to the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like.
  • the selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials).
  • the factors include, but are not limited to, the pharmacokinetic parameters of the antibody that specifically targets WNT16B, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the patient's Immunization status, route of administration, etc.
  • the normal human prostate primary stromal cell line PSC27 and the primary mammary stromal cell line HBF1203 were propagated and passaged in PSCC complete medium.
  • Prostate benign epithelial cell line BPH1, prostate cancer epithelial cell line M12, DU145, PC3, LNCaP and VCaP purchased from ATCC were all in 5% FBS in RPMI-1640 complete medium at 37 ° C, 5% CO 2 Culture in an incubator.
  • Logarithmic growth phase cells were harvested with 0.25% trypsin, centrifuged at 1000 rpm for 2 min, the supernatant was discarded, and the cells were resuspended in freshly disposed cryopreservation. The cells are dispensed into the indicated sterile cryotubes. Then cooled by gradient (4 ° C 10 min, -20 ° C 30 min, -80 ° C 16-18 h), and finally transferred to liquid nitrogen for long-term storage.
  • the frozen cells in liquid nitrogen were taken out and immediately placed in a 37 ° C water bath to allow rapid melting. 2 ml of the cell culture medium was directly added to uniformly suspend the cells. After the cells are attached, replace the new medium.
  • PSC27 cells were grown to 80% (referred to as PSC27-Pre) with 0.6 mM hydrogen peroxide (PSC27-H 2 O 2 ), 100 ⁇ g/ml bleomycin (PSC27). -BLEO), 1 ⁇ M mitoxantrone (PSC27-MIT), 10 ⁇ M satraplatin (Satraplatin/JM216, PSC27-SAT) or 137 Cs ionizing radiation (dosage 10 Gy, PSC27-RAD). HBF1203 cells were plated in culture medium containing 100 ⁇ M cisplatin, 20 ⁇ M carboplatin, 20 ⁇ M doxorubicin or 25 ⁇ M epirubicin. Six hours after the drug treatment, the cells were briefly washed three times with PBS, left in the culture solution for 7 to 10 days, and then subjected to subsequent experiments.
  • Full-length human SFRP2 (GenBank accession number: NM_003013) was cloned between the blank expression vector pCMV6-AC (Origene) cleavage site RsrII and NotI, digested with BamHI and XhoI, and subcloned into pLenti-Puro (Addgene )in.
  • Human WNT16B (GenBank accession number: NM_016087) was cloned in pLenti-CMV/2-Puro-DEST (Invitrogen). The packaging line 293FT was used for cell transfection and lentiviral production.
  • Small hairpin RNAs for knockout of SFRP2 and WNT16B were purchased from GE Healthcare Dharmacon and Thermo Scientific, respectively.
  • shRNA sequence used to knock out SFRP2 (if not specifically indicated in the figure, SFRP2 shRNA1 is used):
  • the shRNA sequence used to knock out WNT16B (when not specifically indicated in the figure, WNT16B shRNA1 is used):
  • WNT16B shRNA1 (#1) sequence:
  • WNT16B shRNA2 (#2) sequence:
  • Mouse monoclonal antibody anti-phospho-Histone H2A.X (Ser139) (clone JBW301, Millipore) and rabbit polyclonal antibody anti-SFRP2 (Santa Cruz), and secondary antibody Alexa 488 (or 594)-conjugated F(ab')2 was added sequentially to slides coated with fixed cells.
  • the nuclei were counterstained with 2 ⁇ g/ml of 4',6-diamidino-2-phenylindole (DAPI). The most representative image was selected from the three observation fields for data analysis and results display.
  • the antibodies E-cadherin (clone HECD-1) and ⁇ -catenin (clone 12F7) used for mouse tissue staining were purchased from abcam.
  • the FV1000 laser scanning confocal microscope (Olympus) was used to obtain confocal fluorescence images of cells.
  • the PSC27 cells were cultured for 3 days with DMEM + 0.5% FBS medium, and then the abundance of the cell population was washed with 1 time PBS. After simple centrifugation, the supernatant was collected and stored as a conditional medium at -80 ° C or used directly. Prostate epithelial cells were cultured in vitro in this conditional medium for 3 days. For the canonical Wnt pathway block, the final concentration of DKK1 in the culture was 10 nM, and the cell response was measured after 48-72 hours of addition.
  • epithelial cell lines are cultured in low serum DMEM (0.5% FBS) ("DMEM"), or in conditioned medium, while mitoxantrone is used to treat cells for 1 to 3 days at concentrations close to each IC 50 values of cell lines, followed by a bright field microscope.
  • DMEM low serum DMEM (0.5% FBS)
  • mitoxantrone is used to treat cells for 1 to 3 days at concentrations close to each IC 50 values of cell lines, followed by a bright field microscope.
  • the total RNA of the growth phase was extracted by the Trizol method. Reverse transcription reaction and cDNA production.
  • the reverse transcription reaction product cDNA was diluted 50-fold as a template.
  • the reaction conditions were: pre-denaturation at 95 ° C for 15 s, then 95 ° C for 5 s, 60 ° C for 31 s, 40 cycles; the melting curve conditions were 95 ° C for 15 s, 60 ° C for 30 s, and 95 ° C for 15 s.
  • the samples were reacted on an ABI ViiA7 (ABI) instrument.
  • the expression of ⁇ -actin was used as an internal reference.
  • the amplification of each gene was analyzed by software analysis, and the corresponding number of domain value cycles were derived.
  • the relative expression of each gene was calculated by the 2- ⁇ Ct method. The peaks and waveforms of the melting surve are analyzed to determine if the resulting amplification product is a specific single-purpose fragment.
  • RIPA cell lysis buffer (Invitrogen) containing 1 mM PMSF (protease inhibitor) was added, and the cells were lysed on ice for 30 min, and the cell lysate was collected with a cell scraper at 1 ° C for 12,000 °C. Centrifuge at rpm for 15 min, take the supernatant, and store at -80 °C.
  • the conventional method was carried out by SDS-PAGE electrophoresis.
  • the nitrocellulose filter was blocked in a blocking solution (TBST (0.1% Tween-20in TBS) containing 5% skim milk powder) for 1 hour at room temperature. Incubate overnight in a primary anti-hybrid solution at 4 °C. Rinse 3 times with TBST at room temperature for 2 minutes each time. HRP-conjugated secondary antibody hybrids prepared in blocking solution were added and incubated for 0.5 hour at room temperature. The filter was rinsed 3 times with PBST at room temperature for 2 min each time.
  • TBST 0.1% Tween-20in TBS
  • the anti-viral vector pBabe-Puro-I ⁇ B ⁇ -Mut (super repressor) containing two IKK phosphorylation mutation sites S32A and S34A encoding the I ⁇ B ⁇ protein sequence was used to transfect the lentiviral packaging cell line PHOENIX.
  • Lentivirus was subsequently used to infect the PSC27 stromal cell line, while 1 ⁇ g/ml puromycin was used to screen for positive clones.
  • 5 ⁇ M of the small molecule inhibitor Bay 11-7082 purchased from Selleck was used for NF- ⁇ B activity control.
  • the stromal cells are then exposed to several different forms of cytotoxicity, and the resulting phenotype is recorded in time to analyze the expression of related genes. The cells treated in this manner are collected and the conditioned medium is collected for various detections of epithelial cells.
  • the human SFRP2 gene (Genbank accession number: NM_003013) was analyzed using the software CONSITE to discover potential core NF- ⁇ B binding sites.
  • ChIP analysis was performed on PSC27 cells (such as p8) with early algebra and PSC27 cells irradiated with gamma rays (10 Gy).
  • the chromosome immobilized in vitro was subjected to sedimentation treatment using mouse monoclonal antibody anti-p65 antibody (F-6, Santa Cruz), and DNA was extracted for amplification.
  • the sequence containing the SFRP2 putative NF- ⁇ B binding site upstream of the 5' end was amplified from the human genomic library and cloned into pCR2.1-TOPO (Invitrogen) and pGL4.22 vector (Promega). Reporter expression vectors carrying multiple NF- ⁇ B binding site mutations were designed and generated by the site-directed mutagenesis (Strategene) method.
  • NF- ⁇ B binding sites and optimized IL-2 minimal promoter are included as the reporter vector for NF- ⁇ B activated transgenic system (NAT system) NAT11-Luc2CP-IRES-nEGFP (Japan Hokkaido University Dr.Hatakeyama friendship) Provided) used as a positive control in the experiment.
  • NAT system NF- ⁇ B activated transgenic system
  • NAT11-Luc2CP-IRES-nEGFP Japan Hokkaido University Dr.Hatakeyama friendship
  • the monoclonal antibody of WNT16B (clone F4-1582, BD Pharmingen) was subjected to column purification by affinity chromatography using Sephadex G25 gel filtration. After one-time chromatographic purification under denaturing conditions, a previously optimized dialysis procedure to aid protein renaturation is used to increase the specific immunological activity of the antibody, while the refolding procedure includes continuous dialysis that gradually reduces the urea concentration. Finally, antibody specificity and affinity as purified products were determined by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance experiments, respectively.
  • ELISA enzyme-linked immunosorbent assay
  • ICF SCID mice (body weight about 25 g) of immunodeficient mice of about 6 weeks old were used for animal experiments.
  • the transplanted tumor was implanted into the mouse by subcapsular transplantation, and the animals were euthanized 8 weeks after the end of the transplantation.
  • mice subcutaneously transplanted under the kidney were fed a standard experimental diet, and after 2 weeks, the chemotherapy drug mitoxantrone (0.2 mg/kg dose) and/or WNT16B antibody (500 ⁇ l, 10 mg/kg) were administered. Dosage) intraperitoneal administration. The time point was the first day of the 3rd, 5th, and 7th week, and the entire course of treatment was administered in 3 cycles, each cycle being 2 weeks. At the end of the course, mouse kidneys were collected for tumor measurement and histological analysis. Each mouse cumulatively received a total of 0.6 mg/kg body weight of mitoxantrone and 30 mg/kg body weight of WNT16B antibody.
  • mitoxantrone was administered to mice by intravenous infusion according to the above steps and sequences, but the dose was reduced to 0.1 mg/kg body weight per time (the total dose received Mito The sputum dose is 0.3 mg/kg body weight) to reduce drug-related toxicity.
  • the WNT16B antibody is administered by intraperitoneal injection with mitoxantrone, and the dose is correspondingly half.
  • the chemotherapy trial was carried out until the end of the 8th week. After the mice were sacrificed, multiple organs including the prostate, lung, colon, and peripheral blood were collected for systematic analysis.
  • Anticancer drugs under clinical conditions can cause significant cellular physiological fluctuations, including DNA damage and increased tumor degenerative responses, including apoptosis, autophagy, and aging procedures.
  • PSC27 primary stromal cell line
  • SFRP2 was significantly up-regulated in PSC27 cells with an average amplitude of 25-fold (P ⁇ 0.01), indicating that stromal cells were under genetic stress.
  • This protein factor can be specifically expressed with high specificity (Fig. 1).
  • SFRP2 showed consistent inducible expression patterns in both prostate and mammary gland-derived stromal cells (Fig. 2A-B; Fig. 3A-B). These data indicate that high expression of SFRP2 is not restricted to certain genotoxic drugs or specific organs, but can be induced in various types of organs by various forms of DNA-damaging drugs.
  • SFRP2 As a soluble factor encoded by the DNA damage secretory program (DDSP), SFRP2 is released into extracellular conditioned medium once it is synthesized by therapeutic induction in fibroblasts. ) (Fig. 2B; Fig. 3B; Fig. 4). Compared to the acute stress response exhibited by fibroblasts after DNA damage (usually the first 24 to 72 hours), SFRP2 upregulation is more easily detected after one week, and this pattern is detected. It is in fact consistent with most of the other factors in the DDSP top list, including MMP1, WNT16B, SPINK1, MMP3, IL-8 and EREG ( Figure 5).
  • SFRP2 is highly expressed in the blood vessels of 85% of human breast cancer patients. Need to determine: Up-regulation of SFRP2 by DNA damage is a common phenomenon in lesions, or in certain cell subpopulations, including some well-defined cells isolated from the primary or metastatic site. system.
  • SFRP2 is more prone to inducible expression in fibroblasts than in slightly reactive epithelial cell lines, even when these differences Types of cell lines were challenged with the same dose of cytotoxicity (Fig. 6A-B).
  • WNT16B Considering the expression characteristics of WNT16B (ie, more prone to up-regulation in stromal cells), this result suggests that there may be some specific SFRP2 regulatory mechanism in non-cancerous tissues surrounding cancer cells, making the protein therapeutically toxic. Under the influence, it shows a very similar expression pattern to WNT16B.
  • Example 2 NF- ⁇ B transcriptional complex mediates high expression of SFRP2 under gene toxic stress conditions
  • NF- ⁇ B signaling pathway as the major transcriptional complex in mammalian cells, can stimulate DDSP and form a senescence-associated secretory phenotype (SASP).
  • SASP senescence-associated secretory phenotype
  • PSC27 I ⁇ B ⁇ PSC27 subline
  • NF- ⁇ B in the control cells can be translocated into the nucleus, showing a markedly increased reporter activity ( ⁇ 10 3 fold increase) with WNT16B and IL8. Up-regulated expression (Figure 9B-C).
  • the human SFRP protein family is a superfamily of five highly conserved members that play a broad role in embryonic development and pathology. Some studies have shown that SFRP2 can inhibit the Wnt/ ⁇ -catenin signaling pathway and thus inhibit tumorigenesis. In contrast, it has also been reported in the literature that SFRP2 can be expressed in solid tumors including glioma and renal cancer by activating the canonical Wnt signaling pathway to induce tumor growth, or by non-classical stimulation of angiogenesis. The promotion of cancer. Once the WNT16B-mediated classical pathway is activated, ⁇ -catenin is stabilized in the cytosol and subsequently enhances TCF/LEF signaling activity by nuclear translocation.
  • SFRP2 To determine the effect of SFRP2, the inventors first chose to knock out SFRP2 with gene-specific shRNAs (Fig. 11A).
  • the transcriptional activity of the downstream factor of TCF/LEF can be enhanced by the conditional culture medium of the injured fibroblast (PSC27-RAD), and this change is mainly mediated by WNT16B; however, once WNT16B is knocked out, the change is immediately Disappeared (P ⁇ 0.05) ( Figure 12).
  • PSC27-SFRP2 conditional medium was used to culture PC3 cancer cells, the TCF/LEF-dependent reporter signal activity remained essentially unchanged, suggesting that SFRP2 itself does not activate this pathway ( Figure 12).
  • the extracellular factor DKK1 can generally antagonize Wnt signaling by binding to the co-receptor LRP6 of the Wnt pathway, thus preventing the formation of the FZD/LRP6 complex and subsequent phosphorylation of LRP6 induced by GSK3.
  • DKK1 In the presence of DKK1, neither WNT16B nor SFRP2 induced classical Wnt signaling with concomitant loss of LRP6 phosphorylation (Fig. 13A).
  • the inventors' data indicate that recombinant DKK1 actually blocks the classical Wnt signal triggered by WNT16B and further enhanced by SFRP2.
  • WNT16B stimulates the canonical Wnt pathway and transduces signals across the membrane, but whether WNT16B recognizes specific Wnt receptors other than LRP6 has not been known.
  • G protein-coupled receptor G-protein-coupled receptors, GPCRs
  • GPCRs G protein-coupled receptors
  • Example 4 SFRP2 expands WNT16B signal intensity and increases the malignant phenotype of prostate cancer cells
  • the DDR program for damage response covers a broad spectrum of secretory factor sets with the potential to alter various characteristics of cancer cells by paracrine.
  • Conditioned broths produced by irradiated PSC27 cells (PSC27-RAD) increased proliferation, migration and invasion activity of malignant prostate epithelial cell lines (Fig. 14A, B, C).
  • SFRP2 was absent, irradiated fibroblasts (PSC27-RAD+shRNA SFRP2 ) caused a relatively mild effect on cancer cells, with a range of approximately 10-12% (P ⁇ 0.05), depending on each cell line.
  • conditional culture medium produced by the release of PSC27 after injury enhanced the viability of PC3 cells exposed to mitoxantrone in the concentration range of 0.1 to 1 ⁇ M, but the WNT16 antibody reduced this protection.
  • the effect of WNT16 antibody was very close to that of XAV939, a Wnt pathway-specific inhibitor, which was used as a positive control in this experiment (Fig. 18B).
  • the present inventors performed a subcapsular transplantation operation on immunodeficient mice by using a recombinant tissue prepared by mixing PC3 cancer cells and PSC26 fibroblasts in a predetermined ratio (4:1) in advance. Two weeks after the vaccination period, after the mice generally absorbed the transplanted tumor, a single dose of mitoxantrone or placebo was administered with WNT16B or non-specific immunoglobulin IgG. Mice were sacrificed 7 days after taking the drug, and the tissues were dissected for immunostaining and analysis.
  • Example 6 Targeting WNT16B under therapeutic conditions can deprive tumors of adaptive resistance obtained from a microenvironment that is activated by injury
  • the inventors have grafted PC3 cancer cells and pre-irradiated PSC27 cells (PSC27-RAD) in vitro.
  • the transplanted tumor was constructed and a relatively longer-term preclinical follow-up was performed.
  • the WNT16B antibody was used for a single injection.
  • the mice were dissected to facilitate tumor volume measurement.
  • the tumor volume of the experimental group (PC3 + PSC27-RAD) harboring PC3 and impaired PSC27 was increased to 587 mm 3 compared to the control PSC27 group (PC3 + PSC27) having an average volume of about 300 mm 3 (Fig. 22).
  • WNT16B antibody treatment did not change the size of the PC3+PSC27 group, but reduced the volume of PC3+PSC27-RAD xenografts to 467 mm 3 , which is a 20.5% reduction (P ⁇ 0.01).
  • the present inventors subsequently sought to accurately mimic the physiological reality in a clinical setting by directly transplanting a tumor containing PSC27 cells in an uninjured or primitive state, followed by an 8-week course of treatment with 3 mitox,
  • the treatment cycle consisted of one week at a time, and the administration of the drug started from week 3 (Fig. 23A).
  • Chemotherapy against the PC3 tumor group significantly reduced tumor volume (59.2%, P ⁇ 0.001), but the WNT16B antibody did not further enhance this effect (Fig. 23B).
  • Epithelial-to-mesenchymal transition is a morphological change of epithelial cancer cells caused by fibroblasts under therapeutic injury, which can promote disease resistance and can be significantly induced by WNT16B.
  • EMT Epithelial-to-mesenchymal transition
  • the cytotoxicity produced by mitoxantrone caused a decrease in the typical E-cadherin expression in the cytoplasm and a simultaneous accumulation of ⁇ -catenin in the nucleus (Fig. 26).
  • a specific antibody targeting WNT16B is used for administration, this trend is clearly suppressed.
  • the same results also appeared in VCaP/PSC27 xenografts.
  • WNT16B and other protein factors produced by the microenvironment under chemotherapeutic conditions may represent a new class of biomarkers that will contribute to real-life clinical diagnosis, efficacy evaluation, and especially as an evaluation of cancer resistance.

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Abstract

La présente invention concerne l'utilisation d'un anticorps ciblant spécifiquement WNT16B dans la préparation d'un médicament anti-tumoral. L'anticorps ciblant spécifiquement WNT16B peut inhiber efficacement l'activité biologique de WNT16B, promouvoir la réponse apoptotique de cellules tumorales, empêcher la tumeur d'acquérir une résistance à partir du micro-environnement activé par une lésion, augmenter la sensibilité de la tumeur aux agents chimiothérapeutiques, et finalement améliorer l'effet thérapeutique d'un traitement anti-tumoral.
PCT/CN2016/073709 2015-03-27 2016-02-06 Utilisation d'anticorps ciblant spécifiquement wnt16b dans la préparation d'un médicament anti-tumoral WO2016155420A1 (fr)

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CN101687005A (zh) * 2004-07-09 2010-03-31 加利福尼亚大学董事会 采用抑制Wnt 16信号转导的试剂治疗癌症的方法

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CN101687005A (zh) * 2004-07-09 2010-03-31 加利福尼亚大学董事会 采用抑制Wnt 16信号转导的试剂治疗癌症的方法

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Title
SUN, YU ET AL.: "Treatment-Induced Damage to the Tumor Microenvironment Promotes Prostate Cancer Therapy Resistance through WNT16B", NATURE MEDICINE, vol. 18, no. 9, 30 September 2012 (2012-09-30), pages 1359 - 1368, XP055319225 *

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