Chimeric CD8-alpha coreceptor compositions and methods of use

Abstract

Chimeric CD8α coreceptor compositions and methods of use therefor are disclosed. In particular, chimeric CD8α homodimer coreceptor compositions, cells containing the chimeric coreceptor, and pharmaceutical compositions containing the coreceptor and / or cells are disclosed. Methods of making and using the coreceptor compositions are also provided.

Description

[Technical Field]

[0001] (Related Applications) This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 371,136, filed August 11, 2022, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure is directed to chimeric CD8α coreceptor compositions and methods of use therefor. In particular, chimeric CD8α homodimer coreceptor compositions, cells comprising chimeric CD8α coreceptors, pharmaceutical compositions comprising chimeric CD8α coreceptors and / or cells, and methods of making and using the same are disclosed.

[0003] (Incorporated by reference to the sequence listing) The Sequence Listing XML associated with this application has been provided electronically in XML file format and is hereby incorporated by reference. The XML file containing the Sequence Listing XML is named "POTH-074_001WO_SeqList_ST26.XML." The XML file is 64,097 bytes, was created on July 24, 2023, and is submitted electronically via the USPTO Patent Center. [Background technology]

[0004] In recent years, adoptive cell therapy, which uses autologous T cells transduced to express a T cell receptor (TCR) or chimeric antigen receptor (CAR), has proven to be a very powerful approach for the treatment of diseases such as cancer. However, challenges remain, including separating cytotoxicity against tumor cells from systemic toxicity. There is a long-standing but unmet medical need for compositions and methods for improving the T cell receptor ("TCR")-mediated cytotoxicity of TCR-T cell therapy ("TCR-T therapy"). The present disclosure provides a solution by providing a chimeric TCR, a CD8α co-receptor, that enhances the cytotoxic activity of TCR-T therapy. Summary of the Invention

[0005] The present disclosure provides a chimeric CD8α coreceptor comprising: a) a truncated CD8α coreceptor comprising a CD8α coreceptor extracellular domain and a CD8α coreceptor transmembrane domain; and b) a CD4 intracellular domain comprising a palmitoylation motif and an Lck-binding domain, wherein the CD4 intracellular domain is fused in-frame to the C-terminus of the truncated CD8α coreceptor.

[0006] In some embodiments, the truncated CD8 coreceptor comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the palmitoylation motif comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the Lck-binding domain comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD4 intracellular domain, comprising the palmitoylation motif and the Lck-binding domain, comprises the amino acid sequence of SEQ ID NO: 2.

[0007] In some embodiments, the CD8α coreceptor comprises: a) a truncated CD8α coreceptor comprising the amino acid sequence of SEQ ID NO: 1; and b) a CD4 intracellular domain comprising a palmitoylation motif comprising the amino acid sequence of SEQ ID NO: 3 and an Lck-binding domain comprising the amino acid sequence of SEQ ID NO: 4, wherein the CD4 intracellular domain is fused in-frame to the C-terminus of the truncated CD8α coreceptor.

[0008] In some embodiments, the CD8α coreceptor comprises: a) a truncated CD8α coreceptor comprising the amino acid sequence of SEQ ID NO: 1; and a CD4 intracellular domain comprising the amino acid sequence of SEQ ID NO: 2, wherein the CD4 intracellular domain is fused in-frame to the C-terminus of the truncated CD8α coreceptor.

[0009] In some embodiments, the chimeric CD8α coreceptor comprises the amino acid sequence of SEQ ID NO:5.

[0010] The present disclosure also provides a polynucleotide comprising a nucleic acid sequence encoding any one of the chimeric CD8α coreceptors of the present disclosure. In some embodiments, the polynucleotide is an mRNA molecule. In some embodiments, the polynucleotide is a DNA molecule. In some embodiments, the polynucleotide further comprises a promoter sequence operably linked to the DNA molecule to produce at least one mRNA molecule encoding the chimeric CD8α coreceptor in a cell.

[0011] The present disclosure also provides a cell comprising a polynucleotide of the present disclosure. In some embodiments, the cell expresses a chimeric CD8α coreceptor. In some embodiments, the cell further expresses a T cell receptor (TCR). In some embodiments, the cell further expresses a chimeric antigen receptor (CAR). In some embodiments, the cell expresses a TCR and a CAR. In some embodiments, the cell is a T cell. In some embodiments, the chimeric CD8α coreceptor is expressed as a homodimer on the plasma membrane of the T cell.

[0012] The present disclosure also provides a pharmaceutical composition comprising any one of the cells of the present disclosure and at least one pharmaceutically acceptable carrier or medicament.

[0013] The present disclosure also provides a method of stimulating T cell receptor (TCR)-mediated cytotoxicity of a T cell population in a subject in need thereof, the method comprising: a) introducing into the T cell population a polynucleotide encoding a TCR and a polynucleotide encoding any one of the chimeric CD8α coreceptors of the present disclosure, wherein a plurality of T cells in the T cell population co-express the TCR and the chimeric CD8α coreceptor on the cell membrane of the T cells, and the chimeric CD8α coreceptor is expressed as a homodimer; and b) administering the T cell population to the subject in need thereof, wherein the T cell population expressing the TCR and the chimeric CD8α coreceptor has a higher level of cytotoxicity compared to a T cell population expressing only the TCR. In some embodiments, the T cell population further expresses a CAR.

[0014] Any of the above aspects may be combined with any other aspect.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein, the singular also includes the plural unless the context clearly dictates otherwise; for example, the terms "a," "an," and "the" are understood to be singular or plural, and the term "or" is understood to be inclusive. As an example, an "element" means one or more elements. Throughout this specification, the word "comprising," or variations such as "comprises" or "comprising," will be understood to imply the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps. About may be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless the context clearly dictates otherwise, all numerical values ​​provided herein are modified by the term "about."

[0016] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. References cited herein are not admitted to be prior art to the claimed invention. In case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the present disclosure will be apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0017] [Figure 1A]1A-1B show schematic diagrams illustrating exemplary chimeric CD8α homodimers (chiCD8-homo-di) and CD8α heterodimers (CD8-hetero-di) and CD8α homodimer (CD8-homo-di) controls. Figure 1A shows a chimeric CD8α homodimer coreceptor comprising a truncated CD8α coreceptor containing only the CD8α coreceptor extracellular domain and CD8α coreceptor transmembrane domain, a CD4 intracellular domain containing a palmitoylation domain and a high-affinity Lck-binding domain, and the CD4 intracellular domain is fused in-frame to the C-terminus of the truncated CD8α coreceptor. [Figure 1B] FIG. 1B shows control wild-type CD8α heterodimeric coreceptor (CD8 hetero-di) and CD8α homodimeric coreceptor (CD8-homo-di). [Figure 2] Figure 2 shows a schematic diagram of a transposon designed for dual expression of a chimeric CD8α coreceptor ("coreceptor") and a TCR receptor consisting of the TCR alpha ("TCRa") and TCR beta ("TCRb") chains. The PGK promoter controls expression of the chimeric CD8α coreceptor. The EF1a promoter controls expression of the TCR receptor: ITR (inverted terminal repeat); Insul (insulator sequence); pA (polyA), iC9 (inducible pro-apoptotic polypeptide); DHFR (dihydrofolate reductase resistance cassette); TCRb (TCR beta chain); TCRa (TCR alpha chain). [Figure 3]Figures 3A-3B show a series of contour plots and graphs depicting flow cytometry sorting and in vitro cytotoxicity data for chimeric CD8α coreceptors compared to TCR controls. Figure 3A shows fluorescence-activated cell sorting (FACS) analysis of TCR+CD8α+ (bottom row) and TCR+CD4+ (top row) T cell populations co-expressing the chimeric CD8α coreceptor (Chi_CD8 homo-di) of the present disclosure, wild-type CD8α coreceptor (CD8 Homo-di), heterodimeric CD8α coreceptor (CD8 Hetero-di), or GFP compared to a mock control. The percentage of the cell population expressing the receptor is indicated in each panel. Figure 3B shows two graphs depicting in vitro cytotoxicity assays using chimeric CD8α coreceptors compared to TCR controls for CD4+ (left) and CD8+ (right) T cells at the indicated ratios. The x-axis shows time (in hours). The y-axis shows the tumor growth percentage (%) normalized to baseline. T cells express coreceptors as indicated in the legend. The data demonstrate superior in vitro cytotoxicity of TCR+chimeric CD8α coreceptor (TCR+chiCD8-homo-di) compared to all other controls (mock, TCR+GFP, TCR+CD8-hetero-di, TCR+CD8-homo-di).

[0018] All documents cited herein, including any cross-referenced or related patents or applications, are hereby incorporated by reference in their entirety for all purposes, unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein, or that it alone, or in any combination with any other reference, teaches, suggests, or discloses any such invention. Furthermore, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall control. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention relates to chimeric coreceptor compositions and methods of use therefor. In particular, the present invention relates to chimeric CD8α homodimeric coreceptor compositions, cells comprising chimeric CD8α coreceptors, pharmaceutical compositions comprising CD8α coreceptors and / or cells, and methods of use thereof.

[0020] The present disclosure provides a chimeric CD8α coreceptor comprising: a) a truncated CD8α coreceptor comprising a CD8α coreceptor extracellular domain and a CD8α coreceptor transmembrane domain; and b) a CD4 intracellular domain comprising a palmitoylation motif and an Lck-binding domain, wherein the CD4 intracellular domain is fused in-frame to the C-terminus of the truncated CD8α coreceptor.

[0021] The present disclosure overcomes problems associated with current technology by providing a chimeric CD8α coreceptor that can be expressed on the surface of T cells and used in immunotherapy, such as for the treatment of immune-related diseases, including cancer and autoimmune disorders, and infections, including, but not limited to, viruses. The present disclosure is based, at least in part, on the discovery that a palmitoylation motif and an Lck-binding domain, which are not present in wild-type CD8α coreceptors, enable the recruitment of factors that improve T cell signaling. Increased T cell signaling leads to increased T cell activation and cytotoxicity, which are advantageous properties for T cell therapy (e.g., TCR-T cells). Accordingly, the present disclosure provides cells and methods for generating cells (e.g., T cells) that express a chimeric CD8α coreceptor. Such cells exhibit increased T cell activation and cytotoxicity against target cells.

[0022] T cell receptor T cell receptors ("TCRs") are molecules localized on the surface of T cells that are responsible for recognizing antigens bound to MHC molecules. During antigen processing, antigens are degraded intracellularly and then delivered to the cell surface in the form of peptides bound to major histocompatibility complex (MHC) molecules (human leukocyte antigens or HLA molecules in humans). T cells recognize these peptide-MHC complexes on the surface of antigen-presenting cells or target tissue cells. Two classes of MHC molecules, MHC class I and MHC class II, deliver peptides from different cellular compartments to the cell surface, where they are recognized by CD8+ and CD4+ T cells, respectively.

[0023] In particular, TCRs are disulfide-linked membrane-anchored heterodimeric proteins consisting of highly variable alpha and beta chains that typically associate with invariant CD3 chain molecules to form a complete, functional TCR. The alpha and beta chains consist of extracellular domains containing constant (C) and variable (V) regions. The constant region is proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the variable region binds to ligands. The variable domains of both the TCR alpha and beta chains each contain three variable regions called complementarity-determining regions (CDRs).

[0024] CD8α coreceptor CD4 and CD8 are transmembrane glycoproteins that function as coreceptors for the T cell receptor (TCR). Binding of CD4 and CD8 to MHC molecules helps stabilize weak T cell receptor (TCR)-pMHC interactions. Meanwhile, the cytoplasmic tails of the CD4 and CD8 coreceptors efficiently recruit the Src kinase Lck to the TCR complex upon coreceptor binding to MHC, thereby enhancing the initiation of TCR signaling and T cell activation.

[0025] To function, CD8 forms a dimer consisting of a pair of CD8 chains. The most common form of CD8 consists of the CD8 α chain and the CD8 β chain. A less common homodimer of the CD8 α chain is also expressed on some cells. A single immunoglobulin-like domain and a long stalk region form the extracellular portion of the CD8 subunit. In its intracellular tail, the α subunit of CD8 contains the Lck-binding site, while the β subunit contains a palmitoylation site.

[0026] The wild-type CD8α coreceptor (UniProt ID No. P01732) has the amino acid sequence of SEQ ID NO: 11. The extracellular and transmembrane domains are shown in bold and underlined font.

[0027] MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT LYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV (SEQ ID NO: 11)

[0028] CD4 consists of a single chain with four immunoglobulin domains (D1-D4) exposed on the extracellular surface of the cell and a short cytoplasmic / intracellular tail. CD4 functional motifs, including the Lck binding site and palmitoylation site, are located within its sole intracellular domain.

[0029] The wild-type CD4 coreceptor (UniProt ID No. P01730) has the amino acid sequence of SEQ ID NO: 12. The intracellular domain is shown in bold font. The Lck-binding domain is shown in bold and italic font. The palmitoylation domain is shown in bold and underlined font.

[0030] MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDS DTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKASS IVYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPLHLTLPQALPQYAGSGNLTLALEAKTGKLHQEVN LVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAKVSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPMALIVLGGVAGLLLFIGLGIFF CVRC RHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPI (SEQ ID NO: 12)

[0031] A. Protein Palmitoylation and Palmitoylation Motifs Protein palmitoylation is a widespread lipid modification in which one or more cysteine ​​thiols on a substrate protein are modified to form a thioester containing a saturated 16-carbon fatty acid palmitoyl group. This lipid modification is readily reversible, a hallmark of protein palmitoylation that allows rapid regulation of the function of many cellular proteins (see, e.g., Guan and Fierke Sci China Chem. 2011 Dec; 54(12): 1888-1897).

[0032] For example, palmitoylation not only serves as a lipid anchor for localizing proteins to the plasma membrane, but also plays a role in shuttling modified proteins between cellular compartments, allowing relocalization of proteins within different regions of the cell or membrane. Palmitoylation motifs are small tetrameric sequences containing cysteine ​​residues at positions 1 and 4 of the tetramer.

[0033] In some embodiments, the palmitoylation motif comprises, consists essentially of, or consists of an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 3. In some embodiments, the palmitoylation motif comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3. In some embodiments, the palmitoylation motif has the amino acid sequence of SEQ ID NO: 3.

[0034] In some embodiments, the palmitoylation motif is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO: 8. In some embodiments, the palmitoylation motif is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO:8.

[0035] B. High-affinity Lck domain T cell signaling begins with ligation of the T cell antigen receptor (TCR) by a cognate peptide and phosphorylation of the receptor's immunoreceptor tyrosine-based activation motif domain by the kinase Lck.

[0036] In some embodiments, the Lck-binding domain (also referred to as a "high-affinity Lck-binding site" or "high-affinity binding domain") comprises, consists essentially of, or consists of an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 4. In some embodiments, the Lck-binding domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4. In some embodiments, the Lck-binding domain has the amino acid sequence of SEQ ID NO: 4.

[0037] In some embodiments, the Lck-binding domain is encoded by a polynucleotide that comprises, consists essentially of, or consists of a nucleic acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO: 9. In some embodiments, the Lck-binding domain is encoded by a polynucleotide that comprises, consists essentially of, or consists of the nucleic acid sequence of SEQ ID NO: 9.

[0038] Exemplary Chimeric CD8α Coreceptors The present disclosure provides chimeric CD8α coreceptors comprising a CD8α extracellular domain and a CD8α transmembrane domain, which allow dimerization of the CD8α coreceptor and the intracellular domain of CD4 (having functional motifs including both an Lck binding site and a palmitoylation site). In some embodiments, the chimeric CD8α coreceptor is heterodimerized. In some embodiments, the chimeric CD8α coreceptor is homodimerized. In some embodiments, the homodimerized chimeric CD8α coreceptor contains two sets of functional motifs and has higher potency in initiating TCR signaling compared to the wild-type CD8α coreceptor.

[0039] In some embodiments, the CD8α extracellular domain and transmembrane domain comprise, consist essentially of, or consist of an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 1. In some embodiments, the CD8α extracellular domain and transmembrane domain comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 1.

[0040] In some embodiments, the CD8α extracellular domain and transmembrane domain are encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO: 6. In some embodiments, the CD8α extracellular domain and transmembrane domain are encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 6.

[0041] In some embodiments, the CD4 intracellular domain comprises, consists essentially of, or consists of an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 2. In some embodiments, the CD4 intracellular domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:2.

[0042] In some embodiments, the CD4 intracellular domain is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO: 7. In some embodiments, the CD4 intracellular domain is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO:7.

[0043] An exemplary chimeric CD8α coreceptor was constructed by fusing the N-terminal region of CD8α (positions 1-203 of SEQ ID NO: 11) to the C-terminal region of CD4 (positions 419-458 of SEQ ID NO: 12).

[0044] An exemplary chimeric CD8α coreceptor was constructed by fusing the wild-type N-terminal CD8α extracellular and transmembrane domains (SEQ ID NO: 1) with the C-terminal wild-type CD4 intracellular domain (SEQ ID NO: 2). Exemplary chimeric CD8α coreceptor amino acid sequence domains and the nucleic acid sequences encoding them are shown in Tables 1 and 2. [Table 1] [Table 2-1] [Table 2-2]

[0045] In some embodiments, the chimeric CD8α coreceptor comprises, consists essentially of, or consists of an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 5. In some embodiments, the chimeric CD8α coreceptor comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:5.

[0046] In some aspects, the chimeric CD8α coreceptor is encoded by a polynucleotide that comprises, consists essentially of, or consists of a nucleic acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO: 10. In some aspects, the chimeric CD8α coreceptor is encoded by a polynucleotide that comprises, consists essentially of, or consists of the nucleic acid sequence of SEQ ID NO: 10.

[0047] The chimeric CD8α coreceptor polypeptide sequence was reverse-translated and codon-optimized for human T cell expression using the GeneArt® tool. The resulting nucleic acid sequence encoding the chimeric CD8α coreceptor (SEQ ID NO: 10) was cloned into a transposon-based vector along with the TCR gene, an inducible suicide gene, and a selection gene.

[0048] In certain embodiments of the present disclosure, the nucleotide sequences encoding the chimeric CD8α coreceptor and TCR are cloned into a transposon that comprises a bidirectional expression cassette for expression of the chimeric CD8α coreceptor and TCR. In some embodiments, the transposon further comprises an expression cassette for expression of the CAR.

[0049] An exemplary transposon containing a nucleotide sequence encoding a chimeric CD8α coreceptor is shown in Figure 2. The transposon contains the following nucleotide sequences from 5' to 3': a left inverted terminal repeat (ITR), a first insulator sequence, a first poly sequence (3'-5'), a chimeric CD8 coreceptor (3'-5'), a PGK promoter (3'-5'), an EF1a promoter, an iCAS9 safety switch, a TCR beta chain, a TCR alpha chain, a DHFR selectable marker (each of the iCAS9 safety switch, TCR beta chain, TCR alpha chain, and DHFR selectable marker are separated by a T2A sequence), a second poly A sequence, a second insulator sequence, and a right ITR.

[0050] Compositions comprising the chimeric CD8α coreceptor of the present disclosure can be incorporated into a cell delivery composition (e.g., a transposon or vector) as described in detail herein, and optionally incorporated into a cell.

[0051] Cells and modified cells of the present disclosure The cells and modified cells of the present disclosure can be mammalian cells. Preferably, the cells and modified cells are human cells. The cells and modified cells of the present disclosure can be immune cells. The immune cells of the present disclosure include lymphoid progenitor cells, T lymphocytes (T cells), T memory stem cells (T SCM cells), central memory T cells (T CM ), or stem cell-like T cells.

[0052] The modified T cells of the present disclosure may be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs. Unlike conventional biologics and chemotherapeutics, the disclosed modified T cells have the ability to rapidly replicate upon antigen recognition, thereby potentially eliminating the need for repeat treatments. To accomplish this, in some embodiments, the modified T cells persist in the patient as a stable population of viable memory T cells not only to drive the initial response but also to prevent potential relapse. Alternatively, in some aspects, the modified T cells do not persist in the patient when this is not desired.

[0053] The disclosed methods can engineer and / or generate a population of engineered T cells, wherein at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, or any percentage therebetween, a plurality of engineered T cells in the population express a chimeric CD8α coreceptor. In some aspects, the chimeric CD8α coreceptor is expressed on the cell surface as a homodimer.

[0054] The methods of the disclosure can modify and / or generate a population of modified T cells, wherein at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, or any percentage therebetween, a plurality of the modified T cells in the population express a TCR.

[0055] The disclosed methods can engineer and / or generate a population of engineered T cells, wherein at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, or any percentage therebetween, a plurality of engineered T cells in the population express a TCR and a chimeric CD8α coreceptor. In some aspects, the chimeric CD8α coreceptor is expressed on the cell surface as a homodimer.

[0056] A plurality of the modified cells of the population comprise a transgene or a sequence encoding the transgene (e.g., a chimeric CD8α coreceptor and a TCR), and at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% of the plurality of cells of the population express the transgene or the sequence encoding the chimeric CD8α coreceptor, and at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% of the modified cells of the population express the sequence encoding the transgene or the TCR.

[0057] Compositions and methods for generating and / or expanding immune cells or immune progenitor cells (e.g., the disclosed modified T cells) and buffers for maintaining or enhancing cell viability and / or levels of stem-like phenotype of immune cells or immune progenitor cells (e.g., the disclosed modified T cells) are disclosed elsewhere herein and in more detail in U.S. Pat. No. 10,329,543 and PCT Publication WO 2019 / 173636.

[0058] The cells and modified cells of the present disclosure can be somatic cells. The cells and modified cells of the present disclosure can be differentiated cells. The cells and modified cells of the present disclosure can be autologous or allogeneic cells. Allogeneic cells are engineered to prevent adverse reactions to engraftment after administration to a subject. Allogeneic cells can be any type of cell. Allogeneic cells can be stem cells or derived from stem cells. Allogeneic cells can be differentiated somatic cells.

[0059] Methods for expressing chimeric CD8α coreceptors The present disclosure provides methods for expressing a chimeric CD8α coreceptor on the cell surface. The methods include: (a) obtaining a cell population; (b) contacting the cell population with a chimeric CD8α coreceptor or a composition comprising a sequence encoding a chimeric CD8α coreceptor under conditions sufficient to translocate the chimeric CD8α coreceptor across the cell membrane of at least one cell in the cell population, thereby generating an engineered cell population; (c) culturing the engineered cell population under conditions suitable for integration of the sequence encoding the chimeric CD8α coreceptor; and (d) expanding and / or selecting at least one cell from the engineered cell population that expresses the chimeric CD8α coreceptor on its cell surface. In some embodiments, the chimeric CD8α coreceptor is a homodimer. In some embodiments, the chimeric CD8α coreceptor is a heterodimer.

[0060] In some aspects, the cell population can include leukocytes and / or CD4+ and CD8+ leukocytes. The cell population can include CD4+ and CD8+ leukocytes in an optimized ratio. In some embodiments, the optimized ratio of CD4+ and CD8+ leukocytes is not naturally occurring in vivo. The cell population can include tumor cells.

[0061] In some embodiments, the conditions sufficient to translocate a chimeric CD8α coreceptor or a sequence encoding a chimeric CD8α coreceptor, a transposon encoding a chimeric CD8α coreceptor, or a vector encoding a chimeric CD8α coreceptor across the cell membrane of at least one cell in the cell population comprise at least one of the following: application of one or more electrical pulses at a particular voltage, a buffer, and one or more cofactors. In some embodiments, the conditions suitable for integration of a sequence encoding a chimeric CD8α coreceptor comprise at least one of a buffer and one or more cofactors.

[0062] The buffer can include PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, human T cell nucleofection buffer, or any combination thereof. The one or more supplemental factors can include (a) recombinant human cytokines, chemokines, interleukins, or any combination thereof, (b) salts, minerals, metabolites, or any combination thereof, (c) cell culture media, (d) inhibitors of cellular DNA sensing, metabolic, differentiation, signal transduction, one or more apoptotic pathways, or combinations thereof, and (e) one or more nucleic acid modifying or stabilizing reagents. Recombinant human cytokines, chemokines, interleukins or any combination thereof include IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, IL14, IL16, IL17, IL18, IL19, IL20, IL22, IL23, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36, GM-CSF, IFN-gamma, IL-1 alpha / IL-1F1, IL-1 beta / IL-1F2, IL-12 p70, IL-12 / IL-35 The proteins may include p35, IL-13, IL-17 / IL-17A, IL-17A / F heterodimer, IL-17F, IL-18 / IL-1F4, IL-23, IL-24, IL-32, IL-32beta, IL-32gamma, IL-33, LAP (TGF-beta 1), lymphotoxin-alpha / TNF-beta, TGF-beta, TNF-alpha, TRANCE / TNFSF11 / RANK L, or any combination thereof.

[0063] The salts, minerals, metabolites, or any combination thereof, can include HEPES, nicotinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solution, ascorbic acid, nucleosides, FBS / FCS, human serum, serum replacement, antibiotics, pH adjusters, Earle's salts, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, Nucleofector PLUS Supplement, KCL, MgCl, NaHPO, NAHPO, sodium lactobionate, mannitol, sodium succinate, sodium chloride, CINa, glucose, Ca(NO), Tris / HCl, KHPO, KHPO, polyethyleneimine, poly-ethylene-glycol, poloxamer 188, poloxamer 181, poloxamer 407, poly-vinylpyrrolidone, Pop313, Crown-5, or any combination thereof. The cell culture medium may include PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC Medium, CTS OpTimizer T Cell Expansion SFM, TexMACS Medium, PRIME-XV T Cell Expansion Medium, ImmunoCult-XF T Cell Expansion Medium, or any combination thereof.

[0064] Inhibitors of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptosis pathways, or combinations thereof, include inhibitors of TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type 1 interferon, proinflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, caspase-1, Pro-IL1B, PI3K, Akt, Wnt3A, glycogen synthase kinase-3β (GSK-3β) (e.g., TWS119), or any combination thereof. Examples of such inhibitors include bafilomycin, chloroquine, quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK, or any combination thereof. Reagents that modify or stabilize one or more nucleic acids include pH adjusting agents, DNA binding proteins, lipids, phospholipids, CaPO4, net neutral charge DNA binding peptides with or without NLS sequences, TREX1 enzyme, or any combination thereof.

[0065] The expansion and selection steps can occur simultaneously or sequentially. Expansion can occur before selection. Expansion can occur after selection, and optionally, a further (i.e., second) selection can occur after expansion. Concurrent expansion and selection can be simultaneous. The expansion and / or selection steps can proceed for a period of 10 to 14 days, inclusive.

[0066] The expansion may include contacting at least one cell of the modified cell population with an antigen to stimulate at least one cell via a TCR and / or CAR, thereby generating an expanded cell population. The antigen may be presented on the surface of a substrate. The substrate may have any form, including, but not limited to, a surface, a well, a bead, or a plurality thereof, and a matrix. The substrate may further include a paramagnetic or magnetic component. The antigen may be presented on the surface of the substrate, the substrate being a magnetic bead, and a magnet may be used to remove or separate the magnetic bead from the modified cell population and the expanded cell population. The antigen may be presented on the surface of a cell or an artificial antigen-presenting cell. The artificial antigen-presenting cell may include, but is not limited to, tumor cells and stem cells.

[0067] In some aspects, where the transposon or vector comprises a selection gene, the selection step comprises contacting at least one cell of the modified cell population with a compound to which the selection gene confers resistance, thereby identifying cells that express the selection gene as surviving the selection and cells that do not express the selection gene as not surviving the selection step.

[0068] The present disclosure provides modifications, extensions of the methods described herein, and compositions comprising selected cell populations.

[0069] The present disclosure provides a cell or cell population, the cell comprising a composition comprising: (a) an inducible transgene construct comprising a sequence encoding an inducible promoter and a sequence encoding a transgene (e.g., a chimeric CD8α coreceptor); and (b) an inducible transgene construct comprising a sequence encoding an inducible promoter and a sequence encoding a TCR and / or CAR, wherein upon integration of the construct of (a) and the construct of (b) into the genomic sequence of the cell, the chimeric CD8α coreceptor and the TCR and / or CAR are expressed on the surface of the cell (e.g., a T cell). Exemplary inducible promoters include, but are not limited to, the PGK promoter and the EF1a promoter. In some aspects, the PGK promoter controls expression of the chimeric CD8α coreceptor. In some aspects, the EF1a promoter controls expression of the TCR.

[0070] Transposon and Vector Compositions The present disclosure provides compositions and methods for delivering a chimeric CD8α coreceptor and / or TCR and / or CAR to a cell or cell population. Non-limiting examples of compositions for delivering the disclosed compositions to a cell or cell population include transposons or vectors. Thus, the present disclosure provides (i) a transposon comprising a chimeric CD8α coreceptor and / or TCR and / or CAR, or (ii) a vector comprising a chimeric CD8α coreceptor and / or TCR and / or CAR.

[0071] A transposon comprising a chimeric CD8α coreceptor and / or TCR and / or CAR of the present disclosure, or a vector comprising a chimeric CD8α coreceptor and / or TCR and / or CAR of the present disclosure, can further comprise a sequence encoding an inducible pro-apoptotic polypeptide (e.g., iC9). Alternatively, or in addition, one transposon or one vector can comprise a chimeric CD8α coreceptor and / or TCR and / or CAR of the present disclosure, and a second transposon or second vector can comprise a sequence encoding an inducible pro-apoptotic polypeptide of the present disclosure. Inducible pro-apoptotic polypeptides are described in more detail herein.

[0072] A transposon comprising a TCR and / or CAR of the present disclosure, or a vector comprising a TCR and / or CAR of the present disclosure, may further comprise a sequence encoding a chimeric CD8α coreceptor. Alternatively, or in addition, one transposon or one vector may comprise a TCR and / or CAR of the present disclosure, and a second transposon or second vector may comprise a sequence encoding a chimeric CD8α coreceptor. Chimeric CD8α coreceptors are described in more detail herein.

[0073] A transposon comprising a chimeric CD8α coreceptor and / or TCR and / or CAR of the present disclosure, or a vector comprising a chimeric CD8α coreceptor and / or TCR and / or CAR of the present disclosure, may further comprise a selection gene. The selection gene may encode a gene product essential for cell viability and survival. The selection gene may encode a gene product essential for cell viability and survival when tested by selective cell culture conditions. The selective cell culture conditions may include a compound deleterious to cell viability or survival, and the gene product confers resistance to the compound. Non-limiting examples of selection genes include neo (confers resistance to neomycin), DHFR (encodes dihydrofolate reductase, conferring resistance to methotrexate), TYMS (encodes thymidylate synthetase), MGMT (encodes O(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encodes aldehyde dehydrogenase 1 family, member A1), FRANCF, RAD51C (encodes RAD51 Paralog C), GCS (encodes glucosylceramide synthase), NKX2.2 (encodes NK2 homeobox 2), or any combination thereof.

[0074] In a preferred embodiment, the selection gene encodes a DHFR mutein enzyme. The DHFR mutein enzyme comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 13. The DHFR mutein enzyme is encoded by a polynucleotide comprising, consists essentially of, or consists of the nucleic acid sequence of SEQ ID NO: 14. The amino acid sequence of the DHFR mutein enzyme may further comprise a mutation at one or more of positions 80, 113, or 153. The amino acid sequence of the DHFR mutein enzyme may comprise one or more of a phenylalanine (F) or leucine (L) substitution at position 80, a leucine (L) or valine (V) substitution at position 113, and a valine (V) or aspartic acid (D) substitution at position 153.

[0075] Transposition System The present disclosure provides transposons or nanotransposons for expressing a chimeric CD8α coreceptor in a cell (e.g., a T cell), or for co-expressing a chimeric CD8α coreceptor and a TCR in a cell (e.g., a T cell). In some embodiments, the cell further comprises a CAR.

[0076] The transposon or nanotransposon of the present disclosure may be a plasmid DNA transposon comprising a sequence encoding a chimeric CD8α coreceptor flanked by two cis-regulatory insulator elements. The transposon or nanotransposon may further comprise a plasmid comprising a sequence encoding a transposase. The sequence encoding the transposase may be a DNA sequence or an RNA sequence. Preferably, the sequence encoding the transposase is an mRNA sequence.

[0077] The transposon or nanotransposon of the present disclosure can be a piggyBac™ (PB) transposon. In some embodiments, when the transposon is a PB transposon, the transposase is a piggyBac™ (PB) transposase, a piggyBac-like (PBL) transposase, or a Super piggyBac™ (SPB) transposase. In some embodiments, the sequence encoding the SPB transposase is an mRNA sequence.

[0078] Nanotransposons are described in more detail in PCT / US2019 / 067758. PB transposons, and non-limiting examples of PB, PBL, and SPB transposases, are described in detail in U.S. Patent No. 6,218,182, U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643, and PCT Publication WO2010 / 099296.

[0079] PB, PBL, and SPB transposases recognize transposon-specific inverted terminal repeats (ITRs) at the ends of the transposon and insert their contents between the ITRs at the sequence 5'-TTAT-3' (TTAT target sequence) within the chromosomal site, or at the sequence 5'-TTAA-3' (TTAA target sequence) within the chromosomal site. The target sequences of the PB or PBL transposons are 5'-CTAA-3', 5'-TTAG-3', 5'-ATAA-3', 5'-TCAA-3', 5'AGTT-3', 5'-ATTA-3', 5'-GTTA-3', 5'-TTGA-3', 5'-TTTA-3', 5'-TTAC-3', 5'-ACTA-3', 5'-AGGG-3', 5'-CTAG-3', 5'-TGAA-3', 5'-AGGT-3', 5'-ATCA-3', 5'-CTCC-3', 5'-TAAA-3', 5'-TCTC-3', 5'TGAA-3', 5'-A AAT-3', 5'-AATC-3', 5'-ACAA-3', 5'-ACAT-3', 5'-ACTC-3', 5'-AGTG-3', 5'-ATAG-3', 5'-CAAA-3', 5'-CACA-3', 5'-CATA-3', 5'-CCAG-3', 5'-CCCA-3', 5'-CGTA-3', 5'-GTCC-3', 5'-TAAG-3', 5'-TCTA-3', 5'-TGAG-3', 5'-TGTT-3', 5'-TTCA-3', 5'-TTCT-3', and 5'-TTTT-3'. The PB or PBL transposon system has no payload limit for the gene of interest that can be included between the ITRs.

[0080] Exemplary amino acid sequences of one or more PB, PBL, and SPB transposases are disclosed in U.S. Patent No. 6,218,185, U.S. Patent No. 6,962,810, and U.S. Patent No. 8,399,643. In preferred embodiments, the PB transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO:15.

[0081] The PB or PBL transposase may comprise or consist of an amino acid sequence having an amino acid substitution at two or more, three or more, or each of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 15. The transposase may be an SPB transposase comprising or consisting of the amino acid sequence of SEQ ID NO: 15, wherein the amino acid substitution at position 30 may be a substitution of valine (V) for isoleucine (I), the amino acid substitution at position 165 may be a substitution of serine (S) for glycine (G), the amino acid substitution at position 282 may be a substitution of valine (V) for methionine (M), and the amino acid substitution at position 538 may be a substitution of lysine (K) for asparagine (N). In preferred embodiments, the SPB transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:16.

[0082] In certain embodiments, where the transposase comprises the above mutations at positions 30, 165, 282, and / or 538, the PB, PBL, and SPB transposases comprise mutations at positions 3, 46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 32 , 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570, and 591, as described in more detail in PCT Publication Nos. WO2019 / 173636 and PCT / US2019 / 049816.

[0083] The PB, PBL, or SPB transposase can be isolated or derived from an insect, vertebrate, crustacean, or urochordate, as described in more detail in PCT Publication Nos. WO2019 / 173636 and PCT / US2019 / 049816. In a preferred embodiment, the PB, PBL, or SPB transposase is isolated or derived from the insect Trichoplusia ni (GenBank Accession No. AAA87375) or Bombyx mori (GenBank Accession No. BAD11135).

[0084] A hyperactive PB or PBL transposase is a transposase that is more active than the naturally occurring variant from which it is derived. In a preferred embodiment, the hyperactive PB or PBL transposase is isolated or derived from Bombyx mori or Xenopus tropicalis. Examples of hyperactive PB or PBL transposases are disclosed in U.S. Patent No. 6,218,185, U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643, and WO2019 / 173636. A list of hyperactive amino acid substitutions is disclosed in U.S. Patent No. 10,041,077.

[0085] In some embodiments, the PB or PBL transposase is integration-deficient. An integration-deficient PB or PBL transposase is a transposase that can excise its corresponding transposon but integrates the excised transposon at a lower frequency than the corresponding wild-type transposase. Examples of integration-deficient PB or PBL transposases are disclosed in U.S. Patent No. 6,218,185, U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643, and WO2019 / 173636. A list of integration-deficient amino acid substitutions is disclosed in U.S. Patent No. 10,041,077.

[0086] In some embodiments, the PB or PBL transposase is fused to a nuclear localization signal. Examples of PB or PBL transposases fused to nuclear localization signals are disclosed in U.S. Patent No. 6,218,185, U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643, and WO2019 / 173636.

[0087] The transposon of the present disclosure can be a Sleeping Beauty transposon. In some embodiments, when the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase (e.g., as disclosed in U.S. Pat. No. 9,228,180) or a hyperactive Sleeping Beauty (SB100X) transposase. In preferred embodiments, the Sleeping Beauty transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 17. In preferred embodiments, the hyperactive Sleeping Beauty (SB100X) transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:18.

[0088] The transposon of the present disclosure can be a Helraiser transposon. Exemplary Helraiser transposons include Helibat1, which comprises or consists of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO: 19. In some embodiments, when the transposon is a Helraiser transposon, the transposase is a Helitron transposase (e.g., as disclosed in WO2019 / 173636). In preferred embodiments, the Helitron transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO: 20.

[0089] The transposon of the present disclosure may be a Tol2 transposon. Exemplary Tol2 transposons, including inverted repeats, sequences near the ends, and a Tol2 transposase, comprise or consist of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO: 21. In some embodiments, when the transposon is a Tol2 transposon, the transposase is a Tol2 transposase (e.g., as disclosed in WO2019 / 173636). In preferred embodiments, the Tol2 transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:22.

[0090] The transposon of the present disclosure can be a TcBuster transposon. In some embodiments, when the transposon is a TcBuster transposon, the transposase is a TcBuster transposase or a hyperactive TcBuster transposase (e.g., as disclosed in WO2019 / 173636). The TcBuster transposase can comprise or consist of a naturally occurring or non-naturally occurring amino acid sequence. In preferred embodiments, the TcBuster transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO: 23. The polynucleotide encoding the TcBuster transposase can comprise or consist of a naturally occurring or non-naturally occurring nucleic acid sequence. In preferred embodiments, the TcBuster transposase is encoded by a polynucleotide comprising or consisting of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO:24.

[0091] In some embodiments, the mutant TcBuster transposase comprises one or more sequence variations when compared to the wild-type TcBuster transposase, as described in more detail in PCT Publications WO2019 / 173636 and PCT / US2019 / 049816.

[0092] Vector System The vectors of the present disclosure may be viral vectors or recombinant vectors. Viral vectors may contain sequences isolated or derived from retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or any combination thereof. Viral vectors may contain sequences isolated or derived from adeno-associated viruses (AAV). Viral vectors may include recombinant AAV (rAAV). Exemplary adeno-associated viruses and recombinant adeno-associated viruses contain two or more inverted terminal repeat (ITR) sequences located in cis adjacent to the sequence encoding the chimeric CD8α coreceptor of the present disclosure. Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, all serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, self-complementary AAV (scAAV) and AAV hybrids containing the genome of one serotype and the capsid of another serotype (e.g., AAV2 / 5, AAV-DJ, and AAV-DJ8). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, rAAV-LK03.

[0093] The vector of the present disclosure can be a nanoparticle. Non-limiting examples of nanoparticle vectors include nucleic acids (e.g., RNA, DNA, synthetic nucleotides, modified nucleotides, or any combination thereof), amino acids (L-amino acids, D-amino acids, synthetic amino acids, modified amino acids, or any combination thereof), polymers (e.g., polymersomes), micelles, lipids (e.g., liposomes), organic molecules (e.g., carbon atoms, sheets, fibers, tubes), inorganic molecules (e.g., calcium phosphate or gold), or any combination thereof. Nanoparticle vectors can be passively or actively transported across cell membranes.

[0094] The cell delivery compositions (e.g., transposons, vectors) disclosed herein can include nucleic acids encoding therapeutic proteins or therapeutic agents. Examples of therapeutic proteins include those disclosed in PCT Publication Nos. WO2019 / 173636 and PCT / US2019 / 049816.

[0095] Inducible Pro-Apoptotic Polypeptides The inducible pro-apoptotic polypeptides disclosed herein are superior to existing inducible polypeptides because the disclosed inducible pro-apoptotic polypeptides are much less immunogenic. The inducible pro-apoptotic polypeptides are recombinant polypeptides and, therefore, are non-naturally occurring. Furthermore, the sequences recombined to produce the inducible pro-apoptotic polypeptides are free of non-human sequences that can be recognized by the host human immune system as "non-self," which results in an immune response in a subject receiving the inducible pro-apoptotic polypeptide, cells comprising the inducible pro-apoptotic polypeptide, or a composition comprising the inducible pro-apoptotic polypeptide or cells comprising the inducible pro-apoptotic polypeptide.

[0096] The present disclosure provides an inducible pro-apoptotic polypeptide comprising a ligand-binding region, a linker, and a pro-apoptotic peptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction site. In certain embodiments, the ligand-binding region can be a multimeric ligand-binding region. In certain embodiments, the pro-apoptotic peptide is a caspase polypeptide. Non-limiting examples of caspase polypeptides include caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, and caspase 14. Preferably, the caspase polypeptide is a caspase 9 polypeptide. The caspase 9 polypeptide can be a truncated caspase 9 polypeptide. The inducible pro-apoptotic polypeptide can be non-naturally derived. When the caspase is caspase 9 or truncated caspase 9, the inducible pro-apoptotic polypeptide may also be referred to as an "iC9 safety switch."

[0097] The inducible caspase polypeptide can comprise (a) a ligand-binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise non-human sequences. In certain embodiments, the inducible caspase polypeptide comprises (a) a ligand-binding region, (b) a linker, and (c) a truncated caspase-9 polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise non-human sequences.

[0098] The ligand-binding region may comprise an FK506 binding protein 12 (FKBP12) polypeptide. The amino acid sequence of the ligand-binding region comprising the FK506 binding protein 12 (FKBP12) polypeptide may comprise a modification at position 36 of the sequence. The modification may be a substitution of valine (V) for phenylalanine (F) at position 36 (F36V). The FKBP12 polypeptide may comprise, consist essentially of, or consist of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:25. The FKBP12 polypeptide may be encoded by a polynucleotide comprising or consisting of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO:26.

[0099] The linker region can comprise, consist essentially of, or consist of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 27. In some embodiments, the linker region can be encoded by a polynucleotide that comprises or consists of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 28. In some embodiments, the nucleic acid sequence encoding the linker does not include any restriction sites.

[0100] A truncated caspase-9 polypeptide may comprise an amino acid sequence that does not include an arginine (R) at position 87 of the sequence. Alternatively, or in addition, a truncated caspase-9 polypeptide may comprise an amino acid sequence that does not include an alanine (A) at position 282 of the sequence. A truncated caspase-9 polypeptide may comprise, consist essentially of, or consist of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:29. In some embodiments, a truncated caspase-9 polypeptide may be encoded by a polynucleotide that comprises or consists of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:30.

[0101] In certain embodiments, when the polypeptide comprises a truncated caspase-9 polypeptide, the inducible pro-apoptotic polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 31. In some embodiments, the inducible pro-apoptotic polypeptide is encoded by a polynucleotide that comprises or consists of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 32.

[0102] The inducible pro-apoptotic polypeptide may be expressed in a cell under the transcriptional control of any promoter known in the art that is capable of initiating and / or regulating expression of the inducible pro-apoptotic polypeptide in that cell.

[0103] Activation of inducible pro-apoptotic polypeptides can be achieved, for example, through chemically induced dimerization (CID) mediated by an inducer to generate conditionally controlled proteins or polypeptides. Not only are the pro-apoptotic polypeptides inducible, but the induction of these polypeptides is also reversible due to degradation of the labile dimerizer or administration of a competitive inhibitor of the monomer.

[0104] In certain embodiments, when the ligand binding region comprises an FKBP12 polypeptide having a substitution of valine (V) for phenylalanine (F) at position 36 (F36V), the inducer is AP1903, a synthetic drug (CAS index name: 2-piperidinecarboxylic acid, 1-[(2S)-1-oxo-2-(3,4,5-trimethoxyphenyl)butyl]-, 1,2-ethanediylbis[imino(2-oxo-2,1-ethanediyl)oxy-3,1-phenylene [(1R )-3-(3,4-dimethoxyphenyl)propylidene]] ester, [2S-[1(R),2R[S[S[1(R),2R]]]]]-(9Cl) CAS Registry Number: 195514-63-7; molecular formula: C78H98N4O20; molecular weight: 1411.65); AP20187 (CAS Registry Number: 195514-80-8 and molecular formula: C82H107N5O20) or an AP20187 analog, such as, for example, AP1510. As used herein, the inducers AP20187, AP1903, and AP1510 may be used interchangeably.

[0105] Inducible pro-apoptotic peptides and methods for inducing these peptides are described in detail in U.S. Patent Publication No. WO2019 / 0225667 and PCT Publication No. WO2018 / 068022.

[0106] Formulations, Dosages, and Modes of Administration The present disclosure provides formulations, dosages, and methods for administration of the compositions described herein.

[0107] The disclosed compositions and pharmaceutical compositions may further comprise at least one of any suitable auxiliary agent, including, but not limited to, a diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, adjuvant, etc. Pharmaceutically acceptable auxiliary agents are preferred. Non-limiting examples and methods for the preparation of such sterile solutions are well known in the art, for example, but not limited to, Gennaro, Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, Pa.) 1990, and "Physician's Desk Reference," 52nd ed., Medical Economics (Montvale, NJ) 1998. Pharmaceutically acceptable carriers may be routinely selected that are suitable for the mode of administration, solubility, and / or stability of the protein scaffold, fragment, or variant composition, as known in the art or described herein.

[0108] Non-limiting examples of pharmaceutical excipients and additives suitable for use include proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars, and polysaccharides or sugar polymers), which may be present singly or in combination, including alone or in combinations ranging from 1 to 99.99% by weight or volume. Non-limiting examples of protein excipients include serum albumins, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, and casein. Representative amino acids / protein components that may also function in a buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. In some embodiments, the amino acid is glycine.

[0109] Non-limiting examples of carbohydrate excipients suitable for use include monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc., disaccharides such as lactose, sucrose, trehalose, cellobiose, etc., polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc., and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), myo-inositol, etc. Preferably, the carbohydrate excipient is mannitol, trehalose, and / or raffinose.

[0110] The composition may also contain a buffer or pH adjuster, and typically, the buffer is a salt prepared from an organic acid or base.Representative buffers include organic acid salts such as citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid salts, tris(tris(hydroxymethyl)aminomethane), tromethamine hydrochloride, or phosphate buffers.Preferred buffers are organic acid salts such as citrate.

[0111] Additionally, the disclosed compositions may include polymeric excipients / additives such as polyvinylpyrrolidone, Ficoll (polymeric sugars), dextrates (e.g., cyclodextrins, e.g., 2-hydroxypropyl-β-cyclodextrin), polyethylene glycol, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "TWEEN 20" and "TWEEN 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

[0112] Many known and developed modes can be used to administer a therapeutically effective amount of a composition or pharmaceutical composition disclosed herein. Non-limiting examples of modes of administration include bolus, buccal, infusion, intra-articular, intrabronchial, intraperitoneal, intracapsular, intrachondral, intracavitary, intracavitary, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intralesional, intramuscular, intramyocardial, intranasal, intraocular, intraosseous, intraperiosteal, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intratumoral, intravenous, intravesical, oral, parenteral, rectal, sublingual, subcutaneous, transdermal, or intravaginal means.

[0113] The compositions of the present disclosure can be prepared for parenteral (subcutaneous, intramuscular, or intravenous) or any other administration, particularly in the form of a liquid solution or suspension. In some embodiments, the compositions can be prepared for vaginal or rectal administration in semi-solid forms, including, but not limited to, creams and suppositories. In some embodiments, the compositions can be prepared for buccal or sublingual administration. Non-limiting examples of formulations for buccal or sublingual administration can include tablets or capsules. In some embodiments, the compositions can be prepared for intranasal administration. Non-limiting examples of formulations for intranasal administration can include powders, nasal sprays, or aerosols. In some embodiments, these formulations further comprise a specific pharmaceutical agent. In some embodiments, the compositions can be prepared for transdermal administration. Non-limiting examples of formulations for transdermal administration can include gels, ointments, lotions, suspensions, or patch delivery systems. In some embodiments, the transdermal composition further comprises a chemical enhancer. In some embodiments, the chemical enhancer may be dimethyl sulfoxide to either modify skin structure or increase drug concentration in transdermal patches (Junginger, et al. In “Drug Permeation Enhancement;” Hsieh, DS, Eds., pp. 59-90 (Marcel Dekker, Inc. New York 1994)). In some embodiments, the suspension or patch delivery system further comprises an oxidizing agent to enable application of protein- and peptide-containing formulations to the skin (WO 98 / 53847). In some embodiments, transdermal administration involves the application of an electric field to create a transient transport pathway (such as electroporation) or to increase the mobility of charged drugs through the skin (such as iontophoresis). In some embodiments, transdermal administration involves the application of ultrasound, such as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402). The above-mentioned publications and patents are incorporated herein by reference in their entireties.

[0114] For parenteral administration, any composition disclosed herein may be formulated as a solution, suspension, emulsion, granules, powder, or lyophilized powder, either associated with a pharmaceutically acceptable parenteral vehicle or provided separately. Formulations for parenteral administration may contain common excipients such as sterile water or saline, polyalkylene glycols (e.g., polyethylene glycol), vegetable oils, hydrogenated naphthalenes, etc. Aqueous or oily suspensions for injection may be prepared using appropriate emulsifiers or wetting agents and suspending agents according to known methods. Injectable preparations may be non-toxic, parenterally administrable diluents such as aqueous solutions, sterile injectable solutions, or suspensions in solvents. Acceptable vehicles or solvents include water, Ringer's solution, isotonic saline, etc., and sterile fixed oils may be used as common solvents or suspension media. For these purposes, any type of fixed oil and fatty acid may be used, including natural, synthetic, or semisynthetic fatty oils or fatty acids, and natural, synthetic, or semisynthetic monoglycerides, diglycerides, or triglycerides. Parenteral administration is known in the art and includes, but is not limited to, conventional injection means as described in U.S. Pat. No. 5,851,198, gas pressurized needleless injection devices, and laser perforation devices as described in U.S. Pat. No. 5,839,446.

[0115] Formulations for oral administration may rely on the combined administration of (i) adjuvants (e.g., resorcinol and nonionic surfactants such as polyoxyethylene oleyl ether and / or n-hexadecyl polyethylene ether) to artificially increase the permeability of the intestinal wall and (ii) enzyme inhibitors (e.g., pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DFF), and trasylol) to inhibit enzymatic degradation. Formulations for the delivery of hydrophilic drugs (including proteins and protein scaffolds) and combinations of at least two surfactants intended for oral, buccal, mucosal, nasal, pulmonary, transvaginal, or rectal administration are described in U.S. Patent No. 6,309,663. The active ingredient compound of the solid dosage form for oral administration can be mixed with at least one additive, including but not limited to sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, and glycerides.These dosage forms can also contain other types of additives, such as inert diluents; lubricants such as magnesium stearate and parabens; preservatives such as sorbic acid, ascorbic acid, and alpha-tocopherol; antioxidants such as cysteine; disintegrants, binders, thickeners, buffers, sweeteners, flavorings, fragrances, etc.

[0116] Tablets and pills can be further processed into enteric-coated preparations. Liquid preparations for oral administration can include emulsions, syrups, elixirs, suspensions, and solution preparations acceptable for medical use. These preparations can contain inert diluents commonly used in the art, such as water. Liposomes have also been described as drug delivery systems for insulin and heparin (U.S. Pat. No. 4,239,754). More recently, microspheres of artificial polymers of mixed amino acids (proteinoids) have been used to deliver pharmaceuticals (U.S. Pat. No. 4,925,673). Additionally, carrier compounds, such as those described in U.S. Pat. Nos. 5,879,681 and 5,871,753, are known in the art and are used to orally deliver bioactive agents.

[0117] For pulmonary administration, the compositions or pharmaceutical compositions described herein are preferably delivered in a particle size effective to reach the lower airways of the lungs or sinuses. The compositions or pharmaceutical compositions can be delivered by any of a variety of inhalation or nasal devices known in the art for administering therapeutic agents by inhalation. These devices capable of depositing aerosolized formulations into a patient's sinus cavities or alveoli include metered-dose inhalers, nebulizers (e.g., jet nebulizers, ultrasonic nebulizers), dry powder generators, sprayers, and the like. All such devices can be used with formulations suitable for administering or dispensing the compositions or pharmaceutical compositions described herein in aerosols. Such aerosols can consist of either solutions (both aqueous and non-aqueous) or solid particles. Additionally, sprays containing the compositions or pharmaceutical compositions described herein can be generated by forcing a suspension or solution of at least one protein scaffold through a nozzle under pressure. In a metered dose inhaler (MDI), the propellant, composition, or pharmaceutical composition described herein, and any excipients or other additives, are contained in a canister as a mixture with a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol, preferably containing particles in the size range of less than about 10 μm, preferably about 1 μm to about 5 μm, and most preferably about 2 μm to about 3 μm. A more detailed description of pulmonary administration, formulations, and related devices is disclosed in PCT Publication WO 2019 / 049816.

[0118] For absorption through mucosal surfaces, the composition comprises an emulsion containing a plurality of submicron particles, a viscous polymer, a bioactive peptide, and an aqueous continuous phase, which promotes absorption through the mucosal surface by achieving mucoadhesion of the emulsion particles (U.S. Pat. No. 5,514,670). Mucous surfaces suitable for application of the emulsions of the present disclosure may include the corneal, conjunctival, oral, sublingual, nasal, vaginal, pulmonary, gastric, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration, such as suppositories, may contain excipients such as polyalkylene glycols, petrolatum, cocoa butter, etc. Formulations for intranasal administration may be solid and may contain excipients such as lactose, or may be aqueous or oily solutions of nasal drops. For buccal administration, excipients may include sugars, calcium stearate, magnesium stearate, pregelatinized starch, etc. (U.S. Pat. No. 5,849,695). A more detailed description of mucosal administration and formulations is disclosed in PCT Publication No. WO2019 / 049816.

[0119] For transdermal administration, the compositions or pharmaceutical compositions disclosed herein are encapsulated in a delivery device such as a liposome or polymer nanoparticle, microparticle, microcapsule, or microsphere (collectively referred to as microparticles unless otherwise specified).Several suitable devices are known in the art, including microparticles made from synthetic polymers such as polyhydroxy acids (e.g., polylactic acid, polyglycolic acid, and copolymers thereof), polyorthoesters, polyanhydrides, polyphosphazenes, or natural polymers such as collagen, polyamino acids, albumin, and other proteins, alginates, and other polysaccharides, and combinations thereof (U.S. Patent No. 5,814,599).A more detailed description of transdermal administration, formulations, and suitable devices is disclosed in PCT Publication WO2019 / 049816.

[0120] It may be desirable to deliver the disclosed compounds to a subject over an extended period of time, for example, from one week to one year from a single administration. Various sustained-release, depot, or implant dosage forms can be utilized. For example, dosage forms can contain, for example, (a) acid addition salts with polybasic acids, such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or disulfonic acid, polygalacturonic acid, and the like; (b) salts with polyvalent metal cations, such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like, or organic cations formed from, for example, N,N'-dibenzyl-ethylenediamine or ethylenediamine; or (c) a combination of (a) and (b), such as zinc tannate, or a pharmaceutically acceptable non-toxic salt of a compound having low solubility in body fluids. Furthermore, the disclosed compounds, or preferably relatively insoluble salts of the disclosed compounds, such as those just described, can be formulated in a gel suitable for injection, such as an aluminum monostearate gel containing, for example, sesame oil. Particularly preferred salts are zinc salts, zinc tannate, and zinc pamoate. Another type of sustained-release depot formulation for injection contains the compound or salt dispersed for encapsulation in a slowly degrading, non-toxic, non-antigenic polymer, such as polylactic acid / polyglycolic acid polymers, as described in U.S. Pat. No. 3,773,919. The disclosed compounds, or preferably relatively insoluble salts thereof, such as those described above, can also be formulated in cholesterol-matrix silastic pellets, particularly for use in animals. Additional sustained-release, depot, or implant formulations, such as gas or liquid liposomes, are known in the literature (U.S. Pat. No. 5,770,222 and "Sustained and Controlled Release Drug Delivery Systems," J.R. Robinson ed., Marcel Dekker, Inc., NY, 1978).

[0121] Suitable dosages are well known in the art.See, for example, Wells et al., eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, California (2000); Nursing 2001 Handbook of Drugs, 21st Edition, Springhouse Corp., Springhouse, Pa., 2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc., Upper Saddle River, NJ. Preferred doses can optionally include about 0.1-99 and / or 100-500 mg / kg / dose, or any range, value, or fraction thereof, or can be to achieve a serum concentration of about 0.1-5000 μg / ml, or any range, value, or fraction thereof, per single or multiple doses. Preferred dose ranges for the compositions or pharmaceutical compositions disclosed herein are from about 1 mg / kg up to about 3, about 6, or about 12 mg / kg of subject body weight.

[0122] Alternatively, the administered dose may vary depending on known factors such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration, the recipient's age, health, and weight, the nature and extent of symptoms, the type of concomitant therapy, the frequency of treatment, and the desired effect. Typically, the dose of active ingredient may be about 0.1 to 100 milligrams per kilogram of body weight. Typically, 0.1 to 50 milligrams, preferably 0.1 to 10 milligrams per kilogram, per administration or in sustained-release form, is effective to obtain the desired results.

[0123] By way of non-limiting example, human or animal treatment can be provided as a single or periodic dose of a composition or pharmaceutical composition disclosed herein. In some embodiments, the dose is about 0.1 to 100 mg / kg per day, or any range, value, or fraction thereof, for at least one of 1 to 40 days, and / or at least one of 1 to 52 weeks, and / or at least one of 1 to 20 years, or any combination thereof, using a single, infusion, or repeated administration.

[0124] Dosage forms suitable for internal administration generally contain from about 0.001 milligrams to about 500 milligrams of active ingredient per unit or container. In these pharmaceutical compositions, the active ingredient is usually present in an amount of from about 0.5 to 99.999% by weight, based on the total weight of the composition.

[0125] An effective amount can include an amount of about 0.001 to about 500 mg / kg (or any effective range or value therein) per single (e.g., bolus), multiple, or continuous administration. In some embodiments, an effective amount achieves a serum concentration of 0.01 to 5000 μg / ml serum concentration per single, multiple, or continuous administration, or any effective range or value therein, as performed and determined using known methods described herein or known in the relevant art.

[0126] In embodiments in which the composition administered to a subject in need thereof comprises modified cells as disclosed herein, the cells are administered in an amount of about 1 x 10 3 ~1×10 15 cells, approximately 1 x 10 4 ~1×10 12 cells, approximately 1 x 10 5 ~1×10 10 cells, approximately 1 x 10 6 ~1×10 9 cells, approximately 1 x 10 6 ~1×10 8 cells, approximately 1 x 10 6 ~1×10 7 cells, or approximately 1 x 10 6 ~25×10 6In one embodiment, the cells are administered in an amount of about 5 x 10 6 ~25×10 6 Each patient is administered one dose.

[0127] A more detailed description of pharmaceutically acceptable excipients, formulations, dosages, and methods of administration of the compositions and pharmaceutical compositions of the present disclosure is disclosed in PCT Publication WO2019 / 049816.

[0128] Methods of Using the Compositions of the Present Disclosure The present disclosure provides methods of using the disclosed compositions or pharmaceutical compositions for the treatment of diseases or disorders in cells, tissues, organs, animals, or subjects known in the art or described herein. In some embodiments, methods of using the disclosed compositions and pharmaceutical compositions include, for example, administering or contacting a cell, tissue, organ, animal, or subject with a therapeutically effective amount of the composition or pharmaceutical composition. In one aspect, the subject is a mammal. Preferably, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0129] The present disclosure provides methods for modulating or treating at least one malignant disease or disorder in a cell, tissue, organ, animal, or subject. Preferably, the malignant disease is cancer. Non-limiting examples of malignant diseases or disorders include leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, B-cell, T-cell, or FAB ALL, acute myeloid leukemia (AML), acute myelogenous leukemia (AML), ... leukemia), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal cancer, pancreatic cancer, nasopharyngeal cancer, malignant histiocytosis, paraneoplastic syndrome / hypercalcemia of malignancies, solid tumors, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head cancer, neck cancer, hereditary non-polypoid cancer, Hodgkin's lymphoma, liver cancer, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, testicular cancer, adenocarcinoma, sarcoma, malignant melanoma, hemangioma, metastatic disease, cancer-related bone resorption, cancer-related bone pain, etc.

[0130] In a preferred embodiment, the treatment of a malignant disease or disorder involves adoptive cell therapy. For example, in one embodiment, the present disclosure provides modified cells expressing at least a chimeric CD8α coreceptor and / or a TCR and / or a CAR, which have been selected and / or expanded for administration to a subject in need thereof. The modified cells can be formulated for storage at any temperature, including room temperature and body temperature. The modified cells can be formulated for cryopreservation and subsequent thawing. The modified cells can be formulated in a pharmaceutically acceptable carrier for direct administration to a subject from sterile packaging. The modified cells can be formulated in a pharmaceutically acceptable carrier. The modified cells can be formulated in a pharmaceutically acceptable carrier at a defined density with one or more agents to inhibit further expansion and / or prevent cell death.

[0131] In some aspects, treating a malignant disease or disorder can comprise administering an effective amount of any of the compositions or pharmaceutical compositions disclosed herein to a cell, tissue, organ, animal, or subject in need of such modulation, treatment, or therapy. Such methods can optionally further comprise a combination administration or combination therapy for treating such a disease or disorder, wherein administering any of the compositions or pharmaceutical compositions disclosed herein further comprises administering at least one chemotherapeutic agent (e.g., alkylating agent, mitotic inhibitor, radiopharmaceutical) prior to, concurrently with, and / or after administering the composition or pharmaceutical composition disclosed herein.

[0132] In some embodiments, the subject does not develop graft versus host (GvH) and / or host versus graft (HvG) after administration. In one embodiment, administration is systemic. Systemic administration can be by any means known in the art and described in detail herein. Preferably, systemic administration is by intravenous injection or infusion. In one embodiment, administration is local. Local administration can be by any means known in the art and described in detail herein. Preferably, local administration is by intratumoral, intraspinal, intraventricular, intraocular, or intraosseous injection or infusion.

[0133] In some embodiments, the therapeutically effective dose is a single dose. In some embodiments, the single dose is one of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or any number in between, produced simultaneously. In some embodiments where the composition is autologous or allogeneic cells, the dose is sufficient to allow the cells to engraft and / or persist for a sufficient time to treat the disease or disorder.

[0134] In one example, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a composition comprising at least a chimeric CD8α coreceptor and / or TCR and / or CAR that specifically binds to an antigen on a tumor cell. In aspects where the composition comprises modified cells or cell populations, the cells or cell populations may be autologous or allogeneic.

[0135] In some aspects of the methods of treatment described herein, treatment may be modified or terminated. Specifically, in aspects in which the composition used in treatment comprises an inducible pro-apoptotic polypeptide, apoptosis may be selectively induced in cells by contacting the cells with an inducer. Treatment may be modified or terminated, for example, in response to signs of recovery or signs of reduced disease severity / progression, signs of disease remission / termination, and / or the occurrence of an adverse event. In some aspects, the method includes administering an inhibitor of the inducer to inhibit modification of the cell therapy, thereby restoring the function and / or effectiveness of the cell therapy (e.g., when signs or symptoms of disease recurrence or increased severity and / or adverse events are resolved).

[0136] Recombinant methods for constructing nucleic acids The isolated nucleic acid compositions of the present disclosure, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methodologies known to those of skill in the art. In some embodiments, oligonucleotide probes that selectively hybridize to the polynucleotides of the present invention under stringent conditions are used to identify the desired sequence in a cDNA or genomic DNA library. The isolation of RNA and the construction of cDNA and genomic libraries are well known to those of skill in the art. (See, e.g., Ausubel, supra, or Sambrook, supra.)

[0137] The isolated nucleic acids of the present disclosure can also be prepared by direct chemical synthesis using known methods (see, for example, Ausubel et al., supra). Chemical synthesis generally produces a single-stranded oligonucleotide, which can be converted into double-stranded DNA by hybridization with a complementary sequence or polymerization with a DNA polymerase using the single strand as a template. Those skilled in the art will recognize that chemical synthesis of DNA can be limited to sequences of about 100 bases or more, but longer sequences can be obtained by ligating shorter sequences.

[0138] Recombinant Expression Cassette The present disclosure further provides recombinant expression cassettes comprising the nucleic acids of the present disclosure. Nucleic acid sequences of the present disclosure, such as cDNA or genomic sequences encoding protein scaffolds of the present disclosure, can be used to construct recombinant expression cassettes that can be introduced into at least one desired host cell. The recombinant expression cassette will typically comprise a polynucleotide of the present disclosure operably linked to a transcription initiation control sequence that directs transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct expression of the nucleic acids of the present disclosure.

[0139] In some aspects, isolated nucleic acids that function as promoters, enhancers, or other elements can be introduced into a suitable location (upstream, downstream, or intron) of a non-heterologous form of a polynucleotide of the disclosure to up- or down-regulate expression of the polynucleotide. For example, endogenous promoters can be modified in vivo or in vitro by mutation, deletion, and / or substitution.

[0140] Expression vectors and host cells The present disclosure also relates to vectors comprising the isolated nucleic acid molecules of the present disclosure, host cells genetically engineered with the recombinant vectors, and the production of at least one protein scaffold by recombinant techniques, which are well known in the art (see, e.g., Sambrook, et al., supra; Ausubel, et al., supra, each of which is incorporated herein by reference in its entirety).

[0141] The polynucleotide can optionally be linked to a vector containing a selectable marker for propagation in a host. Generally, the plasmid vector is introduced into a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into a host cell.

[0142] The DNA insert should be operably linked to a suitable promoter. The expression construct will further contain sites for transcription initiation, termination, and, within the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct will preferably include a translation initiation codon at the beginning and a termination codon (e.g., UAA, UGA, or UAG) appropriately positioned at the end of the mRNA to be translated, with UAA and UAG being preferred for mammalian or eukaryotic expression.

[0143] The expression vector will preferably and optionally include at least one selectable marker, such as, but not limited to, ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / Geneticin (neo gene), DHFR (encodes dihydrofolate reductase and confers resistance to methotrexate), mycophenolic acid, or glutamine synthetase (GS, U.S. Patent Nos. 5,122,464, 5,770,359, 5,827,739), blastocidin (bsd gene), and other selectable markers for eukaryotic cell culture and expression of ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / Geneticin (neo gene), DHFR (encodes dihydrofolate reductase and confers resistance to methotrexate), mycophenolic acid, or glutamine synthetase (GS, U.S. Patent Nos. 5,122,464, 5,770,359, 5,827,739), blastocydin ... Resistance genes for the host cells include genes for resistance to erythromycin (bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / Geneticin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, or tetracycline resistance genes for culturing in E. coli and other bacteria or prokaryotes (the above patents are incorporated herein by reference in their entireties). Appropriate culture media and conditions for the above host cells are known in the art. Suitable vectors will be readily apparent to those skilled in the art. Introduction of the vector construct into the host cell can be affected by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other known methods. Such methods are described in the art, such as Sambrook, supra, Chapters 1-4 and 16-18, and Ausubel, supra, Chapters 1, 9, 13, 15, 16.

[0144] The expression vector will preferably, optionally, include at least one selectable cell surface marker for isolation of cells modified by the compositions and methods of the present disclosure. Selectable cell surface markers of the present disclosure include surface proteins, glycoproteins, or proteins that distinguish a cell or subpopulation of cells from another defined subpopulation of cells. Preferably, the selectable cell surface marker distinguishes cells modified by the compositions or methods of the present disclosure from cells not modified by the compositions or methods of the present disclosure. Such cell surface markers include, for example, but are not limited to, "cluster of designation or classification determinant" proteins (often abbreviated as "CD"), such as truncated or full-length forms of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof. Cell surface markers further include the suicide gene marker RQR8 (Philip B et al. Blood. 2014 Aug 21;124(8):1277-87).

[0145] The expression vector will preferably and optionally include at least one selectable drug resistance marker for isolation of cells modified by the compositions and methods of the present disclosure. The selectable drug resistance markers of the present disclosure may include wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

[0146] At least one protein scaffold of the present disclosure can be expressed in modified forms, such as fusion proteins, and can include not only secretion signals but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of the protein scaffold to improve stability and persistence in host cells during purification or subsequent handling and storage. Peptide moieties can also be added to the protein scaffold of the present disclosure to facilitate purification. Such regions can be removed prior to final preparation of the protein scaffold or at least one fragment thereof. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74, and Ausubel, supra, Chapters 16, 17, and 18.

[0147] Those skilled in the art are familiar with the numerous expression systems available for expressing nucleic acids encoding the proteins of the present disclosure. Alternatively, the nucleic acids of the present disclosure can be expressed in host cells by turning on (manipulating) endogenous DNA encoding the protein scaffolds of the present disclosure in the host cells. Such methods are well known in the art, for example, as described in U.S. Patent Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, which are incorporated herein by reference in their entirety.

[0148] Exemplary cell cultures useful for producing protein scaffolds, specific portions or variants thereof are bacterial, yeast, and mammalian cells known in the art. Mammalian cell systems are often in the form of monolayers of cells, although mammalian cell suspensions or bioreactors can also be used. Several suitable host cell lines capable of expressing intact glycosylated proteins have been developed in the art, including COS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610), and BSC-1 (e.g., ATCC CRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2 / 0-Ag14, 293 cells, HeLa cells, and the like, which are readily available, for example, from the American Type Culture Collection, Manassas, Va. (www.atcc.org). Preferred host cells include cells of lymphoid origin, such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC Accession No. CRL-1580) and SP2 / 0-Ag14 cells (ATCC Accession No. CRL-1851). In a preferred embodiment, the recombinant cell is a P3X63Ab8.653 or SP2 / 0-Ag14 cell.

[0149] Expression vectors for these cells can include one or more expression control sequences, such as, but not limited to, an origin of replication, a promoter (e.g., a late or early SV40 promoter, a CMV promoter (U.S. Pat. Nos. 5,168,062, 5,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alpha promoter (U.S. Pat. No. 5,266,491), or at least one human promoter), an enhancer, and / or processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., the SV40 large T Ag polyA addition site), and a transcription termination sequence. See, e.g., Ausubel et al., supra; Sambrook, et al., supra. Other cells useful for producing the nucleic acids or proteins of the disclosure are known and / or available, for example, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (www.atcc.org) or other known or commercial sources.

[0150] When eukaryotic host cells are used, a polyadenylation or transcription termination sequence is typically incorporated into the vector. An example of a termination sequence is the polyadenylation sequence derived from the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included. An example of a splicing sequence is the VP1 intron derived from SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)). Additionally, gene sequences for regulating replication in host cells may be incorporated into the vector, as is known in the art.

[0151] definition As used throughout this disclosure, the singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes a plurality of such methods, reference to a "dosage" includes a reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0152] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within one or more standard deviations. Alternatively, "about" can mean a range of a given value of up to 20%, or up to 10%, or up to 5%, or up to 1%. Alternatively, particularly with respect to biological systems or processes, the term can mean a value within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold. When particular values ​​are described in this application and claims, unless otherwise specified, the term "about" meaning within an acceptable error range for that particular value should be assumed.

[0153] The present disclosure provides isolated or substantially purified polynucleotide or protein compositions. An "isolated" or "purified" polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an "isolated" polynucleotide does not include sequences that naturally flank the polynucleotide in the genomic DNA of the organism from which the polynucleotide is derived (i.e., sequences located at the 5' and 3' ends of the polynucleotide) (optimal protein-coding sequences). For example, in various embodiments, an isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When a protein of the disclosure or a biologically active portion thereof is recombinantly produced, an optimal culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein chemicals of interest.

[0154] The present disclosure provides fragments and variants of the DNA sequences and proteins of the present disclosure encoded by these DNA sequences. As used throughout this disclosure, the term "fragment" refers to a portion of a DNA sequence or a portion of an amino acid sequence, and thus the protein encoded thereby. Fragments of DNA sequences, including coding sequences, may encode protein fragments that retain the biological activity of the native protein, and thus the DNA recognition or binding activity to target DNA sequences described herein. Alternatively, fragments of DNA sequences that are useful as hybridization probes generally do not encode proteins that retain biological activity or do not retain promoter activity. Thus, fragments of DNA sequences can range from at least about 20 nucleotides, about 50 nucleotides, or about 100 nucleotides, and up to the full-length polynucleotides of the present disclosure.

[0155] The nucleic acids or proteins of the present disclosure can be constructed by a modular approach, including pre-assembling monomer units and / or repeat units in a target vector, which can then be assembled into a final destination vector. The polypeptides of the present disclosure can include repeat monomers of the present disclosure and can be constructed by a modular approach, including pre-assembling repeat units in a target vector, which can then be assembled into a final destination vector. The present disclosure provides polypeptides produced by the present methods, as well as nucleic acid sequences encoding these polypeptides. The present disclosure provides host organisms and cells containing nucleic acid sequences encoding the polypeptides produced by this modular approach.

[0156] "Binding" refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific.

[0157] The term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude others. When used to define compositions and methods, "consisting essentially of" shall mean excluding other elements of any essentially significant element to the combination when used for its intended purpose. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants or inert carriers. "Consisting of" means excluding more than trace elements and substantial method steps of other components. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0158] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0159] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, microRNA, structural RNA, or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs that are modified by processes such as capping, polyadenylation, methylation, and editing, and proteins that are modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[0160] "Modulation" or "regulation" of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression.

[0161] The term "operatively linked" or its equivalents (e.g., "linked operatively") means that two or more molecules are positioned relative to each other so that they can interact to affect a function attributed to one or both molecules or a combination thereof.

[0162] Non-covalently linked components and methods for making and using non-covalently linked components are disclosed. The various components can take a variety of different forms, as described herein. For example, non-covalently linked (i.e., operably linked) proteins can be used to allow for temporary interactions, which avoids one or more problems in the art. The ability of non-covalently linked components, such as proteins, to associate and dissociate allows for functional association only or primarily under circumstances where such association is required for the desired activity. The association can be of sufficient duration to allow for the desired effect.

[0163] The term "nucleic acid" or "oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid can also encompass the complementary strand of a depicted single strand. Nucleic acids of the present disclosure also encompass substantially identical nucleic acids and their complements that retain the same structure or encode the same protein.

[0164] The nucleic acids of the present disclosure may be single-stranded or double-stranded. The nucleic acids of the present disclosure may contain double-stranded sequences even when the majority of the molecule is single-stranded. The nucleic acids of the present disclosure may contain single-stranded sequences even when the majority of the molecule is double-stranded. The nucleic acids of the present disclosure may include genomic DNA, cDNA, RNA, or hybrids thereof. The nucleic acids of the present disclosure may contain a combination of deoxyribonucleotides and ribonucleotides. The nucleic acids of the present disclosure may contain a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. The nucleic acids of the present disclosure may be synthesized to contain unnatural amino acid modifications. The nucleic acids of the present disclosure may be obtained by chemical synthesis or recombinant methods.

[0165] The nucleic acids of the present disclosure, their entire sequences, or portions thereof, may be non-naturally occurring. The nucleic acids of the present disclosure may contain one or more mutations, substitutions, deletions, or insertions that are not naturally occurring, making the entire nucleic acid sequence non-naturally occurring. The nucleic acids of the present disclosure may contain one or more overlapping, inverted, or repeated sequences, the resulting sequence being non-naturally occurring and making the entire nucleic acid sequence non-naturally occurring. The nucleic acids of the present disclosure may contain modified, artificial, or synthetic nucleotides that are not naturally occurring, making the entire nucleic acid sequence non-naturally occurring.

[0166] Given the redundancy in the genetic code, multiple nucleotide sequences may encode any particular protein, and all such nucleotide sequences are contemplated herein.

[0167] As used throughout this disclosure, the term "operably linked" refers to the expression of a gene under the control of a promoter to which it is spatially connected. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene it controls within the gene from which it is derived. Variation in the distance between the promoter and the gene may be accommodated without loss of promoter function.

[0168] As used throughout this disclosure, the term "promoter" refers to a synthetic or naturally occurring molecule that can confer, activate, or enhance expression of a nucleic acid in a cell. A promoter can contain one or more specific transcriptional control sequences to further enhance expression and / or alter its spatial and / or temporal expression. A promoter can also contain distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter can control the expression of genetic components constitutively or differentially with respect to the cell, tissue, or organ in which expression occurs, with respect to the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include a bacteriophage T7 promoter, a bacteriophage T3 promoter, an SP6 promoter, a lac operator-promoter, a tac promoter, an SV40 late promoter, an SV40 early promoter, an RSV-LTR promoter, a CMV IE promoter, an EF-1 alpha promoter, a CAG promoter, an SV40 early promoter, or an SV40 late promoter, and a CMV IE promoter.

[0169] As used throughout this disclosure, the term "substantially complementary" refers to a first sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

[0170] As used throughout this disclosure, the term "substantially identical" refers to when a first and second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids, or, with respect to nucleic acids, when a first sequence is substantially complementary to the complement of a second sequence.

[0171] As used throughout this disclosure, the term "variant," when used to describe a nucleic acid, refers to (i) a portion or fragment of a referenced nucleotide sequence, (ii) a complement of a referenced nucleotide sequence or a portion thereof, (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or its complement, or (iv) a nucleic acid that hybridizes under stringent conditions to a referenced nucleic acid, its complement, or a sequence substantially identical thereto.

[0172] As used throughout this disclosure, the term "vector" refers to a nucleic acid sequence containing an origin of replication. A vector can be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector, preferably a DNA plasmid. A vector can contain a DNA sequence, an RNA sequence, or a combination of amino acids with both DNA and RNA sequences.

[0173] As used throughout this disclosure, the term "variant," when used to describe a peptide or polypeptide, refers to a peptide or polypeptide that differs in amino acid sequence by amino acid insertions, deletions, or conservative substitutions, but retains at least one biological activity. A variant can also refer to a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity.

[0174] Conservative amino acid substitutions, i.e., substitutions of amino acids with different amino acids of similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are typically recognized in the art as involving minor changes. These minor changes can be identified, in part, by considering the hydropathic index of the amino acid, as understood in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. Amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids with hydropathic indexes of ±2 are substituted. The hydrophilicity of an amino acid can also be used to identify substitutions that result in a protein that retains biological function. Considering the hydrophilicity of an amino acid in the context of a peptide allows for calculation of the greatest local average hydrophilicity of the peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. No. 4,554,101, which is incorporated herein by reference in its entirety.

[0175] Substitution of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, e.g., immunogenicity. Substitutions can be made with amino acids with hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Consistent with that observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of the amino acids, particularly their side chains, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0176] As used herein, "conservative" amino acid substitutions may be defined as set forth in Tables A, B, or C below. In some embodiments, fusion polypeptides and / or nucleic acids encoding such fusion polypeptides contain conservative substitutions introduced by modification of a polynucleotide encoding a polypeptide of the present disclosure. Amino acids may be classified according to their physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is the substitution of one amino acid for another amino acid with similar properties. Exemplary conservative substitutions are set forth in Table A. [Table 3]

[0177] Alternatively, conservative amino acids can be grouped as set forth in Table B as described by Lehninger (Biochemistry, Second Edition; Worth Publishers, Inc. NY, NY (1975), pp. 71-77). [Table 4]

[0178] Alternatively, exemplary conservative substitutions are set forth in Table C. [Table 5]

[0179] It should be understood that the polypeptides of the present disclosure are intended to include polypeptides having one or more insertions, deletions, or substitutions of amino acid residues, or any combination thereof, as well as modifications other than insertions, deletions, or substitutions of amino acid residues. A polypeptide or nucleic acid of the present disclosure may contain one or more conservative substitutions.

[0180] As used throughout this disclosure, the term "more than one" of the above amino acid substitutions refers to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more of the listed amino acid substitutions. The term "more than one" may also refer to 2, 3, 4, or 5 of the listed amino acid substitutions.

[0181] The polypeptides and proteins of the present disclosure, their entire sequences, or portions thereof, may be non-naturally occurring. The polypeptides and proteins of the present disclosure may contain one or more mutations, substitutions, deletions, or insertions that are not naturally occurring, rendering the entire amino acid sequence non-naturally occurring. The polypeptides and proteins of the present disclosure may contain one or more overlapping, inverted, or repeated sequences, resulting in a sequence that is not naturally occurring and rendering the entire amino acid sequence non-naturally occurring. The polypeptides and proteins of the present disclosure may contain modified, artificial, or synthetic amino acids that are not naturally occurring, rendering the entire amino acid sequence non-naturally occurring.

[0182] As used throughout this disclosure, "sequence identity" can be determined by using a standalone executable BLAST engine program (bl2seq) for blasting two sequences, which can be obtained from the National Center for Biotechnology Information (NCBI) ftp site using default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250, incorporated herein by reference in its entirety). When used in the context of two or more nucleic acid or polypeptide sequences, the term "identical" or "identity" refers to the percentage of specified residues that are the same over a specified region of each of the sequences. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions at which identical residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences are of different lengths, or if the alignment produces one or more staggered ends and the designated comparison region contains only a single sequence, the residues of the single sequence are included in the denominator of the calculation, but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or by using a computer sequencing algorithm such as BLAST or BLAST 2.0.

[0183] As used throughout this disclosure, the term "endogenous" refers to a nucleic acid or protein sequence that is naturally associated with the target gene or host cell into which it is introduced.

[0184] As used throughout this disclosure, the term "exogenous" refers to a nucleic acid or protein sequence that is not naturally associated with the target gene or host cell into which it is introduced, and includes naturally occurring nucleic acids, e.g., DNA sequences, or non-naturally occurring multiple copies of naturally occurring nucleic acid sequences located in non-naturally occurring genomic locations.

[0185] The present disclosure provides methods for introducing a polynucleotide construct containing a DNA sequence into a host cell. By "introducing," it is intended to present the polynucleotide construct to the cell in such a manner that the construct gains access to the interior of the host cell. The methods of the present disclosure do not depend on a particular method for introducing a polynucleotide construct into a host cell, but only on the polynucleotide construct gaining access to the interior of one cell of the host. Methods for introducing polynucleotide constructs into bacteria, plants, fungi, and animals are known in the art, including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods. [Example]

[0186] Example 1 - Preparation and storage of allogeneic TCR T cells co-expressing chimeric CD8α coreceptors Figure 1A shows a schematic diagram of an exemplary chimeric CD8α homodimeric coreceptor of the present disclosure. The chimeric CD8α homodimeric coreceptor comprises a truncated CD8α coreceptor comprising the CD8α coreceptor extracellular domain and the CD8α coreceptor transmembrane domain, and a CD4 intracellular domain comprising a palmitoylation domain and a high-affinity Lck-binding domain. Figure 2 shows a schematic diagram illustrating the domain orientation of an exemplary chimeric CD8α coreceptor of the present disclosure and a DNA piggyBac nanotransposon-containing sequence encoding the TCR alpha and TCR beta chains of the TCR. The chimeric CD8α coreceptor is operably linked to the PGK promoter, and expression is controlled by the PGK promoter. The iCAS9 safety switch, TCR beta chain, TCR alpha chain, and DHFR selectable marker are operably linked to the EF1a promoter, and expression is controlled by the EF1a promoter. The transposon allows for co-expression of the TCR and chimeric CD8α coreceptor from a single construct.

[0187] Human pan-T cells were collected from healthy human donors by apheresis. Allogeneic TCR-T cells were generated from collected T cells using an automated positive immunomagnetic enrichment protocol for CD4 and CD8 T cells (CliniMACS CD4 MicroBeads and CliniMACS CD8 MicroBeads, respectively) using a CliniMACS Prodigy Instrument (Miltenyi Biotec). Enriched CD4 and CD8 T cells were cryopreserved in a mixture of Hank's balanced salt solution (HBSS, 40%), human serum albumin (HSA, 50%), and dimethyl sulfoxide (DMSO, 10%), then frozen and stored in the vapor phase of liquid nitrogen.

[0188] Frozen enriched CD4 and CD8 T cells were thawed in T cell expansion medium (ImmunoCult™-XF T Cell Expansion Medium, StemCell) and allowed to recover overnight at 37°C in the presence of 5% CO2. T cells were resuspended in supplemented P3 Primary Cell Nucleofector solution before electroporation. The transposons in Figure 2 were introduced into enriched T cells via electroporation. Each electroporation reaction contained mRNA encoding the Super-piggyBac (SPB) transposase; Cas-CLOVER (CC); synthetic, chemically modified gRNAs targeting the TRAC, TRBC1 / 2, and B2M genes; and a DNA piggyBac nanotransposon plasmid encoding the chimeric CD8α coreceptor, the TCR alpha and beta chains of a TCR directed against target NY-ESO (NYESO_TCR_1G4), a dihydrofolate reductase (DHFR) selection cassette, and an inducible caspase-9 (iCasp) safety switch. The contents were pre-loaded into a 100 μl Nucleocuvette vessel. The T cell suspension was added and the vessel was electroporated using the 4D-Nucleofector system according to the manufacturer's instructions.

[0189] Cells were harvested after electroporation in 20 ml of T cell expansion medium (ImmunoCult™-XF T Cell Expansion Medium, StemCell) and cultured in multiple G-Rex 6M vessels (Wilson Wolf Corporation). Cells were incubated overnight. After electroporation and a prolonged period of rest for recovery, cells were activated via TCR in the absence of exogenous cytokines. After activation, fresh culture medium supplemented with methotrexate (MTX) but lacking exogenous cytokines was added to select for DHFR-positive T cells. To ensure sufficient cell expansion, cell cultures were maintained throughout the culture period and routinely replenished with fresh culture medium. Cell harvesting was performed after the cultured cells exited the exponential growth phase and entered the stationary phase.

[0190] Harvested cells were stained with biotinylated CliniMACS® TCR α / β antibody (Miltenyi) and incubated with CliniMACS® Anti-Biotin GMP MicroBeads (Miltenyi) for 30 minutes. The labeled cells were then loaded onto an LS column (Miltenyi) and subjected to immunomagnetic depletion. The transgenic TCR was engineered to mask the epitope bound by the CliniMACS® TCR α / β antibody, thereby labeling and depleting cells expressing endogenous TCRs. Endogenous TCR-depleted cells were cryopreserved in a mixture of HBSS (40%), HSA (50%), and DMSO (10%), then stored in the vapor phase of liquid nitrogen.

[0191] Un-transposed pan T cells lacking transposon DNA during electroporation and without MTX selection were used as controls.

[0192] Example 2 - Co-expression of TCR and chimeric CD8α coreceptor enhances CD4+ and CD8+ MHC1-mediated cytotoxicity Allogeneic TCR-T cells prepared in Example 1 were thawed and resuspended in RPMI 1640 medium (Gibco) supplemented with 10% FBS (Sigma-Aldrich) and 2 mM Glutamax (Gibco) (RPMI-10). After thawing, all cells were stained with orange / propidium iodide (Logos Biosystems) so that viable nucleated cells fluoresce green and nonviable nucleated cells fluoresce red. Viable cells were counted using a LunaFL Dual Fluorescence Cell Counter (Logos Biosystems).

[0193] A37-GFP melanoma cells (HLA-A2 / NY-ESO-1 / GFP) were used as target cells in the T cell cytotoxicity assay. Target cells were thawed in DMEM-10 medium. DMEM-10 medium was prepared by mixing DMEM medium (Gibco) with 10% FBS (Sigma-Aldrich), and the cells were incubated at 37°C and 5% CO. After a 72-hour culture period, fully confluent cultures were counted, pelleted, and 5 x 10 4 The cells were resuspended in DMEM-10 medium at a concentration of 5 × 10 cells / ml. 3 A 100 μl aliquot of target cells was seeded into each well of a Corning Poly-D-lysine treated flat-bottom plate (Corning). Each plate was incubated overnight at 37° C. and 5% CO .

[0194] Allogeneic TCR-T cells were stained using an antibody cocktail consisting of APC-Vb-13.1 antibody (Miltenyi), BV785-hCD4 antibody (Biolegend), APC-Cy7-hCD8 antibody (Biolegend), BV421-hTCR (Biolegend), PE-A2:NYESO Dextramer (Immudex), and Live / Dead Fixed Aqua Dead cell stain Reagent (ThermoFisher). Stained cells were either analyzed for Dextramer+ frequency on a BD LSRFortessa™ Cell Analyzer or sorted to isolate Live / CD4+ / Vb-13.1+ and Live / CD8+ / Vb-13.1+ populations using a Sony SH800S sorter according to the manufacturer's instructions. Figure 3A shows a series of flow cytometry contour plots illustrating the sorting of stained cells.

[0195] Sorted TCR-T cells were pelleted and 2.5 × 10 Live / CD4+ / Vb-13.1+ cells were collected. 5 cells / mL, and 1 × 10 for Live / CD8+ / Vb-13.1+ cells 5The cells were resuspended in RPMI-10 at a concentration of 100 μl / mL. A 100 μl aliquot of the sorted TCR-T cells was seeded in triplicate into Corning poly-D-lysine-treated 96-well plates containing cultured A375 cells. TCR+CD4+ T cells were incubated with A375 target cells at a 5:1 ratio. TCR+CD8+ T cells were cultured with A375 target cells at a 2:1 ratio. Each plate was incubated at 37°C and 5% CO2 for 96 ± 2 hours. TCR-T-mediated cytotoxicity was analyzed by tracking GFP+ A375 target cell proliferation using a Sartorius Incucyte system. Tumor growth percentage was measured at 100 hours. All data were exported to Microsoft Excel and plotted using GraphPad Prism software. FIG. 3B shows the cytotoxicity of T cells co-expressing a TCR and a chimeric CD8α coreceptor, a TCR homodimer, or a TCR heterodimer compared to GFP and mock controls.

[0196] As shown in Figure 3B, coexpression of the chimeric CD8α coreceptor ("TCR+chiCD8-homo-di") resulted in enhanced TCR-mediated cytotoxicity (reduced tumor growth) in both CD8+ and CD4+ T cells compared to the wild-type CD8α homodimeric coreceptor ("TCR+CD8-homo-di"), heterodimeric CD8α coreceptor ("TCR+CD8-h-di"), or controls ("TCR+GFP", "Mock"), with a greater effect observed for CD4+ T cells.

Claims

1. 1. A chimeric CD8α coreceptor, a) a truncated CD8α coreceptor comprising a CD8α coreceptor extracellular domain and a CD8α coreceptor transmembrane domain; b) a CD4 intracellular domain comprising a palmitoylation motif and an Lck-binding domain; A chimeric CD8α coreceptor, wherein the CD4 intracellular domain is fused in-frame to the C-terminus of the truncated CD8α coreceptor.

2. The chimeric CD8α coreceptor of claim 1 , wherein the truncated CD8α coreceptor comprises the amino acid sequence of SEQ ID NO:

1.

3. The chimeric CD8α coreceptor of claim 1, wherein the palmitoylation motif comprises the amino acid sequence of SEQ ID NO:

3.

4. The chimeric CD8α coreceptor of claim 1 , wherein the Lck binding domain comprises the amino acid sequence of SEQ ID NO:

4.

5. The chimeric CD8α coreceptor of claim 1, wherein the CD4 intracellular domain, including the palmitoylation motif and the Lck binding domain, comprises the amino acid sequence of SEQ ID NO:

2.

6. The chimeric CD8α coreceptor of claim 1, wherein the chimeric CD8α coreceptor comprises the amino acid sequence of SEQ ID NO:

5.

7. A polynucleotide comprising a nucleic acid sequence encoding the chimeric CD8α coreceptor of any one of claims 1 to 6.

8. The polynucleotide of claim 7, wherein the polynucleotide is an mRNA molecule.

9. The polynucleotide of claim 7, wherein the polynucleotide is a DNA molecule.

10. The polynucleotide of claim 9, further comprising a promoter sequence operably linked to the DNA molecule to produce at least one mRNA molecule encoding the chimeric CD8α coreceptor in a cell.

11. A cell comprising the polynucleotide according to any one of claims 7 to 10.

12. The cell of claim 11 , wherein the cell expresses the chimeric CD8α coreceptor.

13. The cell of claim 11 , wherein the cell further expresses a T cell receptor (TCR).

14. The cell according to any one of claims 11 to 13, wherein the cell is a T cell.

15. The cell of claim 14, wherein the chimeric CD8α coreceptor is expressed as a homodimer on the plasma membrane of the T cell.

16. A pharmaceutical composition comprising the cells of any one of claims 11 to 15 and at least one pharmaceutically acceptable carrier or medicament.

17. 1. A method of stimulating T cell receptor (TCR)-mediated cytotoxicity of a T cell population in a subject in need thereof, comprising: a) introducing into a population of T cells a polynucleotide encoding a TCR and a polynucleotide encoding a chimeric CD8α co-receptor according to any one of claims 1 to 6, a plurality of T cells in the T cell population co-express the TCR and the chimeric CD8α coreceptor on the plasma membrane of the T cells; introducing said chimeric CD8α coreceptor into a mammalian cell line, wherein said chimeric CD8α coreceptor is expressed as a homodimer; b) administering said population of T cells to said subject in need thereof; wherein the T cell population expressing the TCR and the chimeric CD8α coreceptor has a higher level of cytotoxicity compared to a T cell population expressing only the TCR.

18. 1. A chimeric CD8α coreceptor, a) a truncated CD8α coreceptor comprising the amino acid sequence of SEQ ID NO: 1; b) a CD4 intracellular domain comprising a palmitoylation motif comprising the amino acid sequence of SEQ ID NO: 3 and an Lck-binding domain comprising the amino acid sequence of SEQ ID NO: 4; A chimeric CD8α coreceptor, wherein the CD4 intracellular domain is fused in-frame to the C-terminus of the truncated CD8α coreceptor.

19. 1. A chimeric CD8α coreceptor, a) a truncated CD8α coreceptor comprising the amino acid sequence of SEQ ID NO: 1; b) a CD4 intracellular domain comprising the amino acid sequence of SEQ ID NO: 2; A chimeric CD8α coreceptor, wherein the CD4 intracellular domain is fused in-frame to the C-terminus of the truncated CD8α coreceptor.

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