WO2023212507A1 - Compositions et méthodes pour l'immunothérapie cellulaire - Google Patents

Compositions et méthodes pour l'immunothérapie cellulaire Download PDF

Info

Publication number
WO2023212507A1
WO2023212507A1 PCT/US2023/066048 US2023066048W WO2023212507A1 WO 2023212507 A1 WO2023212507 A1 WO 2023212507A1 US 2023066048 W US2023066048 W US 2023066048W WO 2023212507 A1 WO2023212507 A1 WO 2023212507A1
Authority
WO
WIPO (PCT)
Prior art keywords
construct
motifs
cell
polypeptide
carcinoma
Prior art date
Application number
PCT/US2023/066048
Other languages
English (en)
Inventor
Shihong ZHANG
Aude G. CHAPUIS
Thomas M. Schmitt
Original Assignee
Fred Hutchinson Cancer Center
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fred Hutchinson Cancer Center filed Critical Fred Hutchinson Cancer Center
Publication of WO2023212507A1 publication Critical patent/WO2023212507A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70517—CD8
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • compositions that target a single antigen comprise both CD8 + T cells (which naturally express MHC-I-restricted TCR) and CD4 + T cells (which naturally express MHC-II-restricted TCR) (see, e.g., Sommermeyer et al., Leukemia 30(2): 1888 (2016)). Compositions and methods are needed for new or improved adoptive cell therapies.
  • FIG 1 shows schematic diagrams of certain co-receptor fusion protein constructs according to the present disclosure.
  • Constructs 1-5 a portion of a human CD28 intracellular domain was fused to a truncated human CD8 co-receptor P chain (Constructs 1 (corresponding to Construct G in Table 1), 2 (corresponding to Construct A in Table 1), 3 (corresponding to Construct B in Table 1)), to a truncated human CD8 co-receptor a chain (Construct 5, corresponding to Construct D in Table 1), or to each of a truncated human CD8 co-receptor a chain and a truncated human CD8 co-receptor P chain (Construct 4, corresponding to Construct C in Table 1).
  • Constructs 6-8 a signaling portion of: a human 4-1BB intracellular domain (Construct 6, corresponding to Construct H in Table 1); a human ICOS intracellular domain (Construct 7, corresponding to Construct I in Table 1); or a human 0X40 intracellular domain (Construct 8, corresponding to Construct J in Table 1) was fused to a truncated human CD8 co-receptor P chain.
  • Each construct shown in Figure 1 includes the depicted co-receptor fusion construct(s) with a cognate CD8a chain and/or CD8P chain.
  • Each co-receptor fusion construct includes the extracellular and transmembrane domains of the CD8P or CD8a chain, respectively.
  • Constructs 1, 2, 6, 7, and 8 further comprise a 6-amino acid sequence (HLCCRR; SEQ ID NO.: 10) from the CD8P intracellular domain, located between the CD8P transmembrane domain and the CD28 intracellular domain.
  • Construct 1 includes a dileucine motif ("LL") that is native to a human CD28 intracellular domain.
  • each leucine of the dileucine motif was replaced by a glycine amino acid.
  • Other constructs included constructs in which a native human CD28 diproline motif was altered and each proline in the motif was replaced with an alanine.
  • Figure 2 shows CD8 expression by human primary CD4+ T cells transduced with a MHC-I-restricted TCR and one of the indicated CD8 co-receptor constructs. Cells were sorted on Day 7 following transduction with construct-containing lentivirus and expanded. Left panel: expression of wild-type CD8aP in TCR-transduced cells. Middle panel: expression of CD8a/CD8P-CD28 fusion construct ("Construct 1" in Figure 1). Right panel: expression of CD8a/CD8P-4-lBB fusion construct ("Construct 6" in Figure 1).
  • Figure 3 shows proliferation of human primary CD4+ T cells that were transduced with MHC-I-restricted TCR alone; TCR + Construct 1; or TCR + Construct 6, sorting on Day 7, and stimulation with antigen-expressing MEL275 cells at the indicated effectortarget (E:T) ratios on Day 9 following a Rapid Expansion Protocol (REP).
  • E:T effectortarget
  • FIGS 4A and 4B show production of cytokines by CD4+ T cells transduced to express a MHC-I-restricted (MAGE-Al-278-specific) TCR, either alone (B) or with wild-type or chimeric CD8 co-receptor molecules (A, B), as indicated.
  • A Data from flow cytometry experiments measuring interferon-gamma (shown as "ifny") production by transduced T cells in the absence (bottom row) or presence (top row) of peptide antigen.
  • B Expression of IFN-y and TNFa by cells transduced with (from left to right) TCR alone; TCR + wild-type CD8 co-receptor; TCR + Construct 1 from Figure 1; or TCR + Construct 6 from Figure 1.
  • Figure 5 shows specific killing of tumor cells by T cells, including T cells that express (i) a TCR that specifically binds to an antigen:MHC complex on the tumor cells and (ii) a fusion protein of the present disclosure comprising (a) an extracellular component comprising an extracellular domain (also known as an ectodomain) from CD3( ⁇ , (b) a transmembrane component comprising a transmembrane domain from CD3( ⁇ , and (c) intracellular component comprising (c)(i) a costimulatory domain from CD28 or 4- IBB, and, carboxy -terminal to (c)(i), (c)(ii) an intracellular signaling domain from CD3( ⁇ .
  • An IncuCyte® assay was used to quantitate killing; the Red Object Area on the y-axis represents the presence of tumor cells.
  • Figure 6 shows specific killing of tumor cells (PANC-1) by T cells (2: 1 E:T ratio), including T cells that express (i) a TCR that specifically binds to an antigen:MHC complex on the tumor cells and (ii) a fusion protein comprising (a) an extracellular region from CD3( ⁇ , (b) a transmembrane region from CD3( ⁇ , (c) a costimulatory domain from CD28 or 4-1BB, and (d) an intracellular signaling domain from CD3( ⁇ .
  • An IncuCyte® assay was used to quantitate killing; the Red Object Area on the y-axis represents the presence of tumor cells.
  • Figure 7 shows (top left) IFN-y production, (top right) IL-2 production, and (bottom) proliferation of T cells expressing either (i) a TCR and a wild-type CD8 coreceptor or (ii) a TCR and a CD8-CD28 fusion polypeptide of the present disclosure.
  • Figure 8 shows schematic diagrams of certain fusion protein constructs according to the present disclosure.
  • the parallel horizontal lines across the image represent a cell membrane, with the extracellular portion of the protein shown above the upper horizontal line, and the intracellular portion of the protein shown below the lower horizontal line.
  • Source proteins for the fusion protein components are indicated.
  • the proteins with an extracellular portion marked "CD8a/p” and the construct with an extracellular portion marked “NKG2D” were investigated for function without a cognate co-receptor.
  • Figures 9A-12C provide details of certain fusion protein constructs according to the present disclosure.
  • a group of constructs sharing a structural theme e.g., groups are CD8a/p chimeras, single-stalk fusions, NKG2D immunomodulatory fusion protein, expression constructs encoding a CD8a and a chemokine receptor, CD8/p chimeric mutant, Fas immunomodulatory fusion proteins, PD-1 CD28 immunomodulatory fusion protein (used as a positive control), and wild-type CD8aP control
  • groups are CD8a/p chimeras, single-stalk fusions, NKG2D immunomodulatory fusion protein, expression constructs encoding a CD8a and a chemokine receptor, CD8/p chimeric mutant, Fas immunomodulatory fusion proteins, PD-1 CD28 immunomodulatory fusion protein (used as a positive control), and wild-type CD8aP control
  • the two encoded proteins can function as a pair.
  • the encoded protein can function as a single protein, without associating with a cognate co-protein.
  • Figures 9B-9C, 10B-10C, 1 IB-11C, and 12B-12C further details of the construct designs are provided.
  • Figures 13-16B relate to experiments in which cells were transduced to express a fusion protein-encoding construct along with a TCR.
  • controls included cells transduced with wild-type CD8aP, cells transduced with TCR alone, and cells transduced with an irrelevant TCR (i.e., not specific for the peptide antigen used in the experiment) with wild-type CD8ap.
  • the tested fusion protein constructs (either two-polypeptide or one-polypeptide; for two-polypeptide constructs, the two polypeptides were separated by a P2A self-cleaving peptide sequence ("/P2A/" below) included (see also Figure 13): "E” (full-length CD8a chain /P2A/ truncated CD8P chain (including six CD8P intracellular amino acids H-L-C-C-R-R (SEQ ID NO.: 10)) fused to a CD28 intracellular region comprising a LL-to-GG mutation and partial signaling mutation, discussed herein); "G” (full-length CD8a chain /P2A/ truncated CD8P chain (including six CD8P intracellular amino acids H-L-C-C-R-R (SEQ ID NO.: 10)) fused to a wild-type CD28 intracellular domain); "H” (full-length CD8a chain /P2A/
  • FIGS 14A and 14B show TCR signal activation in Jurkat reporter cells transduced with (i) a TCR and a fusion construct or (ii) a control construct (TCR alone, irrelevant TCR with wild-type CD8aP, or wild-type CD8aP).
  • Jurkat cells were engineered to knock-out endogenous MHC class I and endogenous TCRa and TCRP chains, with a neogreen reporter knocked-in downstream of Nur77. Nur77 gene expression is rapidly upregulated by TCR signaling.
  • the percent reporter-positive of CD3-positive cells (% reporter + of CD3) on the y-axis represents the percentage of transduced Jurkat cells that were reporter-positive after co-culture with antigen peptide- loaded T2 cells (5: 1 E:T ratio).
  • Figure 14A transduced CD8+ Jurkat reporter cells.
  • Figure 14B transduced CD4+ Jurkat reporter cells.
  • FIGS 15A-15D show production of cytokines by CD8+ or CD4+ T cells transduced to express a MHC-I-restricted TCR alone, wild-type CD8aP alone, or the TCR and a fusion protein construct of the present disclosure.
  • Transduced T cells were stimulated with the tumor cell lines ME275 and H1299. After stimulation, the T cells were fixed, permeabilized, and stained for intracellular IFNy and TNFa.
  • Figure 15 A transduced CD8+ T cells following stimulation with ME275 cells.
  • Figure 15B transduced CD8+ T cells following stimulation with H1299 cells.
  • Figure 15C transduced CD4+ T cells following stimulation with ME275 cells.
  • Figure 15D transduced CD4+ T cells following stimulation with H1299 cells.
  • the y-axis in each graph represents the percentage of T cells positive for intracellular JFN-y or TNFa, as indicated.
  • Figures 16A and 16B show specific killing of tumor cells by CD8+ or CD4+ T cells transduced to express MHC class I-restricted TCR alone, wild-type CD8aP alone, or the TCR and the indicated fusion construct.
  • An IncuCyte® assay was used to quantitate killing; the Red Object Area on the y-axis represents the presence of tumor cells (mCherry-positive ME275 tumor cells).
  • Figure 16A presence of tumor cells following co-culture with transduced CD8+ T cells.
  • Figure 16B presence of tumor cells following co-culture with transduced CD4+ T cells.
  • FIGS 17A-17C show transduction of donor T cells with a WT1 -specific TCR, a CD3( ⁇ fusion protein construct, or both.
  • WT1 -specific TCR transduction was evaluated using WT1 tetramers, as shown on the y-axis of each panel.
  • the fusions were designed to co-express GFP.
  • Co-receptor fusion transduction was monitored by GFP, as shown on the x-axis of each panel.
  • the fusion constructs comprise (a) an extracellular component comprising (a) an extracellular component comprising an extracellular domain from CD3( ⁇ , (b) a transmembrane domain from CD3( ⁇ , and (c) intracellular component comprising (i) an intracellular domain from CD28 (28) or 4- IBB (BB), and (ii) an intracellular signaling domain from CD3( ⁇ .
  • Figure 17A T cells from donors 1 and 2, as indicated, were transduced with WTl-specific TCR only.
  • Figure 17B T cells from donor 1 were (top row) transduced with the indicated fusion construct only, or (bottom row) co-transduced with fusion construct and WTl-specific TCR.
  • Figure 17C T cells from donor 2 were transduced with co-receptor fusion constructs only, as indicated, (top row) or co-transduced with co-receptor fusion constructs and WTl-specific TCR (bottom row).
  • Figure 18 shows (A) scheme showing how TCR- and CD8-transduced CD4+ T cells help TCR transduced CD8+ T cells with tumor cell killing.
  • CD4+ T cells from healthy donors were transduced with an HLA-A2 restricted MAGE-A1-278 specific TCR (clone: MOEN) alone or MOEN TCR plus CD8ap.
  • CD4+ T cells transduced with the TCR and CD8aP showed increased tetramer binding capability and functionality, as indicated by the tetramer MFI and cytokine production upon peptide stimulation.
  • Figure 19 shows (A) scheme of CD8a and P chain modification.
  • the longer of the two intracellular portions on CD8a in the middle construct; on CD8P in the right-hand construct
  • the shapes in the longer intracellular portion represent sequence motifs that can be targeted for modification.
  • Figure 20 shows data from a serial killing assay using CD4+ T cells from healthy donors transduced with MAGE-A1 TCR along with different forms of coreceptors as indicated. Transduced cells were sorted, expanded and used for the serial killing assay. MAGE-A1+ HLA-A2+ tumor cell line ME275 were added to T cell culture every 48 hours at 10: 1 E:T ratio. Tumor growth was monitored by the mCherry intensity by Incucyte. Experiment was performed in triplicates, error bars show SEM.
  • Figure 21 shows (A) scheme of an in vitro competitive proliferation assay to identify modified CD8 co-receptors that help T cells proliferate better. (B) Upon stimulation of MAGE-A1+ HLA-A2+ tumor cell line ME275 for 7 days, CD8aP-CD28 coreceptor transduced TCR-T cells proliferated by 3 times in comparison to the other constructs transduced TCR-T cells. Experiment was performed in duplicates.
  • Figure 22 shows schematic diagrams of certain constructs of the present disclosure. Shapes indicate sequence motifs as shown in the key. Open ovals indicate CD28 intracellular motifs for mutation to potentially improve function.
  • FIG 23 shows that CD4+ T cells expressing a TCR and a CD8 co-receptor construct of the present disclosure produce cytokines in response to the TCR peptide (cognate) antigen.
  • engineered (“tether”) CD8 coreceptor- and TCR-transduced and expanded CD4+ T cells were tested for cytokine production capability upon the stimulation of MAGE-A1-278 peptide at various concentrations.
  • CD8aP outperformed CD8aa constructs
  • CD8-CD28 outperformed CD8ap.
  • Figure 24 shows antitumor activity of CD4+ T cells from healthy donors transduced with MAGE-A1 TCR along with one of a number of different forms of co- receptors as indicated. Transduced cells were sorted, expanded and used for the serial killing assay. MAGE-A1+ HLA-A2+ tumor cell line ME275 were added to T cell culture every 48 hours at 10: 1 E:T ratio. Tumor growth was monitored by the mCherry intensity by Incucyte. Experiment was performed in triplicates, error bars show SEM.
  • FIG 25 shows counts of T cells (transduced with TCR and a co-receptor as indicated) by flow cytometry post the serial killing assay for which data is shown in Figure 24.
  • TCR-T cells transduced with wild type CD8aP showed superior proliferation to CD8aa and CD8a/CD4 constructs, while the CD8aP-CD28 constructs further improved proliferation of the TCR-T cells.
  • TCR-T cells co-expressing CD8aP-CD28v2 showed the highest level of proliferation.
  • Figures 26A and 26B show that mutation of CD28 motifs in CD8-CD28 coreceptor fusions can enhance functionality of TCR-T cells.
  • Sixteen different CD8aP- CD28 co-receptors were generated by introducing mutations into CD28 functional motifs (Constructs “BB”-“NN”; see Figures 27-39, Table 3, and elsewhere herein). Constructs A, G, and E were also tested.
  • Transduced and expanded CD4+ T cells were restimulated with ME275 for 5 times in the span of 15 days.
  • TCR-T cells transduced with mutated di-leucine and/or PI3K binding motifs showed enhanced tumor control.
  • the first-listed construct in each row of the figure key corresponds to a dot on the lefthand side of the dot plot in the graph
  • the second-listed construct in each row of the figure key corresponds to a dot on the right-hand side of the dot plot in the graph.
  • Connected dots represent compared constructs that only differ by the wild-type di-leucine or mutant diglycine motif (26A) or the wild-type or mutated PI3K binding site, all other indicated motifs being the same as between the two contracts. Experiment was performed in triplicates.
  • Figures 27-39 show amino acid sequences of two-polypeptide constructs of the present disclosure (“1 protein”; “2 protein”). These constructs are described further in Example 7. The portions of the sequences corresponding to the extracellular, transmembrane, and intracellular domains of each polypeptide are indicated. It will be understood that a signal peptide (shown in these Figures for “2 protein”) may be replaced by a different signal peptide, and/or may be removed in part or in full prior to expression of the two-polypeptide constructs at the cell membrane of a host cell. Accordingly, a signal peptide is optional and, if present, a different signal peptide than the one shown in the Figures can be used.
  • Figure 27 shows amino acid sequences of proteins 1 and 2 of construct BB.
  • Figure 28 shows amino acid sequences of proteins 1 and 2 of construct CC.
  • Figure 29 shows amino acid sequences of proteins 1 and 2 of construct DD.
  • Figure 30 shows amino acid sequences of proteins 1 and 2 of construct EE.
  • Figure 31 shows amino acid sequences of proteins 1 and 2 of construct FF.
  • Figure 32 shows amino acid sequences of proteins 1 and 2 of construct GG.
  • Figure 33 shows amino acid sequences of proteins 1 and 2 of construct HH.
  • Figure 34 shows amino acid sequences of proteins 1 and 2 of construct II.
  • Figure 35 shows amino acid sequences of proteins 1 and 2 of construct JJ.
  • Figure 36 shows amino acid sequences of proteins 1 and 2 of construct KK.
  • Figure 37 shows amino acid sequences of proteins 1 and 2 of construct LL.
  • Figure 38 shows amino acid sequences of proteins 1 and 2 of construct MM.
  • Figure 39 shows amino acid sequences of proteins 1 and 2 of construct
  • the present relates, in part, to polypeptides, such as fusion proteins and other engineered proteins, that are useful to confer to, or improve, a desired activity of a host immune cell, such as an immune cell that targets a diseased or pathogenic cell (e.g., a cancer cell).
  • a host immune cell such as an immune cell that targets a diseased or pathogenic cell (e.g., a cancer cell).
  • Certain embodiments of the polypeptides can, for example, translate an external stimulus (e.g., binding to a ligand) to a preferred (e.g., stimulatory) signal in a host cell, and/or to prevent, reduce, attenuate, modulate, or abrogate an undesired signal in the host cell.
  • a polypeptide comprises an extracellular domain from (e.g, human) CD8P, a transmembrane domain that is optionally from (e.g, human) CD8P, and an intracellular domain comprising a variant of SEQ ID NO: 1
  • the variant can comprise one or more mutation (e.g., one or more substitution mutation), which is optionally a function-reducing or function-abrogating mutation, in: a dileucine motif; a PI3K binding site (YMNM in SEQ ID NO.: 19); an ITK and GADS binding site (PRRP in SEQ ID NO.: 19); a LCK binding site (PYAP in SEQ ID NO.: 19); or any combination thereof.
  • mutation e.g., one or more substitution mutation
  • a function-reducing or function-abrogating mutation in: a dileucine motif; a PI3K binding site (YMNM in SEQ ID NO.: 19); an ITK and GADS binding site (PRRP in SEQ ID NO.: 19); a LCK binding site (PYAP in SEQ ID NO.: 19); or any combination thereof.
  • a polypeptide comprises a mutated dileucine motif (e.g., to a diglycine) and further comprises a mutation in one or more of: a PI3K binding site; an ITK and GADS binding site; or a LCK binding site.
  • polypeptides of the present disclosure can be co-expressed in a host cell (e.g., T cell) with a binding protein that binds to a MHC-1 :antigen complex (e.g., a TCR or a CAR that comprises TCR or TCR-like variable domains), and the host cell has improved antitumor activity as compared to a host cell expressing the binding protein and a reference wild-type CD8 polypeptide.
  • substitutions mutations may be made in SEQ ID NO. : 19 to disrupt (e.g. reduce or abrogate) a function of e.g. a PI3K binding site, ITK and GADS binding site, and a LCK binding site.
  • a substitution mutation can comprise a non-conservative substitution.
  • a substitution mutation can comprise a replacement of an amino acid having a large side chain (e.g. tryptophan or phenylalanine) with an amino acid having a smaller side chain (e.g. alanine or serine), or vice versa. Any mutation or mutations sufficient to disrupt a function of one or more of the aforementioned sites in a CD28 intracellular sequence may be used.
  • a mutation in a PI3K binding site comprises or consists of a Y ⁇ A mutation, e.g., providing the sequence AMNM. In some embodiments, a mutation in a PI3K binding site comprises or consists of a Y ⁇ F mutation, e.g., providing the sequence FMNM. In some embodiments, a mutation in a ITK and GADS binding site comprises or consists of one or more P ⁇ A mutation, e.g., providing the sequence ARRA, or the sequence ARRP, or the sequence PRRA. In some embodiments, a mutation in a LCK binding site comprises or consists of one or more P ⁇ A mutation, e.g., providing the sequence AYYA, or the sequence PYAA, or the sequence AYAP.
  • a mutation reduces, attenuates, or abrogates binding by PI3K, ITK, GADS, or LCK to a disclosed polypeptide. Binding or lack thereof by PI3K, ITK, GADS, or LCK to a disclosed polypeptide can be assessed or inferred by, for example, a Western blot assay, an ELISA, or a dot blot.
  • the extracellular domain and optionally the transmembrane domain is from a CD8P Ml isoform. In some embodiments, the extracellular domain and optionally the transmembrane domain is from a CD8P M2 isoform.
  • the extracellular domain and optionally the transmembrane domain is from a CD8P M3 isoform. In some embodiments, the extracellular domain and optionally the transmembrane domain is from a CD8P M4 isoform. In some embodiments, the extracellular domain and optionally the transmembrane domain is from a CD8P M5 isoform.
  • the polypeptide can be expressed at the surface of a host cell (e.g., a T cell) and associate with a CD8a co-receptor polypeptide (e.g., comprising or consisting of the amino acid sequence set forth in SEQ ID NO.: 114).
  • the polypeptide can confer one or more improved function on the T cell as compared to a reference T cell expressing a (1) CD8P-CD28 fusion polypeptide comprising SEQ ID NO.: 19 (/. ⁇ ., comprising a wild-type CD28 intracellular sequence) and (2) a CD8a co-receptor polypeptide.
  • polynucleotides that encode any one or more of the polypeptides, and vectors that comprise a polynucleotide.
  • a polynucleotide encodes one or more (e.g., two) polypeptides that can function provide a stimulatory signal to a host cell that expresses the same, and/or that can function to prevent, modulate, attenuate, or abrogate a suppressive signal.
  • two or more polypeptides can associate to form a multimer (e.g., a dimer) and confer advantageous functions when expressed at the surface of a host cell.
  • a polypeptide monomer can function at the surface of a host cell.
  • a polynucleotide encodes a fusion polypeptide of the present disclosure and further encodes a CD8a co-receptor polypeptide (e.g, comprising SEQ ID NO. : 114 or SEQ ID NO : 1).
  • disclosed polypeptides may be advantageously employed to improve one or more cellular function, such as in the context of adoptive cell therapy.
  • a stimulatory signal is conferred or improved, and/or a suppressive signal is reduced, prevented, or abrogated, in a host T cell for adoptive therapy against a disease or disorder such as a cancer.
  • a CD4+ T cell can have improved function (e.g., cyotoxic and/or helper function and/or viability) against diseased cells.
  • a CD4+ T cell may further express, or be engineered to further express, an antigen-specific binding protein such as a T cell receptor, which can, in some embodiments, comprise an MHC I- restricted T cell receptor.
  • Such CD4+ T cells may be advantageously utilized in cell therapy, e.g., with or apart from CD8+ effector T cells that also target the diseased cells.
  • a polypeptide that comprises an extracellular domain from CD8P, a transmembrane domain that is optionally from CD8P or CD28, and an intracellular domain comprising a variant of SEQ ID NO.: 19, wherein the variation comprises: (i) a diglycine motif in place of a dileucine motif; (ii) a mutated and optionally non-functional PI3K binding site; (iii) a mutated and optionally nonfunctional ITK and GADS binding site; (iv) a mutated and optionally non-functional LCK binding site; or (v) any combination of (i)-(v).
  • the polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92%, or at least 95% sequence identity to SEQ ID NO.: 19.
  • the intracellular domain of the polypeptide comprises an amino acid sequence according to SEQ ID NO.:205 or 206.
  • the polypeptide comprises Motif 1, Motif 2, Motif 3, and/or Motif 4 of any one of Constructs BB-NN in Table 6 or Table 7, and/or comprising as Motif 2 the amino acid sequence FMNM.
  • polypeptide that comprises, consists essentially of, or consists of the amino acid sequence set forth in any one of SEQ ID NOs.: 192-204. Also provided is a polypeptide that comprises, consists essentially of, or conists of an extracellular domain, a transmembrane domain, and an intracellular domain, as shown under “2 protein size [aa]” in any one of Figures 27-39.
  • polynucleotides that encode a disclosed polypeptide.
  • the polynucleotide further encodes a CD8a co-receptor polypeptide, optionally comprising SEQ ID NO.: 114 or SEQ ID NO.: 1.
  • a polynucleotide can encode a first polypeptide comprising or consisting of any one of SEQ ID NOs.: 192-204 and a second polypeptide comprising or consisting of SEQ ID NO.: 114 or SEQ ID NO.: 1.
  • the polynucleotide comprises or encodes Construct BB, Construct CC, Construct DD, Construct EE, Construct FF, Construct GG, Construct HH, Construct II, Construct JJ, Construct KK, Construe LL, Construct MM, or Construct NN as provided herein.
  • a vector comprises a viral vector such as a lentiviral vector or a retroviral vector.
  • host cells that express a disclosed polypeptide.
  • the host cell further expresses a CD8a co-receptor polypeptide (preferably comprising a CD8a chain, such as a human CD8a chain), optionally comprising SEQ ID NO.: 114.
  • host cells that comprise a polynucleotide or vector as disclosed herein.
  • a host cell expreses a TCR or a CAR, wherein the TCR or CAR comprises a MHC Class I- restricted binding domain.
  • a host cell can be, for example, an immune system cell, such as a human immune system cell.
  • an immune system cell comprises a CD4 + T cell, a CD8 + T cell, a CD4'CD8- double negative T cell, a y6 T cell, a natural killer cell, a natural killer T cell, a dendritic cell, or any combination thereof.
  • a host cell comprises a T cell.
  • a host cell comprises a CD4+ T cell, a CD8+ T cell, or both.
  • compositions comprising a disclosed polypeptide, polynucleotide, vector, or host cell, or any combination thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.
  • Also provided is a method for treating a disease or disorder such as a cancer comprising administering to a subject having the disease or disorder an effective amount of a disclosed polypeptide, polynucleotide, vector, host cell, or composition, or any combination thereof. Also provided is a disclosed polypeptide, polynucleotide, vector, host cell, or composition, or any combination thereof, for use in the preparation of a medicament to treat a disease or disorder, optionally a cancer.
  • a host cell can be, for example, an immune system cell, such as a human immune system cell.
  • an immune system cell comprises a CD4 + T cell, a CD8 + T cell, a CD4'CD8- double negative T cell, a y6 T cell, a natural killer cell, a natural killer T cell, a dendritic cell, or any combination thereof.
  • a host cell comprises a T cell.
  • a host cell comprises a CD4+ T cell, a CD8+ T cell, or both.
  • Certain provided embodiments include a polynucleotide encoding (a) a first polypeptide, wherein the first polypeptide comprises (a)(i) an extracellular component comprising an extracellular domain from a CD8 P-chain (CD8P), or a functional portion or variant thereof that is capable of binding to a MHC Class I molecule, (a)(ii) a transmembrane domain from a CD8P, and (a)(iii) an intracellular component comprising (a)(iii)(l) a CD8P intracellular region amino acid sequence that comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 10 or SEQ ID NO.:9, and (a)(iii)(2) a costimulatory domain or a functional portion or variant thereof, wherein the costimulatory domain or a functional portion or variant thereof is from CD28 (optionally comprising a LL->GG mutation, a partial signaling mutation, and/or a full signaling mutation); and/or
  • the present disclosure provides fusion proteins that comprise: (i) an extracellular component comprising an extracellular domain (or an ectodomain) or functional portion thereof: (i)(l) from a CD8 co-receptor P-chain or a functional portion or variant thereof; (i)(2) from a CD8 co receptor a chain or a functional portion or variant thereof; (i)(3) that comprises an amino acid sequence from a CD8 co-receptor P-chain extracellular domain or a functional portion or variant thereof and an amino acid sequence from a CD8 co-receptor a chain, wherein the extracellular component of (i)(l)-(i)(3) is capable of binding to a MHC class I molecule; (ii) a transmembrane domain, optionally provided that the transmembrane domain is not a transmembrane domain from a CD8 co-receptor a-chain when the extracellular component comprises a full-length extracellular domain from the CD8 co-receptor a chain; and (i) an extra
  • the extracellular component comprises a CD8 co-receptor P-chain, or a functional portion or variant thereof.
  • the co-stimulatory domain comprises a co-stimulatory domain from one or more of CD28, and/or comprises an optionally functional variant of a co-stimulatory domain thereof.
  • fusion proteins that comprise: (i) an extracellular component comprising an extracellular domain (or an ectodomain) from a CD8 co- receptor P-chain or a functional portion or variant thereof, or from a CD8 co receptor a- chain or a functional portion or variant thereof, that is capable of binding to a MHC class I molecule; (ii) a transmembrane domain; and (iii) an intracellular component comprising a co stimulatory domain from a variant sequence of CD28 comprising or consisting of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 19 or 20, provided that: (1) no Tyr residue corresponding to position 12, 27, 30, or 39 of SEQ ID NO: 19 is substituted with Phe when the extracellular component comprises a full length extracellular domain from a CD8 co receptor a chain and the transmembrane domain comprises a transmembrane domain from the CD8 co receptor a chain; and/or (2) one or both of the leucine residues corresponding to
  • fusion proteins that comprise: (i) an extracellular component comprising an amino acid sequence from a CD8a extracellular domain, an amino acid sequence from a CD8P extracellular domain, and an optional amino acid sequence from a CD28 extracellular domain, a transmembrane domain from CD28 or from a CD8 co-receptor, and a CD28 intracellular domain (optionally comprising a LL->GG mutation); (ii) an extracellular component comprising an amino acid sequence from a CD8a extracellular domain and an amino acid sequence from a CD8P extracellular domain, a CD28 transmembrane domain, and a CD28 intracellular domain (optionally comprising a LL->GG mutation); or (iii) an extracellular component comprising an amino acid sequence from a CD8a extracellular domain and an amino acid sequence from a CD8P extracellular domain, a CD28 transmembrane domain, a CD28 intracellular domain (optionally comprising a LL- GG mutation), and a CD8a
  • polypeptides that encode, and/or host cells that express, a first polypeptide and a second polypeptide, wherein: (i) the first polypeptide comprises a CD8a extracellular domain, a transmembrane domain, and a CD8a intracellular domain, and the second polypeptide comprises a CD8P extracellular domain, a transmembrane domain, and an intracellular domain comprising (1) the amino acid sequence HLCCRR and (2) a CD28 intracellular domain comprising a LL-> GG mutation; (ii) the first polypeptide comprises a CD8a extracellular domain, a transmembrane domain, and a CD8a intracellular domain, and the second polypeptide comprises a CD8P extracellular domain, a transmembrane domain, and a CD28 intracellular domain (optionally comprising a LL-> GG mutation); (iii) the first polypeptide comprises a CD8a extracellular domain, a transmembrane domain, and a CD28
  • the first polypeptide comprises a CD8a extracellular domain, a transmembrane domain, and a CD28 intracellular domain (optionally comprising a LL-> GG mutation and/or a mutation that reduces CD28 immune signaling as compared to a wild-type CD28, CD28 intracellular domain, wherein, further optionally, the CD28 intracellular domain comprises the amino acid sequence RSKRSRGGHSDAMNMTARRAGPTRKHYQAYAAPRDFAAYRS), and the second polypeptide comprises a CD8P extracellular domain, a transmembrane domain, and a CD28 intracellular domain (optionally comprising a LL-> GG mutation and/or a mutation that reduces CD28 immune signaling as compared to a wild-type CD28, CD28 intracellular domain, wherein, further optionally, the CD28 intracellular domain comprises the amino acid sequence RSKRSRGGHSDAMNMTARRAGPTRKHYQAYAAPRDFAAYRS); or
  • the first polypeptide comprises a CD8a extracellular domain, a transmembrane domain, and a CD8a intracellular domain
  • the second polypeptide comprises a CD8P extracellular domain, a transmembrane domain, and an intracellular domain comprising (1) the amino acid sequence HLCCRR and (2) a CD28 intracellular domain
  • the first polypeptide comprises a CD8a extracellular domain, a transmembrane domain, and a CD28 intracellular domain (optionally comprising a LL- GG mutation and/or a mutation that reduces or abrogates CD28 immune signaling as compared to a wild-type CD28 intracellular domain
  • the CD28 intracellular domain comprises the amino acid sequence RSKRSRGGHSDAMNMTARRAGPTRKHFQAFAAPRDFAAFRS
  • the second polypeptide comprises a CD8P extracellular domain, a transmembrane domain, and a CD28 intracellular domain (optionally comprising a LL->GG
  • polypeptides of the present disclosure include those fusion proteins and constructs shown and/or described in Figures 9A-13 and 27-39, Tables 1-7, and the Table of Sequences. Accordingly, in any of the presently disclosed embodiments, a polypeptide can comprise an amino acid sequence (e.g., extracellular domain, transmembrane domain, intracellular domain, or any combination thereof) as provided in Tables 1-7 and the Table of Sequences herein and/or shown and/or described in any one or more of Figures 9A-13 and 27-39.
  • amino acid sequence e.g., extracellular domain, transmembrane domain, intracellular domain, or any combination thereof
  • presently disclosed polypeptides and expression constructs can be useful for improving and/or modulating activation and/or one or more therapeutically relevant function of an immune cell (e.g., a T cell) expressing an antigen-specific binding protein, such as, for example, a T cell receptor (TCR).
  • an immune cell e.g., a T cell
  • an antigen-specific binding protein such as, for example, a T cell receptor (TCR).
  • TCR T cell receptor
  • a polypeptide, expression construct, or first and second polypeptide is expressed by a modified host cell (e.g., immune cell, such as, for example, a T cell (e.g., a CD4+ T cell, a CD8+ T cell, or both), NK cell, or NK-T cell) that expresses a binding protein (e.g., a TCR) specific for a target such as an antigen (e.g., tumor associated antigen or an antigen from a pathogen), wherein the polypeptide improves activation or stimulation of the host cell following binding to target, as compared to a reference or unmodified host cell (not expressing the polypeptide, but expressing the binding protein).
  • a modified host cell e.g., immune cell, such as, for example, a T cell (e.g., a CD4+ T cell, a CD8+ T cell, or both), NK cell, or NK-T cell) that expresses a binding protein (e.g.,
  • a host cell expressing an antigen-specific binding protein and a polypeptide (or first and second polypeptides) as disclosed herein kills an antigen-expressing target cell more effectively than does a reference host cell expressing the antigen-specific binding protein and not expressing the polypeptide(s).
  • a modified CD4 + T cell comprises a polypeptide of the instant disclosure (e.g., comprising at least a portion of a CD8 co-receptor ectodomain or extracellular domain and optionally the CD8 transmembrane domain) and/or a first and second polypeptide as provided herein, and optionally a MHC-I-restricted binding protein (e.g., a TCR).
  • host cells that comprise (i) a heterologous polynucleotide that encodes a polypeptide and/or first and second polypeptide.
  • the encoded fusion protein comprises: (a) an extracellular component comprising an extracellular domain from a CD8 co-receptor a-chain; (b) a transmembrane domain from a CD8 co-receptor a-chain; and (c) an intracellular component comprising a co stimulatory domain from CD28, or a functional portion or variant thereof; and (ii) a heterologous polynucleotide encoding a binding protein that specifically binds to an antigen or an antigen:MHC complex.
  • the host cell comprises a human immune system cell; e.g., a CD4 + T cell.
  • polynucleotides that encode the disclosed polypeptides and/or first and second polypeptides, as well as expression vectors that comprise the polynucleotides, and compositions that comprise the polypeptides, polynucleotides, vectors, and/or host cells.
  • a polypeptide, expression construct, and/or first and second polypeptide can be expressed by a host cell, such as an immune cell.
  • methods are provided for treating a disease or condition using a polypeptide, polynucleotide, vector, modified host cell, or cell composition of the present disclosure.
  • the presently disclosed polypeptides and host cells are useful in treating cancer.
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness is to be understood to include any integer within the recited range, unless otherwise indicated.
  • the term “about” means ⁇ 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms “a” and “an” as used herein refer to “one or more" of the enumerated components.
  • a protein domain, region, or module e.g., a binding domain, hinge region, or linker
  • a protein which may have one or more domains, regions, or modules
  • any of the presently disclosed polypeptides can comprise, consist essentially of, or consist of the recited feature(s) (e.g. components, domains, and/or amino acid sequences)
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, z.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.
  • protein or “polypeptide” refers to a polymer of amino acid residues. Proteins apply to naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid and non-naturally occurring amino acid polymers. Variants of proteins, peptides, and polypeptides of this disclosure are also contemplated.
  • variant proteins, peptides, and polypeptides comprise or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to an amino acid sequence of a defined or reference amino acid sequence as described herein.
  • variation of a defined or reference amino acid sequence comprises or consists of one or more conservative amino acid substitutions. It will be understood that the terms “protein” and “polypeptide” are interchangeable herein, unless the context clearly provides otherwise.
  • mutation refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively.
  • a mutation can result in several different types of change in sequence, including substitution, insertion or deletion of nucleotide(s) or amino acid(s).
  • a “conservative substitution” refers to amino acid substitutions that do not significantly affect or alter binding characteristics of a particular protein. Generally, conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Conservative substitutions include a substitution found in one of the following groups: Group 1 : Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3 : Asparagine (Asn or N), Glutamine (Gin or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (He or I), Leucine (Leu or L), Methionine (Met or M), Valine (Vai or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp or W).
  • Group 1 Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T);
  • Group 2 Aspartic
  • amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (c.g, acidic, basic, aliphatic, aromatic, or sulfur-containing).
  • an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Vai, Leu, and He.
  • Other conservative substitutions groups include: sulfur-containing: Met and Cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gin; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gin; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, He, Vai, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.
  • polypeptides may or may not comprise a signal peptide (also known as a leader sequence, leader peptide, or transit peptide).
  • Signal peptides can target newly synthesized polypeptides to their appropriate location inside or outside (including membrane-spanning) the cell.
  • a signal peptide may be removed from the polypeptide during or once localization or secretion is completed.
  • Polypeptides that have a signal peptide can be referred to as a "preprotein” or "precursor protein” and polypeptides having some or all of their signal peptide removed can be referred to as "mature" proteins or polypeptides.
  • a signal peptide native to CD8a isoform 1 comprises amino acids 1-21 of SEQ ID NO.:1.
  • An example of a signal peptide native to CD8P isoform 1 comprises amino acids 1-21 of SEQ ID NO.:6.
  • Certain signal peptide amino acid sequences are also provided in SEQ ID NOs.: 168-172 and 179. Signal peptides are annotated for various precursor protein sequences; e.g., the UniProt database.
  • any suitable naturally occurring or engineered signal peptide can be employed for a polypeptide of the present disclosure (e.g., Polypeptide 1 and/or Polypeptide 2 of any one of Constructs BB-NN).
  • Certain signal peptides and characteristics of these are described in Owji et al., European Journal of Cell Biology 97(6):422-441 (2016); the signal peptides of which are incorporated herein by reference.
  • Certain disclosed amino acid sequences comprise a signal peptide; such a signal peptide will be recognized by those of ordinary skill in the art, and the amino acid sequence resulting from removal of the signal peptide will also be recognized.
  • fusion protein refers to a protein that, in a single chain, has at least two distinct domains, wherein the domains are not naturally found together in a protein.
  • a polynucleotide encoding a fusion protein may be constructed using PCR, recombinantly engineered, or the like, or such fusion proteins can be synthesized.
  • a fusion protein may further contain other components, such as a tag, a linker, or a transduction marker.
  • a fusion protein expressed or produced by a host cell locates to the cell surface, where the fusion protein is anchored to the cell membrane (e.g., via a transmembrane component or domain) and comprises an extracellular component (e.g., capable of associating with a MHC molecule) and an intracellular component (e.g., containing a signaling domain, effector domain, co-stimulatory domain or portions or combinations thereof).
  • a host cell e.g., a T cell
  • the fusion protein is anchored to the cell membrane (e.g., via a transmembrane component or domain) and comprises an extracellular component (e.g., capable of associating with a MHC molecule) and an intracellular component (e.g., containing a signaling domain, effector domain, co-stimulatory domain or portions or combinations thereof).
  • Nucleic acid molecule refers to a polymeric compound including covalently linked nucleotides, which can be made up of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine ring).
  • Purine bases include adenine, guanine, hypoxanthine, and xanthine
  • pyrimidine bases include uracil, thymine, and cytosine.
  • Nucleic acid molecules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA, either of which may be single or double-stranded.
  • the nucleic acid molecule may be the coding strand or non-coding (anti-sense strand).
  • a nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence. Some versions of the nucleotide sequences may also include intron(s) to the extent that the intron(s) would be removed through co- or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence as the result of the redundancy or degeneracy of the genetic code, or by splicing.
  • the polynucleotide (e.g., mRNA) comprises a modified nucleoside, a cap-1 structure, a cap-2 structure, or any combination thereof.
  • the polynucleotide comprises a pseudouridine, a N6-methyladenonsine, a 5-methylcytidine, a 2-thiouridine, or any combination thereof.
  • the pseudouridine comprises N1 -methylpseudouridine.
  • Variants of nucleic acid molecules of this disclosure are also contemplated. Variant nucleic acid molecules are at least 70%, 75%, 80%, 85%, 90%, and are preferably 95%, 96%, 97%, 98%, 99%, or 99.9% identical a nucleic acid molecule of a defined or reference polynucleotide as described herein, or that hybridize to a polynucleotide under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68°C or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42°C. Nucleic acid molecule variants retain the capacity to encode a fusion protein or a binding domain thereof having a functionality described herein, such as specifically binding a target molecule.
  • Percent sequence identity refers to a relationship between two or more sequences, as determined by comparing the sequences. Preferred methods to determine sequence identity are designed to give the best match between the sequences being compared. For example, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment). Further, non-homologous sequences may be disregarded for comparison purposes. The percent sequence identity referenced herein is calculated over the length of the reference sequence, unless indicated otherwise. Methods to determine sequence identity and similarity can be found in publicly available computer programs.
  • Sequence alignments and percent identity calculations may be performed using a BLAST program (e.g., BLAST 2.0, BLASTP, BLASTN, or BLASTX).
  • BLAST program e.g., BLAST 2.0, BLASTP, BLASTN, or BLASTX.
  • the mathematical algorithm used in the BLAST programs can be found in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997.
  • sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. "Default values" mean any set of values or parameters which originally load with the software when first initialized.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated.
  • nucleic acid could be part of a vector and/or such nucleic acid or polypeptide could be part of a composition (e.g., a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide.
  • isolated can, in some embodiments, also describe a polynucleotide, vector, host cell, or composition that is outside of a human body.
  • a polynucleotide, vector, polypeptide, or host cell can be "isolated.”
  • gene means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region ("leader and trailer") as well as intervening sequences (introns) between individual coding segments (exons).
  • a “functional variant” refers to a polypeptide or polynucleotide that is structurally similar or substantially structurally similar to a parent or reference compound of this disclosure, but differs slightly in composition (e.g., one base, atom or functional group is different, added, or removed), such that the polypeptide or encoded polypeptide is capable of performing at least one function of the parent polypeptide with at least 50% efficiency, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent polypeptide.
  • a functional variant of a polypeptide or encoded polypeptide of this disclosure has "similar binding,” “similar affinity” or “similar activity” when the functional variant displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide, such as an assay for measuring binding affinity (e.g., Biacore® or tetramer staining measuring an association (K a ) or a dissociation (KD) constant).
  • binding affinity e.g., Biacore® or tetramer staining measuring an association (K a ) or a dissociation (KD) constant.
  • a “functional portion” or “functional fragment” refers to a polypeptide or polynucleotide that comprises only a domain, portion or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% activity associated with the domain, portion or fragment of the parent or reference compound, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent polypeptide, or provides a biological benefit (e.g., effector function).
  • a biological benefit e.g., effector function
  • a “functional portion” or “functional fragment” of a polypeptide or encoded polypeptide of this disclosure has “similar binding” or “similar activity” when the functional portion or fragment displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide (preferably no more than 20% or 10%, or no more than a log difference as compared to the parent or reference with regard to affinity), such as an assay for measuring binding affinity or measuring effector function (e.g., cytokine release).
  • a functional portion, fragment, or variant of a parent or reference polypeptide preferably retains or substantially retains a native function such that the polypeptide (e.g., fusion protein) of the present disclosure that comprises the functional portion, fragment, or variant is capable of performing the function.
  • the fusion protein is functional to provide a CD28 costimulatory signal upon ligand/target binding; when, e.g., the extracellular component binds to a MHC class I molecule.
  • the native function is preferably retained, substantially retained, or augmented.
  • heterologous or non-endogenous or exogenous refers to any gene, protein, compound, nucleic acid molecule, or activity that is not native to a host cell or a subject, or any gene, protein, compound, nucleic acid molecule, or activity native to a host cell or a subject that has been altered.
  • Heterologous, non-endogenous, or exogenous includes genes, proteins, compounds, or nucleic acid molecules that have been mutated or otherwise altered such that the structure, activity, or both is different as between the native and altered genes, proteins, compounds, or nucleic acid molecules.
  • heterologous, non-endogenous, or exogenous genes, proteins, or nucleic acid molecules may not be endogenous to a host cell or a subject, but instead nucleic acids encoding such genes, proteins, or nucleic acid molecules may have been added to a host cell by conjugation, transformation, transfection, electroporation, or the like, wherein the added nucleic acid molecule may integrate into a host cell genome or can exist as extra-chromosomal genetic material (e.g., as a plasmid or other self-replicating vector).
  • homologous or homolog refers to a gene, protein, compound, nucleic acid molecule, or activity found in or derived from a host cell, species, or strain.
  • a heterologous or exogenous polynucleotide or gene encoding a polypeptide may be homologous to a native polynucleotide or gene and encode a homologous polypeptide or activity, but the polynucleotide or polypeptide may have an altered structure, sequence, expression level, or any combination thereof.
  • a non-endogenous polynucleotide or gene, as well as the encoded polypeptide or activity may be from the same species, a different species, or a combination thereof.
  • endogenous or “native” refers to a polynucleotide, gene, protein, compound, molecule, or activity that is normally present in a host cell or a subject.
  • expression refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene.
  • the process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post- translational modification, or any combination thereof.
  • An expressed nucleic acid molecule is typically operably linked to an expression control sequence (e.g., a promoter).
  • operably linked refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).
  • Unlinked means that the associated genetic elements are not closely associated with one another and the function of one does not affect the other.
  • expression vector refers to a DNA construct containing a nucleic acid molecule that is operably linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host.
  • control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation.
  • the vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself.
  • plasmid "expression plasmid,” “virus” and “vector” are often used interchangeably. Vectors are discussed further herein.
  • the term "introduced” in the context of inserting a nucleic acid molecule into a cell means “transfection", or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell wherein the nucleic acid molecule may be incorporated into the genome of a cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
  • a cell e.g., chromosome, plasmid, plastid, or mitochondrial DNA
  • transiently expressed e.g., transfected mRNA
  • the term "engineered,” “recombinant” or “non-natural” refers to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or has been modified by introduction of an exogenous nucleic acid molecule, wherein such alterations or modifications are introduced by genetic engineering (i.e., human intervention).
  • Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding proteins, fusion proteins or enzymes, or other nucleic acid molecule additions, deletions, substitutions or other functional disruption of a cell’s genetic material. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a polynucleotide, gene or operon.
  • an "engineered” cell refers to a modified cell.
  • more than one heterologous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof.
  • the two or more heterologous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into the host chromosome at a single site or multiple sites, or any combination thereof.
  • the number of referenced heterologous nucleic acid molecules or protein activities refers to the number of encoding nucleic acid molecules or the number of protein activities, not the number of separate nucleic acid molecules introduced into a host cell.
  • construct refers to any polynucleotide that contains a recombinant nucleic acid molecule (or, when the context clearly indicates, a(n, e.g., fusion) protein of the present disclosure).
  • a "transgene” or “transgene construct” refers to a construct that contains two or more genes operably linked in an arrangement that is not found in nature.
  • a (polynucleotide) construct may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into a genome.
  • vector is a nucleic acid molecule that is capable of transporting another nucleic acid molecule.
  • Vectors may be, for example, plasmids, cosmids, viruses, a RNA vector or a linear or circular DNA or RNA molecule that may include chromosomal, non-chromosomal, semi -synthetic or synthetic nucleic acid molecules.
  • Vectors of the present disclosure also include transposon systems (e.g., Sleeping Beauty, see, e.g., Geurts et al., Mol. Ther. 5: 108, 2003: Mates et al., Nat. Genet. 41.753, 2009).
  • Exemplary vectors are those capable of autonomous replication (episomal vector), capable of delivering a polynucleotide to a cell genome (e.g., viral vector), or capable of expressing nucleic acid molecules to which they are linked (expression vectors).
  • the term "host” refers to a cell (e.g., T cell) or microorganism targeted for genetic modification with a heterologous nucleic acid molecule to produce a polypeptide of interest (e.g., a fusion protein of the present disclosure).
  • a host cell may optionally already possess or be modified to include other genetic modifications that confer desired properties related or unrelated to, e.g., biosynthesis of the heterologous protein (e.g., inclusion of a detectable marker; deleted, altered or truncated endogenous TCR; or increased co-stimulatory factor expression).
  • Antigen refers to an immunogenic molecule that provokes an immune response. This immune response may involve, for example, antibody production, activation of specific immunologically-competent cells (e.g., T cells), or both.
  • An antigen immunologically-competent cells
  • An antigen immunologically-competent cell
  • An antigen may be, for example, a peptide, glycopeptide, polypeptide, glycopolypeptide, polynucleotide, polysaccharide, lipid or the like. It is readily apparent that an antigen can be synthesized, produced recombinantly, or derived from a biological sample. Exemplary biological samples that can contain one or more antigens include tissue samples, tumor samples, cells, biological fluids, or combinations thereof. Antigens can be produced by cells that have been modified or genetically engineered to express an antigen. Antigens can be expressed at a cell surface or presented in complex with a MHC molecule.
  • epitope includes any molecule, structure, amino acid sequence or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, T cell receptor (TCR), chimeric antigen receptor, or other binding molecule, domain or protein.
  • a cognate binding molecule such as an immunoglobulin, T cell receptor (TCR), chimeric antigen receptor, or other binding molecule, domain or protein.
  • Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • T cell receptor refers to an immunoglobulin superfamily member (having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail; see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3 rd Ed., Current Biology Publications, p. 4:33, 1997) capable of specifically binding to an antigen peptide bound to a MHC receptor.
  • a TCR can be found on the surface of a cell or in soluble form and generally is comprised of a heterodimer having a and P chains (also known as TCRa and TCRP, respectively), or y and 5 chains (also known as TCRy and TCR5, respectively).
  • each TCR chain contains two immunoglobulin domains: a variable domain (e.g., a- chain variable domain or Va, P-chain variable domain or Vp; typically amino acids 1 to 116 based on Kabat numbering (Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept.
  • variable domains contain complementary determining regions (CDRs) separated by framework regions (FRs) (see, e.g., Jores et al., Proc. Nat'l Acad. Sci. U.S.A. 57:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003).
  • variable region refers to the domain of a TCR a-chain or P-chain (or y-chain and 6-chain for y6 TCRs), or of an antibody heavy or light chain, that is involved in binding to antigen.
  • the variable domains of the a-chain and P-chain (Va and VP, respectively) of a native TCR generally have similar structures, with each domain comprising four generally conserved framework regions (FRs) and three CDRs.
  • FRs generally conserved framework regions
  • VH antibody heavy
  • VL light chains each also generally comprise four generally conserved framework regions (FRs) and three CDRs.
  • CDR complementarity determining region
  • HVR hypervariable region
  • CDR3 is thought to be the main CDR responsible for recognizing processed antigen.
  • CDR1 and CDR2 mainly interact with the MHC.
  • Variable domain sequences can be aligned to a numbering scheme (e.g., Kabat, EU, International Immunogenetics Information System (IMGT) and Aho), which can allow equivalent residue positions to be annotated and for different molecules to be compared using Antigen receptor Numbering And Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298-300).
  • a numbering scheme e.g., Kabat, EU, International Immunogenetics Information System (IMGT) and Aho
  • IMGT International Immunogenetics Information System
  • Aho Antigen receptor Numbering And Receptor Classification
  • a TCR is found on the surface of T cells (or T lymphocytes) and associates with the CD3 complex.
  • the source of a TCR as used in the present disclosure may be from various animal species, such as a human, mouse, rat, rabbit or other mammal.
  • CD3 is a multi-protein complex of six chains that is involved in T cell signaling in response to antigen, (see, Abbas and Lichtman, 2003; Janeway et al., p. 172 and 178, 1999).
  • the complex generally comprises a CD3y chain, a CD36 chain, two CD3s chains (each of which, in general, associates with a cognate CD3y chain or CD36 chain to form a dimer), and a homodimer of CD3( ⁇ chains.
  • the CD3y, CD36, and CD3s chains are related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain.
  • the transmembrane regions of the CD3y, CD36, and CD3s chains are negatively charged, which is thought to allow these chains to associate with positively charged regions of T cell receptor chains.
  • CD3 as used in the present disclosure may be from various animal species, including human, mouse, rat, or other mammals. Accordingly, it will be understood that a functional portion or a variant of a CD3 protein intracellular domain contains one or more ITAM and optionally other sequence features that are involved in signaling. Examples of sequences from human CD3 proteins are provided in SEQ ID NOs:69-77.
  • MHC molecules refer to glycoproteins that deliver peptide antigens to a cell surface.
  • MHC class I molecules are heterodimers consisting of a membrane spanning a chain (with three a domains) and a non-covalently associated P2 microglobulin.
  • MHC class II molecules are composed of two transmembrane glycoproteins, a and P, both of which span the membrane. Each chain has two domains.
  • MHC class I molecules deliver peptides originating in the cytosol to the cell surface, where a peptide:MHC complex is recognized by CD8 + T cells.
  • MHC class II molecules deliver peptides originating in the vesicular system to the cell surface, where they are recognized by CD4 + T cells.
  • An MHC molecule may be from various animal species, including human (/. ⁇ ., HL A molecule), mouse, rat, cat, dog, goat, horse, or other mammals.
  • HLAs corresponding to "class I" MHC present peptides from inside the cell and include, for example, HLA-A, HLA-B, and HLA-C.
  • Alleles include, for example, HLA A*02:01; HLA-A*03:01; HLA-A*l l:01; HLA- B*07:02; HLA-B*40:01; HLA-B*44:02; or HLA-B *44: 03.
  • HLAs corresponding to "class II" MHC present peptides from outside the cell and include, for example, HLA- DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR.
  • a class I MHC comprises an HLA.
  • the HLA comprises HLA-A, HLA-B, and/or HLA-C.
  • the HLA comprises HLA A*02:01; HLA-A*03:01; HLA-A*l l:01; HLA-B*07:02; HLA-B*40:01; HLA-B*44:02; or HLA-B*44:03.
  • CD8 co-receptor includes the cell surface glycoprotein CD8, which is sometimes expressed by T cells as a homodimer comprising two CD8a chains, or as a heterodimer comprising an a chain and a P chain.
  • the CD8 co-receptor is believed to assist in the function of cytotoxic T cells (CD8 + ) and functions through signaling via its cytoplasmic tyrosine phosphorylation pathway (Gao and Jakobsen, Immunol. Today 27:630-636, 2000; Cole and Gao, Cell. Mol. Immunol. 7:81-88, 2004).
  • the CD8 co-receptor binds to an MHC-I protein complex expressed on the surface of an antigen-expressing cell, and that this binding in the context of TCR:antigen-MHC binding initiates or assists in a T cell signaling pathway that produces an immune response (e.g., transcription and expression of cytokines, calcium secretion, cytolytic activity, or the like) against the antigen-expressing cell.
  • an immune response e.g., transcription and expression of cytokines, calcium secretion, cytolytic activity, or the like
  • CD8 beta chain isoforms In humans, eight (8) different CD8 beta chain isoforms are known ("M1"-"M8"; see UniProtKB identifiers P10966-1, 2, 3, 4, 6, 7, 8, and 9); of these, isoforms 1, 2, 4, and 5 are thought to associate with the cell membrane in nature, while isoforms 3, 6, 7, and 8 are believed to associate with extracellular regions or be secreted.
  • the amino acid sequences of these CD8 P-chain isoforms (including with and without leader (i.e., signal peptide) sequences) are incorporated by reference herein. Amino acid sequences from certain CD8 co-receptor P-chains of the present disclosure are shown in SEQ ID NOs:6-17.
  • a(n, e.g., fusion) protein of the present disclosure comprises an extracellular and/or transmembrane component from a CD8 co-receptor P- chain Ml isoform, or functional variant or portion thereof.
  • CD8 alpha chain isoforms are known (see UniProtKB identifiers PO 1732-1, 2, and 3).
  • the amino acid sequences of these CD8 a-chain isoforms are incorporated by reference herein.
  • Amino acid sequences from certain CD8 co-receptor a-chains of the present disclosure are shown in SEQ ID NOs:l-5.
  • Reference to CD8a includes the "canonical" human CD8a protein (NP_001759.3) as well as splice isoform 2, which lacks an internal segment including the transmembrane domain resulting in a secreted protein (RefSeq NP 741969.1), and splice isoform 3, which uses an alternate promoter and 5’ UTR (RefSeq NP 001139345.1).
  • Reference to CD8P includes the "canonical" human CD8P protein (RefSeq NP_004922) as well as isoforms 2-8, corresponding to RefSeq NP742099, RefSeq NP_742100, UniProt Pl 0966-4, RefSeq NP_757362, Uniprot Pl 0966-7, Uniprot P 10966-8, and RefSeq NP 001171571.
  • CD8 polypeptide and “CD8 co-receptor polypeptide” can be used interchangeably, including when the subject polypeptide functions as a protein monomer.
  • a “CD8a” polypeptide can be a wild-type CD8a chain or a fragment thereof (of any isotype or species), as well as an engineered e.g., fusion or chimeric) polypeptide that comprises at least some portion of a CD8a extracellular domain and is capable of binding to a Class I MHC molecule, such as in a manner that is at least substantially similar to the manner in which wild-type CD8a binds to the Class I MHC molecule.
  • a CD8a polypeptide further comprises CD8a transmembrane and/or intracellular amino acid sequence or features.
  • a CD8a polypeptide comprises an intracellular component that can associate with a Lek.
  • a “CD8P” polypeptide can be a wild-type CD8a chain or a fragment thereof (of any isotype or species), as well as an engineered (e.g., fusion or chimeric) polypeptide that comprises at least some portion of a CD8P extracellular domain and is capable of binding to a Class I MHC molecule, such as in a manner that is at least substantially similar to the manner in which wild-type CD8P binds to the Class I MHC molecule.
  • a CD8P polypeptide further comprises CD8P transmembrane and/or intracellular amino acid sequence or features.
  • CD4 refers to an immunoglobulin co-receptor glycoprotein that assists the TCR in communicating with antigen-presenting cells (see, Campbell & Reece, Biology 909 (Benjamin Cummings, Sixth Ed., 2002); UniProtKB P01730).
  • CD4 is found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells, and typically includes four immunoglobulin domains (DI (comprising an Ig-like V-type domain), D2, D3, and D4 (respectively comprising Ig-like C2-type domains 1, 2, and 3)) that are expressed at the cell surface.
  • DI immunoglobulin domains
  • CD4 is recruited, along with the TCR complex, to bind to different regions of the MHCII molecule (CD4 binds MHCII P2, while the TCR complex binds MHCII al/pi).
  • CD4-associated kinase molecules phosphorylate the immunoreceptor tyrosine activation motifs (IT AMs) present on the cytoplasmic domains of CD3. This activity is thought to amplify the signal generated by the activated TCR in order to produce various types of T helper cells.
  • IT AMs immunoreceptor tyrosine activation motifs
  • Examples of human CD4 amino acid sequences are disclosed in UniProt KB entry no. P01730; these amino acid sequences (including with and without leader (i.e., signal peptide) sequences) are incorporated by reference herein. Polypeptides., Polynucleotides, and Vectors
  • the present disclosure provides polypeptides, such as fusion proteins and other engineered proteins, that are useful to confer to, or improve, a desired activity of a host immune cell, such as an immune cell that targets a diseased or pathogenic cell (e.g. a cancer cell).
  • a host immune cell such as an immune cell that targets a diseased or pathogenic cell (e.g. a cancer cell).
  • polynucleotides that encode any one or more of the polypeptides, and vectors that comprise a polynucleotide.
  • a polynucleotide encodes two or more polypeptides that can function when associating as a multimer (e.g., as a dimer) at the surface of a host cell, such as a T cell.
  • a polypeptide can comprise a human amino acid sequence, or can be derived (e.g., engineered) from a human amino acid sequence.
  • a host cell can be a human cell.
  • a polypeptide comprises: (i) an extracellular component that comprising a binding domain that is capable of binding to a MHC Class I molecule; (ii) a transmembrane domain; and (iii) an intracellular component comprising an intracellular sequence from CD28, or an optionally functional portion or variant thereof; e.g. comprising one or more Motif of any one of constructs BB-NN as shown in Table 6 or Table 7.
  • the binding domain comprises an Ig V-like domain (e.g., from a CD8a, a CD8P, or a functional variant thereof), and/or the extracellular component comprises a length of about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, or about 145 amino acids, or of between 145 and 175 amino acids, or of between 145 and 170 amino acids, or of between 145 and 165 amino acids, or of between 145 and 160 amino acids, or of between 145 and 155 amino acids, or of between 145 and 155 amino acids, or of between 145 and 150 amino acids, or of between 150 and 175 amino acids, or of between 150 and 170 amino acids, or of between 150 and 165 amino acids, or of between 150 and 160 amino acids, or of between 150 and 155 amino acids, or of between 155 and 175 amino acids, or of between 150 and 170 amino acids, or of between 150 and 165 amino acids, or of between 150 and 160 amino acids, or of between 150 and
  • Certain embodiments include fusion (also referred-to herein as chimeric) CD8 co-receptor proteins, which can include an intracellular signaling domain from another protein (e.g., CD28) and can translate MHC -binding into a stimulatory signal in a host cell.
  • fusion also referred-to herein as chimeric CD8 co-receptor proteins, which can include an intracellular signaling domain from another protein (e.g., CD28) and can translate MHC -binding into a stimulatory signal in a host cell.
  • Chimeric CD8 co-receptor proteins include those that may function advantageously with a cognate CD8 co-receptor protein (e.g., in some embodiments, a chimeric CD8P protein may function advantageously with a cognate CD8a protein, which itself may be wild-type or engineered (e.g., chimeric); a chimeric CD8a protein can function with a cognate CD8P (or CD8a) protein, which itself may be wild-type or engineered (e.g., chimeric).
  • a chimeric CD8P protein may function advantageously with a cognate CD8a protein, which itself may be wild-type or engineered (e.g., chimeric);
  • a chimeric CD8a protein can function with a cognate CD8P (or CD8a) protein, which itself may be wild-type or engineered (e.g., chimeric).
  • a polypeptide comprises an extracellular domain from CD8P, a transmembrane domain that is optionally from CD8P, and an intracellular domain comprising a variant of SEQ ID NO.: 19, wherein the variation comprises or consists of: (i) a diglycine motif in place of a dileucine motif; (ii) a mutated and optionally non-functional PI3K binding site; (iii) a mutated and optionally non-functional ITK and GADS binding site; (iv) a mutated and optionally nonfunctional LCK binding site; or (v) any combination of (i)-(iv), optionally: (i) and (ii); (i) and (iii); (i) and (iv); (ii) and (iii); (ii) and (iv); (iii) and (iv); (i), (ii), and (ii); (i), (iv); (ii), (i), and (iv); (ii), (
  • a mutation in a PI3K binding site comprises or consists of a Y ⁇ A mutation, e.g., providing the sequence AMNM.
  • a mutation in a PI3K binding site comprises or consists of a Y ⁇ F mutation, e.g., providing the sequence FMNM.
  • a mutation in a ITK and GADS binding site comprises or consists of one or more P ⁇ A mutation, e.g., providing the sequence ARRA, or the sequence ARRP, or the sequence PRRA.
  • a mutation in a LCK binding site comprises or consists of one or more P ⁇ A mutation, e.g., providing the sequence AYYA, or the sequence PYAA, or the sequence AYAP.
  • the polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92%, or at least 95% sequence identity to SEQ ID NO.:7 or SEQ ID NO.:82.
  • the polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92%, or at least 95% sequence identity to SEQ ID NO.: 19.
  • the polypeptide comprises, in the intracellular domain and N-terminal to the variant of SEQ ID NO. : 19, the amino acid sequence of SEQ ID NO.: 10, wherein, optionally, the variant of SEQ ID NO.: 19 is fused directly to the amino acid sequence of SEQ ID NO. : 10.
  • the intracellular domain of the polypeptide comprises an amino acid sequence according to SEQ ID NO.:205 or 206.
  • the variant of SEQ ID NO.: 19 comprises therein Motif 1, Motif 2, Motif 3, and/or Motif 4 of any one of Constructs BB-NN in Table 6 or Table 7, and/or comprising as Motif 2 the amino acid sequence FMNM.
  • the variant of SEQ ID NO.: 19 comprises therein: (A) (i) Motif 1, 2, 3, or 4 of Construct BB in Table 6 or Table 7; (ii) Motifs 1 and 2 of Construct BB in Table 6 or Table 7; (iii) Motifs 1 and 3 of Construct BB in Table 6 or Table 7; (iv) Motifs 1 and 4 of Construct BB in Table 6 or Table 7; (v) Motifs 1, 2, and 3 of of Construct BB in Table 6 or Table 7; (vi) Motifs 1, 2, and 4 of of Construct BB in Table 6 or Table 7; (vii) Motifs 1, 3, and 4 of of Construct BB in Table 6 or Table 7; (viii) Motifs 2 and 3 of Construct BB in Table 6 or Table 7; (ix) Motifs 2 and 4 of Construct BB in Table 6 or Table 7; (x) Motifs 2, 3, and 4 of of Construct BB in Table 6 or Table 7;
  • polypeptide comprising an extracellular domain from CD8P, a transmembrane domain that is optionally from CD8P, and an intracellular domain comprising a variant of SEQ ID NO.: 19, wherein the variant of SEQ ID NO.: 19 comprises: (A)(i) Motif 1, 2, 3, or 4 of Construct BB in Table 6 or Table 7; (ii) Motifs 1 and 2 of Construct BB in Table 6 or Table 7; (iii) Motifs 1 and 3 of Construct BB in Table 6 or Table 7; (iv) Motifs 1 and 4 of Construct BB in Table 6 or Table 7; (v) Motifs 1, 2, and 3 of of Construct BB in Table 6 or Table 7; (vi) Motifs 1, 2, and 4 of of Construct BB in Table 6 or Table 7; (vii) Motifs 1, 3, and 4 of of Construct BB in Table 6 or Table 7; (viii) Motifs 2 and 3 of Construct BB in Table 6 or Table 6
  • the transmembrane domain is from CD8P (e.g., comprises SEQ ID NO.:8) or from CD28; and/or the extracellular domain from CD8P is from a CD8P Ml, M2, M3, M4, or M5 isoform; and/or the extracellular domain from CD8P comprises an Ig V-like domain comprising SEQ ID NO.:68 or comprises amino acids 1-110 of SEQ ID NO.:7.
  • polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs.: 192-204.
  • polypeptide comprising, consisting essentially or, or consisting of: an extracellular domain amino acid sequence, a transmembrane domain amino acid sequence, and an intracellular domain amino acid sequence, as shown under “2 protein size [aa]” in any one of Figures 27-39.
  • two or more CD8 co-receptor proteins are encoded by a same polynucleotide or vector and/or are expressed in a same host cell.
  • a CD8 co-receptor protein e.g., CD8a
  • a chemokine receptor protein e.g., CCR4 or CCR2b
  • two proteins of the present disclosure can be expressed co-ordinately, e.g, by use of an expression construct that drives expression of both proteins in a fusion construct, and the fusion comprises e.g, a cleavable or cleaving amino acid sequence so that the encoded component proteins separate from one another and can express as separate molecules at the host cell surface.
  • the polynucleotide or vector further encodes a binding protein, such as a TCR, and/or the host cell further expresses a binding protein, such as a TCR.
  • a TCR is MHC I-restricted.
  • a polypeptide comprises components (e.g., amino acid sequences or domains) from CD8a and CD8P (of any isoform or combination of isoforms), and, optionally, from one or more additional polypeptides, such as, for example, CD28.
  • additional polypeptides such as, for example, CD28.
  • Binding to or associating with a target or cognate molecule can be assessed by known methods, such as, for example, peptide:MHC multimer/tetramer staining, Western blot, ELISA, analytical ultracentrifugation, spectroscopy, surface plasmon resonance (Biacore®) isothermal titration calorimetry, and biolayer interferometry (see, e.g., Dolton et al., Immunology 146: 11-22, 2015, Scatchard et al., Ann. NY Acad. Sci. 51 :660, 1949; Wilson, Science 20295:2103, 2002; Wolff etal., Cancer Res. 53:2560, 1993; and U.S. Patent Nos.
  • a protein-protein binding interaction can be investigated by immobilizing one binding partner on a bead or plate, and passing the other binding partner thereover in solution, and detecting binding by, e.g., a refractive index or a wavelength shift.
  • Flow cytometry and other cell sorting and imaging techniques may also be used to investigate binding by cell surface-expressed molecules.
  • CD8 co-receptor polypeptides of the present disclosure such as a CD8P-CD28 fusion polypeptide of any one of Constructs BB-NN (and portions or domains or functional variants of same), retain the ability to bind to a MHC complex molecule, such as MHC Class I molecule.
  • CD8 co-receptors will typically include at least one immunoglobulin-like V-type domain that binds to a cognate MHC molecule.
  • V-type domains, and functional variants thereof including those comprising one or more conservative or non-conservative amino acid substitutions relative to a parental or wild-type sequence, are contemplated herein.
  • a functional variant or portion of a CD8a extracellular domain or a CD8P extracellular domain is capable of binding to a MHC Class I molecule.
  • a functional variant or portion of a CD8a or a CD8P extracellular domain comprises an Ig V-like domain, or a functional portion or variant thereof.
  • Sequences comprising an Ig V-like domain can include, for example, amino acids 22- 135 of SEQ ID NO: 1 (see also amino acids 1-114 of SEQ ID NOS:2 and 5), or amino acids 22-132 of SEQ ID NO:6 see also amino acids 1-110 of SEQ ID NO:7), and are set forth in SEQ ID NOs:67 and 68.
  • engineered CD8 coreceptor polypeptides that comprise one or more mutations for enhanced or decreased binding to MHC (see e.g. Devine et al. J. Immunol 177(6):2006, which binding mutants are incorporated herein by reference).
  • a polypeptide comprising an Ig V-like domain comprises or consists of an amino acid sequence having 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%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence set forth in SEQ ID NO.:67 or 68, or a portion or fragment thereof that is capable of binding to a MHC Class I molecule.
  • a CD8 co-receptor polypeptides of the present disclosure comprises, in the extracellular component, a CD8 stalk region.
  • a stalk region refers to the extracellular portion of a mature CD8a or CD8P chain that is not the Ig V-like domain.
  • a stalk region typically comprises a length of from about 30 to about 50 amino acid residues and can, in some embodiments, contain one or more O-linked glycan. See e.g. Kern et al. Immunity 9(4):519-530 (1998).
  • a stalk region has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to, or comprises or consists of, amino acids 112-149 of SEQ ID NO.:7 or amino acids 115-161 of SEQ ID NO.:2.
  • a CD8 co-receptor polypeptide comprises an extracellular portion, component, or domain of sufficient length to allow binding to MHC in the context of an immunological synapse; e.g., a synapse comprising a binding protein (e.g., a TCR) expressed by the CD8- (or fusion protein-) expressing cell in association with an antigen or antigen:MHC complex expressed by a target cell, and optionally further comprising, on the host cell, one or more native costimulatory polypeptide, and, on the target cell, one or more cognate ligand of the one or more costimulatory polypeptide.
  • a binding protein e.g., a TCR
  • transmembrane domain is a portion of a transmembrane protein that can insert into or span a cell membrane.
  • a transmembrane domain may also be referred-to as a "transmembrane component”.
  • Transmembrane domains have a three-dimensional structure that is thermodynamically stable in a cell membrane and generally range in length from about 15 amino acids to about 30 amino acids. In some embodiments, a transmembrane domain has a length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 amino acids.
  • transmembrane domain may comprise an alpha helix, a beta barrel, a beta sheet, a beta helix, or any combination thereof.
  • the transmembrane domain comprises or is derived from a known transmembrane protein (e.g, a CD4 transmembrane domain, a CD8a transmembrane domain, a CD8P transmembrane domain, a CD27 transmembrane domain, a CD28 transmembrane domain, a NKG2D transmembrane domain, a Fas transmembrane domain, a CCR4 transmembrane domain, a CCR2b transmembrane domain, a CD3 (zeta, delta, gamma, or epsilon) transmembrane domain, or any combination thereof).
  • a known transmembrane protein e.g, a CD4 transmembrane domain, a CD8a transmembrane domain, a CD8P transme
  • an amino acid sequence, polypeptide domain, or polypeptide component is "derived from" a source or parent polypeptide when it comprises no more than 5%, 10%, 15%, or 20% variation in amino acid sequence identity as compared to the source or parent polypeptide.
  • the extracellular component of a fusion protein further comprises a linker disposed between a binding component or domain (e.g, an Ig V-like domain) or receptor ectodomain and the transmembrane domain, or between the transmembrane domain and an intracellular component.
  • a linker may be an amino acid sequence having from about two amino acids to about 500 amino acids, which can provide flexibility and room for conformational movement between two regions, domains, motifs, fragments, or modules connected by the linker.
  • a linker of the present disclosure can position a fusion protein or polypeptide away from the surface of a host cell expressing the fusion protein so as to enable proper contact between the host cell and a target cell, binding to MHC or other target or ligand, and subsequent signaling (Patel et al., Gene Therapy 6: 412-419, 1999).
  • Linker length in a fusion or binding protein of the present disclosure may be varied to maximize target (e.g. MHC) recognition based on the selected target molecule, selected binding epitope, or antigen binding domain size and affinity (see, e.g., Guest et al., J. Immunother. 25:203-11, 2005; PCT Publication No. WO 2014/031687).
  • linkers include those having a glycine-serine amino acid chain having from one to about ten repeats of Gly x Ser y , wherein x and y are each independently an integer from 0 to 10, provided that x and y are not both 0.
  • Non-limiting examples of linkers are provided in SEQ ID NOs : 182-184.
  • a linker can have a length of up to and including 4, up to and including 6, up to and including 8, up to and including 10, up to and including 12, up to and including 15, up to and including 20, up to and including 30, up to and including 40, or up to and including 50 amino acids.
  • a linker can have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids.
  • a linker can have a length of one to five amino acids, one to ten amino acids, one to fifteen amino acids, one to twenty amino acids, one to twenty -five amino acids, one to thirty amino acids, five to ten amino acids, five to fifteen amino acids, five to twenty amino acids, five to twenty -five amino acids, five to thirty amino acids, ten to fifteen amino acids, ten to twenty amino acids, ten to twenty-five amino acids, ten to thirty amino acids, fifteen to twenty amino acids, fifteen to twenty-five amino acids, fifteen to thirty amino acids, twenty to twenty-five amino acids, twenty to thirty amino acids, or twenty-five to thirty amino acids, less than thirty amino acids, less than twenty-five amino acids, less than twenty amino acids, less than fifteen amino acids, less than ten amino acids, or less than five amino acids.
  • Linkers of the present disclosure also include immunoglobulin constant regions (/. ⁇ ., CHI, CH2, CH3, or CL, of any isotype) and portions thereof.
  • the linker comprises a CH3 domain, a CH2 domain, or both.
  • the linker comprises a CH2 domain and a CH3 domain.
  • the CH2 domain and the CH3 domain are each a same isotype.
  • the CH2 domain and the CH3 domain are an IgG4 or IgGl isotype.
  • the CH2 domain and the CH3 domain are each a different isotype.
  • the CH2 comprises a N297Q mutation.
  • the linker comprises a human immunoglobulin constant region or a portion thereof. Additional linkers include extracellular domains (or portions thereof, such as hinge or stalk sequences) from CD27, CD28, CD8, CD4, or any combination thereof. In any of the embodiments described herein, a linker may comprise a hinge region or a portion thereof.
  • Hinge regions are flexible amino acid polymers of variable length and sequence (typically rich in proline and cysteine amino acids) and connect larger and less-flexible regions of immunoglobulin proteins.
  • hinge regions connect the Fc and Fab regions of antibodies and connect the constant and transmembrane regions of TCRs.
  • the linker comprises an immunoglobulin constant region or a portion thereof and a hinge region or a portion thereof.
  • the linker comprises a glycine-serine linker of the present disclosure. Hinge regions from CD proteins such as, for example, CD8a and CD8P are also contemplated.
  • a polypeptide of the present disclosure is capable of providing a stimulatory signal to a host cell expressing the polypeptide.
  • a stimulatory signal is typically provided by one or more sequences, domains, or motifs in an intracellular component of a polypeptide, though the extracellular component and/or transmembrane domain may, in some cases, also affect the stimulatory signal.
  • a stimulatory signal is typically initiated by an interaction or association between a polypeptide of the present disclosure and one or more (e.g., extracellular) cognate polypeptides, target molecules, or ligands.
  • “providing” a signal refers to facilitating, relaying, producing, and/or amplifying a signal.
  • a polypeptide can provide a stimulatory signal even if other biomolecules (e.g., other polypeptides) may play a further or downstream role in effecting the signal (e.g., via a signal transduction pathway or transcription of a gene).
  • a portion of a polypeptide e.g., a costimulatory or effector domain
  • a host cell is an immune cell such as, for example, a T cell (e.g., a CD4+ T cell, a CD8+ T cell, or both), and a stimulatory signal activates, or contributes to activation of, the immune cell.
  • a single stimulatory signal may at least partially activate an immune cell, or may contribute to at least partial activation.
  • two or more stimulatory signals e.g., one stimulatory signal from a TCR complex, one co- stimulatory signal
  • Activation of an immune cell can comprise, for example, production of a cytokine, production of an antibody, a cytotoxic activity, a phagocytic activity, proliferation of the immune cell, intracellular mobilization of calcium, activation of a transcription factor, transcription of a gene, or the like, or any combination thereof.
  • a stimulatory signal can improve persistence of a host cell; e.g. of a T cell in an immunosuppressive (e.g., tumor micro-)environment.
  • a polypeptide of the present disclosure comprises an extracellular component from a CD8 polypeptide (e.g., CD8a or CD8P), or a functional portion or variant thereof, and a stimulatory signal is initiated by interaction of the extracellular component with a MHC Class I molecule.
  • a polypeptide e.g., a fusion comprising an extracellular domain from CD8P, a transmembrane domain that is optionally from CD8P, and an intracellular domain comprising a variant of SEQ ID NO.
  • BB-NN can form a complex with a CD8a co-receptor polypeptide wherein the complex is expressed at the surface of a host cell and the complex can provide signal to the host cell when the complex interacts with a MHC Class I molecule.
  • a stimulatory signal comprises or further comprises an effector signal, wherein the effector signal directly or indirectly promotes an immunological response in a cell.
  • An effector signal may be provided by, or to, an effector domain.
  • an "effector domain" is an intracellular portion, component, or domain of a polypeptide (e.g., fusion protein, receptor) that can directly or indirectly promote an immunological response in a cell when receiving an appropriate signal.
  • an effector domain is from a protein or portion thereof or protein complex that receives a signal when bound, or when the protein or portion thereof or protein complex binds directly to a target molecule and triggers a signal from the effector domain.
  • An effector domain may directly promote a cellular response when it contains one or more signaling domains or motifs, such as an Intracellular Tyrosine-based Activation Motif (ITAM), such as those found in costimulatory (also "co-stimulatory", herein) molecules.
  • ITAM Intracellular Tyrosine-based Activation Motif
  • costimulatory also "co-stimulatory”, herein
  • IT AMs are important for T cell activation following ligand engagement by a T cell receptor or by a fusion protein comprising a T cell effector domain.
  • the intracellular component or functional portion thereof comprises an ITAM.
  • Exemplary effector domains include those from, CD3s, CD36, CD3 ⁇ CD25, CD79A, CD79B, CARD11, DAP10, FcRa, FcRp, FcRy, Fyn, HVEM, ICOS, Lek, LAG3, LAT, LRP, NKG2D, N0TCH1, N0TCH2, N0TCH3, N0TCH4, Wnt, R0R2, Ryk, SLAMF1, Slp76, pTa, TCRa, TCRp, TRIM, Zap70, PTCH2, or any combination thereof.
  • a stimulatory signal comprises a costimulatory signal.
  • a costimulatory signal is a secondary signal produced by a T cell costimulatory protein e.g. CD28, ICOS, 4- IBB, 0X40) and promotes e.g. T cell proliferation, survival, or one or more effector function, while a primary stimulatory signal is provided by the TCR complex to the T cell following engagement of the TCR with antigen:MHC.
  • TCR complex signaling and costimulatory signaling may be required for preferred activation of a T cell.
  • a host cell is a T cell (e.g., a CD4+ T cell) and a primary stimulatory signal is provided by the TCR complex e.g., comprising a heterologous MHC Class I-restricted TCR) while a costimulatory signal is provided by a polypeptide of the present disclosure, whether the polypeptide comprises a signaling portion or domain from CD28, ICOS, 4- 1BB, 0X40, or from a different polypeptide e.g., from GITR, TRAF1, or Lek).
  • a costimulatory signal may be provided by a costimulatory domain, or a functional portion or variant thereof.
  • the intracellular component of a polypeptide of the present disclosure comprises a costimulatory domain or a functional portion thereof selected from CD28, 4-1BB (CD137), 0X40 (CD134), ICOS (CD278), CD27, CD2, CD5, ICAM-1 (CD54), LFA-1 (CD1 la/CD18), GITR, CD30, CD40, BAFF-R, HVEM, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, B7-H3 CD94, DAP 12, a ligand that specifically binds with CD83, or a functional variant thereof, or any combination thereof.
  • the intracellular component of a polypeptide e.g., fusion protein) of the present disclosure comprises a costimulatory domain from CD28, or a functional portion or variant thereof (which may optionally include a non-leucine e.g., glycine, serine, cysteine, alanine, valine, isoleucine, or the like) substitution at either one or both of positions 186-187 of the native CD28 protein (e.g., LL- GG; see Nguyen et al., Blood 102:4320, 2003)).
  • a non-leucine e.g., glycine, serine, cysteine, alanine, valine, isoleucine, or the like
  • Non-limiting examples of CD28 amino acid sequences are provided in SEQ ID NOs.: 18-20.
  • a functional variant or portion of a CD28 costimulatory domain retains an ability to: (i) recruit and/or bind a SH2-domain-containing protein; and/or (ii) recruit and/or bind a SH3- domain-containing protein.
  • a functional variant or portion of CD28 can bind to Lek, Tec, Itk, PI3K, Grb, Gads, or any combination thereof.
  • a functional variant or portion of a CD28 costimulatory domain comprises a "PXXP" motif, wherein X can be any one or any two different amino acids (e.g., PRRP; PYAP; see SEQ ID NOS: 19 and 20).
  • a functional variant or portion of a CD28 costimulatory domain comprises a tyrosine at a position corresponding to any one or more of positions 191, 206, 209, 218 of the native full- length human CD28 amino acid sequence.
  • a functional variant or portion of a CD28 costimulatory domain comprises a tyrosine at positions corresponding to positions 206, 209, 218 of the native full-length human CD28 amino acid sequence.
  • a functional variant or portion of a CD28 costimulatory domain comprises a " YXNX" motif, wherein X is any two same or different amino acids (e.g., YMNM). See Ogawa etal., Int. Immunol. 25(12):671 (2013) and Salter et al., Science Signaling 77:eaat6753 (2016).
  • a polypeptide of the present disclosure comprises a CD28 costimulatory domain comprising one or more amino acid mutations (e.g., substitution mutations) that modify (e.g. attenuate, diminish, boost, or increase) a signaling function by the CD28 costimulatory domain.
  • An example of a signaling mutant CD28 sequence is provided in SEQ ID NO.:81 (such a mutant sequence is referred-to herein as a “partial” signaling mutant).
  • Another example of a signaling mutant CD28 sequence is provided in SEQ ID NO.: 108 (such a mutant sequence is referred-to herein as a “full” signaling mutant).
  • costimulatory domain can refer to the portion of a protein that in the e.g., wild-type condition, is involved in producing a costimulatory signal, even if the subject protein contains alterations (e.g. mutations) that reduce or abrogate the signaling function.
  • any of the presently disclosed polypeptides can comprise any one or more (in any arrangement and in any combination) of the presently disclosed costimulatory domains or functional portions or variants thereof.
  • a(n e.g., fusion) protein can comprise an extracellular and/or transmembrane component from a CD8 co-receptor P-chain, or a functional variant or portion thereof, from a CD8 coreceptor a-chain, or a functional variant or portion thereof.
  • a stimulatory or costimulatory signal provided by a presently disclosed polypeptide to a host cell can comprise one or more effect, and/or substantially the same effect(s), as can result from an interaction between a native costimulatory polypeptide of the host cell and one or more cognate molecule; e.g., between CD28 and CD80, or between CD28 and CD86
  • a stimulatory signal can be inferred or recognized by activation or function (e.g., cytotoxic activity, proliferation, or the like) of a host cell.
  • a stimulatory signal can be inferred or recognized by an improved function or increased activation by a host cell (e.g., an immune cell, such as a T cell, for example a CD4+ T cell or a CD8 + T cell) as compared to a reference host cell that does not comprise the polypeptide or polypeptides.
  • a host cell and a reference host cell that each express an antigen-specific T cell receptor may be exposed to the antigen (e.g., pulsed with antigen-peptide, cultured with antigen- presenting cells or antigen-expressing target cells) and a stimulatory signal may be recognized by, for example, increased or improved (e.g., sustained) proliferation of the host cell comprising the polypeptide(s) of the present disclosure, increased or improved production of one or more cytokines (e.g., IFN-y, TNFa, IL-2, or any combination thereof) by the host cell comprising the polypeptide(s) of the present disclosure, increased or improved cytotoxic activity against target cells by the host cell comprising the polypeptide(s) of the present disclosure, increased or improved expression of one or more activation markers, such as Nur77, or the like, as compared to the reference host cell.
  • cytokines e.g., IFN-y, TNFa, IL-2, or any combination thereof
  • a polypeptide or plurality (e.g., a pair) of polypeptides of the present disclosure may be said to provide a stimulatory signal when a host cell expressing the same demonstrates one or more improved function or increased activation, as compared to a reference host cell.
  • a polypeptide may perform a recited function when in association with a further polypeptide.
  • a polypeptide comprising an extracellular component from a CD8a is capable of binding to a MHC Class-I molecule when the polypeptide is comprised in a polypeptide multimer (e.g., homodimer or a heterodimer) with another CD8 polypeptide, such as a CD8P polypeptide or a CD8a polypeptide, including for example a chimeric coreceptor polypeptide of the present disclosure.
  • preferred MHC Class- I-binding preferred provision of a stimulatory signal, association with Lek, or any combination thereof, may occur when a CD8 polypeptide of the present disclosure is comprised in a dimer with a further CD8 polypeptide, which may be wild-type or may be engineered.
  • a polypeptide comprises a variant of a CD28 costimulatory domain comprising one or more amino acid mutations (e.g., substitutions) to reduce (e.g., lower the intensity, duration, and/or frequency) of a stimulatory signal provided thereby, as compared to the stimulatory signal provided by a reference polypeptide comprising a wild-type CD28 costimulatory domain, or comprising a reference CD28 costimulatory domain that does not comprise the one or more amino acid mutations.
  • amino acid mutations e.g., substitutions
  • a reference host cell is a host cell that is identical to (at least according to one or more criteria of interest), or is substantially identical to, the subject host cell with the exception of the identified differences.
  • a host cell may be a CD4+ T cell from a human subject
  • a reference host cell may be a CD4+ T cell from the same human subject (such as, for example, a reference host cell may be obtained from the same blood sample as the host cell).
  • one or more of an extracellular component, a binding domain, a linker, a transmembrane domain, an intracellular component, or a costimulatory domain comprises one or more junction amino acids.
  • junction amino acids or “junction amino acid residues” refer to one or more (e.g., about 2-20) amino acid residues between two adjacent domains, motifs, regions, modules, or fragments of a protein, such as between a binding domain and an adjacent linker, between a transmembrane domain and an adjacent extracellular or intracellular domain, or on one or both ends of a linker that links two domains, motifs, regions, modules, or fragments (e.g., between a linker and an adjacent binding domain or between a linker and an adjacent hinge).
  • junction amino acids may result from the construct design of a fusion protein (e.g., amino acid residues resulting from the use of a restriction enzyme site or self-cleaving peptide sequences during the construction of a polynucleotide encoding a fusion protein).
  • a transmembrane domain of a fusion protein may have one or more junction amino acids at the amino-terminal end, carboxy -terminal end, or both.
  • the present disclosure provides fusion proteins that comprise: (i) an extracellular component comprising an extracellular domain from a CD8 co-receptor P-chain or a functional portion or variant thereof, or from a CD8 co-receptor a-chain or a functional portion or variant thereof, that is capable of binding to a MHC class I molecule; (ii) a transmembrane domain, provided that the transmembrane domain is not a transmembrane domain from a CD8 co-receptor a-chain when the extracellular component comprises a full length extracellular domain from the CD8 co-receptor a-chain; and (iii) an intracellular component comprising a variant of a CD28 co-stimulatory domain.
  • the extracellular component of a polypeptide or fusion protein comprises or is derived from a CD8 co-receptor P-chain, or a functional portion or variant thereof.
  • the CD8 co-receptor P-chain comprises a canonical P-chain, a Ml isoform, a M2 isoform, a M3 isoform, a M4 isoform, a M5 isoform, a M6 isoform, a M7 isoform, or a M8 isoform.
  • the CD8 co-receptor P-chain is a Ml isoform.
  • the extracellular component comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO:7, or comprises or consists of the amino acid sequence set forth in SEQ ID NO:7.
  • the transmembrane domain comprises or consists of a transmembrane domain from a CD4, a CD8P, a CD8a, a CD27, or a CD28, or a functional portion or variant thereof.
  • the transmembrane domain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO:8, or comprises or consists of the amino acid set forth in SEQ ID NO:8.
  • the fusion protein further comprises an amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 10, or a functional portion or variant thereof, disposed between the transmembrane domain and the intracellular component (e.g., costimulatory domain).
  • the extracellular component comprises the amino acid sequence set forth in SEQ ID NO:7 and the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 8.
  • the intracellular component comprises the amino acid sequence set forth in SEQ ID NO.: 10, optionally as set forth in SEQ ID NO.:9.
  • the extracellular component comprises or is derived from a CD8 co-receptor a-chain.
  • the CD8 co-receptor a-chain may comprise or be derived from a canonical a-chain, isoform 2, or isoform 3.
  • the co- stimulatory domain of a fusion protein comprises a co-stimulatory domain from CD28 and/or comprises a functional variant of a costimulatory domain thereof.
  • an intracellular component of a fusion protein comprises a variant of SEQ ID NO. : 19 comprising any one or more of Motifs 1-4 e.g., according to any one of Constructs BB-NN) as set forth in Table 6 or Table 7.
  • the co-stimulatory domain comprises a co-stimulatory domain from CD28, or a functional portion or variant thereof.
  • the co-stimulatory domain comprises or consists of an amino acid sequence having at least 80% at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO: 19.
  • the co-stimulatory domain comprises or consists of an amino acid sequence shown in SEQ ID NO: 19.
  • the co-stimulatory domain comprises a variant of the amino acid sequence shown in SEQ ID NO: 19, wherein one or both of the leucine residues at positions 7 and 8 of SEQ ID NO: 19 is substituted for a different amino acid.
  • the variant of the amino acid sequence shown in SEQ ID NO: 19 comprises a substitution of a glycine for one or both of the leucine residues at positions 7 and 8 of SEQ ID NO: 19.
  • the co- stimulatory domain comprises or consists of an amino acid sequence having at least 80% identity to the amino acid sequence the amino acid sequence shown in SEQ ID NO:20.
  • the co-stimulatory domain comprises or consists of the amino acid sequence shown in SEQ ID NO:20.
  • fusion proteins that comprise: (i) an extracellular component comprising an extracellular domain from a CD8 co-receptor P-chain or a functional portion or variant thereof, or from a CD8 co-receptor a-chain or a functional portion or variant thereof, that is capable of binding to a MHC class I molecule; (ii) a transmembrane domain; and (iii) an intracellular component comprising a variant sequence of CD28 comprising or consisting of an amino acid sequence having at least 80% (i.e.
  • the extracellular component comprises or is derived from a CD8 co-receptor P-chain, or a functional portion or variant thereof.
  • the CD8 co-receptor P-chain comprises a canonical P-chain, a Ml isoform, a M2 isoform, a M3 isoform, a M4 isoform, a M5 isoform, a M6 isoform, a M7 isoform, or a M8 isoform.
  • the CD8 co-receptor P-chain is a Ml isoform.
  • the extracellular component comprises an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:7 , or comprises or consists of the amino acid sequence set forth in SEQ ID NO:7.
  • the transmembrane domain comprises or consists of a transmembrane domain from a CD4, a CD8P, a CD8a, a CD27, or a CD28, or a functional portion or variant thereof.
  • the transmembrane domain comprises an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:8 , or comprises or consists of the amino acid set forth in SEQ ID NO: 8.
  • the fusion protein further comprises the amino acid sequence set forth in SEQ ID NO: 10, or a functional portion or variant thereof, disposed between the transmembrane domain and the intracellular component (e.g. costimulatory domain).
  • the extracellular component comprises the amino acid sequence set forth in SEQ ID NO:7 and the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 8.
  • the intracellular component comprises the amino acid sequence set forth in SEQ ID NO.: 10, optionally as set forth in SEQ ID NO.:9.
  • the extracellular component comprises or is derived from a CD8 co-receptor a-chain.
  • the CD8 co-receptor a-chain comprises a canonical a-chain, isoform2, or isoform 3.
  • the extracellular component comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in set forth in SEQ ID NO: 2.
  • the transmembrane domain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO:3, or comprises or consists of the amino acid sequence set forth in SEQ ID NO:3.
  • the variant sequence of CD28 comprises a substitution of a glycine for one or both of the leucine residues corresponding to positions 7 and 8 of SEQ ID NO: 19.
  • the co-stimulatory domain comprises or consists an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO:20.
  • the co-stimulatory domain comprises or consists of the amino acid sequence shown in SEQ ID NO:20.
  • polypeptide that comprises: (i) an extracellular component from a CD8P; (ii) a transmembrane domain that is optionally from a CD8P; and (iii) an intracellular component comprising (iii)(l) a CD8P intracellular region amino acid sequence comprising or consisting of SEQ ID NO.:9 or SEQ ID NO.: 10 and (iii)(2) a CD28 intracellular region amino acid sequence comprising a CD28 costimulatory domain and, optionally, a LL->GG mutation, wherein the polypeptide is capable of binding to a MHC Class I molecule.
  • polypeptide that comprises: (i) an extracellular component from a CD8P; (ii) a transmembrane domain that is optionally from a CD8P; and (iii) an intracellular component comprising a CD28 costimulatory domain and an optional LL->GG mutation, wherein the polypeptide is capable of binding to a MHC Class I molecule.
  • polypeptide that comprises: (i) an extracellular component from a CD8a; (ii) a transmembrane domain that is optionally from a CD8a; and (iii) an intracellular component comprising a CD28 costimulatory domain and an optional LL->GG mutation, wherein the polypeptide is capable of binding to a MHC Class I molecule.
  • polypeptide that comprises: (i) an extracellular component from a CD8P; (ii) a transmembrane domain that is optionally from a CD8P; and (iii) an intracellular component comprising (1) a CD28 costimulatory domain comprising the amino acid sequence DAMNMTARRAGPTRKHYQAYAAPRDFAAYRS (SEQ ID NO.: 185) and (2) (1) an optional LL- GG mutation, wherein the polypeptide is capable of binding to a MHC Class I molecule.
  • polypeptide that comprises: (i) an extracellular component from a CD8a; (ii) a transmembrane domain that is optionally from a CD8a; and (iii) an intracellular component comprising (1) a CD28 costimulatory domain comprising the amino acid sequence DAMNMTARRAGPTRKHYQAYAAPRDFAAYRS (SEQ ID NO. : 185) and (2) an optional LL- GG mutation, wherein the polypeptide is capable of binding to a MHC Class I molecule.
  • polypeptide that comprises: (i) an extracellular component from a CD8P; (ii) a transmembrane domain that is optionally from a CD8P; and (iii) an intracellular component comprising (iii)(l) a CD8P intracellular region amino acid sequence comprising or consisting of SEQ ID NO.:9 or SEQ ID NO.: 10 and (iii)(2) a(n, optionally wild-type) CD28 costimulatory domain amino acid sequence, wherein the polypeptide is capable of binding to a MHC Class I molecule.
  • polypeptide that comprises: (i) an extracellular component from a CD8a; (ii) a transmembrane domain from a CD28; and (iii) an intracellular component comprising a CD28 costimulatory domain and, optionally, a LL- GG mutation, wherein the polypeptide is capable of binding to a MHC Class I molecule.
  • polypeptide that comprises: (i) an extracellular component from a CD8a; (ii) a transmembrane domain that is optionally from a CD8a; and (iii) an intracellular component comprising a CD28 costimulatory domain comprising the amino acid sequence DAMNMTARRAGPTRKHFQAFAAPRDFAAFRS (SEQ ID NO. : 186) and optionally further comprising a LL- GG mutation, wherein the polypeptide is capable of binding to a MHC Class I molecule.
  • polypeptide that comprises: (i) an extracellular component from a CD8P; (ii) a transmembrane domain that is optionally from a CD8P; and (iii) an intracellular component comprising a CD28 costimulatory domain comprising the amino acid sequence DAMNMTARRAGPTRKHFQAFAAPRDFAAFRS (SEQ ID NO.X) and optionally further comprising a LL- GG mutation, wherein the polypeptide is capable of binding to a MHC Class I molecule.
  • polypeptide that comprises: (i) an extracellular component comprising (i)(l) a CD8a extracellular region amino acid sequence e.g.
  • the CD28 extracellular region amino acid sequence of (i)(3) comprises or consists of amino acids 141-159 of SEQ ID NO.:141.
  • polypeptide that comprises: (i) an extracellular component comprising (i)(l) a CD8a extracellular region amino acid sequence e.g. comprising or consisting of a CD8a Ig V-like domain), (i)(2) a CD8P stalk region amino acid sequence, and (i)(3) a CD28 extracellular region amino acid sequence; (ii) a transmembrane domain from CD28; and (iii) an intracellular component comprising (iii)(l) a CD28 costimulatory domain and an optional LL- GG mutation and (iii)(2) a CD8a intracellular signaling domain, wherein the polypeptide is capable of binding to a MHC Class I molecule.
  • the CD28 extracellular region amino acid sequence of (i)(3) comprises or consists of amino acids 141-159 of SEQ ID NO.: 141.
  • a polypeptide e.g., a fusion protein
  • a referenced component from one source protein e.g. a transmembrane domain, a costimulatory domain, a signaling domain, or the like
  • a polypeptide can comprise additional amino acid sequence or sequences from the said source protein; i.e., the extent to which amino acid sequence from the said source protein is present in the polypeptide or fusion protein may, in certain embodiments, surpass the specifically referenced component or portion thereof, unless the context clearly provides otherwise.
  • any of the presently disclosed fusion proteins, components, or domains can comprise one or more junction amino acids.
  • engineered CD8-containing polypeptides can comprise chimeric sequences or amino acid substitutions derived from different CD8 protein isoforms (e.g., a CD8P fusion protein may comprise amino acid sequences from two or more CD8P isoforms), and/or can comprise one or more amino acid sequence from a CD8P and one or more amino acid sequence from a CD8a.
  • all two-protein combinations of a CD8P-containing protein with a cognate CD8a-containing protein (or of two CD8a-containing proteins) are encompassed, including combinations wherein one or neither of the two proteins is a(n optionally, full-length (with or without signal peptide)) wild-type CD8P or CD8a, respectively.
  • a polypeptide comprises an amino acid sequence having 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%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence set forth in any one of SEQ ID NOs.: 36-42, 83-84, and 90-95.
  • a polypeptide comprises or consists of two or more amino acid sequences, each of the two or more amino acid sequences independently having 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%, or at least 99% identity to, or comprising or consisting of, an amino acid sequence set forth in any one of SEQ ID NOs.: 36-42, 83-84, and 90-95.
  • a polypeptide comprises an amino acid sequence having 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%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence set forth in any one of SEQ ID NOs.: 113, 115-118, 120-127, 136, and ISO- 153.
  • T cell that expresses (i) any one or more of the presently disclosed fusion proteins, polypeptides, or a combination of these and (ii) an antigen-specific T cell receptor (TCR) (e.g., a MHC-I-restricted TCR), is capable of any one or more of the following (l)-(7), as compared to a reference T cell that expresses (ii) and optionally expresses wild-type CD8aP, but does not express (i): (1) increased proliferation (as determined by cell division) in the presence of antigenexpressing cells; (2) increased production of interferon-gamma (IFN-y) in the presence of antigen, wherein the increased production is optionally an increase of 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 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 9
  • the T cell is a CD4+ T cell. In certain embodiments, the T cell is a CD8+ T cell. In certain embodiments, the T cell is a primary human T cell obtained from a donor, such as a healthy donor.
  • polynucleotides that encode any one or more (including, in some contexts, two or more) of the presently disclosed fusion proteins or polypeptides.
  • a polynucleotide can comprise, or be comprised in, an expression construct wherein the polynucleotide is operably linked to an expression control sequence (e.g., a promoter).
  • a polynucleotide or expression construct encodes two or more polypeptides and the nucleic acid sequences encoding the two or more polypeptides may be separated by (or have disposed therebetween) nucleic acid sequences that encode a self-cleaving peptide, a furin cleavage sequence, an internal ribosomal skip element (IRES), or any combination thereof.
  • nucleic acid sequences that encode 2A self-cleaving peptides are provided in SEQ ID NOs.:50-54.
  • Examples of 2A self-cleaving peptide amino acid sequences are provided in SEQ ID NOs.:55-58; in the case of SEQ ID NOs.:55 and 58, a short linker (amino acid sequence G-S-G) is disposed N-terminal to the self-cleaving peptide sequence; any self-cleaving peptide may be provided with a N-terminal GSG linker.
  • a short linker amino acid sequence G-S-G
  • the polynucleotide is codon-optimized for expression in a host cell.
  • Codon optimization can be performed using known techniques and tools, e.g., using the GenScript® OptimiumGeneTM tool; see also Scholten et a!.. Clin. Immunol. 119: 135, 2006).
  • Codon-optimized sequences include sequences that are partially codon-optimized (i.e., one or more codon is optimized for expression in the host cell) and those that are fully codon-optimized.
  • an isolated polynucleotide comprises a polynucleotide that encodes a first fusion protein of the present disclosure, wherein the first encoded fusion protein comprises an extracellular domain from a CD8 co-receptor P-chain, or a functional portion or variant thereof.
  • the isolated polynucleotide further comprises a polynucleotide encoding a second protein, wherein the second encoded protein (which can be a fusion protein, such as a fusion protein of the present disclosure) comprises: (i) a CD8 co-receptor a-chain, or a functional portion or variant thereof; or (ii) an extracellular domain from a CD8 co-receptor a-chain, or a functional portion or variant thereof.
  • the second encoded protein which can be a fusion protein, such as a fusion protein of the present disclosure
  • the second encoded protein comprises: (i) a CD8 co-receptor a-chain, or a functional portion or variant thereof; or (ii) an extracellular domain from a CD8 co-receptor a-chain, or a functional portion or variant thereof.
  • an isolated polynucleotide comprises a polynucleotide that encodes a first fusion protein of the present disclosure, wherein the first encoded fusion protein comprises an extracellular domain from a CD8 co-receptor a-chain, or a functional portion or variant thereof.
  • the isolated polynucleotide further comprises a polynucleotide encoding a second protein (which can be a fusion protein, such as a fusion protein of the present disclosure), wherein the second encoded protein comprises: (i) a CD8 co-receptor a-chain, or a functional portion or variant thereof; (ii) an extracellular domain from a CD8 co-receptor a-chain or a functional portion or variant thereof; or (iii) a CD8 co-receptor P chain, or a functional portion or variant thereof; or (iv) an extracellular domain from a CD8 co- receptor P-chain, or a functional portion or variant thereof.
  • a second protein which can be a fusion protein, such as a fusion protein of the present disclosure
  • a polynucleotide (or vector or host cell, as disclosed herein) comprises a polynucleotide comprising: (a) a polynucleotide encoding a protein comprising at least an extracellular portion of a CD8 co-receptor a chain (which protein may be a wild-type CD8a or any polypeptide or fusion protein as described herein that comprises extracellular amino acid sequence from a CD8a); (b) a polynucleotide encoding a protein comprising at least an extracellular portion of a CD8 co-receptor P chain (which protein may be a wild-type CD8P or any polypeptide or fusion protein as described herein that comprises extracellular amino acid sequence from a CD8P); and (c) a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide of (a) and the polynucleotide of (b).
  • a polynucleotide comprises a polynucleotide that encodes a self-cleaving peptide and is disposed between: (1) a polynucleotide encoding a binding protein (e.g., TCR of the present disclosure) and a polynucleotide of (a); and/or (2) a polynucleotide encoding a binding protein and a polynucleotide of (b).
  • a binding protein e.g., TCR of the present disclosure
  • a polynucleotide can comprise, operably linked inframe: (i) (pnCD8a)-(pnSCPl)-(pnCD8P)-(pnSCP2)-(pnTCR); (ii) (pnCD8P)- (pnSCPl)-(pnCD8a)-(pnSCP2)-(pnTCR); (iii) (pnTCR)-(pnSCPl)-(pnCD8a)- (pnSCP2)-(pnCD8p); (iv) (pnTCR)-(pnSCPl)-(pnCD8p)-(pnSCP2)-(pnCD8a); (v) (pnCD8a)-(pnSCPl)-(pnTCR)-(pnSCP2)-(pnCD8P); or (vi) (pnCD8P)-(pnSCPl)- (pnTCR)--(pnT
  • Constructs BB-NN wherein pnTCR is the polynucleotide encoding a TCR, and wherein pnSCPl and pnSCP2 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and/or the encoded self-cleaving peptides are optionally the same or different (e.g, P2A, T2A, F2A, E2A).
  • the encoded TCR comprises a TCRa chain and a TCRP chain
  • the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide encoding a TCRa chain and the polynucleotide encoding a TCRP chain.
  • the polynucleotide comprises, operably linked in-frame: (i) (pnCD8a)-(pnSCPi)-(pnCD8P)-(pnSCP2)- (pnTCRP)-(pnSCP3)-(pnTCRa); (ii) (pnCD8P)-(pnSCPi)-(pnCD8a)-(pnSCP2)- (pnTCRP)-(pnSCP3)-(pnTCRa); (iii) (pnCD8a)-(pnSCPi)-(pnCD8P)-(pnSCP2)- (pnTCRa)-(pnSCP3)-(pnTCRP); (iv) (pnCD8P)-(pnSCPi)-(pnCD8a)-(pnSCP2)- (pnTCRa)-(pnSCP3)-(pnTCRP); (v) (pnTCRP)-(pn
  • expression constructs are provided, wherein the expression constructs comprise a polynucleotide of the present disclosure operably linked to an expression control sequence (e.g., a promoter).
  • the expression construct is comprised in a vector.
  • An exemplary vector may comprise a polynucleotide capable of transporting another polynucleotide to which it has been linked, or which is capable of replication in a host organism.
  • polynucleotides of the present disclosure may be operatively linked to certain elements of a vector. For example, polynucleotide sequences that are needed to effect the expression and processing of coding sequences to which they are ligated may be operatively linked.
  • Expression control sequences may include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and possibly sequences that enhance protein secretion.
  • Expression control sequences may be operatively linked if they are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
  • Some examples of vectors include plasmids, viral vectors, cosmids, and others. Some vectors may be capable of autonomous replication in a host cell into which they are introduced (e.g.
  • bacterial vectors having a bacterial origin of replication and episomal mammalian vectors
  • other vectors may be integrated into the genome of a host cell or promote integration of the polynucleotide insert upon introduction into the host cell and thereby replicate along with the host genome (e.g., lentiviral vector, retroviral vector).
  • some vectors are capable of directing the expression of genes to which they are operatively linked (these vectors may be referred to as "expression vectors").
  • agents e.g., polynucleotides encoding proteins as described herein
  • each agent may reside in separate or the same vectors, and multiple vectors (each containing a different agent or the same agent) may be introduced to a cell or cell population or administered to a subject.
  • the vector comprises a plasmid vector or a viral vector (e.g., a vector selected from lentiviral vector or a y-retroviral vector).
  • Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as ortho-myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses such as picomavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox).
  • herpesvirus
  • viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.
  • retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).
  • “Retroviruses” are viruses having an RNA genome, which is reverse-transcribed into DNA using a reverse transcriptase enzyme, the reverse-transcribed DNA is then incorporated into the host cell genome.
  • “Gammaretrovirus” refers to a genus of the retroviridae family. Examples of gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses.
  • Lentiviral vector means HIV-based lentiviral vectors for gene delivery, which can be integrative or non-integrative, have relatively large packaging capacity, and can transduce a range of different cell types. Lentiviral vectors are usually generated following transient transfection of three (packaging, envelope and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter the target cell through the interaction of viral surface glycoproteins with receptors on the cell surface. On entry, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is a double-stranded linear viral DNA, which is the substrate for viral integration into the DNA of infected cells.
  • the viral vector can be a gammaretrovirus, e.g., Moloney murine leukemia virus (MLV)-derived vectors.
  • the viral vector can be a more complex retrovirus-derived vector, e.g., a lentivirus-derived vector. HIV-l-derived vectors belong to this category.
  • Other examples include lentivirus vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (ovine lentivirus).
  • TCR transgenes are known in the art and have been previous described, for example, in: U.S. Patent 8,119,772; Walchli et al., PLoS One 6 327939, 2011; Zhao et al., J. Immunol. 174 AMS, 2005; Engels et al., Hum. Gene Ther. 77:1155, 2003; Frecha c/ a/., Mol. Ther. 18A7M3, 2010; and Verhoeyen et al., Methods Mol. Biol. 506:97, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available.
  • viral vectors also can be used for polynucleotide delivery including DNA viral vectors, including, for example adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex viruses (HSVs), including amplicon vectors, replicationdefective HSV and attenuated HSV (Krisky et al., Gene Ther. 5: 1517, 1998).
  • HSVs herpes simplex viruses
  • Other vectors can be used with the compositions and methods of this disclosure.
  • Such vectors include those derived from baculoviruses and a-viruses. (Jolly, D J. 1999. Emerging Viral Vectors, pp 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as sleeping beauty or other transposon vectors).
  • the viral vector may also comprise additional sequences between the two (or more) transcripts allowing for bicistronic or multi ci str onic expression.
  • additional sequences used in viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptide, or any combination thereof.
  • a polynucleotide in each recombinant expression construct includes at least one appropriate expression control sequence (also called a regulatory sequence), such as a leader sequence and particularly a promoter operably (i.e., operatively) linked to the nucleotide sequence encoding the polypeptide of interest.
  • a regulatory sequence also called a regulatory sequence
  • Table 2 provides amino acid sequences of components of these (one- or two- polypeptide) constructs.
  • a polypeptide comprises: (i) an extracellular component comprising an amino acid sequence having 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%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence set forth in any one of SEQ ID NOs.:2, 173, 90, 92, and 7; (ii) a transmembrane domain comprising an amino acid sequence having 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%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence set forth in any one of SEQ ID NOs.:3, 8, and 80; and (iii) an intracellular component comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 9
  • a polypeptide that comprises an extracellular component, a transmembrane domain, and an intracellular component according to Polypeptide 1 of any one of Constructs A-AA in Table 2.
  • a polypeptide is provided that comprises an extracellular component, a transmembrane domain, and an intracellular component according to Polypeptide 2 of any one of Constructs A-AA in Table 2.
  • a polynucleotide or vector that encodes the polypeptide, and/or a host cell that expresses and/or encodes the polypeptide is provided.
  • a polynucleotide or vector encodes or comprise two or more such polypeptides that are different from one another.
  • a polynucleotide or vector encodes: (i) a polypeptide that comprises an extracellular component, a transmembrane domain, and an intracellular component according to Polypeptide 1 of any one of Constructs A-AA in Table 2; and (ii) a polypeptide that comprises an extracellular component, a transmembrane domain, and an intracellular component according to Polypeptide 2 of any one of Constructs A-AA in Table 2.
  • a polynucleotide or vector encodes the first polypeptide and the second polypeptide of any one of Constructs A-AA in Table 2, wherein, optionally, one or both of the encoded polypeptides may not comprise a signal peptide according to Table 2.
  • Table 3 provides amino acid sequences of certain polypeptide constructs (Constructs A-NN (with or
  • Construct Y was used as a positive control and reference in certain experiments described in the Examples. Another construct was generated that encoded wild-type, full-length human CD8a and CD8P separated by a P2A coding sequence; this construct was also used as a control in certain experiments described in the Examples. Table 5 provides certain additional embodiments of polypeptides (Polypeptide
  • a polypeptide is provided, wherein the polypeptide is Polypeptide 1 or Polypeptide 2 of any one of Constructs A-NN in Table 3 or Table 4.
  • a polynucleotide is provided that encodes, and/or a host cell is provided that expresses, the polypeptide.
  • a polynucleotide encodes, and/or a host cell expresses, Polypeptide 1 of any one of Constructs A-NN in Table 3 or Table 4 and Polypeptide 2 of any one of Constructs A-NN in Table 3 or Table 4.
  • a polynucleotide encodes, and/or a host cell expresses, Polypeptide 1 and Polypeptide 2 of any one of Constructs A-NN in Table 3 or Table 4.
  • polypeptides that comprise (i) an extracellular domain from CD8P, or a functional variant thereof, (ii) a transmembrane domain that is optionally from CD8P, and (iii) an intracellular domain comprising a variant of a CD28 intracellular amino acid sequence.
  • the variant can comprise or consist of one or more substitution mutations in an underlined portion of SEQ ID NO.: 19, below:
  • the variation comprises or consists of: (i) a diglycine motif in place of a dileucine motif; (ii) a mutated and optionally non-functional PI3K binding site; (iii) a mutated and optionally non-functional ITK and GADS binding site; (iv) a mutated and optionally non-functional LCK binding site; or (v) any combination of (i)-(v).
  • the variation comprises (i) and any one or more of (ii)-(v).
  • the polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92%, or at least 95% to SEQ ID NO. : 19.
  • the intracellular domain of the polypeptide comprises an amino acid sequence according to SEQ ID NO.:205 or 206.
  • substitutions mutations may be made to disrupt (e.g. reduce or abrogate) a function of e.g. a PI3K binding site, ITK and GADS binding site, and a LCK binding site.
  • a substitution mutation can comprise a nonconservative substitution.
  • a substitution mutation can comprise a replacement of an amino acid having a large side chain (e.g. tryptophan or phenylalanine) with an amino acid having a smaller side chain (e.g. alanine or serine), or vice versa. Any mutation or mutations sufficient to disrupt a function of one or more of the aforementioned sites in a CD28 intracellular sequence may be used.
  • Table 6 summarizes non-limiting examples of variant sequence motifs according to Constructs BB-NN.
  • the positions in SEQ ID NO.: 19 corresponding to the “LL” dileucine motif are referred-to as “Motif 1” in Table 6.
  • the positions in SEQ ID NO.: 19 corresponding to the motif YMNM are referred-to as “Motif 2” in Table 6.
  • the positions in SEQ ID NO.: 19 corresponding to the motif PRRP are referred-to as “Motif 3” in Table 6.
  • the positions in SEQ ID NO. : 19 corresponding to the motif PYAP are referred-to as “Motif 4” in Table 6.
  • the intracellular portions of these CD8P-CD28 fusions can also include, N-terminal to the CD28-derived sequence, the CD8P sequence HLCCRR.
  • the extracellular domain and transmembrane domain of the CD8P-CD28 fusions in Table 6 are from human CD8p.
  • Constructs BB-NN also comprise the wildtype CD8a co-receptor (comprising SEQ ID NO. : 114).
  • a polypeptide comprises Motif 1, Motif 2, Motif 3, and/or Motif 4 in accordance with Table 6.
  • a polypeptide can, as an alternative Motif 2, comprise the sequence FMNM.
  • a polypeptide comprises, consists essentially of, or consists of the amino acid sequence set forth in any one of SEQ ID Nos.: 192-204.
  • a polypeptide comprises, consists essentially of, or consists of an extracellular domain, a transmembrane domain, and an intracellular domain, as shown under “2 protein size [aa]” in any one of Figures 27-39.
  • polynucleotides that encode any one or more of the aforementioned polypeptides.
  • a polynucleotide further encodes a CD8a polypeptide, optionally comprising or consisting of the amino acid sequence set forth in SEQ ID NO. : 114.
  • vectors and host cells that comprise a polynucleotide.
  • host cells encode and optionally express any one or more of the aforementioned polypeptides.
  • a host cell further encodes and optionally expresses a CD8a polypeptide, optionally comprising or consisting of the amino acid sequence set forth in SEQ ID NO.: 114.
  • a host cell can comprise, for example, a T cell, such as a CD4+ T cell, a CD8 + T cell, or both.
  • T host cell can further express a T cell receptor or CAR.
  • the TCR or CAR can comprise an antigenspecific MHC-I-restricted binding domain.
  • compositions that comprise any one of the aforementioned polynucleotides, vectors, host cells, or any combination thereof, and a pharmaceutically acceptable carrier, excipient, or diluent. Also provided are methods of using any one of the aforementioned polynucleotides, vectors, host cells, compositions, or any combination thereof, to treat a disease or disorder, or to manufacture a medicament of treating a disease or disorder.
  • a protein of the present disclosure comprises a protein tag.
  • Protein tags are unique peptide sequences that are affixed or genetically fused to, or are a part of, a protein of interest and can be recognized or bound by, for example, a heterologous or non-endogenous cognate binding molecule or a substrate (e.g., receptor, ligand, antibody, carbohydrate, or metal matrix) or a protein of this disclosure.
  • Protein tags can be useful for detecting, identifying, isolating, tracking, purifying, enriching for, targeting, or biologically or chemically modifying tagged proteins of interest, particularly when a tagged protein is part of a heterogeneous population of cell proteins or cells (e.g., a biological sample like peripheral blood).
  • a protein tag of a protein of this disclosure comprises a Myc tag, His tag, Flag tag, Xpress tag, Avi tag, Calmodulin tag, Polyglutamate tag, HA tag, Nus tag, S tag, SBP tag, Softag, V5 tag, CBP, GST, MBP, GFP, Thioredoxin tag, Strep® tag, or any combination thereof.
  • a polynucleotide encoding any (e.g. fusion) protein (or any two or more of these) of this disclosure can, for example, be inserted into an appropriate vector (e.g., viral vector or non-viral plasmid vector) for introduction into a host cell of interest (e.g., an immune cell, such as a T cell).
  • a polynucleotide or polynucleotides of the present disclosure is/are used to transfect/transduce a host cell (e.g., a T cell) for use in adoptive transfer therapy (e.g., targeting a cancer antigen.
  • adoptive transfer therapy e.g., targeting a cancer antigen.
  • Patent Application Pub. No. US 2004/0087025) as have adoptive transfer procedures using T cells of desired target-specificity (e.g., Schmitt et al., Hum. Gen. 20: 1240, 2009; Dossett c/ a/., Mol. Ther. 17:7 2, 2009; Till et al., Blood 112'.2261, 2008; Wang et al., Hum. Gene Ther. 18:712, 2007; Kuball et al., Blood 109:2331, 2007; US 2011/0243972; US 2011/0189141; Leen etal., Ann. Rev. Immunol.
  • desired target-specificity e.g., Schmitt et al., Hum. Gen. 20: 1240, 2009; Dossett c/ a/., Mol. Ther. 17:7 2, 2009; Till et al., Blood 112'.2261, 2008; Wang et al., Hum. Gene Ther. 18:712, 2007; Kuball
  • host cells comprise a polynucleotide or vector of the present disclosure and can express the encoded protein or proteins.
  • markers can be used to identify, monitor or isolate a host cell transduced with a heterologous polynucleotide encoding a(n e.g. fusion) protein as provided herein.
  • Exemplary markers include green fluorescent protein, an extracellular domain of human CD2, a truncated human EGFR (huEGFRt, (see Wang et al., Blood 118: 1255, 2011), a truncated human CD19 (huCD19t); a truncated human CD34 (huCD34t); or a truncated human NGFR (huNGFRt).
  • an encoded marker comprises EGFRt, CD19t, CD34t, or NGFRt.
  • the host cell is a hematopoietic progenitor cell or a human immune system cell.
  • a "hematopoietic progenitor cell”, as referred to herein, is a cell that can be derived from hematopoietic stem cells or fetal tissue and is capable of further differentiation into mature cells types (e.g., immune system cells).
  • Exemplary hematopoietic progenitor cells include those with a CD24 Lo Lin- CD117 + phenotype or those found in the thymus (referred to as progenitor thymocytes).
  • an "immune system cell” means any cell of the immune system that originates from a hematopoietic stem cell in the bone marrow, which gives rise to two major lineages, a myeloid progenitor cell (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) and a lymphoid progenitor cell (which give rise to lymphoid cells such as T cells, B cells, natural killer (NK) cells, and NK-T cells).
  • a myeloid progenitor cell which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes
  • lymphoid progenitor cell which give rise to lymphoid cells such as T cells, B cells, natural killer (NK) cells, and NK-T cells.
  • Exemplary immune system cells include a CD4 + T cell, a CD8 + T cell, a CD4" CD8" double negative T cell, a y6 T cell, a regulatory T cell, a stem cell memory T cell, a natural killer cell (e.g., a NK cell or a NK-T cell), a B cell, and a dendritic cell.
  • Macrophages and dendritic cells may be referred to as "antigen presenting cells" or "APCs,” which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with a TCR on the surface of a T cell.
  • MHC major histocompatibility complex
  • T cell or "T lymphocyte” is an immune system cell that matures in the thymus and produces T cell receptors (TCRs).
  • T cells can be naive (not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD 127, and CD45RA, and decreased expression of CD45RO as compared to TCM), memory T cells (TM) (antigen-experienced and long-lived), and effector cells (antigen-experienced, cytotoxic).
  • TM can be further divided into subsets of central memory T cells (TCM, increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and decreased expression of CD54RA as compared to naive T cells) and effector memory T cells (TEM, decreased expression of CD62L, CCR7, CD28, CD45RA, and increased expression of CD127 as compared to naive T cells or TCM).
  • TCM central memory T cells
  • TEM effector memory T cells
  • Effector T cells refers to antigen-experienced CD8 + cytotoxic T lymphocytes that have decreased expression of CD62L ,CCR7, CD28, and are positive for granzyme and perforin as compared to TCM.
  • Helper T cells are CD4 + cells that influence the activity of other immune cells by releasing cytokines.
  • CD4 + T cells can activate and suppress an adaptive immune response, and which of those two functions is induced will depend on presence of other cells and signals.
  • T cells can be collected using known techniques, and the various subpopulations or combinations thereof can be enriched or depleted by known techniques, such as by affinity binding to antibodies, flow cytometry, or immunomagnetic selection.
  • Other exemplary T cells include regulatory T cells, such as CD4 + CD25 + (Foxp3 + ) regulatory T cells and Tregl7 cells, as well as Tri, Th3, CD8 + CD28‘, and Qa-1 restricted T cells.
  • enriched or “depleted” with respect to amounts of cell types in a mixture refers to an increase in the number of the "enriched” type, a decrease in the number of the “depleted” cells, or both, in a mixture of cells resulting from one or more enriching or depleting processes or steps.
  • a mixture or composition may contain 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more (in number or count) of the "enriched" cells.
  • Cells subjected to a depleting process can result in a mixture or composition containing 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% percent or less (in number or count) of the "depleted" cells.
  • amounts of a certain cell type in a mixture will be enriched and amounts of a different cell type will be depleted, such as enriching for CD4 + cells while depleting CD8 + cells, or enriching for CD62L + cells while depleting CD62L- cells, or combinations thereof.
  • Cells of T cell lineage refer to cells that show at least one phenotypic characteristic of a T cell, or a precursor or progenitor thereof that distinguishes the cells from other lymphoid cells, and cells of the erythroid or myeloid lineages.
  • Such phenotypic characteristics can include expression of one or more proteins specific for T cells (e.g., CD3 + , CD4 + , CD8 + ), or a physiological, morphological, functional, or immunological feature specific for a T cell.
  • cells of the T cell lineage may be progenitor or precursor cells committed to the T cell lineage; CD25 + immature and inactivated T cells; cells that have undergone CD4 or CD8 linage commitment; thymocyte progenitor cells that are CD4 + CD8 + double positive; single positive CD4 + or CD8 + ; TCRaP or TCR y5; or mature and functional or activated T cells.
  • the immune system cell is a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a y6 T cell, a natural killer cell (e.g., NK cell or NK- T cell), a dendritic cell, a B cell, or any combination thereof.
  • the immune system cell is a CD4+ T cell.
  • the T cell is a naive T cell, a central memory T cell, an effector memory T cell, a stem cell memory T cell, or any combination thereof.
  • a host cell may include any individual cell or cell culture which may receive a vector or the incorporation of nucleic acids or express proteins. The term also encompasses progeny of the host cell, whether genetically or phenotypically the same or different. Suitable host cells may depend on the vector and may include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells. These cells may be induced to incorporate the vector or other material by use of a viral vector, transformation via calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2d ed. (Cold Spring Harbor Laboratory, 1989).
  • methods for preparing a cell, wherein the method comprises introducing into the cell a polynucleotide or vector encoding any protein in accordance with the present disclosure.
  • the vector further comprises a polynucleotide encoding a binding protein.
  • the cell is modified to comprise, or comprises, a heterologous polynucleotide encoding a binding protein.
  • a host cell of the present disclosure can comprise a polynucleotide that encodes a polypeptide comprising an extracellular domain from CD8P, a transmembrane domain that is optionally from CD8P, and an intracellular domain comprising a variant of SEQ ID NO. : 19, wherein the variation comprises: (i)a diglycine motif in place of a dileucine motif; (ii) a mutated and optionally nonfunctional PI3K binding site; (iii) a mutated and optionally non-functional ITK and GADS binding site; (iv) a mutated and optionally non-functional LCK binding site; or (v) any combination of (i)-(iv).
  • a host cell comprises a polynucleotide encoding a polypeptide comprising or consisting of the amino acid sequences set forth in any one of SEQ ID NOs.: 192-204. In certain embodiments, a host cell expresses a polypeptide comprising or consisting of the amino acid sequences set forth in any one of SEQ ID NOs.: 192-204. In some embodiments, a host cell further comprises or expresses a CD8a co-receptor polypeptide, optionally comprising or consisting of SEQ ID NO.: 114. In any of the herein disclosed embodiments, a fusion protein of the present disclosure can associate with a cognate CD8 co-receptor chain (e.g.
  • a second fusion protein that comprises one or more domains or portions of a cognate CD8 co-receptor, to form a homodimer or a heterodimer when expressed at a host cell surface.
  • a polynucleotide of the present disclosure encodes: (i) a fusion protein comprising one or more sequences from (or derived from) a CD8 co-receptor a-chain; and (ii) a fusion protein comprising one or more sequences from (or derived from) a CD8 co-receptor P-chain.
  • a polynucleotide of the present disclosure encodes: (i) a fusion protein comprising one or more sequences from (or derived from) a CD8 co- receptor a-chain; and (ii) a fusion protein comprising one or more sequences from (or derived from) a CD8 co-receptor a-chain.
  • a polynucleotide of the present disclosure encodes (i) a fusion protein comprising one or more sequences from (or derived from) a CD8 co-receptor a-chain; and (ii) a CD8 co-receptor P-chain, or a functional variant or portion thereof.
  • a polynucleotide of the present disclosure encodes (i) a fusion protein comprising one or more sequences from (or derived from) a CD8 co-receptor P-chain; and (ii) a CD8 co-receptor a-chain, or a functional variant or portion thereof.
  • one or both substituent proteins of a co-receptor protein pair comprising (i) a fusion protein of the present disclosure and (ii) a cognate CD8 co- receptor protein or a functional variant or portion of the same (or a second fusion protein comprising one or more (native or variant) sequences from a cognate CD8 co- receptor) is modified to enhance association of the co-receptor pair when the substituent proteins are expressed at a cell surface.
  • a cysteine amino acid is introduced at one or more position in one or both of the substituent proteins such that the substituent proteins can form a cysteine-cysteine bond therebetween.
  • a host cell can further comprise a polynucleotide (endogenous, heterologous, or both) that encodes a binding protein that is capable of specifically binding to an antigen.
  • a binding protein comprises a TCR, which can, in some embodiments, be a MHC-I-restricted TCR or a MHC-II-restricted TCR.
  • a binding protein comprises a CAR.
  • polynucleotide encoding a first protein can be separated from a polynucleotide encoding a second protein (e.g., fusion protein, CD8 co-receptor protein, binding protein, or marker) by a polynucleotide that encodes a selfcleaving polypeptide.
  • a first protein e.g., fusion protein, CD8 co-receptor protein, binding protein, or marker
  • a second protein e.g., fusion protein, CD8 co-receptor protein, binding protein, or marker
  • an encoded self-cleaving polypeptide comprises a 2A peptide from porcine teschovirus-1 (P2A), Thoseaasigna virus (T2A), equine rhinitis A virus (E2A), or foot-and-mouth disease virus (F2A)).
  • P2A porcine teschovirus-1
  • T2A Thoseaasigna virus
  • E2A equine rhinitis A virus
  • F2A foot-and-mouth disease virus
  • CAR Chimeric antigen receptor
  • CARs of the present disclosure include an extracellular portion comprising an antigen binding domain (/. ⁇ ., obtained or derived from an immunoglobulin or immunoglobulin-like molecule, such as a scFv or scTCR derived from an antibody or TCR specific for a cancer antigen, or an antigen-binding domain derived or obtained from a killer immunoreceptor from an NK cell) linked to a transmembrane domain and one or more intracellular signaling domains (optionally containing co-stimulatory domain(s)) (see, e.g., Sadelain et al., Cancer Discov., 3(4):388 (2013); see also Harris and Kranz, Trends Pharmacol. Sci., 37(3): 220 (2016); Stone et al, Cancer Immunol. Immunother., 63(11): 1163 (2014)).
  • an antigen binding domain /. ⁇ ., obtained or derived from an immunoglobulin or immunoglobulin-like molecule, such as
  • a binding protein comprises a CAR comprising an antigen-specific TCR binding domain (see, e.g., Walseng et a!.. Scientific Reports 7: 10713, 2017; the TCR CAR constructs and methods of which are hereby incorporated by reference in their entirety), which can be a MHC-I-restricted TCR binding domain, a MHCII-restricted TCR binding domain, or both.
  • a binding protein e.g. a TCR
  • a binding protein of the instant disclosure specifically binds to a tumor- associated antigen.
  • the tumor-associated antigen is selected from ROR1, EGFR, EGFRvIII, EGP-2, EGP-40, GD2, GD3, HPV E6, HPV E7, Her2, Ll-CAM, Lewis A, Lewis Y, MUC1, MUC16, PSCA, PSMA, CD19, CD20, CD22, CD56, CD23, CD24, CD30, CD33, CD37, CD44v7/8, CD38, CD56, CD123, CA125, c- MET, FcRH5, WT1, folate receptor a, VEGF-a, VEGFR1, VEGFR2, IL-13Ra2, IL- HRa, HER2, MAGE-A1, MAGE-A3, MAGE-A4, SSX-2, PRAME, KRAS (e.g.
  • G12V, G12C, or G12D Merkel Cell polyomavirus T antigen
  • Core Binding Factor protein HA-1 H , PSA, ephrin A2, ephrin B2, an NKG2D, NY-ESO-1, TAG-72, mesothelin, NY-ESO, 5T4, BCMA, FAP, Carbonic anhydrase 9, ERBB2, a BRAF antigen such as a BRAF V600E antigen, and CEA.
  • a tumor- associated antigen is selected from BCMA, CD3, CEACAM6, c-Met, EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, FLT1, KDR, FLT4, CD44v6, CD151, CA125, CEA, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gpl30, Lewis A, Lewis Y, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A (e.g., including MAGE-A1, MAGE-A3, and MAGE-A4), HER2, mesothelin, NY-ESO-1, KRAS (e.g.
  • G12V, G12C, or G12D PSMA, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, HLA, tumor- or pathogen- associated peptide bound to HLA, hTERT peptide bound to HLA, tyrosinase peptide bound to HLA, WT-1 peptide bound to HLA, LTpR, LIFRP, LRP5, MUC1, OSMRp, TCRa, TCRp, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD79a, CD79b, CD80, CD81, CD86, CD123, CD171, CD276, B7H4, TLR7, TLR9, PTCHI, WT-1, HA ⁇ H, Robol, a-fetoprotein (AFP), Frizzled, 0X40, PRAME, and SSX-2.
  • AFP a-fetoprotein
  • Frizzled 0X40, PR
  • a binding protein can comprise a binding domain disclosed in any one of PCT Publication Nos.: WO 2016/022400; WO 2018/170338; WO 2018/090057; WO 2017/112944; WO 2017/193104; WO 2018/058002; or WO 2013/071154; the CDR, framework, variable, and TCR sequences of which are incorporated herein by reference.
  • binding domain refers to a molecule or portion thereof (e.g., peptide, oligopeptide, polypeptide, protein (e.g., a fusion protein)) that possesses the ability to specifically and non-covalently associate, unite, or combine with a target.
  • a binding domain includes any naturally occurring, synthetic, semi -synthetic, or recombinantly produced binding partner for a biological molecule, a molecular complex (i.e., complex comprising two or more biological molecules), or other target of interest.
  • binding domains include, in general, single chain immunoglobulin variable regions (e.g., scTCR, scFv, Fab, TCR variable regions), receptor ectodomains, ligands (e.g., cytokines, chemokines), or synthetic polypeptides selected for their specific ability to bind to a biological molecule, a molecular complex or other target of interest.
  • the binding domain of a binding protein is a scFv, scTCR, or ligand.
  • the binding domain is chimeric, human, or humanized.
  • binding protein e.g., a T cell receptor or a chimeric antigen receptor
  • binding domain or fusion protein thereof
  • K a an affinity or K a (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10 5 M' 1 (which equals the ratio of the on-rate [K on ] to the off rate [K O ff] for this association reaction), while not significantly associating or uniting with any other molecules or components in a sample.
  • Binding proteins or binding domains may be classified as “high-affinity” binding proteins or binding domains or as “low-affinity” binding proteins or binding domains.
  • “High-affinity” binding proteins or binding domains refer to those binding proteins or binding domains having a K a of at least 10 7 M -1 , at least 10 8 M’ 1 , at least 10 9 M at least IO 10 M’ 1 , at least IO 11 M’ 1 , at least 10 12 M -1 , or at least IO 13 M' 1 .
  • “Low-affinity” binding proteins or binding domains refer to those binding proteins or binding domains having a K a of up to 10 7 M’ 1 , up to 10 6 M" or up to 10 5 M' 1 .
  • affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10' 5 M to 10' 13 M).
  • a receptor or binding domain may have "enhanced affinity," which refers to selected or engineered receptors or binding domains with stronger binding to a target antigen than a wild type (or parent) binding domain.
  • enhanced affinity may be due to a K a (equilibrium association constant) for the target antigen that is higher than the wild type binding domain, due to a Kd (dissociation constant) for the target antigen that is less than that of the wild type binding domain, due to an off-rate (koff) for the target antigen that is less than that of the wild type binding domain, or a combination thereof.
  • fusion proteins may be codon-optimized to enhance expression in a particular host cell, such as T cells (Scholten et al., Clin. Immunol. 119 : 135, 2006).
  • a variety of assays are known for identifying binding domains of the present disclosure that specifically bind a particular target, as well as determining binding domain or fusion protein affinities, such as Western blot, ELISA, analytical ultracentrifugation, spectroscopy and surface plasmon resonance (Biacore®) analysis (see, e.g., Scatchard et al., Ann. N.Y. Acad. Sci. 51 :660, 1949; Wilson, Science 295 AQ3, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent). Assays for assessing affinity or apparent affinity or relative affinity are also known.
  • apparent affinity for a fusion protein is measured by assessing binding to various concentrations of tetramers, for example, by flow cytometry using labeled tetramers.
  • apparent KD of a fusion protein is measured using 2-fold dilutions of labeled tetramers at a range of concentrations, followed by determination of binding curves by non-linear regression, apparent KD being determined as the concentration of ligand that yielded half-maximal binding.
  • host cells that comprise: (i) a heterologous polynucleotide that encodes a fusion protein, wherein the encoded fusion protein comprises: (a)an extracellular component comprising an extracellular domain from a CD8 co-receptor a- chain; (b) a transmembrane domain from a CD8 co-receptor a-chain; and (c) an intracellular component comprising a co stimulatory domain from CD28, or a functional portion or variant thereof; and (ii) a heterologous polynucleotide encoding a binding protein that specifically binds to an antigen or an antigen:MHC complex.
  • host cells that comprise: (i) a heterologous polynucleotide that encodes a fusion protein, wherein the encoded fusion protein comprises: (a)an extracellular component comprising an extracellular domain from a CD8 co-receptor P- chain; (b) a transmembrane domain from a CD8 co-receptor a-chain; and (c) an intracellular component comprising a co stimulatory domain from CD28, or a functional portion or variant thereof; and (ii) a heterologous polynucleotide encoding a binding protein that specifically binds to an antigen or an antigen:MHC complex.
  • a binding protein encoded by a host cell e.g., CD4 T cell
  • a binding domain e.g., a CAR or a TCR
  • a host cell e.g., an immune cell
  • a host cell may modified to reduce or eliminate expression of one or more endogenous genes that encode a polypeptide involved in immune signaling or other related activities.
  • exemplary gene knockouts include those that encode PD-1, LAG-3, CTLA4, TIM3, TIGIT, FasL, an HLA molecule, a TCR molecule, or the like.
  • certain endogenously expressed immune cell proteins may be recognized as foreign by an allogeneic host receiving the modified immune cells, which may result in elimination of the modified immune cells (e.g., an HLA allele), or may downregulate the immune activity of the modified immune cells (e.g., PD-1, LAG-3, CTLA4, FasL, Fas, TIGIT, TIM3), or may interfere with, suppress, or counter the activity of a heterologously expressed protein of the present disclosure.
  • the modified immune cells e.g., an HLA allele
  • the modified immune cells e.g., PD-1, LAG-3, CTLA4, FasL, Fas, TIGIT, TIM3
  • a modified cell is a donor cell (e.g., allogeneic) or an autologous cell.
  • a modified cell of this disclosure comprises a chromosomal gene knockout of one or more of a gene that encodes PD-1, LAG-3, CTLA4, TIM3, TIGIT, FasL, Fas, an HLA component e.g., a gene that encodes an al macroglobulin, an a2 macroglobulin, an a3 macroglobulin, a pi microglobulin, or a P2 microglobulin), or a TCR component e.g., a gene that encodes a TCR variable region or a TCR constant region) see, e.g., Torikai et al., Nature Sci. Rep.
  • chromosomal gene knockout refers to a genetic alteration or introduced inhibitory agent in a host cell that prevents e.g., reduces, delays, suppresses, or abrogates) production, by the host cell, of a functionally active endogenous polypeptide product. Alterations resulting in a chromosomal gene knockout can include, for example, introduced nonsense mutations (including the formation of premature stop codons), missense mutations, gene deletion, and strand breaks, as well as the heterologous expression of inhibitory nucleic acid molecules that inhibit endogenous gene expression in the host cell.
  • a chromosomal gene knock-out or gene knock-in is made by chromosomal editing of a host cell.
  • Chromosomal editing can be performed using, for example, endonucleases.
  • endonucleases refers to an enzyme capable of catalyzing cleavage of a phosphodiester bond within a polynucleotide chain.
  • an endonuclease is capable of cleaving a targeted gene thereby inactivating or "knocking out" the targeted gene.
  • An endonuclease may be a naturally occurring, recombinant, genetically modified, or fusion endonuclease.
  • the nucleic acid strand breaks caused by the endonuclease are commonly repaired through the distinct mechanisms of homologous recombination or non-homologous end joining (NHEJ).
  • NHEJ non-homologous end joining
  • a donor nucleic acid molecule may be used for a donor gene "knock-in”, for target gene "knock-out”, and optionally to inactivate a target gene through a donor gene knock in or target gene knock out event.
  • NHEJ is an error- prone repair process that often results in changes to the DNA sequence at the site of the cleavage, e.g., a substitution, deletion, or addition of at least one nucleotide.
  • NHEJ may be used to "knock-out" a target gene.
  • Examples of endonucleases include zinc finger nucleases, TALE-nucleases, CRISPR-Cas nucleases, meganucleases, and megaTALs.
  • a "zinc finger nuclease” refers to a fusion protein comprising a zinc finger DNA-binding domain fused to a non-specific DNA cleavage domain, such as a Fokl endonuclease.
  • Each zinc finger motif of about 30 amino acids binds to about 3 base pairs of DNA, and amino acids at certain residues can be changed to alter triplet sequence specificity (see, e.g., Desjarlais et al., Proc. Natl. Acad. Sci. 90 2256-2260, 1993; Wolfe et al., J. Mol. Biol. 255: 1917-1934, 1999).
  • ZFNs mediate genome editing by catalyzing the formation of a site-specific DNA double strand break (DSB) in the genome, and targeted integration of a transgene comprising flanking sequences homologous to the genome at the site of DSB is facilitated by homology directed repair.
  • DSB DNA double strand break
  • a DSB generated by a ZFN can result in knock out of target gene via repair by non-homologous end joining (NHEJ), which is an error-prone cellular repair pathway that results in the insertion or deletion of nucleotides at the cleavage site.
  • NHEJ non-homologous end joining
  • a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, made using a ZFN molecule.
  • TALEN transcription activator-like effector nuclease
  • a "TALE DNA binding domain” or “TALE” is composed of one or more TALE repeat domains/units, each generally having a highly conserved 33-35 amino acid sequence with divergent 12th and 13th amino acids.
  • the TALE repeat domains are involved in binding of the TALE to a target DNA sequence.
  • the divergent amino acid residues referred to as the Repeat Variable Diresidue (RVD), correlate with specific nucleotide recognition.
  • RVD Repeat Variable Diresidue
  • the natural (canonical) code for DNA recognition of these TALEs has been determined such that an HD (histine-aspartic acid) sequence at positions 12 and 13 of the TALE leads to the TALE binding to cytosine (C), NG (asparagine-glycine) binds to a T nucleotide, NI (asparagine-isoleucine) to A, NN (asparagine-asparagine) binds to a G or A nucleotide, and NG (asparagine-glycine) binds to a T nucleotide.
  • Non-canonical (atypical) RVDs are also known (see, e.g., U.S. Patent Publication No.
  • TALENs can be used to direct site-specific double-strand breaks (DSB) in the genome of T cells.
  • Nonhom ologous end joining (NHEJ) ligates DNA from both sides of a double-strand break in which there is little or no sequence overlap for annealing, thereby introducing errors that knock out gene expression.
  • homology directed repair can introduce a transgene at the site of DSB providing homologous flanking sequences are present in the transgene.
  • a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, and made using a TALEN molecule.
  • CRISPR/Cas nuclease system refers to a system that employs a CRISPR RNA (crRNA)-guided Cas nuclease to recognize target sites within a genome (known as protospacers) via base-pairing complementarity and then to cleave the DNA if a short, conserved protospacer associated motif (PAM) immediately follows 3’ of the complementary target sequence.
  • CRISPR/Cas systems are classified into three types (i.e., type I, type II, and type III) based on the sequence and structure of the Cas nucleases.
  • the crRNA-guided surveillance complexes in types I and III need multiple Cas subunits.
  • Type II system the most studied, comprises at least three components: an RNA-guided Cas9 nuclease, a crRNA, and a trans-acting crRNA (tracrRNA).
  • the tracrRNA comprises a duplex forming region.
  • a crRNA and a tracrRNA form a duplex that is capable of interacting with a Cas9 nuclease and guiding the Cas9/crRNA:tracrRNA complex to a specific site on the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA upstream from a PAM.
  • Cas9 nuclease cleaves a double-stranded break within a region defined by the crRNA spacer. Repair by NHEJ results in insertions and/or deletions which disrupt expression of the targeted locus.
  • a transgene with homologous flanking sequences can be introduced at the site of DSB via homology directed repair.
  • the crRNA and tracrRNA can be engineered into a single guide RNA (sgRNA or gRNA) see, e.g., Jinek et al., Science 337:816-21, 2012).
  • a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, and made using a CRISPR/Cas nuclease system.
  • Exemplary gRNA sequences and methods of using the same to knock out endogenous genes that encode immune cell proteins include those described in Ren et al., Clin. Cancer Res. 23(9):2255-2266 (2017), the gRNAs, CAS9 DNAs, vectors, and gene knockout techniques of which are hereby incorporated by reference in their entirety.
  • Alternative Cas nucleases may be used, including but not limited to, Cas 12, Cas 13, and Cas 14 nucleases, and variants thereof.
  • Cas nucleases disclosed in WO 2019/178427 which is hereby incorporated by reference in its entirety (including the Cas nucleases, CRISPR-Cas systems, and related methods disclosed therein), may be utilized.
  • Exemplary meganucleases include I-Scel, I-Ceul, PI-PspI, Pl-Sce, 1-SceIV, LCsml, I-PanI, LScell, LPpoI, 1-SceIII, LCrel, I- TevI, LTevII and I-TevIII, whose recognition sequences are known (see, e.g., U.S. Patent Nos. 5,420,032 and 6,833,252; Belfort et al., Nucleic Acids Res . 25:3379-3388, 1997; Dujon et al., Gene 82: 115-118, 1989; Perler et al., Nucleic Acids Res.
  • naturally occurring meganucleases may be used to promote site-specific genome modification of a target selected from PD-1, LAG3, TIM3, CTLA4, TIGIT, FasL, an HLA-encoding gene, or a TCR component-encoding gene.
  • a target selected from PD-1, LAG3, TIM3, CTLA4, TIGIT, FasL, an HLA-encoding gene, or a TCR component-encoding gene.
  • an engineered meganuclease having a novel binding specificity for a target gene is used for site-specific genome modification (see, e.g., Porteus et al., Nat. Biotechnol. 23 :967 -73, 2005; Sussman et al., J. Mol. Biol. 342:31- 41, 2004; Epinat et al., Nucleic Acids Res.
  • a chromosomal gene knockout is generated using a homing endonuclease that has been modified with modular DNA binding domains of TALENs to make a fusion protein known as a megaTAL. MegaTALs can be utilized to not only knock-out one or more target genes, but to also introduce (knock in) heterologous or exogenous polynucleotides when used in combination with an exogenous donor template encoding a polypeptide of interest.
  • a chromosomal gene knockout comprises an inhibitory nucleic acid molecule that is introduced into a host cell (e.g., an immune cell) comprising a heterologous polynucleotide encoding an antigen-specific receptor that specifically binds to a tumor associated antigen, wherein the inhibitory nucleic acid molecule encodes a target-specific inhibitor and wherein the encoded target-specific inhibitor inhibits endogenous gene expression (e.g., of PD-1, TIM3, LAG3, CTLA4, TIGIT, Fas, FasL, an HLA component, or a TCR component, or any combination thereof) in the host cell.
  • a host cell e.g., an immune cell
  • a heterologous polynucleotide encoding an antigen-specific receptor that specifically binds to a tumor associated antigen
  • the inhibitory nucleic acid molecule encodes a target-specific inhibitor and wherein the encoded target-specific inhibitor inhibits endogenous gene expression (e.g.
  • chromosomal gene knockout can be confirmed directly by DNA sequencing of the host immune cell following use of the knockout procedure or agent. Chromosomal gene knockouts can also be inferred from the absence of gene expression (e.g., the absence of an mRNA or polypeptide product encoded by the gene) following the knockout.
  • a chromosomal gene knockout comprises a knockout of an HLA component gene selected from an al macroglobulin gene, an a2 macroglobulin gene, an a3 macroglobulin gene, a pi microglobulin gene, or a P2 microglobulin gene.
  • a chromosomal gene knockout comprises a knockout of a TCR component gene selected from a TCR a variable region gene, a TCR P variable region gene, a TCR constant region gene, or a combination thereof.
  • a polynucleotide of the present disclosure e.g, encoding a binding protein and/or a protein such as a CD8 co-receptor polypeptide
  • a heterologous polynucleotide is introduced into a locus encoding an endogenous TCR component, HLA component, PD-1, LAG-3, CTLA4, TIM3, or TIGIT, or a safe harbor locus such as Rosa26, AAVS1, CCR5, or the like.
  • a heterologous polynucleotide encoding a binding protein and/or encoding a CD8 co-receptor polypeptide is introduced into a host cell TRAC locus.
  • a chromosomal knockout of a host cell TRBC locus is introduced.
  • a host cell e.g., modified immune cell
  • the host cell comprises a chromosomal knockout of an endogenous TRBC locus.
  • the present disclosure also provides methods of treating a disease or disorder, wherein the methods comprise administering a host e.g. immune) cell of the present disclosure or a composition comprising the same.
  • a host cell refers to a cell modified to comprise a presently disclosed protein, polynucleotide, or vector, irrespective of whether the host cell is autologous to the subject receiving treatment.
  • host in this context describes the relationship between the cell and the heterologous protein, molecule, or vector, and not the relationship between the cell and the subject receiving treatment.
  • Treatment refers to medical management of a disease, disorder, or condition of a subject (e.g, a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat).
  • a subject e.g, a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat.
  • an appropriate dose or treatment regimen comprising a host cell expressing a polypeptide (e.g. fusion protein) of the present disclosure or multiple such polypeptides, and optionally an adjuvant, is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit.
  • a polypeptide e.g. fusion protein
  • Therapeutic or prophylactic/preventive benefit includes improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease; stabilization of disease state; delay of disease progression; remission; survival; prolonged survival; or any combination thereof.
  • a “therapeutically effective amount” or “effective amount” of a polypeptide, fusion protein or host cell expressing the same refers to an amount of polypeptides or fusion proteins or host cells sufficient to result in a therapeutic effect, including improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease- free status; diminishment of extent of disease, stabilization of disease state; delay of disease progression; remission; survival; or prolonged survival in a statistically significant manner.
  • a therapeutically effective amount refers to the effects of that ingredient or cell expressing that ingredient alone.
  • a therapeutically effective amount refers to the combined amounts of active ingredients or combined adjunctive active ingredient with a cell expressing an active ingredient that results in a therapeutic effect, whether administered serially or simultaneously.
  • a combination may also be a cell expressing more than one active ingredient.
  • pharmaceutically acceptable excipient or carrier or “physiologically acceptable excipient or carrier” refer to biologically compatible vehicles, e.g., physiological saline, which are described in greater detail herein, that are suitable for administration to a human or other non-human mammalian subject and generally recognized as safe or not causing a serious adverse event.
  • statically significant refers to a p-value of 0.050 or less when calculated using the Student’s t-test and indicates that it is unlikely that a particular event or result being measured has arisen by chance.
  • adoptive immune therapy refers to administration of naturally occurring or genetically engineered, disease-antigen-specific immune cells (e.g., T cells).
  • adoptive cellular immunotherapy may be autologous (immune cells are from the recipient), allogeneic (immune cells are from a donor of the same species) or syngeneic (immune cells are from a donor genetically identical to the recipient).
  • the host cell expresses at its cell surface (i) a polypeptide (e.g. a fusion protein) of the present disclosure and (ii) a binding protein specific for an antigen that is associated with or expressed by the disease or condition.
  • the host cell expresses at its cell surface (i) a co-receptor pair comprising (a) a polypeptide (e.g. a fusion protein) and (b) a cognate co-receptor protein or fusion protein comprising a cognate co-receptor domain; and (ii) a binding protein that specifically binds to an antigen that associates with a MHC molecule.
  • a host cell expressing (i) a polypeptide (e.g. a fusion protein) of the present disclosure and (ii) a binding protein is administered as part of a cellular immunotherapy that comprises (e.g., in a same composition or unit dose, or in separate compositions or unit doses) an effector e.g. immune) cell that expresses at its cell surface a binding protein (e.g., CAR or TCR) that specifically binds to an antigen expressed by or otherwise associated with the disease or condition.
  • a binding protein e.g., CAR or TCR
  • the effector e.g immune) cell specifically binds to the same antigen as the host (e.g. immune) cell. In other embodiments, the effector (e.g. immune) cell specifically binds to a different antigen as the host (e.g. immune) cell, provided that the different antigen is also expressed by or otherwise associated with the disease or condition.
  • modified CD4+ T cells are administered to the subject.
  • modified CD8+ T cells are administered to the subject.
  • modified CD4+ T cells and modified CD8+ T cells are administered to the subject.
  • the host cell and/or effector cell are administered to treat a hyperproliferative disorder.
  • hyperproliferative disorder refers to excessive growth or proliferation as compared to a normal or undiseased cell.
  • hyperproliferative disorders include tumors, cancers, neoplastic tissue, carcinoma, sarcoma, malignant cells, pre-malignant cells, as well as non-neoplastic or non-malignant hyperproliferative disorders (e.g., adenoma, fibroma, lipoma, leiomyoma, hemangioma, fibrosis, restenosis, as well as autoimmune diseases such as rheumatoid arthritis, osteoarthritis, psoriasis, inflammatory bowel disease, or the like).
  • proliferative diseases include certain tumors, cancers, neoplastic tissue, carcinoma, sarcoma, malignant cells, pre malignant cells, as well as non-neoplastic or non-malignant disorders.
  • cancer may refer to any accelerated proliferation of cells, including solid tumors, ascites tumors, blood or lymph or other malignancies; connective tissue malignancies; metastatic disease; minimal residual disease following transplantation of organs or stem cells; multi-drug resistant cancers, primary or secondary malignancies, angiogenesis related to malignancy, or other forms of cancer.
  • a cancer treatable according to the presently disclosed methods and uses comprises a carcinoma, a sarcoma, a glioma, a lymphoma, a leukemia, a myeloma, or any combination thereof.
  • cancer comprises a cancer of the head or neck, melanoma, pancreatic cancer, cholangiocarcinoma, hepatocellular cancer, breast cancer including triple-negative breast cancer (TNBC), gastric cancer, non-small-cell lung cancer, prostate cancer, esophageal cancer, mesothelioma, small-cell lung cancer, colorectal cancer, glioblastoma, or any combination thereof.
  • TNBC triple-negative breast cancer
  • a cancer comprises Askin's tumor, sarcoma botryoides, chondrosarcoma, Ewing's sarcoma, PNET, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma protuberans (DFSP), desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, undifferentiated pleomorphic sarcoma,
  • the cancer comprises a solid tumor.
  • the solid tumor is a sarcoma or a carcinoma.
  • the solid tumor is selected from: chondrosarcoma; fibrosarcoma (fibroblastic sarcoma); Dermatofibrosarcoma protuberans (DFSP); osteosarcoma; rhabdomyosarcoma; Ewing’s sarcoma; a gastrointestinal stromal tumor;
  • the solid tumor is selected from a lung carcinoma (e.g., Adenocarcinoma, Squamous Cell Carcinoma (Epidermoid Carcinoma); Squamous cell carcinoma; Adenocarcinoma;
  • a lung carcinoma e.g., Adenocarcinoma, Squamous Cell Carcinoma (Epidermoid Carcinoma); Squamous cell carcinoma; Adenocarcinoma;
  • the solid tumor is an ovarian carcinoma, an ovarian epithelial carcinoma, a cervical adenocarcinoma or small cell carcinoma, a pancreatic carcinoma, a colorectal carcinoma (e.g., an adenocarcinoma or squamous cell carcinoma), a lung carcinoma, a breast ductal carcinoma, or an adenocarcinoma of the prostate.
  • the host cell is an allogeneic cell, a syngeneic cell, or an autologous cell.
  • the host cell will further express or encode an antigen-binding protein such as, for example, a TCR.
  • Subjects that can be treated by the present invention are, in general, human and other primate subjects, such as monkeys and apes for veterinary medicine purposes.
  • the subject may be a human subject.
  • the subjects can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.
  • Cells according to the present disclosure may be administered in a manner appropriate to the disease, condition, or disorder to be treated as determined by persons skilled in the medical art.
  • a cell comprising a cell as described herein is administered intravenously, intraperitoneally, intratumorally, into the bone marrow, into a lymph node, or into the cerebrospinal fluid.
  • compositions will be determined by such factors as the age, size, gender, and condition of the patient; the type and severity of the disease, condition, or disorder; the particular form of the active ingredient; and the method of administration.
  • methods of the present disclosure comprise administering a host cell of the present disclosure.
  • the amount of cells in a composition is at least one cell (for example, one fusion protein-modified CD8 + T cell subpopulation; one fusion protein-modified CD4 + T cell subpopulation) or is more typically greater than 10 2 cells, for example, up to 10 6 , up to 10 7 , up to 10 8 cells, up to 10 9 cells, or more than IO 10 cells.
  • the cells are administered in a range from about 10 6 to about IO 10 cells/m 2 , preferably in a range of about 10 5 to about 10 9 cells/m 2 .
  • the number of cells will depend upon the ultimate use for which the composition is intended as well the type of cells included therein.
  • cells modified to contain a fusion protein and a binding protein specific for a particular antigen will comprise a cell population containing at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of such cells.
  • cells are generally in a volume of a liter or less, 500 mis or less, 250 mis or less, or 100 mis or less.
  • the density of the desired cells is typically greater than 10 4 cells/ml and generally is greater than 10 7 cells/ml, generally 10 8 cells/ml or greater.
  • the cells may be administered as a single infusion or in multiple infusions over a range of time.
  • a clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 cells.
  • Unit doses are also provided herein which comprise a host cell or host cell composition of this disclosure.
  • a unit dose comprises a host cell (i.e., expressing a fusion protein and a binding protein) and an effector immune cell, wherein the host cell and the effector immune cell can each be a CD4 + T cell, a CD8 + T cell, or both.
  • a unit dose comprises: (i) CD4 + T cells that express at their cell surface: (a) a co-receptor pair comprising one or more CD8- derived fusion protein of the present disclosure; and (b) MHC-I-restricted binding protein; and (ii) CD8 + effector immune cells that express at their cell surface a binding protein.
  • a unit dose comprises: (i) CD4 + T cells that express at their cell surface: (a) one or more polypeptide as set forth in any one of Tables 1-7; and (b) MHC-I-restricted binding protein; and (ii) CD8 + effector immune cells that express at their cell surface a binding protein.
  • a unit dose comprises: (i) CD4 + T cells that express at their cell surface: (a) fusion or engineered protein according to the present disclosure; and (b) MHC-I-restricted binding protein; and optionally, (ii) CD8 + effector immune cells that express at their cell surface a binding protein.
  • a unit dose comprises (i) a composition comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified or unmodified CD4 + T cells, combined with (ii) a composition comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified or unmodified CD8 + T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells (i.e., has less than about 50%, less than about 40%, less than about 30%,
  • a unit dose comprises (i) a composition comprising at least about 50% modified or unmodified CD4 + T cells, combined with (ii) a composition comprising at least about 50% modified or unmodified CD8 + T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells.
  • a unit dose comprises (i) a composition comprising at least about 60% modified or unmodified CD4 + T cells, combined with (ii) a composition comprising at least about 60% modified or unmodified CD8 + T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells.
  • a unit dose comprises (i) a composition comprising at least about 70% modified or unmodified CD4 + T cells, combined with (ii) a composition comprising at least about 70% modified or unmodified CD8 + T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells.
  • a unit dose comprises (i) a composition comprising at least about 80% modified or unmodified CD4 + T cells, combined with (ii) a composition comprising at least about 80% modified or unmodified CD8 + T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells.
  • a unit dose comprises (i) a composition comprising at least about 85% modified or unmodified CD4 + T cells, combined with (ii) a composition comprising at least about 85% modified or unmodified CD8 + T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells.
  • a unit dose comprises (i) a composition comprising at least about 90% modified or unmodified CD4 + T cells, combined with (ii) a composition comprising at least about 90% modified or unmodified CD8 + T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells.
  • a unit dose comprises equal, or approximately equal numbers of modified or unmodified CD45RA" CD3 + CD8 + and modified or unmodified CD45RA" CD3 + CD4 + TM cells.
  • compositions that comprise (e.g. fusion) proteins or cells expressing the (e.g. fusion) proteins as disclosed herein and a pharmaceutically acceptable carrier, diluents, or excipient.
  • Suitable excipients include water, saline, dextrose, glycerol, or the like and combinations thereof.
  • compositions comprising fusion proteins or host cells as disclosed herein further comprise a suitable infusion media.
  • suitable infusion media can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), 5% dextrose in water, Ringer's lactate can be utilized.
  • An infusion medium can be supplemented with human serum albumin or other human serum components.
  • compositions may be administered in a manner appropriate to the disease or condition to be treated (or prevented) as determined by persons skilled in the medical art.
  • An appropriate dose and a suitable duration and frequency of administration of the compositions will be determined by such factors as the health condition of the patient, size of the patient (i.e., weight, mass, or body area), the type and severity of the patient's condition, the undesired type or level or activity of the tagged cells, the particular form of the active ingredient, and the method of administration.
  • an appropriate dose and treatment regimen provide the composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (such as described herein, including an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and/or overall survival, or a lessening of symptom severity).
  • a dose should be sufficient to prevent, delay the onset of, or diminish the severity of a disease associated with disease or disorder.
  • Prophylactic benefit of the immunogenic compositions administered according to the methods described herein can be determined by performing pre-clinical (including in vitro and in vivo animal studies) and clinical studies and analyzing data obtained therefrom by appropriate statistical, biological, and clinical methods and techniques, all of which can readily be practiced by a person skilled in the art.
  • Certain methods of treatment or prevention contemplated herein include administering a host cell (which may be autologous, allogeneic or syngeneic) comprising a desired polynucleotide as described herein that is stably integrated into the chromosome of the cell.
  • a host cell which may be autologous, allogeneic or syngeneic
  • a cellular composition may be generated ex vivo using autologous, allogeneic or syngeneic immune system cells (e.g., T cells, antigen-presenting cells, natural killer cells) in order to administer a desired, fusion protein-expressing T-cell composition to a subject as an adoptive immunotherapy.
  • the host cell is a hematopoietic progenitor cell or a human immune cell.
  • the immune system cell is a CD4 + T cell, a CD8 + T cell, a CD4" CD8" double-negative T cell, a y6 T cell, a natural killer cell, a dendritic cell, or any combination thereof.
  • the immune system cell is a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof.
  • the cell is a CD4 + T cell.
  • the cell is a CD8 + T cell.
  • administration of a composition refers to delivering the same to a subject, regardless of the route or mode of delivery. Administration may be effected continuously or intermittently, and parenterally. Administration may be for treating a subject already confirmed as having a recognized condition, disease or disease state, or for treating a subject susceptible to or at risk of developing such a condition, disease or disease state.
  • Co-administration with an adjunctive therapy may include simultaneous and/or sequential delivery of multiple agents in any order and on any dosing schedule (e.g., fusion protein-expressing recombinant (i.e., engineered) host cells with one or more cytokines; immunosuppressive therapy such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low dose of a mycophenolic acid prodrug, or any combination thereof).
  • fusion protein-expressing recombinant i.e., engineered host cells with one or more cytokines
  • immunosuppressive therapy such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low dose of a mycophenolic acid prodrug, or any combination thereof.
  • a plurality of doses of a recombinant host cell as described herein is administered to the subject, which may be administered at intervals between administrations of about two to about four or more weeks.
  • the subject being treated is further receiving immunosuppressive therapy, such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low dose of a mycophenolic acid prodrug, or any combination thereof.
  • immunosuppressive therapy such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low dose of a mycophenolic acid prodrug, or any combination thereof.
  • the subject being treated has received a non-myeloablative or a myeloablative hematopoietic cell transplant, wherein the treatment may be administered at least two to at least three months after the non-myeloablative hematopoietic cell transplant.
  • An effective amount of a pharmaceutical composition refers to an amount sufficient, at dosages and for periods of time needed, to achieve the desired clinical results or beneficial treatment, as described herein.
  • An effective amount may be delivered in one or more administrations. If the administration is to a subject already known or confirmed to have a disease or disease-state, the term "therapeutic amount” may be used in reference to treatment, whereas “prophylactically effective amount” may be used to describe administrating an effective amount to a subject that is susceptible or at risk of developing a disease or disease-state (e.g., recurrence) as a preventative course.
  • a disease or disease-state e.g., recurrence
  • the level of a CTL immune response may be determined by any one of numerous immunological methods described herein and routinely practiced in the art.
  • the level of a CTL immune response may be determined prior to and following administration of any one of the herein described fusion proteins expressed by, for example, a T cell.
  • Cytotoxicity assays for determining CTL activity may be performed using any one of several techniques and methods routinely practiced in the art (see, e.g., Henkart et al., "Cytotoxic T-Lymphocytes" in Fundamental Immunology, Paul (ed.) (2003 Lippincott Williams & Wilkins, Philadelphia, PA), pages 1127-50, and references cited therein).
  • Antigen-specific T cell responses are typically determined by comparisons of observed T cell responses according to any of the herein described T cell functional parameters (e.g., proliferation, cytokine release, CTL activity, altered cell surface marker phenotype, etc.) that may be made between T cells that are exposed to a cognate antigen in an appropriate context (e.g., the antigen used to prime or activate the T cells, when presented by immunocompatible antigen-presenting cells) and T cells from the same source population that are exposed instead to a structurally distinct or irrelevant control antigen.
  • a cognate antigen e.g., the antigen used to prime or activate the T cells, when presented by immunocompatible antigen-presenting cells
  • a response to the cognate antigen that is greater, with statistical significance, than the response to the control antigen signifies antigen-specificity.
  • a biological sample may be obtained from a subject for determining the presence and level of an immune response to a tagged protein or cell as described herein.
  • a "biological sample” as used herein may be a blood sample (from which serum or plasma may be prepared), biopsy specimen, body fluids (e.g., lung lavage, ascites, mucosal washings, synovial fluid), bone marrow, lymph nodes, tissue explant, organ culture, or any other tissue or cell preparation from the subject or a biological source.
  • Biological samples may also be obtained from the subject prior to receiving any immunogenic composition, which biological sample is useful as a control for establishing baseline (i.e., pre-immunization) data.
  • compositions described herein may be presented in unitdose or multi-dose containers, such as sealed ampoules or vials. Such containers may be frozen to preserve the stability of the formulation until.
  • a unit dose comprises a recombinant host cell as described herein at a dose of about 10 7 cells/m 2 to about 10 11 cells/m 2 .
  • the composition may also include sterile aqueous or oleaginous solution or suspension.
  • suitable non-toxic parenterally acceptable diluents or solvents include water, Ringer’s solution, isotonic salt solution, 1,3 -butanediol, ethanol, propylene glycol or polythethylene glycols in mixtures with water.
  • Aqueous solutions or suspensions may further comprise one or more buffering agents, such as sodium acetate, sodium citrate, sodium borate or sodium tartrate.
  • any material used in preparing any dosage unit formulation should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compounds may be incorporated into sustained-release preparation and formulations.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit may contain a predetermined quantity of recombinant cells or active compound calculated to produce the desired effect in association with an appropriate pharmaceutical carrier.
  • an appropriate dosage and treatment regimen provides the active molecules or cells in an amount sufficient to provide therapeutic or prophylactic benefit.
  • a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated subjects as compared to non-treated subjects.
  • Increases in preexisting immune responses to a tumor protein generally correlate with an improved clinical outcome.
  • Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which are routine in the art and may be performed using samples obtained from a subject before and after treatment.
  • Methods according to this disclosure may further include administering one or more additional agents to treat the disease or disorder in a combination therapy.
  • a combination therapy comprises administering a fusion protein (or an engineered host cell expressing the same) with (concurrently, simultaneously, or sequentially) an immune checkpoint inhibitor.
  • a combination therapy comprises administering fusion protein of the present disclosure (or an engineered host cell expressing the same) with an agonist of a stimulatory immune checkpoint agent.
  • a combination therapy comprises administering a fusion protein of the present disclosure (or an engineered host cell expressing the same) with a secondary therapy, such as chemotherapeutic agent, a radiation therapy, a surgery, an antibody, or any combination thereof.
  • immune suppression agent refers to one or more cells, proteins, molecules, compounds or complexes providing inhibitory signals to assist in controlling or suppressing an immune response.
  • immune suppression agents include those molecules that partially or totally block immune stimulation; decrease, prevent or delay immune activation; or increase, activate, or up regulate immune suppression.
  • immunosuppression agents to target include PD-1, PD-L1, PD- L2, LAG3, CTLA4, B7-H3, B7-H4, CD244/2B4, HVEM, BTLA, CD160, TIM3, GAL9, KIR, PVR1G (CD112R), PVRL2, adenosine, A2aR, immunosuppressive cytokines (e.g., IL-10, IL-4, IL-IRA, IL-35), IDO, arginase, VISTA, TIGIT, LAIR1, CEACAM-1, CEACAM-3, CEACAM-5, Treg cells, or any combination thereof.
  • cytokines e.g., IL-10, IL-4, IL-IRA, IL-35
  • IDO arginase
  • VISTA TIGIT
  • LAIR1 CEACAM-1
  • CEACAM-3 CEACAM-5
  • Treg cells or any combination thereof.
  • An immune suppression agent inhibitor may be a compound, an antibody, an antibody fragment or fusion polypeptide e.g., Fc fusion, such as CTLA4-Fc or LAG3-Fc), an antisense molecule, a ribozyme or RNAi molecule, or a low molecular weight organic molecule.
  • a method may comprise administering an engineered host e.g. immune) cell of the present disclosure with one or more inhibitor of any one of the following immune suppression components, singly or in any combination.
  • a modified cell is used in combination with a PD-1 inhibitor, for example a PD-1 -specific antibody or binding fragment thereof, such as pidilizumab, nivolumab (Keytruda, formerly MDX-1106), pembrolizumab (Opdivo, formerly MK-3475), MEDI0680 (formerly AMP-514), AMP-224, BMS-936558 or any combination thereof.
  • a PD-1 inhibitor for example a PD-1 -specific antibody or binding fragment thereof, such as pidilizumab, nivolumab (Keytruda, formerly MDX-1106), pembrolizumab (Opdivo, formerly MK-3475), MEDI0680 (formerly AMP-514), AMP-224, BMS-936558 or any combination thereof.
  • a modified cell of the present disclosure is used in combination with a PD-L1 specific antibody or binding fragment thereof, such as BMS-936559, durvalumab (MEDI4736), atezolizumab (RG7446), avelumab (MSB0010718C), MPDL3280A, or any combination thereof.
  • a modified cell of the present disclosure is used in combination with a LAG3 inhibitor, such as LAG525, IMP321, IMP701, 9H12, BMS-986016, or any combination thereof.
  • a modified cell is used in combination with an inhibitor of CTLA4.
  • a modified cell of the present disclosure is used in combination with a CTLA4 specific antibody or binding fragment thereof, such as ipilimumab, tremelimumab, CTLA4-Ig fusion proteins (e.g., abatacept, belatacept), or any combination thereof.
  • a modified cell of the present disclosure is used in combination with a B7-H3 specific antibody or binding fragment thereof, such as enoblituzumab (MGA271), 376.96, or both.
  • a B7-H4 antibody binding fragment may be a scFv or fusion protein thereof, as described in, for example, Dangaj et al., Cancer Res.
  • a modified cell of the present disclosure is used in combination with an inhibitor of CD244.
  • a modified cell of the present disclosure is used in combination with an inhibitor of BLTA, HVEM, CD 160, or any combination thereof.
  • Anti CD- 160 antibodies are described in, for example, PCT Publication No. WO 2010/084158.
  • a modified cell of the present disclosure is used in combination with an inhibitor of TIM3.
  • a modified cell of the present disclosure is used in combination with an inhibitor of Gal9.
  • a modified cell of the present disclosure is used in combination with an inhibitor of adenosine signaling, such as a decoy adenosine receptor.
  • a modified cell of the present disclosure is used in combination with an inhibitor of A2aR.
  • a modified cell of the present disclosure is used in combination with an inhibitor of KIR, such as lirilumab (BMS-986015).
  • a modified cell of the present disclosure is used in combination with an inhibitor of an inhibitory cytokine (typically, a cytokine other than TGFP) or Treg development or activity.
  • a modified cell of the present disclosure is used in combination with an IDO inhibitor, such as levo-l-methyl tryptophan, epacadostat (INCB024360; Liu et al., Blood 775:3520-30, 2010), ebselen (Terentis et al. , Biochem. 9:591-600, 2010), indoximod, NLG919 (Mautino et al., American Association for Cancer Research 104th Annual Meeting 2013; Apr 6-10, 2013), 1-methyl-tryptophan (l-MT)-tira-pazamine, or any combination thereof.
  • an IDO inhibitor such as levo-l-methyl tryptophan, epacadostat (INCB024360; Liu et al., Blood 775:3520-30, 2010), ebselen (Terentis et al. , Biochem. 9:591-600, 2010), indoximod, NLG919 (Mautino e
  • a modified cell of the present disclosure is used in combination with an arginase inhibitor, such as N(omega)-Nitro-L- arginine methyl ester (L-NAME), N-omega-hydroxy-nor-l-arginine (nor -NOHA), L- NOHA, 2(S)-amino-6-boronohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine (BEC), or any combination thereof.
  • an arginase inhibitor such as N(omega)-Nitro-L- arginine methyl ester (L-NAME), N-omega-hydroxy-nor-l-arginine (nor -NOHA), L- NOHA, 2(S)-amino-6-boronohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine (BEC), or any combination thereof.
  • VISTA such
  • a modified cell of the present disclosure is used in combination with an inhibitor of TIGIT such as, for example, COM902 (Compugen, Toronto, Ontario Canada), an inhibitor of CD155, such as, for example, COM701 (Compugen), or both.
  • a modified cell of the present disclosure is used in combination with an inhibitor of PVRIG, PVRL2, or both.
  • Anti-PVRIG antibodies are described in, for example, PCT Publication No. WO 2016/134333.
  • Anti-PVRL2 antibodies are described in, for example, PCT Publication No. WO 2017/021526.
  • a modified cell of the present disclosure is used in combination with a LAIR1 inhibitor.
  • a modified cell of the present disclosure is used in combination with an inhibitor of CEACAM-1, CEACAM-3, CEACAM-5, or any combination thereof. In certain embodiments, a modified cell of the present disclosure is used in combination with an agent that increases the activity (i.e., is an agonist) of a stimulatory immune checkpoint molecule.
  • a modified cell of the present disclosure can be used in combination with a CD137 (4-1BB) agonist (such as, for example, urelumab), a CD134 (OX-40) agonist (such as, for example, MEDI6469, MEDI6383, or MEDI0562), lenalidomide, pomalidomide, a CD27 agonist (such as, for example, CDX-1127), a CD28 agonist (such as, for example, TGN1412, CD80, or CD86), a CD40 agonist (such as, for example, CP-870,893, rhuCD40L, or SGN-40), a CD122 agonist (such as, for example, IL-2) an agonist of GITR (such as, for example, humanized monoclonal antibodies described in PCT Patent Publication No.
  • a CD137 (4-1BB) agonist such as, for example, urelumab
  • a CD134 (OX-40) agonist such as, for example, MEDI64
  • a method may comprise administering a modified cell of the present disclosure with one or more agonist of a stimulatory immune checkpoint molecule, including any of the foregoing, singly or in any combination.
  • a combination therapy comprises a modified cell of the present disclosure and a secondary therapy comprising one or more of an antibody or antigen binding-fragment thereof that is specific for a cancer antigen expressed by the non-inflamed solid tumor, a radiation treatment, a surgery, a chemotherapeutic agent, a cytokine, RNAi, a further adoptive cell therapy, or any combination thereof.
  • a combination therapy method comprises administering a modified cell and further administering a radiation treatment or a surgery.
  • Radiation therapy is well-known in the art and includes X-ray therapies, such as gamma-irradiation, and radiopharmaceutical therapies. Surgeries and surgical techniques appropriate to treating a given cancer or tumor in a subject are known to those of ordinary skill in the art.
  • a combination therapy method comprises administering a modified (e.g immune) cell of the present disclosure and further administering a chemotherapeutic agent.
  • a chemotherapeutic agent includes, but is not limited to, an inhibitor of chromatin function, a topoisomerase inhibitor, a microtubule inhibiting drug, a DNA damaging agent, an antimetabolite (such as folate antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), a DNA synthesis inhibitor, a DNA interactive agent (such as an intercalating agent), and a DNA repair inhibitor.
  • Illustrative chemotherapeutic agents include, without limitation, the following groups: anti-metabolites/anti-cancer agents, such as pyrimidine analogs (5 -fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2- chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, bus
  • Cytokines are used to manipulate host immune response towards anticancer activity. See, e.g., Floros & Tarhini, Semin. Oncol. 42(4):539-548, 2015. Cytokines useful for promoting immune anticancer or antitumor response include, for example, IFN-a, IL-2, IL-3, IL-4, IL-10, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-24, and GM-CSF, singly or in any combination with the cells or other compositions of this disclosure. In further embodiments, the subject had previously received lymphodepleting chemotherapy prior to receiving the composition or HCT. In certain embodiments, a lymphodepleting chemotherapy comprises a conditioning regimen comprising cyclophosphamide, fludarabine, anti-thymocyte globulin, or a combination thereof.
  • Embodiment 1 A fusion protein comprising: (i)an extracellular component comprising an extracellular domain from a CD8 co-receptor P-chain or a functional portion or variant thereof, or from a CD8 co-receptor a-chain or a functional portion or variant thereof, that is capable of binding to a MHC class I molecule; (ii) a transmembrane domain, provided that the transmembrane domain is not a transmembrane domain from a CD8 co- receptor a-chain when the extracellular component comprises a full length extracellular domain from the CD8 co-receptor a-chain; and (ii) an intracellular component comprising a co-stimulatory domain or a functional portion or variant thereof.
  • Embodiment 3. The fusion protein of Embodiment 2, wherein the CD8 co-receptor P-chain comprises a canonical P-chain, a Ml isoform, a M2 isoform, a M3 isoform, a M4 isoform, a M5 isoform, a M6 isoform, a M7 isoform, or a M8 isoform.
  • Embodiment 4 The fusion protein of Embodiment 3, wherein the CD8 co-receptor P-chain is a Ml isoform.
  • the fusion protein of any one of Embodiments 1-5 wherein the transmembrane domain comprises an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 8, or comprises or consists of the amino acid set forth in SEQ ID NO: 8.
  • Embodiment 8 The fusion protein of any one of Embodiments 1-7, further comprising an amino acid sequence having the amino acid sequence set forth in SEQ ID NOTO, or a functional portion or variant thereof, disposed between the transmembrane domain and the intracellular component.
  • Embodiment 10 The fusion protein of Embodiment 1, wherein the extracellular component comprises or is derived from a CD8 co-receptor a-chain.
  • Embodiment 11 The fusion protein of Embodiment 10, wherein the CD8 co-receptor a-chain comprises a canonical a-chain, an isoform 2, or an isoform 3.
  • the co-stimulatory domain comprises a co-stimulatory domain from one or more of CD28, 4-1BB (CD137), 0X40 (CD134), ICOS (CD278), CD27, CD2, CD5, ICAM-1 (CD54), LFA-1 (CDl la/CD18), GITR, CD30, CD40, BAFF-R, HVEM, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, a ligand that specifically binds with CD83, CD94, DAP12, and/or comprises a functional variant of a co-stimulatory domain thereof.
  • Embodiment 13 a ligand that specifically binds with CD83, CD94, DAP12, and/or comprises a functional variant of a co-stimulatory domain thereof.
  • the fusion protein of Embodiment 12, wherein the co- stimulatory domain comprises a co-stimulatory domain from CD28, or a functional portion or variant thereof.
  • Embodiment 14 The fusion protein of Embodiment 13, wherein the co- stimulatory domain comprises or consists of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 19.
  • Embodiment 15. The fusion protein of Embodiment 13 or 14, wherein the co-stimulatory domain comprises or consists of an amino acid sequence shown in SEQ ID NO: 19.
  • the fusion protein of Embodiment 13 or 14, wherein the co-stimulatory domain comprises a variant of the amino acid sequence shown in SEQ ID NO: 19, wherein one or both of the leucine residues at positions 7 and 8 of SEQ ID NO: 19 is substituted for a different amino acid.
  • Embodiment 17 The fusion protein of Embodiment 16, wherein the variant of the amino acid sequence shown in SEQ ID NO: 19 comprises a substitution of a glycine for one or both of the leucine residues at positions 7 and 8 of SEQ ID NO: 19.
  • the fusion protein of Embodiment 17, wherein the co-stimulatory domain comprises or consists of an amino acid sequence having at least 80% identity to the amino acid sequence the amino acid sequence shown in SEQ ID NO: 20.
  • Embodiment 19 The fusion protein of Embodiment 17 or 18, wherein the co-stimulatory domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 20.
  • Embodiment 20 The fusion protein of any one of Embodiments 12-19, wherein the co-stimulatory domain comprises a co-stimulatory domain from 4-1BB, or a functional portion or variant thereof.
  • Embodiment 21 is any one of Embodiments 12-19, wherein the co-stimulatory domain comprises a co-stimulatory domain from 4-1BB, or a functional portion or variant thereof.
  • the fusion protein of any one of Embodiments 12-22, wherein the co- stimulatory domain comprises a co-stimulatory domain from ICOS, or a functional portion or variant thereof.
  • Embodiment 27 Embodiment 27.
  • the fusion protein of Embodiment 26 wherein the co- stimulatory domain comprises or consists of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO:26.
  • Embodiment 28 The fusion protein of Embodiment 26 or 27, wherein the co-stimulatory domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:26.
  • Embodiment 29 The fusion protein of any one of Embodiments 1-28, further comprising ajunction amino acid.
  • Embodiment 30 The fusion protein of any one of Embodiments 1-28, further comprising ajunction amino acid.
  • a fusion protein comprising: (i) an extracellular component comprising an extracellular domain from a CD8 co-receptor -chain or a functional portion or variant thereof, or from a CD8 co-receptor a-chain or a functional portion or variant thereof, that is capable of binding to a MHC class I molecule; (ii) a transmembrane domain; and (iii) an intracellular component comprising a co-stimulatory domain from one, two, or three of: (a) a variant sequence of CD28 comprising or consisting of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 19 or 20, provided that: (1) no Tyr residue corresponding to position 12, 27, 30, or 39 of SEQ ID NO: 19 is substituted with Phe when the extracellular component comprises a full length extracellular domain from a CD8 co-receptor a-chain and the transmembrane domain comprises a transmembrane domain from the CD8 co-receptor a-chain; and/or
  • Embodiment 31 The fusion protein of Embodiment 30, wherein the extracellular component comprises or is derived from a CD8 co-receptor P-chain, or a functional portion or variant thereof.
  • Embodiment 33 Embodiment 33.
  • Embodiment 34. The fusion protein of any one of Embodiments 30-33, wherein the extracellular component comprises an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 7 , or comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7.
  • Embodiment 35 The fusion protein of any one of Embodiments 30-34, wherein the transmembrane domain comprises or consists of a transmembrane domain from a CD4, a CD8P, a CD8a, a CD27, or a CD28, or a functional portion or variant thereof.
  • the fusion protein of any one of Embodiments 30-35 wherein the transmembrane domain comprises an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 8 , or comprises or consists of the amino acid set forth in SEQ ID NO: 8.
  • Embodiment 37 The fusion protein of any one of Embodiments 30-36, further comprising an amino acid sequence having the amino acid sequence set forth in SEQ ID NOTO , or a functional portion or variant thereof, disposed between the transmembrane domain and the intracellular component.
  • Embodiment 38 is
  • Embodiment 40 The fusion protein of Embodiment 39, wherein the CD8 co-receptor a- chain comprises a canonical a-chain, isoform2, or isoform 3.
  • Embodiment 41 The fusion protein of Embodiment 39 or 40, wherein the extracellular component comprises or consists of an amino acid sequence having at least 80% identity to the amino acid sequence set forth in set forth in SEQ ID NO: 2.
  • Embodiment 43 Embodiment 43.
  • the fusion protein of Embodiment 43, wherein the co-stimulatory domain comprises or consists an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NOTO.
  • the fusion protein of Embodiment 43 or 44, wherein the co-stimulatory domain comprises or consists of the amino acid sequence shown in SEQ ID NOTO.
  • Embodiment 46 Embodiment 46.
  • Embodiment 48 The fusion protein of any one of Embodiments 46 or 47, wherein the co- stimulatory domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 22.
  • Embodiment 49 The fusion protein of any one of Embodiments 30-48, wherein the co-stimulatory domain comprises a co-stimulatory domain from 0X40, or a functional portion or variant thereof.
  • Embodiment 50 The fusion protein of Embodiment 49, wherein the co-stimulatory domain comprises or consists of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO:24.
  • Embodiment 51 The fusion protein of Embodiment 49, wherein the co-stimulatory domain comprises or consists of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO:24.
  • the fusion protein of Embodiment 50 wherein the co-stimulatory domain comprises or consists of the amino acid sequence shown in SEQ ID NO:24.
  • Embodiment 52 The fusion protein of any one of Embodiments 30-51, wherein the co-stimulatory domain comprises a co-stimulatory domain from ICOS, or a functional portion or variant thereof.
  • Embodiment 53 The fusion protein of Embodiment 52, wherein the co-stimulatory domain comprises or consists of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 26.
  • Embodiment 54 is
  • Embodiment 55. The fusion protein of any one of Embodiments 30-54, further comprising a junction amino acid.
  • Embodiment 56. An isolated polynucleotide comprising a polynucleotide that encodes a first fusion protein of any one of Embodiments 1-55, wherein the first encoded fusion protein comprises an extracellular domain from a CD8 co-receptor -chain, or a functional portion or variant thereof.
  • the isolated polynucleotide of Embodiment 56 further comprising a polynucleotide encoding a second protein, wherein the second encoded protein comprises: (i)a CD8 co-receptor a-chain, or a functional portion or variant thereof; or (ii) an extracellular domain from a CD8 co-receptor a-chain, or a functional portion or variant thereof.
  • Embodiment 58 An isolated polynucleotide comprising a polynucleotide that encodes a first fusion protein of any one of Embodiments 1-55, wherein the first encoded fusion protein comprises an extracellular domain from a CD8 co-receptor a-chain, or a functional portion or variant thereof.
  • Embodiment 59 The isolated polynucleotide of Embodiment 58, further comprising a polynucleotide encoding a second protein, wherein the second encoded protein comprises: (i) a CD8 co-receptor a-chain, or a functional portion or variant thereof; (ii) an extracellular domain from a CD8 co-receptor a-chain or a functional portion or variant thereof; (iii) a CD8 co-receptor P chain, or a functional portion or variant thereof; or (iv) an extracellular domain from a CD8 co-receptor P-chain, or a functional portion or variant thereof.
  • Embodiment 60 Embodiment 60.
  • Embodiment 57 or 59 further comprising a polynucleotide encoding a self-cleaving peptide disposed between the first fusion protein and the second protein.
  • Embodiment 61 The isolated polynucleotide of Embodiment 59 or 60, wherein the encoded self-cleaving peptide comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs:55-58.
  • Embodiment 63 Embodiment 63.
  • Embodiment 65 Embodiment 65.
  • Embodiment 64 The isolated polynucleotide of Embodiment 64, wherein the polynucleotide consists of the nucleotide sequence shown in any one of SEQ ID NOs:27-35 and 59-86.
  • Embodiment 66 An expression vector comprising the isolated polynucleotide of any one of Embodiments 56-65 operably linked to an expression control sequence.
  • Embodiment 67 The expression vector of Embodiment 66, wherein the vector is capable of delivering the polynucleotide to a host cell.
  • Embodiment 68 The expression vector of Embodiment 67, wherein the host cell is a hematopoietic progenitor cell or a human immune system cell.
  • Embodiment 69 The isolated polynucleotide of Embodiment 64, wherein the polynucleotide consists of the nucleotide sequence shown in any one of SEQ ID NOs:27-35 and 59-
  • Embodiment 68 wherein the human immune system cell is a CD4 + T cell, a CD8 + T cell, a CD4' CD8' double negative T cell, a y6 T cell, a natural killer cell, a natural killer T cell, a dendritic cell, or any combination thereof.
  • Embodiment 70 The expression vector of Embodiment 68 or 69, wherein the human immune system cell is a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof.
  • Embodiment 71 The expression vector of any one of Embodiments 66-70, wherein the vector is a viral vector.
  • Embodiment 72 The expression vector of any one of Embodiments 66-70, wherein the vector is a viral vector.
  • Embodiment 71 wherein the viral vector is a lentiviral vector or a y-retroviral vector.
  • Embodiment 73 A host cell comprising the polynucleotide of any one of Embodiments 56-65.
  • Embodiment 74 A host cell expressing at its cell surface the fusion protein of any one of Embodiments 1-55.
  • Embodiment 75 The host cell of Embodiment 73 or 74, wherein the host cell is a human immune system cell.
  • Embodiment 76 Embodiment 76.
  • Embodiment 75 wherein the human immune system cell is a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a y8 T cell, a natural killer cell, a natural killer T cell, a dendritic cell, or any combination thereof.
  • Embodiment 77 The host cell of Embodiment 75 or 76, wherein the human immune system cell is a CD4 + T cell.
  • Embodiment 78 The host cell of any one of Embodiments 75-77, wherein the human immune system cell is a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof.
  • Embodiment 79 Embodiment 79.
  • Embodiment 80 The host cell of any one of Embodiments 75-78, further comprising a polynucleotide encoding a binding protein that specifically binds to an antigen or an antigen:MHC complex, wherein the polynucleotide encoding a binding protein is optionally heterologous to the host cell.
  • a host cell comprising: (i) a heterologous polynucleotide that encodes a fusion protein, wherein the encoded fusion protein comprises: (a) an extracellular component comprising an extracellular domain from a CD8 co-receptor a-chain; (b) a transmembrane domain from a CD8 co-receptor a-chain; and (c) an intracellular component comprising a co stimulatory domain from CD28, or a functional portion or variant thereof; and (ii) a heterologous polynucleotide encoding a binding protein that specifically binds to an antigen or an antigen:MHC complex.
  • a heterologous polynucleotide that encodes a fusion protein, wherein the encoded fusion protein comprises: (a) an extracellular component comprising an extracellular domain from a CD8 co-receptor a-chain; (b) a transmembrane domain from a CD8 co-receptor a-chain; and (
  • Embodiment 83. The host cell of Embodiment 81, wherein the human immune system cell comprises a CD4 + T cell.
  • Embodiment 84. The host cell of any one of Embodiments 79-83, wherein the encoded binding protein comprises a TCR or a CAR.
  • Embodiment 86. The host cell of any one of Embodiments 79-85, wherein the binding protein specifically binds to an antigen or antigen:MHC complex that is expressed by or associated with a cancer.
  • Embodiment 87 Embodiment 87.
  • the host cell of Embodiment 86 wherein the antigen is selected from a R0R1, EGFR, EGFRvIII, EGP-2, EGP-40, GD2, GD3, HPV E6, HPV E7, Her2, LI -CAM, Lewis A, Lewis Y, MUC1, MUC16, PSCA, PSMA, CD19, CD20, CD22, CD56, CD23, CD24, CD30, CD33, CD37, CD44v7/8, CD38, CD56, CD123, CA125, c-MET, FcRH5, WT1, folate receptor a, VEGF-a, VEGFR1, VEGFR2, IL-13Ra2, IL-llRa, MAGE-A1, PSA, ephnn A2, ephnn B2, NKG2D, NY-ESO-1, TAG-72, mesothehn, NY-ESO, 5T4, BCMA, FAP, Core Binding Factor protein; Cyclin-Al; Carbonic anhydr
  • Embodiment 88 The host cell of any one of Embodiments 79-87, comprising a chromosomal gene knockout or a mutation of a PD-1 gene; a LAG3 gene; a TIM3 gene; a CTLA4 gene; an HLA component gene; a TCR component gene, or any combination thereof.
  • Embodiment 89 A composition comprising a fusion protein of any one of Embodiments 1-55 and a pharmaceutically acceptable carrier, excipient, or diluent.
  • Embodiment 90 A composition comprising a host cell of any one of Embodiments 79-88, and a pharmaceutically acceptable carrier, excipient, or diluent.
  • Embodiment 91 A unit dose, comprising an effective amount of the host cell of any one of Embodiments 79-88, or of the host cell composition of Embodiment 90.
  • Embodiment 92. The unit dose of Embodiment 91, comprising (i) a composition comprising at least about 30% CD4 + T host cells, combined with (ii) a composition comprising at least about 30% CD8 + T cells, in about a 1:1 ratio.
  • Embodiment 93 The unit dose of Embodiment 92, comprising an effective amount of an effector immune cell comprising a polynucleotide that encodes a binding protein that is capable of specifically binding to an antigen or an antigen:MHC complex.
  • Embodiment 93 wherein the effector immune cell is a T cell, optionally a CD8 + T cell.
  • the unit dose of Embodiment 93 or 94, wherein the binding protein encoded by the effector immune cell comprises a TCR or a CAR.
  • the unit dose of any one of Embodiments 93-95, wherein the binding protein encoded by the effector immune cell is specific for the same or a different antigen as compared to a binding protein encoded by the host cell.
  • Embodiment 97 Embodiment 97.
  • a method of treating a disease or condition in a subject comprising administering to the subject an effective amount of: (i) a host cell of any one of Embodiments 79-88; and/or (ii) a composition of Embodiment 90; and/or (iii) a unit dose of any one of Embodiments 91-96, wherein the disease or condition is characterized by: (a) the presence of the antigen bound by the encoded binding protein of the host cell; and/or (b) the presence of the antigen bound by the encoded binding protein of the effector immune cell.
  • Embodiment 98 The method of Embodiment 97, wherein the disease or condition is a cancer.
  • Embodiment 99 The method of Embodiment 99.
  • Embodiment 98 wherein the cancer comprises a carcinoma, a sarcoma, a glioma, a lymphoma, a leukemia, a myeloma, or any combination thereof.
  • Embodiment 100 The method of Embodiment 98 or 99, wherein the cancer comprises a cancer of the head or neck, melanoma, pancreatic cancer, cholangiocarcinoma, hepatocellular cancer, breast cancer including triple-negative breast cancer (TNBC), gastric cancer, non-small-cell lung cancer, prostate cancer, esophageal cancer, mesothelioma, small-cell lung cancer, colorectal cancer, glioblastoma, or any combination thereof.
  • TNBC triple-negative breast cancer
  • Embodiment 101 The method of any one of Embodiments 98-100, wherein the cancer comprises Askin's tumor, sarcoma botryoides, chondrosarcoma, Ewing's sarcoma, PNET, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma protuberans (DFSP), desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcom
  • Embodiment 102 The method of any one of Embodiments 98-101, wherein the cancer comprises a solid tumor.
  • Embodiment 103 The method of Embodiment 102, wherein the solid tumor is a sarcoma or a carcinoma.
  • Embodiment 104 The method of Embodiment 104.
  • Embodiment 102 or 103 wherein the solid tumor is selected from: chondrosarcoma; fibrosarcoma (fibroblastic sarcoma); Dermatofibrosarcoma protuberans (DFSP); osteosarcoma; rhabdomyosarcoma; Ewing’s sarcoma; a gastrointestinal stromal tumor; Leiomyosarcoma; angiosarcoma (vascular sarcoma);
  • Embodiment 105 The method of Embodiment 102 or 103, wherein the solid tumor is selected from a lung carcinoma (e.g., Adenocarcinoma, Squamous Cell Carcinoma (Epidermoid Carcinoma); Squamous cell carcinoma; Adenocarcinoma; Adenosquamous carcinoma; anaplastic carcinoma; Large cell carcinoma; Small cell carcinoma; a breast carcinoma (e.g., Ductal Carcinoma in situ (non-invasive), Lobular carcinoma in situ (non-invasive), Invasive Ductal Carcinoma, Invasive lobular carcinoma, Non-invasive Carcinoma); a liver carcinoma (e.g., Hepatocellular Carcinoma, Cholangiocarcinomas or Bile Duct Cancer); Large-cell undifferentiated carcinoma, Bronchioalveolar carcinoma);
  • a lung carcinoma e.g., Adenocarcinoma, Squamous Cell Carcinoma (Epidermoid Carcinom
  • Embodiment 106 The method of Embodiment 102 or 103, wherein the solid tumor is an ovarian carcinoma, an ovarian epithelial carcinoma, a cervical adenocarcinoma or small cell carcinoma, a pancreatic carcinoma, a colorectal carcinoma (e.g., an adenocarcinoma or squamous cell carcinoma), a lung carcinoma, a breast ductal carcinoma, or an adenocarcinoma of the prostate.
  • Embodiment 107 The method of any one of Embodiments 97-106, wherein the host cell is allogeneic, syngeneic, or autologous to the subject.
  • Embodiment 108 The method of any one of Embodiments 97-106, wherein the host cell is allogeneic, syngeneic, or autologous to the subject.
  • Embodiment 97-107 comprising administering a plurality of unit doses to the subject.
  • Embodiment 109 The method of Embodiment 108, wherein the plurality of unit doses are administered at intervals between administrations of about two, three, four, five, six, seven, eight, or more weeks.
  • Embodiment 110 The method according to any one of Embodiments 97-109, wherein the unit dose comprises about 10 5 cells/m 2 to about 10 11 cells/m 2 .
  • Embodiment 111 Embodiment 111.
  • Embodiments 97-110 wherein the subject further receives an adjunctive therapy comprising: (i) chemotherapy; (ii) radiation therapy; (iii) an inhibitor of an immune suppression component; (iv) an agonist of a stimulatory immune checkpoint agent; (v) RNAi; (vi) a cytokine; (vii) a surgery; (viii) a monoclonal antibody and/or an antibody-drug conjugate; or (ix) any combination of (i)-(viii), in any order.
  • an adjunctive therapy comprising: (i) chemotherapy; (ii) radiation therapy; (iii) an inhibitor of an immune suppression component; (iv) an agonist of a stimulatory immune checkpoint agent; (v) RNAi; (vi) a cytokine; (vii) a surgery; (viii) a monoclonal antibody and/or an antibody-drug conjugate; or (ix) any combination of (i)-(viii), in any order.
  • Embodiment 112. The
  • Embodiment 163 A polypeptide comprising an extracellular domain from CD8P, a transmembrane domain that is optionally from CD8P, and an intracellular domain comprising a variant of SEQ ID NO. : 19, wherein the variation comprises: (i)a diglycine motif in place of a dileucine motif; (ii) a mutated and optionally non-functional PI3K binding site; (iii) a mutated and optionally non-functional ITK and GADS binding site; (iv) a mutated and optionally non-functional LCK binding site; or (v) any combination of (i)- (v).
  • Embodiment 164 Embodiment 164.
  • Embodiment 163 wherein the polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92%, or at least 95% sequence identity to SEQ ID NO.: 19.
  • Embodiment 165 The polypeptide of Embodiment 163 or 164, wherein the intracellular domain of the polypeptide comprises an amino acid sequence according to SEQ ID NO.:205 or 206.
  • Embodiment 166 The polypeptide of any one of Embodiments 163-165, comprising Motif 1, Motif 2, Motif 3, and/or Motif 4 of any one of Constructs BB-NN in Table 6 or Table 6, and/or comprises as a Motif 2 the sequence FMNM.
  • Embodiment 167 A polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID Nos.: 192-204.
  • Embodiment 168 A polypeptide comprising, consisting essentially or, or consisting of an extracellular domain, a transmembrane domain, and an intracellular domain, as shown under “2 protein size [aa]” in any one of Figures 27-39.
  • Embodiment 169. A polynucleotide encoding the polypeptide of any one of Embodiments 163-168.
  • Embodiment 170 A polynucleotide encoding the polypeptide of any one of Embodiments 163-168.
  • the polynucleotide of Embodiment 169 further encoding a CD8a co-receptor polypeptide, optionally comprising SEQ ID NO.: 114, and wherein, optionally, the polynucleotide comprises or encodes Construct BB, Construct CC, Construct DD, Construct EE, Construct FF, Construct GG, Construct HH, Construct II, Construct JJ, Construct KK, Construe LL, Construct MM, or Construct NN.
  • Embodiment 172 A host cell expressing the polypeptide of any one of Embodiments 163-168. Embodiment 173. The host cell of Embodiment 172, further expressing a CD8a co-receptor polypeptide, optionally comprising SEQ ID NO. : 114. Embodiment 174. A host cell comprising the polynucleotide one any one of Embodiment 169-170 or the vector of Embodiment 171. Embodiment 175. The host cell of any one of Embodiments 172-174, expressing a TCR or a CAR, wherein the TCR or CAR comprises a MHC Class I-restricted binding domain. Embodiment 176.
  • Embodiment 187. The host cell of any one of Embodiments 172-177, wherein the host cell comprises a T cell.
  • Embodiment 177 or 178 comprising a CD4+ T cell, a CD8+ T cell, or both.
  • Embodiment 189. A composition comprising the polypeptide of any one of Embodiments 163-168, the polynucleotide of any one of Embodiments 169-170, the vector of Embodiment 171, or the host cell of any one of Embodiments 172-188, or any combination thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.
  • Embodiment 190 Embodiment 190.
  • a method for treating a disease or disorder such as a cancer comprising administering to a subject having the disease or disorder an effective amount of the polypeptide of any one of Embodiments 163-168, the polynucleotide of any one of Embodiments 169-170, the vector of Embodiments 171, the host cell of any one of Embodiments 172-188, the composition of Embodiment 189, or any combination thereof.
  • Embodiment la A polynucleotide encoding (a) a first polypeptide, wherein the first polypeptide comprises (a)(i) an extracellular component comprising an extracellular domain from a CD8 P-chain (CD8 ), or a functional portion or variant thereof that is capable of binding to a MHC Class I molecule, (a)(ii) a transmembrane domain from a CD8 , and (a)(iii) an intracellular component comprising (a)(iii)(l) a CD8 intracellular region amino acid sequence that comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 10 or SEQ ID NO.: 9, and (a)(iii)(2) a costimulatory domain or a functional portion or variant thereof, wherein, optionally: (1) the costimulatory domain or a functional portion or variant thereof is from one or more of CD28 (optionally comprising a LLD GG mutation, a partial signaling mutation, and/or a full signal
  • Embodiment 2a A fusion protein comprising: (i) an extracellular component comprising an extracellular domain from a CD8 co-receptor -chain or a functional portion or variant thereof, or from a CD8 co-receptor a-chain or a functional portion or variant thereof, that is capable of binding to a MHC class I molecule; (ii) a transmembrane domain, provided that the transmembrane domain is not a transmembrane domain from a CD8 co- receptor a-chain when the extracellular component comprises a full length extracellular domain from the CD8 co-receptor a-chain; and (ii) an intracellular component comprising a co-stimulatory domain or a functional portion or variant thereof.
  • Embodiment 3a The fusion protein of Embodiment 2a, wherein the co-stimulatory domain comprises a costimulatory domain from one or more of CD28, 4-1BB (CD137), 0X40 (CD134), ICOS (CD278), CD27, CD2, CD5, ICAM-1 (CD54), LFA-1 (CDl la/CD18), GITR, CD30, CD40, BAFF-R, HVEM, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, B7-H3, a ligand that specifically binds with CD83, CD94, DAP12, and/or comprises a functional variant of a co-stimulatory domain thereof.
  • Embodiment 4a Embodiment 4a.
  • the fusion protein of Embodiment 3 a wherein the co-stimulatory domain comprises a co-stimulatory domain from CD28, or a functional portion or variant thereof.
  • Embodiment 5a The fusion protein of Embodiment 4a, wherein the co-stimulatory domain comprises or consists of an amino acid sequence having at least 80% identity to, or comprises or consists of, the amino acid sequence shown in SEQ ID NO: 19.
  • Embodiment 6a Embodiment 6a.
  • a fusion protein comprising: (i) an extracellular component comprising an extracellular domain from a CD8 co-receptor P-chain or a functional portion or variant thereof, or from a CD8 co-receptor a-chain or a functional portion or variant thereof, that is capable of binding to a MHC class I molecule; (ii) a transmembrane domain; and (iii) an intracellular component comprising a co-stimulatory domain from one, two, or three of: (a) a variant sequence of CD28 comprising or consisting of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 19 or 20, provided that: (1) no Tyr residue corresponding to position 12, 27, 30, or 39 of SEQ ID NO: 19 is substituted with Phe when the extracellular component comprises a full length extracellular domain from a CD8 co-receptor a-chain and the transmembrane domain comprises a transmembrane domain from the CD8 co-receptor a-chain; and/or
  • Embodiment 7a The fusion protein of any one of Embodiments 2a-6a, wherein the extracellular component comprises a CD8 co-receptor P-chain, or a functional portion or variant thereof.
  • Embodiment 8a The fusion protein of Embodiment 7a, wherein the CD8 co-receptor P-chain comprises a canonical P-chain, a Ml isoform, a M2 isoform, a M3 isoform, a M4 isoform, a M5 isoform, a M6 isoform, a M7 isoform, or a M8 isoform.
  • Embodiment 9a Embodiment 9a.
  • Embodiment 8a wherein the CD8 co-receptor P- chain is a Ml isoform.
  • Embodiment 10a The fusion protein of any one of Embodiments 2a-9a, wherein the extracellular component comprises an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:7 , or comprises or consists of the amino acid sequence set forth in SEQ ID NO:7.
  • Embodiment Ila Embodiment Ila.
  • the fusion protein of any one of Embodiments 2a- 1 la, wherein the transmembrane domain comprises an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 8 , or comprises or consists of the amino acid set forth in SEQ ID NO: 8.
  • Embodiment 13a Embodiment 13a.
  • Embodiment 14a. The fusion protein of any one of Embodiments 2a- 13 a, wherein the extracellular component comprises the amino acid sequence set forth in SEQ ID NO: 7 and the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 8.
  • Embodiment 15a The fusion protein of Embodiment 2a or 6a, wherein the extracellular component comprises or is derived from a CD8 co-receptor a-chain.
  • the fusion protein of Embodiment 15a wherein the CD8 co-receptor a-chain comprises a canonical a-chain, isoform 2, or isoform 3.
  • Embodiment 17a The fusion protein of Embodiment 15a or 16a, wherein the extracellular component comprises or consists of an amino acid sequence having at least 80% identity to the amino acid sequence set forth in set forth in SEQ ID NO: 2.
  • Embodiment 18a The fusion protein of Embodiment any one of Embodiments 1 la- 17a, wherein the transmembrane component comprises an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 3, or comprises or consists of the amino acid sequence set forth in SEQ ID NO:3.
  • Embodiment 19a The fusion protein of any one of Embodiments 2a- 18a, wherein the variant sequence of CD28 comprises a substitution of a glycine for one or both of the leucine residues corresponding to positions 7 and 8 of SEQ ID NO: 19.
  • Embodiment 20a The fusion protein of Embodiment 19a, wherein the co-stimulatory domain comprises or consists of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO:20.
  • Embodiment 21a The fusion protein of Embodiment 19a or 20a, wherein the costimulatory domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 20.
  • Embodiment 22a The fusion protein of any one of Embodiments 2a- 18a, wherein the variant sequence of CD28 comprises a substitution of a glycine for one or both of the leucine residues corresponding to positions 7 and 8 of SEQ ID NO: 19.
  • Embodiment 20a The
  • Embodiment 23a The fusion protein of Embodiment 22a, wherein the co-stimulatory domain comprises or consists of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO:22.
  • Embodiment 24a The fusion protein of of Embodiments 22a or 23 a, wherein the co-stimulatory domain comprises or consists of the amino acid sequence shown in SEQ ID NO:22.
  • Embodiment 25a is
  • Embodiment 26a The fusion protein of Embodiment 25a, wherein the co- stimulatory domain comprises or consists of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO:24.
  • Embodiment 27a The fusion protein of Embodiment 26a, wherein the co-stimulatory domain comprises or consists of the amino acid sequence shown in SEQ ID NO:24.
  • Embodiment 28a is
  • the fusion protein of Embodiment 28a, wherein the co-stimulatory domain comprises or consists of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 26.
  • Embodiment 30a The fusion protein of Embodiment 28a or 29a, wherein the co-stimulatory domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:26.
  • Embodiment 31a The fusion protein of any one of Embodiments 2a-30a, further comprising a junction amino acid.
  • Embodiment 32a An isolated polynucleotide comprising a polynucleotide that encodes a first fusion protein of any one of Embodiments 2a-31a, wherein the first encoded fusion protein comprises an extracellular domain from a CD8 co-receptor P-chain, or a functional portion or variant thereof.
  • Embodiment 33a An isolated polynucleotide comprising a polynucleotide that encodes a first fusion protein of any one of Embodiments 2a-31a, wherein the first encoded fusion protein comprises an extracellular domain from a CD8 co-receptor P-chain, or a functional portion or variant thereof.
  • the isolated polynucleotide of Embodiment 32a further comprising a polynucleotide encoding a second protein, wherein the second encoded protein comprises: (i) a CD8 co-receptor a-chain, or a functional portion or variant thereof; or (ii) an extracellular domain from a CD8 co-receptor a-chain, or a functional portion or variant thereof.
  • Embodiment 34a Embodiment 34a.
  • An isolated polynucleotide comprising a polynucleotide that encodes a first fusion protein of any one of Embodiments 2a-31a, wherein the first encoded fusion protein comprises an extracellular domain from a CD8 co-receptor a-chain, or a functional portion or variant thereof.
  • Embodiment 35a An isolated polynucleotide comprising a polynucleotide that encodes a first fusion protein of any one of Embodiments 2a-31a, wherein the first encoded fusion protein comprises an extracellular domain from a CD8 co-receptor a-chain, or a functional portion or variant thereof.
  • the isolated polynucleotide of Embodiment 34a further comprising a polynucleotide encoding a second protein, wherein the second encoded protein comprises: (i) a CD8 co-receptor a-chain, or a functional portion or variant thereof; (ii) an extracellular domain from a CD8 co-receptor a- chain or a functional portion or variant thereof; (iii) a CD8 co-receptor P chain, or a functional portion or variant thereof; or (iv) an extracellular domain from a CD8 coreceptor -chain, or a functional portion or variant thereof.
  • Embodiment 36a Embodiment 36a.
  • Embodiment 33a or 35a further comprising a polynucleotide encoding a self-cleaving peptide disposed between the first fusion protein and the second protein.
  • Embodiment 37a The isolated polynucleotide of Embodiment 35a or 36a, wherein the encoded self-cleaving peptide comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs:55-58.
  • Embodiment 38a The isolated polynucleotide of any one of Embodiments 32a-37a, wherein the first or second encoded fusion protein comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs:36-42.
  • Embodiment 39a The isolated polynucleotide of any one of Embodiments 32a-38a, wherein any one or more of the polynucleotides encoding the first or second fusion protein is codon-optimized for expression by a host cell, wherein the host cell is optionally a T cell, preferably a CD4 + T cell.
  • Embodiment 40a The isolated polynucleotide of any one of Embodiments 32a-39a, wherein the polynucleotide comprises the nucleic acid sequence shown in any one of SEQ ID NOS:27-35, 43-54, and 59-66.
  • Embodiment 41a Embodiment 41a.
  • Embodiment 42a A polypeptide comprising: (i) an extracellular component from a CD8P; (ii) a transmembrane domain that is optionally from a CD8P; and (iii) an intracellular component comprising (iii)(l) a CD8P intracellular region amino acid sequence comprising or consisting of SEQ ID NO.: 9 or SEQ ID NO.: 10 and (iii)(2) a CD28 intracellular region amino acid sequence comprising a CD28 costimulatory domain and, optionally, a a LL ⁇ GG mutation, wherein the polypeptide is capable of binding to a MHC Class I molecule.
  • Embodiment 43a A polynucleotide comprising (a) a nucleotide sequence encoding the polypeptide of Embodiment 42a.
  • Embodiment 44a The polynucleotide of Embodiment 43a, further comprising (b) a nucleotide sequence encoding a CD8a polypeptide, wherein the CD8a polypeptide is optionally an engineered polypeptide comprising (i) a portion of a CD8P extracellular component, such as a CD8P stalk region amino acid sequence, (ii) a portion of a CD28 extracellular component; (iii) a CD28 transmembrane domain; (iv) an intracellular component comprising a CD28 costimulatory domain and, further optionally, a LL ⁇ GG mutation, wherein, still further optionally, the CD28 costimulatory domain comprises a partial signaling mutant or a full signaling mutant; and/or (v) a intracellular component comprising a costim
  • Embodiment 45a The polynucleotide of Embodiment 44a, further comprising, disposed between the nucleotide sequence of (a) and the nucleotide sequence of (b), (c) a nucleotide sequence encoding any one or more of: a self-cleaving peptide; a furin cleavage sequence; and an internal ribosomal skip element (IRES), wherein, optionally, the nucleotide sequence of (c) separates the nucleotide sequence of (a) from the nucleotide sequence of (b).
  • IRS internal ribosomal skip element
  • a polypeptide comprising: (i) an extracellular component from a CD8P; (ii) a transmembrane domain that is optionally from a CD8P; and (iii) an intracellular component comprising a CD28 costimulatory domain and an optional a LL ⁇ GG mutation, wherein the polypeptide is capable of binding to a MHC Class I molecule.
  • Embodiment 47a A polynucleotide comprising a nucleotide sequence encoding (a) the polypeptide of Embodiment 46a.
  • Embodiment 48a is a polypeptide comprising: (i) an extracellular component from a CD8P; (ii) a transmembrane domain that is optionally from a CD8P; and (iii) an intracellular component comprising a CD28 costimulatory domain and an optional a LL ⁇ GG mutation, wherein the polypeptide is capable of binding to a MHC Class I molecule.
  • the polynucleotide of Embodiment 46a or 47a further comprising (b) a nucleotide sequence encoding a CD8a polypeptide, wherein the CD8a polypeptide is optionally an engineered polypeptide comprising (i) a portion of a CD8P extracellular component, such as a CD8P stalk region amino acid sequence, (ii) a portion of a CD28 extracellular component; (iii) a CD28 transmembrane domain; and/or (iv) an intracellular component comprising a CD28 costimulatory domain and, further optionally, a LL ⁇ GG mutation, wherein, still further optionally, the CD28 costimulatory domain comprises a partial signaling mutant or a full signaling mutant.
  • the CD8a polypeptide is optionally an engineered polypeptide comprising (i) a portion of a CD8P extracellular component, such as a CD8P stalk region amino acid sequence, (ii) a portion of a CD28 extra
  • Embodiment 49a The polynucleotide of Embodiment 48a, further comprising, disposed between the nucleotide sequence of (a) and the nucleotide sequence of (b), (c) a nucleotide sequence encoding any one or more of: a self-cleaving peptide; a furin cleavage sequence; and an internal ribosomal skip element (IRES), wherein, optionally, the nucleotide sequence of (c) separates the nucleotide sequence of (a) from the nucleotide sequence of (b).
  • Embodiment 50a Embodiment 50a.
  • a polypeptide comprising: (i) an extracellular component from a CD8a; (ii) a transmembrane domain that is optionally from a CD8a; and (iii) an intracellular component comprising a CD28 costimulatory domain and an optional LL ⁇ GG mutation, wherein the polypeptide is capable of binding to a MHC Class I molecule.
  • Embodiment 51a A polynucleotide comprising (a) a nucleotide sequence encoding the polypeptide of Embodiment 50a.
  • Embodiment 52a The polynucleotide of Embodiment 51a, further comprising (b) a nucleotide sequence encoding the polypeptide of Embodiment 46.
  • Embodiment 53a The polynucleotide of Embodiment 51a or 52a, further comprising (b) a nucleotide sequence encoding a CD8P polypeptide, , wherein the CD8P polypeptide is optionally an engineered polypeptide comprising (i) a portion of a CD8a extracellular component, such as a CD8a stalk region amino acid sequence, (ii) a portion of a CD28 extracellular component; (iii) a CD28 transmembrane domain; (iv) an intracellular component comprising a CD28 costimulatory domain and, further optionally, a LL ⁇ GG mutation, wherein, still further optionally, the CD28 costimulatory domain comprises a partial signaling mutant or a full signaling mutant; and/or (v) an intracellular component comprising a costimulatory domain from 4-1BB, ICOS, 0X40, GITR, TRAF1, or Lek.
  • the CD8P polypeptide is optionally
  • Embodiment 54a The polynucleotide of Embodiment 52a or 53a, further comprising, disposed between the nucleotide sequence of (a) and the nucleotide sequence of (b), (c) a nucleotide sequence encoding any one or more of: a self-cleaving peptide; a furin cleavage sequence; and an internal ribosomal skip element (IRES), wherein, optionally, the nucleotide sequence of (c) separates the nucleotide sequence of (a) from the nucleotide sequence of (b).
  • IRS internal ribosomal skip element
  • a polypeptide comprising: (i) an extracellular component from a CD8P; (ii) a transmembrane domain that is optionally from a CD8P; and (iii) an intracellular component comprising (1) a CD28 costimulatory domain comprising the amino acid sequence DAMNMTARRAGPTRKHYQAYAAPRDFAAYRS (SEQ ID NO.185) and (2) an optional LL ⁇ GG mutation, wherein the polypeptide is capable of binding to a MHC Class I molecule.
  • Embodiment 56a A polynucleotide comprising (a) a nucleotide sequence encoding the polypeptide of Embodiment 55 a.
  • Embodiment 57a A polynucleotide comprising (a) a nucleotide sequence encoding the polypeptide of Embodiment 55 a.
  • the polynucleotide of Embodiment 56a further comprising (b) a nucleotide sequence encoding a CD8a polypeptide, wherein the CD8a polypeptide is optionally an engineered polypeptide comprising (i) a portion of a CD8P extracellular component, such as a CD8P stalk region amino acid sequence, (ii) a portion of a CD28 extracellular component; (iii) a CD28 transmembrane domain; (iv) an intracellular component comprising a CD28 costimulatory domain and, further optionally, a LL ⁇ GG mutation, wherein, still further optionally, the CD28 costimulatory domain comprises a partial signaling mutant or a full signaling mutant; and/or (v) a intracellular component comprising a costimulatory domain from 4-1BB, ICOS, 0X40, GITR, TRAF1, or Lek.
  • the CD8a polypeptide is optionally an engineered polypeptide comprising (i)
  • Embodiment 58a The polynucleotide of Embodiment 57a, further comprising, disposed between the nucleotide sequence of (a) and the nucleotide sequence of (b), (c) a nucleotide sequence encoding any one or more of: a self-cleaving peptide; a furin cleavage sequence; and an internal ribosomal skip element (IRES), wherein, optionally, the nucleotide sequence of (c) separates the nucleotide sequence of (a) from the nucleotide sequence of (b).
  • IRS internal ribosomal skip element
  • a polypeptide comprising: (i) an extracellular component from a CD8a; (ii) a transmembrane domain that is optionally from a CD8a; and (iii) an intracellular component comprising (1) a CD28 costimulatory domain comprising the amino acid sequence DAMNMTARRAGPTRKHYQAYAAPRDFAAYRS (SEQ ID NO.185) and (2) an optional LL ⁇ GG mutation, wherein the polypeptide is capable of binding to a MHC Class I molecule.
  • Embodiment 60a A polynucleotide comprising (a) a nucleotide sequence encoding the polypeptide of Embodiment 59a.
  • Embodiment 61a A polynucleotide comprising (a) a nucleotide sequence encoding the polypeptide of Embodiment 59a.
  • the polynucleotide of Embodiment 60a further comprising (b) a nucleotide sequence encoding the polypeptide of Embodiment 55.
  • Embodiment 62a The polynucleotide of Embodiment 61a, further comprising, disposed between the nucleotide sequence of (a) and the nucleotide sequence of (b), (c) a nucleotide sequence encoding any one or more of: a self-cleaving peptide; a furin cleavage sequence; and an internal ribosomal skip element (IRES), wherein, optionally, the nucleotide sequence of (c) separates the nucleotide sequence of (a) from the nucleotide sequence of (b).
  • IRS internal ribosomal skip element
  • Embodiment 63a A polypeptide comprising: (i) an extracellular component from a CD8P; (ii) a transmembrane domain that is optionally from a CD8P; and (iii) an intracellular component comprising (iii)(l ) a CD8P intracellular region amino acid sequence comprising or consisting of SEQ ID NO.: 9 or SEQ ID NO.: 10 and (iii)(2) a wild-type CD28 costimulatory domain, wherein the polypeptide is capable of binding to a MHC Class I molecule.
  • Embodiment 64a A polynucleotide comprising (a) a nucleotide sequence encoding the polypeptide of Embodiment 63 a.
  • Embodiment 65a The polynucleotide of Embodiment 64a, further comprising (b) a nucleotide sequence encoding a CD8a polypeptide, wherein the CD8a polypeptide is optionally an engineered polypeptide comprising (i) a portion of a CD8P extracellular component, such as a CD8P stalk region amino acid sequence, (ii) a portion of a CD28 extracellular component; (iii) a CD28 transmembrane domain; (iv) an intracellular component comprising a CD28 costimulatory domain and, further optionally, a LL ⁇ GG mutation, wherein, still further optionally, the CD28 costimulatory domain comprises a partial signaling mutant or a full signaling mutant; and/or (v) a intracellular component comprising a costimulatory domain from 4-1BB, ICOS, 0X40, GITR, TRAF1, or Lek.
  • the CD8a polypeptide is optionally an engineered poly
  • Embodiment 66a The polynucleotide of Embodiment 65a, further comprising, disposed between the nucleotide sequence of (a) and the nucleotide sequence of (b), (c) a nucleotide sequence encoding any one or more of: a self-cleaving peptide; a furin cleavage sequence; and an internal ribosomal skip element (IRES), wherein, optionally, the nucleotide sequence of (c) separates the nucleotide sequence of (a) from the nucleotide sequence of (b).
  • Embodiment 67a Embodiment 67a.
  • a polypeptide comprising: (i) an extracellular component from a CD8P; (ii) a transmembrane domain that is optionally from a CD8P; and (iii) an intracellular component comprising a costimulatory domain from (iii)(l ) a 4-1BB, (iii)(2) an ICOS, (iii)(3), an 0X40, or (iii)(4) a GITR, wherein the polypeptide is capable of binding to a MHC Class I molecule.
  • Embodiment 68a A polynucleotide comprising (a) a nucleotide sequence encoding the polypeptide of Embodiment 67.
  • the polynucleotide of Embodiment 68a further comprising (b) a nucleotide sequence encoding a CD8a polypeptide, wherein the CD8a polypeptide is optionally an engineered polypeptide comprising (i) a portion of a CD8P extracellular component, such as a CD8P stalk region amino acid sequence, (ii) a portion of a CD28 extracellular component; (iii) a CD28 transmembrane domain; (iv) an intracellular component comprising a CD28 costimulatory domain and, further optionally, a LL ⁇ GG mutation, wherein, still further optionally, the CD28 costimulatory domain comprises a partial signaling mutant or a full signaling mutant; and/or (v) a intracellular component comprising a costimulatory domain from 4-1BB, ICOS, 0X40, GITR, TRAF1, or Lek.
  • the CD8a polypeptide is optionally an engineered polypeptide comprising (i
  • Embodiment 70a The polynucleotide of Embodiment 69a, further comprising, disposed between the nucleotide sequence of (a) and the nucleotide sequence of (b), (c) a nucleotide sequence encoding any one or more of: a self-cleaving peptide; a furin cleavage sequence; and an internal ribosomal skip element (IRES), wherein, optionally, the nucleotide sequence of (c) separates the nucleotide sequence of (a) from the nucleotide sequence of (b).
  • Embodiment 71a Embodiment 71a.
  • a polypeptide comprising: (i) an extracellular component from a CD8a; (ii) a transmembrane domain from a CD28; and (iii) an intracellular component comprising a CD28 costimulatory domain and, optionally, a LL ⁇ GG mutation, wherein the polypeptide is capable of binding to a MHC Class I molecule.
  • Embodiment 72a Embodiment 72a.
  • a polypeptide comprising: (i) an extracellular component from a CD8P; (ii) a transmembrane domain from a CD8P; and (iii) an intracellular component comprising (iii)(l ) a CD8P intracellular region amino acid sequence (optionally comprising or consisting of SEQ ID NO.: 9 or 10) and (iii)(2) a signaling domain from Lek, wherein the fusion protein is capable of binding to a MHC Class I molecule.
  • Embodiment 73a Embodiment 73a.
  • a polynucleotide comprising (a) a nucleotide sequence encoding the polypeptide of Embodiment 71a and/or (b) a nucleotide sequence encoding the polypeptide of Embodiment 72a.
  • Embodiment 74a A polynucleotide comprising (a) a nucleotide sequence encoding the polypeptide of Embodiment 71a and/or (b) a nucleotide sequence encoding the polypeptide of Embodiment 72a.
  • the polynucleotide of Embodiment 73 a further comprising, disposed between the nucleotide sequence of (a) and the nucleotide sequence of (b), (c) a nucleotide sequence encoding any one or more of: a self-cleaving peptide; a furin cleavage sequence; and an internal ribosomal skip element (IRES), wherein, optionally, the nucleotide sequence of (c) separates the nucleotide sequence of (a) from the nucleotide sequence of (b).
  • Embodiment 75a Embodiment 75a.
  • a polypeptide comprising: (i) an extracellular component from a CD8a; (ii) a transmembrane domain that is optionally from a CD8 a; and (iii) an intracellular component comprising a CD28 costimulatory domain comprising the amino acid sequence DAMNMTARRAGPTRKHFQAFAAPRDFAAFRS (SEQ ID NO : 186), and optionally further comprising a LL ⁇ GG mutation, wherein the polypeptide is capable of binding to a MHC Class I molecule.
  • Embodiment 76a Embodiment 76a.
  • a polypeptide comprising: (i) an extracellular component from a CD8P; (ii) a transmembrane domain that is optionally from a CD8P; and (iii) an intracellular component comprising a CD28 costimulatory domain comprising the ammo acid sequence DAMNMTARRAGPTRKHFQAFAAPRDFAAFRS (SEQ ID NO.: 186), and optionally further comprising a LL ⁇ GG mutation, wherein the polypeptide is capable of binding to a MHC Class I molecule.
  • Embodiment 77a A polynucleotide encoding (a) the polypeptide of Embodiment 75a and/or (b) the polypeptide of Embodiment 76a.
  • Embodiment 78a The polynucleotide of Embodiment 77a, further comprising, disposed between the nucleotide sequence of (a) and the nucleotide sequence of (b), (c) a nucleotide sequence encoding any one or more of: a self-cleaving peptide; a furin cleavage sequence; and an internal ribosomal skip element (IRES), wherein, optionally, the nucleotide sequence of (c) separates the nucleotide sequence of (a) from the nucleotide sequence of (b).
  • Embodiment 79a Embodiment 79a.
  • a polypeptide comprising: (i) an extracellular component comprising (i)(l) a CD8a extracellular region amino acid sequence (e.g. comprising or consisting of a CD8a Ig V-like domain), (i)(2) a CD8P stalk region amino acid sequence, and (i)(3) a CD28 extracellular region amino acid sequence; (ii) a transmembrane domain from CD28; and (iii) an intracellular component comprising a CD28 costimulatory domain and an optional LL ⁇ GG mutation, wherein the polypeptide is capable of binding to a MHC Class I molecule.
  • Embodiment 80a Embodiment 80a.
  • a polypeptide comprising: (i) an extracellular component comprising (i)(l) a CD8a extracellular region amino acid sequence (e.g. comprising or consisting of a CD8a Ig V-like domain), (i)(2) a CD8P stalk region amino acid sequence, and (i)(3) a CD28 extracellular region amino acid sequence; (ii) a transmembrane domain from CD28; and (iii) an intracellular component comprising (iii)(l) a CD28 costimulatory domain and an optional LL > GG mutation and (iii)(2) a CD8a intracellular region amino acid sequence, wherein the polypeptide is capable of binding to a MHC Class I molecule.
  • Embodiment 81a Embodiment 81a.
  • a polypeptide comprising: (i) an extracellular component from a NKG2D; (ii) a transmembrane domain from a NKG2D; and (iii) an intracellular component comprising a CD28 costimulatory domain and an optional LL ⁇ GG mutation, wherein the polypeptide is capable of binding to a NKG2D ligand.
  • Embodiment 82a A polynucleotide encoding the polypeptide of any one of Embodiments 79a-81a.
  • Embodiment 83a A polynucleotide encoding the polypeptide of any one of Embodiments 79a-81a.
  • a polynucleotide comprising (a) a nucleotide sequence encoding a CD8a polypeptide and (b) a nucleotide sequence encoding a CCR4, wherein the CD8a polypeptide is optionally an engineered polypeptide comprising (i) a portion of a CD8P extracellular component, such as a CD8P stalk region amino acid sequence, (ii) a portion of a CD28 extracellular component; (iii) a CD28 transmembrane domain; (iv) an intracellular component comprising a CD28 costimulatory domain and, further optionally, a LL ⁇ GG mutation, wherein, still further optionally, the CD28 costimulatory domain comprises a partial signaling mutant or a full signaling mutant; and/or (v) a intracellular component comprising a costimulatory domain from 4-1BB, ICOS, 0X40, GITR, TRAF1, or Lek.
  • the CD8a polypeptide is optionally
  • Embodiment 84a A polynucleotide comprising (a) a nucleotide sequence encoding a CD8a and (b) a nucleotide sequence encoding a CCR2b, wherein the CD8a polypeptide is optionally an engineered polypeptide comprising (i) a portion of a CD8P extracellular component, such as a CD8P stalk region amino acid sequence, (ii) a portion of a CD28 extracellular component; (iii) a CD28 transmembrane domain; (iv) an intracellular component comprising a CD28 costimulatory domain and, further optionally, a LL ⁇ GG mutation, wherein, still further optionally, the CD28 costimulatory domain comprises a partial signaling mutant or a full signaling mutant; and/or (v) a intracellular component comprising a costimulatory domain from 4-1BB, ICOS, 0X40, GITR, TRAF1, or Lek.
  • Embodiment 85a The polynucleotide of Embodiment 83a or 84a, further comprising, disposed between the nucleotide sequence of (a) and the nucleotide sequence of (b), (c) a nucleotide sequence encoding any one or more of: a self-cleaving peptide; a furin cleavage sequence; and an internal ribosomal skip element (IRES), wherein, optionally, the nucleotide sequence of (c) separates the nucleotide sequence of (a) from the nucleotide sequence of (b).
  • Embodiment 86a The polynucleotide of Embodiment 83a or 84a, further comprising, disposed between the nucleotide sequence of (a) and the nucleotide sequence of (b), (c) a nucleotide sequence encoding any one or more of: a self-cleaving peptide; a furin cleavage sequence
  • Embodiment 87a A polypeptide comprising: (i) an extracellular component from a Fas; (ii) a transmembrane domain that is optionally from Fas; and (iii) an intracellular component comprising a Lek signaling domain, wherein the polypeptide is capable of binding to a FasL.
  • Embodiment 88a A polypeptide comprising a Fas extracellular component and a transmembrane domain that is optionally from Fas, and does not comprise a functional Fas intracellular signaling domain, wherein the polypeptide is capable of binding to a FasL, and
  • a polypeptide comprising: (i) an extracellular component from a Fas; (ii) a transmembrane domain that is optionally from Fas; and (iii) an intracellular component comprising a CD8a intracellular amino acid sequence, wherein the polypeptide is capable of binding to a FasL and, optionally, associating with a Lek.
  • a polypeptide comprising: (i) an extracellular component from a Fas; (ii) a transmembrane domain that is optionally from a Fas; (iii) an intracellular component comprising a TRAF1 intracellular signaling domain, and, optionally, (iv) a linker amino acid sequence disposed between and connecting the transmembrane domain and the TRAF1 intracellular signaling domain, wherein the polypeptide is capable of binding to a FasL.
  • Embodiment 90a Embodiment 90a.
  • a polypeptide comprising: (i) an extracellular component from a CD3 ⁇ ; (ii) a transmembrane domain that is optionally from CD3 ⁇ ; and (iii) an intracellular component comprising (iii)(a) a CD28 costimulatory domain and (iii)(b) a CD3 ⁇ intracellular signaling domain, wherein, optionally, the extracellular component does not further comprise a target-binding domain (e.g. an antigen-binding domain, such as from an antibody or antigen-binding fragment thereof, a T cell receptor, or a receptor ectodomain).
  • a target-binding domain e.g. an antigen-binding domain, such as from an antibody or antigen-binding fragment thereof, a T cell receptor, or a receptor ectodomain.
  • Embodiment 90a wherein (iii)(a) is disposed between (ii) and (iii)(b).
  • Embodiment 92a A polypeptide comprising: (i) an extracellular component from a CD3 ⁇ ; (ii) a transmembrane domain that is optionally from CD3 ⁇ ; and (iii) an intracellular component comprising (iii)(a) a 4- IBB costimulatory domain and (iii)(b) a CD3 ⁇ costimulatory domain wherein, optionally, the extracellular component does not further comprise a target-binding domain (e.g.
  • Embodiment 93a The polypeptide of Embodiment 92a, wherein (iii)(a) is disposed between (ii) and (iii)(b).
  • Embodiment 94a A polynucleotide comprising a nucleotide sequence encoding the polypeptide of any one of Embodiments 86a-93a.
  • Embodiment 96a Embodiment 96a.
  • a polypeptide comprising or consisting of two or more amino acid sequences, each of the two or more amino acid sequences independently having 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%, or at least 99% identity to, or comprising or consisting of, an amino acid sequence set forth in any one of SEQ ID NOs.: 36-42, 83-97, and 103-105.
  • Embodiment 97a Embodiment 97a.
  • Embodiment 98a A polynucleotide encoding the polypeptide of any one of Embodiments 95a-97a.
  • a polynucleotide encoding (i) a first polypeptide, which is a polypeptide of Embodiment 95a, and a second polypeptide, which is a polypeptide of Embodiment 96a, (ii) a first polypeptide, which is a polypeptide of Embodiment 95a, and a second polypeptide, which is a polypeptide of Embodiment 97a, and/or (iii) a first polypeptide, which is a polypeptide of Embodiment 96a, and a second polypeptide, which is a polypeptide of Embodiment 97a.
  • Embodiment 100a Embodiment 100a.
  • Embodiment 101a The polynucleotide of Embodiment 100a, wherein the first polypeptide and the second polypeptide have 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%, or at least 99% identity to, or comprise or consisting of, the amino acid sequences set forth in SEQ ID NOs.: (a) 1 and 113, respectively; (b) 1 and 116, respectively; (c) 1 and 120, respectively; (d) 1 and 126, respectively; (e) 1 and 128, respectively; (f) 1 and 130, respectively; (g) 1 and 132, respectively; (h) 1 and 134, respectively; (i) 114 and 115, respectively; (j) 114 and 117, respectively; (k) 114 and 121, respectively; (1) 114 and 127, respectively; (m) 114 and 129, respectively; (n) 114 and 131, respectively; (o)
  • Embodiment 102a The polynucleotide of any one of any one of Embodiments 99a-101a, further comprising, disposed between (i) a nucleotide sequence encoding the first polypeptide and (ii) a nucleotide sequence encoding the second polypeptide, (iii) a nucleotide sequence encoding any one or more of: a self-cleaving peptide; a furin cleavage sequence; and an internal ribosomal skip element (IRES), wherein, optionally, the nucleotide sequence of (iii) separates the nucleotide sequence of (i) from the nucleotide sequence of (ii).
  • Embodiment 103a The polynucleotide of any one of any one of Embodiments 99a-101a, further comprising, disposed between (i) a nucleotide sequence encoding the first polypeptide and (ii) a nucleotide
  • a polypeptide comprising: (i) an extracellular component comprising an amino acid sequence having 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%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence set forth in any one of SEQ ID NOs.:2, 173, 90, 92, and 7; (ii) a transmembrane domain comprising an amino acid sequence having 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%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence set forth in any one of SEQ ID NOs.:2, 173, 90, 92, and 7; (ii) a transmembrane domain comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%,
  • Embodiment 105a A polypeptide comprising an extracellular component, a transmembrane domain, and an intracellular component according to Polypeptide 1 of any one of Constructs A-AA in Table 2.
  • Embodiment 106a A polypeptide comprising an extracellular component, a transmembrane domain, and an intracellular component according to Polypeptide 2 of any one of Constructs A-AA in Table 2.
  • Embodiment 107a A polypeptide comprising an extracellular component, a transmembrane domain, and an intracellular component according to Polypeptide 2 of any one of Constructs A-AA in Table 2.
  • Embodiment 108a a polypeptide that comprises an extracellular component, a transmembrane domain, and an intracellular component according to Polypeptide 2 of any one of Constructs A-AA in Table 2.
  • Embodiment 107a The polynucleotide of Embodiment 107a, wherein the polynucleotide encodes the first polypeptide and the second polypeptide of any one of Constructs A-AA in Table 2, wherein, optionally, one or both of the encoded polypeptides does not comprise a signal peptide according to Table 2.
  • Embodiment 109a The polynucleotide of Embodiment 107a, wherein the polynucleotide encodes the first polypeptide and the second polypeptide of any one of Constructs A-AA in Table 2, wherein, optionally, one or both of the encoded polypeptides does not comprise a signal peptide according to Table 2.
  • Embodiment 110a A polynucleotide encoding any two or more polypeptides selected from Types Al- AA in Table 5.
  • Embodiment Illa A polynucleotide encoding any two or more polypeptides selected from Types Al- AA in Table 5.
  • a host cell expressing and/or encoding any two or more polypeptides selected from Types Al-AA in Table 5, wherein, optionally, the host cell comprises an immune cell, wherein, further optionally, the immune cell comprises a T cell, such as a CD4+ T cell, a CD8+ T cell, a CD4' CD8' double negative T cell, a yd T cell, a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof.
  • a T cell such as a CD4+ T cell, a CD8+ T cell, a CD4' CD8' double negative T cell, a yd T cell, a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof.
  • M, N, O, or P respectively; G and M, N, O, or P, respectively; H and M, N, O, or P, respectively; H and M, N, O, or P, respectively; I and M, N, O, or P, respectively; J and M,
  • Embodiment 113a An expression vector comprising the polynucleotide of any one of Embodiments la, 32a-41a, 43a-45a, 47a-49a, 51a-54a, 55a-58a, 60a-62a, 64a-66a, 68a-70a, 73a-74a, 77a-79a, 82a-85a, 94a, 98a-103a, 107a, 108a, 110a, and 112a, operably linked to an expression control sequence, wherein, optionally, the vector is capable of delivering the polynucleotide to a host cell.
  • Embodiment 114a The expression vector of Embodiment 113a, wherein the host cell is a hematopoietic progenitor cell or a human immune system cell.
  • Embodiment 115a The expression vector of Embodiment 114a, wherein the human immune system cell is a CD4 + T cell, a CD8 + T cell, a CD4' CD8' double negative T cell, a yd T cell, a natural killer cell, a natural killer T cell, a dendritic cell, or any combination thereof.
  • Embodiment 116a Embodiment 116a.
  • Embodiment 114a or 115a wherein the human immune system cell is a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof.
  • Embodiment 117a The expression vector of any one of Embodiments 113a-l 16a, wherein the vector is a viral vector.
  • Embodiment 118a The expression vector of Embodiment 117a, wherein the viral vector is a lentiviral vector or a y- retroviral vector.
  • Embodiment 119a is
  • a host cell comprising the polynucleotide of any one of Embodiments la, 32a-41a, 43a-45a, 47a-49a, 51a-54a, 55a-58a, 60a-62a, 64a-66a, 68a- 70a, 73a-74a, 77a-79a, 82a-85a, 94a, 98a-103a, 107a, 108a, 110a, and 112a.
  • Embodiment 120a Embodiment 120a.
  • a host cell expressing at its cell surface the fusion protein of any one of Embodiments 2a-31a or the polypeptide of any one of Embodiments 42a, 46a, 50a, 55a, 59a, 63a, 67a, 71a, 72a, 75a, 76a, 79a, 80a, 81a, 86a, 87-93a, 95-97a, 104-106a, and 109a.
  • Embodiment 121a The host cell of Embodiment I l la, 112a, 119a, or 120a, wherein the host cell is an immune system cell, optionally a human immune system cell.
  • Embodiment 122a The host cell of Embodiment I l la, 112a, 119a, or 120a, wherein the host cell is an immune system cell, optionally a human immune system cell.
  • Embodiment 121a wherein the immune system cell is a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a y6 T cell, a natural killer cell, a natural killer T cell, a dendritic cell, or any combination thereof.
  • Embodiment 123a The host cell of Embodiment 121a or 122, wherein the immune system cell is a CD4 + T cell.
  • Embodiment 124a The host cell of any one of Embodiments 121a-123a, wherein the immune system cell is a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof.
  • Embodiment 125a The host cell of any one of Embodiments 121a-124a, further comprising a polynucleotide encoding a binding protein that specifically binds to an antigen or an antigen:MHC complex, wherein the polynucleotide encoding a binding protein is optionally heterologous to the host cell.
  • Embodiment 126a The host cell of any one of Embodiments 121a-124a, further comprising a polynucleotide encoding a binding protein that specifically binds to an antigen or an antigen:MHC complex, wherein the polynucleotide encoding a binding protein is optionally heterologous to the host cell.
  • a host cell comprising: (i) a heterologous polynucleotide that encodes a fusion protein, wherein the encoded fusion protein comprises: (a) an extracellular component comprising an extracellular domain from a CD8 co-receptor a-chain; (b) a transmembrane domain from a CD8 co-receptor a-chain; and (c) an intracellular component comprising a co stimulatory domain from CD28, or a functional portion or variant thereof; and (ii) a heterologous polynucleotide encoding a binding protein that specifically binds to an antigen or an antigen:MHC complex.
  • Embodiment 127a a heterologous polynucleotide that encodes a fusion protein, wherein the encoded fusion protein comprises: (a) an extracellular component comprising an extracellular domain from a CD8 co-receptor a-chain; (b) a transmembrane domain from a CD8 co-receptor
  • a host cell expressing a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide have 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%, or at least 99% identity to, or comprise or consist of, the amino acid sequences set forth in SEQ ID NOs.: (a) 1 and 113, respectively; (b) 1 and 115, respectively; (c) 1 and 116, respectively; (d) 1 and 117, respectively; (e) 1 and 119, respectively; (f) 1 and 120, respectively; (g) 1 and 121, respectively; (h) 1 and 123, respectively; (i) 1 and any one of 125-135, respectively; (j) 1 and 137, respectively; (k) 1 and 139 respectively; (1) 114 and 113, respectively; (m) 114 and 115, respectively; (n) 114 and 116, respectively; (o) 114
  • Embodiment 128a The host cell of Embodiment 127a, wherein the first polypeptide and the second polypeptide have 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%, or at least 99% identity to, or comprise or consist of, the amino acid sequences set forth in SEQ ID NOs.: (a) 1 and 113, respectively; (b) 1 and 116, respectively; (c) 1 and 120, respectively; (d) 1 and 126, respectively; (e) 1 and 128, respectively; (f) 1 and 130, respectively; (g) 1 and 132, respectively; (h) 1 and 134, respectively; (i) 114 and 115, respectively; (j) 114 and 117, respectively; (k) 114 and 121, respectively; (1) 114 and 127, respectively; (m) 114 and 129, respectively; (n) 114 and 131, respectively; (o) 114 and
  • Embodiment 129a The host cell of any one of Embodiments I l la, 112a, and 119a-128a, wherein the host cell comprises an immune system cell, optionally a human immune system cell.
  • Embodiment 130a The host cell of Embodiment 129a, wherein the immune system cell comprises a CD4 + T cell, a CD8 + T cell, a CD4 CD8- double negative T cell, a y6 T cell, a natural killer cell, a natural killer T cell, a dendritic cell, or any combination thereof.
  • Embodiment 131a The host cell of Embodiment 130a, wherein the immune system cell comprises a CD4 + T cell.
  • Embodiment 132a The host cell of Embodiment 130a, wherein the immune system cell comprises a CD4 + T cell.
  • the host cell of any one of Embodiments 119a-13 la further comprising (1) a binding protein that specifically binds to an antigen or an antigen:MHC complex, and/or (2) a polynucleotide that encodes the binding protein.
  • Embodiment 133a The host cell of any one of Embodiments 125a-132a, wherein the encoded binding protein comprises a TCR or an antigen-binding fragment thereof (e.g., a TCR variable domain, a scTv, or a scTCR).
  • Embodiment 134a Embodiment 134a.
  • Embodiment 135a The host cell of any one of Embodiments 125a-134a, wherein the binding protein specifically binds to an antigen or antigen:MHC complex that is expressed by or associated with a cancer.
  • Embodiment 136a Embodiment 136a.
  • Embodiment 135a wherein the antigen is selected from a R0R1, EGFR, EGFRvIII, EGP-2, EGP-40, GD2, GD3, HPV E6, HPV E7, Her2, LI -CAM, Lewis A, Lewis Y, MUC1, MUC16, PSCA, PSMA, CD 19, CD20, CD22, CD56, CD23, CD24, CD30, CD33, CD37, CD44v7/8, CD38, CD56, CD123, CA125, c-MET, FcRH5, WT1, folate receptor a, VEGF-a, VEGFR1, VEGFR2, IL-13Ra2, IL-l lRa, MAGE-A1, PSA, ephnn A2, ephnn B2, NKG2D, NY-ESO-1, TAG-72, mesothelin, NY-ESO, 5T4, BCMA, FAP, Core Binding Factor protein; Cyclin-Al; Carbonic
  • Embodiment 137a The host cell of Embodiment 135a, wherein the antigen is selected from BCMA, CD3, CEACAM6, c-Met, EGFR, KRAS (e.g.
  • G12V, G12C, or G12D PSMA, RANK, R0R1, TNFRSF4, CD40, CD 137, TWEAK-R, HLA, tumor- or pathogen- associated peptide bound to HLA, hTERT peptide bound to HLA, tyrosinase peptide bound to HLA, WT-1 peptide bound to HLA, LT0R, LIFR0, LRP5, MUC1, OSMR0, TCRa, TCRP, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD79a, CD79b, CD80, CD81, CD86, CD123, CD171, CD276, B7H4, TLR7, TLR9, PTCHI, WT-1, HA 1 - H, Robol, a-fetoprotein (AFP), Frizzled, 0X40, PRAME, a BRAF antigen such as BRAFV600E, and SSX-2.
  • Embodiment 138a The host cell of any one of Embodiments 126a- 137a, comprising a chromosomal gene knockout or a mutation of a PD-1 gene; a LAG3 gene; a TIM3 gene; a CTLA4 gene; an HLA component gene; a TCR component gene; a Fas gene; a FasL gene, a TIGIT gene; or any combination thereof.
  • Embodiment 139a The host cell of any one of Embodiments 126a- 137a, comprising a chromosomal gene knockout or a mutation of a PD-1 gene; a LAG3 gene; a TIM3 gene; a CTLA4 gene; an HLA component gene; a TCR component gene; a Fas gene; a FasL gene, a TIGIT gene; or any combination thereof.
  • a composition comprising: (i) the fusion protein of any one of Embodiments 2a-31a or the polypeptide of any one of Embodiments 42a, 46a, 50a, 55a, 59a, 63a, 67a, 71a, 72a, 75a, 76a, 79a, 80a, 81a, 86a, 87a-93a, and 95a-97a, 104a-106a, and 109a; (ii) the polynucleotide of any one of Embodiments la, 32a-41a, 43a-45a, 47a-49a, 51a-54a, 55a- 58a, 60a-62a, 64a-66a, 68a-70a, 73a-74a, 77a-79a, 82a-85a, 94a, 98a-103a, 107a-108a, 110a, and 112a; (iii) the expression vector of any one of Embodiments 113
  • Embodiment 140a A unit dose, comprising an effective amount of the host cell of any one of Embodiments I l la, 112a, and 119a-138a, or of the host cell composition of Embodiment 139a.
  • Embodiment 141a The unit dose of Embodiment 140a, comprising (i) a composition comprising at least about 30% CD4 + T host cells, combined with (ii) a composition comprising at least about 30% CD8 + T cells, in about a 1:1 ratio.
  • Embodiment 142a Embodiment 142a.
  • Embodiment 141a comprising an effective amount of an effector immune cell comprising a polynucleotide that encodes a binding protein that is capable of specifically binding to an antigen or an antigen:MHC complex.
  • Embodiment 143a The unit dose of Embodiment 142a, wherein the effector immune cell is a T cell, optionally a CD8 + T cell.
  • Embodiment 144a The unit dose of Embodiment 142a or 143 a, wherein the binding protein encoded by the effector immune cell comprises a TCR or a CAR.
  • Embodiment 145a comprising an effective amount of an effector immune cell comprising a polynucleotide that encodes a binding protein that is capable of specifically binding to an antigen or an antigen:MHC complex.
  • Embodiment 143a The unit dose of Embodiment 142a, wherein the effector immune cell is a T cell, optionally a CD8 + T cell.
  • Embodiment 146a The unit dose of any one of Embodiments 142a- 144a, wherein the binding protein encoded by the effector immune cell is specific for the same or a different antigen as compared to a binding protein encoded by the host cell.
  • Embodiment 146a The unit dose of any one of Embodiments 142a- 144a, wherein the binding protein encoded by the effector immune cell is specific for the same or a different antigen as compared to a binding protein encoded by the host cell.
  • a method of treating a disease or condition in a subject comprising administering to the subject an effective amount of: (i) the fusion protein of any one of Embodiments 2a-31a; (ii) the polypeptide of any one of Embodiments 42a, 46a, 50a, 55a, 59a, 63a, 67a, 71a, 72a, 75a, 76a, 79a, 80a, 81a, 86a, 87a-93a, 95a-97a, 104a-106a, and 109a; (iii) the polynucleotide of any one of Embodiments la, 32a-41a, 43a- 45a, 47a-49a, 51a-54a, 55a-58a, 60a-62a, 64a-66a, 68a-70a, 73a-74a, 77a-79a, 82a-85a, 94a, 98a- 103 a, 107a, 108
  • Embodiment 147a The method of Embodiment 146a, wherein the disease or condition is a cancer.
  • Embodiment 148a. The method of Embodiment 147a, wherein the cancer comprises a carcinoma, a sarcoma, a glioma, a lymphoma, a leukemia, a myeloma, or any combination thereof.
  • Embodiment 147a or 148a wherein the cancer comprises a cancer of the head or neck, melanoma, pancreatic cancer, cholangiocarcinoma, hepatocellular cancer, breast cancer including triple-negative breast cancer (TNBC), gastric cancer, non-small-cell lung cancer, prostate cancer, esophageal cancer, mesothelioma, small-cell lung cancer, colorectal cancer, glioblastoma, or any combination thereof.
  • TNBC triple-negative breast cancer
  • gastric cancer non-small-cell lung cancer
  • prostate cancer esophageal cancer
  • mesothelioma small-cell lung cancer
  • colorectal cancer colorectal cancer
  • glioblastoma or any combination thereof.
  • Embodiment 150a Embodiment 150a.
  • the cancer comprises Askin's tumor, sarcoma botryoides, chondrosarcoma, Ewing's sarcoma, PNET, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma protuberans (DFSP), desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, undifferent
  • Embodiment 151a The method of any one of Embodiments 147a-150a, wherein the cancer comprises a solid tumor or a hematological malignancy.
  • Embodiment 152a The method of Embodiment 151a, wherein the solid tumor is a sarcoma or a carcinoma.
  • Embodiment 153a The method of any one of Embodiments 147a-150a, wherein the cancer comprises a solid tumor or a hematological malignancy.
  • Embodiment 152a The method of Embodiment 151a, wherein the solid tumor is a sarcoma or a carcinoma.
  • Embodiment 151a or 152a wherein the solid tumor is selected from: chondrosarcoma; fibrosarcoma (fibroblastic sarcoma); Dermatofibrosarcoma protuberans (DFSP); osteosarcoma; rhabdomyosarcoma; Ewing’s sarcoma; a gastrointestinal stromal tumor; Leiomyosarcoma; angiosarcoma (vascular sarcoma); Kaposi’s sarcoma; liposarcoma; pleomorphic sarcoma; or synovial sarcoma.
  • Embodiment 154a Embodiment 154a.
  • a lung carcinoma e.g., Adenocarcinoma, Squamous Cell Carcinoma (Epidermoid Carcinoma); Squamous cell carcinoma; Adenocarcinoma; Adenosquamous carcinoma; anaplastic carcinoma; Large cell carcinoma; Small cell carcinoma; a breast carcinoma (e.g., Ductal Carcinoma in situ (non-invasive), Lobular carcinoma in situ (non-invasive), Invasive Ductal Carcinoma, Invasive lobular carcinoma, Non-invasive Carcinoma); a liver carcinoma (e.g., Hepatocellular Carcinoma, Cholangiocarcinomas or Bile Duct Cancer); Large-cell undifferentiated carcinoma, Bronchioalveolar carcinoma); an ovarian carcinoma (e.g., Surface epithelial-stromal tumor (Adenocarcinoma) or ovarian epithelial carcinoma (which includes serous)
  • a breast carcinoma e.g., Ductal Carcinoma in situ
  • Embodiment 155a The method of Embodiment 151a or 152a, wherein the solid tumor is an ovarian carcinoma, an ovarian epithelial carcinoma, a cervical adenocarcinoma or small cell carcinoma, a pancreatic carcinoma, a colorectal carcinoma (e.g., an adenocarcinoma or squamous cell carcinoma), a lung carcinoma, a breast ductal carcinoma, or an adenocarcinoma of the prostate.
  • Embodiment 156a The method of any one of Embodiments 146a- 155a, wherein the host cell is allogeneic, syngeneic, or autologous to the subject.
  • Embodiment 157a The method of any one of Embodiments 146a- 155a, wherein the host cell is allogeneic, syngeneic, or autologous to the subject.
  • Embodiment 158a The method of any one of Embodiments 146a- 156a, comprising administering a plurality of unit doses to the subject.
  • Embodiment 158a The method of Embodiment 157a, wherein the plurality of unit doses are administered at intervals between administrations of about two, three, four, five, six, seven, eight, or more weeks.
  • Embodiment 159a The method according to any one of Embodiments 146a- 158a, wherein the unit dose comprises about 10 5 cells/m 2 to about 10 11 cells/m 2 .
  • Embodiment 160a Embodiment 160a.
  • Embodiments 146a- 159a wherein the subject further receives an adjunctive therapy comprising: (i) chemotherapy; (ii) radiation therapy; (iii) an inhibitor of an immune suppression component; (iv) an agonist of a stimulatory immune checkpoint agent; (v) RNAi; (vi) a cytokine; (vii) a surgery; (viii) a monoclonal antibody and/or an antibodydrug conjugate; or (ix) any combination of (i)-(viii), in any order.
  • an adjunctive therapy comprising: (i) chemotherapy; (ii) radiation therapy; (iii) an inhibitor of an immune suppression component; (iv) an agonist of a stimulatory immune checkpoint agent; (v) RNAi; (vi) a cytokine; (vii) a surgery; (viii) a monoclonal antibody and/or an antibodydrug conjugate; or (ix) any combination of (i)-(viii), in any order.
  • Embodiment 162a A method comprising introducing into a host cell the polynucleotide of any one of Embodiments la, 32a-41a, 43a-45a, 47a-49a, 51a-54a, 55a-58a, 60a-62a, 64a- 66a, 68a-70a, 73a-74a, 77a-79a, 82a-85a, 94a, 98a-103a, 107a, 108a, 110a, and 112a or the vector of any one of Embodiments 113a-l 18a, wherein, optionally, the host cell comprises a human T cell, such as for example a CD4+ T cell or a CD8 + T cell.
  • a human T cell such as for example a CD4+ T cell or a CD8 + T cell.
  • Embodiment lb A polypeptide comprising an extracellular domain from CD8P, a transmembrane domain that is optionally from CD8P, and an intracellular domain comprising a variant of SEQ ID NO.: 19, wherein the variation comprises or consists of: (i) a diglycine motif in place of a dileucine motif; (ii) a mutated and optionally non-functional PI3K binding site; (iii) a mutated and optionally non-functional ITK and GADS binding site; (iv) a mutated and optionally non-functional LCK binding site; or (v) any combination of (i)-(iv), optionally: (i) and (ii); (i) and (iii); (i) and (iv); (ii) and (iii); (ii) and (iv); (iii) and (iv); (i), (ii), and (ii); (i), (iv); (ii), (i), and (
  • Embodiment 2b The polypeptide of Embodiment lb, wherein the variant comprises (i) and any one or more of (ii)-(v).
  • Embodiment 3b The polypeptide of Embodiment lb or 2b, wherein the polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92%, or at least 95% sequence identity to SEQ ID NO.: 19.
  • Embodiment 4b The polypeptide of any one of Embodiments lb-3b, comprising, in the intracellular domain and N-terminal to the variant of SEQ ID NO.: 19, the amino acid sequence of SEQ ID NO. : 10, wherein, optionally, the variant of SEQ ID NO.
  • Embodiment 5b The polypeptide of any one of Embodiments lb-4b, wherein the intracellular domain of the polypeptide comprises an amino acid sequence according to SEQ ID NO.:205 or 206.
  • Embodiment 6b The polypeptide of any one of Embodiments lb-5b, comprising, in the variant of SEQ ID NO. : 19, Motif 1 , Motif 2, Motif 3 , and/or Motif 4 of any one of Constructs BB-NN in Table 6 or Table 7, and/or comprising as Motif 2 the amino acid sequence FMNM.
  • Embodiment 7b The polypeptide of any one of Embodiments lb-4b, wherein the intracellular domain of the polypeptide comprises an amino acid sequence according to SEQ ID NO.:205 or 206.
  • Embodiment 6b The polypeptide of any one of Embodiments lb-5b, comprising, in the variant of SEQ ID NO. : 19, Motif 1 , Mo
  • the polypeptide of Embodiment 6b comprising, in the variant of SEQ ID NO.:19: (A)(i) Motif 1, 2, 3, or 4 of Construct BB in Table 6 or Table 7; (ii) Motifs 1 and 2 of Construct BB in Table 6 or Table 7; (iii) Motifs 1 and 3 of Construct BB in Table 6 or Table 7; (iv) Motifs 1 and 4 of Construct BB in Table 6 or Table 7; (v) Motifs 1, 2, and 3 of of Construct BB in Table 6 or Table 7; (vi) Motifs 1, 2, and 4 of of Construct BB in Table 6 or Table 7; (vii) Motifs 1, 3, and 4 of of Construct BB in Table 6 or Table 7; (viii) Motifs 2 and 3 of Construct BB in Table 6 or Table 7; (ix) Motifs 2 and 4 of Construct BB in Table 6 or Table 7; (x) Motifs 2, 3, and 4 of of Construct
  • Embodiment 8b A polypeptide comprising an extracellular domain from CD8P, a transmembrane domain that is optionally from CD8P, and an intracellular domain comprising a variant of SEQ ID NO. : 19, wherein the variant of SEQ ID NO.
  • : 19 comprises: (A) (i) Motif 1, 2, 3, or 4 of Construct BB in Table 6 or Table 7; (ii) Motifs 1 and 2 of Construct BB in Table 6 or Table 7; (iii) Motifs 1 and 3 of Construct BB in Table 6 or Table 7; (iv) Motifs 1 and 4 of Construct BB in Table 6 or Table 7; (v) Motifs 1, 2, and 3 of of Construct BB in Table 6 or Table 7; (vi) Motifs 1, 2, and 4 of of Construct BB in Table 6 or Table 7; (vii) Motifs 1, 3, and 4 of of Construct BB in Table 6 or Table 7; (viii) Motifs 2 and 3 of Construct BB in Table 6 or Table 7; (ix) Motifs 2 and 4 of Construct BB in Table 6 or Table 7; (x) Motifs 2, 3, and 4 of of Construct BB in Table 6 or Table 7; (xi) Motifs 3 and 4 of
  • Embodiment 9b The polypeptide of any one of Embodiments lb-8b, wherein: (i) the transmembrane domain is from CD80 (e.g., comprises or consists of SEQ ID NO.: 8); and/or (ii) the extracellular domain from CD8P is from a CD8P Ml, M2, M3, M4, or M5 isoform; and/or (iii) the extracellular domain from CD8P comprises an Ig V-like domain comprising SEQ ID NO.:68 or comprises amino acids 1-110 of SEQ ID NO : 7.
  • Embodiment 10b Embodiment 10b.
  • Embodiment 11b A polypeptide comprising, consisting essentially or, or consisting of: an extracellular domain amino acid sequence, a transmembrane domain amino acid sequence, and an intracellular domain amino acid sequence, as shown under “2 protein size [aa]” in any one of Figures 27-39.
  • Embodiment 12b. A polynucleotide encoding the polypeptide of any one of Embodiments lb- 1 lb.
  • Embodiment 13b A polynucleotide encoding the polypeptide of any one of Embodiments lb- 1 lb.
  • the polynucleotide of Embodiment 12b further encoding a CD8a coreceptor polypeptide, optionally comprising SEQ ID NO. : 114, and wherein, optionally, the polynucleotide comprises or encodes Construct BB, Construct CC, Construct DD, Construct EE, Construct FF, Construct GG, Construct HH, Construct II, Construct JJ, Construct KK, Construe LL, Construct MM, or Construct NN.
  • Embodiment 14b Embodiment 14b.
  • Construct BB A polynucleotide encoding Construct BB, Construct CC, Construct DD, Construct EE, Construct FF, Construct GG, Construct HH, Construct II, Construct JJ, Construct KK, Construct LL, Construct MM, or Construct NN.
  • Embodiment 15b A polynucleotide encoding Construct BB, Construct CC, Construct DD, Construct EE, Construct FF, Construct GG, Construct HH, Construct II, Construct JJ, Construct KK, Construct LL, Construct MM, or Construct NN.
  • a polynucleotide encoding a first polypeptide and second polypeptide wherein the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 114 and the second polypeptide comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs.:192-204, wherein, optionally, (1) the first polypeptide is encoded by a polynucleotide that is disposed 5’ to a polynucleotide encoding the second polypeptide; (2) the second polypeptide is encoded by a polynucleotide that is discposed 5’ to a polynucleotide encoding the first polypeptide, (3) the polynucleotide further encodes a selfcleaving peptide with an optional N-terminal GSG linker, wherein the self-cleaving peptide is disposed between the first polypeptide and the second polypeptide, or (4) (1) and (3) or (2) and (3).
  • Embodiment 16b The polynucleotide of Embodiment 15b, wherein the first polypeptide is encoded by a polynucleotide that is disposed 5’ to a polynucleotide encoding the second polypeptide.
  • Embodiment 17b The polynucleotide of Embodiment 15b, wherein the second polypeptide is encoded by a polynucleotide that is disposed 5’ to a polynucleotide encoding the first polypeptide).
  • Embodiment 18b The polynucleotide of Embodiment 15b, wherein the second polypeptide is encoded by a polynucleotide that is disposed 5’ to a polynucleotide encoding the first polypeptide.
  • a vector comprising the polynucleotide of any one of Embodiments 12b- 17b, wherein, optionally, the vector comprises a viral vector such as a lentiviral vector or a retroviral vector.
  • Embodiment 19b A host cell expressing the polypeptide of any one of Embodiments lb- 1 lb.
  • Embodiment 20b The host cell of Embodiment 19b, further expressing a CD8a coreceptor polypeptide, optionally comprising SEQ ID NO. : 114.
  • Embodiment 21b A host cell comprising the polynucleotide one any one of Embodiments 12b- 17b or the vector of Embodiment 18b.
  • Embodiment 22b A host cell expressing a polypeptide dimer comprising (i) the polypeptide of any one of Embodiments lb- 1 lb and (ii) a CD8 coreceptor polypeptide, optionally comprising SEQ ID NO. : 114.
  • Embodiment 23b A host cell expressing a polypeptide dimer comprising (i) the polypeptide of any one of Embodiments lb- 1 lb and (ii) a CD8 coreceptor polypeptide, optionally comprising SEQ ID NO. : 114.
  • G12V, G12C, or G12D Merkel Cell polyomavirus T antigen
  • Core Binding Factor protein HA-1 H , PSA, ephrin A2, ephrin B2, an NKG2D, NY-ESO-1, TAG-72, mesothelin, NY-ESO, 5T4, BCMA, FAP, Carbonic anhydrase 9, ERBB2, a BRAF antigen such as a BRAF V600E antigen, BCMA, CD3, CEACAM6, c-Met, EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, FLT1, KDR, FLT4, CD44v6, CD151, CA125, CEA, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gpl30, Lewis A, Lewis
  • G12V, G12C, or G12D PSMA, RANK, R0R1, TNFRSF4, CD40, CD 137, TWEAK- R, HLA, tumor- or pathogen- associated peptide bound to HLA, hTERT peptide bound to HLA, tyrosinase peptide bound to HLA, WT-1 peptide bound to HLA, LTPR, LIFRP, LRP5, MUC1, OSMRP, TCRa, TCRP, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD79a, CD79b, CD80, CD81, CD86, CD123, CD171, CD276, B7H4, TLR7, TLR9, PTCHI, WT-1, HA X -H, Robol, a-fetoprotein (AFP), Frizzled, 0X40, PRAME, or SSX-2.
  • AFP a-fetoprotein
  • Frizzled 0
  • Embodiment 24b The host cell of any one of Embodiments 19b-23b, wherein the host cell comprises a hematopoietic progenitor cell or an immune system cell, optionally a human immune system cell.
  • Embodiment 25b The host cell of Embodiment 24b, wherein the immune system cell comprises a CD4 + T cell, a CD8 + T cell, a CD4’CD8- double negative T cell, a yd T cell, a natural killer cell, a natural killer T cell, a dendritic cell, or any combination thereof.
  • Embodiment 26b Embodiment 26b.
  • Embodiment 27b The host cell of Embodiment 25b or 26b, comprising a CD4+ T cell, a CD8+ T cell, or both.
  • Embodiment 28b is a CD4+ T cell, a CD8+ T cell, or both.
  • a composition comprising the polypeptide of any one of Embodiments lb- 1 lb, the polynucleotide of any one of Embodiments 12b- 17b, the vector of Embodiment 18b, or the host cell of any one of Embodiments 19b-27b, or any combination thereof, and a pharmaceutically acceptable carrier, excipient, or diluent, wherein, optionally, the composition comprises (1) at least about 30% CD4 + T host cells, (2) at least about 30% CD8+ T host cells, or (3) at least about 30% CD4+ T host cells and at least about 30% CD8 + T cells, in about a 1 : 1 ratio.
  • Embodiment 29b A method for treating a disease or disorder such as a cancer, the method comprising administering to a subject having the disease or disorder an effective amount of the polypeptide of any one of Embodiments lb- 1 lb, the polynucleotide of any one of Embodiments 12b- 17b, the vector of Embodiment 18b, the host cell of any one of Embodiments 19b-27b, the composition of Embodiment 28b, or any combination thereof.
  • Embodiment 30b A method for treating a disease or disorder such as a cancer, the method comprising administering to a subject having the disease or disorder an effective amount of the polypeptide of any one of Embodiments lb- 1 lb, the polynucleotide of any one of Embodiments 12b- 17b, the vector of Embodiment 18b, the host cell of any one of Embodiments 19b-27b, the composition of Embodiment 28b, or any combination thereof.
  • Embodiment 31b. The method of Embodiment 29b or the polypeptide, polynucleotide, vector, host cell, composition, or combination for use of Embodiment 30b, wherein the disease or condition is a cancer.
  • Embodiment 32b The method of Embodiment 29b or the polypeptide, polynucleotide, vector, host cell, composition, or combination for use of Embodiment 30b, wherein the disease or condition is a cancer.
  • Embodiment 31 or the polypeptide, polynucleotide, vector, host cell, composition, or combination for use of Embodiment 31b, wherein the cancer comprises a carcinoma, a sarcoma, a glioma, a lymphoma, a leukemia, a myeloma, or any combination thereof.
  • Embodiment 33b Embodiment 33b.
  • Embodiment 31b or 32b or the polypeptide, polynucleotide, vector, host cell, composition, or combination for use of Embodiment 31b or Embodiment 32b, wherein the cancer comprises a cancer of the head or neck, melanoma, pancreatic cancer, cholangiocarcinoma, hepatocellular cancer, breast cancer including triple-negative breast cancer (TNBC), gastric cancer, non-small-cell lung cancer, prostate cancer, esophageal cancer, mesothelioma, small-cell lung cancer, colorectal cancer, glioblastoma, or any combination thereof.
  • TNBC triple-negative breast cancer
  • Embodiment 34b Embodiment 34b.
  • Embodiment 35b The method of Embodiment 31b or the polypeptide, polynucleotide, vector, host cell, composition, or combination for use of Embodiment 3 lb, wherein the cancer comprises a solid tumor or a hematological malignancy.
  • Embodiment 36b The method of Embodiment 35b or the polypeptide, polynucleotide, vector, host cell, composition, or combination for use of Embodiment 35b, wherein the solid tumor is a sarcoma or a carcinoma.
  • Embodiment 37b Embodiment 37b.
  • Embodiment 35b or 36b or the polypeptide, polynucleotide, vector, host cell, composition, or combination for use of Embodiment 35b or Embodiment 36b wherein the solid tumor is selected from: chondrosarcoma; fibrosarcoma (fibroblastic sarcoma); Dermatofibrosarcoma protuberans (DFSP); osteosarcoma; rhabdomyosarcoma; Ewing’s sarcoma; a gastrointestinal stromal tumor; Leiomyosarcoma; angiosarcoma (vascular sarcoma);
  • Embodiment 38b The method of any one of Embodiments 35b-37b or the polypeptide, polynucleotide, vector, host cell, composition, or combination for use of any one of Embodiments 35b-37b, wherein the solid tumor is selected from a lung carcinoma (e.g., Adenocarcinoma, Squamous Cell Carcinoma (Epidermoid Carcinoma); Squamous cell carcinoma; Adenocarcinoma; Adenosquamous carcinoma; anaplastic carcinoma; Large cell carcinoma; Small cell carcinoma; a breast carcinoma (e.g., Ductal Carcinoma in situ (non-invasive), Lobular carcinoma in situ (non-invasive), Invasive Ductal Carcinoma, Invasive lobular carcinoma, Non-invasive Carcinoma); a liver carcinoma (e.g., Adenocarcinoma, Squamous Cell Carcinoma (Epidermoid Carcinoma); Squamous cell carcinoma; Adeno
  • Embodiment 39b The method of Embodiment 35b or 36b, or the polypeptide, polynucleotide, vector, host cell, composition, or combination for use of Embodiment 35b or 36b, wherein the solid tumor is an ovarian carcinoma, an ovarian epithelial carcinoma, a cervical adenocarcinoma or small cell carcinoma, a pancreatic carcinoma, a colorectal carcinoma (e.g., an adenocarcinoma or squamous cell carcinoma), a lung carcinoma, a breast ductal carcinoma, or an adenocarcinoma of the prostate.
  • Embodiment 40b Embodiment 40b.
  • the method of any one of Embodiments 29b-39b or the polypeptide, polynucleotide, vector, host cell, composition, or combination for use of any one of Embodiments 30b-39b comprises administering to the subject a dose of host cells (e.g., T cells) comprising about 10 5 cells/m 2 to about 10 11 cells/m 2 .
  • host cells e.g., T cells
  • an adjunctive therapy comprising: (i) chemotherapy; (ii) radiation therapy; (iii) an inhibitor of an immune suppression component; (iv) an agonist of a stimulatory immune checkpoint agent; (v) RNAi; (vi) a cytokine; (vii) a surgery; (viii) a mono
  • Embodiment 41b or the or the polypeptide, polynucleotide, vector, host cell, composition, or combination for use of Embodiment 41b, wherein the adjunctive therapy is administered to the subject before, concurrently with, or after being administered the host cells or composition.
  • Embodiment 43b A method comprising introducing into a host cell the polynucleotide of any one of Embodiments 12b- 17b or the vector of Embodiment 18b.
  • Embodiment 44b The method of Embodiment 43b, wherein the polynucleotide or vector is introduced into the host cell ex vivo or in vitro.
  • Embodiment 45b The method of Embodiment 41b or the or the polypeptide, polynucleotide, vector, host cell, composition, or combination for use of Embodiment 41b, wherein the adjunctive therapy is administered to the subject before, concurrently with, or after being administered the host cells or composition.
  • Embodiment 43b A method compris
  • Embodiment 43b or 44b wherein host cell comprises a hematopoietic progenitor cell or an immune system cell, optionally a human immune system cell.
  • Embodiment 46b The method of Embodiment 45b, wherein the immune system cell comprises a CD4 + T cell, a CD8 + T cell, a CD4 CD8- double negative T cell, a yd T cell, a natural killer cell, a natural killer T cell, a dendritic cell, or any combination thereof.
  • Embodiment 47b Embodiment 47b.
  • Embodiment 48b The method of Embodiment 46b or 47b, comprising a CD4+ T cell, a CD8+ T cell, or both.
  • MHC-I-restricted antigens Several cancers express MHC-I-restricted antigens.
  • Adoptive T cell therapies have been developed using MHC-I-restricted TCRs that specifically recognize cancer antigens (see, e.g, PCT Publication Nos. WO 2016/022400; WO 2018/170338; WO 2018/090057; WO 2017/112944; WO 2017/193104; WO 2018/058002; and WO 2013/071154).
  • CD8 + T cells which typically naturally express MHC-I-restricted TCR
  • CD4 + T cells which typically naturally express MHC-II-restricted TCR
  • a CD4 + T cell population engineered to express an appropriate MHC-I-restricted TCR may be used.
  • T cells expressing such "cross- MHC-restricted" TCRs perform favorably when also expressing a cognate co-receptor.
  • exogenous costimulatory proteins improved CD4 + T cell responses to MHC-I-antigen in the context of an exogenous MHC-I TCR.
  • single-chain fusion proteins were generated that combined the MHC-I-binding of a CD8 co-receptor complex with costimulatory signaling, thereby effectively tethering costimulatory activity to MHC-I- specific response.
  • fusion proteins are shown schematically in Figures 1 and 8; these and additional constructs are summarized in Figures 9A-12C and Tables 1-3.
  • costimulatory domains from CD28, 4-1BB, ICOS, and 0X40 were fused to CD8P (Ml isotype) and/or CD8a co-receptor chains.
  • CD8P Ml isotype
  • CD8a co-receptor chains In some of the CD8P-containing fusions, a six-amino-acid sequence from the CD8P transmembrane domain (“HLCCRR"; SEQ ID NO. : 10) was included adjacent to the costimulatory domain.
  • HLCCRR six-amino-acid sequence from the CD8P transmembrane domain
  • a native dileucine sequence motif (“LL") was mutated to diglycine (“GG”) in order to improve expression.
  • Tested constructs included Constructs A, B, E, F, G, H, I, J, K, O, Q, S, T, W, and Y (See e.g. Table 1). Constructs comprising costimulatory domains from CD28 or 4-1BB were among those selected for further testing.
  • CD4 + T cells were transduced to express MHC-I-restricted TCR either without CD8 or with a CD8 chimeric fusion construct.
  • Cells were sorted at Day 7 posttransduction and expanded using a rapid expansion protocol (REP), then stimulated at Day 9 post-REP with antigen-expressing MEL-275 cells at various effectortarget ratios.
  • REP rapid expansion protocol
  • inclusion of a chimeric CD8 co-receptor construct improved proliferation of transduced CD4 + T cells in response to antigen (cells encoding a fusion with a CD28 co-stimulatory domain having proliferation more than cells encoding a fusion with a 4-1BB co-stimulatory domain) over CD4 + T cells expressing MHC-I-TCR in the absence of a CD8 co-receptor.
  • proliferation of the fusion-expressing cells was somewhat more consistently tied to E:T ratio.
  • CD4 + T cells were transduced to express MHC-I-restricted TCR either without CD8 or with a CD8 chimeric fusion construct.
  • Cells were sorted at Day 7 post- transduction and expanded using a rapid expansion protocol (REP), and then stimulated at Day 9 post-REP with peptide antigen, or not.
  • Secretion of IFN-y and TNF-a was measured by flow cytometry.
  • the fusion-expressing cells produced more cytokines than cells expressing MHC-I-restricted TCR (alone or with a (heterologous) wild-type CD8 co-receptor).
  • Cells encoding a fusion comprising a CD28 co-stimulatory domain performed best in this assay.
  • Fusion proteins were designed that included (amino-terminal to carboxyterminal direction): a CD3( ⁇ extracellular domain; a CD3( ⁇ transmembrane domain; a costimulatory domain from CD28 or 4-1BB; and a CD3( ⁇ intracellular signaling (effector) domain. See Tables 1-3, Constructs Z and AA. Primary T cells from healthy donors were transduced with lentivirus encoding the fusion construct only, the fusion construct and an antigen-specific TCR, or the TCR only. Expression data is shown in Figures 17A-17C. Transduced T cells were assessed for killing activity against WT-1- expressing cancer cells using IncuCyte® assays. Data are shown in Figures 5 (T cells from Donor 18575; "10" denotes the TCR) and 6 (T cells from Donor 18648; "37” denotes the TCR).
  • CD4 + and CD8 + T cells from healthy donors were selected using CD4 and CD8 selection kits, and separately transduced with lentivirus containing a co- receptor construct and lentivirus containing a HLA-A2 restricted MAGE-A1-278 specific TCR construct. Five to seven days post-transduction, cells were sorted by MAGE-A1-278 tetramer and expanded for seven to nine days prior to downstream analysis.
  • Expanded CD4 + and CD8 + T cells were stimulated with MAGE-A1 and HLA- A2 positive tumor cell lines ME275 and H1299 at an E:T ratio of 5: 1 overnight at 37°C, in the presence of Golgistop and Golgiplug (BD biosciences). After stimulation, cells were fixed, permeabilized, and stained for intracellular IFN-y and TNFa. Intracellular cytokine levels were measured by flow cytometry. Data are shown in Figures 15A-15D. Certain of the tested constructs showed increased production of TNFa (and in for some constructs, also IFNy) by cells (including CD4+ T cells) against Hl 299 and ME275 target cells.
  • CD4 + and CD8 + T cells from healthy donors were selected using CD4 and CD8 selection kits, and separately transduced with lentivirus containing a coreceptor construct and a lentivirus containing a HLA-A2 restricted MAGE-A1-278 specific TCR construct. Five to seven days post-transduction, cells were sorted by MAGE-A1-278 tetramer and expanded for seven to nine days prior to downstream analysis.
  • CD4 + and CD8 + T cells were co-cultured with mCherry-positive ME275 cells at an E:T ratio of 5: 1. Tumor cell killing and control were monitored by an IncuCyte® instrument, which took images every 2 hours over an eleven-day span. Additional ME275 tumor cells were added on day 4 in order to stress the T cells.
  • CD8+ T cells transduced with CD8a/CD8P-CD28 or CD8a/CD8P-41BB fusions performed better than CD8+ T cells transduced with the TCR alone.
  • CD4 + T cells transduced with CD8a/CD8P-CD28 or CD8a/CD8P-41BB fusions performed noticeably better than CD4 + T cells transduced with either the wild-type CD8a/CD8p co-receptors or the TCR alone. It was observed that CD8P fusions comprising the CD8P intracellular amino acid sequence of SEQ ID NO. : 10 demonstrated improved function over those that did not. EXAMPLE 5
  • CD4 + and CD8 + Jurkat reporter cells were separately transduced with lentivirus containing a co-receptor construct and lentivirus containing an HLA-A2-restricted MAGE-A 1-278 specific TCR construct. Comparators were reporter cells transduced only with lentivirus encoding the TCR, with lentivirus encoding the TCR and wild-type CD8aP, and with lentivirus encoding an irrelevant TCR with wild-type CD8ap.
  • the Jurkat reporter cells had MHC class I molecules knocked out, endogenous TCR a and P chains knocked out, and a reporter (neogreen) knocked in downstream of the Nur 77 gene. Nur77 is upregulated after TCR activation.
  • the transduced Jurkat reporter cells were co-cultured with peptide-loaded T2 cells at an E:T ratio of 5: 1.
  • Multiple populations of T2 cells were peptide-loaded in the presence of various concentrations of peptide, as shown on the x-axis (left to right) in Figures 14A and 14B.
  • some, but not all, of the tested constructs provided a higher percentage of reporter-positive transduced cells, as compared to Jurkat cells that contained the TCR construct (alone or with CD8aP), including at lower concentrations of peptide (ug/mL).
  • modification of the CD8a chain produced less robust results as compared to modification of the CD8P chain.
  • Costimulatory molecule genes along with a HLA-A2 restricted MAGE-A1-278 specific TCR were cloned in the lentivirus backbone pRRLSIN.
  • Transfer plasmids encoding the costimulatory molecules along with helper and envelope plasmids were co-transfected into HEK293 cells using the effectene transfection reagent from Qiagen according to the manufacturer’s instruction.
  • Cell culture supernatant containing the lentivirus was collected 48 and 72 hours post transfection. Viral supernatant was concentrated using the Lenti-X concentrator from Takara per manufacturer’s protocol.
  • CD4 and CD8 T cells from healthy donors were selected using the stemcell CD4 or CD8 T cell selection kits and activated with TransAct (Miltenyi) according to manufacturer’s protocols. After 48 hours, concentrated virus was added to activated T cells in the presence of lOug/ml of protamine sulfate. Five to seven days post transduction, cells were sorted by MAGE-A1-278 tetramer and expanded for seven to nine days prior to downstream analysis.
  • Transduced, sorted and expanded T cells were stimulated with tumor cell lines ME275 and H1299, which are positive for MAGE-A1 and HLA-A2, at E:T ratio at 5: 1 overnight at 37°C in the presence of Golgistop and Golgiplug (BD biosciences). After stimulation, cells were fixed, permeabilized and stained for intracellular IFNy, TNFa and IL2. Stained cells were then analyzed by flow cytometry.
  • Tumor cell line ME275 was transduced with mCherry and sorted on mCherry positive cells. Transduced, sorted and expanded T cells were cocultured with mCherry+ tumor cells at 5: 1 E:T ratio in 96 well plates. Images were taken in the incucyte S3 every 2 hours over the span of 11 days. More tumor cells were added to the tested wells on day 4 to future stress the T cells. The intensity of mCherry was measured and analyzed by the Incucyte software. The intensity of the first time point of each well was normalized to 1. Same experimental conditions were measured in duplicates.
  • Jurkat reporter cell with MHC class I molecules knocked out, endogenous TCR alpha and beta chains knocked out and neogreen knocked in downstream of Nur77 was generated in the lab.
  • Jurkat reporter cells were transduced with lentivirus encoding the costimulatory molecules and cocultured with peptide loaded T2 cells at E:T ratio of 5 : 1. After an overnight stimulation, cells were analyzed by flow cytometry. Transduced Jurkat cells are CD3 positive, and stimulated cells are reporter positive.
  • Figure 22 illustrates CD28 intracellular motifs that were optionally mutated to produce “Construct G” (shown as CD8aP-CD28 or CD8aP-CD28wt), “Construct A” (shown as CD8aP-CD28vl) and “Construct E” (shown as CD8aP-CD28v2).
  • Constructs G, A, and E are described elsewhere herein, including in Tables 1-4. These constructs outperformed wild-type CD8aP, CD8aa, and CD8a-CD4 constructs ( Figures 23-25) for cytokine production, T cell proliferation in response to antigen, and antitumor activity in a killing assay.
  • CD8P-CD28 fusions contained mutations in the CD28 intracellular portion.
  • Substitution mutations were introduced into one or more of the motifs shown underlined below in the amino acid sequence of CD28 intracellular domain (SEQ ID NO.: 19)
  • Table 7 summarizes the mutations that were made in the CD28 intracellular portion to generate the CD8P-CD28 fusions according to Constructs BB-NN.
  • the extracellular component and transmembrane domain of these CD8P-CD28 fusions are from human CD8p.
  • the intracellular portions of these CD8P-CD28 fusions alsoinclude, N-terminal to the CD28-derived sequence, the CD8P intracellular sequence HLCCRR (SEQ ID NO.: 10).
  • the positions in SEQ ID NO.: 19 corresponding to the “LL” dileucine motif are referred-to as “Motif 1” in Table 7.
  • the positions in SEQ ID NO.: 19 corresponding to the motif YMNM are referred-to as “Motif 2” in Table 7.
  • the positions in SEQ ID NO.: 19 corresponding to the motif PRRP are referred-to as “Motif 3” in Table 7.
  • the positions in SEQ ID NO. : 19 corresponding to the motif PYAP are referred-to as “Motif 4” in Table 7.
  • Table 7 also shows the motifs present with in the CD8P-CD28 fusions of Constructs E, A, and G.
  • Constructs BB-NN also comprise the wild-type CD8a co-receptor (comprising SEQ ID NO.: 114).
  • CD4+ T cells were transduced with a (1) TCR recognizing an MHC Class I-restricted antigen, (2) a CD8P-CD28 fusion, and (3) a CD8a co-receptor polypeptide (comprising SEQ ID NO.: 114) and expanded, then restimulated with ME275 cells for 5 times in the span of 15 days. Remaining tumor intensity was then measured.
  • Figure 26A shows remaining tumor intensity, wherein the CD8P-CD28 fusion contained the wild-type dileucine (“LL”) motif in SEQ ID NO.: 19, or wherein an otherwise identical CD8P-CD28 fusion contained the mutant diglycine (“GG”) in place of the dileucine.
  • LL wild-type dileucine
  • GG mutant diglycine
  • Figure 26B shows remaining tumor intensity, wherein the CD8P- CD28 fusion contained the wild-type PI3K binding site (“YMNM”) motif in SEQ ID NO.: 19, or wherein an otherwise identical CD8P-CD28 fusion contained the mutant PI3K binding site motif (“AMNM”) in place of the PI3K binding site.
  • YMNM wild-type PI3K binding site
  • AMNM mutant PI3K binding site motif
  • Additional constructs are generated that contain alternative mutations in the CD28 intracellular portion, in one or more of the Motif 1-4 regions as shown in Table 7.
  • the mutations are selected to disrupt function of the moti s).
  • Certain embodiments include, as Motif 2, the amino acid sequence FMNM instead of the wild-type amino acid sequence YMNM.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Toxicology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente divulgation concerne des polypeptides modifiés pour conférer une activité désirée à une cellule immunitaire qui cible une cellule malade. Certains modes de réalisation concernent un polypeptide comprenant un domaine extracellulaire de CD8β, un domaine transmembranaire qui provient éventuellement de CD8β, et un domaine intracellulaire comprenant un variant du NO. ID SEQ : 19, la variation comprenant : (i) un motif diglycine à la place d'un motif dileucine; (ii) un site de liaison PI3K muté et éventuellement non fonctionnel; (iii) un site de liaison ITK et GADS muté et éventuellement non fonctionnel; (iv) un site de liaison LCK muté et éventuellement non fonctionnel; ou (v) toute combinaison de (i)-(iv). Les polypeptides divulgués peuvent traduire un stimulus externe (par exemple, la liaison à un ligand) en un signal désiré (par exemple, de stimulation) chez une cellule hôte, ou prévenir un signal indésirable chez la cellule hôte et peuvent présenter des avantages pour améliorer une thérapie cellulaire adoptive comprenant des lymphocytes T CD4+ exprimant un récepteur spécifique à un antigène.
PCT/US2023/066048 2022-04-26 2023-04-21 Compositions et méthodes pour l'immunothérapie cellulaire WO2023212507A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263335218P 2022-04-26 2022-04-26
US63/335,218 2022-04-26

Publications (1)

Publication Number Publication Date
WO2023212507A1 true WO2023212507A1 (fr) 2023-11-02

Family

ID=88519774

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/066048 WO2023212507A1 (fr) 2022-04-26 2023-04-21 Compositions et méthodes pour l'immunothérapie cellulaire

Country Status (1)

Country Link
WO (1) WO2023212507A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180148508A1 (en) * 2015-06-26 2018-05-31 University Of Southern California Masking chimeric antigen receptor t cells for tumor-specific activation
US20210085721A1 (en) * 2019-05-01 2021-03-25 Pact Pharma, Inc. Compositions and methods for the treatment of cancer using a cd8 engineered t cell therapy

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180148508A1 (en) * 2015-06-26 2018-05-31 University Of Southern California Masking chimeric antigen receptor t cells for tumor-specific activation
US20210085721A1 (en) * 2019-05-01 2021-03-25 Pact Pharma, Inc. Compositions and methods for the treatment of cancer using a cd8 engineered t cell therapy

Similar Documents

Publication Publication Date Title
US11382954B2 (en) Binding proteins specific for RAS neoantigens and uses thereof
AU2018234830B2 (en) High affinity MAGE-A1-specific TCRs and uses thereof
WO2018058002A1 (fr) Tcr spécifiques de l'antigène ha-1 d'histocompatibilité (h) mineure et ses utilisations
US20240165232A1 (en) Chimeric receptor proteins and uses thereof
JP7407701B2 (ja) ｓｔｒｅｐタグ特異的キメラ受容体およびその使用
US20210145882A1 (en) Methods for adoptive cell therapy targeting ror1
WO2023288281A2 (fr) Polypeptides chimériques
WO2023230014A1 (fr) Protéines de liaison et cellules modifiées spécifiques de néoantigènes et leurs utilisations
AU2018314242A1 (en) BRAF-specific TCRs and uses thereof
EP4217387A2 (fr) Immunothérapie ciblant des antigènes sox2
EP4259651A2 (fr) Compositions et méthodes pour l'immunothérapie cellulaire
US20220009992A1 (en) T cell receptors specific for mesothelin and their use in immunotherapy
WO2023212507A1 (fr) Compositions et méthodes pour l'immunothérapie cellulaire
WO2019140278A1 (fr) Immunothérapie ciblant des antigènes du facteur de liaison du noyau
WO2024163371A1 (fr) Protéines de liaison spécifiques pour p53 mutant et leurs utilisations
WO2023220718A1 (fr) Protéines de liaison spécifiques pour des néo-antigènes ras et leurs utilisations
WO2022076353A1 (fr) Compositions et procédés de traitement de la maladie associée à l'expression de mage-a1
KR20220050176A (ko) Wt-1에 특이적인 t-세포 면역요법
EP4031583A1 (fr) Protéines de récepteur chimérique et leurs utilisations

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23797454

Country of ref document: EP

Kind code of ref document: A1