Non-viral delivery of cell therapy constructs

Abstract

The present disclosure provides a transposon-based system for introducing cell therapy products, such as CAR and TCR, into target immune cells. The transposon-based system can carry a larger payload than conventional viral vector-based technology, thereby simplifying multi-gene editing and reducing undesired recombination between homologous sequences in the payload. It also provides a shortened autologous process that can be completed within a few days, a day, or even within a few hours. Even without the activation, enrichment, or proliferation of immune cells, the resulting cell population achieves much higher in vivo therapeutic efficacy than the much longer autologous process using viral vectors.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 346,547, filed May 27, 2022, and U.S. Provisional Patent Application No. 63 / 492,110, filed March 24, 2023, both of which are incorporated by reference in their entireties herein. [Background technology]

[0002] Cell therapy uses enriched or modified human immune cells to target and kill a patient's cancer cells. To increase the ability of immune cells to target and kill specific cancer cells, methods have been developed to engineer immune cells to express constructs that direct the immune cells to specific target cancer cells. Chimeric antigen receptors (CARs) and engineered T cell receptors (TCRs) that contain binding domains that can interact with specific tumor antigens allow immune cells to target and kill cancer cells that express specific tumor antigens.

[0003] The main challenge in preparing CAR or TCR cells is the transfer of coding sequences into target immune cells. Currently, most CAR T cell production relies on transfer by viral vectors. Retroviral genes combined with inducible promoters can increase the transduction rate and generate a relatively large number of CAR-containing T cells.

[0004] Murine leukemia virus (MLV) based vectors are the most commonly used gamma retroviral vectors. However, the use of MLV has been associated with T cell-associated leukemia. Vectors derived from another retroviral family, lentiviruses, have shown better integration efficiency. Lentiviral vectors can target non-dividing cells, which has been a major challenge for MLV-based vectors.

[0005] Viral vectors are commonly used in CAR T cell production, but significant challenges remain. For example, viral vectors are inherently associated with carcinogenic and mutagenic potential. Furthermore, strict regulations are imposed on the use of viruses in current good manufacturing practice (cGMP) laboratories. Also importantly, lentiviral / retroviral transduction is limited by the size of the viral capsid, thus placing a limit on the size of the transgene. Furthermore, the size of the transgene may be limited by the effect of the expression of the gene and / or genes of interest on the physiology of the viral vector itself, including potential genotoxicity to the virus caused by the transgene.

[0006] Yet another important challenge is associated with the highly complex autologous cell engineering and production process, which typically takes at least one week and can take several weeks. During this process, lymphocytes taken from the patient must be transported to a processing center, while the produced cells must be cryopreserved and then transported to the patient for transplantation. This highly complex process inevitably results in high costs and limits clinical applications. Summary of the Invention

[0007] The present disclosure provides, in various embodiments, a transposon-based system for introducing cell therapy products, such as CARs and TCRs, into target immune cells. The transposon-based system can carry larger payloads than conventional viral vector-based technologies and can reduce undesired recombination between homologous sequences in the payload. In certain aspects, the transposon-based system simplifies multi-gene editing to one-step transfection. Also provided is a shortened autologous process that can be completed within a few days or even the same day. In certain aspects, the shortened autologous process can be completed within 4 hours. Even without immune cell activation, enrichment, or proliferation, the resulting cell population achieves much higher in vivo therapeutic efficacy than the much longer autologous process using viral vectors.

[0008] This shortened process can be easily integrated into a sealed automated device and can be performed at a preferred location other than a centralized manufacturing site (e.g., a clinical site), which was not practical for conventional autologous techniques. The two-way shipping required by conventional techniques can be omitted. And, importantly, the freeze / thaw step required for transportation can also be eliminated, further improving both patient care and product quality.

[0009] According to one embodiment of the present disclosure, a transposon is provided that includes a transgene encoding a polypeptide comprising a first chimeric antigen receptor (CAR) and a second CAR, each of the first CAR and the second CAR comprising a single chain fragment (scFv), a transmembrane domain, and an immunoreceptor tyrosine-based activation motif (ITAM).

[0010] In some embodiments, the transposon is a DNA transposon selected from the group consisting of Sleeping Beauty transposon, piggyBac transposon, and Tc Buster transposon, or a retrotransposon such as the R2 transposon. In some embodiments, the transposon is a Sleeping Beauty transposon.

[0011] In some embodiments, the transgene is at least 5000 nucleotides in length. In some embodiments, the transgene is at least 6000, 7000, 8000, 9000, or 10,000 nucleotides in length. In some embodiments, the transgene is more than 10 kb in length.

[0012] In some embodiments, the coding sequences for each ITAM in the transgene are codon-optimized to have no more than 12 consecutive nucleotides of sequence identity to each other, hi some embodiments, the coding sequences for each ITAM in the transgene are codon-optimized to have no more than 9 consecutive nucleotides of sequence identity to each other.

[0013] In some embodiments, the ITAM is a cytoplasmic signaling sequence of a protein selected from the group consisting of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the ITAM is CD3 zeta.

[0014] In another embodiment, a cell is provided that comprises a transposon of the present disclosure. In some embodiments, the cell is a T cell, a NK cell, a NKT cell, a monocyte, a macrophage, a PBMC, or a precursor cell thereof (e.g., an iPSC).

[0015] In some embodiments, the cell further comprises a transposase. In some embodiments, the transposase is selected from the group consisting of piggyBac® transposase, piggy-Bac®-like transposase, Super piggyBac® (SPB) transposase, piggyBat transposase, Sleeping Beauty transposase, hyperactive Sleeping Beauty (SB100X) transposase, Helitron transposase, Tol2 transposase, TcBuster transposase, or hyperactive TcBuster transposase. In some embodiments, the transposase is Sleeping Beauty transposase SB100X.

[0016] Another embodiment provides a method for preparing transfected lymphocytes, the method comprising obtaining a sample comprising T cells from a donor subject, incubating the sample with a transposon to transfect the T cells to produce transfected T cells, and culturing the sample comprising the transfected T cells for less than 96 hours, and then harvesting the T cells to produce a harvested sample, wherein at least 40% of the T cells in the harvested sample are naive T cells. In some embodiments, at least 50% of the T cells in the harvested sample are naive T cells.

[0017] In some embodiments, naive T cells are characterized as CD45RA+ and CCR7+. In some embodiments, naive T cells are characterized as CD62L + , CD27 + and CD28 + It can be further characterized as:

[0018] In some embodiments, the T cells are not activated prior to transfection. In some embodiments, the T cells are not enriched prior to transfection.

[0019] In some embodiments, the transgene is at least 5000 nucleotides in length. In some embodiments, the transgene is at least 6000 nucleotides in length. In some embodiments, the transgene is greater than 10 kb in length. In some embodiments, the transposon comprises a transgene encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0020] In another embodiment, a method for preparing lymphocytes expressing a chimeric antigen receptor (CAR) or a T cell receptor (TCR) is provided, the method comprising obtaining a biological sample comprising lymphocytes from a human subject, introducing into the lymphocytes a transposon comprising a transposase and a transgene encoding a polypeptide comprising a CAR or a TCR, and harvesting the lymphocytes comprising the transposase and the transposon, wherein harvesting of the lymphocytes occurs within 96 hours of obtaining the biological sample.

[0021] In some embodiments, the transposase and transposon are introduced into the lymphocyte by physical delivery methods such as electroporation, nucleofection, lipofection, ultrasound, or magnetofection. In some embodiments, the transposase and transposon are introduced into the lymphocyte by electroporation. The transposase can be delivered to the cell as DNA, mRNA, or protein.

[0022] In some embodiments, lymphocyte harvesting occurs within 48 hours after obtaining the biological sample. In some embodiments, the method further comprises cryopreserving the harvested lymphocytes or injecting the harvested lymphocytes into the patient, wherein the cryopreservation or injection occurs within 48 hours after obtaining the biological sample.

[0023] In some embodiments, the method does not include lymphocyte activation. In some embodiments, the method does not include lymphocyte enrichment. In some embodiments, the method does not include lymphocyte proliferation. In some embodiments, lymphocyte harvesting is performed within 36 hours after obtaining the biological sample.

[0024] In some embodiments, the lymphocytes are NK cells. In some embodiments, the lymphocytes are NKT cells. In some embodiments, the lymphocytes are T cells. In some embodiments, at least 40% of the T cells having a transgene integrated into their genome are naive T cells when harvested. In some embodiments, the naive T cells are CD45RA + and CCR7 + It is.

[0025] In some embodiments, the polypeptide further comprises a second CAR, the first CAR and the second CAR each comprising a single chain fragment (scFv), a transmembrane domain, and an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the polypeptide comprises a total of three or four CARs, each comprising an scFv, a transmembrane domain, and an ITAM. In some embodiments, the transposon comprises two or more transgenes that collectively encode two, three, four, or more CARs.

[0026] In some embodiments, the transposon is a DNA transposon selected from the group consisting of Sleeping Beauty transposon, piggyBac transposon, and TcBuster transposon, or a retrotransposon. In some embodiments, the transposon is a Sleeping Beauty transposon and the transposase is a Sleeping Beauty transposase. In some embodiments, the transgene is at least 5000 nucleotides in length. In some embodiments, the transgene is more than 10 kb in length.

[0027] In some embodiments, the sample subjected to transfection contains at least 25×10 6 In some embodiments, the sample subjected to transfection comprises at least 50×10 cells. 6 In some embodiments, the transfection is performed in a solution having a volume between 0.5 mL and 2 mL. In some embodiments, the transfection transfects at least 40% of the T cells or lymphocytes in the sample. [Brief description of the drawings]

[0028] [Figure 1] Schematic diagram showing three different plasmid constructs: Plasmid 1 (7725 bp with a 4835 bp insert), Plasmid 2 (8203 bp with a 5313 bp insert), and Plasmid 3 (8132 bp with a 5242 bp insert).

[0029] [Diagram 2] Schematic diagram showing two different large payload plasmid constructs: Plasmid 4 (10929 bp with an 8038 bp insert) and Plasmid 5 (12754 bp with a 9863 bp insert).

[0030] [Figure 3A] A plot of CAR expression 8 days after electroporation with Plasmid 4 is shown. [Figure 3B] A plot of CAR expression 8 days after electroporation with Plasmid 4 is shown. [Figure 3C] A plot of CAR expression 8 days after electroporation with Plasmid 4 is shown.

[0031] [Figure 4A] A plot of CAR expression 8 days after electroporation with Plasmid 5 is shown. [Figure 4B]A plot of CAR expression 8 days after electroporation with Plasmid 5 is shown. [Figure 4C] 1 shows a plot of CAR expression 8 days after electroporation with Plasmid 5.

[0032] definition In order that this disclosure may be more readily understood, certain terms are first defined below. Additional definitions for the following terms, as well as other terms, are found throughout the specification.

[0033] As used herein, unless otherwise stated or clear from the context, the term "or" is understood to be inclusive and includes both "or" and "and."

[0034] The term "and / or" as used herein should be interpreted as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include A and B, A or B, A (single), and B (single). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A (single), B (single), and C (single).

[0035] Unless specifically stated or clear from the context, the term "about" refers to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "consisting essentially of" can mean within one or more standard deviations as per the practice of the art. "About" or "consisting essentially of" can mean a range of up to 10% (i.e., ±10%). Thus, "about" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated value. For example, about 5 mg can include any amount between 4.5 mg and 5.5 mg. Additionally, particularly with respect to biological systems or processes, the term can mean a value of up to an order of magnitude or up to 5 times. When a particular value or composition is presented in this disclosure, unless otherwise specified, the meaning of "about" or "consisting essentially of" should be assumed to be within an acceptable error range for that particular value or composition.

[0036] "Administering" refers to the physical introduction of an agent, such as an engineered T cell disclosed herein, into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example, by injection or infusion. The phrase "parenteral administration" refers to a mode of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, for example, orally. Other non-parenteral routes include topical, epidermal, or mucosal routes of administration, for example, intranasal, intravaginal, rectal, sublingual, or topical. Administration can also be, for example, once, multiple times, and / or over one or more extended periods of time.

[0037] The term "allogeneic" refers to any material derived from one individual and then introduced into another individual of the same species, e.g., allogeneic T cell transplantation.

[0038] The term "antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin that specifically binds to an antigen. Generally, an antibody may include at least two heavy (H) chains and two light (L) chains, or antigen-binding molecules thereof, interconnected by disulfide bonds. Each H chain includes a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region includes three constant domains, CH1, CH2, and CH3. Each light chain includes a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region includes one constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), which are embedded in more conserved regions, called framework regions (FRs). Each VH and VL contains three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the Abs can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Generally, human antibodies are tetrameric agents of about 150 kD, composed of two identical heavy (H) chain polypeptides (about 50 kD each) and two identical light (L) chain polypeptides (about 25 kD each) that associate with each other in what is commonly referred to as a "Y-shaped" structure. The heavy and light chains are linked or connected to each other by a single disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions to each other, resulting in the dimers being connected to each other to form a tetramer. Naturally produced antibodies are also glycosylated, for example on their CH2 domains.

[0039] An "antigen-binding molecule," "antigen-binding portion," "antigen-binding fragment," or "antibody fragment" refers to any molecule that contains an antigen-binding portion (e.g., CDR) of the antibody from which the molecule is derived. An antigen-binding molecule may contain an antigen complementarity determining region (CDR). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAbs, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. Peptibodies (i.e., Fc fusion molecules containing a peptide-binding domain) are another example of a suitable antigen-binding molecule. In some embodiments, the antigen-binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen-binding molecule binds to an antigen on a cell involved in a hyperproliferative disease or to a viral or bacterial antigen. In certain embodiments, the antigen-binding molecule is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR).

[0040] The terms "variable region" or "variable domain" are used interchangeably. A variable region typically refers to a portion of an antibody, generally a light or heavy chain, typically about the amino-terminal 110-120 amino acids of the mature heavy chain and about 90-115 amino acids of the mature light chain, which vary widely in sequence between antibodies and are used in the binding and specificity of a particular antibody to its particular antigen. The sequence variability is concentrated in regions called complementarity determining regions (CDRs), while the more highly conserved regions within the variable domain are called framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (eg, non-human primate) framework regions (FR).

[0041] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody or antigen-binding molecule thereof.

[0042] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody or antigen-binding molecule thereof.

[0043] Many definitions of CDRs are in common use: Kabat numbering, Chothia numbering, AbM numbering, or contact numbering. The AbM definition is a compromise between the two used by Oxford Molecular's AbM antibody modeling software. The contact definition is based on an analysis of available complex crystal structures.

[0044] The term "autologous" refers to any material derived from the same individual that is later reintroduced. For example, the engineered autologous cell therapy (eACT™) method described herein involves the collection of lymphocytes from a patient, which are then engineered to express, for example, a CAR construct, and then administered to the same patient.

[0045] "Chimeric antigen receptor" or "CAR" refers to a molecule engineered to contain a binding motif and a means to activate an immune cell (e.g., a T cell, such as a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof) upon antigen binding. CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. In some embodiments, a CAR comprises a binding motif, an extracellular domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain. T cells engineered to express a chimeric antigen receptor may be referred to as CAR T cells. "Extracellular domain" (or "ECD") refers to a portion of a polypeptide that is understood to reside outside the cell membrane, in the extracellular space, when the polypeptide resides in the cell membrane.

[0046] "T cell receptor" or "TCR" refers to the antigen-recognition molecule present on the surface of T cells. During normal T cell development, each of the four TCR genes, α, β, γ, and δ, can rearrange to give rise to a wide variety of TCR proteins.

[0047] The term "heterologous" means derived from any source other than the naturally occurring sequence. For example, the heterologous sequence included as part of the costimulatory protein is an amino acid that does not naturally occur as a wild-type human costimulatory protein, i.e., does not align with the wild-type human costimulatory protein. For example, a heterologous nucleotide sequence refers to a nucleotide sequence other than the nucleotide sequence of the wild-type human costimulatory protein coding sequence.

[0048] The term "identity" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Methods are known for calculating the percent identity between two provided polypeptide sequences. For example, calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). The nucleotides or amino acids at corresponding positions are then compared. If a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account, optionally, the number of gaps and the length of each gap, which may need to be introduced for optimal alignment of the two sequences. Comparison or alignment of sequences and determination of percent identity between two sequences may be accomplished using a mathematical algorithm such as BLAST (Basic Local Alignment Search Tool). In some embodiments, polymer molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%).

[0049] Immune cells for immunotherapy can be derived from any source known in the art. For example, immune cells can be differentiated in vitro from hematopoietic stem cell populations or immune cells can be obtained from a subject. Immune cells can be obtained, for example, from peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. Additionally, immune cells can be derived from one or more immune cell lines available in the art. Immune cells can also be obtained from a unit of blood drawn from a subject using various techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. Further methods of isolating immune cells for immune cell therapy are disclosed in U.S. Patent Application Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0050] A "patient" includes any human suffering from cancer (e.g., lymphoma or leukemia). The terms "subject" and "patient" are used interchangeably herein.

[0051] The term "pharmaceutically acceptable" refers to a molecule or composition that, when administered to a recipient, is not harmful to that recipient or the benefits to that recipient outweigh any harmful effects. With respect to carriers, diluents, or excipients used to formulate the compositions disclosed herein, a pharmaceutically acceptable carrier, diluent, or excipient must be compatible with the other components of the composition and not harmful to the recipient or the benefits to the recipient outweigh any harmful effects. The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in the transport or transportation of a drug from one part of the body to another (e.g., from one organ to another). Each carrier present in a pharmaceutical composition must be "acceptable" in the sense of being compatible with the other components of the formulation and not harmful to the patient or the benefits to the recipient must outweigh any harmful effects. Some examples of materials which may function as pharma- ceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; excipients such as powdered tragacanth, malt, gelatin, talc, cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar, buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, pH buffer solutions, polyesters, polycarbonates, and / or polyanhydrides, and other non-toxic compatible substances used in pharmaceutical formulations.

[0052] The term "pharmaceutical composition" refers to a composition in which an active agent is formulated together with one or more pharma- ceutically acceptable carriers. In some embodiments, the active agent is present in an amount of a unit dose suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant subject or population. In some embodiments, the pharmaceutical compositions may be formulated for administration in solid or liquid form, including, but not limited to, forms adapted for oral administration, e.g., liquids (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and those targeted for systemic absorption, boluses, powders, granules, pastes for application to the tongue, parenteral administration, e.g., as sterile solutions or suspensions, or as sustained release formulations, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, topical administration, e.g., as a cream, ointment, or sustained release patch or spray applied to the skin, lungs, or oral cavity, vaginally or rectally, e.g., as a pessary, cream, or foam, sublingually, ophthalmically, transdermally, or intranasally, to the lungs, and other mucosal surfaces.

[0053] The terms "reduce" and "decrease" are used interchangeably herein and refer to any change that is less than original. "Reduce" and "reduce" are relative terms, requiring a comparison of a before and after measurement. "Reduce" and "reduce" include complete depletion.

[0054] The term "reference" describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control that is an agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested, measured, and / or determined substantially simultaneously with the test, measurement, or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Generally, the reference or control is determined or characterized under conditions or circumstances equivalent to those under evaluation. Where similarity is sufficient to justify reliance on and / or comparison to the selected reference or control.

[0055] The term "abbreviated autologous process" refers to a transposon-based CAR T cell manufacturing process that is completed in less than 7 days, less than 5 days, less than 3 days, less than 1 day, less than 18 hours, less than 12 hours, less than 6 hours, less than 4 hours, or less than 2 hours.

[0056] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dosage" of a therapeutic agent, e.g., an engineered CAR T cell, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from developing a disease or promotes regression of the disease as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of disability or disability due to disease affliction. The ability of a therapeutic agent to promote regression of a disease can be evaluated using a variety of methods known to those of skill in the art, such as by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0057] The terms "transduction" and "transduced" refer to the process by which foreign DNA is introduced into a cell via a viral vector (see Jones et al., "Genetics: principles and analysis," Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

[0058] The terms "transfection" and "transfected" refer to the process by which foreign DNA is introduced into a cell via a non-viral vector. In some embodiments, the non-viral vector is a transposon. In certain embodiments, the transposon is selected from a piggyBac® (PB) transposon, a piggy-Bac®-like transposon, a piggyBac transposon, a Sleeping Beauty transposon, a Helraiser transposon, a Tol2 transposon, or a TcBuster transposon. In certain further aspects, the transposon can be integrated into the genome of the cell by the corresponding transposase. In certain aspects, the transposase can be, but is not limited to, a piggyBac® transposase, a piggy-Bac®-like transposase, a Super piggyBac® (SPB) transposase, a piggyBat transposase, a Sleeping Beauty transposase, a hyperactive Sleeping Beauty (SB100X) transposase, a Helitron transposase, a Tol2 transposase, a TcBuster transposase, or a hyperactive TcBuster transposase. The Helitron transposase can be a Helibatl transposase.

[0059] "Treatment" or "treating" of a subject refers to any type of intervention or process performed on a subject or administration of an active agent to a subject with the goal of reversing, mitigating, ameliorating, inhibiting, slowing or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical manifestation associated with a disease. In one embodiment, "treatment" or "treating" includes partial remission. In another embodiment, "treatment" or "treating" includes complete remission. In some embodiments, treatment may be treatment of a subject who does not show signs of the associated disease, disorder and / or condition and / or who shows only early signs of the disease, disorder and / or condition. In some embodiments, such treatment may be treatment of a subject who shows one or more definitive signs of the associated disease, disorder and / or condition. In some embodiments, treatment may be treatment of a subject who has been diagnosed as suffering from the associated disease, disorder and / or condition. In some embodiments, treatment may be treatment of a subject who is known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the associated disease, disorder and / or condition.

[0060] The term "vector" refers to a recipient nucleic acid molecule that contains or has been modified to incorporate a provided nucleic acid sequence. One type of vector is a "plasmid," which refers to a circular double-stranded DNA molecule into which additional DNA can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors can replicate autonomously in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors contain sequences that direct the expression of inserted genes to which they are operably linked. Such vectors may be referred to herein as "expression vectors." Standard techniques can be used for the manipulation of vectors, for example, as found in Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference.

[0061] Non-viral vehicles As provided, the production of CAR and TCR cells by viral vectors is associated with high processing costs, inherent safety risks, and the size limitations of coding sequences.It is generally recognized that retroviral vectors can only carry payloads less than 6 kilobases (KB) in length, and lentiviral vectors can carry payloads slightly longer than 7.5KB.Such may be sufficient for one normal CAR or TCR coding sequence, but barely enough for a bicistronic one, much less many enhancing elements are required to further improve the efficacy and safety of normal CAR or TCR.

[0062] Many non-viral approaches have been studied. However, the present inventors have demonstrated herein that transposon systems can overcome various challenges faced by viral vector-based systems. First, transposon systems can overcome the physical limitations of viral vectors on payload size. Some tested DNA transposons can support payloads larger than 10 kb, allowing the incorporation of multiple CAR and / or enhancement elements, which may be useful for boosting therapeutic cell products. In certain aspects, transposon-based systems simplify multi-gene editing to one-step transfection.

[0063] Second, when two or more CARs are used together, they are likely to share long strings of similar sequences. For example, the two CARs in a bicistronic CAR may contain the same immunoreceptor tyrosine-based activation motif (ITAM), such as CD3 zeta. Such homology may cause recombination, leading to the deletion or truncation of either or both of the CARs. Such recombination has actually been observed in lentiviral systems. As the complexity of genetic engineering increases, so does the risk of having recombination problems.

[0064] As Example 1 shows, even when the homology between the two copies of CD3 zeta was minimized by codon optimization and sequence wobble, there was still a significant amount of recombination after lentiviral transduction, as represented by the multiple variants of the expressed protein. However, the use of the transposon system in Example 1 resulted in a reduction of such recombination, and a complete elimination of the most damaging recombination (e.g., variants V1 and V15 with large truncations / deletions, see, e.g., Table 1).

[0065] On the other hand, transposon systems can serve as an alternative to viral vectors by offering similar transfection efficiency, cell viability and integration stability (see, for example, Tables 2-8).

[0066] Yet another challenge to conventional viral vector-based autologous cell therapy is the long processing time and high cost associated with it. A typical lentiviral vector-based autologous process takes approximately 7-10 days. Critical steps in such conventional processes include T cell activation, T cell enrichment and proliferation. However, the present inventors have been able to develop a transposon-based process that can be completed within 24 hours without the need for T cell activation, T cell enrichment or proliferation.

[0067] Surprisingly, such a very rapid process achieved higher in vivo efficacy, even at low doses, than the conventional 7-day lentiviral vector-based process at high doses (Examples 4 and 5). The data presented in the Examples (e.g., Tables 9 and 14) suggest that the shortened process resulted in the collection of higher concentrations of naive T cells, which contributes to improved therapeutic efficacy.

[0068] Also importantly, as shown in Tables 12 and 13, transfection of these transposons caused relatively mild cell death, and the transfected T cells were able to proliferate rapidly.

[0069] In Example 8, a comparison was made between Sleeping Beauty and TcBuster, which are different transposon systems. The results show that both transposon systems can generate highly effective CAR-T cells. CAR-T cells prepared using Sleeping Beauty and TcBuster transposon systems or using lentiviral vectors can all effectively inhibit tumor growth in Nalm6 animal models for at least 20 days.

[0070] Based on these successful process developments, we have also demonstrated that 6 ~100×10 6A large-scale process (5×10 in pilot small-scale studies) utilizing 10 cells (scalable to billions of cells) 6 Interestingly, the larger scale not only did not reduce the efficiency or quality of the process, but in fact resulted in significantly improved cell viability and transfection efficiency.

[0071] Thus, according to one embodiment of the present disclosure, a transposon is provided that comprises a transgene encoding one or more polypeptides, including a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In certain aspects, the transgene encodes 2, 3, 4, 5, 6, 7, 8, 9, or 10 different polypeptides, where one or more of the polypeptides are a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0072] In some embodiments, the transgene is at least 500 nucleotides in length. In some embodiments, the transgene is at least 600, 700, 800, 900, 1,000, 1,500, 2,000, 3,000, 4,000, 5,000 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, or 20,000 nucleotides in length. In certain further embodiments, the transgene may be greater than 20,000 nucleotides in length. In some embodiments, the transgene is no greater than 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 20,000, 25,000, or 30,000 nucleotides in length.

[0073] In some embodiments, the transgene comprises two fragments that share homology. In some embodiments, the homology is at least 70% sequence identity. In some embodiments, the homology is at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity. In some embodiments, the two fragments are in different CAR or TCR sequences, respectively.

[0074] An example of such a fragment is an immunoreceptor tyrosine-based activation motif (ITAM). ITAMs are typically found in the cytoplasmic tails of non-catalytic tyrosine phosphorylated receptors, which are cell surface proteins found primarily on immune cells. They are essential components for the initiation of various signaling pathways and the subsequent activation of immune cells. Non-limiting examples of ITAMs include the cytoplasmic signaling sequences of proteins such as TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b and CD66d. A particular example is CD3 zeta.

[0075] Such homology may also be found in other parts of the CAR or TCR sequences, such as, but not limited to, the linker, the extracellular hinge, the transmembrane domain, the intracellular signaling domain, or even the antigen binding sequence.

[0076] In some embodiments, the homologous fragments are codon-optimized to reduce sequence identity without altering the encoded protein. In some embodiments, the homologous fragments are codon-optimized to not share sequence identity of 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, or 8 or more consecutive nucleotides. In some embodiments, the homologous fragments are codon-optimized to not share sequence identity of 15 or more consecutive nucleotides. In some embodiments, the homologous fragments are codon-optimized to not share sequence identity of 12 or more consecutive nucleotides. In some embodiments, the homologous fragments are codon-optimized to not share sequence identity of 10 or more consecutive nucleotides. In some embodiments, the homologous fragments are codon-optimized to not share sequence identity of 9 or more consecutive nucleotides. In some embodiments, the homologous fragments are codon-optimized to not share sequence identity of 8 or more consecutive nucleotides.

[0077] In some embodiments, the transgene encodes a first CAR (or TCR) and a second CAR (or TCR), which may optionally be linked via a protease cleavage site (e.g., an autocleavage site) or a ribosomal skip sequence. In some embodiments, the first CAR and the second CAR each comprise a single chain fragment (scFv), a transmembrane domain, and an immunoreceptor tyrosine-based activation motif (ITAM).

[0078] In some embodiments, the transgene encodes a third CAR (or TCR). In some embodiments, the transgene further encodes a fourth CAR (or TCR). Each of the second, third or third CARs (or TCRs) can be cleaved at a protease cleavage site (e.g., an autocleavage site) or a ribosomal skip sequence to form an individual CAR (TCR).

[0079] In some embodiments, the transposon contains coding sequences for two, three or four CARs (or TCRs), which do not have to be linked. Instead, each coding sequence can have its own promoter or other required regulatory sequences.

[0080] Transposons The transposon used in the present technology can be any transposon known in the art, such as a piggyBac® (PB) transposon, a piggy-Bac®-like transposon, a piggyBat transposon, a Sleeping Beauty transposon, a Helraiser transposon, a Tol2 transposon, or a TcBuster transposon.

[0081] The transposon can be integrated into the genome of the cell by the corresponding transposase. The integration can be transient or stable. The transgene expression can be transient or stable depending on the time of measurement. The transposase can be, but is not limited to, piggyBac® transposase, piggy-Bac®-like transposase, Super piggyBac® (SPB) transposase, piggyBat transposase, Sleeping Beauty transposase, hyperactive Sleeping Beauty (SB100X) transposase, Helitron transposase, Tol2 transposase, TcBuster transposase or hyperactive TcBuster transposase. The Helitron transposase can be Helibatl transposase.

[0082] If the transposon is a piggyBac® transposon, the transposase can be a piggyBac® transposase or a Super piggyBac® transposase. If the transposon is a piggy-Bac®-like transposon, the transposase can be a piggy-Bac®-like transposase. If the transposon is a Sleeping Beauty transposon, the transposase can be a Sleeping Beauty transposase. If the transposon is a Helraiser transposon, the transposase can be a Helitron transposase. If the transposon is a Tol2 transposon, the transposase can be a Tol2 transposase. If the transposon is a TcBuster transposon, the transposase can be a TcBuster transposase or a hyperactive TcBuster transposase.

[0083] In one embodiment, the transposon is a Sleeping Beauty transposon. The Sleeping Beauty transposon may comprise a nucleic acid flanked at either end by an inverted repeat sequence that is recognized by an enzyme having Sleeping Beauty transposase activity. By "recognized" it is meant that the Sleeping Beauty transposase can bind to the inverted repeat sequence and then integrate the transposon flanked by the inverted repeat sequence into the genome of the target cell. Representative inverted repeat sequences that may be found in the Sleeping Beauty transposon of the present method include those disclosed in WO 98 / 40510 and WO 99 / 25817. Of particular interest are inverted repeat sequences that are recognized by transposases that share at least about 80% amino acid identity with SEQ ID NO:1 of WO 99 / 25817.

[0084] In some embodiments, each inverted repeat of the transposon comprises at least one direct repeat. Transposon elements are linear nucleic acid fragments that can be used as linear fragments or circularized, for example, in a plasmid. Alternatively, vectors such as circular plasmids, minicircle plasmids and nanoplasmids can be used. In certain aspects, the backbone encoding the transposon can be anywhere from 100 bp to 4 kb in length. The transposon carrying the gene of interest can be delivered in a variety of DNA formats (plasmids, plasmid alternatives, linear DNA, or ds / ss DNA). In certain embodiments, there are two direct repeats in each inverted repeat. Examples of direct repeats can be found, for example, in WO 98 / 40510. Miniplasmids are supercoiled DNA molecules that are approximately 4,000 bp in length (e.g., 3000 bp to 5000 bp). In one embodiment, the plasmid is a linear plasmid. In one embodiment, the plasmid is a circular plasmid. In one embodiment, the plasmid is a nanoplasmid. In one embodiment, the plasmid is a minicircle plasmid. As demonstrated in the examples, minicircle plasmids are useful for achieving high transfection efficiency.

[0085] The Sleeping Beauty transposon can be integrated into the target genome by the corresponding transposase, such as SB10 and SB100X, described, for example, in WO 2017 / 158029.

[0086] CAR and TCR sequences Examples of CAR and TCR sequences are also provided. CARs contain an antigen-binding portion, typically a single-chain fragment (scFv) derived from an antibody. CARs can be monospecific, bispecific, or multispecific. In certain aspects, the transposon-based system simplifies multigene editing to one-step transfection. Also, the transposon-carried construct (or payload) of the present disclosure can contain two or more CAR molecules. A particular example is a bicistronic CAR, where two CARs are connected via a digestible linker or ribosomal skip sequence. Similarly, a bicistronic TCR can also be included in the payload. In certain aspects, the transposon-carried construct (or payload) of the present disclosure can encode three CARs. In certain aspects, the transposon-carried construct (or payload) of the present disclosure can encode four or more CARs. In certain further aspects, the transposon-carried construct (or payload) of the present disclosure can further encode a polypeptide that enhances the cytotoxicity of T cells expressing one or more CARs.

[0087] In certain embodiments, a bicistronic, tricistronic, or tetracistronic CAR encoding construct comprises a first CAR that targets a first antigen and a second CAR that targets a second antigen. The first and second antigens may be selected from 5T4, alpha fetoprotein, B cell maturation antigen (BCMA), CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD56, CD123, CD138, c-Met, CSPG4, C-type lectin-like molecule 1 (CLL-1), EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, GD3, HER1-HER2 combination, HER2-HER3 combination, HER2 / Neu, HERV-K, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gp120, IL-11R alpha, kappa chain, lambda chain, melanoma associated antigen, mesothelin, MUC-1, mutated p53, mutated ras, prostate specific antigen, ROR1, VEGFR2, or a combination thereof.

[0088] In some embodiments, the first, second, third, or fourth CAR targeted antigen is 2B4 (CD244), 4-1BB, 5T4, A33 antigen, adenocarcinoma antigen, adrenoceptor beta 3 (ADRB3), A-kinase anchoring protein 4 (AKAP-4), alpha fetoprotein (AFP), anaplastic lymphoma kinase (ALK), androgen receptor, B7H3 (CD276), β2-integrin, BAFF, B lymphoma cells, B cell maturation antigen (BCMA), bcr-abl (an oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl)), BhCG, bone marrow stromal cell antigen 2 (BST2), CCCTC binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of ImprintedSites), BST2, C242 antigen, 9-0-acetyl-CA19-9 marker, CA-125, CAEX, calreticulin, carbonic anhydrase 9 (CAIX), C-MET, CCR4, CCR5, CCR8, CD2, CD3, CD4, CD5, CD8, CD7, CD10, CD16, CD19, CD20, CD22, CD23 (IgE receptor), CD24, CD25, CD27, CD28, CD30(TNFRSF8), CD33, CD34, CD38, CD40, CD40L, CD41, CD44, CD44V6, CD49f, CD51, CD52, CD56, CD63, CD70, C D72, CD74, CD79a, CD79b, CD80, CD84, CD96, CD97, CD100, CD123, CD125, CD133, CD137, CD138, CD150, CD152(C TLA-4), CD160, CD171, CD179a, CD200, CD221, CD229, CD244, CD272 (BTLA), CD274 (PDL-1, B7H1), CD279 (PD-1), CD352, CD358, CD300 molecule-like family member f (CD300LF), carcinoembryonic antigen (CEA), claudin 6 (CLDN6), C-type lectin-like molecule-1 (CLL-1 or CLECL1), C-type lectin domain family 12 member A (CLEC12A), cytomegalovirus (CMV)-infected cell antigen, CNT0888, CRTAM (CD355), CS-1 (also called CD2 subset 1, CRACC, CD319, and 19A24), CTLA-4, cyclin B1, chromosome X open reading frame 61 (CXORF61), cytochrome P450 1B1 (CYP1B1), DNAM-1 (CD226), desmoglein 4, DR3, DR5, E-cadherin neoepitope, epidermal growth factor receptor (EGFR), EGF1R, epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), elongation factor 2 variant (EL F2M), endosialin, epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EphA2), ephrin B2, receptor tyrosine-protein kinase erb-B2, 3, 4 (erb-B2, 3, 4), ERBB, ERBB2 (Her2 / neu), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), ETA, ETS translocation mutant gene 6 located on chromosome 12p (ETV6-AML), Fc fragment of IgA receptor (FCAR or CD89), fibroblast activation protein alpha (FAP), FBP, Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor (AChR), fibronectin extra domain B, Fms-like tyrosine kinase 3 (FLT3), folate binding protein (FBP), folate receptor 1, folate receptor alpha, folate receptor beta, Fos-related antigen 1, fucosyl , Fucosyl GM1; GM2, ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(ll)Cer), o-acetyl-GD2 ganglioside (OAcGD2), GITR (TNFRSF18), GM1, ganglioside GM3 (aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(ll)Cer), GP 100, hexasaccharide moiety of GloboH glycoceramide (GloboH), glycoprotein 75, glypican-3 (GPC3), glycoprotein 100 (gplOO), GPNMB, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C group 5, member D (GPRC5D), hepatitis A virus cellular receptor 1 (HAVCR1), human epidermal growth factor receptor 2 (HER-2), HER2 / neu, HER3, HER4, HGF, high molecular weight melanoma-associated antigen (HMWMAA), human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPVE7), heat shock protein 70-2 mutant (mut hsp70-2), human scatter factor receptor kinase, human telomerase reverse transcriptase (hTERT), HVEM, ICOS, insulin-like growth factor receptor 1 (IGF-1 receptor), IGF-I, IgG1, immunoglobulin lambda-like polypeptide 1 (IGLL1), IL-6, interleukin-11 receptor alpha (IL-11Ra), IL-13, interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), insulin-like growth factor I receptor (IGF1-R), integrin α5β1, integrin ανβ3, intestinal carboxylesterase, kappa light chain, KCS1, kinase insert domain receptor (KDR), KIR, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, KIR-L, KG2D ligand, KIT (CD11 7), KLRGI, LAGE-la, LAG3, lymphocyte-specific protein tyrosine kinase (LCK), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), legumain, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Lewis (Y) antigen, LeY, LG, LI cell adhesion molecule (LI-CAM), LIGHT, LMP2, lymphocyte antigen 6 complex, LTBR, ​​locus K9 (LY6K), Ly-6, lymphocyte antigen 75 (LY75), melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2), MAGE, melanoma-associated antigen 1 (MAGE-A1), MAGE-A3 melanoma antigen 1 recognized by T cells (MelanA or MARTI), MelanA / MARTl, mesothelin, MAGEA3, melanoma inhibitor of apoptosis (ML-IAP), melanoma-specific chondroitin sulfate proteoglycan (MCSCP), MORAb-009, MS4A1, Mucin1 (MUCl), MUC2, MUC3, MUC4, MUC5AC, MUC5b, MUC7, MUC16, mucin CanAg, Müllerian inhibitory factor (MIS) receptor type II, neuroblastoma-derived homolog of the v-myc avian myelocytomatosis viral oncogene (MYCN), N-glycolylneuraminic acid, N-acetylglucosaminyltransferase V (NA17), neural cell adhesion molecule (NCAM), NKG2A, NKG2C, NKG2D, NKG2E ligand, NKR-P IA, NPC-1C, NTB-A, mammary differentiation antigen (NY-BR-1), NY-ESO-1, oncofetal antigen (h5T4), olfactory receptor 51E2 (OR51E2), OX40, plasma cell antigen, polySA, proacrosin binding protein sp32 (OY-TESl), p53, p53 mutant, pannexin 3 (PANX3), prostatic acid phosphatase (PAP), paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), PD-1H, platelet-derived growth factor receptor alpha (PDGFR-alpha), PDGFR-beta, PDL192, PEN-5, phosphatidylserine, placenta specific 1 (PLAC1), polysialic acid, prostase, prostate cancer cell, prostein, protease serine 21 (testisin or PRSS21), proteinase 3 (PR1), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), proteasome (prosome, macropain) subunit, beta type, receptor for advanced glycation end products (RAG E-1), RANKL, Ras mutant, Ras homolog family member C (RhoC), RON, receptor tyrosine kinase-like orphan receptor 1 (ROR1), kidney ubiquitous 1 (RU1), kidney ubiquitous 2 (RU2), sarcoma translocation breakpoint, squamous cell carcinoma antigen recognized by T cells 3 (SART3), SAS, SDC1, SLAMF7, sialyl Lewis adhesion molecule (sLe), Siglec-3, Siglec-7, Siglec-9, sonic hedgehog (SHH), sperm protein 17 (SPA17), stage-specific embryonic antigen 4- (SSEA-4), STEAP, sTn antigen, synovial sarcoma, X breakpoint 2 (SSX2), survivin, tumor-associated glycoprotein 72 (TAG72), TCR5y, TCRa, TCRB, TCR gamma alternative reading frame protein (TARP), telomerase, TIGIT TNF-α precursor, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tenascin-C, TGF-beta 2, TGF-β, transglutaminase 5 (TGS5), angiopoietin-binding cell surface receptor 2 (Tie 2), TIM1, TIM2, TIM3, TnAg, TRAIL-R1, TRAIL-R2, tyrosinase-related protein 2 (TRP-2), thyroid-stimulating hormone receptor (TSHR), tumor antigen CTAA16.88, tyrosinase, ROR1, TAG-72, uroplakin 2 (UPK2), VEGF-A, VEGFR-1, vascular endothelial growth factor receptor 2 (VEGFR2), and one of vimentin, Wilms tumor protein (WT1), or X antigen family, member 1A (XAGE1).

[0089] In one embodiment, the first and second antigens are CD19 and CD20, respectively. Exemplary sequences of anti-CD19 and anti-CD20 antibodies and fragments, and dicistronic versions thereof, are provided in Table A. TIFF2025517371000002.tif255160TIFF2025517371000003.tif255160TIFF2025517371000004.tif203161

[0090] The CAR of the present disclosure can comprise, in addition to the antigen binding molecule, a hinge, a transmembrane domain, and / or an intracellular domain. In some embodiments, the intracellular domain can comprise a costimulatory domain and an activation domain.

[0091] The hinge may be an extracellular domain of an antigen-binding system located between the binding motif and the transmembrane domain. The hinge may also be referred to as an extracellular domain or a "spacer". The hinge may contribute to the expression, activity, and / or stability of the receptor. The hinge may also provide flexibility to access the target antigen. In some embodiments, the hinge domain is located between the binding motif and the transmembrane domain.

[0092] In some embodiments, the hinge is, is, or is derived from an immunoglobulin-like hinge domain (e.g., including all or a fragment of an immunoglobulin-like hinge domain). In some embodiments, the hinge domain is from an immunoglobulin or is derived from an immunoglobulin. In some embodiments, the hinge domain is selected from an IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, or IgM hinge, or a fragment thereof.

[0093] In some embodiments, the hinge is a member of the family of antibodies that bind to CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8.alpha, CD8.beta, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD4 9f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated alpha chain), CD79B (B cell antigen receptor complex-associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (K IR2DL1), CD158B1(KIR2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2DL5A), CD158F2(KIR2DL5B), CD158K(KIR3DL2) , CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(SLAMF3), CD244(SLAMF4), CD247(CD3-zeta), CD258(LIGHT), CD268(BAFFR), CD270(TNFSF14), C D272(BTLA), CD276(B7-H3), CD279(PD-1), CD314(NKG2D), CD319(SLAMF7), CD335(NK-p46), CD336(NK-p44), CD337(NK-p30), CD352(SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), inducible T cell costimulatory factor (ICOS), LFA-1 (CD11a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R beta, IL-2R gamma,or is, is from, or is derived from (e.g., includes all or a fragment thereof) IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, activating NK cell receptor, or Toll ligand receptor, or is a fragment or combination thereof;

[0094] In some embodiments, the hinge is, is, or is derived from a CD8 alpha hinge (e.g., including all or a fragment of a CD8 alpha hinge). In some embodiments, the hinge is, is, or is derived from a CD28 hinge. In some embodiments, the hinge is, is, or is derived from a fragment of a CD8 alpha hinge or a fragment of a CD28 hinge, where the fragment is less than the entire hinge. In some embodiments, the fragment of a CD8 alpha hinge or the fragment of a CD28 hinge comprises an amino acid sequence that excludes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids of the CD8 alpha hinge, or at the N-terminus or C-terminus of the CD28 hinge, or both.

[0095] "Transmembrane domain" refers to a domain that has the attribute of being intramembrane (e.g., spanning part or all of a cell membrane) when present in a molecule at the cell surface or cell membrane. Not all amino acids in a transmembrane domain need be present within the membrane. For example, in some embodiments, a transmembrane domain is characterized in that a specified stretch or portion of a protein is located substantially within a membrane. Amino acid or nucleic acid sequences can be analyzed using a variety of algorithms to predict the subcellular localization (e.g., transmembrane localization) of proteins. The programs psort (PSORT.org) and Prosite (prosite.expasy.org) are examples of such programs.

[0096] Transmembrane domains include those of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD45, CD4, CD5, CD7, CD8, CD8 alpha, CD8 beta, CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, 4-1BB / CD1 37, activated NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD276(B7-H3), CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96(Tactile), CD5, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA -1, LFA-1, ligand binding to CD83, LIGHT, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM) protein, SLAM (SLAMF1, CD150;IPO-3), SLAMF4 (CD244, 2B4), SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, truncations, or combinations thereof;

[0097] The intracellular domain (or cytoplasmic domain) comprises one or more signaling domains that upon binding of a target antigen to the binding motif, triggers and / or mediates an intracellular signal that activates, for example, one or more immune cell effector functions (e.g., natural immune cell effector functions). In some embodiments, the signaling domain of the intracellular domain mediates activation of at least one of the normal effector functions of an immune cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity including secretion of cytokines. In some embodiments, the signaling domain of the intracellular domain mediates T cell activation, proliferation, survival, and / or other T cell functions. The intracellular domain can comprise a signaling domain that is an activation domain. The intracellular domain can comprise a signaling domain that is a costimulatory signaling domain.

[0098] Intracellular signaling domains that can transduce signals when antigens bind to immune cells are known. For example, the cytoplasmic sequence of the T cell receptor (TCR) is known to initiate signal transduction after TCR binding to antigen (e.g., Brownlie et al., Nature Rev. Immunol. 13:257-269 (2013)).

[0099] In certain embodiments, suitable signaling domains include, but are not limited to, 4-1BB / CD137, activating NK cell receptor, immunoglobulin proteins, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CD5, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand binding to LFA-1, CD83, LIGHT, LIGHT, LTBR, ​​Ly9 (CD229), Ly108), lymphocyte function-associated antigen-1 (LFA-1, CD1-1) a / CD18), MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1, CD150; IPO-3), SLAMF4 (CD244, 2B4), SLAMF6 (NTB-A, SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, truncations, or combinations thereof.

[0100] CARs can also include costimulatory signaling domains, for example, to enhance signaling efficacy. See U.S. Patent Nos. 7,741,465 and 6,319,494, as well as Krause et al. and Finney et al. (supra), Song et al., Blood 119:696-706 (2012), Kalos et al., Sci Transl. Med. 3:95 (2011), Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016). Signals generated through the TCR alone may be insufficient for full activation of T cells, and secondary or costimulatory signals may increase activation. Thus, in some embodiments, the signaling domain further comprises one or more additional signaling domains (e.g., costimulatory signaling domains) that activate one or more immune cell effector functions (e.g., natural immune cell effector functions described herein). In some embodiments, a portion of such a costimulatory signaling domain may be used so long as that portion transduces an effector function signal. In some embodiments, the cytoplasmic domain described herein comprises one or more cytoplasmic sequences of a T cell co-receptor (or a fragment thereof). Non-limiting examples of such T cell co-receptors include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), MYD88, CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that binds CD83. An exemplary costimulatory protein has the amino acid sequence of a costimulatory protein naturally found on a T cell, the complete natural amino acid sequence of which is set forth in the NCBI Reference Sequence: NP 0.1. In certain examples, the CAR comprises a 4-1BB costimulatory domain. In certain examples, the CAR comprises a CD28 costimulatory domain. In certain examples, the CAR comprises a DAP-10 costimulatory domain.

[0101] In some embodiments, the CAR further comprises an ITAM. Examples of ITAM-containing primary cytoplasmic signaling sequences that are particularly useful in the present disclosure include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the ITAM comprises CD3 zeta.

[0102] Receptor transfection, integration and expression The transposons of the present disclosure can be introduced into target cells, particularly immune cells, to generate genetically engineered cells. The target cells are preferably immune cells, such as T cells, NK cells, monocytes, macrophages, or their precursor cells.

[0103] Integration of the transposon into the genome of the target cell requires the expression of the corresponding transposase. The transposon used in the present technology can be any transposon known in the art, such as piggyBac (registered trademark) (PB) transposon, piggy-Bac (registered trademark)-like transposon, piggyBat transposon, Sleeping Beauty transposon, Helraiser transposon, Tol2 transposon, or TcBuster transposon.

[0104] If the transposon is a piggyBac® transposon, the transposase can be a piggyBac® transposase or a Super piggyBac® transposase. If the transposon is a piggy-Bac®-like transposon, the transposase can be a piggy-Bac®-like transposase. If the transposon is a Sleeping Beauty transposon, the transposase can be a Sleeping Beauty transposase, such as SB10 or SB100X. If the transposon is a Helraiser transposon, the transposase can be a Helitron transposase. If the transposon is a Tol2 transposon, the transposase can be a Tol2 transposase. If the transposon is a TcBuster transposon, the transposase can be a TcBuster transposase or a hyperactive TcBuster transposase.

[0105] The transposase can be introduced into the target cell as a polynucleotide encoding the transposase, regulated by a promoter that allows its expression in the target cell.

[0106] In some embodiments, the transposon and transposase are introduced into the target cell by methods known in the art, such as electroporation, nucleofection, lipofection, ultrasound, and magnetofection. In some embodiments, the transposon and transposase are introduced into the target cell by electroporation. In some embodiments, the molecular copy number ratio of the nucleic acid transposon backbone containing the insert of interest to the nucleic acid encoding the transposase is 1:1. In certain further aspects, the molecular copy number ratio of the nucleic acid transposon backbone containing the insert of interest to the nucleic acid encoding the transposase ranges from 1:1 to 1:32. In some embodiments, the nucleic acid transposon backbone containing the insert of interest is copy number matched to the nucleic acid encoding the transposase. In certain aspects, the ratio of the nucleic acid transposon backbone containing the insert of interest to the nucleic acid encoding the transposase is 1:4, 1:8, or 1:16.

[0107] Once integrated into the target genome, the transgene from the transposon can be transcribed and translated to produce the receptor protein, and as demonstrated in the accompanying experimental examples, such integrated transgenes are highly stable.

[0108] Cell Preparation Process The transposon-based technology of the present disclosure has enabled the inventors to develop a cell therapy process that is significantly shorter than prior art, preferably an autologous cell therapy process, but equally applicable to allogeneic ones.

[0109] In exemplary embodiments, the process involves (1) obtaining and / or enriching a population of lymphocytes obtained from a donor subject, (2) transfecting the population of lymphocytes with a transposon of the present disclosure along with a corresponding transposase, and (3) harvesting the transfected lymphocytes. In some embodiments, the harvested lymphocytes are administered to a patient, such as a donor. In some embodiments, the harvested lymphocytes are cryopreserved.

[0110] In another embodiment, the process involves obtaining a sample comprising lymphocytes (e.g., T cells) from a donor subject, incubating the sample with a transposon to transfect the lymphocytes to produce transfected lymphocytes, culturing the sample comprising the transfected lymphocytes, and then harvesting the lymphocytes to produce a harvested sample, wherein at least 40% of the lymphocytes in the harvested sample are naive cells.

[0111] In some embodiments, at least 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, or 65% of the lymphocytes in the collected sample are naive cells. In some embodiments, at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, or 65% of the T cells in the collected sample are naive T cells.

[0112] In some embodiments, the naive T cells are CD45RA + , CCR7 + , CD62L + , CD27 + , CD28 + , CD127 + , CD132 + , CD25 - , CD44 -, CD45RO - , and HLA-DR - In one embodiment, the naive T cells are characterized by one or more markers such as CD45RA + and CCR7 + In one embodiment, the naive T cells are characterized by CD45RA + , CCR7 + , CD62L + , CD27 + , and CD28 + In one embodiment, the naive T cells are characterized by CD127 + , CD132 + , CD25 - , CD44 - , CD45RO - , and / or HLA-DR - It is additionally characterized by:

[0113] In some embodiments, the entire process from obtaining lymphocytes from a donor to harvesting the transfected lymphocytes is completed within 4 days. In some embodiments, the entire process from obtaining lymphocytes from a donor to harvesting the transfected lymphocytes is completed within 3 days. In some embodiments, the entire process from obtaining lymphocytes from a donor to harvesting the transfected lymphocytes is completed within 2 days. In some embodiments, the entire process from obtaining lymphocytes from a donor to harvesting the transfected lymphocytes is completed within 36 hours. In some embodiments, the entire process from obtaining lymphocytes from a donor to harvesting the transfected lymphocytes is completed within 24 hours. In some embodiments, the entire process from obtaining lymphocytes from a donor to harvesting the transfected lymphocytes is completed within 18 hours. In some embodiments, the entire process from obtaining lymphocytes from a donor to harvesting the transfected lymphocytes is completed within 12 hours. In some embodiments, the entire process from obtaining lymphocytes from a donor to harvesting the transfected lymphocytes is completed within 8 hours. In some embodiments, the entire process from obtaining lymphocytes from a donor to harvesting the transfected lymphocytes is completed within 6 hours. In some embodiments, the entire process from obtaining lymphocytes from a donor to harvesting the transfected lymphocytes is completed within 4 hours.

[0114] In some embodiments, the entire process from obtaining lymphocytes from a donor to transplantation or cryopreservation of the transfected lymphocytes is completed within 4 days. In some embodiments, the entire process from obtaining lymphocytes from a donor to transplantation or cryopreservation of the transfected lymphocytes is completed within 3 days. In some embodiments, the entire process from obtaining lymphocytes from a donor to transplantation or cryopreservation of the transfected lymphocytes is completed within 2 days. In some embodiments, the entire process from obtaining lymphocytes from a donor to transplantation or cryopreservation of the transfected lymphocytes is completed within 36 hours. In some embodiments, the entire process from obtaining lymphocytes from a donor to transplantation or cryopreservation of the transfected lymphocytes is completed within 24 hours. In some embodiments, the entire process from obtaining lymphocytes from a donor to transplantation or cryopreservation of the transfected lymphocytes is completed within 18 hours.

[0115] As observed in the experimental examples, this improved autologous process does not require lymphocyte activation, T cell enrichment or expansion.

[0116] "Lymphocyte activation" or "lymphocyte stimulation" refers to the process of stimulating a population of lymphocytes with one or more stimulatory agents to produce a population of activated lymphocytes. To produce a population of activated lymphocytes, any combination of one or more suitable lymphocyte stimulatory agents may be used, including antibodies or functional fragments thereof that target T cell stimulatory or costimulatory molecules (e.g., anti-CD2 antibodies, anti-CD3 antibodies, anti-CD28 antibodies, or functional fragments thereof), T cell cytokines (e.g., any isolated, wild-type, or recombinant cytokine, such as interleukin-1 (IL-1), interleukin-2, (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6, (IL-6), interleukin-7, (IL-7), interleukin-8, (IL-8), interleukin-9, (IL-9), interleukin-10, (IL-10), interleukin-11, (IL-12), interleukin-12, (IL-13), interleukin-14, (IL-14), interleukin-15, (IL-15), interleukin-16, (IL-17), interleukin-17, (IL-18), interleukin-19, (IL-19), interleukin-20, (IL-20), interleukin-21, (IL-21), interleukin-22, (IL-22), interleukin-23, (IL-23), interleukin-24, (IL-24), interleukin-25, (IL-25), interleukin-26, (IL-26), interleukin-27, (IL-27), interleukin-28, (IL-28), interleukin-29, (IL-29), interleukin-30, (IL-2 The T cell stimulatory or costimulatory molecule may include, but is not limited to, interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 12 (IL-12), interleukin 15 (IL-15), interleukin 21 (IL-21), interleukin 23 (IL-23), tumor necrosis factor alpha (TNFα)), or any other suitable mitogen (e.g., tetradecanoyl phorbol acetate (TPA), phytohemagglutinin (PHA), concanavalin A (conA), lipopolysaccharide (LPS), pokeweed mitogen (PWM)) or a natural ligand for a T cell stimulatory or costimulatory molecule.

[0117] In some embodiments, lymphocyte activation uses an anti-CD3 antibody (or a functional fragment thereof), an anti-CD28 antibody (or a functional fragment thereof), or a combination of an anti-CD3 antibody and an anti-CD28 antibody in stimulating a population of lymphocytes.

[0118] Thus, in one embodiment, in the disclosed process, lymphocytes are not contacted with any exogenous agents (such as those exemplified above) that activate lymphocytes.

[0119] "Lymphocyte enrichment" or "lymphocyte selection" refers to a process in which a subset of lymphocytes is selected in a cell mixture. In certain embodiments, CD3+ T cells may be selected from a cell mixture. In certain embodiments, CD4+ and CD8+ T cells may be selected from a cell mixture. The selection process may be performed using antibodies (e.g., anti-CD3+ antibodies, anti-CD4+ antibodies, anti-CD8+ antibodies) or any other means for the selection of cell subtypes known in the art.

[0120] "Lymphocyte proliferation" refers to the process by which lymphocytes grow in response to nutrients and other conditions (e.g., CO) that allow them to actively divide and grow. 2 ) and incubated at a suitable temperature (e.g., 37° C.). In some embodiments, the process of the present disclosure does not include lymphocyte proliferation. In some embodiments, the process of the present disclosure includes abbreviated lymphocyte proliferation that is 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 36 hours, or 48 hours or less.

[0121] Preferably, in some embodiments, each step is performed in a closed system. Preferably, each step is performed in a culture medium that is free of human or animal serum.

[0122] The transfected lymphocytes generated from this process can be suitably used to treat disease. In some embodiments, the harvested lymphocytes, such as T cells, comprise a sufficient percentage of naive T cells.

[0123] In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the collected lymphocytes are transfected lymphocytes, hi some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the collected lymphocytes have a transgene integrated into their genome.

[0124] In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the harvested T cells are transfected T cells, in some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the harvested T cells have a transgene integrated into the genome of the T cells.

[0125] In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the harvested NK cells are transfected NK cells, in some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the harvested NK cells have a transgene integrated into the genome of the NK cells.

[0126] Various embodiments of the cell manufacturing process of the present disclosure can be scaled up, as shown in Example 8. In some embodiments, the scaled up process uses a sample for transfection, the sample being at least 10×10 6 In some embodiments, the sample contains at least 15×10 cells. 6 cells, 20 x 10 6 cells, 25 x 10 6 cells, 30 x 10 6 cells, 35 x 10 6 cells, 40 x 10 6 cells, 45 x 10 6 cells, 50 x 10 6 cells, 60 x 10 6 cells, 70 x 10 6 cells, 80 x 10 6 cells, 90 x 10 6 cells, 100 x 10 6 cells, 110 x 10 6 cells, 120 x 10 6 cells, 130 x 10 6 cells, 140 x 10 6 cells, 150 x 10 6cells, 200 x 10 6 cells, 250 x 10 6 cells, 300 x 10 6 cells, 400 x 10 6 cells, 500 x 10 6 pieces, 600×10 6 cells, 700 x 10 6 cells, 800 x 10 6 cells, 900 x 10 6 cells, 1000 x 10 6 cells, 1500 x 10 6 Pieces or 2000 x 10 6 Contains cells.

[0127] In some embodiments, transfection (e.g., electroporation) in large scale processes is performed using samples having a volume of at least 0.01 mL, or at least 0.05 mL, 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 12 mL, 15 mL, 18 mL, 20 mL, 22 mL, 25 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, or 100 mL. In some embodiments, transfection (e.g., electroporation) in large scale processes is performed using samples having a volume of 200 mL, 150 mL, 100 mL, 90 mL, 80 mL, 70 mL, 60 mL, 50 mL, 40 mL, 30 mL, 25 mL, 20 mL, 15 mL, 12 mL, 10 mL, 9 mL, 8 mL, 7 mL, 6 mL, 5 mL, 4 mL, 3 mL, 2 mL, 1.9 mL, 1.8 mL, 1.7 mL, 1.6 mL, 1.5 mL, 1.4 mL, 1.3 mL, 1.2 mL, 1.1 mL, 1 mL, 0.9 mL, 0.8 mL, 0.7 mL, 0.6 mL, or 0.5 mL or less.

[0128] In some embodiments, in larger scale processes, at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, or 65% of the lymphocytes in a harvested sample are naive cells.

[0129] In some embodiments, in larger scale processes, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the harvested lymphocytes are transfected lymphocytes, hi some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the harvested lymphocytes have a transgene integrated into their genome.

[0130] In any of the embodiments of the cell preparation process, the harvested cells may be subjected to cell activation (if not activated prior to transfection), selection and / or expansion. In some embodiments, expansion may be performed for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0131] Compositions and Treatments The cells, e.g., allogeneic cells, of the present disclosure can be used to treat a variety of diseases and conditions, particularly cancer. In one embodiment, the cancer may include Wilms' tumor, Ewing's sarcoma, neuroendocrine tumors, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, renal cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and bladder cancer.

[0132] Another embodiment described herein is a method of treating cancer in a subject in need thereof comprising administering an effective amount, e.g., a therapeutically effective amount, of a composition comprising the transfected cells of the present disclosure. Also provided are such compositions comprising the transfected lymphocytes disclosed herein and a pharma- ceutically acceptable excipient.

[0133] The amount and frequency of administration will be determined by factors such as the condition of the patient, the type and severity of the patient's disease, and appropriate dosages may be determined by clinical trials. In some embodiments, the cancer is characterized by expression of an antigen targeted by the CAR or TCR molecule, such as CD19 and / or CD20.

[0134] In other embodiments, methods are provided that include administering a therapeutically effective amount of the modified T cells contemplated herein or a composition comprising the same, alone or in combination with one or more therapeutic agents, to a patient in need thereof. In certain embodiments, the cells of the present disclosure are used to treat a patient at risk of developing cancer. Thus, the present disclosure provides a method for the treatment or prevention of cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the modified T cells of the present disclosure.

[0135] Those skilled in the art will recognize that multiple administrations of the disclosed compositions may be required to affect the desired treatment. For example, the compositions may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a span of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, 10 years, or more.

[0136] In one embodiment, a subject in need thereof is administered an effective amount of the composition to increase the cellular immune response against cancer in the subject. The immune response may include cellular immune responses mediated by cytotoxic T cells, regulatory T cells, and helper T cell responses that can kill infected cells. Thus, a humoral immune response may also be induced that is primarily mediated by helper T cells that can activate B cells leading to antibody production. To analyze the type of immune response induced by the composition of the present disclosure, various techniques that are well described in the art may be used, for example, as described in Current Protocols in Immunology, eds. John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan. M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, NY.

[0137] Methods for administering the cell compositions described herein include any method that is effective to result in either the reintroduction of ex vivo genetically modified immune effector cells that directly express a TCR or CAR in a subject, or the reintroduction of genetically modified precursor cells of immune effector cells that differentiate into mature immune effector cells expressing a TCR or CAR upon introduction into a subject. One method includes transfecting peripheral blood T cells ex vivo with a nucleic acid construct according to the present disclosure and returning the transfected cells to the subject.

[0138] Although the foregoing disclosure has been described in some detail by way of illustration and example for clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the present disclosure that certain changes and modifications can be made without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only, and not by way of limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to produce similar results.

[0139] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, the citation of a reference herein should not be construed as an admission that such reference is prior art to the present disclosure. In the event that any definitions or terms provided in a reference incorporated by reference differ from the terms and explanations provided herein, the terms and definitions shall control. The contents of all references cited throughout this application are expressly incorporated herein by reference. EXAMPLES

[0140] Example 1. Transposon-mediated bicistronic CAR transfection and integration This example tested a transposon system to introduce anti-CD19 and anti-CD20 bicistronic CAR constructs into host cells.

[0141] The bicistronic CAR, referred to herein as KITE-001, is expressed as a single chain precursor that contains, from N- to C-terminus, first signal peptide-anti-CD19 scFv-CD28 hinge and transmembrane domain-CD28 costimulatory domain-CD3ζ-T2A-second signal peptide-anti-CD20 scFv-CD8 hinge and transmembrane domain-4-1BB costimulatory domain-CD3ζ. Self-cleavage at T2A results in the production of two separate CAR molecules.

[0142] The coding sequence of KITE-001 shares a homology region in the CD3ζ domain between the anti-CD19 and anti-CD20 portions. Such homology was suspected to cause recombination and cryptic splicing. Therefore, codon optimization and sequence wobble were performed to reduce the nucleotide homology to less than 9 nucleotides.

[0143] In pilot studies with lentiviral vectors, it was observed that the expressed protein product contained multiple variants. Notably, variant 1 (V1), which contained only anti-CD19, accounted for approximately 1.5%–2% of the total protein product; variant 14 (V14), which contained a frameshift mutation in the anti-CD19 unit and was therefore non-functional, accounted for approximately 0.02–0.05% of the total protein product; variant 15 (V15), which contained a frameshift mutation in the heavy chain of the anti-CD20 scFv and therefore had only anti-CD19 activity, accounted for approximately 0.02–0.05% of the total protein product; and variant 22 (V22), which had a deletion between the anti-CD20 scFv and the CD8 hinge and was therefore only functional for CD19.

[0144] The present example then used the transposon system together with the corresponding transposase to transfect the KITE-001 coding sequence into T cells by electroporation (EP). The transposon system contained the coding sequence flanked by two sets of repeat sequences (CAGTTGAAGTCGGAAGTTTACATACACYTAAG, SEQ ID NO:28, and YCCAGTGGGTCAGAAGTTTACATACACTMART, SEQ ID NO:29). An exemplary transposase sequence is MGKSKEISQDLRKRIVDLHKSGSSLGAISKRLAVPRSSVQTIVRKYKHHGTTQPSYRSGRRRVLSPRDERTLVRKVQINPRTTAKDLVKMLEETGTKVSISTVKRVLYRHNLKGHSARKKPLLQNRHKKARLRFATAHGDKDRTFWRNVLWSDETKIELFGHNDHRYVWRKKGEACKPKNTIPTVKHGGGSIMLWGCFAAGGTGALHKIDGIMDAVQYVDILKQHLKTSVRKLKLGRKWVFQHDNDPKHTSKVVAKWLKDNKVKVLEWPSQSPDLNPIENLWAELKKRVRARRPTNLTQLHQLCQEEWAKIHPNYCGKLVEGYPKRLTQVKQFKGNATKY (SEQ ID NO: 30).

[0145] The transfected cells were then cultured and expanded. Unlike the lentiviral system, variants V1 and V15 were completely undetectable in the transfected cells, and the other variants never had a prevalence above 0.02%. In contrast, variant V22 was detected at significantly higher levels in the transposon-based samples (Table 1). TIFF2025517371000005.tif39128

[0146] The stability of the transposon-based integration was monitored for 3 weeks. As shown in Tables 2 and 3, both the LVV-generated CAR and the non-viral translocated CAR show in vitro stability of CD19 / CD20 expression. TIFF2025517371000006.tif75128TIFF2025517371000007.tif75128

[0147] Example 2. Single CAR transfection efficiency The transfection efficiency of the transposon system was then compared to that using lentiviral vectors using T cells from four different donors. Both systems achieved excellent results in terms of percentage of transfected cells of total viable CD3+ cells (Table 4) and total T cell viability (Table 5). Thus, the results indicate that unlike viral vector-based systems, the transposon system did not require T cell activation to achieve high transfection efficiency. TIFF2025517371000008.tif54150TIFF2025517371000009.tif80150

[0148] Example 3. Transposon-mediated reporter gene or single CAR transfection and integration This example tested a transposon system to introduce one of the following into host cells: a GFP reporter gene, an anti-CD19 CAR construct with a 4-1BB costimulatory region, or an anti-CD19 CAR construct with a CD28 costimulatory region.

[0149] As shown in Table 6, expression of the integrated GFP reporter gene was maintained for more than 21 days. In comparison, the non-integrated expression control (no electroporation, minicircle, or transposase-free plasmid only) declined to less than 1% within 7-10 days. Thus, this data demonstrates that transposase-mediated gene integration was stable. It is noteworthy that transfection efficiency was further improved at all time points when the transposon was packaged in a minicircle plasmid for delivery. TIFF2025517371000010.tif62170

[0150] As shown in Table 7, expression of the integrated anti-CD19 CAR construct with the 4-1BB costimulatory region was maintained for over 21 days. In comparison, the non-integrated expression control, plasmid without transposase alone, declined to less than 1% within 7-10 days. Thus, this data demonstrates that transposon-mediated gene integration for this CAR construct was stable. TIFF2025517371000011.tif70170

[0151] As shown in Table 8, expression of the integrated anti-CD19 CAR construct with CD28 costimulatory region was maintained for over 21 days. In comparison, the non-integrated expression controls, no electroporation or plasmid only, dropped to less than 1% within 7-10 days. Thus, this data demonstrates that transposon-mediated gene integration for this CAR construct was stable. TIFF2025517371000012.tif86170

[0152] Example 4. Development of a transposon-mediated autologous process Conventional viral vector-based autologous processes take seven days or even several weeks from cell acquisition to transplantation. This example attempts to develop an autologous process that can be completed as quickly as within one day.

[0153] Following apheresis, T cells were enriched from harvested peripheral blood mononuclear cells (PMBCs) which were then subjected to electroporation with the transposon constructs (as tested in Example 1). T cells were either harvested at 24 hours or further activated and expanded.

[0154] The effect of the duration of the process was evaluated. As shown in Table 9, TIFF2025517371000013.tif41165

[0155] The process (1 day, 3 days, and 7 days with different cell numbers) was tested in the Nalm6 mouse model of leukemia. Tumor burden was measured after autologous cell transplantation. The results are shown in Table 10. A lentiviral vector-based autologous process (7 days) with the same CAR sequence was used as a control. TIFF2025517371000014.tif81170TIFF2025517371000015.tif84170

[0156] 7-day lentiviral vector-based process (10 6 cells) resulted in a significant reduction in tumor growth. The tumor burden gradually increased and peaked at day 68 (approximately 2.3 × 10 9 photons / sec), then at 89 days, about 1.7 × 10 8 photons / second.

[0157] However, among all transposon-based processes, low doses (2 × 10 5 Only the 7-day process at high doses (10 6 The performance was inferior to the lentiviral vector-based process at high doses (106 Process for 1 day at low doses (2 x 10 cells) 5 A 3-day process at 100x the normal concentration of 100x the normal cells resulted in tumor elimination (<10 cells) at approximately day 20. 6 photons / sec) with no tumor return. Higher doses for 3 and 7 days of treatment performed equally well, whereas lower doses (2 × 10 5 cells) was comparable to the lentiviral vector-based process at higher doses.

[0158] Thus, these results demonstrate that the newly developed 1-day process for transposon-based autologous CAR-T can be used to treat CAR-T with the same dose (10 6 We demonstrate that our method achieved results far superior to those of a 7-day lentiviral vector-based process (10 cells). 6 cells) than a lower dose (2 × 10 5 Even when using fewer than 100 cells per day, the results were still comparable. Such superior results from the newly developed, shortened process may have produced higher quality CAR-T cells with a higher naive T cell population that is advantageous for cancer treatment.

[0159] Example 5. Further testing of the in-house process This example further tested the truncated transposon-based autologous process with other CAR constructs. A construct was used that targets CD19 but contains the 4-1BB costimulatory domain. Similarly, early (day 4) harvest at both high and low doses performed significantly better than late (day 14) harvest.

[0160] Fixed amounts of plasmid DNA (gene cargo, 5 μg) and mRNA (encoding the transposase, 5 μg) were used in each experiment. A comparison is shown in Table 11. TIFF2025517371000016.tif37161

[0161] Electroporation of plasmid DNA can generally result in 30-50% cell death in the first 24 hours. The viability of cells produced by the new process was activated and expanded, and their viability was evaluated at each stage. Table 12 shows that cell viability recovered to 80-90% after 8 days, the same viability level as the cell group without electroporation. Table 13 shows that the surviving cells expanded well with a 30-40 fold increase after the new manufacturing process. TIFF2025517371000017.tif37160TIFF2025517371000018.tif37160

[0162] These data therefore demonstrate that the novel non-viral process is applicable to various types of constructs. Moreover, cell viability can be restored during subsequent expansion, demonstrating the excellent expansion capacity of electroporated T cells.

[0163] Example 6. Evaluation of cells produced from the new process This example assessed CAR-T cells generated from the truncated process using markers specifically associated with the naive state of T cells.

[0164] CAR T cells were manufactured in static bags using a non-viral transposon approach as described in Example 4. Phenotyping data refers to the percentage of T cells expressing CD45RA, CCR7, CD62L, CD27 and CD28 relative to total T cells (Table 14). TIFF2025517371000019.tif25169

[0165] The earlier harvested cells had a higher proportion of the juvenile population, demonstrating the benefit of the accelerated process.

[0166] The effect of pre-transfection T cell state on transfection efficiency was also evaluated. + CCR7 + , CD45RA +CCR7 - , CD45RA - CCR7 + , and CD45RA - CCR7 - Four types of T cells were examined, including: naïve T cells, naïve T cells, and naïve T cells. The cells are thawed with 2 hours of recovery, then electroporated / transfected with DNA transposon and mRNA transposase. One day after transfection, the cells are activated and expanded based on the new process. The harvested cells are labeled as day 0, day 3, and day 7 based on the activation date. As shown in Table 15, naïve T cells received more gene cargo than other subsets. TIFF2025517371000020.tif33170

[0167] Example 7. Vector copy number (VCN) measurement In this example, a method was developed to measure the copy number of a construct integrated into the genome of a target cell.

[0168] Primer sets were designed to target the construct (e.g., CD19 coding sequence), the plasmid backbone region (Amp), and the host reference gene (e.g., CDKN2A). Free unintegrated DNA was digested with Dpn I. VCN experiments were performed using two separate ddPCR reactions, namely, "CD19 vs. host reference gene" and "AMP vs. host reference gene". CD19 VCN=2 * CD19 / host copy; Amp VCN=2 * Amp / host copies (multiplied by the copies per genome of the reference (2 for diploids)). The calculation incorporated was VCN=CD19 VCN-AMP VCN.

[0169] Using this method, the average VCN in cells produced by the above method was determined to be approximately 5.07 (6.07 total DNA-1.0 free undigested DNA).

[0170] Example 8. Comparison with TcBuster In this example, the performance of the transposon system as tested in Example 1 (with LVV) was compared with another non-viral, transposon-based delivery system, TcBuster. All systems used CD19 single CAR with 4-1BB as the costimulatory domain. Frozen transfected cells were thawed overnight for CAR+ percentage measurement assay and in vitro cytotoxicity assay. Results are shown in Tables 16 and 17. TIFF2025517371000021.tif28149TIFF2025517371000022.tif45170

[0171] The tumor-inhibiting efficacy of these CAR cells was tested using the Nalm6 animal model used in Examples 4 and 5. The results are shown in Table 18. TIFF2025517371000023.tif53170

[0172] Thus, these data demonstrate comparable in vitro cytotoxicity and in vivo efficacy of CAR T cells generated from either TcBuster, the transposon system of Example 1, or the LVV system.

[0173] Example 9. Scale-up manufacturing process This example developed a large-scale manufacturing process based on the small-scale pilot study in Example 4.

[0174] For small scale processes, 5×10 6 Cells were used for electroporation in a volume of 100 μL. For large-scale processes, 50 × 10 6 ~100×10 6 Cells were used in a 1 mL volume. In a comparative study, cells harvested from both the small-scale and large-scale processes were subjected to activation (with anti-CD3 antibody) and expansion for up to 7 days.

[0175] There was no significant difference between the processes in terms of cell number fold change (p=08725). However, the large-scale process showed significantly higher viability than the small-scale process using a paired t-test (p=0.0090).

[0176] Finally, transfection efficacy was also evaluated for the large-scale process compared to the small-scale process, which showed significantly higher transfection efficiency (p=0.0115) than the small-scale process using a paired t-test.

[0177] Example 10. Large payload transfection: tricistronic This example describes the separate transfection of three different large payload plasmids utilizing the Sleeping Beauty transposon system, as shown in Figure 1. Three different plasmid constructs (plasmid 1, plasmid 2, and plasmid 3) were electroporated into cells.

[0178] Plasmids 1-3 each individually encode CAR, dominant negative receptor (DNR), and membrane-bound interleukin receptor (mbIL). Plasmid 1 is a 7725 bp long tricistronic construct containing a 4835 bp insert expressed as one mRNA transcript and a single long polypeptide that is post-translationally cleaved at the T2A and P2A sites to provide CAR, DNR, and mbIL. Plasmid 2 is an 8203 bp long tricistronic construct containing a 5313 bp insert expressed as one mRNA transcript and two separate polypeptides. The first polypeptide contains CAR and DNR that is post-translationally cleaved at the T2A site. The second polypeptide is mbIL. Plasmid 3 is an 8132 bp long tricistronic construct containing a 5242 bp insert expressed as two mRNA transcripts and two polypeptides. The first polypeptide contains CAR and DNR that is post-translationally cleaved at the T2A site. The second polypeptide is mbIL. TIFF2025517371000024.tif53170

[0179] Example 11. Large payload transfection: tetracistronic This example describes the separate transfection of two different very large payload plasmids as shown in Figure 2. Two different plasmid constructs (plasmid 4 and plasmid 5) were electroporated into cells with a single pulse. Table 20 shows CAR expression after transfection with plasmid 4 with an 8 kb insert along with transposase. Table 21 shows CAR expression after transfection with plasmid 4 without the addition of transposase. Table 22 shows CAR expression after transfection with plasmid 5 with a 10 kb insert along with transposase. Table 23 shows CAR expression after transfection with plasmid 5 without the addition of transposase. TIFF2025517371000025.tif44128TIFF2025517371000026.tif44128TIFF2025517371000027.tif39128TIFF2025517371000028.tif39128

[0180] Figures 3A-C show that CAR expression was relatively proportional at day 8 after electroporation for each of the four CARs of tetracistronic plasmid 4. Figures 4A-C also show that CAR expression was relatively proportional at day 8 after electroporation for each of the four CARs of tetracistronic plasmid 5.

[0181] Example 12. Stability and restimulation This example describes CAR expression after non-viral transfection of cells with plasmid 4. On day 11 after electroporation, transfected cells were restimulated with anti-CD3 antibody. Table 24 shows CAR expression without restimulation. Table 25 shows CAR expression with restimulation on day 11. The data shows the stability of transgene expression from plasmid 4, a large payload construct. TIFF2025517371000029.tif63149TIFF2025517371000030.tif69150

[0182] Example 13. Promoter evaluation This example describes the evaluation of different promoters in transposon-encoding plasmids. The same gene cassette of interest was designed under different promoters and tested in a non-viral cell culture process to evaluate gene expression kinetics driven by different promoters. The results show that the transposon transfection system works for different promoters and provides CAR expression from single CAR and dual CAR plasmids with various promoters. Furthermore, different promoters provide different levels of CAR expression, providing a selection of CAR expression levels based on the selection of promoters. TIFF2025517371000031.tif40128TIFF2025517371000032.tif79170TIFF2025517371000033.tif80159

[0183] Example 14. Testing transgene expression in nanoplasmid vs. pCDL plasmid This example describes the evaluation of different backbone sizes to compare the expression of genes for anti-CD19 CAR after transfection by electroporation with the transposon encoding anti-CD19 CAR on pCDL plasmid and after transfection by electroporation with the same transposon on nano plasmid.The results in Table 29 show that transfection with nano plasmid achieves higher expression levels for anti-CD19 CAR.The ratio of transposon DNA to transposase mRNA is 1:4. TIFF2025517371000034.tif37167 * * *

[0184] While a number of embodiments have been described, it is apparent that the disclosure and examples may provide other embodiments that utilize or are encompassed by the compositions and methods described herein. It will therefore be appreciated that the scope of the invention should be defined not by the embodiments represented by way of example, but by what can be understood from this disclosure and the appended claims.

Claims

1. 1. A transposon comprising a transgene encoding a polypeptide comprising a first chimeric antigen receptor (CAR) and a second CAR, wherein the first CAR and the second CAR each comprise a single chain fragment (scFv), a transmembrane domain, and an immunoreceptor tyrosine-based activation motif (ITAM).

2. 2. The transposon of claim 1, wherein the transposon is a DNA transposon or a retrotransposon selected from the group consisting of Sleeping Beauty transposon, piggyBac transposon, and Tc Buster transposon.

3. The transposon of claim 2 , wherein the transposon is a Sleeping Beauty transposon or a Tc Buster transposon.

4. The transposon of any one of claims 1 to 3, wherein the transgene is at least 5000 nucleotides in length.

5. The transposon of claim 4, wherein the transgene is at least 6000 nucleotides in length.

6. The transposon of any one of claims 1 to 5, wherein the coding sequences of each ITAM in the transgene are codon-optimized such that they do not share sequence identity with each other for more than 12 consecutive nucleotides.

7. 7. The transposon of claim 6, wherein the coding sequences of each ITAM in the transgene are codon-optimized such that they do not share sequence identity with each other for more than 9 consecutive nucleotides.

8. The transposon of any one of claims 1 to 7, wherein the ITAM is a cytoplasmic signaling sequence derived from a protein selected from the group consisting of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD5, CD22, CD79a, CD79b and CD66d.

9. The transposon of claim 8, wherein the ITAM is derived from CD3ζ, CD3 epsilon, or both.

10. The transposon of any one of claims 1 to 9, wherein the first and second CARs each further comprise an intracellular costimulatory domain.

11. The intracellular costimulatory domain is selected from the group consisting of DAP-10, CD28, OX-40, 4-1BB (CD137), CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulatory factor (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), tumor necrosis factor superfamily member 14, TNFSF14, LIGHT, NKG2C, Ig alpha (CD79a), F c gamma receptor, MHC class 1 molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, CDS, GITR, BAFFR, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R Alpha, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD (CD11d), ITGAE (CD103), ITGAL (CD11a), ITGAM (CD11b), ITGAX (CD11c), I TGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE (RANKL), DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, The transposon according to claim 10, which is a signal transduction region of a protein selected from the group consisting of Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG (Cbp), CD19a, a ligand that specifically binds to CD83, and combinations thereof.

12. The transposon according to claim 2, wherein the intracellular costimulatory domain is a signal transduction region of DAP-10, 4-1BB or CD28.

13. A cell comprising the transposon according to any one of claims 1 to 12.

14. The cell according to claim 13, which is a T cell, a NK cell, a NKT cell, a monocyte, a macrophage, or a precursor cell thereof.

15. The cell of claim 13 or 14, further comprising a heterologous transposase.

16. 16. The cell of claim 15, wherein the heterologous transposase is selected from the group consisting of piggyBac® transposase, piggy-Bac®-like transposase, Super piggyBac® (SPB) transposase, piggyBac transposase, Sleeping Beauty transposase, hyperactive Sleeping Beauty (SB100X) transposase, Helitron transposase, Tol2 transposase, TcBuster transposase, or hyperactive TcBuster transposase.

17. 17. The cell of claim 16, wherein the heterologous transposase is Sleeping Beauty transposase SB100X or piggyBac transposase.

18. 1. A method for preparing transfected lymphocytes, comprising: Obtaining a sample comprising T cells from a donor subject; incubating the sample with a transposon to transfect the T cells and produce transfected T cells; culturing the sample containing the transfected T cells for less than 96 hours, and then harvesting the T cells to produce a harvested sample; The method, wherein at least 40% of the T cells in the collected sample are naive T cells.

19. 20. The method of claim 18, wherein at least 50% of the T cells in the collected sample are naive T cells.

20. The naive T cells are ＋ and CCR7 ＋ 20. The method according to claim 18 or 19, characterized as follows:

21. The naive T cells are ＋ , CD27 ＋ and CD28 ＋ 21. The method of claim 20, further characterized as:

22. The method of any one of claims 18 to 21, wherein the T cells are not activated prior to the transfection.

23. The method of any one of claims 18 to 22, wherein the T cells comprise CD4+ T cells.

24. The method according to any one of claims 18 to 23, wherein the transposon is a DNA transposon or a retrotransposon selected from the group consisting of Sleeping Beauty transposon, piggyBac transposon, and Tc Buster transposon.

25. 25. The method of claim 24, wherein the transposon is a Sleeping Beauty transposon or a Tc Buster transposon.

26. 26. The method of claim 24 or 25, wherein the transgene is at least 5000 nucleotides in length.

27. 27. The method of claim 26, wherein the transgene is at least 6000 nucleotides in length.

28. 28. The method of any one of claims 18 to 27, wherein the transposon comprises a transgene encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

29. 1. A method for preparing lymphocytes expressing a chimeric antigen receptor (CAR) or a T cell receptor (TCR), comprising: Obtaining a biological sample comprising lymphocytes from a human subject; introducing into the lymphocyte a transposon comprising a transposase and a transgene encoding a polypeptide comprising the CAR or the TCR; and harvesting lymphocytes containing the transposase and transposon, The method, wherein said lymphocyte collection is performed within 96 hours after obtaining said biological sample.

30. 30. The method of claim 29, wherein the transposase and transposon are introduced into the lymphocytes by electroporation, nucleofection, lipofection, ultrasound, or magnetofection.

31. 30. The method of claim 29, wherein the transposase and transposon are introduced into the lymphocytes by electroporation.

32. The method according to any one of claims 29 to 31, wherein said lymphocyte collection is carried out within 72 hours after obtaining said biological sample.

33. The method according to any one of claims 29 to 31, wherein said lymphocyte collection is carried out within 48 hours after obtaining said biological sample.

34. The method according to any one of claims 29 to 31, wherein said lymphocyte collection is carried out within 36 hours after obtaining said biological sample.

35. The method according to any one of claims 29 to 31, wherein said lymphocyte collection is carried out within 24 hours after obtaining said biological sample.

36. 36. The method of any one of claims 29 to 35, further comprising cryopreserving the harvested lymphocytes or injecting the harvested lymphocytes into a patient, wherein the cryopreservation or injection occurs within 48 hours of obtaining the biological sample.

37. The method of any one of claims 29 to 36, wherein the method does not involve lymphocyte activation.

38. The method of any one of claims 29 to 37, wherein the method does not include lymphocyte selection or lymphocyte expansion.

39. The method of any one of claims 29 to 38, wherein the lymphocytes are NK cells.

40. The method of any one of claims 29 to 38, wherein the lymphocytes are T cells.

41. 41. The method of claim 40, wherein at least 40% of the T cells having the transgene integrated into their genome are naive T cells when harvested.

42. The naive T cells are ＋ and CCR7 ＋ 42. The method of claim 41 , wherein:

43. 36. The method of any one of claims 29 to 35, wherein the lymphocytes, when harvested, contain more naive T cells compared to a comparable process in which the lymphocyte harvesting is performed 168 hours after obtaining the biological sample.

44. 44. The method of any one of claims 29 to 43, wherein the polypeptide further comprises a second CAR, the first CAR and the second CAR each comprising a single chain fragment (scFv), a transmembrane domain, and an immunoreceptor tyrosine-based activation motif (ITAM).

45. The method according to any one of claims 29 to 44, wherein the transposon is a DNA transposon or a retrotransposon selected from the group consisting of Sleeping Beauty transposon, piggyBac transposon, and Tc Buster transposon.

46. 46. ​​The method of claim 45, wherein the transposon is a Sleeping Beauty transposon or a Tc Buster transposon.

47. The method of any one of claims 29 to 46, wherein the transgene is at least 5000 nucleotides in length.

48. The sample subjected to transfection has at least 25×10 ６ The method of any one of claims 18 to 47, comprising cells.

49. The sample subjected to transfection has at least 50×10 ６ 49. The method of claim 48, comprising cells.

50. 50. The method of claim 48 or 49, wherein the transfection is carried out in a solution having a volume of between 0.1 mL and 100 mL.

51. 51. The method of any one of claims 48 to 50, wherein said transfection transfects at least 40% of T cells or lymphocytes in said sample.

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