Manufacture of cancer cells in a closed and sterile system

Abstract

A method for generating modified cancer cells is provided. The method comprises obtaining a sample comprising unmodified cancer cells; separating unmodified cancer cells from other cell populations in the sample; and modifying the unmodified cancer cells in a cultivation chamber to produce modified cancer cells. The method is performed in a closed and sterile cell culture system. In typical aspects, the method is automated. A system for performing the method on a device and uses of the modified cancer cells are also provided.

Description

[0001] MANUFACTURE OF CANCER CELLS IN A CLOSED AND STERILE SYSTEM

[0002] Field

[0003] The present disclosure relates to methods of generating modified cancer cells. In particular, the present disclosure relates to methods and systems for culturing and isolating modified cancer cells that can then be used for the treatment of patients with cancer.

[0004] Background

[0005] Major histocompatibility complex (MHC) is a cell surface molecule encoded by a large gene family in all vertebrates. The MHC gene family is divided into three classes: class I; class II; and class III. In U.S. Patent No. 9,320,785, cancer cells, while not typically known to express MHCII, were shown to be modified to present a non-self peptide in the context of MHCII to helper T cells and therefore effectively replace the function of APCs. The modified cancer cells are considered bifunctional, because they will also present a tumour-specific antigen in the context of MHCI to cytotoxic T cells. While these modified cancer cells where shown to be useful in autologous cancer cell vaccines, the methods of manufacturing modified cancer cells typically consists of using a large number of devices to perform small steps which can inevitably result in higher error rates and increased time requirements due to the need for manual interventions.

[0006] A need exists for the development of a method and / or system for generating modified cancer cells that provides the public with a useful alternative.

[0007] The background herein is included solely to explain the context of the disclosure. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge as of the priority date.

[0008] Summary

[0009] In an aspect, there is provided a method for generating modified cancer cells, comprising: obtaining a sample comprising unmodified cancer cells; separating the unmodified cancer cells from other cell populations in the sample; and modifying the unmodified cancer cells in a cultivation chamber to produce the modified cancer cells; wherein the method is performed in a closed and sterile cell culture system.

[0010] In an aspect, the modifying the unmodified cancer cells comprises adding an MHCII inducing agent to the unmodified cancer cells within the closed and sterile cell culture system.

[0011] In an aspect, the MHCII inducing agent comprises a cytokine, an MHCII expression construct or an MHCH-expressing cell that will fuse with the cancer cells. In an aspect, the cytokine is selected from interferon-alpha (IFN-a), interferon-beta (IFN- P), interferon-gamma (IFN-y), IL-4, IL-13, IL-23, tumor necrosis factor-alpha (TNF-a) or any combination thereof.

[0012] In an aspect, the cytokine is IFN-y.

[0013] In an aspect, the method further comprises incubating the modified cancer cells with a non-self antigen within the closed and sterile cell culture system.

[0014] In an aspect, the non-self antigen is a non-human antigen.

[0015] In an aspect, the non-self antigen is selected from thyroglobulin, 13-galactosidase, dextran, polylysine, tuberculin derived protein, ovalbumin (OVA), bovine serum albumin (BSA), sheep serum albumin, goat serum albumin, fish serum albumin, or keyhole limpet hemocyanin (KLH).

[0016] In an aspect, the non-self is OVA.

[0017] In an aspect, the non-self is KLH.

[0018] In an aspect, the non-self antigen is BSA.

[0019] In an aspect, the non-self antigen is not BSA.

[0020] In aspect, the non-self antigen is selected from bovine, rabbit, murine, canine, or feline antigen.

[0021] In an aspect, the non-self antigen is not a bovine antigen.

[0022] In an aspect, the unmodified cancer cells are derived from a solid tumor.

[0023] In an aspect, the solid tumour is obtained from a subject during a biopsy procedure or during surgical removal of a tumour.

[0024] In an aspect, the solid tumor is obtained from a patient with breast cancer, colorectal cancer, melanoma, ovarian cancer, pancreatic cancer, gastric cancer or prostate cancer.

[0025] In an aspect, the solid tumour is obtained from a patient with prostate cancer.

[0026] In an aspect, the solid tumour is fragmented, within the closed and sterile system, using enzymes to produce a sample of the unmodified cancer cells and other cell populations.

[0027] In an aspect, the separating is carried out using magnetized antibodies.

[0028] In an aspect, the separating involves direct labeling of the unmodified cancer cells through positive selection thereof.

[0029] In an aspect, the separating involves direct labeling of the other cell populations in the sample through positive selection thereof.

[0030] In an aspect, the separating involves indirect labeling of the unmodified cancer cells through negative selection thereof.

[0031] In an aspect, the other cell populations comprise lymphocytes and fibroblasts. In an aspect, the method further comprises transferring the unmodified cancer cells to the cultivation chamber within the closed and sterile system.

[0032] In an aspect, the method further comprises expanding the unmodified cancer cells within the cultivation chamber within the closed and sterile system.

[0033] In an aspect, the expanding occurs over a period of time and wherein a growth of unmodified cancer cells is determined by a change in pH of a culture medium or a nutrient consumption assay.

[0034] In an aspect, the period of time is up to about 30 days.

[0035] In an aspect, the method further comprises removing the modified cancer cells from the cultivation chamber within the closed and sterile system.

[0036] In an aspect, the removing comprises peeling the modified cancer cells from a surface of the cultivation chamber within the closed and sterile system.

[0037] In an aspect, the peeling is carried out with the use of enzymes supplied to the closed and sterile system.

[0038] In an aspect, the method further comprises producing a pharmaceutical product of the modified cancer cells having a desired volume or cell concentration.

[0039] In an aspect, the cell concentration is about 1 x 105to about 1 x 109modified cancer cells.

[0040] In an aspect, the pharmaceutical product is suitable for cellular therapy of a patient in need thereof.

[0041] In an aspect, the pharmaceutical product is an autologous cancer vaccine.

[0042] In an aspect, the modified cancer cells express both MHCI and MHCII on their cell surface and wherein a cancer antigen is bound to said MHCI and a non-self antigen is bound to said MHCII.

[0043] In an aspect, the method is an automated method.

[0044] In another aspect, there is provided a device for preforming the method described herein in the closed and sterile system.

[0045] In yet another aspect, there is provided an automated computer-controlled system for performing the method described herein on a device.

[0046] The novel features of the present invention will become apparent to those of skill in the art upon examination of the following detailed description of the invention. It should be understood, however, that the detailed description of the invention and the specific examples presented, while indicating certain aspects of the present invention, are provided for illustration purposes only because various changes and modifications within the spirit and scope of the invention will become apparent to those of skill in the art from the detailed description of the invention and claims that follow.

[0047] Brief Description of the Drawings

[0048] The present invention will be further understood from the following description with reference to the Figures, in which:

[0049] Figure 1 shows a schematic of an aspect of the present invention wherein a mixed tumor sample (left) is ready for processing within the closed and sterile system using the methods described herein. A legend (right) is provided to show exemplary markers on the surface of the cells in the sample before and after the cells undergo the methods described herein.

[0050] Figure 2 shows a schematic of an aspect of the present invention wherein the mixed tumor sample of Figure 1 is labelled with anti-CD45 magnetic beads within the closed and sterile system of the present invention to separate unmodified cancer cells from other contaminating populations of cells.

[0051] Figure 3 shows a schematic of an aspect of the present invention wherein the contaminating populations of cells of Figure 2 (right) are removed from the unmodified cancer cells (left) using negative selection (magnetic removal). A purified sample of unmodified cancer cells (left) is provided within the closed and sterile system of the present invention.

[0052] Figure 4 shows a schematic of an aspect of the present invention wherein unmodified cancer cells of Figure 3 are expanded within the closed and sterile system of the present invention.

[0053] Figure 5 shows a schematic of an aspect of the present invention wherein the unmodified cancer cells of Figure 4 are induced to become modified cancer cells expressing MHCII on their cell surface in the closed and sterile system of the present invention.

[0054] Figure 6 shows a schematic of an aspect of the present invention wherein the MHCII expressing modified cancer cells are labeled with anti-MHCH magnetic beads within the closed and sterile system of the present invention.

[0055] Figure 7 shows a schematic of an aspect of the present invention wherein the modified cancer cells are separated from the unmodified cancer cells using positive selection (magnetic removal).

[0056] Figure 8 shows a schematic of an aspect of the present invention wherein the modified cancer cells of Figure 7 are eluted from a column within the closed and sterile system of the present invention. Figure 9 shows a schematic of an aspect of the present invention wherein the modified cancer cells of Figure 8 are prepared for cellular therapy (e.g. prepared as a vaccine) as described herein.

[0057] Detailed Description

[0058] Definitions

[0059] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569- 8). Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the typical materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0060] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. Many patent applications, patents, and publications are referred to herein to assist in understanding the aspects described. Each of these references are incorporated herein by reference in their entirety.

[0061] The terms "isolate", "isolated", and "isolating" as used herein refer to removing cancer cells from the body of a patient afflicted with cancer. The cells may be isolated during a standard biopsy procedure or during surgical removal of the cancer, for example. Unless otherwise specified, these terms do not mean that the cancer cells are purified or free from other cell types.

[0062] As used herein, and as well understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. The terms "therapeutically effective amount", "effective amount" or "sufficient amount" mean a quantity sufficient, when administered to a subject, including a mammal, for example a human, to achieve a desired result, for example an amount effective to treat cancer. Effective amounts of therapeutic agents may vary according to factors such as the disease state, age, sex, and weight of the subject. Dosage or treatment regimes may be adjusted to provide the optimum therapeutic response, as is understood by a skilled person. For example, administration of a therapeutically effective amount of the compositions described herein is sufficient to treat a disease or condition, such as cancer, for example, leukemia.

[0063] Moreover, a treatment regime of a subject with a therapeutically effective amount may consist of a single administration, or alternatively comprise a series of applications. The length of the treatment period depends on a variety of factors, such as the severity of the disease, the age of the subject, the concentration of the cancer vaccine, the responsiveness of the patient to the cancer vaccine, or a combination thereof. It will also be appreciated that the effective dosage of the cancer vaccine used for the treatment may increase or decrease over the course of a particular treatment regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. The cancer vaccine of the present invention may, in aspects, be administered before, during or after treatment with conventional anti-cancer agents, radiotherapy, hormone therapy, biotherapy, and / or surgical tumour resection.

[0064] The term "subject" or “patient” may be used interchangeably. The use of the term “subject” as used herein refers to any member of the animal kingdom, typically a mammal. The term "mammal" refers to any animal classified as a mammal, including humans, other higher primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Typically, the mammal is human.

[0065] The term "autologous" refers to cells obtained from a subject and used to treat that same subject.

[0066] The term "self antigen" or "self peptide" refers to an antigen within the body of a subject that is derived from that specific subject and is usually tolerated by the immune system. The term "non-self antigen" or "non-self peptide" refers to an antigen within the body of a subject that is not derived from that specific subject and is usually identified and attacked by the immune system. A "cancer antigen" or "cancer peptide" refers to an antigen within the body of a subject that is derived from a cancer within the subject. As is understood in the art, cancer antigens are sometimes tolerated by the immune system and are sometimes identified and therefore attacked by the immune system. It will be understood that each cancer cell will present many different cancer antigens in the context of MHCI on its cell surface at any given time, some of which may be tolerated by the immune system and some of which may be identified and attacked by the immune system.

[0067] The cancer cells of the invention may be referred to as "bifunctional", because they express both MHCI and MHCII and are capable of activating both helper T cells and cytotoxic T cells, as will be described. These bifunctional tumour cells, expressing MHCI and MHCII, can also be described as tumour presenting cells (TPCs).

[0068] The term "adjuvant" refers to a compound or mixture that is present in a vaccine and enhances the immune response to an antigen present in the vaccine. For example, an adjuvant may enhance the immune response to a polypeptide present in an autologous cancer cell vaccine as contemplated herein, or to an immunogenic fragment or variant thereof as contemplated herein. An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response. Examples of adjuvants which may be employed include MPL-TDM adjuvant (monophosphoryl Lipid A / synthetic trehalose dicorynomycolate, e.g., available from GSK Biologies). Another suitable adjuvant is the immunostimulatory adjuvant AS021 / AS02 (GSK). These immunostimulatory adjuvants are formulated to give a strong T cell response and include QS- 21 , a saponin from Quillay saponaria, the TL4 ligand, a monophosphoryl lipid A, together in a lipid or liposomal carrier. Other adjuvants include, but are not limited to, nonionic block copolymer adjuvants (e.g., CRL 1005), aluminum phosphates (e.g., AIPO.sub.4), R-848 (a Th1- like adjuvant), imiquimod, PAM3CYS, poly (I :C), loxoribine, BCG (bacille Calmette-Guerin) and Corynebacterium parvum, CpG oligodeoxynucleotides (ODN), cholera toxin derived antigens (e.g., CTA 1-DD), lipopolysaccharide adjuvants, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions in water (e.g., MF59 available from Novartis Vaccines or Montanide ISA 720), keyhole limpet hemocyanins, and dinitrophenol.

[0069] The term "purified" is used herein to encompass compositions that are obtained from a starting material by one or more purification steps that enhance the concentration of the active agent relative to the starting material. For example, in aspects of the present invention, interferon-gamma (IFN-y)-treated cells obtained from a biopsy or surgical tumour sample may be purified by cell-sorting techniques to provide an increased concentration of MHCII positive cells. The purified composition may contain, for example, from about 5% to about 100% MHCII positive cells, and any amount in between, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% MHCII positive cells. The term "purified" also encompasses compositions that contain a significant quantity of active agent in relation to impurities, whether obtained by a purification process or not. The term "purified" should not be construed to connote absolute purity.

[0070] "Parenteral" administration of a composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), intradermal (i.d.), intrasternal injection, or infusion techniques.

[0071] The terms "closed cell sample processing system" and "closed and sterile (cell culture) system" can be used interchangeably.

[0072] The term "closed and sterile system" or "closed cell sample processing system" as used herein refers to any closed system which reduces the risk of cell culture contamination while performing culturing processes such as the introduction of new material, e.g. by modification of the unmodified cancer cells described herein, and performing cell culturing steps such as, but not limited to, proliferation, differentiation, activation, and separation of cells. Such a system can be operated under GMP or GMP-like conditions ("sterile") resulting in cell compositions which are clinically applicable. An exemplarily closed and sterile system is the CliniMACS Prodigy™ (Miltenyi Biotec GmbH, Germany). This system is disclosed in W02009 / 072003 and is incorporated by reference herein. It is noted that provision of this exemplary system is in no way intended to restrict the use of the method of the present invention to the CliniMACS Prodigy™.

[0073] The terms “automated”, "automated method" or "automated process" as used herein refer to any process being automated through the use of devices and / or computers and computer softwares. In some instances the method of the present invention is automated if at least one step of the present method is performed without any human support or intervention. Typically, the method of the present invention is automated if all steps of the method as disclosed herein are performed without human support or intervention other than connecting fresh reagents to the system. In aspects, the automated process is implemented on a closed cell sample processing system such as CliniMACS Prodigy™.

[0074] The term "washing" means the replacement of the medium or buffer in which the cells are kept. The replacement of the supernatant can be in part (example 50% of the medium is removed and 50% fresh medium is added) this often is applied for dilution or feeding purposes, or entirely. Several washing steps can be combined in order to obtain a more profound replacement of the original medium in which the cells are kept. A washing step often involves pelleting the cells by centrifugation forces and removing the supernatant.

[0075] The term "shaking conditions" as used herein refers to any means that allow to keep the cells of the cell culture in suspension. The shaking may be performed by rotating (or sporadic centrifugation) a cultivation chamber of a closed and sterile cell culture system, and wherein said rotation is performed periodically as disclosed herein. The shaking may also be performed e.g. by using a whipping equipment, a propelling device or a flow of liquid (e.g. channels) integrated into the closed and sterile cell culture system used which prevent sedimentation of the cells.

[0076] When introducing elements disclosed herein, the articles “a”, “an”, “the”, and “said” are intended to mean that there may be one or more of the elements.

[0077] The term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives. It will be understood that any embodiments described as “comprising” certain components may also “consist of” or “consist essentially of,” these components, wherein “consisting of’ has a closed-ended or restrictive meaning and “consisting essentially of” means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effects described herein. For example, a composition defined using the phrase “consisting essentially of” encompasses any known pharmaceutically acceptable additive, excipient, diluent, carrier, and the like, suitable for the composition described herein. Typically, a composition consisting essentially of a set of components will comprise less than 5% by weight, typically less than 3% by weight, more typically less than 1 % by weight of non-specified components.

[0078] It will be understood that any component defined herein as being included may be explicitly excluded from the claimed invention by way of proviso or negative limitation, such as any specific compounds or method steps, whether implicitly or explicitly defined herein.

[0079] In addition, all ranges given herein include the end of the ranges and also any intermediate range points, whether explicitly stated or not.

[0080] Finally, terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.

[0081] The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” The word “or” is intended to include “and” unless the context clearly indicates otherwise.

[0082] The phrase “at least one of” is understood to be one or more. The phrase “at least one of...and...” is understood to mean at least one of the elements listed or a combination thereof, if not explicitly listed. For example, “at least one of A, B, and C” is understood to mean A alone or B alone or C alone or a combination of A and B or a combination of A and C or a combination of B and C or a combination of A, B, and C.

[0083] Generally, it is difficult to automate biological processes, especially when multiple processes must be combined in order to generate a complex product such as modified cancer cells expressing MHCII that are capable of presenting both self and non-self antigen. A method of generating modified cancer cells such as these in a closed Good Manufacturing Practices (GMP)-compliant environment (a closed and sterile cell culture system) is described herein that is robust and can lead to equal or even higher amounts of modified cancer cells suitable for clinical application (e.g. injection into a patient in need thereof) compared to manufacturing modified cancer cells without the closed and sterile system. Typically the process is automated, and the automated process provides the above-described advantages in comparison to nonautomated processes.

[0084] Biological samples

[0085] The present invention relates to the processing of biological samples. The biological samples may be fresh or frozen. Typically, the biological samples are fresh samples, such that they have been removed from a subject, typically within about 24 hours prior to use in the methods and systems described herein. In aspects, the biological samples include, but are not limited to, blood, leukapheresis, bone marrow, liposuction, milk, any body fluid, cells from tissue (e. g. cells from various organs), tumor cells, single cells, cell clumps, cell aggregates, and tissue dissected mechanically or in conjunction with enzymes. In typical aspects, the biological samples are tumor samples or blood samples. More typically, the tumour sample is a solid tumour and most typically, the biological samples are tumour samples (e.g. solid tumours) and the cells derived therefrom.

[0086] In most typical aspects, the cells are isolated from an autologous subject, meaning that they will be used to treat the same subject from whom they were derived. Alternatively, the cells could be used in an HLA-matched heterologous subject. Typically the cells are cancer cells and typically the cells are isolated during a biopsy procedure or during surgical tumour removal. The cancer cells may be derived from any type of malignancy and, in an aspect, they are derived from lung cancer, including small cell lung cancer and non-small cell lung cancer (e.g. adenocarcinoma), pancreatic cancer, colon cancer (e.g. colorectal carcinoma, such as, for example, colon adenocarcinoma and colon adenoma), oesophageal cancer, oral squamous carcinoma, tongue carcinoma, gastric carcinoma, liver cancer, nasopharyngeal cancer, hematopoietic tumours of lymphoid lineage (e.g. acute lymphocytic leukemia, B-cell lymphoma, Burkitt's lymphoma), non-Hodgkin's lymphoma (e.g. mantle cell lymphoma), Hodgkin's disease, myeloid leukemia (for example, acute myelogenous leukemia (AML) or chronic myelogenous leukemia (CML)), acute lymphoblastic leukemia, chronic lymphocytic leukemia (CLL), thyroid follicular cancer, myelodysplastic syndrome (MDS), tumours of mesenchymal origin, soft tissue sarcoma, liposarcoma, gastrointestinal stromal sarcoma, malignant peripheral nerve sheath tumour (MPNST), Ewing sarcoma, leiomyosarcoma, mesenchymal chondrosarcoma, lymphosarcoma, fibrosarcoma, rhabdomyosarcoma, melanoma, teratocarcinoma, neuroblastoma, brain tumours, medulloblastoma, glioma, benign tumour of the skin (e.g. keratoacanthoma), breast carcinoma (e.g. advanced breast cancer), kidney carcinoma, nephroblastoma, ovary carcinoma, cervical carcinoma, endometrial carcinoma, bladder carcinoma, prostate cancer, including advanced disease and hormone refractory prostate cancer, testicular cancer, osteosarcoma, head and neck cancer, epidermal carcinoma, multiple myeloma (e.g. refractory multiple myeloma), or mesothelioma. In an aspect, the cancer cells are derived from a solid tumour. In other words, in typical aspects, the solid tumour is taken from a subject diagnosed with any of the aforementioned cancers. Typically, the cancer cells are derived from, or the solid tumour is from a breast cancer, colorectal cancer, melanoma, ovarian cancer, pancreatic cancer, gastric cancer, or prostate cancer. More typically, the cancer cells are derived from, or the solid tumour is from a prostate cancer.

[0087] Methods and Systems for use with the Biological Samples

[0088] As described herein, the method for generating modified cancer cells, cells of which can, for example, be subsequently put to clinical use, can be performed in a single device, using a closed and sterile system with or without programmed software (e.g. with or without automation). Advantageously, the methods and systems described herein allow for a number of cell processing steps to be performed by a single device, all of which can be an automated process, which helps eliminate the requirements for manual cell transfer, in-process controls, related risks to the cellular product, and risk reduction measures. Thus, the methods and systems described herein provide an efficient way to produce cellular therapy products that are ready for direct use, such as for clinical application. In this way, the cellular product is ready for direct transfer into the patient after completion of the methods described herein. In an aspect, the method for generation of modified cancer cells, comprises: obtaining a sample comprising unmodified cancer cells; separating the unmodified cancer cells from other cell populations in the sample; and modifying the unmodified cancer cells in a cultivation chamber to produce the modified cancer cells. The method is performed in a closed and sterile cell culture system. It is noted that while the methods and systems described herein are exemplified for modified cancer cells (e.g. cancer cells expressing MHC II and present self and non-self antigen as described herein), the methods and systems described herein may be applied to a wide range of cell types or other biological samples as described above. In an aspect, lymphocytes in general are specifically excluded from the present invention. In another aspect, CD45+expressing T cells and Macrophages are specifically excluded from the present invention.

[0089] Once the sample is obtained, the sample can be provided in transfer bags or other suited containers which can be connected to the closed system by sterile means. For example, if the closed system device is a CliniMACS Prodigy™ device disclosed in W02009 / 07200, and incorporated herein by reference, the sample can connected via tubing sets to the CliniMACS Prodigy™ device. Typically the sample is a tumor sample, and the cancer cells for use in the methods described herein are derived from the solid tumor. In most typical aspects, the cancer cells are derived from a solid tumour that was obtained during a biopsy procedure or during surgical removal of the tumour. In these aspects, once the tumour sample is connected to the closed system, the tumour sample is typically washed with saline or phosphate bovine saline (PBS) and then subsequently fragmented by adding enzymes like collagenases, trypsin and the like. Such fragmentation allows for subsequent separation of the cells comprising the tumour. If the sample is instead a blood sample, for example, the sample can be centrifugated to remove excess erythrocytes and the cell sample can be washed to avoid cell aggregation, prior to the downstream cell processing steps carried out in the closed and sterile system.

[0090] In typical aspects, the sample comprises unmodified cancer cells which can be modified so as to express MHCII on their cell surface, such that the MHCII presents a non-self antigen to helper T cells and MHCI presents a cancer antigen to cytotoxic T cells of the immune system. Cells thus modified are immunogenic and can be used in an autologous cancer vaccine composition that is effective for treating cancer in the subject. The methods and systems described herein are useful in producing modified (immunogenic) cancer cells that express both MHCI and MHCII on its cell surface, wherein a cancer antigen is bound to said MHCI and a nonself antigen is bound to said MHCII.

[0091] While most cancer cells do not naturally express much if any MHCII on their cell surface, it will be understood that if the cancer cells are derived from antigen-presenting cells, such as a B cell cancer for example, these cells may already express MHCII on their cell surface. It is contemplated that unmodified cancer cells that already express MHCII could be explicitly excluded from the present invention. In other words, it is contemplated that the present invention could encompass cancer cells that are MHCH-negative, MHClI-positive, or both prior to modification according to the present invention. Alternatively, such cells could be included in the invention and it will be understood that, since these cells already express MHCII, incubation with an MHClI-inducing agent is merely optional in order to increase the level of expression.

[0092] Following obtaining of the sample, and in the case of the tumour sample, the fragmentation thereof, the unmodified cancer cells are separated from the other cell populations in the sample. As shown in Figures 1 to 3, this typically occurs by passing the sample of cells through a column to remove the other cell populations. As shown in Figure 1 , typically, the other cell populations comprise lymphocytes, such as tumour infiltrating T cells, macrophages, and fibroblasts. Other non-cancer cells may also be present and they can be removed similarly to the lymphocytes and fibroblasts using the methods described below, or, for example, by microbeads directed to their specific cell surface markers. In this way, the unmodified cancer cells are enriched in the sample and removed from the contaminating, and other unneeded, cellular populations of, for example, lymphocytes, such as tumour infiltrating T cells, macrophages, and fibroblasts (Figure 3).

[0093] Separation of the unmodified cancer cells from the other populations of cells in the sample can be accomplished through different methods, such as for example, using labelled antibodies specific for cell surface marker expression. In typical aspects, the separation is accomplished by magnetic separation of the unmodified cancer cells (Figures 2 and 3). It is understood that other uses of antibodies, such as biotin-conjugates antibodies against lineagespecific antigens, for example, can be used in the methods described herein. Exemplary magnetic separation systems / processes have been described, for example, in EP 0869838 and in US 5691208, each of which are hereby incorporated by reference. In aspects, the magnetic separation can involve the use of magnetically responsive particles or microparticles. Suitable magnetic particles are described in US 4452773, and in the EP 452342, each of which are hereby incorporated by reference.

[0094] In typical aspects, the magnetic separation involves antibodies coupled to magnetic particles can be used for the negative or positive selection of the unmodified cancer cells. In typical aspects, the separating uses negative selection of the unmodified cancer cells (Figure 3). For example, the other populations of cells (e.g. the lymphocytes, macrophages, and fibroblasts) are directly labelled with the magnetized antibodies, such as antibodies directed to CD45+expressing cells (e.g. tumour infiltrating T cells and macrophages; Figure 1), such that passing the sample through a magnetized column within the closed and sterile system allows for only the unmodified cancer cells pass therethrough (See Figure 3). Thus, the unmodified cancer cells are not labelled with the antibodies which can limit any potential side effects of the labeling process, like potential activation of the cells, and would also not require any potential further processing steps to remove the antibodies from the cells of interest (e.g. the unmodified cancer cells). In this way, further additional processing time is not required to recover the unmodified cancer cells, and it results in an enriched sample of the unmodified cancer cells in the flowthrough with retention of the labelled other cell populations in the magnetized portion of the column. In alternative aspects, the separation of the unmodified cancer cells involves positive selection through the use of direct magnetic labeling of the unmodified cancer cells (not shown). As shown in Figures 6 to 8, however, direct labeling is most typically used after the modification of the unmodified cancer cells (i.e. when the cells are transformed into modified cancer cells expressing MHCII on their surface), such that the modified cancer cells may be identified and further enriched through detection of the MHCII molecule.

[0095] After separating the unmodified cancer cells from the other cell populations, the unmodified cancer cells can be transferred to a cultivation chamber within the closed and sterile system. In some aspects, the separation of the unmodified cancer cells described above and the cultivation of the unmodified cancer cells, as described below, can occur in the sample vessel within the closed and sterile system such that there is no requirement to transfer the cells within the closed and sterile system. As shown in Figure 4, in the cultivation chamber, the unmodified cancer cells can be grown or expanded in situ over a period of time in a culture medium. For example, the unmodified cancer cells can be allow to grow for up to 30 days, such as for example, up 24 hours, up to 2 days, up to 3 days, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, up to 10 days, up to 11 days, up to 12 days, up to 13 days, up to 14 days, up to 15 days, up to 16 days, up to 17 days, up to 18 days, up to 19 days, up to 20 days, up to 21 days, up to 22 days, up to 23 days, up to 24 days, up to 25 days, up to 26 days, up to 27 days, up to 28 days, up to 29 days or up to 30 days. The growth of unmodified cancer cells can be determined by any suitable method, such as evaluating changes in pH of the culture medium or any suitable nutrient consumption assay. In aspects, the method further comprises delivering, to the cultivation chamber in the closed and sterile system, nutrient (culture) medium, cytokines, growth factors, serum and other substances necessary for the cultivation of the cells. Nutrient medium can be continually or periodically delivered. The culture medium can be completely or partially exchanged with fresh medium during the cell cultivation process. The culture medium can be enriched with O2, CO2, N2, air or other gases necessary for the growth of the cells. In order to ensure the unmodified cancer cells are cultured in optimal conditions, the culture medium may be sampled during the manufacturing run to monitor critical parameters. In this way, the user of the closed and sterile system is able to take, at any time, a sample of the culture medium into dedicated sampling pouches. Parameters such, glucose, pH etc. can then be measured remotely.

[0096] Following the expansion of the unmodified cancer cells, the unmodified cancer cells can be modified to express MHCII on their cell surface. In other aspects, the unmodified cancer cells are separated from the other cell populations are subjected to the modification process immediately. Thus depending on the time requirements or, for example, the initial yield of unmodified cancer cells from the obtained sample, the user can determine whether or not the unmodified cancer cells should be expanded in situ. In typical aspects, however, the cells are expanded within the cultivation chamber as described above.

[0097] As shown in Figure 5, in order for the unmodified cancer cells to express MHCII on their cell surface, they are incubated with an MHClI-inducing agent. The MHClI-inducing agent can be directly delivered to the cultivation chamber within the closed and sterile system. In aspects, the inducing agents can be provided in solution, coated on the cultivation chamber or coated on a carrier substance present in suspension / solution within the cultivation chamber or on large particles. The MHClI-inducing agent can be, for example, cytokines, chemical agents, and gene constructs. For example, the MHClI-inducing agent may be interferon-gamma (IFN-y), or it may be an MHCII expression vector that is used to transfect or transduce the cancer cells. The MHClI-inducing agent also encompasses a cell expressing MHCII, in that cells that express MHCII could be fused via cell fusion with the cancer cells to render the cancer cells MHCII positive. Examples of such cells include B cells, dendritic cells, macrophages, and monocytes. In another aspect, the MHCII inducing agent may be an agent that activates the MHCII transactivator (CIITA) sequence. Typically, however, the MHClI-inducing agent is a cytokine, such as, for example, interferon-alpha (IFN-a), interferon-beta (IFN-J3), interferon-gamma (IFN- y), IL-4, IL-13, IL-23, or tumor necrosis factor-alpha (TNF-a). Combinations of cytokines may also be used. In a specific aspect, the MHClI-inducing agent is IFN-y. It is understood that the MHClI-inducing agent may also have effects on increasing expression of MHCI on the cancer cells. For example, if IFN-y is used as the MHClI-inducing agent, it may also tend to cause an increase in MHCI on the surface of the cancer cells. Should the user of the system desire, the modified cancer cells may be screened by conventional methods in order to confirm that MHCII is being expressed on the cell surface, after a sample is taken for analysis.

[0098] The modification of the unmodified cancer cells described above can take place in the cultivation chamber under steady or shaking culture conditions. When the modification occurs

Claims

We claim:1 . A method for generating modified cancer cells, comprising: obtaining a sample comprising unmodified cancer cells; separating the unmodified cancer cells from other cell populations in the sample; and modifying the unmodified cancer cells in a cultivation chamber to produce the modified cancer cells; wherein the method is performed in a closed and sterile cell culture system.

2. The method of claim 1 , wherein the modifying the unmodified cancer cells comprises adding an MHCII inducing agent to the unmodified cancer cells within the closed and sterile cell culture system.

3. The method of claim 2, wherein the MHCII inducing agent comprises a cytokine, an MHCII expression construct or an MHClI-expressing cell that will fuse with the cancer cells.

4. The method of claim 3, wherein the cytokine is selected from interferon-alpha (IFN-a), interferon-beta (IFN-|3), interferon-gamma (IFN-y), IL-4, IL-13, IL-23, tumor necrosis factor-alpha (TNF-a) or any combination thereof.

5. The method of claim 3 or 4, wherein the cytokine is IFN-y.

6. The method of any one of claims 1 to 5, further comprising incubating the modified cancer cells with a non-self antigen within the closed and sterile cell culture system.

7. The method of claim 6, wherein the non-self antigen is a non-human antigen.

8. The method of claim 6 or 7, wherein the non-self antigen is selected from thyroglobulin,13-galactosidase, dextran, polylysine, tuberculin derived protein, ovalbumin (OVA), bovine serum albumin (BSA), sheep serum albumin, goat serum albumin, fish serum albumin, or keyhole limpet hemocyanin (KLH).

9. The method of claim 8, wherein the non-self is OVA.

10. The method of claim 8, wherein the non-self is KLH.11 . The method of claim 8, wherein the non-self antigen is BSA.

12. The method of claim 6 or 7, wherein the non-self antigen is not BSA.

13. The method of claim 6 or 7, wherein the non-self antigen is selected from bovine, rabbit, murine, canine, or feline antigen.

14. The method of claim 6 or 7, wherein the non-self antigen is not a bovine antigen.

15. The method of any one of claims 1 to 14, wherein the unmodified cancer cells are derived from a solid tumor.

16. The method of claim 15, wherein the solid tumour is obtained from a subject during a biopsy procedure or during surgical removal of a tumour.

17. The method of claim 15 or 16, wherein the solid tumor is obtained from a patient with breast cancer, colorectal cancer, melanoma, ovarian cancer, pancreatic cancer, gastric cancer or prostate cancer.

18. The method of claim 15 or 16, wherein the solid tumour is obtained from a patient with prostate cancer.

19. The method any one of claims 15 to 18, wherein the solid tumour is fragmented, within the closed and sterile system, using enzymes to produce a sample of the unmodified cancer cells and other cell populations.

20. The method of any one of claims 1 to 19, wherein the separating is carried out using magnetized antibodies.21 . The method of any one of claims 1 to 19, wherein the separating involves direct labeling of the unmodified cancer cells through positive selection thereof.

22. The method of any one of claims 1 to 19, wherein the separating involves direct labeling of the other cell populations in the sample through positive selection thereof.

23. The method of any one of claims 1 to 19, wherein the separating involves indirect labeling of the unmodified cancer cells through negative selection thereof.

24. The method of any one of claims 1 to 23, wherein the other cell populations comprise lymphocytes and fibroblasts.

25. The method of any one of claims 1 to 24, further comprising transferring the unmodified cancer cells to the cultivation chamber within the closed and sterile system.

26. The method of any one of claims 1 to 25, further comprising expanding the unmodified cancer cells within the cultivation chamber within the closed and sterile system.

27. The method of claim 26, wherein the expanding occurs over a period of time and wherein a growth of unmodified cancer cells is determined by a change in pH of a culture medium or a nutrient consumption assay.

28. The method of claim 27, wherein the period of time is up to about 30 days.

29. The method of any one of claims 1 to 28, further comprising removing the modified cancer cells from the cultivation chamber within the closed and sterile system.

30. The method of claim 29, wherein the removing comprises peeling the modified cancer cells from a surface of the cultivation chamber within the closed and sterile system.31 . The method of claim 30, wherein the peeling is carried out with the use of enzymes supplied to the closed and sterile system.

32. The method of any one of claims 1 to 31 , further comprising producing a pharmaceutical product of the modified cancer cells having a desired volume or cell concentration.

33. The method of claim 32, wherein the cell concentration is about 1 x 105to about 1 x 109modified cancer cells.

34. The method of claim 32 or 33, wherein the pharmaceutical product is suitable for cellular therapy of a patient in need thereof.

35. The method of any one of claims 32 to 34, wherein the pharmaceutical product is an autologous cancer vaccine.

36. The method of any one of claims 1 to 35, wherein the modified cancer cells express both MHCI and MHCII on their cell surface and wherein a cancer antigen is bound to said MHCI and a non-self antigen is bound to said MHCII.

37. The method of any one of claims 1 to 36, wherein the method is an automated method.

38. A device for preforming the method according to any one of claims 1 to 37 in the closed and sterile system.

39. An automated computer-controlled system for performing the method according to any one of claims 1 to 36 on a device.

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