CD8 Polypeptides, Compositions, and Methods of Use Thereof

Abstract

The present disclosure relates to T cells capable of co-expressing a T cell receptor ("TCR") together with a CD8 polypeptide, and their use in adoptive cell therapy. The present disclosure further provides modified CD8 sequences, vectors, and methods related thereto.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is an international application under the Patent Cooperation Treaty, which claims priority to U.S. Provisional Patent Application No. 63 / 132,824, filed December 31, 2020, U.S. Provisional Patent Application No. 63 / 247,775, filed September 23, 2021, and German Provisional Patent Application No. 102021100038.6, filed January 4, 2021, the contents of which are incorporated herein by reference in their entireties.

[0002] Reference to Electronically Submitted Sequence Listing An official copy of the Sequence Listing has been submitted electronically via EFS-Web as an ASCII format Sequence Listing having a file named "3000011-022977_Sequence_Listing_Final.txt" created on December 28, 2021, and having a size of 514,610 kilobytes, and is being submitted contemporaneously herewith. The Sequence Listing contained in this ASCII format document is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to T cells capable of co-expressing a T cell receptor ("TCR") together with a CD8 polypeptide, and their use in adoptive cell therapy. The present disclosure further provides modified CD8 sequences, vectors, compositions, transformed T cells, and related methods thereof. [Background technology]

[0004] CD8 and CD4 are transmembrane glycoproteins characteristic of distinct populations of T lymphocytes whose antigen responses are restricted by class I and class II MHC molecules, respectively. They play major roles in both the differentiation and selection of T cells during thymic development and in the activation of mature T lymphocytes in response to antigen-presenting cells. Both CD8 and CD4 are immunoglobulin superfamily proteins. They determine antigen restriction by binding to MHC molecules at interfaces distinct from the regions that present antigenic peptides, but the structural basis for their similar functions seems to be very different. Their sequence similarity is low, and CD4 is expressed on the cell surface as a monomer, whereas CD8 is expressed as an αα homodimer (e.g., FIG. 55C) or αβ heterodimer (e.g., FIG. 55A). In humans, this CD8αα homodimer can functionally substitute for the CD8αβ heterodimer. CD8 contacts an acidic loop within the α3 domain of class I MHC, thereby increasing the avidity of T cells to their targets. CD8 also binds to the cytoplasmic tail of its α chain and the tyrosine kinase p56 lck It is involved in the phosphorylation events that lead to CTL activation via its association with

[0005] It would be desirable to develop methods to produce T cells with enhanced and specific cytotoxic activity for immunotherapy. Summary of the Invention

[0006] In embodiments, the CD8 polypeptides described herein may comprise a CD8α immunoglobulin (Ig)-like domain, a CD8β region, a CD8α transmembrane domain, and a CD8α cytoplasmic domain. In another embodiment, the CD8β region is a CD8β stalk region or domain.

[0007] In embodiments, the CD8 polypeptide described herein comprises (a) an immunoglobulin (Ig)-like domain that comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:1; (b) an immunoglobulin (Ig)-like domain that comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:2; (c) a CD8 beta region comprising at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:3; and (d) a cytoplasmic domain comprising at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:4.

[0008] In embodiments, the CD8 polypeptides described herein have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:5.

[0009] In embodiments, the CD8 polypeptides described herein have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:7.

[0010] In embodiments, a CD8 polypeptide described herein may include a signal peptide having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NO:6, SEQ ID NO:293, or SEQ ID NO:294 fused to the N-terminus or C-terminus of a CD8 polypeptide described herein.

[0011] In embodiments, the CD8 polypeptides described herein may comprise (a) SEQ ID NO:1, which comprises 1, 2, 3, 4, or 5 amino acid substitutions; (b) SEQ ID NO:2, which comprises 1, 2, 3, 4, or 5 amino acid substitutions; (c) SEQ ID NO:3, which comprises 1, 2, 3, 4, or 5 amino acid substitutions; and (d) SEQ ID NO:4, which comprises 1, 2, 3, 4, or 5 amino acid substitutions.

[0012] In embodiments, the CD8 polypeptide described herein may be a CD8α or a modified CD8α polypeptide.

[0013] In embodiments, the disclosure provides nucleic acids encoding the polypeptides described herein.

[0014] In embodiments, a vector may comprise a nucleic acid encoding a CD8 polypeptide as described herein.

[0015] In an embodiment, the vector may comprise a nucleic acid encoding a T cell receptor (TCR) comprising an alpha chain and a beta chain. In another embodiment, the vector may comprise a nucleic acid encoding a CAR-T.

[0016] In embodiments, the TCR alpha and beta chains are selected from the group consisting of SEQ ID NOs: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92.

[0017] In an embodiment, the vector may comprise a nucleic acid encoding a CD8β polypeptide.

[0018] In embodiments, the CD8 β polypeptide may comprise the amino acid sequence of any one of SEQ ID NOs: 8, 9, 10, 11, 12, 13, or 14.

[0019] In an embodiment, the vector may comprise a nucleic acid encoding a 2A peptide or an internal ribosome entry site (IRES) positioned between the nucleic acid encoding the modified CD8 α polypeptide and the nucleic acid encoding the CD8 β polypeptide.

[0020] In an embodiment, the vector may comprise a nucleic acid encoding the 2A peptide positioned between a nucleic acid encoding the TCR alpha chain and a nucleic acid encoding the TCR beta chain.

[0021] In embodiments, the 2A peptide may be selected from P2A (SEQ ID NO: 93), T2A (SEQ ID NO: 94), E2A (SEQ ID NO: 95), or F2A (SEQ ID NO: 96).

[0022] In embodiments, the IRES may be selected from the group consisting of an IRES from a picornavirus, an IRES from a flavivirus, an IRES from a pestivirus, an IRES from a retrovirus, an IRES from a lentivirus, an IRES from an insect RNA virus, and an IRES from a cellular mRNA.

[0023] In embodiments, the vector may further comprise a post-transcriptional regulatory element (PRE) sequence selected from the Woodchuck PRE (WPRE) and variants thereof, the Hepatitis B Virus (HBV) PRE (HPRE), or a combination thereof.

[0024] In embodiments, the vector may further comprise a promoter selected from a cytomegalovirus (CMV) promoter, a phosphoglycerate kinase (PGK) promoter, a myelin basic protein (MBP) promoter, a glial fibrillary acidic protein (GFAP) promoter, a modified MoMuLV LTR containing the myeloproliferative sarcoma virus enhancer (MNDU3), a Ubiquitin C promoter, an EF-1 alpha promoter, a murine stem cell virus (MSCV) promoter, or a combination thereof.

[0025] In embodiments, the vector may be a viral vector or a non-viral vector.

[0026] In embodiments, the vector may be selected from an adenovirus, a poxvirus, an alphavirus, an arenavirus, a flavivirus, a rhabdovirus, a retrovirus, a lentivirus, a herpesvirus, a paramyxovirus, a picornavirus, or a combination thereof.

[0027] In embodiments, the vector may be pseudotyped with an envelope protein of a virus selected from naturally occurring feline endogenous virus (RD114), a chimeric form of RD114 (RD114TR), gibbon ape leukemia virus (GALV), a chimeric form of GALV (GALV-TR), amphotropic murine leukemia virus (MLV 4070A), baculovirus (GP64), vesicular stomatitis virus (VSV-G), avian plague virus (FPV), Ebola virus (EboV), or baboon retrovirus envelope glycoprotein (BaEV), lymphocytic choriomeningitis virus (LCMV), or a combination thereof.

[0028] In embodiments, the vector may further comprise a nucleic acid encoding a T cell receptor (TCR).

[0029] In another embodiment, the vector may further comprise a nucleic acid encoding a chimeric antigen receptor (CAR).

[0030] In embodiments, the isolated nucleic acid may comprise a nucleic acid sequence encoding a T cell receptor comprising an alpha chain and a beta chain, and a CD8 polypeptide comprising an alpha chain and a beta chain. The isolated nucleic acid may comprise a nucleic acid at least 80% identical to a nucleic acid sequence of SEQ ID NO: 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 295, 297, 299, or 301. An isolated nucleic acid can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 295, 297, 299, or 301. In embodiments, the sequences described herein can be isolated or recombinant sequences.

[0031] In an embodiment, the isolated nucleic acid comprises the nucleic acid sequence of SEQ ID NO:267.

[0032] In an embodiment, the isolated nucleic acid comprises the nucleic acid sequence of SEQ ID NO:279.

[0033] In embodiments, the isolated polypeptide may be encoded by a nucleic acid described herein.

[0034] In embodiments, the isolated polypeptide may comprise an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 296, 298, 300, or 302. The amino acid sequence may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 296, 298, 300, or 302. In another embodiment, SEQ ID NO: 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 296, 298, 300, or 302 contains 1, 2, 3, 4, 5, 10, 15, or 20 or more amino acid substitutions or deletions. In yet another embodiment, SEQ ID NO: 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 296, 298, 300, or 302 contains up to 1, 2, 3, 4, 5, 10, 15, or 20 amino acid substitutions or deletions.

[0035] In an embodiment, the isolated polypeptide may comprise the amino acid sequence of SEQ ID NO:268.

[0036] In embodiments, the isolated polypeptide may comprise the amino acid sequence of SEQ ID NO: 280. In embodiments, the cell may be transduced with the vector.

[0037] In embodiments, the cells may include αβ T cells, γδ T cells, natural killer cells, CD4+ / CD8+ cells, or a combination thereof.

[0038] In an embodiment, the αβ T cells may include CD4+ T cells and CD8+ T cells.

[0039] In embodiments, a method of preparing T cells for immunotherapy may include isolating T cells from a blood sample of a human subject, activating the isolated T cells, transducing the activated T cells with a vector, and expanding the transduced T cells.

[0040] In an embodiment, the T cells may be CD4+ T cells.

[0041] In an embodiment, the T cells may be CD8+ T cells.

[0042] In an embodiment, the T cells may be γδ T cells.

[0043] In embodiments, the T cells may be αβ T cells and may express a CD8 polypeptide as described herein.

[0044] In an embodiment, the T cell may be a γδ T cell and may express a modified CD8 polypeptide described herein, such as a modified CD8α polypeptide, or a modified CD8α polypeptide having a CD8β stalk region, such as m1CD8α (Figure 4) and CD8α* (Figure 55B) in constructs no. 11 and no. 12.

[0045] In embodiments, a method of treating a patient having cancer may include administering to the patient a composition comprising a population of expanded T cells, wherein the T cells kill cancer cells that present a peptide on their surface in a complex with an MHC molecule, wherein the peptide is selected from SEQ ID NOs: 98-255, and the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer, bile duct cancer, colorectal cancer, bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain cancer, gastric cancer, prostate cancer, or a combination thereof.

[0046] In embodiments, the composition may further comprise an adjuvant.

[0047] In embodiments, the adjuvant may be selected from anti-CD40 antibodies, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, atezolizumab, interferon-alpha, interferon-beta, CpG oligonucleotides and derivatives, poly(I:C) and derivatives, RNA, sildenafil, microparticle formulations with poly(lactide-co-glycolide) (PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, IL-23, or combinations thereof.

[0048] In embodiments, a method of eliciting an immune response in a patient having cancer may include administering to the patient a composition comprising a population of expanded T cells, wherein the T cells kill cancer cells that present a peptide on their surface in a complex with an MHC molecule, wherein the peptide is selected from SEQ ID NOs: 98-255, and the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer, bile duct cancer, colorectal cancer, bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain cancer, gastric cancer, prostate cancer, or a combination thereof.

[0049] The disclosure further provides a population of engineered T cells that present an exogenous CD8 co-receptor that comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95%, at least 99%, or 100% identical to a polypeptide described herein, e.g., SEQ ID NO: 5, 7, 258, 259, 8, 9, 10, 11, 12, 13, or 14, and a T cell receptor. [Brief description of the drawings]

[0050] [Figure 1] A representative CD8α subunit, e.g., SEQ ID NO: 258 (CD8α1), is shown. In this embodiment, CD8α1 contains five domains: (1) a signal peptide, (2) an Ig-like domain-1, (3) a stalk region, (4) a transmembrane (TM) domain, and (5) a cytoplasmic tail (Cyto) that contains an lck-binding motif. [Diagram 2] 1 shows a sequence alignment between CD8α1 (SEQ ID NO: 258) and m1CD8α (SEQ ID NO: 7). [Diagram 3] A sequence alignment between CD8α2 (sequence number 259) and m2CD8α (sequence number 262) is shown, with the cysteine ​​substitution at position 112 indicated by an arrow. [Figure 4] 1 shows a vector according to an embodiment of the present disclosure. [Diagram 5] Figure 5A: shows the titers of the viral vectors shown in Figure 4. Figure 5B: shows the titers of further viral vectors according to embodiments of the present disclosure. Construct #13, Construct #14, Construct #15, Construct #16, Construct #17, Construct #18, Construct #19, Construct #21, Construct #10n, Construct #11n, and R11KEA (SEQ ID NO:15 and SEQ ID NO:16) (Construct #8) binding to TCR:PRAME-004 (SLLQHLIGL) (SEQ ID NO:147). Note that constructs #10 and #10n are different batches of the same construct (SEQ ID NO:291 and 292), and constructs #11 and #11n are different batches of the same construct (SEQ ID NO:285 and 286). [Figure 6] Shows T cell production. [Figure 7] Figure 7A: Expression of activation markers in CD3+CD8+ cells before and after activation. Figure 7B: Expression of activation markers in CD3+CD4+ cells before and after activation. [Figure 8]Figure 8A: Shows the fold proliferation of cells transduced with various constructs from donor #1. Constructs are as follows: Construct #9b, Construct #10, Construct #11, Construct #12, Construct #1, Construct #2, TCR=R11KEA.WPREwt (TCR with wild type WPRE), NT=non-transduced T cells (as negative control). Note that constructs #9 and #9b are different batches of the same construct (SEQ ID NOs: 287 and 288). Figure 8B: Shows the fold proliferation of cells transduced with various constructs from donor #2. Constructs are as follows: Construct #9b, Construct #10, Construct #11, Construct #12, Construct #1, Construct #2, TCR=R11KEA.WPREwt (TCR with wild type WPRE) (Construct #8), NT=non-transduced T cells (as negative control). [Figure 9-1] Figure 9A: Shows a flow plot of cells transduced with construct number 9. Figure 9B: Shows a flow plot of cells transduced with construct number 10 according to one embodiment of the present disclosure. [Figure 9-2] Figure 9C: Flow plot of cells transduced with construct no. 11. Figure 9D: Flow plot of cells transduced with construct no. 12. [Figure 10] CD8+CD4+% of cells transduced with various constructs for donor #1 and donor #2 are shown. Constructs are as follows: Construct #9b, Construct #10, Construct #11, Construct #12, Construct #1, Construct #2, TCR=R11KEA.WPREwt type (TCR with WPRE), NT=non-transduced T cells (as negative control). [Figure 11] Shown is the Tet% of CD8+CD4+ cells transduced with various constructs: Construct #9b, Construct #10, Construct #11, Construct #12, Construct #1, Construct #2, TCR=R11KEA.WPREwt (TCR with wild type WPRE), NT=non-transduced T cells (as a negative control). [Figure 12]Shown is the Tet MFI (CD8+CD4+Tet+) of cells transduced with various constructs: Construct #9b, Construct #10, Construct #11, Construct #12, Construct #1, Construct #2, TCR=R11KEA.WPREwt (TCR with wild type WPRE), NT=non-transduced T cells (as a negative control). [Figure 13] Shown is the CD8α MFI (CD8+CD4+Tet+) of cells transduced with various constructs: Construct #9b, Construct #10, Construct #11, Construct #12, Construct #1, Construct #2, TCR=R11KEA.WPREwt (TCR with wild type WPRE), NT=non-transduced T cells (as a negative control). [Figure 14] Shown is the CD8+CD4% (of CD3+) of cells transduced with various constructs: Construct #9b, Construct #10, Construct #11, Construct #12, Construct #1, Construct #2, TCR=R11KEA.WPREwt (TCR with wild type WPRE), NT=non-transduced T cells (as a negative control). [Figure 15] Shown is the CD8+Tet+% (of CD3+) of cells transduced with various constructs: Construct #9b, Construct #10, Construct #11, Construct #12, Construct #1, Construct #2, TCR=R11KEA.WPREwt type (TCR with WPRE), NT=non-transduced T cells (as negative control). [Figure 16] Shown is the Tet MFI (CD8+Tet+) of cells transduced with various constructs: Construct #9b, Construct #10, Construct #11, Construct #12, Construct #1, Construct #2, TCR=R11KEA.WPREwt (TCR with wild type WPRE), NT=non-transduced T cells (as a negative control). [Figure 17]Shown is the CD8α MFI (CD8+Tet+) of cells transduced with various constructs: Construct #9b, Construct #10, Construct #11, Construct #12, Construct #1, Construct #2, TCR=R11KEA.WPREwt (TCR with wild type WPRE), NT=non-transduced T cells (as a negative control). [Figure 18] The Tet+% (of CD3+) of cells transduced with various constructs are shown. Constructs are as follows: Construct #9b, Construct #10, Construct #11, Construct #12, Construct #1, Construct #2, TCR=R11KEA.WPREwt (TCR with wild type WPRE), NT=non-transduced T cells (as a negative control). [Figure 19] Shown are the VCN (upper panel) and CD3+Tet+ / VCN (lower panel) of cells transduced with various constructs: Construct #9b, Construct #10, Construct #11, Construct #12, Construct #1, Construct #2, TCR=R11KEA.WPREwt (TCR with wild type WPRE), NT=non-transduced T cells (as negative control). [Figure 20-1] Figures 20A-20C show data demonstrating that constructs (numbers 10, 11, and 12) are comparable to TCR alone in mediating cytotoxicity against target-positive cell lines expressing antigens at different levels (1081 copies of UACC257 per cell, 50 copies of A375 per cell). [Figure 20-2] Same as above [Figure 20-3] Same as above [Figure 21]Figures 21A-21B: Data showing that IFNγ secretion in response to UACC257 is comparable between constructs, but in A375, #10 expression is the highest of all constructs. However, when comparing #9 to #11 expressing wild type and modified CD8 co-receptors, respectively, T cells transduced with #11 induced a stronger cytokine response, measured as IFNγ quantified in the supernatant from the Incucyte plate. Construct #9, #10, #11, #12, #1, #2, #8 = R11KEA TCR only. [Figure 22] An exemplary experimental design for evaluating DC maturation and cytokine secretion by PBMC-derived products in response to UACC257 and A375 targets. N=2. [Figure 23] Figures 23A-23B: Data showing that IFNγ secretion in response to A375 is increased in the presence of iDCs. In triple co-cultures with added iDCs, IFNγ secretion is higher in construct #10 compared to the other constructs. However, when construct #9 is compared to construct #11 expressing wild type and modified CD8 co-receptors, respectively, T cells transduced with #11 induced a stronger cytokine response measured as IFNγ quantified in culture supernatants of triple co-cultures, E:T:iDC::1:1 / 10:1 / 4, using donor D600115. Construct #9, Construct #10, Construct #11, Construct #12, Construct #1, Construct #2, Construct #8 = R11KEA TCR only. [Figure 24] Figures 24A-24B: Data showing that IFNγ secretion in response to A375 is increased in the presence of iDC. In triple co-cultures with iDC, IFNγ secretion was higher in construct #10 compared to other constructs. IFNγ was quantified in culture supernatants of triple cultures, E:T:iDC::1:1 / 10:1 / 4, using donor D150081. Construct #9, Construct #10, Construct #11, Construct #12, Construct #1, Construct #2, Construct #8 = R11KEA TCR only. [Diagram 25]Figures 25A-25B: Data showing that IFNγ secretion in response to UACC257 is increased in the presence of iDCs. In triple co-cultures with iDCs, IFNγ secretion is higher in construct #10 compared to the other constructs. However, when comparing #9 with #11 expressing wild type and modified CD8 co-receptors, respectively, T cells transduced with construct #11 induced a stronger cytokine response measured as IFNγ quantified in culture supernatants of triple co-cultures, E:T:iDC::1:1 / 10:1 / 4, using donor D600115. Construct #9, Construct #10, Construct #11, Construct #12, Construct #1, Construct #2, Construct #8 = R11KEA TCR only. [Figure 26] 1 shows T cell production according to one embodiment of the present disclosure. [Figure 27] Figure 27A: Expression of activation markers before and after activation in CD3+CD8+ cells. Figure 27B: Expression of activation markers before and after activation in CD3+CD4+ cells, according to one embodiment of the present disclosure. [Figure 28] Fold expansion of cells transduced with various constructs is shown. [Figure 29] 29A and 29B: Shows the CD8+CD4+% of cells transduced with various constructs according to one embodiment of the present disclosure. [Diagram 30] 30A and 30B: Shows the Tet% of CD8+CD4+ cells transduced with various constructs according to one embodiment of the present disclosure. [Diagram 31] 31A and 31B: Shows Tet MFI (CD8+CD4+Tet+) of cells transduced with various constructs according to one embodiment of the present disclosure. [Diagram 32] 32A and 32B: Shows the CD8+CD4+% (out of CD3+) of cells transduced with various constructs according to one embodiment of the present disclosure. [Diagram 33] 33A and 33B: Shows the CD8+Tet+% (out of CD3+ cells) of cells transduced with various constructs according to one embodiment of the present disclosure. [Diagram 34]34A and 34B: Shows Tet MFI (CD8+Tet+) of cells transduced with various constructs according to one embodiment of the present disclosure. [Diagram 35] 35A and 35B: Shows the Tet+% (out of CD3+) of cells transduced with various constructs, according to one embodiment of the present disclosure. [Figure 36-1] 36A and 36B: Shows the VCN of cells transduced with various constructs according to one embodiment of the present disclosure. [Figure 36-2] Same as above [Figure 37] 1 shows T cell production according to one embodiment of the present disclosure. [Figure 38] The Tet% of CD8+CD4+ cells transduced with the various constructs is shown. [Figure 39] The Tet MFI of CD8+CD4+Tet+ cells transduced with various constructs is shown. [Diagram 40] The Tet MFI of CD8+Tet+ cells transduced with various constructs is shown. [Diagram 41] The Tet+% of CD3+ cells transduced with the various constructs is shown. [Diagram 42] The vector copy numbers (VCN) of cells transduced with the various constructs are shown. [Diagram 43] The % T cell subsets in cells transduced with the various constructs are shown. FACS analysis was gated on CD3+TCR+. [Figure 44-1] Figure 44A and Figure 44B: % T cell subsets in cells transduced with various constructs. FACS analysis was gated on CD4+CD8+ in Figure 44A and CD4+CD8+TCR+ in Figure 44B. [Figure 44-2] Same as above [Figure 45-1]Figure 45A and Figure 45B: Data showing that constructs #13 and #10 are comparable to TCR alone in mediating cytotoxicity against a UACC257 target positive cell line expressing high levels of antigen (1081 copies per cell). Construct #15 was also effective but showed slower killing compared to constructs #13 and #10. The effector:target ratio used to obtain these results was 4:1. [Figure 45-2] Same as above [Diagram 46] Figure 1 shows that IFNγ secretion in response to construct #13 in the UACC257 cell line was higher compared to construct #10. IFNγ was quantified in the supernatants from the Incucyte plates. The effector:target ratio used to obtain these results was 4:1. [Figure 47] ICI marker frequencies (2B4, 41BB, LAG3, PD-1, TIGIT, TIM3, CD39+CD69+, and CD39-CD69-) are shown. [Figure 48-1] Figures 48A-48G: Increased expression of IFNγ, IL-2, and TNFα by CD4+CD8+ cells transduced with construct #10 (WT signal peptide, CD8β1) compared to other constructs. FACS analysis gated on CD3+CD4+CD8+ cells against UACC257, E:T of 4:1. [Figure 48-2] Same as above [Figure 48-3] Same as above [Figure 48-4] Same as above [Figure 49-1] Figures 49A-49G show increased expression of IFNγ, IL-2, MIP-1β, and TNFα by CD4-CD8+ cells transduced with construct #10 (WT signal peptide, CD8β1) compared to other constructs. FACS analysis gated on CD3+CD4-CD8+ cells against UACC257, E:T of 4:1. [Figure 49-2] Same as above [Figure 49-3] Same as above [Figure 49-4] Same as above [Figure 50-1]Figures 50A-50G: Showing increased expression of IL-2 and TNFα by CD3+TCR+ cells transduced with construct number 10 (WT signal peptide, CD8β1) compared to other constructs. FACS analysis was gated on CD3+TCR+ cells against UACC257, E:T of 4:1. [Figure 50-2] Same as above [Figure 50-3] Same as above [Figure 50-4] Same as above [Figure 51] Figures 51A-C show the results of FACS analysis gated on CD4+CD8+ cells for A375, E:T 4:1. [Figure 52] Figures 52A-C show the results of FACS analysis gated on CD4-CD8+ cells for A375, E:T 4:1. [Figure 53] Figures 53A-C show the results of FACS analysis gated on CD3+TCR+ cells for A375, E:T 4:1. [Figure 54] 1 shows T cell production according to one embodiment of the present disclosure. [Figure 55] 55A-55C: Interaction between peptide / MHC complexes of antigen presenting cells (APCs) and T cells by binding to a complex of TCR and CD8αβ heterodimer (FIG. 55A, e.g., produced by transducing T cells with construct no. 2, no. 3, no. 4, no. 10, no. 13, no. 14, no. 15, no. 16, no. 17, no. 18, or no. 21), a complex of TCR and homodimeric CD8α (CD8αα*) having its stalk region replaced with the CD8β stalk region (FIG. 55B, e.g., produced by transducing T cells with construct no. 11, no. 12, or no. 19), and a complex of TCR and CD8α homodimer (FIG. 55C, e.g., produced by transducing T cells with construct no. 1, no. 5, no. 6, no. 7, or no. 9). [Figure 56]1 shows the level of IL-12 secretion by dendritic cells (DCs) in the presence of CD4+ T cells transduced with construct no. 10 or no. 11 and immature dendritic cells (iDCs) according to one embodiment of the present disclosure. [Figure 57] 1 shows the level of TNF-α secretion by dendritic cells (DCs) in the presence of CD4+ T cells transduced with construct no. 10 or no. 11 and immature dendritic cells (iDCs) according to one embodiment of the present disclosure. [Figure 58] 1 shows the level of IL-6 secretion by dendritic cells (DCs) in the presence of CD4+ T cells transduced with construct no. 10 or no. 11 and immature dendritic cells (iDCs) according to one embodiment of the present disclosure. [Figure 59] 1 shows a scheme for determining the level of cytokine secretion by dendritic cells (DCs) in the presence of PBMCs transduced with various constructs and target cells, according to one embodiment of the present disclosure. [Figure 60] 1 shows the levels of IL-12 secretion by dendritic cells (DCs) in the presence of PBMCs transduced with various constructs and target cells according to one embodiment of the present disclosure. [Figure 61] 1 shows the levels of TNF-α secretion by dendritic cells (DCs) in the presence of PBMCs transduced with various constructs and target cells, according to one embodiment of the present disclosure. [Figure 62] 1 shows the levels of IL-6 secretion by dendritic cells (DCs) in the presence of PBMCs transduced with various constructs and target cells according to one embodiment of the present disclosure. [Figure 63-1] Figures 63A-63C: IFNγ production from transduced CD4+ selected T cells obtained from donor #1 (Figure 63A), donor #2 (Figure 63B), and donor #3 (Figure 63C) according to one embodiment of the present disclosure. Figure 63D: EC50 values ​​(ng / ml) for Figures 63A-63C. [Figure 63-2] Same as above [Figure 64-1]Figures 64A-64C: Showing IFNγ production from transduced PBMCs obtained from donor #4 (Figure 64A), donor #1 (Figure 64B), and donor #3 (Figure 64C) and their respective EC50 values ​​(ng / ml) according to one embodiment of the present disclosure. [Figure 64-2] Same as above [Figure 64-3] Same as above [Figure 64-4] Figure 64D: Comparison of EC50 values ​​(ng / ml) between different donors in Figures 64A-64C. [Figure 65-1] Figures 65A-65C: Show IFNγ production from transduced PBMCs (Figure 65A), CD8+ selected T cells (Figure 65B), and CD4+ selected T cells (Figure 65C) from a single donor according to one embodiment of the disclosure, and their respective EC50 values ​​(ng / ml). [Figure 65-2] Same as above [Figure 65-3] Same as above DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] Modified CD8 Polypeptides The CD8 polypeptides described herein may comprise the general structure of an N-terminal signal peptide (optional), a CD8α immunoglobulin (Ig)-like domain, a CD8β region (domain), a CD8α transmembrane domain, and a CD8α cytoplasmic domain. The modified CD8 polypeptides described herein showed unexpected improvement in the functionality of T cells co-transduced with vectors expressing TCR and CD8 polypeptides.

[0052] The CD8 polypeptides described herein may include the general structure of an N-terminal signal peptide (optional), a CD8α immunoglobulin (Ig)-like domain, a stalk domain or region, a CD8α transmembrane domain, and a CD8α cytoplasmic domain.

[0053] In embodiments, a CD8 polypeptide described herein may comprise: (a) an immunoglobulin (Ig)-like domain that comprises at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:1; (b) a region that comprises at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:2; (c) a transmembrane domain that comprises at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:3; and (d) a cytoplasmic domain that comprises at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to the amino acid sequence of SEQ ID NO:4. The CD8 polypeptides described herein can be co-expressed with a T cell receptor or CAR-T in a T cell and used in methods of adoptive cell therapy (ACT). The T cell can be an αβ T cell or a γδ T cell.

[0054] In another embodiment, the CD8 polypeptide described herein may comprise: (a) at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:1; (b) at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:2; (c) at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:3; and (d) at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:4. The CD8 polypeptides described herein can be co-expressed with a T cell receptor or CAR-T in a T cell and used in methods of adoptive cell therapy (ACT). The T cell can be an αβ T cell or a γδ T cell.

[0055] In another embodiment, the CD8 polypeptide described herein may comprise (a) SEQ ID NO:1, comprising 1, 2, 3, 4, or 5 amino acid substitutions; (b) SEQ ID NO:2, comprising 1, 2, 3, 4, or 5 amino acid substitutions; (c) SEQ ID NO:3, comprising 1, 2, 3, 4, or 5 amino acid substitutions; and (d) SEQ ID NO:4, comprising 1, 2, 3, 4, or 5 amino acid substitutions. In an embodiment, the substitutions are conservative amino acid substitutions. The CD8 polypeptide described herein may be co-expressed with a T cell receptor or CAR-T in T cells and used in methods of adoptive cell therapy (ACT). The T cells may be γδ T cells or γδ (αβ) T cells.

[0056] CD8 is a membrane-bound glycoprotein that functions as a coreceptor for antigen recognition of peptide / MHC class I complexes by the T cell receptor (TCR) and plays an important role in T cell development in the thymus and T cell activation in the periphery. Functional CD8 is a dimeric protein consisting of either two α chains (CD8αα) or an α chain and a β chain (CD8αβ), and surface expression of the β chain may require association with a co-expressed α chain to form the CD8αβ heterodimer. CD8αα and CD8αβ can be differentially expressed on various lymphocytes. CD8αβ is mainly expressed by αβTCR + Expressed on the surface of T cells and thymocytes, CD8αα binds the αβTCR + A subset of γδTCR + Intestinal intraepithelial lymphocytes, NK cells, dendritic cells, and a small fraction of CD4 + It is expressed in T cells.

[0057] For example, the human CD8 gene can express a protein of 235 amino acids. Figure 1 shows the CD8α protein (CD8α1 - SEQ ID NO: 258), which in embodiments is divided into the following domains (starting from the amino terminus and ending at the carboxy terminus of the polypeptide): (1) a signal peptide (amino acids -21 to -1), which may be cleaved in human cells during transport of the receptor to the cell surface and therefore may not constitute part of the mature active receptor, (2) an immunoglobulin (Ig)-like domain (in this embodiment, amino acids 1 to 115), which may assume a structure called the immunoglobulin fold, similar to the structure of the immunoglobulin family of proteins, which are many other molecules involved in regulating the immune system. The crystal structure of the CD8αα receptor in complex with the human MHC molecule HLA-A2 demonstrates how the Ig domain of the CD8αα receptor binds to the ligand; (3) the membrane proximal region (in this embodiment, amino acids 116-160), which may be an extended linker region that allows the CD8αα receptor to "reach" from the surface of the T cell over the top of the MHC to the a3 domain of the MHC to which it binds. The stalk region may be glycosylated and may be inflexible; (4) the transmembrane domain (in this embodiment, amino acids 161-188), which may anchor the CD8αα receptor in the cell membrane and thus is not part of the soluble recombinant protein; (5) the cytoplasmic domain (in this embodiment, amino acids 189-214), which may be involved in the T cell activation cascade of phosphorylation events involving p56 lck It can mediate signaling functions in T cells through its association with

[0058] The CD8α sequence may generally have a sufficient portion of the immunoglobulin domain to be able to bind to MHC. Generally, the CD8α molecule may comprise all or a substantial portion of the immunoglobulin domain of CD8α, e.g., SEQ ID NO: 258, but in embodiments may comprise at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, or 115 amino acids of the immunoglobulin domain. The CD8α molecule of the present disclosure may preferably be a dimer (e.g., CD8αα or CD8αβ), although CD8α monomers may be included within the scope of the present disclosure. In embodiments, the CD8α of the present disclosure may comprise CD8α1 (SEQ ID NO: 258) and CD8α2 (SEQ ID NO: 259).

[0059] CD8 α and β subunits may have similar structural motifs, including an Ig-like domain, a stalk region of 30-40 amino acids, a transmembrane region, and a short cytoplasmic domain of approximately 20 amino acids. CD8 α and β chains have two and one N-linked glycosylation sites, respectively, within the Ig-like domain, which share less than 20% identity within their amino acid sequences. The CD8 β stalk region is 10-13 amino acids shorter than the CD8 α stalk and is highly glycosylated with O-linked carbohydrates. These carbohydrates in the β, but not α, stalk region appear to be highly heterogeneous due to complex sialylation, which may be differentially regulated during thymocyte development and upon T cell activation. Glucan adducts have been shown to play a regulatory role in glycoprotein function and immune responses. Glycans proximal to the transmembrane domain can affect the orientation of adjacent motifs. The unique biochemical properties of the CD8 β chain stalk region may present a plausible candidate to regulate coreceptor function.

[0060] A CD8 polypeptide can be modified such that a CD8α region, such as the stalk region, can be replaced with a CD8β region, in another embodiment, to generate a CD8α-CD8β polypeptide. In embodiments, a modified CD8 polypeptide described herein can have a region that comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:2. The modified CD8 alpha polypeptides described herein may have an immunoglobulin (Ig)-like domain that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. The modified CD8 polypeptides may have a transmembrane domain that comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:3. The modified CD8 polypeptides described herein can have a cytoplasmic tail that comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4. The CD8 polypeptides described herein can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:5. The CD8 polypeptides described herein may include a signal peptide that comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:294 fused to the N-terminus or fused to the C-terminus of the mCD8α polypeptide.The CD8 polypeptides described herein may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:7.

[0061] T cells The T cells may express a modified CD8 polypeptide as described herein. For example, the T cells may co-express a T cell receptor (TCR) and a modified CD8 polypeptide as described herein. The T cells may also express a chimeric antigen receptor (CAR), a CAR analog, or a CAR derivative.

[0062] The T cells, when present in the population, can be αβ T cells, γδ T cells, natural killer T cells, or a combination thereof. + T cells, CD8 + T cells, or CD4 + / CD8 + It may be a T cell.

[0063] T cell receptor The T cells may co-express a T cell receptor (TCR), an antigen binding protein, or both, with a modified CD8 polypeptide described herein, including, but not limited to, those listed in Table 3 (SEQ ID NOs: 15-92).Further, the T cells may be derived from any of the following known methods, including the use of the methods disclosed in U.S. Patent Application Publication No. 2017 / 0267738, U.S. Patent Application Publication No. 2017 / 0312350, U.S. Patent Application Publication No. 2018 / 0051080, U.S. Patent Application Publication No. 2018 / 0164315, U.S. Patent Application Publication No. 2018 / 0161396, U.S. Patent Application Publication No. 2018 / 0162922, U.S. Patent Application Publication No. 2018 / 0273602, U.S. Patent Application Publication No. 2019 / 0016801, U.S. Patent Application Publication No. 2019 / 0002556, U.S. Patent Application Publication No. 2019 / 0135914 No. 10,538,573, U.S. Patent No. 10,626,160, U.S. Patent Application Publication No. 2019 / 0321478, U.S. Patent Application Publication No. 2019 / 0256572, U.S. Patent No. 10,550,182, U.S. Patent No. 10,526,407, U.S. Patent Application Publication No. 2019 / 0284276, U.S. Patent Application Publication No. 2019 / 0016802, U.S. Patent Application Publication No. 2019 / 0016803, U.S. Patent Application Publication No. 2019 / 0016804, U.S. Patent No. 10,583,573, U.S. Patent Application Publication No. 202 No. 2020 / 0339652, U.S. Patent No. 10,537,624, U.S. Patent No. 10,596,242, U.S. Patent Application Publication No. 2020 / 0188497, U.S. Patent No. 10,800,845, U.S. Patent Application Publication No. 2020 / 0385468, U.S. Patent No. 10,527,623, U.S. Patent No. 10,725,044, U.S. Patent Application Publication No. 2020 / 0249233, U.S. Patent No. 10,702,609, U.S. Patent Application Publication No. 2020 / 0254106, U.S. Patent No. 10,800,832, U.S. Patent Application Publication No. 2020 / The T cells may express the TCR and antigen binding proteins described in US Patent Publication No. 0123221, US Patent No. 10,590,194, US Patent No. 10,723,796, US Patent Application Publication No. 2020 / 0140540, US Patent No. 10,618,956, US Patent Application Publication No. 2020 / 0207849, US Patent Application Publication No. 2020 / 0088726, and US Patent Application Publication No. 2020 / 0384028 (the contents of each of these publications and the sequence listings described herein are incorporated by reference in their entirety). The T cells may be αβ T cells, γδ T cells, natural killer T cells. Natural killer cells.In embodiments, the TCRs described herein are single chain TCRs or soluble TCRs.

[0064] Additionally, a TCR that may be co-expressed in a T cell with a modified CD8 polypeptide described herein may be a TCR consisting of an alpha chain (TCRα) and a beta chain (TCRβ). The TCRα and TCRβ chains that may be used in the TCR include R11KEA (SEQ ID NOs: 15 and 16), R20P1H7 (SEQ ID NOs: 17 and 18), R7P1D5 (SEQ ID NOs: 19 and 20), R10P2G12 (SEQ ID NOs: 21 and 22), R10P1A7 (SEQ ID NOs: 23 and 24), R4P1D10 (SEQ ID NOs: 25 and 26), R4P3F9 (SEQ ID NOs: 27 and 28), R4P3H3 (SEQ ID NOs: 29 and 30), R36P3F9 (SEQ ID NOs: 31 and 32), R52 P2G11 (SEQ ID NOs: 33 and 34), R53P2A9 (SEQ ID NOs: 35 and 36), R26P1A9 (SEQ ID NOs: 37 and 38), R26P2A6 (SEQ ID NOs: 39 and 40), R26P3H1 (SEQ ID NOs: 41 and 42), R35P3A4 (SEQ ID NOs: 43 and 44), R37P1C9 (SEQ ID NOs: 45 and 46), R37P1H1 (SEQ ID NOs: 47 and 48), R42P3A9 (SEQ ID NOs: 49 and 50), R43P3F2 (SEQ ID NOs: 51 and 52), R43P3G5 (SEQ ID NOs: 53 and 54), R59P2E7 (SEQ ID NOs: 55 and 56), R11P3D3 (SEQ ID NOs: 57 and 58), R16P1C10 (SEQ ID NOs: 59 and 60), R16P1E8 (SEQ ID NOs: 61 and 62), R17P1A9 (SEQ ID NOs: 63 and 64), R17P1D7 (SEQ ID NOs: 65 and 66), R17P1G3 (SEQ ID NOs: 67 and 68), R17P2B6 (SEQ ID NOs: 69 and 70), R11P3D3KE (SEQ ID NOs: 71 and 303), R39P1C1 2 (SEQ ID NOs: 304 and 74), R39P1F5 (SEQ ID NOs: 75 and 76), R40P1C2 (SEQ ID NOs: 77 and 78), R41P3E6 (SEQ ID NOs: 79 and 80), R43P3G4 (SEQ ID NOs: 81 and 82), R44P3B3 (SEQ ID NOs: 83 and 84), R44P3E7 (SEQ ID NOs: 85 and 86), R49P2B7 (SEQ ID NOs: 87 and 88), R55P1G7 (SEQ ID NOs: 89 and 90), or R59P2A7 (SEQ ID NOs: 91 and 92). The T cell may be an αβ T cell, a γδ T cell, or a natural killer T cell.

[0065] Table 1 shows examples of peptides that the TCR binds when the peptide is in a complex with an MHC molecule (MHC molecules in humans can be referred to as HLA, human leukocyte antigens). [Table 1]

[0066] Tumor-associated antigens (TAA) Tumor-associated antigen (TAA) peptides may be used with the CD8 polypeptide constructs, methods, and embodiments described herein. For example, the T cell receptors (TCRs) described herein may specifically bind to TAA peptides when they bind to human leukocyte antigens (HLA), also known as major histocompatibility complex (MHC) molecules. Human MHC molecules are also called human leukocyte antigens (HLA).

[0067] Tumor associated antigen (TAA) peptides that may be used with the CD8 polypeptides described herein include those listed in Table 3, as well as those listed in U.S. Patent Application Publication Nos. 2016 / 0187351, 2017 / 0165335, 2017 / 0035807, 2016 / 0280759, 2016 / 0287687, 2016 / 0346371, 2016 / 0368965, 2017 / 0 022251, U.S. Patent Application Publication No. 2017 / 0002055, U.S. Patent Application Publication No. 2017 / 0029486, U.S. Patent Application Publication No. 2017 / 0037089, U.S. Patent Application Publication No. 2017 / 0136108, U.S. Patent Application Publication No. 2017 / 0101473, U.S. Patent Application Publication No. 2017 / 0096461, U.S. Patent Application Publication No. 2017 / 0165337, U.S. Patent Application Publication No. 2017 / 0189505, U.S. Patent Application Publication No. 2017 / 0173132, U.S. Patent Application Publication No. 2017 / 02966 40, U.S. Patent Application Publication No. 2017 / 0253633, U.S. Patent Application Publication No. 2017 / 0260249, U.S. Patent Application Publication No. 2018 / 0051080, U.S. Patent Application Publication No. 2018 / 0164315, U.S. Patent Application Publication No. 2018 / 0291082, U.S. Patent Application Publication No. 2018 / 0291083, U.S. Patent Application Publication No. 2019 / 0255110, U.S. Patent No. 9,717,774, U.S. Patent No. 9,895,415, U.S. Patent Application Publication No. 2019 / 024743 ...717,774, U.S. Patent No. 9,895,415, No. 0292520, US Patent Publication No. 2020 / 0085930, US Patent No. 10,336,809, US Patent No. 10,131,703, US Patent No. 10,081,664, US Patent No. 10,081,664, US Patent No. 10,093,715, US Patent No. 10,583,573, and US Patent Publication No. 2020 / 00085930 (the contents of each of these publications, sequences, and sequence listings described herein are incorporated herein by reference in their entirety). The tumor associated antigen (TAA) peptides described herein can be bound to HLA (MHC molecules).A tumor associated antigen (TAA) peptide that binds to HLA can be recognized by a TCR as described herein, optionally co-expressed with a CD8 polypeptide as described herein.

[0068] T cells can be engineered to express chimeric antigen receptors (CARs) that contain ligand binding domains derived from NKG2D, NKG2A, NKG2C, NKG2F, LLT1, AICL, CD26, NKRP1, NKp30, NKp44, NKp46, CD244 (2B4), DNAM-1, and NKp80, or anti-tumor antibodies such as anti-Her2neu or anti-EGFR, and signaling domains obtained from CD3-zeta, Dap10, CD28, 4-IBB, and CD40L. In some examples, the chimeric receptor is selected from the group consisting of MICA, MICB, Her2neu, EGFR, mesothelin, CD38, CD20, CD19, PSA, RON, CD30, CD22, CD37, CD38, CD56, CD33, CD30, CD138, CD123, CD79b, CD70, CD75, CA6, GD2, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CEACAM5, CA-125, MUC-16, 5T4, NaPi2b, ROR1, ROR2, 5T4, PLIF, Her2 / Neu, EGFRvIII, GPMNB, LIV-1, glycolipid F77, fibroblast activation protein, The T cell binds to a protein, PSMA, STEAP-1, STEAP-2, c-met, CSPG4, nectin-4, VEGFR2, PSCA, folate binding protein / receptor, SLC44A4, Cripto, CTAG1B, AXL, IL-13R, IL-3R, SLTRK6, gp100, MART1, tyrosinase, SSX2, SSX4, NYESO-1, epithelial tumor antigen (ETA), MAGEA family genes (MAGE3A, MAGE4A, etc.), KKLC1, mutant ras, βraf, p53, MHC class I chain-related molecule A (MICA), or MHC class I chain-related molecule B (MICB), HPV, or CMV. The T cell can be an αβ T cell, a γδ T cell, or a natural killer T cell.

[0069] T cell culture T cells that can be used for transgene expression, e.g., tumor infiltrating lymphocytes, CD8 + T cells, CD4 + Described herein are T cells and methods for activating, transducing, and / or expanding T cells. T cells can be activated, transduced, and expanded while depleting α- and / or β-TCR positive cells. T cells can be αβ T cells, γδ T cells, or natural killer T cells.

[0070] Methods for ex vivo expansion of populations of engineered γδ T cells for adoptive transfer therapy are described herein. The engineered γδ T cells of the present disclosure may be expanded ex vivo. The engineered T cells described herein may be expanded in vitro without activation by APC or without co-culture with APC and aminophosphate. Methods for transducing T cells are described in U.S. Patent Application Publication No. 2019 / 0175650, published June 13, 2019, the contents of which are incorporated by reference in their entirety. Other methods for transducing and culturing T cells may be used.

[0071] T cells, including γδ T cells, can be isolated from complex samples cultured in vitro. In embodiments, the total PBMC population can be activated and expanded without prior depletion of specific cell populations such as monocytes, αβ T cells, B cells, and NK cells. In embodiments, enriched T cell populations can be generated prior to their specific activation and expansion. In embodiments, activation and expansion of γδ T cells can be performed with or without the presence of natural or engineered antigen presenting cells (APCs). In embodiments, isolation and expansion of T cells from tumor specimens can be performed using T cell mitogens, including antibodies specific for γδ TCR, and other γδ TCR activators, including lectins. In embodiments, isolation and expansion of γδ T cells from tumor specimens can be performed in the absence of immobilized T cell mitogens, including antibodies specific for γδ TCR, and other γδ TCR activators, including lectins.

[0072] T cells, including γδ T cells, may be isolated from a subject, e.g., from leukapheresis of a human subject. In embodiments, γδ T cells are not isolated from peripheral blood mononuclear cells (PBMCs). T cells may be isolated using anti-CD3 and anti-CD28 antibodies, and, optionally, recombinant human interleukin-2 (rhIL-2), e.g., about 50-150 U / mL rhIL-2.

[0073] Isolated T cells can rapidly proliferate in response to contact with one or more antigens. + Some γδ T cells, such as T cells, can rapidly proliferate in vitro in response to contact with several antigens, such as prenyl-pyrophosphate, alkylamines, and metabolites or microbial extracts, during tissue culture. Stimulated T cells can express a number of antigen-presenting, costimulatory, and adhesion molecules that can facilitate isolation of T cells from a complex sample. T cells within a complex sample can be stimulated in vitro with at least one antigen for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or another suitable period of time. Stimulation of T cells with a suitable antigen can expand the T cell population in vitro.

[0074] Activation and expansion of γδ T cells can be performed using activating and costimulatory agents as described herein to induce specific γδ T cell proliferation and persistence populations. In embodiments, activation and expansion of γδ T cells from different cultures can achieve different clonal or mixed polyclonal population subsets. In embodiments, different agonist agents can be used to identify agents that provide specific γδ activating signals. In embodiments, agents that provide specific γδ activating signals can be different monoclonal antibodies (MAbs) directed against γδ TCR. In embodiments, companion costimulatory agents can be used that help induce specific γδ T cell proliferation without induction of cellular energetics and apoptosis. These costimulatory agents can include ligands that bind to receptors expressed on γδ cells, such as NKG2D, CD161, CD70, JAML, DNAX accessory molecule-1 (DNAM-1), ICOS, CD27, CD137, CD30, HVEM, SLAM, CD122, DAP, and CD28. In embodiments, the costimulatory agent can be an antibody specific for a unique epitope on the CD2 and CD3 molecules. CD2 and CD3 can have different conformations when expressed on αβ or γδ T cells. In embodiments, specific antibodies against CD3 and CD2 can result in distinct activation of γδ T cells.

[0075] Non-limiting examples of antigens that may be used to stimulate proliferation of T cells, including γδ T cells, from complex samples in vitro include prenyl pyrophosphates such as isopentenyl pyrophosphate (IPP), alkyl-amines, metabolites of human microbial pathogens, metabolites of commensal bacteria, methyl-3-butenyl-1-pyrophosphate (2M3B1PP), (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), ethyl pyrophosphate (EPP), farnesyl pyrophosphate (FPP), dimethylallyl phosphate (DMAP), dimethylallyl pyrophosphate (DMAPP), ethyl-adenosine triphosphate (EPPPA), geranyl pyrophosphate (GPP), geranylgeranyl pyrophosphate (GGPP ), isopentenyl-adenosine triphosphate (IPPPA), monoethyl phosphate (MEP), monoethyl pyrophosphate (MEPP), 3-formyl-1-butyl-pyrophosphate (TUBAg1), X-pyrophosphate (TUBAg2), 3-formyl-1-butyluridine triphosphate (TUBAg3), 3-formyl-1-butyl-deoxythymidine triphosphate (TUBAg4), monoethyl alkylamines, allyl pyrophosphate, clotoyl pyrophosphate, dimethylallyl-γ-uridine triphosphate, clotoyl-γ-uridine triphosphate, allyl-γ-uridine triphosphate, ethylamine, isobutylamine, sec-butylamine, isoamylamine, and nitrogen-containing bisphosphonates.

[0076] T cell populations, including γδ T cells, can be expanded ex vivo prior to manipulation of the T cells. Non-limiting examples of reagents that can be used to promote expansion of T cell populations in vitro can include anti-CD3 or anti-CD2 antibodies, anti-CD27 antibodies, anti-CD30 antibodies, anti-CD70 antibodies, anti-OX40 antibodies, IL-2, IL-15, IL-12, IL-9, IL-33, IL-18, or IL-21, CD70 (CD27 ligand), phytohaemagglutinin (PHA), concavalin A (ConA), pokeweed (PWM), protein peanut agglutinin (PNA), soybean agglutinin (SBA), Les Culinaris agglutinin (LCA), Pisum Sativum agglutinin (PSA), Helix pomatia agglutinin (HPA), Vicia graminea lectin (VGA), or another suitable mitogen capable of stimulating T cell proliferation. Additionally, T cells can be expanded using MCSF, IL-6, eotaxin, IFN-alpha, IL-7, gamma-inducible protein 10, IFN-gamma, IL-1RA, IL-12, MIP-1 alpha, IL-2, IL-13, MIP-1 beta, IL-2R, IL-15, and combinations thereof.

[0077] The ability of γδ T cells to recognize a broad range of antigens can be enhanced by genetic engineering of γδ T cells. γδ T cells can be engineered to provide universal allogeneic therapy that recognizes antigens of choice in vivo. Genetic engineering of γδ T cells can include stable integration of constructs expressing tumor recognition moieties such as αβ TCR, γδ TCR, chimeric antigen receptors (CARs), combining both antigen binding and T cell activation functions into a single receptor, an antigen-binding fragment thereof, or combining lymphocyte activation domains into isolated γδ T cells, cytokines (e.g., IL-15, IL-12, IL-2, IL-7, IL-21, IL-18, IL-19, IL-33, IL-4, IL-9, IL-23, or IL1β) to enhance T cell proliferation, survival, and function ex vivo and in vivo. Genetic engineering of the isolated γδ T-cell may also involve deleting or disrupting gene expression from one or more endogenous genes in the genome of the isolated γδ T-cell, such as the MHC locus.

[0078] Engineered (or transduced) T cells, including γδ T cells, can be expanded ex vivo without stimulation with antigen-presenting cells or aminobisphosphonates. Antigen-reactive engineered T cells of the present disclosure can be expanded ex vivo and in vivo. In embodiments, active populations of engineered T cells can be expanded ex vivo without antigenic stimulation with antigen-presenting cells, antigenic peptides, non-peptide molecules, or small molecule compounds such as aminobisphosphonates, but using specific antibodies, cytokines, mitogens, or fusion proteins such as IL-17 Fc fusion, MICA Fc fusion, and CD70 Fc fusion. Examples of antibodies that can be used to expand γδ T cell populations include anti-CD3 antibodies, anti-CD27 antibodies, anti-CD30 antibodies, anti-CD70 antibodies, anti-OX40 antibodies, anti-NKG2D antibodies, or anti-CD2 antibodies; examples of cytokines include IL-2, IL-15, IL-12, IL-21, IL-18, IL-9, IL-7, and / or IL-33; examples of mitogens include CD70, which is a ligand for human CD27, phytohemagglutinin (PHA), concavalin A (ConA), pokeweed mitogen (PWM), protein peanut agglutinin (PNA), soybean agglutinin (SBA), les culinaris agglutinin (LCA), pisum sativum agglutinin (PSA), Helix pomatia agglutinin (HPA), Vicia graminea lectin (VGA), or another suitable mitogen capable of stimulating T cell proliferation.

[0079] The population of engineered T cells, including γδ T cells, can be expanded for less than 60 days, less than 48 days, less than 36 days, less than 24 days, less than 12 days, or less than 6 days. In embodiments, the population of engineered T cells can be expanded for about 7 days to about 49 days, about 7 days to about 42 days, about 7 days to about 35 days, about 7 days to about 28 days, about 7 days to about 21 days, or about 7 days to about 14 days. The T cells can be expanded for about 1 to 21 days. For example, the T cells can be expanded for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.

[0080] In an embodiment, the same methodology may be used to isolate, activate, and expand αβ T cells.

[0081] In an embodiment, the same methodology may be used to isolate, activate and expand γδ T cells.

[0082] vector Engineered T cells can be generated using a variety of methods, including those recognized in the literature. For example, a polynucleotide encoding an expression cassette containing a tumor recognition, or another type of recognition moiety, can be introduced into the T cells using a transposon / transposase system, or a viral-based gene transfer system, such as a lentivirus or retrovirus system, or by transfection, electroporation, transduction, lipofection, calcium phosphate (CaPO 4 ), another suitable method such as nanoengineered materials such as ormosil, adenovirus, retrovirus, lentivirus, adeno-associated virus, or another suitable method for stable introduction into T cells. Many viral methods have been used in human gene therapy, such as those described in WO1993 / 020221, the contents of which are incorporated herein in their entirety. Non-limiting examples of viral methods that can be used to engineer T cells can include gamma-retrovirus, adenovirus, lentivirus, herpes simplex virus, vaccinia virus, poxvirus, or adenovirus-associated viral methods. The T cells can be alpha beta T cells or gamma delta T cells.

[0083] Viruses used for transfection of T cells include naturally occurring viruses as well as artificial viruses. Viruses can be either enveloped or non-enveloped viruses. Parvoviruses (such as AAV) are examples of non-enveloped viruses. Viruses can be enveloped viruses. Viruses used for transfection of T cells can be retroviruses, particularly lentiviruses. Viral envelope proteins that can facilitate viral infection of eukaryotic cells can include vesicular stomatitis virus (VSV-G), modified feline endogenous retrovirus (RD114TR) (SEQ ID NO: 97), and HIV-1 derived lentiviral vectors (LV) pseudotyped with envelope glycoproteins (GP) from modified gibbon ape leukemia virus (GALVTR). These envelope proteins can efficiently facilitate the entry of other viruses such as parvoviruses, including adeno-associated viruses (AAV), thereby demonstrating their broad efficiency. For example, other viral envelope proteins that may be used include Moloney murine leukemia virus (MLV) 4070 env (such as described in Merten et al., J. Virol. 79:834-840, 2005, the contents of which are incorporated herein by reference), RD114 env, the chimeric envelope proteins RD114pro or RDpro (which are RD114-HIV chimeras constructed by replacing the R peptide cleavage sequence of RD114 with HIV-1 matrix / capsid (MA / CA), such as described in Bell et al. Experimental Biology and Medicine 2010;235:1269-1276, the contents of which are incorporated herein by reference), baculovirus GP64 env (such as described in Wang et al. J. Virol. 81:10869-10878, 2007, the contents of which are incorporated herein by reference), or GALV env (such as described in Merten et al., J. Virol. 79:834-840, 2005, the contents of which are incorporated herein by reference), or derivatives thereof.

[0084] A single lentiviral cassette can be used to create a single lentiviral vector that expresses at least four individual monomeric proteins of two different dimers from a single multicistronic mRNA such that the dimers are co-expressed on the cell surface. For example, integration of a single copy of a lentiviral vector was sufficient to transduce T cells to co-express TCRαβ and CD8αβ, optionally αβ T cells or γδ T cells.

[0085] The vectors may contain multicistronic cassettes within a single vector capable of expressing more than one, more than two, more than three, more than four, more than five, or more than six genes, and the polypeptides encoded by these genes may interact with each other or form dimers. The dimers may be homodimers, e.g., two identical proteins that form a dimer, or heterodimers, e.g., two structurally different proteins that form a dimer.

[0086] In addition, multiple vectors can be used to transfect cells with the constructs and sequences described herein. For example, a TCR transgene can be on one vector and a CD8 transgene encoding a polypeptide described herein can be on a second vector that is transfected either simultaneously or sequentially using recognized methods. T cell lines can be stably transfected with a CD8 transgene encoding a CD8 polypeptide described herein and then sequentially transfected with a TCR transgene, or vice versa.

[0087] In some embodiments, the transgene may further comprise one or more multicistronic elements, which may be located, for example, between a nucleic acid sequence encoding a TCRα or a portion thereof and a nucleic acid sequence encoding a TCRβ or a portion thereof, between a nucleic acid sequence encoding a CD8α or a portion thereof and a nucleic acid sequence encoding a CD8β or a portion thereof, or between any two nucleic acid sequences encoding TCRα, TCRβ, CD8α, and CD8β. In some embodiments, the multicistronic element may comprise a sequence encoding a ribosome skip element selected from among T2A, P2A, E2A, or F2A, or an internal ribosome entry site (IRES).

[0088] As used herein, the term "self-cleaving 2A peptide" refers to a relatively short peptide (on the order of 20 amino acids in length, depending on the virus of origin) that acts co-translationally by preventing the formation of a normal peptide bond between glycine and the last proline, causing the ribosome to skip to the next codon, and cleaving the nascent peptide between Gly and Pro. After cleavage, the short 2A peptide remains fused to the C-terminus of the "upstream" protein, while a proline is added to the N-terminus of the "downstream" protein. The self-cleaving 2A peptide may be selected from porcine teschovirus-1 (P2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), foot and mouth disease virus (F2A), or any combination thereof (see, e.g., Kim et al., PLOS One 6:e18556, 2011, the contents of which, including 2A nucleic acid and amino acid sequences, are incorporated herein by reference in their entirety). By adding a linker sequence (GSG or SGSG (SEQ ID NO: 266)) before the self-cleaving 2A sequence, this may allow for efficient synthesis of biologically active proteins, such as TCR.

[0089] As used herein, the term "internal ribosome entry site (IRES)" refers to a nucleotide sequence located in a messenger RNA (mRNA) sequence that can initiate translation independent of a 5' cap structure. IRESs are usually located in the 5' untranslated region (5'UTR), but can also be located in other locations in the mRNA. In one embodiment, the IRES may be selected from IRESs from viruses, IRESs from cellular mRNAs, in particular IRESs from picornaviruses such as polio, EMCV, and FMDV, flaviviruses such as hepatitis C virus (HCV), plague viruses such as classical swine fever virus (CSFV), retroviruses such as murine leukemia virus (MLV), lentiviruses such as simian immunodeficiency virus (SIV), and insect RNA viruses such as cricket paralysis virus (CRPV), as well as IRESs from cellular mRNAs, e.g., transcription initiation factors such as eIF4G and DAP5, transcription factors such as c-Myc and NF-κB repressor (NRF), growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), platelet-derived growth factor B (PDGF-B), homeotic genes such as antennapedia, survival proteins such as X-linked inhibitor of apoptosis (XIAP) and Apaf-1, and other cellular mRNAs such as BiP.

[0090] The constructs and vectors described herein are used in conjunction with the methodology described in U.S. Patent Application Publication No. 2019 / 0175650, published June 13, 2019, the contents of which are incorporated by reference in their entirety.

[0091] Non-viral vectors can also be used in conjunction with the sequences, constructs, and cells described herein.

[0092] Cells may be transfected by other means known in the art, including lipofection (liposome-based transfection), electroporation, calcium phosphate transfection, microparticle delivery (e.g., gene gun), microinjection, or a combination thereof. Various methods of transfecting cells are known in the art. See, for example, Sambrook & Russell (Eds.) Molecular Cloning: A Laboratory Manual (3rd Ed.) Volumes 1-3 (2001) Cold Spring Harbor Laboratory Press; Ramamoorth & Narvekar "Non Viral Vectors in Gene Therapy-An Overview." J Clin Diagn Res. (2015) 9(1): GE01-GE06.

[0093] composition The compositions may comprise modified CD8 polypeptides as described herein. Additionally, the compositions described herein may comprise T cells expressing a CD8 polypeptide as described herein. The compositions described herein may comprise T cells expressing a CD8 polypeptide as described herein, and a T cell receptor (TCR), optionally a TCR that specifically binds to one of the TAAs described herein complexed with an antigen-presenting protein, termed HLA in humans, for human leukocyte antigen, e.g., MHC.

[0094] To facilitate administration, the T cells described herein can be made into pharmaceutical compositions or implants suitable for administration in vivo, together with a pharma- ceutically acceptable carrier or diluent. Means for making such compositions or implants are described in the art. See, e.g., Remington's Pharmaceutical Sciences, 16th Ed., Mack, ed. (1980).

[0095] The T cells described herein can be formulated into preparations in semi-solid or liquid form, such as capsules, solutions, infusions, or injections. Means known in the art can be utilized to prevent or minimize release and absorption of the composition or ensure timed release of the composition until it reaches the target tissue or organ. However, desirably, a pharma- ceutical acceptable form is employed that does not prevent the cells from expressing the CAR or TCR. Thus, desirably, the T cells described herein can be made into a pharmaceutical composition including a carrier. The T cells described herein can be formulated with a physiologically acceptable carrier or excipient to prepare a pharmaceutical composition. The carrier and composition can be sterile. Preferred carriers include, for example, balanced salt solutions, preferably Hank's balanced salt solution, or saline. The formulation should be compatible with the mode of administration. Suitable pharma- ceutical acceptable carriers include, but are not limited to, water, salt solutions (e.g., NaCl), saline, buffered saline, and combinations thereof. Pharmaceutical preparations can, if desired, be mixed with auxiliary substances that do not deleteriously react with the T cells, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers. The T cells can be αβ T cells or γδ T cells expressing a CD8 polypeptide as described herein, and optionally a TCR as described herein.

[0096] Compositions of the present invention can be provided in unit dosage form, with each dosage unit, e.g., injection, containing a predetermined amount of the composition alone, or in appropriate combination with other active agents.

[0097] The compositions described herein may be pharmaceutical compositions.The pharmaceutical compositions described herein may further comprise an adjuvant selected from the group consisting of colony stimulating factors, including but not limited to granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, resiquimod, interferon-alpha, or combinations thereof.

[0098] The pharmaceutical compositions described herein may include an adjuvant selected from the group consisting of colony stimulating factors, such as granulocyte-macrophage colony stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, and resiquimod.

[0099] Preferred adjuvants include, but are not limited to, cyclophosphamide, imiquimod, or resiquimod. Even more preferred adjuvants are Montanide IMS1312, Montanide ISA206, Montanide ISA50V, Montanide ISA-51, poly-ICLC (Hiltonol®), and anti-CD40 mAB, or combinations thereof.

[0100] Other examples of useful adjuvants include chemically modified CpG (e.g., CpR, Idera), poly(I:C) and their derivatives (e.g., AmpliGen®, Hiltonol®, dsRNA analogs such as poly-(ICLC), poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA, as well as cyclophosphamide, sunitinib, immune checkpoint inhibitors ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and simiprimab, Bevacizumab®, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF These include immunoreactive small molecules and antibodies such as Trap, ZD2171, AZD2171, anti-CTLA4, other antibodies that target key structures of the immune system (e.g., anti-CD40, anti-TGF beta, anti-TNF alpha receptor), and SC58175, which may act therapeutically and / or as adjuvants. The amounts and concentrations of adjuvants and additives useful in the context of the present invention can be readily determined by one of skill in the art without undue experimentation.

[0101] Other adjuvants include, but are not limited to, anti-CD40, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, atezolizumab, interferon-alpha, interferon-beta, CpG oligonucleotides and derivatives, poly-(I:C) and derivatives, RNA, sildenafil, and particle formulations comprising poly(lactide-co-glycolide) (PLG), polyinosinic acid-polycytidylic acid-poly-l-lysine carboxymethylcellulose (poly-ICLC), virosomes, and / or interleukin-1 (IL-1), IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-18, IL-21, and IL-23. See, e.g., Narayanan et al. J. Med. Chem. (2003) 46(23):5031-5044; Pohar et al. Scientific Reports 7 14598 (2017); Grajkowski et al. Nucleic Acids Research (2005) 33(11):3550-3560; Martins et al. Expert Rev Vaccines (2015) 14(3):447-59).

[0102] The compositions described herein may also include one or more adjuvants. Adjuvants are substances that non-specifically enhance or potentiate immune responses (e.g., immune responses mediated by CD8 positive T cells and helper T (TH) cells to antigens) and are therefore considered useful in the medicaments of the invention. Suitable adjuvants include 1018 ISS, aluminum salts, AMPLIVAX®, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or flagellin-derived TLR5 ligands, FLT3 ligand, GM-CSF, IC30, IC31, imiquimod (ALDARA®), resiquimod, ImuFact Interleukins as IMP321, IL-2, IL-13, IL-21, interferon alpha or beta or pegylated derivatives thereof, IS patch, ISS, ISCOMATRIX, ISCOM, JuvImmune®, LipoVac, MALP2, MF59, monophosphoryl lipid A, Montanide IMS1312, Montanide ISA206, Montanide ISA50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions, OK-432, OM Adjuvants that can be used include, but are not limited to, -174, OM-197-MP-EC, ONTAK, OspA, PepTel® Vector System, poly(lactide-co-glycolide) [PLG]-based dextran microparticles, talactoferrin SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, saponin, mycobacterial extracts, and Aquila's QS21 stimulon derived from synthetic bacterial cell wall mimics, as well as other proprietary adjuvants such as Ribi's Detox, Quil, and Superfos. Adjuvants such as Freund's or GM-CSF are preferred. Several immunological adjuvants specific for dendritic cells (e.g., MF59) and their preparations have already been reported. Cytokines can also be used.Several cytokines have been directly implicated in influencing dendritic cell migration into lymphoid tissues (e.g., TNF-), accelerating the maturation of dendritic cells into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Pat. No. 5,849,589, which is incorporated by reference herein in its entirety), and acting as immune adjuvants (e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha, IFN-beta).

[0103] CpG immunostimulatory oligonucleotides have also been reported to enhance the effect of adjuvants in vaccine settings. Without being bound by theory, CpG oligonucleotides act by activating the innate (non-adaptive) immune system through Toll-like receptors (TLRs), primarily TLR9. CpG-induced TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates, in both prophylactic and therapeutic vaccines. More importantly, it enhances dendritic cell maturation and differentiation, leading to enhanced activation of TH1 cells and potent cytotoxic T lymphocyte (CTL) generation, even in the absence of CD4 T cell help. The TH1 bias induced by TLR9 stimulation is maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA), which normally promote a TH2 bias. CpG oligonucleotides, when formulated or co-administered with other adjuvants or in formulations such as microparticles, nanoparticles, lipid emulsions, or similar formulations, show even greater adjuvant activity, which is especially necessary for inducing strong responses when the antigen is relatively weak. They also accelerate immune responses, allowing antigen doses to be reduced by about two orders of magnitude, with some experiments having antibody responses equivalent to full-dose vaccines without CpG (Krieg, 2006). US Patent No. 6,406,705 (B1) describes the combined use of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens to induce antigen-specific immune responses. The CpG TLR9 antagonist is dSLIM (double stem-loop immunomodulator) by Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical composition of the present invention. Other TLR binding molecules, such as RNA-binding TLR7, TLR8, and / or TLR9, can also be used.

[0104] Treatment and preparation methods The engineered T cells may express a modified CD8 polypeptide as described herein. Additionally, the engineered T cells may express a TCR as described herein. The TCR expressed by the engineered T cells may recognize a TAA bound to an HLA as described herein. The engineered T cells of the present disclosure may be used to treat a subject in need of treatment for a condition, e.g., cancer, as described herein. The T cells may be αβ T cells or γδ T cells expressing a modified CD8 polypeptide, and optionally a TCR as described herein.

[0105] A method of treating a condition (e.g., disease) in a subject with the T cells described herein may include administering to the subject a therapeutically effective amount of the engineered T cells described herein, optionally γδ T cells. The T cells described herein may be administered in a variety of regimens (e.g., timing, concentration, dosage, interval between treatments, and / or formulations). The subject may also be pretreated, for example, with chemotherapy, radiation, or a combination of both, prior to receiving the engineered T cells of the present disclosure. The population of engineered T cells may also be frozen or cryopreserved prior to administration to the subject. A population of engineered T cells may include two or more cells expressing the same, different, or a combination of the same and different tumor recognition moieties. For example, an engineered T cell population may include several different engineered T cells designed to recognize different antigens, or different epitopes of the same antigen. The T cells may be αβ T cells or γδ T cells expressing a CD8 polypeptide described herein, and optionally a TCR described herein.

[0106] The T cells described herein, including αβ T cells and γδ T cells, can be used to treat a variety of conditions. The T cells can be αβ T cells or γδ T cells expressing a CD8 polypeptide, optionally a TCR as described herein. The T cells described herein can be used to treat cancer, including solid tumors and hematological malignancies.Non-limiting examples of cancer include acute lymphoblastic leukemia, acute myeloid leukemia, adrenal cortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, neuroblastoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors, e.g., cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma, breast cancer, bronchial adenoma, Burkitt's lymphoma, cancer of unknown primary, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, disease, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cavity cancer, liposarcoma, liver cancer, lung cancer including non-small cell lung cancer and small cell lung cancer, lymphoma, Leukemia, macroglobulinemia, bone malignant fibrous histiocytoma / osteosarcoma, medulloblastoma, melanoma, mesothelioma, primary occult metastatic squamous cell carcinoma, oral cavity cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndrome, myeloid leukemia, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma / bone malignant fibrous histiocytoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic islet cell cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, These include pineal germinoma, pituitary adenoma, pleuropulmonary blastoma, plasma cell neoplasms, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, skin cancer Merkel cell, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, T-cell lymphoma, throat cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic neoplasm (gestational), carcinoma of unknown primary site, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0107] The T cells described herein may be used to treat infectious diseases. The T cells described herein may be used to treat infectious diseases, which may be caused by a virus. The T cells described herein may be used to treat immune diseases, such as autoimmune diseases. The T cells may be αβ T cells or γδ T cells expressing a CD8 polypeptide, optionally a TCR as described herein.

[0108] Treatment with T cells, optionally γδ T cells, as described herein may be provided to a subject before, during, and after clinical onset of a condition. Treatment may be provided to a subject 1 day, 1 week, 6 months, 12 months, or 2 years after clinical onset of a disease. Treatment may be provided to a subject 1 day, 1 week, 1 month, 6 months, 12 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more after clinical onset of a disease. Treatment may be provided to a subject less than 1 day, 1 week, 1 month, 6 months, 12 months, or 2 years after clinical onset of a disease. Treatment may also include treating humans in clinical trials. Treatment may include administering to a subject a pharmaceutical composition comprising an engineered T cell as described herein. The T cell may be an αβ T cell or a γδ T cell expressing a CD8 polypeptide, optionally a TCR as described herein.

[0109] In embodiments, administration of the engineered T cells of the present disclosure to a subject can modulate the activity of endogenous lymphocytes in the subject's body. In embodiments, administration of the engineered T cells to a subject can provide antigen to endogenous T cells and enhance the immune response. In embodiments, memory T cells are CD4 + In an embodiment, the memory T cells are CD8 + In embodiments, administration of the engineered T cells of the present disclosure to a subject may activate the cytotoxicity of another immune cell. In embodiments, the other immune cell may be a CD8 +In an embodiment, the other immune cells may be natural killer T cells. In an embodiment, administration of the engineered γδ T cells of the present disclosure to a subject may suppress regulatory T cells. In an embodiment, the regulatory T cells may be FOX3 + The regulatory T cells may be Treg cells. In embodiments, the regulatory T cells may be FOX3-Treg cells. Non-limiting examples of cells whose activity can be regulated by the engineered T cells of the present disclosure include hematopoietic stem cells, B cells, CD4, CD8, red blood cells, white blood cells, dendritic cells including dendritic antigen presenting cells, white blood cells, macrophages, memory B cells, memory T cells, monocytes, natural killer cells, neutrophil granulocytes, T helper cells, and T killer cells. The T cells may be αβ T cells or γδ T cells expressing a CD8 polypeptide, optionally a TCR as described herein.

[0110] During most bone marrow transplants, a combination of cyclophosphamide and total body irradiation may be conventionally used to prevent rejection of the transplanted hematopoietic stem cells (HSCs) by the subject's immune system. In an embodiment, ex vivo incubation of donor bone marrow with interleukin-2 (IL-2) may be performed to enhance the generation of killer lymphocytes in the donor bone marrow. Interleukin-2 (IL-2) is a cytokine that may be necessary for the growth, proliferation, and differentiation of wild-type lymphocytes. Current trials of adoptive transfer of γδ T cells into humans may require the co-administration of γδ T cells and interleukin-2. However, both low and high doses of IL-2 may have highly toxic side effects. IL-2 toxicity may manifest in multiple organs / systems, most notably the heart, lungs, kidneys, and central nervous system. In embodiments, the present disclosure provides methods for administering engineered T cells to a subject without co-administration of natural cytokines or modified forms thereof, such as IL-2, IL-15, IL-12, IL-21, etc. In embodiments, the engineered T cells can be administered to a subject without co-administration of IL-2. In embodiments, the engineered T cells can be administered to a subject during a treatment, such as a bone marrow transplant, without co-administration of IL-2.

[0111] In embodiments, the method may further include administering a chemotherapeutic agent. The dose of the chemotherapeutic agent may be sufficient to deplete the patient's T cell population. The chemotherapy may be administered about 5-7 days prior to administration of the T cells. The chemotherapeutic agent may be cyclophosphamide, fludarabine, or a combination thereof. The chemotherapeutic agent may be about 400-600 mg / m of cyclophosphamide. 2 The chemotherapy agent may include administration of about 10-30 mg / m 2 This may include administration on a daily basis.

[0112] In embodiments, the method may further comprise pre-treating the patient with low dose radiation prior to administration of the composition comprising the T cells. The low dose radiation may comprise about 1.4 Gy for 1-6 days, preferably about 5 days, prior to administration of the composition comprising the T cells.

[0113] In an embodiment, the patient may be HLA-A*02.

[0114] In an embodiment, the patient may be HLA-A*06.

[0115] In an embodiment, the method may further include administering an anti-PD1 antibody. The anti-PD1 antibody may be a humanized antibody. The anti-PD1 antibody may be pembrolizumab. The dosage of the anti-PD1 antibody may be about 200 mg. The anti-PD1 antibody may be administered every 3 weeks after administration of the T cells.

[0116] In one embodiment, the dose of T cells is about 0.8 to 1.2 × 10 9 The dose of T cells can be about 0.5 × 10 to about 10 × 10 9 The dose of T cells can be about 1.2-3×10 9 T cells, approximately 3-6 x 10 9 T cells, approximately 10 x 10 9 T cells, approximately 5 x 10 9 T cells, approximately 0.1 x 10 9 T cells, approximately 1 x 10 8 T cells, approximately 5 x 108 T cells, approximately 1.2-6 x 10 9 T cells, approximately 1–6 × 10 9 T cells, or approximately 1–8 × 10 9 The T cells may be

[0117] In some embodiments, the T cells may be administered in three doses. The T cell dose may be escalated from dose to dose. The T cells may be administered by intravenous infusion.

[0118] In embodiments, the CD8 sequences and related products and compositions described herein may be used in autologous or allogeneic methods of adoptive cell therapy. In another embodiment, the CD8 sequences, T cells thereof, and compositions may be used in the methods described, for example, in U.S. Patent Application Publication No. 2019 / 0175650, U.S. Patent Application Publication No. 2019 / 0216852, U.S. Patent Application Publication No. 2019 / 024743, and U.S. Provisional Patent Application No. 62 / 980,844, each of which is incorporated by reference in its entirety.

[0119] The present disclosure also provides modified T cell populations that present an exogenous CD8 polypeptide and a T cell receptor as described herein, where the modified T cell population is activated and expanded by a combination of IL-2 and IL-15. In another embodiment, the modified T cell population is expanded and / or activated by a combination of IL-2, IL-15, and zoledronate. In yet another embodiment, the modified T cell population is activated by a combination of IL-2, IL-15, and zoledronate, while being expanded by a combination of IL-2, IL-15 without zoledronate. The present disclosure further provides for the use of other interleukins during activation and / or expansion, such as IL-12, IL-18, IL-21, and combinations thereof.

[0120] In an aspect, IL-21, histone deacetylase inhibitors (HDACi), or a combination thereof may be utilized in the field of cancer treatment using the methods described herein and / or the ACT process described herein. In an embodiment, the present disclosure provides a method for reprogramming effector T cells to a central memory phenotype, comprising culturing effector T cells with at least one HDACi together with IL-21. Representative HDACi include, for example, trichostatin A, trapoxin B, phenylbutyrate, valproic acid, vorinostat (suberanilohydroxamic acid), belinostat, panobinostat, dacinostat, entinostat, tacedinaline, and mocetinostat.

[0121] Compositions comprising engineered T cells as described herein may be administered for prophylactic and / or therapeutic treatments. In therapeutic applications, the compositions may be administered to a patient already suffering from a disease or condition in an amount sufficient to cure or at least partially halt the symptoms of the disease or condition. Engineered T cells may also be administered to reduce the likelihood of developing, contracting, or worsening the condition. The effective amount of engineered T cell populations for therapeutic use may vary based on the severity and course of the disease or condition, previous therapy, the subject's health, weight, and / or response to drugs, and / or the judgment of the attending physician. The T cells may be αβ T cells or γδ T cells engineered to express a modified CD8 polypeptide as described herein, and optionally a TCR as described herein. T cell therapy has been successful in treating various cancers. Li et al. Signal Transduction and Targeted Therapy 4(35):(2019), incorporated by reference in its entirety.

[0122] Method of administration One or more engineered T cell populations described herein may be administered to a subject in any order or simultaneously. If simultaneous, the multiple engineered T cells may be provided in a single, unified form, such as, for example, an intravenous infusion, or in multiple forms, such as, for example, multiple intravenous infusions, subcutaneous injections, or pills. The engineered T cells may be packaged together or separately in a single package or multiple packages. One or all of the engineered T cells may be administered in multiple doses. If not simultaneous, the timing between multiple administrations may vary from about one week, one month, two months, three months, four months, five months, six months, or up to about one year. In embodiments, the engineered T cells may be expanded in vivo within the subject's body after administration to the subject. The engineered T cells may be frozen to provide cells for multiple treatments with the same cell preparation. The engineered T cells of the present disclosure, and pharmaceutical compositions comprising same, may be packaged as a kit. The kit may include instructions (e.g., written instructions) for the use of the engineered T cells and compositions comprising same.

[0123] A method of treating cancer may include administering to a subject a therapeutically effective amount of engineered T cells, where the administration treats the cancer. In embodiments, the therapeutically effective amount of engineered γδ T cells may be administered for at least about 10 seconds, 30 seconds, 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. In embodiments, the therapeutically effective amount of engineered T cells may be administered for at least 1 week. In embodiments, the therapeutically effective amount of engineered T cells may be administered for at least 2 weeks.

[0124] The engineered T cells, optionally γδ T cells, described herein can be administered before, during, or after the onset of a disease or condition, and the timing of administration of the pharmaceutical composition comprising the engineered T cells can vary. For example, the engineered T cells can be used as a prophylactic and can be administered continuously to a subject with a predisposition to a condition or disease to reduce the likelihood of the onset of the disease or condition. The engineered T cells can be administered to a subject during or as soon as possible after the onset of symptoms. Administration of the engineered T cells can begin immediately, within the onset of symptoms, within the first 3 hours of the onset of symptoms, within the first 6 hours of the onset of symptoms, within the first 24 hours of the onset of symptoms, within 48 hours of the onset of symptoms, or within any period of time from the onset of symptoms. Initial administration can be via any practical route, such as any route described herein, using any formulation described herein. In an embodiment, administration of the engineered T cells of the present disclosure can be intravenous. One or more doses of engineered T cells can be administered as soon as possible after the onset of cancer, infectious disease, immune disease, sepsis, or onset associated with bone marrow transplantation, for the period of time required to treat the immune disease, e.g., from about 24 hours to about 48 hours, from about 48 hours to about 1 week, from about 1 week to about 2 weeks, from about 2 weeks to about 1 month, from about 1 month to about 3 months, etc. For the treatment of cancer, one or more doses of engineered T cells can be administered several years after the onset of cancer, and before or after other treatments. In embodiments, engineered γδ T-cells can be administered for at least about 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years. The duration of treatment may vary from subject to subject.The T cell may be an αβ T cell or a γδ T cell expressing a CD8 polypeptide as described herein, and optionally a TCR as described herein.

[0125] The engineered T cells expressing a CD8 polypeptide as described herein, optionally an αβ T cell or a γδ T cell, are present in a composition at least 1×10 3 At least 2 x 10 cells / ml 3 At least 3 x 10 cells / ml 3 At least 4 x 10 cells / ml 3 At least 5 x 10 cells / ml 3 At least 6 x 10 cells / ml 3 At least 7 x 10 cells / ml 3 At least 8 x 10 cells / ml 3 At least 9 x 10 cells / ml 3 At least 1 x 10 cells / ml 4 At least 2 x 10 cells / ml 4 At least 3 x 10 cells / ml 4 At least 4 x 10 cells / ml 4 At least 5 x 10 cells / ml 4 At least 6 x 10 cells / ml 4 At least 7 x 10 cells / ml 4 At least 8 x 10 cells / ml 4 At least 9 x 10 cells / ml 4 At least 1 x 10 cells / ml 5 At least 2 x 10 cells / ml 5 At least 3 x 10 cells / ml 5 At least 4 x 10 cells / ml 5 At least 5 x 10 cells / ml 5 At least 6 x 10 cells / ml 5 At least 7 x 10 cells / ml 5 At least 8 x 10 cells / ml 5 At least 9 x 10 cells / ml 5 At least 1 x 10 cells / ml 6At least 2 x 10 cells / ml 6 At least 3 x 10 cells / ml 6 At least 4 x 10 cells / ml 6 At least 5 x 10 cells / ml 6 At least 6 x 10 cells / ml 6 At least 7 x 10 cells / ml 6 At least 8 x 10 cells / ml 6 At least 9 x 10 cells / ml 6 At least 1 x 10 cells / ml 7 At least 2 x 10 cells / ml 7 At least 3 x 10 cells / ml 7 At least 4 x 10 cells / ml 7 At least 5 x 10 cells / ml 7 At least 6 x 10 cells / ml 7 At least 7 x 10 cells / ml 7 At least 8 x 10 cells / ml 7 At least 9 x 10 cells / ml 7 At least 1 x 10 cells / ml 8 At least 2 x 10 cells / ml 8 At least 3 x 10 cells / ml 8 At least 4 x 10 cells / ml 8 At least 5 x 10 cells / ml 8 At least 6 x 10 cells / ml 8 At least 7 x 10 cells / ml 8 At least 8 x 10 cells / ml 8 At least 9 x 10 cells / ml 8 cells / ml, at least 1×10 cells / ml, approximately 1×10 3 / ml ~ at least 1 x 10 8 Cells / ml, approximately 1 x 10 5 / ml ~ at least 1 x 10 8 cells / ml, or approximately 1 x 10 6 / ml ~ at least 1 x 10 8 It may be present at 100 cells / ml.

[0126] array The sequences described herein may have about 80%, about 85%, about 90%, about 85%, about 96%, about 97%, about 98%, or about 99%, or 100% identity to any of the sequences of SEQ ID NOs: 1-97, 256-266, 293, and 294. The sequences described herein may have at least 80%, at least 85%, at least 90%, at least 85%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the sequences of SEQ ID NOs: 1-97 and 256-266. A sequence that is "at least 85% identical to a reference sequence" is a sequence that has 85% or more, particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity over its entire length to the entire length of the reference sequence.

[0127] In another embodiment, the disclosure provides a sequence that is at least 80%, at least 85%, at least 90%, at least 85%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to WPREmut1 (SEQ ID NO: 256), or WPRE type 2, such as WPREmut2 (SEQ ID NO: 257). In another aspect, the disclosure provides at least 1, 2, 3, 4, 5, 10, 15, or 20 amino acid substitutions in WPREmut1 (SEQ ID NO: 256), or WPRE type 2, such as WPREmut2 (SEQ ID NO: 257). In another aspect, the disclosure provides up to 1, 2, 3, 4, 5, 10, 15, or 20 amino acid substitutions in WPREmut1 (SEQ ID NO: 256), or WPRE type 2, such as WPREmut2 (SEQ ID NO: 257). In another aspect, the sequence substitutions are conservative substitutions.

[0128] The percentage of identity can be calculated using a global pairwise alignment (e.g., two sequences are compared over their entire length). Methods for comparing the identity of two or more sequences are well known in the art. To find the optimal alignment (including gaps) of two sequences taking into account their entire length, the «needle» program can be used, for example, which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48: 443-453). The needle program is available, for example, at the ebi.ac.uk World Wide Web site and is further described in the following publications (EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp. 276-277). The percentage of identity between two polypeptides according to the invention is calculated using the EMBOSS:needle(global) program with "Gap Open" parameters equal to 10.0, "Gap Extend" parameters equal to 0.5, and the Blosum62 matrix.

[0129] A protein consisting of an amino acid sequence that is "at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical" to a reference sequence may contain mutations, such as deletions, insertions, and / or substitutions, compared to the reference sequence. In the case of substitutions, a protein consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence may correspond to a homologous sequence derived from another species than the reference sequence.

[0130] Amino acid substitutions can be conservative or non-conservative. Preferably, the substitutions are conservative, in which one amino acid is replaced with another amino acid having similar structural and / or chemical properties.

[0131] Conservative substitutions may include those described by Dayhoff in "The Atlas of Protein Sequence and Structure. Vol. 5," Natl. Biomedical Research, the contents of which are incorporated by reference in their entirety. For example, in embodiments, amino acids belonging to one of the following groups may be exchanged for one another and therefore constitute conservative exchanges: Group 1: alanine (A), proline (P), glycine (G), asparagine (N), serine (S), threonine (T); Group 2: cysteine ​​(C), serine (S), tyrosine (Y), threonine (T); Group 3: valine (V), isoleucine (I), leucine (L), methionine (M), alanine (A), phenylalanine (F); Group 4: lysine (K), arginine (R), histidine (H); Group 5: phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H); and Group 6: aspartic acid (D), glutamic acid (E). In embodiments, conservative amino acid substitutions may be selected from T→A, G→A, A→I, T→V, A→M, T→I, A→V, T→G, and / or T→S.

[0132] Conservative amino acid substitutions can include replacing an amino acid with another amino acid of the same class, for example, (1) non-polar: Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, (2) uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln, (3) acidic: Asp, Glu, and (4) basic: Lys, Arg, His. Other conservative amino acid substitutions can also be made, such as: (1) aromatic: Phe, Tyr, His, (2) proton donor: Asn, Gln, Lys, Arg, His, Trp, and (3) proton acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gln (see, e.g., U.S. Pat. No. 10,106,805, which is incorporated by reference in its entirety).

[0133] Conservative substitutions can be made according to Table A. Methods for predicting tolerance to protein modifications can be found, for example, in Guo et al., Proc. Natl. Acad. Sci., USA, 101(25):9205-9210 (2004), the contents of which are incorporated by reference in their entirety. [Table A]

[0134] The sequences described herein may include 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 amino acid or nucleotide mutations, substitutions, or deletions. Any one of SEQ ID NOs: 1-97, 256-266, 293, and 294 may include 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 mutations, substitutions, or deletions. In another embodiment, any one of SEQ ID NOs: 1-97, 256-266, 293, and 294 may include up to 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 mutations, substitutions, or deletions. In an embodiment, the mutations or substitutions may be conservative amino acid substitutions.

[0135] Conservative substitutions in the polypeptides described herein can be those shown in Table B under the heading of "conservative substitutions." If such substitutions result in alterations in biological activity, more substantial changes designated as "exemplary substitutions" in Table B can be introduced and the products screened, if desired. [Table B]

[0136] Unless otherwise specified, all terms used herein have the same meaning as they would to one of ordinary skill in the art.

[0137] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0138] "Activated" as used herein broadly refers to the state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. The term "activated T cells" refers, inter alia, to T cells that are proliferating.

[0139] "Antibody" as used herein refers broadly to antibodies or immunoglobulins of any isotype, fragments of antibodies, including but not limited to Fab, Fab', Fab'-SH, (Fab')2Fv, scFv, bivalent scFv, and Fd fragments that retain specific binding to an antigen, chimeric antibodies, humanized antibodies, single chain antibodies, and fusion proteins containing antigen-specific targeting regions of antibodies and non-antibody proteins. Antibodies are organized into five classes--IgG, IgE, IgA, IgD, and IgM.

[0140] "Antigen" or "antigenic" as used herein refers broadly to a peptide or a portion of a peptide that can be bound by an antibody and can induce an animal to produce an antibody that can bind to an epitope of that antigen. An antigen can have one epitope or more than one epitope. A specific reaction as referred to herein refers to an antigen reacting in a highly selective manner with its corresponding antibody and not with the multitude of other antibodies that may be elicited by other antigens.

[0141] As used herein, "chimeric antigen receptor" or "CAR" or "CARs" broadly refers to a genetically engineered receptor that transfers antigen specificity to cells, such as T cells, NK cells, macrophages, and stem cells. A CAR can include at least one antigen-specific targeting region (ASTR), a hinge or stalk domain, a transmembrane domain (TM), one or more costimulatory domains (CSDs), and an intracellular activation domain (IAD). In certain embodiments, the CSD is optional. In another embodiment, the CAR is a bispecific CAR, specific for two different antigens or epitopes. After the ASTR specifically binds to the target antigen, the IAD activates intracellular signaling. For example, the IAD can redirect T cell specificity and reactivity to a selected target in a non-MHC-restricted manner, taking advantage of the antigen-binding properties of antibodies. Non-MHC-restricted antigen recognition gives CAR-expressing T cells the ability to recognize antigens independent of antigen processing, thus bypassing a major mechanism of tumor escape. Furthermore, when expressed in a T cell, the CAR advantageously does not dimerize with the endogenous T cell receptor (TCR) alpha and beta chains.

[0142] As used herein, "cytotoxic T lymphocytes" (CTLs) refer broadly to T lymphocytes that express CD8 on their surface (e.g., CD8+ T cells). Such cells may preferably be antigen-experienced "memory" T cells (TM cells).

[0143] As used herein, "effective amount", "therapeutically effective amount", or "effective amount" refers broadly to the amount of a drug, or the combined amount of two drugs, that when administered to a mammal or other subject to treat a disease is sufficient to affect such treatment for the disease. A "therapeutically effective amount" will vary depending on the drug, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0144] As used herein, "genetically modified" refers broadly to methods of introducing exogenous nucleic acid into a cell, whether or not the exogenous nucleic acid is integrated into the genome of the cell. As used herein, "genetically modified cells" refers broadly to cells containing exogenous nucleic acid, whether or not the exogenous nucleic acid is integrated into the genome of the cell.

[0145] "Immune cells" as used herein broadly refer to white blood cells (leukocytes) derived from hematopoietic stem cells (HSCs) produced in the bone marrow "Immune cells" include, but are not limited to, lymphocytes (T cells, B cells, natural killer (NK) (CD3-CD56+) cells) and bone marrow derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). "T cells" include all types of immune cells that express CD3, including T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), T regulatory cells (Tregs) and gamma-delta T cells, as well as NK T cells (CD3+ and CD56+). One of skill in the art will understand that as used throughout this disclosure, T cells and / or NK cells can include only T cells, only NK cells, or both T cells and NK cells. In certain exemplary embodiments and aspects provided herein, the T cells are activated and transduced. Additionally, T cells are provided in certain exemplary composition embodiments and aspects provided herein. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells, NK-T cells, γδ T cells, and neutrophils, which are cells capable of mediating a cytotoxic response.

[0146] "Individual," "subject," "host," and "patient," as used interchangeably herein, refer broadly to mammals, including, but not limited to, humans, murines (e.g., rats, mice), lagomorphs (e.g., rabbits), non-human primates, dogs, cats, and ungulates (e.g., horses, cows, sheep, pigs, goats).

[0147] As used herein, "peripheral blood mononuclear cells" or "PBMCs" refer broadly to any peripheral blood cell that has a round nucleus. PBMCs include lymphocytes, such as T cells, B cells, and NK cells, as well as monocytes.

[0148] "Polynucleotide" and "nucleic acid," as used interchangeably herein, refer broadly to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the terms include, but are not limited to, single-, double-, or multistranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified non-natural, or derivatized nucleotide bases.

[0149] "T cells" or "T lymphocytes," as used herein, broadly refer to thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. Exemplary populations of T cells suitable for use in certain embodiments include, but are not limited to, helper T cells (HTL, CD4+ T cells), cytotoxic T cells (CTL, CD8+ T cells), CD4+CD8+ T cells, CD4-CD8- T cells, natural killer T cells, T cells expressing αβTCR (αβ T cells), T cells expressing γδTCR (γδ T cells), or any other subset of T cells. Other exemplary T cell populations suitable for use in certain embodiments include, but are not limited to, T cells expressing one or more of the following markers: CD3, CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L, CD127, CD197, and HLA-DR, and can be further isolated by positive or negative selection techniques, if desired.

[0150] In the present invention, the term "homology" refers to the degree of identity (see percentage identity above) between two amino acid sequences, e.g., peptide or polypeptide sequences. Said "homology" is determined by comparing two sequences aligned under optimal conditions for the sequences to be compared. Such sequence homology can be calculated, for example, by creating an alignment using the ClustalW algorithm. Publicly available sequence analysis software, more specifically Vector NTI, GENETYX, or other tools, are provided by public databases.

[0151] The terms "sequence homology" or "sequence identity" are used interchangeably herein. For the purposes of the present invention, it is defined here that in order to determine the percentage of sequence homology or sequence identity of two amino acid sequences or two nucleotide sequences, the sequences are aligned for optimal comparison purposes. To optimize the alignment between the two sequences, gaps may be introduced into either of the two sequences being compared. Such alignment can be performed over the entire length of the sequences being compared. Alternatively, alignment can be performed over a shorter length, for example, over about 5, about 10, about 20, about 50, about 100 or more nucleotides or amino acids. Sequence identity is the percentage of identical matches between the two sequences over the reported aligned region.

[0152] The comparison of sequences and the determination of the percent sequence identity between two sequences can be accomplished using a mathematical algorithm. Those skilled in the art will recognize the fact that several different computer programs are available for aligning two sequences and determining the identity between two sequences (Kruskal, JB (1983) An overview of sequence comparison. In D. Sankoff and JB Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, Addison Wesley). The percent sequence identity between two amino acid sequences or between two nucleotide sequences can be determined using the Needleman and Wunsch algorithm for alignment of two sequences (Needleman, SB and Wunsch, CD (1970) J. Mal. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by algorithms. The Needleman-Wunsch algorithm is implemented in the computer program NEEDLE. For the purposes of the present invention, the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, Longden, and Bleasby, Trends in Genetics 16, (6) 276-277, emboss.bioinformatics.nl / ). For amino acid sequences, EBLOSUM62 is used for the substitution matrix. For nucleotide sequences, EDNAFULL is used. The arbitrary parameters used are a gap open penalty of 10 and a gap extension penalty of 0.5. The skilled person will understand that all of these different parameters will give slightly different results, but the overall percentage identity of two sequences will not change significantly when using different algorithms.

[0153] After alignment by the program NEEDLE described above, the percentage of sequence identity between the query sequence and the sequence of the present invention is calculated as follows: The number of corresponding positions in the alignment that show identical amino acids or identical nucleotides in both sequences is divided by the total length of the alignment after subtracting the total number of gaps in the alignment. Identity as defined herein can be obtained from NEEDLE by using the NOBRIEF option and is displayed as "longest-identity" in the program output. The nucleotide and amino acid sequences of the present invention can further be used as "query sequences" to perform searches against sequence databases, for example to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J.Mal.Biol.215:403-10. To obtain nucleotide sequences homologous to the polynucleotides of the present invention, BLAST nucleotide searches can be performed with the NBLAST program, score=100, word length=12. To obtain amino acid sequences homologous to the polypeptides of the invention, BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST, Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0154] As used herein, "T cell receptor (TCR)" refers broadly to a protein receptor on T cells consisting of a heterodimer of alpha (α) and beta (β) chains, although in some cells the TCR consists of gamma and delta (γ / δ) chains. TCRs can be engineered on any cell that contains a TCR, including helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, or gamma delta T cells.

[0155] The TCR is generally found on the surface of T lymphocytes (or T cells) where it is generally responsible for the recognition of antigens bound to major histocompatibility complex (MHC) molecules. It is a heterodimer consisting of alpha and beta chains in 95% of T cells, while 5% of T cells have a TCR consisting of gamma and delta chains. The association of the TCR with antigen and MHC leads to the activation of that T lymphocyte through a series of biochemical events mediated by associated enzymes, co-receptors, and specialized accessory molecules. In immunology, the CD3 antigen (CD stands for cluster of differentiation) is a protein complex consisting of four different chains (CD3-γ, CD3δ, and twice CD3ε) in mammals that associate with the T cell receptor (TCR) and a molecule known as the ζ chain to generate an activation signal within the T lymphocyte. The TCR, ζ chain, and CD3 molecule together constitute the TCR complex. The CD3-γ, CD3δ, and CD3ε chains are cell surface proteins closely related to the immunoglobulin superfamily that contain a single extracellular immunoglobulin domain. The transmembrane regions of the CD3 chains are negatively charged, a property that allows these chains to associate with the positively charged TCR chains (TCRα and TCRβ). The intracellular tail of the CD3 molecule contains a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM for short, which is essential for the signaling capacity of the TCR.

[0156] "Treatment," "treating," and the like, as used herein, refer broadly to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that a disease or its symptoms are completely or partially prevented, and / or may be therapeutic, in that a disease and / or side effects resulting from the disease are partially or completely cured. "Treatment," as used herein, encompasses any treatment of a disease in a mammal, e.g., a human, and includes (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it, (b) inhibiting the disease, e.g., arresting its onset, and (c) relieving the disease, e.g., causing regression of the disease.

[0157] The ability of dendritic cells (DCs) to activate and proliferate antigen-specific CD8+ T cells may depend on the DC maturation stage, and DCs may need to receive a "licensing" signal related to IL-12 production to induce cytolytic immune responses. In particular, the provision of signals via CD40 ligand (CD40L)-CD40 interactions on CD4+ T cells and DCs may be considered important for DC licensing and induction of cytotoxic CD8+ T cells, respectively. DC licensing may result in upregulation of costimulatory molecules, increased survival rate of DCs, and better cross-presentation ability. This process can be mediated through CD40 / CD40L interactions [SR Bennet et al., "Help for cytotoxic T-cell responses is mediated by CD40 signalling," Nature 393(6684):478-480(1998), SP Schoenberger et al., "T-cell help for cytotoxic T-cell help is mediated by CD40-CD40L interactions," Nature 393(6684):480-483(1998)], but CD40 / CD40L-independent mechanisms also exist (CD70, LTβR). In addition, a direct interaction between CD40L expressed on DCs and CD40 expressed on CD8+ T cells has also been suggested, providing a possible explanation for the generation of helper-independent CTL responses [S.Johnson et al., "Selected Toll-like receptor ligands and viruses promote helper-independent cytotoxic T-cell priming by upregulating CD40L on dendritic cells," Immunity 30(2):218-227(2009)].

[0158] Example 1 Exemplary Nucleic Acid and Amino Acid Sequences [Table 2] We found that various CD8 elements in the vectors resulted in surprising increases in expression and activity. For example, construct #10 was observed to have a lower viral titer than constructs #9b, #11, and #12 (Figure 5A), yet at the lowest viral volume concentration, e.g., 1.25 μl / cell 10 6 T cells transduced with construct no. 10 expressing the CD8αβ heterodimer and TCR generated higher CD8+CD4+TCR+ cells (56.7%, FIG. 9B) than those transduced with construct no. 9b expressing CD8α and TCR (42.3%, FIG. 9A), construct no. 11 expressing the CD8αCD8β stalk with CD8α transmembrane and intracellular domains and TCR (51.6%, FIG. 9C), and construct no. 12 expressing the CD8αCD8β stalk with neural adhesion molecule 1 (NCAM1) transmembrane and intracellular domains and TCR (14.9%, FIG. 9D).

[0159] The vector can include any one of the nucleic acid sequences of SEQ ID NOs: 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 295, 297, 299, or 301.

[0160] T cells can be transduced to express the nucleic acid of SEQ ID NO: 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 295, 297, 299, or 301.

[0161] Some elements of the constructs in Table 2 are described in Table 3. [Table 3-1] [Table 3-2] [Table 3-3]

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

Table 3-13

Table 3-14

Table 3-15

Table 3-16

Table 3-17

Table 3-18

Table 3-19

Table 3-20

Table 3-21

Table 3-22

Table 3-23

Table 3-24

Table 3-25

Table 3-26

Table 3-27

Table 3-28

Table 3-29

Table 3-30

Table 3-31

Table 3-32

Table 3-33

Table 3-34

Table 3-35

Table 3-36

Table 3-37

Table 3-38

Table 3-39

Table 3-40

Table 3-41

Table 3-42

[0162] The constructs of Table 2 can be an assembly of individual components described in Table 3. The inventors have found that the combination, order, and inclusion of transcription enhancers from Table 3 as described in Table 2 results in unexpected improvements in transfection efficiency, expression levels, and induction of cytotoxic T cell activity, such as IL-12 secretion, IFN-γ secretion, TNF-α secretion, Granzyme A secretion, MIP-1a secretion, IP-10 secretion, Granzyme B secretion, and combinations thereof.

[0163] Tumor-associated antigens (TAA) In an MHC class I-dependent immune response, not only must peptides be able to bind to specific MHC class I molecules expressed by tumor cells, but they must also subsequently be recognized by T cells bearing specific T cell receptors (TCRs).

[0164] In order for a protein to be recognized by T lymphocytes as a tumor-specific or associated antigen and used in therapy, certain prerequisites must be met. The antigen should be expressed mainly by tumor cells and not expressed or expressed in relatively low amounts by normal healthy tissues. In a preferred embodiment, the peptide should be over-presented by tumor cells compared to normal healthy tissues. Furthermore, it is desirable that the respective antigen is not only present in the tumor type, but also in high concentration (e.g., copy number of the respective peptide per cell). Tumor-specific and tumor-associated antigens are often derived from proteins that are directly involved in the transformation of normal cells into tumor cells due to their function, for example in cell cycle control or suppression of apoptosis. In addition, downstream targets of proteins that directly cause transformation may be upregulated and therefore indirectly tumor-associated. Such indirect tumor-associated antigens may also be targets for vaccination approaches. Singh-Jasuja et al.Cancer Immunol.Immunother.53(2004):187-195. Epitopes are present in the amino acid sequence of an antigen, making the peptide an "immunogenic peptide", and are derived from tumor-associated antigens, which elicit T cell responses both in vitro and in vivo.

[0165] Any peptide that can bind to MHC molecule can function as T cell epitope. For induction of T cell response, TAA must be presented to T cell with corresponding TCR, and host must not have immunological tolerance to this particular epitope. Exemplary tumor-associated antigens (TAA) that can be used with the CD8 polypeptide described herein are disclosed herein. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0166] Example 2 CD8α molecule A CD8α homodimer (CD8αα) can be composed of two α subunits held together by two disulfide bonds at the stalk region. Figure 1 shows a CD8α polypeptide, e.g., SEQ ID NO: 258 (CD8α1), which contains five domains: (1) one signal peptide (-21 to -1), e.g., SEQ ID NO: 6, (2) one Ig-like domain-1 (1 to 115), e.g., SEQ ID NO: 1, (3) one stalk region (116 to 160), e.g., SEQ ID NO: 260, (4) one transmembrane (TM) domain (161 to 188), e.g., SEQ ID NO: 3, and (5) one cytoplasmic tail (Cyto) (189 to 214) containing an lck-binding motif, e.g., SEQ ID NO: 4. Another example of a CD8α subunit, for example, CD8α2 (SEQ ID NO: 259), differs from CD8α1 at position 112, where CD8α2 contains a cysteine ​​(C) and CD8α1 contains a tyrosine (Y).

[0167] Modified CD8 Polypeptides Unlike CD8α polypeptides, e.g., CD8α1 (SEQ ID NO:258) and CD8α2 (SEQ ID NO:259), modified CD8α polypeptides, e.g., m1CD8α (SEQ ID NO:7) and m2CD8α (SEQ ID NO:262), may contain additional regions, such as sequence extensions from a CD8β polypeptide. In an embodiment, SEQ ID NO:2 or a variant thereof is used with a CD8α polypeptide. In other embodiments, a portion of a CD8α polypeptide, e.g., SEQ ID NO:260, is removed or not included in the modified CD8 polypeptides described herein. Figure 2 shows a sequence alignment between CD8α1 (SEQ ID NO:258) and m1CD8α (SEQ ID NO:7). Figure 3 shows a sequence alignment between CD8α2 (SEQ ID NO:259) and m2CD8α (SEQ ID NO:262), with cysteine ​​substitutions indicated by arrows. The stalk region is boxed.

[0168] The modified CD8-expressing cells showed improved functionality in terms of cytotoxicity and cytokine responses compared to the original CD8-expressing T cells transduced with the TCR.

[0169] Example 3 Lentiviral Vectors The lentiviral vector used herein contains several elements that enhance vector function, including a central polypurine tract (cPPT) for improved replication and nuclear import, a promoter from murine stem cell virus (MSCV) (SEQ ID NO: 263) that reduces vector silencing in some cell types, a woodchuck hepatitis virus post-transcriptional response element (WPRE) (SEQ ID NO: 264) for improved transcription termination, and the backbone was a deleted 3'-LTR self-inactivating (SIN) vector design that improves safety, sustained gene expression, and anti-silencing properties. Yang et al. Gene Therapy (2008) 15, 1411-1423.

[0170] In an embodiment, the vector, construct, or sequence described herein comprises a mutant form of WPRE. In an embodiment, the sequence or vector described herein comprises a mutation in WPRE type 1, e.g., WPREmut1 (SEQ ID NO: 256), or WPRE type 2, e.g., WPREmut2 (SEQ ID NO: 257). Construct No. 9 and Construct No. 9b represent two LV production batches with a construct containing the same SEQ ID NO: 257 as WPREmut2, with the difference between Construct No. 9 and Construct No. 9b being titers consistent with Table 4 (Table 5). In an embodiment, the WPRE mutant comprises at most 1 mutation, at most 2 mutations, at most 3 mutations, at least 4 mutations, or at most 5 mutations. In an embodiment, the vector, construct, or sequence described herein does not comprise a WPRE. In an aspect, the WPRE sequence described in U.S. Patent Application Publication No. 2021 / 0285011 (the contents of which are incorporated by reference in their entirety) may be used with the vector, sequence, or construct described herein.

[0171] In embodiments, the vectors, constructs, or sequences described herein do not include an X protein promoter. The WPRE variants described herein do not express the X protein. The WPRE promotes accumulation of mRNA, theorized to promote export of mRNA from the nucleosome to the cytoplasm to promote translation of the transgene mRNA.

[0172] Lentiviral vectors with different designs were generated to obtain optimal co-expression levels of TCRαβ, mCD8α (e.g., m1CD8α (SEQ ID NO: 7) and m2CD8α (SEQ ID NO: 262)), and CD8β (e.g., any one of CD8β1-7 (SEQ ID NO: 8-14)) in transduced CD4+ T cells, CD8+ T cells, and / or γδ T cells. T cells can be transduced with two separate lentiviral vectors (2-in-1), e.g., one expressing TCRα and TCRβ and the other expressing mCD8α and CD8β for co-expression of TCRαβ and CD8αβ heterodimers, or one expressing TCRα and TCRβ and the other expressing mCD8α for co-expression of TCRαβ and mCD8α homodimers. Alternatively, T cells can be transduced with a single lentiviral vector (4-in-1) co-expressing TCRα, TCRβ, mCD8α, and CD8β for co-expression of the TCRαβ and CD8αβ heterodimer. In the 4-in-1 vector, the nucleotides encoding the TCR α chain, TCR β chain, mCD8 α chain, and CD8 β chain can be shuffled in various orders, for example, from 5' to 3' direction: TCR α-TCR β-mCD8 α-CD8 β, TCR α-TCR β-CD8 β-mCD8 α, TCR β-TCR α-mCD8 α-CD8 β, TCR β-TCR α-CD8 β-mCD8 α, mCD8 α-CD8 β-TCR α-TCR β, mCD8 α-CD8 β-TCR β-TCR α, CD8 β-mCD8 α-TCR α-TCR β, and CD8 β-mCD8 α-TCR β-TCR α. The various 4-in-1 vectors thus generated may be used to transduce CD4+ T cells, CD8+ T cells, and / or γδ T cells, followed by measuring TCRαβ / mCD8α / CD8β co-expression levels in the transduced cells using techniques known in the art, e.g., flow cytometry. Similarly, T cells may be transduced with a single lentiviral vector (3-in-1) co-expressing TCRα, TCRβ, and mCD8α (e.g., m1CD8α and m2CD8α) for co-expression of TCRαβ and mCD8α homodimers.In the 3-in-1 vector, the nucleotides encoding the TCR α, TCR β and mCD8α chains can be shuffled in various orders, for example TCR α-TCR β-mCD8α, TCR β-TCR α-mCD8α, mCD8α-TCR α-TCR β and mCD8α-TCR β-TCR α. The various 3-in-1 vectors thus generated can be used to transduce CD4+ T cells, CD8+ T cells and / or γδ T cells, followed by measuring the TCR αβ / mCD8α co-expression levels of the transduced cells using techniques known in the art.

[0173] To generate lentiviral vectors co-expressing TCRαβ and mCD8α and / or CD8β, nucleotides encoding a furin linker (GSG or SGSG (SEQ ID NO: 266))-2A peptide may be placed between the TCRα and TCRβ chains, between the mCD8α and CD8β chains, and between the TCR and CD8 chains to allow highly efficient gene expression. The 2A peptide may be selected from P2A (SEQ ID NO: 93), T2A (SEQ ID NO: 94), E2A (SEQ ID NO: 95), or F2A (SEQ ID NO: 96).

[0174] Lentiviral vectors may also contain a post-transcriptional regulatory element (PRE) such as WPRE (SEQ ID NO: 264), WPREmut1 (SEQ ID NO: 256), or WPREmut2 (SEQ ID NO: 257) to enhance transgene expression by increasing both nuclear and cytoplasmic mRNA levels. One or more regulatory elements including mouse RNA transport element (RTE), simian retrovirus type 1 (SRV-1) constitutive transport element (CTE), and human heat shock protein 70 5' untranslated region (Hsp70 5'UTR) may also be used and / or in combination with WPRE to increase transgene expression. WPREmut1 and WPREmut2 do not express the X protein but still act to enhance translation of the transgene mRNA.

[0175] Lentiviral vectors can be pseudotyped with RD114TR (e.g., SEQ ID NO: 97), a chimeric glycoprotein that includes the extracellular and transmembrane domains of feline endogenous virus (RD114) fused to the cytoplasmic tail (TR) of murine leukemia virus. Other viral envelope proteins can also be used, such as VSV-G env, MLV4070A env, RD114 env, chimeric envelope protein RD114pro, baculovirus GP64 env, or GALV env, or derivatives thereof. RD114TR variants that include at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% relative to SEQ ID NO: 97 are also provided.

[0176] For example, Figure 4 shows exemplary vectors including two 4-in-1 vectors, e.g., constructs no. 10 and no. 2, co-expressing TCR (TCR alpha and TCR beta chains), CD8 alpha, and CD8 beta, three 3-in-1 vectors expressing TCR and CD8 alpha, e.g., constructs no. 1 and no. 9, two 3-in-1 vectors expressing TCR and m1CD8 alpha (SEQ ID NO: 7), e.g., constructs no. 11 and no. 12, and construct no. 8, expressing only TCR. To improve transcription termination, wild-type WPRE (WPRE) (SEQ ID NO: 264) is included in constructs no. 1, no. 2, and no. 8, and WPREmut (SEQ ID NO: 257) is included in constructs no. 9, no. 10, no. 11, and no. 12.

[0177] Further exemplary constructs (constructs no. 13-19 and no. 21-26) are described in Table 2 above. In particular, constructs no. 13, no. 14, and no. 16 are 4-in-1 constructs co-expressing TCR, CD8α, and CD8β3 with various signal peptide combinations (SEQ ID NO: 6 [WT CD8α signal peptide], SEQ ID NO: 293 [WT CD8β signal peptide], and SEQ ID NO: 294 [S19 signal peptide]) with different element orders. Constructs no. 15 and no. 17 are 4-in-1 constructs co-expressing TCR, CD8α, and CD8β5. Construct no. 15 contains WT CD8α signal peptide (SEQ ID NO: 6) and WT CD8β signal peptide (SEQ ID NO: 293), while construct no. 17 contains S19 signal peptide (SEQ ID NO: 294) at the N-terminus of both CD8α and CD8β5. Construct number 21 is a 4-in-1 construct co-expressing TCR, CD8α, and CD8β2, including the WT CD8α signal peptide (SEQ ID NO: 6) and the WT CD8β signal peptide (SEQ ID NO: 293). Construct number 18 is a variant of construct number 10, in which the WT signal peptides of CD8α and CD8β1 (SEQ ID NO: 6 and 293, respectively) are replaced with the S19 signal peptide (SEQ ID NO: 294). Construct number 19 is a variant of construct number 11, in which the WT CD8α signal peptide (SEQ ID NO: 6) is replaced with the S19 signal peptide (SEQ ID NO: 294). Construct number 22 is a variant of construct number 11, in which the CD4 transmembrane and intracellular domains are fused to the C-terminus of the CD8β stalk sequence in place of the CD8α transmembrane and intracellular domains. Construct number 25 is a variant of construct number 22 in which the CD8β stalk sequence (SEQ ID NO:2) is replaced with the CD8α stalk sequence (SEQ ID NO:260).

[0178] Example 4 Vector Screening (Constructs #1, #2, #8, #9, #10, #11, and #12) Viral titer Figure 5A shows the viral titers of constructs #1, #2, #8, #9, #10, #11, and #12. Table 5 shows the viral titers and lentiviral P24 ELISA data for constructs #9, #10, #11, and #12. [Table 5]

[0179] For construct 12, NCAMfu refers to the NCAMFusion protein expressing a modified CD8a extracellular domain and neural cell adhesion molecule 1 (CD56) intracellular domain.

[0180] In Table 5, the WPREmut2 portion refers to SEQ ID NO:257.

[0181] T cell manufacturing activation FIG. 6 shows PBMCs (approximately 9×10 cells) obtained from two donors (donor no. 1 and donor no. 2) on day +0. 8 Figure 7 shows that 100 pieces of PBMCs (100 pieces) were thawed and rested. Cells were activated in anti-CD3 and anti-CD28 antibody coated bags (AC290) in the presence of serum. Activation markers, such as CD25, CD69, and human low density lipoprotein receptor (H-LDL-R), were present on CD8+ and CD4+ cells and subsequently measured. Figure 7A shows that CD3+CD8+CD25+ cell %, CD3+CD8+CD69+ cell %, and CD3+CD8+H-LDL-R+ cell % are increased after activation (Post-A) compared to before activation (Pre-A). Similarly, Figure 7B shows that CD3+CD4+CD25+ cell %, CD3+CD4+CD69+ cell %, and CD3+CD4+H-LDL-R+ cell % are increased after activation (Post-A) compared to before activation (Pre-A). These results confirm the activation of PBMCs.

[0182] Transduction FIG. 6 shows that on day +1, activated PBMCs were cultured with viral vectors, e.g., constructs #1, #2, #8, #9, #10, #11, and #12, in G-Rex® 6-well plates in the absence of serum at approximately 5×10 cells. 6 The amount of virus used for transduction is shown in Table 6. [Table 6]

[0183] Proliferation Figure 6 shows that on day +2, transduced PBMCs expanded in the presence of serum. On day +6, cells were harvested and cryopreserved for subsequent analysis, e.g., FACS-Dextramer and vector copy number (VCN). Figures 8A and 8B show the fold expansion on day +6 of the transduced T cell products obtained from donor #1 and donor #2, respectively. Cell viability is greater than 90% on day +6.

[0184] Characterization of T cell products Cell counts, FACS-dextramers, and vector copy numbers (VCN) were determined. Tetramer panels may include live / dead cells, CD3, CD8α, CD8β, CD4, and peptide / MHC tetramers, such as PRAME-004 (SLLQHLIGL) (SEQ ID NO: 147) / MHC tetramers. FACS analysis was gated on live singlets, followed by CD3+, followed by CD4+CD8+, followed by CD4+CD8+tetramer (Tet)+ and CD8+Tet+.

[0185] Figures 9A, 9B, 9C, and 9D show representative flow plots of cells obtained from donor #1, showing CD8, CD4, and PRAME-004 / MHC tetramer (Tet)% of cells transduced with constructs #9b, #10, #11, or #12, respectively.

[0186] FIG. 10 shows the results of in vitro cell culture of 1×10 cells using constructs no. 1, no. 2, no. 8 (TCR), no. 9, no. 10, no. 11, or no. 12. 6 The % CD8+CD4+ cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with 1.25 μl, 2.5 μl, or 5 μl per donor are shown. The results show that transduction with vectors expressing CD8α and TCR with wild type WPRE (construct #1) and WPREmut2 (construct #9) results in a higher % CD8+CD4+ than transduced with cell constructs #10, #11, or #12. Construct #8 (TCR only) serves as a negative control. Figure 11 shows the % CD8+CD4+ cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with 1.25 μl, 2.5 μl, or 5 μl per donor. The results show that transduction with vectors expressing CD8α and TCR with wild type WPRE (construct #1) and WPREmut2 (construct #9) results in a higher % CD8+CD4+ than transduced with cell constructs #10, #11, or #12. Construct #8 (TCR only) serves as a negative control. Figure 11 shows the % CD8+CD4+ cells from 1×10 cells transduced with constructs #1, #2, #8 (TCR), #9, #10, #11, and #12. 6 Shown are the Tet% of CD8+CD4+ cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with 1.25 μl, 2.5 μl, or 5 μl per donor. These results show that the Tet% of CD8+CD4+ cells appears comparable between cells transduced with constructs #9, #10, and #11, and higher than cells transduced with construct #12. FACS analysis was gated on live singlets, followed by CD3+, followed by CD4+CD8+, followed by CD4+CD8+Tet+.

[0187] FIG. 12 shows the results of in vitro cell culture of 1×10 cells using constructs no. 1, no. 2, no. 8 (TCR), no. 9, no. 10, no. 11, and no. 12. 6 Shown are the Tet MFIs of CD8+CD4+Tet+ cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with 1.25 μl, 2.5 μl, or 5 μl per donor. These results show that the tetramer MFI in CD4+CD8+Tet+ differs between donors. Figure 13 shows the Tet MFIs of CD8+CD4+Tet+ cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with 1.25 μl, 2.5 μl, or 5 μl per donor. 6Shown are CD8α MFI of CD8+CD4+Tet+ cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with 1.25 μl, 2.5 μl, or 5 μl per donor. These results show that the CD8α MFI in cells transduced with vectors expressing CD8α and TCR with wild-type WPRE (construct #1) and WPREmut2 (construct #9) is higher than cells transduced with other constructs. 5 μl / 10 6 The transduction volume is 1.25 μl / 10 6 and 2.5 μl / 106 appear to produce better results. FACS analysis was gated on live singlets, followed by CD3+, followed by CD4+CD8+, followed by CD4+CD8+Tet+, followed by Tet MFI / CD8α MFI.

[0188] FIG. 14 shows the results of in vitro cell culture of 1×10 cells using constructs no. 1, no. 2, no. 8 (TCR), no. 9, no. 10, no. 11, or no. 12. 6 Shown are CD8 frequencies (% of CD8+CD4− cells among CD3+) in cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with 1.25 μl, 2.5 μl, or 5 μl per cell. These results show no difference in CD8 frequency between constructs. Non-transduced (NT) serves as a negative control. FIG. 15 shows the CD8 frequencies of 1×10 cells transduced with constructs #1, #2, #8 (TCR), #9, #10, #11, or #12. 6 Shown are the % CD8+Tet+ cells (of CD3+) from donor #1 (upper panel) and donor #2 (lower panel) transduced with 1.25 μl, 2.5 μl, or 5 μl per donor. These results show that the frequency of CD8+Tet+ (of CD3+) in cells transduced with constructs #9, #11, and #12 is higher than those transduced with construct #10. FACS analysis was gated on live singlets, followed by CD3+, followed by CD8+CD4−, followed by CD8+Tet+.

[0189] FIG. 16 shows the results of in vitro cell culture of 1×10 cells using constructs no. 1, no. 2, no. 8 (TCR), no. 9, no. 10, no. 11, and no. 12. 6 Shown are the Tet MFIs of CD8+Tet+ cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with 1.25 μl, 2.5 μl, or 5 μl per donor. These results show that the tetramer MFI of CD8+tet+ cells differs between donors. Figure 17 shows the Tet MFIs of CD8+tet+ cells transduced with constructs #1, #2, #8 (TCR), #9, #10, #11, and #12 from 1×10 cells. 6 CD8α MFI of CD8+Tet+ cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with 1.25 μl, 2.5 μl, or 5 μl per donor are shown. These results show that the CD8α MFI of CD8+Tet+ is comparable between cells transduced with different constructs. FACS analysis was gated on live singlets, followed by CD3+, followed by CD4+CD8+, followed by CD4+CD8+Tet+, followed by Tet MFI / CD8α MFI.

[0190] FIG. 18 shows the results of in vitro cell culture of 1×10 cells using constructs #1, #2, #8 (TCR), #9, #10, #11, or #12. 6 Shown are the Tet+% of CD3+ cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with 1.25 μl, 2.5 μl, or 5 μl per donor. These results show that the frequency of CD3+Tet+ in cells transduced with construct #9 or #11 is higher than those transduced with construct #10 or #12. The % Tet+CD3+ cells in cells transduced with construct #10 (WPREmut2) was 1.0% higher than that transduced with construct #2 (wild type WPRE) at 1×10 cells. 6 This appears to be higher than cells transduced at 5 μl per cell. FACS analysis was gated on live singlets, followed by CD3+, followed by CD3+, followed by Tet+.

[0191] FIG. 19 (upper panel) shows the results of in vitro cell culture of 1×10 cells using constructs #1, #2, #8 (TCR), #9, #10, #11, or #12. 6 Figure 19 (lower panel) shows vector copy numbers (VCN) from donor #1 transduced with 1.25 μl, 2.5 μl, or 5 μl per cell. These results show a higher VCN in cells transduced with constructs #11 or #12 (potentially due to higher titers) than cells transduced with constructs #9 or #10. Figure 19 (lower panel) shows the VCN of 1×10 cells transduced with constructs #1, #2, #8 (TCR), #9, #10, #11, or #12. 6 Shown are CD3+Tet+ / VCN from donor #1 transduced with 1.25 μl, 2.5 μl, or 5 μl per donor. These results show that CD3+Tet+ / VCN in cells transduced with construct #9 is higher than in cells transduced with construct #10, #11, or #12.

[0192] In summary, these results show: (1) a higher % CD8+CD4+ cells obtained by cells transduced with vectors expressing CD8α and TCR with wild-type WPRE (construct no. 1) and WPREmut2 (construct no. 9) than cells transduced with construct no. 10, no. 11, or no. 12; (2) the % CD8+CD4+Tet+ cells was comparable between cells transduced with the different constructs; and (3) a dose-dependent increase in % tetramer, e.g., at 1×10 cells. 6 5 μl per piece, 1.25 μl per piece and 1 x 10 cells 6(4) comparable % CD8+ cells between cells transduced with different constructs; (5) higher CD8+Tet+ frequency in cells transduced with constructs no. 9, no. 11, or no. 12 than in cells transduced with construct no. 10; (6) higher CD3+Tet+ frequency in cells transduced with constructs no. 9 or no. 11 than in cells transduced with construct no. 10 or no. 12; (7) higher VCN in cells transduced with constructs no. 11 or no. 12 than in cells transduced with constructs no. 9 or no. 10; and (8) higher CD3+tet+ / VCN in cells transduced with construct no. 9 than in cells transduced with constructs no. 10, no. 11, or no. 12.

[0193] T cell products transduced with viral vectors expressing a transgenic TCR and a modified CD8 co-receptor demonstrated superior cytotoxicity and increased cytokine production against target-positive cell lines.

[0194] Example 5 Tumor death assay Figures 20A-C show data demonstrating that constructs (numbers 10, 11, and 12) are comparable to TCR alone in mediating cytotoxicity against target-positive cell lines expressing antigen at different levels (UACC257 at 1081 copies per cell, A375 at 50 copies per cell). [Table 7]

[0195] Construct #9 loses tumor control over time against the low target antigen expressing A375 cell line.

[0196] Example 6 IFNγ secretion assay IFNγ secretion was measured in UACC257 and A375 cell lines. IFNγ secretion in response to UACC257 cell line was comparable between constructs. However, in A375 cell line, construct no. 10 showed higher IFNγ secretion than other constructs. IFNγ was quantified in the supernatant from Incucyte plates. Figure 21A-B.

[0197] FIG. 22 shows an exemplary experimental design to assess dendritic cell (DC) maturation and cytokine secretion by PBMC-derived T cell products in response to exposure to the target-positive tumor cell lines UACC257 and A375.

[0198] IFNγ secretion in response to A375 is increased in the presence of immature DCs (iDCs). In triple cocultures with iDCs, IFNγ secretion is higher with construct no. 10 compared to the other constructs. However, when comparing no. 9 with no. 11 expressing wild-type and modified CD8 co-receptors, respectively, T cells transduced with no. 11 induced a stronger cytokine response, measured as IFNγ quantified in the culture supernatant of the triple coculture, E:T:iDC::1:1 / 10:1 / 4, using donor D600115. Figures 23A-B

[0199] IFNγ secretion in response to A375 is increased in the presence of iDC. In triple cocultures with added iDC, IFNγ secretion was higher with construct no. 10 compared to the other constructs. IFNγ quantified in supernatants from DC cocultures D150081, E:T:iDC::1:1 / 10:1 / 4. Figures 24A-B

[0200] IFNγ secretion in response to UACC257 is increased in the presence of iDCs. In triple cocultures supplemented with iDCs, IFNγ secretion is higher with construct no. 10 compared to the other constructs. However, when comparing no. 9 with no. 11 expressing wild type and modified CD8 co-receptors, respectively, T cells transduced with construct no. 11 induced a stronger cytokine response, measured as IFNγ quantified in the culture supernatant of triple cocultures, E:T:iDC::1:1 / 10:1 / 4, using donor D600115 (Figure 25A-B). These results demonstrate that T cell products co-expressing transgenic TCR and CD8 co-receptors (αβ heterodimer or modified CD8α homodimer) are able to license DCs in the microenvironment through antigen cross-presentation, thus holding the potential to mount stronger anti-tumor responses and modulate the tumor microenvironment.

[0201] Example 7 Vector screening (constructs no. 13 to 21) Viral titer Figure 5B shows the viral titers of constructs no. 10, no. 10n (new batch), no. 11, no. 11n (new batch), no. 13 to no. 21, and TCR alone as a control.

[0202] T cell manufacturing activation Figure 26 shows that on day +0, PBMCs obtained from two HLA-A02+ donors (donor number 1 and donor number 2) were thawed and rested. Cells were activated in bags (AC290) coated with anti-CD3 and anti-CD28 antibodies in the absence of serum. Activation markers, such as CD25, CD69, and human low-density lipoprotein receptor (H-LDL-R), were subsequently measured in CD8+ and CD4+ cells. Figure 27A shows that CD3+CD8+CD25+ cell %, CD3+CD8+CD69+ cell %, and CD3+CD8+H-LDL-R+ cell % are increased after activation (Post-A) compared to before activation (Pre-A). Similarly, Figure 27B shows that the percentage of CD3+CD4+CD25+ cells, CD3+CD4+CD69+ cells, and CD3+CD4+H-LDL-R+ cells are increased after activation (Post-A) compared to before activation (Pre-A), confirming the activation of PBMCs.

[0203] Transduction FIG. 26 shows that on day +1, activated PBMCs were cultured with viral vectors, e.g., constructs #8, #10, #10n, #11, #11n, and #13- #21, in G-Rex® 24-well plates in the absence of serum at approximately 2×10 cells. 6 The amount of virus used for transduction is shown in Table 8. [Table 8]

[0204] Proliferation Figure 26 shows that on day +2, transduced PBMCs expanded in the absence of serum. On day +6, cells were harvested and cryopreserved for subsequent analysis, e.g., FACS-dextramer (tetramer?) and vector copy number (VCN). Figure 28 shows the fold expansion of the transduced T cell product on day +6. Cell viability is greater than 90% on day +6.

[0205] Characterization of T cell products Cell counts, FACS-dextramers, and vector copy numbers (VCN) were determined. Tetramer panels may include live / dead cells, CD3, CD8α, CD8β, CD4, and peptide / MHC tetramers, such as PRAME-004 (SLLQHLIGL) (SEQ ID NO: 147) / MHC tetramers. FACS analysis was gated on live singlets, followed by CD3+, followed by CD4+CD8+, followed by CD4+CD8+tetramer (Tet)+ and CD8+Tet+.

[0206] Figures 29A and 29B show the results of culturing 1 × 10 cells using constructs No. 10, No. 10n, No. 11, and No. 13 to No. 21. 6 Figures 30A and 30B show the % CD8+CD4+ cells transduced with 0.3 μl, 1.1 μl, 3.3 μl, 10 μl, or 30 μl per cell. These results show comparable frequencies of CD8+CD4+ cells obtained by transduction with all vectors tested. Construct #8 (TCR only) serves as a negative control. ... 6 The Tet% of CD8+CD4+ cells transduced with 0.3 μl, 1.1 μl, 3.3 μl, 10 μl, or 30 μl per cell is shown. The results show that there was a trend towards a higher frequency of CD4+CD8+tet+ in the CD8β1 isoform (constructs #10 and #18) compared to the CD8β3 isoform (construct #16) and the CD8β5 isoform (constructs #15 and #17). FACS analysis was gated on live singlets, followed by CD3+, followed by CD4+CD8+, followed by CD4+CD8+Tet+.

[0207] Figures 31A and 31B show the results of culturing 1 × 10 cells using constructs No. 10, No. 10n, No. 11, and No. 13 to No. 21. 6The Tet MFI of CD8+CD4+Tet+ cells transduced with 0.3 μl, 1.1 μl, 3.3 μl, 10 μl, or 30 μl per cell is shown. The results show a trend for higher tetramer MFI in the CD4+CD8+Tet+ population for the CD8β1 isoform (constructs #10 and #18) compared to the CD8β3 isoform (construct #16) and the CD8β5 isoform (constructs #15 and #17).

[0208] Figures 32A and 32B show the results of culturing 1 × 10 cells using constructs No. 10, No. 10n, No. 11, and No. 13 to No. 21. 6 Figures 33A and 33B show CD8 frequencies (CD8+CD4-% of CD3+) in cells transduced with 0.3 μl, 1.1 μl, 3.3 μl, 10 μl, or 30 μl per cell. These results show no difference in CD8 frequencies between constructs. ... 6 The % CD8+Tet+ cells (out of CD3+) transduced with 0.3 μl, 1.1 μl, 3.3 μl, 10 μl, or 30 μl per cell are shown. These results show that the frequency of CD8+Tet+ (out of CD3+) in cells transduced with construct #10 is slightly higher than those transduced with the other constructs. FACS analysis was gated on live singlets, followed by CD3, followed by CD8+CD4-, followed by Tet+.

[0209] Figures 34A and 34B show the results of culturing 1 × 10 cells using constructs No. 10, No. 10n, No. 11, and No. 13 to No. 21. 6 Figure 1 shows the Tet MFI of CD8+Tet+ cells transduced with 0.3 μl, 1.1 μl, 3.3 μl, 10 μl, or 30 μl per cell. These results show that the tetramer MFI of CD8+tet+ cells was comparable between CD8β1 (constructs #18 and #10), CD8β5 (constructs #15 and #17), and CD8β3 (construct #16) isoforms, while construct #21 displayed a lower tetramer MFI.

[0210] Figures 35A and 35B show the results of culturing 1 × 10 cells using constructs No. 10, No. 10n, No. 11, and No. 13 to No. 21. 6 Shown is the Tet+% of CD3 cells transduced with 0.3 μl, 1.1 μl, 3.3 μl, 10 μl, or 30 μl per cell. These results show that the frequency of CD3+Tet+ in cells transduced with construct #10 (CD8β1) is higher compared to those transduced with CD8β3 (construct #16) and CD8β5 (constructs #15 and #17). FACS analysis was gated on live singlets followed by CD3+ followed by Tet+.

[0211] Figures 36A and 36B show the results of culturing 1 × 10 cells using constructs No. 10, No. 10n, No. 11, and No. 13 to No. 21. 6 Vector copy numbers (VCN) are shown for cells transduced with 0.3 μl, 1.1 μl, 3.3 μl, 10 μl, or 30 μl per cell. These results demonstrate comparable abilities of all constructs to integrate and express the CD8 / TCR genes.

[0212] In summary, these results showed that (1) viral vectors carrying CD8β1, CD8β3, and CD8β5 isoforms had good transduction titers, (2) all constructs could be successfully manufactured (e.g., high viability, proliferation fold ranging from 6 to 12), (3) the frequency of CD3+tet+ among CD8β isoforms: CD8β1 (construct no. 10) was higher than CD8β3 (construct no. 16) and CD8β5 (construct no. 15 and no. 17), and construct no. 21 showed the lowest value, (4) the frequency of CD3+tet+ in construct no. 11 and no. 19 (m1CD8α (sequence number 7)) showed the highest value, and (5) saturation in CD3+tet+%, CD8+tet+%, and CD4+CD8+tet+% was observed at 10 μl / e6. Optimal vector doses range from 3.3 to 10 μl / e6 for all constructs.

[0213] Example 7 Mid-scale vector screening (constructs no. 13 to no. 19) T cell manufacturing Activation / Transduction Figure 37 shows that on day +0, PBMCs from four HLA-A02+ donors were thawed and rested. Cells were activated in bags coated with anti-CD3 and anti-CD28 antibodies (AC290) in the absence of serum. On day +1, activated PBMCs were transfected with viral vectors, e.g., constructs no. 8, no. 10n, no. 11n, and no. 13-no. 19, in G-Rex® 6-well plates in the absence of serum at approximately 7x10 cells. 6 The amount of virus used for transduction is shown in Table 9. [Table 9]

[0214] Proliferation Figure 37 shows that on day +2, transduced PBMCs expanded in the absence of serum. On day +7, cells were harvested and cryopreserved for subsequent analysis, e.g., FACS-tetramer and vector copy number (VCN). The fold expansion on day +7 was comparable for all constructs (approximately 30-fold expansion). Cell viability is greater than 90% on day +7.

[0215] Characterization of T cell products Cell counts, FACS-dextramers, and vector copy numbers (VCN) were determined. Tetramer panels may include live / dead cells, CD3, CD8α, CD8β, CD4, and peptide / MHC tetramers, such as PRAME-004 (SLLQHLIGL) (SEQ ID NO: 147) / MHC tetramers. FACS analysis was gated on live singlets, followed by CD3+, followed by CD4+CD8+, followed by CD4+CD8+tetramer (Tet)+ and CD8+Tet+.

[0216] Similar to the results described in Example 6, comparable frequencies of CD8+CD4+ cells were obtained with constructs no. 10n, no. 11n, no. 13 to no. 19 at 1×10 cells. 6The results were obtained by transduction with 2.5 μl or 5.0 μl per 1×106 cells. Construct #8 (TCR only) serves as a negative control. Figure 38 shows the Tet% of CD8+CD4+ cells transduced with constructs #10n, #11n, #13-#19 at 2.5 μl or 5.0 μl per 1×106 cells. Similar to the results described in Example 6, these results show that there was a trend towards a higher frequency of CD4+CD8+tet+ in the CD8β1 isoform (construct #10n) compared to the CD8β3 isoform (constructs #13, #14, #16) and the CD8β5 isoform (constructs #15 and #17). FACS analysis was gated on live singlets, followed by CD3+, followed by CD4+CD8+, followed by Tet+.

[0217] FIG. 39 shows the results of culturing 1×10 cells using constructs No. 10n, No. 11n, and No. 13 to No. 19. 6 The Tet MFI of CD8+CD4+Tet+ cells transduced at 2.5 μl or 5.0 μl per cell is shown. The results show a higher tetramer MFI in the CD4+CD8+Tet+ population in the CD8β1 isoform (construct no. 10n) compared to the CD8β3 isoform (construct no. 13) and the CD8β5 isoform (construct no. 15 and no. 17).

[0218] Similar to the results described in Example 6, the results showed that among the constructs, constructs no. 10n, no. 11n, no. 13 to no. 19 were used to culture 1×10 cells. 6 The results show that there is no difference in CD8 frequency (CD8+CD4-% of CD3+) in cells transduced with 2.5 μl or 5.0 μl of cells per cell (data not shown). 6 Comparable frequencies of CD8+Tet+ (among CD3+) in cells transduced at 2.5 μl or 5.0 μl per cell (data not shown). FACS analysis was gated on live singlets, followed by CD3, followed by CD8+CD4-, followed by Tet+.

[0219] FIG. 40 shows the results of culturing 1×10 cells using constructs No. 10n, No. 11n, and No. 13 to No. 19. 6 Figure 1 shows the Tet MFI of CD8+Tet+ cells transduced with 2.5 μl or 5.0 μl per cell. These results show that the tetramer MFI of CD8+tet+ cells was comparable between CD8β1 (constructs #18 and #10) and CD8β5 (construct #15) isoforms, whereas CD8β3 (constructs #13, #14, and #16) displayed a lower tetramer MFI.

[0220] FIG. 41 shows the results of culturing 1×10 cells using constructs No. 10n, No. 11n, and No. 13 to No. 19. 6 The Tet% of CD3+ cells transduced with 2.5 μl or 5.0 μl per cell is shown. These results show that the frequency of CD3+Tet+ in cells transduced with construct #10 (CD8β1) is slightly higher compared to those transduced with CD8β3 (constructs #13, #14, and #16) and CD8β5 (construct #15). FACS analysis was gated on live singlets followed by CD3+ followed by Tet+. A slightly higher total CD3+tet+ cell number was observed in PBMCs transduced with construct #10CD8β1 compared to those transduced with CD8β3 (constructs #13, #14, and #16) and CD8β5 (construct #15) (data not shown).

[0221] FIG. 42 shows the results of culturing 1×10 cells using constructs No. 10n, No. 11n, and No. 13 to No. 19. 6 The vector copy number (VCN) of cells transduced with 2.5 μl or 5.0 μl per cell is shown. These results are for 1×10 cells 6 Figure 2 shows that in PBMC products derived with each individual construct at vector doses of 2.5 μl or 5.0 μl per cell, vector copies remained below 5.

[0222] Figure 43 shows the T cell subset % in cells transduced with constructs #10, #11, #13, and #15 for each donor. Construct #8 (TCR only) and non-transduced cells were used as controls. These results show that the TCR only condition has few naive cells compared to the other constructs, consistent with a lower fold expansion. Figures 44A and 44B show the T cell subset % in cells transduced with constructs #10, #11, #13, and #15 for each donor. Construct #8 (TCR only) and non-transduced cells were used as controls. FACS analysis was gated on CD4+CD8+ in Figure 44A and CD4-CD8+TCR+ in Figure 44B. These results show donor-to-donor variability between the frequencies of T cell memory subsets, but little difference in the frequencies of T naive and Tcm between constructs.

[0223] In summary, these results indicate that (1) viability and fold expansion were comparable between all constructs at day 7; (2) a slightly higher frequency of CD3+tet+ was observed with CD8β1 (construct no. 10) compared to CD8β3 (constructs no. 13, no. 14, and no. 16) and CD8β5 (constructs no. 15 and no. 17); and (3) a median cell count of 2.5–5 ul / 10 6 (3) fewer than 5 vector copies per cell in the majority of constructs at individual doses; and (4) donor-to-donor variability in the frequencies of T cell memory subsets; in general, construct no. 10 has fewer naive but more Tcm cells than the other β isoform constructs.

[0224] Example 8 Tumor Death Assay - Constructs #10, #11, #13, and #15 Figures 45A and 45B show data showing that constructs #13 and #10 are comparable to TCR alone in mediating cytotoxicity against a UACC257 target positive cell line expressing high levels of antigen (1081 copies per cell). Construct #15 was also effective but showed slower killing compared to constructs #13 and #10. The effector:target ratio used to generate these results was 4:1. Similar results were obtained with an effector:target ratio of 2:1 (data not shown).

[0225] Example 9 IFNγ Secretion Assay - Constructs #10, #11, #13, and #15 IFNγ secretion was measured in the UACC257 cell line. FIG. 46 shows that IFNγ secretion in response to UACC257 cell line was higher with construct #13 compared to construct #10. IFNγ was quantified in the supernatants from Incucyte plates. The effector:target ratio used to generate these results was 4:1. Similar results were obtained with an effector:target ratio of 2:1 (data not shown).

[0226] Example 10 ICI marker expression - constructs no. 10, no. 11, no. 13, and no. 15 ICI marker frequencies (2B4, 41BB, LAG3, PD-1, TIGIT, TIM3, CD39+CD69+, and CD39-CD69-) were measured. Figure 47 shows that construct number 15 has higher expression of LAG3, PD-1, and TIGIT compared to other constructs, followed by construct number 10.

[0227] Example 11 Cytokine Expression - Constructs #10, #11, #13, and #15 Expression of various cytokines was measured in UACC257 cells co-cultured with PBMCs transduced with constructs #10, #11, #13, and #15 at an E:T ratio of 4:1. Figures 48A-48G show increased expression of IFNγ, IL-2, and TNFα by CD4+CD8+ cells transduced with construct #10 (WT signal peptide, CD8β1) compared to other constructs. FACS analysis gated on CD3+CD4+CD8+ cells versus UACC257, E:T of 4:1. Figures 49A-49G show increased expression of IFNγ, IL-2, MIP-1β, and TNFα by CD4-CD8+ cells transduced with construct #10 (WT signal peptide, CD8β1) compared to other constructs. FACS analysis was gated on CD3+CD4-CD8+ cells vs. UACC257, E:T 4:1. Figures 50A-50G show increased expression of IL-2 and TNFα by CD3+TCR+ cells transduced with construct #10 (WT signal peptide, CD8β1) compared to other constructs. MIP-1β expression is highest with construct #11 (similar results when gated on CD4+CD8+ cells). FACS analysis was gated on CD3+TCR+ cells vs. UACC257, E:T 4:1.

[0228] Expression of various cytokines was measured in A375 cells co-cultured with PBMCs transduced with constructs #10, #11, #13, and #15 at an E:T ratio of 4:1. Figures 51A-51C show the results of FACS analysis gated on CD4+CD8+ cells for A375, E:T 4:1. Figures 52A-52C show the results of FACS analysis gated on CD4-CD8+ cells for A375, E:T 4:1. Figures 53A-53C show the results of FACS analysis gated on CD3+TCR+ cells for A375, E:T 4:1. Overall, results were more variable when cells were co-cultured with A375+RFP, but similar trends were observed compared to activation with UACC257+RFP.

[0229] Example 12 Large-scale vector screening (constructs #10, #11, #13, #16, #18, #19) T cell manufacturing Activation / Transduction Figure 54 shows that on day +0, PBMCs from three HLA-A02+ donors were thawed and rested. Cells were activated in anti-CD3 and anti-CD28 antibody coated bags (AC290) in the absence of serum. On day +1, activated PBMCs were transfected with viral vectors, e.g., constructs no. 8, no. 10n, no. 11n, no. 13, no. 16, no. 18, and no. 19 in G-Rex® 100 cell culture vessels at approximately 5x10 cells in the absence of serum. 7 The amount of virus used for transduction is shown in Table 10. [Table 10]

[0230] Proliferation Figure 54 shows that on day +2, transduced PBMCs expanded in the absence of serum. On day +7, cells were harvested and cryopreserved for subsequent analysis, e.g., FACS-tetramer and vector copy number (VCN). The fold expansion on day +7 was comparable for all constructs (approximately 30-fold expansion). Cell viability is greater than 90% on day +7.

[0231] Characterization of T cell products Cell counts, FACS-dextramers, and vector copy numbers (VCN) were determined. Tetramer panels may include live / dead cells, CD3, CD8α, CD8β, CD4, and peptide / MHC tetramers, such as PRAME-004 (SLLQHLIGL) (SEQ ID NO: 147) / MHC tetramers. FACS analysis was gated on live singlets, followed by CD3+, followed by CD4+CD8+, followed by CD4+CD8+tetramer (Tet)+ and CD8+Tet+.

[0232] Tumor death assays and cytokine expression in the presence and absence of autologous immature dendritic cells were also measured.

[0233] The results are consistent with the previous examples and are summarized in Table 11. [Table 11]

[0234] Example 13 DC Licensing with CD4 Cells Expressing Constructs of the Disclosure 59 shows a scheme for determining the level of cytokine secretion by dendritic cells (DCs) in the presence of PBMCs transduced with constructs of the present disclosure and in the presence of target cells, e.g., UACC257 cells. Briefly, on day 0, PBMCs (n=3) were thawed and rested, followed by monocyte isolation and autologous immature DC (iDC) generation in the presence of IL-4 and GM-CSF, on days 2 and 4-5, DCs were fed in the presence of IL-4 and GM-CSF, and on day 6, iDCs (+DCs) were cultured in a 1:1 ratio using constructs #13, #16, #10n, #18, #11n, or #19 (effector) and UACC257 cells (target). PBMCs were co-cultured at a ratio of 1 / 10:1 / 4 with PBMCs transduced with no iDCs (-DC), PBMCs transduced with TCR only, PBMCs without transduction (NT), PBMCs treated with iDCs and LPS, and iDCs only serving as controls, and on day 7 (after 24 hours of co-culture), supernatants from the co-cultures were collected and subsequently subjected to cytokine profiling using Multiplex, including, for example, IL-12, IL-6, and TNF-α.

[0235] Secretion of proinflammatory cytokines was increased in triplicate cultures of autologous immature dendritic cells, UACC257 tumor cell line, and CD4+ T cell products expressing CD8αβ heterodimer and TCR (construct no. 10) compared to those expressing CD8α* homodimer and TCR (construct no. 11), in which the stalk region is replaced by the CD8β stalk region.

[0236] To determine the ability of CD4+ T cells expressing construct #10 or #11 to license DCs, bulk PBMCs were transduced with construct #10 or #11 followed by selection of CD8+ and CD4+ cells from the products. PBMCs, CD8+CD4- selected products, or CD4+CD8+ selected products were triple-cultured with UACC257 tumor cell line in the presence or absence of autologous immature dendritic cells (iDCs) for 24 hours followed by cytokine quantification of IL-12, TNF-α, and IL-6 using Multiplex with iDCs alone or LPS added as control, N=4-7, mean ± SD, P values ​​based on two-way ANOVA.

[0237] In the presence of immature dendritic cells (iDCs) and UACC257 cells, CD4+ T cells expressing construct no. 10 (CD4+CD8+ T cells) performed better than CD4+ T cells expressing construct no. 11 by inducing higher levels of IL-12 (Figure 56), TNF-α (Figure 57), and IL-6 (Figure 58) secreted by dendritic cells (DCs). Meanwhile, the levels of IL-12, TNF-α, and IL-6 were comparable between CD8+ T cells expressing construct no. 10 and no. 11 (CD8+CD4- T cells). These results suggest that CD4+ T cells expressing CD8αβ heterodimer and TCR (construct no. 10) may be a better product in DC licensing than CD4+ T cells expressing CD8α* homodimer and TCR (construct no. 11). Negative controls included cytokine levels obtained in the absence of (1) iDC (-iDC), in the presence of (2) non-transduced T cells (NT) + UACC257 cells, and in the presence of (3) T cells transduced with TCR only (TCR) + UACC257 cells. Positive controls included cytokine levels obtained from iDC treated with lipopolysaccharide (LPS), which can activate DC.

[0238] Example 14 Assessment of DC maturation and cytokine secretion by PBMC products in response to UACC257 targeting Figure 60 shows the IL-12 secretion levels induced by co-culturing PBMCs transduced with constructs of the present disclosure in the presence or absence of iDCs and target cells, e.g., UACC257 cells. For example, co-culturing PBMCs transduced with constructs no. 10 and 13 in the presence of iDCs (+DCs) and UACC257 increased IL-12 secretion compared to co-culturing PBMCs transduced with TCR alone. Increased IL-12 secretion suggests (1) polarization toward Th1 cell-mediated immunity, including TNF-α production (see Figure 61), (2) T cell proliferation, (3) IFN-γ production, and (4) cytolytic activity of cytotoxic T lymphocytes (CTLs).

[0239] Figure 61 shows the TNF-α secretion levels induced by co-culturing PBMCs transduced with constructs of the present disclosure in the presence or absence of iDCs and target cells, e.g., UACC257 cells. For example, co-culturing PBMCs transduced with constructs no. 10 and 13 in the presence of iDCs (+DCs) and UACC257 increased TNF-α secretion compared to co-culturing PBMCs transduced with TCR alone.

[0240] Increased IL-6 secretion (in addition to IL-12, TNF-α) may signify dendritic cell maturation and may be augmented by CD40-CD40L interactions between CD4+ T cells and DCs. DC maturation and subsequent cytokine secretion may help regulate the proinflammatory environment.

[0241] Figure 62 shows the IL-6 secretion levels induced by co-culturing PBMCs transduced with constructs of the present disclosure in the presence or absence of iDCs and target cells, e.g., UACC257 cells. For example, co-culturing PBMCs transduced with constructs no. 10 and 13 in the presence of iDCs (+DCs) and UACC257 increased IL-6 secretion compared to co-culturing PBMCs transduced with TCR alone.

[0242] These results indicate that PBMC products containing CD4+ T cells co-expressing a transgenic TCR and CD8 coreceptor (CD8αβ heterodimer or CD8α homodimer) can license DCs in the microenvironment through antigen cross-presentation and modulate the tumor microenvironment, for example, by increasing IL-12, IL-6, and TNF-α secretion.

[0243] Table 12 shows a comparison between the constructs based on manufacturability and functionality. [Table 12]

[0244] Note: "+++" = best response, "++" = excellent response, "+" = average response, "+ / -" = poor response.

[0245] Table 13 shows the comparison and ranking of the constructs (lower numbers are better). [Table 13] *Time delay here refers to, for example, any delay from GMP vector manufacturing, or any delay due to an incomplete data set, which may add to the delay in implementing the construct in clinical trials.

[0246] In summary, manufacturability, e.g., in terms of viability, fold expansion, transgene expression, and vector copy number, may be similarly superior between cells transduced with constructs no. 10, no. 11, no. 13, or no. 19, as rank 1, and functionality, e.g., in terms of cell killing, cytokine secretion, DC licensing, and 3D spheroid formation ability, of cells transduced with constructs no. 10 and no. 13, as rank 1, may be superior to those transduced with constructs no. 11 and no. 19, as ranks 1-3.

[0247] Example 15 EC50 assay To determine the efficacy of T cells transduced with constructs of the present disclosure, such as constructs no. 10 and no. 11, against target cells, the EC50 was determined based on the level of IFNγ produced by the transduced cells in the presence of PRAME peptide-pulsed T2 cells.

[0248] For example, to compare the EC50 of CD4+ selected T cells transduced with construct #10 (CD8αβ-TCR), construct #11 (m1CD8α-TCR), or construct #8 (TCR only), the CD4+ selected products (TCR+ normalized) were co-cultured with PRAME peptide-pulsed T2 cells at defined concentrations and an E:T ratio of 1:1 for 24 hours. IFNγ levels were quantified in the supernatants after 24 hours. Figures 63A-63C show the IFNγ levels produced by transduced CD4+ selected T cells obtained from donors #1, #2, and #3, respectively. In general, CD4+ selected T cells transduced with construct #10 were more sensitive to PRAME antigen than those transduced with construct #11 (m1CD8αTCR+CD4 T cells), as shown by the lower EC50 values ​​(ng / ml) of CD4+ selected T cells transduced with construct #10 than those transduced with construct #11 (Figure 63D). No response was observed among TCR+CD4+ cells (Figures 63A-63D). These results suggest that CD8αβ heterodimers may confer increased avidity to CD8αβTCR+CD4+ T cells compared to m1CD8a homodimers, resulting in better efficacy against target cells.

[0249] Similar experiments were performed using PBMCs obtained from donors #1, #3, and #4. Briefly, PBMC products (TCR+non-normalized) were co-cultured with PRAME peptide-pulsed T2 cells at defined concentrations with an E:T ratio of 1:1 for 24 hours. IFNγ levels were quantified in the supernatants after 24 hours. Figures 64A-64C show IFNγ levels produced by transduced PBMCs obtained from donors #4, #1, and #3, respectively. Donor-to-donor variability was observed in the EC50 values. For example, donor #3 (Figures 64C and 64D) shows a lower EC50 for PBMCs transduced with construct #10 compared to those transduced with TCR alone, whereas donors #1 (Figure 64B) and #4 (Figure 64A) show comparable EC50s between construct #10 and TCR alone (Figure 64D). Thus, the increased avidity and efficacy observed in CD4+ selected T cell products expressing TCR and CD8αβ heterodimers compared to those expressing TCR alone may be obtained, but to a lesser extent than when using PBMC products.

[0250] To compare the EC50 of different T cell products obtained from the same donor, PBMC products, CD8+ selected products, and CD4+ selected products obtained from a single donor were co-cultured with PRAME peptide-pulsed T2 cells (TCR+ normalized) at defined concentrations at an E:T ratio of 1:1 for 24 hours. IFNγ levels were quantified in the supernatants after 24 hours. Figures 65A-65C show the IFNγ levels produced by the PBMC products (Figure 65A), CD8+ selected products (Figure 65B), and CD4+ selected products (Figure 65C), respectively. Consistently, the EC50 of CD4+ selected T cells transduced with construct #10 was lower than those transduced with construct #11 or TCR only (Figure 65C), while the EC50 of transduced PBMC and CD8+ selected T cells was comparable between construct #10 and TCR only transduction. Thus, the increased avidity and efficacy observed in CD4+ selected T cell products expressing TCR and CD8αβ heterodimers compared to those expressing TCR and m1CD8α homodimers, or those expressing TCR alone, may be obtained, but to a lesser extent than when using PBMC or CD8+ selected T cell products.

[0251] All references cited herein are incorporated by reference as if each reference was specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such reference by virtue of prior invention.

[0252] It will be understood that each of the above-mentioned elements, or two or more together, may find useful application in other types of methods different from the types described above. Without further analysis, the foregoing will sufficiently clarify the gist of the present disclosure so that others, by applying their current knowledge, may readily adapt it to various applications without omitting features that, in the light of the prior art, fairly constitute essential features of the general or specific embodiments of the present disclosure as set forth in the appended claims. The above-mentioned embodiments are presented only as examples, and the scope of the present disclosure should be limited only by the scope of the following claims.

Claims

1. 1. An isolated nucleic acid comprising: (a) a T cell receptor (TCR) comprising an α chain and a β chain, and a CD8 polypeptide comprising an α chain and a β chain; or (b) a TCR comprising an alpha chain and a beta chain, and a CD8 polypeptide comprising an alpha chain without a beta chain; The method comprises the steps of: the CD8 α chain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:5, and the CD8 β chain comprises SEQ ID NO:9, 11, 12, or 13; Isolated nucleic acid.

2. 2. An isolated polypeptide encoded by the nucleic acid of claim 1.

3. A vector comprising the nucleic acid of claim 1.

4. The vector of claim 3, further comprising a nucleic acid encoding a 2A peptide or an internal ribosome entry site (IRES) located between the nucleic acid encoding the CD8 α chain and the nucleic acid encoding the CD8 β chain.

5. The vector of claim 3 or 4, further comprising a nucleic acid encoding a 2A peptide or an IRES, positioned between the nucleic acid encoding the TCR alpha chain and the nucleic acid encoding the TCR beta chain.

6. The vector of claim 5, wherein the 2A peptide is P2A (SEQ ID NO: 93), T2A (SEQ ID NO: 94), E2A (SEQ ID NO: 95), or F2A (SEQ ID NO: 96).

7. 7. The vector of any one of claims 3 to 6, wherein the vector further comprises a post-transcriptional regulatory element (PRE) sequence selected from a woodchuck PRE (WPRE), a woodchuck PRE (WPRE) variant 1, a woodchuck PRE (WPRE) variant 2, or a Hepatitis B virus (HBV) PRE (HPRE).

8. The post-transcriptional regulatory element (PRE) sequence is Woodchuck PRE (WPRE) variant 1 comprising the amino acid sequence of SEQ ID NO: 256; or 8. The vector of claim 7, which is a Woodchuck PRE (WPRE) mutant 2 comprising the amino acid sequence of SEQ ID NO:

257.

9. 9. The vector according to any one of claims 3 to 8, further comprising a promoter selected from a cytomegalovirus (CMV) promoter, a phosphoglycerate kinase (PGK) promoter, a myelin basic protein (MBP) promoter, a glial fibrillary acidic protein (GFAP) promoter, a modified MoMuLV LTR containing myeloproliferative sarcoma virus enhancer (MNDU3), a Ubiquitin C promoter, an EF-1 alpha promoter, or a murine stem cell virus (MSCV) promoter.

10. The vector according to any one of claims 3 to 9, wherein the vector is a viral vector or a non-viral vector.

11. An isolated T cell transduced with the nucleic acid of claim 1.

12. 12. The T cell of claim 11 , wherein the cell is an αβ T cell, a γδ T cell, and / or a natural killer T cell. a) the αβ T cells are CD4+ T cells or CD8+ T cells, or b) the γδ T cells are Vγ9Vδ2 T cells; T cells.

13. A composition comprising a T cell according to claim 11 or 12.

14. The composition of claim 13, further comprising an adjuvant selected from anti-CD40 antibodies, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, atezolizumab, interferon-alpha, interferon-beta, CpG oligonucleotides and derivatives, poly(I:C) and derivatives, RNA, sildenafil, microparticle formulations having poly(lactide-co-glycolide) (PLG), virosomes, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), interleukin-21 (IL-21), interleukin-23 (IL-23), and combinations thereof.

15. 1. An in vitro method of preparing T cells, comprising: Isolating T cells from a blood sample of a human subject; activating the isolated T cells; Transducing the activated T cells with a nucleic acid according to claim 1 or a vector according to any one of claims 3 to 10, and and expanding the transduced T cells.

16. 16. The method of claim 15, wherein the blood sample comprises peripheral blood mononuclear cells (PMBCs).

17. 17. The method of claim 15 or 16, wherein the activating comprises contacting the T cells with an anti-CD3 antibody and an anti-CD28 antibody.

18. The method of any one of claims 15 to 17, wherein the T cells are CD4+ T cells, CD8+ T cells, γδ T cells or αβ T cells.

19. The nucleic acid described in claim 1, wherein the CD8 α chain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:

5.

20. The nucleic acid described in claim 1, wherein the CD8 α chain comprises an amino acid sequence having at least 98% identity to SEQ ID NO:

5.

21. The nucleic acid of claim 1, wherein the CD8 α chain comprises an amino acid sequence having 100% identity to SEQ ID NO:5.