Chimeric CD8-alpha coreceptor compositions and methods of use

Abstract

Chimeric CD8 alpha co-receptor compositions and methods of use thereof are disclosed. In particular, chimeric CD8 [alpha] homodimer co-receptor compositions, cells comprising chimeric co-receptors, and pharmaceutical compositions comprising the co-receptors and / or cells are disclosed. Methods of making and using the co-receptor compositions are also provided.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority and benefit of U.S. Provisional Application No. 63 / 371,136, filed on August 11, 2022, the entire content of which is incorporated herein by reference. Technical field

[0003] The present disclosure relates to chimeric CD8α co - receptor compositions and methods of using the same. Specifically, chimeric CD8α homodimer co - receptor compositions, cells comprising the chimeric CD8α co - receptor, pharmaceutical compositions comprising the chimeric CD8α co - receptor and / or cells, and methods for their preparation and use are disclosed.

[0004] Sequence listing incorporated by reference

[0005] The sequence listing XML associated with this application is provided electronically in XML file format and is hereby incorporated by reference into this specification. The name of the XML file containing the sequence listing XML is "POTH - 074_001WO_SeqList_ST26.xml". The XML file is 64,097 bytes, created on July 24, 2023, and is being submitted electronically via the USPTO Patent Center. Background art

[0006] In recent years, adoptive cell therapies using autologous T cells transduced to express a T - cell receptor (TCR) or a chimeric antigen receptor (CAR) have proven to be very effective methods for treating diseases such as cancer. However, challenges still remain, including decoupling cytotoxicity against tumor cells from systemic toxicity. There has long been a felt but unmet medical need for compositions and methods for improving the cytotoxicity of T - cell receptor (“TCR”)-mediated TCR - T cell therapy (“TCR - T therapy”). The present disclosure provides a solution by providing chimeric TCRs, CD8α co - receptors that enhance the cytotoxic activity of TCR - T therapy. Summary of the invention

[0007] The present disclosure provides a chimeric CD8α co - receptor comprising: a) a truncated CD8α co - receptor comprising a CD8α co - receptor extracellular domain and a CD8α co - receptor transmembrane domain; and b) a CD4 intracellular domain comprising a palmitoylation motif and an Lck - binding domain; wherein the CD4 intracellular domain is in - frame fused to the C - terminus of the truncated CD8α co - receptor.

[0008] In some aspects, the truncated CD8 coreceptor comprises the amino acid sequence of SEQ ID NO:1. In some aspects, the palmitoylation motif comprises the amino acid sequence of SEQ ID NO:3. In some aspects, the Lck binding domain comprises the amino acid sequence of SEQ ID NO:4. In some aspects, the CD4 intracellular domain comprising the palmitoylation motif and the Lck binding domain comprises the amino acid sequence of SEQ ID NO:2.

[0009] In some aspects, the CD8α coreceptor comprises: a) a truncated CD8α coreceptor that comprises the amino acid sequence of SEQ ID NO:1, and b) a CD4 intracellular domain that comprises a palmitoylation motif and an Lck binding domain, the palmitoylation motif comprising the amino acid sequence of SEQ ID NO:3 and the Lck binding domain comprising the amino acid sequence of SEQ ID NO:4; wherein the CD4 intracellular domain is in-frame fused to the C-terminus of the truncated CD8α coreceptor.

[0010] In some aspects, the CD8α coreceptor comprises: a) a truncated CD8α coreceptor that comprises the amino acid sequence of SEQ ID NO:1; and a CD4 intracellular domain that comprises the amino acid sequence of SEQ ID NO:2; wherein the CD4 intracellular domain is in-frame fused to the C-terminus of the truncated CD8α coreceptor.

[0011] In some aspects, the chimeric CD8α coreceptor comprises the amino acid sequence of SEQ ID NO:5.

[0012] The present disclosure also provides a polynucleotide that comprises a nucleic acid sequence encoding any one of the chimeric CD8α coreceptors of the present disclosure. In some aspects, the polynucleotide is an mRNA molecule. In some aspects, the polynucleotide is a DNA molecule. In some aspects, the polynucleotide further comprises a promoter sequence that is operably linked to the DNA molecule to produce at least one mRNA molecule encoding the chimeric CD8α coreceptor in a cell.

[0013] The present disclosure also provides a cell comprising the polynucleotide of the present disclosure. In some aspects, the cell expresses the chimeric CD8α co-receptor. In some aspects, the cell further expresses a T cell receptor (TCR). In some aspects, the cell further expresses a chimeric antigen receptor (CAR). In some aspects, the cell expresses both a TCR and a CAR. In some aspects, the cell is a T cell. In some aspects, the chimeric CD8α co-receptor is expressed as a homodimer on the cell membrane of the T cell.

[0014] 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 drug.

[0015] The present disclosure also provides a method of stimulating 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 co-receptors of the present disclosure, wherein a plurality of T cells in the T cell population co-express the TCR and the chimeric CD8α co-receptor on the cell membrane of the T cell, and wherein the chimeric CD8α co-receptor 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 co-receptor 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.

[0016] Any aspect among the above aspects can be combined with any other aspect.

[0017] 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. In this specification, unless the context clearly dictates otherwise, the singular forms also include the plural forms; for example, the terms "a / an" and "the" are understood to be singular or plural, and the term "or" is understood to be inclusive. For example, "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 the 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 can 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 indicates otherwise, all numerical values provided herein are modified by the term "about".

[0018] Although methods and materials similar or equivalent to those described herein can be used to practice or test 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. The 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 DESCRIPTION OF THE DRAWINGS

[0019] Figure 1A-1B A schematic diagram depicting an exemplary chimeric CD8α homodimer (chiCD8-homo-di) and CD8α heterodimer (CD8-hetero-di) and a CD8α homodimer (CD8-homo-di) control is shown. Figure 1A A chimeric CD8α homodimer coreceptor is shown, the chimeric CD8α homodimer coreceptor comprising a truncated CD8α coreceptor that includes only the extracellular domain and transmembrane domain of the CD8α coreceptor; a CD4 intracellular domain that includes a palmitoylation domain and a high-affinity Lck-binding domain, wherein the CD4 intracellular domain is in-frame fused to the C-terminus of the truncated CD8α coreceptor. Figure 1BShows the control wild-type CD8α heterodimeric coreceptor (CD8hetero-di) and CD8α homodimeric coreceptor (CD8-homo-di).

[0020] Figure 2 Shows a schematic representation of a transposon designed for dual expression of a chimeric CD8α coreceptor (“coreceptor”) and a TCR receptor comprising a TCRα (“TCRa”) chain and a TCRβ (“TCRb”) chain. The PGK promoter controls the expression of the chimeric CD8α coreceptor. The EF1a promoter controls the 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 (TCRβ chain); TCRa (TCRα chain).

[0021] Figure 3A-3B Shows a series of contour plots and graphs depicting flow cytometry sorting and in vitro cytotoxicity data of the chimeric CD8α coreceptor compared to a TCR control. 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 of the present disclosure (Chi_CD8homo-di), wild-type CD8α coreceptor (CD8Homo-di), heterodimeric CD8α coreceptor (CD8Hetero-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 the chimeric CD8α coreceptor compared to a TCR control in CD4+ (left) and CD8+ (right) T cells at the indicated ratios. The x-axis depicts the time elapsed (in hours). The y-axis depicts the percentage of tumor growth normalized to baseline (%). The T cells express the coreceptor as indicated in the legend. This data shows that the in vitro cytotoxicity of TCR+ chimeric CD8α coreceptor (TCR+chiCD8-homo-di) is superior to all other controls (mock, TCR+GFP, TCR+CD8-hetero-di, TCR+CD8-homo-di).

[0022] 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 does not admit that it is prior art with respect to any invention disclosed or claimed herein, or that it alone, or in any combination with any one or more other references, teaches, suggests, or discloses any such invention. Further, 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 the term in this document shall govern. Detailed Description

[0023] The present invention relates to chimeric co-receptor compositions and methods of using the same. Specifically, the present invention relates to chimeric CD8α homodimer co-receptor compositions, cells comprising the chimeric CD8α co-receptor, pharmaceutical compositions comprising the CD8α co-receptor and / or cells, and methods of using the same.

[0024] The present disclosure provides a chimeric CD8α co-receptor comprising: a) a truncated CD8α co-receptor comprising a CD8α co-receptor extracellular domain and a CD8α co-receptor transmembrane domain; and b) a CD4 intracellular domain comprising a palmitoylation motif and an Lck binding domain; wherein the CD4 intracellular domain is in-frame fused to the C-terminus of the truncated CD8α co-receptor.

[0025] The present disclosure overcomes problems associated with the current art by providing a chimeric CD8α co-receptor that can be expressed on the surface of T cells and used in immunotherapy, such as for treating immune-related diseases, including cancer and autoimmune disorders, and infections, including but not limited to viruses. The present disclosure is at least in part based on the discovery that a palmitoylation motif and an Lck binding domain not present in the wild-type CD8α co-receptor allow recruitment of factors that improve T cell signaling. The increased T cell signaling results in increased T cell activation and cytotoxicity, which are advantageous properties of T cell therapeutics, such as TCR-T cells. Accordingly, the present disclosure provides cells and methods for generating cells (e.g., T cells) that express the chimeric CD8α co-receptor. Such cells exhibit increased T cell activation and cytotoxicity against target cells.

[0026] T cell receptor

[0027] The T cell receptor (“TCR”) is a molecule located on the surface of T cells that is responsible for recognizing antigens bound to MHC molecules. During antigen processing, antigens are degraded inside the cell and then carried 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 the molecules are recognized by CD8+ and CD4+ T cells, respectively.

[0028] Specifically, the TCR is a disulfide-linked membrane-anchored heterodimeric protein that typically consists of highly variable α and β chains that associate with invariant CD3 chain molecules to form a fully functional TCR. The α and β chains consist of extracellular domains that contain constant (C) and variable (V) regions. The constant region is close to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the variable region binds to the ligand. The variable domains of both the TCR α and β chains each have three variable regions called complementarity-determining regions (CDRs).

[0029] CD8α coreceptor

[0030] CD4 and CD8 are transmembrane glycoproteins that act as co-receptors for the T cell receptor (TCR). The binding of CD4 and CD8 to MHC molecules helps to stabilize weak T cell receptor (TCR)-pMHC interactions. At the same time, the cytoplasmic tails of the CD4 and CD8 co-receptors effectively recruit Lck (Src kinase) to the TCR complex when the co-receptors bind to MHC, thereby enhancing TCR signaling and the initiation of T cell activation.

[0031] To function, CD8 forms a dimer consisting of a pair of CD8 chains. The most common form of CD8 consists of CD8α and CD8β chains. The less common homodimer of the CD8α chain is also expressed on some cells. A single immunoglobulin-like domain and a stalk region form the extracellular portion of the CD8 subunit. In its intracellular tail, the α subunit of CD8 contains a Lck binding site, while the β subunit contains a palmitoylation site.

[0032] The wild-type CD8α co-receptor (UniProt ID number P01732) has the amino acid sequence of SEQ ID NO:11. The extracellular and transmembrane domains are shown in bold and underlined font.

[0033]

[0034] CD4 consists of a single chain that has four immunoglobulin domains (D1 to D4) exposed on the extracellular surface of the cell and a short cytoplasmic / intracellular tail. The CD4 functional motifs (including the Lck binding site and the palmitoylation site) are within its only intracellular domain.

[0035] The wild-type CD4 co-receptor (UniProt ID number 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.

[0036]

[0037]

[0038] A. Protein palmitoylation and palmitoylation motifs

[0039] Protein palmitoylation is a widespread lipid modification in which one or more cysteine thiols on the modified substrate protein are formed into a thioester with the saturated 16-carbon fatty acid palmitoyl group. This lipid modification is readily reversible, which is a characteristic of protein palmitoylation that allows for the rapid regulation of the functions of many cellular proteins (see, e.g., Guan and Fierke, Sci China Chem. December 2011; 54(12):1888-1897.).

[0040] For example, palmitoylation not only serves as a lipid anchor to localize proteins to the cell membrane, but also transports the modified proteins between cellular compartments, thereby allowing the proteins to be re-localized within the cell or to different regions of the membrane. The palmitoylation motif is a small tetrameric sequence that contains cysteine residues at positions 1 and 4 of the tetramer.

[0041] In some aspects, the palmitoylation motif comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:3, consists essentially of or consists of said amino acid sequence. In some aspects, the palmitoylation motif comprises the amino acid sequence of SEQ ID NO:3, consists essentially of or consists of said amino acid sequence. In some aspects, the palmitoylation motif has the amino acid sequence of SEQ ID NO:3.

[0042] In some aspects, the palmitoylation motif is encoded by a polynucleotide comprising a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:8, consisting essentially of or consisting of the nucleic acid sequence. In some aspects, the palmitoylation motif is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:8, consisting essentially of or consisting of the nucleic acid sequence.

[0043] B. High-affinity Lck domain

[0044] T cell signaling begins with the ligation of the T cell antigen receptor (TCR) by a cognate peptide and phosphorylation of the immunoreceptor tyrosine-based activation motif domain of the receptor by the kinase Lck.

[0045] In some aspects, the Lck binding domain (also referred to as the "high-affinity Lck binding site" or "high-affinity binding domain") comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:4, consisting essentially of or consisting of the amino acid sequence. In some aspects, the Lck binding domain comprises the amino acid sequence of SEQ ID NO:4, consisting essentially of or consisting of the amino acid sequence. In some aspects, the Lck binding domain has the amino acid sequence of SEQ ID NO:4.

[0046] In some aspects, the Lck binding domain is encoded by a polynucleotide comprising a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:9, consisting essentially of or consisting of the nucleic acid sequence. In some aspects, the Lck binding domain is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:9, consisting essentially of or consisting of the nucleic acid sequence.

[0047] Exemplary chimeric CD8α coreceptor

[0048] The present disclosure provides a chimeric CD8α coreceptor, the chimeric CD8α coreceptor comprising a CD8α extracellular domain and a CD8α transmembrane domain, which permits 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 aspects, the chimeric CD8α coreceptor is heterodimeric. In some aspects, the chimeric CD8α coreceptor is homodimeric. In some aspects, the homodimerized chimeric CD8α coreceptor contains two sets of functional motifs and has higher potency at initiating TCR signaling relative to the wild-type CD8α coreceptor.

[0049] In some aspects, the CD8α extracellular and transmembrane domains comprise an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:1, consisting essentially of or consisting of the amino acid sequence. In some aspects, the CD8α extracellular and transmembrane domains comprise the amino acid sequence of SEQ ID NO:1, consisting essentially of or consisting of the amino acid sequence.

[0050] In some aspects, the CD8α extracellular and transmembrane domains are encoded by a polynucleotide comprising a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:6, consisting essentially of or consisting of the nucleic acid sequence. In some aspects, the CD8α extracellular and transmembrane domains are encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:6, consisting essentially of or consisting of the nucleic acid sequence.

[0051] In some aspects, the CD4 intracellular domain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:2, consisting essentially of or consisting of the amino acid sequence. In some aspects, the CD4 intracellular domain comprises the amino acid sequence of SEQ ID NO:2, consisting essentially of or consisting of the amino acid sequence.

[0052] In some aspects, the CD4 intracellular domain is encoded by a polynucleotide comprising a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:7, consisting essentially of or consisting of the nucleic acid sequence. In some aspects, the CD4 intracellular domain is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:7, consisting essentially of or consisting of the nucleic acid sequence.

[0053] An exemplary chimeric CD8α co-receptor 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).

[0054] An exemplary chimeric CD8α co-receptor 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). Tables 1 and 2 show the amino acid sequence domains of the exemplary chimeric CD8α co-receptor and the nucleic acid sequences encoding them.

[0055] Table 1: Exemplary Amino Acid Sequences of Chimeric CD8α Co-Receptors

[0056]

[0057] Table 2: Exemplary Nucleic Acid Sequences of Chimeric CD8α Co-Receptors

[0058]

[0059] In some aspects, the chimeric CD8α co-receptor comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:5, consisting essentially of or consisting of the amino acid sequence. In some aspects, the chimeric CD8α co-receptor comprises the amino acid sequence of SEQ ID NO:5, consisting essentially of or consisting of the amino acid sequence.

[0060] In some aspects, the chimeric CD8α co-receptor is encoded by a polynucleotide comprising a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:10, consisting essentially of or consisting of the nucleic acid sequence. In some aspects, the chimeric CD8α co-receptor is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:10, consisting essentially of or consisting of the nucleic acid sequence.

[0061] The chimeric CD8α co-receptor polypeptide sequence was back-translated by a tool and codon-optimized for human T cell expression. The resulting nucleic acid sequence encoding the chimeric CD8α co-receptor (SEQ ID NO:10) was cloned into a transposon vector together with the TCR gene, an inducible suicide gene, and a selectable gene. In certain embodiments of the present disclosure, the nucleotide sequences encoding the chimeric CD8α co-receptor and the TCR are cloned into a transposon that contains a bidirectional expression cassette for expressing the chimeric CD8α co-receptor and the TCR. In some embodiments, the transposon further contains an expression cassette for expressing a CAR.

[0062] An exemplary transposon containing the nucleotide sequence encoding the chimeric CD8α co-receptor of the present disclosure is shown in

[0063] In the 5' to 3' direction, the transposon contains the following nucleotide sequences: left inverted terminal repeat (ITR), first insulator sequence, first multi-sequence (3'-5'), chimeric CD8 co-receptor (3'-5'), PGK promoter (3'-5'), EF1a promoter, iCAS9 safety switch, TCRβ chain, TCRα chain, DHFR selectable marker (each of the iCAS9 safety switch, TCRβ chain, TCRα chain, and DHFR selectable marker is separated by a T2A sequence), second polyA sequence, second insulator sequence, and right ITR. Figure 2 Compositions containing the chimeric CD8α co-receptor of the present disclosure can be incorporated into cell delivery compositions (e.g., transposons or vectors) as described in detail herein and optionally can be incorporated into cells.

[0064] 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 can include lymphoid progenitors, T lymphocytes (T cells), stem cell memory T cells (T

[0065] Cells and modified cells of the present disclosure

[0066] cells), central memory T cells (T SCM cells), or stem cell-like T cells. CM )

[0067] The modified T cells of the present disclosure can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs. Different from traditional biologics and chemotherapeutic agents, the disclosed modified T cells have the ability to rapidly proliferate upon antigen recognition, thereby potentially obviating the need for repeated treatments. To achieve this, in some embodiments, the modified T cells not only drive an initial response but also persist in the patient as a stable population of viable memory T cells to prevent potential relapse. Alternatively, in some aspects, the modified T cells do not persist in the patient when not desired.

[0068] The methods of the present 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 of the plurality of modified T cells in the population express a chimeric CD8α co-receptor. In some aspects, the chimeric CD8α co-receptor is expressed on the cell surface as a homodimer.

[0069] The methods of the present 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 of the plurality of modified T cells in the population express a TCR.

[0070] The methods of the present 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 of the plurality of modified T cells in the population express a TCR and a chimeric CD8α co-receptor. In some aspects, the chimeric CD8α co-receptor is expressed on the cell surface as a homodimer.

[0071] Multiple modified cells of the population comprise a transgene or a sequence encoding a transgene (e.g., chimeric CD8α co-receptor and TCR), wherein 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 multiple cells of the population express the transgene or the sequence encoding the chimeric CD8α co-receptor, and wherein 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 cell population express the transgene or the sequence encoding the TCR.

[0072] 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 the cell viability level and / or stem-like phenotype of immune cells or immune progenitor cells (e.g., the disclosed modified T cells) are disclosed elsewhere herein and are more particularly disclosed in U.S. Patent No. 10,329,543 and PCT Publication No. WO 2019 / 173636.

[0073] 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 cells or allogeneic cells. Allogeneic cells are engineered to prevent adverse reactions to transplantation after administration to a subject. Allogeneic cells can be any type of cell. Allogeneic cells can be stem cells or can be derived from stem cells. Allogeneic cells can be differentiated somatic cells.

[0074] Methods of expressing a chimeric CD8α coreceptor

[0075] The present disclosure provides methods for expressing a chimeric CD8α co-receptor on the cell surface. The methods comprise: (a) obtaining a cell population; (b) contacting the cell population with a composition comprising the chimeric CD8α co-receptor or a sequence encoding the chimeric CD8α co-receptor, under conditions sufficient to effect transfer of the chimeric CD8α co-receptor across the cell membrane of at least one cell in the cell population, thereby generating a modified cell population; (c) culturing the modified cell population under conditions suitable for integration of the sequence encoding the chimeric CD8α co-receptor; and (d) amplifying and / or selecting from the modified cell population at least one cell that expresses the chimeric CD8α co-receptor on the cell surface. In some embodiments, the chimeric CD8α co-receptor is a homodimer. In some embodiments, the chimeric CD8α co-receptor is a heterodimer.

[0076] In some aspects, the cell population can comprise white blood cells and / or CD4+ and CD8+ white blood cells. The cell population can comprise CD4+ and CD8+ white blood cells in an optimized ratio. In some embodiments, the optimized ratio of CD4+ and CD8+ white blood cells is not naturally occurring in vivo. The cell population can comprise tumor cells.

[0077] In some aspects, the conditions sufficient to effect transfer of the chimeric CD8α co-receptor or a sequence encoding the chimeric CD8α co-receptor, a transposon encoding the chimeric CD8α co-receptor, or a vector encoding the chimeric CD8α co-receptor 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 specified voltage, a buffer, and one or more supplementary factors. In some aspects, the conditions suitable for integration of the sequence encoding the chimeric CD8α co-receptor comprise at least one of a buffer and one or more supplementary factors.

[0078] The buffer can include PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, human T cell nucleofection buffer, or any combination thereof. The one or more supplementary factors can include: (a) recombinant human cytokines, chemokines, interleukins, or any combination thereof; (b) salts, minerals, metabolites, or any combination thereof; (c) cell culture medium; (d) inhibitors of cell DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathways, or a combination thereof; and (e) reagents that modify or stabilize one or more nucleic acids. The recombinant human cytokines, chemokines, interleukins, or any combination thereof can 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-γ, IL-1α / IL-1F1, IL-1β / IL-1F2, IL-12p70, IL-12 / IL-35p35, IL-13, IL-17 / IL-17A, IL-17A / F heterodimer, IL-17F, IL-18 / IL-1F4, IL-23, IL-24, IL-32, IL-32β, IL-32γ, IL-33, LAP(TGF-β1), lymphotoxin-α / TNF-β, TGF-β, TNF-α, TRANCE / TNFSF11 / RANK L, or any combination thereof.

[0079] Salts, minerals, metabolites, or any combination thereof may include HEPES, niacinamide, 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 Salt, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, NucleofectorPLUS supplement, KCL, MgCl2, Na2HPO4, NAH2PO4, sodium lactate, mannitol, sodium succinate, sodium chloride, CINa, glucose, Ca(NO3)2, Tris / HCl, K2HPO4, KH2PO4, polyethyleneimine, poly-ethylene-glycol, Poloxamer 188, Poloxamer 181, Poloxamer 407, poly-vinyl-pyrrolidone, 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.

[0080] Inhibitors of cell DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathways, or combinations thereof include inhibitors of TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type I interferon, pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNApol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, caspase 1, Pro-IL1B, PI3K, Akt, Wnt3A; inhibitors of glycogen synthase kinase-3β (GSK-3β) (e.g., TWS119); or any combination thereof. Examples of such inhibitors may 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 modifiers, DNA-binding proteins, lipids, phospholipids, CaPO4, net neutral charge DNA-binding peptides with or without NLS sequences, TREX1 enzymes, or any combination thereof.

[0081] The amplification and selection steps can occur simultaneously or sequentially. Amplification can occur before selection. Amplification can occur after selection, and optionally, an additional (i.e., second) selection can occur after amplification. Simultaneous amplification and selection can be concurrent. The amplification and / or selection steps can be carried out for a period of 10 to 14 days, including the end values.

[0082] Amplification can include contacting at least one cell of the modified cell population with an antigen to stimulate the at least one cell via a TCR and / or CAR, thereby generating an amplified cell population. The antigen can be presented on the surface of a substrate. The substrate can be in any form, including but not limited to a surface, a well, a bead, or a plurality thereof, and a matrix. The substrate can further comprise a paramagnetic or magnetic component. The antigen can be presented on the surface of a substrate, wherein the substrate is a magnetic bead, and wherein a magnet can be used to remove or separate the magnetic bead from the modified and amplified cell population. The antigen can be presented on the surface of a cell or an artificial antigen-presenting cell. Artificial antigen-presenting cells can include but are not limited to tumor cells and stem cells.

[0083] In some aspects where the transposon or vector contains a selection gene, the selection step includes contacting at least one cell in the modified cell population with a compound to which the selection gene confers resistance, thereby identifying cells expressing the selection gene as surviving the selection, and identifying cells that fail to express the selection gene as failing to survive the selection step.

[0084] The present disclosure provides a composition comprising the modified, amplified, and selected cell population of the methods described herein.

[0085] The present disclosure provides a cell or cell population, wherein the cell comprises a composition comprising: (a) an inducible transgenic construct comprising a sequence encoding an inducible promoter and a sequence encoding a transgenic (e.g., a chimeric CD8α co-receptor), and (b) an inducible transgenic 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 co-receptor 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 the expression of the chimeric CD8α co-receptor. In some aspects, the EF1a promoter controls the expression of the TCR.

[0086] Transposon and vector compositions

[0087] The present disclosure provides compositions and methods for delivering chimeric CD8α co - receptors and / or TCRs and / or CARs to a cell or cell population. Non - limiting examples of compositions for delivering the compositions of the present disclosure to a cell or cell population include transposons or vectors. Accordingly, the present disclosure provides: (i) a transposon comprising a chimeric CD8α co - receptor and / or TCR and / or CAR; or (ii) a vector comprising a chimeric CD8α co - receptor and / or TCR and / or CAR.

[0088] A transposon comprising the chimeric CD8α co - receptor and / or TCR and / or CAR of the present disclosure or a vector comprising the chimeric CD8α co - receptor and / or TCR and / or CAR of the present disclosure may further comprise a sequence encoding an inducible pro - apoptotic polypeptide (e.g., iC9). Alternatively or additionally, one transposon or one vector may comprise the chimeric CD8α co - receptor and / or TCR and / or CAR of the present disclosure, and a second transposon or second vector may comprise a sequence encoding the inducible pro - apoptotic polypeptide of the present disclosure. Inducible pro - apoptotic polypeptides are described in more detail herein.

[0089] A transposon comprising the TCR and / or CAR of the present disclosure or a vector comprising the TCR and / or CAR of the present disclosure may further comprise a sequence encoding a chimeric CD8α co - receptor. Alternatively or additionally, one transposon or one vector may comprise the TCR and / or CAR of the present disclosure, and a second transposon or second vector may comprise a sequence encoding a chimeric CD8α co - receptor. Chimeric CD8α co - receptors are described in more detail herein.

[0090] A transposon comprising the chimeric CD8α co - receptor and / or TCR and / or CAR of the present disclosure or a vector comprising the chimeric CD8α co - receptor and / or TCR and / or CAR of the present disclosure may further comprise a selectable gene. The selectable gene may encode a gene product that is essential for cell viability and survival. When challenged with selective cell culture conditions, the selectable gene may encode a gene product that is essential for cell viability and survival. The selective cell culture conditions may comprise a compound that is harmful to cell viability or survival, and wherein the gene product confers resistance to the compound. Non - limiting examples of selectable genes include neo (conferring resistance to neomycin), DHFR (encoding dihydrofolate reductase and conferring resistance to methotrexate), TYMS (encoding thymidylate synthase), MGMT (encoding O(6)-methylguanine - DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encoding aldehyde dehydrogenase 1 family, member A1), FRANCF, RAD51C (encoding RAD51 Paralog C), GCS (encoding glucosylceramide synthase), NKX2.2 (encoding NK2 homeobox 2), or any combination thereof.

[0091] In a preferred aspect, a gene encoding a DHFR mutant protease is selected. The DHFR mutant protease comprises the amino acid sequence of SEQ ID NO: 13, consists essentially of or consists of said amino acid sequence. The DHFR mutant protease is encoded by a polynucleotide which comprises the nucleic acid sequence of SEQ ID NO: 14, consists essentially of or consists of said nucleic acid sequence. The amino acid sequence of the DHFR mutant protease may further comprise a mutation at one or more of positions 80, 113 or 153. The amino acid sequence of the DHFR mutant protease may comprise one or more of a substitution of phenylalanine (F) or leucine (L) at position 80, a substitution of leucine (L) or valine (V) at position 113, and a substitution of valine (V) or aspartic acid (D) at position 153.

[0092] Transposon system

[0093] The present disclosure provides a transposon or minitransposon for expressing a chimeric CD8α co-receptor in a cell (e.g., a T cell) or co-expressing a chimeric CD8α co-receptor and a TCR in a cell (e.g., a T cell). In some embodiments, the cell further comprises a CAR.

[0094] The transposon or minitransposon of the present disclosure may be a plasmid DNA transposon which comprises a sequence encoding a chimeric CD8α co-receptor flanked by two cis-regulatory insulator elements. The transposon or minitransposon 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.

[0095] The transposon or minitransposon of the present disclosure may be a piggyBac TM (PB) transposon. In some aspects, when the transposon is a PB transposon, the transposase is a piggyBac TM (PB) transposase, a piggyBac-like (PBL) transposase or a SuperpiggyBac TM (SPB) transposase. In some aspects, the sequence encoding the SPB transposase is an mRNA sequence.

[0096] The minitransposon is described in more detail in PCT / US2019 / 067758. Non-limiting examples of PB transposons and 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 No. WO 2010 / 099296.

[0097] The PB, PBL, and SPB transposases recognize transposon-specific inverted terminal repeats (ITRs) at the ends of transposons and insert the contents between ITRs at the sequence 5'-TTAT-3' (TTAT target sequence) within a chromosomal locus or at the sequence 5'-TTAA-3' (TTAA target sequence) within a chromosomal locus. The target sequences for the PB or PBL transposons can include or consist of: 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'-AAAT-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 limitation on the gene of interest included between the ITRs.

[0098] Exemplary amino acid sequences of one or more of the 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 a preferred aspect, the PB transposase comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:15, or consists of said amino acid sequence.

[0099] ​The PB or PBL transposase can comprise, or consist of, an amino acid sequence having amino acid substitutions 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 can 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 can be a valine (V) substitution for isoleucine (I), the amino acid substitution at position 165 can be a serine (S) substitution for glycine (G), the amino acid substitution at position 282 can be a valine (V) substitution for methionine (M), and the amino acid substitution at position 538 can be a lysine (K) substitution for asparagine (N). In a preferred aspect, the SPB transposase comprises, or consists of, an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:16.

[0100] In certain aspects where the transposase comprises the above-described mutations at positions 30, 165, 282, and / or 538, the PB, PBL, and SPB transposases can further comprise amino acid substitutions at one or more of positions 3, 46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570, and 591 of the sequence of SEQ ID NO:15 or SEQ ID NO:16, which are described in more detail in PCT Publication No. WO 2019 / 173636 and PCT / US2019 / 049816.

[0101] The PB, PBL, or SPB transposase can be isolated from or derived from an insect, vertebrate, crustacean, or urochordate, as described in more detail in PCT Publication No. WO2019 / 173636 and PCT / US2019 / 049816. In a preferred aspect, the PB, PBL, or SPB transposase is isolated from or derived from the insect Trichoplusia ni (GenBank accession number AAA87375) or the silkworm Bombyx mori (GenBank accession number BAD11135).

[0102] A hyperactive PB or PBL transposase is a transposase that is more active than the naturally occurring variant from which it is derived. In preferred aspects, the hyperactive PB or PBL transposase is isolated from 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 WO 2019 / 173636. A list of hyperactive amino acid substitutions is disclosed in U.S. Patent No. 10,041,077.

[0103] In some aspects, 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 WO 2019 / 173636. A list of integration-deficient amino acid substitutions is disclosed in U.S. Patent No. 10,041,077.

[0104] In some aspects, the PB or PBL transposase is fused to a nuclear localization signal. Examples of PB or PBL transposases fused to a nuclear localization signal 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 WO 2019 / 173636.

[0105] The transposon of the present disclosure can be a Sleeping Beauty transposon. In some aspects, when the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase (e.g., as disclosed in U.S. Patent No. 9,228,180) or a hyperactive Sleeping Beauty (SB100X) transposase. In preferred aspects, the Sleeping Beauty transposase comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:17, or consists of the amino acid sequence. In preferred aspects, the hyperactive Sleeping Beauty (SB100X) transposase comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:18, or consists of the amino acid sequence.

[0106] The transposons of the present disclosure can be Helraiser transposons. Exemplary Helraiser transposons include Helibat1, which comprises a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 19, or consists of said nucleic acid sequence. In some aspects, when the transposon is a Helraiser transposon, the transposase is a Helitron transposase (e.g., as disclosed in WO 2019 / 173636). In a preferred aspect, the Helitron transposase comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 20, or consists of said amino acid sequence.

[0107] The transposons of the present disclosure can be Tol2 transposons. Exemplary Tol2 transposons (including inverted repeats, sub-terminal sequences and Tol2 transposase) comprise a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 21, or consists of said nucleic acid sequence. In some aspects, when the transposon is a Tol2 transposon, the transposase is a Tol2 transposase (e.g., as disclosed in WO 2019 / 173636). In a preferred aspect, the Tol2 transposase comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 22, or consists of said amino acid sequence.

[0108] The transposon of the present disclosure can be the TcBuster transposon. In some aspects, when the transposon is the TcBuster transposon, the transposase is the TcBuster transposase or the hyperactive TcBuster transposase (e.g., as disclosed in WO 2019 / 173636). The TcBuster transposase can comprise a naturally occurring amino acid sequence or a non-naturally occurring amino acid sequence, or consist of the naturally occurring amino acid sequence or the non-naturally occurring amino acid sequence. In a preferred aspect, the TcBuster transposase comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:23, or consists of the amino acid sequence. The polynucleotide encoding the TcBuster transposase can comprise a naturally occurring nucleic acid sequence or a non-naturally occurring nucleic acid sequence, or consist of the naturally occurring nucleic acid sequence or the non-naturally occurring nucleic acid sequence. In a preferred aspect, the TcBuster transposase is encoded by a polynucleotide that comprises a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:24, or consists of the nucleic acid sequence.

[0109] In some aspects, when compared to the wild-type TcBuster transposase, the mutant TcBuster transposase comprises one or more sequence variations, as described in more detail in PCT Publication No. WO 2019 / 173636 and PCT / US2019 / 049816.

[0110] Vector system

[0111] The vectors of the present disclosure can be viral vectors or recombinant vectors. The viral vectors can comprise sequences isolated or derived from retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or any combination thereof. The viral vectors can comprise sequences isolated or derived from adeno-associated virus (AAV). The viral vectors can comprise recombinant AAV (rAAV). Exemplary adeno-associated viruses and recombinant adeno-associated viruses comprise two or more inverted terminal repeat (ITR) sequences located in cis adjacent to the sequence encoding the chimeric CD8α co-receptor 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.

[0112] The vectors of the present disclosure can be nanoparticles. 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., polymer vesicles), 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. The nanoparticle vectors can transport across cell membranes passively or actively.

[0113] The cell delivery compositions (e.g., transposons, vectors) disclosed herein can comprise nucleic acids encoding therapeutic proteins or therapeutic agents. Examples of therapeutic proteins include those disclosed in PCT Publication No. WO 2019 / 173636 and PCT / US2019 / 049816.

[0114] Inducible pro-apoptotic polypeptide

[0115] The inducible pro-apoptotic polypeptides disclosed herein are superior to existing inducible polypeptides because the inducible pro-apoptotic polypeptides of the present disclosure have much lower immunogenicity. The inducible pro-apoptotic polypeptides are recombinant polypeptides and are thus non-naturally occurring. In addition, the sequences recombined to produce the inducible pro-apoptotic polypeptides do not contain non-human sequences that can be recognized by the host human immune system as "non-self", which would thus induce an immune response in a subject against: the inducible pro-apoptotic polypeptides, cells comprising the inducible pro-apoptotic polypeptides, compositions comprising the inducible pro-apoptotic polypeptides, or cells comprising the inducible pro-apoptotic polypeptides.

[0116] 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 non-human sequences. In some aspects, the non-human sequences comprise restriction sites. In some aspects, the ligand-binding region can be a multimeric ligand-binding region. In some aspects, 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 occurring. When the caspase is caspase 9 or truncated caspase 9, the inducible pro-apoptotic polypeptide can also be referred to as an "iC9 safety switch".

[0117] 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 some aspects, 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.

[0118] The ligand-binding region can comprise a FK506 binding protein 12 (FKBP12) polypeptide. The amino acid sequence of the ligand-binding region comprising the FK506 binding protein 12 (FKBP12) polypeptide can comprise a modification at position 36 of the sequence. The modification can be a valine (V) substitution for phenylalanine (F) at position 36 (F36V). The FKBP12 polypeptide can comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:25, consisting essentially of or consisting of the amino acid sequence. The FKBP12 polypeptide can be encoded by a polynucleotide comprising a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:26, or consisting of the nucleic acid sequence.

[0119] The linker region may comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:27, consisting essentially of or consisting of said amino acid sequence. In some aspects, the linker region may be encoded by a polynucleotide comprising a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:28, or consisting of said nucleic acid sequence. In some aspects, the nucleic acid sequence encoding the linker does not contain restriction sites.

[0120] The truncated caspase-9 polypeptide may comprise an amino acid sequence that does not contain arginine (R) at position 87 of the sequence. Alternatively or additionally, the truncated caspase-9 polypeptide may comprise an amino acid sequence that does not contain alanine (A) at position 282 of the sequence. The truncated caspase-9 polypeptide may comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:29, consisting essentially of or consisting of said amino acid sequence. In some aspects, the truncated caspase-9 polypeptide may be encoded by a polynucleotide comprising a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:30, or consisting of said nucleic acid sequence.

[0121] In certain aspects, when the polypeptide comprises the truncated caspase-9 polypeptide, the inducible pro-apoptotic polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:31, consisting essentially of or consisting of said amino acid sequence. In some aspects, the inducible pro-apoptotic polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:32, or consisting of said nucleic acid sequence.

[0122] The inducible pro-apoptotic polypeptide may be expressed in the cell under the transcriptional regulation of any promoter known in the art capable of initiating and / or regulating the expression of the inducible pro-apoptotic polypeptide in the cell.

[0123] Activation of the inducible pro-apoptotic polypeptide can be accomplished, for example, by chemically induced dimerization (CID) mediated by an inducer to produce a conditionally controlled protein or polypeptide. Due to degradation of the labile dimer or administration of a monomeric competitive inhibitor, the pro-apoptotic polypeptide is not only inducible, but the induction of these polypeptides is also reversible.

[0124] In some aspects, when the ligand-binding region comprises an FKBP12 polypeptide having a valine (V) substitution for phenylalanine (F) at position 36 (F36V), the inducer can comprise 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 analogue such as AP1510. As used herein, the inducers AP20187, AP1903, and AP1510 can be used interchangeably.

[0125] Inducible pro-apoptotic peptides and methods of inducing these peptides are described in detail in U.S. Patent Publication No. WO 2019 / 0225667 and PCT Publication No. WO 2018 / 068022.

[0126] Formulations, dosages, and modes of administration

[0127] The present disclosure provides formulations, dosages, and methods for administering the compositions described herein.

[0128] The disclosed compositions and pharmaceutical compositions may further comprise at least one of any suitable adjuvants, such as but not limited to diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, etc. Preferably, the adjuvants are pharmaceutically acceptable. Non-limiting examples and methods for preparing such sterile solutions are well known in the art, such as but not limited to Gennaro, editor, Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, Pa.), 1990 and "Physician's Desk Reference", 52nd Edition, Medical Economics (Montvale, N.J.), 1998. Pharmaceutically acceptable carriers suitable for the mode of administration, solubility and / or stability of protein scaffolds, fragments or variant compositions well known in the art or as described herein can be routinely selected.

[0129] Non-limiting examples of suitable pharmaceutical excipients and additives include proteins, peptides, amino acids, lipids and carbohydrates (e.g., sugars, including monosaccharides, disaccharides, trisaccharides, tetrasaccharides and oligosaccharides; derived sugars, such as sugar alcohols, aldonic acids, esterified sugars, etc.; and polysaccharides or sugar polymers), which may be present alone or in combination, alone or in combination accounting for 1-99.99% by weight or volume. Non-limiting examples of protein excipients include serum albumin, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, etc. Representative amino acid / protein components that can also play a role in buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, etc. In some aspects, the amino acid is glycine.

[0130] Non-limiting examples of suitable carbohydrate excipients 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 sugar alcohols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (sorbitol), inositol, etc. Preferably, the carbohydrate excipient is mannitol, trehalose and / or raffinose.

[0131] The composition may also include a buffer or a pH regulator; generally, a buffer is a salt prepared from an organic acid or base. Representative buffers include salts of organic acids such as citrate, ascorbate, gluconate, carbonate, tartrate, succinate, acetate or phthalate; Tris (tris(hydroxymethyl)aminomethane); tromethamine hydrochloride or phosphate buffer. A preferred buffer is a salt of an organic acid such as citrate.

[0132] Additionally, the disclosed compositions may include polymeric excipients / additives such as polyvinylpyrrolidone, polysucrose (a polymeric sugar), dextrans (e.g., cyclodextrins such as 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).

[0133] Many known and developed modes can be used to administer a therapeutically effective amount of the compositions or pharmaceutical compositions disclosed herein. Non-limiting examples of modes of administration include bolus injection, oral, infusion, intra-articular, intra-bronchial, intraperitoneal, intra-capsular, intra-cartilaginous, intracavitary, intraspinal, intracerebellar, intraventricular, intra-colonic, intra-cervical, intra-gastric, intra-hepatic, intrathecal, intramuscular, intra-myocardial, intranasal, intra-ocular, intra-medullary, intra-osseous, intra-pelvic, intra-pericardial, intra-peritoneal, intra-pleural, intra-prostatic, intra-pulmonary, intra-rectal, intra-renal, intra-retinal, intra-spinal canal, intra-vaginal, intravenous, intra-bladder, oral, parenteral, rectal, sublingual, subcutaneous, transdermal or vaginal routes.

[0134] 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 a semi-solid form (including but not limited to creams and suppositories). In some embodiments, the compositions can be prepared for oral or sublingual administration. Non-limiting examples of formulations for oral 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 drops or aerosols. In some embodiments, these formulations further include certain agents. In some embodiments, the compositions can be prepared for transdermal administration. Non-limiting examples of formulations for transdermal administration can include gels, ointments, emulsions, suspensions or patch delivery systems. In some embodiments, the transdermal administration compositions further include chemical enhancers. In some embodiments, the chemical enhancer can be dimethyl sulfoxide to modify the skin structure or increase the drug concentration in the transdermal patch (Junginger et al. "Drug Permeation Enhancement"; edited by Hsieh, D.S., pp. 59-90 (Marcel Dekker, Inc. New York 1994)). In some embodiments, the suspension or patch delivery system further includes an oxidizing agent capable of applying a formulation containing proteins and peptides to the skin (WO 98 / 53847). In some embodiments, transdermal administration includes applying an electric field to create transient transport pathways (such as electroporation) or increasing the mobility of charged drugs through the skin (such as iontophoresis). In some embodiments, transdermal administration includes applying ultrasound, such as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402). The above publications and patents are incorporated herein by reference in their entirety.

[0135] For parenteral administration, any composition disclosed herein can be formulated as a solution, suspension, emulsion, granule, powder, or lyophilized powder associated with a pharmaceutically acceptable parenteral vehicle or provided alone. Formulations for parenteral administration may contain sterile water or saline, polyalkylene glycols (such as polyethylene glycol), oils of vegetable origin, hydrogenated naphthalene, etc. as common excipients. According to known methods, aqueous or oily suspensions for injection can be prepared by using appropriate emulsifying or wetting agents and suspending agents. The agents for injection can be non-toxic, non-oral diluents, such as aqueous solutions, sterile injection solutions, or suspensions in solvents. As available vehicles or solvents, water, Ringer's solution, isotonic saline, etc. are permitted; as common solvents or suspending solvents, sterile non-volatile oils can be used. For these purposes, any kind of non-volatile oil and fatty acid can be used, including natural or synthetic or semi-synthetic fatty oils or fatty acids, natural or synthetic or semi-synthetic glycerol monoesters or glycerol di-esters or glycerol tri-esters. Parenteral administration is known in the art and includes but is not limited to conventional injection methods, such as the pneumatic needleless injection device described in U.S. Patent No. 5,851,198, and the laser perforation device described in U.S. Patent No. 5,839,446.

[0136] Formulations for oral administration may rely on (i) the co-administration of adjuvants (e.g., resorcinol and non-ionic surfactants such as polyoxyethylene oleyl ether and / or cetyl polyethylene ether) to artificially increase the permeability of the intestinal wall, and (ii) the co-administration of enzyme inhibitors (e.g., trypsin inhibitor, diisopropyl fluorophosphate (DFF), and aprotinin) to inhibit enzymatic degradation. Combinations of at least two surfactants for the delivery of hydrophilic agents (including proteins and protein scaffolds) and intended for oral, buccal, mucosal, nasal, pulmonary, vaginal transmucosal, or rectal administration are described in U.S. Patent No. 6,309,663. The active ingredient compounds in solid dosage forms for oral administration can be mixed with at least one additive, which includes but is not limited to sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, arginine salts, chitin, chitosan, pectin, tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, and glycerol esters. These dosage forms may also contain other types of additives, such as inactive diluents; lubricants, such as magnesium stearate and parabens; preservatives, such as sorbic acid, ascorbic acid, and α-tocopherol; antioxidants, such as cysteine; disintegrants; binders; thickeners; buffers; sweeteners; flavoring agents; fragrances, etc.

[0137] Tablets and pills can be further processed into enteric-coated formulations. Liquid formulations for oral administration can include pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, and solution formulations. These formulations 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. Patent No. 4,239,754). Recently, microspheres of synthetic polymers of mixed amino acids (proteinoids) have been used to deliver drugs (U.S. Patent No. 4,925,673). Additionally, carrier compounds, such as those described in U.S. Patent No. 5,879,681 and U.S. Patent No. 5,871,753, are known in the art and are used for oral delivery of bioactive agents.

[0138] For pulmonary administration, preferably, the compositions or pharmaceutical compositions described herein are delivered in a particle size effective to reach the lower airways of the lung or sinuses. The compositions or pharmaceutical compositions can be delivered by any of a variety of inhalation devices or nasal devices known in the art for administering therapeutic agents by inhalation. Such devices capable of depositing the nebulized formulation in the patient's sinus cavities or alveoli include metered-dose inhalers, nebulizers (e.g., jet nebulizers, ultrasonic nebulizers), dry powder generators, atomizers, etc. All such devices can be used to formulate the compositions or pharmaceutical compositions described herein for administration in an aerosol or to dispense the compositions or pharmaceutical compositions described herein. Such aerosols can contain solutions (both aqueous and non-aqueous) or solid particles. Additionally, a spray including 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, the compositions or pharmaceutical compositions described herein, and any excipients or other additives are contained in the canister as a mixture including a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol, preferably containing particles in a size range 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 No. WO 2019 / 049816.

[0139] For absorption through the mucosal surface, the composition comprises an emulsion comprising a plurality of submicron particles, a mucoadhesive macromolecule, a bioactive peptide, and an aqueous continuous phase, which promotes absorption through the mucosal surface by achieving adhesion of the emulsion particles (U.S. Patent No. 5,514,670). Mucosal surfaces suitable for the emulsion applications of the present disclosure may include corneal, conjunctival, oral, sublingual, nasal, vaginal, pulmonary, gastric, intestinal, and rectal administration routes. Formulations for vaginal or rectal administration, such as suppositories, may contain, for example, polyalkylene glycols, petrolatum (vaseline), cocoa butter, etc. as excipients. Formulations for intranasal administration may be solid and contain, for example, lactose as an excipient, or may be an aqueous or oily solution of a nasal drop. For oral administration, excipients may include sugars, calcium stearate, magnesium stearate, pregelatinized starch, etc. (U.S. Patent No. 5,849,695). A more detailed description of mucosal administration and formulations is disclosed in PCT Publication No. WO 2019 / 049816.

[0140] For transdermal administration, the compositions or pharmaceutical compositions disclosed herein are encapsulated in a delivery device, such as liposomes or polymeric nanoparticles, microparticles, microcapsules, or microspheres (collectively referred to as microparticles unless otherwise specified). Many suitable devices are known in the art, including microparticles prepared from synthetic polymers such as polyhydroxy acids (e.g., polylactic acid, polyglycolic acid, and their copolymers), polyorthoesters, polyanhydrides, polyphosphazenes, or natural polymers such as collagen, polyamino acids, albumin, and other proteins, alginic acid, 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 No. WO 2019 / 049816.

[0141] It may be desirable to deliver the disclosed compounds to a subject over an extended period of time, e.g., a period of from one week to one year commencing from a single administration. A variety of sustained release, depot, or implantable dosage forms may be utilized. For example, the dosage form may contain a pharmaceutically acceptable non-toxic salt of the compound having low solubility in body fluids, e.g., (a) an acid addition salt with a polybasic acid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or di-sulfonic acid, polygalacturonic acid, etc.; (b) a salt with a polyvalent metal cation such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, etc., or a salt with an organic cation formed by, e.g., N,N'-dibenzylethylenediamine or ethylenediamine; or (c) a combination of (a) and (b), e.g., zinc tannate. Additionally, the disclosed compound or preferably a relatively insoluble salt of the disclosed compound (such as the relatively insoluble salts just described) may be formulated in a gel (e.g., aluminum monostearate gel) with, for injection, e.g., sesame oil. Particularly preferred salts are zinc salts, zinc tannate salts, pamoate salts, etc. Another type of sustained release depot formulation for injection will contain the compound or salt dispersed for encapsulation in a slowly degradable, non-toxic, non-antigenic polymer such as, for example, the polylactic acid / polyglycolic acid polymer described in U.S. Patent No. 3,773,919. The relatively insoluble salt of the compound or preferably the disclosed compound (such as the relatively insoluble salts described above) may also be formulated into cholesterol matrix silicone rubber pellets, particularly for animals. Additional sustained release, depot, or implantable formulations, e.g., gaseous or liquid liposomes, are known in the literature (U.S. Patent No. 5,770,222 and “Sustained and Controlled Release Drug Delivery Systems”, edited by J.R. Robinson, Marcel Dekker, Inc., New York, 1978).

[0142] Appropriate dosages are well known in the art. See, e.g., Wells et al., eds., Pharmacotherapy Handbook, 2d ed., Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia - Tarascon Pocket Pharmacopoeia 2000, hardcover ed., Tarascon Publishing, Loma Linda, Calif. (2000); Nursing 2001 Handbook of Drugs, 21st ed., Springhouse Corp., Springhouse, Pa., 2001; Health Professional's Drug Guide 2001, eds. Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle River, N.J. Preferred dosages can optionally include from about 0.1 mg / kg / dose - 99 mg / kg / dose and / or 100 mg / kg / dose - 500 mg / kg / dose, or any range, value or fraction thereof, or a serum concentration of from about 0.1 μg / ml - 5000 μg / ml serum concentration per single or multiple doses, or any range, value or fraction thereof. The preferred dosage range for the compositions or pharmaceutical compositions disclosed herein is from about 1 mg / kg to about 3 mg / kg, about 6 mg / kg or about 12 mg / kg of the subject's body weight.

[0143] Alternatively, the dosage administered can vary depending on known factors such as the pharmacodynamic properties of the particular agent and its mode and route of administration; the age, health status and weight of the recipient; the nature and degree of the symptoms; the type of concurrent treatment; the frequency of treatment and the desired effect. Generally, the dosage of the active ingredient can be from about 0.1 milligram to 100 milligrams per kilogram of body weight. Each administration is usually from 0.1 milligram to 50 milligrams per kilogram and preferably from 0.1 milligram to 10 milligrams per kilogram or can be effectively obtained in a sustained release form to achieve the desired result.

[0144] As a non-limiting example, treatment of a human or animal can be provided as a single or periodic dose of the compositions or pharmaceutical compositions disclosed herein. In some embodiments, a single infusion or repeated doses are used, on at least one day of days 1 - 40 and / or at least one week of weeks 1 - 52 and / or at least one year of years 1 - 20 or any combination thereof, at a dose of about 0.1 mg / kg to 100 mg / kg per day or any range, value, or fraction thereof.

[0145] Dosage forms suitable for internal administration generally contain from about 0.001 milligrams to about 500 milligrams of the active ingredient per unit or container. In these pharmaceutical compositions, the active ingredient will generally be present in an amount of about 0.5 - 99.999% by weight, based on the total weight of the composition.

[0146] An effective amount can comprise an amount of about 0.001 mg / kg to about 500 mg / kg (or any effective range or value therein) per single (e.g., bolus), multiple, or continuous administration. In some embodiments, the effective amount achieves a serum concentration of 0.01 μg / ml - 5000 μg / ml (or any effective range or value therein) per single, multiple, or continuous administration, as accomplished and determined using known methods described herein or known in the relevant art.

[0147] In aspects where the composition administered to a subject in need contains modified cells as disclosed herein, the following cells can be administered: between about 1x10 3 cells and 1x10 15 cells, between about 1x10 4 cells and 1x10 12 cells, between about 1x10 5 cells and 1x10 10 cells, between about 1x10 6 cells and 1x10 9 cells, between about 1x10 6 cells and 1x10 8 cells, between about 1x10 6 cells and 1x10 7 cells, or between about 1x10 6 cells and about 25x10 6 cells. In one aspect, between about 5x10 6 cells and 25x10 6 cells are administered.

[0148] A more detailed description of pharmaceutically acceptable excipients, formulations, dosages, and methods of administration of the disclosed compositions and pharmaceutical compositions is disclosed in PCT Publication No. WO 2019 / 049816.

[0149] Methods of using the compositions of the present disclosure

[0150] The present disclosure provides methods of using the disclosed compositions or pharmaceutical compositions to treat a disease or disorder in a cell, tissue, organ, animal, or subject, as known in the art or as described herein. In some embodiments, the methods of using the disclosed compositions and pharmaceutical compositions include, for example, administering a therapeutically effective amount of the composition or pharmaceutical composition to a cell, tissue, organ, animal, or subject or contacting the 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.

[0151] The present disclosure provides a method 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; chronic myelogenous 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 / malignant hypercalcemia; solid tumors; bladder cancer; breast cancer; colorectal cancer; endometrial cancer; head cancer; neck cancer; hereditary non-polyposis cancer; Hodgkin's lymphoma; liver cancer; lung cancer; non-small cell lung cancer; ovarian cancer; pancreatic cancer; prostate cancer; renal cell cancer; testicular cancer; adenocarcinoma; sarcoma; malignant melanoma; hemangioma; metastatic disease; cancer-related bone resorption; cancer-related bone pain; and the like.

[0152] In preferred aspects, the treatment of a malignant disease or disorder involves adoptive cell therapy. For example, in one aspect, the present disclosure provides modified cells that express at least a chimeric CD8α co-receptor 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 a sterile package. The modified cells can be formulated in a pharmaceutically acceptable carrier. The modified cells can be formulated in a pharmaceutically acceptable carrier at a specified density with one or more reagents to inhibit further expansion and / or prevent cell death.

[0153] In some aspects, the treatment of a malignant disease or disorder can involve administering to a cell, tissue, organ, animal, or subject in need of such modulation, treatment, or therapy an effective amount of any composition or pharmaceutical composition disclosed herein. Such methods can optionally further comprise co-administration or combination therapy for treating such disease or disorder, wherein the administration of any composition or pharmaceutical composition disclosed herein further comprises administering at least one chemotherapeutic agent (e.g., an alkylating agent, a mitotic inhibitor, a radiopharmaceutical) before, simultaneously with, and / or after the administration of the composition or pharmaceutical composition disclosed herein.

[0154] In some aspects, the subject does not exhibit graft-versus-host (GvH) and / or host-versus-graft (HvG) after administration. In one aspect, the administration is systemic. Systemic administration can be in any manner known in the art and described in detail herein. Preferably, systemic administration is by intravenous injection or infusion. In one aspect, the administration is local. Local administration can be in any manner known in the art and described in detail herein. Preferably, local administration is by intratumoral injection or infusion, intraspinal injection or infusion, intraventricular injection or infusion, intravitreal injection or infusion, or intramedullary injection or infusion.

[0155] In some aspects, the therapeutically effective dose is a single dose. In some aspects, 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 therebetween of doses that are manufactured simultaneously. In some aspects, in the case where the composition is autologous or allogeneic cells, the dose is an amount sufficient to effect cell engraftment and / or persist for a sufficient time to treat the disease or disorder.

[0156] In one instance, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a composition comprising at least a chimeric CD8α co-receptor and / or TCR and / or CAR that specifically binds to an antigen on a tumor cell. In aspects where the composition comprises a modified cell or cell population, the cell or cell population can be autologous or allogeneic.

[0157] In some aspects of the treatment methods described herein, treatment can be modified or terminated. Specifically, in aspects where the composition for treatment comprises an inducible pro-apoptotic polypeptide, apoptosis can be selectively induced in the cells by contacting the cells with an inducer. Treatment can be modified or terminated in response to, for example, signs of recovery or decreased signs of disease severity / progression, signs of disease remission / cessation, and / or the occurrence of an adverse event. In some aspects, the method comprises a step of administering an inhibitor of the inducer to inhibit the alteration of the cell therapy, thereby restoring the function and / or efficacy of the cell therapy (e.g., when signs or symptoms of the disease recur or increase in severity and / or an adverse event is resolved).

[0158] Recombinant methods of constructing nucleic acids

[0159] The isolated nucleic acid compositions of the present disclosure can be obtained from biological sources using any number of cloning methods known to those of skill in the art, such as RNA, cDNA, genomic DNA, or any combination thereof. In some embodiments, oligonucleotide probes that selectively hybridize to the polynucleotides of the present invention under stringent conditions are used to identify desired sequences in cDNA or genomic DNA libraries. The isolation of RNA and the construction of cDNA and genomic libraries are well known to those of skill in the art. (See, for example, Ausubel, supra; or Sambrook, supra).

[0160] The isolated nucleic acids of the present disclosure can also be prepared by direct chemical synthesis by known methods (see, for example, Ausubel et al., supra). Chemical synthesis generally produces single-stranded oligonucleotides, which can be converted to double-stranded DNA by hybridization with a complementary sequence or by polymerization using a DNA polymerase with the single-strand as a template. Those of skill in the art will recognize that although chemical synthesis of DNA can be limited to sequences of about 100 bases or more, longer sequences can be obtained by ligating shorter sequences.

[0161] Recombinant expression cassette

[0162] The present disclosure further provides recombinant expression cassettes comprising the nucleic acids of the present disclosure. The nucleic acid sequences of the present disclosure, such as cDNA or genomic sequences encoding the 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 generally comprise a polynucleotide of the present disclosure operably linked to a transcriptional initiation regulatory sequence that will direct transcription of the polynucleotide in the intended host cell. Both heterologous promoters and non-heterologous (i.e., endogenous) promoters can be used to direct the expression of the nucleic acids of the present disclosure.

[0163] In some aspects, isolated nucleic acids that act as promoters, enhancers, or other elements can be introduced into appropriate positions (upstream, downstream, or within introns) of the non-heterologous form of the polynucleotides of the present disclosure to upregulate or downregulate the expression of the polynucleotides of the present disclosure. For example, the endogenous promoter can be altered in vivo or in vitro by mutation, deletion, and / or substitution.

[0164] Expression vectors and host cells

[0165] 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 well known in the art. See, for example, Sambrook et al., supra; Ausubel et al., supra, each of which is incorporated herein by reference in its entirety.

[0166] The polynucleotide can optionally be ligated to a vector containing a selectable marker for propagation in the host. Generally, plasmid vectors are introduced into precipitates (such as calcium phosphate precipitates) or complexes with charged lipids. If the vector is viral, it can be packaged in vitro using an appropriate packaging cell line and then transduced into the host cell.

[0167] The DNA insert should be operably linked to an appropriate promoter. The expression construct will further contain sites for transcriptional initiation, termination, and a ribosome binding site for translation within the transcribed region. The coding portion of the mature transcript expressed by the construct will preferably include a translation initiation at the start and a termination codon (e.g., UAA, UGA, or UAG) appropriately located at the end of the mRNA to be translated, where UAA and UAG are preferably used for mammalian or eukaryotic cell expression.

[0168] The expression vector will preferably but optionally include at least one selectable marker. Such markers include, for example but not limited to, ampicillin, bleomycin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB), G418 / geneticin (neo gene), DHFR (encoding dihydrofolate reductase and conferring resistance to methotrexate), mycophenolic acid or glutamine synthetase (GS, U.S. Patent Nos. 5,122,464; 5,770,359; 5,827,739), blasticidin (bsd gene), resistance genes for eukaryotic cell culture and ampicillin, bleomycin (Shbla 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 culture in Escherichia coli and other bacteria or prokaryotes (the above patents are hereby incorporated by reference in their entirety). Suitable media and conditions for the above host cells are known in the art. Suitable vectors will be readily apparent to the person skilled in the art. The vector construct can be introduced into the host cell 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; Ausubel, supra, Chapters 1, 9, 13, 15, 16.

[0169] The expression vector will preferably but optionally include at least one selectable cell surface marker for isolating cells modified by the compositions and methods of the present disclosure. The selectable cell surface markers of the present disclosure comprise surface proteins, glycoproteins or proteomes that distinguish a cell or cell subset from another defined cell subset. Preferably, the selectable cell surface marker will distinguish those cells modified by the compositions or methods of the present disclosure from those cells not modified by the compositions or methods of the present disclosure. Such cell surface markers include, for example but not limited to, "cluster of name" or "class determinant" proteins (commonly abbreviated as "CD"), such as truncated or full-length forms of CD19, CD271, CD34, CD22, CD20, CD33, CD52 or any combination thereof. The cell surface marker further includes the suicide gene marker RQR8 (Philip B et al. Blood. August 21, 2014; 124(8):1277-87).

[0170] The expression vector will preferably but optionally include at least one selectable drug resistance marker for isolating cells modified by the compositions and methods of the present disclosure. The selectable drug resistance markers of the present disclosure can include wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

[0171] At least one protein scaffold of the present disclosure can be expressed in a modified form, such as a fusion protein, and can include not only a secretion signal 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 during subsequent processing and storage. Similarly, a peptide moiety can be added to the protein scaffolds of the present disclosure to facilitate purification. Such regions can be removed prior to the 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; Ausubel, supra, Chapters 16, 17, and 18.

[0172] Those skilled in the art are knowledgeable about numerous expression systems that can be used to express 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 (by manipulation) the endogenous DNA containing the protein scaffold encoding 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.

[0173] Exemplary cell cultures that can be used to produce protein scaffolds, specific portions or variants thereof are bacteria, yeast, and mammalian cells known in the art. Mammalian cell systems will typically be in the form of a cell monolayer, although mammalian cell suspensions or bioreactors can also be used. Many suitable host cell lines capable of expressing fully glycosylated proteins have been developed in the art and include 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, etc., which can be readily obtained from, for example, 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 number CRL-1580) and SP2 / 0-Ag14 cells (ATCC accession number CRL-1851). In a preferred aspect, the recombinant cells are P3X63Ab8.653 or SP2 / 0-Ag14 cells.

[0174] Expression vectors for these cells can include one or more of the following expression control sequences, such as but not limited to an origin of replication; a promoter (e.g., a late or early SV40 promoter, CMV promoter (U.S. Patent Nos. 5,168,062; 5,385,839), HSVtk promoter, pgk (phosphoglycerate kinase) promoter, EF-1α promoter (U.S. Patent No. 5,266,491), or at least one human promoter); an enhancer and / or processing information sites, such as a ribosome binding site, an RNA splicing site, a polyadenylation site (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 that can be used to produce the nucleic acids or proteins of the present 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.

[0175] When using eukaryotic host cells, a polyadenylation or transcription terminator sequence is typically incorporated into the vector. An example of a terminator sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences for precise splicing of transcripts may also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague et al., Journal of Virology (J. Virol.) 45:773-781 (1983)). Additionally, gene sequences that control replication in the host cell can be incorporated into the vector as known in the art.

[0176] Definitions

[0177] As used throughout this disclosure, unless the context clearly dictates otherwise, the singular forms "a", "and", and "the" include plural referents. Thus, for example, reference to "a method" includes a plurality of such methods and reference to "a dose" includes reference to one or more doses known to those of ordinary skill in the art and their equivalents, etc.

[0178] The terms "about" or "approximately" mean within an acceptable error range of 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 measuring system. For example, "about" can mean within one or more standard deviations. Alternatively, "about" can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, especially for biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of the value. When a particular value is described in this application and the claims, unless otherwise stated, it should be assumed that the term "about" means within an acceptable error range of the particular value.

[0179] The present disclosure provides isolated or substantially purified polynucleotide or protein compositions. An "isolated" or "purified" polynucleotide or protein or a biologically active portion thereof is substantially or essentially free of components that normally accompany or interact with the polynucleotide or protein as found in its natural environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material, or when produced by recombinant techniques is substantially free of culture medium, or when chemically synthesized is substantially free of chemical precursors or other chemicals. Preferably, an "isolated" polynucleotide is free of sequences (preferably protein-coding sequences) that naturally flank the polynucleotide in genomic DNA of the organism from which the polynucleotide is derived (i.e., sequences located at the 5' and 3' ends of the polynucleotide). For example, in various aspects, an isolated polynucleotide may contain fewer than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences of polynucleotides in genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes protein preparations having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When the proteins or biologically active portions thereof of the present disclosure are produced recombinantly, the culture medium preferably represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or chemicals other than the protein of interest.

[0180] The present disclosure provides fragments and variants of the disclosed DNA sequences and proteins encoded by these DNA sequences. As used throughout the present disclosure, the term "fragment" refers to a portion of a DNA sequence or a portion of an amino acid sequence and thus a protein encoded thereby. A fragment of a DNA sequence that contains a coding sequence may encode a protein fragment that retains the biological activity of the native protein and thus retains the DNA recognition or binding activity to a target DNA sequence as described herein. Alternatively, a fragment of a DNA sequence that is used as a hybridization probe generally does not encode a protein that retains biological activity or does not retain promoter activity. Thus, the range of fragments of a DNA sequence can be at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotides of the present disclosure.

[0181] The nucleic acids or proteins of the present disclosure can be constructed by a modular method that includes pre-assembling monomeric units and / or repeating units in a target vector, which can then be assembled into a final destination vector. The polypeptides of the present disclosure can contain the repeating monomers of the present disclosure and can be constructed by a modular method, i.e., by pre-assembling repeating units in a target vector, which can then be assembled into a final destination vector. The present disclosure provides polypeptides produced by this method and nucleic acid sequences encoding these polypeptides. The present disclosure provides host organisms and cells containing nucleic acid sequences encoding polypeptides produced by this modular method.

[0182] "Binding" refers to sequence-specific non-covalent interactions between macromolecules (e.g., between a protein and a nucleic acid). Not all components of the binding interaction need to be sequence-specific (e.g., contacts with phosphate residues in the DNA backbone), as long as the interaction as a whole is sequence-specific.

[0183] The term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. When used to define compositions and methods, "consisting essentially of" shall mean excluding other elements that have any significant meaning for the intended purpose. Thus, a composition consisting essentially of the elements defined herein will not exclude trace contaminants or inert carriers. "Consisting of" shall mean excluding other components in amounts greater than trace elements and substantial method steps. Aspects defined by each of these transitional terms are within the scope of the present disclosure.

[0184] 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 can include splicing of the mRNA in a eukaryotic cell.

[0185] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. The gene product can be the direct transcriptional product of the 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 the mRNA. Gene products also include RNAs modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation, and glycosylation.

[0186] "Modulation" or "regulation" of gene expression refers to a change in gene activity. Regulation of expression can include, but is not limited to, gene activation and gene inhibition.

[0187] The term "operatively linked" or its equivalent (e.g., "linked operatively") means that two or more molecules are positioned relative to each other such that they are able to interact to affect the function attributable to one or both of the molecules or a combination thereof.

[0188] Non-covalently linked components and methods of preparing and using non-covalently linked components are disclosed. As described herein, the various components can take a variety of different forms. For example, non-covalently linked (i.e., operatively linked) proteins can be used to allow transient interactions, thereby avoiding one or more problems in the art. The ability of non-covalently linked components (such as proteins) to associate and dissociate achieves functional association only or primarily when such association is required for the desired activity. The linkage can have a duration sufficient to allow the desired effect.

[0189] 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 the depicted single strand. The 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.

[0190] The nucleic acids of the present disclosure can be single-stranded or double-stranded. Even when most of the molecule is single-stranded, the nucleic acids of the present disclosure can contain double-stranded sequences. Even when most of the molecule is double-stranded, the nucleic acids of the present disclosure can contain single-stranded sequences. The nucleic acids of the present disclosure can include genomic DNA, cDNA, RNA, or hybrids thereof. The nucleic acids of the present disclosure can contain a combination of deoxyribonucleotides and ribonucleotides. The nucleic acids of the present disclosure can contain a combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. The nucleic acids of the present disclosure can be synthesized to contain non-natural amino acid modifications. The nucleic acids of the present disclosure can be obtained by chemical synthesis methods or by recombinant methods.

[0191] The nucleic acids of the present disclosure or their complete sequences or any part thereof can be non-naturally occurring. The nucleic acids of the present disclosure can contain one or more non-naturally occurring mutations, substitutions, deletions, or insertions such that the entire nucleic acid sequence is non-naturally occurring. The nucleic acids of the present disclosure can contain one or more duplicated, inverted, or repeated sequences such that the resulting sequence is not naturally occurring and the entire nucleic acid sequence is non-naturally occurring. The nucleic acids of the present disclosure can contain non-naturally occurring modified, artificial, or synthetic nucleotides such that the entire nucleic acid sequence is non-naturally occurring.

[0192] Given the redundancy in the genetic code, multiple nucleotide sequences can encode any particular protein. All such nucleotide sequences are considered herein.

[0193] As used throughout this disclosure, the term "operably linked" refers to the expression of a gene under the control of a promoter that is spatially linked thereto. The promoter can be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene can be substantially the same as the distance between the promoter and the gene that the promoter controls in the gene from which it is derived. Variations in the distance between the promoter and the gene can be accommodated without loss of promoter function.

[0194] As used throughout this disclosure, the term "promoter" refers to a synthetic or naturally-derived molecule that is capable of conferring, activating, or enhancing the expression of a nucleic acid in a cell. A promoter can contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. A promoter can also contain distal enhancer or repressor elements that can be located up to several thousand base pairs from the transcription start site. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter can regulate the expression of a genetic component constitutively or differentially with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage at which expression occurs or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include the bacteriophage T7 promoter, the bacteriophage T3 promoter, the SP6 promoter, the lac operon-promoter, the tac promoter, the SV40 late promoter, the SV40 early promoter, the RSV-LTR promoter, the CMVIE promoter, the EF-1α promoter, the CAG promoter, the SV40 early promoter or the SV40 late promoter, and the CMV IE promoter.

[0195] As used throughout this disclosure, the term "substantially complementary" means that a first sequence 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 the two sequences hybridize under stringent hybridization conditions.

[0196] As used throughout this disclosure, the term "substantially identical" means that the first sequence and the second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical in 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, in the case of nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence.

[0197] As used throughout this disclosure, the term "variant", when used to describe a nucleic acid, means: (i) a portion or fragment of the recited nucleotide sequence; (ii) the complement of the recited nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to the recited nucleic acid or its complement; or (iv) a nucleic acid that hybridizes to the recited nucleic acid, its complement, or a sequence that is substantially identical thereto under stringent conditions.

[0198] As used throughout this disclosure, the term "vector" means a nucleic acid sequence that contains an origin of replication. A vector can be a viral vector, a phage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be an extrachromosomal self-replicating vector and is preferably a DNA plasmid. A vector can contain a combination of amino acids and a DNA sequence, an RNA sequence, or both a DNA and an RNA sequence.

[0199] As used throughout this disclosure, the term "variant", when used to describe a peptide or polypeptide, means a peptide or polypeptide that differs in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. A variant can also mean a protein having an amino acid sequence that is substantially identical to the recited protein having an amino acid sequence and that retains at least one biological activity.

[0200] Conservative substitutions of amino acids, i.e., substituting an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions) are generally considered in the art to typically involve minor changes. As understood in the art, these minor changes can be identified in part by considering the hydrophilicity index of the amino acids. Kyte et al., Journal of Molecular Biology (J. Mol. Biol.) 157:105-132 (1982). The hydrophilicity index of an amino acid is based on considerations of its hydrophobicity and charge. Amino acids with similar hydrophilicity indices can be substituted and still retain protein function. In one aspect, amino acids with a hydrophilicity index of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substituents that will result in a protein retaining its biological function. Consideration of amino acid hydrophilicity in the context of a peptide allows calculation of the maximal local average hydrophilicity of the peptide, which is a useful measure that has been reported to be well correlated with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, which is incorporated herein by reference in its entirety.

[0201] 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 whose hydrophilicity values are within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are affected by the specific side chain of the amino acid. Consistent with this observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and in particular the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0202] As used herein, "conservative" amino acid substitutions can be defined as shown in Table A, B, or C below. In some aspects, fusion polypeptides and / or nucleic acids encoding such fusion polypeptides include conservative substitutions introduced by modifying the polynucleotide encoding the polypeptides of the present disclosure. Amino acids can be classified according to physical properties and their contribution to secondary and tertiary protein structure. A conservative substitution is one amino acid replacing another with similar properties. Exemplary conservative substitutions are listed in Table A.

[0203] Table A - Conservative Substitutions I

[0204]

[0205] Alternatively, conservative amino acids can be grouped as described by Lehninger (Biochemistry, 2nd ed.; Worth Publishers, Inc. NY, N.Y. (1975), pp. 71-77), as listed in Table B.

[0206] Table B - Conservative Substitutions II

[0207]

[0208] Alternatively, exemplary conservative substitutions are listed in Table C.

[0209] Table C - Conservative Substitutions III

[0210] Original residue Exemplary substitutions Ala (A) ValLeuIleMet Arg (R) LysHis Asn (N) Gln Asp (D) Glu Cys (C) SerThr Gln (Q) Asn Glu (E) Asp Gly (G) AlaValLeuPro His (H) LysArg Ile (I) LeuValMetAlaPhe Leu (L) IleValMetAlaPhe Lys (K) ArgHis Met (M) LeuIleValAla Phe (F) TrpTyrIle Pro (P) GlyAlaValLeuIle Ser (S) Thr Thr (T) Ser Trp (W) TyrPheIle Tyr (Y) TrpPheThrSer Val (V) IleLeuMetAla

[0211] It should be understood that the polypeptides of the present disclosure are intended to include polypeptides with one or more insertions, deletions, or substitutions of amino acid residues or any combination thereof, as well as polypeptides modified in addition to insertions, deletions, or substitutions of amino acid residues. The polypeptides or nucleic acids of the present disclosure may contain one or more conservative substitutions.

[0212] As used throughout the present disclosure, the previously mentioned amino acid substitutions of "more than one" refer 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 recited amino acid substitutions. The term "more than one" may refer to 2, 3, 4, or 5 of the recited amino acid substitutions.

[0213] The polypeptides and proteins of the present disclosure or their complete sequences or any part thereof may be non-naturally occurring. The polypeptides and proteins of the present disclosure may contain one or more non-naturally occurring mutations, substitutions, deletions, or insertions such that the entire amino acid sequence is non-naturally occurring. The polypeptides and proteins of the present disclosure may contain one or more duplicated, inverted, or repeated sequences, the resulting sequences of which are not naturally occurring, such that the entire amino acid sequence is non-naturally occurring. The polypeptides and proteins of the present disclosure may contain non-naturally occurring modified, artificial, or synthetic amino acids such that the entire amino acid sequence is non-naturally occurring.

[0214] As used throughout this disclosure, "sequence identity" can be determined by using the stand-alone executable BLAST engine program, which is used to blast two sequences (bl2seq), which can be retrieved from the National Center for Biotechnology Information (NCBI) ftp site, using the default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; which is incorporated herein by reference in its entirety). When used in the context of two or more nucleic acid or polypeptide sequences, the terms "identical" or "identity" refer to the percentage of identical specific residues in the designated regions of each sequence. The percentage can be calculated by: optimally aligning the two sequences, comparing the two sequences in the designated region, determining the number of positions at which the same residues occur in the two sequences to yield the number of matching positions, dividing the number of matching positions by the total number of positions in the designated region, and multiplying the result by 100 to obtain the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the designated comparison region includes only a single sequence, the residues of the single sequence are included in the denominator but not in the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or by using computer sequence algorithms such as BLAST or BLAST 2.0.

[0215] 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 the host cell into which it is introduced.

[0216] 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 the host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid, such as a DNA sequence or a naturally occurring nucleic acid sequence located at a non-naturally occurring genomic location.

[0217] This disclosure provides methods for introducing a polynucleotide construct comprising a DNA sequence into a host cell. "Introducing" is intended to present the polynucleotide construct to the cell in such a manner that the construct enters the interior of the host cell. The methods of this disclosure do not depend on the particular method used to introduce the polynucleotide construct into the host cell, only on the polynucleotide construct entering the interior of one cell of the host. Methods for introducing polynucleotide constructs into bacteria, plants, fungi, and animals are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0218] Examples

[0219] Example 1 - Preparation and storage of allogeneic TCR T cells co-expressing a chimeric CD8α co-receptor

[0220] Figure 1A A schematic representation of an exemplary chimeric CD8α homodimer co-receptor of the present disclosure is shown. The chimeric CD8α homodimer co-receptor comprises a truncated CD8α co-receptor, the truncated CD8α co-receptor comprising a CD8α co-receptor extracellular domain and a CD8α co-receptor transmembrane domain; and a CD4 intracellular domain, the CD4 intracellular domain comprising a palmitoylation domain and a high-affinity Lck binding domain. Figure 2 A schematic diagram depicting the domain orientation of a DNA piggyBac nanoparticle transposon containing sequences encoding an exemplary chimeric CD8α co-receptor of the present disclosure, as well as the TCRα and TCRβ chains of the TCR is shown. The chimeric CD8α co-receptor is operably linked to a PGK promoter and its expression is controlled by the PGK promoter. The iCAS9 safety switch, TCRβ chain, TCRα chain, and DHFR selectable marker are operably linked to an EF1a promoter and their expression is controlled by the EF1a promoter. The transposon allows for co-expression of the TCR and the chimeric CD8α co-receptor from a single construct.

[0221] Human pan T cells were collected from healthy human donors by apheresis. Allogeneic TCR-T cells were generated from T cells collected using an automated positive immunomagnetic enrichment protocol targeting CD4 and CD8 T cells (CliniMACS CD4 microbeads and CliniMACS CD8 microbeads, respectively) using a CliniMACS Prodigy instrument (Miltenyi Biotec). The 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%) before being frozen and stored in the liquid nitrogen phase.

[0222] The cryopreserved enriched CD4 and CD8 T cells were thawed in T cell expansion medium (Immuno Cult TM -XF T cell expansion medium, StemCell) and recovered overnight at 37 °C in the presence of 5% CO2. Prior to electroporation, the T cells were resuspended in supplemented P3 primary cell Nucleofector solution. By electroporation Figure 2The transposon was introduced into the enriched T cells. 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 nanoparticle transposon plasmid encoding a chimeric CD8α co-receptor, TCRα and β chains of a TCR specific for the 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 container. T cell suspension was added and the container was electroporated using a 4D-Nucleofector system according to the manufacturer's instructions.

[0223] After electroporation, the cells were recovered in 20 ml of T cell expansion medium (ImmunoCult TM -XF T cell expansion medium, StemCell Technologies) for a total culture in multiple G-Rex 6M containers (Wilson Wolf Corporation). The cells were incubated overnight. After electroporation and an extended recovery quiescence period, the cells were activated via the TCR in the absence of exogenous cytokines. After activation, fresh medium supplemented with methotrexate (MTX) but lacking exogenous cytokines was added to select for DHFR-positive T cells. The cell culture was maintained throughout the culture period and routinely supplemented with fresh medium to ensure sufficient cell expansion. Cell harvest was performed after the cultured cells exited the exponential growth phase and entered the stationary phase.

[0224] The harvested cells were stained with biotinylated TCRα / β antibody (Miltenyi) and incubated with anti-biotin GMP microbeads (Miltenyi) for 30 minutes. Then, the labeled cells were loaded onto an LS column (Miltenyi) and subjected to immunomagnetic depletion. Since the transgenic TCR was engineered to mask the epitope recognized by the TCRα / β antibody, cells expressing the endogenous TCR were labeled and depleted. The cells depleted of endogenous TCR were cryopreserved in a mixture of HBSS (40%), HSA (50%), and DMSO (10%) before storage in the liquid nitrogen vapor phase.

[0225] Un-transposed pan T cells lacking transposon DNA during electroporation and without MTX selection were used as controls.

[0226] Example 2 - Co-expression of TCR and chimeric CD8α coreceptor enhances CD4+ and CD8+ MHC1-mediated cytotoxicity

[0227] The 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 Biosystem) such that viable nucleated cells fluoresced green and non-viable nucleated cells fluoresced red. Live cells were counted by using a LunaFL dual-fluorescence cell counter (Logos Biosystem).

[0228] A37-GFP melanoma cells (HLA-A2+ / NY-ESO-1+ / GFP+) were used as target cells in the T cell cytotoxicity assay. The 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% CO2. After a 72-hour culture period, the confluent cultures were counted, pelleted, and resuspended in DMEM-10 medium at a concentration of 5x10 4 cells / ml. 100 μl aliquots of 5x10 3 cells of target cells were plated in each well of a Corning poly-D-lysine-treated flat-bottom plate (Corning). Each plate was incubated at 37 °C and 5% CO2 overnight.

[0229] An antibody cocktail consisting of the following was used to stain the allogeneic TCR-T cells: APC-Vb-13.1 antibody (Miltenyi Biotec), BV785-hCD4 antibody (Biolegend), APC-Cy7-hCD8 antibody (Biolegend), BV421-hTCR (Biolegend), PE-A2:NYESODextramer (Immudex), and Live / Dead Fixable Aqua Dead Cell Stain Reagent (Thermo Fisher). On a BD LSRFortessa TMAnalyze the Dextramer+ frequency of stained cells in a cell analyzer or perform sorting using a Sony SH800S sorter according to the manufacturer's instructions to isolate live / CD4+ / Vb-13.1+ and live / CD8+ / Vb-13.1+ populations. Figure 3A A series of flow cytometry contour plots depicting the sorting of stained cells are shown.

[0230] Pellet the sorted TCR-T cells and resuspend them at a concentration of 2.5x10 5 cells / mL for live / CD4+ / Vb-13.1+ cells and 1x10 5 cells / mL for live / CD8+ / Vb-13.1+ cells in RPMI-10. Aliquot 100 μl of the sorted TCR-T cells in triplicate and plate them into a Corning poly-D-lysine-treated 96-well plate containing cultured A375 cells. Incubate the TCR+CD4+ T cells with A375 target cells at a ratio of 5:1. Incubate the TCR+CD8+ T cells with A375 target cells at a ratio of 2:1. Incubate each plate at 37 °C and 5% CO2 for 96 ± 2 hours. Analyze TCR-T-mediated cytotoxicity by tracking the growth of GFP+ A375 target cells using a Sartorius Incucyte system. Measure the percentage of tumor growth for 100 hours. Export all data to Microsoft Excel and plot using GraphPad Prism software. Figure 3B Cytotoxicity of T cells co-expressing TCR and chimeric CD8α co-receptor, TCR homodimer, or TCR heterodimer is shown compared to GFP and mock controls. As Figure 3B shown, co-expression of the chimeric CD8α co-receptor (“TCR+chiCD8-homo-di”) enhances TCR-mediated cytotoxicity (reduced tumor growth) compared to the wild-type CD8α homodimer co-receptor (“TCR+CD8-homo-di”), heterodimeric CD8α co-receptor (“TCR+CD8-h-di”), or control (“TCR+GFP”; “mock”) in both CD8+ and CD4+ T cells, with a greater effect observed for CD4+ T cells.

Claims

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

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

1.

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

3.

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

4.

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

2.

6. The chimeric CD8α coreceptor according to claim 1, wherein said 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 according to any one of claims 1 to 6.

8. The polynucleotide according to claim 7, wherein said polynucleotide is an mRNA molecule.

9. The polynucleotide according to claim 7, wherein said polynucleotide is a DNA molecule.

10. The polynucleotide according to claim 9, which further comprises a promoter sequence, said promoter sequence being operably linked to said DNA molecule to produce at least one mRNA molecule encoding said chimeric CD8α coreceptor in a cell.

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

12. The cell according to claim 11, wherein said cell expresses said chimeric CD8α coreceptor.

13. The cell according to claim 11, wherein said cell further expresses a T cell receptor (TCR).

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

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

16. A pharmaceutical composition, comprising the cell according to any one of claims 11 to 15 and at least one pharmaceutically acceptable carrier or drug.

17. A method of stimulating the cytotoxicity of a T cell population mediated by a T cell receptor (TCR) in a subject in need thereof, said method comprising: a) introducing into a T cell population a polynucleotide encoding a TCR and a polynucleotide encoding the chimeric CD8α coreceptor 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 co-receptor on the cell membrane of the T cells, and the chimeric CD8α co-receptor 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 co-receptor has a higher level of cytotoxicity compared to a T cell population expressing only the TCR.

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

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

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