Patent Application
20190212325
Present invention relates to the field of pharmacology and, in particular, to the assessment of anti-tumor drugs activity.
The microenvironment (ME) critically affects tumor cell survival, proliferation and drug resistance (1). The role of the ME has become even more important with the discovery and development of cancer drugs that specifically target components of the ME (2). This has become an obstacle for the ex vivo testing of drugs that treat cancer. The absence of the factors that constitute the ME in these ex vivo assays will influence the perceived efficacy of many drugs. The result of this is a lack of correlation between the ex vivo results and the clinical effectiveness of these drugs.
Several groups have developed microenvironment-mimicking assays, to varying degrees of success, to try and overcome some of these obstacles. Those methods, disclosed in the prior art, include adding cellular elements (3-5), adding components of the extracellular matrix (6, 7) and/or the addition of cocktails of soluble cytokines/chemokines or molecules derived from non-tumor cells present in the tumor microenvironment (8, 9). While these attempts have had some success, the present invention introduces a new method for mimicking components of the microenvironment that have not previously been attempted.
Chronic Lymphocytic Leukemia (CLL) is a prototype of a disease with an extremely heterogeneous biology and clinical course; it is the most frequent adult hematologic malignancy (10). This pronounced clinical heterogeneity is linked to both cell-intrinsic and cell-extrinsic mechanisms of the disease. The best known amongst cell-intrinsic mechanisms concerns genomic aberrations, epigenetic modifications and micro-RNAs. CLL behavior and outcome, including chemorefractoriness, also depends on stimuli present in the leukemic ME (11). The critical role of external stimulation is shown by the remarkable therapeutic activity of drugs interfering with CLL-ME interactions, namely inhibitors of signaling through the clonotypic B cell receptors (BcR); these novel drugs (Ibrutinib and Idelalisib) constitute a major paradigm shift in CLL treatment (12, 13).
Multiple Myeloma (MM) is a malignant disease characterized by proliferation of clonal plasmatic cells in the bone marrow (BM). MM is still an incurable disease, being the second most frequent hematological malignancy in the USA and Europe. MM cells have a strong BM microenvironment dependence, which confers anti-apoptotic and pro-survival signals and resistance to drugs. MM tumor cells have an elevated proliferation rate promoted by soluble factors and molecular interactions within the particular BM microenvironment.
Here we show that the addition of an Artificial Environment (AE) to ex vivo assays using isolated primary tumor cells, had a profound effect on viability and proliferation of said tumor cells. This constitutes a novel method for improving the responsiveness to drugs, particularly anti-tumor drugs, of primary tumor cells, in ex vivo assays.
Tumor samples extracted from cancer patients contain the tumor cells in the context of a highly heterogeneous environment. This environment may represent 99% of the sample extracted from the patient, for example in hematological tissue samples such as bone marrow, peripheral blood, or lymph node. To study these primary tumor cells, it is common practice to remove most of this heterogeneous environment, concentrating the remaining tumor cells that are being studied. In hematological samples this is commonly performed by a density gradient isolating the fraction containing the leukocytes, which corresponds roughly to only 1% of the entire sample. The other major fractions of the density gradient commonly discarded are the plasma fraction, and the erythrocyte fraction. We refer to both of these discarded fractions as the Artificial Environment (AE). These discarded fractions could be quite relevant to the leukocytes and discarding them may lead to artifacts in the leukocyte's biology. We have investigated the effect of these discarded fractions on the activity of compounds on primary tumor cells, by performing a density gradient isolation and by adding Artificial Environment fractions isolated therein from the same or different patient samples to primary tumors cells.
The disclosed method of the present invention adds Artificial Environment to primary tumor cells. The present invention has several advantages over that of the prior art. The activity of compounds on primary cells is artificially altered in assays which use isolated primary cells ex vivo. The method of the present invention, in which the assay of potential anti-tumor drugs is also included, adds an Artificial Environment that compensates for the lack of a microenvironment in isolated cells. In some prior assays, the whole sample has been used, but it is generally greatly diluted.
Adding AE to whole sample assays would compensate for artifacts imposed by the initial dilution of the ME of the sample. Another advantage is that the methods of present invention enhance the viability of a primary tumor cell population. An additional advantage is that the method of the present invention promotes the proliferation of a primary tumor cell population, to levels that have not been previously reported (14). These advantages allow for the ex vivo analysis of the cellular response of the primary tumor cell population to a large number of variables, including different incubation times and testing of many drug compositions, including drugs that inhibit proliferation. Moreover, another advantage of the present invention is the capacity to analyze the primary tumor cells in a manner that more closely mimics the in vivo microenvironment of a patient. This is particularly important in the case of drugs targeting the microenvironment e.g. ibrutinib and idelalisib, whose assessment will likely require microenvironment-mimicking assays. Investigating the effect of these compounds and their combinations with other compounds, with the Artificial Environment, prevents the major artifacts actually existing with the assays based on isolated primary tumor cells. Methods of the present invention afford, therefore, the advantage of improving the ability of an ex vivo test to determine if a drug or drugs would be beneficial to a patient (or patients).
The invention presented here relates to a method for producing an artificial environment (AE) of primary cell populations, particularly an artificial tumor environment of primary tumor cell populations and its use in an ex vivo method to test the cellular responsiveness of primary tumor cell populations to a drug or combination of drugs. The test of the cellular responsiveness comprises incubation of the primary tumor cells with the artificial tumor environment and the drug or drugs to be tested and analyze the response of the primary tumor cell populations. The incubation of the primary tumor cells with the artificial tumor environment, enhances the viability of said tumor cells and/or induces greater levels of tumor cell proliferation and, consequently, increases the sensitivity and accuracy of the test with regard to the drug/s assayed.
Described herein is also the process for obtaining components of AE from peripheral blood (PB), or bone marrow (BM), or lymph nodes (LN) samples. Said components are used to stabilize primary tumor cell populations. These selected components which constitute the AE are isolated and/or mixed components from a sample selected from: peripheral blood, or bone marrow, or lymph nodes obtained from a donor (
Reference will now be made in detail to the preferred embodiments of the invention, examples of which are provided hereto. Although the disclosure herein refers to certain illustrated embodiments, it is to be understood that these embodiments are presented by way of example and not by way of limitation. The intent of the following detailed description, although discussing exemplary embodiments, is to be construed to cover all modifications, alternatives and equivalents of the embodiments as may fall within the spirit and scope of the invention as defined by the appended claims. The disclosed methods may be utilized in conjunction with various medical procedures that are conventionally used in the art.
The present invention relates to a method for producing an artificial tumor environment of primary tumor cells and its use in an ex vivo method to test the cellular responsiveness of primary tumor cell populations to a drug or combination of drugs. This method is characterized for enhancing and/or promoting the proliferation of a primary tumor cell population in ex vivo assays. Isolated primary tumor cells cannot be duly analyzed because they lack certain microenvironmental elements which are critical in promoting the survival and proliferation of a tumor in vivo.
We have first defined the method of incorporating the AE for ex vivo analysis for the evaluation of responsiveness of primary tumor cells isolated from patients diagnosed with Chronic Lymphocytic Leukemia (CLL), to the presence of anti-tumor drugs in the assay media. Present disclosure, although centered on CLL, represents a technical invention that could be applied to any primary tumor cell population, whether it is from a hematological malignancy or a solid tumor. Included here we also demonstrate the effect of AE on primary tumor cell populations from other hematological malignancies, including Acute Myeloid Leukemia (AML), Acute Lymphoid Leukemia (ALL), Myelodysplastic Syndrome (MDS), non-Hodgkin's Lymphoma (NHL) and Multiple Myeloma (MM)
The present invention also discloses the use of the AE in ex vivo assays for determine the effect of a drug (analysis and quantification of the drug effect) on primary tumor cells isolated from patient samples.
Microenvironment—is the environment in which the tumor exists, including surrounding blood vessels, immune cells, fibroblasts, stromal cells, the extracellular matrix (ECM), soluble factors (e.g. tumor derived exosomes, signaling molecules. growth factors, micro RNA, chemokines, cytokines and any soluble molecule derived from tumor or non-tumor cells), all of which affect tumor cell dynamics.
Artificial Environment—(AE, also referred to as AE fraction) Fraction or mixture of fractions isolated from a peripheral blood, or bone marrow, or lymph node sample from a donor after density gradient centrifugation excluding leukocyte fraction (AE leukocyte-free). Residual leukocytes could still remain in the AE. AE can be only the plasma fraction, only the erythrocyte fraction, or a combination of the two fractions at any ratio (e.g. 1:1, 1:2, 2:1, etc.).
AE Leukocyte-free: Fraction or sample without leukocytes, or with a residual number of leukocytes, defined as less than 100 leukocytes per μl of AE.
Donor—Refers to a person who provides a biological sample. The person can be healthy or have been diagnosed with a disease (e.g. cancer). The biological sample can be, for example, blood, bone marrow, lymph node or tumor biopsy.
Ex vivo—experimentation or measurements done in or on tissue or cell/s from an organism in an external environment with the minimum alteration of natural conditions. In the present invention refers to primary human cells in vitro, from patients suffering a disease or normal individuals, where the cells can be either recently extracted, cryopreserved, or frozen, to preserve their state.
In some embodiments, these cells are thawed for the in vitro evaluation of drug effects. For the purposes of present patent specification, the term “Ex vivo” and “In vitro” are used indistinctly.
Hematological Malignancies—(HM) Also called hematological neoplasms, refers to a group of diseases, defined according to the World Health Organization classification, that includes Hodgkin's lymphoma, Non-Hodgkin's lymphoma (e.g., B cell lymphoma, diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia), chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, multiple myeloma, or acute lymphocytic leukemia.
Solid Tumors—consists of neoplasms or lesions formed by abnormal growth of body tissue cells other than blood, bone marrow or lymphatic cells. Solid tumors include, but are not limited to, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, carcinoma of the fallopian tubes, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, metastatic lesions of said cancers, or combinations thereof.
Sample—Also called patient sample, primary tumor cells, isolated tumor cells, primary cells, all refer to primary cells that have been extracted from a patient and have been isolated for analysis. A sample can be primary cells from a healthy individual, a patient diagnosed with cancer, or a patient diagnosed with another hematological disorder. A sample may or may not have been cryopreserved. Cryopreserved samples have to be thawed before use.
ExviTech—Automated and proprietary flow cytometry platform that enable pharmacological characterization of drugs and drug combination treatments in the context of microenvironmental stimuli, directly on patient samples to assess response (15).
Primary tumor cells—Refers to tumor cells taken directly from living tissue (e.g. bone marrow, peripheral blood, lymph nodes, spleen, or tumor biopsy), isolated and established for ex vivo growth. Primary tumor cells may have been previously extracted and cryopreserved and thawed before use, or may be recently extracted and used without cryopreservation.
Primary cell population—Refers to cells (non-diseased) taken directly from living tissue (e.g. bone marrow, peripheral blood, lymph nodes, spleen, or tumor biopsy) that are established for ex vivo growth.
Stromal cell population representative of a cell type found in the in vivo tumor microenvironment. The tumor microenvironment is composed of several non-tumor cell types that can include, but is not limited to, fibroblastic reticular cells, nurse-like cells, mesenchymal stem cells, follicular dendritic cells, endothelial cells and pericytes. The stromal cells can be primary cells, or cells derived from primary stromal cells including, but not limited to, HS-5, HS27a, UE6E7T-2, NK-Tert, HMEC-1, KUSA-H1, M210B4, ST-2, and KUM-4.
Density Gradient Centrifugation—Refers to a process that separates a blood, or bone marrow, or sample into fractions of discrete components, e.g. plasma, erythrocytes, granulocytes and monocytes. Blood, or bone marrow, or lymph node samples are layered over a gradient medium (e.g. Ficoll-Paque™, Lymphoprep™, Histopaque-1077™, or other similar mediums) and after centrifugation the isolated fractions of each component can be removed (
Plasma fraction or Serum fraction—Plasma obtained from a blood, or bone marrow, or lymph node sample after density gradient centrifugation. This is the top most fraction, as seen in
Erythrocyte fraction—AE comprising mainly erythrocytes. When a sample of peripheral blood, bone marrow or lymph node is separated into various component parts by density gradient centrifugation, this is the bottom fraction as see in
Soluble factors—Refers non-cellular components of the tumor microenvironment that can include tumor derived exosomes, signaling molecules, growth factors, chemokines, cytokines, micro RNA, and any soluble molecule derived from tumor or non-tumor cells (9, 16, 17). Examples of these can include, but are not limited to, Interleukin (IL)-2, IL-4, IL-6, IL-8, IL-10, IL-13, IL-15, IL-21, soluble cluster of differentiation (sCD) 40 ligand, Immunoglobulin (Ig) M, CpG (short synthetic single-stranded DNA molecules containing non-methylated CpG dinucleotides motifs), B cell activating factor (BAFF), a Proliferation Inducing Ligand (APRIL), Tumor derived exosomes (tumor cell-derived vesicles that are present in the tumor microenvironment).
Types of Drugs—chemotherapeutic or cytotoxic drug, medicines that contain chemicals which are toxic to cells, preventing their replication or growth (e.g. busulfan, cyclophosphamide, mitoxantrone, daunorubicin, doxorubicin, melphalan, vincristine, vinblastine and chlorambucil); targeted anti-cancer therapy drug, drugs designed to interfere with specific molecules necessary for tumor growth and progression (e.g. imatinib, dasatinib, nilotinib); oncolytic drug, a virus that invades tumors and replicates itself, thereby killing cancer and spurring an immune response to multiply its effect (e.g. Imlygic and Peptichemio); immunotherapy drug, treats disease by inducing, enhancing, or suppressing an immune response (e.g. Alentuzumab, Ofatumumab, Rituximab); cytokine therapy, a broad category of small proteins that are important in cell signalling that can be used to treat disease (e.g. interferons (IFNα, IFNβ, IFNγ and IFNλ) and interleukins (IL-2, IL-8, IL-21); tumor microenvironment drugs, drugs that target the microenvironment rather than the tumor (e.g. Ibrutinib and idelalisib); activator of a costimulatory molecule, agents that activate co-stimulatory molecules that are crucial to the development of an effective immune response (e.g. Cluster of differentiation (CD)28, OX40, CD137, CD27, Herpesvirus-entry mediator (HVEM)); inhibitor of an inhibitory molecule, drugs that evokes a response by silencing inhibitors of a system (e.g. antibodies to programmed cell death 1 (PD1) and Lymphocyte-activation gene 3 (LAG-3)).
Parameters that may be measured—viability, measurement of live cells; proliferation, the formation or development of cells; apoptosis, the process of programmed cell death; cell depletion, loss of cells through mechanisms that are not necrosis or apoptosis; changes in surface marker expression, monitoring the appearance or disappearance of cellular surface markers such as the CD markers; changes in cytokine/chemokine production, measuring levels of these molecules that are produced by a cell population.
3D Cell Culture—an artificially-created environment in which biological cells are permitted to grow or interact with their surroundings in all three dimensions (e.g. spheroid constructs, extracellular matrix gels, synthetic scaffolds, rotary cell culture systems, or on low/non-adherent culture plastics (15, 18, 19).
Circulating Tumor Cells—cells that break away from a tumor and move through a cancer patient's bloodstream.
Whole sample (e.g. whole peripheral blood, whole bone marrow or whole lymph node)—The use of the sample in its entirety. No components have been removed or isolated from the sample. As an example, lymphocytes isolated from a bone marrow sample are not considered whole sample.
The use of AE to test of the responsiveness of primary tumor cells populations to a drug or combination of drugs method disclosed in the present invention has several advantages over that of the prior art that are described herein. One advantage is that the method of present invention enhances the viability of a primary tumor cell population, thus prolonging the duration of the tests and the corresponding assessment of drug long term activity. Another advantage is that the method promotes the proliferation of a primary tumor cell population, to levels that have not been previously reported (14). These advantages allow the use of a large number of variables in an ex vivo analysis of the cellular response of the primary tumor cell population, including different incubation times and many drug compositions such as drugs that inhibit cell proliferation. Another advantage is the capacity to analyze the primary tumor cell population in a manner that more closely mimics the in vivo microenvironment of the primary tumor cell population in a patient. This is particularly important in the case of drugs targeting the microenvironment e.g. Ibrutinib and Idelalisib, whose assessment requires microenvironment-mimicking assays. Combined with previously mentioned features and advantages of the method, the present invention also affords the advantage of improving the ability of an ex vivo test to determine if a drug or drugs would be beneficial to a patient (or patients) in the treatment of primary tumors. In a particular embodiment, this test can be used determine the effectiveness of a given treatment in a patient.
In an embodiment, AE (e.g. plasma fraction only, erythrocyte fraction only or both, but always excluding leukocyte fraction, AE leukocyte-free) is added to ex vivo assays that test the cellular responsiveness of primary cells to a drug or drugs. In another embodiment, AE (e.g. plasma fraction only, erythrocyte fraction only or both, but always excluding leukocyte fraction, AE leukocyte-free) is added to ex vivo assays that test the cellular responsiveness of primary tumor cells to a drug or drugs. In an embodiment, the AE contains a residual number of leukocytes less than 100 per μl of AE. In another embodiment, the residual leukocytes present in an AE preparation are labeled to distinguish them from primary tumor cells to be tested. In one embodiment, the primary tumor cells are incubated in an incubation mixture comprising primary tumor cells, AE and a drug or drugs. In another embodiment, the primary tumor cells are incubated in an incubation mixture comprising AE, one or more soluble factors (e.g. cytokines/chemokines or molecules derived from non-tumor cells) and a drug or drugs. In another embodiment, the primary tumor cells are incubated in an incubation mixture comprising AE, a co-culture of an additional cell population that is representative of a cell type found in the in vivo tumor microenvironment, and a drug or drugs. In a further embodiment, the primary tumor cells are incubated in an incubation mixture comprising AE, one or more soluble factors (e.g. cytokines/chemokines or molecules derived from non-tumor cells), and a co-culture of an additional cell population that is representative of a cell type found in the in vivo tumor microenvironment, and a drug or drugs.
In an embodiment, AE is added to ex vivo assay systems that contain components from other species. These components include, but are not limited to, fetal bovine serum, calf bovine serum, or components derived from these. In one embodiment, AE is added to ex vivo assays that contain culture media supplemented with different nutrients. These media include, but are not limited to RPMI-1640, AIM-V, DMEM and IMDM. In a further embodiment, AE is added to assay systems comprised of ex vivo 3D cell culture constructs built to mimic the microenvironment architecture of solid tumors. This is achieved for example by culturing primary tissues or established cell lines within spheroids, extracellular matrix gels, synthetic scaffolds, rotary cell culture systems, or on low/non-adherent culture plastics (18-20).
In an embodiment, the incubation mixtures are incubated for a period of 2 hours to 14 days prior to the analysis of the effect of the drug or drugs on the primary tumor cells. In an embodiment, the soluble factors added to the incubation mixture are comprised from a list including, but not limited to tumor derived exosomes, signaling molecules, growth factors, chemokines, cytokines and any soluble molecule derived from tumor or non-tumor cells (9, 16, 17). Examples of these can include, but are not limited to, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, IL-15, IL-21, sCD40L, IgM, CpG, BAFF, or APRIL.
In an embodiment, the additional cell population that is representative of a cell type found in the in vivo tumor microenvironment added to the incubation mixture is comprised from a list including, but not limited to either primary cells or cell lines derived from: fibroblastic reticular cells, nurse-like cells, mesenchymal stem cells, follicular dendritic cells, endothelial cells, and pericytes (5, 21, 22). Cell lines derived from primary stromal cells can include, but are not limited to, HS-5, HS27a, UE6E7T-2, NK-Tert, HMEC-1, KUSA-H1, M210B4, ST-2, and KUM-4.
In a preferred embodiment, the AE fractions are collected from several donors, all different from the patient providing the primary tumor cells, wherein all plasma fractions have been pooled together and erythrocytes from compatible blood types have been pooled, reducing the interpatient donor variability in the AE. In another embodiment, the AE fractions are comprised of fractions from a single donor, different from the patient providing the primary tumor cells. In a further embodiment, the AE fractions are comprised of fractions obtained from the same patient that provides the primary tumor cells.
In a preferred embodiment, the AE is comprised of fractions obtained from donors/patients with the same type of cancer as the patient from which the primary tumor cells to be tested have been extracted (e.g. to test a primary tumor sample from a patient diagnosed with CLL, the AE used in the assays would be comprised of plasma fractions and serum fractions obtained from one or more patients also diagnosed with CLL). In another embodiment, the AE is comprised of fractions obtained from healthy donors. In a further embodiment, the AE is comprised of fractions obtained from donors diagnosed with a cancer different from the cancer of the primary tumor cells to be tested.
In an embodiment, the AE corresponding to a primary cell sample is preserved separately, whereby plasma fraction can be frozen at −80° C. and the erythrocyte fraction is preserved in CPDA bags for up to 45 days at 4° C.
In a preferred embodiment, the primary cell sample has been cryopreserved prior the test. In another embodiment, the primary cell sample has been freshly extracted. In a particular embodiment, the cryopreserved primary cell sample is thawed together with the AE. In a preferred embodiment, the cryopreserved primary cell sample is cryopreserved together with the AE. Primary cell samples can have been cryopreserved for any length of time after freezing. In one embodiment, a primary cell sample is cryopreserved from 5 days to 1 year after freezing. In another embodiment, a primary cell sample is cryopreserved from 1 year to 20 years after freezing. The reconstitution of the cryopreserved primary cell sample with the AE provides a sample more similar to the native environment of said primary cells and increase viability and/or proliferation and/or maintaining of the pharmacological properties of the primary cells.
In one embodiment, the primary tumor sample has been extracted from an adult cancer patient. In another embodiment, the primary tumor sample has been extracted from a pediatric patient. In an embodiment, the primary tumor sample has been extracted from a cancer patient that has not received a prior treatment for the cancer. In another embodiment, the primary tumor sample has been extracted from a cancer patient that has received one or more previous treatments for the cancer. In a further embodiment, the primary tumor sample has been extracted from a patient with Minimal Residual Disease (MRD).
In a specific embodiment, the primary tumor cells are obtained from a patient diagnosed with a hematological malignancy. Hematological malignancies include, but are not limited to, Hodgkin's lymphoma, Non-Hodgkin's lymphoma (e.g., B cell lymphoma, diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia), chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, multiple myeloma, or acute lymphocytic leukemia. In a preferred embodiment, the primary tumor cells from a patient diagnosed with a hematological malignancy is selected from a group consisting of peripheral blood, bone marrow, lymph node, and spleen.
In another embodiment, the primary tumor cells are extracted from a patient diagnosed with a solid tumor. Solid tumors include, but are not limited to, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, carcinoma of the fallopian tubes, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, metastatic lesions of said cancers, or combinations thereof. In a preferred embodiment, the primary tumor cells from a solid tumor are obtained via a biopsy of the solid tumor. In another embodiment, the solid tumor cells are circulating tumor cells extracted via a peripheral blood sample. In an embodiment, the drug or drugs tested in an incubation mixture containing AE, consist of one or more therapeutic agent or agents chosen from, but not limited to, the following categories: chemotherapy, a targeted anti-cancer therapy, an oncolytic drug, a cytotoxic agent, an immune-based therapy, a cytokine, drugs targeting the microenvironment, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, or a cellular immunotherapy.
In one embodiment, the ex vivo analysis of the cellular response of the primary tumor cell population includes, but is not limited to, measuring one or more of the following: viability, proliferation, apoptosis, cell depletion, changes in surface marker expression, changes in cytokine/chemokine production, and analysis of these parameters on one or more sub-populations of cells identified within the primary tumor cell population.
In an embodiment, AE (e.g. plasma fraction only, erythrocyte fraction only or both) is added to ex vivo assays that test the cellular responsiveness of primary cells, cells that are not tumor cells, to a drug or drugs. The primary cells may be extracted from patients diagnosed with other hematological disorders, including but not limited to, autoimmune diseases, blood coagulation disorders, and anemia. In a particular embodiment, primary tumor cells are extracted from a patient after treatment of the disorder has been started. The ex vivo assay that test the cellular responsiveness of primary tumor cells can be used to determine if a prescribed treatment is effective to said patient. In an embodiment, the patient sample is obtained 5, 6, 7, 8, 9, or 10 days after initiation of treatment. In a particular embodiment, the sample from the patient corresponds to a lymph node biopsy obtained after being treated with idelalisib 7 days after the start of treatment.
In an embodiment, AE (e.g. plasma fraction only, erythrocyte fraction only or both) is added to ex vivo assays that test the cellular responsiveness of primary cells, either healthy or pathological, to a drug or drugs that are in development.
In an embodiment, AE (e.g. plasma fraction only, erythrocyte fraction only or both) is added to ex vivo assays that test the cellular responsiveness of primary cells to a drug or drugs being tested in a clinical trial. In this embodiment, results could provide information on the whether the drug is potentially beneficial to a patient, and if the patient would be a good candidate for the clinical trial.
The PB, BM and LN samples used in this document were from adult patients over 18 years of age. All patients gave informed written consent for study participation using protocols approved by the ethical committees of each hospital. Samples were provided by Spanish Centers from the Programa Español de Tratamientos en Hematologia (PETHEMA) group and Hospital San Raffaelle, Milan, Italy.
The components of the AE described in this invention are prepared as follows: Histopaque 1077 (Sigma) is added to a conical centrifuge tube. The peripheral blood (PB), or bone marrow (BM), or lymph nodes (LN) samples from donors are carefully layered onto the Histopaque-1077 solution and centrifuge at 1500 rpm for 40 minutes. The upper layer, containing the plasma fraction is removed first. The middle layers containing the mononuclear cells (MNC), Histopaque-1077 and granulocytes layers (leucocyte fraction) are removed and discarded. The bottom layer containing the red blood cells (RBC) or erythrocyte fraction is then removed (
The middle sections are excluded to remove the gradient media and the leukocyte population as well. The exclusion of the leukocytes allows for the use of an AE preparation in tests of primary cell samples from other donors without the worry of Major Histocompatability Complex (MHC) interactions. The MHC receptors on some of the leukocyte populations (e.g. T cells and NK cells) may cause them to attack primary cells from non-autologous samples. Thus it is necessary to exclude the leukocyte population from the AE preparation. However, the method described is in practice an enrichment process. All fractions may contain a residual number of leukocytes, but not enough to have a measurable MHC effect on the primary cells populations being tested. Using an AE composed of plasma and erythrocytes adds factors to an ex vivo assay system that can impact the viability of primary cells and their ability to proliferate. These AE factors also may affect how a drug interacts with the primary cells in terms of efficacy and potency.
In case of using only the plasma fraction, the AE is prepared as follows: The whole PB, BM or LN sample is centrifuged at 2500 rpm for 15 minutes at 4° C. The upper layer containing the plasma fraction is removed, aliquoted in small fractions and stored at −80° C. until use.
The role of AE in thawing previously cryopreserved samples was investigated. Cryopreservation methods commonly rely on a density gradient to isolate the leukocytes, and only the leukocyte fraction is cryopreserved. As a consequence, when these cells are thawed the result is isolated leukocytes devoid of its AE. The activity of compounds on these thawed cells could thus be artificially modified by the absence of the AE. We have investigated whether we can add back the AE of these cryopreserved cells, and whether the freeze/thaw process alters the compound activity of both cytotoxic and antiproliferative drugs. Fresh BM AML samples were divided into two parts; in one part, we evaluated the dose response curves of several compounds in its AE as fresh samples. In the other part, we performed a density gradient isolating the leukocyte fraction that was cryopreserved, the plasma fraction that was frozen at −20° C., and the erythrocyte-granulocyte fraction that was preserved in CPDA from blood bags at 4° C. After 2 weeks, the cryopreserved vials were thawed, and the plasma and erythrocyte fractions from each patient were mixed in a 1:1 ratio and added to the thawed primary cells. We repeated the same evaluation of the activity of these compounds and compared the results in fresh vs frozen samples of the same patients with reconstituted AE.
Eight concentrations of seven different drugs were tested and incubated for 24 hours and 48 hours, and the results of the viability prior to incubation were also obtained. After incubation, red blood cells were lysed and leukocytes stained using a combination of monoclonal antibodies to identify leukemic cells. Annexin V was also included to exclude apoptotic cells from the analysis. Area under the curve (AUC) was calculated for each drug and each condition.
Similar results were observed with other hematological malignancies such as ALL.
For all assays testing primary tumor cell populations, the Vybrant® CFDA SE Cell Tracer Kit (Molecular Probes) is used to measure cell proliferation. The CFDA SE (component A) is dissolved in dimethylsufoxide (component B) at a concentration of 5 mM as stock solution and kept at −20° C. until further use. In this example, freshly extracted and isolated CLL cells are adjusted to 10×106 cells/ml in phosphate buffer saline (PBS, Sigma). CFDA is added to a 1 ml cell suspension of primary CLL cells, to a final concentration of 5 μM. After addition of CFDA, cells are vortexed and incubated at room temperature for 10 min with continuous shaking and light protected. At the end of the incubation period, the cells are resuspended in media and kept on ice for 5 min. The cells are washed twice with media and maintained at 4° C. until use. This step is performed prior all other steps in the experimental preparation.
The CFDA labeled primary CLL cells are then dispensed into 96-well plates. Cells are stimulated with the cytokine combination of 1 μg/ml Oligodeoxy-ribonucleotides containing CpG motifs (ODN2006; Miltenyi Biotec, Bergisch Gladbach, Germany) plus 50 ng/ml Interleukin-2 (IL-2, Peprotech, London, United Kingdom). No stimulation is also used as a control of both viability and proliferation. After 96 h of incubation the plates are washed. Then 20 μl of a combination of Annexin-V CF Blue (Immunostep, Salamanca, Spain), CD19 PC7 (Clone J4.119, Beckman Coulter) and CD45-APC (clon HI30, Immunostep, Salamanca, Spain) is added. After 15 minutes of incubation at room temperature in the dark, a wash step is performed and the pellet is resuspended in 80 μl of buffer for analysis in Vivia's ExviTech platform.
For analysis, the antibodies CD19 and CD45 are used to identify the primary CLL cells, Annexin-V staining discerns between apoptotic and live cells to test viability, and CFDA allows for proliferation to be monitored.
A flow cytometry based screening process is used to test for the optimal conditions for primary Tumor cells from patient diagnosed with CLL. The conditions tested are summarized in Table 1. The ratio of HS-5 stromal cells to B-cells in each assay is indicated.
Three cryopreserved CLL primary tumor samples are tested with and without the presence of HS-5 stromal cells (none, 1:10 and 1:100). Plates that incorporated HS-5 (23) cells are plated at either 102 or 103 cells/60 μl/well in 96-well plates and incubated for 24 hours to allow cells to adhere. The CLL primary tumor cells are labeled with CFDA. Any residual leukocytes that are present in the AE will not be labeled with CFDA and in this manner can be differentiated from the primary tumor cell population being tested.
The AE, the plasma and erythrocyte fractions, are isolated from 6 healthy donors, three from PB and three from BM, and are tested separately (samples were not pooled). The AE is added to the appropriate wells at 1 μl/well. Three cytokine pairs are also tested: 1 μg/ml CD40 Ligand (sCD40L, Peprotech, London, United Kingdom) plus 25 ng/ml IL-2 (Peprotech, London, United Kingdom), 1 μg/ml sCD40L plus 1 μg/ml CpG (ODN2006 Miltenyi Biotec, Bergisch Gladbach, Germany) and 1 μg/ml CpG plus 50 ng/ml IL-2. No stimulation is also used as a control of both viability and proliferation. Replicate plates for each condition are incubated for 2 time points, 72 hours and 96 hours, each in triplicate.
The experiment tested both viability and proliferation of the malignant B-cell population in the CLL patient samples. The overall results are shown in
The present methods were used to evaluate AE derived from the PB of both healthy donors and CLL patients. Conditions for this experiment were set using 1 μg/ml CpG plus 50 ng/ml IL-2 to stimulate the samples both without HS-5 co-culture and with a 1:10 HS-5 co-culture, each at 72-hour and 96-hour incubation times. Cryopreserved primary CLL tumor cells from a representative patient sample is shown in
The present methods were used to evaluate the effect of the different component of the AE from CLL or normal samples in CLL cryopreserved samples. Four CLL frozen samples were seeded with 1 μg/ml CpG plus 50 ng/ml IL-2 (grey circles) together with controls (black circles) and both proliferation (X-axis, Median proliferation+/−SD) and viability (Y-axis, median viability+/−SD) were tested (
These conditions show a clear difference between adding AE composed of plasma and erythrocytes from CLL PB (column 1) vs NPB (column 2). The AE from the CLL PB plus cytokines increased proliferation (see circles) while the AE from normal patients (NPB) had little effect on proliferation, even in the presence of cytokines. In addition, for the CLL derived AE it seems to be relevant to add AE composed of plasma and erythrocytes (columns 1 and 5) rather than only the plasma fraction (columns 3 and 7), as when only the plasma was used both proliferation (X-axis) and viability (Y-axis) are diminished. There appears to be a saturating effect with the amount of AE that is added as well. Adding higher concentrations as in columns 5-8 showed no marked improvement over what is seen in columns 1-4. These experiments demonstrate the importance of AE to improve proliferation and viability, especially with AE derived from CLL PB, where some of the components seem to increase these parameters in comparison with NPB.
The media that the primary CLL tumor cells are maintained in during the incubation process contains 10% Fetal Bovine Serum (FBS). To determine the effect of FBS on the assay system, three CLL samples were tested in four different serum conditions for the incubation media, 10% FBS, 20% FBS, 10% Human Serum (HS), or 20% HS. The CLL tumor samples were tested either without HS-5 co-culture, or with co-culture at ratios of 1:10 or 1:100, and were stimulated with 1 μg/ml CpG plus 50 ng/ml IL-2. The AE was derived from 6 PB samples from CLL patients. The plasma and erythrocyte fractions from the 6 samples were mixed in a 1:1 ratio, and added to the wells at 1 μl/well. Results show that HS significantly increases the viability of the tumor cells over FBS in all conditions (
The present experiment evaluates the effect of viability and proliferation of 24 cytokines combinations (Table 2) that cover different cell signaling. The cytokines used were: CpG (1 μg/ml), IL-2 (50 ng/ml), sCD40L (1 μg/ml), B cell activating factor (BAFF, 10 ng/ml), IgM+IgG (BCR, 10 μg/ml), IL-21 (Interleukin-21, 50 ng/ml). When included, stromal cells, HS-5 were used at a dilution of 1:100. The AE for these experiments was plasma and erythrocytes, obtained from PB of 3 patients with CLL. The AE fractions from each sample were mixed in equal parts prior to adding to the culture media. The media used contained the AE (1.6%) plus human serum (HS) 10%. 20 CLL cryopreserved primary tumor samples were processed, and tested with each of the cytokine combinations. Both proliferation (X-axis) and viability (Y-axis) were tested as previously described for each cytokine combination. The numbers indicated in Table 2 correspond to each combination represented in
Each data point of the dot plot in
Cryopreserved Peripheral Blood (PB) mononuclear cells were provided from 15 CLL patients in need of treatment. The AE for these experiments was plasma and erythrocytes, obtained from PB of 3 patients with CLL. The AE fractions from each sample were mixed in equal parts prior to adding to the culture media. Cryopreserved CLL primary tumor cells to be tested were cultured at a ratio of 100:1 on confluent layers of HS-5 cell line in AIM-V (AIM-V®+Albumax® (BSA 1X)) supplemented with 1.6% AE, 10% (vol/vol) Human Serum, 2% HEPES, 1% antibiotic, and 1% L-glutamine 200 mM. Cells were stimulated with the following pair cytokine combination: 1 μg/ml CpG ODN plus 50 ng/ml IL-2 for 96 hours at 37° C. in humidified air containing 5% CO2. CLL cell viability (assessed by Annexin V staining) and proliferation (assessed by the CFDA dye) is tested with 8 concentrations of Ibrutinib (N=15) and Idelalisib (N=21) that cover the range of activities across patients.
AML and MDS are both associated with malignancies in multipotential hemopoietic stem cells that can be identified by their expression of CD34+. These cells can prove to be difficult to keep viable ex vivo, and have very low levels of proliferation. An appropriate source of serum, media or AE should be used to avoid CD34+ cell death and allow proliferation with specific cytokines. In these experiments, we tested cryopreserved primary cells (mononuclear) obtained from BM from a Myelodysplastic Syndrome (MDS) patient (
The Control (CON) was incubated in RPMI+20% FBS. Viability was assessed by Annexin V and absolute CD34+ cell counts by our ExviTech® platform. For analysis, the antibodies CD45-APC (Clon H130, Immunostep) and CD34-PerCP (Clon581, Biolegend) are used to identify the primary CD34+ cells. The upper panel in
As shown for CLL samples, the addition of different cytokine combinations and/or AE could improve the viability or proliferation of B-Non Hodgkin's Lymphoma (B-NHL) primary samples (
For analysis, the antibodies CD19-PE (clone HIB19, e-Bioscience), CD10-PE-Cya7(clone HI10a, Biolegend), CD45PO (clone HI30, Invitrogen) and CD20 APC (clone L27, Becton Dickinson) are used to identify the primary lymphoma cells. The median response for the 4 B-NHL samples is shown for each condition tested, where each data point of the dot plots represents one combination in Table 3. Results for the 72-hour incubation are shown in
Bone marrow samples obtained from MM patients were evaluated to determine the best conditions for preserving viability and proliferation rates. Different sample treatments were analyzed to determine how they affect the viability and/or proliferation of the tumor cells. Additionally, each sample was treated differently prior to its use in the assay. The conditions are as follows:
Four types of culture medium were tested, including RPMI 1640, AIMV, DMEM and IMDM. The addition of two human stromal cell lines, HS27a and HS5, at different ratios—1:1, 1:10 and 1:100—were tested as well.
Patient whole BM was obtained and prepared as indicated above for each sample type. The Vybrant® CFDA SE Cell Tracer Kit (Molecular Probes) was used to measure cell proliferation as it has been disclosed in Example 3. After the CFDA protocol, all samples were resuspented in Culture Medium+Zell Shield (Labclinics)+L-Glu (Lonza)+20% FBS. Condition A samples stayed as is, condition B samples had different concentrations of autologous MM plasma plus RBCs added and condition C samples received 20% autologous plasma. The final cell concentration ranged from 20 cells/μl to 70 cells/μl. Alternatively, the autologous plasma can be replaced with a pool of three MM plasmas.
The CFDA labeled plasmatic cells were dispensed into 96-well plates (60 μl/well) and incubated at 37° C. with 5% CO2 during 72 or 96 hours. For analysis, MM cells (assessed by monoclonal antibodies) were tested for both viability (Annexin-V staining) and proliferation (CFDA).
After 96 hours of culture, the CFDA staining in conjunction with the immunophenotyping allowed for the analysis of both viability (Annexin-V negative) and proliferation (CFDA peaks) of the plasmatic cells, following the previously described methods using Vivia's ExviTech platform.
As the number of MM cells detected after 96 hours of incubation was too low in just a single well, a pooled well assay was performed, obtaining sample from 4 wells that have been treated in an identical manner. Thus, a significantly higher number of cells can be evaluated, increasing the reliability of the data. According to this, and based on the initial cell count, calculation of the sample volume was done for plating between 1000 and 4000 live pathological MM cells per well. During acquisition, sample from four wells was obtained for each treatment, thus obtaining a minimum of 4000 and a maximum of 16000 live plasmatic cells. The results of these experiments indicate that the use of patient's whole bone marrow plus 20% of autologous plasma, or a pool of at least 3 MM plasmas (Condition C from above) and the use of IMDM culture medium provided the best conditions to achieve better MM cells viability and higher proliferation rates (data not shown). Additionally, the presence of either of the two human stromal cell lines, HS27a and HS5, appeared to have a negative effect on both viability and proliferation.
A follow up experiment was conducted to analyze the effect of two cytokines, IL-6 (Interleukin-6) and CpG in this experimental setup. Table 4 shows the specific conditions assayed in each sample. Experimental procedures were as stated above. When cytokines were included (marked as X), they were added at the beginning of the incubation period.
The sample type refers to the different sample treatments outlined at the beginning of this example. All samples were assayed in duplicate (except sample 5). The numbers correspond to the samples in
The result indicates that the presence of cytokines does not provide any advantage in the viability of the cells nor in percentage of proliferation of the cells. Sample 1 even displayed higher levels of viability in the absence of IL-6 and CpG. Sample 7, which did not include stromal cells, displayed the highest level of viability and proliferation, which corresponds with previous results. Here again, cytokines did not have any effect on the outcome. It can be concluded that IL-6 and CpG do not appear to affect viability and proliferation of MM cells in ex vivo assays. The presence of autologous plasma, minus stromal cells and IL-6 offers the best conditions so far for MM cells. Other cytokines might have a different outcome.
Some drugs used to treat tumor cells preferentially target cells that are actively proliferating, acting as anti-proliferative agents. An example of this is the use of lenalidomide to treat patients diagnosed with MM. The efficacy of lenalidomide is difficult to assess in ex vivo assays against MM primary tumor cells, as these cells have very low levels of proliferation under the conditions that have traditionally been used. In this example, we tested BM samples from 10 patients diagnosed with MM in our ex vivo assay. AE (plasma only) was added to the culture medium to increase levels of proliferation and lenalidomide was tested across a concentration range of 0.03 to 30 μM.
Whole BM from 10 MM patients was cultured in IMDM medium supplemented with 1% antibiotic, 1% L-Glutamine plus an additional 20% of autologous plasma (AE), for 96 hours. Samples were processed and analyzed as outlined in Example 12. MM cell viability (Annexin V) and proliferation (CFDA) were tested against 4 concentrations of Lenalidomide.
The survival index of the MM cells is displayed on the Y-axis, and the X-axis represents the lenalidomide drug concentration. This figure demonstrates that there is a clear difference in the sensitivity of lenalidomide between NP and PR MM cell populations. It also shows that there is a large level of interpatient variability. For sample 1 (
It can be concluded that the addition of the AE to the culture medium can change how cells react to a drug in an ex vivo setting. This may help to eventually correlate the ex vivo effect of drugs like lenalidomide to clinical outcomes specifically for each particular patient.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/076690 | 11/4/2016 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62250687 | Nov 2015 | US |
