Non-viral delivery of cell therapy constructs

Abstract

The present disclosure provides transposon-based systems for introducing cell therapeutic products, such as CAR and TCR, into target immune cells. The transposon-based system can carry a larger payload, simplify multi-gene editing, and reduce undesired recombination between homologous sequences in the payload compared to conventional viral vector-based techniques. Also provided are shortened autologous processes that can be completed within several days, one day, or even several hours. Even if immune cells are not activated, enriched or expanded, the resulting cell population achieves a higher in vivo therapeutic efficacy than a longer autologous process using a viral vector.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 346,547 filed on May 27, 2022 and U.S. Provisional Patent Application No. 63 / 492,110 filed on March 24, 2023, both of which are hereby incorporated by reference in their entirety. Background Art

[0003] Cell therapy uses enriched or modified human immune cells to target and kill cancer cells in a patient. 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 immune cells to specific target cancer cells. Chimeric antigen receptors (CARs) and engineered T cell receptors (TCRs) that include binding domains capable of interacting with specific tumor antigens allow immune cells to target and kill cancer cells that express specific tumor antigens.

[0004] The main challenge in making CAR or TCR cells is to transfer the coding sequence to the target immune cells. Currently, the vast majority of CAR T cell production relies on the transfer of viral vectors. Combining retroviral genes with inducible promoters can increase the transduction rate and produce relatively large numbers of CAR-containing T cells.

[0005] The murine leukemia virus (MLV) based vector is the most commonly used gamma retroviral vector. However, the use of MLV has been associated with T cell related leukemias. Vectors from another retroviral family, lentiviruses, have shown better integration efficiency. Lentiviral vectors are able to target non-dividing cells, which has been a major challenge for MLV based vectors.

[0006] Viral vectors are commonly used for CAR T cell production, but significant challenges remain. For example, viral vectors are inherently associated with carcinogenic and mutagenic potential. In addition, the use of viruses in current good manufacturing practice (cGMP) laboratories is strictly regulated. Equally important, lentiviral / retroviral transduction is limited by the size of the viral capsid, thus placing a limit on the size of the transgene. In addition, the size of the transgene may be limited by the effects of the gene of interest and / or multi-gene expression on the physiology of the viral vector itself, including potential genotoxicity to the virus caused by the transgene.

[0007] However, another major challenge is the highly complex autologous cell engineering and production process, which usually takes at least a week and can take up to several weeks. During this process, lymphocytes collected from the patient must be transported to a processing center, and the produced cells must be cryopreserved and then transported back to the patient for transplantation. This highly complex process inevitably leads to high costs and limited clinical applications. Summary of the invention

[0008] In various embodiments, the present disclosure provides a transposon-based system for introducing cell therapy products, such as CAR and TCR, into target immune cells. Compared with conventional viral vector-based technologies, the transposon-based system can carry a larger payload and can reduce undesired recombination between homologous sequences in the payload. In some aspects, the transposon-based system simplifies multi-gene editing to a one-step transfection. A shortened autologous process that can be completed within a few days or even within the same day is also provided. In some aspects, the shortened autologous process can be completed within 4 hours. Even without immune cell activation, enrichment or amplification, the resulting cell population obtains a higher in vivo therapeutic efficacy than a longer autologous process using a viral vector.

[0009] This shortened process can be easily introduced into a closed automated device and performed at a preferred location (e.g., a clinical site) rather than at a centralized manufacturing site. This is impractical for conventional autologous techniques. Two modes of transportation required by conventional techniques can be skipped. Equally important, the freezing / thawing steps required for transportation can also be eliminated, which will further improve both patient care and product quality.

[0010] According to one embodiment of the present disclosure, a transposon comprising a transgene encoding a polypeptide is provided, the 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).

[0011] In some embodiments, the transposon is a DNA transposon selected from the group consisting of a Sleeping Beauty transposon, a piggyBac transposon, and a Tc Buster transposon, or a retrotransposon, such as an R2 transposon. In some embodiments, the transposon is a Sleeping Beauty transposon.

[0012] 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 greater than 10 kb in length.

[0013] In some embodiments, the coding sequence of each ITAM in the transgene is codon optimized so that they do not have 12 consecutive nucleotides or longer sequence identity with each other. In some embodiments, the coding sequence of each ITAM in the transgene is codon optimized so that they do not have 9 consecutive nucleotides or longer sequence identity with each other.

[0014] In some embodiments, the ITAM is a cytoplasmic signaling sequence of a protein selected from the group consisting of: TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the ITAM is CD3ζ.

[0015] In another embodiment, a cell comprising a transposon of the present disclosure is also provided. 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 (eg, iPSC).

[0016] In some embodiments, the cell further comprises a transposase. In some embodiments, the transposase is selected from the group consisting of: Transposase, piggy- Like transposase, Super (SPB) transposase, ppiggyBat 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.

[0017] Another embodiment provides a method for preparing transfected lymphocytes, the method comprising obtaining a sample containing T cells from a donor subject; incubating the sample with a transposon to transfect the T cells, thereby producing transfected T cells; and culturing the sample containing the transfected T cells for less than 96 hours before 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.

[0018] In some embodiments, the naive T cells are characterized as CD45RA+ and CCR7+. In some embodiments, the naive T cells are further characterized as CD62L+. + 、CD27 + and CD28 + .

[0019] In some embodiments, the T cells are not activated prior to transfection. In some embodiments, the T cells are not enriched prior to transfection.

[0020] In some embodiments, the transgenic gene is at least 5000 nucleotides in length. In some embodiments, the transgenic gene is at least 6000 nucleotides in length. In some embodiments, the transgenic gene is greater than 10 kb in length. In some embodiments, the transposon comprises a transgenic gene encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0021] 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: collecting a biological sample comprising lymphocytes from a human subject; introducing a transposase and a transposon into the lymphocytes, the transposon comprising a transgene encoding a polypeptide comprising a CAR or a TCR; and harvesting the lymphocytes comprising the transposase and the transposon, wherein the harvesting of the lymphocytes occurs within 96 hours of collecting the biological sample.

[0022] In some embodiments, the transposase and the transposon are introduced into the lymphocyte by a physical delivery method, such as electroporation, nucleofection, lipofection, ultrasound, or magnetofection. In some embodiments, the transposase and the transposon are introduced into the lymphocyte by electroporation. The transposase can be delivered to the cell as DNA, mRNA, or protein.

[0023] In some embodiments, the lymphocytes are harvested within 48 hours after the biological sample is collected. In some embodiments, the method further comprises cryopreserving the harvested lymphocytes, or injecting the harvested lymphocytes into a patient, wherein the cryopreservation or the injection occurs within 48 hours after the biological sample is collected.

[0024] 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 expansion. In some embodiments, the harvesting of the lymphocytes occurs within 36 hours after the biological sample is collected.

[0025] 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, when harvested, at least 40% of the T cells that have the transgene integrated into their genome are naive T cells. In some embodiments, the naive T cells are CD45RA + and CCR7 + .

[0026] In some embodiments, the polypeptide further comprises a second CAR, and the first CAR and the second CAR each comprise a single-chain fragment (scFv), a transmembrane domain, and an activation motif (ITAM) based on immunoreceptor tyrosine. In some embodiments, the polypeptide comprises a total of three or four CARs, each of which comprises scFv, a transmembrane domain, and an ITAM. In some embodiments, the transposon comprises two or more transgenes, which together encode two, three, four, or more CARs.

[0027] In some embodiments, the transposon is a DNA transposon selected from the group consisting of a Sleeping Beauty transposon, a piggyBac transposon, and a 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 greater than 10 kb in length.

[0028] In some embodiments, the sample undergoing transfection contains at least 25×10 6 In some embodiments, the sample undergoing transfection contains at least 50×10 6 In some embodiments, the transfection is performed in a solution having a volume of 0.5 mL to 2 mL. In some embodiments, the transfection results in at least 40% of the T cells or lymphocytes in the sample being transfected. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a schematic diagram depicting three different plasmid constructs: Plasmid 1 (7725 bp, including a 4835 bp insert), Plasmid 2 (8203 bp, including a 5313 bp insert), and Plasmid 3 (8132 bp, including a 5242 bp insert).

[0030] Figure 2 is a schematic diagram depicting two different large payload plasmid constructs: Plasmid 4 (10929 bp, including 8038 bp insert) and Plasmid 5 (12754 bp, including 9863 bp insert).

[0031] FIG. 3A to FIG. 3C A graph showing CAR expression at day 8 after electroporation of plasmid 4 is shown.

[0032] FIG. 4A to FIG. 4C A graph showing CAR expression at day 8 after electroporation of plasmid 5 is shown. DETAILED DESCRIPTION

[0033] definition

[0034] In order to more easily understand the present disclosure, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout the specification.

[0035] Unless otherwise stated or obvious from the context, as used herein, the term "or" is to be construed as inclusive and encompasses both "or" and "and."

[0036] As used herein, the term "and / or" should be considered 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 in phrases such as "A and / or B" herein is intended to include A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: 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 (alone); B (alone); and C (alone).

[0037] Unless otherwise specified or apparent from the context, the term "about" refers to a value or composition within an acceptable error range of a particular value or composition as determined by a person of ordinary skill in the art, which will depend in part on how to measure or determine the value or composition, i.e., the limitations of the measurement system. For example, according to the practice of the art, "about" or "consisting essentially of" may mean within one or more than one standard deviation. "About" or "consisting essentially of" may mean a range of up to 10% (i.e., ± 10%). Therefore, "about" may be understood as being 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 value. For example, about 5 mg may include any amount between 4.5 mg and 5.5 mg. In addition, particularly with respect to a biological system or process, these terms may mean up to an order of magnitude or up to 5 times of a certain value. When a specific value or composition is provided in the present disclosure, unless otherwise stated, the meaning of "about" or "consisting essentially of should be assumed to be within an acceptable error range for the specific value or composition.

[0038] "Administering" refers to the use of any of the various methods and delivery systems known to those skilled in the art to physically introduce a medicament into a subject, such as modified T cells disclosed herein. Exemplary routes of administration for preparations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration (e.g., by injection or infusion). The phrase "parenteral administration" means a mode of administration (usually by injection) in addition to enteral and topical administration, and includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, the preparation is administered via a non-parenteral route (e.g., oral). Other non-parenteral routes include topical, epidermal or mucosal routes of administration, such as intranasal, vaginal, rectal, sublingual or topical. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0039] The term "allogeneic" refers to any material derived from one individual and subsequently introduced into another individual of the same species, such as an allogeneic T cell transplant.

[0040] The term "antibody" (Ab) includes, but is not limited to, glycoprotein immunoglobulins that specifically bind to antigens. Generally speaking, an antibody may include at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or their antigen-binding molecules. 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 a constant domain CL. The VH region and the VL region can be further subdivided into hypervariable regions (called complementary determining regions (CDRs)), with more conservative regions (called framework regions (FRs)) interspersed therebetween. Each VH and VL includes three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains include binding domains that interact with antigens. The constant region of Ab can mediate the binding of immunoglobulins 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 speaking, human antibodies are tetrameric agents of about 150 kD, which are composed of two identical heavy (H) chain polypeptides (each about 50 kD) and two identical light (L) chain polypeptides (each about 25 kD), which are associated with each other into a structure commonly referred to as a "Y-shaped" structure. The heavy chain and the light chain are linked or connected to each other by a single disulfide bond; the other two disulfide bonds connect the heavy chain hinge regions to each other, so that the dimers are connected to each other and form a tetramer. Naturally produced antibodies are also glycosylated, for example, glycosylated on the CH2 domain.

[0041] "Antigen binding molecule", "antigen binding portion", "antigen binding fragment", "antibody fragment" refers to any molecule comprising an antigen binding portion (e.g., CDR) of an antibody from which the molecule is derived. Antigen binding molecules may include antigen complementary determining regions (CDRs). 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 by antigen binding molecules. Peptibodies (i.e., Fc fusion molecules comprising peptide binding domains) are another example of suitable antigen binding molecules. In some embodiments, antigen binding molecules bind to antigens on tumor cells. In some embodiments, antigen binding molecules bind to antigens on cells involved in hyperproliferative diseases or viral or bacterial antigens. In certain embodiments, the antigen binding molecule is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR).

[0042] The term "variable region" or "variable domain" is used interchangeably. The variable region generally refers to a part of an antibody, generally a part of a light chain or a heavy chain, generally about 110 to 120 amino acids at the amino terminal in a mature heavy chain and about 90 to 115 amino acids in a mature light chain, which are widely different in sequence between antibodies and are used for the binding and specificity of a specific antibody to its specific antigen. The variability of the sequence is concentrated in those regions called complementary determining regions (CDRs), while the more highly conserved regions in the variable domains are called framework regions (FRs). It is not desirable to be bound by any particular mechanism or theory, and it is believed that the CDRs of light and heavy chains are primarily responsible for the interaction and specificity of antibodies with antigens. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region includes rodent or mouse CDRs and human framework regions (FRs). In specific embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region includes rodent or mouse CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0043] 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.

[0044] 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.

[0045] Many definitions of CDRs are commonly used: Kabat numbering, Chothia numbering, AbM numbering, or Contact numbering. The AbM definition is a compromise between the two definitions used by Oxford Molecular's AbM antibody modeling software. The Contact definition is based on analysis of available complex crystal structures.

[0046] The term "autologous" refers to any material that originates from the same individual and is later reintroduced into that individual. For example, the engineered autologous cell therapy (eACT) described herein TM ) method involves collecting lymphocytes from a patient, then engineering them to express, for example, a CAR construct, and subsequently administering them back to the same patient.

[0047] "Chimeric antigen receptor" or "CAR" refers to a molecule engineered to include a binding motif and a means of activating immune cells (e.g., T cells, such as naive T cells, central memory T cells, effector memory T cells, or a combination thereof) after antigen binding. CAR is also referred to as an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor. In some embodiments, CAR comprises a binding motif, an extracellular domain, a transmembrane domain, one or more co-stimulatory domains, and an intracellular signaling domain. T cells that have been genetically engineered to express a chimeric antigen receptor can be referred to as CAR T cells. "Extracellular domain" (or "ECD") refers to a portion of a polypeptide, which, when present in a cell membrane, is understood to be located outside the cell membrane, in the extracellular space.

[0048] "T cell receptor" or "TCR" refers to an antigen recognition molecule present on the surface of a T cell. During normal T cell development, each of the four TCR genes, α, β, γ, and δ, can rearrange, resulting in a highly diverse set of TCR proteins.

[0049] The term "heterologous" refers to any source other than a naturally occurring sequence. For example, a heterologous sequence included as part of a costimulatory protein is an amino acid that does not occur naturally, i.e., is not identical to a wild-type human costimulatory protein. For example, a heterologous nucleotide sequence refers to a nucleotide sequence that is different from a wild-type human costimulatory protein coding sequence.

[0050] The term "identity" refers to the overall correlation between polymeric molecules, such as nucleic acid molecules (for example, DNA molecules and / or RNA molecules) and / or polypeptide molecules. The method for calculating the percentage identity between two provided polypeptide sequences is known. For example, the calculation of the percentage identity of the two sequences can be performed by comparing two nucleic acids or polypeptide sequences for the best comparison purpose (for example, a room can be introduced in one or both of the first sequence and the second sequence for the best comparison, and different sequences can be ignored for the purpose of comparison). Then the nucleotides or amino acids at the corresponding positions are compared. When the position in the first sequence is occupied by the same residue (for example, nucleotides or amino acids) as the corresponding position in the second sequence, the molecules are the same at this position. The percentage identity between the two sequences is a function of the number of identical positions shared by these sequences, and optionally considering the number of rooms and the length of each room, it may be necessary to introduce a room for the best comparison of the two sequences. The comparison or comparison of sequences and the determination of the percentage identity between the two sequences can be realized using a mathematical algorithm such as BLAST (basic local alignment search tool). In some embodiments, polymer molecules are considered "homologous" to each other 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% identical).

[0051] The immune cells of immunotherapy can be from any source known in the art. For example, immune cells can be differentiated from a hematopoietic stem cell population in vitro, or immune cells can be obtained from a subject. Immune cells can be obtained from, for example, peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In addition, immune cells can be derived from one or more immune cell lines available in the art. A variety of techniques known to technicians (such as FICOLL TM Immune cells are obtained from a unit of blood collected from a subject by separation and / or apheresis. Additional methods for isolating immune cells for immune cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0052] "Patient" includes any person suffering from cancer (eg, lymphoma or leukemia). The terms "subject" and "patient" are used interchangeably herein.

[0053] The term "pharmaceutically acceptable" refers to a molecule or composition that is harmless to the recipient when applied to the recipient, or any harmful effect is outweighed by the beneficial effect to the recipient. In terms of the carrier, diluent or excipient used to prepare the composition as disclosed herein, the pharmaceutically acceptable carrier, diluent or excipient must be compatible with the other ingredients of the composition and harmless to the recipient, or any harmful effect must be outweighed by the beneficial effect to the recipient. The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle that involves carrying or transporting an agent from one part of the body to another part (e.g., from one organ to another), such as a liquid or solid filler, diluent, excipient or solvent encapsulating material. Each carrier present in the pharmaceutical composition must be "acceptable" in the following sense: compatible with the other ingredients of the preparation and harmless to the patient, or any harmful effect must be outweighed by the beneficial effect to the recipient. Some examples of materials that can serve as pharmaceutically 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; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, 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; ethanol; pH buffered solutions; polyesters, polycarbonates, and / or polyanhydrides; and other nontoxic compatible substances employed in pharmaceutical formulations.

[0054] The term "pharmaceutical composition" refers to a composition in which an active agent is formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that, when administered to a relevant subject or population, shows a statistically significant probability of achieving a predetermined therapeutic effect. In some embodiments, the pharmaceutical composition can be formulated for administration in solid or liquid form, including but not limited to forms suitable for the following: oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those targeted for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue; parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection, as, for example, sterile solutions or suspensions, or sustained-release formulations; topical application, such as as a cream, ointment, or controlled-release patch, or a spray applied to the skin, lungs, or mouth; intravaginal or rectal administration, for example, as a vaginal suppository, cream, or foam; sublingual administration; ophthalmic administration; transdermal administration; or nasal, pulmonary, and other mucosal surfaces.

[0055] The terms "reduce" and "reduction" are used interchangeably herein and indicate any change that is less than the original value. "Reduce" and "reduction" are relative terms requiring a comparison between before and after measurement. "Reduce" and "reduction" include complete depletion.

[0056] The term "reference" describes a standard or control relative to which a comparison is made. For example, in some embodiments, an agent of interest, an animal, an individual, a colony, a sample, a sequence or a value is compared with a reference or control as an agent, an animal, an individual, a colony, a sample, a sequence or a value. In some embodiments, a reference or control is tested, measured and / or measured substantially simultaneously with a test, measurement or determination of interest. In some embodiments, a reference or control is a historical reference or control, which is optionally embodied in a tangible medium. Generally speaking, a reference or control is measured or characterized under conditions or circumstances comparable to the conditions or circumstances of the assessment. When there are enough similarities to prove that dependence and / or comparison to the selected reference or control is correct.

[0057] 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.

[0058] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a therapeutic agent (e.g., engineered CAR T cells) is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of disease or promotes disease regression (as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic disease periods, or the prevention of disorders or disabilities resulting from the disease). The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by measuring the activity of the agent in an in vitro assay.

[0059] The terms "transduction" and "transduced" refer to the process of introducing foreign DNA into cells 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 papillomavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

[0060] 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 (PB) Transposon, piggy- In some other aspects, the transposon can be integrated into the genome of the cell by a corresponding transposase. In some aspects, the transposase can be Transposase, piggy- Like transposase, Super (SPB) transposase, ppiggyBat transposase, Sleeping Beauty transposase, Hyperactive Sleeping Beauty (SB100X) transposase, Helitron transposase, Tol2 transposase, TcBuster transposase or Hyperactive TcBuster transposase, but not limited thereto. Helitron transposase may be Helibat1 transposase.

[0061] "Treatment" or "treatment" of a subject refers to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, with the purpose of reversing, alleviating, improving, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of symptoms, complications or conditions or biochemical indicators associated with a disease. In one embodiment, "treatment" includes partial relief. In another embodiment, "treatment" or "treatment" includes complete relief. In some embodiments, treatment may be treatment of subjects who do not show signs of the relevant disease, disorder and / or condition and / or subjects who only show early signs of the disease, disorder and / or condition. In some embodiments, such treatment may be treatment of subjects who show one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment may be treatment of subjects who have been diagnosed with the relevant disease, disorder and / or condition. In some embodiments, treatment may be treatment of subjects who are known to have one or more susceptibility factors that are statistically associated with an increased risk of the development of the relevant disease, disorder and / or condition.

[0062] The term "vector" refers to a receptor nucleic acid molecule modified to include or incorporate a provided nucleic acid sequence. One type of vector is a "plasmid", which refers to a circular double-stranded DNA molecule to which additional DNA can be connected. Another type of vector is a viral vector, in which additional DNA segments can be connected to a viral genome. Some vectors can replicate autonomously in the host cells they are introduced into (e.g., bacterial vectors with bacterial replication origins, and additional mammalian vectors). Other vectors (e.g., non-additional mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, and thus replicated with the host genome. In addition, some vectors include sequences that guide the expression of inserted genes operably connected to them. Such vectors may be referred to as "expression vectors" herein. Standard techniques may be used for vector engineering, for example, as present in Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), the document is incorporated herein by reference.

[0063] Non-viral vectors

[0064] As provided, the production of CAR and TCR cells with viral vectors is accompanied by high processing costs, inherent safety risks, and restrictions on the size of the coding sequence. It is generally believed 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. This may be sufficient for a conventional CAR or TCR coding sequence, but it is hardly enough for a bicistronic coding sequence, not to mention the enhancement elements that are urgently needed to further improve the efficacy and safety of conventional CARs or TCRs.

[0065] Many non-viral methods are under study. However, the inventors here demonstrate that the transposon system can overcome the various challenges faced by viral vector-based systems. First, the transposon system can overcome the physical limitations of viral vectors on payload size. Several tested DNA transposons can support payloads greater than 10kb, allowing the integration of multiple CARs and / or enhancing elements, which may help enhance therapeutic cell products. In some aspects, the transposon-based system simplifies multi-gene editing to a one-step transfection.

[0066] Second, when two or more CARs are used together, they are likely to share a long string of similar sequences. For example, the two CARs in a bicistronic CAR may include the same immunoreceptor tyrosine-based activation motif (ITAM), such as CD3ζ. Such homology can cause recombination, resulting in the deletion or truncation of either or both CARs. Such recombination has indeed been observed in lentiviral systems. With the increase in the complexity of genetic engineering, the risk of recombination problems will increase.

[0067] As shown in Example 1, even when the homology between the two copies of CD3ζ was minimized by codon optimization and sequence wiggling, there was still a lot of recombination after lentiviral transduction, as indicated by the expression of multiple variants of the protein. However, the use of the transposon system in Example 1 resulted in a reduction in such recombination and completely eliminated the most disruptive recombination (e.g., variants V1 and V15 with large truncations / deletions; see, e.g., Table 1).

[0068] At the same time, the transposon system can serve as an alternative to viral vectors by providing similar transfection efficiency, cell viability, and integration stability (see, e.g., Tables 2 to 8).

[0069] Another challenge of conventional autologous cell therapy based on viral vectors is the long processing time and high cost associated with them. A typical autologous process based on lentiviral vectors takes about 7-10 days. The key steps of such conventional methods include T cell activation, T cell enrichment and amplification. However, the inventors were able to develop a transposon-based method that can be completed within 24 hours without the need for T cell activation, T cell enrichment or amplification.

[0070] Surprisingly, even at lower doses, such greatly accelerated processes achieved greater in vivo efficacy than conventional 7-day lentiviral vector-based processes at higher doses (Examples 4 and 5). The data presented in the Examples (e.g., Tables 9 and 14) indicate that the shortened process results in a higher concentration of naive T cells being harvested, which contributes to improved therapeutic efficacy.

[0071] Equally important, as shown in Tables 12 and 13, transfection of these transposons resulted in relatively modest cell death, and the transfected T cells were able to expand rapidly.

[0072] In Example 8, a comparison was made between Sleeping Beauty and another transposon system, TcBuster. The results showed that both transposon systems were able to generate highly effective CAR-T cells. In the Nalm6 animal model, CAR-T cells prepared with the Sleeping Beauty transposon system and the TcBuster transposon system or with lentiviral vectors were able to effectively inhibit tumor growth for at least 20 days.

[0073] Based on these successful process developments, the inventors were also able to develop a large-scale process that utilizes 50×10 6 Up to 100×10 6 cells (applicable to billions of cells) (compared to 5 × 10 6 Interestingly, not only did the larger scale not reduce the efficiency or quality of the process, but it actually significantly improved cell viability and transfection efficiency.

[0074] Therefore, according to one embodiment of the present disclosure, a transposon is provided, the transposon comprising a transgene encoding one or more polypeptides, the polypeptide comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some aspects, the transgene encodes two, three, four, five, six, seven, eight, nine or ten different polypeptides, wherein one or more polypeptides are chimeric antigen receptors (CAR) or T cell receptors (TCR).

[0075] In some embodiments, the transgenic gene is at least 500 nucleotides in length. In some embodiments, the transgenic gene 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 other embodiments, the transgenic gene may be greater than 20,000 nucleotides in length. In some embodiments, the transgenic gene is no longer than 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 20,000, 25,000, or 30,000 nucleotides in length.

[0076] In some embodiments, the transgene includes 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 each in a different CAR sequence or TCR sequence.

[0077] The embodiment of such fragments is an activation motif (ITAM) based on immunoreceptor tyrosine. ITAM is usually found in the cytoplasmic tail of non-catalytic tyrosine phosphorylation receptors, which are cell surface proteins mainly found on immune cells. It is a component that starts multiple signal transduction pathways and subsequently activates immune cells. Non-limiting examples of ITAM include cytoplasmic signal transduction sequences of proteins, such as TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b and CD66d. A specific example is CD3 ζ.

[0078] Such homology may also be found in other parts of the CAR sequence or TCR sequence, such as linkers, extracellular hinges, transmembrane domains, intracellular signaling domains, or even antigen binding sequences, without limitation.

[0079] In some embodiments, homologous fragments are codon optimized to reduce sequence identity without changing the encoded protein. In some embodiments, homologous fragments are codon optimized so that they do not share 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9 or 8 continuous nucleotides or longer sequence identities. In some embodiments, homologous fragments are codon optimized so that they do not share 15 continuous nucleotides or longer sequence identities. In some embodiments, homologous fragments are codon optimized so that they do not share 12 continuous nucleotides or longer sequence identities. In some embodiments, homologous fragments are codon optimized so that they do not share 10 continuous nucleotides or longer sequence identities. In some embodiments, homologous fragments are codon optimized so that they do not share 9 continuous nucleotides or longer sequence identities. In some embodiments, homologous fragments are codon optimized so that they do not share 8 continuous nucleotides or longer sequence identities.

[0080] In some embodiments, transgenic encoding the first CAR (or TCR) and the second CAR (or TCR), which can be optionally cascaded via a protease cleavage site (e.g., a self-cleavage site) or a ribosome skipping sequence. In some embodiments, the first CAR and the second CAR each include a single-chain fragment (scFv), a transmembrane domain, and an activation motif (ITAM) based on immunoreceptor tyrosine.

[0081] 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 CAR, the third CAR, or the third CAR (or TCR) can be cut at a protease cleavage site (e.g., a self-cleavage site) or a ribosomal skipping sequence to form a separate CAR (TCR).

[0082] In some embodiments, the transposon includes two, three or four CAR (or TCR) coding sequences, but they do not have to be cascaded. Instead, each coding sequence can have its own promoter or other required regulatory sequences.

[0083] Transposon

[0084] The transposon used in the present technology can be any transposon known in the art, such as (PB) Transposon, piggy- transposon, piggyBat transposon, Sleeping Beauty transposon, Helaiser transposon, Tol2 transposon or TcBuster transposon.

[0085] The transposon can be integrated into the cell's genome by the corresponding transposase. This integration can be transient or stable. Depending on the time of measurement, transgene expression can be transient or stable. The transposase can be Transposase, piggy- Like transposase, Super (SPB) transposase, piggyBat transposase, Sleeping Beauty transposase, Hyperactive Sleeping Beauty (SB100X) transposase, Helitron transposase, Tol2 transposase, TcBuster transposase or Hyperactive TcBuster transposase, but not limited thereto. Helitron transposase may be Helibat1 transposase.

[0086] When the transposon is When a transposon is present, the transposase can be Transposase or Super Transposase. When the transposon is piggy- When the transposon is a piggy- When the transposon is a Sleeping Beauty transposon, the transposase may be a Sleeping Beauty transposase. When the transposon is a Helaizer transposon, the transposase may be a Helitron transposase. When the transposon is a Tol2 transposon, the transposase may be a Tol2 transposase. When the transposon is a TcBuster transposon, the transposase may be a TcBuster transposase or a hyperactive TcBuster transposase.

[0087] In one embodiment, the transposon is a Sleeping Beauty transposon. The Sleeping Beauty transposon may include a nucleic acid flanked by inverted repeat sequences that are recognized by an enzyme having Sleeping Beauty transposase activity. "Recognized" means that the Sleeping Beauty transposase is able to 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 repeats that can be found in the Sleeping Beauty transposon of the subject method include those disclosed in WO 98 / 40510 and WO 99 / 25817. Of particular interest are inverted repeat sequences recognized by a transposase that has at least about 80% amino acid identity with SEQ ID NO: 1 of WO 99 / 25817.

[0088] In some embodiments, each reverse repeat of a transposon includes at least one forward repeat. A transposon element is a linear nucleic acid fragment that can be used as a linear fragment or is cyclized, such as in a plasmid. Alternatively, a carrier such as a circular plasmid, a mini-circle plasmid, and a nanoplasmid can be used. In some aspects, the backbone length of the encoding transposon can be 100bp to 4kb. The transposon carrying a gene of interest can be delivered in various DNA forms (plasmid, plasmid surrogate, linear DNA, or ds / ssDNA). In certain embodiments, two forward repeats are present in each reverse repeat sequence. For example, examples of forward repeat sequences can be found in WO 98 / 40510. Miniplasmids are supercoiled DNA molecules with a length of about 4,000bp (for example, between 3000bp and 5000bp). In one embodiment, a plasmid is a linear plasmid. In one embodiment, a plasmid is a circular plasmid. In one embodiment, a plasmid is a nanoplasmid. In one embodiment, a plasmid is a mini-circle plasmid. As shown in the examples, minicircle plasmids facilitate high transfection efficiencies.

[0089] The Sleeping Beauty transposon can be integrated into the target genome via the corresponding transposase, such as SB10 and SB100X described in, for example, WO 2017 / 158029.

[0090] CAR sequence and TCR sequence

[0091] Examples of CAR sequences and TCR sequences are also provided.CAR includes an antigen binding portion, which is typically a single-chain fragment (scFv) derived from an antibody.CAR can be monospecific, bispecific or multispecific.In some aspects, the transposon-based system simplifies multi-gene editing to a one-step transfection.In addition, the construct (or payload) carried by the transposon disclosed herein can include two or more CAR molecules.Specific examples are bicistronic CARs, in which two CARs are connected by digestible linkers or ribosomal skipping sequences.Similarly, bicistronic TCRs can also be included in the payload.In some aspects, the construct (or payload) carried by the transposon disclosed herein can encode three CARs.In some aspects, the construct (or payload) carried by the transposon disclosed herein can encode four or more CARs.In some other aspects, the construct (or payload) carried by the transposon disclosed herein can further encode a polypeptide that enhances the cytotoxic potential of T cells expressing one or more CARs.

[0092] In certain embodiments, the bicistronic, tricistronic, or tetracistronic CAR encoding construct includes a first CAR targeting a first antigen and a second CAR targeting a second antigen. The first and second antigens can 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-11α, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-1, mutated p53, mutated ras, prostate-specific antigen, ROR1, VEGFR2, or a combination thereof.

[0093] In some embodiments, the first, second, third, or fourth CAR targeting antigen is one of the following: 2B4 (CD244), 4-1BB, 5T4, A33 antigen, adenocarcinoma antigen, adrenergic receptor beta 3 (ADRB3), A kinase anchoring protein 4 (AKAP-4), alpha-fetoprotein (AFP), anaplastic lymphoma kinase (ALK), androgen receptor, B7H3 (CD276), beta 2-integrin, BAFF, B lymphoma cells, B cell maturation antigen (BCMA), bcr-abl (oncogene fusion consisting of breakpoint cluster region (BCR) and Aberson murine leukemia virus oncogene homolog 1 (Abl) protein), BhCG, bone marrow stromal cell antigen 2 (BST2), CCCTC-binding factor (zinc finger protein)-like (BORIS or imprinting site regulator brother), 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 (TNF RSF8), CD33, CD34, CD38, CD40, CD40L, CD41, CD44, CD44V6, CD49f, CD51, CD52, CD56, CD63, CD70, CD72, CD74, CD79a, CD79b, CD80, CD84, CD 96. CD97, CD100, CD123, CD125, CD133, CD137, CD138, CD150, CD152(CTLA-4), CD160, CD171, CD179a, CD200, CD221, CD229, CD244, CD272(B TLA), CD274 (PDL-1, B7H1), CD279 (PD-1), CD352, CD358, CD300 molecule-like family member f (CD300LF), carcinoembryonic antigen (CEA), occludin 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 known as CD2 subclass 1, CRACC, CD319 and 19A24), CTLA-4, cyclin B l, chromosome X open reading frame 61 (CXORF61), cytochrome P450 1B1(CYP1B1), DNAM-1(CD226), desmoglein 4, DR3, DR5, E-cadherin neo-epitope, epidermal growth factor receptor (EGFR), EGF1R, epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), mucin-like hormone receptor-like 2 containing an EGF-like module (EMR2), elongation factor 2 mutant (ELF2M), 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 variant 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 (TNFRSF 18), GM1, ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), GP 100, hexose portion of globoH glycoceramide (GloboH), glycoprotein 75, phosphatidylinositol proteoglycan 3 (GPC3), glycoprotein 100 (gp100), 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 (HPV E7), heat shock protein 70-2 mutant (muthsp70-2), 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 α (IL-1 lRa), 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 (CD117), KLRGI, LAGE-la, LAG3, lymphocyte-specific protein tyrosine kinase (LCK), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), legumin, 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 K 9 (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 recognized by T cells 1 (MelanA or MARTI), MelanA / MART1, mesothelin, MAGE A3, melanoma cell apoptosis inhibitor (ML-IAP), melanoma-specific chondroitin sulfate proteoglycan (MCSCP), MORAb-009, MS4A1, mucin 1 (MUCl), MUC2, MUC3, MUC4, MUC5AC, MUC5b, MUC7, MUC16, mucin CanAg, Müllerian inhibitory substance (MIS) type II receptor, v-myc avian myelocytic tumor viral oncogene neuroblastoma-derived homolog (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, breast differentiation antigen (NY-BR-1), NY-ESO-1, carcinoembryonic antigen (h5T4), olfactory receptor 51E2 (OR51E2), OX40, plasma cell antigen, poly SA, acrosome protease binding protein sp32 (OY-TESl), p53, p53 mutant, pan-linked protein 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-α), PDGFR-β, PDL192, PEN-5, phosphatidylserine, placenta-specific 1 (PLAC1), polysialic acid, prostate enzyme, prostate cancer cell, prostaglandin, protease serine 21 (testosterone or PRSS21), protease 3 (PR1), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), proteasome (pro, megalin factor) subunit beta type, receptor for advanced glycation end products (RAGE-1), RANKL, Ras mutant, Ras homolog family member C (RhoC), RON, receptor tyrosine kinase-like orphan receptor 1 (RO R1), renal ubiquitin 1 (RU1), renal ubiquitin 2 (RU2), sarcoma translocation breakpoints, 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, TCRγ alternate reading frame protein (TARP), telomerase, TIGIT, TNF-α precursor, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7 related (TEM7R), tenascin C, TGFβ2, TGF-β, transglutaminase 5 (TGS5), angiopoietin binding cell surface receptor 2 (Tie 2), TIM1, TIM2, TIM3, Tn Ag, TRAIL-R1, TRAIL-R2, tyrosinase-related protein 2 (TRP-2), thyroid stimulating hormone receptor (TSHR), tumor antigen CTAA16.88, tyrosinase, ROR1, TAG-72, urokinase 2 (UPK2), VEGF-A, VEGFR-1, vascular endothelial growth factor receptor 2 (VEGFR2), and vimentin, Wilms tumor protein (WT1), or X antigen family member 1A (XAGE1).

[0094] In one embodiment, the first antigen and the second antigen are CD19 and CD20, respectively. Table A provides sequence examples of anti-CD19 and anti-CD20 antibodies and fragments, as well as their bicistronic forms.

[0095] Table A. Example antigen binding sequences

[0096]

[0097]

[0098]

[0099]

[0100]

[0101] In addition to the antigen binding molecules, the CAR of the present disclosure may include a hinge, a transmembrane domain and / or an intracellular domain. In some embodiments, the intracellular domain may include a costimulatory domain and an activation domain.

[0102] The hinge can be an extracellular domain of an antigen binding system located between the binding motif and the transmembrane domain. The hinge can also be referred to as an extracellular domain or a "spacer". The hinge can contribute to receptor expression, activity and / or stability. The hinge can also provide flexibility in accessing the target antigen. In some embodiments, the hinge domain is located between the binding motif and the transmembrane domain.

[0103] In some embodiments, the hinge is, is from or is derived from an immunoglobulin-like hinge domain (e.g., comprising all or a fragment thereof). In some embodiments, the hinge domain is from or is derived from an immunoglobulin. In some embodiments, the hinge domain is selected from the hinge of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE or IgM or a fragment thereof.

[0104] In some embodiments, the hinge is, is from, or is derived from (e.g., includes all or fragments of) CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8.α., CD8.β., 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), CD4 9d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex associated α chain), CD79B (B cell antigen receptor complex associated β chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), C D158A(KIR2DL1), CD158B1(KIR2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2DL5A), CD158F2(KIR2DL5B), CD158K(K IR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(SLAMF3), CD244(SLAMF4), CD247(CD3-ζ), CD258(LIGHT), CD268(BAFFR), CD270(TNFSF 14), CD272 (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 co-stimulator (ICOS), LFA-1 (CD11a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2Rβ, IL-2Rγ,IL-7Rα, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fcγ receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, activated NK cell receptor, or Toll ligand receptor, or fragments or combinations thereof.

[0105] In some embodiments, the hinge is, is derived from or is derived from the hinge of CD8α (e.g., including all or fragments thereof). In some embodiments, the hinge is, is derived from or is derived from the hinge of CD28. In some embodiments, the hinge is, is derived from or is derived from a fragment of a CD8α hinge or a fragment of a CD28 hinge, wherein the fragment is any fragment less than the whole. In some embodiments, a fragment of a CD8α hinge or a fragment of a CD28 hinge is included in 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 at the N-terminus or C-terminus or both of the CD8α hinge or CD28 hinge.

[0106] A "transmembrane domain" refers to a domain that has the property of being present in a membrane when present in a molecule on the cell surface or cell membrane (e.g., spanning a portion or all of a cell membrane). It is not necessary for every amino acid in a transmembrane domain to be present in the membrane. For example, in some embodiments, a transmembrane domain is characterized in that a specified fragment or portion of a protein is substantially located in the membrane. A variety of algorithms can be used to analyze amino acid or nucleic acid sequences to predict protein subcellular localization (e.g., transmembrane localization). Programs psort (PSORT.org) and Prosite (prosite.expasy.org) are examples of such programs.

[0107] The transmembrane domain can be derived from any membrane-bound or transmembrane protein: such as α, β or ζ chain, CD28, CD3ε, CD3δ, CD3γ, CD45, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, 4-1BB / CD137 , activated NK cell receptor, immunoglobulin, 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), CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, inducible T cell co-stimulator (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 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecules (SLAM proteins), 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, truncated forms or combinations thereof.

[0108] The intracellular domain (or cytoplasmic domain) comprises one or more signaling domains, which cause and / or mediate intracellular signals after the target antigen binds to the binding motif, such as intracellular signals that activate 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 the activation of at least one normal effector function of an immune cell. The effector function of a T cell can be, for example, cytolytic activity or auxiliary activity, including the 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 may include a signaling domain as an activation domain. The intracellular domain may include a signaling domain that is a co-stimulatory signaling domain.

[0109] It is known that intracellular signaling domains can transduce signals when antigens bind to immune cells. For example, it is known that the cytoplasmic sequence of the T cell receptor (TCR) triggers signal transduction after TCR binds to antigen (see, e.g., Brownlie et al., Nature Rev. Immunol. 13: 257-269 (2013)).

[0110] In certain embodiments, suitable signaling domains include, but are not limited to, 4-1BB / CD137, activating NK cell receptors, immunoglobulins, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3δ, CD3ε, CD3γ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8α, CD8β, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, inducible T cell co-stimulator (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), Ly108, lymphocyte function-associated antigen-1 (LFA-1; CD1-1a / 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 molecules (SLAM proteins), 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, truncated forms or combinations thereof.

[0111] CAR can also include a co-stimulatory signaling domain, for example, to increase signaling efficacy. See U.S. Patents 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). The signal generated by TCR alone may not be sufficient to fully activate T cells, and secondary or co-stimulatory signals can increase activation. Therefore, in some embodiments, the signaling domain also includes one or more additional signaling domains (e.g., co-stimulatory signaling domains), which 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 can be used, as long as the portion transduces an effector function signal. In some embodiments, the cytoplasmic domain described herein includes one or more cytoplasmic sequences of a T cell co-receptor (or its fragment). 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 bound to CD83. Exemplary costimulatory proteins have amino acid sequences of costimulatory proteins naturally found on T cells, and the complete native amino acid sequences of the costimulatory proteins are described in NCBI reference sequences: NP 0.1. In some cases, CAR includes a 4-1BB costimulatory domain. In some cases, CAR includes a CD28 costimulatory domain. In some cases, CAR includes a DAP-10 costimulatory domain.

[0112] In some embodiments, CAR also includes ITAM. Examples of primary cytoplasmic signaling sequences containing ITAM that are particularly useful in the present disclosure include those derived from TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b and CD66d. In some embodiments, ITAM includes CD3 ζ.

[0113] Receptor transfection, integration and expression

[0114] The transposon of the present disclosure can be introduced into target cells, particularly immune cells, to generate engineered cells. The target cells are preferably immune cells, such as T cells, NK cells, monocytes, macrophages or their precursor cells.

[0115] Integration of the transposon into the genome of the target cell requires expression of the corresponding transposase. The transposon used in the present technology can be any transposon known in the art, such as (PB) Transposon, piggy- transposon, piggyBat transposon, Sleeping Beauty transposon, Helaiser transposon, Tol2 transposon or TcBuster transposon.

[0116] When the transposon is When a transposon is present, the transposase can be Transposase or Super Transposase. When the transposon is piggy- When the transposon is a piggy- When the transposon is a Sleeping Beauty transposon, the transposase may be a Sleeping Beauty transposase, such as SB10 or SB100X. When the transposon is a Helaizer transposon, the transposase may be a Helitron transposase. When the transposon is a Tol2 transposon, the transposase may be a Tol2 transposase. When the transposon is a TcBuster transposon, the transposase may be a TcBuster transposase or a hyperactive TcBuster transposase.

[0117] The transposase can be introduced into the target cell as a polynucleotide encoding the transposase, under the control of a promoter that allows its expression in the target cell.

[0118] In some embodiments, the transposon and transposase are introduced into the target cell by methods known in the art, such as electroporation, nuclear transfection, 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 some other aspects, the molecular copy number ratio of the nucleic acid transposon backbone containing the insert of interest to the nucleic acid encoding the transposase is between 1:1 and 1:32. In some embodiments, the nucleic acid transposon backbone containing the insert of interest matches the nucleic acid encoding the transposase in copy number. In some 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.

[0119] Once integrated into the target genome, the transposon transgene can be transcribed and translated to produce the receptor protein. As shown in the accompanying experimental examples, such integrated transgenes are highly stable.

[0120] Cell preparation process

[0121] The presently disclosed transposon-based technology enables the inventors to develop a cell therapy process that is significantly shorter than conventional techniques. The process is preferably an autologous cell therapy process, but is also applicable to allogeneic cell therapy processes.

[0122] In exemplary embodiments, the process entails (1) collecting and / or enriching a lymphocyte population obtained from a donor subject; (2) transfecting the lymphocyte population with a transposon of the present disclosure and 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.

[0123] In another embodiment, the process entails obtaining a sample comprising lymphocytes (e.g., T cells) from a donor subject; incubating the sample with a transposon to transfect the lymphocytes, thereby producing transfected lymphocytes; and culturing the sample comprising the transfected lymphocytes prior to harvesting the lymphocytes to produce a harvest sample, wherein at least 40% of the lymphocytes in the harvest sample are naive cells.

[0124] 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 lymphocytes in the harvested sample are immature 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 T cells in the harvested sample are naive T cells.

[0125] In some embodiments, one or more markers, such as CD45RA + 、CCR7 + 、CD62L + 、CD27 + 、CD28 + 、CD127 + 、CD132 + 、CD25 - 、CD44 -、CD45RO - and HLA-DR - To characterize naive T cells. In one embodiment, CD45RA + and CCR7 + Characterization of naive T cells. In one embodiment, CD45RA + 、CCR7 + 、CD62L + 、CD27 + and CD28 + To characterize naive T cells. In one embodiment, CD127 + 、CD132 + 、CD25 - 、CD44 - 、CD45RO - and / or HLA-DR - To characterize naive T cells.

[0126] In some embodiments, the whole process from obtaining lymphocytes from a donor to harvesting transfected lymphocytes is completed within 4 days. In some embodiments, the whole process from obtaining lymphocytes from a donor to harvesting transfected lymphocytes is completed within 3 days. In some embodiments, the whole process from obtaining lymphocytes from a donor to harvesting transfected lymphocytes is completed within 2 days. In some embodiments, the whole process from obtaining lymphocytes from a donor to harvesting transfected lymphocytes is completed within 36 hours. In some embodiments, the whole process from obtaining lymphocytes from a donor to harvesting transfected lymphocytes is completed within 24 hours. In some embodiments, the whole process from obtaining lymphocytes from a donor to harvesting transfected lymphocytes is completed within 18 hours. In some embodiments, the whole process from obtaining lymphocytes from a donor to harvesting transfected lymphocytes is completed within 12 hours. In some embodiments, the whole process from obtaining lymphocytes from a donor to harvesting transfected lymphocytes is completed within 8 hours. In some embodiments, the entire process from obtaining lymphocytes from a donor to harvesting transfected lymphocytes is completed within 6 hours. In some embodiments, the entire process from obtaining lymphocytes from a donor to harvesting transfected lymphocytes is completed within 4 hours.

[0127] In some embodiments, the entire process from obtaining lymphocytes from a donor to implanting or cryopreserving transfected lymphocytes is completed within 4 days. In some embodiments, the entire process from obtaining lymphocytes from a donor to implanting or cryopreserving transfected lymphocytes is completed within 3 days. In some embodiments, the entire process from obtaining lymphocytes from a donor to implanting or cryopreserving transfected lymphocytes is completed within 2 days. In some embodiments, the entire process from obtaining lymphocytes from a donor to implanting or cryopreserving transfected lymphocytes is completed within 36 hours. In some embodiments, the entire process from obtaining lymphocytes from a donor to implanting or cryopreserving transfected lymphocytes is completed within 24 hours. In some embodiments, the entire process from obtaining lymphocytes from a donor to implanting or cryopreserving transfected lymphocytes is completed within 18 hours.

[0128] As observed in the experimental examples, this improved autologous process does not require lymphocyte activation, T cell enrichment or expansion.

[0129] "Lymphocyte activation" or "lymphocyte stimulation" refers to the process of stimulating a lymphocyte population with one or more stimulatory agents to produce an activated lymphocyte population. Any combination of one or more suitable lymphocyte stimulators can be used to produce an activated lymphocyte population, including but not limited to antibodies or functional fragments thereof targeting T cell stimulatory molecules or co-stimulatory molecules (e.g., anti-CD2 antibodies, anti-CD3 antibodies, anti-CD28 antibodies or their functional fragments), T cell cytokines (e.g., any isolated, wild-type or recombinant cytokines, such as interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-7), interleukin 7 (IL-8), interleukin 8 (IL-9), interleukin 10 (IL-11), interleukin 12 (IL-13), interleukin 14 (IL-15), interleukin 16 (IL-17), interleukin 18 (IL-19), interleukin 19 (IL-22), interleukin 20 (IL-23), interleukin 21 (IL-24), interleukin 22 (IL-25), interleukin 23 (IL-26), interleukin 24 (IL-27), interleukin 25 (IL-28), interleukin 26 (IL-29), interleukin 27 (IL-30), interleukin 28 (IL-31), interleukin 29 (IL-32), interleukin 30 (IL-33), interleukin 31 (IL-34), interleukin 32 (IL-35), interleukin 33 (IL-36), interleukin 34 (IL-37), interleukin 3 (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 α (TNFα)) or any other suitable mitogen (e.g., tetradecanoylphorbol acetate (TPA), phytohemagglutinin (PHA), concanavalin A (conA), lipopolysaccharide (LPS), pokeweed mitogen (PWM)) or natural ligands of T cell stimulatory or co-stimulatory molecules.

[0130] In some embodiments, lymphocyte activation employs anti-CD3 antibodies (or functional fragments thereof), anti-CD28 antibodies (or functional fragments thereof), or a combination of anti-CD3 antibodies and anti-CD28 antibodies to stimulate a lymphocyte population.

[0131] Thus, in one embodiment, in the disclosed methods, lymphocytes are not contacted with any exogenous agents that activate lymphocytes, such as those exemplified above.

[0132] "Lymphocyte enrichment" or "lymphocyte selection" refers to the process of selecting lymphocyte subsets in a cell mixture. In some aspects, CD3+T cells can be selected from a cell mixture. In some aspects, CD4+T cells and CD8+T cells can be selected from a cell mixture. The selection process can be performed with antibodies (e.g., anti-CD3+ antibodies, anti-CD4+ antibodies, anti-CD8+ antibodies) or any other method known in the art for selecting cell subtypes.

[0133] "Lymphocyte expansion" refers to a process in which lymphocytes are incubated at a suitable temperature (e.g., 37° C.) and provided with sufficient nutrients and other conditions (e.g., CO2) to allow active division and proliferation. In some embodiments, the process of the present disclosure does not include lymphocyte expansion. In some embodiments, the method of the present disclosure includes a brief lymphocyte expansion that is no longer than 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 36 hours, or 48 hours.

[0134] Preferably, in some embodiments, each step is performed in a closed system. Preferably, each step is performed in a culture medium that does not contain human or animal serum.

[0135] The transfected lymphocytes generated by the present method may be suitable for treating a disease. In some embodiments, the harvested lymphocytes, such as T cells, include a sufficient percentage of naive T cells.

[0136] In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the harvested lymphocytes are transfected lymphocytes. In 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.

[0137] 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 T cell genome.

[0138] 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 NK cell genome.

[0139] As shown in Example 8, various embodiments of the presently disclosed cell manufacturing process can be scaled up. In some embodiments, the scaled-up process uses a sample for transfection that includes at least 10×10 6 In some embodiments, the sample includes at least 15×10 6 cells, 20×10 6 cells, 25×10 6 cells, 30×10 6 cells, 35×10 6 cells, 40×10 6 cells, 45×10 6 cells, 50×10 6 cells, 60×10 6 cells, 70×10 6 cells, 80×10 6 cells, 90×10 6 cells, 100×10 6 cells, 110×10 6 cells, 120×10 6 cells, 130×10 6 cells, 140×10 6 cells, 150×10 6 cells, 200×10 6 cells, 250×10 6 cells, 300×10 6 cells, 400×10 6 cells, 500×10 6 , 600×10 6 cells, 700×10 6 cells, 800×10 6 cells, 900×10 6 cells, 1000×10 6 cells, 1500×10 6 cells or 2000×10 6 cells.

[0140] In some embodiments, transfection (e.g., electroporation) in a large-scale process is performed with a sample 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 a large-scale process is performed with a sample volume of no greater than 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.

[0141] 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.

[0142] In some embodiments, in a larger scale process, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the harvested lymphocytes are transfected lymphocytes. In 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.

[0143] In any embodiment of the cell preparation process, harvesting cells can be performed for cell activation (if not activated before transfection), selection and / or expansion. In some embodiments, expansion can be performed for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

[0144] Composition and processing

[0145] Cells (e.g., allogeneic cells) disclosed herein can be used to treat various diseases and disorders, particularly cancer. In one embodiment, cancer can include Wilm's tumor, Ewing's sarcoma, neuroendocrine tumors, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, bile duct cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid carcinoma, 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.

[0146] 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 a transfected cell of the present disclosure. Also provided is a composition comprising a transfected lymphocyte disclosed herein and a pharmaceutically acceptable excipient.

[0147] The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, although appropriate dosages can be determined through clinical trials. In certain embodiments, the cancer is characterized by expression of an antigen targeted by a CAR or TCR molecule, such as CD19 and / or CD20.

[0148] In other embodiments, methods are provided, comprising administering a therapeutically effective amount of the modified T cells contemplated herein, or a composition comprising the T cells, 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 treating or preventing cancer, comprising administering a therapeutically effective amount of the modified T cells of the present disclosure to a subject in need thereof.

[0149] One of ordinary skill in the art will recognize that multiple administrations of the disclosed compositions may be required to achieve the desired therapy. For example, the composition may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or more 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.

[0150] In one embodiment, an effective amount of the composition is administered to a subject in need to increase a cellular immune response to the subject's cancer. The immune response may include a cellular immune response mediated by cytotoxic T cells capable of killing infected cells, regulatory T cells, and a helper T cell response. A humoral immune response may also be induced, which is primarily mediated by helper T cells capable of activating B cells to result in antibody production. A variety of techniques can be used to analyze the type of immune response induced by the compositions of the present disclosure, which are fully described in the art; for example, Current Protocols in Immunology, edited by John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober, (2001) John Wiley & Sons, NY, NY.

[0151] Methods for administering the cell compositions described herein include any of the following methods: it is effective to cause the reintroduction of in vitro genetically modified immune effector cells that directly express engineered TCR or CAR in a subject, or reintroduce genetically modified source progenitor cells of immune effector cells that differentiate into mature immune effector cells expressing engineered TCR or CAR when introduced into a subject. One method includes transfecting ex vivo peripheral blood T cells with a nucleic acid construct according to the present disclosure, and returning the transfected cells to the subject.

[0152] Although the foregoing disclosure has been described in considerable detail by way of illustration and example for the purpose of clear understanding, it will be apparent to one of ordinary skill in the art from the teachings of the present disclosure that certain changes and modifications may be made thereto 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. One of ordinary skill in the art will readily recognize that a variety of non-critical parameters may be changed or modified to produce similar results.

[0153] 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 specifically and individually indicated to be incorporated by reference. However, the references cited herein should not be construed as admitting that such references are prior art of the present disclosure. To the extent that any definition or term provided in the references incorporated by reference is different from the term and discussion provided herein, the present term and definition shall prevail. The contents of all references cited in this application are expressly incorporated herein by reference.

[0154] Example

[0155] Example 1. Transposon-mediated bicistronic CAR transfection and integration

[0156] This example tests the transposon system for introducing anti-CD19 and anti-CD20 bicistronic CAR constructs into host cells.

[0157] The bicistronic CAR, referred to herein as KITE-001, is expressed as a single-chain precursor, including from N-terminus to C-terminus a first signal peptide-anti-CD19 scFv-CD28 hinge and transmembrane domain-CD28 co-stimulatory domain-cd3ζ-T2A-a second signal peptide-anti-CD20 scFv-CD8 hinge and transmembrane domain-4-1BB co-stimulatory domain-cd3ζ. Self-cleavage at T2A results in the production of two separate CAR molecules.

[0158] The coding sequence of KITE-001 shares a region of homology between the anti-CD19 portion and the anti-CD20 portion at the CD3ζ domain. Such homology is suspected to lead to recombination and cryptic splicing. Therefore, codon optimization and sequence wiggling were performed to reduce the nucleotide homology to less than 9 nucleotides.

[0159] In preliminary studies conducted with lentiviral vectors, it was observed that the expressed protein products included many variants. Specifically, variant 1 (V1), which accounted for about 1.5%-2% of all protein products, only included anti-CD19. Variant 14 (V14), which accounted for about 0.02-0.05% of all protein products, contained a frameshift mutation in the anti-CD19 unit and was therefore non-functional; variant 15 (V15), which accounted for about 0.02-0.05% of all protein products, contained a frameshift mutation in the heavy chain of the anti-CD20 scFv and therefore had only anti-CD19 activity; variant 22 (V22), which accounted for about 0.02-0.05% of all protein products, had a deletion between the anti-CD20 scFv and CD8 hinge regions and was therefore only functional for CD19.

[0160] Then, this example uses a transposon system with a corresponding transposase to transfect the KITE-001 coding sequence into T cells by electroporation (EP). The transposon system includes a coding sequence flanked by two sets of repeat sequences (CAGTTGAAGTCGGAAGTTTACATACACYTAAG, SEQ ID NO: 28, and YCCAGTGGGTCAGAAGTTTACATACACTMART, SEQ ID NO: 29). An example of a transposase sequence is

[0161]

[0162] The transfected cells were then cultured and expanded. Unlike the lentiviral system, variants V1 and V15 were completely undetectable from the transfected cells, and no other variants were found to have a prevalence greater than 0.02%. In contrast, significantly higher levels of variant V22 were detected in transposon-based samples (Table 1).

[0163] Table 1. Percentage of variants

[0164] Variants Lentivirus Transposon Not translated 0.13％ 0.0048％ V1 0.40％ 0％ V14 0.026％ 0.015％ V15 0.048％ 0％ V22 0.026％ 0.15％

[0165] The stability of transposon-based integration was monitored for three weeks. As shown in Tables 2 and 3, both LVV-generated and non-viral transposed CARs showed in vitro stability of CD19 / CD20 expression.

[0166] Table 2. Full CAR expression (LVV)

[0167]

[0168] Table 3. Total CAR expression (non-viral)

[0169]

[0170]

[0171] Example 2. Single CAR transfection efficiency

[0172] The transfection efficiency of the transposon system was then compared with that of the lentiviral vector using T cells from four different donors. Both systems achieved superior results in terms of the percentage of transfected cells of total viable CD3+ cells (Table 4) and total T cell viability (Table 5). Therefore, this result indicates that, unlike viral vector-based systems, the transposon system of the present invention does not require T cell activation to achieve high transfection efficiency.

[0173] Table 4. CAR+ percentage of live CD3+ cells

[0174] Transposon Control (no EP) Study EP scale Enlarging the EP scale Day 1 0.81±0.32 36.20±19.95 47.66±11.35 Day 4 0.2±0.099 58.55±14.56 73.90±9.48 Day 7 0.16±0.095 38.26±3.87 54.95±13.28

[0175] Lentivirus NTD LVV-TD Day 0 0±0 0±0 Day 3 0.050±0.038 61.73±4.07 Day 7 0.020±0.0052 77.83±2.99

[0176] Table 5. T cell viability

[0177] Transposon Control (no EP) Study EP scale Enlarging the EP scale Day 0 94.55±1.92 88.45±2.27 93.78±1.67 Day 1 92.23±1.48 73.8±2.81 79.45±3.10 Day 4 94.65±1.82 81.25±4.34 85.63±1.63 Day 7 92.53±0.90 86.9±4.29 89.3±1.01

[0178] Lentivirus NTD LVV-TD Day 0 85.25±2.49 84.00±3.39 Day 1 62.13±10.89 65.5±12.03 Day 3 82.38±2.63 81.00±4.06 Day 4 86.00±6.04 86.25±3.34 Day 5 87.25±3.70 91.5±1.66 Day 6 92.00±5.062 92.75±1.09 Day 7 92.25±3.11 92±2.24

[0179] Example 3. Transposon-mediated reporter gene or single CAR transfection and integration

[0180] This example tests a transposon system for introducing one of a GFP reporter gene, an anti-CD19 CAR construct with a 4-1BB co-stimulatory region, or an anti-CD19 CAR construct with a CD28 co-stimulatory region into host cells.

[0181] As shown in Table 6, expression of the integrated GFP reporter gene was maintained for more than 21 days. In comparison, non-integrated expression controls (no electroporation, minicircles, or plasmids without transposase) dropped below 1% within 7-10 days. Therefore, this data demonstrates that transposase-mediated gene integration is stable. It is noteworthy that when the transposon was included in the minicircle plasmid for delivery, transfection efficiency was further improved at all time points.

[0182] Table 6. Percentage of GFP-expressing cells in the total number of live CD3+ T cells

[0183]

[0184] As shown in Table 7, expression of the integrated anti-CD19 CAR construct with the 4-1BB co-stimulatory region was maintained for more than 21 days. In comparison, the non-integrated expression control, plasmid only without transposase, dropped below 1% within 7-10 days. Therefore, this data demonstrates that transposon-mediated gene integration of the CAR construct is stable.

[0185] Table 7. Percentage of CD3 T cells expressing anti-CD19 CAR with 4-1B Costim

[0186]

[0187]

[0188] As shown in Table 8, expression of the integrated anti-CD19 CAR construct with CD28 co-stimulatory region was maintained for more than 21 days. As a comparison, the non-integrated expression control, no electroporation or plasmid only, dropped below 1% within 7-10 days. Therefore, this data demonstrates that transposon-mediated genetic integration of the CAR construct is stable.

[0189] Table 8. Percentage of CD3 T cells expressing anti-CD19 CAR with CD28Costim

[0190]

[0191] Example 4. Research progress on transposon-mediated autologous processes

[0192] Conventional autologous processes based on viral vectors require 7 days or even weeks from cell acquisition to implantation. This example attempts to develop an autologous process that can be completed within one day.

[0193] After isolation, T cells were enriched from collected peripheral blood mononuclear cells (PMBCs) and then electroporated with the transposon constructs (as tested in Example 1). T cells were harvested at 24 hours or further activated and expanded.

[0194] The impact of the duration of this process was evaluated. As shown in Table 9,

[0195] Table 9. Basic features of transposon-mediated autologous processes

[0196]

[0197] These processes were tested using a Nalm6 leukemia mouse model (1 day, 3 days, and 7 days, with different cell counts). Tumor burden was measured after autologous cell transplantation and the results are listed in Table 10. An autologous process based on a lentiviral vector with the same CAR sequence (7 days) was used as a control.

[0198]

[0199]

[0200] A 7-day process based on lentiviral vectors (10 6 The tumor burden gradually reached a peak on day 68 (approximately 2.3×10 9 photons / s), and then dropped to about 1.7×10 8 Photons / second s.

[0201] However, in all transposon-based processes, only low doses (2 × 10 5 The 7-day course of the high-dose (10 6 Specifically, high doses (10 6 cells) for 1 day and low dose (2×10 5 The 3-day process of 6 Photons / s) achieved tumor elimination, and there was no tumor recurrence at all. The high doses treated for 3 days and 7 days also performed well, while the low dose (2×10 photons / s) treated for 1 day 5 cells) was comparable to high-dose lentiviral vector-based treatments.

[0202] Therefore, these results indicate that the newly developed 1-day approach to autologous transposon-based CAR-T achieves results far superior to those achieved with the same dose (10 6 cells) based on a 7-day lentiviral vector-based approach. Even with the dose (2×10 5 cells) is lower than the 7-day lentiviral vector-based method (10 6This superior result from the newly developed shortened process is expected to produce high-quality CAR-T cells with a higher population of naive T cells that are beneficial for cancer treatment.

[0203] Example 5. Further testing of autologous processes

[0204] This embodiment further tests the shortened autologous process based on transposon with other CAR constructs. Using a construct targeting CD19 but including a 4-1BB co-stimulatory domain. Similarly, early (4th day) harvests at high and low doses are much better than late (14th day) harvests.

[0205] In each experiment, fixed amounts of plasmid DNA (gene cargo, 5 μg) and mRNA (encoding transposase, 5 μg) were used. The comparison is shown in Table 11.

[0206] Table 11. Transfection efficiency of each construct

[0207]

[0208] Electroporation of plasmid DNA usually results in 30-50% cell death within the first 24 hours. The viability of cells produced by the new method is activated and amplified, and their viability is assessed at each stage. Table 12 shows that cell viability has recovered to 80-90% after 8 days, the same level of viability as the non-electroporated cell group. Table 13 shows that live cells grow well after the new production process, expanding 30-40 times.

[0209] Table 12. T cell activity on different days

[0210]

[0211] Table 13. Fold expansion on different days

[0212]

[0213] Thus, these data suggest that the novel nonviral process is applicable to a variety of construct types. Furthermore, cell viability could be restored in subsequent expansion, demonstrating the superior growth potential of electroporated T cells.

[0214] Example 6. Evaluation of cells produced by the new process

[0215] This example evaluates CAR-T cells generated by the shortened process, specifically using markers associated with the naive state of the T cells.

[0216] CAR T cells were produced in static bags using the non-viral transposon method described in Example 4. Phenotypic data refer to the percentage of T cells expressing CD45RA, CCR7, CD62L, CD27 and CD28 relative to total T cells (Table 14).

[0217] Table 14. Percentage of naive T cells (CD45RA + CCR7 + CD62L + CD27 + CD28 + )

[0218] date Starting Materials Day 0 after transfection Day 3 Day 7 ％ 52.5 59.3 34.6 27.2

[0219] Cells harvested earlier had a higher number of juvenile populations, demonstrating the benefit of the accelerated process.

[0220] The effect of T cell status before transfection on transfection efficiency was also evaluated. Four types of T cells were examined, including CD45RA + CCR7 + 、CD45RA + CCR7-, CD45RA-CCR7 + and CD45RA - CCR7 - . Cells were recovered 2 hours after thawing and then electroporated / transfected with DNA transposon and mRNA transposase. One day after transfection, cells were activated and expanded according to the new method. Based on the day of activation, the harvested cells were labeled as day 0, day 3, and day 7. As shown in Table 15, naive T cells received more gene cargo than other subunits.

[0221] Table 15. Transfection efficiency of T cell subunits

[0222]

[0223] Example 7: Vector Copy Number (VCN) Measurement

[0224] This example develops a method to measure the copy number of a construct integrated into the genome of a target cell.

[0225] Primer sets were designed to target constructs (e.g., CD19 coding sequences), plasmid backbone regions (Amp), and host reference genes (e.g., CDKN2A). Unintegrated free DNA was digested with Dpn I. VCN experiments were performed with two independent ddPCR reactions—"CD19 versus host reference gene" and "AMP versus host reference gene" CD19 VCN = 2*CD19 / host copy number; AmpVCN = 2*Amp / host copy number (multiplied by the copy number of the reference genome (diploid is 2)). Integration calculations are VCN = CD19VCN-AMP VCN.

[0226] Using this method, the average VCN in cells generated using the method described above was determined to be approximately 5.07 (6.07 total DNA minus 1.0 free undigested DNA).

[0227] Example 8. Comparison with TcBuster

[0228] This example compares the performance of the transposon system (and LVV) tested in Example 1 with TcBuster (another non-viral transposon-based delivery system). All systems use CD19 single CAR, including 4-1BB as a co-stimulatory domain. Frozen transfected cells were thawed overnight for CAR+ percentage measurement and in vitro cytotoxicity assay. The results are shown in Tables 16 and 17.

[0229] Table 16. Percentage of transfected T cells

[0230] Transposon only TcBuster Example 1 LVV transduction Donor #1 0.055 27 18.2 53.7 Donor #2 0.54 20 12.9 52.7 Donor #3 0.034 30.2 15.6 57.8

[0231] Table 17. Cytotoxicity

[0232]

[0233] The Nalm6 animal model used in Examples 4 and 5 was used to test the tumor suppressive efficacy of these CAR cells. The results are shown in Table 18.

[0234]

[0235] Therefore, these data demonstrate comparable in vitro cytotoxicity and in vivo efficacy of CAR T cells made by TcBuster, the transposon system of Example 1, or the LVV system.

[0236] Example 9. Scaling up the manufacturing process

[0237] This example develops a large-scale manufacturing process based on the small-scale pilot study in Example 4.

[0238] In a small-scale process, 5 × 10 cells were used in electroporation in a 100 μL volume. 6 In large-scale processes, 50 × 10 cells were used in 1 mL volume. 6 Up to 100×10 6 In a comparative study, cells harvested from both the small-scale process and the large-scale process were activated (with anti-CD3 antibodies) and expanded for up to 7 days.

[0239] There was no significant difference between the two processes in terms of cell number fold change (p=08725). However, using a paired t-test, the large-scale process showed significantly higher viability than the small-scale process (p=0.0090).

[0240] Finally, the transfection efficiency of the large-scale process compared to the small-scale process was also assessed. Using a paired t-test, the large-scale process showed significantly higher transfection efficiency than the small-scale process (p=0.0115).

[0241] Example 10. Large Payload Transfection: Tricistronic

[0242] This example describes the use of the Sleeping Beauty transposon system to separately transfect three different large payload plasmids, such as Figure 1 Three different plasmid constructs (plasmid 1, plasmid 2 and plasmid 3) were electroporated into cells.

[0243] Plasmids 1-3 encode CAR, dominant negative receptor (DNR) and membrane-bound interleukin receptor (mbIL) individually. Plasmid 1 is a three cistron construct with a length of 7725bp, including an insert of 4835bp, expressed as an mRNA transcript and a single long polypeptide, which is cut at the T2A site and P2A site after translation to provide CAR, DNR and mbIL. Plasmid 2 is a three cistron construct with a length of 8203bp, including a 5313bp insert expressed as an mRNA transcript and two independent polypeptides. The first polypeptide includes CAR and the DNR cut after translation at the T2A site. The second polypeptide is mbIL. Plasmid 3 is a three cistron construct with a length of 8132bp, including an insert of 5242bp expressed as two mRNA transcripts and two polypeptides. The first polypeptide includes CAR and the DNR cut after translation at the T2A site. The second polypeptide is mbIL.

[0244] Table 19. Transfection efficiency

[0245]

[0246]

[0247] Example 11. Large Payload Transfection: Tetracistronic

[0248] This example describes the separate transfection of two different very large payload plasmids, e.g. Figure 2 As shown. 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 8kb transposase insert. 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 10kb transposase insert. Table 23 shows CAR expression after transfection with plasmid 5 without the addition of transposase.

[0249] Table 20: 8 kb insert (plasmid 4)

[0250]

[0251] Table 21: No transposase (plasmid 4)

[0252]

[0253] Table 22: 10 kb insert (plasmid 5)

[0254]

[0255] Table 23: No transposase (plasmid 5)

[0256]

[0257] FIG. 3A to FIG. 3C The relative proportions of CAR expression for each of the four CARs of tetracistronic plasmid 4 are shown at day 8 after electroporation. FIG. 4A to FIG. 4C It is shown that CAR expression of each of the four CARs of tetracistronic plasmid 5 on day 8 after electroporation is also relatively proportional.

[0258] Example 12. Stability and restimulation

[0259] This example describes CAR expression following non-viral transfection of cells with plasmid 4. At day 11 after electroporation, transfected cells were restimulated with anti-CD3 antibodies. Table 24 shows CAR expression without restimulation. Table 25 shows CAR expression upon restimulation at day 11. The data show the stability of transgene expression from the large payload construct plasmid 4.

[0260] Table 24: CAR expression without restimulation

[0261]

[0262] Table 25: CAR expression upon restimulation on day 11

[0263]

[0264] Example 13. Promoter Evaluation

[0265] This example describes the evaluation of different promoters in transposon encoding plasmids. The same gene of interest cassette was designed under different promoters and tested in non-viral cell culture to evaluate the gene expression dynamics driven by different promoters. The results show that the transposon transfection system works on different promoters, providing CAR expression from single CAR and dual CAR plasmids with different promoters. In addition, different promoters provide different levels of CAR expression, thereby selecting the CAR expression level based on the choice of promoter.

[0266] Table 26: Plasmids used for promoter evaluation

[0267] CAR Promoter Plasmid 6 Single CAR 1 Plasmid 7 Dual CAR 2 Plasmid 8 Dual CAR 1 Plasmid 9 Dual CAR 3 Plasmid 10 Dual CAR 4

[0268] Table 27: CAR expression using the Sleeping Beauty transposon system

[0269]

[0270] Table 28: CAR expression without Sleeping Beauty transposase

[0271]

[0272] Example 14. Transgenic expression test of nanoplasmid and pCDL plasmid

[0273] This example describes the evaluation of different backbone sizes, comparing the expression of the anti-CD19 CAR gene after electroporation transfection with a transposon encoding anti-CD19 CAR on a pCDL plasmid and the expression after electroporation transfection with the same transposon on a nanoplasmid. The results of Table 29 show that higher expression levels of anti-CD19 CAR were achieved by transfection with a nanoplasmid. The ratio of transposon DNA to transposase mRNA was 1:4.

[0274] Table 29: Anti-CD19CAR expression nanoplasmid vs. pCDL plasmid

[0275] time pCDL plasmid 8ug Nanoplasmid 5.6ug Nanoplasmid 8ug Non-EP Day 1 11.8 12.8 22.5 0.43 Day 4 53.9 68.9 77.9 2.07 Day 6 39 53.3 65.2 1.55 Day 8 30.8 44.9 52 0.81

[0276] ***

[0277] Although multiple embodiments have been described, it is apparent that the present disclosure and embodiments may provide other embodiments utilizing or being encompassed by the compositions and methods described herein. Therefore, it should be understood that the scope of the present disclosure will be defined by what can be understood from the present disclosure and the appended claims, rather than by the embodiments that have been represented by way of example.

Claims

1. A transposon comprising a transgene encoding a polypeptide, wherein the polypeptide comprises 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. The transposon according to claim 1, wherein the transposon is a DNA transposon selected from the group consisting of: Sleeping Beauty transposon, piggyBac transposon and Tc Buster transposon or a retrotransposon.

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

4. The transposon according to any preceding claim, 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 according to any preceding claim, wherein the coding sequence of each ITAM in the transgene is codon optimized such that they do not share sequence identity of 12 consecutive nucleotides or more with each other.

7. The transposon of claim 6, wherein the coding sequence of each ITAM in the transgene is codon-optimized so that they do not have a sequence identity of 9 consecutive nucleotides or longer with each other.

8. The transposon according to any preceding claim, wherein the ITAM is a cytoplasmic signaling sequence derived from a protein selected from the group consisting of: TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b and CD66d.

9. The transposon of claim 8, wherein the ITAM is derived from CD3ζ, CD3ε, or both.

10. The transposon of any preceding claim, wherein the first CAR and the second CAR each further comprise an intracellular co-stimulatory domain.

11. The transposon of claim 10, wherein the intracellular co-stimulatory domain is a signaling region of a protein 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 co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), tumor necrosis factor superfamily member 14, TNFSF14, LIGHT), NKG2C, Igα (CD79a), Fcγ receptor, MHC Class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, CDS, GITR, BAFFR, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD (CD11d), ITGAE (CD103), ITGAL (CD11a), ITGAM (CD11b), ITGAX (CD11c) , ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE (RANKL), DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG (Cbp), CD19a, ligands that specifically bind to CD83, and combinations thereof.

12. The transposon of claim 2, wherein the intracellular co-stimulatory domain is a signaling region of DAP-10, 4-1BB or CD28.

13. A cell comprising the transposon according to any one of claims 1 to 12. 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. The cell of claim 15, wherein the heterologous transposase is selected from the group consisting of: Transposase, Like transposase, Super (SPB) transposase, piggyBac transposase, Sleeping Beauty transposase, Hyperactive Sleeping Beauty (SB100X) transposase, Helitron transposase, Tol2 transposase, TcBuster transposase, or Hyperactive TcBuster transposase.

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

18. 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, thereby producing transfected T cells; as well as culturing the sample comprising the transfected T cells for less than 96 hours prior to harvesting the T cells to produce a harvest sample, wherein at least 40% of the T cells in the harvested sample are naive T cells.

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

20. The method of claim 18 or 19, wherein the naive T cells are characterized by CD45RA + and CCR7 + .

21. The method of claim 20, wherein the naive T cells are further characterized as CD62L + 、CD27 + and CD28 + .

22. The method according to any one of claims 18 to 21, wherein the T cells are not activated prior to 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 selected from the group consisting of a Sleeping Beauty transposon, a piggyBac transposon and a Tc Buster transposon or a retrotransposon.

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

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

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

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. A method for preparing a lymphocyte expressing a chimeric antigen receptor (CAR) or a T cell receptor (TCR), the method comprising: collecting a biological sample comprising lymphocytes from a human subject; introducing a transposase and a transposon into the lymphocyte, wherein the transposon comprises a transgene encoding a polypeptide comprising the CAR or the TCR; and harvesting lymphocytes containing the transposase and the transposon, Lymphocyte harvesting occurred within 96 hours of biological sample collection.

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

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

32. The method of any one of claims 29 to 31, wherein the lymphocyte harvesting occurs within 72 hours of collection of the biological sample.

33. The method of any one of claims 29 to 31, wherein the lymphocyte harvesting occurs within 48 hours of collection of the biological sample.

34. The method of any one of claims 29 to 31, wherein the lymphocyte harvesting occurs within 36 hours of collection of the biological sample.

35. The method of any one of claims 29 to 31, wherein the lymphocyte harvesting occurs within 24 hours of collection of the biological sample.

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 after the biological sample is collected.

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

38. The method according to any one of claims 29 to 37, wherein the method does not comprise 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. The method of claim 40, wherein at least 40% of the T cells that have the transgene integrated into their genome are naive T cells when harvested.

42. The method of claim 41, wherein the naive T cells are CD45RA + and CCR7 + .

43. The method of any one of claims 29 to 35, wherein the lymphocytes when harvested comprise more naive T cells compared to a comparable process wherein the lymphocyte harvest occurs 168 hours after collection of the biological sample.

44. The method of any one of claims 29 to 43, wherein the polypeptide further comprises a second CAR, and 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).

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

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

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

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

49. The method of claim 48, wherein the sample subjected to transfection comprises at least 50×10 6 cells.

50. The method of claim 48 or 49, wherein the transfection is performed in a solution having a volume of 0.1 mL to 100 mL.

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

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