CN105339000A - Highly pure ovarian cancer stem cells for active autoimmune therapy - Google Patents

Abstract

The present invention provides cancer stem cells for stimulating an immune response against cancer (such as ovarian cancer). The present invention provides methods for preparing and purifying cancer stem cells.

Description

Highly pure ovarian cancer stem cells for active autoimmune therapy

Cross References to Related Applications

This application claims the benefit of U.S. Provisional Patent Application 61/778,225, filed March 12, 2013, under 35 U.S.C. §119(e). This application is also a continuation-in-part of International Application PCT/US2013/053850, filed August 6, 2013, which under 35 U.S.C. §119(e) requires U.S. Provisional Patent Application 61/ 683,477 and the benefit of U.S. Provisional Patent Application 61/718,643, filed October 25, 2012. The entire content of said application is incorporated herein by reference.

technical field

The present invention relates to ovarian cancer stem cells, immunogenic compositions derived from said stem cells, and methods of making and using said stem cells.

Background technique

Ovarian cancer is thought to originate in the epithelium lining the ovary and thus has the histological features of adenocarcinoma. Ovarian cancer stem cells have been previously described as "subpopulations" or "side populations" (SP) selected from the main population (MP) in the SKOV3 and A224 cell lines, which express stem cell marker genes (Oct4 and Nanog ), transporter genes (ABCG2, ABCC4, ABCB1) and CD markers (CD44, CD24, CD177), have the possibility of differentiation into cancer, and have different tissue structures, indicating the pluripotent characteristics of stem cells. Isolation of such cells was achieved using the exclusion of the fluorescent DNA-binding dye Hoechst 33342.

Specific cancer stem cell populations may be the source of neoplasia and may be the source of recurrence of already treated cancers. In addition, subpopulations of cancer stem cells in tissues that when exposed to certain signals may restart the growth cycle and generate cells that can reconstitute tumors. Cancer stem cell niches are dormant until the correct signaling triggers re-entry into the proliferative cycle. Re-entry signals can originate from local events, such as trauma, cellular damage, microbial attack (viruses, bacteria, or fungi), or be mediated by local growth factors, cytokines, or intercellular communication. Additionally, hormones can regulate stem cells in tissue-specific niches. Defects or mutations in the stem cell niche may lead to perturbations of the above functions. Neoplasms can arise from such perturbations, and these perturbations include random mutations that affect the control of the cell cycle. Cancer-causing mutations vary between individuals. Such variability is observed between those with one type of cancer (such as one breast cancer patient versus another) and between different types of cancer (such as ovarian cancer versus melanoma). In ovarian cancer, damage to the TP53 gene has been identified in most cases, however this mutation is not always reflected in the tumor cell phenotype.

Discordant markers have also been associated in various proportions with tumor cells and have been generally successfully used to trigger immune responses. Such therapies using tumor-associated antigens employ various proteins or peptides, such as CA125, Her-2, Muc-1, Neu, NY-ESO-1, or heat shock proteins (HSPs) derived from tumors.

Autoimmune therapy uses the patient's own tumor tissue to sensitize the immune system and attach itself to the cancer cells. Administration of lysates or whole-cell approaches, alone or with adjuvants, has been used to enhance the immune response to tumors.

Contents of the invention

Disclosed herein are ovarian cancer (OV) cancer stem cells (CSCs), OV-CSC cell lines, and immunogenic compositions comprising OV-CSC-loaded dendritic cells for use in the treatment of ovarian cancer.

Specifically provided herein is an immunogenic composition comprising dendritic cells activated ex vivo by a tumor antigen derived from a purified ovarian cancer (OV) cancer stem cell (CSC) population disclosed herein. In one embodiment, said tumor antigen comprises a cell extract of said OV-CSC. In another embodiment, said tumor antigen comprises a lysate of said OV-CSC. In another embodiment, the tumor antigen comprises intact OV-CSC. In another embodiment, said tumor antigen comprises messenger RNA transfected into said dendritic cells ex vivo.

In another embodiment, said intact OV-CSCs are rendered non-proliferative. In another embodiment, said intact OV-CSCs are rendered non-proliferative by irradiation. In yet another embodiment, said intact OV-CSC is rendered non-proliferative by exposing said OV-CSC to a nuclear cross-linking agent or a protein cross-linking agent.

In one embodiment, the immunogenic composition further comprises a pharmaceutically acceptable carrier and/or excipient. In another embodiment, the immunogenic composition further comprises an adjuvant. In another embodiment, the adjuvant is granulocyte macrophage colony stimulating factor.

In yet another embodiment, the immunogenic composition comprises activated dendritic cells and OV-CSCs. In another embodiment, the OV-CSC is in the form of OV-CSC spheroids, early OV-CSC, mixed OV-CSC or EMT-OV-CSC.

Also provided is a method of treating ovarian cancer in a subject in need thereof comprising administering to the subject an immunogenic composition disclosed herein. In one embodiment, the immunogenic composition is administered in multiple doses, each dose comprising about 5-20 x ¹⁰⁶ cells. In another embodiment, the dose comprises about ¹⁰ x 106 cells. In another embodiment, the dose is administered weekly for 2-5 doses, followed by monthly administration for 3-6 doses. In yet another embodiment, the subject receives 6-10 doses of the immunogenic composition.

Also provided is the use of the immunogenic composition disclosed herein, the OV-CSC disclosed herein or the OV-CSC cell line disclosed herein in the manufacture of a medicament for the treatment of ovarian cancer.

Also provided is a use of an immunogenic composition disclosed herein, an OV-CSC disclosed herein, or an OV-CSC cell line disclosed herein for the treatment of ovarian cancer.

Further provided herein is a method for preparing a population of ovarian cancer (OV) cancer stem cells (CSCs), the method comprising: obtaining a sample of OV; wherein the defined medium is serum-free and supplemented with at least one growth factor acting via the mitogen-activated protein kinase (MAPK) pathway, whereby OV-CSC spheroids are formed; wherein at least 80% of the cells in the OV-CSC spheroid population express two or more of the following biomarkers: EpCAM, CA-125, MUC-1, CD117, He-4 , ALDH, CD133, CD24 and Ki-67. In another embodiment, at least 80% of the cells in the OV-CSC spheroid population further express one or more of the following biomarkers; CA19-9, HER2/neu, NCAM, ganglioside CD2, estrogen Hormone receptor alpha, vimentin, CK8, CK18, AFP, testosterone, TGFβR, EGFR, TAG-72, CD46, CD44, ABCG2, Slug/Snail, nestin, and TP53. In another embodiment, at least 90% of the cells in the OV-CSC spheroid population express two or more of the following biomarkers: EpCAM, CA-125, MUC-1, CD117, He-4, ALDH, CD133, CD24, and Ki-67.

In another embodiment, the method further comprises culturing the OV-CSC spheroids on an adhesive substrate in a defined medium, wherein the defined medium is serum-free and supplemented with at least one growth factor acting via said MAPK pathway, thereby forming a population of early OV-CSCs, wherein at least 80% of the cells in said early OV-CSC population express two or more of the following biomarkers : EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD17 and Ki-67. In another embodiment, at least 80% of the cells in the early OV-CSC population further express one or more of the following biomarkers: CA-125, MUC-1, TGFβR, and CD24. In yet another embodiment, at least 90% of the cells in the early stage OV-CSC population express two or more of the following biomarkers: EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD17, and Ki-67.

In another embodiment, the method further comprises culturing the OV-CSC spheroids on an adhesive substrate in a defined medium, wherein the defined medium contains serum, thereby forming a mixed A population of OV-CSCs, wherein at least 80% of the cells in the mixed OV-CSC population express two or more of the following biomarkers: EpCAM, CA-125, MUC-1, CD117, CK8, CK18, and Ki -67. In another embodiment, said defined medium further comprises at least one growth factor acting via said MAPK pathway. In yet another embodiment, the defined medium is a low calcium defined medium. In another embodiment, at least 80% of the cells in the mixed OV-CSC population further express one or more of the following biomarkers: CA19-9, HER2/neu, NCAM, ganglioside CD2, estrogen Receptor α, Testosterone, TGFβR, EGFR, TAG-72, CD46, He-4, ALDH, CD133, CD44, ABCG2, Nestin, and TP53. In yet another embodiment, at least 90% of the cells in the mixed OV-CSC population express two or more of the following biomarkers: EpCAM, CA-125, MUC-1, CD117, CK8, CK18, and Ki-67.

In another embodiment, the method further comprises culturing the OV-CSC spheroids on an adhesive substrate in a defined medium, wherein the defined medium contains serum and is supplemented with at least one A growth factor acting via the MAPK pathway, thereby forming a population of epithelial to mesenchymal transition (EMT)-OV-CSCs, wherein at least 80% of the cells in the EMT-OV-CSC population express Two or more of the following biomarkers: NCAM, Slug/Snail, CD24, and Twist. In another embodiment, at least 80% of the cells in the EMT-OV-CSC population further express one or more of the following biomarkers: CA-125, MUC-1, CD133, Nanog, CD117, N-calcium Cohesin, CD44, and Vimentin. In another embodiment, at least 90% of the cells in said EMT-OV-CSC population express two or more of the following biomarkers: NCAM, Slug/Snail, CD24, and Twist.

In another embodiment, the method further comprises culturing said OV-CSC spheroids, said mixed OV-CSC or EMT-OV-CSC on an adhesive substrate in a defined medium, wherein said The defined medium is serum-free and supplemented with at least one growth factor acting via the MAPK pathway, thereby forming a population of early OV-CSCs, wherein at least 80% of the early OV-CSC populations are Cells express two or more of the following biomarkers: EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD17, and Ki-67. In another embodiment, at least 80% of the cells in the early OV-CSC population further express one or more of the following biomarkers: CA-125, MUC-1, TGFβR, and CD24. In yet another embodiment, at least 90% of the cells in the early stage OV-CSC population express two or more of the following biomarkers: EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD17, and Ki-67.

In another embodiment, the method further comprises culturing said OV-CSC spheroids, said early OV-CSC or EMT-OV-CSC on an adhesive substrate in a defined medium, wherein said The defined medium contains serum and is supplemented with at least one growth factor acting via the MAPK pathway, thereby forming a mixed OV-CSC population wherein at least 80% of the cells in the mixed OV-CSC population express Two or more of the following biomarkers: AFP, CK7, CK19, EpCAM, E-cadherin, Nanog, FoxA2 HNF4a, and ABCG2. In another embodiment, said defined medium further comprises at least one growth factor acting via said MAPK pathway. In yet another embodiment, the defined medium is a low calcium defined medium. In another embodiment, at least 80% of the cells in the mixed OV-CSC population further express one or more of the following biomarkers: CA19-9, HER2/neu, NCAM, ganglioside CD2, estrogen Receptor α, Testosterone, TGFβR, EGFR, TAG-72, CD46, He-4, ALDH, CD133, CD44, ABCG2, Nestin, and TP53. In yet another embodiment, at least 90% of the cells in the mixed OV-CSC population express two or more of the following biomarkers: EpCAM, CA-125, MUC-1, CD117, CK8, CK18, and Ki-67.

In another embodiment, the method further comprises culturing said OV-CSC spheroids, said early OV-CSCs or mixed OV-CSCs on an adhesive substrate in a defined medium, wherein said A defined medium containing a serum source and supplemented with at least one growth factor acting via said MAPK pathway, thereby forming a population of EMT-OV-CSCs, wherein at least 80% of said EMT-OV-CSC populations are Cells express two or more of the following biomarkers: NCAM, Slug/Snail, CD24, and Twist. In another embodiment, at least 80% of the cells in the EMT-OV-CSC population further express one or more of the following biomarkers: CA-125, MUC-1, CD133, Nanog, CD117, N-calcium Cohesin, CD44, and Vimentin. In another embodiment, at least 90% of the cells in said EMT-OV-CSC population express two or more of the following biomarkers: NCAM, Slug/Snail, CD24, and Twist.

In one embodiment, the defined medium is any medium described in Table 2; any medium from the combination of Table 2 and Table 3; the combination of Table 2, Table 3 and Table 4 or any medium from the combination of Table 2 and Table 4.

In one embodiment, the growth factor is one or more of fibroblast growth factor (FGF), epidermal growth factor (EGF), or activin A. In another embodiment, the FGF is basic FGF (bFGF). In another embodiment, the defined medium is not supplemented with Activin A. In yet another embodiment, the defined medium is supplemented with an agonist of activin A in an amount effective to prevent spontaneous differentiation of OV stem cells. In another embodiment, the culture medium comprises an antagonist of activin A, and the antagonist is follistatin or an antibody that specifically binds activin A.

In another embodiment, the medium is not supplemented with antioxidants. In another embodiment, the antioxidant is superoxide dismutase, catalase, glutathione, putrescine, or beta-mercaptoethanol. In yet another embodiment, the medium is supplemented with glutathione.

In another embodiment, the adhesive substrate is configured for adhesion to anchorage-dependent cells (anchorage dependent cells) (such as fibroblasts) and optionally collect the cells. In another embodiment, the non-adhesive substrate is an ultra-low adhesion polystyrene surface. In yet another embodiment, the adhesive substrate comprises a surface coated with an RGD tripeptide motif-rich protein.

Also provided is a purified population of OV-CSC cells prepared by any of the methods disclosed herein. In certain embodiments, the OV-CSCs are OV-CSC spheroids, early OV-CSCs, mixed OV-CSCs, or EMT-OV-CSCs.

Also provided is an OV-CSC cell line prepared by any of the methods disclosed herein. In certain embodiments, the OV-CSCs are OV-CSC spheroids, early OV-CSCs, mixed OV-CSCs, or EMT-OV-CSCs.

Also provided is a method of stimulating an immune response against ovarian cancer in a subject in need thereof comprising administering an immunogenic composition disclosed herein, an OV-CSC cell disclosed herein, or an OV-CSC cell disclosed herein Tie.

Also provided is a use of the OV-CSC cell disclosed herein or the OV-CSC cell line disclosed herein in the preparation of a medicament for treating ovarian cancer.

Also provided is a use of the OV-CSC cells disclosed herein or the OV-CSC cell line disclosed herein for the treatment of ovarian cancer.

Description of drawings

Figure 1. Ovarian cancer (OV) cancer stem cells (OV-CSCs) derived from resected tumors (solid boxes and arrows) or from small samples such as needle biopsies or peritoneal lavages (dashed boxes and arrows). ) flow diagram of the process of isolation, amplification, and harvesting into spheroids. Following spheroid generation, the pathway to generate OV-CSC subpopulations is a common one.

Figure 2 depicts an established tumor cell line (OVCAR3) that produced irregular cell conglomerates in non-adherent conditions, thus indicating a higher degree of differentiation (20× difference).

Figure 3 depicts patient-derived ovarian cancer stem cells producing typical spheroids in non-adherent conditions, thus indicating the presence and expansion of cancer stem cells.

Figures 4A-D depict patient-derived ovarian cancer stem cell cultures labeled for cancer stem cell biomarkers EpCAM and NCAM (Figure 4A). Cells were seeded in medium containing 5% serum, 10 ng/mL bFGF, and 10 ng/mL EGF. Figure 4B depicts a red channel image of the cells of Figure 4A, reflecting NCAM positive cells. Figure 4C depicts a green channel image of the cells of Figure 4A, demonstrating the loss of EpCAM in a phenomenon associated with the epithelial to mesenchymal transition (EMT) of the cells. Figure 4D depicts a blue channel image for bisbenzimide nuclear staining.

Figures 5A-D depict patient-derived ovarian stem cell cultures labeled for EMT biomarkers Slug/Snail and CD117 (Figure 5A). Cells were seeded in medium containing 5% serum, 10 ng/mL bFGF and 10 ng/mL EGF. Figure 5B depicts a red channel image indicating that a majority of the cells of Figure 5A are positive for Slug/Snail. Figure 5C depicts a green channel image indicating that the cells of Figure 5A are positive for CD117. Figure 5D depicts the blue channel image used for bisbenzidine nuclear staining.

Figures 6A-C depict patient-derived ovarian cancer stem cell cultures tagged for the cancer stem cell biomarker Nestin (Figure 6A). Cells were seeded in medium containing 5% serum, 10 ng/mL bFGF, and 10 ng/mL EGF. Figure 6B depicts the red channel image, which indicates that most of the cells are positive for Nestin. This phenomenon is associated with EMT. Figure 6C depicts the blue channel image used for bisbenzidine nuclear staining.

Figure 7 depicts patient-derived ovarian cancer stem cell cultures on gelatin in medium containing 5% FBS (phase contrast, 10×).

Figure 8 depicts gelatin containing 5% FBS, 10 ng/mL bFGF and 10 Patient-derived ovarian cancer stem cell cultures in media with ng/mL EGF (phase contrast, 10×).

Figure 9 depicts patient-derived ovarian cancer stem cell cultures on gelatin in serum-free medium (phase contrast, 10×).

Figure 10 depicts patient-derived ovarian cancer stem cell cultures on gelatin in serum-free medium containing 10 ng/mL bFGF and 10 ng/mL EGF (phase contrast, 10×).

Figure 11 depicts in the presence of 10 ng/mL bFGF, 10 ng/mL EGF and 5 Small colonies of patient-derived ovarian cancer stem cells in serum-free medium with ng/mL activin A (phase contrast, 40×). Small cells with large nuclei representing about 90% of the cell size can be observed in dense colonies, indicating very early cancer stem cells (embryonic stem cell-like, "early" OV-CSCs).

Figures 12A-D depict patient-derived ovarian cancer stem cell cultures labeled for ovarian cancer biomarkers CA125 and MUC-1 (Figure 12A). Cells were seeded in a medium containing 10 ng/mL bFGF and 10 ng/mL EGF in serum-free medium. Figure 12B depicts a red channel image of the cells of Figure 12A showing that more than 90% of the cells were positive for CA125. Figure 12C depicts the green channel image of the cells of Figure 12A, indicating that the majority of cells are positive for MUC-1 at various intensities. Figure 12D depicts a blue channel image for bisbenzidine nuclear staining.

Figures 13A-D depict patient-derived ovarian cancer stem cell cultures labeled for the ovarian cancer biomarker CK8 and the proliferation marker Ki67 (Figure 13A). Cells were seeded in a medium containing 10 ng/mL bFGF and 10 ng/mL EGF in serum-free medium. Figure 13B depicts a red channel image of the cells of Figure 13A, demonstrating intensive proliferation (positive for Ki67 labeling). Figure 13C depicts a green channel image of the cells of Figure 13A showing that the majority of cells are positive for CK8. Figure 13D depicts a blue channel image for bisbenzidine nuclear staining.

14A-D depict patient-derived ovarian cancer stem cell cultures labeled for the cancer stem cell biomarkers EpCAM and NCAM. Cells were seeded in 10 ng/mL bFGF and 10 ng/mL EGF in serum-free medium. Figure 14B depicts a red channel image of the cells of Figure 14A showing faint peri-nuclear expression of NCAM in some cells. Figure 14C depicts a green channel image of the cells of Figure 14A, which depicts that the majority of cells are positive for EpCAM and indicates the epithelial nature of the cells. Figure 14D depicts a blue channel image for bisbenzidine nuclear staining.

Figures 15A-D depict patient-derived ovarian cancer stem cell cultures labeled for cancer stem cell biomarkers CD44 and Nestin (Figure 15A). Cells were seeded in 10 ng/mL bFGF and 10 ng/mL EGF in serum-free medium. Figure 15C depicts the green channel image of the cells of Figure 15A, indicating that the majority of cells are positive for CD44. Figure 15B depicts a red channel image of the cells of Figure 15A, indicating some nestin-positive cells predominantly located in the perinuclear region. Figure 15D depicts a blue channel image for bisbenzidine nuclear staining.

Figures 16A-C depict patient-derived ovarian cancer stem cell cultures labeled for the cancer stem cell biomarker CD133 (Figure 16A). Cells were seeded in 10 ng/mL bFGF and 10 ng/mL EGF in serum-free medium. Figure 16B depicts the green channel image of the cells of Figure 16A, indicating that the majority of cells are positive for CD133. Figure 16C depicts a blue channel image for bisbenzidine nuclear staining.

detailed description

The present invention provides a cell population obtained from human ovarian cancer (OV) tumors, the cell population mainly composed of high-purity cancer stem cells. In embodiments, the purity of the cell population is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% cancer stem cells. These cancer stem cells are ovarian cancer progenitors and have the ability to continuously regenerate themselves and differentiate to a certain level. The present invention also relates to a method of producing a purified population of OV-derived stem cells for further use as a source of antigens for autoimmune therapy of cancer.

Testing and screening implementations are also contemplated. The present invention uses highly purified OV stem cell populations for genetic analysis to identify unique changes that drive the formulation of personalized medicines. The present invention provides a novel cell line modified in vitro, wherein the modification enhances the immunostimulatory properties of OV. OV cell lines are an improvement over similar techniques using crude tumor preparations, as they provide superior antigen signal-to-noise ratios. Cell lines lack contaminating cell populations, such as fibroblasts, that could alter or impair in vitro or in vivo applications. Exemplary cell lines of the invention are also useful in the manufacture of medicaments for the treatment of OV.

As used herein, the term "derived from" in the case of a peptide derived from one or more cancer cells encompasses any method of obtaining the peptide from a cancer cell or population of cancer cells. Cancer cells can be destroyed, for example, by a homogenizer or by osmotic disruption, yielding a crude extract. The peptides, oligopeptides, and polypeptides of the crude extract can be exposed to dendritic cells, followed by processing of the peptides by the dendritic cells. The term "derived from" also encompasses intact cancer cells, where the cancer cells are viable, or where the cancer cells have been treated with radiation but are still metabolically active, or where the cancer cells have been treated with a cross-linking agent and thus The peptide is still included. "Derived from" also includes mixtures of cancer cell fragments, free cancer cell proteins, and irradiated cancer cells (which are thus derived from cancer cells). "Derived from" also includes isolating or amplifying messenger RNA from cancer cells or cancer stem cells for ex vivo transfection of dendritic cells to enable antigen presentation.

"Administering" in its application to a human, mammal, mammalian subject, animal, veterinary subject, placebo subject, research subject, experimental subject, cell, tissue, organ or biological fluid refers to (but is not limited to) contacting an exogenous ligand, reagent, placebo, small molecule, medicament, therapeutic agent, diagnostic agent or composition with the subject, cell, tissue, organ or biological fluid, etc. . "Administering" can refer to, for example, therapy, pharmacokinetic, diagnostic, research, placebo, and experimental procedures. Administration can refer to in vivo treatment of a human or animal subject. Cell treatment encompasses contacting a reagent with a cell, as well as contacting a reagent with a fluid, wherein the fluid contacts the cell. "Administering" also encompasses in vitro and ex vivo treatment of a cell by a reagent, diagnostic, binding composition, or by another cell.

An "effective amount" encompasses, but is not limited to, an amount that can ameliorate, reverse, alleviate, prevent or diagnose at least one symptom or sign of a medical condition or disorder. Unless otherwise expressly or dictated by context, an "effective amount" is not limited to the minimum amount sufficient to achieve the desired result, nor is it limited to the optimal amount sufficient to achieve the desired result.

Without any limitation implied, the severity of a disease or condition and the ability of a treatment to prevent, treat or alleviate the disease or condition (to achieve a desired result) can be measured by biomarkers or by clinical parameters. Biomarkers include blood counts; metabolite levels in serum, urine, or cerebrospinal fluid; tumor cell counts; cancer stem cell counts; tumor levels. Tumor levels can be determined by Response Assessment Guidelines for Solid Tumors (Response Evaluation Criteria In Solid Tumors; RECIST) guidelines to determine (Eisenhauer et al. (2009) Eur. J. Cancer 45:228-247). Expression markers encompass genetic expression of mRNA or gene amplification, expression of antigens, and expression of polypeptides. Clinical parameters including progression-free survival survival; PFS), 6-month PFS, disease-free survival (disease-free survival; DFS), time to progress (time to progression; TTP), time to distant metastasis (time to distant metastasis; TDM) and overall survival without any implied limitation.

A "labeled" composition is detectable, directly or indirectly, by spectroscopic, photochemical, biochemical, immunochemical, isotopic or chemical methods. For example, suitable labels include ³² P, ³³ P, ³⁵ S, ¹⁴ C, ³ H, ¹²⁵ I, stable isotopes, epitope-tagging fluorescent dyes, electron-dense reagents, substrates or enzymes, such as in ELISA Those used in the assays, or fluorettes (disclosed in US 6,747,135, the entire disclosure of fluorettes therein is incorporated herein by reference).

Accordingly, disclosed herein is a method for preparing a purified spheroid population of cancer stem cells or a spheroid-derived single cell preparation comprising obtaining an OV biopsy; dissociating the cells of the biopsy; The dissociated cells are cultured in vitro in a defined medium supplemented with at least one growth factor acting through the mitogen-activated protein kinase (MAPK) pathway to obtain OV Populations of purified spheroids or single cell preparations of stem cells. At least about 50%, at least about 60%, at least about 70%, or at least about 80% of the cancer stem cells in the purified OV-CSC population express one or more of the following biomarkers: ATP-binding cassette subfamily G member 2 (ATP -binding cassette sub-family G member 2; ABCG2; GenBank accession number AAG52982.1), CD133, CD24, CD44, CD46, CD117, cytokeratin 18 (CK18), cytokeratin 8 (CK8), alpha-fetoprotein (alpha fetoprotein; AFP), epithelial cell adhesion molecule (EpCAM; GenBank accession number NP_002345.2), Ki-67, Nanog (GenBank accession number NM_024865.2, NP_079141.20), N-cadherin, neural cell adhesion Adjunct molecule (NCAM; CD56), Oct3/4 (Genbank accession number NP_002692.2; NP_976034.4; NP_001167002.1; NP_068812.10), Slug (SNAI2)/Snail (SNAI1) (Slug/Snail), Twist, Vimentin, cancer antigen-125 (CA-125), cell surface associated mucin 1 (mucin 1 cell surface associated; MUC-1), human epididymis protein (He-4), aldehyde dehydrogenase (ALDH), cancer antigen 19-9 (CA19-9), human epidermal growth factor receptor 2 (HER2/neu), nerve Ganglioside CD2, estrogen receptor alpha, testosterone, transforming growth factor beta receptor (TGFβR), Sox2, epidermal growth factor receptor (EGFR), tumor-associated glycoprotein 72 (TAG-72), nestin, and tumor proteins p53 (TP53). OV-CSCs do not express substantially any of the following: carcinoembryonic antigen (CEA), follicle stimulating hormone (FSH), alpha human chorionic gonadotropin (αHCG), beta human chorionic gonadotropin (βHCG), and desmin. As used herein, the term "substantially" refers to a cell or population of cells in which the indicated marker is expressed on less than 20% of the cells. In other embodiments, the biomarker is expressed on less than 15% of the cells, less than 10% of the cells, or less than 5% of the cells. A flow diagram for forming the disclosed cell populations is presented in FIG. 1 .

As used herein, the term "spheroid" refers to spherical aggregates of cancer stem cells formed by culturing cancer cells in serum-free medium. The ability to form spheroids is a hallmark of cancer stem cells.

In certain embodiments, at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the cells in a population of OV-CSC spheroids express two or more of the following biomarkers: EpCAM, CA-125, MUC-1, CD117, He-4, ALDH, CD133, CD24, Ki-67, CA19-9, HER2/neu, NCAM, ganglioside CD2, estrogen receptor alpha, vimentin, CK8 , CK18, AFP, Testosterone, TGFβR, EGFR, TAG-72, CD46, CD44, ABCG2, Slug/Snail, Nestin, and TP53. In other embodiments, at least about 80% of the cells in the OV-CSC spheroid population express two or more of the following biomarkers: AFP, CK7, CK19, EpCAM, E-cadherin, Ov1, and OV6. In another embodiment, at least about 90% of the cells in the OV-CSC spheroid population express two or more of the following biomarkers: EpCAM, CA-125, MUC-1, CD117, He-4, ALDH , CD133, CD24 and Ki-67.

The spheroid population can be further expanded into one of three different subpopulations by varying culture conditions such as media composition and substrates. Bulk tumors, spheroids, early stage, mixed, and EMT The characteristics of the OV-CSC population are presented in Table 1.

Table 1. Summary of Conditions Used to Generate OV Cell Populations from Bulk OV Tumors

*OV = ovarian cancer; **CSC = cancer stem cells; *** EMT = epithelial to mesenchymal transition

Furthermore, as disclosed in Table 1, early OV-CSC, mixed OV-CSC can be obtained from OV-CSC spheroids, early OV-CSC, mixed OV-CSC or EMT-OV-CSC by changing the medium and conditions. or any of the EMT-OV-CSC populations.

In one embodiment, OV-CSC spheroids are further cultured on an adhesive substrate in the presence of activin A, FGF, and serum-free medium (selection medium) to obtain colonies with smaller cells (in Referred to herein as "early" OV-CSC populations), which have characteristics of embryonic stem cells, and at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the cells in the early OV-CSC population express Two or more of the following biomarkers: EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD117, and Ki-67. In another embodiment, at least about 80% of the cells in the early OV-CSC population express two or more of the following biomarkers: EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD117, Ki- 67, CA-125, MUC-1, TGFβR, and CD24. In another embodiment, at least about 90% of the cells in the early OV-CSC population express two or more of the following biomarkers: EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD117, and Ki- 67.

In another embodiment, the OV-CSC spheroids are further cultured on an adhesive substrate under low calcium conditions in serum containing (expansion medium) to obtain colonies mixed with a monolayer in which the cells have uneven shape. The medium optionally includes EGF. These cells are referred to herein as "mixed" OV-CSC populations, which have a mixed differentiation profile, and at least about 50%, at least about 60%, at least about 70%, or at least about 80% of said mixed OV-CSC population cells express two or more of the following biomarkers: EpCAM, CA-125, MUC-1, CD117, CK8, CK18, and Ki-67. In another embodiment, at least about 80% of the cells in the mixed OV-CSC population express two or more of the following biomarkers: EpCAM, CA-125, MUC-1, CD117, CK8, CK18, Ki- 67, CA19-9, HER2/neu, NCAM, ganglioside CD2, estrogen receptor α, testosterone, TGFβR, EGFR, TAG-72, CD46, He-4, ALDH, CD133, CD44, ABCG2, nestin and TP53. In another embodiment, at least about 90% of the cells in the mixed OV-CSC population express two or more of the following biomarkers: EpCAM, CA-125, MUC-1, CD117, CK8, CK18, and Ki- 67.

In yet another embodiment, the OV-CSC spheroids are further cultured on an adhesive substrate in the presence of FGF and serum-containing medium (expansion medium) to obtain single cells of axis-shaped or irregular-shaped cells. layer, which is referred to herein as mesenchymal-like OV-CSC or "EMT-OV-CSC" (cancer stem cells of epithelial to mesenchymal transition [EMT]). In this population, spheroids had undergone an EMT process characterized by loss of expression of at least one or all of the epithelial cell biomarkers CK8, CK18, and EpCAM. As used herein, loss of expression of a biomarker refers to undetectable expression, or expression in 40% (or less) of cells, expression in 30% (or less) of cells, expression in 20% (or less) ), or expressed in 10% (or less) of cells. Additionally, the EMT process is characterized by increased expression of at least one or all of the mesenchymal cell biomarkers Slug/Snail, Twist, CD44, NCAM, N-cadherin, and vimentin to at least 30% in a population expressing the biomarker of interest. %, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the cells.

In one embodiment, at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the cells in the EMT-OV-CSC population express two or more of the following biomarkers: NCAM, Slug /Snail, CD24, and Twist. In yet another embodiment, at least about 80% of the cells in the EMT-OV-CSC population express two or more of the following biomarkers: NCAM, Slug/Snail, CD24, Twist, CA-125, MUC- 1. N-cadherin, CD44, vimentin, CD33, Nanog and CD117. In yet another embodiment, at least about 90% of the cells in the EMT-OV-CSC population express two or more of the following biomarkers: NCAM, Slug/Snail, CD24, and Twist.

In certain embodiments of the population of cells, the cells express one or more of the indicated biomarkers. In other embodiments, the cell expresses two or more, three or more, four or more, five or more, six or more of the indicated biomarkers seven or more, eight or more, nine or more, or ten or more. In yet other embodiments the cells express 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 of the indicated biomarkers , 18, 19, 20, 21, 22, 23, 24 or 25.

Expression of a biomarker by a single cell, a population of cells, or a population of cells located in a specific structure such as a monolayer or spheroid can be determined by measuring the expression of the polypeptide form of the biomarker or the mRNA form of the biomarker. Polypeptide expression can be measured using labeled antibodies, while nucleic acid expression can be measured by hybridization techniques, available to the skilled artisan. Biomarkers that are not polypeptides or nucleic acids, such as oligosaccharides or small molecule metabolites, can also be measured by methods available to the skilled artisan.

Also disclosed herein are methods for obtaining pure isolated OV stem cell populations from ovarian tumor samples of various sizes (1 mg to several grams). Tumor samples can be fresh or frozen, dissociated by mechanical and/or enzymatic treatment, or directly incubated with minimal mechanical fragmentation.

Also disclosed herein, a non-adhesive substrate is any biocompatible material with anti-fouling properties or a coating with anti-fouling properties (reduces accumulation of cells on wetted surfaces) coated on common culture surfaces. coating. Coatings can be applied using coating agents such as aminosilanes. If there is a non-adhesive or anti-bioadhesive substrate, this substrate can be used for about 0-25 days, such as 0-21 days, 5-20 days, 5-10 days, 10-20 days, or between Any time period between zero and 25 days.

In another embodiment of the method using an adhesive substrate, the adhesive substrate may be an RGD (Arg-Gly-Asp) tripeptide motif-rich substrate (e.g., collagen, gelatin, MATRIGEL®). Adhesive substrates are surfaces configured to adhere to and collect anchorage-dependent cells. Furthermore, the substrate may be an adhesive substrate configured to adhere to anchorage-dependent cells that are fibroblasts and collect the cells. RGD peptides can also be grafted onto a polymeric backbone such as polystyrene, hyaluronan, polylactic acid, or combinations thereof. The backbone can further carry proteoglycans. Proteoglycans can carry growth factors such as fibroblast growth factor (FGF), epidermal growth factor (EGF), activin A or follistatin.

Non-adhesive substrates allow rapid and efficient enrichment of cancer stem cells in culture media. Non-adherent substrates can be used when a sufficiently large sample (such as a surgically resected tumor) is provided so that purification of OV-CSCs can begin immediately. If the sample is extremely small (such as needle aspirate or peritoneal lavage) and non-adherent culture is not feasible, then adherent culture can be used for initial expansion followed by a purification step on a non-adherent substrate, This is followed by another amplification under adherent conditions. Alternative treatments are detailed in Figure 1 (dotted lines and boxes) and below.

In certain culture embodiments, a first-phase culture is provided on an adherent substrate, followed by a second-phase culture on a non-adherent substrate. Also provided is a first stage culture on a non-adherent substrate followed by a second stage culture on an adherent substrate. Phases can be for example half day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days Etc or any range thereof like 2-4 days or 8-10 days etc. Alternatively, cycles can be repeated, such as adherent culture, followed by non-adherent culture, followed by adherent culture, etc. In another embodiment, cycles can be repeated, such as non-adherent culture, followed by adherent culture, followed by non-adherent culture, and so on.

In another embodiment, the defined medium is supplemented with at least one growth factor acting via the mitogen-activated protein kinase (MAPK) pathway. In one embodiment, the growth factor is one or both of FGF and EGF, or an analog thereof. In one embodiment, the FGF is basic fibroblast growth factor (bFGF). In another embodiment, the defined medium is supplemented with Activin A. In another embodiment, the defined medium is not supplemented with Activin A. Also disclosed is a defined medium supplemented with an activin A agonist in an amount effective to prevent spontaneous differentiation of OV stem cells. In other embodiments, the defined medium consists of bone morphogenic protein; BMP) 2, 4 or 7, or all supplements.

Also provided is an OV-CSC cell line unique to each patient obtained from a patient's primary ovarian tumor, said cell line (a) carrying the characteristics of self-regenerative and pluripotent stem cells and the ability to differentiate and (b) carry a unique genomic cancer signature in a majority of the cells (eg, greater than 50%).

The invention encompasses nucleic acids, gene products, polypeptides, and peptide fragments, where identity can reasonably be established by trivial name alone. Also encompassed are nucleic acids, gene products, polypeptides, and peptide fragments based on a particular GenBank accession number, wherein said nucleic acids, polypeptides, etc. have at least 50% sequence identity, at least 60% sequence identity, or at least 60% sequence identity to said GenBank accession number. %, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity, wherein the biochemical function or physiological function is at least partially in common or alternatively with the functional irrelevant.

A method is provided wherein an immune response to cancer is stimulated in a subject with one of the compositions disclosed herein. The immune response stimulated comprises one or more of a CD4 ⁺ T cell response, a CD8 ⁺ T cell response, and a B cell response. In certain embodiments, the CD4 ⁺ T cell response, CD ⁺ T cell response, or B cell response can be analyzed by ELISPOT, by intracellular cytokine staining (ICS) analysis, by tetramer analysis, or by analysis according to ordinary skill in the art. It is measured by assays known to the practitioner to detect antigen-specific antibody production. The immune response may comprise survival time, such as 2-year overall survival (OS), and wherein the 2-year overall survival is at least 60%. Immune responses in patients can also be assessed by endpoints used in oncology clinical trials including response on target (RECIST criteria), overall survival, progression-free survival (PFS), disease-free survival, appearance of distant Time of cancer metastasis, 6-month PFS, 12-month PFS, etc.

Also disclosed herein are dendritic cells stimulated ex vivo with OV-CSCs or antigens derived from said OV-CSCs for use in ovarian cancer therapy. Contemplated herein are immunogenic compositions, such as vaccine compositions, comprising dendritic cells ex vivo loaded with (exposed to) OV-CSCs. In certain embodiments, the dendritic cells and tumor cells are from the same human subject (autologous), although embodiments where the dendritic cells and OV cells are from different subjects are also within the scope of the invention.

Dendritic cells can be loaded with OV tumor cell antigens comprising whole cells, cell lysates, cell extracts, irradiated cells or any protein derivatives of OV tumor cells such as OV-CSCs. Dendritic cell immunogenic compositions can be prepared and administered to human subjects by one or more routes of administration as known to those of ordinary skill in the art.

In certain embodiments, OV-CSC cells are irradiated or otherwise treated to prevent cell division prior to loading with dendritic cells. Alternatives to radiation include nucleic acid cross-linking agents that prevent cell division. Also provided is a method of using an OV-CSC population as disclosed above as a source of antigen for autoimmune therapy, e.g., wherein radiated energy (e.g., gamma, UV, X), temperature (e.g., heat or cold) or chemical (e.g., cytostatic, aldehyde, alcohol) methods or a combination thereof to inactivate OV-CSCs. In other embodiments, OV-CSCs are used as a source of antigen for ex vivo activation of dendritic cells.

The present invention provides prepared OV cells (OV-CSC), provides DC loaded with prepared OV cells, and provides an immunogenic composition (or vaccine) comprising dendritic cells loaded with prepared OV cells. Without implying any limitation, the immunogenic composition of the invention may comprise DC loaded with OV spheroids, DC loaded with a cell population comprising spheroids, loaded with spheroids derived and loaded on DC DC loaded with cell populations previously expanded on an adherent surface, DC loaded with spheroids homogenized or sonicated prior to loading on DC, DC loaded with homogenized or sonicated prior to loading on DC The expanded cell population of DC et al. In other embodiments, DCs are loaded with early OV-CSCs, mixed OV-CSCs, or EMT-OV-CSCs.

Also disclosed herein is a population of OV-CSCs capable of stimulating an effective immune response against cells expressing at least one OV-specific antigen, wherein the population of OV-CSCs is contacted with at least one dendritic cell, wherein the OV- The CSC population is treated with said dendritic cells in vivo or ex vivo, and wherein an effective immune response occurs in said subject in response to administration of said at least one dendritic cell to the subject.

The immunostimulatory amount of the disclosed compositions is, but is not limited to, an amount that achieves a measurable increase in ELISPOT assay results, a measurable increase in ICS assay results, a measurable increase in tetramer assay results amount, increases the antigen-specific CD4+ T cell population in the blood by a measurable amount, increases the antigen-specific CD8+ T cell population in the blood by a measurable amount, or wherein increases by at least 10% when compared to a suitable control, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5 times, 2.0 times, 3.0 times, etc. A suitable control may be a control composition in which the dendritic cells are not loaded with OV cells or with peptides derived from OV cells.

The present invention also provides medicaments, reagents and kits (including diagnostic kits), wherein the medicaments, reagents and kits comprise dendritic cells (DC), antibodies or antigens. Also provided are methods for administering a composition comprising at least one dendritic cell and at least one antigen; methods for stimulating antibody formation; methods for stimulating antibody-dependent cellular cytotoxicity methods for stimulating complement-dependent cytotoxicity; and methods for determining patient suitability, for determining patient inclusion or exclusion criteria in clinical trials or general medical treatment settings, and for predicting response to Methods and kits for the reaction of the drug or reagent. The pharmaceutical compositions, reagents and related methods of the invention encompass CD83 positive dendritic cells, wherein CD83 is induced by loading with IFN-γ treated or untreated cancer cells. In the CD83 aspect of the invention, said CD83 is induced by at least 2%, at least 3%, at least 4%, 6%, 7%, 8%, 9%, 10%, etc. On the other hand, DC reagents or DC-associated approaches were excluded in which CD83 on dendritic cells failed to be detectably induced by loading with IFN-γ.

In one embodiment, a kit is provided comprising a method for generating OV-CSC spheroids, early stage OV-CSC, pooled OV-CSC and/or EMT-OV-CSC from a tumor sample according to the methods disclosed herein and/or reagents used to characterize the OV-CSC spheroids, early stage OV-CSCs, mixed OV-CSCs and/or EMT-OV-CSCs; and reagents used to generate and/or characterize the OV-CSCs Instructions for CSC spheroids, early stage OV-CSCs, hybrid OV-CSCs and/or EMT-OV-CSCs. In another embodiment, the kit additionally or alternatively comprises a method for isolating dendritic cells, for loading said dendritic cells with OV-CSCs and/or for administering to a subject a DC-OV combination reagents and instructions.

Tumor Sample Processing

Ovarian cancer (OV) stem cell populations of the invention may be derived from fresh or frozen samples of patient tumors. The tumor sample can be a biopsy or lavage of the peritoneal cavity containing OV cells. Isolation of OV stem cells from needle biopsies and from lavage fluid.

Tumor samples can be shipped in a universal buffered medium such as RPMI, DMEM, F12, Williams, or a combination at a pH of approximately 7.4 (+/- 0.6) that contains protein at a concentration of 0% to 100% The source (such as animal or human serum) either contains albumin at a concentration of 0% to 0.5% or contains a macromolecule that ensures physiological osmolarity. Examples of natural or artificial macromolecules are (but not limited to) hyaluronan, dextran, polyvinyl alcohol. Antibiotics (eg, penicillin, streptomycin, gentramicyn) and antifungals (eg, amphotericin B, FUNGIZONE® (Life Technologies, Carlsbad, CA)) can be used in the medium An optional combination to provide antimicrobial properties and reduce the risk of contamination during transport.

Tumor samples can be kept below metabolic activity by reducing the temperature of the medium to 2°C to 30°C, thus allowing survival to be maintained for a limited period of time (between 0 and 72 hours) prior to treatment. Packaging (eg, insulated packaging) may be used to ensure proper temperature control during transport.

Solid tumor tissue is subsequently processed into smaller (less than 1 mm in any dimension) fragments by mechanical dissociation using a sharp blade or tissue grinder device.

The solid tissue is optionally further processed by enzymatic dissociation. A variety of enzymes can be used to isolate single cells. Non-specific proteolytic enzymes such as trypsin and pepsin can be used successfully. Specific enzymes targeting minimal cell membrane damage, including collagenase, dispase, elastase, hyaluronidase, or combinations thereof, can be used in the disclosed methods. Deoxyribonucleases (DNAse) can be used to degrade free DNA from cellular debris that is responsible for the undesired viscosity of cell preparations. After dissociation, through 50-100 Cells in suspension are washed from excess enzyme and debris by leaching through a μm mesh and repeated centrifugation in buffered saline (PBS, HBSS) or cell culture medium.

Cell culture conditions and spheroid production

The single cell suspensions described above are transferred to culture conditions that promote isolation, expansion and differentiation of stem cells and/or suppression of normal cells. This is achieved through consistency in physical conditions, chemical environment, and manipulation.

The cell suspension is exposed to a non-adherent (anti-bioadhesive) substrate that does not allow the cells to attach. Mature cells are usually anchorage-dependent and are rapidly eliminated when not provided with the correct adherent substrate. Anti-bioadhesive substrates can be commercial products such as ultra-low adhesion flasks (Corning, Corning, NY); polymers with naturally hydrophobic properties (polyvinyl, polyethylene, polypropylene, fluoropolymers); or coatings with natural carbohydrate polymers (such as agar, starch, etc.).

Cancer stem cells will aggregate and/or clonally expand into a spheroid form containing cancer stem cells of high purity. Cancer stem cell aggregate cultures with readily identifiable spheroid structures of various sizes are shown in FIG. 3 . Mature cells will remain detached and non-adherent. Differential gravity separation can be used to select for larger spheroids from single cells by simply allowing timed vertical settling or short, low-force centrifugation (less than 100 x G). The described selection method was designed to achieve the following: (a) elimination of anchorage-dependent cells, typically mature normal cells; (b) promotion of anchorage-independent cloning of small aggregates or spheroids of immature stem cells expansion; (c) promoting local autocrine activity due to clonal expansion of said stem cells; and (d) eliminating autocrine sources of activin A secreted by normal fibroblasts or ovarian cells.

The cells' ability to form spheres is due in part to cell surface proteins called integrins. Homophilic integrins expressed on the cell surface ensure that cells of the same type "keep together". Spheroids are formed directly from enzymatic digests as a single cell suspension at the beginning of culture, or can be formed at any time from frozen samples or existing attached cultures. Seeding with an enzymatic digest produces this spherical form incorporating cells with specific surface properties.

For example, fibroblasts were not incorporated into the spheroids and were removed from the culture during gravity feeding. The medium used lacks adhesion-promoting molecules in order to prevent non-specific aggregation of cells that are not homophilic and to prevent adhesion to the surface of the culture vessel. Such cell adhesion molecules (CAMs) are commonly found in animal or human serum. Therefore, serum-free media compositions are suitable for culturing non-adherent spheroids.

In serum-free medium culture, supplements to the medium may include any hormones, nutrients, minerals and vitamins required to support growth and maintenance or other desired aspects of cell physiology and function. In some examples, stem cell proliferation can be stimulated and maintained by adding or modulating the amount of growth factors with mitogenic activity, such as FGF family and EGF.

Spheroids (spheroids) of cells, including cancer stem cell spheroids, can be characterized for biomarker expression by means of fixation and staining with labeled antibodies, followed by confocal microscopy. Biomarkers can also be measured by other immunochemical methods, such as flow cytometry. Spheroids can be prepared, for example, from suspensions obtained from fresh tumors or from cells adapted to grow as adherent cells. The morphology of the spheroids (eg, large and irregular versus tiny and dense) can be influenced by medium choice.

In another embodiment, the cell population that adheres to the anti-bioadhesive coating can be based on the combination of protein kinase B (AKT) and focal adhesion kinase (focal adhesion). kinase (FAK) signaling mediated sonic hedgehog (sonic hedgehog) signaling abnormal activation to isolate. These phenomena can be enhanced by membrane modifications induced by enzymes like metalloproteases or enzymes for dissociation (trypsin/collagenase). Such cell populations may be associated with rapidly proliferating and invasive tumors. Methods for assessing normal or abnormal activation of sonic hedgehog signaling are available and known to those of ordinary skill in the art.

Media used in cell culture

The defined medium for isolation of OV stem cells promotes cell viability and is specially formulated for selection. The medium is rich in carbohydrates and lipids, but has minimal amounts of protein (0.1%-3% albumin or 1%-5% serum). It contains no more than 1.5 mMol of total calcium and contains no inorganic iron compounds; instead, iron is fully bound to transporters such as transferrin. The medium had an excess of essential and non-essential amino acids as well as essential lipids (α-linolenic acid and linoleic acid) (Table 4). Optionally, the medium does not contain activin A, and may contain an activin A receptor blocker, such as follistatin. Also optionally, the medium does not contain antioxidants, such as superoxide dismutase (SOD) or catalase; but contains thiolic antioxidants, such as glutathione.

In certain embodiments, the medium contains low levels (0.1-1.5 mM) of calcium, such as 10-150 mg/L form of calcium chloride.

As depicted in Table 2, the medium is based on a basic formulation, such as supplemented with protein (in some formulations), amino acids, antioxidants, high-energy substrates (glucose, galactose, L-glutamine), vitamins ( B12), hormones (thyroid hormone, insulin) and growth factors (FGF, EGF) in DMEM, F12, Williams, RPMI, Lebovitz.

The protein may be albumin at a concentration of 0.1%-0.5%, fetal bovine serum (FBS) at a concentration of 0.5%-20%. Proteins can be replaced by macromolecules such as dextran, hyaluronan, polyvinyl alcohol at concentrations ranging from 0.1% to 0.5%. The composition of such media is listed in Table 2, Table 3 and Table 4. Supplements are added to the medium and mixed for feeding the cell culture.

Media can be replaced on a three day weekly schedule (eg, Monday-Wednesday-Friday), or more often, eg, every other day or daily if expansion is rapid. Continuous-feed or micro-batch-feed bioreactors can be used during the expansion phase.

The medium contains growth factors such as FGF and EGF that act via the MAPK pathway. The concentration of these growth factors can vary from 0.1 to 100 ng/mL, usually around 10 ng/mL.

Table 3. Lineage Stem Cell Supplements (50 mL Units for Rehydration in 1 L Basal Medium)

Table 4. Lipid Mixtures

components concentration: µg/mL Linolenic acid 10 Linoleic acid 10 tocopheryl acetate 50 cholesterol 100

Lipid mixtures were made from o/w emulsions using Pluronic F68, Phosphatidylcholine, Tween 80, Cyclodextrin, or combinations thereof.

In one embodiment, the medium contains about 0.1 to 100 ng/mL, about 0.5-50 ng/mL, about 1-40 ng/mL, about 2-30 ng/mL, about 3-20 ng/mL, about 5-15 ng/mL, about 6-14 ng/mL, about 7-13 ng/mL, about 8-12 ng/mL, about 9-11 ng/mL, or about 10 ng/mL supplemented with FGF. In other embodiments, FGF is present at about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 11 ng/mL, about 12 ng/mL, About 12 ng/mL, about 14 ng/mL, or about 15 ng/mL were present in the medium.

In another embodiment, the medium contains about 0.1 to 100 ng/mL, about 0.5-50 ng/mL, about 1-40 ng/mL, about 2-30 ng/mL, about 3-20 ng/mL, about 5-15 ng/mL, about 6-14 ng/mL, about 7-13 ng/mL, about 8-12 ng/mL, about 9-11 ng/mL, or about 10 ng/mL supplemented with EGF. In other embodiments, EGF is present at about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 11 ng/mL, about 12 ng/mL, About 12 ng/mL, about 14 ng/mL, or about 15 ng/mL were present in the medium.

Also provided are media not supplemented with either or both of superoxide dismutase (SOD) or catalase. Using antioxidants can have positive or negative consequences. Cancer stem cells are much more tolerant to free radicals and sugar catabolism than normal cells. Therefore, in suboptimal media (e.g., high density, infrequent media replacement, high concentrations of metabolites in the media), normal sensitive cells are most likely to be eliminated first. By not including antioxidants in the culture medium, it is possible to select for a population of cells that are more likely to be derived from cancerous tumors than normal cells that are more resistant. Thus, in certain embodiments, antioxidants, such as catalase and SOD inhibitors, are added to the medium, and in other embodiments, these compounds are omitted from the medium.

In an alternative approach, activin/follistatin can be used to isolate very early stage cancer stem cells. Addition of Activin A can select for Activin A-resistant OV stem cell subsets. Follistatin is used to block activin A receptors and prevent spontaneous differentiation of OV stem cells, especially when there are large numbers of endogenous activin A-secreting cells such as fibroblasts and normal ovarian cells. The use of follistatin has no effect if the cells are insensitive to activin A or are in a highly purified OV stem cell population that can secrete follistatin endogenously.

Activin A is a protein that is itself a member of the transforming growth factor-beta (TGF-beta) superfamily. When added or included in the culture medium, activins help maintain stem cell pluripotency and self-regeneration. However, activin A promotes the maturation and differentiation of susceptible immature ovarian and cancer cells. Therefore, the initial goal was to rapidly expand tumors in vitro by creating the correct autocrine environment in culture, which also sustains the proliferation of cancer stem cells. Although activin A may select for a subpopulation of very immature cancer stem cells, such conditions applied early in manufacturing would greatly delay expansion given the very low concentration of liver cancer stem cells overall. For example, "rapid expansion" is an expansion that results in significantly higher per day numbers of media and cells (reflected by increased confluency) in the culture vessel with obvious signs of depletion (eg, changes in pH).

For rapid expansion, Activin A is preferably omitted and not added, as it will slow down the growth of the culture. For some applications, it is of interest to obtain a population of very early stem cells, and the use of Activin A will select for that population. Accordingly, in one embodiment, activin A-containing expansion is induced and a first composition comprising cultured cells activated by activin A is administered to a subject, followed by isolation of the pair in activin A-free cultures. activin A-insensitive cells, and administering this second composition comprising activin A-free cultured cells to the subject.

In one embodiment, the medium contains about 0.01 to 10 ng/mL, about 0.05-9 ng/mL, about 0.1-8 ng/mL, about 0.5-7 ng/mL, about 1-6 ng/mL, about 1-5 ng/mL supplemented with Activin A. In other embodiments, activin A is present at about 0.5 ng/mL, about 0.7 ng/mL, about 0.9 ng/mL, about 1 ng/mL, about 1.25 ng/mL, about 1.5 ng/mL, about 1.75 ng/mL, about 2 ng/mL, about 2.25 ng/mL, about 2.5 ng/mL, about 2.75 ng/mL, about 3 ng/mL, about 3.5 ng/mL, about 4 ng/mL, about 4.5 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL or about 10 ng/mL present in the medium.

Embodiments are also disclosed wherein the culture medium is supplemented with an activin A antagonist such as, but not limited to, follistatin or an antibody that specifically binds activin A.

In another embodiment, the medium contains about 0.1 to 100 ng/mL, about 0.5-50 ng/mL, about 1-40 ng/mL, about 2-30 ng/mL, about 3-20 ng/mL, about 5-15 ng/mL, about 6-14 ng/mL, about 7-13 ng/mL, about 8-12 ng/mL, about 9-11 ng/mL, or about 10 ng/mL supplemented with follistatin. In other embodiments, follistatin is present at about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 11 ng/mL, about 12 ng/mL mL, about 12 ng/mL, about 14 ng/mL, or about 15 ng/mL is present in the medium.

The combination of mitogens (eg, FGF/EGF), activin A, and adhesion substrates can cause increased proliferation of normal cells such as fibroblasts or stellate cells. Thus, generation of very early OV stem or progenitor cells that are insensitive to activin A in an enriched environment or "stroma" composed of cells with trophoblast or enveloping properties (e.g., fibroblasts, stellate cells) Conditions for cell expansion. It was gradually observed that during subsequent days to weeks of cell culture, OV colonies develop along the matrix and spatially replace it. The medium used in this method was a combination of formulations described in Tables 2, 3 and 4.

There is a relationship between FGF, EGF and Activin A and "very early stage" OV cancer stem cells. FGF and EGF cause proliferation of OV stem cells, including very early stage OV stem cells, in any state of differentiation. Activin A allows (allows) very early stage OV stem cells that are insensitive to Activin A to proliferate exclusively in the presence of Activin A in the cell culture medium. If OV stem cells become sensitized, proliferation will be stopped or reduced by Activin A.

Insensitivity to FGF and EGF is uncommon, and no natural blockers exist. Insensitivity to activin A may be mediated by follistatin, a natural blocker of activin receptors. Follistatin can be secreted by the same tumor cells or by cells surrounding the tumor. Activin A is normally secreted by cells surrounding tumors, so tumor expansion likely depends on surrounding cells (inhibition) and the tumor (promotion of expansion). Lack of receptors for activin A, a hallmark of very early undifferentiated cancer stem cells, prevents surrounding tissue from taking hold of the tumor.

The in vitro culture medium will contain embryonic stem cell-like colonies. These colonies may be surrounded by stromal cells, which may be normal fibroblasts, differentiated tumor cells, or mesenchymal transformed tumor cells.

The present invention provides methods for preparing OV-CSCs, wherein the total culture time (including the time required for manipulations such as changing media, reseeding, centrifugation, and settling) is less than five months, less than four months, less than three months month, less than two months, less than one month, less than 150 days, less than 120 days, less than 90 days, less than 60 days, less than 30 days or less than 150 days (+/-20 days), less than 120 days (+/-20 days days), less than 90 days (+/-20 days), less than 60 days (+/-20 days), less than 30 days (+/-20 days). In an exclusive embodiment, the invention may exclude any method for producing cancer stem cells and any population of cancer stem cells produced by said method, wherein the time required for manipulation is greater than the time-frame disclosed above . The time in adherent culture indicated by one of the time periods above is also provided. The time in non-adherent culture, itself one of the above time periods, is also provided. In addition, the combined time in adherent culture and in non-adherent culture identified by one of the time periods above is provided.

Epithelial to mesenchymal transition ( EMT )

Tumors derived from epithelial cells are known to regress or dedifferentiate to a mesenchymal state. The epithelial cell phenotype is immobile, contributes to volumetric growth of tumors confined to the tissue of origin, and is generally more differentiated. When EMT occurs, cells gain mobility and produce adjacent tissue invasion and distant cancer metastasis. Transformed cells also acquire a stem cell-like phenotype, with the ability to replicate and differentiate to give rise to new tumors (metastases) in host tissues with characteristics of the tumor of origin (primary). Through EMT, tumor cells acquire additional immunosuppressive capacity, drug pumping, and radiation resistance.

Media compositions and physical selection methods promote the phenomenon of EMT in vitro. An advantage of using an EMT-transformed population as an immunogen is the prevention of tumor recurrence. The antigenicity of EMT cancer cells may enable the immune system to recognize and destroy mobile cancer cells that cause metastasis. During cancer metastasis, these cells migrate in very low numbers, inoculate host tissue, revert to an epithelial phenotype (MTE transition), grow and form new tumors with features similar to the primary tumor. The conditions necessary to cause EMT in vitro are spheroid formation in serum-free medium, stimulation with bFGF, stimulation with BMP2, 4, or 7, and subsequent seeding on cells containing the RGD (Arg-Gly-Asp) peptide motif (e.g., collagen , gelatin, etc.) on adhesive substrates.

EMT-OV-CSC subsets were obtained by culturing OV spheroids or early OVs or mixed OVs under the culture conditions as described in Table 1 and Figure 1.

As used herein, the term "OV-CSC" may generally refer to OV-CSC spheroids, early OV-CSC, mixed OV-CSC or EMT-OV-CSC.

Obtained from small tumor sources OV-CSC

Alternative methods of OV stem cell selection are used when sample cell numbers are low. For example, the few viable cells obtained from a tumor after enzymatic dissociation are less than ¹⁰ x 106 viable cells. For the purposes of the present invention, small samples are those obtained from, for example, needle biopsies or peritoneal lavages, in contrast to samples obtained from resected tumors (which are generally not considered small samples and weigh at least 0.5 to 5-10 grams). samples obtained from the material. Core biopsies were performed with 18- or 16- or 14-gauge needles, yielding 5-50 mg samples. A relatively new procedure called vacuum-assisted biopsy is also performed with an 11-gauge needle and a vacuum assisted device (VAD). An 11 gauge probe paired with a vacuum assist typically yields 94 mg of each core sample. A 14 gauge needle with vacuum assist usually yields 37 mg, but only 17 mg when paired with an automated biopsy gun. In this alternative approach depicted in Figure 1 (dotted arrows and boxes), cells obtained from tumor samples are transferred to RGD-enriched (Arg-Gly-Asp)-containing compounds before or after dissociation. (e.g., collagen, gelatin, or MATRIGEL®) in the presence of selective (serum-free) medium as described herein. The described selection method was designed to (a) promote the initial clonal expansion of individual cancer stem cells present in lower numbers and (d) promote local autocrine activity due to the clonal expansion of said stem cells.

Adhesion substrates are RGD-rich proteins such as collagen or gelatin. Substrates can be constructed by attaching the protein or peptide to various materials such as polystyrene polycarbonate, cycloolefin copolymers, or glass. RGD peptides can be grafted onto polymeric backbones such as hyaluronic acid, polylactic acid, and combinations. Such polymers can be further enhanced with carrier ends (eg, heparan sulfate, chondroitin sulfate, keratin sulfate, etc.) of growth factors such as proteoglycans.

Cell culture surfaces can be applied directly or with coating agents such as aminosilanes. A coating is a compound that has adhesive properties (substrate) for cells and is applied on top of the growth vessel material. It can be a natural compound such as collagen or gelatin, and can also be built from more synthetic polymers with the groups/terminals mentioned. Coating agents (glues, such as silanes) can be used to improve the adhesion of the coating to culture vessel materials such as glass. Silanes can be used alone if they contain desired groups or end groups.

Using this method and formulation, large numbers of cells can be obtained in a relatively short period of time. Starting with a few milligrams, cultures of tissue samples such as needle biopsies containing 10 ³ to 10 ⁶ cells can be expanded to approximately 10 ⁸ cells in 3-4 weeks.

Expansion of OV stem cell cultures and generation of subpopulations

As another characteristic of stem cells, OV-CSCs can reproduce and expand indefinitely.

Furthermore, OV-CSCs can be partially or fully differentiated. If stem cell expansion conditions are removed, OV stem cells can slow or cease proliferation and change morphology and phenotype to a more differentiated cell type. The morphology can become flattened, epithelioid, or stellate with multiple nuclei, which is characteristic of more mature ovarian cells or stellate cells.

Adherent cultures can be dissociated in single cell suspensions and transferred to non-adherent (anti-bioadhesion) conditions to remove anchorage-dependent differentiated cells. After 2-3 days, stem cells tend to aggregate and expand clonally into small spheroids that can be separated from single cells based on differential sedimentation. Spheroids can be reseeded in adherent conditions and propagated further. If the culture is populated with differentiated cells or normal cells such as fibroblasts, this method will purify the culture from 1%-30% stem cell content to 90%-99% stem cell content. The method can be repeated as many times as necessary to restore the purity of the stem cells.

The size of the smaller spheroids is generally between 0.1 mm and 2 mm. The size distribution is generally between 10 cells and 10,000 cells in terms of the number of cells per smaller spheroid. Smaller spheroids can be spherical or ovoid in shape and can also occur as agglomerates of spherical or ovoid structures.

Patient-specific OV-CSC cell lines can be used to identify genomic mutations responsible for neoplastic transformation when compared to normal tissue from the same patient. Genomic mutations may not be expressed at every stage of differentiation. Some regulatory proteins, or transcription factors, are only transiently expressed and can disappear during maturation, resulting in cells that are deformed but have normal proteins. Identifying the cell population that maximally expresses the mutation and exposing this population to the immune system can be a major advantage of using cancer stem cells as a source of antigen for immunotherapy.

By identifying genomic mutations, personalized agents for cancer therapy can be generated, such as small molecules, DNA sequences, antisense RNA, or combinations.

Such cell lines can further be used to generate screening plates (eg 96 wells) for drug discovery. Multiple lineages from various patients can be combined in a single culture plate to account for inter-subject variability.

Ovarian cancer stem cells can maintain some properties of the tissue of origin, such as secreting proteins, growth factors, and hormones (functional tumor). Given the immortality of cell lines, these properties can be exploited to produce "own" proteins (e.g. albumin, transforming growth factor (TGF), insulin, glucagon, DOPA, etc.) that can be used in the same patient. Cells can be introduced into small bioreactors and secreted products collected, purified and stored for use on the same patient. This approach is particularly advantageous in that patients will not develop immune resistance as with more traditional biosimilars.

loaded dendritic cells

Individual OV-CSC cells obtained from patients can be used to generate antigens for immunotherapy. The advantage of using purified stem cell lines is the better signal to noise ratio. More mature cells from tumors may have compensatory mechanisms that can mask antigenicity and may not be recognized by the immune system. As an antigen source, OV-CSCs can be used in a live form, a mitotically inactive form, an inactive form or a fragmented form. Various methods can be used to modify cells for optimal antigen exposure: radiative energy (eg, gamma, UV, X), temperature (eg, heat or cold), or chemical (eg, cytostatic, aldehyde, alcohol) or a combination.

In exemplary embodiments, the present invention encompasses reagents and methods for activating dendritic cells (DCs) using one or more immune adjuvants such as GM-CSF; toll-like receptor (TLR) agonists , such as CpG-oligonucleotides (TLR9), imiquimod (TLR7), poly(I:C) (TLR3), glucopyranosyl lipid A (TLR4), murein (TLR2), flagella protein (TLR5); and adjuvants such as CD40 agonists such as CD40 ligands; or cytokines, interferon-γ, prostaglandin E2, etc. See eg US Patent 7,993,659; US 7,993,648; US 7,935,804, each of which is incorporated herein by reference in its entirety for the disclosure regarding activated DC. The present invention contemplates ex vivo treatment of DCs with one or more of the above adjuvant agents, or additionally or alternatively, administration of said adjuvant to a human, animal or veterinary subject.

The immune system encompasses cellular immunity, humoral immunity, and complement responses. Cellular immunity includes a network of cells and events involving dendritic cells, CD8 ⁺ T cells (cytotoxic T cells; cytotoxic lymphocytes), and CD4 ⁺ T cells (helper T cells). Dendritic cells (DCs) acquire polypeptide antigens, where these antigens can be acquired from the outside of the DC or biosynthesized inside the DC by infectious organisms. DCs process polypeptides, produce peptides about ten amino acids in length, transfer the peptides to MHC class I or MHC class II to form complexes, and shuttle the complexes to the surface of the DCs. When DCs bearing MHC class I/peptide complexes contact CD8 ⁺ T cells, the result is activation and proliferation of said CD8 ⁺ T cells. Regarding the role of MHC class II, when DCs bearing MHC class II/peptide complexes contact CD4 ⁺ T cells, the result is activation and proliferation of said CD4 ⁺ T cells. Although dendritic cells presenting antigens to T cells can "activate" said T cells, activated T cells may not be able to mount an effective immune response. An effective immune response by CD8 ⁺ T cells often requires pre-stimulation of DCs by one or more of a variety of interactions. These interactions include direct contact of CD4 ⁺ T cells with DCs (by means of contacting the CD40 ligand of said CD4 ⁺ T cells with the DC CD40 receptor) or direct contact of TLR agonists with toll-like receptors (TLRs) of dendritic cells. )one.

Humoral immunity refers to B cells and antibodies. B cells become transformed into plasma cells, and the plasma cells express and secrete antibodies. naïve B cells B cells) differ in that they do not express the marker CD27, whereas antigen-specific B cells express CD27. The secreted antibodies can then bind to tumor antigens residing on the surface of tumor cells. The result is that infected cells, or tumor cells, become tagged with antibodies. Where the antibody binds to an infected or tumor cell, the bound antibody mediates killing of the infected or tumor cell, wherein the killing is by NK cells. Although NK cells are not configured to recognize a specific target antigen in the manner that T cells are configured to recognize a target antigen, the ability of NK cells to bind to the constant region of an antibody enables NK cells to specifically kill labeled Antibody cells. Recognition of antibodies by NK cells is mediated by Fc receptors (of NK cells) that bind to the Fc portion of the antibody. This type of killing is known as antibody-dependent cellular cytotoxicity (ADCC). NK cells can also kill cells independent of ADCC mechanisms, where such killing requires absence or insufficient expression of MHC class I in target cells.

Without wishing to be bound by any particular mechanism, the invention encompasses administration of cancer stem cell antigens or administration of dendritic cells loaded with cancer stem cell antigens, wherein said antigenic stimulus specifically recognizes one or more of said cancer stem cell antigens production of antibodies of the individual, and wherein the antibodies mediate ADCC. The phrase antigen-loaded refers to the ability of dendritic cells to capture live cells, capture necrotic cells, capture dead cells, capture polypeptides or capture peptides, and the like. Tumor antigens include OC-CSC cell extracts, OC-CSC lysates or intact OC-CSC cells. In another embodiment, the tumor antigen comprises messenger RNA transfected into dendritic cells ex vivo.

The present invention encompasses capture by cross-presentation. Also contemplated is the use of antigen presenting cells other than dendritic cells, such as macrophages or B cells.

The technique of "delayed hypersensitivity" can be used to distinguish between immune responses that are predominantly cellular or predominantly humoral. A positive signal in delayed-type hypersensitivity indicates a cellular response.

The invention provides compositions and methods for inactivating tumor cells, for example, by radiation, nucleic acid cross-linking agents, polypeptide linkers, or combinations of these. Crosslinking is the attachment of two chains of a polymer molecule by a bridge made up of elements, groups or compounds that join certain carbon atoms in the chains by primary chemical bonds. Crosslinking occurs in nature in substances consisting of polypeptide chains joined by disulfide bonds involving two cysteine residues, as in keratin or insulin, the trivalent pyridinoline and pyrrole of mature collagen Cross-linking, and cross-linking in blood clots, which involves covalent ε-(γ-glutamyl groups between the γ-carboxy-amine groups of glutamine residues and the ε-amino groups of lysine residues ) lysine cross-linking.

Cross-linking can be achieved artificially in proteins, by adding chemical substances (cross-linking agents) or by exposing the polymer to high-energy radiation. Cross-linking with fixatives and heat-induced aggregation has been shown to enhance immune responses and completely inhibit proliferation. Substances that can be used to cross-link proteins on the OV-CSC surface and thus prevent OV-CSC proliferation include, but are not limited to, 10% neutral buffered formalin, 4% paraformaldehyde, 1% glutaraldehyde , 0.25-5 mM dimethyl suberimide (dimethyl suberimidate), ice-cold 100% acetone or 100% methanol. Alternatively, a combination of 1% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffered saline can also be used.

Both formaldehyde and glutaraldehyde have been shown to induce the activation of type 1 and type 2 T helper cells. Specifically, it was also shown that heat-induced antigen aggregation enhances the in vivo priming of cytotoxic T lymphocytes. Antigen cross-linking by 3,3'-dithiobis(sulfosuccinimidyl propionate) causes increased binding of antigen to dendritic cells, and the cross-linked antigen passes through the proteasomal pathway for antigen presentation deal with. In addition, formalin-fixed ovarian cancer tumor cells have been used in clinical trials without evidence of proliferation.

In one embodiment, whole OV-CSCs are immobilized with a cross-linking agent and then used as a source of antigen in combination with dendritic cells.

In another embodiment, the nucleic acids of the cells are crosslinked. An exemplary nucleic acid alkylating agent is β-alanine, N-(acridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester. Exemplary cross-linking agents such as psoralen, often combined with ultraviolet (UVA) radiation, have the ability to cross-link DNA but leave proteins unmodified. For example, the nucleic acid targeting compound may be 4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen (S-59). Cells can be inactivated with 150 µM psoralen S-59 and 3 J/cm ² UVA light (FX 1019 Radiation Unit, Baxter Fenwal, Round Lake, IL). Inactivation using S-59 with UV light is known as photochemical treatment, where treatment conditions can be adjusted or titrated such that cross-linked DNA completely prevents cell division but wherein damage to polypeptides (including polypeptide antigens) is minimized degree. Cells can be suspended in 5 mL of saline containing 0, 1, 10, 100, and 1000 nM psoralen S-59. Samples may be subjected to UVA radiation at a dose of approximately ² J/cm2. Each sample can then be transferred to a 15 mL test tube, centrifuged and the supernatant removed, and then washed with 5 mL of saline, centrifuged and the supernatant removed, and the final pellet suspended in 0.5 mL of saline. See US Patent Nos. 7,833,775 and 7,691,393, the entire disclosures regarding cell inactivation are incorporated herein by reference.

For any cell preparation treated with a cross-linking agent, the ability to divide can be determined by a skilled artisan by incubating or culturing in a standard medium for at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least two months , at least three months, at least four months, etc. to test. Cell division can be assessed by staining that reveals chromosomes and reveals whether cell division has occurred. Cell division can also be measured by counting cells. Therefore, where the number of cells in a culture plate remains stable over a period of two weeks, one month, two months, etc., it is reasonable to conclude that the cells are unable to divide.

In one embodiment, the dendritic cell immunogenic composition is administered subcutaneously (SC). In other embodiments, each dose is in the range of about 5-20 million loaded DCs, repeated in a series of 6-10 doses. In certain embodiments, doses are administered every five days, every week, every 10 days, every other week, or every other two weeks for two, three, four, five, or six doses, followed by every two weeks, every three weeks , doses are administered every four weeks, every month, every five weeks, or every six weeks, followed by additional doses of two, three, four, five, or six doses, for a total of 6-10 doses. In one embodiment, the first four injections are given weekly for one month, and the subsequent 4 injections are given once a month thereafter. In an alternative embodiment, dosing is weekly for 3 weeks, followed by monthly for 5 months, for a total of 8 dosings.

Each dose contains about 5-20 × 10 ⁶ loaded DC, about 5-17 × 10 ⁶ loaded DC, about 6-16 × 10 ⁶ loaded DC, about 7-15 × 10 ⁶ loaded DC DC, about 7-14×10 ⁶ loaded DC, about 8-13×10 ⁶ loaded DC, about 8-12×10 ⁶ loaded DC, or about 9-11×10 ⁶ loaded DC. In additional embodiments, each dose comprises about ⁸ x 106 loaded DC, about ⁹ x 106 loaded DC, about ¹⁰ x 106 loaded DC, about ¹¹ x 106 loaded DC Or about 12 x 10 ⁶ load DC. Loaded DCs comprise a mixture of DCs and residual OV-CSCs not absorbed by the DCs. The dose administered contains a mixture of these cells and the dose reflects this mixture.

In another embodiment, the loaded DCs are administered with a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable excipients (eg vehicles, adjuvants, carriers or diluents) described herein are well known to those skilled in the art and are readily available to the public. Preferably, the pharmaceutically acceptable carrier or excipient is a carrier or excipient that is chemically inert to the loaded DC, and that has no harmful side effects or toxicity under the conditions of use.

The choice of excipient or carrier will be determined in part by the particular therapeutic composition and by the particular method used to administer the composition. The formulations described herein are exemplary only, and in no way limiting.

Physiologically acceptable carriers are often aqueous pH buffered solutions. Examples of physiologically acceptable carriers include, but are not limited to, saline; solvents; dispersion media; cell culture media; aqueous buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight ( less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

In some exemplary embodiments, an adjuvant is administered with each dose. In certain embodiments, the dose of cells is suspended in a carrier containing an adjuvant. In an alternative exemplary embodiment, an adjuvant is administered but not with each individual dose. In other exemplary embodiments, no adjuvant is present at all. In one embodiment, the adjuvant is GM-CSF.

Without limitation, dendritic cells (e.g., autologous or allogeneic dendritic cells) in the form of cell lysates, acid eluates, cell extracts, partially purified antigen, purified antigen, The cancer stem cell antigen is contacted in the form of isolated antigen, partially purified peptide, purified peptide, isolated peptide, synthetic peptide, or any combination thereof. The dendritic cells are then administered to a subject, eg, a subject with OV or a control subject without OV. In an exemplary embodiment, dendritic cells are contacted, injected into, or administered to a subject by one or more of the following routes: subcutaneous, intraperitoneal, intranodal, Intramuscular, intravenous, intranasal, by inhalation, orally, by administration into the intestinal lumen, and the like. Alternatively, the immunogenic composition can be administered directly to the site of tumor or cancer metastasis.

Example

Example 1. Generation of cancer stem cell lines from ovarian tumor samples

Ovarian tumor samples from 3 patients were obtained during the surgical procedure after informed consent was properly obtained. Samples were transported to the tissue processing facility in antibiotic-containing medium and maintained at a temperature of 4°C-10°C during transport.

Samples were minced with a scalpel blade in a sterile Petri dish (Petri dish), and the fragments were subsequently exposed under continuous agitation to a solution containing collagenase-I (3000 UI to 6000 Ul per gram of tissue depending on tissue consistency). UI) solution.

Dissociated cells were washed in culture medium, counted and frozen in aliquots for later use in RPMI medium containing 15% FBS and 10% DMSO.

A typical aliquot contains 10-30 x 10 ⁶ cells and the expected viability upon thawing is 40% to 75%. Aliquots can be used for later purification and expansion of cancer stem cells.

Example 2. for OV-CSC Optimal Media Composition for Isolation and Expansion

Three commercially available ovarian cancer cell lines were compared to cryopreserved tumor cells dissociated from 3 different samples obtained from ovarian cancer patients.

The spherogenic properties of cancer stem cells are used to isolate cancer stem cells. Two different media consisting of a literature-described composition (Lit-M) and a proprietary formulation (CSC-M) as shown in Table 5 were used to assess the spheroidizing properties of the tumor lineages.

OVCAR-3, SKOV-3, and TOV-21G commercial cell lines as well as enzymatically digested tumors from patients were thawed from the mother cell bank. After enumeration and viability assessment, 50,000 viable cells per square centimeter from each lineage were seeded in appropriately labeled ultra-low adhesion flasks (Corning).

Cultures were grown for 7 days, and medium was replaced every two days or on a Monday-Wednesday-Friday schedule. To feed the cultures, the cell suspension was collected in 50 mL tubes, centrifuged at 150 rcf for 3 minutes, and the supernatant was replaced with fresh CSC-M or Lit-M medium.

After 7 days, the cells were transferred to normal cell culture flasks, and the medium was changed to Lit-E and CSC-E (expansion) formulations (Table 5). CSC-E medium is an expansion medium and contains serum; CSC-M is a serum-free selection medium. Both CSC-E and CSC-M are basal media based on Tables 2-4. Spheroids were first dissociated with a collagenase/hyaluronidase mixture for 5-10 minutes and gently pipetted, followed by centrifugation to remove enzyme and replace with medium. Cells were expanded for 2 passages following the M-W-F feeding schedule, each reseeded at a density of 10,000 cells/cm2. Cells were finally transferred to 96-well culture plates at a density of 5,000-10,000 cells/well for immunocytochemical analysis. After 2 days, plates were fixed with 4% paraformaldehyde and stained for the following markers: Nanog, Nestin, Sox2, CD133, CD117, NCAM, EpCAM, Slug/Snail, CD24, CA-125, ALDH, CD46 , CEA, He-4, Muc-1, CD44, and HER-2.

Table 5. Medium formulations used in OV-CSC purification and expansion

At the end of 7 days of growth in ultra-low adhesion conditions, none of the commercial cell lines formed typical cancer stem cell spheroids in Lit-M or CSC-M medium. Instead, it formed loose cell conglomerates with irregular shapes (Fig. 2). The patient's tumor formed a typical dense spherical structure that reconstituted a stem cell spheroid that gradually expanded over a 7-day period (Fig. 3).

After transferring the cells to adherent conditions, the culture expanded slowly and reconstituted into a monolayer of epithelial cells. Some cells showed a rapidly growing small cuboidal cell phenotype, while some formed large epithelial cells with vacuolated cytoplasm or small spindle-shaped reconstituted mesenchymal cells. Commercial cell lines expanded faster in CSC-E medium compared to Lit-E conditions. Patient lineages expanded more slowly in the same conditions and were impaired at passaging, exhibiting cytoplasmic vacuoles, detachment from substrate, and massive cell death.

In this experiment, we found that commercial cell lines have a more differentiated and homogeneous phenotype suitable for high concentrations of FBS (15%), but may not contain cancer stem cells, as indicated by the lack of typical spheroid-forming properties. Instead, patient samples contained enough cancer stem cells to produce the expected dense spheroids. In the case of transfer to media containing 15% serum, patient lineages were compromised compared to commercial lineages and it was not possible to perform correct immunocytochemical characterization immediately after spheroid inoculation.

Table 6. Summary of ICC scores for patient-derived ovarian cancer cell lines

Reactivity % Positive Rating: += 1%-25%, ++ = 26%-50%, +++ = 51%-75%, ++++ = 76%-100%. Intensity Rating: 1 = Mild, 2 = Moderate, 3 = High.

As indicated by the ICC results (Table 6), cultures exhibited an EMT phenotype (epithelial to mesenchymal transition): low EpCAM, high NCAM (Fig. 4A-D), Slug/Snail (Slg/Snl) , CD117 (Fig. 5A-D) and nestin (Fig. 6A-C).

In summary, 1) commercial cell lines are not equivalent to fresh patient samples in terms of CSC content; 2) OV-CSCs are sensitive to media composition and physical manipulation and are not resistant to passage in the conditions tested; 3) proprietary cultures basal preparations (CSC-M and CSC-E) favored the growth of OV-CSCs; and 4) the tested culture conditions favored EMT.

Example 3 : Single Seed Expansion of Tumor Cells

Typical in vitro cell culture is a cyclic process in which cells are seeded at a lower density, allowed to grow until reaching confluence, then dissociated and seeded again at a lower density on a larger surface (passaging). Our previous observations led to the conclusion that OV-CSCs are sensitive to physical and enzymatic manipulation during passaging as manifested by differentiation and massive cell death and epithelial to mesenchymal transition (EMT). To address this phenomenon, in subsequent experiments, we tested the possibility of single-passage expansion of OV-CSCs to provide active specific immunotherapy (active specific immunotherapy; ASI) the cells are seeded on the final surface at a low density of the desired total cell number. In common cell culture conditions, single-shot expansion is avoided to save culture space in the incubator and cell proliferation is stimulated by periodic enzymatic dissociation and avoidance of contact inhibition.

In this method, cells are seeded at various low densities of 5000, 10,000, and 15,000 cells/cm2 and allowed to expand for 4 days. Cultures were fed on a Monday-Wednesday-Friday feeding schedule with CSC-E medium (Table 5) containing only 5% instead of 15% FBS in the initial formulation. At the end, cells were counted and analyzed for their phenotype.

Cell counts in the various conditions did not exhibit significant differences in doubling time. Immuno-cytochemistry (ICC) revealed the same profile regardless of the initial seeding density (Table 7).

Table 7. Overview of ICC Scoring

Reactivity % Positive Rating: += 1%-25%, ++ = 26%-50%, +++ = 51%-75%, ++++ = 76%-100%

Intensity Rating: 1 = Mild, 2 = Moderate, 3 = High.

To avoid cell loss or differentiation by mechanical or enzymatic dissociation, a method can be used to expand the cells to the desired number by initially seeding at low density and allowing the culture to grow until it reaches Confluency was performed without repeated passaging. Additionally, as in previous experiments (Table 6) at higher concentrations (15%) of serum, media containing 5% FBS caused EMT according to the ICC profile (Table 7).

Example 4. Determining cell culture conditions for selection of early stage ovarian cancer stem cells

As shown in previous experiments, FBS-containing media caused differentiation and EMT of cancer stem cells. This phenomenon can be caused by factors contained in serum, such as bone morphogenetic protein (BMP4). In cultures containing FBS, or by addition of BMP4, cells appeared larger, with an epithelial appearance and shorter doubling rate typical for well differentiated carcinomas. If growth factors are added to this medium, EMT is observed. In vivo, ovarian cancer expands almost exclusively in the peritoneal cavity, where abundant growth factors secreted by peritoneal cells can accelerate tumor expansion. Such growth factors include FGF, EGF, VEGF, HGF, PDGF, Activin A, and TGF[beta].

Subsequent experiments investigated the medium and substrate compositions that allow rapid expansion of ovarian cancer stem cells.

Patient-derived ovarian tumor cells were seeded on plain tissue culture plastic, 1% gelatin, or MATRIGEL ^® (1:60). For each substrate condition, various media formulations were used: CSC-E modified with 5% FBS; CSC-M (Table 5); commercial medium KGM-GOLD™ formulated for epithelial cells (keratocytes) ( Lonza); and a serum-free medium formulation (STEMBLAST ^® ) for human embryonic stem cell culture. Each condition was tested with or without the addition of the growth factors bFGF (10 ng/mL) and EGF (10 ng/mL).

KGM-GOLD™ Basal Medium formulated with low calcium for epithelial cell expansion. STEMBLAST ^® is a serum-free formulation containing 5 ng/mL Activin A for the culture of embryonic stem cells. MATRIGEL ^® is a complex extracellular matrix containing laminin, collagen and proteoglycan.

Morphological analysis and growth performance were performed after 1 week of culture in these conditions and are summarized in Table 8.

Table 8. Performance of Patient-Derived Ovarian Cancer Cultures in Various Media, Substrate, and Growth Factor Conditions

MATRIGEL ^® inhibits the growth of tumor cells in all media or growth factor conditions. Tissue culture plastic (plasma treated) showed less adherence of cells upon initial seeding, however did support adequate expansion of tumor cells in all conditions. Gelatin (0.1%) coating allows very good initial adhesion and supports rapid expansion of tumor cells.

Regardless of substrate or presence of growth factors, KGM-GOLD™ did not support rapid tumor cell expansion, resulting in ovarian cancer cell senescence with large cell bodies and vacuolated cytoplasm.

CSC-E medium containing 5% FBS supported rapid expansion of cells only in the presence of bFGF and EGF (Figures 7 and 8), whereas the serum-free version supported rapid expansion regardless of the presence of growth factors (Figures 9 and 10 ). Animal sera appear to contain factors that inhibit the rapid expansion of ovarian tumor cells, an effect that is compensated by the addition of growth factors (FGF, EGF) that are themselves ligands for receptor tyrosine kinases. receptor tyrosine kinase Other ligands of tyrosine kinase (RTK) (such as VEGF, HGF, PDGF) can have the same effect on tumor cell expansion. The morphology of cells grown in serum-free medium supplemented with FGF and EGF was of the small cuboidal type, packed in dense and uniform colonies similar to embryonic stem cell cultures.

STEMBLAST ^® supports good expansion only in the presence of growth factors. The combination of EGF, FGF and Activin A in STEMBLAST ^® resulted in a morphology very similar to embryonic stem cell cultures, with small cell bodies and large nuclei occupying most of the cells with minimal cytoplasm (Figure 11).

Immunocytological analysis of common ovarian and cancer stem cell markers in serum-free medium revealed a cancer stem cell phenotype and negligible EMT.

The ovarian cancer epithelial cell marker, the marker CA125, was found on more than 90% of the cells (Fig. 12B). Another ovarian cancer epithelial cell marker, MUC-1, was found in approximately 60% of cells with various expression intensities (Fig. 12C). The ovarian cancer cell keratin marker CK8 was present in more than 80% of the cells (Fig. 13B).

The culture conditions promoted the expansion of cancer stem cells, as indicated by the presence of high percentages (better than 75%) of cells expressing EpCAM (Fig. 14C), CD44 (Fig. 15C) and CD133 (Fig. 16). The cultures exhibited rapid expansion by high expression of the Ki67 proliferative marker (Figure 13B) on most cells.

We conclude that serum-free media with minimal amounts of RTK ligands or low concentrations of animal serum in media supplemented with RTK ligands (eg, FGF, EGF) can optimally maintain and expand ovaries A population of cancer stem cells. Increasing the amount of serum and decreasing RTK ligands will result in CSC differentiation. Increasing serum concentrations and increasing RTK ligand concentrations will cause EMT. Serum-free medium containing RTK ligands and activin A can maintain an embryonic stem cell-like very early stem cell state.

Although the presence of the gelatin (collagen) coating increased initial cell adhesion, it did not significantly alter the expansion rate or phenotype of the cells.

These experiments demonstrate our ability to isolate, purify and expand CSCs from ovarian tumors and manipulate their differentiation or EMT. In contrast to the diluted antigenicity of bulky tumors, the cell populations described here can be used as a high-quality source of antigens for whole-cell active autoimmune therapies.

Example 5. Generation of Loaded Dendritic Cell Compositions

The source of antigens is autologous tumor cells derived from continuously proliferating self-regenerative cells derived from fresh tumor tissue of the patient. These cells have the characteristics of cancer stem cells. At all times in the surgical and lesion setting, a strict aseptic protocol was followed for handling biopsies to ensure that samples were sterile.

A pathologist obtains fresh tissue from a biopsy of a patient's tumor. Using a sterile scalpel and forceps, the samples were cut into 10 mm sections and transferred to transport tubes containing transport medium, which were processed promptly to avoid drying of the samples. Samples were shipped to the manufacturing facility by overnight courier within 48 days of surgical resection.

In the manufacturing facility, samples were dissociated into single cell suspensions in a clean room and placed in cell culture conditions designed to enrich and proliferate OV-CSCs. During processing of tumor samples, normal cells such as lymphocytes, stromal cells, and connective tissue are eliminated. After completing the expansion and purification steps, the enriched proliferating OV-CSCs (tumor cells, TCs) were inactivated by irradiation and placed in a vapor-phase liquid nitrogen storage tank. Depending on the quantity and quality of the tumor sample, this process can take up to eight weeks.

Once the tumor cell product has passed quality assurance, the patient is told to undergo a procedure called leukapheresis (usually a six-liter procedure). This procedure involves filtering the blood to collect peripheral blood mononuclear cells (PBMCs). The collected PBMCs are shipped by overnight courier to the manufacturing facility for further purification by countercurrent density centrifugation known as elutriation. Elutriation is the process of purifying monocytes from other lymphocytes to enrich for cells that can become antigen presenting cells or dendritic cells. To generate dendritic cells, elutriated monocytes were incubated with the cytokines GM-CSF and interleukin-4 (IL-4) for six days.

On day 6, purified tumor cell product was removed from frozen storage, thawed and combined with dendritic cells for 18-24 hours. This process produces an "antigen load" of DCs. The final product is entirely DC, or may contain some residual irradiated TC (considered to be permissible) and is referred to as DC-TC. The combined dendritic cell/tumor cell mixture was collected, cryopreserved to preserve dendritic cell viability, and stored in vapor phase liquid nitrogen.

After completion of quality control analysis and release of autologous cell therapy product, the batch is shipped to the treatment facility under vapor phase liquid nitrogen conditions. Upon arrival, the cell therapy product is stored under gaseous liquid nitrogen conditions until ready to be administered.

Example 6. Ovapuldencel-T Compared GM-CSF Autologous peripheral blood mononuclear cells in patients with ovarian cancer II phase, double-blind, randomized, clinical trial

This study is ovapuldencel-T (autologous dendritic cells loaded with irradiated autologous OV-CSC in GM-CSF) versus autologous peripheral blood mononuclear cells in GM-CSF (MC) in patients with stage III or IV Phase II, double-blind, randomized, single-centre trial as a component of maintenance or secondary therapy after debulking surgery and adjuvant chemotherapy in patients with stage epithelial ovarian, fallopian tube, or primary peritoneal carcinoma. The aim of the study was to compare overall survival (OS) from the date of randomization in patients treated with ovapuldencel-T with those treated with autologous blood mononuclear cells in the GM-CSF control group. According to the method of Example 5, ovapuldencel-T was prepared.

Female subjects 18 years or older who were recently diagnosed with stage III or IV ovarian cancer, candidates for surgical debulking and chemotherapy, were candidates for the study. Performance status at the time of enrollment for ASI treatment must be an ECOG score of 0 or 1.

Inclusion criteria included histologic diagnosis of epithelial ovarian, fallopian tube, or primary peritoneal carcinoma; advanced (metastatic, stage III, or IV) epithelial ovarian, fallopian tube, or primary peritoneal carcinoma and surgical debulking to obtain Candidate for fresh live tumor tissue (for establishment of short-lived tumor cell lines); age > 18 years old; each patient must be aware of the neoplastic nature of her disease process and must voluntarily consent to the manipulation of tumor tissue for the establishment of tumor cell lines; and the patient must have the ability to move to a treatment center for ASI treatment administration and will.

Exclusion criteria included ECOG performance status greater than 2; known positive for hepatitis B or C or HIV; pregnant or lactating females; underlying cardiac disease associated with abnormal myocardial function requiring active medical treatment, or associated with atherosclerosis Unstable angina associated with sclerotic cardiovascular disease, or under treatment for arterial or venous peripheral vascular disease; diagnosis of any other invasive cancer considered life-threatening within the next five years, and/or for cancers other than ovarian Anticancer therapy (such as continuation of hormone therapy for prostate or breast cancer diagnosed more than five years ago); potentially life-threatening active infection or other active medical condition, including active blood clotting or bleeding diathesis; active CNS while on treatment Nervous system cancer metastasis; known autoimmune disease, immunodeficiency, or disease process involving the use of immunosuppressive therapy; tumors with low likelihood of malignant disease; or receiving another investigational drug within 28 days of the first dose.

Approximately 99 patients with stage III or IV epithelial ovarian, fallopian tube, or intraperitoneal cancer for whom cell lines have been successfully established and who were eligible for ASI treatment at randomization and who The aforementioned patients have consented to participate in trials and treatments using ASI.

Following recovery from debulking surgery and prior to initiation of chemotherapy, patients will undergo leukapheresis to obtain PBMCs from which autologous dendritic or monocytes are derived. Following leukapheresis, patients will be treated with a planned six cycles of primary adjuvant chemotherapy consisting of taxanes and platinum chemotherapeutic agents, according to the standard of care for such patients.

Based on elevated blood CA-125 and/or other tumor markers and/or disease detection by physical examination or imaging, patients will be stratified as: (1) Platinum resistant, based on progression during adjuvant chemotherapy or detectable disease at the end of adjuvant therapy; or (2) platinum sensitive with no evidence of disease (NED) at the end of adjuvant therapy.

After stratification, patients will be randomized in a 2:1 ratio to ovapuldencel-T or MC treatment groups. This approach yields two clinically similar patient populations, standardizes therapy schedules, maximizes cell line use, and incorporates practice standards for maintenance or secondary adjuvant therapy:

Patients with platinum-resistant disease will be treated with secondary therapy according to their managing physician and will receive ovapuldencel-T or MC at the same time.

Platinum-sensitive patients with NED will receive maintenance therapy (usually paclitaxel for up to one year) and will receive ovapuldencel-T or MC at the same time according to their managing physician.

Patients randomized into the ovapuldencel-T arm will receive autologous dendritic cells pulsed with autologous irradiated OV-CSC cells to generate patient-specific therapy. Depending on the timing of maintenance or secondary therapy, ovapuldencel-T will be administered in 500 micrograms of GM-CSF once a week for three weeks, and then every three to four weeks when subsequent maintenance or secondary therapy is given, between four and Up to a total of 8 doses of vaccine therapy are given over six months.

Depending on the timing of maintenance or secondary therapy, patients randomized into the MC arm will receive autologous monocytes given in 500 micrograms of GM-CSF once a week for three weeks, followed by subsequent maintenance or secondary therapy Therapy was given every three to four weeks for up to a total of eight doses of ASI therapy over four to six months.

Patients experiencing progressive disease while on study may continue ASI in combination with any other standard systemic therapy as prescribed by their managing oncologist until all 8 treatment doses have been administered.

The primary study objective was to compare overall survival (OS) from the date of randomization in patients treated with ovapuldencel-T with those treated with autologous blood mononuclear cells in the GM-CSF control group.

The primary endpoint of this trial was death from any cause, measured as overall survival (OS) from the date of randomization. Progression-free survival (PFS) was a secondary endpoint and was calculated as the time from the date of randomization to treatment until subjective tumor progression or death. Progression was defined subjectively by the treating physician and was based on tumor marker levels (eg, CA-125) and/or imaging. Secondary, PFS and OS were defined from the date of volume reduction surgery.

All patients eligible for study participation will be followed for up to 5 years from the date of randomization or until death, whichever occurs first.

Kaplan-Meier curve curve) will display the survival time for each treatment group. A log rank test will be used to analyze OS to test the null hypothesis of no treatment difference. Cox regression model (Cox regression model) and Wald tests will be used to estimate hazard ratios associated with treatment and to identify the significance of potential predictors and their effects, if any, on treatment differences. Analyzes based on intent-to-treat population will be considered primary analyses, and analyzes based on per-protocol population will be considered sensitivity analyses. Subgroup analyzes of OS endpoints will mimic the plans laid out for the primary endpoint in platinum-resistant and platinum-sensitive patients, respectively.

Unless otherwise indicated, all numbers expressing amounts of ingredients, properties (such as molecular weights), reaction conditions, etc. used in the specification and claims are to be understood as being modified in all instances by the term "about". As used herein, the terms "about" and "approximately" mean within 10% to 15%, preferably within 5% to 10%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

Unless otherwise indicated herein or clearly contradicted by context, the terms "a/an", "the" and similar referents are used in the context of describing the invention, especially in the context of the following claims should be construed to cover both the singular and the plural. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (eg, "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limiting. Each group member may be referred to and claimed individually or in any combination with other members of said group or other elements found herein. It is contemplated that one or more members of a group may be included in or deleted from a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified, thereby satisfying all Markush groups as used in the appended claims. group) written description.

Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations of those described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, the invention encompasses any combination of the above-described elements in all possible variations thereof unless otherwise indicated herein or otherwise clearly contradicted by context.

Specific embodiments disclosed herein can be further limited in the claims using consisting of or consisting essentially of language. When used in a claim, the transitional term "consisting of" excludes any element, step or ingredient not specified in the claim, whether as filed or added under amendment. The transition term "consisting essentially of" limits the scope of a claim to the specified materials or steps and those materials or steps that do not materially affect the basic and novel characteristics. Embodiments of the invention so claimed are inherently or expressly described and implemented herein.

Furthermore, extensive reference has been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications is individually incorporated herein by reference in its entirety.

In conclusion, it is to be understood that the embodiments of the invention disclosed herein illustrate the principles of the invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example and not limitation, alternative configurations of the present invention may be utilized in light of the teachings herein. Accordingly, the invention is not limited to what is precisely shown and described.

Accordingly, while there has been shown and described and indicated the substantially novel features of the invention as applied to exemplary embodiments and/or aspects thereof, it is to be understood that those skilled in the art may, without departing from the spirit of the invention and/or the claims, Various omissions, reconfigurations and substitutions and changes in the form and details of the exemplary embodiments, disclosures and aspects thereof are made without limitation. For example, all combinations of those elements and/or method steps which perform substantially the same function in substantially the same way to achieve the same results are expressly intended to be within the scope of the invention. Furthermore, it should be recognized that structures and/or elements and/or method steps shown and/or described in connection with any disclosed form or embodiment may be incorporated as a matter of general design choice into any other disclosed or described or suggested form or In the implementation plan. Accordingly, no limitation of the scope of the invention is intended. All such modifications are intended to come within the scope of the claims appended hereto.

All publications, patents, patent applications, references, and sequence listings cited in this specification are hereby incorporated by such reference as if fully set forth herein.

An abstract is provided to comply with 37 CFR §1.72(b) to allow the reader to quickly ascertain the nature and gist of the technical disclosure. The Abstract is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims.

Claims (74)

CLAIMS 1. An immunogenic composition comprising dendritic cells activated ex vivo by tumor antigens derived from a purified ovarian cancer (OV) cancer stem cell (CSC) population. 2. The immunogenic composition of claim 1, wherein the tumor antigen comprises a cellular extract of the OV-CSC. 3. The immunogenic composition of claim 1, wherein the tumor antigen comprises a lysate of the OV-CSC. 4. The immunogenic composition of claim 1, wherein the tumor antigen comprises intact OV-CSC. 5. The immunogenic composition of claim 4, wherein the intact cells are rendered non-proliferative. 6. The immunogenic composition of claim 5, wherein the intact cells have been rendered non-proliferative by irradiation. 7. The immunogenic composition of claim 4, wherein the intact cells are rendered non-proliferative by exposing the cells to a nuclear cross-linking agent. 8. The immunogenic composition according to any one of claims 1 to 7, further comprising a pharmaceutically acceptable carrier and/or excipient. 9. The immunogenic composition of any one of claims 1 to 8, further comprising an adjuvant. 10. The immunogenic composition according to claim 9, wherein the adjuvant is granulocyte macrophage colony stimulating factor. 11. The immunogenic composition of any one of claims 1-10, wherein the composition comprises activated dendritic cells and OV-CSCs. 12. The immunogenic composition of any one of claims 1-11, wherein the OV-CSCs are in the form of OV-CSC spheroids. 13. The immunogenic composition of any one of claims 1-11, wherein the OV-CSC is in the early OV-CSC form. 14. The immunogenic composition of any one of claims 1-11, wherein the OV-CSCs are in the form of mixed OV-CSCs. 15. The immunogenic composition of any one of claims 1-11, wherein the OV-CSC is in the form of an EMT-OV-CSC. 16. A method of treating ovarian cancer in a subject in need thereof, comprising administering to the subject the immunogenic composition of any one of claims 1-15. 17. The method of claim 16, wherein the immunogenic composition is administered in multiple doses, each dose comprising about 5-20 x ¹⁰⁶ cells. 18. The method of claim 17, wherein the dose comprises about ¹⁰ x 106 cells. 19. The method of any one of claims 16-18, wherein the dose is administered weekly for 2-5 doses, followed by monthly administration for 3-6 doses. 20. The method of any one of claims 16-19, wherein the subject receives 6-10 doses of the immunogenic composition. 21. Use of the immunogenic composition according to any one of claims 1 to 15 for the manufacture of a medicament for the treatment of ovarian cancer. 22. Use of an immunogenic composition according to any one of claims 1 to 15 for the treatment of ovarian cancer. 23. A method for preparing a population of ovarian cancer (OV) cancer stem cells (CSC), the method comprising: Obtain OV samples; dissociating the cells of the sample, and The dissociated cells are cultured in vitro on a non-adherent substrate in a defined medium that is serum-free and supplemented with at least one mitogen-activated protein kinase ( MAPK) pathway acting growth factors, thereby forming OV-CSC spheroids; wherein at least 80% of the cells in the OV-CSC spheroid population express two or more of the following biomarkers: EpCAM, CA-125, MUC-1, CD117, He-4, ALDH, CD133, CD24, and Ki-67. 24. The method of claim 24, wherein at least 80% of the cells in the OV-CSC spheroid population further express one or more of the following biomarkers: CA19-9, HER2/neu, NCAM, ganglion Aside CD2, Estrogen Receptor α, Vimentin, CK8, CK18, AFP, Testosterone, TGFβR, EGFR, TAG-72, CD46, CD44, ABCG2, Slug/Snail, Nestin, and TP53. 25. The method of claim 24, wherein at least 90% of the cells in the OV-CSC spheroid population express two or more of the following biomarkers: EpCAM, CA-125, MUC-1, CD117 , He-4, ALDH, CD133, CD24 and Ki-67. 26. The method of claim 24, further comprising: The OV-CSC spheroids are cultured on an adhesive substrate in a defined medium that is serum-free and supplemented with at least one growth agent acting via the MAPK pathway factor, thereby forming a population of early OV-CSC, wherein at least 80% of the cells in said early OV-CSC population express two or more of the following biomarkers: EpCAM, CD133, CD44, Nanog, Sox2, Oct3 /4, CD17, and Ki-67. 27. The method of claim 27, wherein at least 80% of the cells in the early stage OV-CSC population further express one or more of the following biomarkers: CA-125, MUC-1, TGFβR, and CD24. 28. The method according to claim 27, wherein at least 90% of the cells in the early OV-CSC population express two or more of the following biomarkers: EpCAM, CD133, CD44, Nanog, Sox2, Oct3/ 4. CD17 and Ki-67. 29. The method of claim 24, further comprising: The OV-CSC spheroids are cultured on an adhesive substrate in a defined medium containing serum, thereby forming a population of mixed OV-CSCs, wherein the mixed OV- At least 80% of the cells in the CSC population express two or more of the following biomarkers: EpCAM, CA-125, MUC-1, CD117, CK8, CK18, and Ki-67. 30. The method of claim 30, wherein the defined medium further comprises at least one growth factor acting via the MAPK pathway. 31. The method of claim 30, wherein at least 80% of the cells in the mixed OV-CSC population further express one or more of the following biomarkers: CA19-9, HER2/neu, NCAM, gangliosides lipid CD2, estrogen receptor α, testosterone, TGFβR, EGFR, TAG-72, CD46, He-4, ALDH, CD133, CD44, ABCG2, nestin, and TP53. 32. The method of claim 30, wherein at least 90% of the cells in the mixed OV-CSC population express two or more of the following biomarkers: EpCAM, CA-125, MUC-1, CD117, CK8, CK18 and Ki-67. 33. The method of claim 24, further comprising: culturing said OV-CSC spheroids on an adhesive substrate in a defined medium containing serum and supplemented with at least one growth factor acting via said MAPK pathway, A population of epithelial to mesenchymal transition (EMT)-OV-CSCs is thus formed, wherein at least 80% of the cells in the EMT-OV-CSC population express two or more of the following biomarkers: NCAM, Slug/Snail, CD24, and Twist. 34. The method according to claim 34, wherein at least 80% of the cells in the EMT-OV-CSC population further express one or more of the following biomarkers: CA-125, MUC-1, CD133, Nanog, CD117, N-cadherin, CD44, and vimentin. 35. The method of claim 34, wherein at least 90% of cells in the EMT-OV-CSC population express two or more of the following biomarkers: NCAM, Slug/Snail, CD24, and Twist. 36. The method of any one of claims 24, 30, or 34, further comprising: The OV-CSC spheroids, the mixed OV-CSC or EMT-OV-CSC were cultured on an adhesive substrate in a defined medium, wherein the defined medium was serum-free and supplemented with There is at least one growth factor acting via the MAPK pathway, thereby forming a population of early OV-CSCs, wherein at least 80% of the cells in the early OV-CSC population express two or more of the following biomarkers Each: EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD17, and Ki-67. 37. The method of claim 37, wherein at least 80% of the cells in the early OV-CSC population further express one or more of the following biomarkers: EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4 , CD17 and Ki-67. 38. The method of claim 37, wherein at least 90% of the cells in the early OV-CSC population express one or more of the following biomarkers: EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD17 and Ki-67. 39. The method of any one of claims 24, 27, or 34, further comprising: The OV-CSC spheroids, the early OV-CSC or EMT-OV-CSC are cultured on an adhesive substrate in a defined medium containing a serum source and supplemented with at least one growth factor acting via said MAPK pathway, thereby forming a population of mixed OV-CSCs, wherein at least 80% of the cells in said mixed OV-CSC population express two or more of the following biomarkers : AFP, CK7, CK19, EpCAM, E-cadherin, Nanog, FoxA2HNF4a and ABCG2. 40. The method of claim 40, wherein said defined medium further comprises at least one growth factor acting via said MAPK pathway. 41. The method of claim 40, wherein at least 80% of the cells in the mixed OV-CSC population further express one or more of the following biomarkers: CA19-9, HER2/neu, NCAM, ganglioside lipid CD2, estrogen receptor α, testosterone, TGFβR, EGFR, TAG-72, CD46, He-4, ALDH, CD133, CD44, ABCG2, nestin, and TP53. 42. The method of claim 40, wherein at least 90% of the cells in the mixed OV-CSC population express two or more of the following biomarkers: AFP, CK7, CK19, EpCAM, E-cadherin protein, Nanog, FoxA2HNF4a, and ABCG2. 43. The method of any one of claims 24, 27, or 30, further comprising: The OV-CSC spheroids, the early OV-CSC or mixed OV-CSC are cultured on an adhesive substrate in a defined medium containing serum and supplemented with at least one A growth factor acting via the MAPK pathway, thereby forming a population of EMT-OV-CSCs, wherein at least 80% of the cells in the EMT-OV-CSC population express two or more of the following biomarkers : NCAM, Slug/Snail, CD24 and Twist. 44. The method according to claim 44, wherein at least 80% of the cells in the EMT-OV-CSC population further express one or more of the following biomarkers: CA-125, MUC-1, CD133, Nanog, CD117, N-cadherin, CD44, and vimentin. 45. The method of claim 44, wherein at least 90% of cells in the EMT-OV-CSC population express two or more of the following biomarkers: NCAM, Slug/Snail, CD24, and Twist. 46. The method of any one of claims 24-46, wherein the defined medium is any medium described in Table 2. 47. The method of any one of claims 24-46, wherein the defined medium is any medium from the combination of Table 2 and Table 3. 48. The method of any one of claims 24-46, wherein the defined medium is any medium from the combination of Table 2, Table 3 and Table 4. 49. The method of any one of claims 24-46, wherein the defined medium is any medium from the combination of Table 2 and Table 4. 50. The method of claim 24, wherein the growth factor is one or more of fibroblast growth factor (FGF), epidermal growth factor (EGF), or activin A. 51. The method of claim 51, wherein the FGF is basic FGF (bFGF). 52. The method of any one of claims 24-52, wherein the defined medium is not supplemented with activin-A. 53. The method of any one of claims 24-52, wherein the defined medium is supplemented with an agonist of activin A in an amount effective to prevent spontaneous differentiation of OV stem cells. 54. The method of claim 54, wherein the culture medium further comprises an antagonist of activin A, and the antagonist is follistatin or an antibody that specifically binds to activin A. 55. The method of any one of claims 24-55, wherein the culture medium is not supplemented with antioxidants. 56. The method of claim 56, wherein the antioxidant is superoxide dismutase, catalase, glutathione, putrescine, or beta-mercaptoethanol. 57. The method of any one of claims 24-55, wherein the medium is supplemented with glutathione. 58. The method of any one of claims 24 to 58, wherein the adhesive substrate is configured for adhering to anchorage-dependent cells and collecting the cells. 59. The method of claim 59, wherein the anchorage-dependent cells are fibroblasts. 60. The method of any one of claims 24-58, wherein the non-adhesive substrate is an ultra-low adhesion polystyrene surface. 61. The method of claim 61, wherein the adhesive substrate comprises a surface coated with a protein rich in RGD tripeptide motifs. 62. A population of purified OV-CSC cells prepared by the method of any one of claims 24-58. 63. The population of claim 63, wherein the purified OV-CSC cells are OV-CSC spheroids. 64. The population of claim 63, wherein the purified OV-CSC cells are early stage OV-CSCs. 65. The population of claim 63, wherein the purified OV-CSC cells are mixed OV-CSCs. 66. The population of claim 63, wherein the purified OV-CSC cells are EMT-OV-CSC. 67. An OV-CSC cell line prepared by the method of any one of claims 1-29. 68. The OV-CSC cell line of claim 68, wherein the OV-CSC is an OV-CSC spheroid. 69. The OV-CSC cell line of claim 68, wherein the OV-CSC is an early stage OV-CSC. 70. The OV-CSC cell line of claim 68, wherein the OV-CSC is a mixed OV-CSC. 71. The OV-CSC cell line of claim 68, wherein the OV-CSC is an EMT-OV-CSC. 72. A method of stimulating an immune response against ovarian cancer in a subject in need thereof, comprising administering to said subject the immunogenic composition of any one of claims 1-15 . The OV-CSC cell according to any one of claims 63-67 or the OV-CSC cell line according to any one of claims 68-72. 73. The OV-CSC cell according to any one of claims 63 to 67 or the OV-CSC cell line according to any one of claims 68 to 72 in the preparation of a medicament for treating ovarian cancer the use of. 74. Use of the OV-CSC cell according to any one of claims 63 to 67 or the OV-CSC cell line according to any one of claims 68 to 72 for the treatment of ovarian cancer.