Prepared dehydration, drying and / or freeze-drying culture vessel for T cell activation and methods of use thereof

Abstract

The present invention provides a container comprising dehydration, drying and / or lyophilization agents for isolating and / or stimulating T cells for therapeutic use, a method of making the container, and a method of using the container for T cell-based therapy.

Description

Cross-references to related applications

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

[0002] References to sequence lists

[0003] This application hereby incorporates, by reference, the entire contents of the sequence list XML document named "JBI6816WOPCT1_Sequence_Listing.xml". The XML file containing the sequence list of this application was created on July 16, 2024 and is 3,237 bytes in size. Background Technology

[0004] Chimeric antigen receptor (CAR) T-cell therapy is a form of T-cell transfer therapy in which T cells are harvested from a patient and modified to express antigen receptors that are not normally expressed. T-cell stimulation (also known as activation) is required for gene insertion (also known as transduction) events and subsequent cell expansion, allowing for the attainment of appropriate dose levels. Successful stimulation / activation of primary T cells occurs via a co-stimulatory response to CD3 and CD28 receptors on the cell surface. This is typically achieved by localizing anti-CD3 and anti-CD28 antibodies onto a polymer matrix or beads formulated into a suspension and then added to the cell solution in the culture medium during CAR-T treatment. The use of functional antibodies has historically been employed to stimulate T cells; however, the process is complex and requires significant preparation time due to the insolubility of anti-CD3.

[0005] There remains a need in the art for compositions and methods that can be engineered to optimize and simplify CAR-T therapy. This disclosure addresses this unmet need. Summary of the Invention

[0006] The present invention partially provides a container comprising a vacuum-sealable internal chamber, wherein the inner surface of the internal chamber is coated with at least one dehydrating, drying, and / or lyophilizing agent capable of separating and / or stimulating T cells.

[0007] In some examples, the container is a flexible bag.

[0008] In some examples, at least one of the dehydrating, drying, and / or lyophilizing reagents 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, drying, and / or lyophilization, wherein the solution comprises a buffer selected from the group consisting of trehalose, PBS, and water.

[0009] In some examples, the device includes at least one additional dehydrating, drying, and / or lyophilizing agent for stimulating or expanding a T cell population. In some examples, at least one additional dehydrating, drying, and / or lyophilizing agent comprises IL-2.

[0010] In some examples, the container contains no air.

[0011] In some examples, the container is a fluid bag that includes at least one fluid port.

[0012] In some examples, the container is a fluorinated ethylene propylene (FEP) fluid bag with high surface energy.

[0013] In some examples, the volume of solution added to the container is such that at least 85% of the container's inner surface area can be coated with the solution, but the internal volume is not filled.

[0014] In some examples, the container does not contain polymer beads.

[0015] In some examples, the container is stable when stored at 2°C to 8°C for at least 1 day. In other examples, the container is stable when stored at 2°C to 8°C for at least 6 months.

[0016] This invention partially provides a method for stimulating or expanding a population of T cells, wherein the method includes applying a mixture containing the population of T cells and a culture medium for culturing the T cells to a container comprising a vacuum-sealable internal chamber, wherein the inner surface of the internal chamber is coated with at least one dehydrating, drying, and / or lyophilizing agent capable of stimulating the T cells. In some examples, the T cells are chimeric antigen receptor (CAR) T cells.

[0017] The present invention partially provides a method for expanding a T cell population by stimulating or amplifying the T cell population, wherein the method includes applying a mixture containing the T cell population and a culture medium for culturing the T cells to a container comprising a vacuum-sealable internal chamber, wherein the inner surface of the internal chamber is coated with at least one dehydrating, drying and / or lyophilizing agent capable of stimulating the T cells.

[0018] This invention partially provides a method for treating, preventing, reducing, or eliminating a disease or condition in a subject of need, wherein the method includes administering a therapeutically effective amount of a population of T cells expanded by means of stimulation or expansion of a T cell population, wherein the method includes applying a mixture containing the T cell population and a culture medium for culturing T cells to a container comprising a vacuum-sealable internal chamber, wherein the inner surface of the internal chamber is coated with at least one dehydrating, drying, and / or lyophilizing agent capable of stimulating T cells. In some examples, the disease or condition is cancer, a cancer-related disease or condition, an infection-related disease or condition, an autoimmune disease or condition, fibrosis, an organ transplant-related disease or condition, a tissue transplant-related disease or condition, or a cell transplant-related disease or condition, or any combination thereof.

[0019] This invention partially provides a method for improving the effectiveness of immunotherapy in subjects in need, wherein the method includes administering a therapeutically effective amount of a population of T cells expanded by means of stimulation or expansion of the T cell population, wherein the method includes applying a mixture containing the T cell population and a culture medium for culturing the T cells to a container comprising a vacuum-sealable internal chamber, wherein the inner surface of the internal chamber is coated with at least one dehydrating, drying, and / or lyophilizing agent capable of stimulating T cells. In some examples, the subject suffers from at least one of the following groups: cancer, infection-related disease or condition, autoimmune disease or condition, fibrosis, organ transplant-related disease or condition, tissue transplant-related disease or condition, or cell transplant-related disease or condition, or any combination thereof.

[0020] The present invention partially provides a method for producing a container comprising a vacuum-sealable internal chamber, wherein the inner surface of the internal chamber is coated with at least one dehydrating, drying, and / or lyophilizing agent capable of stimulating T cells, the method comprising the following steps:

[0021] a) Obtain a vacuum-sealable container containing an internal chamber;

[0022] b) Deposit a mixture of at least one reagent and a buffer solution into the internal chamber of the container;

[0023] c) Coat at least 85% of the inner surface of the internal chamber of the container with the mixture to form a reagent coating;

[0024] d) Freeze the container until a completely uniform temperature is reached within the reagent coating; and

[0025] e) Dehydrate, dry and / or freeze-dry the reagent coating.

[0026] In some examples, the container includes a flexible bag.

[0027] In some examples, the container is a FEP fluid bag with high surface energy.

[0028] In some examples, at least one reagent to be dehydrated, dried, and / or lyophilized into the internal chamber of the container contains an anti-CD3 antibody, an anti-CD28 antibody, or a combination thereof.

[0029] In some examples, the buffer contains at least one lyophilization protectant. In some examples, the buffer contains water, PBS, trehalose, surfactants, amino acids, polymers, sugars, or any combination thereof.

[0030] In some examples, at least one additional reagent for stimulating or expanding the T cell population is applied to the container prior to dehydration, drying, and / or lyophilization. In some examples, at least one additional reagent is IL-2.

[0031] In some examples, the reagent mixture is applied to the container without introducing air into it.

[0032] In some examples, the method also includes removing any excess air before dehydration, drying, and / or freeze-drying.

[0033] In some examples, the bag is a fluid bag that includes at least one fluid port.

[0034] In some examples, the method also includes adding a filter to the fluid port prior to dehydration, drying, and / or freeze-drying to allow vapor to escape while preventing contamination during dehydration, drying, and / or freeze-drying.

[0035] In some examples, the volume of reagent solution added to the container is such that at least 85% of the container's inner surface area can be coated with the solution, but the internal volume is not filled.

[0036] In some examples, polymer beads are not introduced into the container.

[0037] In some examples, the container is stable when stored at 2°C to 8°C for at least 1 day. In other examples, the container is stable when stored at 2°C to 8°C for at least 6 months.

[0038] The present invention partially provides a method for isolating a population of T cells, wherein the method comprises applying a mixture containing a population of T cells and a culture medium for culturing T cells to a container comprising a vacuum-sealable internal chamber, wherein the inner surface of the internal chamber is coated with at least one dehydrating, drying and / or lyophilizing agent capable of isolating and / or stimulating T cells. Attached Figure Description

[0039] The specific embodiments of the invention will be better understood when read in conjunction with the accompanying drawings, which illustrate preferred embodiments of the invention. The drawings show currently preferred embodiments for illustrative purposes. However, it should be understood that the invention is not limited to the precise arrangement and means of the embodiments shown in the drawings.

[0040] Figures 1A to 1C An exemplary container of the present invention is depicted. Figure 1A A line drawing of an exemplary container is shown. Figure 1B and Figure 1C This is an image showing a prototype cell culture bag for T cell activation in a closed container. Figure 1B An exemplary cell culture bag containing lyophilized reagents is depicted, wherein trehalose is used as a solvent. Figure 1C An exemplary cell culture bag containing lyophilized reagents is depicted, wherein PBS is used as a solvent.

[0041] Figure 2 An exemplary cell culture system incorporating an exemplary container of the present invention is described.

[0042] Figure 3 The CAR-T method (TransAct) is described. ™ This study presents exemplary experimental results for a generic drug product using a method and a culture bag containing lyophilized reagents for T-cell activation. Product doubling is indicated by the number of cell doublings observed during the process based on initial seeding parameters. Product doubling is quantified at the initial time of the manufacturing device (initial) and at least six months (6+ months) after the manufacturing device. TransAct ™ The culture bags (devices) containing lyophilized reagents showed a very comparable product multiplication rate over the duration of this study.

[0043] Figure 4 Exemplary experimental results from a study of a generic drug product using the CAR-T method are depicted. Transduction efficiency indicates the number of T cells expressing the exogenous CAR construct relative to the entire population. Transduction efficiency was quantified at the initial time of device fabrication (initial) and at least six months (6+ months) after device fabrication. The CAR% from the population prepared in culture bags (devices) containing lyophilized reagents was compared with that of conventional TransAct. ™ Significant increase.

[0044] Figure 5 Exemplary experimental results illustrating the viability of cell populations at harvest are depicted. Viability was quantified at the initial time of device fabrication (initial) and at least six months (6+ months) after device fabrication. The prepared culture bags (devices) containing lyophilized reagents exhibited higher viability at harvest than conventional TransAct. ™ Higher vitality indicates TransAct ™The polymer matrix contained within may lead to harmful interactions with T cells over time.

[0045] Figure 6A to Figure 6D Exemplary experimental results are depicted, showing cell viability (harvest viability) (Figure 6A) and cell growth (fold expansion) using different formulations in culture bags. Figure 6B ), transduction efficiency (percentage of CAR+ cells) (Figure 6C) and total viable CAR+ cells ( Figure 6D For CAR construct 1, PBS had the highest CAR% at 40%. For CAR construct 2, TransAct... ™ The CAR% values ​​for PBS and trehalose were all within very close range. For CAR construct 3, TransAct... ™ PBS and water all had very similar CAR%. Overall, this culture bag demonstrates comparable efficacy to TransAct in inducing transduction. ™ Similar methods are effective. In all CAR constructs, all vector solutions are generated to work with TransAct. ™ The device has considerable performance to stimulate T cells and promote transduction, resulting in considerable transgene expression.

[0046] Figure 7 A representative schematic diagram illustrating the simultaneous selection and activation of T cells using this culture bag is depicted.

[0047] Figure 8 A representative schematic diagram illustrating the general methodological steps for using this culture bag is depicted. A basal bag represents a culture bag containing 10 μg (1×) of anti-CD3 antibody and anti-CD28 antibody per bag. A high-density bag represents a culture bag containing 20 μg (2×) of anti-CD3 antibody and anti-CD28 antibody per bag.

[0048] Figure 9 Exemplary experimental results are described, demonstrating that the culture bag effectively enriches CD3+ T cells from apheresis blood components.

[0049] Figures 10A to 10C Exemplary experimental results are depicted, showing the total viability of cells using basal bag (1×) and high bag (2×) formulations. Figure 10A ), cell growth (product multiplication) Figure 10B ) and cell viability ( Figure 10C These results demonstrate that cells can be compared with standard methods (Transactin). ™ They grow at similar rates.

[0050] Figure 11Exemplary experimental results are depicted, showing the activation profile of T cells cultured in a culture bag. LDLr expression was quantified before cells were added to the culture bag (day 0) and two days after cells were added to the culture bag (day 2).

[0051] Figure 12A and Figure 12B Exemplary experimental results were described, demonstrating that the CD3 content of the batch of apheresis blood components contained in the culture dish continuously decreased over a 45-minute period, thus indicating that the culture dish was isolating CD3+ cells from the apheresis blood components. Figure 12A ) Quantitative LDLr expression after selection and activation ( Figure 12B These results demonstrate that the culture bag captures CD3+ cells from bulk apheresis blood components. Detailed Implementation

[0052] This disclosure is based in part on the development of a closable or sealable cell culture container pre-coated with at least one dehydrating, drying, and / or lyophilized reagent. In some examples, the dehydrating, drying, and / or lyophilized reagent is a reagent for activating T cells for use in immunotherapies (e.g., adoptive T-cell therapy, chimeric antigen receptor (CAR) T-cell therapy). In some examples, the cell culture container is a cell culture bag comprising an inner surface coated with at least one dehydrating, drying, and / or lyophilized reagent for activating T cells and allowing the regulated flow of one or more solutions (e.g., cell culture medium and a solution containing T cells for culturing, isolating, stimulating, and / or expanding). Exemplary cell culture bags containing lyophilized reagents are shown in... Figures 1A to 1C middle. Figure 1B An exemplary bag is depicted in which trehalose is used as a solvent. Figure 1C An exemplary bag in which PBS is used as a solvent is depicted.

[0053] Therefore, some examples of this disclosure include cell culture bags for isolating, stimulating, and / or expanding T cells, wherein the bag contains at least one dehydrated, dried, and / or lyophilized antibody for T cell isolation and / or stimulation. In some examples, the at least one dehydrated, dried, and / or lyophilized antibody for T cell isolation and / or stimulation is an anti-CD3 antibody, an anti-CD28 antibody, or a combination thereof.

[0054] Some examples of this disclosure include methods for improving the effectiveness of T cell isolation and / or stimulation for immunotherapy, and methods for isolating and / or stimulating T cells in a container containing at least one dehydrated, dried, and / or lyophilized reagent for activating T cells for use in immunotherapy.

[0055] Some examples of this disclosure include methods for preparing T cells for administration to subjects in need using a container. In some examples, the subjects have undergone organ transplantation, tissue transplantation, cell transplantation, allogeneic transplantation, intestinal transplantation, reconstructive transplantation, and have cancer, cancer-related diseases or conditions, or any combination thereof.

[0056] definition

[0057] Unless otherwise defined, 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 pertains. However, any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of some examples of this disclosure.

[0058] As used herein, each of the following terms has the meaning associated with it in this section.

[0059] This article uses the articles “a” and “an” to refer to the grammatical object of one or more articles (i.e., at least one). By way of example, “element” refers to one or more elements.

[0060] As used herein, when referring to measurable values ​​such as quantity, time interval, etc., “about” means to cover a variation of ±20% or, in some cases, ±10%, or, in some cases, ±5%, or, in some cases, ±1%, or, in some cases, ±0.1% from the specified value, because such variation is suitable for performing the disclosed method.

[0061] The terms “patient,” “subject,” “individual,” etc., are used interchangeably herein and refer to any animal with a complement system, in some examples mammals and in some examples human, including those who require treatment or are susceptible to the condition or its sequelae. Individuals may include, for example, dogs, cats, pigs, cattle, sheep, goats, horses, rats, monkeys, and mice, as well as humans.

[0062] The term "abnormal," when used in the context of an organism, tissue, cell, or component thereof, refers to those organisms, tissues, cells, or components thereof that differ from those exhibiting "normal" (expected / homeostatic) characteristics in at least one observable or detectable feature (e.g., age, treatment, time of day, etc.). A feature that is normal or expected for one cell type, tissue type, or subject may be abnormal for different cell or tissue types.

[0063] "Disease" is a health state of a subject in which the subject is unable to maintain homeostasis, and in which the subject's health continues to deteriorate if the disease does not improve.

[0064] In contrast, a subject's "symptom" represents a state of health that allows them to maintain homeostasis, but in a way that is less favorable than their state of health without the symptom. Without treatment, the symptom does not necessarily lead to a further decline in the subject's health.

[0065] As used in this article, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system.

[0066] The disease or condition is considered to have "mitigated" if the severity of the signs or symptoms of the disease or condition, the frequency with which the patient experiences such signs or symptoms, or both decrease.

[0067] As used herein, “activation” refers to a state in which T cells have been adequately stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. The term “activated T cell” specifically refers to T cells that are undergoing cell division.

[0068] As used herein, the terms “inhibit” and “inhibition” mean a reduction, inhibition, attenuation, or blocking of activity or function by at least about 10% relative to a control. In some examples, activity is inhibited or blocked by at least about 50% compared to a control. In some examples, activity is inhibited or blocked by at least about 75%. In some examples, activity is inhibited or blocked by at least about 95%.

[0069] As used herein, the term “self” means any material derived from the same individual that is later reintroduced into that individual.

[0070] "Allogeneic" refers to any material derived from a different animal of the same species as the individual into which the material was introduced. Two or more individuals are said to be allogeneic when the genes at one or more loci are not identical. In some examples, allogeneic material from individuals of the same species may be genetically different enough to interact antigenically.

[0071] As used herein, the phrase “diseases associated with allogeneic responsive cell expression” includes, but is not limited to, diseases associated with allogeneic responsive cell expression or symptoms associated with cells expressing an allogeneic response, which includes, for example, allogeneic transplant rejection, immune rejection, chronic allogeneic rejection, implant rejection, graft rejection, inflammation, inflammation caused by local ischemia / reperfusion, infection, immune response to allogeneic grafts, and any combination thereof.

[0072] "Heterogeneous" refers to grafts originating from animals of different species.

[0073] As used in this article, “endogenous” means any substance that originates from or is produced within an organism, cell, tissue, or system.

[0074] As used herein, the term “exogenous” means any substance introduced from or produced outside of an organism, cell, tissue, or system.

[0075] As used herein, the term "immune cells" includes cells of hematopoietic origin that play a role in the immune response. Immune cells include lymphocytes, such as B cells and T cells; natural killer cells; and myeloid cells, such as monocytes, macrophages, dendritic cells, eosinophils, mast cells, basophils, and granulocytes.

[0076] As used herein, the term "immune response" includes T-cell-mediated and / or B-cell-mediated immune responses regulated by T-cell co-stimulation. The term also includes immune responses indirectly achieved through T-cell activation, such as antibody production (humoral response), and immune responses indirectly achieved through the activation of cytokine-responsive cells, such as macrophages.

[0077] As used herein, the term “T-cell immune response” refers to the activation of antigen-specific T cells, as measured by the proliferation or expression of molecules on the cell surface or the secretion of proteins such as cytokines.

[0078] As used herein, the term "T cell" refers to a lymphocyte (e.g., a leukocyte) that plays a role 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 assist 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 (CTL), natural killer T cells (NK cells), γδ T cells, and mucosa-associated inertial T cells (MAIT).

[0079] As used herein, the term “TCR” refers to “T cell receptor”. T cell receptors are molecules on the surface of T lymphocytes (“T cells”). In this example, the receptor is an αβ-TCR receptor, meaning that the T cell receptor contains an alpha (α) chain and a beta (β) chain, which is typically expressed as part of a complex with the CD3 chain molecule.

[0080] As used herein, the term "B cell" refers to a cell produced in the bone marrow of an animal that expresses membrane-bound antibodies specific to an antigen. Upon interaction with the antigen, it differentiates into plasma cells that produce antibodies specific to the antigen or into memory B cells. "B cell" and "B lymphocyte" are used interchangeably. Primary and activated B cells are within the scope of this invention.

[0081] As used herein, the term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain, the cytoplasmic signaling domain comprising a functional signaling domain derived from a stimulating molecule as defined below. In one example, the stimulating molecule is a ζ-chain associated with a T-cell receptor complex. In one example, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulating molecule as defined below. In one example, the co-stimulating molecule is selected from 41BB (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, the intracellular signaling domain comprising a functional signaling domain derived from a stimulating molecule. In one example, the CAR comprises a chimeric fusion protein containing an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, the 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 another example, the CAR comprises a chimeric fusion protein containing an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, the intracellular signaling domain comprising two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In yet another example, the CAR comprises a chimeric fusion protein containing an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, the intracellular signaling domain comprising at least two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one example, the CAR includes an optional leader sequence at the N-terminus (N-ter) of the CAR fusion protein. In one example, the CAR also includes 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 cell processing and CAR localization to the cell membrane.

[0082] The portion of the CAR composition containing an antibody or an antibody fragment thereof can be present in various forms, wherein the antigen-binding domain is expressed as part of a continuous polypeptide chain, including, for example, single-domain antibody fragments (sdAbs), single-chain antibodies (scFvs), and humanized antibodies (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 the CAR composition of the present invention comprises an antibody fragment. In one example, the CAR comprises an antibody fragment having an scFv.

[0083] As used in this article, a “signal transduction domain” is a functional part of a protein that functions by transmitting information within the cell to regulate cellular activity via defined signal transduction pathways by generating a second messenger, or to act as an effector in response to such a messenger.

[0084] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. Antibodies are typically tetramers of immunoglobulin molecules.

[0085] The term "antibody fragment" refers to at least a portion of a complete antibody or its recombinant variants, and specifically to an antigen-binding domain, such as an antigen-determining variable region, 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 (VL or VH), the Camelidae VHH domain, and multispecific antibodies formed from antibody fragments. The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are continuously linked via a short, flexible polypeptide linker and are capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the complete antibody from which it originates. Unless otherwise specified, as used herein, an scFv may have VL and VH variable regions in either order, for example, relative to the N-terminus and C-terminus of a polypeptide, the scFv may contain VL-connector-VH or may contain VH-connector-VL.

[0086] As used in this article, “antibody heavy chain” refers to the larger of two polypeptide chains that exist in the antibody molecule in their naturally occurring conformation, and it usually determines the grade to which the antibody belongs.

[0087] As used in this article, "antibody light chain" refers to the smaller of the two polypeptide chains that exist in the antibody molecule in their naturally occurring conformation. κ and λ light chains refer to the two main isotypes of antibody light chains.

[0088] As used herein, the term "recombinant antibody" means an antibody generated using recombinant DNA technology, such as an antibody expressed by a phage or yeast cell expression system. The term should also be understood to mean an antibody generated by synthesizing a DNA molecule encoding the antibody and expressing the antibody protein, or specifying the amino acid sequence of the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequencing technologies available and well known in the art.

[0089] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response may involve antibody production, or activation of specific immune-active cells, or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can be used as an antigen. Furthermore, antigens can be derived from recombinant DNA or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or a portion of a protein encoding an immune response therefore encodes an antigen as used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It will be 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 a polypeptide that elicits the desired immune response. Furthermore, those skilled in the art will understand that an antigen does not necessarily need to be encoded by a "gene". It will be apparent that antigens can be generated synthetically or derived from biological samples, or can be macromolecules other than polypeptides. Such biological samples may include, but are not limited to, tissue samples, cells, or fluids containing other biological components.

[0090] As used herein, “antigen-presenting cell” or “APC” refers to immune system cells, such as helper cells (e.g., B cells, dendritic cells, etc.), that display exogenous antigens on their surface that are complexed with HLA I, HLA II, MHCI, MHC II, or MHC III complexes. For example, T cells can recognize these complexes using their T cell receptors (TCRs). APCs process the antigens and present them to T cells.

[0091] As used herein, the term "binding agent" refers to a molecule that binds to a specific antigen or target. Binding agents may include proteins, peptides, nucleic acids, carbohydrates, lipids, or low molecular weight compounds. In some examples, the binding agent comprises a full-length antibody. In some examples, the binding agent is an antigen-binding fragment of an antibody. In some examples, the binding agent comprises an alternative protein scaffold or artificial scaffold (e.g., a non-immunoglobulin backbone). In some examples, the binding agent is a fusion protein containing antigen-binding sites. In some examples, the binding agent is a bispecific molecule containing at least two antigen-binding sites. In some examples, the binding agent is a multispecific molecule containing at least three antigen-binding sites.

[0092] The term "binding" refers to the interaction between molecules, including, for example, the formation of 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 comprise 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 interaction 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 for that epitope. The ratio (koff / kon) of the dissociation rate (koff) of a binding molecule (e.g., an antibody) to the association rate (kon) of a monovalent antigen is the dissociation constant KD, which is inversely proportional to affinity. The lower the KD value, the higher the affinity of the antibody. The KD value varies with different antibody-antigen complexes and depends on both kon and koff. The dissociation constant KD of antibodies provided herein can be determined using any of the methods provided herein or any other methods well known to those skilled in the art. The affinity at a binding site does not always reflect the true strength of the interaction between the antibody and the antigen. When a complex antigen containing multiple repeating antigenic determinants (such as a multivalent antigen) comes into contact with an antibody containing multiple binding sites, the interaction between the antibody and the antigen at one site increases the probability of a reaction at a second site. The strength of this multiple interaction between a multivalent antibody and an antigen is called affinity.

[0093] Terms related to binding molecules as described herein, such as “binding,” “specific binding,” and similar terms, are used interchangeably herein and refer to binding molecules that specifically bind antigen-binding domains of antigens, such as peptides. Binding molecules or antigen-binding domains that bind or specifically bind antigens can be identified, for example, by immunoassay, Octet... ® Biacore ®Or other techniques known to those skilled in the art may be used for identification. In some examples, such as those determined using experimental techniques like enzyme-linked immunosorbent assay (ELISA), a binding molecule or antigen-binding domain binds or specifically binds an antigen when it binds to the antigen with a higher affinity than to any cross-reactive antigen. Typically, the specific or selective reaction will be at least twice the background signal or noise, and can be more than 10 times the background signal or noise. See, for example, Fundamental Immunology, pp. 332-336 (edited by Paul, 2nd edition, 1989) for a discussion of binding specificity. In some examples, the binding of a binding molecule or antigen-binding domain to a degree less than about 10% of its binding to a specific target antigen may occur, for example, as determined by fluorescence activated cell sorting (FACS) analysis. A binding molecule or antigen-binding domain that binds to an antigen includes a binding molecule or antigen-binding domain capable of binding an antigen with sufficient affinity such that the binding molecule can be used as, for example, a therapeutic agent and / or diagnostic agent targeting the antigen. In some examples, the binding molecule or antigen-binding domain that binds to the antigen has a dissociation constant (KD) less than or equal to 1 μM, 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 some examples, the binding molecule or antigen-binding domain binds to a conserved antigenic epitope in antigens from different species.

[0094] The term “differentiation cluster 3ε” or “CD3ε” refers to the known protein also known as the “T cell surface glycoprotein CD3ε chain” or “T3E”. CD3ε, together with CD3-γ, CD3-δ, and CD3-ζ, and the T cell receptor α / β and γ / δ heterodimers, forms the T cell receptor-CD3 complex. This complex plays a crucial role in coupling antigen recognition to several intracellular signal transduction pathways. The CD3 complex mediates signal transduction, leading to T cell activation and proliferation. CD3 is required for immune responses. The amino acid sequence of the full-length CD3ε is shown in SEQ ID NO: 1. The amino acid sequence of the extracellular domain (ECD) of CD3ε is shown in SEQ ID NO: 2. Throughout this specification, “CD3ε specific” or “specifically binding CD3ε” or “anti-CD3 antibody” refers to an antibody that specifically binds to the CD3ε polypeptide, including antibodies that specifically bind to the CD3ε extracellular domain (ECD).

[0095] Human CD3ε:

[0096] MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKP EDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO:1);

[0097] Human CD3ε extracellular domain:

[0098] DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD (SEQ ID NO: 2).

[0099] The term "stimulus" refers to a primary response induced by the binding of a stimulating molecule (e.g., the TCR complex, BCR complex, etc.) to its homologous ligand, thereby mediating signal transduction events (such as, but not limited to, signal transduction via the TCR complex and / or BCR complex, etc.). Stimulus can mediate alterations in the expression of certain molecules, such as downregulation of TGF-β and / or reorganization of cytoskeleton structure.

[0100] The terms “antibody,” “immunoglobulin,” or “Ig” are used interchangeably herein and in the broadest sense, and specifically cover, for example, polyclonal antibodies, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, and full-length monoclonal antibodies), antibody compositions having multiple or single epitope specificity, recombinant antibodies, single-domain (e.g., VHH) antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human antibody versions having full-length heavy and / or light chains. Antibodies also include antibody fragments (and / or polypeptides containing antibody fragments) that retain the binding characteristics of their parent antibody. Non-limiting examples of antibody fragments include antigen-binding and / or effector regions of antibodies (e.g., Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single-chain antibody molecules, double-variable-domain antibodies, single-variable-domain linear antibodies, V regions, multispecific antibodies formed from antibody fragments, F(ab)2, Fd, Fc, biantibodies, di-diabody, disulfide-linked Fv (dsFv), single-domain antibodies (e.g., nanobodies), or other fragments (e.g., fragments consisting of variable regions of non-covalently coupled heavy and light chains). Generally, the variable (V) region domain can be any suitable arrangement of immunoglobulin heavy (VH) and / or light (VL) variable domains. For example, antibodies also... This includes tetrameric antibodies, which comprise 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 can be dimer and contain VHH-VHH, VH-VH, VH-VL, or VL-VL dimers that bind to NKG2A. If desired, VH and VL can be covalently coupled directly or via 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 antibody fragment is a peptide containing one or more complementarity-determining regions (CDRs) of an antibody. CDRs (also known as “minimum recognition units” or “hypervariates”) can be obtained by constructing polynucleotides encoding one or more CDRs of interest.Such polynucleotides are prepared, for example, by using polymerase chain reaction to synthesize variable regions using mRNA from antibody-producing cells as templates (see, for example, Larrick et al., “Methods: A Companion to Methods in Enzymology”, Vol. 2, p. 106 (1991); Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies”, in Monoclonal Antibodies Production, Engineering and Clinical Application, Ritter et al. (eds.), p. 166, Cambridge University Press (1995); Ward et al., “Genetic Manipulation and Expression of Antibodies”, in Monoclonal Antibodies: Principles and Applications, Birch et al. (eds.), p. 137, Wiley-Liss, Inc. (1995)). Antibody fragments may be incorporated, for example, into single-domain antibodies, large antibodies, small antibodies, intracellular antibodies, bivalent antibodies, trivalent antibodies, tetravalent antibodies, variable domains (v-NARs) of neoantigen receptors, and double single-chain Fv regions (see, for example, Hollinger and Hudson, Nature Biotechnology, Vol. 23, No. 9: pp. 1126–1136, 2005). In some examples, antibodies containing VH and / or VL contain light chain and / or heavy chain constant regions, such as one or more constant regions, including one or more IgG1, IgG2, IgG3, and / or IgG4 constant regions. In some examples, antibodies may include epitope-binding fragments of any of the above. The antibodies described herein may be any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules.

[0101] As used herein, the term "stimulatory molecule" refers to a molecule expressed by a cell that provides a primary cytoplasmic signaling sequence that modulates the initial activation of the TCR and / or BCR complex in a stimulatory manner on at least some examples of cellular signaling pathways. In one example, the primary signal is initiated, for example, by the binding of the TCR and / or BCR complex to a peptide-loaded human leukocyte antigen (HLA) I, HLA II, major histocompatibility complex (MHC) I, MHC II, or MHC III molecule, and this leads to the mediating of cellular responses, including but not limited to proliferation, activation, differentiation, etc. The primary cytoplasmic signaling sequence acting in a stimulatory manner may contain a signaling motif called an immune receptor tyrosine-based activation motif or ITAM. Examples of ITAM-containing primary cytoplasmic signaling sequences particularly useful in this invention include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), and CD66d. In the specific CAR of this invention, the cytoplasmic signaling sequence derived from CD3-ζ is derived from non-human species, such as mice, rodents, monkeys, apes, etc.

[0102] The terms "effective amount" and "pharmaceutical effective amount" refer to a dosage of medicine sufficient to provide the desired biological outcome. The outcome can be a reduction and / or mitigation of signs, symptoms, or causes of a disease or condition, or any other desired alteration of a biological system. In any individual case, the appropriate effective amount can be determined by a person skilled in the art using routine laboratory methods.

[0103] As used in this article, the term "therapeutic" means treatment. Therapeutic effects are achieved through the reduction, suppression, relief, or eradication of the disease state.

[0104] "Therapeutic treatment" is treatment administered to a subject who is exhibiting signs of disease or symptom in order to reduce or eliminate those signs.

[0105] As used in this article, “treating a disease or condition” means reducing the frequency and / or severity of the signs and / or symptoms of a disease or condition experienced by a patient.

[0106] As used in this article, the term “prevention” means the prevention or protective treatment of a disease or disease state.

[0107] As used herein, the phrases “biological sample,” “sample,” or “sample” are intended to include any sample containing cells, tissues, or body fluids in which the expression of nucleic acids or peptides can be detected. A biological sample may contain any biological material suitable for detecting a desired biomarker and may include cellular and / or non-cellular material obtained from an individual. Examples of such biological samples include, but are not limited to, blood, lymph, bone marrow, biopsies, and smears. Samples that are essentially liquid are referred to herein as “body fluids.” Biological samples can be obtained from a patient using a variety of techniques, including, for example, obtaining body fluids by scraping or wiping an area or by using a needle. Methods for collecting various bodily samples are well known in the art.

[0108] As used in this article, “immunoassay” refers to any binding assay that uses antibodies capable of specifically binding to target molecules to detect and quantify the binding of target molecules.

[0109] The term “specifically binding” as used in this article with respect to peptides (e.g., TCRs or TCR chains) refers to peptides that recognize and bind to a specific target molecule, but substantially do not recognize or bind to other molecules in the sample. In some cases, the terms “specifically binding” or “specifically binding” are used to mean that recognition and binding depend on the presence of a specific structure on the target molecule (e.g., antigenic determinants or epitopes).

[0110] A gene's "coding region" consists of nucleotide residues in the gene's coding strand and nucleotides in the gene's non-coding strand, which are homologous to or complementary to the coding region of the mRNA molecule produced by gene transcription. The "coding region" of the mRNA molecule also consists of nucleotide residues that match or encode stop codons in the anticodon region of the transfer RNA molecule during mRNA translation. Therefore, the coding region can include nucleotide residues containing codons for amino acid residues not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in the protein output signal sequence).

[0111] As used herein, "complementarity" refers to nucleic acids, and is a broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is well known that adenine residues in the first nucleic acid region can form specific hydrogen bonds ("base pairing") with residues in the second nucleic acid region. If the residue is thymine or uracil, then the residue is antiparallel to the first region. Similarly, it is known that cytosine residues in the first nucleic acid strand can pair with bases in the second nucleic acid strand. If the residue is guanine, then the residues in the second nucleic acid strand are antiparallel to the first strand. If, when the two regions are arranged in an antiparallel manner, at least one nucleotide residue in the first region can pair with a base in the second region, then the first region of the nucleic acid is complementary to the second region of the same or different nucleic acids. In some examples, the first region contains a first part and the second region contains a second part, such that, when the first and second parts are arranged in an antiparallel manner, at least about 50%, and / or at least about 75%, or at least about 90%, or at least about 95% of the nucleotide residues in the first part can pair with the nucleotide residues in the second part. In some examples, all nucleotide residues in the first part are able to pair with the nucleotide residues in the second part.

[0112] "Homology" refers to the sequence similarity or identity between two polypeptides or two nucleic acid molecules. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if every position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage 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 comparison positions multiplied by 100. For example, if six out of ten positions in two sequences are matching or homologous, then the two sequences are 60% homologous. Generally, two sequences are compared to give the maximum homology.

[0113] As used herein, a “variant” is a nucleic acid or peptide sequence that differs sequentially from a reference nucleic acid or peptide sequence, but retains the essential biological characteristics of the reference molecule. Changes in the nucleic acid variant sequence may not alter the amino acid sequence of the peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions, and truncations. Changes in the peptide variant sequence are generally limited or conserved, so the sequences of the reference peptide and the variant are very similar overall and identical in many regions. Variants and the reference peptide may differ in amino acid sequence by any combination of one or more substitutions, additions, or deletions. Variants of nucleic acids or peptides may be naturally occurring, such as allelic variants, or may be unknown naturally occurring variants. Non-naturally occurring variants of nucleic acids and peptides may be prepared by mutagenesis or by direct synthesis. In various examples, the variant sequence has 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%, or at least 85% identity with the reference sequence.

[0114] "Encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) to serve as a template for the synthesis of other polymers and macromolecules in biological processes. These polymers and macromolecules have defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences and the resulting biological properties. Therefore, if the transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, then that gene encodes that protein. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing, and the non-coding strand, which serves as a template for the transcription of a gene or cDNA, can be said to encode that protein, or other products of that gene or cDNA.

[0115] Unless otherwise specified, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. The phrase "nucleotide sequence" encoding a protein or RNA may also include introns to such an extent that the nucleotide sequence encoding that protein may contain introns in some forms.

[0116] "Separated" means altered or removed from its natural state. For example, nucleic acids or peptides that are naturally present in a living subject under normal conditions are not "separated," but the same nucleic acids or peptides that are partially or completely separated from their natural state are "separated." Separated nucleic acids or proteins may exist in a substantially purified form or may exist in non-natural environments, such as host cells.

[0117] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to that of naturally occurring nucleotides. Unless otherwise stated, a specific nucleic acid sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as explicitly stated sequences. Specifically, degenerate codon substitution can be achieved by producing a sequence in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue (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. Probes8:91-98 (1994)).

[0118] In the context of some examples in this disclosure, the following abbreviations for common nucleic acid bases are used: “A” for adenosine, “C” for cytosine, “G” for guanosine, “T” for thymidine, and “U” for uridine.

[0119] As used herein, the term "polynucleotide" is defined as a nucleotide chain. Furthermore, nucleic acids are polymers of nucleotides. Therefore, as used herein, nucleic acids and polynucleotides are interchangeable. Those skilled in the art will generally know that nucleic acids are polynucleotides, which can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant methods, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using common cloning techniques and PCR, as well as synthetic methods.

[0120] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short and long chains; short chains are generally referred to in the art, for example, as peptides, oligopeptides, and oligomers, while long chains are generally referred to in the art, and there are many types of long chains. “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0121] As used in this article, “recombinant DNA” is defined as DNA produced by linking DNA fragments from different sources.

[0122] As used in this article, the term "recombinant polypeptide" is defined as a polypeptide produced using recombinant DNA methods.

[0123] As used in this article, "conjugated" means that one molecule is covalently attached to a second molecule.

[0124] As used herein, "operably linked" or "operably linked" can mean that gene expression is controlled by a promoter to which it is spatially linked. The promoter can be located at the 5' (upstream) or 3' (downstream) of the gene it controls. The distance between the promoter and the gene can be approximately the same as the distance between the promoter and the genes it controls within the gene from which it originates. As is known in the art, variations in this distance can be accommodated without loss of promoter function.

[0125] As used herein, the term “promoter” is defined as a DNA sequence that is recognized by the cell’s synthetic mechanism or an introduced synthetic mechanism and is required to initiate specific transcription of a polynucleotide sequence.

[0126] As used herein, the term "regulation" can refer to any method that alters substrate levels or activity. Non-limiting examples of protein regulation include affecting expression (including transcription and / or translation), affecting folding, affecting degradation or protein turnover, and affecting protein localization. Non-limiting examples of enzyme regulation also include affecting enzyme activity. "Regulator" refers to a molecule whose activity includes affecting substrate levels or activity. Regulators can be direct or indirect. Regulators can be used to activate, inhibit, or otherwise regulate their substrates.

[0127] As used in this article, "vector" can refer to a nucleic acid sequence containing an origin of replication. Vectors can be plasmids, bacteriophages, bacterial artificial chromosomes, or yeast artificial chromosomes. Vectors can be DNA or RNA vectors. Vectors can be self-replicating extrachromosomal vectors or vectors integrated into the host genome.

[0128] As used herein, “substantially purified” cells are cells that are substantially free of other cell types. Substantially purified cells also refer to cells that have been isolated from other cell types and are typically associated with them in their native state. In some cases, a substantially purified cell population refers to a homogeneous cell population. In other cases, the term simply refers to cells that have been isolated from other cells and are naturally associated with them in their native state. In some examples, the cells are cultured in vitro. In other examples, the cells are not cultured in vitro.

[0129] Scope: Throughout this disclosure, various examples of the invention may be presented in a scope format. It should be understood that the scope format is merely for convenience and brevity and should not be construed as a fixed limitation on the scope of the invention. Therefore, the scope description should be considered to have all possible sub-scopes explicitly disclosed and the various numerical values ​​within those scopes. For example, a scope such as 1 to 6 should be considered to have explicitly disclosed sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the various numerical values ​​within those scopes, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the scope.

[0130] describe

[0131] In some examples and such Figure 1A , Figure 1B and Figure 1C As shown, the present invention provides a sealable or sealable container 10 for holding at least one reagent. The container 10 includes an inner surface forming one or more internal chambers 14 for holding or storing a sample containing the at least one reagent. The container 10 can be of any size and shape, and can also be rigid, flexible, resilient, or any combination thereof. For example, the container 10 can be sized to hold 1,000 mL, 500 mL, 250 mL, 100 mL, or 50 mL of fluid. In some examples, the container 10 is a sealable flexible bag, such as VueLife. ® “AC” series bags. In some examples, container 10 has a plurality of internal chambers 14 fluidly connected to each other via a permeable or semipermeable membrane and / or at least one valve.

[0132] In some examples, the inner surface of the internal chamber 14 is smooth. In some examples, the inner surface of the internal chamber 14 includes one or more grooves, ridges, channels, recesses, protrusions, cavities, etc., thereby creating one or more regions with altered surface profiles within the internal chamber 14. In some examples, the altered surface profile can increase the total surface area within the internal chamber 14. In some examples, the altered surface profile can guide or influence the flow of liquid or gas within the internal chamber 14. In some examples, the internal chamber 14 includes at least one microfluidic structure configured to guide a fluid sample through it.

[0133] In some examples, container 10 includes one or more ports 12 fluidly connected to an internal chamber 14. Ports 12 may be connected to any type of tubing or conduit suitable for guiding liquid or gas samples into and / or out of the internal chamber 14 of container 10. Ports 12 and / or connected tubing may also include any type of valve or Luer lock to open or close the internal chamber 14 of container 10 to prevent 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 creating a complete or partial vacuum (i.e., negative pressure) within the internal chamber 14 of container 10. Therefore, container 10 may include one or more liquid or gas flow paths into and out of the internal chamber 14. For example, container 10 may include an inlet port 12a and an outlet port 12b, each connected to the internal chamber 14, such that fluid is configured to flow into container 10 through inlet port 12a, into the internal chamber 14, and subsequently out of container 10 through outlet port 12b.

[0134] As considered herein, container 10 may be made of any suitable disposable or sterilizable material, such as polyvinyl chloride (PVC), ethylene vinyl acetate, polypropylene, fluorinated ethylene propylene (FEP), or any other suitable material.

[0135] In some examples, container 10 is made of an impermeable material. In some examples, container 10 is made of a semi-permeable or porous material. In some examples, container 10 is made of a gas-permeable but liquid-impermeable material. For example, the material may include a pore size of less than 0.2 μm to maintain the sterility of the contents while allowing 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 between 10 μm and 1000 μm. In some examples, the material of container 10 may have a thickness between 100 μm and 500 μm. In some examples, the inner surface of container 10 may have high surface energy. In some examples, the inner surface of container 10 may carry a charge. In some examples, the inner surface of container 10 may be modified with hydroxyl or carboxyl groups or other chemical groups that cause changes in the adhesive properties of one or more molecules.

[0136] Cell culture system

[0137] In some examples, container 10 may be incorporated into a cell culture system comprising multiple components. In one example, the cell culture system is an automated system. In another example, the cell culture system is a microfluidic system. For example, the container may be included in or incorporated into a commercial cell culture and manufacturing system, including but not limited to the Miltenyi Biotech CliniMACSProdigy Platform.

[0138] In one example, the cell culture system according to the invention includes at least one cell culture module comprising a container 10 that acts as a bioreactor. In the context of this application, the term bioreactor refers to a vessel intended for receiving cells, including but not limited to variations of cell proliferation flasks, centrifuge vessels, cell separation vessels, cell differentiation vessels, cell seeding vessels, sample vessels, etc.

[0139] In one example, the cell culture module is a closed system, meaning that a closed, sterile environment can be maintained within the closed cell culture module. In one example, the cell culture system also includes at least one pump for pumping liquids within the closed cell culture module. In some examples, the system also includes at least one additional tool module. In the context of this application, the term "tool module" means any tool or instrument used to manipulate or monitor any one or more components of the cell culture system (such as cell cultures grown in the bioreactor of the cell culture system) or other components included in the cell culture system (such as culture media and enzymes).

[0140] Such tool modules include monitoring tool modules for monitoring processes and cell cultures in bioreactors. These monitoring modules are cell imaging devices (e.g., including microscopes and cameras), or any kind of sensor technology device such as pH and temperature sensors. Other 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 a closed cell culture module and / or tool module relative to each other. Additional tools include harvesting modules, such as cell washing / cell concentration devices (e.g., centrifuges). In some examples of closed cell culture modules, the system includes a peristaltic pump.

[0141] In some examples, the cell culture system includes at least two units: a cell maintenance unit for the appropriate storage of cell culture intermediates, final products, and process fluids; and a processing unit (or cell processing unit) for cell growth and cell processing. In some examples, the cell culture system includes additional units, such as additional storage units, for example, for cryopreservation of cells. Within each unit, the environmental physical conditions are individually adjustable, such as, for example, temperature and humidity.

[0142] For example, the temperature can be adjusted to set the processing unit to a temperature of, for example, 37°C, the cell maintenance unit to a temperature of 4°C, and the storage unit to a temperature of -196°C, etc.

[0143] In one example, one or more containers (e.g., 10) of the present invention, as part of a closed cell culture module, are held within a cell processing unit. In one example, a cell maintenance unit provides standard freezer temperatures to allow for the appropriate storage of temperature-sensitive liquids (such as culture media or enzyme solutions) and the preservation of cell-based final products or cell intermediates (such as samples for quality control purposes). In one example, those components of a closed cell culture system requiring refrigeration temperatures are housed within the cell maintenance unit.

[0144] Now for reference Figure 2 An exemplary cell culture system 200 is shown, incorporated into any exemplary container. In some examples, the cell culture system 200 may be automated. The cell culture system 200 may include a cell separator 201. The cell separator 201 may be any suitable cell separator, including, for example, a centrifuge, a magnet-based separator, a size exclusion separator, and / or an antibody affinity column separator. The cell culture system 200 may include a container 203, which may be implemented as one of the exemplary containers described herein for dehydrating, drying, and / or lyophilizing reagents (e.g., Figure 1A , Figure 1B and Figure 1C(Container 10). The cell culture system 200 may include one or more pumps and / or gravity drain pipes (e.g., 202, 204, 207, 209, 211) for transferring products between devices within the cell culture system 200. Although individual pumps are shown, it should be understood that pumps 202, 204, 207, 209, 211 may be implemented using as few as a single pump.

[0145] T cells separated by cell separator 201 can be provided to container 203 via pump and / or gravity drain pipe 204. Activated T cells from container 203 can be transferred from container 203 to one or both of gene editing device or vessel 205 and attenuated vessel 206 via pump or gravity drain pipe 204. Gene editing device or vessel may include, for example, an electroporator and / or a bioreactor. Attenuated vessel may be, for example, a bioreactor. Cells can be transferred from gene editing device or vessel 205 and / or attenuated vessel 206 to cell expansion vessel or device via pump or gravity device 207. After expansion, cells are provided to washing or impurity removal device 210 via pump or gravity drain pipe 209, which may include, for example, a cell washing device, a cell concentration device, and / or a centrifuge. Finally, cells are provided to preparation device 212 via pump or gravity drain pipe 211. The preparation apparatus 212 may include means for mixing, pumping and filling vials and / or bags, as well as storage units for storing vials and / or bags, such as cryopreservation units.

[0146] Dehydrated, dried and / or lyophilized reagents

[0147] As considered herein, the inner surface within the internal chamber 14 of container 10 includes at least one dehydrating, drying, and / or lyophilizing agent. The dehydrating, drying, and / or lyophilizing agent may adhere directly to or bind 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 the binding of the dehydrating, drying, and / or lyophilizing agent. The agent may be introduced into the internal chamber 14 via a liquid or gas sample delivered through inlet port 12a, and subsequently lyophilized within the internal chamber 14 of container 10. The binding of the agent to the inner surface of the internal chamber 14 may occur prior to dehydration, drying, and / or lyophilization, or as a result of dehydration, drying, and / or lyophilization.

[0148] In some examples, the internal chamber 14 may have a mixture or concentrate of the at least one lyophilized reagent on its inner surface. In some examples, the mixture or concentrate of the at least one lyophilized reagent on the inner surface may be homogeneous or substantially homogeneous. For example, a substantially homogeneous mixture or concentrate may have… > 80% > 85%, > 90%, > 92%, >94%, > 96%, > 98% or > 99% uniformity. In other examples, the mixture or concentrate of the at least one lyophilized reagent on the inner surface can be variable. For example, the inner chamber 14 may have a concentration gradient of the lyophilized reagent across the inner surface, such as a higher concentration near the bottom of the container 10 and a lower concentration near the top of the container 10. In some examples, the inner chamber 14 may have multiple inner surface regions (e.g., 2, 3, 4, 5, 6, 7, or 8 inner surface regions), wherein a first region has a different concentration of lyophilized reagent compared to a second region. In some examples, each region has a different concentration of lyophilized reagent. Similarly, a first region may have a first concentration of a first lyophilized reagent, and a second region may have a second concentration of a second lyophilized reagent.

[0149] In one example, the lyophilized reagent is a reagent used to stimulate T cells. In another example, the lyophilized reagent is a reagent used to isolate T cells. In some examples, the inner surface is coated with a combination of at least two, three, four, five, six, seven, eight, nine, ten, or more than ten different reagents for isolating and / or stimulating T cells. In one example, two or more agents are lyophilized in the inner chamber of a container. In one example, each of the two or more reagents is lyophilized in the inner chamber of a container at a 1:1 ratio. In one example, each of the two or more reagents is lyophilized in the inner chamber of a container at a ratio ranging from 100:1 to 1:100 and all integer values ​​therebetween. In one example, two or more agents are lyophilized in the inner chamber of a container. In one example, each of the three or more reagents is lyophilized in the inner chamber of a container at a 1:1:1 ratio. In one example, each of three or more reagents is freeze-dried in the inner chamber of a container at a ratio ranging from 1:100:1 to 1:1:100 to 100:1:1 and all integer values ​​therebetween.

[0150] In one example, at least one reagent to be dehydrated, dried, and / or lyophilized is suspended in a buffer solution in a container prior to dehydration, drying, and / or lyophilization. In one example, the buffer solution protects the reagent from degradation or inactivation during dehydration, drying, and / or lyophilization. In one example, the buffer solution contains at least one lyophilization protectant. Exemplary lyophilization protectants include, but are not limited to, surfactants (poloxam, polysorbate, etc.), amino acids (arginine, serine, histidine, etc.), polymers (PEG, PVP, dextran, etc.), and sugars (sucrose, etc.). In one example, the buffer solution contains PBS. In one example, the buffer solution contains trehalose. In one example, the buffer solution contains water.

[0151] In some examples, at least one dehydrating, drying, and / or lyophilizing agent is a peptide, protein, antibody, nucleic acid molecule, or a combination thereof. In one example, the cell culture apparatus includes a surface coated with at least one dehydrating, drying, and / or lyophilized antibody for isolating and / or stimulating T cells.

[0152] In some examples, the reagents used to isolate and / or stimulate T cells are not attached to the polymer beads. Therefore, in some examples, the polymer beads are not introduced into the container.

[0153] The peptide reagents of the present invention can be post-translational modified prior to dehydration, drying, and / or lyophilization. For example, post-translational modifications falling within the scope of some examples of this disclosure include signal peptide cleavage, glycosylation, acetylation, isoprenelation, proteolysis, myristoylation, protein folding, and proteolytic processing. Some modification or processing events require the introduction of additional biological machinery. For example, processing events such as signal peptide cleavage and core glycosylation can be examined by adding canine microsomal membrane or Xenopus laevis egg extract (US Patent 6,103,489) to a standard translation reaction.

[0154] Dehydrated, dried, and / or lyophilized peptide reagents may include non-natural amino acids formed through post-translational modification or by introducing non-natural amino acids during translation. Various methods can be used to introduce non-natural amino acids during protein translation. For example, specialized tRNAs, such as repressive tRNAs, have been used in the process of site-directed non-natural amino acid substitution (SNAAR). In SNAAR, unique codons are required on both the mRNA and the repressive tRNA, whose role is to target the non-natural amino acid to a unique site during protein synthesis (described in WO 90 / 05785). However, the repressive tRNA must not be recognized by aminoacyl-tRNA synthetases present in the protein translation system. In some cases, non-natural amino acids can be formed using chemical reactions following aminoacylation of the tRNA molecule, which specifically modify the natural amino acid without significantly altering the functional activity of the aminoacylated tRNA. These reactions are called post-aminoacylation modifications. For example, the ε-amino group of a lysine residue linked to its homologous tRNA (tRNALYS) can be modified using amine-specific photoaffinity labeling.

[0155] Some examples of dehydrated, dried, and / or lyophilized peptide reagents disclosed herein can be prepared using chemical methods. For example, peptides can be synthesized using solid-phase techniques (Roberge JY et al. (1995) Science 269: 202-204), cleaved from a resin, and purified by preparative high-performance liquid chromatography. Automated synthesis can be achieved, for example, using an ABI 431 A peptide synthesizer (Perkin Elmer) according to the manufacturer's instructions. Alternatively, peptides can be prepared via recombinant methods or by cleavage from longer polypeptides. The composition of the peptide can be confirmed by amino acid analysis or sequencing.

[0156] In some examples, the present invention includes peptide variants. Variants of peptides according to some examples of this disclosure may be (i) variants in which one or more amino acid residues are substituted with conserved or non-conserved amino acid residues (preferably conserved amino acid residues) and such substituted amino acid residues may or may not be residues encoded by the genetic code, (ii) variants in which one or more modified amino acid residues are present, such as variants of residues modified by attachment of substituents, (iii) variants in which the polypeptide is a variant of an alternative splice variant of a polypeptide of some examples of this disclosure, (iv) fragments of the polypeptide, and / or (v) variants in which the polypeptide is fused with another polypeptide, such as a leader sequence or secretion sequence or a sequence for purification (e.g., a His tag) or a sequence for detection (e.g., an Sv5 epitope tag). Fragments include polypeptides produced via proteolytic cleavage (including multisite proteolysis) of the original sequence. Variants may be post-translational or chemically modified. Such variants are considered to be within the scope of those skilled in the art in accordance with the teachings herein.

[0157] The dehydrated, dried and / or lyophilized peptide reagents of the present invention can 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, benzenesulfonic acid and toluenesulfonic acid).

[0158] In one example, at least one dehydrated, dried, and / or lyophilized peptide reagent is a conjugate containing an antigen-binding region that binds to a T-cell receptor (TCR). In some examples, the conjugate contains at least one antigen-binding region that binds to CD3, CD28, or a combination of CD3 and CD28. In some examples, the conjugate 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 IgG1 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 contains an IgG heavy chain. In some examples, the antibody contains an IgG1 heavy chain. In some examples, the antibody contains an IgG2 heavy chain. In some examples, the antibody contains an IgG4 heavy chain. In some examples, the antibody contains a κ light chain. In some examples, the antibody contains a κ light chain constant region. In some examples, the antibody contains a λ light chain. In some examples, the antibody contains a λ light chain constant region. In some examples, the antibody is an antibody fragment containing an antigen-binding 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 disulfide-linked sc(Fv)2. In some examples, the antibody is a Fab, Fab', or F(ab)2 antibody. In some examples, the antibody is a bispecific antibody. 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 a trivalent antibody. In some examples, the antibody is a tetravalent antibody.

[0159] In one example, the dehydrating, drying, and / or lyophilizing reagent contains anti-CD3 antibodies and / or anti-CD28 antibodies, including any and all clones thereof. In one example, the dehydrating, drying, and / or lyophilizing reagent contains a combination of anti-CD3 antibodies, anti-CD28 antibodies, and / or any and all clones thereof. In one example, the anti-CD3 antibody is OKT3-αCD3. In one example, the anti-CD28 antibody is 15e8-CD28. In some examples, the dehydrating, drying, and / or lyophilizing reagent contains a combination of anti-CD3 antibodies and anti-CD28 antibodies. In some examples, the dehydrating, drying, and / or lyophilizing reagent contains a combination of anti-CD3 antibodies and anti-CD28 antibodies, along with one or more additional reagents. In some examples, one or more additional reagents are IL-2 or other cytokines.

[0160] cell

[0161] In some examples, the container of the present invention is used to culture or amplify a specific cell type. In some examples, the cells cultured in the device can be any suitable cell type. In some examples, the cultured cells are used in a method of introducing cells into a recipient. In some examples, the cells are autologous, allogeneic, syngeneic, or allogeneic relative to the recipient. In some examples, the cells are derived from stem cells or precursor cells. In some examples, the stem cells or precursor cells from which the modified cells are derived are autologous, allogeneic, syngeneic, or allogeneic relative to the recipient.

[0162] In one example, the cell is an immune cell. Exemplary immune cells that can be isolated, stimulated, and / or expanded or cultured as described herein include, but are not limited to, T cells (including cytotoxic T cells, helper T cells, regulatory T cells, TCR-carrying T cells, allogeneic responsive T cells, allogeneic specific T cells, T cells carrying allogeneic reactive TCRs, and γδ 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, etc.

[0163] The disclosed compositions and methods are applicable to the isolation and / or stimulation of T cells for use in basic research and treatment 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 target cancer cells, treat autoimmune diseases, and / or treat fibrosis.

[0164] In some examples, the T cell source is obtained from the subject prior to expansion. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In some examples of this disclosure, many T cell lines available in the art can be used. In some examples of this disclosure, T cells can be obtained using many techniques known to those skilled in the art (such as Ficoll). ™ Cells are obtained from units of blood collected from a subject. In one example, cells from an individual's circulating blood are obtained via apheresis. Apheresis products typically contain lymphocytes (including T cells), monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one example, cells collected via apheresis may be washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing steps.

[0165] In another example, by lysing red blood cells and clearing monocytes, for example by PERCOLL ™ T cells are isolated from peripheral blood lymphocytes by gradient centrifugation or countercurrent centrifugation. Specific T cell subsets, such as CD3, can be further isolated using positive or negative selection techniques. + CD28 + CD4 + CD8 + CD45RA + and CD45RO + T cells.

[0166] In one example, T cells are isolated by culturing them for a sufficient period of time to positively select the desired T cells. In one example, this period is approximately 30 minutes. In another example, the period ranges from 30 minutes to 36 hours or longer, and all integer values ​​in between. In yet another example, the period is at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours. In yet another example, the period is from 10 hours to 24 hours. In one example, the incubation period is 24 hours. Compared to other cell types, longer incubation times can be used to isolate T cells in any situation where a small number of T cells are present, such as isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals. Furthermore, using longer incubation times can increase the capture efficiency of CD8+ T cells. Therefore, by simply shortening or lengthening the time that allows T cells to bind to CD3 / CD28 antibodies, T cell subsets can be preferentially selected or eliminated at the start of culture or at other points in the process. Additionally, by increasing or decreasing the ratio of anti-CD3 antibodies and / or anti-CD28 antibodies on the surface, T cell subsets can be preferentially selected or eliminated at the start of culture or at other desired time points. Those skilled in the art will recognize that multi-round selection can also be used in the context of this invention. In some examples, it may be desirable to perform a selection procedure and use “unselected” cells during activation and expansion. “Unselected” cells may also undergo further rounds of selection.

[0167] Enriching a T cell population through negative selection can be accomplished using a combination of antibodies targeting surface markers specific to the negatively selected cells. One approach is selection via negative immunoadhesion, which uses a mixture of monoclonal antibodies targeting cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells through negative selection, the monoclonal antibody mixture typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some examples, it may be desirable to enrich or positively select regulatory T cells that typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in some examples, regulatory T cells are eliminated by anti-C25 conjugated beads or other similar selection methods.

[0168] To separate a desired cell population through positive or negative selection, the concentrations of cells and the selection antibody can be varied. In some examples, it may be desirable to significantly reduce the volume of the selection antibody and cells mixed together (i.e., increase the cell concentration) to ensure maximum contact between the cells and the selection antibody. For example, in one example, a concentration of 2 billion cells / ml is used. In another example, a concentration of 1 billion cells / ml is used. In yet another example, a concentration greater than 100 million cells / ml is used. In yet another example, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells / ml are used. In yet another example, cell concentrations of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells / ml are used. In other examples, concentrations of 125 million or 150 million cells / ml can be used. Using high concentrations can produce increased cell yield, cell activation, and cell expansion. Furthermore, using high cell concentrations allows for more efficient capture of cells that weakly express target antigens of interest. Such cell populations may have therapeutic value and will be desirable. For example, using high cell concentrations allows for more efficient selection of CD8+ T cells that typically have weak CD28 expression.

[0169] In relevant examples, it might be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and the selection antibody, the interaction between the selection antibody and the cells is minimized. This selects cells that express high levels of the desired antigen that will bind to the surface-attached antibody. For example, CD4+ T cells express higher levels of CD28 and are captured more effectively than diluted concentrations of CD8+ T cells. In one example, the cell concentration used was 5 × 10⁻⁶. 6 Quantities / ml. In other examples, the concentration used may be approximately 1 × 10⁻⁶. 5 From 1×10⁻¹ / ml 6 The number of units per ml, and any integer values ​​in between.

[0170] In some examples, cells can be incubated for varying lengths of time at 2°C to 10°C or at room temperature. In some examples, cells can be incubated on a rotor at different speeds.

[0171] In the context of this invention, it is also contemplated to collect blood samples or apheresis products from subjects at a time prior to the potential need for cell amplification as described herein. Thus, the source of cells to be amplified can be collected at any point in time when necessary, and the desired cells (such as T cells) can be isolated and frozen for later use in T-cell therapy for many diseases or conditions that will benefit from T-cell therapy (such as those described herein). In one example, the blood sample or apheresis component is taken from a generally healthy subject. In some examples, the blood sample or apheresis component is taken from a generally healthy subject who is at risk of developing a disease but has not yet developed one, and the cells of interest are isolated and frozen for later use. In some examples, T cells can be amplified, frozen, and used later. In some examples, samples are collected from the patient shortly after a diagnosis of a specific disease as described herein, but before any treatment. In another example, prior to many related treatments, cells are isolated from a subject's blood sample or apheresis components, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressants (such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506), antibodies or other immunoablation agents (such as CAMPATH, anti-CD3 antibodies, cyclophosphamide, fludarabine, cyclosporine, FK506, rapamycin, mycophenolate mofetil, steroids, FR901228), and radiation. These drugs inhibit calcium-dependent phosphatase calmodulin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important for growth factor-induced signaling (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). In another example, cells are isolated from a patient and frozen for later use in conjunction with (e.g., before, during, or after) bone marrow or stem cell transplantation, using chemotherapeutic agents such as fludarabine in T-cell ablation therapy, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In yet another example, cells are isolated prior to treatment and can be frozen for later use after B-cell ablation therapy, such as agents that respond to CD20, such as rituximab (Rituxan).

[0172] In some examples of this disclosure, T cells are obtained directly from the patient after treatment. In this regard, it has been observed that the quality of T cells obtained shortly after certain cancer treatments, particularly those with drugs that impair the immune system, may be optimal or improved for their ex vivo expansion capacity during the patient's normal recovery period from treatment. Similarly, these cells can be in a preferred state for enhanced engraftment and in vivo expansion after ex vivo manipulation using the methods described herein. Therefore, in the context of some examples of this disclosure, the collection of blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery period is considered. Furthermore, in some examples, mobilization (e.g., mobilization with GM-CSF) and conditioning protocols may be used to generate conditions in the subject that favor the reproliferation, recycling, regeneration, and / or expansion of specific cell types, particularly during a defined time window after treatment. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0173] T cells can be activated and expanded. Typically, the T cells of this invention are expanded by contacting a surface with a ligand attached to an agent that stimulates a signal associated with the CD3 / TCR complex and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. Specifically, T cell populations can be stimulated as described herein, such as by contacting them in a container with an anti-CD3 antibody or its antigen-binding fragment, or an anti-CD28 antibody or its antigen-binding fragment, or a combination thereof. For co-stimulating helper molecules on the surface of the T cells, a ligand binding to the helper molecule is used. For example, a T cell population can be contacted with anti-CD3 and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation. Anti-CD3 and anti-CD28 antibodies are used to stimulate the proliferation of CD4+ T cells or CD8+ T cells. Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besançon, France), which can be used as well as other methods known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):1319-1328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0174] In some examples, T cells expand before genetic modification. In other examples, T cells expand after genetic modification.

[0175] In some examples, the primary stimulatory signal and co-stimulatory signal of T cells can be provided in different ways. For example, the agent providing each signal can be in solution or conjugated to a surface. When conjugated to a surface, the agent can be conjugated to the same surface (i.e., in the "cis" form) or to a separate surface (i.e., in the "trans" form). Alternatively, one agent can be conjugated to a surface while another agent is in solution. In one example, the agent providing the co-stimulatory signal binds to the cell surface, and the agent providing the primary activation signal is in solution or conjugated to a surface. In some examples, both agents can be in solution. In another example, the agent can be in a soluble form and then cross-linked to a surface, such as a cell expressing an Fc receptor or an antibody or other binding agent that will bind to the agent. In this regard, see, for example, artificial antigen-presenting cells (aAPCs) in U.S. Patent Application Publications 2004 / 0101519 and 2006 / 0034810, and consider using these cells for activation and expansion of T cells in some examples of this disclosure.

[0176] In one example, the two agents are dehydrated, dried, and / or lyophilized in the internal chamber of the container. By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or its antigen-binding fragment, and the agent providing the co-stimulatory signal is an anti-CD28 antibody or its antigen-binding fragment; and the two agents are co-dehydrated, co-dried, and / or co-lyophilized in the internal chamber of the container. In one example, for CD4+ T cell expansion and T cell growth, each antibody is used in a 1:1 ratio dehydrated, dried, and / or lyophilized in the internal chamber of the container. In some examples of some examples of this disclosure, a certain ratio of anti-CD3:CD28 antibodies dehydrated, dried, and / or lyophilized in the internal chamber of the container is used, such that an increase in T cell expansion is observed compared to expansion observed using a 1:1 ratio. In one particular example, an increase of approximately 1 to approximately 3 times is observed compared to expansion observed using a 1:1 ratio. In one example, the ratio of CD3:CD28 antibodies dehydrated, dried, and / or lyophilized in the inner chamber of the container ranges from 100:1 to 1:100 and all integer values ​​between therebetween. In one example of some examples of this disclosure, more anti-CD28 antibodies are dehydrated, dried, and / or lyophilized in the inner chamber of the container than anti-CD3 antibodies, i.e., the CD3:CD28 ratio is less than 1. In some examples of the invention, the ratio of anti-CD28 antibodies to anti-CD3 antibodies dehydrated, dried, and / or lyophilized in the inner chamber of the container is greater than 2:1. In one example, antibodies dehydrated, dried, and / or lyophilized in the inner chamber of the container at a CD3:CD28 ratio of 1:100 are used. In another example, antibodies dehydrated, dried, and / or lyophilized in the inner chamber of the container at a CD3:CD28 ratio of 1:75 are used. In yet another example, antibodies dehydrated, dried, and / or lyophilized in the inner chamber of the container at a CD3:CD28 ratio of 1:50 are used. In another example, antibodies are dehydrated, dried, and / or lyophilized in the inner chamber of the container using a CD3:CD28 ratio of 1:30. In a preferred example, antibodies are dehydrated, dried, and / or lyophilized in the inner chamber of the container using a CD3:CD28 ratio of 1:10. In another example, antibodies are dehydrated, dried, and / or lyophilized in the inner chamber of the container using a CD3:CD28 ratio of 1:3. In yet another example, antibodies are dehydrated, dried, and / or lyophilized in the inner chamber of the container using a CD3:CD28 ratio of 3:1.

[0177] Those skilled in the art will readily recognize that any cell concentration can be used. For example, the target cells may be very rare in a sample, comprising only 0.01% of the sample, or the entire sample (i.e., 100%) may contain the target cells of interest. Therefore, any cell number is possible within the context of some examples of this disclosure. For instance, in one example, a concentration of approximately 2 billion cells / ml is used. In another example, a concentration greater than 100 million cells / ml is used. In yet another example, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells / ml are used. In yet another example, cell concentrations of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells / ml are used. In other examples, concentrations of 125 million or 150 million cells / ml can be used. Using high concentrations can produce increased cell yield, cell activation, and cell expansion. Furthermore, using high cell concentrations allows for more efficient capture of cells that may weakly express the target antigen, such as CD28-negative T cells. In some cases, such cell populations may have therapeutic value and would be desirable. For example, using high cell concentrations allows for more efficient selection of CD8+ T cells that typically have weaker CD28 expression.

[0178] In some examples of this disclosure, cells may be cultured for several hours (about 3 hours) to about 14 days or any integer value of hours in between. In another example, cells may be cultured for 21 days. In one example of the invention, T cells are cultured for about 8 days. In another example, T cells are cultured for 2 to 3 days. Several stimulation cycles may also be required, so that the culture time of T cells may be 60 days or longer. Suitable conditions for T cell culture include appropriate culture media (e.g., minimum essential medium or RPMI 1640 or X-vivo 15, (Lonza)) that may contain factors essential for proliferation and viability, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ and TNF-α, or any other additives known to those skilled in the art for cell growth. Other additives for cell growth include, but are not limited to, surfactants, plasma protein powder, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. Culture media may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, ImmunoCult-XF and X-Vivo 20 from StemCell, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, serum-free or supplemented with adequate serum (or plasma) or a defined set of hormones, and / or a sufficient amount of cytokines for T cell growth and expansion. Antibiotics such as penicillin and streptomycin should be included only in the experimental culture, not in the cell culture to be infused to the subject. Target cells should be maintained under conditions necessary to support growth, such as a suitable temperature (e.g., 37°C) and atmosphere (e.g., air with 5% CO2).

[0179] T cells exposed to different stimulation times can exhibit different characteristics. For example, typical blood or isolated peripheral blood mononuclear cell products have a larger population of helper T cells (TH, CD4+) than cytotoxic or repressive T cell populations (TC, CD8+). T cell populations generated by stimulating CD3 and CD28 receptors in vitro can be predominantly composed of TH cells until approximately day 8-9, after which they increasingly include a larger population of TC cells. Therefore, depending on the therapeutic objective, infusing a subject with a population of T cells primarily composed of TH cells may be advantageous. Similarly, if an antigen-specific subset of TC cells has been isolated, expanding that subset to a greater extent may be beneficial.

[0180] Furthermore, apart from CD4 and CD8 markers, other phenotypic markers also differed significantly, but were largely reproducible during cell expansion. Therefore, this reproducibility allows for the customization of activated T cell products for specific purposes.

[0181] CAR T cells

[0182] 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 some examples disclosed herein, the CAR typically includes an antigen-binding domain, a transmembrane domain, and an intracellular domain. In some examples, the CAR includes an antigen-binding domain that binds to tumor-associated antigens or tumor-specific antigens.

[0183] In various examples, CAR can be any CAR molecule, including but not limited to “first-generation,” “second-generation,” “third-generation,” “fourth-generation,” or “fifth-generation” CARs (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 Mech. 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).

[0184] The "first-generation" CAR used in this invention comprises an antigen-binding domain fused to a transmembrane domain, such as a single-stranded variable fragment (scFv), which is fused to a cytoplasmic / intracellular domain of the T-cell receptor chain. The "first-generation" CAR typically has an intracellular domain derived from the CD3ζ chain, which is the primary signaling medium from the endogenous T-cell receptor (TCR). The "first-generation" CAR can provide de novo antigen recognition and induce activation of both CD4+ and CD8+ T cells via its CD3ζ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation.

[0185] The "second-generation" CAR used in this invention comprises an antigen-binding domain, such as a single-stranded variable fragment (scFv), fused to an intracellular signaling domain capable of activating T cells, and a co-stimulatory domain designed to enhance T cell potency and persistence (Sadelain et al., Cancer Discov. 3:388-398 (2013)). Thus, CAR design can combine antigen recognition with signal transduction, both physiologically performed by two separate complexes: the TCR heterodimer and the CD3 complex. The "second-generation" CAR includes intracellular domains of various co-stimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40, etc.) derived from the cytoplasmic tail of the CAR to provide additional signals to the cell.

[0186] "Second-generation" CARs provide co-stimulation (e.g., via CD28 or 4-1BB domains) and activation (e.g., via CD3ζ signaling domains). Preclinical studies have indicated that "second-generation" CARs can enhance the antitumor activity of T cells. For example, robust efficacy of "second-generation" CAR-modified T cells has been demonstrated in clinical trials targeting CD19 molecules in patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL) (Davila et al., Oncoimmunol. 1(9):1577-1583 (2012)).

[0187] The “third-generation” CAR provides multiple co-stimulation (e.g., by including CD28 and 4-1BB domains) and activation (e.g., by including the CD3ζ activation domain).

[0188] The “fourth generation” CAR provides co-stimulation (e.g., through the CD28 or 4-1BB domains) and activation (e.g., through the CD3ζ signaling domain and constitutive or inducible chemokine components).

[0189] The "fifth generation" CAR provides co-stimulation (e.g., via the CD28 or 4-1BB domain) and activation (e.g., via the CD3ζ signaling domain, constitutive or inducible chemokine components, and intracellular domains of cytokine receptors (e.g., IL-2Rβ).

[0190] In various examples, CARs can be contained in multivalent CAR systems, such as dual CAR or "tandem CAR" systems. Multivalent CAR systems include systems or cells containing multiple CARs and systems or cells containing bivalent / bispecific CARs that target more than one antigen.

[0191] In one example, the scFv portion of the CAR of the present invention is encoded by a transgene whose sequence has been codon-optimized for expression in mammalian cells. In another example, the entire CAR construct of the present invention is encoded by a transgene whose entire sequence has been codon-optimized for expression in mammalian cells. Codon optimization refers to the discovery that the frequency of synonymous codons (i.e., codons encoding the same amino acids) in encoding DNA is biased across different species. Such codon degeneracy allows the same polypeptide to be encoded by multiple nucleotide sequences.

[0192] In some examples, T cells modified to express recombinant TCRs or CARs contain antibodies or fragments thereof engineered to enhance 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 the following: 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 the heavy chain variable domain (VH), light chain variable domain (VL), and variable domain (VHH) of camel-derived nanobodies, and alternative scaffolds known in the art to serve as antigen-binding domains, such as recombinant fibronectin domains, etc. In some cases, it is advantageous for the target-specific binding domain to originate from the same species from which the CAR will ultimately be used. For example, for use in humans, it may be advantageous for the CAR's target-specific binding domain to contain human or humanized residues of the target-specific binding domain of an antibody or fragment thereof. Thus, in one example, the target-specific binding domain partially contains a humanized antibody or fragment thereof.

[0193] In some examples, the non-human antibody is humanized, where specific sequences or regions of the antibody are modified to increase its similarity to naturally occurring human antibodies or fragments thereof. In one example, the target-specific binding domain portion is humanized.

[0194] In some examples, T cells modified to express recombinant TCRs or CARs include an extracellular receptor, a transmembrane domain, and an intracellular signaling domain. In one example, the extracellular receptor is linked to the intracellular signaling domain. In another example, the extracellular receptor is linked to the intracellular signaling domain via the transmembrane domain.

[0195] In some examples, T cells modified to express recombinant TCRs or CARs contain recombinant DNA constructs that encode sequences for TCRs or CARs.

[0196] The nucleic acid sequence encoding the desired molecule can be obtained using recombination methods known in the art, such as by screening a library from cells expressing the gene, obtaining the gene from a vector known to contain the gene, or isolating it directly from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be synthesized rather than cloned.

[0197] The nucleic acid sequence encoding the desired molecule can be obtained using recombination methods known in the art, such as by screening a library from cells expressing the gene, obtaining the gene from a vector known to contain the gene, or isolating it directly from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be synthesized rather than cloned.

[0198] In some examples, to assess peptide expression, the expression vector to be introduced into the cell may also contain a selection marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells attempting to be transfected or infected by a viral vector. In other examples, the selection marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selection marker and the reporter gene may be side-linked with appropriate regulatory sequences to enable their expression in the host cell. Useful selection markers include, for example, antibiotic resistance genes, such as neo.

[0199] Reporter genes are used to identify potential transfected cells and evaluate the function of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed in the recipient organism or tissue, and encodes a polypeptide whose expression is manifested as an easily detectable property (e.g., enzyme activity). Reporter gene expression is measured at an appropriate time after DNA is introduced into the recipient cell. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well-known and can be prepared using known techniques or are commercially available. Generally, constructs exhibiting the highest level of reporter gene expression are identified as promoters. Such promoter regions can be linked to reporter genes and used to evaluate the ability of drugs to regulate promoter-driven transcription.

[0200] Methods for introducing and expressing genes into cells are known in the art. In the case of expression vectors, the vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0201] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0202] Biological approaches to introducing polynucleotides of interest into host cells 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 may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patents 5,350,674 and 5,585,362.

[0203] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery medium in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0204] In the case of using non-viral delivery systems, an exemplary delivery medium is liposomes. Consider using lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another example, nucleic acids can associate with lipids. Lipid-associated nucleic acids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linker molecules associated with both liposomes and oligonucleotides, embedded in liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in lipids as a suspension, contained in or complexed with micelles, or otherwise associated with lipids. Lipids, lipid / DNA, or lipid / expression vector-related compositions are not limited to any particular structure in solution. For example, they can exist in bilayer structures, such as micelles or “collapsed” structures. They can also simply be dispersed in solution, possibly forming aggregates of varying sizes or shapes. Lipids are fatty substances that can be naturally occurring or synthetic. For example, lipids include fat droplets that are naturally present in the cytoplasm, as well as compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0205] Suitable lipids are available from commercial sources. For example, dimyristicophosphatidylcholine (“DMPC”) is available from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) is available from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) is available from Calbiochem-Behring; dimyristicophosphatidylglycerol (“DMPG”) and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, AL). The lipids in stock in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. “Liposome” is a general term encompassing various monolayer and multilayer lipid mediators formed by the production of closed lipid bilayers or aggregates. Liposomes can be characterized as vesicle structures with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They spontaneously form when phospholipids are suspended in excess aqueous solution. Lipid components undergo self-rearrangement before the formation of a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions exhibiting structures in solution that differ from normal vesicle structures are also included. For example, lipids can present as micellar structures or simply as heterogeneous aggregates of lipid molecules. Lipid-transfected amine-nucleic acid complexes have also been considered.

[0206] Regardless of the method used to introduce exogenous nucleic acids into host cells, various assays can be performed to confirm the presence of recombinant DNA sequences in the host cells. Such assays include, for example, "molecular biology" assays known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; and "biochemical" assays, such as those used to detect the presence or absence of specific peptides by means of immunological methods (ELISA and Western blotting) or by assays described herein to identify agents falling within the scope of this invention.

[0207] RNA can be introduced into target cells using any of a variety of different methods, such as commercially available methods, including but not limited to electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg, Germany), cationic liposome-mediated transfection using lipid transfection, polymer encapsulation, peptide-mediated transfection, or biological ballistic particle delivery systems such as “gene guns” (see, for example, Nishikawa et al. Hum Gene Ther., 12(8):861-70 (2001).

[0208] Methods for preparing containers

[0209] Some examples of this disclosure relate in part to a method of producing a container containing at least one dehydrating, drying, and / or lyophilizing reagent for isolating and / or stimulating at least one cell of interest (e.g., T cells, CAR cells, CAR T cells, T cells expressing T cell receptors, etc.). In one example, one or more steps of the method of producing the container are performed in a biosafety cabinet (BSC) to maintain sterility.

[0210] In one example, the method includes the following ordered steps:

[0211] a) Deposit one or more reagents to be dehydrated, dried and / or lyophilized in a cell culture vessel;

[0212] b) Deposit an appropriate amount of buffer solution into the container to form a reagent mixture;

[0213] c) Ensure that ≥85% of the inner surface of the container is wetted by the reagent mixture to form a reagent coating;

[0214] d) Freeze the container until a completely uniform temperature is reached within the reagent coating;

[0215] e) Remove the container from the freezer and transfer it to a dehydrator, dryer, and / or lyophilizer to allow the reagent coating to be dehydrated, dried, and / or lyophilized.

[0216] In one example, one or more reagents to be dehydrated, dried, and / or lyophilized are deposited into the cell culture vessel at a concentration of approximately 1 mg / ml. In another example, one or more reagents to be dehydrated, dried, and / or lyophilized are deposited into the cell culture vessel in an amount that fills the surface area of ​​the bag but not its volume. For example, for a 30 ml bag, a total volume of 10 ml will coat the surface area of ​​the bag without filling it. The cell culture vessel can be filled and subsequently lyophilized while lying flat or upright. In some examples, excess air is neither introduced nor removed from the vessel during preparation. In some examples, a microfilter is added at the top port of the vessel to allow vapor to escape while preventing contamination during dehydration, drying, and / or lyophilization.

[0217] In one example, the cell culture container includes a fluid bag. In one example, the cell culture container includes an AC-grade fluid bag. In one example, the cell culture container includes an SG32 AC bag. In one example, using a syringe, appropriate volumes and concentrations of each reagent are deposited into the container using the integrated port on the container. In one example, 100 μL each of anti-CD3 antibody and anti-CD28 antibody are deposited into the container. In one embodiment, approximately 5 μg to 200 μg each of anti-CD3 antibody and anti-CD28 antibody are deposited into the container. In another example, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, or 200 μg of anti-CD3 antibody and anti-CD28 antibody are deposited into the container. In some embodiments, 20 μg of anti-CD3 antibody and 50 μg of anti-CD28 antibody are deposited into the container. In some embodiments, 10 μg of anti-CD3 antibody and 10 μg of anti-CD28 antibody are deposited into the container. In some embodiments, 20 μg of anti-CD3 antibody and 20 μg of anti-CD28 antibody are deposited into the container.

[0218] In one example, the buffer comprises water, PBS, trehalose, or a combination thereof. In another example, the buffer is injected into the container via an integrated port on the container.

[0219] In one example, the container is prepared in a manner that prevents air from being introduced into it, thereby eliminating the need to remove excess air. In another example, excess air, including air bubbles, is removed from the container. For example, a syringe, pump, or other device may be used to remove excess air from the container.

[0220] In some examples, at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or the entire inner surface area of ​​the container is wetted by the reagent mixture.

[0221] In one example, the prepared container is placed in a -20°C freezer. In another example, the container is kept in the freezer until a completely homogeneous temperature is reached within the freezing solution. In one example, a thermocouple is used to track the temperature. In yet another example, the container is placed in the freezer for at least approximately 12 hours to ensure the solution reaches a homogeneous temperature.

[0222] In one example, the frozen container is removed from the freezer and placed on dry ice to prevent thawing during freeze-drying preparation. In another example, a pre-installed Luerlock fluid port is removed and a 0.22 μm PES filter is installed. In yet another example, the container is transferred to a dehydrator, dryer, and / or freeze dryer on dry ice to prevent thawing.

[0223] In one example, the container is placed in a dehydrator, dryer, and / or freeze dryer for a sufficient amount of time to undergo dehydration, drying, or freeze drying. In one example, the moisture content of the container is tracked via the dehydration, drying, or freeze drying unit. In one example, the container remains in the dehydrator, dryer, and / or freeze dryer for at least approximately 72 hours to ensure complete removal of moisture.

[0224] In one example, the container holding the dehydrated, dried, and / or lyophilized reagent is removed from the dehydrator, dryer, and / or lyophilizer and transferred to the BSC. In another example, the PES filter is replaced with a new fluid path Luer lock port.

[0225] In one example, the prepared dehydrated, dried, and / or lyophilized reagent containers are stored at 2°C to 8°C until use. In one example, the prepared dehydrated, dried, and / or lyophilized reagent containers are stable at 2°C to 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 more than 1 year. In some examples, the prepared dehydrated, dried and / or lyophilized reagent containers, when removed from storage at 2°C to 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 more, are capable of being used to isolate and / or stimulate T cells.

[0226] How to use

[0227] Some examples of this disclosure relate in part to a method of stimulating at least one cell of interest (e.g., T cells, CAR cells, CAR T cells, T cells expressing T cell receptors, etc.). Some examples of this disclosure provide methods for improving the effectiveness of immunotherapies (e.g., adoptive T cell therapy, CAR-T cell therapy).

[0228] Some examples of this disclosure also provide methods for isolating T cells. In some examples, T cells can be isolated from a bulk sample by first applying a bulk sample to a container and then isolating or capturing CD3+ cells from the bulk sample. In one example, the method includes isolating a population of T cells from a blood sample obtained by apheresis. In some examples, the container can be used to isolate T cells, stimulate T cells, or isolate and stimulate T cells simultaneously. T cell isolation and stimulation can be performed alone or in combination.

[0229] Some examples of this disclosure also provide methods for preventing or treating various diseases or conditions. In some examples, the method includes administering a therapeutically effective amount of the cells described herein. In one example, the method includes administering a therapeutically effective amount of at least one stimulated T cell of the present invention.

[0230] In some examples, the disease or condition includes cancer, cancer-related disease or condition, GvHD, autoimmune disease or condition, organ transplantation-related disease or condition, tissue transplantation-related disease or condition, cell transplantation-related disease or condition, allogeneic transplantation-related disease or condition, intestinal transplantation-related disease or condition, reconstructive transplantation-related disease or condition, or any combination thereof.

[0231] Some examples of this disclosure provide methods for administering an effective amount of at least one stimulated T cell (e.g., T cell, CAR cell, CAR T cell, T cell expressing T cell receptor, etc.) to a subject.

[0232] In one example, the present invention provides a method for preventing or treating a disease or condition. Furthermore, some examples of this disclosure provide nucleic acid molecules and compositions and cells comprising nucleic acid molecules, and their use in medicaments or methods for preventing, reducing, and / or eliminating diseases or conditions. Some examples of this disclosure include cell therapy (CAR T-cell therapy).

[0233] In some examples, the present invention includes a type of cell therapy in which cells are genetically engineered to express chimeric antigen receptors (CARs), and CAR cells (e.g., CAR immune cells, CAR T cells, CAR B cells, etc.) are infused into recipients in need. Unlike antibody therapies, CAR-modified cells are capable of replicating in vivo, producing long-term persistence. In various examples, the cells are administered to patients and persist in the patients for at least four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three months, two years, three years, four years, or five years after administration.

[0234] In one example, CAR T-cell therapy can induce an active or passive immune response, or alternatively, can be attributed to a direct versus an indirect immune response. In one example, CAR T cells exhibit specific pro-inflammatory cytokine secretion and potent cytolytic activity in response to cells expressing antigens.

[0235] In the case of in vitro immunization, at least one of the following occurs in vitro before the cells are administered to mammals: i) cell expansion, ii) introduction of nucleic acid encoding CAR into the cells, or iii) cell cryopreservation.

[0236] In vitro procedures are well known in the art and are discussed more fully below. In short, cells are isolated from mammals (e.g., humans) and genetically modified (i.e., transduced or transfected in vitro) using a CAR-expressing vector disclosed herein. The CAR-modified cells can be administered to mammalian recipients to provide therapeutic benefits. The mammalian recipient can be human and the CAR-modified cells can be autologous relative to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or allogeneic relative to the recipient.

[0237] In one example, in vitro culture and expansion of cells includes: (1) collecting progenitor cells from mammals (e.g., from peripheral blood harvests or bone marrow explants); and (2) stimulating and expanding such cells in vitro in a container in a medium suitable for T cell expansion. In addition to cell growth factors, other factors such as flt3L, IL-1, IL-3, and c-kit ligands may also be used for cell culture and expansion. In another example, in vitro culture and expansion of cells includes: (1) collecting progenitor cells from mammals (e.g., from peripheral blood harvests or bone marrow explants), (2) stimulating and expanding such cells in vitro in a container in a medium suitable for T cell expansion, and (3) isolating activated T cells from the cell mixture in the container.

[0238] In some examples, the CAR-modified cells of the present invention are used to treat diseases, conditions and symptoms, including but not limited to autoimmune diseases or symptoms (e.g., lupus), inflammatory diseases or symptoms (e.g., allergies and asthma), diseases or symptoms caused by transplantation, allogeneic transplant rejection, immune rejection, chronic allogeneic rejection, implantation rejection, transplant rejection or any combination thereof, inflammation, inflammation caused by ischemia / reperfusion, infection, immune response to allogeneic grafts or any combination thereof.

[0239] Some examples of this disclosure provide methods for preventing disease or recurrence of symptoms, methods including administering at least one genetically engineered cell of the present invention to a subject in need. In one example, these methods include administering multiple effective amounts of at least one genetically engineered cell of the present invention to a subject in need. In one example, these methods include administering an effective amount of at least one genetically engineered cell of the present invention in combination with an effective amount of another therapy to a subject in need.

[0240] Some examples of the compositions and genetically engineered cells disclosed herein can be administered alone or as pharmaceutical compositions in combination with diluents and / or other components, such as IL-2 or other cytokines or cell populations.

[0241] In another example, some of the compositions of this disclosure are administered to the patient in conjunction with transplantation (e.g., bone marrow transplantation, organ transplantation, etc.) (e.g., before, during, or after transplantation). In some examples, after transplantation, the subject receives an infusion of expanded immune cells of some of the compositions of this disclosure. In other examples, the expanded cells are administered before or after surgery.

[0242] Subjects considering the administration of the compositions and pharmaceutical compositions of the present invention include, but are not limited to, humans and other primates and mammals, including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats and dogs.

[0243] The dosage of the above treatments to be administered to patients will vary depending on the exact characteristics of the condition being treated and the recipient of the treatment. Doses for human administration can be scaled according to accepted practices in the field. Strategies for T-cell dosing and timing have been discussed (Ertl et al., 2011, Cancer Res, 71:3175-81; Junghans, 2010, Journal of Translational Medicine, 8:55).

[0244] Some examples of pharmaceutical compositions disclosed herein may comprise a combination of the composition described herein 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, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In one example, some examples of compositions disclosed herein are formulated for intravenous administration.

[0245] Some of the pharmaceutical compositions disclosed herein can be administered in a manner suitable for treating (or preventing) a disease. Although the appropriate dosage can be determined through clinical trials, the amount and frequency of administration will be determined based on factors such as the patient's condition and the type and severity of the patient's disease.

[0246] When referring to an "immunely effective dose" or a "therapeutic dose," the precise amount of some example compositions of this disclosure to be administered can be determined by a physician taking into account individual differences in the patient's (subject's) age, weight, degree of infection or metastasis, and condition. Generally speaking, it can be said that a pharmaceutical composition containing the cells described herein can be administered in quantities of 10... 4 One to 10 9 Cells / kg body weight, in some cases 10 5 One to 10 6 Administered at doses of cells per kg body weight (including all integer values ​​within those ranges). The cell composition may also be administered multiple times at these doses. Cells can be administered using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a particular patient can be readily determined by a physician in the medical field by monitoring the patient's symptoms and adjusting treatment accordingly.

[0247] In various examples, it may be desirable to administer activated cells to the subject, followed by a re-drawing of blood (or apheresis) according to some examples of this disclosure, activation of the cells derived therefrom, and re-infusion of these activated and expanded T cells into the patient. This process can be performed multiple times every few weeks. In various examples, cells can be activated from blood draws ranging from 10 cc to 400 cc. In various examples, cells can be activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.

[0248] The subject compositions can be administered in any convenient manner, including via aerosol inhalation, injection, ingestion, infusion, implantation, or transplantation. The compositions described herein can be administered to a patient via arterial, subcutaneous, intradermal, intranodal, intramedullary, intramuscular, intravenous (iv) injection, or intraperitoneal administration. In one example, some example T-cell compositions of this disclosure are administered to a patient via intradermal or subcutaneous injection. In one example, some example cell compositions of this disclosure are administered via intravenous injection. Cell compositions can...

[0249] In various examples of some examples of this disclosure, cells activated and expanded using the apparatus and methods described herein are administered to a patient in conjunction with a number of related therapeutic modalities (e.g., before, during, or after the related therapeutic modalities). In some examples, the apparatus and methods of the present invention are used for the isolation, stimulation, and / or expansion of cells that can be used in treatment regimens in combination with chemotherapy, radiation, immunosuppressants, antibodies, immunoablation agents, steroids, cytokines, and surgery. In one example, cell compositions of some examples of this disclosure are administered to a patient in conjunction with bone marrow transplantation and cell ablation therapy (e.g., before, during, or after bone marrow transplantation and cell ablation therapy).

[0250] In one example, a subject may undergo leukapheresis, in which leukocytes are collected, enriched, or removed in vitro to select and / or isolate cells of interest (e.g., T cells). These cell isolates can be expanded using the methods of the present invention. Subjects in need may subsequently undergo additional treatment (e.g., transplantation, chemotherapy, administration of CAR T cells, etc.). In various examples, subjects receive an infusion of expanded CAR T cells, some of the examples of this disclosure. In one example, the expanded cells are administered before or after surgery.

[0251] Implementation Plan

[0252] The present invention provides the following non-limiting embodiments.

[0253] Implementation scheme 1 is a container comprising a vacuum-sealable internal chamber, wherein the inner surface of the internal chamber is coated with at least one dehydrating, drying and / or lyophilizing agent capable of separating and / or stimulating T cells.

[0254] Implementation scheme 2 is the container according to implementation scheme 1, wherein the container is a flexible bag.

[0255] Implementation scheme 3 is a container according to implementation scheme 1 or 2, wherein the at least one dehydrating, drying and / or lyophilizing reagent comprises an anti-CD3 antibody, an anti-CD28 antibody or a combination thereof.

[0256] Implementation scheme 4 is a container according to any one of implementation schemes 1 to 3, wherein one or more of the anti-CD3 antibody and the anti-CD28 antibody are mixed with a solution prior to dehydration, drying and / or lyophilization, wherein the solution contains a buffer selected from the group consisting of trehalose, PBS and water.

[0257] Embodiment 5 is a container according to any one of Embodiments 1 to 4, wherein the device contains at least one additional dehydrating, drying and / or lyophilizing reagent for stimulating or expanding a population of T cells.

[0258] Embodiment 6 is the container according to Embodiment 5, wherein the at least one additional dehydrating, drying and / or lyophilizing reagent comprises IL-2.

[0259] Implementation scheme 7 is a container according to any one of implementation schemes 1 to 6, wherein the container is free of air.

[0260] Embodiment 8 is a container according to any one of embodiments 1 to 7, wherein the container is a fluid bag including at least one fluid port.

[0261] Embodiment 9 is a container according to any one of Embodiments 1 to 8, wherein the container is a fluorinated ethylene propylene (FEP) fluid bag with high surface energy.

[0262] Embodiment 10 is a container according to any one of embodiments 1 to 9, wherein the volume of the solution added to the container is such that at least 85% of the inner surface area of ​​the container can be coated by the solution, but the internal volume is not filled.

[0263] Embodiment 11 is a container according to any one of embodiments 1 to 10, wherein the container does not contain polymer beads.

[0264] Implementation scheme 12 is a container according to any one of implementation schemes 1 to 11, wherein the container is stable when stored at 2°C to 8°C for at least 1 day.

[0265] Implementation scheme 13 is a container according to any one of implementation schemes 1 to 11, wherein the container is stable when stored at 2°C to 8°C for at least 6 months.

[0266] Embodiment 14 is a method for stimulating or expanding a population of T cells, wherein the method comprises applying a mixture containing a population of T cells and a culture medium for culturing T cells into a container according to any one of Embodiments 1 to 13.

[0267] Implementation scheme 15 is the method according to implementation scheme 14, wherein the T cell is a chimeric antigen receptor (CAR) T cell.

[0268] Implementation scheme 16 is a T cell population that is expanded by the method described in accordance with implementation scheme 14 or 15.

[0269] Implementation scheme 17 is a method for treating, preventing, reducing or eliminating a disease or condition in a subject in need, wherein the method includes administering a therapeutically effective amount of the T cell population according to implementation scheme 16.

[0270] Implementation scheme 18 is the method according to implementation scheme 17, wherein the disease or condition is cancer, cancer-related disease or condition, infection-related disease or condition, autoimmune disease or condition, fibrosis, organ transplant-related disease or condition, tissue transplant-related disease or condition, or cell transplant-related disease or condition, or any combination thereof.

[0271] Implementation scheme 19 is a method for improving the effectiveness of immunotherapy in subjects in need, wherein the method includes administering a therapeutically effective amount of the T cell population as described in implementation scheme 16.

[0272] Implementation scheme 20 is the method according to implementation scheme 17, wherein the subject suffers from at least one of the following groups: cancer, infection-related disease or condition, autoimmune disease or condition, fibrosis, organ transplant-related disease or condition, tissue transplant-related disease or condition, or cell transplant-related disease or condition, or any combination thereof.

[0273] Implementation scheme 21 is a method for producing a container according to any one of embodiments 1 to 13, the method comprising the steps of: a) obtaining a vacuum-sealable container comprising an internal chamber;

[0274] b) Deposit a mixture of at least one reagent and a buffer solution into the internal chamber of the container;

[0275] c) Coat at least 85% of the inner surface of the internal chamber of the container with the mixture to form a reagent coating;

[0276] d) Freeze the container until a completely uniform temperature is reached within the reagent coating; and

[0277] e) Dehydrate, dry and / or freeze-dry the reagent coating.

[0278] Implementation scheme 22 is the method according to implementation scheme 21, wherein the container includes a flexible bag.

[0279] Implementation scheme 23 is the method according to implementation scheme 21 or 22, wherein the container is an FEP fluid bag with high surface energy.

[0280] Implementation scheme 24 is a method according to any one of implementation schemes 21 to 23, wherein at least one reagent to be dehydrated, dried and / or freeze-dried into the internal chamber of the container comprises an anti-CD3 antibody, an anti-CD28 antibody or a combination thereof.

[0281] Implementation scheme 25 is a method according to any one of implementation schemes 21 to 24, wherein the buffer solution contains at least one lyophilization protectant.

[0282] Implementation scheme 26 is a method according to any one of implementation schemes 21 to 25, wherein the buffer comprises water, PBS, trehalose, surfactant, amino acid, polymer, sugar or any combination thereof.

[0283] Implementation scheme 27 is a method according to any one of implementation schemes 21 to 26, wherein at least one additional reagent for stimulating or expanding a population of T cells is applied to the container prior to dehydration, drying and / or lyophilization.

[0284] Implementation scheme 28 is the method according to implementation scheme 27, wherein at least one additional reagent is IL-2.

[0285] Implementation scheme 29 is a method according to any one of implementation schemes 21 to 28, wherein the reagent mixture is applied to the container without introducing air into the container.

[0286] Implementation scheme 30 is the method according to any one of implementation schemes 21 to 29, wherein the method further includes removing any excess air prior to dehydration, drying and / or freeze-drying.

[0287] Implementation scheme 31 is a method according to any one of implementation schemes 21 to 30, wherein the container is a fluid bag including at least one fluid port.

[0288] Implementation scheme 32 is a method according to any one of implementation schemes 21 to 31, wherein the method further includes adding a filter to the fluid port prior to dehydration, drying and / or freeze-drying to allow vapor to escape while preventing contamination during dehydration, drying and / or freeze-drying.

[0289] Embodiment 33 is the method according to any one of embodiments 21 to 32, wherein the volume of the reagent solution added to the container is such that at least 85% of the inner surface area of ​​the container can be coated by the solution, but the internal volume is not filled.

[0290] Implementation scheme 34 is the method according to any one of implementation schemes 21 to 33, wherein polymer beads are not introduced into the container.

[0291] Implementation scheme 35 is the method according to any one of implementation schemes 21 to 34, wherein the container is stable when stored at 2°C to 8°C for at least 1 day.

[0292] Implementation scheme 36 is the method according to any one of implementation schemes 21 to 35, wherein the container is stable when stored at 2°C to 8°C for at least 6 months.

[0293] Embodiment 37 is a method for isolating a population of T cells, wherein the method includes applying a mixture containing the population of T cells and a culture medium for culturing T cells into a container according to any one of Embodiments 1 to 13.

[0294] Experimental Examples

[0295] The invention is described in further detail with reference to the following experimental embodiments. Unless otherwise specified, these embodiments are provided for illustrative purposes only and are not intended to be limiting. Therefore, the invention should not in any way be construed as limited to the following embodiments, but should be construed as covering any and all variations that become apparent as a result of the teachings provided herein.

[0296] Without further description, it is believed that those skilled in the art can implement and utilize the embodiments of this disclosure and practice the claimed methods using the previously described and the following illustrative examples. Therefore, the following working examples specifically point to preferred embodiments of this disclosure and should not be construed as limiting the remainder of the invention in any way.

[0297] Example 1: Scheme for preparing culture bags containing lyophilized reagents

[0298] An exemplary culture bag containing lyophilized reagents is shown in Figure 1B and Figure 1C The culture bags prepared can be fabricated according to the following exemplary scheme:

[0299] Material :

[0300] 1. Fluid supply bag. Although this scheme uses 32AC, any "AC" specified bag should meet the requirements, but the volume will need to be scaled proportionally to the working volume of the bag specified by the manufacturer.

[0301] *Note: Saint Gobain AC Vue Life bags were used for preliminary experiments because they have been modified to promote increased protein and cell adhesion compared to standard FEP culture bags.

[0302] 2. Functional aCD3- liquid, 200 μg / mL. Available from multiple manufacturers.

[0303] 3. Functional aCD28- liquid, 100 μg / mL. Available from multiple manufacturers.

[0304] 4. Phosphate-buffered saline, cell culture grade.

[0305] 5. Biomolecular-level water

[0306] 6. Trehalose, 2% w / v solution, cell culture grade.

[0307] 7.0.22μm PES Luerlock Filter

[0308] equipment :

[0309] 1. Biosafety cabinet, Class 2

[0310] 2. Freezer, -20℃

[0311] 3. Freeze dryer; LabConco Freezone and Lyostar 3 are examples.

[0312] 4. Single-use disposable Luer lock syringe

[0313] method :

[0314] 1. Place the fluid supply bag, antibody, buffer solution, and syringe in a biosafety cabinet (BSC) to maintain sterility.

[0315] 2. Use the integrated port on the bag to deposit both aCD3 and aCD28 into the fluid supply bag.

[0316] a.*Note: For SG32 AC bags, assuming no antibody loss during lyophilization, depositing 100 μL of antibody each will yield 0.22 μg antibody / cm³ for aCD3 and aCD28, respectively. 2 and 0.11 μg antibody / cm 2 .

[0317] 3. Then deposit an appropriate volume of buffer (water / PBS / trehalose) into the fluid supply bag via the port (e.g., 10 mL for an SG32 AC bag).

[0318] 4. If necessary, remove any excess air (including air bubbles) from the bag so that at least 85% of the bag’s surface is wetted with the antibody mixture.

[0319] 5. Immediately place the prepared bag into a -20°C freezer.

[0320] 6. Freeze the bag until the temperature in the freezing solution is completely uniform.

[0321] a.*Note: Use a thermocouple to track the temperature, or alternatively freeze for at least about 12 hours to ensure the solution reaches the homogeneous point.

[0322] 7. Remove the bag from the freezer and place it on dry ice to prevent thawing.

[0323] 8. Place the bag into the BSC, remove one pre-installed Luer lock fluid port, and install a 0.22μm PES filter.

[0324] 9. Transfer the bag to a freeze dryer on dry ice to prevent thawing.

[0325] 10. Place the bag in the vacuum chamber and allow freeze-drying to occur.

[0326] a.*Note: The moisture content of the bag is tracked via the freeze-drying unit, or alternatively, the bag is left in a vacuum chamber for at least approximately 72 hours to ensure complete removal of moisture.

[0327] 11. Remove the bag from the vacuum chamber and transfer it to the BSC. Dry ice is not needed at this point.

[0328] 12. Replace the PES filter with a new fluid path Luer lock port.

[0329] 13. Store the bag in a refrigerator at 2°C to 8°C until use.

[0330] Example 2: Device

[0331] Successful stimulation of primary T cells occurs through a co-stimulatory response via CD3 and CD28 receptors. T cell stimulation (also known as activation) is required for gene insertion (also known as transduction) events and subsequent cell expansion, allowing for the attainment of appropriate dose levels. This is typically achieved by targeting anti-CD3 and anti-CD28 antibodies onto a polymer matrix or beads. Many commercial reagents are available for this purpose. However, these reagents can be expensive and require complex manipulation by highly skilled individuals. This paper describes a series of prototype devices that eliminate many of these complex steps and reduce costs.

[0332] This device relies on the deposition of CD3 antibody, CD28 antibody, or a combination of CD3 and CD28 antibodies onto the surface of a cell culture vessel via lyophilization. Typical stimulatory events primarily depend on the use of Miltenyi's TransAct. ™ A reagent is used to stimulate T cells. This method requires the operator to deposit both T cells and cell culture medium into a culture bag. The operator then calculates the TransAct added to the culture bag. ™ The volume. In contrast, the container prepared in this disclosure ( Figure 1B and Figure 1CThis device already possesses functional antibodies deposited internally, enabling a "grab and run" strategy where the only requirements are culture medium and cells. No further computation or reagent addition is needed. Therefore, this device can provide an alternative approach to stimulating CD3, CD28, or a combination of CD3 and CD28 domains, requiring less manipulation, and can be performed in a functionally closed manner, while maintaining significantly lower costs, longer shelf life at room temperature, potentially allowing for direct assembly onto single-use tube sets, and potentially improving the quality of drug products.

[0333] Example 3: Development of Culture Bags

[0334] Preliminary experimental results using the prepared apparatus show that TransAct ™ The apparatus used for preparation showed a very considerable product multiplication during the duration of the study. Figure 3 ). Figure 3 The CAR-T method (TransAct) is described. ™ This study presents exemplary experimental results for a generic drug product using a method and a culture bag containing lyophilized reagents for T-cell activation. Product doubling is indicated by the number of cell doublings observed during the process based on initial seeding parameters. Product doubling is quantified at the initial time of the manufacturing device (initial) and at least six months (6+ months) after the manufacturing device. TransAct ™ The culture bags (devices) containing lyophilized reagents showed a very comparable product multiplication rate over the duration of this study.

[0335] Figure 4 Exemplary experimental results from a study of a generic drug product using the CAR-T method are depicted. Transduction efficiency indicates the number of T cells expressing the exogenous CAR construct relative to the entire population. Transduction efficiency was quantified at the initial time of device fabrication (initial) and at least six months (6+ months) after device fabrication. The CAR% from the population prepared in culture bags (devices) containing lyophilized reagents was compared with that of conventional TransAct. ™ Significantly increased. These data demonstrate that the CAR% of the cell population cultured in the prepared device is significantly higher than that of the conventional TransAct. ™ Significantly increased ( Figure 4 ).

[0336] Figure 5Exemplary experimental results illustrating the viability of cell populations at harvest are depicted. To determine the stability of stored culture bags over time, two independent experiments were conducted using culture bags prepared on the day of their use (initial) and culture bags used at least six months (6+ months) after their preparation and storage. Viability was quantified at the initial time of device manufacturing (initial) and at least six months (6+ months) after device manufacturing. The prepared culture bags (devices) containing lyophilized reagents exhibited higher viability at harvest than conventional TransAct. ™ Higher vitality indicates TransAct ™ The polymer matrix contained within may lead to harmful interactions with T cells over time. These data demonstrate that, compared to traditional TransAct... ™ Compared to that, they are more vigorous at harvest time. Figure 5 Lyophilized reagents offer advantages over other conventional methods because lyophilization is a common technique used across multiple industries, including chemical, food, and pharmaceutical, to increase shelf life / stability.

[0337] Preliminary experiments using the prepared device also showed equivalent results for various formulations and different CAR constructs. Specifically, different experimental buffers (such as PBS, water, and trehalose) and different CAR constructs (Figures 6A to 6B) were used. Figure 6D ), vitality (Figure 6A), cell growth ( Figure 6B ), transduction efficiency (Figure 6C) and total viable CAR+ cells ( Figure 6D The results are comparable. For CAR construct 1, PBS has the highest CAR%, at 40%. For CAR construct 2, TransAct... ™ The CAR% values ​​for PBS and trehalose were all within very close range. For CAR construct 3, TransAct... ™ PBS and water all have very similar CAR percentages. In all CAR constructs, all vector solutions are generated to be compatible with TransAct. ™ The device exhibits comparable performance in stimulating T cells and promoting transduction, resulting in substantial transgene expression. Overall, this culture bag demonstrates comparable efficacy to TransAct in inducing transduction. ™ Similar results are effective. These results demonstrate that the product cells are compatible with CAR T therapy and that the device is an effective solution for conventional methods.

[0338] This device allows for the simultaneous selection and activation of T cells. Figure 7For T cell activation, monoclonal antibodies against CD3 (primary signaling) and CD28 (secondary signaling) were generated. The antibodies against CD3 and CD28 bind to CD3 and CD28, respectively, thereby replicating primary and secondary signaling. The concentration of antibodies added to the device has a dose-dependent effect on viability and cell growth. Figure 8 , Figure 9 and Figures 10A to 10C ). Figure 8 A representative schematic diagram illustrating the general methodological steps for using this culture bag is depicted. A basal bag represents a culture bag containing 10 μg (1×) of anti-CD3 antibody and anti-CD28 antibody per bag. A high-density bag represents a culture bag containing 20 μg (2×) of anti-CD3 antibody and anti-CD28 antibody per bag. Figure 9 Exemplary experimental results are described, demonstrating that the culture bag effectively enriches CD3+ T cells from apheresis blood components. Figures 10A to 10C Exemplary experimental results are depicted, showing the total viability of cells using basal bag (1×) and high bag (2×) formulations. Figure 10A Product doubling ( Figure 10B ) and cell viability ( Figure 10C These results demonstrate that cells can be compared with standard methods (Transactin). ™ They grow at similar rates.

[0339] Preliminary experiments were also conducted to monitor low-density lipoprotein receptor (LDLr) expression. The amount of LDLr on the surface increased when T cells were activated. Therefore, this metric was used to quantify the number of activated or inactivated T cells by measuring LDLr before and 2 days after adding cells to the culture bag. Figure 11 These results showed that LDLr started at 16% for all cells on day 0 and increased to over 95% for all experimental buffers. These results demonstrate that all cells were activated. Finally, preliminary experimental results were used to monitor the cellular composition of bulk apheresis blood components. Figure 12A and Figure 12B These results demonstrate that the CD3 content of the bulk apheresis blood components contained in the culture dish continuously decreased over a 45-minute period, indicating that the culture dish was separating CD3+ cells from the apheresis blood components. Figure 12A ) Quantitative LDLr expression after selection and activation ( Figure 12B These results demonstrate that the culture bag can capture CD3+ cells from bulk apheresis blood components.

[0340] These data demonstrate that this device is a potential alternative to traditional activation methods. The device can be integrated into automated systems and compatible with technologies such as Dynabeads.™ and TransAct ™ It is cheaper and easier to use compared to traditional methods.

[0341] Every patent, patent application, and patent publication cited herein is incorporated herein by reference in its entirety. While the invention has been disclosed with reference to specific examples, it will be apparent to those skilled in the art that other examples and variations of the invention can be devised without departing from its true spirit and scope. The appended claims are intended to be construed as encompassing all such examples and equivalent variations.

Claims

1. A bag comprising a vacuum sealable interior chamber, wherein the interior chamber's interior surface is coated with at least one dehydrated, dried, and / or lyophilized reagent capable of isolating and / or stimulating T cells.

2. The bag of claim 1, wherein the at least one dehydrated, dried, 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 is mixed with a solution prior to dehydrating, drying, and / or lyophilizing, 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, dried, and / or lyophilized reagent for isolating, stimulating, and / or expanding a population of T cells.

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

6. The bag of claim 1, wherein the bag is a fluorinated ethylene propylene (FEP) fluid bag having a 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 filled.

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

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

10. A population of T cells 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 the population of T cells of claim 10, wherein the disease or disorder is a 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 mixture of at least one reagent and a buffer into an interior chamber of a vacuum sealable bag; b) coating at least 85% of an interior surface of the interior chamber of the bag with the mixture to form a reagent coating; c) freezing the bag until a fully uniform temperature is achieved within the reagent coating; and d) dehydrating, drying, and / or lyophilizing the reagent coating.

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

14. The method of claim 12, wherein the at least one reagent to be dehydrated, dried, 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 isolating, stimulating, and / or expanding a population of T cells is applied to the bag prior to dehydration, drying, and / or lyophilization.

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

19. The method of claim 12, wherein the reagent mixture is applied to the bag without air being introduced into the bag when the bag is free of air.

20. The method of claim 12, wherein the method further comprises adding a filter to a fluid port to allow vapor to escape while preventing contamination during dehydration, drying, and / or lyophilization prior to dehydration, drying, 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 internal surface area of the bag is capable of being coated by the solution, but the internal volume is not filled.

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

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