CN105339000A - High purity ovarian cancer stem cells for active autologous immune therapy - Google Patents

Abstract

The disclosure provides cancer stem cells, for use in stimulating immune response against a cancer, such as ovarian carcinoma. Methods for preparing and purifying the cancer stem cells are provided.

Description

For the high-purity ovarian cancer stem cell of active autoimmune therapy

the cross reference of related application

The application requires the rights and interests of the U.S. Provisional Patent Application 61/778,225 that on March 12nd, 2013 submits to according to 35U.S.C. § 119 (e).The application is also the part continuation application of International Application Serial No. PCT/US2013/053850 that on August 6th, 2013 submits to, described international application requires according to 35U.S.C. § 119 (e) U.S. Provisional Patent Application 61/683 that on August 15th, 2012 submits to, the rights and interests of the U.S. Provisional Patent Application 61/718,643 that on October 25th, 477 and 2012 submits to.The full content of described application is incorporated herein by reference.

Technical field

The present invention relates to ovarian cancer stem cell, be derived from the method for the immunogenic composition of described stem cell and manufacture and the described stem cell of use.

Background technology

Ovarian cancer is considered to derive from the epithelium being arranged in ovary internal layer, and therefore has adenocarcinoma tissue feature.Ovarian cancer stem cell had previously been described as and had been selected from main group (mainpopulation in SKOV3 and A224 cell line; MP) " subgroup " or " side group " (sidepopulation; SP), it expresses stem cell marker genes (Oct4 and Nanog), transporter gene (ABCG2, ABCC4, ABCB1) and CD labelling (CD44, CD24, CD177), there is the probability being divided into cancer, there is different tissues structure, show the multipotency feature of stem cell.The separation of this kind of cell uses the eliminating of Fluorescent DNA binding dyes Hoechst33342 to realize.

Particular cancers stem cell population may be anything superfluous or useless source, and may be the source of the cancer return of having treated.In addition, cancer stem cell subgroup in the tissue may restart growth cycle when being exposed in some signal and produce the cell can rebuilding tumor.Cancer stem cell microhabitat (niche) is dormancy, until correct intracellular signaling triggers enter proliferating cycle again.Entering signal can derive from local event again, as wound, cell injury, microorganism attack (virus, antibacterial or fungus), or is mediated by tropic growth factors, cytokine or cell-cell communication.In addition, hormone can regulate the stem cell in tissue specificity microhabitat.The defect of stem cell microhabitat or sudden change may cause the disturbance of function above.Anything superfluous or useless may be produced by this kind of disturbance, and these disturbances comprise the random mutation of the control affecting cell cycle.The sudden change of cancer is caused to change between individuals.Between those people of cancer suffering from a type (as a patients with mastocarcinoma contrasts another patients with mastocarcinoma) and between dissimilar cancer (as ovarian cancer contrast melanoma) observe this kind of variability.In ovarian cancer, in most of situation, identified the damage to TP53 gene, but this sudden change is not reflected in tumor cell phenotype all the time.

Also in various ratio, make inconsistent labelling be associated with tumor cell, and successfully use it for triggering immune reaction substantially.The therapy of this kind of use tumor associated antigen adopts various protein or peptide, as CA125, Her-2, Muc-1, Neu, NY-ESO-1 or the heatshock protein (HSP) being derived from tumor.

Autoimmune therapy adopts the tumor tissues of patient self to make immune system responsive and to be attached cancerous cell.Separately or give lysate or full cellular processes together with adjuvant, for strengthening the immunoreation to tumor.

Summary of the invention

Disclose ovarian cancer (OV) cancer stem cell (CSC), OV-CSC cell line herein and be used for the treatment of the immunogenic composition comprising OV-CSC support type dendritic cell of ovarian cancer.

Specifically provide a kind of immunogenic composition herein, it comprises the dendritic cell by the tumor antigen ex vivo activation being derived from purified ovarian cancer (OV) cancer stem cell (CSC) colony disclosed herein.In one embodiment, described tumor antigen comprises the cell extract of described OV-CSC.In another embodiment, described tumor antigen comprises the lysate of described OV-CSC.In another embodiment, described tumor antigen comprises complete OV-CSC.In another embodiment, described tumor antigen comprises ex vivo transfection to the messenger RNA in described dendritic cell.

In another embodiment, described complete OV-CSC is endowed non-proliferative.In another embodiment, described complete OV-CSC is endowed non-proliferative by radiation.In another embodiment again, described complete OV-CSC is endowed non-proliferative by being exposed in core cross-linking agent or protein cross agent by described OV-CSC.

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

In another embodiment again, immunogenic composition comprises by the dendritic cell that activates and OV-CSC.In another embodiment, described OV-CSC is OV-CSC spheroid, early stage OV-CSC, mixing OV-CSC or EMT-OV-CSC form.

Also provide a kind of method for the treatment of ovarian cancer in experimenter in need, it comprises and gives immunogenic composition disclosed herein to described experimenter.In one embodiment, described immunogenic composition gives with multiple dosage form, and each dosage comprises about 5-20 × 10 ⁶individual cell.In another embodiment, described dosage comprises about 10 × 10 ⁶individual cell.In another embodiment, described dosage gives weekly once, continues 2-5 dosage, then monthly gives once, continues 3-6 dosage.In another embodiment again, described experimenter accepts the immunogenic composition of 6-10 dosage.

Immunogenic composition disclosed herein, OV-CSC disclosed herein or OV-CSC cell line disclosed herein purposes in the medicine for the preparation for the treatment of ovarian cancer is also provided.

Immunogenic composition disclosed herein, OV-CSC disclosed herein or OV-CSC cell line disclosed herein is also provided to be used for the treatment of the purposes of ovarian cancer.

Be provided for the method for the colony of preparing ovarian cancer (OV) cancer stem cell (CSC) herein further, described method comprises: obtain OV sample; The cell dissociated described in In vitro culture in the culture medium making the cell dissociation of described sample and determine at composition on non-adhesive substrate, the culture medium that wherein said composition is determined is serum-free and is supplemented with the somatomedin that at least one works via mitogen-activated protein kinase (MAPK) path, forms OV-CSC spheroid thus; In wherein said OV-CSC spheroid colony at least 80% the following biomarker of cellular expression in two or more: EpCAM, CA-125, MUC-1, CD117, He-4, ALDH, CD133, CD24 and Ki-67.In another embodiment, in described OV-CSC spheroid colony the cell of at least 80% to express in following biomarker further one or more; CA19-9, HER2/neu, NCAM, ganglioside CD2, estrogen receptor alpha, Vimentin, CK8, CK18, AFP, testosterone, TGF β R, EGFR, TAG-72, CD46, CD44, ABCG2, Slug/Snail, nestin (nestin) and TP53.In another embodiment, in described OV-CSC spheroid colony at least 90% the following biomarker of cellular expression in two or more: EpCAM, CA-125, MUC-1, CD117, He-4, ALDH, CD133, CD24 and Ki-67.

In another embodiment, described method is included in further in the culture medium that adhesiveness substrate is determined at composition and cultivates described OV-CSC spheroid, the culture medium that wherein said composition is determined is serum-free and is supplemented with the somatomedin that at least one works via described MAPK path, form the colony of early stage OV-CSC thus, in wherein said early stage OV-CSC colony at least 80% the following biomarker of cellular expression in two or more: EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD17 and Ki-67.In another embodiment, in described early stage OV-CSC colony the cell of at least 80% to express in following biomarker further one or more: CA-125, MUC-1, TGF β R and CD24.In another embodiment again, in described early stage OV-CSC colony at least 90% the following biomarker of cellular expression in two or more: EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD17 and Ki-67.

In another embodiment, described method is included in further in the culture medium that adhesiveness substrate is determined at composition and cultivates described OV-CSC spheroid, the culture medium that wherein said composition is determined contains serum, thus formed mixing OV-CSC colony, in wherein said mixing OV-CSC colony at least 80% the following biomarker of cellular expression in two or more: EpCAM, CA-125, MUC-1, CD117, CK8, CK18 and Ki-67.In another embodiment, the culture medium that described composition is determined comprises the somatomedin that at least one works via described MAPK path further.In another embodiment again, the culture medium that described composition is determined is the culture medium that the composition of low calcium is determined.In another embodiment, in described mixing OV-CSC colony the cell of at least 80% to express in following biomarker further one or more: CA19-9, HER2/neu, NCAM, ganglioside CD2, estrogen receptor alpha, testosterone, TGF β R, EGFR, TAG-72, CD46, He-4, ALDH, CD133, CD44, ABCG2, nestin and TP53.In another embodiment again, in described mixing OV-CSC colony at least 90% the following biomarker of cellular expression in two or more: EpCAM, CA-125, MUC-1, CD117, CK8, CK18 and Ki-67.

In another embodiment, described method is included in further in the culture medium that adhesiveness substrate is determined at composition and cultivates described OV-CSC spheroid, the culture medium that wherein said composition is determined contains serum and is supplemented with the somatomedin that at least one works via described MAPK path, form the colony of (EMT)-OV-CSC that epithelial cell changes to mesenchymal cell thus, in wherein said EMT-OV-CSC colony at least 80% the following biomarker of cellular expression in two or more: NCAM, Slug/Snail, CD24 and Twist.In another embodiment, in described EMT-OV-CSC colony the cell of at least 80% to express in following biomarker further one or more: CA-125, MUC-1, CD133, Nanog, CD117, N-cadherins, CD44 and Vimentin.In another embodiment, in described EMT-OV-CSC colony at least 90% the following biomarker of cellular expression in two or more: NCAM, Slug/Snail, CD24 and Twist.

In another embodiment, described method is included in further in the culture medium that adhesiveness substrate is determined at composition and cultivates described OV-CSC spheroid, described mixing OV-CSC or EMT-OV-CSC, the culture medium that wherein said composition is determined is serum-free and is supplemented with the somatomedin that at least one works via described MAPK path, form the colony of early stage OV-CSC thus, in wherein said early stage OV-CSC colony at least 80% the following biomarker of cellular expression in two or more: EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD17 and Ki-67.In another embodiment, in described early stage OV-CSC colony the cell of at least 80% to express in following biomarker further one or more: CA-125, MUC-1, TGF β R and CD24.In another embodiment again, in described early stage OV-CSC colony at least 90% the following biomarker of cellular expression in two or more: EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD17 and Ki-67.

In another embodiment, described method is included in further in the culture medium that adhesiveness substrate is determined at composition and cultivates described OV-CSC spheroid, described early stage OV-CSC or EMT-OV-CSC, the culture medium that wherein said composition is determined contains serum and is supplemented with the somatomedin that at least one works via described MAPK path, form the colony of mixing OV-CSC thus, wherein said mixing OV-CSC colony at least 80% the following biomarker of cellular expression in two or more: AFP, CK7, CK19, EpCAM, E-cadherins, Nanog, FoxA2HNF4a and ABCG2.In another embodiment, the culture medium that described composition is determined comprises the somatomedin that at least one works via described MAPK path further.In another embodiment again, the culture medium that described composition is determined is the culture medium that the composition of low calcium is determined.In another embodiment, in described mixing OV-CSC colony the cell of at least 80% to express in following biomarker further one or more: CA19-9, HER2/neu, NCAM, ganglioside CD2, estrogen receptor alpha, testosterone, TGF β R, EGFR, TAG-72, CD46, He-4, ALDH, CD133, CD44, ABCG2, nestin and TP53.In another embodiment again, in described mixing OV-CSC colony at least 90% the following biomarker of cellular expression in two or more: EpCAM, CA-125, MUC-1, CD117, CK8, CK18 and Ki-67.

In another embodiment, described method is included in the culture medium that adhesiveness substrate is determined at composition further cultivates described OV-CSC spheroid, described early stage OV-CSC or mixing OV-CSC, the culture medium that wherein said composition is determined contains serum origin and is supplemented with the somatomedin that at least one works via described MAPK path, form the colony of EMT-OV-CSC thus, in wherein said EMT-OV-CSC colony at least 80% the following biomarker of cellular expression in two or more: NCAM, Slug/Snail, CD24 and Twist.In another embodiment, in described EMT-OV-CSC colony the cell of at least 80% to express in following biomarker further one or more: CA-125, MUC-1, CD133, Nanog, CD117, N-cadherins, CD44 and Vimentin.In another embodiment, in described EMT-OV-CSC colony at least 90% the following biomarker of cellular expression in two or more: NCAM, Slug/Snail, CD24 and Twist.

In one embodiment, the culture medium that described composition is determined is any culture medium described in table 2; Come from any culture medium of the combination of table 2 and table 3; Come from any culture medium of the combination of table 2, table 3 and table 4; Or come from any culture medium of combination of table 2 and table 4.

In one embodiment, described somatomedin is one or more in fibroblast growth factor (FGF), epidermal growth factor (EGF) or activin A.In another embodiment, described FGF is basic FGF (bFGF).In another embodiment, the culture medium that described composition is determined is not supplemented with activin A.In another embodiment again, the culture medium that described composition is determined is with the agonist effectively preventing the amount of OV stem cell Spontaneous Differentiation to be supplemented with activin A.In another embodiment, described culture medium comprises the antagonist of activin A, and described antagonist is follistatin or the antibody with activin A specific binding.

In another embodiment, described culture medium is not supplemented with antioxidant.In another embodiment, described antioxidant is superoxide dismutase, catalase, glutathion, putrescine or beta-mercaptoethanol.In another embodiment again, described culture media supplemented has glutathion.

In another embodiment, described adhesiveness substrate is configured for and adheres on anchorage dependence cell (anchoragedependentcell) (as fibroblast) and cell as described in optionally collecting.In another embodiment, described non-adhesive substrate is ultralow adhesiveness polystyrene surface.In another embodiment again, described adhesiveness substrate comprises the surface being coated with the protein being rich in RGD tri-peptide motif.

Also provide a kind of colony of purified OV-CSC cell, it is prepared by any one in method disclosed herein.In certain embodiments, described OV-CSC is OV-CSC spheroid, early stage OV-CSC, mixing OV-CSC or EMT-OV-CSC.

Also provide a kind of OV-CSC cell line, it is prepared by any one in method disclosed herein.In certain embodiments, described OV-CSC is OV-CSC spheroid, early stage OV-CSC, mixing OV-CSC or EMT-OV-CSC.

Also be provided in the immunoreactive method of experimenter's moderate stimulation in need for ovarian cancer, it comprises and gives immunogenic composition disclosed herein, OV-CSC cell disclosed herein or OV-CSC cell line disclosed herein.

OV-CSC cell disclosed herein or the purposes of OV-CSC cell line disclosed herein in the medicine for the preparation for the treatment of ovarian cancer are also provided.

OV-CSC cell disclosed herein or OV-CSC cell line disclosed herein is also provided to be used for the treatment of the purposes of ovarian cancer.

Accompanying drawing explanation

Fig. 1 is from the tumor of excision (solid line boxes and arrow) or ovarian cancer (OV) cancer stem cell (OV-CSC) is separated, increases and is gathered the flow chart of the process of glomeration body from the small sample (dashed rectangle and arrow) of such as syringe needle biopsy body or peritoneal lavage thing.After generation spheroid, the path producing OV-CSC subgroup is common path.

Fig. 2 is depicted in the tumor cell line (OVCAR3) of the establishment producing irregular cell agglomerated thing in non-adhesive condition, therefore shows comparatively differentiated degree (difference 20 ×).

Fig. 3 is depicted in non-adhesive condition the ovarian cancer stem cell being derived from patient producing typical spheroid, therefore shows existence and the amplification of cancer stem cell.

The ovarian cancer stem cell culture (Fig. 4 A) being derived from patient identified is carried out in Fig. 4 A-D description for cancer stem cell biomarker EpCAM and NCAM.Cell is seeded in the culture medium containing 5% serum, 10ng/mLbFGF and 10ng/mLEGF.The red channel image of Fig. 4 B depiction 4A cell, reflection NCAM positive cell.The green channel images of Fig. 4 C depiction 4A cell, shows that EpCAM to change the disappearance in the phenomenon that (EMT) be associated to mesenchymal cell with the epithelial cell of cell.Fig. 4 D describes the blue channel image being used for two benzimide (bisbenzimide) nuclear staining.

The dry carcinoma cell culture of the ovary being derived from patient (Fig. 5 A) identified is carried out in Fig. 5 A-D description for EMT biomarker Slug/Snail and CD117.Cell is seeded in the culture medium containing 5% serum, 10ng/mLbFGF and 10ng/mLEGF.Fig. 5 B describes red channel image, and the major part of its indicator diagram 5A cell is positive to Slug/Snail.Fig. 5 C describes green channel images, and its indicator diagram 5A cell is positive to CD117.Fig. 5 D describes the blue channel image being used for two benzimide nuclear staining.

The ovarian cancer stem cell culture (Fig. 6 A) being derived from patient identified is carried out in Fig. 6 A-C description for cancer stem cell biomarker nestin.Cell is seeded in the culture medium containing 5% serum, 10ng/mLbFGF and 10ng/mLEGF.Fig. 6 B describes red channel image, and its instruction most cells is positive to nestin.This phenomenon is associated with EMT.Fig. 6 C describes the blue channel image being used for two benzimide nuclear staining.

Fig. 7 is depicted on gelatin containing the ovarian cancer stem cell culture (difference, 10 ×) being derived from patient in the culture medium of 5%FBS.

Fig. 8 is depicted on gelatin containing the ovarian cancer stem cell culture (difference, 10 ×) being derived from patient in the culture medium of 5%FBS, 10ng/mLbFGF and 10ng/mLEGF.

Fig. 9 is depicted in the ovarian cancer stem cell culture (difference, 10 ×) being derived from patient on gelatin in serum-free medium.

Figure 10 is depicted on gelatin containing the ovarian cancer stem cell culture (difference, 10 ×) being derived from patient in the serum-free medium of 10ng/mLbFGF and 10ng/mLEGF.

Figure 11 is depicted in the ovarian cancer stem cell microcolony (difference, 40 ×) being derived from patient in the serum-free medium containing 10ng/mLbFGF, 10ng/mLEGF and 5ng/mL activin A.The minicell with macronucleus of representative about 90% cell size can be observed in fine and close colony, show pole early-stage cancer stem cell (embryonic stem cell sample, " in early days " OV-CSC).

The ovarian cancer stem cell culture (Figure 12 A) being derived from patient identified is carried out in Figure 12 A-D description for ovarian cancer biomarker CA125 and MUC-1.Cell is seeded in the serum-free medium containing 10ng/mLbFGF and 10ng/mLEGF.The red channel image of Figure 12 B depiction 12A cell, it shows that the cell more than 90% is positive to CA125.The green channel images of Figure 12 C depiction 12A cell, its instruction most cells is positive to MUC-1 with various intensity.Figure 12 D describes the blue channel image being used for two benzimide nuclear staining.

Figure 13 A-D describes the ovarian cancer stem cell culture (Figure 13 A) being derived from patient identified ovarian cancer biomarker CK8 and proliferation marker Ki67.Cell is seeded in the serum-free medium containing 10ng/mLbFGF and 10ng/mLEGF.The red channel image of Figure 13 B depiction 13A cell, shows intensive propagation (being positive to Ki67 labelling).The green channel images of Figure 13 C depiction 13A cell, it shows that most cells is positive to CK8.Figure 13 D describes the blue channel image being used for two benzimide nuclear staining.

The ovarian cancer stem cell culture being derived from patient identified is carried out in Figure 14 A-D description for cancer stem cell biomarker EpCAM and NCAM.Cell is seeded in the serum-free medium containing 10ng/mLbFGF and 10ng/mLEGF.The red channel image of Figure 14 B depiction 14A cell, it shows that the faint core week (peri-nuclear) of NCAM in some cells is expressed.The green channel images of Figure 14 C depiction 14A cell, it is described most cells and is positive to EpCAM, and shows the epithelial cell character of described cell.Figure 14 D describes the blue channel image being used for two benzimide nuclear staining.

The ovarian cancer stem cell culture (Figure 15 A) being derived from patient identified is carried out in Figure 15 A-D description for cancer stem cell biomarker CD44 and nestin.Cell is seeded in the serum-free medium containing 10ng/mLbFGF and 10ng/mLEGF.The green channel images of Figure 15 C depiction 15A cell, instruction most cells is positive to CD44.The red channel image of Figure 15 B depiction 15A cell, indicates some to be mainly positioned at the cell of the nestin-positive in core week.Figure 15 D describes the blue channel image being used for two benzimide nuclear staining.

The ovarian cancer stem cell culture (Figure 16 A) being derived from patient identified is carried out in Figure 16 A-C description for cancer stem cell biomarker CD133.Cell is seeded in the serum-free medium containing 10ng/mLbFGF and 10ng/mLEGF.The green channel images of Figure 16 B depiction 16A cell, instruction most cells is positive to CD133.Figure 16 C describes the blue channel image being used for two benzimide nuclear staining.

Detailed description of the invention

The invention provides a kind of cell colony obtained from human ovarian carcinoma (OV) tumor, described cell colony forms primarily of high-purity cancer stem cell.In embodiments, the purity of cell colony 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 cell.These cancer stem cells are ovarian cancer CFU-GM, and the ability that there is continuous autosynthesis and break up to certain level.The invention still further relates to a kind of method that generation is derived from the purified colony of the stem cell of OV, for the antigenic source being used as cancer autoimmune therapy further.

Also contain test and screening embodiment.The present invention uses high-purity O V stem cell population to carry out genetic analysis to identify the uniqueness change driving individualized medicine allocation.The invention provides a kind of novel cell lines through external modification, wherein this immunostimulation feature of modifying enhancing OV.OV cell line is the improvement to the similar techniques using rough antineoplastic agents, because it provides excellent antigen signal to noise ratio.Cell line lacks pollutant cell colony (as fibroblast), and described cell colony can change or weaken application in external or body.Exemplary cells system of the present invention is also for the manufacture of the medicine being used for the treatment of OV.

As used herein, term " is derived from " any method containing when being derived from the peptide of one or more cancerous cell and obtain described peptide from the colony of cancerous cell or cancerous cell.Cancerous cell can such as destroy by homogenizer or by osmotic bursting, produces crude extract.The peptide of crude extract, oligopeptide and polypeptide can be exposed in dendritic cell, then by peptide described in described dendritic cell process.Term " is derived from " also contains complete cancerous cell, wherein said cancerous cell is alive, or wherein said cancerous cell has metabolic activity by radiation treatment, or therefore wherein said cancerous cell still comprises described peptide with cross-linking agent process." be derived from " mixture also comprising cancerous cell fragment, free cancer cell proteins matter and the cancerous cell (therefore it be derived from cancerous cell) through overshoot." be derived from " also comprise from cancerous cell or cancer stem cell be separated or amplification messenger RNA for ex vivo transfection dendritic cell to make it possible to carry out antigen presentation (antigenpresentation).

" give " its be applied to the mankind, mammal, mammalian subject, animal, veterinary experimenter, placebo subjects, research experimenter, experimental subjects, cell, tissue, organ or biofluid time refer to that (but being not limited to) makes exogenous part, reagent, placebo, micromolecule, medicament, therapeutic agent, diagnostic agent or compositions contact described experimenter, cell, tissue, organ or biofluid etc." give " to refer to such as therapy, pharmacokinetics, diagnosis, research, placebo and experimental technique.Give the interior therapeutic that can refer to the mankind or animal subjects.Cell process is contained makes reagent and cells contacting, and makes reagent and fluid contact, wherein said fluid and cells contacting." give " also to contain by reagent, diagnosis, binding compositions or carry out external and ex vivo treatment cell by another cell.

" effective dose " is contained (but being not limited to) can improve, reverse, relax, prevent or at least one symptom of diagnostic medicine condition of illness or disease or the amount of sign.Unless in addition clear and definite or specified by context, otherwise " effective dose " is not limited to the minimum flow being enough to realize results needed, is also not limited to the optimal amount being enough to realize results needed.

Or the seriousness of disease or disease can be measured by clinical parameter and treat in order to prevention, the ability treating or relax described disease or disease (realizing results needed) by biomarker, and implicit any restriction.Biomarker comprises blood counting; Metabolite level in serum, urine or cerebrospinal fluid; Tumor cell counts; Cancer stem cell counts; Tumor levels.Tumor levels can react assessment level (ResponseEvaluationCriteriaInSolidTumors by entity tumor; RECIST) criterion measures (people (2009) Eur.J.Cancer45:228-247 such as Eisenhauer).Presentation markup contains the expression of the genetic expression of mRNA or gene amplification, the expression of antigen and polypeptide.Clinical parameter comprises progresson free survival phase (progression-freesurvival; PFS), 6 months PFS, without disease survival period (disease-freesurvival; DFS), there is the time (timetoprogression of progress; TTP), there is the time (timetodistantmetastasis of remote cancerometastasis; TDM) and total survival rate, implicit any restriction.

Compositions through " mark " directly or indirectly detects by spectroscopy, photochemistry, biochemistry, immunochemistry, isotope or chemical method.For example, the label be suitable for comprises ³²p, ³³p, ³⁵s, ¹⁴c, ³h, ¹²⁵i, stable isotope, epitope tag fluorescent dye, electron-dense reagent, substrate or enzyme, such as use in enzyme immunoassay those, or fluorette(is disclosed in the U.S. 6,747, in 135, the wherein disclosed full content about fluorette is incorporated herein by reference).

Therefore, the open colony for the preparation of the purified spheroid of cancer stem cell or be derived from the method for single cell preparation of spheroid herein, described method comprises and obtains OV biopsy body; Make the cell dissociation of described biopsy body; The cell dissociated described in In vitro culture in the culture medium that substrate is determined at composition, the somatomedin that the culture media supplemented that wherein said composition is determined has at least one to work via mitogen-activated protein kinase (MAPK) path, to obtain colony or the single cell preparation of the purified spheroid of OV stem cell.In purified OV-CSC colony at least about 50%, at least about 60%, at least about 70% or at least about 80% cancer stem cell to express in following biomarker one or more: ATP is in conjunction with box subfamily G member 2(ATP-bindingcassettesub-familyGmember2; ABCG2; Gene bank accession number AAG52982.1), CD133, CD24, CD44, CD46, CD117, CK18 (CK18), CK8 (CK8), alpha fetal protein (alphafetoprotein; AFP), epithelial cell adhesion molecule (EpCAM; Gene bank accession number NP_002345.2), Ki-67, Nanog(gene bank accession number NM_024865.2, NP_079141.20), N-cadherins, nerve cell adhesion molecule (NCAM; CD56), Oct3/4(gene bank 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), MUC-1 (mucin1cellsurfaceassociated that cell surface is relevant; MUC-1), human epididymal albumen (He-4), aldehyde dehydrogenase (ALDH), cancer antigen 19-9(CA19-9), ErbB-2 (HER2/neu), ganglioside CD2, estrogen receptor alpha, testosterone, transforming growth factor β receptor (TGF β R), Sox2, EGF-R ELISA (EGFR), tumor-associated glycoprotein 72(TAG-72), nestin and oncoprotein p53(TP53).OV-CSC do not express in fact following in any one: carcinoembryonic antigen (CEA), FSH (FSH), α mankind's chorionic gonadotropic hormone (α HCG), β mankind's chorionic gonadotropic hormone (β HCG) and desmin (desmin).As used herein, be marked at the cell that the cell being less than 20% is expressed or cell colony indicated by term " in fact " refers to wherein.In other embodiments, biomarker is expressed being less than on the cell of 15%, the cell being less than 10% or the cell being less than 5%.The flow chart of the cell colony disclosed in formation presents in FIG.

As used herein, term " spheroid " refers to the cancer stem cell sphere aggregates formed by cultivating cancerous cell in serum-free medium.The ability forming spheroid is the feature of cancer stem cell.

In certain embodiments, in OV-CSC spheroid colony at least about 50%, at least about 60%, at least about 70% or at least about two or more in the following biomarker of cellular expression of 80%: 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, TGF β R, EGFR, TAG-72, CD46, CD44, ABCG2, Slug/Snail, nestin and TP53.In other embodiments, in OV-CSC spheroid colony at least about two or more in the following biomarker of cellular expression of 80%: AFP, CK7, CK19, EpCAM, E-cadherins, Ov1 and OV6.In another embodiment, in OV-CSC spheroid colony at least about two or more in the following biomarker of cellular expression of 90%: EpCAM, CA-125, MUC-1, CD117, He-4, ALDH, CD133, CD24 and Ki-67.

Spheroid colony can increase into the one in three kinds of different subgroups further by change condition of culture (as culture medium composition and substrate).The feature of block tumor, spheroid, early stage, mixing and EMTOV-CSC colony presents in Table 1.

table 1. for being produced the general introduction of the condition of OV cell colony by block OV tumor

* OV=ovarian cancer; * CSC=cancer stem cell; * * EMT=epithelial cell changes to mesenchymal cell

In addition, as disclosed in table 1, can by change culture medium and condition cause OV-CSC spheroid, early stage OV-CSC, mixing OV-CSC or EMT-OV-CSC obtain early stage OV-CSC, mix in OV-CSC or EMT-OV-CSC colony any one.

In one embodiment, on adhesiveness substrate, at activin A, OV-CSC spheroid is cultivated further to obtain that there is the colony (being called as " in early days " OV-CSC colony in this article) compared with minicell under the existence of FGF and serum-free medium (Selective agar medium), it has the feature of embryonic stem cell, and at least about 50% in early stage OV-CSC colony, at least about 60%, at least about 70% or at least about two or more in the following biomarker of cellular expression of 80%: EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD117 and Ki-67.In another embodiment, in early stage OV-CSC colony at least about two or more in the following biomarker of cellular expression of 80%: EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD117, Ki-67, CA-125, MUC-1, TGF β R and CD24.In another embodiment, in early stage OV-CSC colony at least about two or more in the following biomarker of cellular expression of 90%: EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD117 and Ki-67.

In another embodiment, on adhesiveness substrate, under low calcium condition, containing cultivating OV-CSC spheroid in serum (amplification culture medium) further to obtain the colony mixed with monolayer, wherein cell has uneven form.Culture medium optionally comprises EGF.These cells are called as " mixing " OV-CSC colony in this article, it has mixing differentiation overview, and in described mixing OV-CSC colony at least about 50%, at least about 60%, at least about 70% or at least about two or more in the following biomarker of cellular expression of 80%: EpCAM, CA-125, MUC-1, CD117, CK8, CK18 and Ki-67.In another embodiment, to mix in OV-CSC colony at least about two or more in the following biomarker of cellular expression of 80%: EpCAM, CA-125, MUC-1, CD117, CK8, CK18, Ki-67, CA19-9, HER2/neu, NCAM, ganglioside CD2, estrogen receptor alpha, testosterone, TGF β R, EGFR, TAG-72, CD46, He-4, ALDH, CD133, CD44, ABCG2, nestin and TP53.In another embodiment, to mix in OV-CSC colony at least about two or more in the following biomarker of cellular expression of 90%: EpCAM, CA-125, MUC-1, CD117, CK8, CK18 and Ki-67.

In another embodiment again, on adhesiveness substrate, FGF and containing the existence of blood serum medium (amplification culture medium) under cultivate further OV-CSC spheroid to obtain the monolayer of axle shape or irregular shape cell, it is called as mesenchymal cell sample OV-CSC or " EMT-OV-CSC " (epithelial cell changes the cancer stem cell of [EMT] to mesenchymal cell) in this article.In this colony, spheroid once went through EMT process, it is characterized in that at least one in epithelial cell biomarker CK8, CK18 and EpCAM or all expression deletions.As used herein, the expression deletion of biomarker refers to undetectable expression, or at 40%(or less) cells, at 30%(or less) cells, at 20%(or less) cells or at 10%(or less) cells.In addition, the feature of EMT process is that at least one or all expression of mesenchymal cell biomarker Slug/Snail, Twist, CD44, NCAM, N-cadherins and Vimentin are increased to the cell of in the colony of expressing object biomarker at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%.

In one embodiment, in EMT-OV-CSC colony at least about 50%, at least about 60%, at least about 70% or at least about two or more in the following biomarker of cellular expression of 80%: NCAM, Slug/Snail, CD24 and Twist.In another embodiment again, at least about two or more in the following biomarker of the cellular expression of 80% in EMT-OV-CSC colony: NCAM, Slug/Snail, CD24, Twist, CA-125, MUC-1, N-cadherins, CD44, Vimentin, CD33, Nanog and CD117.In another embodiment again, at least about two or more in the following biomarker of the cellular expression of 90% in EMT-OV-CSC colony: NCAM, Slug/Snail, CD24 and Twist.

In some embodiment of cell colony, one or more in the biomarker indicated by described cellular expression.In other embodiments, in the biomarker indicated by described cellular expression two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more or ten or more.1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 or 25 in the biomarker again described in other embodiment indicated by cellular expression.

Single cell, cell colony or the biomarker of cell colony being arranged in ad hoc structure (as monolayer or spheroid) express can by the polypeptide form of biomarker as described in measuring or as described in the expression of mRNA form of biomarker measure.Expression of polypeptides can use the antibody of mark to measure, and expression of nucleic acid can be measured by hybridization technique, can for those skilled in the art.And the biomarker of non-polypeptide or nucleic acid (as oligosaccharide or small molecule metabolites) also can by measuring for the method for those skilled in the art.

Also disclose from the ovarian tumor sample of all size (1mg is to some grams) herein and obtain the pure method through being separated OV stem cell population.Tumor sample can be fresh or freezing, dissociated by machinery and/or enzymatic treatment, or directly cultivate when having bottom line mechanical debris.

Also open herein, non-adhesive substrate is any biocompatible materials with antibiont attachment characteristic or the coating with antibiont attachment characteristic (reducing cell gathering on wetting surface) be coated on common culture surface.Coating can use smears (as amino silane) to apply.If there is non-adhesive or antibiont tack substrate, this substrate so can be used to continue about 0-25 days, as 0-21 days, 5-20 days, 5-10 days, 10-20 days, or any time section between zero and 25 days.

In another embodiment of method using adhesiveness substrate, described adhesiveness substrate can be rich in RGD(Arg-Gly-Asp) substrate (such as, collagen protein, gelatin, MATRIGEL) of three peptide motifs.Adhesiveness substrate is configured for adhere on anchorage dependence cell and collect the surface of described cell.In addition, described substrate can be configured for adhere to this as fibroblastic anchorage dependence cell collects the adhesiveness substrate of described cell.RGD peptide also can be grafted on polymerism main chain, and described polymerism main chain is as polystyrene, hyaluronan, polylactic acid or its combination.Main chain can carry proteoglycan further.Proteoglycan can carry somatomedin, as fibroblast growth factor (FGF), epidermal growth factor (EGF), activin A or follistatin.

Non-adhesive substrate can make culture medium fast and efficiently concentrating cancer stem cell.Non-adhesive substrate can use, can start immediately to make the purification of OV-CSC providing enough large sample (such as with the tumor of modus operandi excision) time.If sample minimum (as syringe needle aspirate or peritoneal lavage thing) and non-adhesive are cultivated infeasible, so adhesiveness can be used to cultivate to primary amplification, then on non-adhesive substrate, carry out purification step, under adhesiveness condition, carry out another amplification subsequently.In FIG (dotted line and square frame) hereafter describing substituting processing method in detail.

Cultivate in embodiment at some, adhesiveness substrate provides the first stage to cultivate, then on non-adhesive substrate, carries out second stage cultivation.Also be provided on non-adhesive substrate and carry out first stage cultivation, then on adhesiveness substrate, carry out second stage cultivation.Stage can be such as half a 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 its any scope, as 2-4 days or 8-10 days etc.In addition, circulation can repeat, and as adhesiveness is cultivated, then non-adhesive is cultivated, then adhesiveness cultivation etc.In another embodiment, circulation can repeat, and as non-adhesive is cultivated, then adhesiveness is cultivated, then non-adhesive cultivation etc.

In another embodiment, the somatomedin that the culture media supplemented that composition is determined has at least one to work via mitogen-activated protein kinase (MAPK) path.In one embodiment, somatomedin is one or two or its analog in FGF and EGF.In one embodiment, FGF is basic fibroblast growth factor (bFGF).In another embodiment, the culture media supplemented that composition is determined has activin A.In another embodiment, the culture medium that composition is determined is not supplemented with activin A.The also open culture medium determined with the composition effectively preventing the amount of OV stem cell Spontaneous Differentiation to be supplemented with activin A agonist.In other embodiments, the culture medium determined of composition is by bone morphogenetic protein (bonemorphogenicprotein; BMP) one in 2,4 or 7 or all supplement.

Also provide a kind of obtain from patient's Primary ovarian cancer to each patient, there is unique OV-CSC cell line, described cell line (a) carries the autosynthesis of stem cell and the feature of versatility and differentiation capability; And (b) in most cells (as more than 50%), carry unique gene group cancer characteristic indication.

Nucleic acid, gene outcome, polypeptide and fragments of peptides are contained in the present invention, and wherein identity reasonably can be established separately through popular name (trivialname).Also contain based on the nucleic acid of specific gene storehouse accession number, gene outcome, polypeptide and fragments of peptides, wherein said nucleic acid, polypeptide etc. have with the sequence iden of the nucleic acid, polypeptide etc. at least 50% of described gene bank number, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, the sequence iden of at least 99% or 100%, wherein biochemical function or physiologic function share at least in part or have nothing to do with function alternatively.

There is provided a kind of wherein with in compositions disclosed herein in the immunoreactive method of experimenter's moderate stimulation to cancer.The immunoreation stimulated comprises CD4 ⁺t cell response, CD8 ⁺t cell response and B cell response in one or more.In certain embodiments, CD4 ⁺t cell response, CD ⁺t cell response or B cell are replied and can being analyzed by ELISPOT, being analyzed by intracellular cytokine dyeing (ICS), analyzed by the tetramer or measuring by producing according to analysis detectable antigens specific antibody known to persons of ordinary skill in the art.Immunoreation can comprise the time-to-live, as 2 years total survival rate (overallsurvival; And wherein said 2 years total survival rates are at least 60% OS).Immunoreation in patient terminal used in also can being tested by oncological clinical is assessed, and described terminal comprises target response (RECIST criterion), total survival rate, progresson free survival phase (PFS), without disease survival period, occurs time, 6 months PFS of remote cancerometastasis, 12 months PFS etc.

The also dendritic cell that stimulates in vitro of open OV-CSC or the antigen that is derived from described OV-CSC herein, it is in ovarian cancer therapy.Contain herein and comprise the immunogenic composition that in vitro load has the dendritic cell of (being exposed to) OV-CSC, as vaccine combination.In certain embodiments, dendritic cell and tumor cell are from identical human experimenter's (autologous), although dendritic cell and OV cell are also in scope of the present invention from the embodiment of different experimenter.

Dendritic cell load can have OV tumor-cell antigen, described antigen comprise the full cell of OV tumor cell (as OV-CSC), cell lysate, cell extract, through the cell of overshoot or any protein derivatives.Dendritic cell vaccination Immunogenic Compositions can be prepared, and given to human experimenter by one or more route of administration as known to persons of ordinary skill in the art.

In certain embodiments, OV-CSC cell with dendritic cell load before through overshoot or otherwise process to prevent cell division.The replacement scheme of radiation comprises the fissional nucleic acid crosslinking agent of prevention.Also providing a kind of uses OV-CSC colony as disclosed to be used as the method for the antigenic source for autoimmune therapy, such as wherein by radiant (such as, γ, UV, X), temperature (such as, hot or cold) or chemistry (such as, cell growth inhibition, aldehyde, ethanol) method or its combination make OV-CSC inactivation.In other embodiments, OV-CSC is used as the antigenic source of dendritic cell ex vivo activation.

The invention provides prepared OV cell (OV-CSC), provide load to have the DC of prepared OV cell, and providing package contains the immunogenic composition (or vaccine) of the dendritic cell of OV cell prepared by load.When not implicit any restriction, immunogenic composition of the present invention can comprise that load has the DC of OV spheroid, load has the DC of the cell colony comprising spheroid, load has and is derived from spheroid and the DC of the cell colony increased on adhesiveness surface before load is on DC, load has the DC of the spheroid carrying out homogenization or sound wave process before load is on DC, load has the amplifying cells colony carrying out homogenization or sound wave process before load is on DC DC etc.In other embodiments, DC load has early stage OV-CSC, mixing OV-CSC or EMT-OV-CSC.

A kind of OV-CSC colony that can stimulate effective immune response for the cell of expressing at least one OV specific antigen is also disclosed herein, wherein said OV-CSC colony contacts with at least one dendritic cell, by in described dendritic cell body or ex vivo treatment, and wherein in described experimenter, there is effective immune response in response to described at least one dendritic cell to the administration of experimenter in wherein said OV-CSC colony.

The immunostimulation amount of disclosed compositions is that (but being not limited to) realizes following amount: make ELISPOT analysis result increase measurable amount, ICS analysis result is made to increase measurable amount, tetramer analysis result is made to increase measurable amount, antigenic specificity CD4+T cell colony in blood is made to increase measurable amount, antigenic specificity CD8+T cell colony in blood is made to increase measurable amount, or wherein increase at least 10% compared with suitable contrast time, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5 doubly, 2.0 doubly, 3.0 times etc.Suitable contrast can be reference composition, and wherein the non-load of dendritic cell has OV cell or non-load to have the peptide being derived from OV cell.

The present invention also provides medicine, reagent, test kit (comprising diagnostic kit), and wherein said medicine, reagent and test kit comprise dendritic cell (dendriticcell; DC), antibody or antigen.Also be provided for the method for the compositions comprising at least one dendritic cell and at least one antigen; The method formed for stimulating antibody; For stimulating antibody dependent cellular cytotoxicity (antibody-dependentcytotoxicity; ADCC) method; For stimulating the method for CDC; And for determining patient compliance, for determining patient selection under clinical trial or general medicine treatment situation or getting rid of criterion and for predicting method to the reaction of described medicine or reagent and test kit.Pharmaceutical composition of the present invention, reagent and correlation technique contain CD83 Protein-Positive Dendritic Cells, and wherein CD83 is by inducing with through IFN-γ process or undressed cancerous cell load.In in CD83 of the present invention, described CD83 is induced at least 2%, at least 3%, at least 4%, 6%, 7%, 8%, 9%, 10% etc.On the other hand, get rid of DC reagent or DC correlation technique, the CD83 wherein in dendritic cell by not inducing with IFN-γ load with detecting.

In one embodiment, provide a kind of test kit, it comprises all reagent for generating OV-CSC spheroid, early stage OV-CSC, mixing OV-CSC and/or EMT-OV-CSC according to method cause tumor sample disclosed herein; And/or for characterizing the reagent of described OV-CSC spheroid, early stage OV-CSC, mixing OV-CSC and/or EMT-OV-CSC; And for generating and/or characterize the description of described OV-CSC spheroid, early stage OV-CSC, mixing OV-CSC and/or EMT-OV-CSC.In another embodiment, test kit additionally or alternatively comprises for separating of dendritic cell, for by dendritic cell described in OV-CSC load and/or reagent from DC-OV compositions to experimenter and description for giving.

tumor sample process

Ovarian cancer of the present invention (OV) stem cell population can derive from the fresh or freezing sample of patient tumors.Tumor sample can be biopsy body or the lavation thing of peritoneal cavity containing OV cell.From irrigating solution, OV stem cell is separated from the neutralization of syringe needle biopsy body.

Can be about 7.4(+/-0.6 at pH) universal buffering culture medium in transport tumor sample, described universal buffering culture medium is as RPMI, DMEM, F12, Williams or combination, and it contains protein source (as animals or humans serum) with the concentration of 0% to 100% or contain albumin or containing the macromole guaranteeing physiology's osmotic pressure with the concentration of 0% to 0.5%.Natural or artificial macromolecular example is (but being not limited to) hyaluronan, dextran, polyvinyl alcohol.Antibiotic (as penicillin (penicillin), streptomycin (streptomycin), gentamycin (gentramicyn)) and antifungal can be used in the medium (as amphotericin B, FUNGIZONE (LifeTechnologies, Carlsbad, CA) optional combination provide antimicrobial property also to reduce the risk during transportation polluted.

Tumor sample can keep below metabolic activity state by culture medium temperature is reduced to 2 DEG C to 30 DEG C, therefore allows the rate of surviving before treatment to continue the limited time (between 0 and 72 hour).Packaging (such as, Thermoinsulating packaging) can be used to guarantee, and correct temperature during transportation controls.

Subsequently by use sharp-pointed blade or tissue grinder apparatus to carry out fragment that solid tumor tissue to be processed into less (being less than 1mm in any dimension) by mechanical dissociation.

To dissociate further processing entities tissue optionally by enzymatic.Multiple enzyme may be used for being separated single cell.Can successfully use non-specific proteolysis enzyme, as trypsin and pepsin.The enzyme-specific that can to use with MIN cell membrane damage in disclosed method be target, comprises Collagenase, Bacillus polymyxa Neutral proteinase, Elastase, hyaluronidase or its combination.Deoxyribonuclease (DNAse) may be used for making the free DNA degradation from cell debris, and described cell debris is the reason causing the non-required viscosity of cell preparation.After dissociating, from excessive enzyme and fragment, wash out the cell in suspension by repeated centrifugation via 50-100 μm of screen cloth leaching and in buffer saline (PBS, HBSS) or cell culture medium.

cell culture condition and spheroid produce

Single cell suspension as described above is transferred in the condition of culture promoting the separation of stem cell, amplification and differentiation and/or Normocellular suppression.This is by physical condition, unanimously the realizing of chemical environment and manipulation.

Cell suspending liquid is exposed in non-adhesive (antibiont tack) substrate not allowing cellular attachment.Mature cell normally anchorage dependence, and to be eliminated fast when not providing correct adhesiveness substrate.Antibiont tack substrate can adopt commercial product, as ultralow adhesiveness flask (Corning, Corning, NY); There is the polymer (polyvinyl, polyethylene, polypropylene, fluoropolymer) of native hydrophobic characteristic; Or there is the coating of natural carbohydrate polymers (as agar, starch etc.).

Cancer stem cell will be assembled and/or increase into the spheroid form containing high-purity cancer stem cell with clone.Show the cancer stem cell aggregation culture with the spheroid structure that can easily identify of all size in figure 3.Mature cell is separated keeping and non-adhesive.Can usage variance Gravity Separation, by allow simply timing vertical sedimentation or in short-term low-force centrifugal (being less than 100 × G) select the larger spheroid from single cell.Described system of selection is designed to below realization: (a) eliminates and be generally ripe Normocellular anchorage dependence cell; B () promotes the clonal expansion of the ripe little coagulation block of stem cell in the end of anchorage independence or spheroid; C () promotes the local autocrine activity produced due to the clonal expansion of described stem cell; (d) autocrine eliminating the activin A secreted by normal fibroblast or gonad cell is originated.

Cell is the cell cortex protein owing to being called as integrin (integrin) with forming the part ability of spheroid.The same preferendum integrin that cell surface is expressed guarantees that the cell of identical type " keeps together ".Spheroid is directly formed by the enzymic digestion thing as single cell suspension when cultivating and just starting, or can be formed by freezing sample or existing attachment culture at any time.The inoculation of enzymic digestion thing produces this spherical form being incorporated to the cell with particular surface property.

For example, fibroblast is not incorporated in spheroid, and removes from culture during gravity charging.Used medium lacks the molecule promoting to adhere to, to prevent from not having with the non-specific coalescent of the cell addicted to characteristic and to prevent from adhering to culture vessel surface.This kind of cell adhesion molecule (CAM) sees in animals or humans serum usually.Therefore, the culture media composition of serum-free is suitable for cultivating non-adhesive spheroid.

In serum-free medium is cultivated, the supplement of culture medium can comprise support growth and maintain or cell physiological and function other needed for any hormone, nutrient, the minerals and vitamins of aspect needs.In some instances, can by adding or regulating the amount of the somatomedin (as FGF family and EGF) with mitogenic activity stimulate and maintain stem cells hyperplasia.

The spheroid (spheroid) (comprising cancer stem cell spheroid) of cell can by means of fixing and with labeled antibody dyeing, then carrying out the sign of biomarker expression aspect with confocal microscopy.Biomarker also can pass through other immuno-chemical method, and such as flow cytometry is measured.Spheroid can such as with the suspension obtained by fresh or with being suitable for preparing with the cell of adherent cell form growth.The form of spheroid (such as large and irregular contrast is small and fine and close) can be subject to the impact that culture medium is selected.

In another embodiment, adhering to cell colony in antibiont tack coating can based on by Protein Kinase B (AKT) and focal adhesion kinases (focaladhesionkinase; FAK) abnormal activation of sound hedgehog (sonichedgehog) intracellular signaling that intracellular signaling mediates is separated.These phenomenons can by by as metalloproteases enzyme or for the enzyme (trypsin/Collagenase) that dissociates induce film modifiedly to strengthen.This kind of cell colony may be associated with rapid multiplication and invasive tumor.For assessment of the normal of sound hedgehog intracellular signaling or abnormal activation method can those of ordinary skill in the art available and known.

culture medium used in cell culture

The culture medium determined for separating of the composition of OV stem cell promotes cell survival rate, and through allocating for selection especially.Culture medium is rich in carbohydrate and lipid, but has the protein (0.1%-3% albumin or 1%-5% serum) of minimum tolerance.It contains the total calcium being no more than 1.5mMol, not containing inorganic iron compound; On the contrary, ferrum is combined completely with the transporter of such as transferrins.Culture medium has excessive required and nonessential aminoacid and required lipid (alpha-linolenic acid and linoleic acid) (table 4).Optionally, culture medium not containing activin A, and can contain activin A receptor blocking agent, as follistatin.In addition optionally, culture medium does not contain antioxidant, as superoxide dismutase (SOD) or catalase; But containing thiol antioxidants (thiolicantioxidant), as glutathion.

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

As in table 2 describe, culture medium is basic components, as being supplemented with DMEM, F12, Williams, RPMI, Lebovitz of protein (in some formula), aminoacid, antioxidant, high energy substrate (glucose, galactose, L-glutaminate), vitamin (B12), hormone (thyroxin, insulin) and somatomedin (FGF, EGF).

Protein can be concentration is the albumin of 0.1%-0.5%, the hyclone (FBS) of 0.5%-20%.Protein can be replaced by the macromole of concentration in 0.1% to 0.5% scope (as dextran, hyaluronan, polyvinyl alcohol).The composition of this kind of culture medium is listed in table 2, table 3 and table 4.Supplement to be added in culture medium and mixing for feeder cell culture.

Culture medium can by one Wednesday sky schedule (such as, Monday-Wednesday-Friday) replace, if or amplification be fast, so more frequently, such as every other day or every day replace.Continuous feed or micro-batch of charging bioreactor can be used in the amplification phase.

Culture medium contains the somatomedin worked via MAPK path, as FGF and EGF.The concentration of these somatomedin can change between 0.1 to 100ng/mL, about 10ng/mL usually.

table 3.lineage stem cells supplement (50mL unit is used for rehydration in 1L basal medium)

table 4.lipid mixture

Component	Concentration: μ g/mL
		Linolenic acid	10
Linoleic acid	10
		Tocopherol acetate	50
Cholesterol	100

Lipid mixture is made by using PluronicF68, phosphatidylcholines, Tween80, cyclodextrin or its o/w emulsion combined.

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

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

One or two the culture medium be not supplemented with in superoxide dismutase (SOD) or catalase is also provided.Use antioxidant can have front or negative results.The toleration of cancer stem cell to free radical and sugar decomposition metabolism is more much higher than normal cell.Therefore, in suboptimum culture medium (high-concentration metabolite as in the replacement of high density, infrequently culture medium, culture medium), first most probable eliminates normal sensitive cells.By not comprising antioxidant in the medium, can select probably to derive from carcinoma, having more the cell colony of resistance than normal cell.Therefore, in certain embodiments, in culture medium, add antioxidant (as catalase and SOD inhibitor), and in other embodiments, in described culture medium, omit these compounds.

In an alternative method, activin/follistatin can be used to be separated pole early-stage cancer stem cell.The interpolation of activin A can select activin A resistance OV stem cell subgroup.Follistatin is for blocking activin A receptor and preventing the Spontaneous Differentiation of OV stem cell, especially when there is cell (as fibroblast and the normal ovarian cell) of a large amount of endogenous excretion activin A.If cell is insensitive or be in and can in the high-purity O V stem cell population of endogenous excretion follistatin, so use follistatin without effect to activin A.

Activin A is originally as the protein of transforming growth factor-β (TGF-β) superfamily member.When interpolation or when comprising in the medium, activin contributes to maintaining stem cell versatility and autosynthesis.But activin A promotes the last mature ovarian cell of susceptibility and the maturation of cancerous cell and differentiation.Therefore, initial target carrys out rapid amplifying tumor in vitro by producing correct autocrine environment in culture, and it also maintains the propagation of cancer stem cell.Although activin A can select the cancer stem cell subgroup that extremely end is ripe, in view of the extremely low concentration of hepatic cancer stem cell on the whole, amplification greatly will be postponed in early stage the applied this kind of condition of manufacture.For example, " rapid amplifying " causes having in culture vessel the culture medium of apparent consumption sign (change of such as pH) and the amplification of cell number obvious every day higher (reflected by converging of increasing).

It for rapid amplifying, preferably omits and does not add activin A, because will slow down culture growth.For some application, pay close attention to and obtain extremely early stage stem cell population, and use activin A will select described cell colony.Therefore, in one embodiment, cause the amplification containing activin A and comprise the first compositions through cultured cell activated by activin A to experimenter, then be separated the insensitive cell of activin A in without the culture of activin A, and comprise this second compositions through cultured cell without activin A to described experimenter.

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

Also open wherein culture media supplemented has the embodiment of activin A antagonist, and described antagonist is as (but being not limited to) follistatin or the antibody with activin A specific binding.

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

The increase that the combination of mitogen (such as, FGF/EGF), activin A and adhesiveness substrate can cause normal cell (as fibroblast or sternzellen) to breed.Therefore, in the enrichment environment be made up of the cell (such as, fibroblast, sternzellen) with nourishing or parcel characteristic or " substrate (stroma) ", produce the condition promoted activin A insensitive OV stem cell or progenitor cells amplification extremely in early days.Little by little observe in the process of cell culture a few days to a few weeks subsequently, OV colony is along substrate development and replace described substrate in space.For the combination that the culture medium in the method is the formula described in table 2,3 and 4.

Relation is there is at FGF, EGF with between activin A and " extremely early stage " OV cancer stem cell.FGF and EGF causes the OV stem cell under any differentiation state (comprising extremely early stage OV stem cell) propagation.When activin A is in cell culture medium, activin A is allowed (permission) exclusively breeds the insensitive extremely early stage OV stem cell of activin A.If OV stem cell becomes responsive, so propagation will be stopped by activin A or reduce.

Be not common to the insensitivity of FGF and EGF, and there is not natural blocker.Can be mediated by follistatin the insensitivity of activin A, described follistatin is the natural blocker of activin acceptor.Follistatin can by same tumor cell or by the emiocytosis around tumor.Activin A is usually by the emiocytosis around tumor, and therefore encircling cell (suppression) and described tumor (promoting amplification) are likely depended in the amplification of tumor.The receptor (feature of extremely early stage undifferentiated carcinoma disease stem cell) lacking activin A can prevent surrounding tissue from controlling tumor.

In-vitro culture medium will containing embryonic stem cell sample colony.These colonies can by stromal cell around, described stromal cell can be normal fibroblast, the tumor cell that changes of the tumor cell of differentiation or mesenchymal cell.

The invention provides the method for the preparation of OV-CSC, wherein the total incubation time time needed for manipulation of centrifugal and sedimentation (comprise as changed culture medium, inoculate) be less than five months, be less than four months, be less than three months, be less than two months, be less than one month, be less than 150 days, be less than 120 days, be less than 90 days, be less than 60 days, be less than 30 days or be less than 150 days (+/-20 days), be less than 120 days (+/-20 days), be less than 90 days (+/-20 days), be less than 60 days (+/-20 days), be less than 30 days (+/-20 days).In exclusiveness embodiment, the present invention can get rid of for the preparation of any method of cancer stem cell with by any cancer stem cell colony prepared by described method, wherein manipulates the time that the required time is greater than time limit disclosed above (time-frame).Also provide by the time be in adherent cultures of one of time limit instruction above.This time be in non-adhesive culture as one of time limit above is also provided.In addition, the adherent cultures that is in identified by one of time limit is above provided to neutralize the assembly time be in non-adhesive culture.

epithelial cell is to the transformation (EMT) of mesenchymal cell

Knownly derive from epithelial tumor regression or be instead divided into mesenchymal cell state.Epithelial phenotype is immotile, contributes to being limited to the volume growth of tumor of tissue of rising, and usually more breaks up.When EMT occurs, cell obtains mobility, and produces adjacent tissue infiltration and remote cancerometastasis.The cell changed also obtains stem-like cell phenotype, has copying and breaks up the ability to cause new tumor (cancerometastasis) in the host tissue with (constitutional) tumoral character that rises.By EMT, tumor cell obtains other immunosuppression capability, Teat pipette and radiation resistance.

Culture media composition and physics system of selection promote external EMT phenomenon.The colony using EMT to change is prophylaxis of tumours recurrence as immunogenic advantage.The antigenicity of EMT cancerous cell can make immune system to identify and destroy the mobile cancerous cell causing cancerometastasis.In the process of cancerometastasis, these cells move with pole low number, and inoculation host tissue, reverts to epithelial cell phenotype (MTE transformation), grow and form the new tumor with the feature similar with primary tumor.Cause the necessary condition of external EMT to be form spheroid in serum-free medium, stimulate with bFGF, stimulate with BMP2,4 or 7, be seeded in subsequently containing RGD(Arg-Gly-Asp) on the adhesiveness substrate of peptide motif (such as, collagen protein, gelatin etc.).

EMT-OV-CSC subgroup is obtained by cultivating OV spheroid or early stage OV or mixing OV under the condition of culture described in such as table 1 and Fig. 1.

As used herein, term " OV-CSC " generally can refer to OV-CSC spheroid, early stage OV-CSC, mixing OV-CSC or EMT-OV-CSC.

oV-CSC is obtained from little tumor source

When sample cell number is less, use the alternative method that OV stem cell is selected.For example, after enzymatic dissociates, a small amount of living cells obtained from tumor is less than 10 × 10 ⁶individual living cells.For purposes of the present invention, contrast with the sample obtained from the tumor of excising (it is not usually considered as small sample and weight is at least 0.5 to 5-10 gram), small sample refers to the sample obtained from such as syringe needle biopsy body or peritoneal lavage thing.Core biopsy 18 or 16 or 14 specification syringe needles carry out, and produce 5-50mg sample.The relatively new program being called as vacuum assisted biopsy also uses 11 specification syringe needles and vacuum auxiliary device (vacuumassisteddevice; VAD) carry out.11 gage probes and vacuum auxiliary device match and usually obtain each core sample of 94mg.14 specification syringe needles and vacuum aided obtain 37mg usually, but only obtain 17mg when matching with automatic biopsy gun.Depicted in figure 1 in this alternative method (dotted arrow and square frame), before or after dissociating, the cell obtained from tumor sample is transferred to containing being rich in RGD(Arg-Gly-Asp) compound (such as, collagen protein, gelatin or MATRIGEL) adhesiveness substrate in, and under being in the existence of selection described herein (serum-free) culture medium.Described system of selection is designed to the local autocrine activity that (a) promotes to increase due to described stem cell clone with the amplification of the initial clones of the indivedual cancer stem cells existed compared with low number and (d) promotion.

Adhesiveness substrate is the protein being rich in RGD, as collagen protein or gelatin.Substrate can build by described protein or peptide being attached on various material (as polystyrene polycarbonate, cyclic olefine copolymer or glass).RGD peptide can be grafted on polymerism main chain (as hyaluronic acid, polylactic acid and combination).This base polymer can use the carrier end of somatomedin (as proteoglycan) (such as, heparin sulfate, chondroitin sulfate, keratin sulfate etc.) to strengthen further.

Cell culture surface can directly use or use the smears as amino containing silane to be used.Coating has adhesiveness characteristic (substrate) to cell and is applied to the compound on growing container material top.It can be native compound, as collagen protein or gelatin, and can by have mentioned group/end have more synthetic polymer to build.Smears (glue, as silane) may be used for the adhesiveness improving coating and culture vessel material (such as glass).If silane contains required group or end group, so can be used alone.

Make in this way and formula, a large amount of cell to be obtained in the relatively short time period.From several milligrams, tissue samples is (as contained 10 ³to 10 ⁶the syringe needle biopsy body of individual cell) culture can increase to about 10 in 3-4 week ⁸individual cell.

OV the amplification of stem cell culture and the generation of subgroup

As another feature of stem cell, OV-CSC can Immortalization and amplification.

In addition, OV-CSC can partially or completely break up.If remove expansion of stem cells condition, so propagation can be slowed down or stop to OV stem cell, and form is become the cell type more broken up with character mutation.Form can become flat, class epithelium or have the starlike of multiple core, and it is the feature of more mature ovarian cell or sternzellen.

Adherent cultures can dissociate and be transferred in non-adhesive (antibiont tack) condition to remove anchorage dependence noble cells in single cell suspension.After 2-3 days, stem cell tends to assemble and increases into globule with cloning, and described spheroid can be separated with single cell based on differential subsidence.Spheroid can to inoculate in adhesiveness condition and to breed further.If culture is occupied by noble cells or normal cell (as fibroblast), so this method will make described culture stem cell content be purified to 90%-99% stem cell content from 1%-30%.Described method can repeat to recover the many number of times required for stem cell purity.

Compared with the size of globule generally between 0.1mm and 2mm.With regard to each cell number compared with globule, size distribution is generally between 10 cells and 10,000 cell.Can be spherical or avette compared with the shape of globule, and also can occur by agglomerated form that is spherical or oval structures.

Patient-specific OV-CSC cell line may be used for the genome mutation causing tumor sex reversal when being identified in compared with the normal structure from same patient.Genome mutation can not be expressed in each stage of differentiation.Some Function protein or transcription factor are only temporarily express, and can during maturing disappear, and generation is out of shape but is had the cell of normal protein matter.Identifying the cell colony of expressing sudden change to greatest extent and this colony be exposed in immune system can be use cancer stem cell as the major advantage of immunotherapy antigenic source.

By identifying genome mutation, the individualized preparation for treatment of cancer can be produced, such as micromolecule, DNA sequence, antisense RNA or combination.

This kind of cell line can be further used for producing the sifting motion cultivation plate (such as 96 holes) for drug discovery.Multiple pedigrees from various patient can combine the variability solved between experimenter in single culture plate.

Ovarian cancer carcinoma stem cell can keep rising some characteristics of tissue, as secretory protein, somatomedin and hormone (functioning tumour).In view of the immortal feature of cell line, these characteristics can be adopted produce " self " protein (such as albumin, transforming growth factor (TGF), insulin, glycemic element, DOPA etc.) that may be used for same patient.Cell can be incorporated in small-sized biological reactor, and collects secretory product, and purification also stores to use same patient.This method is particularly advantageous, is the immune repellence that patient will not develop as more traditional biological analog.

loaded dendritic cell

The indivedual OV-CSC cells obtained from patient may be used for producing the antigen for immunotherapy.The advantage of purified stem line is used to be good signal to noise ratio.More mature cells from tumor can have can cover antigenic compensatory mechanism, and can can't help immune system recognition.As antigenic source, OV-CSC can use by the form of the form of the form of living, mitosis inactive form, non-live or fragmentation.Various method may be used for modified cells for optimum antigen-exposed: radiant (such as, γ, UV, X), temperature (such as, hot or cold) or chemistry (such as, cell growth inhibition, aldehyde, ethanol) or combination.

In an exemplary embodiment, reagent for dendritic cell activated (DC) and method are contained in the present invention, and it uses one or more immunological adjuvants, as GM-CSF; Toll sample receptor (TLR) agonist, such as CpG-oligonucleotide (TLR9), imiquimod (TLR7), poly-(I:C) (TLR3), glycopyranosyl lipid A (TLR4), murein (TLR2), flagellin (TLR5); And adjuvant, such as, as CD40 agonist, CD40L; Or cytokine, interferon-γ, PGE2 etc.See such as United States Patent (USP) 7,993,659; US7,993,648; US7,935,804, the full content about activation DC disclosed in wherein each is incorporated herein by reference.The present invention is contained with the one or more ex vivo treatment DC in adjuvant reagent above, or additionally or alternatively, gives described adjuvant to human experimenter, animal subjects or veterinary experimenter.

Immune system contains cellular immunity, humoral immunity and complementary reaction.Cellular immunity comprises and relates to dendritic cell, CD8 ⁺t cell (cytotoxic T cell; Cytotoxic lymphocyte) and CD4 ⁺the cell of T cell (helper T cell) and the network of event.Dendritic cell (DC) obtains polypeptide antigen, and wherein these antigens can from the outside acquisition of described DC or by the inner side biosynthesis of infectious organisms at described DC.DC process polypeptide, produce length be about ten amino acid whose peptides, described peptide is transferred to form complex in I class MHC or II class MHC, and described complex is shuttled back and forth move to described DC surface on.When the DC with I class MHC/ peptide complexes contacts CD8 ⁺during T cell, result is described CD8 ⁺the activation of T cell and propagation.About the effect of II class MHC, when the DC with II class MHC/ peptide complexes contacts CD4 ⁺during T cell, result is described CD4 ⁺the activation of T cell and propagation.Although " can activate " described T cell to the dendritic cell of T cell antigen-presenting, the T cell of activation may not set up effective immune response.CD8 ⁺the effective immune response of T cell usually needs to stimulate DC in advance by one or more in multiple interaction.These interact to comprise and make CD4 ⁺t cell directly contacts DC(by means of making described CD4 ⁺the CD40L contact DCCD40 receptor of T cell) or make one of TLR agonist toll sample receptor (TLR) directly contacting dendritic cell.

Humoral immunity refers to B cell and antibody.B cell becomes and changes plasma cell into, and described plasma cell expression and secretion antibody.The difference of B progenitor cells (na veBcell) is its not presentation markup CD27, and antigen-specific b cells expresses CD27.Secreted antibody can subsequently with reside in the tumor antigen on tumor cell surface and be combined.Result is that the cell that infects or tumor cell become and be marked with antibody.When the cell of antibody and infection or tumor cell in conjunction with, in conjunction with antibody-mediated cell or the tumor cell killing infection, wherein killing is undertaken by NK cell.Although NK cell is not configured for be configured for T cell identify that the mode of target antigen carrys out identification specificity target antigen, the ability that NK cell is combined with antibody constant region makes NK cell can kill the cell being marked with antibody specifically.The identification of NK cell antagonist is receptor-mediated by (the NK cell) Fc be combined with antibody Fc portion.Such killing is called as antibody dependent cellular toxicity (ADCC).NK cell also can kill the cell not relying on ADCC mechanism, and wherein this killing needs the expression of I class MHC to lack in target cell or deficiency.

When not wishing by any specific mechanism constraint, the present invention is contained and is given cancer stem cell antigen or give the dendritic cell that load has cancer stem cell antigen, the generation of the one or more antibody in cancer stem cell antigen described in wherein said antigenic stimulus specific recognition, and wherein said antibody-mediated ADCC.Phrase load has antigen to refer to, and dendritic cell is caught living cells, catches non-viable non-apoptotic cell, catches dead cell, catches polypeptide or is caught the ability of peptide etc.Tumor antigen comprises OC-CSC cell extract, OC-CSC lysate or complete OC-CSC cell.In another embodiment, tumor antigen comprises ex vivo transfection to the messenger RNA in dendritic cell.

The present invention is contained and is caught by cross presentation.Also contain the antigen-presenting cell using not dendritic cell, as macrophage or B cell.

" delayed hypersensitivity " technology may be used for distinguishing the immunoreation relating generally to cellular immunity or relate generally to humoral immunity.The positive signal indicator cells reaction of delayed hypersensitivity.

The invention provides the compositions and method that make tumor cell inactivation, it is such as undertaken by radiation, nucleic acid crosslinking agent, polypeptide linker or these combination.Crosslinked is that two of polymer molecule chains are attached by bridge, and described bridge is formed by being engaged the element of some carbon atom in described chain, group or compound by main chemical bond.Be cross-linked and occur in the material be made up of the polypeptide chain of the disulfide bond joint by relating to two cysteine residues at occurring in nature; as in keratin or insulin; trivalent pyridinoline and the pyrroles of ripe collagen protein are cross-linked; and crosslinked in clot, it relates to covalency ε-(γ-glutamyl) lysine cross-links of the γ-carboxyl-between amine groups and the epsilon-amino of lysine residue in glutamine residue.

Be cross-linked and can realize to manually in protein, by interpolation chemical substance (cross-linking agent) or by carrying out high energy radiation to described polymer.The crosslinked enhancing immunoreation and complete Inhibit proliferaton with fixative and thermoinducible gathering are shown.May be used for making the protein cross on OV-CSC surface and the material therefore preventing OV-CSC from breeding includes, but is not limited to 10% neutral formalin buffer, 4% paraformaldehyde, 1% glutaraldehyde, 0.25-5mM suberoyl imidic acid dimethyl ester (dimethylsuberimidate), ice-cold 100% acetone or 100% methanol.In addition, 1% glutaraldehyde and the combination of 4% paraformaldehyde in 0.1M phosphate buffered solution can also be used.

Show that formaldehyde and glutaraldehyde both induce the activation of 1 type and 2 type t helper cells.Exactly, also show that thermoinducible antigen aggregation strengthens the interior initiation of body of cytotoxic T lymphocyte.Cause the combination of antigen and dendritic cell to increase by the antigen crosslinking of 3,3'-dithiobis (propanoic acid sulfosuccinimide ester), and crosslinked antigen process via the proteasome path for antigen presentation.In addition, the fixing ovarian cancer tumor cell of formalin in clinical trial, and without propagation evidence.

In one embodiment, fix whole OV-CSC with cross-linking agent, and subsequently used as the antigenic source combined with dendritic cell.

In another embodiment, the nucleic acid of cell is made to be cross-linked.Exemplary core dialkylaminobenzoic acid agent is Beta-alanine, N-(acridine-9-base), 2-[two (2-chloroethyl) is amino] ethyl ester.Usually have with the Exemplary cross linking agents (as psoralen (psoralen)) of ultraviolet (UVA) radiating composite and DNA is cross-linked but the not modified ability of retaining protein.For example, nucleic acid target compound can be 4'-(4-amino-2-oxa-) butyl-4,5', 8-trimethylpsoralen (S-59).Can with 150 μMs of psoralen S-59 and 3J/cm ²uVA light (FX1019 radiation appliance, BaxterFenwal, RoundLake, IL) makes cell inactivation.Use the inactivation of S-59 and UV light to be called as photochemical treatment, wherein treatment conditions can be conditioned or titration one-tenth makes the DNA be cross-linked reach to prevent cell division completely but wherein to the degree that the damage of polypeptide (comprising polypeptide antigen) minimizes.Can by cell suspension in the 5mL saline containing 0,1,10,100 and 1000nM psoralen S-59.Sample can at roughly 2J/cm ²dosage under carry out UVA radiation.Each sample can be transferred in 15mL test tube subsequently, centrifugal and remove supernatant, and use the water washing of 5mL salt subsequently, centrifugal and remove supernatant, and final precipitation is suspended in 0.5mL saline.See United States Patent (USP) 7,833,775 and 7,691,393, the wherein disclosed full content about cell inactivation is incorporated herein by reference.

For any cell preparation with cross-linking agent process, splitting ability can by those skilled in the art by cultivating or cultivate at least one week, at least two weeks, at least three weeks, at least surrounding, testing at least five weeks, at least two months, at least three months, at least four months etc. in standard medium.Cell division can be assessed by disclosing chromosome and disclosing the dyeing whether cell division occur.Cell division also can be measured by counting cell.Therefore, the cell number in culture plate keeps stable within the period of two weeks, one month or two months etc., the conclusion that cell cannot divide can reasonably be drawn.

In one embodiment, subcutaneous (SC) gives dendritic cell vaccination Immunogenic Compositions.In other embodiments, each dosage, in the scope of about 500 ten thousand-2 thousand ten thousand support type DC, repeats with the form of a series of 6-10 dosage.In certain embodiments, every five days, weekly, every 10 days, give dosage week about or every two weeks, continue two, three, four, five or six dosage, then every two weeks, every three weeks, often surrounding, every month, every five weeks or every 6 weeks give dosage, continue the extra dose of two, three, four, five or six dosage, to reach 6-10 dosage altogether.In one embodiment, front four injections give weekly, continue one month, and within one month subsequently, once give rear 4 injections.In an alternative embodiment, administration continues 3 weeks weekly, within one month subsequently, once continues 5 months, to reach 8 administrations altogether.

Each dosage comprises about 5-20 × 10 ⁶individual support type DC, about 5-17 × 10 ⁶individual support type DC, about 6-16 × 10 ⁶individual support type DC, about 7-15 × 10 ⁶individual support type DC, about 7-14 × 10 ⁶individual support type DC, about 8-13 × 10 ⁶individual support type DC, about 8-12 × 10 ⁶individual support type DC or about 9-11 × 10 ⁶individual support type DC.In further embodiment, each dosage comprises about 8 × 10 ⁶individual support type DC, about 9 × 10 ⁶individual support type DC, about 10 × 10 ⁶individual support type DC, about 11 × 10 ⁶individual support type DC or about 12 × 10 ⁶individual support type DC.The mixture of remaining OV-CSC that support type DC comprises DC and do not absorbed by described DC.The dosage given comprises the mixture of these cells, and described dosage reflects this mixture.

In another embodiment, support type DC is given together with pharmaceutically acceptable carrier or excipient.Pharmaceutically acceptable excipient described herein (such as mediator, adjuvant, carrier or diluent) is that those skilled in the art knows and the public can easily obtain.Preferably, pharmaceutically acceptable carrier or excipient are to the chemically inert carrier of support type DC or excipient, and are under conditions of use without carrier or the excipient of harmful side effect or toxicity.

The selection of excipient or carrier will partly decide by specific therapeutic composition and by the ad hoc approach being used for giving described compositions.Preparation described herein is only exemplary, and not in any limiting sense.

The upper acceptable carrier of physiology is usually aqueous pH buffer solution.The example of the upper acceptable carrier of physiology includes, but is not limited to saline; Solvent; Disperse medium; Cell culture medium; Aqueous buffer solution, as phosphate, citrate and other organic acid; Antioxidant, comprises ascorbic acid; Low-molecular-weight (being less than about 10 residues) polypeptide; Protein, as serum albumin, gelatin or immunoglobulin; Hydrophilic polymer, as polyvinylpyrrolidone; Aminoacid, as glycine, glutamine, agedoite, arginine or lysine; Monosaccharide, disaccharide and other carbohydrate, comprise glucose, mannose or dextrin; Chelating agen, as EDTA; Sugar alcohol, as mannitol or Sorbitol; Form the counter ion counterionsl gegenions of salt, as sodium; And/or non-ionic surface active agent, as TWEEN, Polyethylene Glycol (PEG) and PLURONICS.

In some example embodiments, give adjuvant with each dosage simultaneously.In certain embodiments, cell dosage is suspended in the carrier containing adjuvant.In alternative exemplary embodiment, give adjuvant, but give together with not single with each dosage.In other exemplary, not there is adjuvant.In one embodiment, adjuvant is GM-CSF.

In the case of unrestricted, make dendritic cell (such as, autologous or allogeneic dendritic cells) and the cancer stem cell antigen contact in cell lysate, sour eluent, cell extract, partially purified antigen, the antigen of purification, the antigen be separated, partially purified peptide, the peptide of purification, the peptide be separated, synthetic peptide or its any combining form.Subsequently to experimenter, the experimenter such as suffering from OV or the contrast experimenter without OV give described dendritic cell.In an exemplary embodiment, make dendritic cell contact with experimenter by one or more in following approach, be expelled in experimenter or give: subcutaneous, intraperitoneal, tuberosity interior (intranodal), intramuscular, intravenous, intranasal, suction, oral, by being applied to enteral chamber etc.In addition, immunogenic composition directly can be administered to the site of tumor or cancerometastasis.

Embodiment

embodiment 1. generates cancer stem cell system by ovarian tumor sample

After correct acquisition informed consent, during operative procedure, obtain the oophoroma sample from 3 patients.By in sample transport to described organized processing facility and under during transportation maintaining 4 DEG C of-10 DEG C of temperature in containing antibiotic culture medium.

In sterile petri dish (Petridish), mince sample with knife blade, with continuous stirring fragment be exposed to subsequently and depend on tissue consistency containing Collagenase-I(, organize 3000UI to 6000UI for every gram) solution in.

Wash the cell, the counting and freezing with aliquot form that dissociate in the medium, for using in the RPMI culture medium containing 15%FBS and 10%DMSO after a while.

Typical case's aliquot contains 10-30 × 10 ⁶individual cell, and expection survival rate when thawing is 40% to 75%.Aliquot may be used for the purification and amplification after a while of cancer stem cell.

embodiment 2. is separated for OV-CSC and the best medium of amplification forms

By three kinds of commercially available ovarian cancer cell lines and freezen protective by 3 the different sample solutions obtained from ovarian cancer patients from tumor cell compared with.

Glomeration body (spherogenic) characteristic of cancer stem cell is used to be separated cancer stem cell.Use two kinds of different culture medias that the compositions (Lit-M) described by document as shown in table 5 and proprietary formula (CSC-M) form to assess the glomeration bulk properties of tumor lineage.

Thaw from blast cell storehouse by OVCAR-3, SKOV-3 and TOV-21G business cell line and from the enzymic digestion tumor of patient.After counting and survival rate assessment, by from every square centimeter 50 of each pedigree, 000 seeded with living celis is in the ultralow adhesiveness flask (Corning) appropriately identified.

Make culture grow 7 days, and every two days or press Monday-Wednesday-Friday schedule replace culture medium.In order to feed culture, collecting cell suspension in 50mL test tube, with 150rcf centrifugal 3 minutes, and replace supernatant by fresh CSC-M or Lit-M culture medium.

After 7 days, cell is transferred in ordinary cells culture flask, and culture medium is changed over Lit-E and CSC-E(amplification) formula (table 5).CSC-E culture medium is amplification culture medium and contains serum; CSC-M is serum-free Selective agar medium.CSC-E and CSC-M is both based on the basal medium of table 2-4.First with Collagenase/hyaluronic acid enzymatic mixture, spheroid is dissociated 5-10 minute and imbibition lightly, dezymotize by centrifugal making a return journey and replace by culture medium subsequently.Feed schedule according to M-W-F and make cell amplification 2 generation, at every turn all with 10, the density of 000 cells/square cm inoculates.Finally by cell with 5,000-10, the density of 000 cells/well is transferred in 96 well culture plates for immunocytochemical assay.After 2 days, fix culture plate with 4% paraformaldehyde, and dye for following labelling: 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.for the culture medium prescription in OV-CSC purification and amplification

At the end of growths in 7 days in ultralow adhesiveness condition, business cell line does not form exemplary cancers stem cell spheroid in Lit-M or CSC-M culture medium.Replace, its formation has erose loose cell agglomerated thing (Fig. 2).Patient tumors forms typical tight spherical structure, its stem cell spheroid (Fig. 3) increased gradually in 7 days of having recombinated.

After being transferred to by cell in adhesiveness condition, culture slowly increases, and reassembles into epithelial cell monolayer.The little cuboid cell phenotype that the display of some cells grows fast, and some are configured as the cytoplasmic large epithelial cell or little fusiformis restructuring mesenchymal cell that have and occur vacuole.Compared with Lit-E condition, business cell line increases comparatively fast in CSC-E culture medium.Patient's pedigree increases comparatively slow in the same terms, impaired when going down to posterity, and shows Cytoplasm vacuole, departs from and a large amount of cell death from substrate.

In this experiment, we find that business cell line has and are suitable for high concentration FBS(15%) more differentiation and uniform phenotype, but shown by the typical glomeration bulk properties of shortage, it may not contain cancer stem cell.Replace, clinical samples contains the cancer stem cell of the fine and close spheroid be enough to desired by generation.When transferring in the culture medium containing 15% serum, patient's pedigree is impaired compared with business pedigree, and can not carry out correct immunocytochemistry sign immediately after spherical inoculation.

table 6.be derived from the ICC scoring general introduction of the ovarian cancer cell line of patient

The positive grade of reactive %: +=1%-25%, ++=26%-50%, +++=51%-75%, +++ +=76%-100%.Strength grade: 1=is slight, 2=moderate, 3=height.

As indicated in ICC result (table 6), EMT phenotype (epithelial cell is to the transformation of mesenchymal cell) shown by culture: low EpCAM, high NCAM(Fig. 4 A-D), Slug/Snail(Slg/Snl), CD117(Fig. 5 A-D) and nestin (Fig. 6 A-C).

In a word, 1) in CSC content, business cell line inequivalence is in fresh clinical samples; 2) OV-CSC to culture medium composition and physical manipulation responsive, and do not tolerate in institute's test condition and go down to posterity; 3) proprietary culture medium preparation (CSC-M and CSC-E) contributes to the growth of OV-CSC; And the condition of culture 4) tested is conducive to EMT.

embodiment 3: the single inoculation amplification of tumor cell

Typical case's Cell culture invitro is cyclic process, wherein with comparatively low-density inoculating cell, allows its growth until reach and converge, makes it subsequently to dissociate, and is again seeded on larger surface with comparatively low-density and (goes down to posterity).We draw to draw a conclusion at previous observation: OV-CSC has sensitivity in period of going down to posterity to physics and enzymatic manipulation, and this is embodied by the transformation (EMT) to mesenchymal cell of differentiation and a large amount of cell death and epithelial cell.In order to solve this phenomenon, in experiment subsequently, we test OV-CSC single to go down to posterity the probability of amplification, with providing active specific immunotherapy (activespecificimmunotherapy; ASI) cell is seeded on final surface by the low-density of required total cell number.In usual cell condition of culture, single is avoided to increase with the culture space of saving in incubator and dissociated by periodicity enzymatic and avoid contact inhibition to stimulate cellular proliferation.

In this approach, with the various low-density inoculating cells of 5000,10,000 and 15,000 cells/square cm, and allow its amplification 4 days.With Monday-Wednesday-Friday feeds schedule and to be used in initial formulation only containing 5% but not the CSC-E culture medium (table 5) of 15%FBS feeds culture.At the end of, cell is counted, and analyzes its phenotype.

Cell counting in various condition does not show significant difference in the doubling time.No matter Initial seeding density, immunocytochemistry (immuno-cytochemistry; ICC) identical overview (table 7) is disclosed.

table 7.iCC marks general introduction

The positive grade of reactive %: +=1%-25%, ++=26%-50%, +++=51%-75%, +++ +=76%-100%

Strength grade: 1=is slight, 2=moderate, 3=height.

Dissociate cause differentiation in order to avoid Cells Depletion or by machinery or enzymatic, can use a kind of method by cell amplification to required number, described method by first allowing culture to grow until reach and converge and do not repeat to go down to posterity to carry out with low-density inoculation before final collection.In addition, as the serum of higher concentration (15%) in earlier experiments (table 6), according to ICC overview (table 7), the culture medium containing 5%FBS causes EMT.

embodiment 4. determines the cell culture condition selecting early ovarian cancer stem cell

As shown in earlier experiments, the culture medium containing FBS causes cancer stem cell to break up and EMT.This phenomenon can by the factor contained in serum, as bone morphogenetic protein (BMP4) causes.Containing in the culture of FBS, or by adding BMP4, cell seems larger, has for typical epithelial cell outward appearance and shorter multiplication rate differentiated carcinoma.If add somatomedin in this culture medium, so observe EMT.In vivo, ovarian cancer is almost complete to increase in abdominal cavity, and the raised growth factor of wherein peritoneal cell secretion can accelerate tumor amplification.This kind of somatomedin comprises FGF, EGF, VEGF, HGF, PDGF, activin A and TGF β.

Experimentation subsequently allows culture medium and the substrate composition of ovarian cancer stem cell rapid amplifying.

The ovarian tumor cell being derived from patient is seeded in normal tissue and cultivates plastics, 1% gelatin or MATRIGEL ^?(1:60) on.For each substrate condition, use various culture medium prescription: with the CSC-E of 5%FBS modification; CSC-M(table 5); For the commercial medium KGM-GOLD (Lonza) that epithelial cell (keratocyte) is allocated; And for hESC cultivate serum-free culture based formulas (STEMBLAST ^?).When add or do not add somatomedin bFGF(10ng/mL) and EGF(10ng/mL) test each condition.

The low calcium allotment of KGM-GOLD basal medium is increased for epithelial cell.STEMBLAST ^?it is the serum-free formula containing 5ng/mL activin A for cultivating embryonic stem cell.MATRIGEL ^?it is the compound cells epimatrix containing laminin，LN, collagen protein and proteoglycan.

Cultivate in these conditions after 1 week and carry out morphological analysis and growth performance, and summarize in table 8.

table 8.be derived from the performance of ovarian cancer culture in various culture medium, substrate and somatomedin condition of patient

MATRIGEL ^?the growth of inhibition tumor cell in all culture medium or somatomedin condition.The less adhesiveness of tissue culturing plastic's (through plasma treatment) showed cell when initial inoculation, but the abundant amplification really all supporting tumor cell in all conditions.Gelatin (0.1%) coating allows extremely good initial adherence, and supports the rapid amplifying of tumor cell.

Though substrate how or somatomedin whether exist, KGM-GOLD does not support the rapid amplifying of tumor cell, causes the ovarian cancer cell occurred together with the Cytoplasm occurring vacuole with maxicell body old and feeble.

CSC-E culture medium containing 5%FBS is the rapid amplifying (Fig. 7 and 8) of sustenticular cell under bFGF and EGF exists only, no matter and whether somatomedin exists, serum-free version all supports rapid amplifying (Fig. 9 and 10).Animal serum seems containing the factor suppressing ovarian tumor cell rapid amplifying, and described effect compensates by adding this somatomedin (FGF, EGF) as the part of receptor tyrosine kinase.Receptor tyrosine kinase (receptortyrosinekinase; RTK) other part (as VEGF, HGF, PDGF) can have phase same-action to tumor cell proliferation.The form of the cell grown in the serum-free medium being supplemented with FGF and EGF has little cube type, is filled in and is similar in the intensive and uniform colony of embryonic stem cell cultures.

STEMBLAST ^?only under somatomedin exists, support good amplification.STEMBLAST ^?middle EGF, FGF cause the form extremely similar with embryonic stem cell cultures with combining of activin A, have minicell body and occupy the most macronucleus of cell and MIN Cytoplasm (Figure 11).

In serum-free medium, cancer stem cell phenotype and insignificant EMT are disclosed for the immunocyte Epidemiological Analysis of common ovary and stem cell cancer marker.

Ovarian cancer epithelial cell marker is being found, labelling CA125(Figure 12 B) more than on the cell of 90%.Another kind of ovarian cancer epithelial cell marker MUC-1 sees (Figure 12 C) in the cell of about 60% with various expression intensity.Ovarian cancer cell cytokeratin markers CK8 is present in (Figure 13 B) in the cell more than 80%.

Express EpCAM(Figure 14 C by existing with high percentage ratio (being better than 75%)), CD44(Figure 15 C) and CD133(Figure 16) cell indicated by, condition of culture promotes the amplification of cancer stem cell.Culture shows rapid amplifying by the high level expression of Ki67 proliferative labelling (Figure 13 B) on most cells.

We draw to draw a conclusion: have the serum-free medium of RTK part of minimum tolerance or the low concentration animal serum in the culture medium being supplemented with RTK part (as FGF, EGF) and can optimally to maintain and increase the colony of ovarian cancer stem cell.The amount and the minimizing RTK part that increase serum will cause CSC to break up.Increase serum-concentration and increase RTK ligand concentration and will cause EMT.Serum-free medium containing RTK part and activin A can maintain the extremely early stage stem cell state of embryonic stem cell sample.

Although the existence of gelatin (collagen protein) coating increases initial cell adhesiveness, it also insignificantly changes rate of amplification or the phenotype of cell.

These experiments show that we are separated from oophoroma, purification and amplification CSC and manipulate it and break up or the ability of EMT.Compared with the antigenicity of the dilution of block tumor, cell colony described herein can be used as the high-quality antigenic source of full cytoactive autoimmune therapy.

embodiment 5. produces support type dendritic cell compositions

Antigenic source is the autologous tumor cell of the autosynthesis cell from the continuous propagation being derived from patient's fresh tumor tissue.These cells have the feature of tumor stem cell.Operation and pathological changes environment institute if having time in, strictly observe aseptic scheme to dispose biopsy body to guarantee that sample is aseptic.

Pathologist obtains flesh tissue from the biopsy body of patient tumors.Use sterile scalpel and tweezers, sample cut into 10mm section and transfer in the transport test tube containing transport medium, process is to avoid sample dry rapidly.After excision within 48, by overnight courier by manufacturing facility described in sample transport.

In manufacturing facility, in toilet, sample is dissociated into single cell suspension, and is placed in the cell culture condition being designed to make OV-CSC enrichment and propagation.During process tumor sample, eliminate normal cell, as lymphocyte, stromal cell and connective tissue.After completing amplification and purification step, made the propagation OV-CSC(tumor cell of enrichment by radiation, TC) inactivation, and be placed in gas phase liquid nitrogen storage tank.Depend on quantity and the quality of tumor sample, this process may need up to eight weeks.

Once tumor cell product is by the quality assurance, patient is apprised of the program (usual six lift sequences) that will carry out being called as Leukapheresis.This process need filtering blood is to collect peripheral blood mononuclear cell (PBMC).By overnight courier, collected PBMC being transported in manufacturing facility, being further purified with the adverse current density centrifugation by being called as elutriation.Elutriation is to make the process of the cell enrichment that can become antigen-presenting cell or dendritic cell by other lymphocyte purifying monocytes.In order to generate dendritic cell, the mononuclear cell of institute's elutriation is cultivated six days with cytokine GM-CSF together with interleukin 4 (IL-4).

At the 6th day, from keep in cold storage, shift out purified tumor cell product, thaw and combine 18-24 hour with dendritic cell.This process produces " antigen load " of DC.End product is DC completely, maybe can be considered as allowing containing the TC(through overshoot of some remnants) and be called as DC-TC.Collect the dendritic cell/tumor cell mixture of combination, freezen protective to keep the survival rate of dendritic cell, and is stored in gas phase liquid nitrogen.

, under gas phase liquid nitrogen condition, described batch to be transported in treatment facility after discharging autogenous cell therapy product at finished goods matter check analysis.After arrival, under cell therapy product being stored in gas phase liquid nitrogen condition, until prepare administration.

the II phase of autologous peripheral blood mononuclear cell in the patient suffering from ovarian cancer in embodiment 6.Ovapuldencel-T contrast GM-CSF, double blinding, random, clinical trial

This research is that load in ovapuldencel-T(GM-CSF has the autologous dendritic cell of the autologous OV-CSC through overshoot) contrast GM-CSF(MC) in autologous peripheral blood mononuclear cell in the patient suffering from III phase or IV phase epithelial cell ovary, fallopian tube or Primary peritoneal carcinoma as to maintain after subtracting long-pending operation and adjuvant chemotherapy or II phase of component of secondary therapy, double blinding, random, single centre are tested.The object of research is from the date of random packet, compare total survival rate (OS) of patient and the patient treated with autoblood mononuclear cell in GM-CSF matched group treated with ovapuldencel-T.Method according to embodiment 5 prepares ovapuldencel-T.

Nearest diagnosis has 18 years old or larger female subjects of III phase or IV phase ovarian cancer (its be operation subtract sum accept chemotherapeutic candidate) to be the candidate of described research.Performance state when registering ASI treatment must be that ECOG must be divided into 0 or 1.

Selected criterion comprises the histodiagnosis of epithelial cell ovary, fallopian tube or Primary peritoneal carcinoma; Late period (transitivity, III phase or IV phase) epithelial cell ovary, fallopian tube or Primary peritoneal carcinoma and to be that operation subtracts long-pending with the candidate obtaining new fresh and alive tumor tissues (for establishing short-term tumor cell line); Age >18 year; Each patient must know the neoplasia character of her lysis and must agree to voluntarily manipulate for establishment tumor cell line to tumor tissues; And patient must have and moves to therapeutic community and treat to carry out ASI the ability and wish that give.

Eliminating criterion comprises ECOG performance state and is greater than 2; Known to Type B or C type hepatitis or HIV-positive; Pregnancy or breast-feeding female; Need initiatively therapeutic treatment with the abnormal potential heart disease that is associated of myocardial function or the unstable angina with Atherosclerotic cardiovascular disease association, or under the treatment being in tremulous pulse or venous peripheral blood vessel disease; Be regarded as the diagnosis of life-threatening other invasive cancer any in 5 years afterwards, and/or carry out anti-cancer therapies (as proceeding for the carcinoma of prostate of diagnosis before more than 5 years or the hormonotherapy of breast carcinoma) for the cancer except ovary; The remarkable life-threatening active infection of possibility or other active medicinal condition of illness, comprise active blood condensation or hemorrhagic diathesis; Active central nervous system's cancerometastasis during treatment; Known autoimmune disease, immunodeficiency or relate to and use the lysis of immunosuppressive therapy; The tumor of low malignant disease probability; Or another kind of research medicine was accepted in 28 days of the first dosage.

The patient that roughly 99 are suffered from III phase or IV phase epithelial cell ovary, fallopian tube or intraperitoneal cancer will be recruited, successfully cell line is established to described patient, and described patient meets ASI when random packet treats condition, and described patient has agreed to the test and the treatment that participate in use ASI.

Patient from after subtracting long-pending surgery recovery and before chemotherapy, will carry out Leukapheresis to obtain PBMC to patient, autologous dendritic or mononuclear cell are derived from described PBMC.After Leukapheresis, according to the nursing standard of this kind of patient, patient will be carried out to the one-level adjuvant chemotherapy of 6 circulations according to plan, described chemotherapy comprises taxane and platinum chemotherapeutant.

According to the blood CA-125 raised and/or other tumor marker and/or the disease detection of being undertaken by physical examination or imaging, patient will be classified to: (1) platinum resistance, based on the process during adjuvant chemotherapy or the detectable disease at the end of complementary therapy; Or (2) platinum is responsive, does not have disease evidence (NED) at the end of complementary therapy.

After the fractionation, patient will with 2:1 ratio random packet in ovapuldencel-T or MC treatment group.This method produces two PATIENT POPULATION similar clinically, carries out standardization to therapy arrangement of time, maximizes the use of cell line, and is incorporated to the practice standard about maintenance or secondary complementary therapy:

The patient suffering from platinum resistance disease will manage doctor according to it and use secondary therapy for treating, and will accept ovapuldencel-T or MC treatment simultaneously.

The platinum sensitive patients with NED will manage doctor according to it and accept maintenance therapy (normally paclitaxel continued up to a year), and will accept ovapuldencel-T or MC treatment simultaneously.

Random packet impacts the autologous dendritic cell of sensitization (pulse) to produce patient-specific therapy by accepting with the autologous OV-CSC cell through overshoot to the patient in ovapuldencel-T group.Depend on the arrangement of time of maintenance or secondary therapy, ovapuldencel-T will be given in 500 microgram GM-CSF, continue three weeks weekly, and subsequently when giving follow-up maintenance or secondary therapy every three to surrounding once, in four to six months, give the vaccine therapy of 8 dosage at most altogether.

Depend on the arrangement of time of maintenance or secondary therapy, random packet will be received in the autologous monocyte given in 500 microgram GM-CSF to the patient in MC group, continue three weeks weekly, and subsequently when giving follow-up maintenance or secondary therapy every three to surrounding once, in four to six months, give the ASI therapy of 8 dosage at most altogether.

While research the patient of experience PD can continue ASI and with managed other modular system Sex therapy any that oncologist specifies by it and be combined, until given all 8 therapeutic doses.

Main goal in research is from the date of random packet, compare total survival rate (OS) of patient and the patient treated with autoblood mononuclear cell in GM-CSF matched group treated with ovapuldencel-T.

The Primary Endpoint of this test is the death due to any reason, and its tolerance is the total survival rate (OS) from the date of random packet.The progresson free survival phase, (PFS) was secondary endpoints, and with from random packet with carrying out date for the treatment of until the time form of subjective tumour progression or death calculates.Progress is subjectively defined by treating physician, and is based on tumor marker level (such as CA-125) and/or imaging.Secondary strategic point, PFS and OS is defined as from subtracting the date of long-pending operation.

There are all patients participating in research qualification to continue following the trail of from the date of random packet maximum 5 years or until dead, with being as the criterion of first occurring.

Kaplan-Meier curve (Kaplan-Meiercurve) is by the time-to-live of each treatment group of display.Log-Rank Test (logranktest) analyzes OS to test the null hypothesis without treatment difference by being used for.Cox regression model (Coxregressionmodel) and Grindelwald inspection (Waldtest) will be used for estimating and treat the Hazard ratio that is associated and identify that potential predictor and its (if existence) are on the significance of impact for the treatment of difference.Analysis based on the intention for the treatment of colony will be regarded as Main Analysis, will be regarded as sensitive analysis based on the analysis by scheme colony.The subgroup analysis of OS terminal will imitate the plan of arranging for the Primary Endpoint of platinum resistant patients and platinum sensitive patients respectively.

Except as otherwise noted, otherwise all numbers being expressed as component, character (as molecular weight), reaction condition etc. used be in the specification and claims interpreted as all being modified by term " about " in all cases.As used herein, term " about " and " roughly " mean in 10% to 15%, preferably in 5% to 10%.Therefore, be contrary unless indicated, otherwise the numerical parameter of setting forth in description and appended claims may depend on attempt to be obtained by the present invention needed for character and the approximation changed.Minimally, and do not attempt restriction equivalent principle to the application of Claims scope, each numerical parameter should at least be explained by applying the generally technology of rounding up according to the number of reported significant digits.Although numerical range and the parameter of setting forth broad range of the present invention are approximations, the numerical value of setting forth in instantiation is as far as possible accurately report.But any numerical value is inherently containing some error that must be caused by the standard deviation found in other thermometrically value of its point.

Unless indicate in addition herein or obvious and contradicted by context, otherwise the term " (a/an) " that (when especially at following claims) is used under description situation of the present invention, " described " and similar indicant should be interpreted as containing odd number and plural number.Describing of value scope herein is only intended to serve as the shorthand method individually mentioning each independent value be in described scope.Unless instruction in addition herein, otherwise each indivedual value is incorporated in this description, as it is individually enumerated in this article.Unless instruction or in addition obvious and contradicted by context in addition herein, otherwise all methods described herein can be undertaken by any suitable order.The use of any and all examples provided herein or exemplary language (such as, " as "), only intends to illustrate the present invention better, and does not cause restriction to the otherwise required scope of the invention.Any language in this description should not be interpreted as indicating the key element of any failed call to be required for putting into practice for the present invention.

The grouping of substituting key element of the present invention disclosed herein or embodiment should not be construed as restriction.Each group member can individually or to mention and requirement with any combining form of other member in described group or other key element of finding herein.Expection, the one or more members in group can be included in group or by group for the reason of convenience and/or patentability and delete.When any this kind of comprise or delete occur time, this description be considered to as with revising containing group, therefore meet the written description of all Ma Kuxi groups (Markushgroup) used in appended claims.

There is described herein certain embodiments of the present invention, comprise the present inventor known for carrying out optimal mode of the present invention.Certainly, after reading the above description, the change of embodiment described by these will become apparent for those of ordinary skill in the art.The present inventor expects that those skilled in the art adopt this kind of change in due course, and the present inventor intend be different from herein specifically described alternate manner to put into practice the present invention.Therefore, the present invention includes as by applicable law allow, in appended claims all modifications of theme that describes and equivalent.In addition, unless instruction herein in addition or in addition obviously and contradicted by context, otherwise the present invention contain key element as described above with its any combination of likely version.

Specific embodiments disclosed herein can use in detail in the claims by ... composition or substantially by ... composition language limits further.Time in for claims, and though as submit to or add according to amendment, transitional term " by ... composition " gets rid of unspecified any key element, step or composition in claims.The scope of claims to be limited to specified material or step and not to affect in fact those materials or the step of fundamental sum novel feature by transitional term " substantially by ... composition ".The embodiment of the present invention of requirement like this describes inherently or clearly and is implemented in herein.

In addition, at this description a large amount of referenced patent and printed publication in the whole text.Each mode individually quoted in full in list of references quoted above and printed publication is incorporated herein.

Finally, embodiment of the present invention disclosed herein should be understood and principle of the present invention is described.Other amendment that can adopt is in scope of the present invention.Therefore, unrestricted as an example, alternate configuration of the present invention can be utilized according to content of teaching herein.Therefore, the invention is not restricted to as the content of accurately showing and describe.

Therefore, although shown and described and point out to be applied to the basic novel feature of the present invention of illustrative embodiments of the invention and/or its aspect, should understand those skilled in the art can carry out when not departing from the spirit of the present invention and/or claims its exemplary, disclosure and in form and details various omissions, reconfigure and replace and change.For example, intend to make to carry out identical function in fact with same way in fact to be clearly in scope of the present invention to all combinations of those key elements and/or method step of realizing identical result.In addition, will be appreciated that the structure and/or key element that to be combined with any disclosed form or embodiment and to show and/or describe and/or method step can as general design alternative situation to be incorporated in any form that other discloses or describe or advises or embodiment.Therefore, be intended that and do not limit the scope of the invention.All this kind of amendments are all intended to be in the scope of appended claims.

All open, the patent quoted in this description, patent application, list of references and sequence table are all incorporated herein, as being set forth in completely herein in this mode quoted.

There is provided summary to meet 37CFR § 1.72 (b), with the character and the main points that allow reader to determine rapidly technology disclosure.Described summary is submitted to when having following understanding: it is explained not being used in or limits scope or the implication of claims.

Claims (74)

1. an immunogenic composition, it comprises the dendritic cell by the tumor antigen ex vivo activation being derived from purified ovarian cancer (OV) cancer stem cell (CSC) colony.

2. immunogenic composition according to claim 1, wherein said tumor antigen comprises the cell extract of described OV-CSC.

3. immunogenic composition according to claim 1, wherein said tumor antigen comprises the lysate of described OV-CSC.

4. immunogenic composition according to claim 1, wherein said tumor antigen comprises complete OV-CSC.

5. immunogenic composition according to claim 4, wherein said intact cell is endowed non-proliferative.

6. immunogenic composition according to claim 5, wherein said intact cell is endowed non-proliferative by radiation.

7. immunogenic composition according to claim 4, wherein said intact cell is endowed non-proliferative by being exposed in core cross-linking agent by described cell.

8. the immunogenic composition according to claim arbitrary in claim 1 to 7, it comprises pharmaceutically acceptable carrier and/or excipient further.

9. the immunogenic composition according to claim arbitrary in claim 1 to 8, it comprises adjuvant further.

10. immunogenic composition according to claim 9, wherein said adjuvant is granulocyte macrophage colony stimulating factor.

11. immunogenic compositions according to claim arbitrary in claim 1 to 10, wherein said compositions comprises by the dendritic cell that activates and OV-CSC.

12. immunogenic compositions according to claim arbitrary in claim 1 to 11, wherein said OV-CSC is OV-CSC spheroid form.

13. immunogenic compositions according to claim arbitrary in claim 1 to 11, wherein said OV-CSC is early stage OV-CSC form.

14. immunogenic compositions according to claim arbitrary in claim 1 to 11, wherein said OV-CSC is in mixing OV-CSC form.

15. immunogenic compositions according to claim arbitrary in claim 1 to 11, wherein said OV-CSC is EMT-OV-CSC form.

16. 1 kinds of methods for the treatment of ovarian cancer in experimenter in need, it comprises the immunogenic composition giving according to claim arbitrary in claim 1 to 15 to described experimenter.

17. methods according to claim 16, wherein said immunogenic composition gives with multiple dosage form, and each dosage comprises about 5-20 × 10 ⁶individual cell.

18. methods according to claim 17, wherein said dosage comprises about 10 × 10 ⁶individual cell.

19. methods according to claim arbitrary in claim 16 to 18, wherein said dosage gives weekly once, continues 2-5 dosage, then monthly gives once, lasting 3-6 dosage.

20. methods according to claim arbitrary in claim 16 to 19, wherein said experimenter accepts the immunogenic composition of 6-10 dosage.

The purposes of 22. immunogenic compositions according to claim arbitrary in claim 1 to 15 in the medicine for the preparation for the treatment of ovarian cancer.

23. immunogenic compositions according to claim arbitrary in claim 1 to 15 are used for the treatment of the purposes of ovarian cancer.

24. 1 kinds of methods for the preparation of the colony of ovarian cancer (OV) cancer stem cell (CSC), described method comprises:

Obtain OV sample;

Make the cell dissociation of described sample, and

The cell dissociated described in In vitro culture in the culture medium that non-adhesive substrate is determined at composition, the culture medium that wherein said composition is determined is serum-free and is supplemented with the somatomedin that at least one works via mitogen-activated protein kinase (MAPK) path, forms OV-CSC spheroid thus;

In wherein said OV-CSC spheroid colony at least 80% the following biomarker of cellular expression in two or more: EpCAM, CA-125, MUC-1, CD117, He-4, ALDH, CD133, CD24 and Ki-67.

25. methods according to claim 24, in wherein said OV-CSC spheroid colony, to express in following biomarker further one or more for the cell of at least 80%: CA19-9, HER2/neu, NCAM, ganglioside CD2, estrogen receptor alpha, Vimentin, CK8, CK18, AFP, testosterone, TGF β R, EGFR, TAG-72, CD46, CD44, ABCG2, Slug/Snail, nestin and TP53.

26. methods according to claim 24, in wherein said OV-CSC spheroid colony at least 90% the following biomarker of cellular expression in two or more: EpCAM, CA-125, MUC-1, CD117, He-4, ALDH, CD133, CD24 and Ki-67.

27. methods according to claim 24, it comprises further:

Adhesiveness substrate in the culture medium determined at composition cultivate described OV-CSC spheroid, the culture medium that wherein said composition is determined is serum-free and is supplemented with the somatomedin that at least one works via described MAPK path, form the colony of early stage OV-CSC thus, in wherein said early stage OV-CSC colony at least 80% the following biomarker of cellular expression in two or more: EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD17 and Ki-67.

28. methods according to claim 27, in wherein said early stage OV-CSC colony, to express in following biomarker further one or more for the cell of at least 80%: CA-125, MUC-1, TGF β R and CD24.

29. methods according to claim 27, in wherein said early stage OV-CSC colony at least 90% the following biomarker of cellular expression in two or more: EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD17 and Ki-67.

30. methods according to claim 24, it comprises further:

Adhesiveness substrate in the culture medium determined at composition cultivate described OV-CSC spheroid, the culture medium that wherein said composition is determined contains serum, thus formed mixing OV-CSC colony, in wherein said mixing OV-CSC colony at least 80% the following biomarker of cellular expression in two or more: EpCAM, CA-125, MUC-1, CD117, CK8, CK18 and Ki-67.

31. methods according to claim 30, the culture medium that wherein said composition is determined comprises the somatomedin that at least one works via described MAPK path further.

32. methods according to claim 30, in wherein said mixing OV-CSC colony, to express in following biomarker further one or more for the cell of at least 80%: CA19-9, HER2/neu, NCAM, ganglioside CD2, estrogen receptor alpha, testosterone, TGF β R, EGFR, TAG-72, CD46, He-4, ALDH, CD133, CD44, ABCG2, nestin and TP53.

33. methods according to claim 30, in wherein said mixing OV-CSC colony at least 90% the following biomarker of cellular expression in two or more: EpCAM, CA-125, MUC-1, CD117, CK8, CK18 and Ki-67.

34. methods according to claim 24, it comprises further:

Adhesiveness substrate in the culture medium determined at composition cultivate described OV-CSC spheroid, the culture medium that wherein said composition is determined contains serum and is supplemented with the somatomedin that at least one works via described MAPK path, form the colony of (EMT)-OV-CSC that epithelial cell changes to mesenchymal cell thus, in wherein said EMT-OV-CSC colony at least 80% the following biomarker of cellular expression in two or more: NCAM, Slug/Snail, CD24 and Twist.

35. methods according to claim 34, in wherein said EMT-OV-CSC colony, to express in following biomarker further one or more for the cell of at least 80%: CA-125, MUC-1, CD133, Nanog, CD117, N-cadherins, CD44 and Vimentin.

36. methods according to claim 34, in wherein said EMT-OV-CSC colony at least 90% the following biomarker of cellular expression in two or more: NCAM, Slug/Snail, CD24 and Twist.

37. according to the method in claim 24,30 or 34 described in arbitrary claim, and it comprises further:

Adhesiveness substrate in the culture medium determined at composition cultivate described OV-CSC spheroid, described mixing OV-CSC or EMT-OV-CSC, the culture medium that wherein said composition is determined is serum-free and is supplemented with the somatomedin that at least one works via described MAPK path, form the colony of early stage OV-CSC thus, in wherein said early stage OV-CSC colony at least 80% the following biomarker of cellular expression in two or more: EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD17 and Ki-67.

38. according to method according to claim 37, and in wherein said early stage OV-CSC colony, to express in following biomarker further one or more for the cell of at least 80%: EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD17 and Ki-67.

39. according to method according to claim 37, in wherein said early stage OV-CSC colony at least 90% the following biomarker of cellular expression in one or more: EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD17 and Ki-67.

40. according to the method in claim 24,27 or 34 described in arbitrary claim, and it comprises further:

Adhesiveness substrate in the culture medium determined at composition cultivate described OV-CSC spheroid, described early stage OV-CSC or EMT-OV-CSC, the culture medium that wherein said composition is determined contains serum origin and is supplemented with the somatomedin that at least one works via described MAPK path, thus formed mixing OV-CSC colony, in wherein said mixing OV-CSC colony at least 80% the following biomarker of cellular expression in two or more: AFP, CK7, CK19, EpCAM, E-cadherins, Nanog, FoxA2HNF4a and ABCG2.

41. methods according to claim 40, the culture medium that wherein said composition is determined comprises the somatomedin that at least one works via described MAPK path further.

42. methods according to claim 40, in wherein said mixing OV-CSC colony, to express in following biomarker further one or more for the cell of at least 80%: CA19-9, HER2/neu, NCAM, ganglioside CD2, estrogen receptor alpha, testosterone, TGF β R, EGFR, TAG-72, CD46, He-4, ALDH, CD133, CD44, ABCG2, nestin and TP53.

43. methods according to claim 40, in wherein said mixing OV-CSC colony at least 90% the following biomarker of cellular expression in two or more: AFP, CK7, CK19, EpCAM, E-cadherins, Nanog, FoxA2HNF4a and ABCG2.

44. according to the method in claim 24,27 or 30 described in arbitrary claim, and it comprises further:

Adhesiveness substrate is cultivated in the culture medium determined at composition described OV-CSC spheroid, described early stage OV-CSC or mixing OV-CSC, the culture medium that wherein said composition is determined contains serum and is supplemented with the somatomedin that at least one works via described MAPK path, form the colony of EMT-OV-CSC thus, in wherein said EMT-OV-CSC colony at least 80% the following biomarker of cellular expression in two or more: NCAM, Slug/Snail, CD24 and Twist.

45. methods according to claim 44, in wherein said EMT-OV-CSC colony, to express in following biomarker further one or more for the cell of at least 80%: CA-125, MUC-1, CD133, Nanog, CD117, N-cadherins, CD44 and Vimentin.

46. methods according to claim 44, in wherein said EMT-OV-CSC colony at least 90% the following biomarker of cellular expression in two or more: NCAM, Slug/Snail, CD24 and Twist.

47. methods according to claim arbitrary in claim 24 to 46, the culture medium that wherein said composition is determined is any culture medium described in table 2.

48. methods according to claim arbitrary in claim 24 to 46, the culture medium that wherein said composition is determined is any culture medium of the combination coming from table 2 and table 3.

49. methods according to claim arbitrary in claim 24 to 46, the culture medium that wherein said composition is determined is any culture medium of the combination coming from table 2, table 3 and table 4.

50. methods according to claim arbitrary in claim 24 to 46, the culture medium that wherein said composition is determined is any culture medium of the combination coming from table 2 and table 4.

51. methods according to claim 24, wherein said somatomedin is one or more in fibroblast growth factor (FGF), epidermal growth factor (EGF) or activin A.

52. methods according to claim 51, wherein said FGF is basic FGF (bFGF).

53. methods according to claim arbitrary in claim 24 to 52, the culture medium that wherein said composition is determined is not supplemented with activin A.

54. methods according to claim arbitrary in claim 24 to 52, the culture medium that wherein said composition is determined is with the agonist effectively preventing the amount of OV stem cell Spontaneous Differentiation to be supplemented with activin A.

55. methods according to claim 54, wherein said culture medium comprises the antagonist of activin A further, and described antagonist is follistatin or the antibody with activin A specific binding.

56. methods according to claim arbitrary in claim 24 to 55, wherein said culture medium is not supplemented with antioxidant.

57. methods according to claim 56, wherein said antioxidant is superoxide dismutase, catalase, glutathion, putrescine or beta-mercaptoethanol.

58. methods according to claim arbitrary in claim 24 to 55, wherein said culture media supplemented has glutathion.

59. methods according to claim arbitrary in claim 24 to 58, wherein said adhesiveness substrate is configured for and adheres on anchorage dependence cell and collect described cell.

60. methods according to claim 59, wherein said anchorage dependence cell is fibroblast.

61. methods according to claim arbitrary in claim 24 to 58, wherein said non-adhesive substrate is ultralow adhesiveness polystyrene surface.

60. methods according to claim 61, wherein said adhesiveness substrate comprises the surface being coated with the protein being rich in RGD tri-peptide motif.

The colony of 63. 1 kinds of purified OV-CSC cells, it is prepared by the method according to claim arbitrary in claim 24 to 58.

64. colonies according to claim 63, wherein said purified OV-CSC cell is OV-CSC spheroid.

65. colonies according to claim 63, wherein said purified OV-CSC cell is early stage OV-CSC.

66. colonies according to claim 63, wherein said purified OV-CSC cell is mixing OV-CSC.

67. colonies according to claim 63, wherein said purified OV-CSC cell is EMT-OV-CSC.

68. 1 kinds of OV-CSC cell lines, it is prepared by the method according to claim arbitrary in claim 1 to 29.

69. OV-CSC cell lines according to claim 68, wherein said OV-CSC is OV-CSC spheroid.

70. OV-CSC cell lines according to claim 68, wherein said OV-CSC is early stage OV-CSC.

71. OV-CSC cell lines according to claim 68, wherein said OV-CSC is mixing OV-CSC.

72. OV-CSC cell lines according to claim 68, wherein said OV-CSC is EMT-OV-CSC.

73. 1 kinds in the immunoreactive method of experimenter's moderate stimulation in need for ovarian cancer, it comprises the immunogenic composition from claim arbitrary in claim 1 to 15 to described experimenter, the OV-CSC cell according to claim arbitrary in claim 63 to 67 or the OV-CSC cell line according to claim arbitrary in claim 68 to 72 that give according to.

The 74. OV-CSC cells according to claim arbitrary in claim 63 to 67 or the purposes of the OV-CSC cell line according to claim arbitrary in claim 68 to 72 in the medicine for the preparation for the treatment of ovarian cancer.

The 75. OV-CSC cells according to claim arbitrary in claim 63 to 67 or the OV-CSC cell line according to claim arbitrary in claim 68 to 72 are used for the treatment of the purposes of ovarian cancer.