DE102021100038A1 - MODIFIED CD8 POLYPEPTIDES, COMPOSITIONS AND METHODS OF USE THEREOF - Google Patents

Abstract

The present disclosure relates to T cells capable of co-expressing T cell receptors ("TCR") along with CD8 polypeptides and their use in adoptive cellular therapy. The present disclosure also provides modified CD8 sequences, vectors and related methods.

Description

BACKGROUND OF THE INVENTION

field of invention

The present disclosure relates to T cells capable of co-expressing T cell receptors ("TCR") along with CD8 polypeptides and their use in adoptive cellular therapy. The present disclosure also provides modified CD8 sequences, vectors and related methods.

State of the art

CD8 and CD4 are transmembrane glycoproteins characteristic of diverse populations of T lymphocytes whose antigenic responses are restricted by class I and class II MHC molecules, respectively. They play an important role both in 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 proteins of the immunoglobulin superfamily. They determine antigenic restriction by binding to MHC molecules at an interface distinct from the region presenting the antigenic peptide, but the structural basis for their similar functions appears to be very different. Their sequence similarity is low and while CD4 is expressed as a monomer on the cell surface, CD8 is expressed as an αα homodimer or αβ heterodimer. In humans, this CD8αα homodimer can functionally replace the CD8αβ heterodimer. CD8 contacts an acidic loop in the α3 domain of the MHC class I molecule, increasing the T cell's avidity for its target. CD8 is also involved in the phosphorylation events leading to CTL activation through the association of its cytoplasmic end of the α-chain with the tyrosine kinase p56 ^lck .

It is desirable to develop methods for producing T cells with specific cytotoxic activity for immunotherapy.

SHORT SUMMARY

In one embodiment, 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 CD8 βStalk region or domain.

In another embodiment, the presently described CD8 polypeptides (a) an immunoglobulin (Ig)-like domain comprising at least about 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 1, (b) a CD8β region comprising at least about 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 2, (c) a transmembrane domain comprising at least about 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 3, and (d) a cytoplasmic domain comprising at least about 80%, at least 85%, at least 90% %, at least 95% or 100% sequence identity with the amino acid sequence of SEQ ID NO:4.

In one embodiment, the CD8 polypeptides described herein have at least about 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:5.

In one embodiment, the CD8 polypeptides described herein have at least about 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:7.

In one embodiment, the presently described CD8 polypeptides may include a signal peptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 6 attached to the N-terminus or fused to the C-terminus of the CD8 polypeptides described herein.

In another embodiment, the CD8 polypeptides described herein can be (a) SEQ ID NO: 1 comprising having one, two, three, four or five amino acid substitutions; (b) SEQ ID NO: 2 comprising having one, two, three, four or five amino acid substitutions; (c) SEQ ID NO: 3 comprising having one, two, three, four or five amino acid substitutions, and (d) SEQ ID NO: 4 comprising having one, two, three, four or five amino acid substitutions.

In one embodiment, the CD8 polypeptides described herein are CD8α or modified CD8α polypeptides.

In one embodiment, the disclosure provides nucleic acids encoding polypeptides described herein.

In one embodiment, a vector may comprise a nucleic acid encoding the CD8 polypeptides described herein.

In one embodiment, the vector may comprise a nucleic acid encoding a T cell receptor (TCR) comprising an α chain and a β chain. In another embodiment, the vector may comprise a nucleic acid encoding a CAR-T.

In one embodiment, the TCR α chain and the TCR β chain of SEQ ID NO: 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 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 72, 73 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 can be selected.

In one embodiment, the vector may comprise a nucleic acid encoding a CD8β polypeptide.

In one embodiment, the CD8β polypeptide may comprise the amino acid sequence of any one of SEQ ID NO: 8-14.

In one embodiment, the vector may comprise a nucleic acid encoding a 2A peptide positioned between the nucleic acid encoding the modified CD8α polypeptide and the nucleic acid encoding a CD8β polypeptide.

In one embodiment, the vector may comprise a nucleic acid encoding a 2A peptide positioned between the TCR α chain-encoding nucleic acid and the TCR β chain-encoding nucleic acid.

In one embodiment, the 2A peptide can be selected from P2A (SEQ ID NO: 93), T2A (SEQ ID NO: 94), E2A (SEQ ID NO: 95), or F2A (SEQ ID NO: 96).

In one embodiment, the vector may further comprise a post-transcriptional regulatory element (PRE) sequence selected from a Woodchuck PRE (WPRE), a hepatitis B virus (HBV) PRE (HPRE), or a combination thereof.

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

In one embodiment, the vector can be a viral vector or a non-viral vector.

In one embodiment, the vector can be selected from, for example, adenoviruses, poxviruses, alphaviruses, arenaviruses, flaviruses, rhabdoviruses, retroviruses, lentiviruses, herpesviruses, paramyxoviruses, picornaviruses, or a combination thereof.

In one embodiment, the vector may be pseudotyped with a coat protein of a virus derived from feline native endogenous virus (RD114), a chimeric version of RD114 (RD114TR), gibbon simian leukemia virus (GALV), a chimeric version of GALV ( GALV-TR), amphotropic murine leukemia virus (MLV 4070A), baculovirus (GP64), vesicular stomatitis virus (VSV-G), avian influenza virus (FPV), Ebola virus (EboV), or baboon retrovirus envelope glycoprotein (BaEV ), lymphocytic choriomeningitis virus (LCMV), or a combination thereof.

In one embodiment, the vector may further comprise a nucleic acid encoding a T cell receptor (TCR).

In another embodiment, the vector may also comprise a nucleic acid encoding a chimeric antigen receptor (CAR).

In one embodiment, a cell can be transduced with the vector.

In one embodiment, the cell may comprise an αβ T cell, a γδ T cell, a natural killer cell, a CD4+ /CD8+ cell, or a combination thereof.

In one embodiment, the αβ T cells may include CD4+ T cells and CD8+ T cells.

In one embodiment, a method for producing T cells for immunotherapy may include isolating T cells from a blood sample from a human subject, activating the isolated T cells, transducing the activated T cells with the vector, and expanding the transduced T cells include.

In one embodiment, the T cell can be a CD4+ T cell.

In one embodiment, the T cell can be a CD8+ T cell.

In one embodiment, the T cell can be a γδ T cell.

In one embodiment, the T cells can be an αβ T cell and express a CD8 polypeptide as described herein.

In one embodiment, the T cells may be a γδ T cell and express a modified CD8 polypeptide as described herein, for example a modified CD8α polypeptide or a modified CD8α polypeptide having a CD8β stalk region.

In one embodiment, a method of treating a patient who has cancer can comprise administering to the patient a composition comprising the population of expanded T cells, wherein the T cells kill cancer cells expressing a peptide in a complex with a Presenting an MHC molecule on the surface, wherein the peptide is selected from SEQ ID NO: 98-255, wherein 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.

In one embodiment, the composition may also include an adjuvant.

In one embodiment, the adjuvant may be anti-CD40 antibody, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, atezolizumab, interferon-alpha, interferon-beta, CpG oligonucleotides and derivatives, poly-(I:C) and derivatives, RNA, sildenafil, particulate formulations with poly(lactidecoglycolide) (PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15 , IL-21, IL-23 or combinations thereof.

In one embodiment, a method of eliciting an immune response in a patient having cancer may comprise administering to the patient a composition comprising the population of expanded T cells, wherein the T cells kill cancer cells expressing a peptide in a A complex presenting on the surface an MHC molecule, wherein the peptide is selected from SEQ ID NO: 98-255, wherein 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, stomach cancer, prostate cancer, or a combination thereof.

The disclosure further contemplates a population of modified T cells presenting an exogenous CD8 co-receptor encoding a polypeptide described herein, e.g., at least 80%, at least 85%, at least 90%, or at least 95%, at least 99% amino acid or 100% to SEQ ID NO: 5, 7, 258 or 259 and a T cell receptor.

character list

• shows a representative CD8α subunit, e.g. B. SEQ ID NO: 258 (CD8α1), in accordance with an embodiment of the present disclosure. In this embodiment, CD8α1 contains five domains: (1) signal peptide, (2) Ig-like domain-1, (3) a stalk region, (4) transmembrane (TM) domain, and (5) a cytoplasmic terminal (cyto) region, the includes an Ick-binding motif.
• Figure 1 shows a sequence alignment between CD8α1 (SEQ ID NO:258) and m1CD8α (SEQ ID NO:7).
• shows a sequence alignment between CD8α2 (SEQ ID NO: 259) and m2CD8α (SEQ ID NO: 262), in which the cysteine substitution at position 112 is indicated by an arrow.
• Figure 1 shows vectors in accordance with some embodiments of the present disclosure.
• shows the titers of the in illustrated viral vectors according to an embodiment of the present disclosure.
• Figure 1 shows the production of T cells in accordance with an embodiment of the present disclosure.
• Figure 12 shows the expression of activation markers before and after activation in CD3+CD8+ cells according to an embodiment of the present disclosure.
• Figure 12 shows the expression of activation markers before and after activation in CD3+CD4+ cells according to an embodiment of the present disclosure.
• Figure 12 shows the magnitude of expansion of cells transduced with various donor 1 constructs.
• Figure 12 shows the magnitude of expansion of cells transduced with various donor 2 constructs.
• Figure 12 shows flow diagrams of cells transduced with Constructs #9 according to an embodiment of the present disclosure.
• Figure 12 shows flow charts of cells transduced with Constructs #10 according to an embodiment of the present disclosure.
• Figure 12 shows flow diagrams of cells transduced with Constructs #11 according to an embodiment of the present disclosure.
• Figure 12 shows flow diagrams of cells transduced with Constructs #12 according to an embodiment of the present disclosure.
• Figure 12 shows % CD8+CD4+ of cells transduced with various constructs according to an embodiment of the present disclosure.
• Figure 12 shows % Tet of CD8+CD4+ from cells transduced with various constructs according to an embodiment of the present disclosure.
• Figure 12 shows the Tet-MFI (CD8+CD4+Tet+) of cells transduced with various constructs in accordance with an embodiment of the present disclosure.
• Figure 12 shows the CD8α MFI (CD8+CD4+Tet+) of cells transduced with various constructs according to an embodiment of the present disclosure.
• Figure 12 shows % CD8+CD4 (of CD3+) of cells transduced with various constructs according to an embodiment of the present disclosure.
• Figure 12 shows % CD8+Tet+ (of CD3+) of cells transduced with various constructs according to an embodiment of the present disclosure.
• Figure 12 shows the Tet-MFI (CD8+Tet+) of cells transduced with various constructs in accordance with an embodiment of the present disclosure.
• Figure 12 shows the CD8α MFI (CD8+Tet+) of cells transduced with various constructs according to an embodiment of the present disclosure.
• Figure 12 shows % Tet+ (from CD3+) of cells transduced with various constructs in accordance with an embodiment of the present disclosure.
• Figure 12 shows VCN (upper panel) and CD3+Tet+/VCN (lower panel) of cells transduced with various constructs in accordance with an embodiment of the present disclosure.
• Figure 12 presents data showing that the constructs (#10, 11 and 12) are effective in mediating cytotoxicity against target-positive cell lines expressing antigen at different levels (UACC257 at 1081 copies per cell and A375 at 50 copies per cell), are comparable to pure TCRs.
• presents data showing that IFNγ secretion in response to UACC257 is comparable between constructs, however, #10 expression at A375 is highest among all constructs. However, when comparing #9 with #11, which express wild-type and modified CD8 co-receptor sequences, respectively, T cells transduced with #11 induced a stronger cytokine response as measured than IFNγ, quantitated in the supernatants of Incucyte plates. #9 = CD8α.TCR.WPREmut2; #10 = CD8βα.TCR.WPREmut2; #11 = CD8αCD8βst.TCR.WPREmut2; #12 = CD8α CD8βst.NCAMfu.TCR.WPREmut2; #1 = CD8α.TCR.WPRE ^wt ; No. 2 = CD8βα.TCR.WPRE ^wt ; #8= R11KEA TCR only.
• shows an exemplary experimental setup to assess DC maturation and cytokine secretion by PBMC-derived products in response to UACC257 and A375 targets. N=2.
• present data showing that IFNγ secretion increases in response to A375 in the presence of iDCs. In the tri-cocultures with iDCs, IFN _γ secretion is higher in #10 compared to the other constructs. However, when comparing #9 with #11, which express wild-type and modified CD8 co-receptor sequences, respectively, T cells transduced with #11 induced a stronger cytokine response as measured than IFNγ, quantitated in the culture supernatants of three-way co-cultures using donor D600115, E:T:iDC::1:1/10:1/4. #9 = CD8α.TCR.WPREmut2; #10 = CD8βα.TCR.WPREmut2; #11 = CD8αCD8βst.TCR.WPREmut2; #12 = CD8α CD8βst.NCAMfu.TCR.WPREmut2; #1 = CD8α.TCR.WPRE ^wt ; No. 2 = CD8βα.TCR.WPRE ^wt ; #8= R11KEA TCR only.
• Figure 12 presents data showing that IFNγ secretion increases in response to A375 in the presence of iDCs. In the triple co-cultures with iDCs, IFNγ secretion was higher in #10 compared to the other constructs. IFNγ quantified in the culture supernatants of three-way co-cultures using donor _D150081 , E:T:iDC::1:1/10:1/4. #9 = CD8α.TCR.WPREmut2; #10 = CD8βα.TCR.WPREmut2; #11 = CD8αCD8βst.TCR.WPREmut2; #12 = CD8α CD8βst.NCAMfu.TCR.WPREmut2; #1 = CD8α.TCR.WPRE ^wt ; No. 2 = CD8βα.TCR.WPRE ^wt ; #8= R11KEA TCR only.
• Figure 12 presents data showing that IFNγ secretion increases in response to UACC257 in the presence of iDCs. In the triple co-cultures with iDCs, IFNγ secretion was higher in #10 compared to the other constructs. However, when comparing #9 with #11, which express wild-type and modified CD8 co-receptor sequences, respectively, T cells transduced with #11 induced a stronger cytokine response as measured than IFNγ, quantitated in the culture supernatants of three-way co-cultures using donor D600115, E:T:iDC::1:1/10:1/4. #9 = CD8α.TCR.WPREmut2; #10 = CD8βα.TCR.WPREmut2; #11 = CD8αCD8βst.TCR.WPREmut2; #12 = CD8α CD8βst.NCAMfu.TCR.WPREmut2; #1 = CD8α.TCR.WPRE ^wt ; No. 2 = CD8βα.TCR.WPRE ^wt ; #8= R11KEA TCR only.

DETAILED DESCRIPTION

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 demonstrate an unexpected improvement in the functionality of T cells cotransduced with a vector expressing a TCR and a CD8 polypeptide.

The CD8 polypeptides described herein may comprise 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.

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

In another embodiment, the CD8 polypeptides described herein may (a) have at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with 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 with 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 in a T cell with a T cell receptor or CAR-T and used in adoptive cell therapy (ACT) methods. The T cell can be an αβ T cell or a γδ T cell.

In another embodiment, the CD8 polypeptides described herein can be (a) SEQ ID NO: 1 comprising having one, two, three, four or five amino acid substitutions; (b) SEQ ID NO: 2 comprising having one, two, three, four or five amino acid substitutions; (c) SEQ ID NO: 3 comprising having one, two, three, four or five amino acid substitutions, and (d) SEQ ID NO: 4 comprising having one, two, three, four or five amino acid substitutions. In one embodiment, the substitutions are conservative amino acid substitutions. The CD8 polypeptides described herein can be co-expressed in a T cell with a T cell receptor or CAR-T and used in adoptive cell therapy (ACT) methods. The T cell can be an αβ T cell or a γδ T cell.

CD8 is a membrane-anchored glycoprotein that acts as a co-receptor for antigen recognition of the peptide/MHC class I complexes by T cell receptors (TCR) and plays an important role in thymic T cell development and T cell activation plays in the periphery. Functional CD8 is a dimeric protein composed of either two α-chains (CD8αα) or one α-chain and one β-chain (CD8αβ), and surface expression of the β-chain may require its association with the co-expressed α-chain, to form the CD8αβ heterodimer. CD8αα and CD8αβ can be differentially expressed on a variety of lymphocytes. CD8αβ is predominantly expressed on the surface of αβTCR ⁺ T cells and thymocytes, and CD8α on a subset of αβTCR ⁺ , γδTCR ⁺ intestinal intraepithelial lymphocytes, NK cells, dendritic cells and a small fraction of CD4 ⁺ T cells.

For example, the human CD8 gene can express a 235 amino acid protein. Figure 12 shows a CD8α protein (CD8α1 - SEQ ID NO:258)) divided in one aspect into the following domains (starting at the amino terminus and ending at the carboxy terminus of the polypeptide): (1) signal peptide (amino acids -21 to -1) , which in human cells can be cleaved off during transport of the receptor to the cell surface and therefore may not be part of the mature, active receptor; (2) Immunoglobulin (Ig)-like domain (in this embodiment, amino acids 1-115), which can adopt a structure termed the immunoglobulin fold, which is similar to many other molecules involved in regulation of the immune system, the Immunoglobulin family of proteins. The crystal structure of the CD8αα receptor in complex with the human MHC molecule HLA-A2 has revealed how the Ig domain of the CD8αα receptor binds the ligand; (3) membrane-proximal region (in this embodiment, amino acids 116-160), which may be an extended linker region that allows the CD8αα receptor to move from the surface of the T cell across the top of the MHC to the a3 domain of the MHC to "grab" where it binds. The stalk region can be glycosylated and inflexible; (4) transmembrane domain (in this embodiment, amino acids 161-188), which can anchor the CD8αα receptor in the cell membrane and is therefore not part of the soluble recombinant protein; and (5) cytoplasmic domain (in this embodiment, amino acids 189-214), which may mediate signaling function in T cells through its association with p56 ^lck , which may be involved in the T cell activation cascade of phosphorylation events.

CD8α sequences can generally have a sufficient portion of the immunoglobulin domain to be able to bind to MHC. In general, CD8α molecules may encode all or a substantial part of the immunoglobulin domain of CD8α, e.g. B. SEQ ID NO: 258, but in one aspect they can be 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 molecules of the present disclosure may preferably be dimers (e.g., CD8αα or CD8αβ), although CD8α monomers may be encompassed within the scope of the present disclosure. In one aspect, CD8α of the present disclosure may include CD8α1 (SEQ ID NO:258) and CD8α2 (SEQ ID NO:259).

CD8α and β subunits may share similar structural motifs, such as an Ig-like domain, a 30-40 amino acid stalk region, a transmembrane region, and a short cytoplasmic domain of approximately 20 amino acids. CD8α and β chains have two and one N-linked glycosylation site, respectively, in the Ig-like domains where they share <20% identity in their amino acid sequences. The CD8ß stalk region is 10-13 amino acids shorter than the CD8α stalk and is highly glycosylated with O-linked carbohydrates. These carbohydrates on the β but not the α stalk region appear to be quite heterogeneous due to complex sialylations that may be differentially regulated during the developmental stages of thymocytes and upon T cell activation. Glycan adducts have been shown to play a regulatory role in glycoprotein functions and immune responses. Glycans proximal to transmembrane domains can influence the orientation of neighboring motifs. The unique biochemical properties of the stalk region of the CD8β chain could represent a plausible candidate for modulation of co-receptor function.

The CD8 polypeptide may be modified in which the CD8α region, e.g. B. a stalk region that can be replaced by the CD8β region. In another aspect to produce a CD8α-CD8β polypeptide. In one embodiment, the modified CD8 polypeptides described herein may have a region that is 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 CD8α polypeptides described herein may have an immunoglobulin (Ig)-like domain of 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 with the amino acid sequence of SEQ ID NO: 1. Modified CD8 polypeptides may have a transmembrane domain that is 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 may have a cytoplasmic tail that is 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 may be 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 comprise a signal peptide which is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 6 fused to the N-terminus or fused to the C-terminus of the mCD8α polypeptide. The CD8 polypeptides described herein may be 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.

T cells

T cells can express the modified CD8 polypeptides described herein. For example, a T cell can co-express a T cell receptor (TCR) and modified CD8 polypeptides described herein. T cells can also express a chimeric antigen receptor (CAR), CAR analogs, or CAR derivatives.

The T cell can be an αβ T cell, γδ T cell, natural killer T cell, or a combination thereof when present in a population. The T cell can be a CD4+ T cell, CD8+ T cell, or a CD4+/CD8+ T cell.

T cell receptors

A T cell may co-express a T cell receptor (TCR), an antigen binding protein, or both with modified CD8 polypeptides described herein, including but not limited to those listed in Table 2 (SEQ ID NOs: 13-90). . Furthermore, a T cell can express TCRs and antigen binding proteins described in US Patent Application Publication No. 2017/0267738; US Patent Application Publication No. 2017/0312350; US Patent Application Publication No. 2018/0051080; US Patent Application Publication No. 2018/0164315; US Patent Application Publication No. 2018/0161396; US Patent Application Publication No. 2018/0162922; US Patent Application Publication No. 2018/0273602; US Patent Application Publication No. 2019/0016801; US Patent Application Publication No. 2019/0002556; US Patent Application Publication No. 2019/0135914; US patent 10,538,573 ; US patent 10,626,160 ; US patent application publication number 2019/0321478 ; US patent application publication number 2019/0256572 ; US patent 10,550,182 ; US patent 10,526,407 ; US patent application publication number 2019/0284276 ; US Patent Application Publication No. 2019/0016802; US Patent Application Publication No. 2019/0016803; US Patent Application Publication No. 2019/0016804; U.S. Patent 10,583,573 ; US patent application publication number 2020/0339652 ; US patent 10,537,624 ; US patent 10,596,242 ; US patent application publication number 2020/0188497 ; US patent 10,800,845 ; US patent application publication number 2020/0385468 ; US patent 10,527,623 ; US patent 10,725,044 ; US patent application publication number 2020/0249233 ; U.S. Patent 10,702,609 ; US patent application publication number 2020/0254106 ; US patent 10,800,832 ; US patent application publication number 2020/0123221 ; US patent 10,590,194 ; U.S. Patent 10,723,796 ; US patent application publication number 2020/0140540 ; US patent 10,618,956 ; US patent application publication number 2020/0207849 ; US patent application publication number 2020/0088726 ; and US patent application publication number 2020/0384028 are described; the contents of each of these publications and the sequence listings described therein are incorporated herein by reference in their entirety. The T cell can be an αβ T cell, γδ T cell, natural killer T cell. Natural killer cell. In one embodiment, the TCRs described herein are single chain TCRs or soluble TCRs.

Furthermore, the TCRs that can be coexpressed with the modified CD8 polypeptides described herein in a T cell can be TCRs composed of an alpha chain (TCR _α ) and a beta chain (TCRβ). The TCRα chains and TCRß chains that can be used in TCRs can be selected from R11KEA (SEQ ID NO: 15 and 16), R20P1H7 (SEQ ID NO: 17 and 18), R7P1D5 (SEQ ID NO: 19 and 20) , R10P2G12 (SEQ ID NO: 21 and 22), R10P1A7 (SEQ ID NO: 23 and 24), R4P1D10 (SEQ ID NO: 25 and 26), R4P3F9 (SEQ ID NO: 27 and 28), R4P3H3 (SEQ ID NO : 29 and 30), R36P3F9 (SEQ ID NO: 31 and 32), R52P2G11 (SEQ ID NO: 33 and 34), R53P2A9 (SEQ ID NO: 35 and 36), R26P1A9 (SEQ ID NO: 37 and 38), R26P2A6 (SEQ ID NO: 39 and 40), R26P3H1 (SEQ ID NO: 41 and 42), R35P3A4 (SEQ ID NO: 43 and 44), R37P1C9 (SEQ ID NO: 45 and 46), R37P1H1 (SEQ ID NO: 47 and 48), R42P3A9 (SEQ ID NO: 49 and 50), R43P3F2 (SEQ ID NO: 51 and 52), R43P3G5 (SEQ ID NO: 53 and 54), R59P2E7 (SEQ ID NO: 55 and 56), R11P3D3 (SEQ ID NO: 57 and 58), R16P1C10 (SEQ ID NO: 59 and 60), R16P1E8 (SEQ ID NO: 61 and 62), R17P1A9 (SEQ ID NO: 63 and 64), R17P1D7 (SEQ ID NO: 65 and 66), R17P1G3 (SEQ ID NO: 67 and 68), R17P2B6 (SEQ ID NO: 69 and 70), R11P3D3KE ( SEQ ID NO: 71 and 72), R39P1C12 (SEQ ID NO: 73 and 74), R39P1F5 (SEQ ID NO: 75 and 76), R40P1 C2 (SEQ ID NO: 77 and 78), R41P3E6 (SEQ ID NO: 79 and 80), R43P3G4 (SEQ ID NO: 81 and 82), R44P3B3 (SEQ ID NO: 83 and 84), R44P3E7 (SEQ ID NO: 85 and 86), R49P2B7 (SEQ ID NO: 87 and 88), R55P1G7 ( SEQ ID NO: 89 and 90), or R59P2A7 (SEQ ID NO: 91 and 92). The T cell can be an αβ T cell, γδ T cell or a natural T killer cell.

Table 1 shows examples of the peptides to which TCRs bind when the peptide is in a complex with an MHC molecule. (In humans, MHC molecules are also referred to as HLA, human leukocyte antigens). Table 1: T cell receptor and peptides TCR designation Peptide (SEQ ID NO:) R20P1H7, R7P1D5, R10P2G12 KVLEHWRV (SEQ ID NO: 215) R10P1A7 KIQEILTQV (SEQ ID NO: 123) R4P1D10, R4P3F9, R4P3H3 FLLDGSANV (SEQ ID NO: 238) R36P3F9, R52P2G11, R53P2A9 ILQDGQFLV (SEQ ID NO: 193) R26P1A9, R26P2A6, R26P3H1, R35P3A4, R37P1C9, R37P1H1, R42P3A9, R43P3F2, R43P3G5, R59P2E7 KVLEYVIKV (SEQ ID NO: 202) R11KEA, R11P3D3, R16P1C10, R16P1E8, R17P1A9, R17P1D7, R17P1G3, R17P2B6, R11P3D3KE SLLQHLIGL (SEQ ID NO: 147) R39P1C12, R39P1F5, R40P1C2, R41P3E6, R43P3G4, R44P3B3, R44P3E7, R49P2B7, R55P1G7, R59P2A7 ALSVLRLAL (SEQ ID NO: 248)

Tumor-Associated Antigens (TAA)

Tumor-associated antigen (TAA) peptides can be used with the CD8 polypeptide constructs, methods, and embodiments described herein. For example, the T cell receptors (TCRs) described herein can specifically bind to the TAA peptide when bound to a human leukocyte antigen (HLA). This is also known as the major histocompatibility complex (MHC) molecule. Human MHC molecules are also known as human leukocyte antigens (HLA).

Tumor-associated antigen (TAA) peptides that can be used with the CD8 polypeptides described herein include, but are not limited to, those listed in Table 3 and the TAA peptides described in US Patent Application Publication No. 2016/0187351; US Patent Application Publication No. 2017/0165335; US Patent Application Publication No. 2017/0035807; US Patent Application Publication No. 2016/0280759; US Patent Application Publication No. 2016/0287687; US Patent Application Publication No. 2016/0346371; US Patent Application Publication No. 2016/0368965; US Patent Application Publication No. 2017/0022251; US Patent Application Publication No. 2017/0002055; US Patent Application Publication No. 2017/0029486; US Patent Application Publication No. 2017/0037089; US Patent Application Publication No. 2017/0136108; US Patent Application Publication No. 2017/0101473; US Patent Application Publication No. 2017/0096461; US Patent Application Publication No. 2017/0165337; US Patent Application Publication No. 2017/0189505; US Patent Application Publication No. 2017/0173132; US Patent Application Publication No. 2017/0296640; US Patent Application Publication No. 2017/0253633; US Patent Application Publication No. 2017/0260249; US Patent Application Publication No. 2018/0051080; US patent application publication number 2018/0164315 ; US patent application publication number 2018/0291082 ; US patent application publication number 2018/0291083 ; US patent application publication number 2019/0255110 ; US patent 9,717,774 ; U.S. Patent 9,895,415; US patent application publication number 2019/0247433 ; US patent application publication number 2019/0292520 ; US patent application publication number 2020/0085930 ; US patent 10,336,809 ; US patent 10,131,703 ; US patent 10,081,664 ; U.S. Patent 10,093,715 ; U.S. Patent 10,583,573 ; and US Patent Application Publication No. 2020/00085930; the contents of each of these publications and the sequence listings described therein are incorporated herein by reference in their entirety. The tumor-associated antigen (TAA) peptides described herein can be bound to an HLA (MHC molecule). The tumor-associated antigen (TAA) peptides bound to an HLA can be recognized by a TCR as described herein, which is optionally co-expressed with CD8 polypeptides as described herein.

T cells can be engineered to express a chimeric antigen receptor (CAR) comprising a ligand binding domain derived from NKG2D, NKG2A, NKG2C, NKG2F, LLT1, AICL, CD26, NKRP1, NKp30, NKp44, NKp46, CD244 (2B4 ), DNAM-1 and NKp80, or an anti-tumor antibody such as anti-Her2neu or anti-EGFR and a signaling domain derived from CD3-ζ, Dap 10, CD28, 4-IBB and CD40L. In some examples, the chimeric receptor binds 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, EGFRvlll, GPMNB, LIV -1, GlycolipidF77, Fibroblast Activating 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 (such as MAGE3A. MAGE4A), KKLC1, mutated 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, γδ T cell or a natural T killer cell.

Cultivation of T cells

Methods for activating, transducing and/or expanding T cells, e.g. B. tumor infiltrating lymphocytes, CD8 + T cells, CD4 + T cells and T cells that can be used for transgene expression are described herein. T cells can be activated, transduced and expanded, while α- and/or β-TCR positive cells are depleted. The T cell can be an αβ T cell, γδ T cell or a natural T killer cell.

Methods for the ex vivo expansion of a population of modified γδ T cells for adoptive transfer therapy are described herein. Modified γδ T cells of the disclosure can be expanded ex vivo. Modified T cells as described herein can be expanded in vitro without activation by APCs or without co-culture with APCs and aminophosphates. Methods for transducing T cells are described in U.S. Patent Application No. 2019/0175650 which was published on June 13, 2019, the contents of which are incorporated by reference in their entirety. Other methods of transducing and culturing T cells can be used.

T cells, including γδ T cells, can be isolated from a complex sample that is cultured in vitro. In one embodiment, the entire 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 one embodiment, enriched T cell populations can be generated prior to their specific activation and expansion. In one embodiment, activation and expansion of γδ T cells can be performed with or without the presence of native or engineered antigen presenting cells (APCs). In one embodiment, isolation and expansion of T cells from tumor samples can be performed using immobilized T cell mitogens comprising antibodies specific for γδ TCR and other γδ TCR activating agents, including lectins. In one embodiment, the isolation and expansion of γδ T cells from tumor specimens can be performed in the absence of γδ T cell mitogens, including γδ TCR specific antibodies and other γδ TCR activating agents, including lectins.

T cells, including γδ T cells, can be derived from leukapheresis in a subject, e.g. B. a human being isolated. In one embodiment, γδ T cells are not isolated from peripheral blood mononuclear cells (PBMC). The T cells can be isolated using anti-CD3 and anti-CD28 antibodies, optionally with recombinant human interleukin-2 (rhIL-2), e.g. B. between about 50 and 150 U/mL rhIL-2.

The isolated T cells can expand rapidly in response to exposure to one or more antigens. Some γδ T cells, such as Cells such as Vγ9Vδ2+ T cells can rapidly expand in vitro in response to exposure to some antigens such as prenyl pyrophosphates, alkylamines and metabolites or microbial extracts during tissue culture. Stimulated T cells can have numerous antigen presenting, costimulatory, and adhesion molecules that can facilitate the 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 other suitable length of time. Stimulating T cells with an appropriate antigen can expand the T cell population in vitro.

Activation and expansion of γδ T cells can be performed with the activating and costimulating agents described herein to induce specific γδ T cell proliferation and persistence populations. In one embodiment, different clonal or mixed polyclonal population subsets can be achieved by activating and expanding γδ T cells from different cultures. In one embodiment, different agonist substances can be used to identify agents that deliver specific γδ-activating signals. In one embodiment, various monoclonal antibodies (MAbs) directed against the γδ TCRs can be substances that deliver specific γδ-activating signals. In one embodiment, adjunctive co-stimulatory substances can be used to help trigger specific γδ T cell proliferation without inducing cell energy and apoptosis. These costimulatory agents may include ligands that bind to receptors expressed on γδ cells, such as. B. NKG2D, CD161, CD70, JAML, DNAX accessory molecule-1 (DNAM-1), ICOS, CD27, CD137, CD30, HVEM, SLAM, CD122, DAP and CD28. In one embodiment, costimulatory substances can be antibodies specific for unique epitopes on CD2 and CD3 molecules. CD2 and CD3 may have different conformational structures when expressed on αβ or γδ T cells. In one embodiment, specific antibodies against CD3 and CD2 can result in marked activation of γδ T cells.

Non-limiting examples of antigens that can be used to stimulate expansion of T cells, including γδ T cells, from a complex sample in vitro can include prenyl pyrophosphates such as isopentenyl pyrophosphate (IPP), alkylamines, human microbial metabolites 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 (TUBAg 1), X-pyrophosphate (TUBAg 2), 3-formyl-1-butyl-uridine triphosphate (TUBAg 3 ), 3-formyl-1-butyl-deoxythymidine triphosphate (TUBAg 4), monoethylalkylamines, allyl pyrophosphate, crotoyl pyrophosphate, dimethylal lyl-γ-uridine triphosphate, crotoyl-γ-uridine triphosphate, allyl-γ-uridine triphosphate, ethylamine, isobutylamine, sec-butylamine, isoamylamine and nitrogenous bisphosphonates.

A population of T cells, including γδ T cells, can be expanded ex vivo prior to modification of the T cells. A non-limiting example of reagents that can be used to promote expansion of a T cell population in vitro can be anti-CD3 or anti-CD2, anti-CD27, anti-CD30, anti-CD70, anti-OX40 antibodies , IL-2, IL-15, IL-12, IL-9, IL-33, IL-18 or IL-21, CD70 (CD27 Ligand), Phytohemagglutinin (PHA), Concavalin A (ConA), Pokeweed (PWM ), Peanut Agglutinin (PNA), Soybean Agglutinin (SBA), Lentil 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. Furthermore, the T cells with MCSF, IL-6, eotaxin, IFN-alpha, IL-7, gamma-induced protein 10, IFN-gamma, IL-1RA, IL-12, MIP-1alpha, IL-2, IL-13 , MIP-1beta, IL-2R, IL-15 and combinations thereof.

The ability of γδ T cells to recognize a wide range of antigens can be improved by genetic modification of γδ T cells. The γδ T cells can be engineered to enable universal allogeneic therapy that recognizes an antigen of choice in vivo. Genetic modification of γδ T cells can allow stable integration of a construct expressing a tumor-recognizing entity, such as e.g. B. αβ TCR, γδ TCR, chimeric antigen receptor (CAR) combining both antigen-binding and T-cell activating functions in a single receptor, an antigen-binding fragment thereof, or a lymphocyte-activating domain into the genome of the isolated T-cell(s). ), a cytokine (e.g. IL-15, IL-12, IL-2, IL-7, IL-21, IL-18, IL-19, IL-33, IL-15, IL-12, IL-2 , IL1β) to enhance T cell proliferation, survival and function ex vivo and in vivo. The genetic modifi cation of the isolated γδ T cell can also be the deletion or disruption of gene expression of one or more endogenous genes in the genome of the isolated γδ T cells, such as. the MHC locus (loci).

Modified (or transduced) T cells, including γδ T cells, can be expanded ex vivo without stimulation by an antigen presenting cell or an aminobisphosphonate. Antigen-reactive modified T cells of the present disclosure can be expanded ex vivo and in vivo. In one embodiment, an active population of modified T cells can be generated ex vivo without antigen stimulation by an antigen presenting cell, an antigenic peptide, a non-peptide molecule or a small molecule such as e.g. B. an aminobisphosphonate, but using certain 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 in expanding a γδ T cell population include anti-CD3, anti-CD27, anti-CD30, anti-CD70, anti-OX40, anti-NKG2D, or anti-CD2 antibodies, examples of cytokines may include IL-2, IL-15, IL-12, IL-21, IL-18, IL-9, IL-7 and/or IL-33, and examples of mitogens Can CD70, the ligand for human CD27, phytohemagglutinin (PHA), conavalin A (ConA), pokeweed mitogen (PWM), peanut agglutinin (PNA), soybean agglutinin (SBA), lentil 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.

A population of modified T cells, including γδ T cells, can be expanded in 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 one embodiment, a population of modified T cells can grow from about 7 days to about 49 days, from about 7 days to about 42 days, from about 7 days to about 35 days, from about 7 days to about 28 days, from about 7 days to about 21 days or from about 7 days to about 14 days. The T cells can be expanded between about 1 and 21 days. For example, the T cells may be expanded for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days will.

In one embodiment, the same methodology can be used to isolate, activate, and expand αβ T cells.

In one embodiment, the same methodology can be used to isolate, activate, and expand γδ T cells.

vectors

Modified T cells can be generated by a variety of methods, including methods recognized in the literature. For example, a polynucleotide encoding an expression cassette containing tumor recognition or some other type of recognition moiety can be stably introduced into the T cell by a transposon/transposase system or a viral-based gene delivery system, such as a lentiviral or retroviral system , or other suitable method such as transfection, electroporation, transduction, lipofection, calcium phosphate (CaPO ₄ ), nanoengineered substances such as Ormosil, viral delivery methods including adenoviruses, retroviruses, lentiviruses, adeno-associated viruses, or other suitable method. A number of viral methods have been used for human gene therapy, such as those in WO 1993/020221 described procedures, the content of which is hereby incorporated in its entirety. Non-limiting examples of viral methods that can be used to generate T cells could include γ-retroviral, adenoviral, lentiviral, herpes simplex virus, vaccinia virus, pox virus, or adenovirus-associated viral methods. The T cells can be αβ T cells or γδ T cells.

Viruses used for transfection of T cells include both naturally occurring viruses and artificial viruses. Viruses can be either an enveloped or non-enveloped virus. Parvoviruses (such as AAVs) are examples of non-enveloped viruses. The viruses can be enveloped viruses. The viruses used for the transfection of T cells can be retroviruses and in particular lentiviruses. The viral envelope proteins that can promote viral infection of eukaryotic cells can be HIV-1-derived lentiviral vectors (LVs) transfected with envelope glycoproteins (GPs) from vesicular stomatitis virus (VSV-G), the modified endogenous feline retrovirus (RD114TR ) (SEQ ID NO: 97), and modified gibbon simian leukemia virus (GALVTR). These envelope proteins can efficiently promote the entry of other viruses, such as parvoviruses, including adeno-associated viruses (AAV), demonstrating their broad effectiveness. For example, other viral envelope proteins ver including Moloney murine leukemia virus (MLV) 4070 env (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 Coat protein RD114pro or RDpro (which is an RD114-HIV chimera modified by replacing the R-peptide cleavage sequence of RD114 with the HIV-1 matrix/capsid (MA/CA) cleavage sequence as described in Bell et al. Described in Experimental Biology and Medicine 2010;235:1269-1276; the contents of which are incorporated herein by reference), Baculovirus GP64 env (as described in Wang et al. J. Virol. 81:10869-10878, 2007; the contents of which are incorporated by reference incorporated herein), or GALV env (as described in Merten et al. J. Virol. 79:834-840, 2005; the contents of which are incorporated herein by reference), or derivatives thereof.

A single lentiviral cassette can be used to produce a single lentiviral vector expressing at least four individual monomeric proteins from 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 the lentiviral vector was sufficient to transform T cells to co-express TCRαβ and CD8αβ, optionally αβ T cells or γδ T cells.

Vectors can comprise a multicistronic cassette 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, the polypeptides encoded by these genes interacting with one another or dimers can form. The dimers can be homodimers, e.g. B. two identical proteins forming a dimer, or heterodimers, e.g. B. two structurally different proteins forming a dimer.

In addition, several vectors can be used to transfect cells with the constructs and sequences described herein. For example, the TCR transgene may be on one vector and the CD8 transgene encoding a polypeptide described herein on a second, transfected either simultaneously or sequentially using established methods. A T cell line can be stably transfected with a CD8 transgene encoding a CD8 polypeptide described herein and then sequentially transfected with a TCR transgene or visa verse.

The constructs and vectors described herein were made using US patent application publication no 2019/0175650 methodology described, published on June 13, 2019, the contents of which are incorporated by reference in their entirety.

Non-viral vectors can also be used with the sequences, constructs and cells described herein.

The cells can be transfected by other means known in the art, including lipofection (liposome-based transfection), electroporation, calcium phosphate transfection, biolistic particle delivery (e.g., gene guns), microinjection, or combinations thereof. Various methods for transfecting cells are known in the prior art. See e.g. B., 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.

compositions

The compositions may comprise the modified CD8 polypeptides described herein. Furthermore, the compositions described herein may comprise a T cell expressing the CD8 polypeptides described herein. The compositions described herein may comprise a T cell expressing CD8 polypeptides described herein and a T cell receptor (TCR), optionally a TCR, which specifically binds any of the TAAs described herein complexed with an antigen presenting protein , e.g. MHC, referred to as HLA - human leukocyte antigen - in humans.

In order to facilitate administration, the T cells described herein can be processed with pharmaceutically acceptable carriers or diluents into a pharmaceutical composition or implant suitable for administration in vivo. The means of making such a composition or implant are described in the art. See e.g. B., Remington's Pharmaceutical Sciences, 16th Ed., Mack, ed. (1980).

The T-cells described herein can be formulated into a semi-solid or liquid form preparation, e.g. B. a capsule, solution, infusion or injection.

Means known in the art can be used to prevent or minimize the release and absorption of the composition until it reaches the target tissue or organ, or to provide a time-limited release of the composition. However, it is desirable that a pharmaceutically acceptable form that does not prevent the cells from expressing the CAR or TCR is used. Desirably, the T cells described herein can be made into a pharmaceutical composition containing a carrier. The T cells described herein can be formulated with a physiologically acceptable carrier or excipient to produce a pharmaceutical composition. The carrier and composition can be sterile. Preferred carriers include e.g. B. Balanced Salt Solution, preferably Hanks' Balanced Salt Solution, or normal saline. The formulation should suit the mode of administration. Suitable pharmaceutically acceptable carriers include, but are not limited to: Water, saline solutions (e.g., NaCl), saline, buffered saline, and combinations thereof. If desired, excipients, e.g. B. lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts to influence the osmotic pressure, buffers that do not react deleteriously with the T-cells. The T cells may be αβ T cells or γδ T cells expressing CD8 polypeptides as described herein, optionally a TCR as described herein.

A composition of the present invention may be provided in unit dosage form, each dosage unit, e.g. an injection, containing a predetermined amount of the composition, alone or in suitable combination with other active ingredients.

The compositions described herein can be a pharmaceutical composition. The pharmaceutical composition 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 a combination thereof.

The pharmaceutical composition described herein may contain an adjuvant selected from the group consisting of colony stimulating factors, e.g. granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod and resiquimod.

Preferred adjuvants include cyclophosphamide, imiquimod, or resiquimod, among others. Even more preferred adjuvants are Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, poly-ICLC (Hiltonol®), and anti-CD40 mAB or combinations thereof.

Other examples of useful adjuvants include, but are not limited to, modified CpGs (e.g., CpR, Idera), dsRNA analogs such as poly(I:C), and derivatives thereof (e.g., AmpliGen®, Hiltonol®, poly(ICLC), poly(IC-R), poly(I:C12U)), non-CpG bacterial DNA or RNA, and immunoactive small molecules and antibodies such as cyclophosphamide, sunitinib, immune checkpoint inhibitors including ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and cemiplimab , Bevacizumab®, Celebrex, NCX-4016, Sildenafil, Tadalafil, Vardenafil, Sorafenib, Temozolomide, Temsirolimus, XL-999, CP-547632, Pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodies targeting key structures of the immune system (e.g. anti-CD40, anti-TGFbeta, anti-TNFalpha receptor), and SC58175, which may act therapeutically and/or as an adjuvant. The amounts and concentrations of the adjuvants and additives useful in the context of the present invention can be readily determined by those skilled in the art without undue experimentation.

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 particulate formulations containing poly(lactide-co-glycolide) (PLG), polyinosine-polycytidylic acid-poly-I-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. B. 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.

The composition described herein may also contain one or more adjuvants. Adjuvants are substances that non-specifically enhance or potentiate the immune response (e.g., CD8-positive T-cell, and T-helper (TH) cell-mediated immune responses against an antigen) and thus are considered useful in a medicament of the present invention. Suitable adjuvants include, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin, or TLR5 ligand derived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod (ALDARA®), Resiquimod, ImuFact IMP321, Interleukins such as IL-2, IL-13, IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, IS Patch, ISS, ISCOMATRIX, ISCOMs, Juvlmmune, LipoVac, MALP2, MF59, Monophosphoryl Lipid A, Montanid IMS 1312, Montanid ISA 206, Montanid ISA 50V, Montanid ISA-51, water-in-oil and oil-in-water emulsions, OK-432, OM-174 , OM-197-MP-EC, ONTAK, OspA, PepTel® Vectorsystem, Poly(lactide co-glycolide) [PLG]-based and dextran microparticles, Talactoferrin, SRL172, Virosomes and other virus-like particles, YF-17D, VEGF trap , R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon derived from saponin, extracts of mycobacteria and synthetic bacterial cell wall mimics and proprietary adj uvantie such as Ribi's Detox, Quil or Superfos. Adjuvants such as Freund's or GM-CSF are preferred. Various immunological adjuvants (e.g. MF59) specific for dendritic cells and their generation have been described previously. Cytokines can also be used. Various cytokines have been directly implicated in influencing the migration of dendritic cells to 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) ( US Pat. No. 5,849,589 incorporated by reference in its entirety) and function as an immunoadjuvant (e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha, IFN-beta).

It has also been described that CpG immunostimulatory oligonucleotides enhance the effect of adjuvants in a vaccine composition. Without being bound by theory, CpG oligonucleotides act by activating the innate (non-adaptive) immune system via Toll-like receptors (TLR), primarily TLR9. CpG-triggered 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 increased activation of TH1 cells and potent generation of cytotoxic T lymphocytes (CTL), even in the absence of CD4 T cell help. TH1 imprinting 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 TH2 imprinting. CpG oligonucleotides exhibit even greater adjuvant activity when formulated with other adjuvants or co-administered in formulations such as microparticles, nanoparticles, lipid emulsions, or similar formulations that are particularly necessary to induce a strong response when the antigens are relatively are weak. They also accelerate the immune response and allow the antigen dose to be reduced by about two orders of magnitude, while the antibody responses to the full-dose vaccine without CpG are comparable in some experiments (War, 2006). in the U.S. Patent No. 6,406,705 B1 describes the combined use of CpG oligonucleotides, non-nucleic acid adjuvants and an antigen to generate an antigen-specific immune response. A CpG TLR9 antagonist is dSLIM (double stem loop immunomodulator) from Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical composition of the present invention. Other TLR-binding molecules such as RNA-binding TLR 7, TLR 8 and/or TLR 9 can also be used.

Methods of treatment and preparation

Modified T cells can express the modified CD8 polypeptides described herein. Furthermore, the modified T cells can express a TCR as described herein. The TCR expressed by the modified T cells can recognize a TAA linked to an HLA, as described herein. Modified T-cells of the present disclosure can be used to treat a subject who, due to a condition, e.g. B. a cancer disease described herein, needs treatment. The T cells may be αβ T cells or γδ T cells expressing a modified CD8 polypeptide, optionally a TCR as described herein.

A method of treating a condition (e.g., a condition) in a subject having T cells as described herein may comprise administering to the subject a therapeutically effective amount of modified T cells, optionally γδ T cells, as described herein. Those described here T cells can be administered according to a variety of schedules (e.g., timing, concentration, dosage, spacing between treatments, and/or formulation). A subject can also z. B. be preconditioned with chemotherapy, radiation or a combination of both before receiving modified T cells of the present disclosure. A population of modified T cells can also be frozen or cryopreserved prior to administration to a subject. A population of modified T cells may contain two or more cells expressing identical, different, or a combination of identical and different tumor recognition units. For example, a population of modified T cells may contain several different modified 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 as described herein, optionally a TCR as described herein.

The T cells described herein, including αβ T cells and γδ T cells, can be used to treat a variety of diseases. The T cells may 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 cancers include: acute lymphoblastic leukemia, acute myeloid leukemia, adrenal cortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendectomy, astrocytoma, neuroblastoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors such as e.g. B. Cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, optic pathway glioma and hypothalamic glioma, breast cancer, bronchial adenomas, Burkitt's lymphoma, carcinoma of unknown primary origin, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, Childhood cancers, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial carcinoma, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumors, gallbladder cancer, gastric cancer, gastrointestinal Carcinoid tumor, gastrointestinal stromal tumor, glioma, hairy cell leukemia, head and neck cancer, cardiac cancer, hepatocellular (liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, kidney cancer, larynx cancer, lip and oral cavity cancer , liposarcoma, liver cancer, lung cancer, such as B. non-small cell and small cell lung cancer, lymphoma, leukemia, macroglobulinemia, malignant fibrous histiocytoma of bone / osteosarcoma, medulloblastoma, melanoma, mesothelioma, metastatic squamous cell cancer with occult primary tumor, oral cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndromes, myeloid leukemia, nasal cavities - and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma/malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial carcinoma, ovarian germ cell tumor, pancreatic cancer, pancreatic islet cell cancer, paranasal sinuses - and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, astrocytoma of the epiphysis, germinoma of the epiphysis, pituitary adenoma, pleuropulmonary blastoma, plasma cell neoplasia, primary central nervous system lymphoma, prostate cancer, rectal cancer inoma, renal cell carcinoma, renal pelvis and ureteral transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, skin cancer, Merkel cell skin carcinoma, small bowel cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, T-cell lymphoma, pharyngeal cancer, thymoma, thymus carcinoma, thyroid cancer, trophoblastic Tumor (pregnancy tumor), cancers of unknown primary site, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

The T cells described here can be used to treat an infectious disease. The T cells described herein can be used to treat an infectious disease, where an infectious disease can be caused by a virus. The T cells described herein can be used to treat an immune disease, such as. B. an autoimmune disease can be used. The T cells may be αβ T cells or γδ T cells expressing a CD8 polypeptide, optionally a TCR as described herein.

Treatment with the T cells described herein, optionally with γδ T cells, can be administered to the subject before, during and after the clinical onset of the disease. Treatment can be granted 1 day, 1 week, 6 months, 12 months or 2 years after the clinical onset of the disease. Treatment may last more than 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 granted after the clinical onset of the disease. Treatment can be for less than 1 day, 1 week, 1 month, 6 months, 12 months or 2 years after the clinical onset of the disease. Treatment may also involve treating a human in a clinical trial. Treatment may include administering to a subject a pharmaceutical composition comprising modified T cells as described herein. The T cells may be αβ T cells or γδ T cells expressing a CD8 polypeptide, optionally a TCR as described herein.

In one embodiment, administration of modified T cells of the present disclosure to a subject can modulate the activity of endogenous lymphocytes in the subject's body. In one embodiment, administration of modified T cells to a subject can deliver an antigen to an endogenous T cell and enhance an immune response. In one embodiment, the memory T cell can be a CD4+ T cell. In one embodiment, the memory T cell can be a CD8+ T cell. In one embodiment, administration of modified T cells of the present disclosure to a subject can activate cytotoxicity of another immune cell. In one embodiment, the other immune cell can be a CD8+ T cell. In one embodiment, the other immune cell can be a natural killer T cell. In one embodiment, administration of modified γδ T cells of the present disclosure to a subject can suppress a regulatory T cell. In one embodiment, the regulatory T cell can be a FOX3+ Treg cell. In one embodiment, the regulatory T cell can be a FOX3 Treg cell. Non-limiting examples of cells whose activity can be modulated by modified T cells of the present disclosure can include: hematopoietic stem cells; B cells; CD4; CD8; Red blood cells; White blood cells; dendritic cells, including dendritic antigen presenting cells; leukocytes; macrophages; memory B cells; memory T cells; monocytes; natural killer cells; neutrophilic 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.

In most bone marrow transplants, a combination of cyclophosphamide with total body irradiation can be used conventionally to prevent rejection of the hematopoietic stem cells (HSC) in the transplant by the subject's immune system. In one embodiment, incubation of donor bone marrow with interleukin-2 (IL-2) can be performed ex vivo to promote the formation of killer lymphocytes in the donor marrow. Interleukin-2 (IL-2) is a cytokine that may be necessary for the growth, proliferation, and differentiation of wild-type lymphocytes. Current studies of adoptive transfer of γδ T cells into humans may require co-administration of γδ T cells and interleukin-2. However, both low and high doses of IL-2 can cause highly toxic side effects. IL-2 toxicity can manifest itself in multiple organs/systems, most significantly the heart, lungs, kidneys and central nervous system. In one embodiment, the disclosure provides a method of administering modified T cells to a subject without co-administering a native cytokine or modified versions thereof, such as IL-2, IL-15, IL-12, IL-21. In one embodiment, modified T cells can be administered to a subject without the co-administration of IL-2. In one embodiment, administration of modified T cells to a subject during a procedure, e.g. B. a bone marrow transplant, without the concomitant administration of IL-2.

In one embodiment, the methods can further comprise administering a chemotherapeutic agent. The dosage of the chemotherapy drug may be sufficient to deplete the patient's T cell population. Chemotherapy can be administered approximately 5-7 days prior to T cell administration. The chemotherapy drug can be cyclophosphamide, fludarabine, or a combination thereof. The chemotherapeutic agent can include a dosage of about 400-600 mg/m ² /day of cyclophosphamide. The chemotherapeutic agent may include a dosage of about 10-30 mg/m ^{2 /} day of fludarabine.

In one embodiment, the methods may further comprise pretreating the patient with low dose radiation prior to administering the composition comprising T cells. The low dose irradiation may include about 1.4 Gy for 1-6 days, preferably about 5 days, prior to administration of the T cell-comprising composition.

In one embodiment, the patient may be HLA-A*02.

In one embodiment, the patient may be HLA-A*06.

In one embodiment, the methods can further comprise administering an anti-PD1 antibody. The anti-PD1 antibody can be a humanized antibody. The anti-PD1 antibody can be pembrolizumab. The dosage of the anti-PD1 antibody can be about 200 mg. The anti-PD1 antibody can be administered every 3 weeks after T cell administration.

In one embodiment, the dosage of the T cells can be between about 0.8-1.2 x 10 ⁹ T cells. The dosage of the T cells can be from about 0.5×10 ⁸ to about 10×10 ⁹ T cells. The dosage of T cells can be about 1.2-3x10 ⁹ T cells, about 3-6x10 ⁹ T cells, about 10x10 ⁹ T cells, about 5x10 ⁹ T cells, about 0.1 × 10 ⁹ TT cells, about 1 × 10 ⁸ T cells, about 5 × 10 ⁸ T cells, about 1.2-6 × 10 ⁹ T cells, about 1-6 × 10 ⁹ T- cells or about 1-8 x 10 ⁹ T cells.

In one embodiment, the T cells can be administered in 3 doses. T cell doses can escalate with each dose. The T cells can be administered by intravenous infusion.

In one embodiment, the CD8 sequences described herein and associated products and compositions can be used for autologous or allogeneic methods of adoptive cell therapy. Alternatively, CD8 sequences, T cells thereof, and compositions can be used, for example, in methods disclosed in US Patent Application Publication 2019/0175650 , the publication of the US patent application 2019/0216852 , the publication of the US patent application 2019/024743 and US provisional patent application 62/980,844 , each of which is incorporated by reference in its entirety.

The disclosure also provides a population of modified T cells displaying an exogenous CD8 polypeptide as described herein and a T cell receptor, wherein the population of modified T cells activates and expands with a combination of IL-2 and IL-15 becomes. In another embodiment, the modified T cell population is expanded and/or activated with a combination of IL-2, IL-15 and zoledronate. In another embodiment, the modified T cell population is activated with a combination of IL-2, IL-15 and zoledronate and expanded with a combination of IL-2, IL-15 and no zoledronate. The disclosure further contemplates the use of other interleukins during activation and/or expansion, such as IL-12, IL-18, IL-21, and combinations thereof.

In one aspect, IL-21, a histone deacetylase inhibitor (HDACi), or combinations thereof, can be used in the field of cancer treatment with the methods described herein and/or with the ACT methods described herein. In one embodiment, the present disclosure provides methods for reprogramming effector T cells to a central memory phenotype, comprising culturing the effector T cells with at least one HDACi along 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.

Compositions comprising modified T cells can be administered for prophylactic and/or therapeutic treatments. In therapeutic uses, pharmaceutical compositions can be administered to a person already suffering from a disease or condition in an amount sufficient to cure or at least partially reverse the symptoms of the disease or condition. A modified T cell can also be administered to reduce the likelihood that a condition will develop, contract, or worsen. The effective amount of a population of modified T cells for therapeutic use may vary depending on the severity and course of the disease or condition, previous therapy, the patient's medical condition, their weight and/or response to the drugs, and/or the judgment of the patient treating physician vary. The T cells can be αβ T cells or γδ T cells modified to express the modified CD8 polypeptides described herein and optionally a TCR described herein. T-cell therapy has proven itself in the treatment of various types of cancer. Li et al. Signal Transduction and Targeted Therapy 4(35): (2019), the contents of which are incorporated in their entirety by reference.

Method of Administration

One or more modified T cell populations described herein can be administered to a patient in any order or simultaneously. When concurrently, the multiply modified T cell can be assembled in a single, uniform form, e.g. as an intravenous injection, or in several forms, e.g. B. administered as multiple intravenous infusions, subcutaneous injections or pills. Modified T cells can be packaged together or separately, in a single package or in multiple packages packaging. Any or all of the modified T cells can be administered in multiple doses. If not simultaneously, the timing between the multiple doses can vary by up to a week, a month, two months, three months, four months, five months, six months, or about a year. In one embodiment, the modified T cells are capable of expanding in the body in vivo after administration to a subject. Modified T cells can be frozen to provide cells for multiple treatments with the same cell preparation. Modified T cells of the present disclosure and pharmaceutical compositions comprising them can be packaged as a kit. A kit may include instructions (e.g., written instructions) about the use of modified T cells and compositions comprising them.

A method of treating a cancer can include administering to a subject a therapeutically effective amount of modified T cells, which administration treats the cancer. In one embodiment, the therapeutically effective amount of modified γδ T cells can last 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 will. In one embodiment, the therapeutically effective amount of modified T cells can be administered for at least one week. In one embodiment, the therapeutically effective amount of the modified T cells can be administered for at least two weeks.

The modified 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 a pharmaceutical composition comprising a modified T cell can vary. For example, modified T cells can be used as prophylaxis and administered continuously to subjects with a predisposition to the condition or disease to reduce the likelihood of the disease or condition occurring. Modified T cells can be administered to a subject during or as soon as possible after the onset of symptoms. Modified T cell administration can be administered immediately during symptom onset, within the first 3 hours after symptom onset, within the first 6 hours after symptom onset, within the first 24 hours after symptom onset, within Be initiated 48 hours after symptom onset or within any time period after symptom onset. Initial administration can be by any practicable route, e.g. B. in any way described herein with any formulation described herein. In one embodiment, administration of modified T cells of the present disclosure may be intravenous administration. One or more doses of modified T cells can be administered as soon as practically practicable after the onset of cancer, infectious disease, immune disease, sepsis, or with bone marrow transplantation, for a period of time necessary to treat the immune disease, such as 10 days . B. 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. For the treatment of cancer, one or more doses of modified T cells can be administered years after the onset of the cancer and before or after other treatments. In one embodiment, modified γδ T cells can last 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 length of treatment may vary for each patient. The T cells may be αβ T cells or γδ T cells expressing a CD8 polypeptide as described herein, optionally a TCR as described herein.

Modified T cells expressing a CD8 polypeptide as described herein, optionally αβ T cells or γδ T cells, may be present in a composition in an amount of at least 1×10 ³ cells/ml, at least 2×10 ³ cells/ml , at least 3×10 ³ cells/ml, at least 4×10 ³ cells/ml, at least 5×10 ³ cells/ml, at least 6×10 ³ cells/ml, at least 7×10 ³ cells/ml, at least 8×10 ³ cells/mL, at least 9×10 ³ cells/mL, at least 1×10 ⁴ cells/mL, at least 2×10 ⁴ cells/mL, at least 3×10 ⁴ cells/mL, at least 4×10 ⁴ cells/mL, at least 5×10 ⁴ cells/ml, at least 6×10 ⁴ cells/ml, at least 7×10 ⁴ cells/ml, at least 8×10 ⁴ cells/ml, at least 9×10 ⁴ cells/ml, at least 1×10 ⁵ cells/ml, at least 2×10 ⁵ cells/ml, at least 3×10 ⁵ cells/ml, at least 4×10 ⁵ cells/ml, at least 5×10 ⁵ cells/ml, at least 6×10 ⁵ cells/ml, at least 7×10 ⁵ cells/ml, at least 8×10 ⁵ cells/ml, at least 9×10 ⁵ cells/ml, at least 1×10 ⁶ cells/ml, at least 2x10 ⁶ cells/mL, at least 3x10 ⁶ cells/mL, at least 4x10 ⁶ cells/mL, at least 5x10 ⁶ cells/mL, at least 6x10 ⁶ cells/mL, at least 7x10 ⁶ cells /ml, at least 8×10 ⁶ cells/ml, at least 9×10 ⁶ cells/ml, at least 1×10 ⁷ cells/ml, at least 2×10 ⁷ cells/ml, at least 3×10 ⁷ cells/ml, at least 4 ×10 ⁷ cells/mL, at least 5×10 ⁷ cells/mL, at least 6×10 ⁷ cells/mL, at least 7×10 ⁷ cells/mL, at least 8×10 ⁷ cells/mL, at least 9×10 ⁷ cells/mL ml, at least 1×10 ⁸ cells/ml, at least 2×10 ⁸ cells/ml, at least 3×10 ⁸ cells/ml, at least 4×10 ⁸ cells/ml, at least 5×10 ⁸ cells/ml, at least 6× 10 ⁸ cells/mL, at least 7x10 ⁸ cells/mL, at least 8x10 ⁸ cells/mL, at least 9x10 ⁸ cells/mL, at least 1x10 ⁹ cells/mL, or more, of about 1x10 ³ cells/mL to about at least 1 ×10 ⁸ cells/ml, from about 1×10 ⁵ cells/ml to about at least 1×10 ⁸ cells/ml, or from about 1×10 ⁶ cells/ml to about at least 1×10 ⁸ cells/ml.

sequences

The sequences described herein can have about 80%, about 85%, about 90%, about 85%, about 96%, about 97%, about 98% or about 99% or 100% identity with the sequence of SEQ ID NO: 1 - 97 and 256 - 266. The sequences described herein can be 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 the sequence of SEQ ID NO: 1 - 97 and 256 - 266. A sequence "at least 85% identical to a reference sequence" is a sequence that is 85% or more, in particular 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, over its entire length , 98%, 99% or 100% sequence identity with the entire length of the reference sequence.

In another embodiment, the disclosure provides sequences that are 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% with WPREmut1 (SEQ ID NO: 256) or WPRE version 2, e.g. B. WPREmut2 (SEQ ID NO: 257) are identical.

Percent identity can be calculated using a global pairwise alignment (e.g. the two sequences are compared over their entire length). Methods for comparing the identity of two or more sequences are known in the art. The program "needle" which uses the global Needleman-Wunsch alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimal alignment (including gaps) of two sequences considering their total length , can be used for example. For example, the Needle program is available on the ebi.ac.uk website and is further described in the following publication (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 is calculated according to the invention with the program EMBOSS needle (global) with a "Gap Open" parameter equal to 10.0, a "Gap Extend" parameter equal to 0.5 and a Blosum62 matrix.

Proteins that consist 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 include substitutions compared to the reference sequence. In the case of substitutions, the 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 can correspond to a homologous sequence, derived from a species other than the reference sequence.

"Amino acid substitutions" can be conservative or non-conservative. Preferably, substitutions are conservative substitutions, replacing one amino acid with another amino acid having similar structural and/or chemical properties.

Conservative substitutions may include those suggested 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 one embodiment, amino acids belonging to any of the following groups can be substituted for each other, thus forming a conservative substitution: 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 In one embodiment, a conservative amino acid substitution can be selected from T→A, G→A, A→1, T→V, A→M, T→I, A→V, T→G and/or T→S.

A conservative amino acid substitution may involve the substitution of one amino acid for 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 as follows: (1) aromatic: Phe, Tyr, His; (2) proton donor: Asn, Gin, Lys, Arg, His, Trp; and (3) proton acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gln (see, e.g., U.S. Patent No. 10 106 805 , the contents of which are incorporated by reference in their entirety).

Conservative substitutions can be made according to Table 1. Methods for predicting tolerance to protein modifications can be found, for example, in Guo et al., Proc. national Acad. Sci., USA, 101(25):9205-9210 (2004), the contents of which are incorporated by reference in their entirety. Table A: Conservative amino acid substitution Conservative amino acid substitution amino acid Substitutions (others are known in the art) Ala Ser, Gly, Cys argument Lys, Gln, His asn Gln, His, Glu, Asp asp Glu, Asn, Gln Cys Ser, Met, Thr equation Asn, Lys, Glu, Asp, Arg glue Asp, Asn, Eqn Gly Pro, Ala, Ser His Asn, Gln, Lys Ile Leu, Val, Met, Ala leu Ile, Val, Met, Ala Lys Arg, Gln, His mead Leu, Ile, Val, Ala, Phe Phe Met, Leu, Tyr, Trp, His ser Thr, Cys, Ala Thr Ser, Val, Ala trp Tyr, Phe Tyr Trp, Phe, His Val Ile, Leu, Met, Ala, Thr

The sequences described herein may contain 1, 2, 3, 4, 5, 10, 15, 20, 25 or 30 amino acid or nucleotide mutations, substitutions, deletions. Each sequence of SEQ ID NO: 1-97 and 265-266 may contain 1, 2, 3, 4, 5, 10, 15, 20, 25 or 30 mutations, substitutions or deletions. The mutations or substitutions are conservative amino acid substitutions.

Conservative amino acid substitutions in the polypeptides described herein may be those listed in Table B under the heading "Conservative Substitutions". If such substitutions result in a change in biological activity, more substantial changes, referred to in Table B as "exemplary substitutions," can be introduced and the products reviewed as necessary. Table B: Amino acid substitution Amino acid substitution Original residue (naturally occurring amino acid) Conservative Substitutions Exemplary Substitutions Ala (A) Val Val; leu; Ile Arg (R) Lys Lys; Gln; asn Asn (N) equation Gln; His; Asp, Lys; argument Asp (D) glue Glu; asn Cys (C) ser ser; Ala Gln (Q) asn Asn; glue Glu (E) asp asp; equation Gly (G) Ala Ala His (H) argument Asn; Gln; Lys; argument Ile (I) leu leu; Val; mead; ala; Phe; norleucine Leu (L) Ile norleucine; ile; Val; mead; ala; Phe Lys (K) argument arg; Gln; asn Mead (M) leu leu; Phe; Ile Phe (F) Tyr leu; Val; ile; ala; Tyr Per (P) Ala Ala Ser (S) Thr Thr Thr (T) ser ser Trp (W) Tyr Tyr; Phe Tire (Y) Phe trp; Phe; Thr; ser Val (V) leu ile; leu; mead; Phe; ala; norleucine

Unless otherwise specified, all terms used herein have the same meaning as they would to a person skilled in the art.

As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents 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 the disclosure pertains.

"Activation," as used herein, refers broadly to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation may also be associated with induced cytokine production and detectable effector functions. The term “activated T cells” refers, among other things, to T cells that are proliferating.

"Antibody" as used herein refers broadly to antibodies or immunoglobulins of any isotype, fragments of antibodies that retain specific binding to an antigen, including but not limited to Fab, Fab', Fab'-SH -, (Fab') ₂ Fv, scFv, divalent scFv and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies and fusion proteins comprising an antigen-specific target region of an antibody and a non-antibody protein. Antibodies are divided into five classes - IgG, IgE, IgA, IgD and IgM.

"Antigen" or "antigen (adj.)", as used herein, refers broadly to a peptide or part of a peptide that can be bound by an antibody that is additionally capable of targeting an animal to produce a To cause antibody that can bind to an epitope of this antigen. An antigen can have one epitope or more than one epitope. The specific reaction to that here is referred to means that the antigen reacts in a highly selective manner with its corresponding antibody and not with the variety of other antibodies that other antigens may elicit.

"Chimeric antigen receptor" or "CAR" or "CARs" as used herein refers broadly to genetically engineered receptors that confer antigenic specificity on cells, e.g. B. T cells, NK cells, macrophages and stem cells graft. CARs can include at least one antigen-specific targeting region (ASTR), a hinge or stalk region, a transmembrane domain (TM), one or more costimulatory domains (CSDs), and an intracellular activating 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 a target antigen, the IAD activates intracellular signaling. For example, the IAD can redirect T cell specificity and reactivity to a chosen target in a non-MHC-restricted manner by utilizing the antigen-binding properties of antibodies. Non-MHC-restricted antigen recognition gives T cells expressing the CAR the ability to recognize an antigen independently of antigen processing, thus bypassing an important tumor escape mechanism. Furthermore, when expressed in T cells, CARs have the advantage of not dimerizing with the endogenous T cell receptor (TCR) alpha and beta chains.

"Cytotoxic T lymphocyte" (CTL), as used herein, generally refers to a T lymphocyte that expresses CD8 on its surface (e.g., a CD8+ T cell). Such cells may preferably be "memory" T cells ( _TM cells) that are antigen-experienced.

"Effective amount," "therapeutically effective amount," or "effective amount," as used herein, refers broadly to the amount of an active ingredient, or combined amounts of two active ingredients, that when administered to a mammal or other subject to treat a disease sufficient to effect such treatment of the disease. The "therapeutically effective amount" depends on the active ingredient(s), the disease and its severity, as well as the age, weight, etc. of the subject being treated.

"Genetically engineered" as used herein refers broadly to methods of introducing exogenous nucleic acids into a cell, whether or not the exogenous nucleic acids are integrated into the genome of the cell. "Genetically engineered cell" as used herein refers broadly to cells containing exogenous nucleic acids, whether or not the exogenous nucleic acids are integrated into the genome of the cell.

"Immune cells" as used herein refers broadly to white blood cells (leukocytes) derived from hematopoietic stem cells (HSC) produced in the bone marrow. "Immune cells" include, without limitation, lymphocytes (T cells, B cells, natural killer (NK) (CD3-CD56+) cells) and cells derived from myeloid cells (neutrophils, eosinophils, basophils, monocytes, macrophages , dendritic cells). “T cells” include all types of immune cells that express CD3, including helper T cells (CD4+ cells), cytotoxic T cells (CD8+ cells), T regulatory cells (Treg) and gamma delta T cells, and NK -T cells (CD3+ and CD56+). One skilled in the art will understand that, as used throughout the disclosure, the terms "T cells and/or NK cells" can include only T cells, only NK cells, or both T cells and NK cells . In certain embodiments and aspects presented herein, T cells are activated and transduced. In addition, T cells are included in certain embodiments and aspects of the composition presented herein. A "cytotoxic cell" includes CD8+ T cells, natural killer (NK) cells, NK T cells, γδ T cells, and neutrophils, which are cells capable of mediating cytotoxic responses.

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

"Peripheral blood mononuclear cells" or "PBMCs" as used herein refers broadly to all peripheral blood cells having a round nucleus. PBMCs include lymphocytes such as B. T-cells, B-cells and NK-cells, as well as monocytes.

"Polynucleotide" and "nucleic acid", as used interchangeably herein, refer broadly to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term does not include, but is not limited to, single, double or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids or a polymer containing purine and pyrimidine bases or other natural, chemically or biochemically modified bases - contains natural or derivatized nucleotide bases.

"T cell" or "T lymphocyte" as used herein broadly refers to thymocytes, naïve T lymphocytes, immature T lymphocytes, mature T lymphocytes, quiescent T lymphocytes or activated T lymphocytes. Illustrative populations of T cells suitable for use in certain embodiments include, but are not limited to, T helper cells (HTL; CD4+ T cell), a cytotoxic T cell (CTL; CD8+ T cell ), CD4+CD8+ T cell, CD4-CD8- T cell natural killer T cell, T cells expressing αβ-TCR (αβ T cells), T cells expressing γδ-TCR ( γδ T cells), or any other subset of T cells. Other exemplary populations of T cells suitable for use in certain embodiments include, but are not limited to, T cells that express 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, if desired, by positive or negative selection techniques.

In the present invention, the term "homologous" refers to the degree of identity (see percentage identity above) between the sequences of two amino acid sequences, i. H. peptide or polypeptide sequences. The previously mentioned “homology” is obtained by comparing two sequences that, under optimal conditions, align with the sequences that need to be compared. Such sequence homology can be calculated by forming an alignment using, for example, the ClustalW algorithm. Generally available sequence analysis software, more specifically, Vector NTI, GENETYX or other tools are provided through public databases.

The terms "sequence homology" or "sequence identity" are used interchangeably herein. For purposes of the present invention, it is defined herein that 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. In order to optimize the alignment between the two sequences, gaps can be inserted in each of the two sequences to be compared. Such an alignment can be carried out over the full length of the sequences to be compared. Alternatively, the alignment can also 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 in the specified aligned region.

A comparison of sequences and the determination of the percentage sequence identity between two sequences can be performed using a mathematical algorithm. Those skilled in the art know that there are various computer programs to align two sequences and to determine the identity between two sequences (Kruskal, J.B. (1983) An overview of sequence comparison. In D. Sankoff and J.B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, Addison Wesley). Percent sequence identity between two amino acid sequences or between two nucleotide sequences can be determined using the Needleman and Wunsch algorithm for two-sequence alignment. (Needleman, S.B. and Wunsch, C.D. (1970) J. Mal. Biol. 48 , 443-453). Both amino acid sequences and nucleotide sequences can be aligned with the algorithm. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purposes of this invention, the program NEEDLE 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. EDNAFULL is used for the nucleotide sequence. The optional parameters used are a gap opening penalty of 10 and a gap lengthening penalty of 0.5. Those skilled in the art will understand that all of these different parameters produce slightly different results, but that the overall percent identity of two sequences is not significantly altered using different algorithms.

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

"T cell receptor (TCR)" as used herein refers broadly to a protein receptor on T cells composed of a heterodimer of an alpha (α) and beta (β) chain, although the TCR consists of gamma and delta (γ/δ) chains in some cells. The TCR can be modified on any cell that comprises a TCR, including a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a natural killer T cell, or a gamma Delta T cell.

A TCR is generally located on the surface of T lymphocytes (or T cells) and is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. It is a heterodimer composed of an alpha and beta chain in 95% of T cells, while 5% of T cells have TCRs composed of gamma and delta chains. The interaction of the TCR with antigen and MHC leads to the activation of its T lymphocyte through a series of biochemical events mediated by associated enzymes, co-receptors and special helper molecules. In immunology, the CD3 antigen (CD stands for differentiation cluster) is a protein complex that in mammals consists of four different chains (CD3-γ, CD35 and twice CD3ε) that combine with molecules known as the T-cell receptor ( TCR) and ζ chain are known to generate an activation signal in T lymphocytes. The TCR, ζ chain and CD3 molecule together form the TCR complex. The CD3-γ, CD3δ, and CD3ε chains are highly related cell-surface proteins of the immunoglobulin superfamily that contain a single extracellular immunoglobulin domain. The transmembrane region of CD3 chains is negatively charged, a property that allows these chains to associate with the positively charged TCR chains (TCRα and TCRβ). The intracellular ends of the CD3 molecules contain a single conserved motif that is essential for the signaling capacity of the TCR as the immune receptor tyrosine-based activation motif, or ITAM for short.

"Treatment," "treating," and the like, as used herein, refer broadly to achieving a desired pharmacological and/or physiological effect. The effect may be prophylactic in the sense of completely or partially preventing a disease or a symptom thereof and/or therapeutic in the sense of partially or completely curing a disease and/or an undesirable effect attributable to the disease. "Treatment" as used herein includes any treatment of a disease in a mammal, e.g. in a human, and comprises: (a) preventing the disease from occurring in a subject who may be predisposed to the disease but who has not yet been diagnosed with it; (b) inhibition of the disease, e.g. B. stopping their development; and (c) alleviating the disease, e.g. B. Causing regression of the disease.

EXAMPLE 1

Exemplary nucleic acid and amino acid sequences

2 CD8β Region

3 CD8α Transmembrane domäne

4 CD8α zytoplasmatisch er Schwanz

5 m1CD8α (signallos)

6 Signalpeptid

7 m1CD8α

8 CD8β1

9 CD8β2

10 CD8β3

11 CD8β4

12 CD8β5

13 CD8β6

14 CD8β7

15 R11 KEA alpha-Kette

16 R11 KE beta-Kette

17 R20P1H7 alpha-Kette

18 R20P1 H7 beta-Kette

19 R7P1D5 alpha-Kette

20 R7P1D5 beta-Kette

21 R10P2G12 alpha-Kette

22 R10P2G12 beta-Kette

23 R10P1A7 alpha-Kette

24 R10P1A7 beta-Kette

25 R4P1D10 alpha-Kette

26 R4P1D10 beta-Kette

27 R4P3F9 alpha-Kette

28 R4P3F9 beta-Kette

29 R4P3H3 alpha-Kette

30 R4P3H3 beta-Kette

31 R36P3F9 alpha-Kette

32 R36P3F9 beta-Kette

33 R52P2G11 alpha-Kette

34 R52P2G11 beta-Kette

35 R53P2A9 alpha-Kette

36 R53P2A9 beta-Kette

37 R26P1A9 alpha-Kette

38 R26P1A9 beta-Kette

39 R26P2A6 alpha-Kette

40 R26P2A6 beta-Kette

41 R26P3H1 alpha-Kette

42 R26P3H1 beta-Kette

43 R35P3A4 alpha-Kette

44 R35P3A4 beta-Kette

45 R37P1C9 alpha-Kette

46 R37P1 C9 beta-Kette

47 R37P1H1 alpha-Kette

48 R37P1 H1 beta-Kette

49 R42P3A9 alpha-Kette

50 R42P3A9 beta-Kette

51 R43P3F2 alpha-Kette

52 R43P3F2 beta-Kette

53 R43P3G5 alpha-Kette

54 R43P3G5 beta-Kette

55 R59P2E7 alpha-Kette

56 R59P2E7 beta-Kette

57 R11P3D3 alpha-Kette

58 R11 P3D3 beta-Kette

59 R16P1C10 alpha-Kette

60 R16P1C10 beta-Kette

61 R16P1E8 alpha-Kette

62 R16P1E8 beta-Kette

63 R17P1A9 alpha-Kette

64 R17P1A9 beta-Kette

65 R17P1D7 alpha-Kette

66 R17P1D7 beta-Kette

67 R17P1G3 alpha-Kette

68 R17P1G3 beta-Kette

69 R17P2B6 alpha-Kette

70 R17P2B6 beta-Kette

71 R11P3D3KE alpha-Kette

72 R11P3D3KE beta-Kette

73 R39P1C12 alpha-Kette

74 R39P1C12 beta-Kette

75 R39P1F5 alpha-Kette

76 R39P1 F5 beta-Kette

77 R40P1C2 alpha-Kette

78 R40P1C2 beta-Kette

79 R41P3E6 alpha-Kette

80 R41 P3E6 beta-Kette

81 R43P3G4 alpha-Kette

82 R43P3G4 beta-Kette

83 R44P3B3 alpha-Kette

84 R44P3B3 beta-Kette

85 R44P3E7 alpha-Kette

86 R44P3E7 beta-Kette

87 R49P2B7 alpha-Kette

88 R49P2B7 beta-Kette

89 R55P1G7 alpha-Kette

90 R55P1 G7 beta-Kette

91 R59P2A7 alpha-Kette

92 R59P2A7 beta-Kette

93 P2A

94 T2A

95 E2A

96 F2A

97 RD114TR

256 WPREmut1

257 WPREmut2

258 CD8α1

259 CD8α2

260 CD8α Stalk

261 CD8α Ig-ähnliche Domäne-2

262 m2CD8α

263 MSCV Promoter

264 WPRE

265 Furin-Konsensus

266 Linker

Table 2 Representative protein and DNA sequences SEQ ID NO: description sequence 1 CD8α Ig-like domain-1

2 CD8β region

3 CD8α transmembrane domain

4 CD8α cytoplasmic tail

5 m1CD8α (no signal)

6 signal peptide

7 m1CD8α

8th CD8β1

9 CD8β2

10 CD8β3

11 CD8β4

12 CD8β5

13 CD8β6

14 CD8β7

15 R11 KEA alpha chain

16 R11 KE beta chain

17 R20P1H7 alpha chain

18 R20P1 H7 beta chain

19 R7P1D5 alpha chain

20 R7P1D5 beta chain

21 R10P2G12 alpha chain

22 R10P2G12 beta chain

23 R10P1A7 alpha chain

24 R10P1A7 beta chain

25 R4P1D10 alpha chain

26 R4P1D10 beta chain

27 R4P3F9 alpha chain

28 R4P3F9 beta chain

29 R4P3H3 alpha chain

30 R4P3H3 beta chain

31 R36P3F9 alpha chain

32 R36P3F9 beta chain

33 R52P2G11 alpha chain

34 R52P2G11 beta chain

35 R53P2A9 alpha chain

36 R53P2A9 beta chain

37 R26P1A9 alpha chain

38 R26P1A9 beta chain

39 R26P2A6 alpha chain

40 R26P2A6 beta chain

41 R26P3H1 alpha chain

42 R26P3H1 beta chain

43 R35P3A4 alpha chain

44 R35P3A4 beta chain

45 R37P1C9 alpha chain

46 R37P1 C9 beta chain

47 R37P1H1 alpha chain

48 R37P1 H1 beta chain

49 R42P3A9 alpha chain

50 R42P3A9 beta chain

51 R43P3F2 alpha chain

52 R43P3F2 beta chain

53 R43P3G5 alpha chain

54 R43P3G5 beta chain

55 R59P2E7 alpha chain

56 R59P2E7 beta chain

57 R11P3D3 alpha chain

58 R11 P3D3 beta chain

59 R16P1C10 alpha chain

60 R16P1C10 beta chain

61 R16P1E8 alpha chain

62 R16P1E8 beta chain

63 R17P1A9 alpha chain

64 R17P1A9 beta chain

65 R17P1D7 alpha chain

66 R17P1D7 beta chain

67 R17P1G3 alpha chain

68 R17P1G3 beta chain

69 R17P2B6 alpha chain

70 R17P2B6 beta chain

71 R11P3D3KE alpha chain

72 R11P3D3KE beta chain

73 R39P1C12 alpha chain

74 R39P1C12 beta chain

75 R39P1F5 alpha chain

76 R39P1 F5 beta chain

77 R40P1C2 alpha chain

78 R40P1C2 beta chain

79 R41P3E6 alpha chain

80 R41 P3E6 beta chain

81 R43P3G4 alpha chain

82 R43P3G4 beta chain

83 R44P3B3 alpha chain

84 R44P3B3 beta chain

85 R44P3E7 alpha chain

86 R44P3E7 beta chain

87 R49P2B7 alpha chain

88 R49P2B7 beta chain

89 R55P1G7 alpha chain

90 R55P1 G7 beta chain

91 R59P2A7 alpha chain

92 R59P2A7 beta chain

93 P2A

94 T2A

95 E2A

96 F2A

97 RD114TR

256 WPREmut1

257 WPREmut2

258 CD8α1

259 CD8α2

260 CD8α Stalk

261 CD8α Ig-like domain-2

262 m2CD8α

263 MSCV Promoter

264 WPRE

265 Furin consensus

266 leftist

Tumor-Associated Antigens (TAA)

In an MHC class I-dependent immune response, the peptides must not only be able to bind to specific MHC class I molecules that are expressed by tumor cells, they must then also be absorbed by T cells that carry a specific T cell receptor (TCR).

In order for the proteins to be recognized by the T-lymphocytes as a tumor-specific or tumor-associated antigen, and thus to be able to be used in therapy, certain requirements must be met. The antigen should be expressed mainly by tumor cells and not, or only in comparatively small amounts, by healthy normal tissues. In a preferred embodiment, the peptide should be over-presented by tumor cells compared to normal healthy tissue. Furthermore, it is desirable if the antigen in question is not only present in one type of tumor, but also in a high concentration (e.g. number of copies of the corresponding peptide per cell). Tumor-specific and tumor-associated antigens are often derived from proteins that are directly involved in the transformation of a normal cell into a tumor cell because of their function, for example in cell cycle control or the suppression of apoptosis. In addition, downstream targets of the proteins directly responsible for transformation can be upregulated and are thus indirectly tumor associated. Such indirectly tumor-associated antigens can also be targets of a vaccination approach. Singh-Jasuja et al. Cancer Immunol. immunother. 53 (2004): 187-195. Epitopes are present in the antigen's amino acid sequence, making the peptide an "immunogenic peptide" and because it is derived from a tumor-associated antigen, it elicits a T-cell response, both in vitro and in vivo.

Any peptide that can bind to an MHC molecule can serve as a T cell epitope. For induction of a T cell response, the TAA must be presented to a T cell with a corresponding TCR and the host must not be immunologically tolerant to that particular epitope. Exemplary tumor-associated antigens (TAA) that can be used with the CD8 polypeptides described herein are disclosed herein. Table 3 TAA peptide sequences SEQ ID NO: amino acid sequence SEQ ID NO: amino acid sequence SEQ ID NO: amino acid sequence 98 YLYDSETKNA 151 LLWGHPRVALA 204 SLLNQPKAV 99 HLMDQPLSV 152 VLDGKVAW 205 KMSELQTYV 100 GLLKKINSV 153 GLLGKVTSV 206 ALLEQTGDMSL 101 FLVDGSSAL 154 KMISAIPTL 207 VIIKGLEEITV 102 FLFDGSANLV 155 SMOOTHGLAT 208 KQFEGTVEI 103 FLYKIIDEL 156 TLNTLDINL 209 KLQEEIPVL 104 FILDSAETTL 157 VIIKGLEEI 210 GLAEFQENV 105 SVDVSPPKV 158 YLEDGFAYV 211 NVAEIVIHI 106 VADKIHSV 159 KIWEELSVLEV 212 ALAGIVTNV 107 IVDDLTINL 160 LLIPFTIFM 213 NLLIDDKGTIKL 108 GLLEELVTV 161 ISLDEVAVSL 214 VLMQDSRLYL 109 TLDGAAVNQV 162 KISDFGLATV 215 KVLEHWRV 110 SVLEKEIYSI 163 KLIGNIHGNEV 216 LLWGNLPEI 111 LLDPKTIFL 164 ILLSVLHQL 217 SLMEKNQSL 112 YTFSGDVQL 165 LDSEALLTL 218 KLLAVIHEL 113 YLMDDFSSL 166 VLQENSSDYQSNL 219 ALGDKFLLRRV 114 KVWSDVTPL 167 HLLGEGAFAQV 220 FLMKNSDLYGA 115 LLWGHPRVALA 168 SLVENIHVL 221 KLIDHQGLYL 116 KIWEELSVLEV 169 YTFSGDVQL 222 GPGIFPPPPPQP 117 LLIPFTIFM 170 SLSEKSPEV 223 ALNESLVEC 118 FLIENLLAA 171 AMFPDTIPRV 224 GLAALAVHL 119 LLWGHPRVALA 172 FLIENLLAA 225 LLLEAVWHL 120 FLLEREQLL 173 FTAEFLEKV 226 SIIEYLPTL 121 SLAETIFIV 174 ALYGNVQQV 227 TLHDQVHLL 122 TLLEGISRA 175 LFQSRIAGV 228 SLLMWITQC 123 KIQEILTQV 176 ILAEEPIYIRV 229 FLLDKPQDLSI 124 VIFEGEPMYL 177 FLLEREQLL 230 YLLDMPLWYL 125 SLFESLEYL 178 LLLPLELSLA 231 GLLDCPIFL 126 SLLNQPKAV 179 SLAETIFIV 232 VLIEYNFSI 127 GLAEFQENV 180 AILNVDEKNQV 233 TLYNPERTITV 128 KLLAVIHEL 181 RLFEEVLGV 234 AVPPPPSSV 129 TLHDQVHLL 182 YLDEVAFML 235 KLQELNKV 130 TLYNPERTITV 183 CLOTHES DEPLFL 236 KLMDPGSLPPL 131 KLQEKIQEL 184 KLFEKSTGL 237 ALIVSLPYL 132 SVLEKEIYSI 185 SLLEVNEASSV 238 FLLDGSANV 133 RVIDDSLVVGV 186 GVYDGREHTV 239 ALDPSGNQLI 134 VLFGELPAL 187 GLYPVTLVGV 240 ILIKHLVKV 135 GLVDIMVHL 188 ALLSSVAEA 241 VLLDTILQL 136 FLNAIE VALLEY 189 TLLEGISRA 242 HLIAEIHTA 137 ALLQALMEL 190 SLEESEEL 243 SMNGGVFAV 138 ALSSSQAEV 191 ALYVQAPTV 244 MLAEKLLQA 139 SLITGQDLLSV 192 KLIYKDLVSV 245 YMLDIFHEV 140 QLIEKNWLL 193 ILQDGQFLV 246 ALWLPTDSATV 141 LLDPKTIFL 194 SLLDYEVSI 247 GLASS RILDA 142 RLHDENILL 195 LLGDSSFFL 248 ALSVLRLAL 143 YTFSGDVQL 196 VIFEGEPMYL 249 SYVKVLHHL 144 GLPSATTTV 197 ALSYILPYL 250 VYLPKIPSW 145 GLLPSAESIKL 198 FLFVDPELV 251 NYEDHFPLL 146 KTASINQNV 199 SEWGSPHAAVP 252 VYIAELEKI 147 SLLQHLIGL 200 ALSELERVL 253 VHFEDTGKTLLF 148 YLMDDFSSL 201 SLFESLEYL 254 VLSPFILTL 149 LMYPYIYHV 202 KVLEYVIKV 255 HLLEGSVGV 150 KVWSDVTPL 203 VLLNEILEQV

EXAMPLE 2

CD8α molecules

The CD8α homodimer (CD8αα) can consist of two α-subunits held together by two disulfide bonds at the stalk regions. shows a CD8α polypeptide, e.g. B. SEQ ID NO: 258 (CD8α1) comprising five domains: (1) a signal peptide (from -21 to -1), e.g. B. SEQ ID NO: 6, (2) an Ig-like domain-1 (from 1 to 115), eg SEQ ID NO: 1, (3) a stalk region (from 116 to 160), e.g. B., SEQ ID NO: 260, (4) a transmembrane (TM) domain (from 161-188), e.g. B. SEQ ID NO: 3, and (5) a cytoplasmic tail (cyto) comprising an Ick binding motif (from 189 to 214), e.g. B. SEQ ID NO: 4. Another example of a CD8α subunit, e.g. B. CD8α2 (SEQ ID NO: 259), differs from CD8α1 at position 112 where CD8α2 contains a cysteine (C) while CD8α1 contains a tyrosine (Y).

Modified CD8 Polypeptides

Unlike the CD8α polypeptide, e.g. B. CD8α1 (SEQ ID NO: 258) and CD8α2 (SEQ ID NO: 259), a modified CD8α polypeptide, e.g. B. m1CD8α (SEQ ID NO: 7) and m2CD8α (SEQ ID NO: 262), contain additional regions such. B. Sequence segments from a CD8ß polypeptide. In one embodiment, SEQ ID NO: 2 or variants thereof are used with a CD8α polypeptide. In other embodiments, a portion of a CD8α polypeptide, e.g. B. SEQ ID NO: 260, removed or not included in the modified CD8 polypeptides described herein. Figure 1 shows a sequence alignment between CD8α1 (SEQ ID NO:258) and m1CD8α (SEQ ID NO:7). shows a sequence alignment between CD8α2 (SEQ ID NO: 259) and m2CD8α (SEQ ID NO: 262), in which the cysteine substitution is indicated by an arrow. The stalk regions are shown inside the boxes.

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

EXAMPLE3

Lentiviral Viral Vectors

The lentiviral vectors used herein contain several elements that enhance the function of the vector, including a central polypurine tract (cPPT) for enhanced replication and nuclear import, a murine stem cell virus (MSCV) promoter (SEQ ID NO: 263), which reduces silencing of the vector in some cell types, a post-transcriptional responsive element (WPRE) of Woodchuck hepatitis virus (SEQ ID NO: 264) for improved transcriptional 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, the content of which is fully incorporated by reference.

In one embodiment, the vectors, constructs, or sequences described herein comprise mutant forms of WPRE. In one embodiment, the sequences or vectors described herein comprise mutations in WPRE version 1, e.g. WPREmut1 (SEQ ID NO: 256), or WPRE version 2, e.g. B. WPREmut2 (SEQ ID NO: 257). #9 and #9b represent two LV production lots with the same construct containing SEQ ID NO: 257 as WPREmut2, the difference between #9 and #9b being the titer consistent with Table 4. In one embodiment, the WPRE mutants comprise at most one mutation, at most two mutations, at most three mutations, at least four mutations, or at most five mutations. In one embodiment, the vectors, constructs, or sequences described herein do not contain a WPRE.

In one embodiment, the vectors, constructs, or sequences described herein do not include an X protein promoter.

To achieve optimal coexpression levels of TCRaβ, mCD8α (e.g. m1CD8α (SEQ ID NO: 7) and m2CD8α (SEQ ID NO: 262)) and CD8β (e.g. one of CD8β1-7 (SEQ ID NO: 8-14 )) in the transduced CD4+ T cells, CD8+ T cells and/or γδ T cells, lentiviral vectors with different designs were generated. T cells can be transduced with two separate lentiviral vectors (2-in-1), e.g. B. one expressing TCRα and TCRβ and the other expressing mCD8α and CD8β for co-expression of TCRaβ and CD8αβ heterodimers, or one expressing TCRα and TCRβ and the other expressing mCD8α for co-expression of TCRaβ and mCD8α homodimers. Alternatively, T cells can be transduced with a single lentiviral vector (4-in-1) co-expressing TCRα, TCRβ, mCD8α and CD8β to co-express TCRaβ and CD8αβ heterodimers. In the 4-in-1 vector, the nucleotides encoding TCRα chain, TCRβ chain, mCD8α chain and CD8β chain can be mixed in different orders, e.g. B. from 5' to 3' direction, TCRα-TCRβ-mCD8α-CD8β, TCRα-TCRβ-CD8β-mCD8α, TCRβ-TCRα-mCD8α-CD8β, TCRβ-TCRα-CD8β-mCD8α, mCD8α-CD8β-TCRα- TCRβ, mCD8α-CD8β-TCRβ-TCRα, CD8β-mCD8α-TCRα-TCRβ and CD8β-mCD8α-TCRβ-TCRα. Various 4-in-1 vectors so generated can be used to transduce CD4+ T cells, CD8+ T cells and/or γδ T cells, followed by measurement of TCRαβ/mCD8α/CD8β coexpression levels of the transduced cells Using techniques known in the art, e.g. B. flow cytometry. Similarly, T cells can be transduced with a single lentiviral vector (3-in-1) co-expressing TCRα, TCRβ and mCD8α (e.g. m1CD8α and m2CD8α) to co-express TCRaβ and mCD8α homodimers. In the 3-in-1 vector, the nucleotides encoding the TCRα chain, the TCRβ chain and the mCD8α chain can be mixed in different orders, e.g. B. TCRα-TCRβ-mCD8α, TCRβ-TCRα-mCD8α, mCD8α-TCRα-TCRβ and mCD8α-TCRβ-TCRα. Various 3-in-1 vectors so generated can be used to transduce CD4+ T cells, CD8+ T cells and/or γδ T cells, followed by measurement of TCRαβ/mCD8α coexpression levels of the transduced cells with in the Technique known techniques.

To generate lentiviral vectors co-expressing TCRaβ and mCD8α and/or CD8β, a nucleotide encoding furin-linker (GSG or SGSG (SEQ ID NO: 266))-2A peptide can be inserted between TCRα chain and TCRβ- chain, between mCD8α chain and CD8β chain and between a TCR chain and a CD8 chain to enable highly efficient gene expression. The 2A peptide can be selected from P2A (SEQ ID NO: 93), T2A (SEQ ID NO: 94), E2A (SEQ ID NO: 95) or F2A (SEQ ID NO: 96).

Lentiviral viral vectors may also contain a post-transcriptional regulatory element (PRE), such as. B. WPRE (SEQ ID NO: 264), WPREmut1 (SEQ ID NO: 256) or WPREmut2 (SEQ ID NO: 257) to enhance expression of the transgene by increasing nuclear and cytoplasmic mRNA levels. One or more regulatory elements such as the mouse RNA transport element (RTE), the constitutive transport element (CTE) of simian retrovirus type 1 (SRV-1) and the 5' untranslated region of human heat shock protein 70 (Hsp70 5'UTR). also and/or in combination with WPRE to increase transgene expression.

Lentiviral vectors can be pseudotyped with RD114TR (eg, SEQ ID NO: 97), which is a chimeric glycoprotein comprising an extracellular and transmembrane domain of feline endogenous virus (RD114) that is conjugated to the cytoplasmic tail (TR) of murine leukemia virus is fused. Other viral envelope proteins such as VSV-G env, MLV 4070A env, RD114 env, the chimeric envelope protein RD114pro, the baculovirus GP64 env or GALV env or derivatives thereof can also be used. RD114TR variants comprising at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% of SEQ ID NO: 97 are also contemplated.

For example, FIG. B. Constructs #10 and #2 co-expressing TCR (TCRα chain and TCRβ chain), CD8α and CD8β; three 3-in-1 vectors expressing TCR and CD8α, e.g. constructs #1 and #9, two 3-in-1 vectors expressing TCR and m1CD8α (SEQ ID NO:7), e.g. B. constructs #11 and #12, and construct #8, which only expresses TCR. To improve transcription termination, wild-type WPRE (WPRE) (SEQ ID NO: 264) is included in constructs #1, #2, and #8; WPREmut (SEQ ID NO:257) is contained in constructs #9, #10, #11, and #12.

EXAMPLE 4

vector screening

viral titers

Figure 4 shows viral titers of constructs #1, #2, #8, #9, #10, #11 and #12. Table 4 shows viral titers and lentiviral P24 ELISA data for constructs # 9, #10, #11, and #12. Table 4 No of constructs description titer Lentiviral P24 9 CD8α.TCR.WPREmut2 5.40×10 ⁹ 6556 9b CD8α.TCR.WPREmut2 9.80×10 ⁹ 16196 10 MSCV-PTE.CD8βα.TCR.WPREmut2 6.40×10 ⁹ 9525 11 CD8aCD8βstalk.TCR.WPREmut2 1.30×10 ¹⁰ 16797 12 CD8αCD8βstalk.NCAMfu.TCR.WPREmut2 1.20×10 ¹⁰ 17996

For construct 12, NCAMfu refers to the NCAM fusion protein expressing a modified extracellular CD8a and an intracellular neural cell adhesion molecule 1 (CD56) domain.

In Table 4, the WPREmut2 part refers to SEQ ID NO: 257.

T cell production

activation

shows that on day +0 PBMCs (ca. 9 x 10 ⁸ cells) from two donors (donor #1 and donor #2) were thawed and rested. Cells were activated in bags (AC290) coated with anti-CD3 and anti-CD28 antibodies in the presence of serum. activation markers, e.g. B. CD25, CD69 and human low-density lipoprotein receptor (H-LDL-R) in CD8 + and CD4 + cells were then measured. shows that the percentage of CD3+CD8+CD25+ cells, the percentage of CD3+CD8+CD69+ cells and the percentage of CD3+CD8+H-LDL-R+ cells after activation (post-A) compared to which increases before activation (Pre-A). Similarly shows , that the percentage of CD3+CD4+CD25+ cells, the percentage of CD3+CD4+CD69+ cells and the Pro percentage of CD3+CD4+H-LDL-R+ cells after activation (post-A) compared to before activation (pre-A). These results support the activation of PBMCs.

transduction

shows that day +1 activated PBMCs with viral vectors, e.g. constructs #1, #2, #8, #9, #10, #11 and #12, in G-Rex® 6-well plates at approximately 5 x 10 ⁶ cells/ Well transduced in the absence of serum. The amounts of virus used for transduction are listed in Table 5. Table 5 constructs Volume of virus/1 x 10 ⁶ cells #9, #10, #11, #12 1.25µl, 2.5µl, 5µl number 1 1.25 µl No. 2 5 µl No. 8 (TCR) 2.5 µl

expansion

shows that PBMCs transduced on day +2 were expanded in the presence of serum. On day +6, cells were isolated for subsequent analyses, e.g. B. FACS dextramer and vector copy number (VCN), harvested and cryopreserved. and show the expansion of transduced T-cells from donor #1 and donor #2, respectively, on day +6. Cell viability is greater than 90% on day +6.

Characterization of T cell products

Cell numbers, FACS dextramers and vector copy numbers (VCN) were determined. Tetramer panels can include live/dead cells, CD3, CD8α, CD8β, CD4 and peptide/MHC tetramers, e.g. B. PRAME-004 (SLLQHLIGL) (SEQ ID NO: 147)/MHC tetramers. FACS analysis was gated for live singlets, followed by CD3+, followed by CD4+CD8+, followed by CD4+CD8+Tetramer(Tet)+ and CD8+Tet+.

, , and Figure 1 shows representative flow diagrams of cells obtained from Donor #1 and indicate the percentage of CD8, CD4 and PRAME-004/MHC tetramer (Tet) of cells transfected with Construct #9, #10, #1 11 and #12 were transduced, respectively.

shows the percentage of CD8+CD4+ cells from donor #1 (upper panel) and donor #2 (lower panel) infected with construct #1, #2, #8 (TCR), #9, #10, #11 or #12 were transduced at 1.25 µl, 2.5 µl or 5 µl per 1 x 10 ⁶ cells. These results show that transduction of CD8α and TCR expressing vectors with wild-type WPRE (construct #1) and WPREmut2 (construct #9) yielded a higher percentage of CD8+CD4+ cells than transduction with the constructs #10, #11, or #12. Construct #8 (TCR only) serves as a negative control. shows the Tet percentage of CD8+CD4+ cells from donor #1 (upper panel) and donor #2 (lower panel) infected with constructs #1, #2, #8 (TCR), # 9, #10, #11 and #12 were transduced at 1.25 µl, 2.5 µl or 5 µl per 1 x 10 ⁶ cells. These results show that the percent Tet of CD8+CD4+ cells in cells transduced with constructs #9, #10, and #11 appears to be comparable and greater than in cells transduced with construct #12. FACS analysis was gated for live singlets followed by CD3+, followed by CD4+CD8+ and followed by CD4+CD8+Tet+.

shows the Tet-MFI of CD8+CD4+Tet+ cells from donor #1 (upper panel) and donor #2 (lower panel) infected with construct #1, #2, #8 (TCR), #9, #10, #11 or #12 were transduced at 1.25 µl, 2.5 µl or 5 µl per 1 x 10 ⁶ cells. These results show that the tetramer MFI on CD4+CD8+Tet+ varies between donors. shows the CD8α MFI of CD8+CD4+Tet+ cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with construct #1, #2, #8 (TCR), #9, #10, #11 or #12 at 1.25 µl, 2.5 µl or 5 µl per 1 x 10 ⁶ cells. These results indicate a higher CD8α MFI in cells transduced with vectors encoding CD8α and TCR Wild-type WPRE (construct #1) and WPREmut2 (construct #9) express than in cells transduced with the other constructs. A transduction volume of 5 µl/10 ⁶ seems to give better results than 1.25 µl/10 ⁶ and 2.5 µl/10 ⁶ . FACS analysis was gated for live singlets followed by CD3+, followed by CD4+CD8+, followed by CD4+CD8+Tet+ and followed by Tet MFI/CD8α MFI.

Figure 12 shows CD8 frequencies (% CD8+CD4- of CD3+) in cells from donor #1 (upper panel) and donor #2 (lower panel) infected with construct #1, #2, #8 ( TCR), #9, #10, #11 or #12 at 1.25 µl, 2.5 µl or 5 µl per 1 x 10 ⁶ cells. These results show no difference in CD8 frequencies between the constructs. The non-transduction (NT) serves as a negative control. shows the percentage of CD8+Tet+ (from CD3+) cells from donor #1 (upper panel) and donor #2 (lower panel) infected with construct #1, #2, #8 (TCR), #9, #10, #11, or #12 were transduced at 1.25 µl, 2.5 µl, or 5 µl per 1 x 10 ⁶ cells. These results show higher frequency of CD8+Tet+ (from CD3+) in cells transduced with construct #9, #11 and #12 than in cells transduced with construct #10. FACS analysis was gated for live singlets followed by CD3+, followed by CD8+CD4- and followed by CD8+Tet+.

shows the Tet-MFI of CD8+Tet+ cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with construct #1, #2, #8 (TCR), # 9, #10, #11, or #12 at 1.25 µl, 2.5 µl, or 5 µl per 1 x 10 ⁶ cells. These results show that the tetramer MFI of CD8+tet+ cells varies between donors. shows the Tet-MFI of CD8+Tet+ cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with construct #1, #2, #8 (TCR), # 9, #10, #11, or #12 at 1.25 µl, 2.5 µl, or 5 µl per 1 x 10 ⁶ cells. These results demonstrate that the CD8α MFI of CD8+Tet+ is comparable among cells transduced with different constructs. FACS analysis was gated for live singlets followed by CD3+, followed by CD4+CD8+, followed by CD4+CD8+Tet+ and followed by Tet MFI/CD8α MFI.

shows the percentage of Tet+ cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with construct #1, #2, #8 (TCR), #9, # 10, #11, or #12 at 1.25 µL, 2.5 µL, or 5 µL per 1 x 10 ⁶ cells. These results show higher frequency of CD3+Tet+ (from CD3+) in cells transduced with construct #9 or #11 than in cells transduced with construct #10 or #12. Percentage more Tet+CD3+ cells appear in cells transduced with construct #10 (WPREmut2) than in cells transduced with construct #2 (wild-type WPRE) at 5 µl per 1 x 10 ⁶ cells . FACS analysis was gated to live singlets followed by CD3+, followed by CD3+ and followed by Tet+.

(upper panel) shows the vector copy number (VCN) of cells from donor #1 infected with construct #1, #2, #8 (TCR), #9, #10, #11, or # 12 were transduced at 1.25 µl, 2.5 µl or 5 µl per 1 x 10 ⁶ cells. These results show a higher VCN for cells transduced with construct #11 or #12 (possibly due to higher titers) than for cells transduced with construct #9 or #10. (lower panel) shows CD3+Tet+/VCN of cells from donor #1 infected with construct #1, #2, #8 (TCR), #9, #10, #11, or #1. 12 were transduced at 1.25 µl, 2.5 µl or 5 µl per 1 x 10 ⁶ cells. These results indicate higher CD3+Tet+/VCN in cells transduced with construct #9 than in cells transduced with construct #10, #11, or #12.

Taken together, these results demonstrate (1) higher percentage of CD8+CD4+ cells obtained by transducing cells with CD8α and TCR expressing vectors with wild-type WPRE (construct #1) and WPREmut2 (construct #9) than those transduced with construct #10, #11, or #12; (2) the percentage of CD8+CD4+Tet+ cells, which was comparable between cells transduced with different constructs; (3) dose-dependent percentage increase in tetramer, e.g. B., 5 µl per 1 x 10 ⁶ cells showed better results than 1.25 µl and 2.5 µl per 1 x 10 ⁶ cells; (4) the comparable percentage of CD8+ cells between cells transduced with different constructs; (5) higher frequencies of CD8+Tet+ in cells transduced with construct #9, #11, or #12 than those transduced with construct #10; (6) higher frequencies of CD3+Tet+ in cells transduced with construct #9 or #11 than in cells transduced with construct #10 or #12; (7) higher VCN in cells transduced with construct #11 or #12 than in cells transduced with construct #9 or #10; and (8) higher CD3+Tet+/VCN in cells transduced with construct #9 than in cells transduced with construct #10, #11, or #12.

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

EXAMPLE 5

Tumor Death Assay

represent data showing that the constructs (#10, #11, and #12) are effective in mediating cytotoxicity against target-positive cell lines expressing antigen at different levels (UACC257 at 1081 copies per cell and A375 at 50 copies per cell) are comparable to "TCR only". Table 6 tumor cell line antigen positivity UACC257 high A375 low MCF7 negative

Construct #9 loses tumor control over time against the A375 cell line, which expresses little target antigen.

EXAMPLE 6

IFNγ Secretion Assay

IFNγ secretion was measured in cell lines UACC257 and A375. The IFNγ secretion in response in the UACC257 cell line was comparable between the constructs. However, in the A375 cell line, construct #10 showed higher IFNγ secretion than the other constructs. IFNγ is quantitated in the supernatants of Incucyte plates. 21A-B .

Figure 1 shows an exemplary experimental setup to assess 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.

IFNγ secretion is increased in response to A375 in the presence of iDCs. In the tri-cultures with iDCs, IFNγ secretion is higher in #10 compared to the other constructs. However, when comparing #9 with #11, which express wild-type and modified CD8 co-receptor sequences, respectively, T cells transduced with #11 induced a stronger cytokine response as measured as IFNγ, quantitated in the culture supernatants of three-way co-cultures using donor D600115, E:T:iDC::1:1/10:1/4. 23A-B .

IFNγ secretion is increased in response to A375 in the presence of iDCs. In the tri-cultures with iDCs, IFNγ secretion was higher in #10 compared to the other constructs. IFNγ-quantified in the supernatants of DC co-cultures D150081, E:T:iDC::1:1/10:1/4. 24A-B

IFNγ secretion in response to UACC257 increases in the presence of iDCs. In the tri-cultures with iDCs, IFNγ secretion is higher in #10 compared to the other constructs. However, when comparing #9 with #11, which express wild-type and modified CD8 co-receptor sequences, respectively, T cells transduced with #11 induced a stronger cytokine response as measured as IFNγ, quantitated in the culture supernatants of three-way co-cultures using donor D600115, E:T:iDC::1:1/10:1/4. 25A-B . These results demonstrate that T cell products co-expressing a transgenic TCR and CD8 co-receptor (αβ heterodimer or modified CD8α homodimer) are able to license DCs in the microenvironment through antigen cross-presentation and therefore have the potential have to show a stronger anti-tumor response and modulate the tumor microenvironment.

All references cited in this specification are incorporated herein by reference as if each reference was shown to be specifically and individually incorporated by reference. The citation of each clue is intended for disclosure thereof prior to the filing date and should not be construed as a permission that the present disclosure may not pre-date this reference in accordance with prior inventions.

It is understood that any of the elements described above, or two or more elements together, also find useful application in other types of methods other than the type described above. Thus, without further investigation, the foregoing will disclose the essence of the present disclosure sufficiently completely that others, using current knowledge, may readily adapt the latter for various applications without omitting features which, from the point of view of the prior art, are largely essential characteristics of the generic or specific Embodiments of this disclosure set forth in the appended claims. The above embodiments are presented by way of example only; the scope of the present disclosure is to be limited only by the following claims.

QUOTES INCLUDED IN DESCRIPTION

This list of the documents cited by the applicant was generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

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Claims (10)

A polypeptide comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:5. polypeptide after claim 1 , wherein the polypeptide is at least 90% identical to the amino acid sequence of SEQ ID NO: 5. polypeptide after claim 1 , wherein the polypeptide is at least 95% identical to the amino acid sequence of SEQ ID NO: 5 and optionally 100% identical to the amino acid sequence of SEQ ID NO: 5. The polypeptide according to any one of Claims 1 until 3 , wherein the polypeptide further comprises a signal peptide. polypeptide after claim 4 wherein the signal peptide is at least 90% identical to the amino acid sequence of SEQ ID NO: 6 and optionally 100% identical to the amino acid sequence of SEQ ID NO: 6. Nucleic acid encoding the polypeptide of any one of Claims 1 until 5 coded. Vector comprising the nucleic acid according to claim 6 . T cell following the vector claim 7 is transduced and wherein the T cell is optionally an αβ T cell, γδ T cell or natural killer T cell. Population of modified T cells presenting an exogenous CD8 co-receptor encoding the polypeptide of any one of Claims 1 until 5 and a T cell receptor. A composition comprising a cell according to any one of Claims 8 or 9 .

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