WO2025022288A1 - Prepared dehydrated, desiccated and/or lyophilized culture containers for t cell activation and methods of use thereof - Google Patents

Abstract

The invention provides a container comprising dehydrated, desiccated and/or lyophilized reagents for isolation and/or stimulation of T cells for therapeutic use, methods of making the container and methods of use thereof for T cell based therapy.

Description

PREPARED DEHYDRATED, DESICCATED AND/OR LYOPHILIZED CULTURE CONTAINERS FOR T CELL ACTIVATION AND METHODS OF USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63/514,963, filed July 21, 2023, the disclosures of which are hereby incorporated by reference herein in its entirety.

REFERENCE TO A SEQUENCE LISTING

[0002] The present application hereby incorporates by reference the entire contents of the sequence listing xml document named “JBI6816WOPCTl_Sequence_Listing.xml”. The xml file containing the Sequence Listing of the present application was created on July 16, 2024 and is 3,237 bytes in size.

BACKGROUND OF THE INVENTION

[0003] Chimeric antigen receptor (CAR) T cell therapy is a form of T cell transfer therapy in which T cells are harvested from the patient and modified to express antigen receptors that are not typically expressed. Stimulation (aka activation) of T-cells is required for the gene insertion (aka transduction) event to occur and for subsequent cell expansion allowing for proper dose levels to be achieved. Successful stimulation/activation of primary T cells occurs through a co -stimulatory response via the CD3 and CD28 receptors on the cell surface. This is typically achieved through localization of anti-CD3 and anti-CD28 antibodies on polymeric matrices or beads which are formulated in a suspension and then added to a solution of cells in culture media during CAR-T processing. Use of functional antibodies has historically been used to stimulate T cells, however, the process is complex and requires a significant amount of prep time due to the insoluble nature of anti-CD3.

[0004] There remains a need in the art for compositions and methods that can be engineered to optimize and streamline CAR-T therapy. The present disclosure meets this unmet need.

SUMMARY OF THE INVENTION

[0005] The invention provides, in part, a container comprising a vacuum sealable interior chamber, wherein an inner surface of the interior chamber is coated with at least one dehydrated, desiccated, and/or lyophilized reagent capable of isolating and/or stimulating a T cell.

[0006] In some examples, the container is a flexible bag.

[0007] In some examples, at least one dehydrated, desiccated, and/or lyophilized reagent comprises an anti-CD3 antibody, an anti-CD28 antibody or a combination thereof. In some examples, one or more of the anti-CD3 antibody, and the anti-CD28 antibody are mixed with a solution prior to dehydration, desiccation and/or lyophilization, wherein the solution comprises a buffer selected from the group consisting of trehalose, PBS, and water.

[0008] In some examples, the device comprises at least one additional dehydrated, desiccated and/or lyophilized reagent for the stimulation or expansion of a T cell population. In some examples, at least one additional dehydrated, desiccated and/or lyophilized reagent comprises IL-2.

[0009] In some examples, the container is devoid of air.

[0010] In some examples, the container is a fluid bag comprising at least one fluid port.

[0011] In some examples, the container is a fluorinated ethylene propylene (FEP) fluid bag with high surface energy.

[0012] In some examples, the volume of solution added to the container is such that at least 85% of the interior surface area of the container can be coated with the solution, but the interior volume is not fdled.

[0013] In some examples, the container does not comprise polymeric beads.

[0014] In some examples, the container is stable when stored at 2-8°C for at least 1 day.

In some examples, the container is stable when stored at 2-8°C for at least 6 months.

[0015] The invention provides, in part, a method of stimulating or expanding a T cell population, wherein the method comprises applying a mixture containing a T cell population and a media for culturing T cells to a container comprising a vacuum sealable interior chamber, wherein an inner surface of the interior chamber is coated with at least one dehydrated, desiccated, and/or lyophilized reagent capable of stimulating a T cell. In some examples, the T cells are chimeric antigen receptor (CAR) T cells.

[0016] The invention provides, in part, a T cell population expanded by the method of stimulating or expanding a T cell population, wherein the method comprises applying a mixture containing a T cell population and a media for culturing T cells to a container comprising a vacuum sealable interior chamber, wherein an inner surface of the interior chamber is coated with at least one dehydrated, desiccated, and/or lyophilized reagent capable of stimulating a T cell. [0017] The invention provides, in part, a method of treating, preventing, reducing, or eliminating a disease or disorder in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of a T cell population expanded by the method of stimulating or expanding a T cell population, wherein the method comprises applying a mixture containing a T cell population and a media for culturing T cells to a container comprising a vacuum sealable interior chamber, wherein an inner surface of the interior chamber is coated with at least one dehydrated, desiccated, and/or lyophilized reagent capable of stimulating a T cell. In some examples, the disease or disorder is cancer, a disease or disorder associated with cancer, a disease or disorder associated with infection, an autoimmune disease or disorder, fibrosis, a disease or disorder associated with organ transplantation, a disease or disorder associated with tissue transplantation, or a disease or disorder associated with cell transplantation, or any combination thereof.

[0018] The invention provides, in part, a method of improving the effectiveness of an immunotherapy in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of a T cell population expanded by the method of stimulating or expanding a T cell population, wherein the method comprises applying a mixture containing a T cell population and a media for culturing T cells to a container comprising a vacuum sealable interior chamber, wherein an inner surface of the interior chamber is coated with at least one dehydrated, desiccated, and/or lyophilized reagent capable of stimulating a T cell. In some examples, the subject has at least one selected from the group consisting of cancer, a disease or disorder associated with infection, an autoimmune disease or disorder, fibrosis, a disease or disorder associated with organ transplantation, a disease or disorder associated with tissue transplantation, or a disease or disorder associated with cell transplantation, or any combination thereof.

[0019] The invention provides, in part, a method of generating a container comprising a vacuum sealable interior chamber, wherein an inner surface of the interior chamber is coated with at least one dehydrated, desiccated, and/or lyophilized reagent capable of stimulating a T cell, the method comprising the steps of: a) obtaining a vacuum sealable container comprising an interior chamber; b) depositing a cocktail of at least one reagent and a buffer into the interior chamber of the container; c) coating at least 85% of the inner surface of the interior chamber of the container with the cocktail to form a reagent coating; d) freezing the container until a completely homogenous temperature is reached within the reagent coating; and e) dehydrating, desiccating, and/or lyophilizing the reagent coating.

[0020] In some examples, the container comprises a flexible bag.

[0021] In some examples, the container is a FEP fluid bag with high surface energy.

[0022] In some examples, at least one reagent to be dehydrated, desiccated and/or lyophilized into the interior chamber of the container comprises an anti-CD3 antibody, an anti-CD28 antibody or a combination thereof.

[0023] In some examples, the buffer comprises at least one lyoprotectant. In some examples, the buffer comprises water, PBS, trehalose, a surfactant, an amino acid, a polymer, a sugar or any combination thereof.

[0024] In some examples, at least one additional reagent for the stimulation or expansion of a T cell population is applied to the container prior to dehydration, desiccation and/or lyophilization. In some examples, at least one additional reagent is IL-2.

[0025] In some examples, the reagent cocktail is applied to the container without introducing air into the container.

[0026] In some examples, the method further comprises removing any excess air prior to dehydration, desiccation and/or lyophilization.

[0027] In some examples, the bag is a fluid bag comprising at least one fluid port.

[0028] In some examples, the method further comprises adding a fdter to the fluid port prior to dehydration, desiccation and/or lyophilization to allow vapor to escape, while preventing contamination during dehydration, desiccation, and/or lyophilization.

[0029] In some examples, the volume of reagent solution added to the container is such that at least 85% of the interior surface area of the container can be coated with the solution, but the interior volume is not filled.

[0030] In some examples, polymeric beads are not introduced into the container.

[0031] In some examples, the container is stable when stored at 2-8°C for at least 1 day.

In some examples, the container is stable when stored at 2-8°C for at least 6 months.

[0032] The invention provides, in part, a method of isolating a T cell population, wherein the method comprises applying a mixture containing a T cell population and a media for culturing T cells to a container comprising a vacuum sealable interior chamber, wherein an inner surface of the interior chamber is coated with at least one dehydrated, desiccated, and/or lyophilized reagent capable of isolating and/or stimulating a T cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The following detailed description of preferred examples of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings examples which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the examples shown in the drawings.

[0034] Figure 1A through Figure 1C depict exemplary containers of the present invention. Figure 1A depicts a line drawing of an exemplary container. Figure IB and Figure 1C are images showing a prototype cell culture bag for T-Cell Activation in a closed container. Figure IB depicts an exemplary prepared cell culture bag containing lyophilized reagents in which trehalose was used as the solvent. Figure 1C depicts an exemplary prepared cell culture bag containing lyophilized reagents in which PBS was used as the solvent.

[0035] Figure 2 depicts an exemplary cell culture system incorporating the exemplary containers of the present invention.

[0036] Figure 3 depicts exemplary experimental results from a generic drug product study of a CAR-T process (the TransAct™ process) and a T cell activation process using prepared culture bags containing lyophilized reagents). Product doublings indicates the number of cell doublings observed within the process based on the initial seeding parameters. Product doublings were quantified at the initial time the device was made (Initial) and at least six months after the device was made (6+ months). Both the TransAct™ and prepared culture bags containing lyophilized reagents (Device) had very comparable product doublings over the duration of this study.

[0037] Figure 4 depicts exemplary experimental results from a generic drug product study of a CAR-T process. Transduction efficiency indicates the number of T cells expressing an exogenous CAR construct in relation to the entire population. Transduction efficiencies were quantified at the initial time the device was made (Initial) and at least six months after the device was made (6+ months). The population from the prepared culture bags containing lyophilized reagents (Device) had a significant increase in CAR% over that of traditional TransAct™.

[0038] Figure 5 depicts exemplary experimental results showing the viability of cell population at harvest. Viability was quantified at the initial time the device was made (Initial) and at least six months after the device was made (6+ months). The prepared culture bags containing lyophilized reagents (Device) had higher viability at harvest over that of traditional TransAct™ indicating that the polymer matrix contained within TransAct™ may lead to deleterious interactions with T cells over time.

[0039] Figure 6A through Figure 6D depict exemplary experimental results showing the viability (harvest viability) (Figure 6A), cell growth (fold expansion) (Figure 6B), transduction efficiency (Percent of CAR+ cells) (Figure 6C), and total viable CAR+ cells (Figure 6D) of cells using various formulations of the culture bag. For CAR construct 1, PBS has the highest CAR % with 40%. For CAR Construct 2, the CAR% of TransAct™, PBS and trehalose are all within a very close range. For CAR Construct 3, TransAct™, PBS, and water all have very comparable CAR%. Overall, the culture bag proved to be effective at inducing transduction similar to that of TransAct™. Across all of the CAR constructs, all the carrying solutions resulted in a device that stimulated T cells in a comparable performance to that of TransAct™ and facilitated transduction that led to comparable transgene expression.

[0040] Figure 7 depicts a representative schematic of simultaneous selection and activation of T cells using the culture bag.

[0041] Figure 8 depicts a representative schematic demonstrating the general method steps of using the culture bag. Base bag denotes the culture bag with 10 pg (lx) of both anti- CD3 and anti-CD28 antibody concentrations per bag. High bag denotes the culture bag with 20 pg (2x) of both anti-CD3 and anti-CD28 antibody concentrations per bag.

[0042] Figure 9 depicts exemplary experimental results demonstrating that the culture bag effectively enriched CD3+ T cells from apheresis.

[0043] Figure 10A through Figure 10C depict exemplary experimental results showing the total viability (Figure 10A), cell growth (product doublings) (Figure 10B), and cell viability (Figure 10C) of cells using the base bag (lx) and high bag (2x) formulations of the culture bag. These results demonstrate that cells grow at similar rates to standard methods (Transact™).

[0044] Figure 11 depicts exemplary experimental results showing the activation profile of T cells cultured in the culture bag. LDLr expression was quantified prior to cells being added to the culture bag (Day 0) and two days after cells were added to the culture bag (Day 2).

[0045] Figure 12A and Figure 12B depict exemplary experimental results demonstrating that over the course of 45 mins, the CD3 content of bulk apheresis contained within the culture vessel continually decreased, therefore the culture vessel was isolating CD3+ cells from the apheresis (Figure 12A). LDLr expression was quantified post selection and post activation (Figure 12B). These results demonstrate that the culture bag captured CD3+ cells from bulk apheresis. DETAILED DESCRIPTION

[0046] The present disclosure is based, in part, on the development of closable or sealable cell culture containers that have been pre-coated with at least one dehydrated, desiccated and/or lyophilized reagent. In some examples the dehydrated, desiccated and/or lyophilized reagent is a reagent for activating T cells for use in immunotherapy (e.g., adoptive T-cell therapy, chimeric antigen receptor (CAR) T cell therapy). In some examples, the cell culture containers are cell culture bags comprising an inner surface that is coated with at least one dehydrated, desiccated and/or lyophilized reagent for activating T cells and which allows for regulated flow through of one or more solution (e.g., cell culture media and a solution comprising T cells for culturing, isolation, stimulation and/or expansion.) Exemplary cell culture bags comprising lyophilized reagents are shown in Figure 1A - Figure 1C. Figure IB depicts an exemplary bag in which trehalose was used as the solvent. Figure 1C depicts an exemplary bag in which PBS was used as the solvent.

[0047] Therefore, some examples of this disclosure include cell culture bags for isolation, stimulation and/or expansion of T cells, wherein the bag comprises at least one dehydrated, desiccated and/or lyophilized antibody for T cell isolation and/or stimulation. In some examples, at least one dehydrated, desiccated and/or lyophilized antibody for T cell isolation and/or stimulation is an anti-CD3 antibody, an anti-CD28 antibody, or a combination thereof. [0048] Some examples of this disclosure include methods of improving the effectiveness of T cell isolation and/or stimulation for immunotherapy, as well as methods of isolating and/or stimulating T cells in a container comprising at least one dehydrated, desiccated and/or lyophilized reagent for activating T cells for use in immunotherapy.

[0049] Some examples of this disclosure include methods of use of the container for preparation of T cells for administration to a subject in need thereof. In some examples, the subject has had an organ transplantation, tissue transplantation, cell transplantation, allotransplantation, intestinal transplantation, reconstructive transplantation, cancer, a disease or disorder associated with cancer, or any combination thereof.

Definitions

[0050] Unless defined otherwise, 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 invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of some examples of this disclosure. [0051] As used herein, each of the following terms has the meaning associated with it in this section.

[0052] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0053] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0054] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, in some examples a mammal, and in some examples a human, having a complement system, including a human in need of therapy for, or susceptible to, a condition or its sequelae. The individual may include, for example, dogs, cats, pigs, cows, sheep, goats, horses, rats, monkeys, and mice and humans.

[0055] The term “abnormal” when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the “normal” (expected/homeostatic) respective characteristic. Characteristics which are normal or expected for one cell, tissue type, or subject, might be abnormal for a different cell or tissue type.

[0056] A “disease” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject’s health continues to deteriorate.

[0057] In contrast, a “disorder” in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject’s state of health.

[0058] The term “cancer” as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. [0059] A disease or disorder is “alleviated” if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a patient, or both, is reduced.

[0060] “Activation”, as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are undergoing cell division. [0061] The terms “inhibit” and “inhibition,” as used herein, means to reduce, suppress, diminish or block an activity or function by at least about 10% relative to a control value. In some examples, the activity is suppressed or blocked by at least about 50% compared to a control value. In some examples, the activity is suppressed or blocked by at least about 75%. In some examples, the activity is suppressed or blocked by at least about 95%.

[0062] As used herein, the term “autologous” is meant to refer to any material derived from the same individual to whom it is later to be re-introduced into the individual.

[0063] “Allogeneic” refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some examples, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.

[0064] The phrase “disease associated with expression of expression of alloresponsive cells” as used herein includes, but is not limited to, a disease associated with expression of alloresponsive cells or condition associated with cells which express alloresponse including, e.g., an allograft rejection, an immune rejection, a chronic allogeneic rejection, an engraftment rejection, a transplant rejection, an inflammation, an inflammation caused by ischemia/reperfusion, an infection, an immune response to an allograft, and any combination thereof.

[0065] “Xenogeneic” refers to a graft derived from an animal of a different species.

[0066] As used herein “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.

[0067] As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0068] As used herein, the term “immune cell” includes cells that are of hematopoietic origin and that play a role in the immune response. Immune cells include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, dendritic cells, eosinophils, mast cells, basophils, and granulocytes.

[0069] As used herein, the term “immune response” includes T cell mediated and/or B cell mediated immune responses that are influenced by modulation of T cell co-stimulation. The term immune response further includes immune responses that are indirectly effected by T cell activation such as antibody production (humoral responses) and the activation of cytokine responsive cells such as macrophages.

[0070] As used herein, the term “T cell immune response” refers to activation of antigen specific T cells as measured by proliferation or expression of molecules on the cell surface or secretion of proteins such as cytokines.

[0071] As used herein, the term “T cell” refers to a lymphocyte (e.g., white blood cell) that functions in cell-mediated immunity. In some examples, the presence of a T cell receptor (TCR) on the cell surface distinguishes T cells from other lymphocytes. As is known in the art, T cells typically do not present antigens, and rely on other lymphocytes (e.g., natural killer cells and B cells) to aid in antigen presentation. Types of T cells include: T helper cells (TH cells), Memory T cells (Tcm, Tem, or Temra), Regulatory T cells (Treg), Cytotoxic T cells (CTLs), Natural killer T cells (NK cells), gamma delta T cells, and Mucosal associated invariant T cells (MAIT).

[0072] As used herein, the term “TCR” refers to “T cell receptor.” A T cell receptor is a molecule on the surface of T lymphocytes (“T cells”). In examples, the receptor is an a[3-TCR receptor, meaning that the T cell receptor comprises an alpha (a) and beta ( ) chain, which is typically expressed as part of a complex with CD3 chain molecules.

[0073] As used herein, the term “B cell” refers to a cell produced in the bone marrow of an animal expressing membrane-bound antibody specific for an antigen. Following interaction with the antigen it differentiates into a plasma cell producing antibodies specific for the antigen or into a memory B cell. “B cell” and “B lymphocyte” is used interchangeably. Naive as well as activated B cells are within the scope of the invention. [0074] As used herein, the term “chimeric antigen receptor” or alternatively a “CAR” refers to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain comprising a functional signaling domain derived from a stimulatory molecule as defined below. In one example, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In one example, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In one example, the costimulatory molecule is chosen from 4 IBB (i.e., CD137), CD3, and/or CD28. In one example, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one example, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one example, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain comprising two functional signaling domains derived from one or more co-stimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one example, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more co- stimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one example the CAR comprises an optional leader sequence at the aminoterminus (N-ter) of the CAR fusion protein. In one example, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the scFv domain during cellular processing and localization of the CAR to the cellular membrane.

[0075] The portion of the CAR composition comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv) and a humanized antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one example, the antigen binding domain of a CAR composition of the invention comprises an antibody fragment. In one example, the CAR comprises an antibody fragment that comprises a scFv.

[0076] As used herein, a “signaling domain” is the functional portion of a protein which acts by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers.

[0077] The term “antibody,” as used herein, refers to a protein, or polypeptide sequence derived from an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be polyclonal or monoclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources. Antibodies are typically tetramers of immunoglobulin molecules.

[0078] The term “antibody fragment” refers to at least one portion of an intact antibody, or recombinant variants thereof, and refers to the antigen binding domain, e.g., an antigenic determining variable region of an intact antibody, that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, and multi-specific antibodies formed from antibody fragments. The term “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0079] An “antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.

[0080] An “antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa and lambda light chains refer to the two major antibody light chain isotypes.

[0081] By the term “recombinant antibody” as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art.

[0082] The term “antigen” or “Ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that some examples of this disclosure include, but are not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample, or might be macromolecule besides a polypeptide. Such a biological sample can include, but is not limited to a tissue sample, a cell or a fluid with other biological components.

[0083] An “antigen presenting cell” or “APC” as used herein, means an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays foreign antigens complexed with HLA I, HLA II, MHC I, MHC II, or MHC III complexes on their surfaces. For example, T cells may recognize these complexes using their T-cell receptors (TCRs). APCs process antigens and present them to T-cells.

[0084] The term “binding agent” as used herein refers to a molecule that binds a specific antigen or target. A binding agent may comprise a protein, peptide, nucleic acid, carbohydrate, lipid, or small molecular weight compound. In some examples, a binding agent comprises a full-length antibody. In some examples, a binding agent is an antigen binding fragment of an antibody. In some examples, a binding agent comprises an alternative protein scaffold or artificial scaffold (e.g., a non-immunoglobulin backbone). In some examples, a binding agent is a fusion protein comprising an antigen-binding site. In some examples, a binding agent is a bispecific molecule comprising at least two antigen-binding sites. In some examples, a binding agent is a multispecific molecule comprising at least three antigen-binding sites. [0085] The terms “binds” or “binding” refer to an interaction between molecules including, for example, to form a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment forthat epitope. The ratio of dissociation rate (koff) to association rate (kon) of a binding molecule (e.g., an antibody) to a monovalent antigen (koff/kon) is the dissociation constant KD, which is inversely related to affinity. The lower the KD value, the higher the affinity of the antibody. The value of KD varies for different complexes of antibody and antigen and depends on both kon and koff. The dissociation constant KD for an antibody provided herein can be determined using any method provided herein or any other method well known to those skilled in the art. The affinity at one binding site does not always reflect the true strength of the interaction between an antibody and an antigen. When complex antigens containing multiple, repeating antigenic determinants, such as a polyvalent antigen, come in contact with antibodies containing multiple binding sites, the interaction of antibody with antigen at one site will increase the probability of a reaction at a second site. The strength of such multiple interactions between a multivalent antibody and antigen is called the avidity.

[0086] In connection with the binding molecules described herein terms such as “bind to,” “that specifically bind to,” and analogous terms are also used interchangeably herein and refer to binding molecules of antigen binding domains that specifically bind to an antigen, such as a polypeptide. A binding molecule or antigen binding domain that binds to or specifically binds to an antigen can be identified, for example, by immunoassays, Octet®, Biacore®, or other techniques known to those of skill in the art. In some examples, a binding molecule or antigen binding domain binds to or specifically binds to an antigen when it binds to an antigen with higher affinity than to any cross-reactive antigen as determined using experimental techniques, such as enzyme linked immunosorbent assay (ELISA). Typically, a specific or selective reaction will be at least twice background signal or noise and may be more than 10 times background. See, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989) for a discussion regarding binding specificity. In certain examples, the extent of binding of a binding molecule or antigen binding domain to a “non-target” protein is less than about 10% of the binding of the binding molecule or antigen binding domain to its particular target antigen, for example, as determined by fluorescence activated cell sorting (FACS) analysis. A binding molecule or antigen binding domain that binds to an antigen includes one that is capable of binding the antigen with sufficient affinity such that the binding molecule is useful, for example, as a therapeutic and/or diagnostic agent in targeting the antigen. In certain examples, a binding molecule or antigen binding domain that binds to an antigen has a dissociation constant (KD) of less than or equal to IpM, 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. In certain examples, a binding molecule or antigen binding domain binds to an epitope of an antigen that is conserved among the antigen from different species.

[0087] The term “Cluster of Differentiation 3 a” or “CD3a” refers to a known protein which is also called “T-cell surface glycoprotein CD3 epsilon chain,” or “T3E.” CD3s. together with CD3-gamma, -delta and -zeta, and the T-cell receptor alpha/beta and gamma/delta heterodimers, forms the T-cell receptor-CD3 complex. This complex plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. The CD3 complex mediates signal transduction, resulting in T cell activation and proliferation. CD3 is required for the immune response. The amino acid sequence of a full length CD3s is shown in SEQ ID NO: 1. The amino acid sequence of the extracellular domain (ECD) of CD3s is shown in SEQ ID NO: 2. Throughout the specification, “CD3a- specific” or “specifically binds CD3a” or ”anti-CD3s antibody” refers to antibodies that bind specifically to the CD3s polypeptide, including antibodies that bind specifically to the CD3s extracellular domain (ECD);

[0088] Human CD3 epsilon:

[0089] MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCP QYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPED ANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVT RGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NOT); [0090] Human CD3 epsilon extracellular domain:

[0091] DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDK NIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD (SEQ ID NO:2).

[0092] By the term “stimulation,” is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR complex, BCR complex, etc.) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR complex and/or BCR complex, etc. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-J3, and/or reorganization of cytoskeletal structures, and the like.

[0093] The term “antibody,” “immunoglobulin,” or “Ig” is used interchangeably herein, and is used in the broadest sense and specifically covers, for example polyclonal antibodies, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full-length monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, recombinantly produced antibodies, single domain (e g., VHH) antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human versions of antibodies having full-length heavy and/or light chains. Antibodies also include antibody fragments (and/or polypeptides that comprise antibody fragments) that retain binding characteristics of their parental antibodies. Non-limiting examples of antibody fragments include antigenbinding regions and/or effector regions of the antibody, e.g, Fab, Fab’, F(ab’)2, Fv, scFv, (SCFV)2, single chain antibody molecule, dual variable domain antibody, single variable domain, linear antibody, V region, a multispecific antibody formed from antibody fragments, F(ab)2, Fd, Fc, diabody, di-diabody, disulfide-linked Fvs (dsFv), single-domain antibody (e.g., nanobody) or other fragments (e.g., fragments consisting of the variable regions of the heavy and light chains that are non-covalently coupled). In general terms, a variable (V) region domain may be any suitable arrangement of immunoglobulin heavy (VH) and/or light (VL) variable domains. For example, antibodies also include tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, and an antibody heavy chain monomer. Thus, for example, the V region domain may be dimeric and contain VHH- VHH, VH-VH, VH-VL, or VL-VL dimers that bind NKG2A. If desired, the VH and VL may be covalently coupled either directly or through a linker to form a single chain Fv (scFv). For ease of reference, scFv proteins are referred to herein as included in the category “antibody fragments.” Another form of an antibody fragment is a peptide comprising one or more complementarity determining regions (CDRs) of an antibody. CDRs (also termed “minimal recognition units” or “hypervariable regions”) can be obtained by constructing polynucleotides that encode one or more CDRs of interest. Such polynucleotides are prepared, for example, by using the polymerase chain reaction to synthesize the variable region using mRNA of antibody-producing cells as a template (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology, 2: 106 (1991); Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166, Cambridge University Press (1995); and Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137, Wiley-Liss, Inc. (1995)). Antibody fragments may be incorporated, for example, into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, variable domains of new antigen receptors (v-NAR), and bis-single chain Fv regions (see, e.g., Hollinger and Hudson, Nature Biotechnology, 23(9): 1126-1136, 2005). In some examples, antibodies comprising a VH and/or VL contain a light chain and/or a heavy chain constant region, such as one or more constant regions, including one or more IgGl, IgG2, IgG3 and/or IgG4 constant regions. In some examples, antibodies can include epitopebinding fragments of any of the above. The antibodies described herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) of immunoglobulin molecule.

[0094] A “stimulatory molecule,” as the term is used herein, means a molecule expressed by a cell that provide the primary cytoplasmic signaling sequence(s) that regulate primary activation of the TCR and/or BCR complex in a stimulatory way for at least some examples of the cell signaling pathway. In one example, the primary signal is initiated by, for instance, binding of a TCR and/or BCR complex with a human leukocyte antigen (HLA) I, HLA II, major histocompatibility complex (MHC) I, MHC II, or MHC III molecule loaded with peptide, and which leads to mediation of a cell response, including, but not limited to, proliferation, activation, differentiation, and the like. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences that are of particular use in the invention include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as “ICOS”) and CD66d. In a specific CAR of the invention, the cytoplasmic signaling sequence derived from CD3-zeta is derived from a non-human species, e.g., mouse, rodent, monkey, ape and the like.

[0095] The terms “effective amount” and “pharmaceutically effective amount” refer to a sufficient amount of an agent to provide the desired biological result. That result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system. An appropriate effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. [0096] The term “therapeutic” as used herein means a treatment. A therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.

[0097] A “therapeutic treatment” is a treatment administered to a subject who exhibits signs of disease or disorder, for the purpose of diminishing or eliminating those signs.

[0098] As used herein, “treating a disease or disorder” means reducing the frequency and/or severity of a sign and/or symptom of the disease or disorder is experienced by a patient.

[0099] The term “prophylaxis” as used herein means the prevention of or protective treatment for a disease or disease state.

[00100] The phrase “biological sample”, “sample” or “specimen” as used herein, is intended to include any sample comprising a cell, a tissue, or a bodily fluid in which expression of a nucleic acid or polypeptide can be detected. The biological sample may contain any biological material suitable for detecting the desired biomarkers, and may comprise cellular and/or non-cellular material obtained from the individual. Examples of such biological samples include but are not limited to blood, lymph, bone marrow, biopsies and smears. Samples that are liquid in nature are referred to herein as “bodily fluids.” Biological samples may be obtained from a patient by a variety of techniques including, for example, by scraping or swabbing an area or by using a needle to obtain bodily fluids. Methods for collecting various body samples are well known in the art.

[00101] As used herein, an “immunoassay” refers to any binding assay that uses an antibody capable of binding specifically to a target molecule to detect and quantify the target molecule.

[00102] By the term “specifically binds,” as used herein with respect to a polypeptide (e.g., a TCR or TCR chain), is meant a polypeptide which recognizes and binds to a specific target molecule, but does not substantially recognize or bind other molecules in a sample. In some instances, the terms “specific binding” or “specifically binding,” is used to mean that the recognition and binding is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the target molecule.

[00103] A “coding region” of a gene consists of the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the gene. A “coding region” of a mRNA molecule also consists of the nucleotide residues of the mRNA molecule which are matched with an anti -codon region of a transfer RNA molecule during translation of the mRNA molecule or which encode a stop codon. The coding region may thus include nucleotide residues comprising codons for amino acid residues which are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).

[00104] “Complementary” as used herein to refer to a nucleic acid, refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (“base pairing”) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. In some examples, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and or at least about 75%, or at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. In some examples, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

[00105] “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared X 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. Generally, a comparison is made when two sequences are aligned to give maximum homology.

[00106] “Variant” as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential biological properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis. In various examples, the variant sequence is at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, at least 85% identical to the reference sequence.

[00107] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting there from. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[00108] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[00109] ‘ ‘Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in its normal context in a living subject is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural context is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. [00110] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[00111] In the context of some examples of this disclosure, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[00112] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[00113] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[00114] The term “recombinant DNA” as used herein is defined as DNA produced by joining pieces of DNA from different sources.

[00115] The term “recombinant polypeptide” as used herein is defined as a polypeptide produced by using recombinant DNA methods.

[00116] As used herein, “conjugated” refers to covalent attachment of one molecule to a second molecule.

[00117] “Operably linked” or “operatively linked” as used herein may mean that expression of a gene is under the control of a promoter with which it is spatially connected. A promoter may be positioned 5' (upstream) or 3' (downstream) of a gene under its control. The distance between the promoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function.

[00118] The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[00119] The term “regulating” as used herein can mean any method of altering the level or activity of a substrate. Non-limiting examples of regulating with regard to a protein include affecting expression (including transcription and/or translation), affecting folding, affecting degradation or protein turnover, and affecting localization of a protein. Non-limiting examples of regulating with regard to an enzyme further include affecting the enzymatic activity. “Regulator” refers to a molecule whose activity includes affecting the level or activity of a substrate. A regulator can be direct or indirect. A regulator can function to activate or inhibit or otherwise modulate its substrate.

[00120] “Vector” as used herein may mean a nucleic acid sequence containing an origin of replication. A vector may be a plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be either a self-replicating extrachromosomal vector or a vector which integrates into a host genome. [00121] As used herein, a “substantially purified” cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some examples, the cells are cultured in vitro. In other examples, the cells are not cultured in vitro.

[00122] Ranges: throughout this disclosure, various examples of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Description

[00123] In some examples and as illustrated in Figures 1A, IB and 1C, the present invention provides a closable or sealable container 10 for holding at least one reagent. Container 10 includes an inner surface forming one or more interior chambers 14 for holding or storing a sample containing the at least one reagent. Container 10 may be any size and shape, and further may be rigid, flexible, elastic, or any combination thereof. For example, container 10 may be sized to contain 1,000 mb, 500 mb, 250 mb, 100 mb, or 50 mb of fluid. In some examples, container 10 is a sealable, flexible bag, such as a Vue Life® “AC” Series Bag. In some examples, container 10 has a plurality of interior chambers 14 fluidly connected to each other via a permeable or semipermeable membrane and/or at least one valve.

[00124] In some examples, the inner surface of interior chamber 14 is smooth. In some examples, the interior surface of interior chamber 14 includes one or more grooves, ridges, channels, recesses, protrusions, pockets or the like, thereby creating one or more regions within interior chamber 14 that have an altered surface profile. In some examples, the altered surface profile may expand the total surface area within interior chamber 14. In some examples, the altered surface profile may direct or influence flow of a liquid or gas within interior chamber 14. In some examples, interior chamber 14 includes at least one microfluidic structure configured to direct flow of a fluid sample therethrough.

[00125] In some examples, container 10 includes one or more ports 12 fluidly connected to interior chamber 14. Ports 12 may be connected to any type of tubing or conduit suitable for directing flow of a liquid or gas sample into and/or out of interior chamber 14 of container 10. Ports 12 and/or the connected tubing may further include any type of valve or luer-lock to open or close interior chamber 14 of container 10 from the flow of gas or liquid. In some examples, at least one port 12 is fluidly connected to a cell culture system. In some examples, at least one port 12 is fluidly connected to a vacuum pump for generating a full or partial vacuum (i.e. negative pressure) within interior chamber 14 of container 10. Accordingly, container 10 may include one or more liquid or gas flow paths into and out of interior chamber 14. For example, container 10 may include an inlet port 12a and an outlet port 12b each connected to interior chamber 14, such that fluid is configured to flow into container 10 through inlet port 12a, into interior chamber 14, and subsequently out of container 10 through outlet port 12b.

[00126] As contemplated herein, container 10 may be constructed of any suitable single use or sterilizable material, such as polyvinyl chloride (PVC), ethylene vinyl acetate, polypropylene, fluorinated ethylene propylene (FEP) or any other appropriate material. [00127] In some examples, container 10 is composed of an impermeable material. In some examples, container 10 is composed of a semi-permeable material or a porous material. In some examples, container 10 is composed of a material that is gas permeable and liquid impermeable. For example, the material may include a pore size of less than 0.2pm to maintain sterility of the contents while permitting gas exchange within container 10. The material of container 10 may have any desired thickness. In some examples, the material of container 10 may have a thickness of between 10 - 1000 pm. In some examples, the material of container 10 may have a thickness of between 100 - 500 pm. In some examples, the inside surface of container 10 may have a high surface energy. In some examples, the inside surface of container 10 may carry an electrical charge. In some examples, the inside surface of container 10 may be modified with hydroxyl or carboxyl groups, or other chemical groups that result in a change in adhesion properties of one or more molecules.

Cell Culture System [00128] In some examples, container 10 can be incorporated into a cell culture system comprising multiple components. In one example, the cell culture system is an automated system. In one example, the cell culture system is a microfluidic system. For example, the container can be included or incorporated into commercial cell culture and manufacturing systems, including but not limited to, Miltenyi Biotech CliniMACS Prodigy Platform.

[00129] In one example, the cell culture system according to the invention comprises at least one cell culture module comprising container 10 which functions as a bioreactor. The term bioreactor in the context of this application refers to vessels intended for the take-up of cells, which include but are not limited to variations of cell proliferation flasks, centrifugation vessels, cell isolation vessels, cell differentiation vessels, cell seeding vessels, sample vessels, etc.

[00130] In one example, the cell culture module is a closed system, which means that within the closed cell culture module a closed sterile environment can be maintained. In one example, the cell culture system further comprises at least one pump for pumping liquids within the closed cell culture module. In some examples, the system further comprises at least one additional tool module. In the context of this application, the term "tool module" refers to any tool or instrument, which manipulates or monitors in any way anyone or more than one of the components of the cell culture system such as the cell cultures grown in bioreactors of the cell culture system or other components, which are comprised in the cell culture system such as culture media and enzymes etc.

[00131] Such tool modules include monitoring tool modules for monitoring the process and the cell cultures in the bioreactors, such monitoring modules being, a cell imaging device (e.g. comprising a microscope and a camera), or any kind of sensor technology device such as a pH and temperatures sensors etc. Further possible tool modules include manipulator tool modules such as, shakers, peristaltic pumps, actuators for opening and closing valves, actuators or moving mechanisms for displacing modules or other components of the closed cell culture module and/or the tool modules relative to each other. Yet further tools include harvesting modules such as a cell wash/cell concentration device (e.g. a centrifuge). In some examples of the closed cell culture module, the system comprises a peristaltic pump.

[00132] In some examples the cell culture system comprises at least two units, a cell maintenance unit for proper storage of cell cultivation intermediates, final products as well as for storage of process fluids and a processing unit (or cell processing unit) for cell growth and cell processing. In some examples the cell culture system comprises additional units, for example an additional storage unit such as for the cryo-preservation of cells. In each unit the ambient physical conditions are adjustable individually such as for example temperature and humidity.

[00133] For example, the temperature is regulated to set the processing unit e.g. at a temperature of 37°C, the cell maintenance unit at a temperature of 4°C and a storage unit at a temperature of -196°C etc.

[00134] In one example, one or more containers of the invention (e.g., 10), which are part of the closed cell culture module, are kept in the cell processing unit. In one example, the cell maintenance unit provides standard refrigerator temperatures to allow proper storage of temperature sensitive liquids such as culture media or enzyme solutions as well as preservation of final cell-based products or cell intermediates such as samples for quality control purposes. In one example, those components of the closed cell culture system requiring refrigerated temperatures will be housed in the cell maintenance unit.

[00135] Referring now to Figure 2, an exemplary cell culture system 200 incorporating any of the exemplary containers is shown. In some examples, cell culture system 200 can be automated. Cell culture system 200 may include a cell separator 201. Cell separator 201 can be any suitable cell separator, including, for example, a centrifugation separator, a magnetbased separator, a size exclusion-based separator, and/or an antibody affinity column separator. The cell culture system 200 may include container 203, which can be implemented as one of exemplary containers described herein with a dehydrated, desiccated, and/or lyophilized reagent (e.g. container 10 of Figures 1A, IB and 1C). The cell culture system 200 can comprise one or more pumps and/or gravity drains (e.g., 202, 204, 207, 209, 211) that transfer product between devices in the cell culture system 200. Although separate pumps are shown, it will be understood that as few as a single pump can be used to implement pumps 202, 204, 207, 209, 211.

[00136] T-cells separated by the cell separator 201 can be provided to the container 203 by a pump and/or gravity drain 204. Activated T-cells from the container 203 can be transferred from container 203 by pump or gravity drain 204 to one or both of a gene editing device or vessel 205 and attenuation vessel 206. The gene editing device or vessel can comprise, for example, an electroporator and/or bioreactor. The attenuation vessel can be, for example, a bioreactor. The cells can be transferred from the gene editing device or vessel 205 and/or attenuation vessel 206 to a cell expansion vessel or device via pump or gravity device 207. After expansion, the cells are provided via pump or gravity drain 209 to a washing or impurity removal device 210, which can comprise, for example, a cell washing device, a cell concentration device, and/or a centrifuge. Lastly, the cells are provided via pump or gravity drain 211 to a formulation device 212. Formulation device 212 can comprise devices for mixing, pumping, and fdling vials and/or bags and storage units such as cryopreservation units for storing the vials and/or bags.

Dehydrated, Desiccated and/or Lyophilized Reagents

[00137] As contemplated herein, the inner surface within interior chamber 14 of container 10 includes at least one dehydrated, desiccated, and/or lyophilized reagent. The dehydrated, desiccated, and/or lyophilized reagent may adhere or bind directly to at least a portion of the inner surface, or the inner surface may include a coating of one or more materials to aid or enhance binding of the dehydrated, desiccated, and/or lyophilized reagent. The reagent may be introduced into interior chamber 14 via a liquid or gas sample delivered through inlet port 12a, and then subsequently lyophilized within interior chamber 14 of container 10. Binding of the reagent to the inner surface of interior chamber 14 may occur prior to dehydration, desiccation, and/or lyophilization, or as a result of dehydration, desiccation, and/or lyophilization.

[00138] In some examples, interior chamber 14 may have a mixture or concentration of the at least one lyophilized reagent on the inner surface. In some examples, the mixture or concentration of the at least one lyophilized reagent on the inner surface can be uniform or substantially uniform. For example, the substantially uniform mixture or concentration may have a uniformity that is > 80%, > 85%, > 90%, > 92%, > 94%, > 96%, > 98%, or > 99%. In other examples, the mixture or concentration of the at least one lyophilized reagent on the inner surface can be variable. For example, inner chamber 14 may have a concentration gradient of lyophilized reagent across the inner surface, such as a higher concentration near the bottom of container 10, and a lower concentration near the top of container 10. In some examples, interior chamber 14 may have a plurality of inner surface regions (e.g. 2, 3, 4, 5, 6, 7 or 8 inner surface regions) where a first region has a different concentration of lyophilized reagent as compared to a second region. In some examples, each region has a different concentration of lyophilized reagent. Likewise, a first region may have a first lyophilized reagent of a first concentration, and second region may have a second lyophilized reagent of a second concentration.

[00139] In one example, the lyophilized reagent is a reagent for stimulation of T cells. In one example, the lyophilized reagent is a reagent for isolation of T cells. In some examples, the inner surface is coated with a combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 different reagents for isolation and/or stimulation of T cells. In one example, two or more agents are lyophilized in the interior chamber of the container. In one example, each of two or more reagent is lyophilized in the interior chamber of the container at a 1: 1 ratio. In one example, each of two or more reagent is lyophilized in the interior chamber of the container at a ratio in a range from 100: 1 to 1 : 100 and all integer values there between. In one example, two or more agents are lyophilized in the interior chamber of the container. In one example, each of three or more reagents are lyophilized in the interior chamber of the container at a 1: 1: 1 ratio. In one example, each of three or more reagent is lyophilized in the interior chamber of the container at a ratio in a range from 1: 100: 1 to 1: 1: 100 to 100: 1: 1 and all integer values there between.

[00140] In one example, at least one reagent to be dehydrated, desiccated and/or lyophilized in a container is suspended in a buffer solution prior to dehydration, desiccation and/or lyophilization. In one example, the buffer solution protects the reagent from degradation or inactivation during dehydration, desiccation and/or lyophilization. In one example, the buffer solution comprises at least one lyoprotectant. Exemplary lyoprotectants include, but are not limited to, surfactants (poloxamers, solysorbates, etc.), amino acids (arginine, serine, histidine, etc.), polymers (PEG, PVP, dextran, etc.) and sugars (sucrose, etc.). In one example, the buffer solution comprises PBS. In one example, the buffer solution comprises trehalose. In one example, the buffer solution comprises water.

[00141] In some examples, at least one dehydrated, desiccated and/or lyophilized reagent is a peptide, a protein, an antibody, a nucleic acid molecule, or a combination thereof. In one example, the cell culture device comprises a surface coated with at least one dehydrated, desiccated and/or lyophilized antibody for isolating and/or stimulating T cells.

[00142] In some examples, the reagents for isolating and/or stimulating T cells are not attached to polymeric beads. Therefore in some examples, polymeric beads are not introduced into the container.

[00143] The peptidic reagent of the invention can be post-translationally modified prior to dehydration, desiccation and/or lyophilization. For example, post-translational modifications that fall within the scope of some examples of this disclosure include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events require introduction of additional biological machinery. For example, processing events, such as signal peptide cleavage and core glycosylation, are examined by adding canine microsomal membranes or Xenopus egg extracts (U.S. Pat. No. 6,103,489) to a standard translation reaction. [00144] The dehydrated, desiccated and/or lyophilized peptidic reagent may include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation. A variety of approaches are available for introducing unnatural amino acids during protein translation. By way of example, special tRNAs, such as tRNAs which have suppressor properties, suppressor tRNAs, have been used in the process of site-directed non-native amino acid replacement (SNAAR). In SNAAR, a unique codon is required on the mRNA and the suppressor tRNA, acting to target a non-native amino acid to a unique site during the protein synthesis (described in WO 90/05785). However, the suppressor tRNA must not be recognizable by the aminoacyl tRNA synthetases present in the protein translation system. In certain cases, a non-native amino acid can be formed after the tRNA molecule is aminoacylated using chemical reactions which specifically modify the native amino acid and do not significantly alter the functional activity of the aminoacylated tRNA. These reactions are referred to as post-aminoacylation modifications. For example, the epsilon-amino group of the lysine linked to its cognate tRNA (tRNALYS), could be modified with an amine specific photoaffinity label.

[00145] The dehydrated, desiccated and/or lyophilized peptidic reagent of some examples of this disclosure may be made using chemical methods. For example, peptides can be synthesized by solid phase techniques (Roberge J Y et al (1995) Science 269: 202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography. Automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer. The peptide may alternatively be made by recombinant means or by cleavage from a longer polypeptide. The composition of a peptide may be confirmed by amino acid analysis or sequencing.

[00146] In some examples, the invention includes peptidic variants. Variants of the peptides according to some examples of this disclosure may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the polypeptide is an alternative splice variant of the polypeptide of some examples of this disclosure, (iv) fragments of the polypeptides and/or (v) one in which the polypeptide is fused with another polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification (for example, His-tag) or for detection (for example, Sv5 epitope tag). The fragments include polypeptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post-translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.

[00147] The dehydrated, desiccated and/or lyophilized peptidic reagents of the invention may be converted into pharmaceutical salts by reacting with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, etc., or organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benezene sulfonic acid, and toluenesulfonic acids.

[00148] In one example, at least one dehydrated, desiccated and/or lyophilized peptidic reagent is a binding agent comprising an antigen binding region that binds a T cell receptor (TCR). In some examples, the binding agent comprises at least one antigen binding region that binds to CD3, CD28, or a combination of CD3 and CD28. In some examples, the binding agent is an antibody or a binding domain derived from an antibody. In some examples, the antibody is a recombinant antibody. In some examples, the antibody is a monoclonal antibody. In some examples, the antibody is a chimeric antibody. In some examples, the antibody is a humanized antibody. In some examples, the antibody is a human antibody. In some examples, the antibody is an IgG antibody. In some examples, the antibody is an IgG I antibody. In some examples, the antibody is an IgG2 antibody. In some examples, the antibody is an IgG3 antibody. In some examples, the antibody is an IgG4 antibody. In some examples, the antibody comprises an IgG heavy chain. In some examples, the antibody comprises an IgGl heavy chain. In some examples, the antibody comprises an IgG2 heavy chain. In some examples, the antibody comprises an IgG4 heavy chain. In some examples, the antibody comprises a kappa light chain. In some examples, the antibody comprises a kappa light chain constant region. In some examples, the antibody comprises a lambda light chain. In some examples, the antibody comprises a lambda light chain constant region. In some examples, the antibody is an antibody fragment comprising an antigenbinding site. In some examples, the antibody is an scFv. In some examples, the antibody is a disulfide-linked scFv. In some examples, the antibody is a di sulfide -linked sc(Fv)2. In some examples, the antibody is a Fab, Fab’, or a F(ab)2 antibody. In some examples, the antibody is a diabody. In some examples, the antibody is a nanobody. In some examples, the antibody is a monospecific antibody. In some examples, the antibody is a bispecific antibody. In some examples, the antibody is a trispecific antibody. In some examples, the antibody is a multispecific antibody. In some examples, the antibody is a monovalent antibody. In some examples, the antibody is a multivalent antibody. In some examples, the antibody is a bivalent antibody. In some examples, the antibody is atrivalent antibody. In some examples, the antibody is a tetravalent antibody.

[00149] In one example, the dehydrated, desiccated and/or lyophilized reagent comprises an anti-CD3, and/or an anti-CD28 antibody, including any and all clones thereof. In one example, the dehydrated, desiccated and/or lyophilized reagent comprises a combination of an anti-CD3, an anti-CD28 antibody, and/or any and all clones thereof. In one example, the anti-CD3 antibody is OKT3-aCD3. In one example, the anti-CD28 antibody is 15e8-CD28. In some examples, the dehydrated, desiccated and/or lyophilized reagent comprises a combination of an anti-CD3 antibody and an anti-CD28 antibody. In some examples, the dehydrated, desiccated and/or lyophilized reagent comprises a combination of an anti-CD3 antibody and an anti-CD28 antibody and one or more additional reagent. In some examples, one or more additional reagent is IL-2 or other cytokines.

Cells

[00150] In certain examples, the container of the invention is used to culture or expand a specific cell type. In some examples the cells that are cultured in the device may be of any suitable cell type. In certain examples, the cultured cell is used in a method where the cell is introduced into a recipient. In certain examples, the cell is autologous, allogeneic, syngeneic or xenogeneic with respect to recipient. In certain examples the cell is derived from a stem cell or precursor cell. In some examples, the stem cell or precursor cell from which the modified cell is derived is autologous, allogeneic, syngeneic or xenogeneic with respect to recipient.

[00151] In one example, the cell is an immune cell. Exemplary immune cells that may isolated, stimulated and/or expanded or cultured as described herein include, but are not limited to, T cells (including killer T cells, helper T cells, regulatory T cells, T cell bearing TCRs, alloresponsive T cell, allo-specific T cell, T cell bearing alloreactive TCR, and gamma delta T cells), B cells, antigen presenting cells (APCs), NK cells, NK T cells, CAR T cells, and TCR-expressing T cells, dendritic cells (DCs), macrophages, Langerhans cells, and the like.

[00152] The disclosed compositions and methods can be applied to isolation and/or stimulation of T cells for use in basic research and therapy, in the fields of cancer, stem cells, acute and chronic infections, fibrosis and autoimmune diseases, including the use of genetically modified T cells to kill a target cancer cell, treat an autoimmune disease, and/or to treat fibrosis.

[00153] In some examples, prior to expansion a source of T cells is obtained from a subject. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some examples of this disclosure, any number of T cell lines available in the art, may be used. In some examples of this disclosure, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Fico 11™ separation. In one example, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one example, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.

[00154] In another example, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3⁺, CD28⁺, CD4⁺, CD8⁺, CD45RA⁺, and CD45RO⁺T cells, can be further isolated by positive or negative selection techniques.

[00155] In one example, T cells are isolated by culturing the cells for a time period sufficient for positive selection of the desired T cells. In one example, the time period is about 30 minutes. In a further example, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further example, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another example, the time period is 10 to 24 hours. In one example, the incubation time period is 24 hours. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immune- compromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3/CD28 antibodies, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process.

Additionally, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on the surface, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points. The skilled artisan would recognize that multiple rounds of selection can also be used in the context of this invention. In certain examples, it may be desirable to perform the selection procedure and use the “unselected” cells in the activation and expansion process. “Unselected” cells can also be subjected to further rounds of selection.

[00156] Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is selection via negative immunoadherence that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD1 lb, CD16, HLA-DR, and CD8. In certain examples, it may be desirable to enrich for or positively select for regulatory T cells which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain examples, T regulatory cells are depleted by anti-C25 conjugated beads or other similar method of selection.

[00157] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and selection antibodies can be varied. In certain examples, it may be desirable to significantly decrease the volume in which selection antibodies and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and selection antibodies. For example, in one example, a concentration of 2 billion cells/ml is used. In one example, a concentration of 1 billion cells/ml is used. In a further example, greater than 100 million cells/ml is used. In a further example, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In yet another example, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further examples, concentrations of 125 or 150 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest. Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.

[00158] In a related example, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and selection antibodies, interactions between the selection antibodies and cells is minimized. This selects for cells that express high amounts of desired antigens to be bound to the surface adherent antibodies. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations. In one example, the concentration of cells used is 5 X 10⁶/ml. In other examples, the concentration used can be from about 1 X 10⁵/ml to 1 X 10⁶/ml, and any integer value in between.

[00159] In some examples, the cells may be incubated for varying lengths of time at either 2-10 °C or at room temperature. In some examples, the cells may be incubated on a rotator at varying speeds.

[00160] Also contemplated in the context of the invention is the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein. In one example a blood sample or an apheresis is taken from a generally healthy subject. In certain examples, a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain examples, the T cells may be expanded, frozen, and used at a later time. In certain examples, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments. In a further example, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson et al., hnmun. 73:316-321, 1991; Bierer et al., Curr. Opin. hnmun. 5:763-773, 1993). In a further example, the cells are isolated for a patient and frozen for later use in conjunction with (e.g., before, simultaneously or following) bone marrow or stem cell transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another example, the cells are isolated prior to and can be frozen for later use for treatment following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. [00161] In some examples of this disclosure, T cells are obtained from a patient directly following treatment. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of some examples of this disclosure to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery phase. Further, in certain examples, mobilization (for example, mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[00162] The T cells can be activated and expanded. Generally, the T cells of the invention are expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD28 antibody, or antigen-binding fragment thereof, or a combination thereof, in a container. For co -stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besangon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328, 1999; Garland et al., J. Immunol Meth. 227(1 -2):53-63, 1999).

[00163] In certain examples, the T cells are expanded prior to genetic modification. In certain examples, the T cells are expanded following genetic modification.

[00164] In certain examples, the primary stimulatory signal and the co-stimulatory signal for the T cell may be provided by different protocols. For example, the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in “cis” formation) or to separate surfaces (i.e., in “trans” formation). Alternatively, one agent may be coupled to a surface and the other agent in solution. In one example, the agent providing the co-stimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain examples, both agents can be in solution. In another example, the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents. In this regard, see for example, U.S. Patent Application Publication Nos. 2004/0101519 and 2006/0034810 for artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding T cells in some examples of this disclosure.

[00165] In one example, the two agents are dehydrated, desiccated and/or lyophilized in the interior chamber of the container. By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof and the agent providing the co-stimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof; and both agents are co-dehydrated, co-desiccated and/or co-lyophilized in the interior chamber of the container. In one example, a 1 : 1 ratio of each antibody dehydrated, desiccated and/or lyophilized in the interior chamber of the container for CD4+ T cell expansion and T cell growth is used. In certain examples of some examples of this disclosure, a ratio of anti CD3:CD28 antibodies dehydrated, desiccated and/or lyophilized in the interior chamber of the container is used such that an increase in T cell expansion is observed as compared to the expansion observed using a ratio of 1 : 1. In one particular example an increase of from about 1 to about 3 fold is observed as compared to the expansion observed using a ratio of 1: 1. In one example, the ratio of CD3:CD28 antibody dehydrated, desiccated and/or lyophilized in the interior chamber of the container ranges from 100: 1 to 1 : 100 and all integer values there between. In one example of some examples of this disclosure, more anti-CD28 antibody is dehydrated, desiccated and/or lyophilized in the interior chamber of the container than anti-CD3 antibody, i.e., the ratio of CD3:CD28 is less than one. In certain examples of the invention, the ratio of anti CD28 antibody to anti CD3 antibody dehydrated, desiccated and/or lyophilized in the interior chamber of the container is greater than 2: 1. In one example, a 1: 100 CD3:CD28 ratio of antibody dehydrated, desiccated and/or lyophilized in the interior chamber of the container is used. In another example, a 1:75 CD3:CD28 ratio of antibody dehydrated, desiccated and/or lyophilized in the interior chamber of the container is used. In a further example, a 1:50 CD3:CD28 ratio of antibody dehydrated, desiccated and/or lyophilized in the interior chamber of the container is used. In another example, a 1:30 CD3:CD28 ratio of antibody dehydrated, desiccated and/or lyophilized in the interior chamber of the container is used. In one preferred example, a 1 : 10 CD3:CD28 ratio of antibody dehydrated, desiccated and/or lyophilized in the interior chamber of the container is used. In another example, a 1:3 CD3:CD28 ratio of antibody dehydrated, desiccated and/or lyophilized in the interior chamber of the container s is used. In yet another example, a 3: 1 CD3:CD28 ratio of antibody dehydrated, desiccated and/or lyophilized in the interior chamber of the container is used.

[00166] Those of ordinary skill in the art can readily appreciate any cell concentration may be used. For example, the target cell may be very rare in the sample and comprise only 0.01% of the sample or the entire sample (i.e., 100%) may comprise the target cell of interest. Accordingly, any cell number is within the context of some examples of this disclosure. For example, in one example, a concentration of about 2 billion cells/ml is used. In another example, greater than 100 million cells/ml is used. In a further example, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In yet another example, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further examples, concentrations of 125 or 150 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells. Such populations of cells may have therapeutic value and would be desirable to obtain in certain examples. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.

[00167] In some examples of this disclosure, the cells may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. In another example, the cells may be cultured for 21 days. In one example of the invention the T cells are cultured for about eight days. In another example, the T cells are cultured for 2-3 days. Several cycles of stimulation may also be desired such that culture time of T cells can be 60 days or more. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-y, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF[3, and TNF-a or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, ImmunoCult-XF from StemCell, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% CO2).

[00168] T cells that have been exposed to varied stimulation times may exhibit different characteristics. For example, typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (TH, CD4+) that is greater than the cytotoxic or suppressor T cell population (TC, CD8+). Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of TC cells. Accordingly, depending on the purpose of treatment, infusing a subject with a T cell population comprising predominately of TH cells may be advantageous. Similarly, if an antigen-specific subset of TC cells has been isolated it may be beneficial to expand this subset to a greater degree.

[00169] Further, in addition to CD4 and CD8 markers, other phenotypic markers vary significantly, but in large part, reproducibly during the course of the cell expansion process. Thus, such reproducibility enables the ability to tailor an activated T cell product for specific purposes.

CAR T cells

[00170] In one example, the cell comprises a T cell modified to express a recombinant T cell receptor (TCR) or a chimeric antigen receptor (CAR). In certain examples disclosed herein, a CAR generally comprises an antigen binding domain, a transmembrane domain and an intracellular domain. In some examples, the CAR comprises an antigen binding domain that binds to a tumor-associated antigen or a tumor-specific antigen.

[00171] In various examples, the CAR can be any CAR molecule including, but not limited to, a “first generation,” “second generation,” “third generation,” “fourth generation” or “fifth generation” CAR (see, for example, Sadelain et al., Cancer Discov. 3(4):388-398 (2013); Jensen et al., Immunol. Rev. 257: 127-133 (2014); Sharpe et al., Dis. Model Meeh. 8(4):337-350 (2015); Brentjens et al., Clin. Cancer Res. 13:5426-5435 (2007); Gade et al., Cancer Res. 65:9080-9088 (2005); Maher et al., Nat. Biotechnol. 20:70-75 (2002); Kershaw et al., J. Immunol. 173:2143-2150 (2004); Sadelain et al., Curr. Opin. Immunol. (2009); Hollyman et al., J. Immunother. 32: 169-180 (2009)).

[00172] ‘ ‘First generation” CARs for use in the invention comprise an antigen binding domain, for example, a single -chain variable fragment (scFv), fused to a transmembrane domain, which is fused to a cytoplasmic/intracellular domain of a T cell receptor chain. “First generation” CARs typically have the intracellular domain from the CD3^-chain. which is the primary transmitter of signals from endogenous T cell receptors (TCRs). “First generation” CARs can provide de novo antigen recognition and cause activation of both CD4+ and CD8+ T cells through their CD3^ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation.

[00173] ‘ ‘Second-generation” CARs for use in the invention comprise an antigen binding domain, for example, a single -chain variable fragment (scFv), fused to an intracellular signaling domain capable of activating T cells and a co-stimulatory domain designed to augment T cell potency and persistence (Sadelain et al., Cancer Discov. 3:388-398 (2013)). CAR design can therefore combine antigen recognition with signal transduction, two functions that are physiologically borne by two separate complexes, the TCR heterodimer and the CD3 complex. “Second generation” CARs include an intracellular domain from various co-stimulatory molecules, for example, CD28, 4-1BB, ICOS, 0X40, and the like, in the cytoplasmic tail of the CAR to provide additional signals to the cell.

[00174] ‘ ‘Second generation” CARs provide both co-stimulation, for example, by CD28 or

4-1BB domains, and activation, for example, by a CD3^ signaling domain. Preclinical studies have indicated that “Second Generation” CARs can improve the anti-tumor activity of T cells. For example, robust efficacy of “Second Generation” CAR modified T cells was demonstrated in clinical trials targeting the CD 19 molecule in patients with chronic lymphoblastic leukemia (CLL) and acute lymphoblastic leukemia (ALL) (Davila et al., Oncoimmunol. 1(9): 1577-1583 (2012)).

[00175] ‘ ‘Third generation” CARs provide multiple co-stimulation, for example, by comprising both CD28 and 4-1BB domains, and activation, for example, by comprising a CD3^ activation domain.

[00176] ‘ ‘Fourth generation” CARs provide co-stimulation, for example, by CD28 or 4-

1BB domains, and activation, for example, by a CD3^ signaling domain in addition to a constitutive or inducible chemokine component. [00177] “Fifth generation” CARs provide co-stimulation, for example, by CD28 or 4-1BB domains, and activation, for example, by a CD3^ signaling domain, a constitutive or inducible chemokine component, and an intracellular domain of a cytokine receptor, for example, IL-2R[3.

[00178] In various examples, the CAR can be included in a multivalent CAR system, for example, a DualCAR or “TandemCAR” system. Multivalent CAR systems include systems or cells comprising multiple CARs and systems or cells comprising bivalent/bispecific CARs targeting more than one antigen.

[00179] In one example, the scFv portion of a CAR of the invention is encoded by a transgene whose sequence has been codon optimized for expression in a mammalian cell. In one example, the entire CAR construct of the invention is encoded by a transgene whose entire sequence has been codon optimized for expression in a mammalian cell. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences.

[00180] In some examples, the T cell modified to express a recombinant TCR or CAR comprises an antibody or a fragment thereof engineered for enhanced binding to at least one target of interest. The target-specific binding domain can be any domain that binds to a specific target including, but not limited to, target-specific binding domains derived from any one or more of monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, HLA I molecules, HLA II molecules, MHC I molecules, MHC II molecules, and fragments thereof, including, but not limited to, single-domain antibodies, such as a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain (VHH) of camelid derived nanobody, and to alternative scaffolds known in the art that function as antigen binding domains, such as recombinant fibronectin domains, and the like. In some instances, it is beneficial for the target-specific binding domain to be derived from the same species in which the CAR will ultimately be used in. For example, for use in humans, it may be beneficial for the target-specific binding domain of the CAR to comprise human or humanized residues for the target-specific binding domain of an antibody or a fragment thereof. Thus, in one example, the target-specific binding domain portion comprises a humanized antibody or a fragment thereof.

[00181] In some examples, a non-human antibody is humanized, where specific sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in a human or fragment thereof. In one example, the target-specific binding domain portion is humanized.

[00182] In some examples, the T cell modified to express a recombinant TCR or CAR comprises an extracellular receptor, a transmembrane domain, and an intracellular signaling domain. In one example, the extracellular receptor is connected to the intracellular signaling domain. In one example, the extracellular receptor is connected to the intracellular signaling domain through the transmembrane domain to the intracellular signaling domain.

[00183] In some examples, the T cell modified to express a recombinant TCR or CAR comprises a recombinant DNA construct comprising sequences encoding the TCR or CAR. [00184] The nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.

[00185] The nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.

[00186] In some examples, in order to assess the expression of a peptide, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other examples, the selectable marker may be carried on a separate piece of DNA and used in a cotransfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.

[00187] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5 ’ flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter- driven transcription.

[00188] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[00189] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.

[00190] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[00191] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[00192] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another example, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[00193] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20°C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

[00194] Regardless of the method used to introduce exogenous nucleic acids into a host cell, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.

[00195] RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al.

Hum Gene Then, 12(8):861-70 (2001).

Methods of Making a Container

[00196] Some examples of this disclosure relates, in part, to a method of generating a container comprising at least one dehydrated, desiccated and/or lyophilized reagent for isolating and/or stimulating at least one cell of interest (e.g., a T cell, CAR cell, CAR T cell, T cell receptor-expressing T cell, etc.). In one example, one or more steps of the method of generating a container are performed in a bio-safety cabinet (BSC) to maintain sterility.

[00197] In one example, the method comprises the ordered steps of: a) depositing one or more reagent to be dehydrated, desiccated and/or lyophilized into the cell culture container; b) depositing an appropriate amount of buffer into the container to form a reagent cocktail; c) ensuring that > 85% of the interior surface of the container is wetted by the reagent cocktail to form a reagent coating; d) freezing the container until a completely homogenous temperature is reached within the reagent coating; e) removing the container from the freezer and transferring to a dehydrator, desiccator, and/or lyophilizer to allow for dehydration, desiccation and/or lyophilization of the reagent coating.

[00198] In one example, one or more reagent to be dehydrated, desiccated and/or lyophilized are deposited into the cell culture container at a concentration of about Img/ml. In one example, one or more reagent to be dehydrated, desiccated and/or lyophilized are deposited into the cell culture container at a volume that fills surface area of the bag, but does not fill the volume of the bag. For example, for a 30 ml bag, 10 ml total volume would coat the surface area of the bag without filling the bag. The cell culture container can be filled and subsequently lyophilized while lying flat or standing on end. In some examples, excess air is neither introduced nor removed from the container during the preparation process. In some examples, a microfilter is added at a top port of the container to allow vapor to escape, while preventing contamination during dehydration, desiccation, and/or lyophilization.

[00199] In one example, the cell culture container comprises a fluid bag. In one example, the cell culture container comprises an AC class fluid bag. In one example, the cell culture container comprises a SG32 AC bag. In one example, a syringe is used to deposit an appropriate volume and concentration of each reagent into the container using the integrated port on the container. In one example, 100 pL of each of an anti-CD3 and an anti-CD28 antibody are deposited into the container. In one embodiment, about 5pg-200pg of each of an anti-CD3 and an anti-CD28 antibody are deposited into the container. In a further example, 10 pg, 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg, 110 pg, 120 pg, 130 pg, 140 pg, 150 pg, 160 pg, 170 pg, 180 pg, 190 pg, or 200 pg of each of an anti-CD3 and an anti-CD28 antibody are deposited into the container. In some embodiments, 20 pg of anti- CD3 antibody and 50 pg of anti-CD28 antibody is deposited into the container. In some embodiments, 10 pg of anti-CD3 antibody and 10 pg of anti-CD28 antibody is deposited into the container. In some embodiments, 20 pg of anti-CD3 antibody and 20 pg of anti-CD28 antibody is deposited into the container.

[00200] In one example, the buffer comprises water, PBS, trehalose or a combination thereof. In one example, the buffer in injected into the container via the integrated port on the container.

[00201] In one example, the container is prepared in such a way as to prevent introduction of air into the container, thereby eliminating the need to remove excess air. In another example, excess air, including bubbles, is removed from the container. For example, a syringe, pump or other device can be used to remove excess air from the container.

[00202] In some examples, at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or the entire interior surface area of the container is wetted by the reagent cocktail.

[00203] In one example, the prepared container is placed into a -20 °C freezer. In one example, the container remains in the freezer until completely homogenous temperature is reached within the frozen solution. In one example, a thermocouple is used to track the temperature. In one example, the container is left in the freezer for at least about 12 hours to ensure that the solution reaches a homogenous temperature.

[00204] In one example, the frozen container is removed from the freezer and placed on dry ice to prevent thawing during the preparation for lyophilization. In one example, one preinstalled luer-lock fluid port is removed and a 0.22 pm PES filter is installed. In one example, the container is transported to the dehydrator, desiccator, and/or lyophilizer on dry ice to prevent thawing.

[00205] In one example, the container is placed into the dehydrator, desiccator, and/or lyophilizer for a sufficient amount of time for dehydration, desiccation, or lyophilization to occur. In one example, the water content of the container is tracked via the dehydration, desiccation, or lyophilization unit. In one example, the container remains in the dehydrator, desiccator, and/or lyophilizer for at least about 72 hours to ensure complete removal of moisture.

[00206] In one example, the container containing the dehydrated, desiccated and/or lyophilized reagent(s) is removed from the dehydrator, desiccator, and/or lyophilizer and transported to the BSC. In one example, the PES filter is replaced with a new fluid path luer- lock port.

[00207] In one example, the prepared dehydrated, desiccated and/or lyophilized reagent containers are stored at 2-8 °C until use. In one example, the prepared dehydrated, desiccated and/or lyophilized reagent containers are stable at 2-8 °C for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year or for more than 1 year. In some examples, the prepared dehydrated, desiccated and/or lyophilized reagent containers are able to be used to isolate and/or stimulate T cells when removed from storage after being stored at 2-8 °C for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year or for more than 1 year.

Methods of Use [00208] Some examples of this disclosure relates, in part, to a method of stimulating at least one cell of interest (e.g., a T cell, CAR cell, CAR T cell, T cell receptor-expressing T cell, etc.). Some examples of this disclosure provide methods of improving the effectiveness of immunotherapy (e.g., adoptive T-cell therapy, CAR-T cell therapy).

[00209] Some examples of this disclosure also provide methods of isolating T cells. In some examples, T cells can be isolated from a bulk sample by first administering a bulk sample to the container and then isolating or capturing CD3+ cells from the bulk sample. In one example, the method comprises isolating a population of T cells from a blood sample obtained from apheresis. In some examples, the container can be used to isolate T cells, stimulate T cells, or both isolate and stimulate T cells simultaneously. Isolation and stimulation of T cells can be performed individually or in combination.

[00210] Some examples of this disclosure also provide methods of preventing or treating various disease or disorder. In some examples, the method comprises an administration of a therapeutically effective of cells described herein. In one example, the method comprises an administration of a therapeutically effective of at least one stimulated T cell of the invention. [00211] In some examples, the disease or disorder comprises cancer, disease or disorder associated with cancer, GvHD, an autoimmune disease or disorder, disease or disorder associated with organ transplantation, disease or disorder associated with tissue transplantation, disease or disorder associated with cell transplantation, disease or disorder associated with allotransplantation, disease or disorder associated with intestinal transplantation, disease or disorder associated with reconstructive transplantation, or any combination thereof.

[00212] Some examples of this disclosure provide methods of administering an effective amount of at least one stimulated T cell (e.g., T cell, CAR cell, CAR T cell, T cell receptorexpressing T cell, etc.) to a subject.

[00213] In one example, the invention provides methods for preventing or treating a disease or disorder. Furthermore, some examples of this disclosure provide nucleic acid molecules and compositions and cells comprising thereof as well as their use in medicaments or methods for preventing, reducing, and/or eliminating a disease or disorder. Some examples of this disclosure include cellular therapy (CAR T cell therapy).

[00214] In some examples, the invention includes a type of cellular therapy where cells are genetically engineered to express a chimeric antigen receptor (CAR) and the CAR cell (e.g., CAR immune cell, CAR T cell, CAR B cell, etc.) is infused to a recipient in need thereof. Unlike antibody therapies, CAR-modified cells are able to replicate in vivo resulting in long- term persistence. In various examples, the cell is administered to the patient, and persists in the patient for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen month, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty-two months, twenty-three months, two years, three years, four years, or five years after administration of the cell to the patient.

[00215] In one example, CAR T cell therapy may induce an active or a passive immune response, or alternatively may be due to a direct vs indirect immune response. In one example, the CAR T cells exhibit specific proinflammatory cytokine secretion and potent cytolytic activity in response to cells expressing the antigen.

[00216] With respect to ex vivo immunization, at least one of the following occurs in vitro prior to administering the cell into a mammal: i) expansion of the cells, ii) introducing a nucleic acid encoding a CAR to the cells or iii) cryopreservation of the cells.

[00217] Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (e.g., a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed herein. The CAR-modified cell can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient may be a human and the CAR-modified cell can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient.

[00218] In one example, ex vivo culture and expansion of cells comprises: (1) collecting progenitor cells from a mammal (e.g., from peripheral blood harvest or bone marrow explants); and (2) stimulating and expanding such cells ex vivo in a container in media appropriate for expansion of T cells. In addition to the cellular growth factors, other factors such as flt3L, IL-1, IL-3 and c-kit ligand, can be used for culturing and expansion of the cells. In another example, ex vivo culture and expansion of cells comprises: (1) collecting progenitor cells from a mammal (e.g., from peripheral blood harvest or bone marrow explants), (2) stimulating and expanding such cells ex vivo in a container in media appropriate for expansion of T cells and (3) isolating the activated T cells from the mixture of cells in the container.

[00219] In some examples, the CAR-modified cells of the invention are used in the treatment of diseases, disorders and disorders including, but not limited to, autoimmune disease or disorder (e.g., lupus), inflammatory disease or disorder (e.g., allergies and asthma), disease or disorder caused by transplantation, an allograft rejection, an immune rejection, a chronic allogeneic rejection, an engraftment rejection, a transplant rejection, or any combination thereof, an inflammation, an inflammation caused by ischemia/reperfusion, an infection, an immune response to an allograft, or any combination thereof.

[00220] Some examples of this disclosure provide methods for preventing relapse of disease or disorder, the methods comprising administering to a subject in need thereof at least one genetically engineered cell of the invention. In one example, the methods comprise administering to the subject in need thereof multiple effective amounts of at least one genetically engineered cell of the invention. In one example, the methods comprise administering to the subject in need thereof an effective amount of at least one genetically engineered cell of the invention in combination with an effective amount of another therapy. [00221] The compositions and genetically engineered cells of some examples of this disclosure may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components, such as IL-2, or other cytokines or cell populations.

[00222] In a further example, the compositions of some examples of this disclosure are administered to a patient in conjunction with (e.g., before, simultaneously or following) transplantation (e.g., bone marrow transplantation, organ transplantation, etc.). In certain examples, following the transplant, subjects receive an infusion of the expanded immune cells of some examples of this disclosure. In an additional example, expanded cells are administered before or following surgery.

[00223] Subjects to which administration of the compositions and pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.

[00224] The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art-accepted practices. Strategies for T cell dosing and scheduling have been discussed (Ertl et al, 2011, Cancer Res, 71:3175-81; Junghans, 2010, Journal of Translational Medicine, 8:55).

[00225] Pharmaceutical compositions of some examples of this disclosure may comprise a composition as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of some examples of this disclosure are in one example formulated for intravenous administration.

[00226] Pharmaceutical compositions of some examples of this disclosure may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages may be determined by clinical trials.

[00227] When “an immunologically effective amount” or “therapeutic amount” is indicated, the precise amount of the compositions of some examples of this disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the cells described herein may be administered at a dosage of 10⁴ to 10⁹ cells/kg body weight, in some instances 10⁵ to 10⁶ cells/kg body weight, including all integer values within those ranges. The cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[00228] In various examples, it may be desired to administer activated cells to a subject and then subsequently redraw blood (or have an apheresis performed), activate cells therefrom according to some examples of this disclosure, and reinfuse the patient with these activated and expanded cells. This process can be carried out multiple times every few weeks. In various examples, cells can be activated from blood draws of from lOcc to 400cc. In various examples, cells are activated from blood draws of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or lOOcc.

[00229] The administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one example, the T cell compositions of some examples of this disclosure are administered to a patient by intradermal or subcutaneous injection. In one example, the cell compositions of some examples of this disclosure are administered by i.v. injection. The compositions of cells may [00230] In various examples of some examples of this disclosure, cells activated and expanded using the device and methods described herein, are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities. In some examples, the device and methods of the invention are used for isolation, stimulation and/or expansion of cells that may be used in a treatment regimen in combination with chemotherapy, radiation, immunosuppressive agents, antibodies, immunoablative agents, steroids, cytokines, and surgery. In one example, the cell compositions of some examples of this disclosure are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation and cell ablative therapy.

[00231] In one example, subjects may undergo leukapheresis, wherein leukocytes are collected, enriched, or depleted ex vivo to select and/or isolate the cells of interest, e.g., T cells. These cell isolates may be expanded by methods of the invention. Subjects in need thereof may subsequently undergo additional treatment (e.g., transplantation, chemotherapy, administration of CAR T cells, etc). In various examples, subjects receive an infusion of the expanded CAR T cells of some examples of this disclosure. In one example, expanded cells are administered before or following surgery.

EMBODIMENTS

[00232] This invention provides the following non-limiting embodiments.

[00233] Embodiment 1 is a container comprising a vacuum sealable interior chamber, wherein an inner surface of the interior chamber is coated with at least one dehydrated, desiccated, and/or lyophilized reagent capable of isolating and/or stimulating a T cell.

[00234] Embodiment 2 is the container of embodiment 1, wherein the container is a flexible bag.

[00235] Embodiment 3 is the container of embodiment 1 or 2, wherein the at least one dehydrated, desiccated, and/or lyophilized reagent comprises an anti-CD3 antibody, an anti- CD28 antibody or a combination thereof.

[00236] Embodiment 4 is the container of any one of embodiments 1 through 3, wherein one or more of the anti-CD3 antibody, and the anti-CD28 antibody are mixed with a solution prior to dehydration, desiccation and/or lyophilization, wherein the solution comprises a buffer selected from the group consisting of trehalose, PBS, and water. [00237] Embodiment 5 is the container of any one of embodiments 1 through 4, wherein the device comprises at least one additional dehydrated, desiccated and/or lyophilized reagent for the stimulation or expansion of a T cell population.

[00238] Embodiment 6 is the container of embodiment 5, wherein the at least one additional dehydrated, desiccated and/or lyophilized reagent comprises IL-2.

[00239] Embodiment 7 is the container of any one of embodiments 1 through 6, wherein the container is devoid of air.

[00240] Embodiment 8 is the container of any one of embodiments 1 through 7, wherein the container is a fluid bag comprising at least one fluid port.

[00241] Embodiment 9 is the container of any one of embodiments 1 through 8, wherein the container is a fluorinated ethylene propylene (FEP) fluid bag with high surface energy. [00242] Embodiment 10 is the container of any one of embodiments 1 through 9, the volume of solution added to the container is such that at least 85% of the interior surface area of the container can be coated with the solution, but the interior volume is not filled.

[00243] Embodiment 11 is the container of any one of embodiments 1 through 10, wherein the container does not comprise polymeric beads.

[00244] Embodiment 12 is the container of any one of embodiments 1 through 11, wherein the container is stable when stored at 2-8°C for at least 1 day.

[00245] Embodiment 13 is the container of any one of embodiments 1 through 11, wherein the container is stable when stored at 2-8°C for at least 6 months.

[00246] Embodiment 14 is a method of stimulating or expanding a T cell population, wherein the method comprises applying a mixture containing a T cell population and a media for culturing T cells to a container of any one of embodiments 1 through 13.

[00247] Embodiment 15 is the method of embodiment 14, wherein the T cells are chimeric antigen receptor (CAR) T cells.

[00248] Embodiment 16 is a T cell population expanded by the method of embodiment 14 or 15.

[00249] Embodiment 17 is a method of treating, preventing, reducing, or eliminating a disease or disorder in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of a T cell population of embodiment 16.

[00250] Embodiment 18 is the method of embodiment 17, wherein the disease or disorder is cancer, a disease or disorder associated with cancer, a disease or disorder associated with infection, an autoimmune disease or disorder, fibrosis, a disease or disorder associated with organ transplantation, a disease or disorder associated with tissue transplantation, or a disease or disorder associated with cell transplantation, or any combination thereof.

[00251] Embodiment 19 is a method of improving the effectiveness of an immunotherapy in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of a T cell population of embodiment 16.

[00252] Embodiment 20 is the method of embodiment 17, wherein the subject has at least one selected from the group consisting of cancer, a disease or disorder associated with infection, an autoimmune disease or disorder, fibrosis, a disease or disorder associated with organ transplantation, a disease or disorder associated with tissue transplantation, or a disease or disorder associated with cell transplantation, or any combination thereof.

[00253] Embodiment 21 is a method of generating a container of any one of embodiments 1 through 13, the method comprising the steps of: a) obtaining a vacuum sealable container comprising an interior chamber; b) depositing a cocktail of at least one reagent and a buffer into the interior chamber of the container; c) coating at least 85% of the inner surface of the interior chamber of the container with the cocktail to form a reagent coating; d) freezing the container until a completely homogenous temperature is reached within the reagent coating; and e) dehydrating, desiccating, and/or lyophilizing the reagent coating.

[00254] Embodiment 22 is the method of embodiment 21, wherein the container comprises a flexible bag.

[00255] Embodiment 23 is the method of embodiment 21 or 22, wherein the container is a FEP fluid bag with high surface energy.

[00256] Embodiment 24 is the method of any one of embodiment 21 through 23, wherein at least one reagent to be dehydrated, desiccated and/or lyophilized into the interior chamber of the container comprises an anti-CD3 antibody, an anti-CD28 antibody or a combination thereof.

[00257] Embodiment 25 is the method of any one of embodiment 21 through 24, wherein the buffer comprises at least one lyoprotectant.

[00258] Embodiment 26 is the method of any one of embodiment 21 through 25, wherein the buffer comprises water, PBS, trehalose, a surfactant, an amino acid, a polymer, a sugar or any combination thereof. [00259] Embodiment 27 is the method of any one of embodiment 21 through 26, wherein at least one additional reagent for the stimulation or expansion of a T cell population is applied to the container prior to dehydration, desiccation and/or lyophilization.

[00260] Embodiment 28 is the method of embodiment 27, wherein at least one additional reagent is IL-2.

[00261] Embodiment 29 is the method of any one of embodiment 21 through 28, wherein the reagent cocktail is applied to the container without introducing air into the container.

[00262] Embodiment 30 is the method of any one of embodiment 21 through 29, wherein the method further comprises removing any excess air prior to dehydration, desiccation and/or lyophilization.

[00263] Embodiment 31 is the method of any one of embodiment 21 through 30, wherein the container is a fluid bag comprising at least one fluid port.

[00264] Embodiment 32 is the method of any one of embodiment 21 through 31, wherein the method further comprises adding a fdter to the fluid port prior to dehydration, desiccation and/or lyophilization to allow vapor to escape, while preventing contamination during dehydration, desiccation, and/or lyophilization.

[00265] Embodiment 33 is the method of any one of embodiment 21 through 32, wherein the volume of reagent solution added to the container is such that at least 85% of the interior surface area of the container can be coated with the solution, but the interior volume is not fdled.

[00266] Embodiment 34 is the method of any one of embodiment 21 through 33, wherein polymeric beads are not introduced into the container.

[00267] Embodiment 35 is the method of any one of embodiment 21 through 34, wherein the container is stable when stored at 2-8°C for at least 1 day.

[00268] Embodiment 36 is the method of any one of embodiment 21 through 35, wherein the container is stable when stored at 2-8°C for at least 6 months.

[00269] Embodiment 37 is a method of isolating a T cell population, wherein the method comprises applying a mixture containing a T cell population and a media for culturing T cells to a container of any one of embodiments 1 through 13.

EXPERIMENTAL EXAMPLES

[00270] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[00271] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the examples of this disclosure and practice the claimed methods. The following working examples therefore, specifically point out the preferred examples of the disclosure, and are not to be construed as limiting in any way the remainder of the invention.

Example 1 : Protocol for Preparation of Culture Bags Containing Lyophilized Reagents [00272] Exemplary prepared culture bags containing lyophilized reagents are shown in Figure IB and Figure 1C. The prepared culture bag can be prepared according to the following exemplary protocol:

Materials:

1. Fluid Supply Bag. Although this protocol uses the 32AC, any ‘AC’ designated bag should suffice but volumes would need to be scaled proportionally to the working volume of the bag, stipulated by the manufacturer.

*Note: Saint Gobain AC Vue Life bags are used for the preliminary experiments as they have been modified to promote increased protein and cell adhesion over standard FEP culture bags.

2. Functional aCD3- liquid, 200 pg/mL. Available from multiple manufacturers.

3. Functional aCD28- liquid, 100 pg/mL. Available from multiple manufacturers.

4. Phosphate Buffered Saline, cell culture grade.

5. Bio-molecular grade water

6. Trehalose, 2% w/v solution, cell culture grade.

7. 0.22 pm PES luer-lock filter

Equipment:

1. Biosafety Cabinet, Level 2

2. Freezer, -20 °C

3. Lyophilizer; LabConco Freezone and Lyostar 3 are examples

4. Single use disposable luer-lock syringes

Method:

1. Place fluid supply bag, antibodies, buffers, and syringes into a bio-safety cabinet (BSC) to maintain sterility. . Deposit both the aCD3 and aCD28 into the fluid supply bag using the integrated port on the bag. a. *Note: For an SG32 AC bag, assuming that no antibody is lost during the lyophilization process, depositing 100 pL of each antibody would result in 0.22 and 0.11 pg of antibody per cm² for aCD3 and aCD28, respectively.

3. Then deposit an appropriate volume of buffer (either water/PBS/trehalose) into the fluid supply bag via the port (e.g., 10 mb for a SG32 AC bag).

4. If necessary, remove any excess air, including bubbles, from the bag so that at least 85% of the surface of the bag is wetted by the antibody cocktail.

5. Immediately place the prepared bag into a -20 °C freezer.

6. Allow the bag to freeze until completely homogenous temperature is reached within the frozen solution. a. *Note: Use thermocouples to track temperature, or alternatively freeze for at least about 12 hours to ensure that the solution reaches this homogenous point.

7. Remove the bag from the freezer and place on dry ice to prevent thawing.

8. Place bag into BSC and remove one pre-installed luer-lock fluid port and install a 0.22 pm PES filter.

9. Transport bag to lyophilizer on dry ice to prevent thawing.

10. Place bag into vacuum chamber and allow the lyophilization to occur. a. *Note: Track water content of the bag via the lyophilization unit or alternatively leave the bag in the vacuum chamber for at least about 72 hours to ensure complete removal of moisture.

11. Remove bag from vacuum chamber and transport to BSC. At this point, no dry ice is necessary.

12. Replace PES filter with a new fluid path luer-lock port.

13. Store bags in 2-8 °C fridge until use.

Example 2: Device

[00273] Successful stimulation of primary T cells occurs through a co-stimulatory response via the CD3 and CD28 receptors. Stimulation (aka activation) of T-cells is required for the gene insertion (aka transduction) event to occur and for subsequent cell expansion allowing for proper dose levels to be achieved. This is typically achieved through localization of anti-CD3 and anti-CD28 antibodies on polymeric matrices or beads. There are many commercial reagents available to achieve this. However, these reagents can be costly and require complex operations from highly skilled individuals. Described herein is a series of prototype devices that remove many of these complex steps and can reduce cost.

[00274] The device relies on deposition of CD3, CD28, or a combination of CD3 and CD28 antibodies to the surface of a cell culture container by lyophilization. A typical stimulation event primarily relies upon the use of Miltenyi’s TransAct™ reagent for stimulation of T-cells. This method requires an operator to deposit both T cells and cell culture media into a culture bag. The operator would then calculate the volume of TransAct™ to add to the culture bag. In contrast, the prepared containers (Figure IB and 1C) of this disclosure, already having the functional antibodies deposited inside, can provide a ‘grab and go’ strategy where the only requirement is media and cells. No further calculations or reagent additions are needed. Thus, the device can provide an alternative approach to stimulation of the CD3, CD28, or a combination of CD3 and CD28 domain that requires less manipulations which can be executed in a functionally closed manner, while also maintaining a much lower cost, longer shelflife at room temperature, thereby allowing for possible direct assembly onto a disposable tubing set, and possible improvement to the quality of the drug product.

Example 3: Development of the Culture Bag

[00275] Results from preliminary experiments performed using prepared devices show that both the TransAct™ and the prepared device had very comparable product doublings over the duration of the study (Figure 3). Figure 3 depicts exemplary experimental results from a generic drug product study of a CAR-T process (the TransAct™ process) and a T cell activation process using prepared culture bags containing lyophilized reagents). Product doublings indicates the number of cell doublings observed within the process based on the initial seeding parameters. Product doublings were quantified at the initial time the device was made (Initial) and at least six months after the device was made (6+ months). Both the TransAct™ and prepared culture bags containing lyophilized reagents (Device) had very comparable product doublings over the duration of this study.

[00276] Figure 4 depicts exemplary experimental results from a generic drug product study of a CAR-T process. Transduction efficiency indicates the number of T cells expressing an exogenous CAR construct in relation to the entire population. Transduction efficiencies were quantified at the initial time the device was made (Initial) and at least six months after the device was made (6+ months). The population from the prepared culture bags containing lyophilized reagents (Device) had a significant increase in CAR% over that of traditional TransAct™. These data demonstrate that the population of cells cultured in the prepared devices had a significant increase in CAR% over that of traditional TransAct™ (Figure 4). [00277] Figure 5 depicts exemplary experimental results showing the viability of cell population at harvest. To determine the stability of the culture bags stored overtime, two independent experiments were performed using a culture bag that was made the same day it was used (Initial) and a culture bag that was used after it had been made and stored for at least six months (6+ months). Viability was quantified at the initial time the device was made (Initial) and at least six months after the device was made (6+ months). The prepared culture bags containing lyophilized reagents (Device) had higher viability at harvest over that of traditional TransAct™ indicating that the polymer matrix contained within TransAct™ may lead to deleterious interactions with T cells over time. These data demonstrate a higher viability at harvest over that of traditional TransAct™ (Figure 5). The lyophilized reagents provide advantages over other traditional devices because lyophilization is a common technique used to increase shelf-life/stability across multiple industries, including chemical, food, and pharmaceutical.

[00278] Results from preliminary experiments performed using prepared devices also show comparable results for various formulations of the device and different CAR constructs. Specifically, viability (Figure 6A), cell growth (Figure 6B), transduction efficiency (Figure 6C), and total viable CAR+ cells (Figure 6D) were comparable using different experimental buffers such as PBS, water, and trehalose and using different CAR constructs (Figure 6A through Figure 6D). For CAR construct 1, PBS has the highest CAR % with 40%. For CAR Construct 2, the CAR% of TransAct™, PBS and trehalose are all within a very close range. For CAR Construct 3, TransAct™, PBS, and water all have very comparable CAR%. Across all of the CAR constructs, all the carrying solutions resulted in a device that stimulated T cells in a comparable performance to that of TransAct™ and facilitated transduction that led to comparable transgene expression. Overall, the culture bag proved to be effective at inducing transduction similar to that of TransAct™. These results demonstrate that the product cells are compatible with CAR T therapies and that the device is an effective solution for traditional methods.

[00279] This device allows for the simultaneous selection and activation of T cells (Figure 7). For T cell activation, monoclonal antibodies for CD3 (primary signaling) and CD28 (secondary signaling) were created. The antibodies for CD3 and CD28 bind to CD3 and CD28 respectively thereby replicating primary and secondary signaling. The concentration of antibody added to the device had a dose dependent effect on viability and cell growth (Figure 8, Figure 9, and Figure 10A through Figure 10C). Figure 8 depicts a representative schematic demonstrating the general method steps of using the culture bag. Base bag denotes the culture bag with 10 pg (lx) of both anti-CD3 and anti-CD28 antibody concentrations per bag. High bag denotes the culture bag with 20 pg (2x) of both anti-CD3 and anti-CD28 antibody concentrations per bag. Figure 9 depicts exemplary experimental results demonstrating that the culture bag effectively enriched CD3+ T cells from apheresis. Figure 10A through Figure 10C depict exemplary experimental results showing the total viability (Figure 10A), product doublings (Figure 10B), and cell viability (Figure IOC) of cells using the base bag (lx) and high bag (2x) formulations of the culture bag. These results demonstrate that cells grow at similar rates to standard methods (Transact™).

[00280] Preliminary experiments were also performed to monitor low density lipoprotein receptor (LDLr) expression. When T cells become activated the amount of LDLr increases on the surface. This metric is therefore used to quantify the number of T cells that were activated or not by measuring LDLr before the cells were added to the culture bag and then 2 days afterwards (Figure 11). These results show that LDLr started at 16% for all cells on Day 0 and increased to upwards of 95% for all of the experimental buffers. These results demonstrate that the cells were all activated. Lastly, results from preliminary experiments were performed to monitor the cell composition of bulk apheresis (Figure 12A and Figure 12B). These results demonstrate that over the course of 45 mins, CD3 content of bulk apheresis contained within the culture vessel continually decreased, therefore the culture vessel was isolating CD3+ cells from the apheresis (Figure 12A). LDLr expression was quantified post selection and post activation (Figure 12B). These results demonstrate that the culture bag captured CD3+ cells from bulk apheresis. These results showed that the culture bags were able to capture CD3+ cells from apheresis.

[00281] These data demonstrate that this device is a potential alternative to traditional activation methods. This device can be integrated into automated systems sand is cheaper and easier to use compared to the traditional methods such as Dynabeads™ and TransAct™.

[00282] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific examples, it is apparent that other examples and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such examples and equivalent variations.

Claims

CLAIMS What is claimed is:

1. A bag comprising a vacuum sealable interior chamber, wherein an inner surface of the interior chamber is coated with at least one dehydrated, desiccated, and/or lyophilized reagent capable of isolating and/or stimulating a T cell.

2. The bag of claim 1, wherein the at least one dehydrated, desiccated, and/or lyophilized reagent comprises an anti-CD3 antibody, an anti-CD28 antibody or a combination thereof.

3. The bag of claim 2, wherein one or more of the anti-CD3 antibody, and the anti-CD28 antibody are mixed with a solution prior to dehydration, desiccation and/or lyophilization, wherein the solution comprises a buffer selected from the group consisting of trehalose, PBS, and water.

4. The bag of claim 1, wherein the device comprises at least one additional dehydrated, desiccated and/or lyophilized reagent for the isolation, stimulation, and/or expansion of a T cell population.

5. The bag of claim 4, wherein the at least one additional dehydrated, desiccated and/or lyophilized reagent comprises IL-2.

6. The bag of claim 1, wherein the bag is a fluorinated ethylene propylene (FEP) fluid bag with high surface energy.

7. The bag of claim 1, wherein the volume of solution added to the bag is such that at least 85% of the interior surface area of the bag can be coated with the solution, but the interior volume is not fdled.

8. The bag of claim 1, wherein the bag is stable when stored at 2-8°C for at least 1 day.

9. A method of isolating, stimulating, and/or expanding a T cell population, wherein the method comprises applying a mixture containing a T cell population and a media for culturing T cells to a bag of any one of claims 1-8.

10. A T cell population expanded by the method of claim 9.

11. A method of treating, preventing, reducing, or eliminating a disease or disorder in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of a T cell population of claim 10, wherein the disease or disorder is cancer, a disease or disorder associated with cancer, a disease or disorder associated with infection, an autoimmune disease or disorder, fibrosis, a disease or disorder associated with organ transplantation, a disease or disorder associated with tissue transplantation, or a disease or disorder associated with cell transplantation, or any combination thereof.

12. A method of manufacturing a bag, the method comprising: a) depositing a cocktail of at least one reagent and a buffer into an interior chamber of a vacuum sealable bag; b) coating at least 85% of the inner surface of the interior chamber of the bag with the cocktail to form a reagent coating; c) freezing the bag until a completely homogenous temperature is reached within the reagent coating; and d) dehydrating, desiccating, and/or lyophilizing the reagent coating.

13. The method of claim 12, wherein the bag is a FEP fluid bag with high surface energy.

14. The method of claim 12, wherein at least one reagent to be dehydrated, desiccated and/or lyophilized into the interior chamber of the bag comprises an anti- CD3 antibody, an anti-CD28 antibody or a combination thereof.

15. The method of claim 12, wherein the buffer comprises at least one lyoprotectant.

16. The method of claim 12, wherein the buffer comprises water, PBS, trehalose, a surfactant, an amino acid, a polymer, a sugar or any combination thereof.

17. The method of claim 12, wherein at least one additional reagent for the isolation, stimulation, and/or expansion of a T cell population is applied to the bag prior to dehydration, desiccation and/or lyophilization.

18. The method of claim 17, wherein at least one additional reagent is IL- 2.

19. The method of claim 12, wherein the reagent cocktail is applied to the bag while the bag is devoid of air without introducing air into the bag.

20. The method of claim 12, wherein the method further comprises adding a filter to the fluid port prior to dehydration, desiccation and/or lyophilization to allow vapor to escape, while preventing contamination during dehydration, desiccation, and/or lyophilization.

21. The method of claim 12, wherein the volume of reagent solution added to the bag is such that at least 85% of the interior surface area of the bag can be coated with the solution, but the interior volume is not filled.

22. The method of claim 12, wherein the bag is stable when stored at 2- 8°C for at least 1 day.