Methods to kill cancer cells or inhibit the growth of cancer cells

Abstract

In certain embodiments, provided herein is a composition comprising an isolated heavy chain-hyaluronan / pentraxin 3 ("HC-HA / PTX3") complex for use in a method for killing cancer cells. Also provided herein is a method for inhibiting tumor cancer cell regrowth in an individual in need thereof, comprising contacting the area surrounding the tumor after a surgical procedure with an isolated HC-HA / PTX3 complex. Also provided herein are combinations and kits for use in carrying out the method.

Description

[Technical Field]

[0001] cross reference

[0001] This application claims priority and benefit to U.S. Provisional Application No. 62 / 975,599, filed on 12 February 2020, the entire contents of which are incorporated herein by reference. [Background technology]

[0002]

[0002] All publications, patents, and patent applications referenced in this specification are incorporated herein by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference. [Overview of the project] [Means for solving the problem]

[0003]

[0003] Disclosed herein is a method for directly killing cancer cells in a subject where the killing of cancer cells is required, comprising the step of contacting cancer cells with an isolated HC-HA / PTX3 complex. In some embodiments, the cancer cells originate from or are contained within a solid tumor. In some embodiments, the cancer cells originate from a cancer selected from the group consisting of liver cancer, pancreatic cancer, bladder cancer, prostate cancer, lung cancer, non-small cell lung cancer, ovarian cancer, breast cancer, melanoma, gastric cancer, colon cancer, colorectal cancer, central nervous system (CNS) cancer, bone cancer, lymphoma, skin cancer, head and neck cancer, kidney cancer, testicular cancer, uterine cancer, cervical cancer, esophageal cancer, thyroid cancer, salivary gland cancer, adrenal cancer, and gastrointestinal cancer. In some embodiments, the cancer is inoperable. In some embodiments, the CNS cancer is a glioma or metastatic cancer. In some embodiments, the glioma is a glioblastoma pleomorphic or anaplastic astrocytoma. In some embodiments, the cancer is lung cancer, breast cancer, colon cancer, or skin cancer. In some embodiments, colon cancer is adenocarcinoma. In some embodiments, skin cancer is melanoma. In some embodiments, cancer is prostate cancer. In some embodiments, the contact step includes injecting the HC-HA / PTX3 complex into the tumor, surrounding tissue, or a combination thereof. In some embodiments, the contact step is performed before, during, or after surgical excision, cryoexcision, or radiofrequency ablation of cancer cells. In some embodiments, the contact step includes applying HC-HA / PTX3 to the excision margin of surgical excision of cancer cells or within any remaining portion of the tumor. In some embodiments, the isolated HC-HA / PTX3 complex is a native HC-HA / PTX3 complex, a reconstituted HC-HA / PTX3 complex, or a combination thereof. In some embodiments, the native HC-HA / PTX3 complex is isolated from fetal supporting tissue. In some embodiments, the reconstituted HC-HA / PTX3 complex comprises heavy chain 1 (HC1) and heavy chain 2 (HC2) of inter-α inhibitors (IαI), hyaluronic acid (HA), and PTX3. In some embodiments, the reconstituted HC-HA / PTX3 complex comprises HC1, HC2, HA, PTX3, and TSG-6.In some embodiments, HC-HA / PTX3 is included in a composition comprising a pharmaceutically acceptable diluent, excipient, vehicle, or carrier. In some embodiments, the method further includes a step of administering a further therapeutic agent. In some embodiments, the therapeutic agent is selected from the group consisting of chemotherapeutic agents, analgesics, anti-inflammatory agents, steroids, immunotherapies, cell therapies, radiotherapy, targeted drug therapies, and antibiotics. In some embodiments, the step of administering the therapeutic agent is performed before contacting the cancer cells with the HC-HA / PTX3 complex. In some embodiments, the step of administering the therapeutic agent is performed after contacting the cancer cells with the HC-HA / PTX3 complex. In some embodiments, the step of administering the therapeutic agent is performed simultaneously with contacting the cancer cells with the HC-HA / PTX3 complex. In some embodiments, angiogenesis is reduced or inhibited. In some embodiments, cancer cell death is by apoptosis or necrosis.

[0004]

[0004] Disclosed herein are methods for directly inhibiting the proliferation of cancer cells in subjects requiring inhibition of cancer cell proliferation, comprising the step of contacting cancer cells with an isolated HC-HA / PTX3 complex. In some embodiments, the cancer cells originate from or are contained within a solid tumor. In some embodiments, the cancer cells originate from a cancer selected from the group consisting of liver cancer, pancreatic cancer, bladder cancer, prostate cancer, lung cancer, non-small cell lung cancer, ovarian cancer, breast cancer, melanoma, gastric cancer, colon cancer, colorectal cancer, central nervous system (CNS) cancer, bone cancer, lymphoma, skin cancer, head and neck cancer, kidney cancer, testicular cancer, uterine cancer, cervical cancer, esophageal cancer, thyroid cancer, salivary gland cancer, adrenal cancer, and gastrointestinal cancer. In some embodiments, the cancer is inoperable. In some embodiments, the CNS cancer is a glioma or metastatic cancer. In some embodiments, the glioma is glioblastoma multiforme or anaplastic astrocytoma. In some embodiments, the cancer is lung cancer, breast cancer, colon cancer, or skin cancer. In some embodiments, colon cancer is adenocarcinoma. In some embodiments, skin cancer is melanoma. In some embodiments, cancer is prostate cancer. In some embodiments, the contact step includes injecting the HC-HA / PTX3 complex into the tumor, surrounding tissue, or a combination thereof. In some embodiments, the contact step is performed before, during, or after surgical excision, cryoexcision, or radiofrequency ablation of cancer cells. In some embodiments, the contact step includes applying HC-HA / PTX3 to the excision margin of surgical excision of cancer cells. In some embodiments, the isolated HC-HA / PTX3 complex is a native HC-HA / PTX3 complex, a reconstituted HC-HA / PTX3 complex, or a combination thereof. In some embodiments, the native HC-HA / PTX3 complex is isolated from fetal supporting tissue. In some embodiments, the reconstituted HC-HA / PTX3 complex comprises heavy chain 1 (HC1) and heavy chain 2 (HC2) of inter-α inhibitors (IαI), hyaluronic acid (HA), and PTX3. In some embodiments, the reconstituted HC-HA / PTX3 complex comprises HC1, HC2, HA, PTX3, and TSG-6. In some embodiments, it comprises a pharmaceutically acceptable diluent, excipient, vehicle, or carrier.In further embodiments, HC-HA / PTX3 is included in a composition comprising the administration of a therapeutic agent. In some embodiments, the therapeutic agent is selected from the group consisting of chemotherapeutic agents, analgesics, anti-inflammatory agents, steroids, immunotherapies, cell therapies, radiotherapy, targeted drug therapies, and antibiotics. In some embodiments, the step of administering the therapeutic agent is performed before contacting cancer cells with the HC-HA / PTX3 complex. In some embodiments, the step of administering the therapeutic agent is performed after contacting cancer cells with the HC-HA / PTX3 complex. In some embodiments, the step of administering the therapeutic agent is performed simultaneously with contacting cancer cells with the HC-HA / PTX3 complex. In some embodiments, angiogenesis is reduced or inhibited. In some embodiments, proliferation is inhibited in cells expressing CD44 or RHAMM.

[0005]

[0005] Methods for directly reducing the metabolic activity of cancer cells in subjects requiring a reduction in the metabolic activity of cancer cells are described herein, comprising the step of contacting cancer cells with an isolated HC-HA / PTX3 complex. In some embodiments, the cancer cells originate from or are contained within a solid tumor. In some embodiments, the cancer cells originate from a cancer selected from the group consisting of liver cancer, pancreatic cancer, bladder cancer, prostate cancer, lung cancer, non-small cell lung cancer, ovarian cancer, breast cancer, melanoma, gastric cancer, colon cancer, colorectal cancer, central nervous system (CNS) cancer, bone cancer, lymphoma, skin cancer, head and neck cancer, kidney cancer, testicular cancer, uterine cancer, cervical cancer, esophageal cancer, thyroid cancer, salivary gland cancer, adrenal cancer, and gastrointestinal cancer. In some embodiments, the cancer is inoperable. In some embodiments, the CNS cancer is a glioma or metastatic cancer. In some embodiments, the glioma is glioblastoma multiforme or anaplastic astrocytoma. In some embodiments, the cancer is lung cancer, breast cancer, colon cancer, or skin cancer. In some embodiments, colon cancer is adenocarcinoma. In some embodiments, skin cancer is melanoma. In some embodiments, cancer is prostate cancer. In some embodiments, the contact step includes injecting the HC-HA / PTX3 complex into the tumor, surrounding tissue, or a combination thereof. In some embodiments, the contact step is performed before, during, or after surgical excision, cryoexcision, or radiofrequency ablation of cancer cells, or within any remaining portion of the tumor. In some embodiments, the contact step includes applying HC-HA / PTX3 to the excision margin of surgical excision of cancer cells. In some embodiments, the isolated HC-HA / PTX3 complex is a native HC-HA / PTX3 complex, a reconstituted HC-HA / PTX3 complex, or a combination thereof. In some embodiments, the native HC-HA / PTX3 complex is isolated from fetal supporting tissue. In some embodiments, the reconstituted HC-HA / PTX3 complex comprises heavy chain 1 (HC1) and heavy chain 2 (HC2) of inter-α inhibitors (IαI), hyaluronic acid (HA), and PTX3. In some embodiments, the reconstituted HC-HA / PTX3 complex comprises HC1, HC2, HA, PTX3, and TSG-6.In some embodiments, HC-HA / PTX3 is included in a composition comprising a pharmaceutically acceptable diluent, excipient, vehicle, or carrier. In further embodiments, HC-HA / PTX3 is included in a composition comprising administering a therapeutic agent. In some embodiments, the therapeutic agent is selected from the group consisting of chemotherapeutic agents, analgesics, anti-inflammatory agents, steroids, immunotherapies, cell therapies, radiotherapy, targeted drug therapies, and antibiotics. In some embodiments, the step of administering the therapeutic agent is performed before contacting cancer cells with the HC-HA / PTX3 complex. In some embodiments, the step of administering the therapeutic agent is performed after contacting cancer cells with the HC-HA / PTX3 complex. In some embodiments, the step of administering the therapeutic agent is performed simultaneously with contacting cancer cells with the HC-HA / PTX3 complex. In some embodiments, angiogenesis is reduced or inhibited. In some embodiments, metabolic activity is reduced in cells expressing CD44 or RHAMM.

[0006]

[0006] A method for killing cancer cells is described herein, comprising the step of contacting the resection margin or any portion of the tumor with an isolated HC-HA / PTX3 complex before, during, or after surgical resection, cryoexcision, or radiofrequency ablation of the tumor, thereby killing cancer cells at the resection margin. In some embodiments, the tumor is a cancer selected from the group consisting of liver cancer, pancreatic cancer, bladder cancer, prostate cancer, lung cancer, non-small cell lung cancer, ovarian cancer, breast cancer, melanoma, gastric cancer, colon cancer, colorectal cancer, central nervous system (CNS) cancer, bone cancer, lymphoma, skin cancer, head and neck cancer, kidney cancer, testicular cancer, uterine cancer, cervical cancer, esophageal cancer, thyroid cancer, salivary gland cancer, adrenal cancer, and gastrointestinal cancer. In some embodiments, the cancer is inoperable. In some embodiments, the CNS cancer is a glioma or metastatic cancer. In some embodiments, the glioma is glioblastoma multiforme. In some embodiments, the cancer is lung cancer, breast cancer, colon cancer, or skin cancer. In some embodiments, colon cancer is adenocarcinoma. In some embodiments, skin cancer is melanoma. In some embodiments, the tumor is prostate cancer. In some embodiments, the HC-HA / PTX3 complex is a native HC-HA / PTX3 complex, a reconstituted HC-HA / PTX3 complex, or a combination thereof. In some embodiments, the native HC-HA / PTX3 complex is isolated from fetal supporting tissue. In some embodiments, the reconstituted HC-HA / PTX3 complex comprises heavy chain 1 (HC1) and heavy chain 2 (HC2) of an inter-α inhibitor (IαI), hyaluronic acid (HA), and PTX3. In some embodiments, the reconstituted HC-HA / PTX3 complex comprises HC1, HC2, HA, PTX3, and TSG-6. In some embodiments, HC-HA / PTX3 is included in a composition comprising a pharmaceutically acceptable diluent, excipient, vehicle, or carrier. In further embodiments, the reconstituted HC-HA / PTX3 complex comprises administering a therapeutic agent. In some embodiments, the therapeutic agent is selected from the group consisting of chemotherapeutic agents, analgesics, anti-inflammatory agents, steroids, immunotherapy, cell therapy, radiotherapy, targeted drug therapies, and antibiotics. In some embodiments, the step of administering the therapeutic agent is performed before the cancer cells are brought into contact with the HC-HA / PTX3 complex.In some embodiments, the step of administering the therapeutic agent is performed after contacting the cancer cells with the HC-HA / PTX3 complex. In some embodiments, the step of administering the therapeutic agent is performed simultaneously with contacting the cancer cells with the HC-HA / PTX3 complex. In some embodiments, cancer cell death is by apoptosis or necrosis.

[0007]

[0007] Disclosed herein are methods for inhibiting tumor cancer cell regrowth in individuals requiring inhibition of tumor cancer cell regrowth, comprising the step of contacting the region surrounding the tumor after a surgical procedure with an isolated heavy-chain hyaluronan / pentraxin 3 (HC-HA / PTX3) complex, thereby inhibiting cancer cell regrowth in the region surrounding the tumor. In some embodiments, the surgical procedure includes surgical resection, cryoexcision, or radiofrequency ablation of the tumor. In some embodiments, the surgical procedure includes chemotherapy, immunotherapy, or targeted therapy. In some embodiments, the region surrounding the tumor includes the resection margin. In some embodiments, the region surrounding the tumor is the peritumoral region. In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is a cancer selected from the group consisting of liver cancer, pancreatic cancer, bladder cancer, prostate cancer, lung cancer, non-small cell lung cancer, ovarian cancer, breast cancer, melanoma, stomach cancer, colon cancer, colorectal cancer, central nervous system (CNS) cancer, bone cancer, lymphoma, skin cancer, head and neck cancer, kidney cancer, testicular cancer, uterine cancer, cervical cancer, esophageal cancer, thyroid cancer, salivary gland cancer, adrenal cancer, and gastrointestinal cancer. In some embodiments, the cancer is inoperable. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is glioblastoma multiforme. In some embodiments, the cancer is skin cancer. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the area surrounding the tumor is brought into contact with approximately 10 to 100 micrograms. In some embodiments, the HC-HA / PTX3 complex is a native HC-HA / PTX3 complex, a reconstituted HC-HA / PTX3 complex, or a combination thereof. In some embodiments, the native HC-HA / PTX3 complex is isolated from fetal supporting tissue. In some embodiments, the reconstituted HC-HA / PTX3 complex contains heavy chain 1 (HC1) and heavy chain 2 (HC2) of inter-α inhibitors (IαI), hyaluronic acid (HA), and PTX3. In some embodiments, the reconstituted HC-HA / PTX3 complex contains HC1, HC2, HA, PTX3, and tumor necrosis factor α-stimulating gene 6 (TSG-6).In some embodiments, hyaluronan (HA) is high molecular weight hyaluronan (HMW HA). In some embodiments, hyaluronan (HA) is low molecular weight hyaluronan (LMW HA). In some embodiments, the HC-HA / PTX3 complex is cryopreserved. In some embodiments, the HC-HA / PTX3 complex contains living cells. In some embodiments, the method further includes a step of administering a therapeutic agent. In some embodiments, the therapeutic agent is selected from the group consisting of chemotherapeutic agents, analgesics, anti-inflammatory agents, steroids, immunotherapy, cell therapy, radiotherapy, targeted drug therapies, and antibiotics. In some embodiments, the step of administering the therapeutic agent is performed before contacting the area surrounding the tumor with the HC-HA / PTX3 complex. In some embodiments, the step of administering the therapeutic agent is performed after contacting the area surrounding the tumor with the HC-HA / PTX3 complex. In some embodiments, the step of administering the therapeutic agent is performed simultaneously with contacting the area surrounding the tumor with the HC-HA / PTX3 complex. In some embodiments, the method inhibits tumor cell regrowth by killing cancer cells. In some embodiments, cancer cell death occurs by apoptosis or necrosis. In some embodiments, the method inhibits tumor cell regrowth by inhibiting cancer cell proliferation. In some embodiments, the method inhibits tumor cell regrowth by inhibiting the metabolic activity of cancer cells.

[0008]

[0008] Disclosed herein are methods for killing cancer cells in a tumor in an individual requiring the elimination of cancer cells in a tumor, comprising the step of contacting the tumor or the region surrounding the tumor with an isolated heavy-chain hyaluronan / pentraxin 3 (HC-HA / PTX3) complex before, during, or after a surgical procedure, thereby killing the cancer cells. In some embodiments, the surgical procedure includes surgical resection, cryoexcision, or radiofrequency ablation of the tumor. In some embodiments, the surgical procedure includes chemotherapy, immunotherapy, or targeted therapy. In some embodiments, the region surrounding the tumor includes the resection margin. In some embodiments, the region surrounding the tumor is the peritumoral region. In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is a cancer selected from the group consisting of liver cancer, pancreatic cancer, bladder cancer, prostate cancer, lung cancer, non-small cell lung cancer, ovarian cancer, breast cancer, melanoma, stomach cancer, colon cancer, colorectal cancer, central nervous system (CNS) cancer, bone cancer, lymphoma, skin cancer, head and neck cancer, kidney cancer, testicular cancer, uterine cancer, cervical cancer, esophageal cancer, thyroid cancer, salivary gland cancer, adrenal cancer, and gastrointestinal cancer. In some embodiments, the cancer is inoperable. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is glioblastoma multiforme. In some embodiments, the cancer is skin cancer. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the area surrounding the tumor is brought into contact with approximately 10 to 100 micrograms. In some embodiments, the HC-HA / PTX3 complex is a native HC-HA / PTX3 complex, a reconstituted HC-HA / PTX3 complex, or a combination thereof. In some embodiments, the native HC-HA / PTX3 complex is isolated from fetal supporting tissue. In some embodiments, the reconstituted HC-HA / PTX3 complex contains heavy chain 1 (HC1) and heavy chain 2 (HC2) of inter-α inhibitors (IαI), hyaluronic acid (HA), and PTX3. In some embodiments, the reconstituted HC-HA / PTX3 complex contains HC1, HC2, HA, PTX3, and tumor necrosis factor α-stimulating gene 6 (TSG-6).In some embodiments, the HC-HA / PTX3 complex is cryopreserved. In some embodiments, the HC-HA / PTX3 complex contains living cells. In some embodiments, hyaluronan (HA) is high molecular weight hyaluronan (HMW HA). In some embodiments, hyaluronan (HA) is low molecular weight hyaluronan (LMW HA). In some embodiments, the contact step includes injecting HC-HA / PTX3 directly into the tumor. In some embodiments, the method further includes the step of administering a therapeutic agent. In some embodiments, the therapeutic agent is selected from the group consisting of chemotherapeutic agents, analgesics, anti-inflammatory agents, steroids, immunotherapy, cell therapy, radiotherapy, targeted drug therapies, and antibiotics. In some embodiments, the step of administering the therapeutic agent is performed before contacting the cancer cells with the HC-HA / PTX3 complex. In some embodiments, the step of administering the therapeutic agent is performed after contacting the cancer cells with the HC-HA / PTX3 complex. In some embodiments, the step of administering the therapeutic agent is performed simultaneously with contacting the cancer cells with the HC-HA / PTX3 complex. In some embodiments, cancer cell death occurs through apoptosis or necrosis. [Brief explanation of the drawing]

[0009] [Figure 1]

[0009] Figure 1A shows the aggregation of LNCaP cells after culturing in RPMI medium.

[0010] Figure 1B shows the uniform distribution of PC-3 cells after culturing in RPMI medium. [Figure 2A]

[0011] Figures 2A to 2D show the morphology and cellular metabolic activity of LNCaP after treatment with a series of doses of purified BTGel or HC-HA / PTX3. [Figure 2B] Figures 2A to 2D show the morphology and cellular metabolic activity of LNCaP after treatment with a series of doses of purified BTGel or HC-HA / PTX3. [Figure 2C] Figures 2A to 2D show the morphology and cellular metabolic activity of LNCaP after treatment with a series of doses of purified BTGel or HC-HA / PTX3. [Figure 2D] Figures 2A - 2D are diagrams showing the morphology and cell metabolic activity of LNCaP by treatment with a series of doses of purified BTGel or HC - HA / PTX3. [Figure 3A]

[0012] Figures 3A - 3D are diagrams showing the morphology and metabolic activity of PC - 3 by treatment with a series of doses of purified BTGel or HC - HA / PTX3. [Figure 3B] Figures 3A - 3D are diagrams showing the morphology and metabolic activity of PC - 3 by treatment with a series of doses of purified BTGel or HC - HA / PTX3. [Figure 3C] Figures 3A - 3D are diagrams showing the morphology and metabolic activity of PC - 3 by treatment with a series of doses of purified BTGel or HC - HA / PTX3. [Figure 3D] Figures 3A - 3D are diagrams showing the morphology and metabolic activity of PC - 3 by treatment with a series of doses of purified BTGel or HC - HA / PTX3. [Figure 4A]

[0013] It is a diagram showing WST - 1 assay data in LNCaP cells after treatment with HA using UC extract, HC - HA / PTX3, and HA in aqueous UC extract. [Figure 4B]

[0014] It is a diagram showing WST - 1 assay data in PC - 3 cells after treatment with HA using UC extract, HC - HA / PTX3, and HA in aqueous UC extract. [Figure 5]

[0015] Figure 5A is a diagram showing the morphology of LNCaP cells after treatment with HA. Figure 5B is a diagram showing the morphology of LNCaP cells after treatment with HC - HA / PTX3. Figure 5C is a diagram showing the morphology of LNCaP cells after treatment with umbilical cord extract (UCE). [Figure 6]

[0016] Figure 6A is a diagram showing the morphology of PC - 3 cells after treatment with HA. Figure 6B is a diagram showing the morphology of PC - 3 cells after treatment with HC - HA / PTX3. Figure 6C is a diagram showing the morphology of PC - 3 cells after treatment with umbilical cord extract (UCE). [Figure 7]

[0017] This figure shows that LNCaP cells grown on laminin and type IV collagen exhibit greater cell aggregation than cells grown on other surfaces. [Figure 8A]

[0018] This figure shows bright-field images of PrEC prostate cell line morphology taken at 10× and 20× magnification. [Figure 8B]

[0019] This figure shows bright-field images of the morphology of the PNT2 prostate cell line taken at 10× and 20× magnification. [Figure 9]

[0020] This figure shows representative bright-field microscope images (scale bar 50 μm) of primary normal human prostate cells after 48 hours of incubation with various concentrations of HC-HA / PTX3 and HMW-HA. [Figure 10]

[0021] Figure 10A shows the metabolic activity (%) evaluated by the WST-1 assay in normal human primary prostate epithelial cells (PrEC) after 48-hour incubation with various concentrations (0.78, 3.125, 6.25, 12.5, 25, 50, 100 μg / ml) of HC-HA / PTX3 and HA. Figure 10B shows the metabolic activity (%) evaluated by the WST-1 assay in normal human prostate cell line PNT2 after 48-hour incubation with various concentrations (0.78, 3.125, 6.25, 12.5, 25, 50, 100 μg / ml) of HC-HA / PTX3 and HA. The p-value was calculated by a two-sided t-test against the untreated sample. [Figure 11]

[0022] Figure 11A shows a comparative analysis of metabolic activity (%) evaluated in normal primary prostate epithelial cells (PrEC) and cell line (PNT2), as well as prostate cancer cell lines: PC3 and LNCaP, after 48-hour incubation with HC-HA / PTX3 at various concentrations (0.78, 3.125, 6.25, 12.5, 25, 50, and 100 μg / ml). Figure 11B shows a comparative analysis of logarithmic metabolic activity evaluated in normal primary prostate epithelial cells (PrEC) and cell line (PNT2), as well as prostate cancer cell lines: PC3 and LNCaP, after 48-hour incubation with HC-HA / PTX3 at various concentrations (0.78, 3.125, 6.25, 12.5, 25, 50, and 100 μg / ml). [Figure 12]

[0023] Figure 12A shows a comparative analysis of metabolic activity (%) evaluated in normal primary prostate epithelial cells (PrEC) and cell line (PNT2), as well as prostate cancer cell lines: PC3 and LNCaP, after 48 hours of incubation with various concentrations of HA. Figure 12B shows a comparative analysis of metabolic activity on a logarithmic scale evaluated in normal primary prostate epithelial cells (PrEC) and cell line (PNT2), as well as prostate cancer cell lines: PC3 and LNCaP, after 48 hours of incubation with various concentrations of HA. [Figure 13]

[0024] This figure shows representative bright-field microscope images (scale bar 50 μm) of A375 (melanoma) cells after 48 hours of incubation with various concentrations of HC-HA / PTX3 and HMW-HA. [Figure 14]

[0025] This figure shows representative bright-field microscope images (scale bar 50 μm) of HT-29 (colon cancer) cells after 48 hours of incubation with various concentrations of HC-HA / PTX3 and HMW-HA. [Figure 15]

[0026] This figure shows representative bright-field microscope images (scale bar 50 μm) of A549 (lung cancer) cells after 48 hours of incubation with various concentrations of HC-HA / PTX3 and HMW-HA. [Figure 16]

[0027] This figure shows representative bright-field microscope images (scale bar 50 μm) of MCF-7 (breast cancer) cells after 48 hours of incubation with various concentrations of HC-HA / PTX3 and HMW-HA. [Figure 17A]

[0028] Figures 17A-17D show the metabolic activity (%) evaluated by the WST-1 assay in four human cancer cell lines: A375 (Figure 17A), HT-29 (Figure 17B), MCF-7 (Figure 17C), and A-549 (Figure 17D) after 48 hours of incubation with various concentrations of HC-HA / PTX3 and HA. [Figure 17B] Figures 17A-17D show the metabolic activity (%) evaluated by the WST-1 assay in four human cancer cell lines: A375 (Figure 17A), HT-29 (Figure 17B), MCF-7 (Figure 17C), and A-549 (Figure 17D) after 48 hours of incubation with various concentrations of HC-HA / PTX3 and HA. [Figure 17C] Figures 17A-17D show the metabolic activity (%) evaluated by the WST-1 assay in four human cancer cell lines: A375 (Figure 17A), HT-29 (Figure 17B), MCF-7 (Figure 17C), and A-549 (Figure 17D) after 48 hours of incubation with various concentrations of HC-HA / PTX3 and HA. [Figure 17D] Figures 17A-17D show the metabolic activity (%) evaluated by the WST-1 assay in four human cancer cell lines: A375 (Figure 17A), HT-29 (Figure 17B), MCF-7 (Figure 17C), and A-549 (Figure 17D) after 48 hours of incubation with various concentrations of HC-HA / PTX3 and HA. [Figure 18A]

[0029] This figure shows representative bright-field microscope images (scale bar 50 μm) of limbal niche cells (LNCs) after treatment at various time points with different concentrations of HC-HA / PTX3: 15-30 minutes, 1 hour, 5 hours, 24 hours, and 48 hours, respectively. [Figure 18B]

[0030] This figure shows representative bright-field microscope images (scale bar 50 μm) of LNCs (limbal niche cells) after treatment at various time points with different concentrations of HMW-HA: 15-30 minutes, 1 hour, 5 hours, 24 hours, and 48 hours, respectively. [Figure 18C]

[0031] This figure shows a representative bright-field microscope image (scale bar 50 μm) of LNCs (limbal niche cells) after 48 hours of incubation with 100 μg / ml HC-HA / PTX3 and HMW-HA. [Figure 19]

[0032] This figure shows the metabolic activity (%) evaluated in limbal niche cells by the WST-1 assay after 48 hours of incubation with various concentrations of HC-HA / PTX3 and HA. [Figure 20A]

[0033] Figures 20A and 20B show representative bright-field microscope images (scale bar 50 μm) of HTM (human trabecular mesh) cells after treatment at various time points with different concentrations of HC-HA / PTX3 (Figure 20A) and HMW-HA (Figure 20B). [Figure 20B] Figures 20A and 20B show representative bright-field microscope images (scale bar 50 μm) of HTM (human trabecular mesh) cells after treatment at various time points with different concentrations of HC-HA / PTX3 (Figure 20A) and HMW-HA (Figure 20B). [Figure 21]

[0034] This figure shows the metabolic activity (%) evaluated in human trabecular mesh cells by the WST-1 assay after 48 hours of incubation with various concentrations of HC-HA / PTX3 and HA. [Figure 22]

[0035] Figure 22A shows representative bright-field microscope images (scale bar 50 μm) of human corneal fibroblast (HCF) cells after treatment with various concentrations of HC-HA / PTX3 at various time points. Figure 22B shows representative bright-field microscope images (scale bar 50 μm) of human corneal fibroblast (HCF) cells after treatment with various concentrations of HMW-HA at various time points. [Figure 23]

[0036] This figure shows the metabolic activity (%) evaluated in human corneal fibroblast cells by the WST-1 assay after 48 hours of incubation with various concentrations of HC-HA / PTX3 and HA. [Figure 24]

[0037] Figure 24A shows a comparative analysis of metabolic activity (%) in three types of normal human primary mesenchymal cells: HCF, HTM, and LNC, evaluated by the WST-1 assay after 48 hours of incubation with various concentrations of HC-HA / PTX3. Figure 24B shows a comparative analysis of metabolic activity (%) in three types of normal human primary mesenchymal cells: HCF, HTM, and LNC, evaluated by the WST-1 assay after 48 hours of incubation with various concentrations of HA. [Figure 25]

[0038] This figure shows a representative bright-field microscope image (scale bar 50 μm) illustrating the transient effect of HC-HA / PTX3 (100 μg / ml) on the morphology of LNC and HCF cells, and the absence of a corresponding effect in HTM cells. [Figure 26A]

[0039] This figure shows bright-field images of A375 cell morphology after treatment with HC-HA / PTX3 and HA, compared to untreated cells. [Figure 26B]

[0040] This figure shows the BrdU cell proliferation assay curve using A375 cells. [Figure 26C]

[0041] This figure shows the semi-logarithmic scale BrdU cell proliferation assay curve using A375 cells. [Figure 27A]

[0042] This figure shows bright-field images of PrEC cell morphology at two magnifications (10× and 20×) after treatment with HA or HC-HA / PTX3. [Figure 27B]

[0043] This figure shows the BrdU cell proliferation assay curve after HC-HA / PTX3 treatment. [Figure 27C]

[0044] This figure shows the BrdU cell proliferation assay curve after HA treatment. [Figure 28A]

[0045] This figure shows bright-field images of PNT2 cell morphology after or after treatment with HC-HA / PTX3 and HMW-HA. [Figure 28B]

[0046] This figure shows the BrdU cell proliferation assay curves of PNT2 cells after treatment with HC-HA / PTX3 or HA. [Figure 29A]

[0047] This figure shows bright-field images of PC3 cell morphology after or after treatment with HC-HA / PTX3 and HMW-HA. [Figure 29B]

[0048] This figure shows the BrdU cell proliferation assay curves in PC3 cells after treatment with HC-HA / PTX3 or HA. [Figure 30A]

[0049] This figure shows bright-field images of LNCaP cell morphology after or after treatment with HC-HA / PTX3 and HMW-HA. [Figure 30B]

[0050] This figure shows the BrdU cell proliferation assay curve in LNCaP cells after treatment with HC-HA / PTX3 or HA. [Modes for carrying out the invention]

[0010] Specific technical terms

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in the field to which the claimed subject matter pertains. All patents, patent applications, published applications and publications, websites and other publicly available materials referenced throughout this disclosure are incorporated by reference in their entirety unless otherwise specified. If a term herein has multiple definitions, the definition in this section shall prevail. Where URLs or other such identifiers or addresses are referenced, it is understood that such identifiers may change and certain information on the Internet may shift, but equivalent information is publicly known and readily accessible, for example, by searching the Internet and / or appropriate databases. References thereto demonstrate the availability and public dissemination of such information.

[0011]

[0052] As used herein, ranges and quantities may be expressed as "approximately" a specific value or range. "Approximately" may also include an exact quantity or value. Thus, "approximately 5 μg" means "approximately 5 μg" and also means "5 μg". In some embodiments, the term "approximately" includes an amount that is expected to be within experimental error. In some embodiments, the term "approximately" refers to 20%, 10%, or 5% of the value + / - value.

[0012]

[0053] As used herein, HC-HA / PTX3 complex or isolated HC-HA / PTX3 refers to native HC-HA / PTX3, reconstituted HC-HA / PTX3, or a combination thereof. As used herein, reconstituted HC-HA / PTX3 (rcHC-HA / PTX3) complex is an HC-HA / PTX3 complex formed by the in vitro assembly of the complex's component molecules. In some embodiments, the rcHC-HA / PTX3 complex comprises HA, IαI HC1 and HC2, and PTX3. In some embodiments, the rcHC-HA / PTX3 complex comprises HA, IαI HC1 and HC2, PTX3, and TSG-6. Processes for assembling rcHC-HA / PTX3 include reconstitution by the synthesis of molecules by in vitro synthesis, using purified native proteins or molecules isolated from biological sources, recombinant proteins produced by recombinant methods, or molecules isolated by in vitro synthesis. In some cases, the purified native protein used in the assembly of rcHC-HA / PTX3 is a protein in a complex with other proteins (i.e., a multimer, a multi-chain protein, or another complex). In some cases, PTX3 is purified from cells as a multimer (e.g., a homomultimer) and used in the assembly of the rcHC-HA / PTX3 complex.

[0013]

[0054] As used herein, purified natural HC-HA / PTX3 (nHC-HA / PTX3) complex refers to an HC-HA / PTX3 complex purified from a biological source such as cells, tissues, or biofluids. In some embodiments, HC-HA / PTX3 is isolated from fetal supporting tissue such as the placenta, amnion, chorion, umbilical cord, or amniotic membrane of the umbilical cord. In some embodiments, HC-HA / PTX3 is isolated from the amnion. In some embodiments, the natural HC-HA / PTX3 complex comprises HA, IαI HC1, and PTX3. Such complexes are generally assembled in vivo in a subject or ex vivo in cells, tissues, or biofluids derived from a subject including humans or other animals.

[0014]

[0055] As used herein, “hyaluronan,” “hyaluronic acid,” or “hyaluronate” (HA) are used interchangeably and refer to substantially non-sulfated linear glycosaminoglycans (GAGs) having repeating disaccharide units of D-glucuronic acid and N-acetylglucosamine (D-glucuronosyl-N-acetylglucosamine).

[0015]

[0056] As used herein, the terms “high molecular weight” or “HMW,” such as “high molecular weight hyaluronan” (HMW HA), are intended to refer to HA having a weight-average molecular weight greater than about 500 kilodaltons (kDa), for example, about 500 kDa to about 10,000 kDa, about 800 kDa to about 8,500 kDa, about 1100 kDa to about 5,000 kDa, or about 1400 kDa to about 3,500 kDa. In some embodiments, HMW HA has a weight-average molecular weight of 3000 kDa or more. In some embodiments, HMW HA has a weight-average molecular weight of 3000 kDa. In some embodiments, HMW HA is Healon® having a weight-average molecular weight of about 3000 kDa. In some embodiments, HMW HA has a molecular weight of about 500 kDa to about 10,000 kDa. In some embodiments, HMW HA has a molecular weight of approximately 800 kDa to approximately 8,500 kDa. In some embodiments, HMW HA has a molecular weight of approximately 3,000 kDa.

[0016]

[0057] As used herein, the terms “low molecular weight” or “LMW,” such as low molecular weight hyaluronan (LMW HA), are intended to refer to HA having a weight-average molecular weight of less than 500 kDa, for example, less than about 400 kDa, less than about 300 kDa, less than about 200 kDa, about 200-300 kDa, or about 1-300 kDa.

[0017]

[0058] As used herein, pentraxin 3 or PTX3 protein or polypeptide refers to any PTX3 protein, including but not limited to recombinantly produced proteins, synthetically produced proteins, native PTX3 proteins, and PTX3 proteins extracted from cells or tissues. PTX3 includes, but not limited to, naturally produced or artificially produced dimers, trimers, tetramers, pentamers, hexamers, tetramers, octamers, and other multimeric forms of PTX3 (e.g., homomultimers).

[0018]

[0059] As used herein, “hyaluronan-binding protein,” “HA-binding protein,” or “HABP” refers to any protein that specifically binds to HA.

[0060] As used herein, “link module” means a hyaluronan-binding domain.

[0019]

[0061] As used herein, “biological activity” refers to the in vivo activity of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex or the physiological response induced by in vivo administration of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex or a composition or mixture containing the nHC-HA / PTX3 or rcHC-HA / PTX3 complex. Therefore, biological activity encompasses the therapeutic and pharmaceutically active effects of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex and its compositions and mixtures.

[0020]

[0062] As used herein, the terms “subject,” “individual,” and “patient” are interchangeable. None of these terms should be construed as requiring supervision by a medical professional (e.g., a doctor, nurse, medical assistant, janitor, or hospice staff). As used herein, “subject” refers to any animal, including mammals (e.g., humans or non-human animals) and non-mammals. In one embodiment of the methods and compositions provided herein, the mammal is a human.

[0021]

[0063] As used herein, the terms “to treat,” “to treat,” or “to cure,” and other grammatical equivalents include alleviating, reducing, or improving one or more symptoms of a disease or condition; improving, preventing, or reducing the appearance, severity, or frequency of one or more additional symptoms of a disease or condition; improving or preventing the underlying metabolic causes of one or more symptoms of a disease or condition; reducing a disease or condition, causing a regression of a disease or condition, reducing a condition caused by a disease or condition, or preventing and / or therapeutically inhibiting symptoms of a disease or condition, such as preventing the onset of a disease or condition. In non-limiting examples, for prophylactic benefits, the rcHC-HA / PTX3 complex, natural HC-HA / PTX3 complex, or composition disclosed herein is administered to an individual at risk of developing a particular disorder, who is prone to developing a particular disorder, or who reports one or more physiological symptoms of a disorder.

[0022]

[0064] As used herein, “placenta” refers to the organ that connects the developing fetus to the mother’s uterine wall, enabling the uptake of nutrients, elimination of waste products, and gas exchange through the mother’s blood supply. The placenta consists of three layers. The innermost placental layer surrounding the fetus is called the amnion. The allantois is the middle layer of the placenta (derived from the hindgut of the embryo), and blood vessels originating from the umbilicus cross this membrane. The chorion, the outermost layer of the placenta, is in contact with the endometrium. The chorion and allantois fuse to form the chorioallantoic membrane.

[0023]

[0065] As used herein, “chorionic membrane” refers to the membrane formed by the extraembryonic mesoderm and two layers of trophoblast. The chorion consists of two layers: an outer layer formed by the trophoblast and an inner layer formed by the parietal mesoderm, with the amnion in contact with the latter. The trophoblast consists of the trophoblastic cell layer or Langhans layer, which is the inner layer of cuboidal or prism-shaped cells, and the trophoblastic syncytial layer, which is the outer layer of richly nucleated protoplasm without cell boundaries. The vascularized amnion adheres to the inner layer of the chorion.

[0024]

[0066] As used herein, “amnion-chorionic” refers to a product containing the amnion and chorion. In some embodiments, the amnion and chorion are not separated (i.e., the amnion is naturally attached to the inner layer of the chorion). In some embodiments, the amnion is initially separated from the chorion but later binds to the chorion during processing.

[0025]

[0067] As used herein, “umbilical cord” refers to the organ that connects the developing fetus to the placenta. The umbilical cord consists of Wharton’s jelly, a jelly-like substance that is mostly composed of mucopolysaccharides. It contains one vein that carries oxygenated, nutrient-rich blood to the fetus, and two arteries that carry deoxygenated, nutrient-depleted blood.

[0026]

[0068] As used herein, “placental amniotic membrane” (PAM) refers to the amniotic membrane derived from the placenta. In some embodiments, the PAM is substantially isolated.

[0069] As used herein, “umbilical amniotic membrane” (UCAM) means the amniotic membrane derived from the umbilical cord. UCAM is a translucent membrane. UCAM has several layers: an epithelial layer, a basement membrane, a compact layer, a fibroblast layer, and a spongy layer. It lacks blood vessels or a direct blood supply. In some embodiments, UCAM contains Wharton's jelly. In some embodiments, UCAM contains blood vessels and / or arteries. In some embodiments, UCAM contains Wharton's jelly as well as blood vessels and / or arteries.

[0027]

[0070] As used herein, the terms “purified” and “isolated” mean a substance (e.g., an nHC-HA / PTX3 complex) that is substantially or essentially free of components that normally accompany it in its natural state. In some embodiments, “purified” or “isolated” means that a substance (e.g., an nHC-HA / PTX3 complex) does not contain about 50% or more of components that normally accompany it in its natural state, for example, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of components that normally accompany it in its natural state.

[0028] Treatment methods

[0071] In certain embodiments, methods for treating an individual in need of treatment are disclosed herein, including methods for directly killing cancer cells, directly inhibiting the proliferation of cancer cells, reducing the metabolic activity of cancer cells, or a combination thereof, the method comprising the step of administering the individual an nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is used to directly kill cancer cells. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is used to directly inhibit the proliferation of cancer cells. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is used to reduce the metabolic activity of cancer cells. In some embodiments, the cells are derived from or contained within a solid tumor.

[0029]

[0072] In some embodiments, a method for treating an individual in need of treatment, comprising the step of administering to the individual an nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein by any suitable method. In some embodiments, the individual in need has cancer. In some embodiments, the individual in need has inoperable cancer. In some embodiments, the individual in need has inoperable cancer selected from the group consisting of pancreatic cancer, prostate cancer, and glioblastoma pleomorphoni. In some embodiments, the individual has a solid tumor. In some embodiments, the individual has cancer selected from the group consisting of liver cancer, pancreatic cancer, bladder cancer, prostate cancer, lung cancer, non-small cell lung cancer, ovarian cancer, breast cancer, melanoma, gastric cancer, colon cancer, colorectal cancer, central nervous system (CNS) cancer, bone cancer, lymphoma, skin cancer, head and neck cancer, kidney cancer, testicular cancer, uterine cancer, cervical cancer, esophageal cancer, thyroid cancer, salivary gland cancer, adrenal cancer, and gastrointestinal cancer.

[0030]

[0073] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is administered to the individual before, during, or after a surgical procedure. In some embodiments, the surgical procedure includes tumor resection. In some embodiments, the surgical procedure includes surgical resection, cryoexcision, or radiofrequency ablation of the tumor. In some embodiments, the surgical procedure includes chemotherapy, immunotherapy, or targeted therapy.

[0031]

[0074] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is administered for up to 1 day, up to 2 days, up to 3 days, up to 5 days, or more than 5 days after tumor resection. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is administered for 1 week, 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 1 year, 2 years, 3 years, 4 years, 5 years, or more than 5 years after tumor resection. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is administered as a single dose or over time after tumor resection, for example, daily, multiple times per week, weekly, bi-weekly, monthly, or less frequently. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is administered as a single dose or over time after tumor resection, for example, daily, multiple times per week, weekly, bi-weekly, monthly, or more frequently.

[0032]

[0075] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is administered before tumor resection. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is administered for up to 1 day, up to 2 days, up to 3 days, up to 5 days, or more than 5 days before tumor resection. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is administered as a single dose or over time, for example, daily, multiple times per week, weekly, bi-weekly, monthly, or less frequently, before tumor resection. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is administered as a single dose or over time, for example, daily, multiple times per week, weekly, bi-weekly, monthly, or more frequently, before tumor resection.

[0033]

[0076] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is administered to the individual during or after surgical resection, cryoexcision, or radiofrequency ablation of the tumor. In some examples, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is administered to the resection margin (e.g., the area of ​​apparent tissue around the surgically removed tumor) after surgical resection (e.g., whole or partial), cryoexcision, or radiofrequency ablation of the tumor.

[0034]

[0077] In some embodiments, a method for treating an individual in need of treatment, comprising the step of administering an nHC-HA / PTX3 or rcHC-HA / PTX3 complex to the individual by any suitable route of administration. The suitable method of administration depends on the disease or condition being treated. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is administered locally to the site of treatment. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is injected into the tumor, the tissue surrounding the tumor, or both. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is injected into the tumor. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is applied to the area surrounding the tumor after the tumor has been surgically removed or treated with cryoexcision or radiofrequency ablation. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is administered systemically. Exemplary methods of administering nHC-HA / PTX3 or rcHC-HA / PTX3 complexes provided herein include, but are not limited to, parenteral, enteral, subcutaneous, percutaneous, transdermal, intradermal, intravenous, topical, inhalation, or implantation.

[0035]

[0078] Isolated HC-HA / PTX3 complexes are demonstrated herein to directly kill cancer cells. In certain embodiments, the use of isolated HC-HA / PTX3 complexes, comprising preparations or compositions containing HC-HA / PTX3, for killing cancer cells is provided herein. In some embodiments, the use of isolated HC-HA / PTX3 complexes for killing cancer cells in solid tumors is provided herein. In some embodiments, the use of isolated HC-HA / PTX3 complexes for killing cancer cells in the region surrounding a tumor after the tumor has been surgically removed or treated with cryoexcision or radiofrequency ablation is provided herein. In some embodiments, the use of isolated HC-HA / PTX3 complexes for localized cancer cell death in subjects where cancer cell death is required is provided herein. In some embodiments, the use of isolated HC-HA / PTX3 complexes for systemic cancer cell death in subjects where cancer cell death is required is provided herein.

[0036]

[0079] Isolated HC-HA / PTX3 complexes are demonstrated herein to inhibit the proliferation of cancer cells. In certain embodiments, the use of isolated HC-HA / PTX3 complexes, comprising preparations or compositions containing HC-HA / PTX3, for inhibiting the proliferation of cancer cells is provided herein. In some embodiments, the use of isolated HC-HA / PTX3 complexes for inhibiting the proliferation of cancer cells in solid tumors is provided herein. In some embodiments, the use of isolated HC-HA / PTX3 complexes for inhibiting the proliferation of cancer cells in the region surrounding a tumor after the tumor has been surgically removed or treated with cryoexcision or radiofrequency ablation is provided herein. In some embodiments, the use of isolated HC-HA / PTX3 complexes for local inhibition of cancer cell proliferation in subjects where inhibition of cancer cell proliferation is required is provided herein. In some embodiments, the use of isolated HC-HA / PTX3 complexes for systemic inhibition of cancer cell proliferation in subjects where inhibition of cancer cell proliferation is required is provided herein. In some embodiments, growth is inhibited or reduced by 5-95%, 10-90%, 20-80%, 30-70%, 40-60%, 50-95%, 65-85%, or 75-95%. In some embodiments, growth is inhibited or reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%. In some embodiments, growth is inhibited or reduced by at least 5%. In some embodiments, growth is inhibited or reduced by at least 10%. In some embodiments, growth is inhibited or reduced by at least 50%.

[0037]

[0080] Isolated HC-HA / PTX3 complexes are demonstrated herein to reduce the metabolic activity of cancer cells. In certain embodiments, the use of isolated HC-HA / PTX3 complexes, comprising preparations or compositions containing HC-HA / PTX3, for reducing metabolic activity in cancer cells is provided herein. In some embodiments, the use of isolated HC-HA / PTX3 complexes for reducing metabolic activity of cancer cells in solid tumors is provided herein. In some embodiments, the use of isolated HC-HA / PTX3 complexes for reducing metabolic activity of cancer cells in the region surrounding a tumor after the tumor has been surgically removed or treated with cryoexcision or radiofrequency ablation is provided herein. In some embodiments, the use of isolated HC-HA / PTX3 complexes for locally reducing the metabolic activity of cancer cells in subjects where a reduction in the metabolic activity of cancer cells is required is provided herein. In some embodiments, the use of isolated HC-HA / PTX3 complexes for systemically reducing the metabolic activity of cancer cells in subjects where a reduction in the metabolic activity of cancer cells is required is provided herein. In some embodiments, metabolic activity is reduced by 5-95%, 10-90%, 20-80%, 30-70%, 40-60%, 50-95%, 65-85%, or 75-95%. In some embodiments, metabolic activity is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%. In some embodiments, metabolic activity is reduced by at least 5%. In some embodiments, metabolic activity is reduced by at least 10%. In some embodiments, metabolic activity is reduced by at least 50%.

[0038]

[0081] In certain embodiments, the use of isolated HC-HA / PTX3 complexes, including preparations or compositions containing HC-HA / PTX3, for increasing cancer cell death is provided herein. In some embodiments, cancer cell death is increased by about 10% to about 25%, about 10% to about 50%, and about 20% to about 90%. In some embodiments, cancer cell death is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more than 95%. In some embodiments, cancer cell death is induced by apoptosis. In some embodiments, cancer cell death is induced by necrosis.

[0039]

[0082] In some embodiments, cancer cells originate from or are located within a solid tumor. In some embodiments, the solid tumor is liver cancer, pancreatic cancer, bladder cancer, prostate cancer, lung cancer, non-small cell lung cancer, ovarian cancer, breast cancer, melanoma, stomach cancer, colon cancer, colorectal cancer, central nervous system (CNS) cancer, bone cancer, lymphoma, skin cancer, head and neck cancer, kidney cancer, testicular cancer, uterine cancer, cervical cancer, esophageal cancer, thyroid cancer, salivary gland cancer, adrenal cancer, or gastrointestinal cancer. In some embodiments, cancer cells originate from a humoral tumor. In some embodiments, the humoral tumor is lymphoma or leukemia. In some embodiments, the CNS cancer is glioma or metastatic cancer. In some embodiments, the glioma is glioblastoma pleomorphicum or anaplastic astrocytoma. In some embodiments, the colon cancer is adenocarcinoma, carcinoid tumor, primary colorectal lymphoma, stromal tumor, or leiomyosarcoma. In some embodiments, the skin cancer is melanoma, basal cell carcinoma, or squamous cell carcinoma.

[0040] Method for producing isolated nHC-HA / PTX3 complexes

[0083] In some embodiments, isolated natural HC-HA / PTX3 (nHC-HA / PTX3) complexes are used in the methods provided herein.

[0041]

[0084] In some embodiments, the isolated nHC-HA / PTX3 complex is isolated from amniotic tissue. In some embodiments, the isolated nHC-HA / PTX3 complex is isolated from the amniotic membrane or umbilical cord. In some embodiments, the isolated nHC-HA / PTX3 complex is isolated from fresh, frozen, or pre-frozen placental amniotic membrane (PAM), fresh, frozen, or pre-frozen umbilical cord amniotic membrane (UCAM), fresh, frozen, or pre-frozen placenta, fresh, frozen, or pre-frozen umbilical cord, fresh, frozen, or pre-frozen chorionic membrane, fresh, frozen, or pre-frozen amniotic-chorionic membrane, or any combination thereof. Such tissues may be obtained from any mammal, such as humans, non-human primates, cattle, or pigs, for example, but are not limited to these.

[0042]

[0085] In some embodiments, nHC-HA / PTX3 is purified by any suitable method. In some embodiments, the nHC-HA / PTX3 complex is purified by centrifugation (e.g., ultracentrifugation, gradient centrifugation), chromatography (e.g., ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), tangential flow filtration (TFF), gel filtration, or differential solubility, ethanol precipitation, or any other available technique for protein purification (see, for example, Scopes, Protein Purification Principles and Practice 2nd Edition, Springer-Verlag, New York, 1987; Higgins, SJ and Hames, BD (eds.), Protein Expression: A Practical Approach, Oxford Univ Press, 1999; and Deutscher, MP, Simon, MI, and Abelson, JN (eds.), Guide to Protein Purification: Methods in Enzymology (Methods in Enzymology Series, Vol. 182), Academic Press, 1997).

[0043]

[0086] In some embodiments, nHC-HA / PTX3 is isolated from the extract. In some embodiments, the extract is prepared from an amniotic membrane extract. In some embodiments, the extract is prepared from an umbilical cord extract. In some embodiments, the umbilical cord extract contains umbilical cord stroma and / or Wharton's jelly. In some embodiments, the nHC-HA / PTX3 complex is contained in an extract prepared by ultracentrifugation. In some embodiments, the nHC-HA / PTX3 complex is contained in an extract prepared by ultracentrifugation using a CsCl / 4~6M guanidine HCl gradient. In some embodiments, the extract is prepared by at least two ultracentrifugations. In some embodiments, the extract is prepared by more than two ultracentrifugations (i.e., nHC-HA / PTX3 2 ndIn some embodiments, the extract is prepared by ultracentrifugation at least four times (i.e., nHC-HA / PTX3 4). th In some embodiments, the nHC-HA / PTX3 complex comprises small molecule leucine-rich proteoglycans. In some embodiments, the nHC-HA / PTX3 complex comprises HC1, HA, PTX3 and / or small molecule leucine-rich proteoglycans.

[0044]

[0087] In some embodiments, ultracentrifugation is performed on an extract prepared by extraction in an isotonic solution. In some embodiments, the isotonic solution is PBS. For example, in some embodiments, tissue is homogenized in PBS to produce a homogenized sample. The homogenized sample is then separated into soluble and insoluble parts by centrifugation. In some embodiments, ultracentrifugation is performed on the soluble part of the tissue extracted in PBS. In such embodiments, the nHC-HA / PTX3 purified by ultracentrifugation of the tissue extracted in PBS is called the nHC-HA / PTX3 soluble complex. In some embodiments, the nHC-HA soluble complex contains small molecule leucine-rich proteoglycans. In some embodiments, the nHC-HA / PTX3 soluble complex contains HC1, HA, PTX3 and / or small molecule leucine-rich proteoglycans.

[0045]

[0088] In some embodiments, ultracentrifugation is performed on extracts prepared by direct guanidine HCl extraction (e.g., 4-6 M GnHCl) of amniotic membrane and / or umbilical cord tissue. In some embodiments, the GnHCl extract tissue is then centrifuged to produce GnHCl-soluble and GnHCl-insoluble portions. In some embodiments, ultracentrifugation is performed on the GnHCl-soluble portion. In such embodiments, the nHC-HA / PTX3 purified by ultracentrifugation of the tissue extracted with guanidine HCl is called the nHC-HA / PTX3 insoluble complex. In some embodiments, the nHC-HA insoluble complex contains small molecule leucine-rich proteoglycans. In some embodiments, the nHC-HA / PTX3 insoluble complex contains HC1, HA, PTX3 and / or small molecule leucine-rich proteoglycans.

[0046]

[0089] In some embodiments, ultracentrifugation is performed on an extract prepared by further guanidine HCl extraction of the insoluble portion of the tissue extracted with PBS. For example, in some embodiments, the tissue is homogenized in PBS to produce a homogenized sample. The homogenized sample is then separated into soluble and insoluble portions by centrifugation. The insoluble portion is then further extracted in guanidine HCl (e.g., 4-6 M GnHCl) and centrifuged to produce guanidine HCl-soluble and insoluble portions. In some embodiments, ultracentrifugation is performed on the guanidine HCl-soluble portion. In such embodiments, the nHC-HA / PTX3 purified by ultracentrifugation of the tissue extracted with guanidine HCl is called the nHC-HA / PTX3 insoluble complex. In some embodiments, the nHC-HA insoluble complex contains small molecule leucine-rich proteoglycans. In some embodiments, the nHC-HA / PTX3 insoluble complex contains HC1, HA, PTX3 and / or small molecule leucine-rich proteoglycans.

[0047]

[0090] In some embodiments, a method for purifying an isolated nHC-HA / PTX3 extract includes the steps of (a) dissolving the isolated extract (e.g., prepared by the soluble or insoluble methods described herein) in CsCl / 4-6M guanidine HCl at an initial density of 1.35 g / ml to produce a CsCl mixture; (b) centrifuging the CsCl mixture at 125,000 × g for 48 hours at 15°C to produce a first purified extract, and pooling the HA-containing fraction to adjust it to an initial density of 1.40 g / ml for a second ultracentrifugation at 125,000 × g for 48 hours at 15°C; and (c) pooling the purified fraction and dialyzing it against distilled water to remove CsCl and guanidine HCl to produce a dialysate. In some embodiments, a method for purifying an isolated extract further comprises the steps of (d) mixing the dialysate with 3 volumes of 95% (v / v) ethanol containing 1.3% (w / v) potassium acetate at 0°C for 1 hour to produce a first dialysate / ethanol mixture; (e) centrifuging the first dialysate / ethanol mixture at 15,000 × g to produce a second purified extract; and (f) extracting the second purified extract. In some embodiments, a method for purifying an isolated extract further comprises the steps of (g) washing the second purified extract with ethanol (e.g., 70% ethanol) to produce a second purified extract / ethanol mixture; (h) centrifuging the second purified extract / ethanol mixture to produce a third purified extract; and (i) extracting the third purified extract. In some embodiments, a method for purifying the isolated extract further includes (j) washing the third purified extract with ethanol (e.g., 70% ethanol) to produce a third purified extract / ethanol mixture, (k) centrifuging the third purified extract / ethanol mixture to produce a fourth purified extract, and (l) extracting the fourth purified extract. In some embodiments, the purified extract contains an nHC-HA / PTX3 complex.

[0048]

[0091] In some embodiments, the nHC-HA / PTX3 complex is purified by immunoaffinity chromatography. In some embodiments, an anti-HC1 antibody, an anti-HC2 antibody, or both are generated and attached to a fixed support. In some embodiments, the unpurified HC-HA complex (i.e., mobile phase) passes through the support. In certain cases, the HC-HA complex binds to an antibody (e.g., by interaction of (a) anti-HC1 antibody and HC1, (b) anti-HC2 antibody and HC2, (c) anti-PTX antibody and PTX3, (d) anti-SLRP antibody and SLRP, or (e) any combination thereof). In some embodiments, the support is washed to remove any unbound or loosely bound molecules (e.g., with PBS). In some embodiments, the support is then washed with a solution that allows elution of the nHC-HA / PTX3 complex from the support (e.g., 1% SDS, 6M guanidine-HCl, or 8M urea).

[0049]

[0092] In some embodiments, the nHC-HA / PTX3 complex is purified by affinity chromatography. In some embodiments, HABP is generated and attached to a stationary support. In some embodiments, the unpurified nHC-HA / PTX3 complex (i.e., mobile phase) passes through the support. In certain cases, the nHC-HA / PTX3 complex binds to the HABP. In some embodiments, the support is washed (e.g., with PBS) to remove any unbound or loosely bound molecules. In some embodiments, the support is then washed with a solution that allows the HC-HA complex to be eluted from the support.

[0050]

[0093] In some embodiments, the nHC-HA / PTX3 complex is purified by HABP affinity chromatography and immunoaffinity chromatography using an antibody against an anti-HC1 antibody, an anti-HC2 antibody, an anti-PTX3 antibody, SLRP or a combination of SLRPs, or any combination of these antibodies.

[0051]

[0094] In some embodiments, the nHC-HA / PTX3 complex is purified from the insoluble fraction described herein using one or more antibodies. In some embodiments, the nHC-HA / PTX3 complex is purified from the insoluble fraction described herein using an anti-SLRP antibody.

[0052]

[0095] In some embodiments, the nHC-HA / PTX3 complex is purified from the soluble fraction described herein. In some embodiments, the nHC-HA / PTX3 complex is purified from the soluble fraction described herein using an anti-PTX3 antibody.

[0053]

[0096] In some embodiments, the nHC-HA / PTX3 complex contains small leucine-rich proteoglycans (SLRPs). In some embodiments, the nHC-HA / PTX3 complex contains class I, class II, or class III SLRPs. In some embodiments, the small leucine-rich proteoglycan is selected from class I SLRPs such as decorin and biglycan. In some embodiments, the small leucine-rich proteoglycan is selected from class II SLRPs such as fibromodulin, lumican, PRELP (proline-arginine-rich terminal leucine-rich protein), keratocan, and osteoadherin. In some embodiments, the small leucine-rich proteoglycan is selected from class III SLRPs such as epiphycan and osteoglycin. In some embodiments, the small leucine-rich proteoglycan is selected from bikunin, decorin, biglycan, and osteoadherin. In some embodiments, the small leucine-rich protein contains glycosaminoglycans. In some embodiments, the small molecule leucine-rich proteoglycan contains keratan sulfate.

[0054] Method for producing rcHC-HA / PTX3 complex

[0097] In some embodiments, the rcHC-HA / PTX3 complex is used in the method provided herein. Such reconstituted HC-HA / PTX3 complex may or may not contain SLRP.

[0055]

[0098] In some embodiments, a method for producing a reconstituted HC-HA / PTX3 complex includes the steps of (a) contacting hyaluronan (HA) with IαI and TSG-6 to form an HC-HA complex pre-bound to TSG-6, and (b) contacting the HC-HA complex with pentraxin 3 (PTX3) under suitable conditions for forming an rcHC-HA / PTX3 complex. An rcHC-HA / PTX3 complex produced by such a method is provided herein. In some embodiments, HC1 of IαI forms a covalent bond with HA. In some embodiments, steps (a) and (b) of the method are performed sequentially. In some embodiments, the method includes the step of contacting the HC-HA complex pre-bound to TSG-6 with PTX3. In some embodiments, hyaluronan (HA) is high molecular weight hyaluronan (HMW HW). In some embodiments, hyaluronan (HA) is low molecular weight hyaluronan (LMW HW).

[0056]

[0099] In some embodiments, a method for producing a reconstituted HC-HA / PTX3 complex comprises the steps of (a) contacting high molecular weight hyaluronane (HMW HA) with IαI and TSG-6 to form an HC-HA complex pre-bound to TSG-6, and (b) contacting the HC-HA complex with pentraxin 3 (PTX3) under suitable conditions for forming an rcHC-HA / PTX3 complex. An rcHC-HA / PTX3 complex produced by such a method is provided herein. In some embodiments, HC1 of IαI forms a covalent bond with HA. In some embodiments, steps (a) and (b) of the method are performed sequentially. In some embodiments, the method includes contacting the HC-HA complex pre-bound to TSG-6 with PTX3.

[0057]

[0100] In some embodiments, the IαI protein and the TSG-6 protein are contacted with HMW HA in a molar ratio of approximately 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, or 20:1 (IαI:TSG-6). In some embodiments, the IαI:TSG-6 ratio is in the range of approximately 1:1 to approximately 20:1, for example, approximately 1:1 to approximately 10:1, for example, approximately 1:1 to approximately 5:1, for example, approximately 1:1 to approximately 3:1. In some embodiments, the IαI:TSG-6 ratio is 3:1 or greater. In some embodiments, the IαI:TSG-6 ratio is 3:1.

[0058]

[0101] In certain cases, TSG-6 interacts with IαI to form covalent complexes with IαI's HC1 and HC2 (e.g., HC1·TSG-6 and HC2·TSG-6). In certain cases, in the presence of HA, HC is transferred to HA to form rcHC-HA.

[0059]

[0102] In some embodiments, the step of contacting hyaluronan (HA) with IαI and TSG-6 is carried out for at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, or at least 24 hours or longer. In some embodiments, the step of contacting HA with IαI and TSG-6 is carried out for at least 2 hours or longer. In some embodiments, the step of contacting HA with IαI and TSG-6 is carried out for at least 2 hours. In some embodiments, the step of contacting HA with IαI and TSG-6 is carried out at 37°C. In some embodiments, the step of contacting immobilized HA with IαI and TSG-6 is carried out in 5 mM MgCl2 in PBS. In some embodiments, hyaluronan (HA) is high molecular weight hyaluronan (HMW HW). In some embodiments, hyaluronan (HA) is low molecular weight hyaluronan (LMW HW).

[0060]

[0103] In some embodiments, the step of contacting high molecular weight hyaluronane (HMW HA) with IαI and TSG-6 is carried out for at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, or at least 24 hours or longer. In some embodiments, the step of contacting HMW HA with IαI and TSG-6 is carried out for at least 2 hours or longer. In some embodiments, the step of contacting HMW HA with IαI and TSG-6 is carried out for at least 2 hours. In some embodiments, the step of contacting HMW HA with IαI and TSG-6 is carried out at 37°C. In some embodiments, the step of contacting immobilized HMW HA with IαI and TSG-6 is carried out in 5 mM MgCl2 in PBS.

[0061]

[0104] In some embodiments, the step of contacting PTX3 with the HC-HA complex is carried out for at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, or at least 24 hours or longer. In some embodiments, the step of contacting PTX3 with the HC-HA complex is carried out for at least 2 hours or longer. In some embodiments, the step of contacting PTX3 with the HC-HA complex is carried out for at least 2 hours. In some embodiments, the step of contacting PTX3 with the HC-HA complex is carried out at 37°C. In some embodiments, the step of contacting PTX3 with the HC-HA complex is carried out in 5 mM MgCl2 in PBS.

[0062]

[0105] In some embodiments, the method includes the step of contacting hyaluronan (HA) with pentraxin 3 (PTX3) protein, an inter-α inhibitor (IαI) protein containing heavy chain 1 (HC1) and heavy chain 2 (HC2), and tumor necrosis factor α-stimulating gene 6 (TSG-6) under suitable conditions for forming an HC-HA / PTX3 complex. In some embodiments, the step of contacting HA with PTX3, IαI, and TSG-6 is carried out for at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, or at least 24 hours or longer. In some embodiments, the step of contacting HA, PTX3, IαI, and TSG-6 is carried out at 37°C. In some embodiments, the step of contacting HA, PTX3, IαI, and TSG-6 is carried out in 5 mM MgCl2 in PBS. In some embodiments, hyaluronan (HA) is high molecular weight hyaluronan (HMW HW). In some embodiments, hyaluronan (HA) is low molecular weight hyaluronan (LMW HW).

[0063]

[0106] In some embodiments, the method includes the step of contacting high molecular weight hyaluronan (HMW HA) with pentraxin 3 (PTX3) protein, inter-α inhibitor (IαI) protein containing heavy chain 1 (HC1) and heavy chain 2 (HC2), and tumor necrosis factor α-stimulating gene 6 (TSG-6) under suitable conditions for forming an HC-HA / PTX3 complex. In some embodiments, the step of contacting HMW HA with PTX3, IαI, and TSG-6 is carried out for at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, or at least 24 hours or longer. In some embodiments, the step of contacting HMW HA, PTX3, IαI, and TSG-6 is carried out at 37°C. In some embodiments, the step of contacting HMW HA, PTX3, IαI, and TSG-6 is carried out in 5 mM MgCl2 in PBS.

[0064]

[0107] In some embodiments, the method includes sequentially contacting hyaluronan (HA) with pentraxin 3 (PTX3) protein, inter-α inhibitor (IαI) proteins including heavy chain 1 (HC1) and heavy chain 2 (HC2), and tumor necrosis factor α-stimulating gene 6 (TSG-6) in any order, under conditions suitable for forming an HC-HA / PTX3 complex. In some embodiments, the step of contacting HMW HA with PTX3, IαI, and TSG-6 is carried out for at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, or at least 24 hours or longer. In some embodiments, the step of contacting HA, PTX3, IαI, and TSG-6 is carried out at 37°C. In some embodiments, the step of contacting HA, PTX3, IαI, and TSG-6 is carried out in 5 mM MgCl2 in PBS. In some embodiments, hyaluronan (HA) is high molecular weight hyaluronan (HMW HW). In some embodiments, hyaluronan (HA) is low molecular weight hyaluronan (LMW HW).

[0065]

[0108] In some embodiments, the method includes sequentially contacting high molecular weight hyaluronan (HMW HA) with pentraxin 3 (PTX3) protein, inter-α inhibitor (IαI) protein comprising heavy chain 1 (HC1) and heavy chain 2 (HC2), and tumor necrosis factor α-stimulating gene 6 (TSG-6) in any order, under conditions suitable for forming an HC-HA / PTX3 complex. In some embodiments, the step of contacting HMW HA with PTX3, IαI, and TSG-6 is carried out for at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, or at least 24 hours or longer. In some embodiments, the step of contacting HMW HA, PTX3, IαI, and TSG-6 is carried out at 37°C. In some embodiments, the step of contacting HMW HA, PTX3, IαI, and TSG-6 is carried out in 5 mM MgCl2 in PBS.

[0066]

[0109] In some embodiments, a method for producing an rcHC-HA / PTX3 complex further comprises the addition of one or more small molecule leucine-rich proteoglycans (SLRPs). In some embodiments, a method for generating a reconstituted HC-HA / PTX3 complex comprises (a) contacting hyaluronan (HA) with IαI and TSG-6 with HA to form an HC-HA complex pre-bound to TSG-6; (b) contacting the HC-HA complex with pentraxin 3 (PTX3); and (c) contacting the HC-HA complex with one or more SLRPS under suitable conditions for forming an rcHC-HA / PTX3 complex. In some embodiments, a method for producing a reconstituted HC-HA / PTX3 complex includes the steps of (a) contacting high molecular weight hyaluronane (HMW HA) with IαI and TSG-6 with HA to form an HC-HA complex pre-bound to TSG-6; (b) contacting the HC-HA complex with pentraxin 3 (PTX3); and (c) contacting the HC-HA complex with one or more SLRPS under suitable conditions for forming an rcHC-HA / PTX3 complex. An rcHC-HA / PTX3 complex produced by such a method is provided herein. In some embodiments, HC1 of IαI forms a covalent bond with HA. In some embodiments, the method includes the step of contacting the HC-HA complex pre-bound to TSG-6 with PTX3. In some embodiments, steps (a), (b), and (c) of the method are performed sequentially. In some embodiments, steps (a), (b), and (c) of the method are performed simultaneously. In some embodiments, step (a) of the method is performed, followed by steps (b) and (c) of the method in sequence. In some embodiments, step (a) of the method is performed, followed by steps (b) and (c) of the method simultaneously.

[0067]

[0110] In some embodiments, the SLRP is selected from class I, class II, or class II SLRPs. In some embodiments, the SLRP is selected from class I SLRPs such as decorin and biglycan. In some embodiments, the small molecule leucine-rich proteoglycan is selected from class II SLRPs such as fibromodulin, lumican, PRELP (proline arginine-rich terminal leucine-rich protein), keratocan, and osteoadherin. In some embodiments, the small molecule leucine-rich proteoglycan is selected from class III SLRPs such as epiphycan and osteoglycin. In some embodiments, the small molecule leucine-rich proteoglycan is selected from bikunin, decorin, biglycan, and osteoadherin. In some embodiments, the small molecule leucine-rich protein includes glycosaminoglycans. In some embodiments, the small molecule leucine-rich proteoglycan includes keratan sulfate.

[0068]

[0111] PTX3

[0112] In some embodiments, PTX3 for use in the method is isolated from a cell or a group of cells (e.g., a tissue extract). Exemplary cells suitable for PTX3 expression include, but are not limited to, mammalian cells, primate cells, human cells, rodent cells, insect cells, bacteria, and yeast, as well as, but are not limited to, plant cells including, but are not limited to, algae, angiosperms, gymnosperms, ferns, and mosses. In some embodiments, PTX3 for use in the method is isolated from human cells. In some embodiments, PTX3 for use in the method is isolated from cells stimulated with one or more inflammatory cytokines to upregulate PTX3 expression. In some embodiments, the inflammatory cytokine is IL-1 or TNF-α.

[0069]

[0113] In some embodiments, PTX3 for use in the method is isolated from amniotic cells. In some embodiments, PTX3 for use in the method is isolated from amniotic cells derived from the umbilical cord. In some embodiments, amniotic cells are stimulated with one or more inflammatory cytokines to upregulate PTX3 expression. In some embodiments, the inflammatory cytokine is IL-1 or TNF-α.

[0070]

[0114] In some embodiments, PTX3 for use in the method is isolated from umbilical cord cells. In some embodiments, umbilical cord cells are stimulated with one or more inflammatory cytokines to upregulate PTX3 expression. In some embodiments, the inflammatory cytokine is IL-1 or TNF-α.

[0071]

[0115] In some embodiments, PTX3 for use in the method is isolated from amniotic epithelial cells. In some embodiments, PTX3 for use in the method is isolated from umbilical cord epithelial cells. In some embodiments, amniotic epithelial cells or umbilical cord epithelial cells are stimulated with one or more inflammatory cytokines to upregulate PTX3 expression. In some embodiments, the inflammatory cytokine is IL-1 or TNF-α.

[0072]

[0116] In some embodiments, PTX3 for use in the method is isolated from amniotic stromal cells. In some embodiments, PTX3 for use in the method is isolated from umbilical cord stromal cells. In some embodiments, amniotic stromal cells or umbilical cord stromal cells are stimulated with one or more inflammatory cytokines to upregulate PTX3 expression. In some embodiments, the inflammatory cytokine is IL-1 or TNF-α.

[0073]

[0117] In some embodiments, the PTX3 used in the method is a native PTX3 protein isolated from cells. In some embodiments, cells are stimulated with one or more inflammatory cytokines to upregulate PTX3 expression. In some embodiments, the inflammatory cytokine is IL-1 or TNF-α.

[0074]

[0118] In some embodiments, PTX3 is prepared by recombinant technology. In some embodiments, PTX3 is expressed from a recombinant expression vector. In some embodiments, the nucleic acid encoding PTX3 is operably ligated to a constitutive promoter. In some embodiments, the nucleic acid encoding PTX3 is operably ligated to an inductive promoter. In some embodiments, PTX3 is expressed in transgenic animals. In some embodiments, PTX3 is a recombinant protein. In some embodiments, PTX3 is a recombinant protein isolated from cells. In some embodiments, PTX3 is a recombinant protein produced in cell-free extracts.

[0075]

[0119] In some embodiments, PTX3 is purified from the amnion, umbilical cord, amniotic membrane of the umbilical cord, chorion, amniotic fluid, or a combination thereof. In some embodiments, PTX3 is purified from amnion cells. In some embodiments, the amnion cells are amnion epithelial cells. In some embodiments, the amnion cells are umbilical cord epithelial cells. In some embodiments, the amnion cells are amnion stromal cells. In some embodiments, the amnion cells are umbilical cord stromal cells. In some embodiments, the amnion cells are stimulated with one or more inflammatory cytokines to upregulate PTX3 expression. In some embodiments, the inflammatory cytokines are IL-1 or TNF-α.

[0076]

[0120] In some embodiments, PTX3 is not isolated from cells or groups of cells (e.g., tissue extracts).

[0121] In some embodiments, PTX3 comprises a fragment of PTX3 sufficient to promote the formation of an rcHC-HA / PTX3 complex. Variants of PTX3 for use in the provided method include variants having amino acid modifications, which are amino acid substitutions, deletions, or insertions. In some embodiments, such modifications enhance one or more properties of the PTX3 polypeptide, for example, one or more therapeutic properties of the rcHC-HA / PTX3 complex (e.g., anti-inflammatory, anti-immune, anti-angiogenic, anti-scarring, anti-adhesion, regenerative, or other therapeutic activities described herein).

[0077]

[0122] In some embodiments, the PTX3 protein is obtained from a commercial source. An example commercial source of PTX3 is, but is not limited to, PTX3 (catalog number 1826-TS; R&D Systems, Minneapolis, MN).

[0078]

[0123] In some embodiments, the PTX3 protein used in the method is a multimeric protein. In some embodiments, the PTX3 protein used in the method is a homomultimer. In some embodiments, the homomultimer is a dimer, trimer, tetramer, hexamer, pentamer, or octamer. In some embodiments, the PTX3 homomultimer is a trimer, tetramer, or octamer. In certain embodiments, the PTX3 homomultimer is an octamer. In some embodiments, the multimerizing domain is modified to improve the multimerization of the PTX3 protein. In some embodiments, the multimerizing domain is replaced with a heterologous multimerizing domain (e.g., an Fc multimerizing domain or a leucine zipper) that improves the multimerization of PTX3 when fused with PTX3.

[0079]

[0124] TSG-6

[0125] In some embodiments, TSG-6 for use in the method is isolated from a cell or a group of cells (e.g., a tissue extract). Exemplary cells suitable for TSG-6 expression include, but are not limited to, mammalian cells, primate cells, human cells, rodent cells, insect cells, bacteria, and yeast, as well as, but are not limited to, plant cells including, but are not limited to, algae, angiosperms, gymnosperms, ferns, and mosses. In some embodiments, TSG-6 for use in the method is isolated from human cells. In some embodiments, TSG-6 for use in the method is isolated from cells stimulated with one or more inflammatory cytokines to upregulate TSG-6 expression. In some embodiments, the inflammatory cytokine is IL-1 or TNF-α.

[0080]

[0126] In some embodiments, TSG-6 for use in the method is isolated from amniotic cells. In some embodiments, TSG-6 for use in the method is isolated from amniotic cells derived from the umbilical cord. In some embodiments, TSG-6 for use in the method is isolated from amniotic cells stimulated with one or more inflammatory cytokines to upregulate TSG-6 expression. In some embodiments, the inflammatory cytokine is IL-1 or TNF-α.

[0081]

[0127] In some embodiments, TSG-6 for use in the method is isolated from umbilical cord cells. In some embodiments, TSG-6 for use in the method is isolated from umbilical cord cells stimulated with one or more inflammatory cytokines to upregulate TSG-6 expression. In some embodiments, the inflammatory cytokine is IL-1 or TNF-α.

[0082]

[0128] In some embodiments, TSG-6 for use in the method is isolated from amniotic epithelial cells. In some embodiments, TSG-6 for use in the method is isolated from umbilical cord epithelial cells. In some embodiments, TSG-6 for use in the method is isolated from amniotic epithelial cells or umbilical cord epithelial cells stimulated with one or more inflammatory cytokines to upregulate TSG-6 expression. In some embodiments, the inflammatory cytokine is IL-1 or TNF-α.

[0083]

[0129] In some embodiments, TSG-6 for use in the method is isolated from amniotic stromal cells. In some embodiments, TSG-6 for use in the method is isolated from umbilical cord stromal cells. In some embodiments, TSG-6 for use in the method is isolated from amniotic stromal cells or umbilical cord stromal cells stimulated with one or more inflammatory cytokines to upregulate TSG-6 expression. In some embodiments, the inflammatory cytokine is IL-1 or TNF-α.

[0084]

[0130] In some embodiments, the TSG-6 used in the method is a native TSG-6 protein isolated from cells. In some embodiments, cells are stimulated with one or more inflammatory cytokines to upregulate TSG-6 expression. In some embodiments, the inflammatory cytokine is IL-1 or TNF-α.

[0085]

[0131] In some embodiments, TSG-6 is prepared by recombinant technology. In some embodiments, TSG-6 is expressed from a recombinant expression vector. In some embodiments, the nucleic acid encoding TSG-6 is operably ligated to a constitutive promoter. In some embodiments, the nucleic acid encoding TSG-6 is operably ligated to an inductive promoter. In some embodiments, TSG-6 is expressed in transgenic animals. In some embodiments, TSG-6 is a recombinant protein. In some embodiments, TSG-6 is a recombinant protein isolated from cells. In some embodiments, TSG-6 is a recombinant protein produced in cell-free extracts.

[0086]

[0132] In some embodiments, TSG-6 is purified from the amnion, chorion, amniotic fluid, or a combination thereof. In some embodiments, TSG-6 is purified from amnion cells. In some embodiments, the amnion cells are amnion epithelial cells. In some embodiments, the amnion epithelial cells are umbilical cord epithelial cells. In some embodiments, the amnion cells are amnion stromal cells. In some embodiments, the amnion cells are umbilical cord stromal cells. In some embodiments, the amnion cells are umbilical cord stromal cells. In some embodiments, the amnion cells are stimulated with one or more inflammatory cytokines to upregulate TSG-6 expression. In some embodiments, the inflammatory cytokines are IL-1 or TNF-α.

[0087]

[0133] In some embodiments, TSG-6 is not isolated from cells or groups of cells (e.g., tissue extracts).

[0134] In some embodiments, TSG-6 includes a fragment of TSG-6 that is sufficient to promote or catalyze the transfer of IαI HC1 to HA. In some embodiments, TSG-6 includes a link module of TSG-6.

[0088]

[0135] In some embodiments, TSG-6 includes affinity tags. Exemplary affinity tags include, but are not limited to, hemagglutinin tags, polyhistidine tags, myc tags, FLAG tags, and glutathione-S-transferase (GST) tags. Such affinity tags are well known in the art for their use in purification. In some embodiments, such affinity tags are incorporated into the TSG-6 polypeptide as a fusion protein or via a chemical linker. In some embodiments, TSG-6 includes affinity tags, and unbound TSG-6 is removed from the rcHC-HA / PTX3 complex by affinity purification.

[0089]

[0136] In some embodiments, the TSG-6 protein is obtained from a commercial source. An example commercial source of TSG-6 is, but is not limited to, TSG-6 (catalog number 2104-TS R&D Systems, Minneapolis, MN).

[0090]

[0137] IαI

[0138] In some embodiments, IαI includes an HC1 chain. In some embodiments, IαI includes both HC1 and HC2 chains. In some embodiments, IαI includes both HC1 and HC2 chains as well as bicinnamin. In some embodiments, IαI includes both HC1 and HC2 chains as well as bicinnamin, linked by chondroitin sulfate chains.

[0091]

[0139] In some embodiments, IαI is isolated from a biological sample. In some embodiments, the biological sample is a mammalian biological sample. In some embodiments, the mammal is a human. In some embodiments, the biological sample is a blood, serum, plasma, liver, amnion, chorionic, or amniotic fluid sample. In some embodiments, the biological sample is a blood, serum, or plasma sample. In some embodiments, the biological sample is a blood sample. In some embodiments, the biological sample is a serum sample. In some embodiments, the biological sample is a plasma sample. In some embodiments, IαI is purified from human blood, plasma, or serum. In some embodiments, IαI is isolated from human serum. In some embodiments, IαI is not isolated from serum. In some embodiments, IαI for use in the method is produced in amniotic cells. In some embodiments, IαI for use in the method is produced in umbilical cord cells. In some embodiments, IαI for use in the method is produced in umbilical cord-derived amniotic cells. In some embodiments, IαI for use in the method is produced in amniotic epithelial cells. In some embodiments, IαI for use in the method is produced in umbilical cord epithelial cells. In some embodiments, IαI for use in the method is produced in amniotic stromal cells. In some embodiments, IαI for use in the method is produced in umbilical cord stromal cells. In some embodiments, IαI for use in the method is produced in hepatocytes. In some embodiments, IαI is prepared by recombinant technology.

[0092]

[0140] In some embodiments, IαI HC1 is isolated from a biological sample. In some embodiments, the biological sample is a mammalian biological sample. In some embodiments, the mammal is a human. In some embodiments, the biological sample is a blood, serum, plasma, liver, amnion, chorionic, or amniotic fluid sample. In some embodiments, the biological sample is a blood, serum, or plasma sample. In some embodiments, the biological sample is a blood sample. In some embodiments, the biological sample is a serum sample. In some embodiments, the biological sample is a plasma sample. In some embodiments, IαI HC1 is purified from human blood, plasma, or serum. In some embodiments, IαI is isolated from human serum. In some embodiments, IαI HC1 is not purified from serum. In some embodiments, IαI HC1 is prepared by recombinant technology. In some embodiments, IαI HC1 is purified from hepatocytes. In some embodiments, IαI HC1 is purified from amnion cells. In some embodiments, IαI HC1 is purified from amnion epithelial cells or umbilical cord epithelial cells. In some embodiments, IαI HC1 is purified from amniotic stromal cells or umbilical cord stromal cells.

[0093]

[0141] In some embodiments, IαI HC2 is isolated from a biological sample. In some embodiments, the biological sample is a mammalian biological sample. In some embodiments, the mammal is a human. In some embodiments, the biological sample is a blood, serum, plasma, liver, amnion, chorionic, or amniotic fluid sample. In some embodiments, the biological sample is a blood, serum, or plasma sample. In some embodiments, the biological sample is a blood sample. In some embodiments, the biological sample is a serum sample. In some embodiments, the biological sample is a plasma sample. In some embodiments, IαI HC2 is purified from human blood, plasma, or serum. In some embodiments, IαI HC2 is isolated from human serum. In some embodiments, IαI HC2 is isolated from human serum. In some embodiments, IαI HC2 is not isolated from serum. In some embodiments, IαI HC2 is prepared by recombinant technology. In some embodiments, IαI HC2 is purified from hepatocytes. In some embodiments, IαI HC2 is purified from amnion cells. In some embodiments, IαI HC2 is purified from amnion epithelial cells or umbilical cord epithelial cells. In some embodiments, IαI HC2 is purified from amniotic stromal cells or umbilical cord stromal cells.

[0094]

[0142] HA

[0143] In some embodiments, HA is purified from cell, tissue, or fluid samples. In some embodiments, HA is obtained from commercial suppliers (e.g., Sigma Aldrich or Advanced Medical Optics, Irvine, CA (e.g., Healon)). In some embodiments, HA is obtained as a powder from commercial suppliers. In some embodiments, HA is expressed in cells. Exemplary cells suitable for HA expression include, but are not limited to, mammalian cells, primate cells, human cells, rodent cells, insect cells, bacteria, and yeast, as well as plant cells including, but are not limited to, algae, angiosperms, gymnosperms, ferns, and mosses. In some embodiments, HA is expressed in human cells. In some embodiments, HA is expressed in transgenic animals. In some embodiments, HA is obtained from cells expressing hyaluronansynthase (e.g., HAS1, HAS2, and HAS3). In some embodiments, the cells contain a recombinant expression vector expressing HA synthase. In certain cases, HA synthase elongates hyaluronan by repeatedly adding glucuronic acid and N-acetylglucosamine to the newly synthesized polysaccharide, while the hyaluronan is pushed through the cell membrane into the extracellular space.

[0095]

[0144] In some embodiments, the HA used in the method is high molecular weight (HMW) HA. In some embodiments, the weight-average molecular weight of HMW HA exceeds about 500 kilodaltons (kDa), for example, about 500 kDa to about 10,000 kDa, about 800 kDa to about 8,500 kDa, about 1100 kDa to about 5,000 kDa, or about 1400 kDa to about 3,500 kDa. In some embodiments, the weight-average molecular weight of HMW HA is about 3000 kDa.

[0096]

[0145] Additional ingredients

[0146] In some embodiments, one or more additional components are added to form the rcHC-HA / PTX3 complex. In some embodiments, small molecule leucine-rich proteoglycans (SLRPs) are added to form the rcHC-HA / PTX3 complex. In some embodiments, the SLRP is a class I, class II, or class II SLRP. In some embodiments, the SLRP is selected from among class I SLRPs such as decorin and biglycan. In some embodiments, the SLRP is selected from among class II SLRPs such as fibromodulin, lumican, PRELP (proline-arginine-rich terminal leucine-rich protein), keratocan, and osteoadherin. In some embodiments, the SLRP is selected from among class III SLRPs such as epiphycan and osteoglycin. In some embodiments, the SLRP is selected from among bikunin, decorin, biglycan, and osteoadherin. In some embodiments, the SLRP contains glycosaminoglycans. In some embodiments, the SLRP contains keratan sulfate.

[0097]

[0147] In some embodiments, HMW HA is immobilized by any suitable method. In some embodiments, HMW HA is immobilized on a solid support, such as a culture dish, beads, column, or other suitable surface, such as the surface of an implantable medical device or a part thereof, or a surface to be connected to or bonded to an implantable medical device described herein later. In some embodiments, HMW HA is immobilized directly on a solid support by chemical bonding or the like. In some embodiments, HMW HA is indirectly bonded to a solid support via a linker or intermediate protein. Numerous heterobifunctional crosslinking reagents used to form covalent bonds between amino and thiol groups and to introduce thiol groups into proteins are known to those skilled in the art. In some embodiments, HMW HA is immobilized directly on a solid support by crosslinking to the solid support. In some embodiments, HMW HA is immobilized directly on a solid support without crosslinking to the solid support. In some embodiments, HMW HA is immobilized directly on a solid support as a coating agent. In some embodiments, HMW HA is immobilized on a Covalink(trademark)-NH surface. In some embodiments, HMW HA is immobilized directly on a solid support as a coating agent. In some embodiments, HMW HA is fixed to the Covalink™-NH surface for approximately 16 hours at 4°C.

[0098]

[0148] In some embodiments, the method includes the step of immobilizing HMW HA on a solid surface by direct binding to a solid support (i.e., without intermediate proteins). In some embodiments, the solid support is washed to remove unbound HMW HA before contacting the immobilized HA with IαI, TSG-6, and PTX3. In some embodiments, the solid support is washed with a washing solution of 8M GnHCl and PBS to remove unbound HMW HA before contacting the immobilized HA with IαI, TSG-6, and PTX3.

[0099]

[0149] In some embodiments, the method includes the step of immobilizing HA on a solid surface via an intermediate protein or linker. In some embodiments, the linker is a peptide linker. In some embodiments, the intermediate protein is an HA-binding protein (HABP). In some embodiments, the HABP is first bound to the solid support (e.g., by crosslinking, chemical bonding, or via a chemical linker). In some embodiments, the solid support containing the HABP is then brought into contact with HA (e.g., HMW HA) to immobilize the HA to the solid support by binding of the HABP to the HA. In some embodiments, before contacting the immobilized HMW HA with IαI, TSG-6, and PTX3, the solid support is washed to remove unbound HMW HA. In some embodiments, before contacting the immobilized HA with IαI, TSG-6, and PTX3, the solid support is washed with a washing solution of 8M GnHCl and PBS to remove unbound HMW HA.

[0100]

[0150] In some embodiments, the method includes the step of immobilizing HA on a solid surface by binding a peptide linker to a solid support and HA to the peptide linker. In some embodiments, the peptide linker includes a protease cleavage site.

[0101]

[0151] In some embodiments, the method includes the step of fixing HA to a solid surface via the bonding of a cleavable chemical linker, such as a disulfide chemical linker, but is not limited thereto.

[0102]

[0152] In some embodiments, the HABP selected for use in the method is a HABP that is dissociated from HA after the formation of the rcHC-HA / PTX3 complex. In some embodiments, the HABP is non-covalently bound to HA. In some embodiments, the method further includes the step of dissociating the rcHC-HA / PTX3 complex from the HABP using one or more dissociating agents. Dissociating agents for breaking non-covalent interactions (e.g., guanidine hydrochloride, urea, and various surfactants, e.g., SDS) are known in the art. In some embodiments, the dissociating agent is urea. In some embodiments, the dissociating agent is guanidine hydrochloride. In some embodiments, the dissociating agent is about 4M to about 8M guanidine-HCl. In some embodiments, the dissociating agent is about 4M, about 5M, about 6M, about 7M, or about 8M guanidine-HCl. In some embodiments, the dissociating agent is about 4M to about 8M guanidine-HCl in PBS at pH 7.5.

[0103]

[0153] In some embodiments, such a dissociating agent is used to dissociate the rcHC-HA / PTX3 complex from an intermediate HABP. The HABP used in the method is typically selected so that its binding affinity to HA is strong enough to allow assembly of the rcHC-HA / PTX3 complex, but it is dissociated from the rcHC-HA / PTX3 complex with a suitable dissociating agent. In some embodiments, the dissociating agent is guanidine hydrochloride.

[0104]

[0154] Exemplary HABPs for use with the methods provided herein include, but are not limited to, HAPLIN1, HAPLIN2, HAPLIN3, HAPLIN4, aggrecan, versican, neurocan, brevican, phosphacan, TSG-6, CD44, stabilin-1, stabilin-2, or a portion thereof sufficient to bind to HA (e.g., their link modules). In some embodiments, HABP is versican. In some embodiments, HABP is a recombinant protein. In some embodiments, HABP is a recombinant mammalian protein. In some embodiments, HABP is a recombinant human protein. In some embodiments, HABP is a recombinant versican protein or a portion thereof sufficient to bind to HA. In some embodiments, HABP is a recombinant aggrecan protein or a portion thereof sufficient to bind to HA. In some embodiments, HABP is native HABP or a portion thereof sufficient to bind to HA. In some embodiments, native HABP is isolated from mammalian tissue or cells. In some embodiments, HABP is isolated from bovine nasal cartilage (e.g., HABP from Seikagaku containing the HA-binding domains of aggrecan and link proteins).

[0105]

[0155] In some embodiments, HABP includes a link module of HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican, neurocan, brevican, phosphacan, TSG-6, CD44, stabilin-1, or stabilin-2. In some embodiments, HABP includes a link module of versican. In some embodiments, HABP including a link module is a recombinant protein. In some embodiments, HABP including a link module of versican is a recombinant protein.

[0106]

[0156] In some embodiments, the intermediate protein, such as HABP, contains a proteolytic cleavage sequence that is recognized and hydrolyzed by a site-specific protease such as furin, 3C protease, caspase, matrix metalloproteinase, or TEV protease. In such embodiments, the assembled rcHC-HA / PTX3 complex is released from the solid support by contacting the immobilized complex with a protease that cleaves a specific cleavage sequence.

[0107]

[0157] In some embodiments, the rcHC-HA / PTX3 complex is purified. In some embodiments, the rcHC-HA / PTX3 complex is purified by any suitable method or combination of methods. The embodiments described below are not intended to be exclusive and are illustrative only.

[0108]

[0158] In some embodiments, the rcHC-HA / PTX3 complex is purified by chromatography (e.g., ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), tangential flow filtration (TFF), gel filtration, centrifugation (e.g., gradient centrifugation), differential solubility, ethanol precipitation, or any other available technique for protein purification.

[0109]

[0159] In some embodiments, the rcHC-HA / PTX3 complex is purified by immunoaffinity chromatography. In some embodiments, antibodies are produced against components of the rcHC-HA / PTX3 complex (e.g., anti-HC1, anti-PTX3, antibodies against one or more SLRPs of the rcHC-HA / PTX3 complex, e.g., anti-bikunin, anti-decolin, anti-biglycan, or anti-osteoadherin) and attached to a solid support. In some embodiments, the unpurified rcHC-HA / PTX3 complex (i.e., mobile phase) passes through the support. In certain cases, the rcHC-HA / PTX3 complex binds to the antibody. In some embodiments, the support is washed to remove any unbound or loosely bound molecules (e.g., with PBS). In some embodiments, the support is then washed with a solution that allows elution of the rcHC-HA / PTX3 complex from the support (e.g., 1% SDS, 6M guanidine-HCl, or 8M urea). In some embodiments, the dissociating agent is removed from the dissociated rcHC-HA / PTX3 complex. In some embodiments, the dissociating agent is removed from the dissociated rcHC-HA / PTX3 complex by methods including, but not limited to, ion exchange chromatography, dialysis, tangential flow filtration (TFF), gel filtration chromatography, ultrafiltration, or dialysis.

[0110]

[0160] In some embodiments, the rcHC-HA / PTX3 complex is purified by affinity chromatography. In some embodiments, HABP is used to bind to the rcHC-HA / PTX3 complex for purification and is attached to a stationary support. In some embodiments, the unpurified rcHC-HA / PTX3 complex (i.e., mobile phase) passes through the support. In certain cases, the rcHC-HA / PTX3 complex binds to the HABP. In some embodiments, the support is washed (e.g., with PBS) to remove any unbound or loosely bound molecules. In some embodiments, the support is then washed with a solution (e.g., a dissociator) that allows the rcHC-HA / PTX3 complex to be eluted from the support. In some embodiments, the dissociator is removed from the dissociated rcHC-HA / PTX3 complex by methods including, but not limited to, ion exchange chromatography, dialysis, tangential flow filtration (TFF), gel filtration chromatography, ultrafiltration, or dialysis.

[0111]

[0161] In some embodiments, the rcHC-HA / PTX3 complex is purified by a combination of HABP affinity chromatography and immunoaffinity chromatography using antibodies against one or more components of the rcHC-HA / PTX3 complex.

[0112]

[0162] In some embodiments, one or more components of the rcHC-HA / PTX3 complex disclosed herein include an affinity tag (e.g., a fusion protein of PTX3 or HC1 with the affinity tag). Exemplary affinity tags incorporated into one or more components of the rcHC-HA / PTX3 complex include, but are not limited to, hemagglutinin tags, polyhistidines, myc tags, FLAG tags, or glutathione-S-transferase sequences in some embodiments. In some embodiments, the ligand of the affinity tag is attached to a solid support. In some embodiments, the unpurified rcHC-HA / PTX3 complex passes through the support. In certain cases, the rcHC-HA / PTX3 complex binds to the ligand. In some embodiments, the support is washed (e.g., with PBS) to remove any unbound or loosely bound molecules. In some embodiments, the support is then washed with a solution that allows the rcHC-HA / PTX3 complex disclosed herein to be eluted from the support. In some embodiments, the eluent is removed from the dissociated rcHC-HA / PTX3 complex by methods including, but not limited to, ion exchange chromatography, dialysis, tangential flow filtration (TFF), gel filtration chromatography, ultrafiltration, or dialysis.

[0113]

[0163] In some embodiments, PTX3, TSG-6, and / or HC1 are conjugated to a label. “Label” refers to a detectable compound or composition that is directly or indirectly conjugated to a polypeptide to produce a labeled polypeptide. In some embodiments, the label is detectable on its own (e.g., radioisotope labeling or fluorescent labeling) or, in the case of enzymatic labeling, catalyzes a chemical change in a detectable substrate compound composition. Non-limiting examples of labels include fluorescence-generating moieties, dyes, fluorescent tags, green fluorescent proteins, or luciferases.

[0114]

[0164] Method for determining the activity of nHC-HA / PTX3 and rcHC-HA / PTX3 complexes

[0165] The properties of the nHC-HA / PTX3 and rcHC-HA / PTX3 complexes provided herein are determined by any suitable method, including in vitro and in vivo methods. Exemplary in vitro methods provided herein include, but are not limited to, cell culture methods for determining the ability of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex to promote macrophage binding to an immobilized nHC-HA / PTX3 or rcHC-HA / PTX3 complex, to inhibit or reduce macrophage aggregation, to promote neutrophil apoptosis, to promote macrophage phagocytosis by apoptotic neutrophils, and to promote M2 polarization of stimulated macrophages. In some embodiments, macrophages used in the assay are stimulated, for example, by exposure to LPS or IFN-γ. In some embodiments, gene or protein expression in the stimulated macrophages is determined after contact with the nHC-HA / PTX3 or rcHC-HA / PTX3 complex. In such a method for determining the activity of an nHC-HA / PTX3 or rcHC-HA / PTX3 complex, a suitable control is used for comparison. In some embodiments, the control is untreated with the nHC-HA / PTX3 or rcHC-HA / PTX3 complex (i.e., a negative control).

[0115]

[0166] In some embodiments, the activity of the rcHC-HA / PTX3 complex is compared to the activity of the native HC-HA / PTX3 complex. In some embodiments, the native HC-HA / PTX3 is isolated from the amniotic membrane.

[0116]

[0167] In some embodiments, gene expression in treated macrophages is determined by PCR, RT-PCR, Northern blotting, Western blotting, dot blotting, immunohistochemistry, chromatography, or other suitable method for detecting proteins or nucleic acids. In some embodiments, the expression levels of IL-10, IL-12, IL-23, LIGHT, and SPHK1 are determined.

[0117] Pharmaceutical composition

[0168] In certain embodiments, pharmaceutical compositions comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein are disclosed herein. In certain embodiments, pharmaceutical compositions comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex produced by the method provided herein are disclosed herein. In some embodiments, the pharmaceutical composition is formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and adjuvants that facilitate the treatment of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex into a preparation suitable for pharmaceutically acceptable use. The suitable formulation depends on the selected route of administration. Any of the well-known techniques, carriers, and excipients may be used appropriately as understood in the art.

[0118]

[0169] In certain embodiments, pharmaceutical compositions comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein are disclosed herein. In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises adjuvants, excipients, preservatives, absorption retarders, fillers, binders, adsorbents, buffers and / or solubilizers. Exemplary pharmaceutical compositions formulated to contain the nHC-HA / PTX3 or rcHC-HA / PTX3 complex provided herein include, but are not limited to, liquids, suspensions, emulsions, syrups, granules, powders, ointments, tablets, capsules, pills, tinctures, transdermal systems, ointments, lotions, creams, pastes, foams, gels, or aerosols.

[0119]

[0170] Dosage form

[0171] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are administered as an aqueous suspension. In some embodiments, the aqueous suspension comprises water, Ringer's solution and / or an isotonic sodium chloride solution. In some embodiments, the aqueous suspension comprises a sweetener or flavoring agent, a coloring agent or pigment, and, if necessary, an emulsifier or suspending agent, together with a diluent, water, ethanol, propylene glycol, glycerin, or a combination thereof. In some embodiments, the aqueous suspension comprises a suspending agent. In some embodiments, the aqueous suspension comprises sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum and / or gum arabic. In some embodiments, the aqueous suspension comprises a dispersant or wetting agent. In some embodiments, the aqueous suspension includes naturally occurring phospholipids, such as lecithin, or condensation products of alkylene oxides and fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxide and long-chain aliphatic alcohols, such as heptadecaethylene-oxycetanol, or condensation products of ethylene oxide and partial esters derived from fatty acids and hexitol, such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide and partial esters derived from fatty acids and hexitol anhydride, such as polyethylene sorbitan monooleate. In some embodiments, the aqueous suspension includes a preservative. In some embodiments, the aqueous suspension includes ethyl p-hydroxybenzoate or n-propyl. In some embodiments, the aqueous suspension includes a sweetener. In some embodiments, the aqueous suspension includes sucrose, saccharin, or aspartame.

[0120]

[0172] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are administered as an oily suspension. In some embodiments, the oily suspension is formulated by suspending the active ingredient in a vegetable oil (e.g., peanut oil, olive oil, sesame oil, or coconut oil) or mineral oil (e.g., liquid paraffin). In some embodiments, the oily suspension contains a thickener (e.g., beeswax, solid paraffin, or cetyl alcohol). In some embodiments, the oily suspension contains a sweetener (e.g., one of the above). In some embodiments, the oily suspension contains an antioxidant (e.g., butylhydroxyanisole or alpha-tocopherol).

[0121]

[0173] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated for parenteral injection (e.g., by injection or infusion including intra-arterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreous, epidural, and / or subcutaneous injection). In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are administered as sterile solutions, suspensions, or emulsions.

[0122]

[0174] In some embodiments, the formulation for parenteral administration includes an aqueous and / or non-aqueous (oily) sterile injection solution of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein, which in some embodiments contains an antioxidant, a buffer, a bacteriostatic agent and / or a solute to make the formulation isotonic with the blood of the recipient to whom the formulation is intended, and / or an aqueous and / or non-aqueous sterile suspension, which in some embodiments contains a suspending agent and / or a thickening agent. In some embodiments, the formulation for parenteral administration includes an agent to increase the solubility of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein in order to enable the preparation of a suitable stabilizer or a highly concentrated solution.

[0123]

[0175] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are administered as oil-in-water microemulsions in which the active ingredient is dissolved in an oily phase. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are dissolved in fatty oils (e.g., sesame oil), synthetic fatty acid esters (e.g., ethyl oleate or triglycerides), or liposomes. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are dissolved in a mixture of soybean oil and / or lecithin. In some embodiments, the oil is introduced into a mixture of water and glycerol and processed to form a microemulsion.

[0124]

[0176] In some embodiments, the composition formulated for parenteral administration is administered as a single bolus shot. In some embodiments, the composition formulated for parenteral administration is administered by a continuous intravenous delivery device (e.g., a Deltec CADD-PLUS® model 5400 intravenous pump).

[0125]

[0177] In some embodiments, the formulation for injection is provided in unit dosage form, for example, in ampoules or multi-dose containers with preservatives added. In some embodiments, the formulation for injection is stored in powder form or lyophilized (freeze-dried) state, requiring only the addition of a sterile liquid carrier, for example, saline or sterile pyrogen-free water, immediately before use.

[0126]

[0178] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated for topical administration. Topical formulations include, but are not limited to, ointments, creams, lotions, solutions, pastes, gels, films, sticks, liposomes, and nanoparticles. In some embodiments, topical formulations are administered by the use of patches, bandages, or wound dressings.

[0127]

[0179] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated as compositions in the form of solids, crosslinked gels, or liposomes. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated as insoluble crosslinked hydrogels.

[0128]

[0180] In some embodiments, the topical formulation includes a gelling (or thickening) agent. Suitable gelling agents include cellulose, cellulose derivatives, cellulose ethers (e.g., carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose), guar gum, xanthan gum, locust bean gum, alginates (e.g., alginic acid), silicates, starch, tragacanth, carboxyvinyl polymer, carrageenan, paraffin, petrolatum, acacia gum (acacia gum), agar, magnesium aluminum silicate, sodium alginate, sodium stearate, fucus vesiculosus, bentonite, carbomer, carrageenan, carbopol, xanthan gum, cellulose, crystalline cellulose (MCC), ceratonia, dextrose, ferceleran, gelatin, ghati gum, guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, and bee. This includes, but is not limited to, honey, corn starch, wheat starch, rice starch, potato starch, gelatin, karaya gum, polyethylene glycol (e.g., PEG200-4500), tragacanth gum, ethylcellulose, ethyl hydroxyethylcellulose, ethyl methylcellulose, methylcellulose, hydroxyethylcellulose, hydroxyethyl methylcellulose, hydroxypropylcellulose, poly(hydroxyethyl methacrylate), oxypolygelatin, pectin, polygerin, povidone, propylene carbonate, methyl vinyl ether / maleic anhydride copolymer (PVM / MA), poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate), hydroxypropylcellulose, hydroxypropyl methylcellulose (HPMC), sodium carboxymethylcellulose (CMC), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), or combinations thereof.

[0129]

[0181] In some embodiments, the topical formulations disclosed herein include emollients. Moulients include castor oil esters, cocoa butter esters, safflower oil esters, cottonseed oil esters, corn oil esters, olive oil esters, cod liver oil esters, almond oil esters, avocado oil esters, palm oil esters, sesame oil esters, squalene esters, kukui oil esters, soybean oil esters, acetylated monoglycerides, ethoxylated glyceryl monostearate, hexyl laurate, isohexyl laurate, isohexyl palmitate, isopropyl palmitate, methyl palmitate, decyl oleate, isodecyl oleate, hexadecyl stearate, decyl stearate, isopropyl isostearate, methyl isostearate, diisopropyl adipate, diisohexyl adipate, dihexyldecyl adipate, diisopropyl sebacate, lauryl lactate, myristyl lactate, and This includes, but is not limited to, cetyl lactate, oleyl myristate, oleyl stearate, and oleyl oleate, pelargonic acid, lauric acid, myristic acid, palmitic acid, stearic acid, isostearic acid, hydroxystearic acid, oleic acid, linoleic acid, lithinolic acid, arachidonic acid, behenic acid, erucic acid, lauryl alcohol, myristyl alcohol, cetyl alcohol, hexadecyl alcohol, stearyl alcohol, isostearyl alcohol, hydroxystearyl alcohol, oleyl alcohol, ricinoleyl alcohol, behenyl alcohol, erucyl alcohol, 2-octyldodecanyl alcohol, lanolin and lanolin derivatives, beeswax, whale wax, myristyl myristate, stearyl stearate, carnauba wax, candelilla wax, lecithin, and cholesterol.

[0130]

[0182] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated using one or more natural polymers. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated using natural polymers such as fibronectin, collagen, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparan sulfate, and chondroitin sulfate. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated using polymer gels composed of natural polymers. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated using polymer gels composed of natural polymers such as, but are not limited to, fibronectin, collagen, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparan sulfate, chondroitin sulfate, and combinations thereof. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated using a crosslinked polymer. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated using an uncrosslinked polymer. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated using both an uncrosslinked polymer and a crosslinked polymer. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated using a crosslinked hyaluronan gel. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated using an insoluble crosslinked HA hydrogel. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated using an uncrosslinked hyaluronan gel. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated using a collagen matrix.In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated using a fibrin matrix. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated using a fibrin / collagen matrix.

[0131]

[0183] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated for administration to a tumor or associated tissue. Formulations suitable for administration to a tumor include, but are not limited to, liquids, suspensions (e.g., aqueous suspensions), ointments, gels, creams, liposomes, niosomes, pharmacosomes, nanoparticles, or combinations thereof. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein for injection into solid tumors are administered by injection into the tumor, surrounding tissue, or combination thereof. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are administered concurrently with the excision of cancer cells or tumors. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are administered to the excision margin of cancer cells or tumors. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered.

[0132]

[0184] As used herein, “depot preparation” is a controlled-release formulation implanted in a tumor or associated tissue (e.g., at the resection margin) (e.g., subcutaneously, intramuscularly, intravitreously, or subconjunctivally). In some embodiments, the depot preparation is formulated by forming a microencapsulation matrix (also known as a microencapsulation matrix) of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein in a biodegradable polymer. In some embodiments, the depot preparation is formulated by encapsulating the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein within liposomes or microemulsifications.

[0133]

[0185] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated for rectal or vaginal administration. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are administered as suppositories. In some embodiments, compositions suitable for rectal administration are prepared by mixing the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore dissolves rectally to release the drug. In some embodiments, compositions suitable for rectal administration are prepared by mixing the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein with cocoa butter, glycerin gelatin, hydrogenated vegetable oil, a mixture of polyethylene glycols of various molecular weights, or fatty acid esters of polyethylene glycol.

[0134]

[0186] In certain embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes described herein are optionally incorporated within controlled-release particles, lipid complexes, liposomes, nanoparticles, microspheres, microparticles, nanocapsules, or other agents that enhance or facilitate topical delivery to the skin. Examples of conventional microencapsulation processes for pharmaceutical preparations are described in U.S. Patent No. 3,737,337, which is incorporated herein by reference for such disclosure.

[0135]

[0187] Dosage

[0188] The amount of pharmaceutical composition administered depends in part on the individual being treated. When a pharmaceutical composition is administered to a human subject, the daily dose is usually determined by the prescribing physician, and the dose generally varies according to the individual's age, sex, diet, weight, overall health and response, the severity of the individual's symptoms, the exact disease or condition being treated, the severity of the disease or condition being treated, the time of administration, the route of administration, the properties of the composition, the rate of elimination, the combination of drugs, and the discretion of the prescribing physician.

[0136]

[0189] In some cases, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are administered directly to the tumor and / or surrounding tissue by local injection. In some cases, the tumor is not surgically removable, or is "inoperable." In some embodiments, the inoperable tumor is inaccessible, or the patient has a medical condition that limits their ability to tolerate surgery. In some cases, the tumor is located in a delicate area, such as the spinal cord, brain, or other tissue, where surgical removal could cause significant damage to surrounding tissue. In some cases, for example with certain brain cancers, the tumor has infiltrated or invaded surrounding tissue, making surgical extraction without damaging the surrounding tissue impossible. Inoperable tumors may arise from central nervous system (CNS) cancers such as glioblastoma pleomorphism, breast cancer, pancreatic cancer, or bladder cancer, without limitation. In some cases, the cancer has numerous secondary tumors or metastases in other parts of the body. The number of secondary tumors may be too large to be safely removed. Metastatic cancers include, without limitation, bladder cancer, breast cancer, colon cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, stomach cancer, thyroid cancer, liver cancer, or uterine cancer.

[0137]

[0190] In some embodiments, the administered dose of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is about 0.001 to about 1000 mg. In some embodiments, the amount of nHC-HA / PTX3 or rcHC-HA / PTX3 complex administered is in the range of about 0.5 to about 50 mg. In some embodiments, the amount of nHC-HA / PTX3 or rcHC-HA / PTX3 complex administered is about 0.001 to about 7 g. In some embodiments, the amount of nHC-HA / PTX3 or rcHC-HA / PTX3 complex administered is about 0.01 to about 7 g. In some embodiments, the amount of nHC-HA / PTX3 or rcHC-HA / PTX3 complex administered is about 0.02 to about 5 g. In some embodiments, the amount of nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is about 0.05 to about 2.5 g. In some embodiments, the amount of nHC-HA / PTX3 or rcHC-HA / PTX3 administered is approximately 0.1 to 1 g.

[0138]

[0191] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered before, during, or after the onset of a disease or condition. In some embodiments, combination therapy is administered before, during, or after the onset of a disease or condition. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered as combination therapy before, during, or after the onset of a disease or condition. In some embodiments, the timing of administration of a composition containing the nHC-HA / PTX3 or rcHC-HA / PTX3 disclosed herein varies. Therefore, in some examples, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used as a prophylactic agent and is continuously administered to subjects prone to developing a condition or disease in order to prevent the onset of the disease or condition. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered to subjects during or as soon as possible after the onset of symptoms. In some embodiments, administration of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is initiated within the first 48 hours of symptom onset, preferably within the first 48 hours of symptom onset, more preferably within the first 6 hours of symptom onset, and most preferably within 3 hours of symptom onset. In some embodiments, the initial dose is by any practical route, such as intravenous injection, bolus injection, infusion over 5 minutes to about 5 hours, pills, capsules, transdermal patches, oral delivery, or a combination thereof. The nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is preferably administered as soon as it becomes feasible after the onset of the disease or condition is detected or suspected, for a duration necessary for treating the disease, such as about 1 month to about 3 months. In some embodiments, the duration of treatment varies for each subject, and the duration is determined using known criteria. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex or formulation containing the complex disclosed herein is administered for at least two weeks, preferably about one month to about five years, and more preferably about one month to about three years.

[0139]

[0192] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered once daily in a single dose. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered more than once daily in multiple doses. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered twice daily. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered three times daily. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is administered four times daily. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered more than four times daily.

[0140]

[0193] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered in a single dose. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered in a single dose in conjunction with tumor resection, cryoexcision, or radiofrequency ablation.

[0141]

[0194] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are administered for prophylactic and / or therapeutic purposes. In therapeutic applications, in some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are administered to an individual already suffering from a disease or condition in an amount sufficient to cure or at least partially inhibit the symptoms of the disease or condition. The effective amount for this use depends on the severity and course of the disease or condition, previous therapies, the individual's health status, weight, and response to the drug, and the judgment of the treating physician.

[0142]

[0195] In prophylactic applications, in some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are administered to individuals at risk of specific disorders. Such amounts are defined as “prophylactic effective doses or doses.” In such use, the exact amount also depends on the individual’s health status, body weight, and other physical parameters of the individual.

[0143]

[0196] If the individual's condition does not improve, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein may be administered chronically, i.e., for an extended period including the individual's entire lifespan, to improve, or otherwise control or limit the symptoms of the individual's disease or condition.

[0144]

[0197] In some embodiments, if the individual's condition improves, the physician may, at their discretion, continue administering the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein, or temporarily reduce the dose of the administered drug or temporarily discontinue it for a certain length of time (i.e., a “drug-free period”). In some embodiments, the length of the drug-free period may vary from 2 days to 1 year, including, but are not limited to, 2, 3, 4, 5, 6, 7, 10, 12, 15, 20, 28, 35, 50, 70, 100, 120, 150, 180, 200, 250, 280, 300, 320, 350, or 365 days. In some embodiments, the dose reduction during drug-free periods ranges from 10% to 100%, including, but are not limited to, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0145]

[0198] Once the individual's condition improves, a maintenance dose is administered as needed. In some embodiments, the dose, frequency, or both are then reduced symptomatically to a level at which the improved disease, disorder, or condition is maintained. In some embodiments, the individual requires long-term intermittent treatment upon any relapse of symptoms.

[0146]

[0199] In some embodiments, the pharmaceutical compositions described herein are unit dosage forms suitable for single doses of precise amounts. In unit dosage forms, the formulation is divided into unit doses containing an appropriate amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein. In some embodiments, the unit dose is in the form of packaging containing individual amounts of the formulation. Non-limiting examples include packaged tablets or capsules, and powders in vials or ampoules. In some embodiments, aqueous suspension compositions are packaged in single-dose non-reusable containers. In some embodiments, multi-dose reusable containers are used, in which case preservatives are typically included in the composition. In some embodiments, formulations for parenteral injection are provided in unit dosage forms including, but not limited to, ampoules or multi-dose containers with added preservatives.

[0147]

[0200] In some embodiments, the area surrounding the tumor is brought into contact with at least or about 1 microgram (ug), 10 ug, 20 ug, 30 ug, 40 ug, 50 ug, 60 ug, 70 ug, 80 ug, 90 ug, 100 ug, 200 ug, 300 ug, 400 ug, 500 ug, 600 ug, 700 ug, 800 ug, 900 ug, 1000 ug, or more than 1000 ug of HC-HA / PTX3 complex. In some embodiments, the area surrounding the tumor is brought into contact with at least or about 1 milligram (mg), 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, or more than 500 mg of HC-HA / PTX3 complex. In some embodiments, the area surrounding the tumor is divided into approximately 1-10, 1-20, 1-40, 1-60, 1-80, 1-100, 1-150, 1-200, 10-20, 10-40, 10-60, 10-80, 10-100, 10-150, 10-200, 20-40, 20-60, 20-80, 20-100, 20-150, and 20-2 Contact with HC-HA / PTX3 complexes in the range of 00, 40-60, 40-80, 40-100, 40-150, 40-200, 60-80, 60-100, 60-150, 60-200, 80-100, 80-150, 80-200, 100-150, 100-200, or 150-200 micrograms (ug). In some embodiments, the region surrounding the tumor is approximately 1-10, 1-20, 1-40, 1-60, 1-80, 1-100, 1-150, 1-200, 10-20, 10-40, 10-60, 10-80, 10-100, 10-150, 10-200, 20-40, 20-60, 20-80, 20-100, 20-150, 20- Contact with HC-HA / PTX3 complexes in the range of 200, 40-60, 40-80, 40-100, 40-150, 40-200, 60-80, 60-100, 60-150, 60-200, 80-100, 80-150, 80-200, 100-150, 100-200, or 150-200 milligrams (mg).

[0148]

[0201] Appropriate daily doses for the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are, for example, about 0.01 to 100 mg. In large mammals, including but not limited to humans, indicated daily doses range from about 10 ug to about 100 mg, or about 0.5 mg to about 100 mg, conveniently administered in divided doses or extended-release forms, including up to four doses per day. Appropriate unit dosage forms for oral administration contain about 1 to 50 mg of the active ingredient. Appropriate doses for injection into tumors and / or surrounding tissues range from about 0.1 to about 100 mg per injection. The aforementioned ranges are merely suggestions, as there are many variable factors relating to individual treatment regimes and significant deviations from these recommendations are not uncommon. In some embodiments, the dosage can be varied depending on several variable factors, not limited to the activity of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex used, the disease or condition being treated, the mode of administration, the needs of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[0149]

[0202] In some embodiments, the toxicity and therapeutic efficacy of such treatment regimens are LD 50 (A lethal dose for 50% of the population) and ED 50 This is determined by standard pharmaceutical procedures in cell culture or experimental animals, including, but not limited to, determining the dose that is therapeutically effective in 50% of the population. In some embodiments, the dose-to-toxicity ratio is the therapeutic index, LD50. 50 and ED 50 This is expressed as a ratio. nHC-HA / PTX3 or rcHC-HA / PTX3 complexes exhibiting a high therapeutic index are preferred. In some embodiments, data obtained from cell culture assays and animal studies are used to formulate various dosages for use in humans. The dosages of nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are preferably ED with minimal toxicity. 50 It is within a range of circulating concentrations, including [specific concentrations]. In some embodiments, the dose varies within this range depending on the dosage form used and the route of administration utilized.

[0150]

[0203] In some embodiments, the pharmaceutical composition of nHC-HA / PTX3 or rcHC-HA / PTX3 complex is packaged as a product containing packaging material, the pharmaceutical composition which is effective for prevention and / or treatment of a disease or condition, and a label indicating that the pharmaceutical composition is used to treat a disease or condition. In some embodiments, the pharmaceutical composition is packaged in unit dosage forms containing an amount of the pharmaceutical composition for single or multiple doses. In some embodiments, the packaged composition contains a lyophilized powder of the pharmaceutical composition which is reconstituted (e.g., with water or saline) before administration.

[0151]

[0204] Medical devices

[0205] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 composites disclosed herein are assembled directly on the surface of an implantable medical device or formulated as coatings for implantable medical devices. Methods for covalent bonding of hyaluronane to surfaces such as metal, polymer, ceramic, silica, and composite surfaces are well known in the Art and, in some embodiments, are used in conjunction with the methods provided herein for assembling nHC-HA / PTX3 or rcHC-HA / PTX3 composites on such surfaces (see, for example, U.S. Patents 5,356,433, 5,336,518, 4,613,665, 4,810,784, 5,037,677, and 8,093,365). In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 composites are assembled directly on the surface or a portion thereof of an implantable medical device. In some embodiments, an nHC-HA / PTX3 or rcHC-HA / PTX3 complex produced according to the method provided herein is purified and then directly bonded to the surface or part thereof of an implantable medical device. In some embodiments, an nHC-HA / PTX3 or rcHC-HA / PTX3 complex produced according to the method provided herein is purified and then formulated as a coating agent for bonding to the medical device or part thereof. In some embodiments, the coating agent is applied directly to the surface or to a pre-treated or coated surface, and the pre-treatment or coating is designed to aid in the adhesion of the coating agent to the substrate. In some embodiments, an nHC-HA / PTX3 or rcHC-HA / PTX3 complex produced according to the method provided herein is purified and then bonded to the medical device or part thereof coated with a substance that facilitates the bonding of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex. For example, in some embodiments, the medical device or part thereof is coated with an adhesive polymer that provides functional groups on its surface for the covalent bonding of hyaluronane of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex.In some embodiments, coupling agents such as carbodiimides, but not limited to these, are used to bond the nHC-HA / PTX3 or rcHC-HA / PTX3 complex to a polymer coating. In some embodiments, photoimmobilization is used to covalently bond the nHC-HA / PTX3 or rcHC-HA / PTX3 complex produced according to the methods provided herein to a medical device or a part thereof. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex produced according to the methods provided herein is bonded to a medical device or a part thereof using spacer molecules containing photochemical or thermochemically reactive groups.

[0152]

[0206] In some embodiments, coating formulations containing nHC-HA / PTX3 or rcHC-HA / PTX3 complexes are applied to a substrate, for example, by dip coating. Other methods of application include, but are not limited to, spraying, washing, vapor deposition, brushing, rolling, curtaining, spin coating, and other methods known in the art.

[0153]

[0207] Examples of implantable medical devices include, but are not limited to, bone implants, wound drains, shunts, urethral implants, metal or plastic implants, stents, stent grafts, grafted blood vessels, pellets, wafers, implantable drug pumps, drug delivery systems, microparticles, nanoparticles, and microcapsules.

[0154]

[0208] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 composites disclosed herein are conjugated to or assembled directly on microcapsules. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 composites are combined with a material used to form the microcapsules to produce microcapsules containing the nHC-HA / PTX3 or rcHC-HA / PTX3 composites. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 composites are used to coat the inner surface of the microcapsules. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 composites are used to coat the outer surface of the microcapsules. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 composites are used to coat both the inner and outer surfaces of the microcapsules.

[0155] combination

[0209] In some embodiments, the compositions and methods described herein are used in combination with a second, further, or additional therapeutic agent in addition to a natural or reconstituted HC-HA / PTX3 complex. In some embodiments, the compositions and methods described herein are used in combination with two or more therapeutic agents. In some embodiments, the compositions and methods described herein are used in combination with one or more therapeutic agents. In some embodiments, the compositions and methods described herein are used in combination with two, three, four, five, six, seven, eight, nine, ten or more therapeutic agents.

[0156]

[0210] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex and the second therapeutic agent disclosed herein are administered in the same dosage form. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex and the second therapeutic agent disclosed herein are administered in separate dosage forms.

[0157]

[0211] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein and the second therapeutic agent are administered simultaneously (e.g., concurrently, essentially simultaneously, or within the same therapeutic protocol).

[0158]

[0212] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein and the second therapeutic agent are administered sequentially. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered before or after the second therapeutic agent. In some embodiments, the interval between the administration of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein and the second activator ranges from several minutes to several hours, depending on the characteristics of each drug, such as the potency, solubility, bioavailability, plasma half-life, and kinetic profile. In some embodiments, the circadian rhythm of the target molecule concentration determines the optimal administration interval. In some embodiments, the timing between the administration of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex and the second activator disclosed herein may be about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 1 month, or longer.

[0159]

[0213] In some embodiments, co-administration of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein results in a lower required dose of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex than the required dose when the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is administered alone. In some embodiments, co-administration of a second therapeutic agent results in a lower required dose of the second agent than the required dose when the second agent is administered alone. Methods for experimentally determining the therapeutically effective doses of drugs and other agents for use in combination therapy regimens are known and described in the art. For example, the use of metronomic administration, i.e., providing more frequent and lower doses to minimize toxic side effects, has been well described in the art. Combination therapy further includes periodic treatments initiated and terminated at various times to assist in the clinical management of the individual.

[0160]

[0214] In some embodiments, the combination therapy of nHC-HA / PTX3 or rcHC-HA / PTX3 complex and one or more additional therapeutic agents is modified. In some embodiments, the combination therapy is modified so that the amount of nHC-HA / PTX3 or rcHC-HA / PTX3 complex is increased relative to the amount of the second therapeutic agent. In some embodiments, the combination therapy is modified so that the amount of nHC-HA / PTX3 or rcHC-HA / PTX3 complex is decreased relative to the amount of the second therapeutic agent. In some embodiments, the combination therapy is modified so that the amount of the second therapeutic agent is increased relative to the amount of nHC-HA / PTX3 or rcHC-HA / PTX3 complex. In some embodiments, the combination therapy is modified so that the amount of the second therapeutic agent is decreased relative to the amount of nHC-HA / PTX3 or rcHC-HA / PTX3 complex.

[0161]

[0215] In some embodiments, the second therapeutic agent is selected from cytotoxic agents, analgesics, anti-inflammatory agents, antibiotics, antimicrobial agents, anti-angiogenic agents, chemotherapeutic agents, antineoplastic agents, immunotherapies, or radiotherapy. In some embodiments, the second therapeutic agent is a chemotherapeutic agent. In some embodiments, the second therapeutic agent is alkylating agent, antimetabolites, epipodophyllotoxin, antineoplastic enzymes, topoisomerase inhibitors, procarbazine, mitoxantrone, platinum coordination complexes, bio-response modifiers and growth inhibitors, hormone / anti-hormone therapeutic agents, hematopoietic growth factors, aromatase inhibitors, anti-estrogens, anti-androgens, corticosteroids, gonadrelin agonists, microtubule activators, nitrosourea, lipid or protein kinase targeting agents, immunomodulators (IMiDs), protein or lipid phosphatase targeting agents, anti-angiogenic agents, Akt inhibitors, IGF-I inhibitors, FGF3 modulators, mTOR inhibitors, Smac mimetic agents, HDAC inhibitors, cell differentiation-inducing agents, bradykinin The antimicrobial agent is selected from 1 receptor antagonists, angiotensin II antagonists, cyclooxygenase inhibitors, heparanase inhibitors, lymphokine inhibitors, cytokine inhibitors, IKK inhibitors, P38MAPK inhibitors, HSP90 inhibitors, multikinase inhibitors, bisphosphonates, rapamycin derivatives, anti-apoptotic pathway inhibitors, apoptotic pathway agonists, PPAR agonists, RAR agonists, Ras isoform inhibitors, telomerase inhibitors, protease inhibitors, metalloproteinase inhibitors, aminopeptidase inhibitors, SHIP activators-AQX-MN100, Humax-CD20 (ofatumumab), CD20 antagonists, IL2-diphtheria toxin fusions, or combinations thereof. In some embodiments, the antimicrobial agent is an antiviral, antibacterial, or antifungal agent.Non-limiting and exemplary antimicrobial agents include aminoglycosides, beta-lactams, quinolones or fluoroquinolones, macrolides, sulfonamides, sulfamethaxozole, tetracyclines, streptogramin, oxazolidinones (e.g., linezolid), clindamycin, lincomycin, rifamycin, glycopeptides, polymxin, lipopeptide antibiotics, as well as pharmacoagulably accepted sodium salts, pharmacoagulably accepted calcium salts, pharmacoagulably accepted potassium salts, lipid preparations, and those classified as derivatives and / or analogs of the above. Exemplary classes of some innate peptides or proteins include transferrin, lactoferrin, defensins, phospholipases, lysozyme, cathelicidine, serprosidine, bactericidal permeability-enhancing proteins, amphiphilic alpha-helix peptides, and other synthetic antimicrobial proteins. In some embodiments, antimicrobial agents are antiseptics.

[0162]

[0216] In some embodiments, the second therapeutic agent is ARRY-797, dacarbazine (DTIC), actinomycin C2, C3, D, and F1, cyclophosphamide, melphalan, estramustine, meitansinol, rifamycin, streptovaricin, doxorubicin, daunorubicin, epirubicin, idarubicin, detrubicin, carminomycin, esorubicin, mitoxantrone, bleomycin A, A2, and B, camptothecin, irinotecan, topotecan, 9-aminocamptothecin, 10,11-methylenedioxycamptothecin 9-nitrocamptothecin, bortezomib, temozolomide, TAS103, NPI0052, combretastatin, combretastatin A-2, combretastatin A-4, calicheamicin, neocalsinostatin, epotilon A, B, C, and semi-synthetic variants, herceptin, rituxan, CD40 antibody, asparaginase, interleukin, interferon, leuprolide, and pegasparagase, 5-fluorouracil, fluorodeoxyuridine, ptorafur, 5'-deoxyfluorouridine, UF T, MITC, S-1 capecitabine, diethylstilbestrol, tamoxifen, toremifine, tolmudex, thimitax, flutamide, fluoxymesterone, bicalutamide, finasteride, estradiol, trioxyfen, dexamethasone, leuproelin acetate, estramustine, droxyfen, medroxyprogesterone, megesterol acetate, aminoglutethimide, testolactone, testosterone, diethylstilbestrol Strol, hydroxyprogesterone, mitomycin A, B and C, porphyromycin, cisplatin, carboplatin, oxaliplatin, tetraplatin, platinum-DACH, ormaplatin, thalidomide, lenalidomide, CI-973, telomestatin, CHIR258, Rad001, SAHA, tubacin, 17-AAG, sorafenib, JM-216, podophyllotoxin, epipodophyllotoxin, etoposide, teniposide, tarceva, ilesa, imatinib, miltefosine, perifosine, aminopterin, methotrexate,Selected from metopterin, dichloromethotrexate, 6-mercaptopurine, thioguanine, azathioprine, allopurinol, cladribine, fludarabine, pentostatin, 2-chloroadenosine, deoxycytidine, cytosine arabinoside, cytarabine, azacitidine, 5-azacitosine, gemcitabine, 5-azacitosine arabinoside, vincristine, vinblastine, vinorelbine, leulosine, leulosidine and vindesine, paclitaxel, taxotere and / or docetaxel.

[0163]

[0217] In some embodiments, the second therapeutic agent is niacin, fibrates, statins, Apo-A1 mimetic polypeptides (e.g., DF-4, Novartis), apoA-I transcription upregulators, ACAT inhibitors, CETP modulators, glycoprotein (GP) IIb / IIIa receptor antagonists, P2Y12 receptor antagonists, Lp-PLA2 inhibitors, antitumor necrosis factor (TNF) agents, interleukin-1 (IL-1) receptor antagonists, interleukin-2 (IL-2) receptor antagonists, interleukin-6 (IL-6) receptor antagonists Antagonists, interleukin-12 (IL-12) receptor antagonists, interleukin-17 (IL-17) receptor antagonists, interleukin-23 (IL-23) receptor antagonists, cytotoxic agents, antimicrobial agents, immunomodulators, antibiotics, T-cell costimulatory blockers, B-cell depletion agents, immunosuppressants, anti-lymphocyte antibodies, alkylating agents, antimetabolites, plant alkaloids, terpenoids, topoisomerase inhibitors, antitumor antibiotics, monoclonal antibodies, hormone therapies (e.g., aromatase inhibitors), or combinations thereof.

[0164]

[0218] In some embodiments, the second activator is an anti-TGF-β antibody, an anti-TGF-β receptor blocking antibody, an anti-TNF antibody, an anti-TNF receptor blocking antibody, an anti-IL-1β antibody, an anti-IL-1β receptor blocking antibody, an anti-IL-2 antibody, an anti-IL-2 receptor blocking antibody, an anti-IL-6 antibody, an anti-IL-6 receptor blocking antibody, an anti-IL-12 antibody, an anti-IL-12 receptor blocking antibody, an anti-IL-17 antibody, an anti-IL-17 receptor blocking antibody, an anti-IL-23 antibody, or an anti-IL-23 receptor blocking antibody.

[0165]

[0219] In some embodiments, the second activator is alefacept, ephalizumab, methotrexate, acitretin, isotretinoin, hydroxyurea, mycophenolate mofetil, sulfasalazine, 6-thioguanine, dovonex, tacronex, betamethasone, tazarotene, hydroxychloroquine, sulfasalazine, etanercept, adalimumab, infliximab, abatacept, rituximab, trastuzumab, anti-CD45 monoclonal antibody AHN-12 (NCI), iodine-131 anti-B1 antibody (Corixa Corp.), anti-CD66 monoclonal antibody BW250 / 183 (NCI, Southampton General Hospital), anti-CD45 monoclonal antibody (NCI, Baylor College of Medicine), antibody anti-anb3 integrin (NCI), BIW-8962 (BioWa (BioInvent International Inc.), Antibody BC8 (NCI), Antibody muJ591 (NCI), Indium In111 Monoclonal Antibody MN-14 (NCI), Yttrium Y90 Monoclonal Antibody MN-14 (NCI), F105 Monoclonal Antibody (NIAID), Monoclonal Antibody RAV12 (Raven Biotechnologies), CAT-192 (Human Anti-TGF-Beta-1 Monoclonal Antibody, Genzyme), Antibody 3F8 (NCI), 177Lu-J591 (Weill Medical College of Cornell University), TB-403 (BioInvent International) AB), Anakinra, Azathioprine, Cyclophosphamide, Cyclosporine A, Leflunomide, d-Penicillamine, Amitriptyline, or Nortriptyline, Chlorambucil, Nitrogen Mustard, Plasteron, LJP394 (Abetimus sodium), LJP1082 (La Jolla Pharmaceutical), Eculizumab, Belibumab, RhuCD40L (NIAID), Epratuzumab, Sirolimus, Tacrolimus, Pimecrolimus, Thalidomide, Antithymocyte globulin - Horse (Atgam, Pharmacia Upjohn), Antithymocyte globulin - Rabbit (Thymoglobulin, Genzyme),Muromonab-CD3 (FDA Office of Orphan Products Development), basiliximab, daclizumab, riluzole, cladribine, natalizumab, interferon beta-1b, interferon beta-1a, tizanidine, baclofen, mesalazine, asacol, pentasa, mesalamine, valsalazid, olsalazine, 6-mercaptopurine, AIN457 (anti-IL-17 monoclonal antibody, Novartis), theophylline, D2E7 (Knoll Human anti-TNFmAb (manufactured by Pharmaceuticals), mepolizumab (anti-IL-5 antibody, SB240563), canakinumab (anti-IL-1 beta antibody, NIAMS), anti-IL-2 receptor antibody (daclizumab, NHLBI), CNTO328 (anti-IL-6 monoclonal antibody, Centocor), ACZ885 (fully human anti-interleukin-1 beta monoclonal antibody, Novartis), CNTO1275 (fully human anti-IL-12 monoclonal antibody, Centocor), (3S)-N-hydroxy-4-({4-[(4-hydroxy-2-butynyl)oxy]phenyl}sulfonyl)-2,2-dimethyl-3-thiomorpholine carboxamide (aplatastat), golimumab (CNTO148), onercept, BG9924 (Biogen Idec), certolizumab pegol (CDP870, UCB) Pharma), AZD9056 (AstraZeneca), AZD5069 (AstraZeneca), AZD9668 (AstraZeneca), AZD7928 (AstraZeneca), AZD2914 (AstraZeneca), AZD6067 (AstraZeneca), AZD3342 (AstraZeneca), AZD8309 (AstraZeneca), [(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazine-2-ylcarbonyl)amino]propanoyl}amino)butyl]boronic acid (bortezomib), AMG-714 (anti-IL-15 human monoclonal antibody, Amgen), ABT-874 (anti-IL-12 monoclonal antibody, Abbott) Labs), MRA (tocilizumab, anti-IL-6 receptor monoclonal antibody, Chugai Pharmaceutical), CAT-354 (human anti-interleukin-13 monoclonal antibody,Cambridge Antibody Technology (MedImmune), aspirin, salicylic acid, gentisic acid, choline magnesium salicylate, choline salicylate, choline magnesium salicylate, choline salicylate, magnesium salicylate, sodium salicylate, diflunisal, carprofen, fenoprofen, fenoprofen calcium, flurbiprofen, ibuprofen, ketoprofen, nabutone, ketrolacto, ketrolactromethamine, naproxen, oxaprozin, diclofe Nak, etodolac, indomethacin, sulindac, tolmetin, meclofenamic acid, sodium meclofenamate, mefenamic acid, piroxicam, meloxicam, celecoxib, rofecoxib, valdecoxib, parecoxib, etoricoxib, lumiracoxib, CS-502 (Sankyo), JTE-522 (Japan Tobacco Inc.), L-745, 337 (Almirall), NS398 (Sigma), betamethasone (Celeston), prednisone (Deltazone), alclomethasone, aldosterone, amcinonide, beclomethasone, be Tametasone, budesonide, ciclesonide, clobetasol, clobetasol, crocortol, cloprednol, cortisone, cortibazole, deflazacort, deoxycorticosterone, desonide, dexomethasone, dexoxycorton, dexamethasone, diflorasone, diflucortolone, difluprednate, fluchlorolone, fludrocortisone, fludroxicortide, flumetasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin, fludrocortolone, fluorometholone, fluperolon, flup Redniden, fluticasone, formocortal, formoterol, halcinonide, halomethasone, hydrocortisone, hydrocortisone aceponate, hydrocortisone buteplat, hydrocortisone butyrate, loteprednol, medrisone, meprednisone, methylprednisolone, methylprednisolone aceponate, mometasone furoate, paramethasone, prednicarbart, prednisone, rimexolone, thixocortol, triamcinolone, urobetasol, cisplatin, carboplatin, oxaliplatin, mechloretamine,Cyclophosphamide, Chlorambucil, Vincristine, Vinblastine, Vinorelbine, Vindesine, Azathioprine, Mercaptopurine, Fludarabine, Pentostatin, Cladribine, 5-Fluorouracil (5FU), Phloxuridine (FUDR), Cytosine Arabinoside, Methotrexate, Trimethoprim, Pyrimethamine, Pemetrexed, Paclitaxel, Docetaxel, Etoposide, Teniposide, Irinotecan, Topotecan, Amsacrin, Etoposide, Etoposide Phosphate, Teniposide, Dactinomycin, Doxo Rubicin, daunorubicin, barurubicin, idarubicin, epirubicin, bleomycin, plicamycin, mitomycin, trastuzumab, cetuximab, rituximab, bevacizumab, finasteride, goserelin, aminoglutethimide, anastrozole, letrozole, borozole, exemestane, 4-androsten-3,6,17-trione ("6-oxo"); 1,4,6-androstatriene-3,17-dione (ATD), formestan, testolactone, fadrozole, or combinations thereof.

[0166]

[0220] In some embodiments, the second therapeutic agent is an antibiotic. In some embodiments, the second therapeutic agent is an antibacterial agent. In some embodiments, the second therapeutic agent is amikacin, gentamicin, kanamycin, neomycin, netylmycin, streptomycin, tobramycin, paromomycin, geldanmycin, harbimycin, loracalbef, ertapenem, doripenem, imipenem, cilastatin, meropenem, cefadroxil, cefazolin, cephalothin, cephalexin, cefaclor, cephamandol, cefoxitin, cefprozil, cefuroxime, cefixime, ce Fujinil, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftoviprol, teicoplanin, vancomycin, azithromycin, clarithromycin, dilithromycin, erythromycin, roxithromycin, troleandmycin, telithromycin, spectinomycin, aztreonam, amoxicillin, ampicillin, azurocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin Mezlocillin, methicillin, nafcillin, oxacillin, penicillin, piperacillin, ticarcillin, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, mafenide, prontosil, sulfacetamide, sulfamethizol, sulfanimilamide, sulfa Sulfsalazine, sulfsioxazole, trimethoprim, demeclocycline, doxycycline, minocycline, oxtetracycline, tetracycline, arsphenamine, chloramphenicol, clindamycin, lincomycin, ethambutol, fosfomycin, fusidic acid, furazolidone, isoniazid, linezolid, metronidazole, mupirocin, nitrofurantoin, platensimycin, pyrazinamide,These include quinuspristin / dalfopristin, rifampin, tinidazole, and combinations thereof.

[0167]

[0221] In some embodiments, the second therapeutic agent is radiotherapy. In some embodiments, the second therapeutic agent is selected from X-ray therapy or proton therapy. In some embodiments, the radiotherapy may be external beam or close-range beam therapy.

[0168]

[0222] In some embodiments, the second therapeutic agent is a targeted therapy. Targeted therapy targets specific genes, proteins, or tissue environments that contribute to cancer growth and survival. In some embodiments, the targeted therapy comprises one or more monoclonal antibodies. In some embodiments, the targeted therapy comprises small molecules, such as, without limitation, angiogenesis inhibitors as described herein. In some embodiments, the targeted therapy comprises one or more monoclonal antibodies and one or more small molecules as described herein.

[0169]

[0223] combination with cells

[0224] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are co-administered with cells, multiple cells, or tissues.

[0170]

[0225] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is co-administered with therapeutic cells. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is co-administered with tissue grafts. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is co-administered with stem cell grafts. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is co-administered with organ grafts. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is co-administered with immune cells.

[0171]

[0226] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are administered concurrently with tumor resection, cryoexcision, or radiofrequency ablation (e.g., simultaneously, essentially simultaneously, or within the same treatment protocol). In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are administered before or after tumor resection, cryoexcision, or radiofrequency ablation. In some embodiments, the time between the administration of the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein and tumor resection, cryoexcision, or radiofrequency ablation ranges from several minutes to several hours, depending on the characteristics of each drug, such as the potency, solubility, bioavailability, plasma half-life, and kinetic profile. In some embodiments, the circadian rhythm of the target molecule concentration determines the optimal dosing interval. In some embodiments, the timing between the administration of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex and the second activator disclosed herein is less than about one hour, less than one day, less than one week, or less than one month.

[0172]

[0227] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 conjugates disclosed herein are co-administered with tumor resection, cryoexcision, or radiofrequency ablation and immunosuppressants. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 conjugates disclosed herein are co-administered with tumor resection, cryoexcision, or radiofrequency ablation and calcineurin inhibitors (e.g., cyclosporine or tacrolimus), mTOR inhibitors (sirolimus, everolimus), antiproliferative agents (azathioprine or mycophenolic acid), corticosteroids (e.g., prednisolone or hydrocortisone), monoclonal anti-IL-2Rα receptor antibodies (e.g., basiliximab or daclizumab), polyclonal anti-T cell antibodies (e.g., anti-thymocyte globulin (ATG) or anti-lymphocyte globulin (ALG)), chemotherapeutic agents, analgesics, anti-inflammatory agents, steroids, and antibiotics or combinations thereof.

[0173]

[0228] In some embodiments, tissues are coated with the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein. In some embodiments, multiple stem cells are coated with the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein. In some embodiments, organs are coated with the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein. In some embodiments, coating tissues with the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein prevents the tissues from being acted upon by the host immune system.

[0174]

[0229] In some embodiments, an organ, tissue, or a group of stem cells is contacted with the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein. In some embodiments, an organ, tissue, or a group of stem cells is contacted with a composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein. In some embodiments, the composition has a pH of about 7.0 to about 7.5. In some embodiments, the composition has a pH of 7.4. In some embodiments, the composition further comprises potassium, magnesium, and raffinose. In some embodiments, the composition further comprises at least one of adenosine, glutathione, allopurinol, and hydroxyethyl starch. In some embodiments, the composition is a UW solution supplemented with the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein.

[0175]

[0230] In some embodiments, an organ, tissue, or a group of stem cells is brought into contact with the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein for about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 24 hours, about 36 hours, or about 48 hours. In some embodiments, the contact is made at a temperature that protects tissue and vascular conditioning (e.g., below ambient temperature). In some embodiments, the contact is made at 4°C.

[0176]

[0231] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are co-administered with immune cells to a target requiring them. In some embodiments, the immune cells are homogeneous to the target requiring them. In some embodiments, the immune cells are autogenic to the target requiring them. In some embodiments, the immune cells are genetically modified before administration to the target requiring them. In some embodiments, the immune cells are modified to contain a chimeric antigen receptor (CAR T-cell therapy). [Examples]

[0177]

[0232] The following examples are included for illustrative purposes only and are not intended to limit the scope of the claimed subject matter. Example 1: Determining the viability and metabolic activity of prostate cancer cell lines after exposure to cryopreserved AM and UC extracts or purified HC-HA / PTX3(AM).

[0233] Published data (Alvim et al., "The potential risk of tumor progression after use of dehydrated human amnion / chorion membrane allograft in a positive margin resection model," Ther Adv Uro 2019, Vol. 11: 1-10) show that mice treated with dehydrated human amnion and chorion allograft products exhibit faster tumor growth after partial resection compared to untreated mice. Experiments were conducted to determine the viability and metabolic activity of prostate cancer cell lines after exposure to cryopreserved AM and UC extracts or purified HC-HA / PTX3(AM).

[0178]

[0234] The extracts of cryopreserved AM and UC or purified HC-HA / PTX3 after more than two runs of ultracentrifugation were examined to determine whether they inhibit the growth of human prostate cancer cell lines, namely PC-3 and LNCaP, and whether they reduce their overall cell metabolic activity.

[0179]

[0235] For initial feasibility, the effects of purified HC-HA / PTX3 and purified BTGEL (i.e., fragmented AM and UC) were tested using the WST-1 assay. The total cell metabolic activity was quantified using the WST-1 assay. Amplification of LNCaP and PC-3

[0236] Purchased cryopreserved PC-3 (ATCC® CRL-1435™, lot number 70004013) and LNCaP (LNCaP clone FGC ATCC® CRL-1740, lot number 64207637) were received and stored in liquid nitrogen. Each vial of LNCaP and PC-3 (1 ml, cell amount not shown) was centrifuged at 200×g for 5 minutes. The storage medium was removed and the cells were resuspended in 1 ml of RPMI1640 complete medium. A total of 0.5 ml was transferred to 100 mm culture dishes (two culture dishes for each cell line). The cells were incubated at 37 °C with 5% CO2. After 3 days, the medium was changed and the cells were incubated for a further 4 days, at which time the cells were at 70% confluence. It was observed that LNCaP tended to form aggregates (Figure 1A), while PC-3 grew as a uniformly distributed single cell layer (Figure 1B). The cells were recovered by 0.25% trypsin-EDTA (catalog number 25200-056, Fisher Scientific) and counted using a hemocytometer. The total cells of LNCaP and PC-3 were 5.92×10 6 and 2.88×10 6 respectively.

[0180]

[0237] The cells were resuspended in RPMI1640 complete medium containing 5% DMSO as follows: LNCaP: 10 tubes, 5.4×10 5 / Tube, 0.5ml per tube (approx. 1.08 x 10) 6 / ml), liquid nitrogen tank: S1R4B5, and PC-3: 10 tubes, 2.6 x 10 5 / Tube, 0.5ml per tube (approx. 5.2 x 10 5 ( / ml), Liquid nitrogen tank: S1R4B5.

[0181] Effects of rBTGel and HC-HA / PTX3 on the cellular metabolic activity of LNCaP and PC-3

[0238] 2.9 × 10⁻¹⁰ from amplification (above) 5 individual LNCaP cells or 2.9 × 10⁶ 5 Individual PC-3 cells were subcultured in a single 100 mm culture dish and cultured for 4 days. The cells were harvested (LNCaP: total cells were 1.24 × 10⁶). 6 The number of cells was 91%; PC-3: Total cells were 4.08 × 10⁶ 6 The number of cells was 1 × 10¹⁶, and the viability was 97%; therefore, PC-3 cells grow faster than LNCaP cells, consistent with reports that the cell doubling times for LNCaP and PC-3 are approximately 60 hours and 33 hours, respectively), in a 96-well plate. 4 / cm 2 or 3.2 × 10 4Seeded at 0 / ml (one plate of LNCaP and one plate of PC-3). After overnight incubation, both LNCaP and PC-3 cells (n=3) were treated with rBTGel (donor number BTR161857; HA: 175 μg / ml; protein: 165 μg / ml) or HC-HA / PTX3 (pooled fractions 3-9 from two ultracentrifuges in AM / PBS, donor 2 for stability verification, DI: TGLP17E002) (e.g., 11.1 μl of 10× dose added to 100 μl medium to obtain the exact dose). After 48 hours of processing, cell morphology (Figures 2A and 2B and 3A and 3B) was recorded by microscopic imaging and then used in the WST-1 assay for cellular metabolic activity (catalog number 10008883, Cayman Chemical Company, Ann Arbor, MI) according to the manufacturer's instructions (OD450 or OD450-OD670) (Figures 2C and 2D and 3C and 3D).

[0182]

[0239] Morphologically, LNCaP grew as small and large cell clusters. After 48 hours of cell treatment, higher doses of purified BTgel (50 and 100 μg / ml) transformed most spindle-shaped cells into round cells, although most cells remained bound and existed as clusters. Based on the lower cell density, cell death may also have occurred. Similar morphological changes occurred with much lower doses of HC-HA / PTX3 (≥6.25 μg / ml). These data suggest that both purified BTgel and HC-HA / PTX3 inhibit the cellular metabolic activity of LNCaP. Purified BTgel was prepared in saline (approximately 154 mM NaCl), while HC-HA / PTX3 was thoroughly dialyzed with distilled water and contained undetectable salts. Therefore, lyophilized BTgel, when added to cell culture medium, may have increased the salt concentration in the medium, and if BTgel increased the salt concentration by approximately 30 mM or more, it may have potentially reduced cell proliferation. When purified BTGel was added at concentrations of 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, and 100 μg / ml, it increased the salt concentrations in the culture medium by 6.2, 12.4, 24.7, 49.5, 98.9, 197.9, 395.8, and 791.5 mM, respectively (the formula used to obtain the salt concentration is dose / 175*154 / 0.111; the HA concentration of BTGel is 175 μg / ml, the saline solution contains 154 mM NaCl, and the total volume of medium in each well is 0.111 ml). Therefore, inhibition by purified BTGel above 6.25 μg / ml may be caused by salt, but there should be no concern about the salt effect of HC-HA / PTX3. WST-1 assay data showed that LNCaP metabolic activity was inhibited when purified BTGel doses were 25 μg / ml or higher (p<0.05) (Figure 2C). Due to concerns about the salt effect, it is inconclusive whether purified BTGel can inhibit LNCaP cellular metabolic activity at these doses. In contrast, HC-HA / PTX3 at doses of 6.25 μg / ml or higher inhibited LNCaP metabolic activity (40-85%) (Figures 2C and 2D).

[0183]

[0240] Morphologically, PC-3 cells grew more uniformly distributed than LNCaP cells. However, after 48 hours of cell treatment, higher doses (50 and 100 μg / ml) of purified BTGel resulted in more rounded cells. Based on the lower cell density, cell death may also have occurred. Similar morphological changes occurred with much lower doses of HC-HA / PTX3 (≥12.5 μg / ml). These data suggest that both purified BTGel and HC-HA / PTX3 inhibit the proliferation (viability) of PC-3. However, as mentioned above, inhibition by purified BTGel above 6.25 μg / ml may have been caused by salt, but there should be no concern about a salt effect from HC-HA / PTX3. WST-1 assay data showed that PC-3 proliferation was inhibited when the dose of purified BTGel was ≥6.25 μg / ml (p<0.05) (Figure 3C). Due to concerns about the salt effect, it remains inconclusive whether purified BTGel can inhibit PC-3 proliferation at these doses. In contrast, HC-HA / PTX3 at doses of 3.125 μg / ml or higher inhibits PC-3 proliferation (8-100%) (Figures 3C and 3D).

[0184]

[0241] Both treatment groups demonstrated a dose-dependent effect that significantly reduced the metabolic effects of PC-3 and LNCaP prostate cancer cells. Based on morphology, cell death associated with higher WST-1 readings appeared to occur at higher concentrations.

[0185]

[0242] These data showed that HC-HA / PTX3 does not promote PC-3 and LNCaP carcinogenesis and can be used after prostatectomy. Furthermore, rBTGEL was shown to have anticancer effects. The following examples deal with the effect of UC extract, which has no potential salt effect, on the proliferation of prostate cancer cells.

[0186]

[0243] conclusion

[0244] Purified BTGel at concentrations of 6.25 μg / ml or higher and HC-HA / PTX3 at concentrations of 3.125 μg / ml or higher inhibit PC-3 proliferation. Purified BTGel at concentrations of 25 μg / ml or higher and HC-HA / PTX3 at concentrations of 6.25 μg / ml or higher inhibit LNCaP proliferation. Due to concerns about the salt effect, it is inconclusive whether amniotic membrane and umbilical cord (AMUC) or salts in purified BTGel can inhibit proliferation at these doses.

[0187]

[0245] Since PC-3 cells are known to have a more rapid doubling time, lower concentrations of rBTGEL and HC-HA / PTX3 may be required to inhibit PC-3 compared to LNCaP. Therefore, the difference between negative controls and treatment groups is greater in PC-3.

[0188]

[0246] Therefore, despite published data indicating that certain fetal support products are associated with faster tumor recurrence and growth, it is demonstrated here that AM and UC products do not promote cancer recurrence after prostatectomy.

[0189] Example 2: Determine the metabolic activity of prostate cancer cell lines after exposure to cryopreserved UC extract, HA, and purified HC-HA / PTX3(AM).

[0247] The results of Example 1 show that HC-HA / PTX3 reduces the metabolic activity of both prostate cell lines at a low concentration of 6.25 μg / ml in both cell types. In addition, rBTGEL was shown to inhibit activity at concentrations of 25 μg / ml and above. The following study was conducted to rule out the possibility that the salt concentration in higher doses of rBTGel was complicating the interpretation of the data.

[0190]

[0248] The same WST-1 assay was used to test UC extracts in water. In addition, HC-HA / PTX3 was tested and compared with the UC results. HA was used as a control group. A series of doses were tested based on the μg / ml of HA.

[0191]

[0249] In a 96-well plate (100 µl / well) (n=3 for each group), PC3 and LNCaP cells were divided into 1 × 10⁶ cells. 4 / cm 2 Cells were plated. After overnight incubation, cells were treated with UC extracted with water at a series of doses (defined by HA concentration: 0, 1.56, 3.125, 6.25, 12.5, 25, 50, 100, 200, and 400 μg / ml, n=3), purified HC-HA / PTX3 (pooled fractions 3-9 from two ultracentrifuges in AM / PBS, donor 2 for stability verification, DI: TGLP17E002, prepared on 17 / 09 / 19) and HA. After 48 hours of treatment, cell morphology was recorded, and then cells were measured for cellular metabolic activity by WST-1 (catalog number 10008883, Cayman Chemical Company, Ann Arbor, MI) according to the manufacturer's instructions (OD450 or OD450-OD670).

[0192]

[0250] WST-1 assay data showed that metabolic activity in LNCaP cells was significantly inhibited by UC extract (≧100 μg / ml) and HC-HA / PTX3 (≧6.25 μg / ml) (p<0.05), but not by HA (see Figure 4A). Similarly, metabolic activity in PC-3 cells was significantly inhibited by UC extract (≧200 μg / ml) and HC-HA / PTX3 (≧1.56 μg / ml), but not by HA (see Figure 4B). Microscopic examination revealed significant cell death in both LNCaP (Figures 5A-5C) and PC3 (Figures 6A-6C) cells treated with HC-HA / PTX3 at concentrations of ≧25 μg / ml.

[0193]

[0251] There was clear inhibition of metabolic activity by HC-HA / PTX3, as determined by WST-1 and morphological evaluation. Based on morphology, when exposed to higher concentrations of HC-HA / PTX3, both PC3 and LNCaP cells became rounder, less adherent to the matrix, and fewer cells. The difference was that LNCaP cells aggregated as clusters with small-diameter, rounded cells at higher HC-HA / PTX3 concentrations, while PC3 cells did not aggregate.

[0194]

[0252] WST-1 assay data showed that metabolic activity in LNCaP cells was significantly inhibited by UC extract (≥100 μg / ml) and HC-HA / PTX3 (≥6.25 μg / ml) (p<0.05), but not by HA. Similarly, metabolic activity in PC-3 cells was significantly inhibited by UC extract (≥200 μg / ml) and HC-HA / PTX3 (≥1.56 μg / ml), but not by HA. Microscopic examination revealed significant cell death in both LNCaP and PC3 cells treated with HC-HA / PTX3 at concentrations of ≥25 μg / ml.

[0195]

[0253] Consideration

[0254] There was clear inhibition of metabolic activity by HC-HA / PTX3, as determined by WST-1 and morphological evaluation. Based on morphology, when exposed to higher concentrations of HC-HA / PTX3, both PC3 and LNCaP cells became rounder, less adherent to the matrix, and had fewer cells. The difference was that at higher HC-HA / PTX3 concentrations, LNCaP cells aggregated as clusters with small-diameter rounded cells, while PC3 cells did not aggregate. It remains unclear why LNCaP aggregated while PC3 did not when exposed to HC-HA / PTX3, but LNCaP cells were shown to have a faster aggregation rate than PC3. This may be due to their binding dependence, with LNCaP and PC3 being anchorage-dependent and independent, respectively. In the case of LNCaP, binding to the surface is necessary for viability and proliferation, while detachment induces cell death through the anoikis process. The literature compared the effects of LNCaP grown on various coating reagents (poly-l-lysine, poly-l-ornithine, human placental type IV collagen, fibronectin, and laminin) and showed that laminin and type IV collagen promoted cell aggregation (Figure 7, taken at 24 hours). This aggregation is similar in form to that observed in the experiments provided herein and may suggest that HC-HA / PTX3 reduces the cell surface binding of LNCaP. It is well known that various substrate properties, including surface charge, topography, hydrophobicity / hydrophilicity, surface chemistry, and surface energy, can influence cell behavior, and that modified cell-substrate interactions can affect the generation of intracellular signals. In fact, liquid overlay techniques are commonly used in this field to induce aggregation / spheroids by culturing cells on non-adherent surfaces, and therefore, cell-cell interactions are more pronounced than those established between cells and surfaces. As a result, cells aggregate, leading to spheroid formation in 1–3 days for most cell lines.

[0196]

[0255] conclusion

[0256] WST-1 assay data showed that metabolic activity in LNCaP cells was significantly inhibited by UC extract (≥100 μg / ml) and HC-HA / PTX3 (≥6.25 μg / ml) (p<0.05), but not by HA. Similarly, metabolic activity in PC-3 cells was significantly inhibited by UC extract (≥200 μg / ml) and HC-HA / PTX3 (≥1.56 μg / ml), but not by HA. Microscopic examination revealed significant cell death in both LNCaP and PC3 cells treated with HC-HA / PTX3 at concentrations of ≥25 μg / ml.

[0197]

[0257] These data demonstrated that the inhibition of metabolic activity in prostate cells by UC extracts was independent of the salt effect. Furthermore, it was demonstrated here that AM and UC products did not promote cancer recurrence after prostatectomy.

[0198] Example 3: Determine the effects of HC-HA / PTX3 and HA on the morphology and metabolic activity of normal prostate cells / cell lines and compare them with those of prostate cancer cell lines.

[0258] The prostatic epithelium consists of two histologically distinct layers: the secretory luminal layer and the basal cell layer. Human normal prostatic epithelial basal and luminal cells were used. Human normal prostatic epithelial basal cells (PrECs) were obtained from Clonetics-BioWhittaker, Inc. (Walkersville, MD, USA) and cultured in basic prostatic epithelial medium (both from Clonetics) using a PrEgM Bullet Kit containing supplements and growth factors (BPE, hydrocortisone, hEGF, epinephrine, insulin, triiodothyronine, transferrin, gentamicin / amphotericin B, and retinoic acid).

[0199]

[0259] The human normal prostatic lumen PNT2 cell line was purchased from Sigma (catalog 95012613). The cell line was established by immortalizing normal adult prostatic epithelial cells by transfection with a plasmid containing the SV40 genome with a missing origin of replication. PNT2 cells were cultured at 37°C in a humidified incubator under a 5% CO2 atmosphere in air in RPMI1640 medium (RPMI1640 complete medium) supplemented with 10% fetal bovine serum (FBS), L-glutamine (2 mM), penicillin (100 U / ml), and streptomycin (100 μg / ml). Cells were harvested at a concentration of 70-80% using EDTA solution of 0.25% (w / v) trypsin-0.53 mM and stored in aliquots in 95% complete medium and 5% DMSO in a liquid nitrogen vapor phase.

[0200] [Table 1]

[0201]

[0260] The cells were revived in their respective growth media. Then, for the WST-1 assay, 3.2 × 10⁶ cells were placed in a 96-well plate. 4 Cells were plated overnight at 37°C / ml (i.e., 3200 cells + 100 ul / well) (n=3 for each assay). Purified HC-HA / PTX3 [AM2P(F3~9)[TGAM17H008]; stock amount = 67 μg] and HMW-HA (Healon; catalog number CE0344; lot number UP30583; stock concentration = 10 mg / ml) were serially diluted in their respective culture media to obtain the concentrations mentioned above. Cells were treated with a series of doses of purified HC-HA / PTX3 or HA and maintained at 37°C for 48 hours. Cell morphology was recorded under a microscope before treatment and after treatment at 24 and 48 hours (bright-field at 10× and 20× magnification). The metabolic activity of each cell line was measured over 48 hours using WST-1 (catalog number 10008883, Cayman Chemical Company, Ann Arbor, MI) according to the manufacturer's instructions (OD450 or OD450-OD670). A. Amplification of normal human prostate cells / cell lines

[0261] Human normal prostatic epithelial basal cells (PrECs) grow faster than human normal prostatic luminal cells (PNT2s). The doubling time for PrECs is 18-24 hours, while for PNT2s it is longer, approximately 36-48 hours.

[0202]

[0262] Bright-field images of PrEC (Figure 8A) and PNT2 (Figure 8B) prostate cell lines taken at 10× and 20× magnification show the morphology of normal prostate cells. B. Effects of HC-HA / PTX3 and HMW-HA on normal human prostate cell morphology

[0263] PrEC cells typically proliferated rapidly and adhered well to surfaces. The cells usually adhered well to each other.

[0203]

[0264] After 24 hours of HC-HA / PTX3 treatment: At 6.25 μg / ml, cells lost cell-cell adhesion; at 12.5 μg / ml, the loss of cell-cell adhesion increased; at 25 μg / ml, cells gradually began to change their morphology; at 50 μg / ml, cells gradually became rounder and lost cell-cell adhesion; at 100 μg / ml, cells became rounder and smaller in size but did not die. After 48 hours of HC-HA / PTX3 treatment: At 3.13 μg / ml, cells began to dissociate; at 25 μg / ml, cells completely dissociated from each other and did not grow into aggregates; at 50 μg / ml, cells were stressed, became smaller in size, and became even smaller and rounder; at 100 μg / ml, cells died one after another, and the number of cells decreased dramatically (Figure 9).

[0204]

[0265] No significant effect of HA treatment was observed. C. Effects of HC-HA / PTX3 and HA on human normal prostate cell metabolism

[0266] The metabolic activity of PrEC and PNT2 was significantly reduced in a dose-dependent manner after treatment with HC-HA / PTX3. In contrast, HA had no significant effect on cellular metabolism (Figures 10A and 10B). Between these two types of normal prostate cells, PrEC (Figure 10A) was highly sensitive to very low concentrations of HC-HA / PTX3 (1.56 and 3.125 μg / ml). The same low concentrations of HC-HA / PTX3 (1.56 and 3.13 μg / ml) did not affect the cellular metabolic activity of PNT2 (Figure 10B).

[0205]

[0267] Metabolic activity (%) was assessed in normal human primary prostate epithelial cells (PrEC) and normal human prostate cell line (PNT2) using the WST-1 assay after 48-hour incubation with various concentrations (0.78, 3.125, 6.25, 12.5, 25, 50, 100 μg / ml) of HC-HA / PTX3 or HA. p-values ​​were calculated by a two-sided t-test against untreated samples. Comparative analysis of cellular metabolic activity in normal and cancerous prostate cells / cell lines treated with D.HC-HA / PTX3 and HA.

[0268] The effects of various concentrations of HC-HA / PTX3 (Figures 11A and 11B) and HA (Figures 12A and 12B) on the metabolic activity of both normal prostate cells (PrEC and PNT2) and prostate cancer cell lines (LNCaP and PC3) were compared. Both normal and cancer cell types responded to HC-HA / PTX3 and HMW-HA in a similar manner. At 25 μg / ml of HC-HA / PTX3, both PrEC and PNT2 cell lines showed lower sensitivity compared to cancer cell lines (LNCaP and PC3). There were no significant differences in the metabolic activity of PrEC, PNT2, PC3, and LNCaP cells after treatment with HMW-HA.

[0206]

[0269] Figures 11A–11B and 12A–12B show a comparative analysis of metabolic activity (%) evaluated after 48-hour incubation with various concentrations (0.78, 3.125, 6.25, 12.5, 25, 50, 100 μg / ml) of HC-HA / PTX3 and HA in normal primary prostate epithelial cells (PrEC) and cell line (PNT2), as well as prostate cancer cell lines: PC3 and LNCaP, using the WST-1 assay. Semi-log regression analysis demonstrated that HC-HA / PTX3 linearly inhibited the metabolic activity of all types of prostate cells in a dose-dependent manner, while HA had no significant effect* (p<0.05), ** (p<0.01), *** (p<0.001).

[0207]

[0270] Overall, HC-HA / PTX3 inhibited the cellular metabolic activity of all types of human prostate cells / cell lines, including normal and cancerous cells, in a dose-dependent manner, while HMW-HA did not have a significant effect. At concentrations up to 25 μg / ml, the PNT2 cell line showed low sensitivity to HC-HA / PTX3 (cellular metabolic activity: 60%, p<0.001), while the metabolic activity of cancer cell lines (both PC3 and LNCaP) was reduced to less than 25% (p<0.001).

[0208] Example 4: Determine the effects of HC-HA / PTX3 and HA on the morphology and metabolic activity of selected cancer epithelial cell lines.

[0271] The above example demonstrated that HC-HA / PTX3 controls the metabolic activity of two prostate cancer cell lines (PC-3 and LNCaP). In this study, the effects of HC-HA / PTX3 and HMW-HA (as a control) on the morphology and metabolic activity of four different human cancer cell lines: A-375 (melanoma), A549 (lung cancer), MCF-7 (breast cancer), and HT-29 (colon adenocarcinoma) were tested by the WST-1 assay. The concentrations of HC-HA / PTX3 and HA were selected based on the results shown in Example 1.

[0209]

[0272] In Example 1, the HC-HA / PTX3 response curve was observed to plateau at concentrations of 50 μg / ml or higher. There was no significant change in metabolic activity between the 50 μg / ml and 100 μg / ml concentrations of HC-HA / PTX3. Therefore, the maximum concentration of HC-HA / PTX3 was maintained at 100 μg / ml.

[0210]

[0273] The cells were thawed in 5 ml of pre-warmed fresh complete culture medium, resuspended, and then centrifuged at 250 g for 5 minutes. The supernatant was carefully removed, the cell pellet was resuspended in 10 ml of pre-warmed fresh complete culture medium, seeded onto 100 mm culture plates, and maintained at 37°C. After 5 days of culture, A-549, HT-29, and A357 cells had a colonization density of approximately 70-80%, while MCF-7 cells grew slowly. The cell plates were removed from the incubator, the medium was carefully removed, and then washed twice with 3-5 ml of D-PBS. Cells were then collected from each 100 mm plate using 5 ml of 0.25% trypsin-0.53 mM EDTA. After 5-10 minutes, 5 ml of fresh culture medium was added to neutralize the effect of the trypsin-EDTA, and the cells were centrifuged at 250 g for 5 minutes. After centrifugation, the supernatant was carefully removed, and the cell pellet was resuspended in 2 ml of fresh culture medium. For each cell line, 10 μl of the cell suspension was mixed with 10 μl of trypan blue to stain dead cells, and the number of viable cells was counted under a microscope using a hemocytometer. In this study, four 96-well plates, one for each cell line, were used. The number of cells to seed was determined based on previous examples (number of seeded cells: 1 × 10⁶ in Examples 1 and 2). 4 / cm 2 ). The well area of ​​each 96-well plate is 0.32 cm². 2 Considering that, 3.2 × 10 4 / ml of cells were required. 3200 cells in 100 μl of culture medium were seeded in each well. The cells were treated in triplicate with HC-HA / PTX3 and HA (as a control) at the following concentrations: 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100 (μg / ml). Two untreated samples were used: untreated without WST-1 reagent and untreated with WST-1 reagent (as a control for the WST-1 assay). To avoid pipetting errors, the cells were diluted to a final volume of 8 ml in culture medium as shown in Table 2.

[0211]

Table 2

[0212]

[0274] After overnight incubation at 37 °C, cell morphology was captured under a bright-field microscope at magnifications of 10× and 20×, respectively. Purified HC-HA / PTX3 [AM2P (F3-9); purified on February 2, 2018; stock amount = 25.78 μg] and HMW-HA (Healon; catalog number CE0344; lot number UP30583; expiration date: December 2018; stock concentration = 10 mg / ml) were serially diluted in their respective culture media to obtain the required concentrations (i.e., 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100 μg / ml). The cells were treated with a series of doses of purified HC-HA / PTX3 or HA and maintained in an incubator at 37 °C for 48 hours. Before treatment and after 24 hours and 48 hours of treatment, cell morphology was recorded under the microscope (bright-field at 10× and 20× magnifications). The metabolic activity of each cell line was measured by WST-1 (catalog number 10008883, Cayman Chemical Company, Ann Arbor, MI) at 48 hours according to the manufacturer's instructions (OD450 or OD450 - OD670).

[0213]

[0275] Effect of HC-HA / PTX3 or HMW-HA on human cancer epithelial cell morphology: a. A-375 (melanoma): Normally, the cells grow fast and adhere well to the surface.

[0214] i. HC-HA / PTX3 treatment: After 24 hours: At 25 (μg / ml): The cells tended to become spindle-shaped. At 50 (μg / ml): The cells changed to spindle shape. At 100 (μg / ml): The cells became round and died. After 48 hours: At 6.25 (μg / ml): The cells showed slightly loose cell-cell adhesion. At 12.5 (μg / ml): The cells further lost cell-cell adhesion. The cells tended to grow individually rather than in clumps. At 25 (μg / ml): >80% of the cells shrank and were spindle-shaped, and the remaining cells were round. At 50 (μg / ml): >60% of the cells died and looked round, and the remaining cells were spindle-shaped. At 100 (μg / ml): All the cells looked round and died. See Figure 13.

[0215] ii. HA treatment: No significant effect of HA was observed. iii. The results showed that as the concentration of HC-HA / PTX3 increased, A-375 cells significantly changed their morphology in a dose-dependent manner according to the following steps:

[0276] Normal epithelial cells adhere to each other > The cells lose cell-cell junctions and change to spindle shape. Cell shrinkage > The cells change to round and die. a. HT-29 (colorectal cancer): The cells are usually round and grow in aggregates. Each aggregate looks like a ball.

[0216] i. HC-HA / PTX3 treatment: After 24 hours: At 50 (μg / ml): The cells lost adhesion and detached. At 100 (μg / ml): The cells looked like round beads and did not survive well. After 48 hours: At 1.56 (μg / ml): Cell-cell adhesion was gradually lost. At 6.25 (μg / ml): <10 cells adhered to each other in each clump. The cells further lost cell-cell adhesion. At 25 (μg / ml): The cells tended to grow individually rather than in aggregates. At 50 (μg / ml): The cells grew individually. At 100 (μg / ml): The individual cells were circular and did not survive well. See Figure 14.

[0217] ii. HA treatment: No significant effect of HA was observed. iii. As the concentration of HC-HA / PTX3 increased, HT-29 cells underwent significant morphological changes in a dose-dependent manner, following the steps below:

[0277] >20 cells adhere to each other. It looks like a bunch of grapes. >Intercellular adhesion gradually weakens. ><10 cells adhere to each other in each cluster.> The cells gradually separate from the cluster. Instead of aggregates, intercellular and cell-matrix adhesion decreases. >Single round cells do not survive well. a. A549 (lung cancer): Typically, the cells are epithelial and proliferate rapidly.

[0218] i. HC-HA / PTX3 treatment: 24 hours later: At 25 μg / ml: Cell morphology changed slightly to a spindle shape. At 50 μg / ml: Cells tended to become more spindle-shaped. At 100 μg / ml: Not all cells died. Dead cells were round. 48 hours later: At 12.5 μg / ml: Cell morphology changed slightly to a spindle shape. At 50 μg / ml: Cells tended to become more spindle-shaped. At 100 μg / ml: Not all cells died. Dead cells were round. See Figure 15.

[0219] ii. HA treatment: No significant effect of HA was observed. iii. Under HC-HA / PTX3 treatment, A-549 cells underwent significant morphological changes in the following steps:

[0278] Cells proliferate. Cell-cell adhesion. >Cells are round and dot-like. Dead a. MCF-7 (breast cancer): Typically, the cells are epithelial. They grow slowly. They adhere to each other. They grow in aggregates.

[0220] i. HC-HA / PTX3 treatment: After 24 hours: No effect up to 50 μg / ml. At 100 μg / ml: Round, bead-like cells were killed. After 48 hours, the effect was the same. See Figure 16.

[0221] ii. HA treatment: No significant effect of HA was observed. iii. Under HC-HA / PTX3 treatment, MCF-7 cells underwent significant morphological changes in the following steps:

[0279] Cells grow as aggregates > The cells dissociated. They died.

[0222]

[0280] Effects of HC-HA / PTX3 or HMW-HA on the metabolic activity of cancer cell lines:

[0281] A375:Cell metabolism was significantly reduced in a dose-dependent manner. After 48 hours of treatment with HC-HA / PTX3, cell metabolism changed significantly from 1.56 μg / ml onwards. No significant effect of HA was observed. (Figure 17A)

[0282] HT-29: HT-29 cellular metabolism was significantly reduced in a dose-dependent manner. After 48 hours of treatment with HC-HA / PTX3, cellular metabolic activity changed significantly from 6.25 μg / ml onwards. No significant effect of HA was observed. (Figure 17B)

[0283] A549: Cellular metabolism was significantly reduced in a dose-dependent manner. After 48 hours of treatment with HC-HA / PTX3, cellular metabolic activity changed from 0.78 μg / ml onwards. No significant effect of HA was observed. (Figure 17D)

[0284] MCF-7 showed minimal sensitivity to HC-HA / PTX3. A significant effect of HC-HA / PTX3 was observed at a high concentration (50 μg / ml), but the effect was not as strong as that observed in other cell lines such as A375 and HT-29. (Figure 17C)

[0285] HC-HA / PTX3 inhibited cell aggregation, intercellular junction, cell morphology, and cell adhesion, and thus the cellular metabolic activity of all four cancer cell types tested, in a dose-dependent manner. In contrast, hyaluronan had no significant effect on the cellular morphology and metabolic activity of all four cell types. These effects occurred quite rapidly, e.g., within 24 hours, which strongly suggests that HC-HA / PTX3, upon binding to CD44, induces changes in cytoskeleton / membrane interactions, which is specific and different from HMW HA.

[0223]

[0286] Of the four cell lines, A375 and HT-29 were more sensitive to HC-HA / PTX3 than A549 and MCF-7. Since MCF-7 does not significantly express CD44, the differences among these four tumor cell lines may have been related to the degree of CD44 expression.

[0224]

[0287] Changes in cell-cell and substrate adhesion differed between A375 and HT-29 upon contact with HC-HA / PTX3. For A375, morphological changes were observed after detachment: cells first became spindle-shaped and then rounded. In contrast, HT-29 showed no morphological changes; the cells remained round from the beginning. Under high concentrations of HC-HA / PTX3, they gradually lost cell-cell junctions. These results suggest that HC-HA / PTX3 may regulate cell adhesion signaling through different pathways in various types of cancer.

[0225]

[0288] The main phenomenon observed in these cancer cell lines was inhibition of cellular metabolic activity, which led to changes in cell shape and ultimately to cell death. Example 5: Determine the effects of HC-HA / PTX3 and HA on mesenchymal cells.

[0226]

[0289] In Examples 2 and 4, HC-HA / PTX3 was found to affect the morphology and metabolic activity of normal prostate epithelial cells in a similar manner to that of tumor cells. To address whether such effects apply to normal mesenchymal cells, we investigated whether HC-HA / PTX3 exerts similar effects on the morphology and metabolic activity of a range of mesenchymal cells.

[0227]

[0290] Three types of normal mesenchymal primary cells were used: (a) limbal niche cells (LNC), (b) human trabecular mesh (HTM), and (c) human corneal fibroblasts (HCF). The cells were cultured in their respective growth media. LNCs were cultured in HSCM medium on 5% Matrigel (serum-free). HTM cells were cultured in ESCM medium on 5% Matrigel containing 5% FBS. HCF cells were cultured in DMEM only on plastic containing 10% FBS. The cells were cultured in each well of a 6-well plate. For LNC and HTM cells, the wells were first coated with 1000 μl of 5% Matrigel and incubated at 37°C for 1 hour. Then, the cells were seeded into each well with 1000 μl of culture medium. After the cells had achieved sufficient density, they were trypsinized and then centrifuged briefly at 200 g for 5 minutes. The medium was removed and the cell pellet was replenished with fresh culture medium. Selected wells of a 96-well plate were coated with 50 μl of 5% Matrigel and incubated at 37°C for 1 hour. LNC and HTM cells were then seeded into the Matrigel-coated 96-well plates and maintained at 37°C for overnight incubation. In each well, 3200 cells were seeded in 100 μl of culture medium. The following day, cells were treated with a series of doses (defined by HA concentration, e.g., 0, 1.56, 3.125, 6.25, 12.5, 25, 50, 100 μg / ml, n=3) of purified HC-HA / PTX3 and HA. The concentrations of HC-HA / PTX3 and HA were calculated by serial dilution as performed in Example 3. Cell morphology was recorded after 15–30 minutes, 1 hour, 5 hours, 24 hours, and 48 hours of treatment, respectively. After 48 hours of incubation, cellular metabolic activity was measured using WST-1 (catalog number 10008883, Cayman Chemical Company, Ann Arbor, MI) according to the manufacturer's instructions (OD450 or OD450-OD670).

[0228]

[0291] Changes in the morphology of limbal niche cells (LNC) were observed within 15 minutes using 25 - 100 μg / ml of HC-HA / PTX3. However, after a longer incubation (1 hour) with HC-HA / PTX3, the cells returned to their original shape. The cell morphology did not change further (see Fig. 18A).

[0229]

[0292] Cells treated with a high dose of HMW-HA (100 μg / ml) further shrank and became thread-like (Fig. 18B). LNC became flattened but relatively shorter when treated with 100 μg / ml of HC-HA / PTX3 for 48 hours. Fig. 18C shows representative bright-field microscope images (scale bar 50 μm) of LNC (limbal niche cells) after 48-hour incubation with 100 μg / ml of HC-HA / PTX3 or HMW-HA.

[0230]

[0293] LNC was metabolically quite resistant to HC-HA / PTX3. After 48-hour incubation, the overall cell metabolic activity remained at about 75%, indicating little effect of HC-HA / PTX3 on LNC. However, with a gradual increase in the HC-HA / PTX3 concentration, the cell metabolic activity rapidly decreased. A significant effect of HC-HA / PTX3 started at a concentration of 12.5 μg / ml. No significant change in the metabolic activity of LNC was detected during treatment with HMW-HA. Fig. 19 shows the metabolic activity (%) evaluated by the WST-1 assay in limbal niche cells after 48-hour incubation with various concentrations (1.56, 3.125, 6.25, 12.5, 25, 50, 100 μg / ml) of HC-HA / PTX3 and HA. The p-values were calculated by two-sided Student's t-test against untreated samples.

[0231]

[0294] Human trabecular mesh (HTM) cells are more resistant to HC-HA / PTX3 compared to LNC cells. No morphological changes were observed in HTM cells within a short timeframe. Cell morphology did not change during 48-hour incubation with various concentrations of HC-HA / PTX3. 5% FBS in the culture medium may make cells more resistant to HC-HA / PTX3. Cell morphology did not change during treatment with HMW-HA. Figures 20A and 20B show representative bright-field microscopy images (scale bar 50 μm) of HTM (human trabecular mesh) cells after treatment with various concentrations of HC-HA / PTX3 (Figure 20A) and HMW-HA (Figure 20B) at various time points: 15–30 minutes, 1 hour, 5 hours, 24 hours, and 48 hours, respectively.

[0232]

[0295] HTM cells were highly resistant to HC-HA / PTX3, as observed in their cell morphology (Figure 20A). After 48 hours of incubation, overall cellular metabolic activity remained above 75%, indicating little effect of HC-HA / PTX3 on HTM cells. However, a significant decrease in cellular metabolic activity (p=0.04) was observed at the highest concentration of HC-HA / PTX3 (100 μg / ml). No significant changes in the metabolic activity of HTM cells were detected during treatment with HMW-HA (Figure 20B).

[0233]

[0296] Figure 21 shows the metabolic activity (%) evaluated in human trabecular mesh cells by the WST-1 assay after 48-hour incubation with various concentrations (1.56, 3.125, 6.25, 12.5, 25, 50, 100 μg / ml) of HC-HA / PTX3 and HA. The p-value was calculated by a two-sided Student's t-test against the untreated sample. As shown, HC-HA / PTX3 and HMW-HA had little effect on HTM.

[0234]

[0297] The effect of HC-HA / PTX3 on the morphology of human corneal fibroblasts (HCFs) was nearly the same as that on LNCs. Cells became rounded within 15–30 minutes of treatment with 100 μg / ml HC-HA / PTX3, but recovered to their original shape by 1 hour. Subsequently, cell morphology remained unchanged even with longer incubation (48 hours) with higher concentrations of HC-HA / PTX3. HMW-HA had no significant effect on HCF cell morphology. Figures 22A and 22B show representative bright-field microscopy images (scale bar 50 μm) of human corneal fibroblast (HCF) cells after treatment at various time points: 15–30 minutes (Figure 22A) and HMW-HA (Figure 22B), respectively, after treatment for 15–30 minutes, 1 hour, 5 hours, 24 hours, and 48 hours.

[0235]

[0298] HC-HA / PTX3 inhibited the cellular metabolic activity of HCF cells in a dose-dependent manner, while HMW-HA did not have a significant effect. Significant inhibition was observed at concentrations of 12.5 μg / ml and above. Figure 23 shows the metabolic activity (%) evaluated in human corneal fibroblast cells by the WST-1 assay after 48-hour incubation with various concentrations (1.56, 3.125, 6.25, 12.5, 25, 50, 100 μg / ml) of HC-HA / PTX3 and HA. The p-value was calculated by a two-sided Student's t-test against the untreated sample.

[0236]

[0299] Overall, despite significant dose-dependent inhibition of cellular metabolic activity by HC-HA / PTX3, normal human mesenchymal primary cells showed greater resistance to HC-HA / PTX3 compared to epithelial cells (normal and cancerous) (Table 3). HMW-HA had no significant effect on the cellular morphology or metabolic activity of primary mesenchymal cells. Of the three mesenchymal cell types, HTM was more robust to high concentrations (100 μg / ml) of HC-HA / PTX3 (metabolic activity: 84.8%, p=0.04), but the same concentrations of HC-HA / PTX3 were able to more effectively suppress the metabolic activity of HCF (50%, p=0.01) and LNC (61.4%, p=0.04). Figures 24A and 24B show a comparative analysis of metabolic activity (%) in three types of normal human primary mesenchymal cells: HCF, HTM, and LNC, evaluated by the WST-1 assay after 48-hour incubation with HC-HA / PTX3 (Figure 24A) and HA (Figure 24B) at various concentrations (1.56, 3.125, 6.25, 12.5, 25, 50, and 100 μg / ml). The p-values ​​were calculated using a two-sided Student's t-test against the untreated samples. * indicates p<0.05.

[0237]

[0300] LNCs and HCFs were highly sensitive to HC-HA / PTX3, and their cell morphology changed very rapidly with high doses of HC-HA / PTX3 (within 15–30 minutes), but this change did not last longer. Within one hour, the cells recovered to their normal shape, and thereafter, cell morphology was no longer affected, even with long incubation (48 hours) with high concentrations of HC-HA / PTX3. In contrast, HC-HA / PTX3 failed to alter HTM cell morphology at high concentrations, both for shorter and longer incubation periods. Representative bright-field microscopy images (scale bar 50 μm) (Figure 25) show morphological abnormalities and rapid recovery of mesenchymal cells (LNCs and HCFs) during short-term loading with HC-HA / PTX3 (100 μg / ml). HTM cells do not respond to HC-HA / PTX3 like other normal mesenchymal cells.

[0238] [Table 3]

[0239] Example 6: Determine the role of HC-HA / PTX3 in suppressing cell proliferation in normal and cancer cells.

[0301] In Example 4, the inventors observed that the metabolic activity (measured by WST-1) and morphology of A375 (melanoma) cells were sensitive to HC-HA / PTX3. As the concentration of HC-HA / PTX3 increased, metabolic activity was inhibited, cell morphology changed from epithelial to spindle-shaped, then to rounded, and intercellular and cell-matrix adhesion was lost. Since cellular metabolic activity is directly proportional to the rate of cell proliferation, the role of HC-HA / PTX3 in inhibiting cell proliferation is determined by quantifying the DNA content of the proliferating cells. 5-bromo-2'-deoxyuridine (BrdU) incorporated into cellular DNA during cell proliferation is detected using an anti-BrdU antibody with the BrdU Cell Proliferation Assay Kit (catalog no. 6813; Cell Signaling Technology, USA).

[0240]

[0302] Considering the results in Example 4, where A375 cells showed significant morphological changes starting from 25 μg / ml of HC-HA / PTX3 after 24 hours of treatment, cells were treated for 24 hours with the following concentrations of HC-HA / PTX3: 0, 25, 50, and 100 μg / ml. 100 μg / ml of HA was used as a control due to the lack of significant effects on cell morphology and metabolic activity as shown in Example 5. Based on this pilot study, the proliferation assay protocol was optimized and applied to determine the effects of HC-HA / PTX3 on human prostate cells (normal and cancerous). The test groups are shown in Table 4.

[0241]

[0303] A375 cells were seeded in a 96-well plate (3200 cells / well in 100 μl of culture medium) and incubated overnight. Cells were treated with HC-HA / PTX3 and HA at the aforementioned concentrations for 48 hours. 10 μl of 10×BrdU solution and cells were placed in an incubator for 4 hours. The medium was removed, and 100 μl / well of fixation / denaturation solution was added and left for 30 minutes. The solution was removed, and 100 μl / well of 1× detection antibody solution was added and left for 1 hour. The solution was removed, the plates were thoroughly washed three times with wash buffer, and 100 μl / well of 1× HRP conjugate secondary antibody solution was added at room temperature for 30 minutes. The solution was removed, the plates were thoroughly washed three times with wash buffer, and TMB substrate was added at room temperature for 30 minutes. 100 μL / well of stop solution was added, and absorbance was read at 450 nm.

[0242] [Table 4]

[0243]

[0304] The antiproliferative activity of HC-HA / PTX3 and HA was studied in A375 (melanoma) cells. 48-hour incubation with various concentrations of HC-HA / PTX3 (25, 50, and 100 μg / ml) and 100 μg / ml of HA showed the same effects on A375 cell morphology as observed in Example 4 (Figure 26A). BrdU incorporation assays revealed that HC-HA / PTX3 significantly inhibited A375 cell growth in a dose-dependent manner (p<0.005), while HA did not make a significant contribution (Figure 26B). Statistical significance (p-value) was calculated using Student's t-test. Semi-log linear regression analysis determined that HC-HA / PTX3 has a linear inhibitory effect on A375 cell growth (R 2 =0.9681). (Figure 26C) Cell proliferation analysis of prostate cell lines using the BrdU Cell Proliferation Assay Kit (catalog number 6813; Cell Signaling Technology, USA).

[0244] [Table 5]

[0245]

[0305] PrEC, PNT2, PC-3, and LNCaP cells were measured in 3.2 × 10⁶ well plates. 3 Cells were seeded at 100 μg / well and incubated overnight. The cells were then treated in triplicate for 48 hours with five concentrations of HC-HA / PTX3 (1.56, 3.13, 6.25, 12.5, and 25 μg / ml) and 100 μg / ml of HMW-HA, as shown in Table 5. Finally, 10 μM BrdU was added to the wells and the cells were incubated for 4 hours. The medium was removed, and 100 μl / well of fixation / denaturation solution was added for 30 minutes. The solution was removed, 1× detection antibody solution was added, and 100 μl / well of 1× HRP conjugate secondary antibody solution was added at room temperature for 30 minutes. The solution was removed, the cells were washed three times appropriately with wash buffer, and TMB substrate was added at room temperature for 30 minutes at 100 μl / well. 100 μL / well of stop solution was added, and absorbance was read at 450 nm.

[0246]

[0306] Cell morphological analysis of PrEC cells by bright-field phase-contrast microscopy (Figure 27A) and BrdU incorporation assay demonstrate the antiproliferative effect of HC-HA / PTX3 on PrEC cells. HA does not inhibit cell proliferation. In this experiment, the maximum concentration of HC-HA / PTX3 used was 25 μg / ml. At HC-HA / PTX3 concentrations of 12.5 and 25 μg / ml, the OD values ​​were negative, indicating the absence of cells in the wells. Due to deviations in the analytical procedure, these data may not reflect the effect attributable to HC-HA / PTX3 exposure.

[0247]

[0307] Figures 27A and 27B show that HC-HA / PTX3 inhibited PrEC cell proliferation in a dose-dependent manner, while HMW-HA (Figure 27C) did not have a significant effect on cell proliferation, as detected by the BrdU Cell Proliferation Assay Kit #6813 (Cell Signaling, USA). Figure 27A shows bright-field images of PrEC cell morphology at two magnifications (10× and 20×), and Figures 27B and 27C show BrdU cell proliferation assay curves. Statistical significance (p-value) was calculated using Student's t-test.

[0248]

[0308] Bright-field phase-contrast microscopy and cell morphology analysis using the BrdU integration assay demonstrated the antiproliferative effect of HC-HA / PTX3 on PNT2 cells. HA did not inhibit cell proliferation. Normally, cells grow in aggregates. As the concentration of HC-HA / PTX3 increased, it inhibited cell-cell adhesion (Figure 28A). In the BrdU assay, no significant effect of HC-HA / PTX3 was observed due to a high standard error (Figure 28B). The p-value was greater than 0.05. The BrdU data showed similarity to the previously observed WST-1 data.

[0249]

[0309] As detected by BrdU Cell Proliferation Assay Kit #6813 (Cell Signaling, USA), HC-HA / PTX3 inhibited PNT2 cell proliferation in a dose-dependent manner, while HMW-HA did not have a significant effect on cell proliferation. PNT2 cells proliferated to 3.2 × 10⁶ in a 96-well plate. 3 Cells were seeded in individual wells and incubated overnight. The cells were then treated in triplicate for 48 hours with five concentrations of HC-HA / PTX3 (1.56, 3.13, 6.25, 12.5, and 25 μg / ml) and two concentrations of HMW-HA (25 and 100 μg / ml). Finally, 10 μM BrdU was added to the wells, and the cells were incubated for 4 hours.

[0250]

[0310] As observed in Example 7, HC-HA / PTX3 had an antiproliferative effect on the PC-3 prostate cancer cell line, but HA did not. The BrdU assay showed that HC-HA / PTX3 significantly inhibited PC-3 proliferation at a concentration of 25 μg / ml (p=0.04). The BrdU data showed similarity to the WST-1 data.

[0251]

[0311] As detected by BrdU Cell Proliferation Assay Kit #6813 (Cell Signaling, USA), HC-HA / PTX3 inhibited PC3 cell proliferation in a dose-dependent manner, while HMW-HA did not have a significant effect on cell proliferation. PC3 cells were proliferated in a 96-well plate at a rate of 3.2 × 10⁶ 3 Cells were seeded in 100 μg / wells and incubated overnight. The cells were then treated in triplicate for 48 hours with five concentrations of HC-HA / PTX3 (1.56, 3.13, 6.25, 12.5, and 25 μg / ml) and two concentrations of HMW-HA (25 and 100 μg / ml). Finally, 10 μM BrdU was added to the wells and the cells were incubated for 4 hours. Figure 29A shows bright-field images of PC3 cell morphology. Figure 29B shows the BrdU cell proliferation assay curve.

[0252]

[0312] Under treatment with HC-HA / PTX3, the cell morphology of LNCaP cells changed in a dose-dependent manner. Surprisingly, despite being cancer cells, LNCaP cells did not proliferate as rapidly as PC-3, PNT2, or PrEC cells. This observation was well supported by BrdU data for all cell types. Under untreated conditions, the OD value of LNCaP cells was 0.5, while for the other cells it was >0.75. Interestingly, in the case of LNCaP cells, even at high concentrations of HC-HA / PTX3, the cells continued to grow in aggregates, but their morphology changed, thus representing a high effect of HC-HA / PTX3 on cell-matrix binding rather than cell-cell adhesion. High standard errors at concentrations of HC-HA / PTX3 of 3.13 and 12.5 μg / ml, as well as 100 μg / ml of HA, represented erroneous graphs. Due to a pipetting error in the blank sample, the OD value was negative at 25 μg / ml HC-HA / PTX3 (see Figure 30A). Sufficient cells were visualized in the target well (see Figure 30B).

[0253]

[0313] As detected by BrdU Cell Proliferation Assay Kit #6813 (Cell Signaling, USA), HC-HA / PTX3 inhibited LNCaP cell proliferation in a dose-dependent manner, while HMW-HA did not have a significant effect on cell proliferation. LNCaP cells proliferated to 3.2 × 10⁶ in a 96-well plate. 3 Cells were seeded in 100 μg / wells and incubated overnight. Cells were then treated in triplicate for 48 hours with five concentrations of HC-HA / PTX3 (1.56, 3.13, 6.25, 12.5, and 25 μg / ml) and two concentrations of HMW-HA (25 and 100 μg / ml). Finally, 10 μM BrdU was added to the wells and the cells were incubated for 4 hours. Figure 30A shows bright-field images of LNCaP cell morphology. Figure 30B shows the BrdU cell proliferation assay curve.

[0254]

[0314] BrdU assays on four types of prostatic epithelial cells / cell lines (both normal and cancerous) suggested that HC-HA / PTX3 inhibited cell proliferation in a dose-dependent manner, while HMW-HA did not. Due to technical failures, the PrEC and LNCaP results do not reflect the same effects observed in the WST-1 assay data. However, corresponding cell morphological images supported the similarity of the effects of HC-HA / PTX3 and HA on prostatic cells as observed in previous experiments.

[0255]

[0315] While preferred embodiments are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided merely as examples. Numerous variations, modifications, and substitutions are now possible. It should be understood that various alternatives to the embodiments described herein may be used in the practice of the methods described. The following claims define the scope of the embodiments, and the methods and structures within these claims, as well as their equivalents, are intended to be encompassed thereby. This specification includes the disclosure of the following inventions. [Item 1] A method for inhibiting tumor cancer cell regrowth in an individual requiring inhibition of tumor cancer cell regrowth, comprising the step of contacting the region surrounding a tumor after a surgical procedure with an isolated heavy-chain hyaluronan / pentraxin 3 (HC-HA / PTX3) complex, thereby inhibiting cancer cell regrowth in the region surrounding the tumor. [Item 2] The method according to Item 1, wherein the surgical procedure includes surgical excision, cryoexcision, or radiofrequency ablation of the tumor. [Item 3] The method described in Item 1, wherein the surgical procedure includes chemotherapy, immunotherapy, or targeted therapy. [Item 4] The method according to Item 1, wherein the region surrounding the tumor includes the resection margin. [Item 5] The method described in Item 1, wherein the area surrounding the tumor is the peritumoral region. [Item 6] The method according to Item 1, wherein the tumor is a solid tumor. [Item 7] The method according to Item 1, wherein the tumor is a cancer selected from the group consisting of liver cancer, pancreatic cancer, bladder cancer, prostate cancer, lung cancer, non-small cell lung cancer, ovarian cancer, breast cancer, melanoma, stomach cancer, colon cancer, colorectal cancer, central nervous system (CNS) cancer, bone cancer, lymphoma, skin cancer, head and neck cancer, kidney cancer, testicular cancer, uterine cancer, cervical cancer, esophageal cancer, thyroid cancer, salivary gland cancer, adrenal cancer, and gastrointestinal cancer. [Item 8] The method described in Item 7 for cancer that is inoperable. [Item 9] The method described in Item 7, where the cancer is pancreatic cancer. [Item 10] The method described in Item 7, wherein the cancer is prostate cancer. [Item 11] The method described in Item 7, wherein the cancer is glioblastoma multiforme. [Item 12] The method described in Item 7, where the cancer is skin cancer. [Item 13] The method described in Item 7, where the cancer is colon cancer. [Item 14] The method described in Item 7, where the cancer is lung cancer. [Item 15] The cancer is breast cancer, as described in Item 7. [Item 16] The method described in Item 1, wherein the area surrounding the tumor is brought into contact with approximately 10 micrograms to 100 milligrams. [Item 17] The method described in any one of Items 1 to 16, wherein the HC-HA / PTX3 complex is a natural HC-HA / PTX3 complex, a reconstituted HC-HA / PTX3 complex, or a combination thereof. [Item 18] The method described in Item 17, wherein the natural HC-HA / PTX3 complex is isolated from fetal supporting tissue. [Item 19] The method according to Item 17, wherein the reconstituted HC-HA / PTX3 complex comprises heavy chain 1 (HC1) and heavy chain 2 (HC2) of an inter-α inhibitor (IαI), hyaluronic acid (HA), and PTX3. [Item 20] The method according to Item 17, wherein the reconstituted HC-HA / PTX3 complex contains HC1, HC2, HA, PTX3, and tumor necrosis factor alpha-stimulating gene 6 (TSG-6). [Item 21] The method according to Item 1, wherein hyaluronan (HA) is high molecular weight hyaluronan (HMW HA). [Item 22] The method according to Item 1, wherein hyaluronan (HA) is low molecular weight hyaluronan (LMW HA). [Item 23] The method described in Item 1, wherein the HC-HA / PTX3 complex is cryopreserved. [Item 24] The method described in Item 1, wherein the HC-HA / PTX3 complex contains living cells. [Item 25] The method according to Item 1, further comprising the step of administering a therapeutic agent. [Item 26] The method according to Item 25, wherein the therapeutic agent is selected from the group consisting of chemotherapy drugs, analgesics, anti-inflammatory drugs, steroids, immunotherapy, cell therapy, radiotherapy, targeted drug therapies, and antibiotics. [Item 27] ​​The method of Item 25, wherein the step of administering the therapeutic agent is performed before the area surrounding the tumor comes into contact with the HC-HA / PTX3 complex. [Item 28] The method of Item 25, wherein the step of administering the therapeutic agent is performed after the area surrounding the tumor has been brought into contact with the HC-HA / PTX3 complex. [Item 29] The method of Item 25, wherein the step of administering the therapeutic agent is performed simultaneously with bringing the area surrounding the tumor into contact with the HC-HA / PTX3 complex. [Item 30] The method described in any one of Items 1 to 29, which inhibits tumor cell regrowth by killing cancer cells. [Item 31] The method described in Item 30, wherein cancer cells are killed by apoptosis or necrosis. [Item 32] The method of any one of items 1 to 29, which inhibits tumor cell regrowth by inhibiting the proliferation of cancer cells. [Item 33] The method according to any one of items 1 to 29, which inhibits tumor cell regrowth by inhibiting the metabolic activity of cancer cells. [Item 34] A method for killing cancer cells in a tumor in an individual requiring the elimination of cancer cells in a tumor, comprising the step of contacting the tumor or the area surrounding the tumor with an isolated heavy-chain hyaluronan / pentraxin 3 (HC-HA / PTX3) complex before, during, or after a surgical procedure, thereby killing the cancer cells. [Item 35] The method described in Item 34, wherein the surgical procedure includes surgical excision, cryoexcision, or radiofrequency ablation of a tumor. [Item 36] The method described in Item 34, wherein the surgical procedure includes chemotherapy, immunotherapy, or targeted therapy. [Item 37] The method of Item 34, wherein the region surrounding the tumor includes the resection margin. [Item 38] The method described in Item 34, wherein the area surrounding the tumor is the peritumoral region. [Item 39] The method of Item 34, wherein the tumor is a solid tumor. [Item 40] The method according to Item 34, wherein the tumor is a cancer selected from the group consisting of liver cancer, pancreatic cancer, bladder cancer, prostate cancer, lung cancer, non-small cell lung cancer, ovarian cancer, breast cancer, melanoma, stomach cancer, colon cancer, colorectal cancer, central nervous system (CNS) cancer, bone cancer, lymphoma, skin cancer, head and neck cancer, kidney cancer, testicular cancer, uterine cancer, cervical cancer, esophageal cancer, thyroid cancer, salivary gland cancer, adrenal cancer, and gastrointestinal cancer. [Item 41] Cancer is inoperable, as described in Item 34. [Item 42] The method described in Item 34, wherein the cancer is pancreatic cancer. [Item 43] The method described in Item 34, wherein the cancer is prostate cancer. [Item 44] The method described in Item 34, where the cancer is glioblastoma pleomorphoni. [Item 45] The method described in Item 34, where cancer is skin cancer. [Item 46] The method described in Item 34, wherein the cancer is colon cancer. [Item 47] The method described in Item 34, wherein the cancer is lung cancer. [Item 48] The cancer is breast cancer, as described in Item 34. [Item 49] The method described in Item 34, which involves bringing the area surrounding the tumor into contact with approximately 10 micrograms to 100 milligrams. [Item 50] The method according to any one of items 34-49, wherein the HC-HA / PTX3 complex is a natural HC-HA / PTX3 complex, a reconstituted HC-HA / PTX3 complex, or a combination thereof. [Item 51] The method described in Item 50, wherein the natural HC-HA / PTX3 complex is isolated from fetal supporting tissue. [Item 52] The method according to Item 50, wherein the reconstituted HC-HA / PTX3 complex comprises heavy chain 1 (HC1) and heavy chain 2 (HC2) of an inter-α inhibitor (IαI), hyaluronic acid (HA), and PTX3. [Item 53] The method according to Item 50, wherein the reconstituted HC-HA / PTX3 complex comprises HC1, HC2, HA, PTX3, and tumor necrosis factor alpha-stimulating gene 6 (TSG-6). [Item 54] The method described in Item 34, wherein the HC-HA / PTX3 complex is cryopreserved. [Item 55] The method described in Item 34, wherein the HC-HA / PTX3 complex contains living cells. [Item 56] The method according to Item 34, wherein hyaluronan (HA) is high molecular weight hyaluronan (HMW HA). [Item 57] The method according to Item 34, wherein hyaluronan (HA) is low molecular weight hyaluronan (LMW HA). [Item 58] The method of Item 34, wherein the contact step includes injecting HC-HA / PTX3 directly into the tumor. [Item 59] The method according to Item 34, further comprising the step of administering a therapeutic agent. [Item 60] The method according to Item 59, wherein the therapeutic agent is selected from the group consisting of chemotherapy drugs, analgesics, anti-inflammatory drugs, steroids, immunotherapy, cell therapy, radiotherapy, targeted drug therapy, and antibiotics. [Item 61] The method according to Item 59, wherein the step of administering the therapeutic agent is performed before the cancer cells come into contact with the HC-HA / PTX3 complex. [Item 62] The method according to Item 59, wherein the step of administering the therapeutic agent is performed after the cancer cells have been brought into contact with the HC-HA / PTX3 complex. [Item 63] The method according to Item 59, wherein the step of administering the therapeutic agent is performed simultaneously with the contact of cancer cells with the HC-HA / PTX3 complex. [Item 64] The method described in Item 34, wherein cancer cells are killed by apoptosis or necrosis.

Claims

1. A composition for use in inhibiting cancer cell regrowth, comprising an isolated inter-alpha inhibitor (IαI) heavy chain 1 (HC1)-hyaluronic acid (HA) / pentraxin 3 (HC-HA / PTX3) complex, which is administered to an individual requiring administration of the composition by contacting the area surrounding the tumor before, during, or after a surgical procedure, thereby inhibiting cancer cell regrowth.

2. The composition according to claim 1, wherein the surgical procedure includes surgical excision, cryoexcision, or radiofrequency ablation of a tumor.

3. The composition according to claim 1, wherein the surgical procedure includes surgical excision.

4. The composition according to claim 1, wherein the region surrounding the tumor includes the resection margin.

5. The composition according to claim 1, wherein the region surrounding the tumor includes a tumor-peritumoral region.

6. The composition according to claim 1, wherein the tumor is a solid tumor.

7. The composition according to claim 1, wherein the tumor is a cancer selected from the group consisting of liver cancer, pancreatic cancer, bladder cancer, prostate cancer, lung cancer, non-small cell lung cancer, ovarian cancer, breast cancer, melanoma, stomach cancer, colon cancer, colorectal cancer, central nervous system (CNS) cancer, bone cancer, lymphoma, skin cancer, head and neck cancer, kidney cancer, testicular cancer, uterine cancer, cervical cancer, esophageal cancer, thyroid cancer, salivary gland cancer, adrenal cancer, glioblastoma multiforme, and gastrointestinal cancer.

8. The composition according to claim 1, wherein the area surrounding the tumor is brought into contact with 10 micrograms to 100 milligrams.

9. The composition according to any one of claims 1 to 8, wherein the isolated HC-HA / PTX3 complex is a natural HC-HA / PTX3 complex, a recombinant HC-HA / PTX3 complex, or a combination thereof.

10. The composition according to claim 9, wherein a natural HC-HA / PTX3 complex is isolated from fetal supporting tissue.

11. The composition according to claim 9, wherein the recombinant HC-HA / PTX3 complex comprises heavy chain 1 (HC1) and heavy chain 2 (HC2) of an inter-α inhibitor (IαI), hyaluronic acid (HA), pentraxin 3 (PTX3), and tumor necrosis factor α-stimulating gene 6 (TSG-6).

12. The composition according to claim 10, wherein the natural HC-HA / PTX3 complex is cryopreserved.

13. The composition according to claim 1, wherein HA is high molecular weight HA.

14. The composition according to any one of claims 1 to 10, wherein when the composition is administered to the area surrounding the tumor, cancer cell regrowth is inhibited, and this inhibition of cancer cell regrowth is due to apoptosis or necrosis of cancer cells.

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