Engineered cells expressing prostate-specific membrane antigen (PSMA) or its modified forms and related methods

Abstract

Cells expressing prostate-specific membrane antigen (PSMA) or a modified form thereof, such as engineered cells, are provided. In some embodiments, the cells further contain a genetically engineered recombinant receptor, such as a chimeric antigen receptor, that specifically binds to the antigen. The present disclosure also provides methods for detecting, identifying, selecting or targeting cells expressing PSMA, for example, in combination with administering such cells to a subject, including methods for adoptive cell therapy, or in combination with methods for making engineered cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 483,313, filed April 7, 2017, entitled “ENGINEERED CELLS EXPRESSING PROSTATE-SPECIFIC MEMBRANE ANTIGEN (PSMA) OR A MODIFIED FORM THEREOF AND RELATED METHODS”; and U.S. Provisional Application No. 62 / 483,313, filed August 30, 2017, entitled “ENGINEERED CELLS EXPRESSING PROSTATE-SPECIFIC MEMBRANE ANTIGEN (PSMA) OR A MODIFIED FORM THEREOF AND RELATED METHODS”. U.S. Provisional Application No. 62 / 552,354, filed on September 7, 2017, entitled “ENGINEERED CELLS EXPRESSING PROSTATE-SPECIFIC MEMBRANE ANTIGEN (PSMA) OR A MODIFIED FORM THEREOF AND RELATED METHODS”; U.S. Provisional Application No. 62 / 555,635, filed on November 7, 2017, entitled “ENGINEERED CELLS EXPRESSING PROSTATE-SPECIFIC MEMBRANE ANTIGEN (PSMA) OR A MODIFIED FORM THEREOF AND RELATED METHODS”; and U.S. Provisional Application No. 62 / 555,635, filed on November 7, 2017, entitled “ENGINEERED CELLS EXPRESSING PROSTATE-SPECIFIC MEMBRANE ANTIGEN (PSMA) OR A MODIFIED FORM THEREOF AND RELATED METHODS”. and U.S. Provisional Application No. 62 / 582,913, filed January 19, 2018, entitled “ENGINEERED CELLS EXPRESSING PROSTATE-SPECIFIC MEMBRANE ANTIGEN (PSMA) OR A MODIFIED FORMTHEREOF AND RELATED METHODS,” the contents of which are incorporated by reference in their entireties.

[0003] Incorporation by Reference into the Sequence Listing

[0004] This application is submitted with a sequence listing in electronic format. The sequence listing is provided as a 59.3 kilobyte file named 735042010640SeqList.txt, created on April 6, 2018. The information in the electronic sequence listing is incorporated by reference in its entirety. Technical Field

[0005] The present disclosure relates in some aspects to engineered cells, such as engineered T cells, that express prostate-specific membrane antigen (PSMA), typically modified PSMA. The cells also contain a genetically engineered recombinant receptor, such as a chimeric antigen receptor, that specifically binds to the antigen. The present disclosure also provides methods for detecting, identifying, selecting, or targeting cells that express PSMA, for example, in conjunction with methods for administering such cells to a subject, including adoptive cell therapy, or in conjunction with methods for making engineered cells. Background Art

[0006] Various strategies can be used to treat disease or illness (such as cancer or tumor), including giving cell therapy. In addition, strategy can be used to engineer immune cells to express genetically engineered recombinant receptors such as chimeric antigen receptors (CAR), and give a composition containing such cells to a subject. Improved strategies are needed, for example, to improve the ability to monitor, detect or regulate engineered cells in combination with such therapies after being given to a subject. Compositions, cells and methods meeting such needs are provided. Summary of the Invention

[0007] Provided herein are engineered cells comprising prostate-specific membrane antigen (PSMA) or a modified form thereof and a recombinant receptor. Also provided herein are engineered cells comprising nucleic acids encoding prostate-specific membrane antigen (PSMA) or a modified form thereof; and nucleic acids encoding recombinant receptors. In some of any of the provided embodiments, the recombinant receptor is a chimeric receptor and / or a recombinant antigen receptor, such as a chimeric antigen receptor (CAR).

[0008] In some embodiments, PSMA or a modified form thereof is expressed on the surface of a cell. In some embodiments, PSMA or a modified form thereof comprises an extracellular portion and a transmembrane domain. In some embodiments, PSMA or a modified form thereof, optionally the extracellular portion, is capable of being recognized by a PSMA targeting molecule or portion thereof.

[0009] In some embodiments, the PSMA-targeting molecule or portion thereof: is capable of binding to PSMA and / or a modified form thereof, and / or is capable of binding to the active site of PSMA and / or the active site of a modified form of PSMA, and / or is capable of being cleaved by PSMA and / or by a modified form of PSMA; and / or is an antagonist, selective antagonist, inverse agonist, selective inverse agonist, agonist, selective agonist, inhibitor, and / or selective inhibitor of PSMA and / or its modified forms.

[0010] In some embodiments, PSMA or a modified form thereof comprises an N-acetylated alpha-linked acid dipeptidase (NAALADase) domain, and / or comprises one or more active site residues and / or residues involved in PSMA substrate binding and / or PSMA catalytic activity, which, with reference to the position of the amino acid sequence set forth in SEQ ID NO: 23, are optionally residues at positions 210, 257, 269, 272, 377, 387, 387, 424, 424, 425, 433, 436, 453, 517, 518, 519, 552, 553, 534, 535, 536, 552, 553, 628, 666, 689, 699, and / or 700.

[0011] In some embodiments, PSMA or a modified form thereof is human PSMA or a modified form thereof. In some embodiments, PSMA or a modified form thereof is wild-type PSMA, optionally wild-type human PSMA. In some embodiments, PSMA or a modified form thereof comprises the amino acid sequence set forth in SEQ ID NO: 23, or its extracellular and / or transmembrane domain, or an amino acid sequence that exhibits at least or about 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 23, or its extracellular and / or transmembrane domain.

[0012] In some embodiments, the PSMA or modified form thereof is a modified PSMA that contains one or more amino acid modifications compared to wild-type or unmodified PSMA.

[0013] In some embodiments, the wild-type or unmodified PSMA is human PSMA and / or comprises the amino acid sequence set forth in SEQ ID NO: 23, or an amino acid sequence that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0014] In some embodiments, the one or more amino acid modifications comprise one or more amino acid substitutions, deletions, and / or insertions. In some embodiments, the modified PSMA (i) exhibits reduced endogenous signaling; (ii) exhibits increased cell surface expression; and / or (iii) exhibits reduced cellular internalization compared to wild-type or unmodified PSMA.

[0015] In some embodiments, with reference to the position in the amino acid sequence set forth in SEQ ID NO: 23, the modified PSMA includes at least one amino acid substitution corresponding to W2G or does not include W2 or does not include any residue at position 2. In some embodiments, the modified PSMA includes the amino acid sequence set forth in SEQ ID NO: 24, or a fragment thereof, or an amino acid sequence that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 24, or a fragment thereof, and includes the at least one amino acid substitution.

[0016] In some embodiments, the modified PSMA comprises a deletion of one or more N-terminal amino acid residues in the intracellular portion compared to wild-type or unmodified PSMA. In some embodiments, the modified PSMA comprises a deletion of up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 N-terminal amino acid residues compared to wild-type or unmodified PSMA. In some embodiments, with reference to the positions in the amino acid sequence set forth in SEQ ID NO: 23, the modified PSMA comprises a deletion of a contiguous amino acid sequence at the N-terminus starting at residues at positions 2, 3, 4, or 5 and up to positions 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 compared to wild-type or unmodified PSMA. In some embodiments, with reference to the positions in the amino acid sequence set forth in SEQ ID NO: 23, the modified PSMA comprises a deletion of the residues at positions 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3.

[0017] In some embodiments, PSMA or a modified form thereof comprises the amino acid sequence set forth in SEQ ID NO: 52, or a fragment thereof; or an amino acid sequence that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 52, or a fragment thereof, and comprises a deletion of one or more N-terminal amino acid residues and optionally contains a methionine as the first residue or a methionine start codon.

[0018] In some embodiments, PSMA or a modified form thereof comprises the amino acid sequence set forth in SEQ ID NO: 25, or a fragment thereof; or an amino acid sequence that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 25, or a fragment thereof, and comprises a deletion of the one or more N-terminal amino acid residues.

[0019] In some embodiments, PSMA or a modified form thereof is encoded by the nucleic acid sequence set forth in SEQ ID NO: 26 or 53, or a fragment thereof; or a nucleic acid sequence that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 26 or 53, or a fragment thereof, and optionally contains a nucleic acid encoding methionine as the first residue or a methionine start codon.

[0020] In some embodiments, PSMA or a modified form thereof is encoded by a nucleic acid sequence that is modified to be CpG-free and / or codon-optimized. In some embodiments, PSMA or a modified form thereof is encoded by the following: a nucleic acid sequence set forth in SEQ ID NO: 27, or a fragment thereof; or a nucleic acid sequence that exhibits at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 27, or a fragment thereof, and optionally contains a nucleic acid encoding a methionine as the first residue or a methionine start codon.

[0021] In some embodiments, the modified PSMA includes all or substantially all of the transmembrane domain of wild-type or unmodified PSMA; or the modified PSMA includes a transmembrane domain that has the same or at least the same number of amino acids as the transmembrane domain of wild-type or unmodified PSMA.

[0022] In some embodiments, the PSMA-targeting molecule is or includes a small molecule, a ligand, an antibody or antigen-binding fragment thereof, an aptamer, a peptide, or a conjugate thereof.

[0023] In some embodiments, the PSMA targeting molecule is or includes a small molecule. In some embodiments, the small molecule is selected from 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid (DCFPyL), N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine ​​(DCFBC), (aminostyryl)pyridinium (ASP) dye, 2-(3-[1-carboxy-5-[(5-iodo-pyridine-3-carbonyl)-amino]-pentyl]-ureido)-glutaric acid (YC-VI-11), 2-[3-[1-carboxy-5-(4-iodo-benzoylamino)-pentyl]-ureido]-glutaric acid (DCIBzL), or YC-7), 1-(3-carboxy-4-(3-hydroxy-6-oxo-6H-xanthen-9-yl)phenylamino)-9,16,24-trioxo-1-thioxo-2,8,17,23,25-pentaazaoctacosane-7,22,26,28-tetracarboxylic acid (YC-36), Glu-NH-CO-NH-Lys(Ahx)-HBED-CC(PSMA-HBED-CC), 9-(4-fluoro-3-[hydroxymethyl]butyl)guanine (FHBG), Glu-urea-Lys-(Ahx)-[(HBED-CC)](PSMA-11), PSMA-617, 2-(phosphonomethyl) 1,3-Dicarboxypropyl)ureido]glutaric acid (DUPA), PSMAN, N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid, MIP-1072, MIP-1095, MIP-1404, MIP-1405, and N -[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-L-glutamate (ZJ43), (S)-2-(4-iodobenzylphosphonylmethyl)-glutaric acid (GPI-18431), 2-[(3-{4-[(2-amino-4-hydroxy-pteridin-6-ylmethyl)-amino]-benzoylamino}-3-carboxy-propyl)-hydroxy-phosphorylmethyl]-glutaric acid (MPE), (2S,3'S)-{[(3'-amino-3'-carboxy-propyl)-hydroxyphosphoryl]methyl}-glutaric acid (EPE) and (2S)-2-{[(2-carboxy-ethyl)-hydroxy-phosphoryl]methyl}-glutaric acid (SPE).In some embodiments, the PSMA-targeting molecule is or includes 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid (DCFPyL) or N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine ​​(DCFBC).

[0024] In some embodiments, the PSMA targeting molecule is or comprises an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof optionally has a binding site that specifically binds to PSMA. In some embodiments, the antibody or antigen-binding fragment thereof is selected from J591, DFO-J591, CYT-356, J415, 3 / A12, 3 / F11, 3 / E7, D2B, 107-1A4, YPSMA-1, YPSMA-2, 3E6, 2G7, 24.4E6, GCP-02, GCP-04, GCP-05, J533, E99, 1G9, 3C6, 4.40, 026, D7-Fc, D7-CH3, 4D4, A5, and antigen-binding fragments and derivatives thereof, or comprises CDR3, V H and / or V L , and / or compete for binding to PSMA or bind to the same PSMA epitope as any of the foregoing.

[0025] In some embodiments, the PSMA-targeting molecule is or includes an aptamer or a conjugate thereof. In some embodiments, the aptamer includes A9, A10, A10g, A10-3.2, or SZT101, or a conjugate thereof.

[0026] In some embodiments, the recombinant receptor is or includes a chimeric receptor and / or a recombinant antigen receptor. In some embodiments, the recombinant receptor is capable of binding to a target antigen that is associated with a cell or tissue of a disease or disorder, is specific for a cell or tissue of a disease, disorder or condition, and / or is expressed on a cell or tissue of a disease or disorder. In some embodiments, the disease, disorder or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer. In some embodiments, the target antigen is a tumor antigen.

[0027] In some embodiments, the target antigen is selected from αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), cancer-testis antigen, cancer / testis antigen 1B (CTAG,Also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclins, cyclin A2, CC motif chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), truncated epidermal growth factor protein (tEGFR), epidermal growth factor receptor type III mutant (EGFRvIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor-like protein 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), glypican (GPC3), G protein-coupled receptor 5D (GPCR5D), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimer, human high score melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22Rα), IL-13 receptor alpha 2 (IL-13Rα2), kinase insert domain receptor (KDR), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, leucine-rich repeat-containing protein 8 family member A (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer cell group 2 member D (NKG2D) ligand, melanin A (MART-1), neural cell adhesion molecule (NCAM), carcinoembryonic antigen, preferentially expressed melanoma antigen (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein (TPBG, also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-related protein 1 (TRP1, also known as TYRP1 or gp75), tyrosinase-related protein 2 (TRP2,Also known as dopachrome tautomerase, dopachrome delta isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor 1 (WT-1), pathogen-specific or pathogen-expressed antigens, or antigens associated with a universal tag, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens. In some embodiments, the target antigen is selected from ROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2 / neu (receptor tyrosine kinase erbB2), L1-CAM, CD19, CD20, CD22, mesothelin, CEA and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, erb-B2, erb-B3, erb-B4, erbB dimer, EGFR vIII, folate binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, GD2, GD3, G protein-coupled receptor 5D (GPCR5D), HMW-MAA, IL-22R-α, IL-13R-α2, kinase insert domain receptor (KDR), kappa light chain, Lewis Y, L1 cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, preferentially expressed melanoma antigen (PRAME), survivin, TAG72, B7-H6, IL-13 receptor α2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250 / CAIX, HLA-A1, MAGE A1, HLA-A2, NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7 / 8, avb6 integrin, 8H9, NCAM, VEGF receptor, 5T4, fetal AchR, NKG2D ligand, CD44v6, dual antigen, cancer-testis antigen, mesothelin, murine CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp 100, carcinoembryonic antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), Her2 / neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms tumor 1 (WT-1), cyclins, cyclin A2, CCL-1, CD138, pathogen-specific antigens, and antigens associated with universal tags.

[0028] In some embodiments, the recombinant receptor is or includes a functional non-TCR antigen receptor or TCR or an antigen-binding fragment thereof. In some embodiments, the recombinant receptor is a chimeric antigen receptor (CAR).

[0029] In some embodiments, the recombinant receptor includes an extracellular domain comprising an antigen binding domain. In some embodiments, the antigen binding domain is or includes an antibody or an antibody fragment thereof, the antibody fragment being optionally a single-chain fragment. In some embodiments, the fragment includes an antibody variable region connected by a flexible linker. In some embodiments, the fragment includes an scFv.

[0030] In some embodiments, the recombinant receptor further comprises a spacer and / or a hinge region.

[0031] In some embodiments, the recombinant receptor includes an intracellular signaling region. In some embodiments, the intracellular signaling region includes an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or includes a primary signaling domain, a signaling domain capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain containing an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling domain is or includes the intracellular signaling domain of a CD3 chain, optionally a CD3-zeta (CD3ζ) chain, or a signaling portion thereof.

[0032] In some embodiments, the recombinant receptor further comprises a transmembrane domain disposed between the extracellular domain and the intracellular signaling region.

[0033] In some embodiments, the intracellular signaling region further comprises a costimulatory signaling region. In some embodiments, the costimulatory signaling region comprises an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof. In some embodiments, the costimulatory signaling region comprises an intracellular signaling domain of CD28, 4-1BB or ICOS or a signaling portion thereof. In some embodiments, the costimulatory signaling region is located between the transmembrane domain and the intracellular signaling region.

[0034] In some embodiments, the cell comprises one or more polynucleotides comprising a nucleic acid encoding PSMA or a modified form thereof and a nucleic acid encoding a recombinant receptor. In some embodiments, the one or more polynucleotides are one polynucleotide, and the nucleic acid encoding PSMA or a modified form thereof and the nucleic acid encoding the recombinant receptor are operably linked to the same promoter and are optionally separated by an internal ribosome entry site (IRES) or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping (which is optionally T2A, P2A, E2A, or F2A).

[0035] In some embodiments, the cell comprises a nucleic acid encoding PSMA or a modified form thereof and a nucleic acid encoding a CAR within one polynucleotide, the polynucleotide comprising, in 5' to 3' order: i) a nucleic acid encoding a signal peptide; ii) a nucleic acid encoding a CAR comprising an scFv; a spacer; a transmembrane domain; a co-stimulatory signaling region, and an intracellular signaling domain of the CD3-zeta (CD3ζ) chain, or an intracellular region of a signaling portion thereof; iii) a nucleic acid sequence encoding a self-cleaving peptide or a peptide that causes ribosome skipping (which is optionally T2A, P2A, E2A, or F2A); and iv) a nucleic acid encoding PSMA or a modified form thereof, the PSMA or modified form thereof optionally comprising the amino acid sequence shown in SEQ ID NO: 52 or a fragment thereof; or exhibiting an amino acid sequence identical to SEQ ID NO: 52. NO:52 or a fragment thereof that has at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and comprises an amino acid sequence of a deletion of the one or more N-terminal amino acid residues.

[0036] In some embodiments, the nucleic acid encoding PSMA or a modified form thereof and the nucleic acid encoding the recombinant receptor are operably linked to two different promoters.

[0037] In some embodiments, the nucleic acid encoding the recombinant receptor is present downstream of the nucleic acid encoding PSMA or a modified form thereof.

[0038] In some embodiments, the one or more polynucleotides comprise two different polynucleotides, the nucleic acid encoding PSMA or a modified form thereof and the nucleic acid encoding the recombinant receptor are operably linked to two different promoters, and / or the nucleic acid encoding PSMA or a modified form thereof and the nucleic acid encoding the recombinant receptor are present or inserted at different locations within the genome of the cell.

[0039] In some embodiments, the cell is an immune cell; the cell is a T cell, optionally selected from CD4+ T cells and subtypes thereof and CD8+ T cells and subtypes thereof; the cell is a NK cell; and / or the cell is derived from a multipotent or pluripotent cell, which is optionally an iPSC.

[0040] In some embodiments, the cell is a T cell selected from the following: central memory T cells, effector memory T cells, naive T cells, stem cell central memory T cells, effector T cells and regulatory T cells; and / or the cell comprises a plurality of cells, the plurality of cells containing at least 50% of a population of the following cells selected from the group consisting of: CD4+ T cells, CD8+ T cells, central memory T cells, effector memory T cells, naive T cells, stem cell central memory T cells, effector T cells and regulatory T cells. In some embodiments, the cell is a regulatory T cell.

[0041] In some embodiments, the engineered cells provided herein further comprise recombinant FOXP3 or a variant thereof.

[0042] Also provided are polynucleotides comprising a first nucleic acid encoding a prostate-specific membrane antigen (PSMA) or a modified form thereof and a second nucleic acid encoding a recombinant receptor. In some embodiments of any of the provided embodiments, the recombinant receptor is a chimeric receptor and / or a recombinant antigen receptor, such as a chimeric antigen receptor (CAR). In some embodiments, the nucleic acid encoding PSMA or a modified form thereof and the nucleic acid encoding the recombinant receptor are operably linked to the same promoter and are optionally separated by an internal ribosome entry site (IRES) or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping (which is optionally T2A, P2A, E2A, or F2A).

[0043] In some embodiments, the nucleic acid encoding PSMA or a modified form thereof and the nucleic acid encoding the recombinant receptor are operably linked to two different promoters.

[0044] In some embodiments, the nucleic acid encoding the recombinant receptor is present downstream of the nucleic acid encoding PSMA or a modified form thereof.

[0045] Also provided are polynucleotide sets comprising a first polynucleotide comprising a nucleic acid encoding a prostate-specific membrane antigen (PSMA) or a modified form thereof and a second polynucleotide comprising a nucleic acid encoding a recombinant receptor. Also provided are compositions comprising such polynucleotide sets. In some of any of the provided embodiments, the recombinant receptor is a chimeric receptor and / or a recombinant antigen receptor, such as a chimeric antigen receptor (CAR).

[0046] In some embodiments of the polynucleotide, set of polynucleotides, or composition comprising a set of polynucleotides provided herein, the nucleic acid encoding PSMA or a modified form thereof and the nucleic acid encoding the recombinant receptor are each independently operably linked to a promoter.

[0047] In some embodiments, the encoded PSMA or its modified form is capable of being expressed on the surface of a cell. In some embodiments, the encoded PSMA or its modified form comprises an extracellular portion and a transmembrane domain.

[0048] In some embodiments, PSMA or a modified form thereof, optionally an extracellular portion, is capable of being recognized by a PSMA targeting molecule or portion thereof.

[0049] In some embodiments, the PSMA-targeting molecule or portion thereof: is capable of binding to PSMA and / or a modified form thereof, and / or is capable of binding to the active site of PSMA and / or the active site of a modified form of PSMA, and / or is capable of being cleaved by PSMA and / or by a modified form of PSMA; and / or is an antagonist, selective antagonist, inverse agonist, selective inverse agonist, agonist, selective agonist, inhibitor, and / or selective inhibitor of PSMA and / or its modified forms.

[0050] In some embodiments, the encoded PSMA or modified form thereof comprises an N-acetylated alpha-linked acid dipeptidase (NAALADase) domain and / or comprises one or more active site residues and / or residues involved in PSMA substrate binding and / or PSMA catalytic activity, which are optionally residues at positions 210, 257, 269, 272, 377, 387, 387, 424, 424, 425, 433, 436, 453, 517, 518, 519, 552, 553, 534, 535, 536, 552, 553, 628, 666, 689, 699, and / or 700 with reference to the position of the amino acid sequence set forth in SEQ ID NO: 23.

[0051] In some embodiments, the encoded PSMA or its modified form is human PSMA or its modified form. In some embodiments, the encoded PSMA or its modified form is wild-type PSMA, optionally wild-type human PSMA. In some embodiments, the encoded PSMA or its modified form comprises the amino acid sequence set forth in SEQ ID NO: 23, or its extracellular and / or transmembrane domain, or an amino acid sequence that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 23, or its extracellular and / or transmembrane domain.

[0052] In some embodiments, the encoded PSMA or modified form thereof is a modified PSMA that contains one or more amino acid modifications compared to wild-type or unmodified PSMA. In some embodiments, the wild-type or unmodified PSMA is human PSMA and / or comprises the amino acid sequence set forth in SEQ ID NO: 23, or an amino acid sequence that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0053] In some embodiments, the one or more amino acid modifications comprise one or more amino acid substitutions, deletions, and / or insertions. In some embodiments, the encoded modified PSMA (i) exhibits reduced endogenous signaling; (ii) exhibits increased cell surface expression; and / or (iii) exhibits reduced cellular internalization compared to wild-type or unmodified PSMA.

[0054] In some embodiments, with reference to positions in the amino acid sequence set forth in SEQ ID NO: 23, the encoded modified PSMA includes at least one amino acid substitution corresponding to W2G or does not include W2 or does not include any residue at position 2. In some embodiments, the encoded modified PSMA includes the amino acid sequence set forth in SEQ ID NO: 24, or a fragment thereof, or an amino acid sequence that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 24, or a fragment thereof, and includes the at least one amino acid substitution.

[0055] In some embodiments, the encoded modified PSMA comprises a deletion of one or more N-terminal amino acid residues in the intracellular portion compared to wild-type or unmodified PSMA. In some embodiments, the encoded modified PSMA comprises a deletion of up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 N-terminal amino acid residues compared to wild-type or unmodified PSMA. In some embodiments, with reference to the position in the amino acid sequence set forth in SEQ ID NO: 23, the encoded modified PSMA comprises a deletion of a contiguous amino acid sequence at the N-terminus starting at residues at positions 2, 3, 4, or 5 and up to positions 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. In some embodiments, the encoded modified PSMA comprises a deletion of residues at positions 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3, with reference to the positions in the amino acid sequence set forth in SEQ ID NO: 23.

[0056] In some embodiments, the encoded PSMA or modified form thereof comprises the amino acid sequence set forth in SEQ ID NO: 52, or a fragment thereof; or an amino acid sequence that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 52, or a fragment thereof, and comprises a deletion of one or more N-terminal amino acid residues and optionally contains a methionine as the first codon or a methionine start codon.

[0057] In some embodiments, the encoded PSMA or modified form thereof comprises the amino acid sequence set forth in SEQ ID NO: 25, or a fragment thereof; or an amino acid sequence that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 25, or a fragment thereof, and comprises a deletion of the one or more N-terminal amino acid residues.

[0058] In some embodiments, PSMA or a modified form thereof is encoded by the nucleic acid sequence set forth in SEQ ID NO: 26 or 53, or a fragment thereof; or a nucleic acid sequence that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 26 or 53, or a fragment thereof, and optionally contains a nucleic acid encoding methionine as the first residue or a methionine start codon.

[0059] In some embodiments, PSMA or a modified form thereof is encoded by a nucleic acid sequence that is modified to be CpG-free and / or codon-optimized. In some embodiments, PSMA or a modified form thereof is encoded by the following: a nucleic acid sequence set forth in SEQ ID NO: 27, or a fragment thereof; or a nucleic acid sequence that exhibits at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 27, or a fragment thereof, and optionally contains a nucleic acid encoding a methionine as the first residue or a methionine start codon.

[0060] In some embodiments, the encoded modified PSMA includes all or substantially all of the transmembrane domain of wild-type or unmodified PSMA; or the encoded modified PSMA includes a transmembrane domain having the same or at least the same number of amino acids as the transmembrane domain of wild-type or unmodified PSMA.

[0061] In some embodiments, the PSMA-targeting molecule is or includes a small molecule, a ligand, an antibody or antigen-binding fragment thereof, an aptamer, a peptide, or a conjugate thereof.

[0062] In some embodiments, the PSMA targeting molecule is or includes a small molecule. In some embodiments, the small molecule is selected from 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid (DCFPyL), N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine ​​(DCFBC), (aminostyryl)pyridinium (ASP) dye, 2-(3-[1-carboxy-5-[(5-iodo-pyridine-3-carbonyl)-amino]-pentyl]-ureido)-glutaric acid (YC-VI-11), 2-[3-[1-carboxy-5-(4-iodo-benzoylamino)-pentyl]-ureido]-glutaric acid (DCIBzL), or YC-7), 1-(3-carboxy-4-(3-hydroxy-6-oxo-6H-xanthen-9-yl)phenylamino)-9,16,24-trioxo-1-thioxo-2,8,17,23,25-pentaazaoctacosane-7,22,26,28-tetracarboxylic acid (YC-36), Glu-NH-CO-NH-Lys(Ahx)-HBED-CC(PSMA-HBED-CC), 9-(4-fluoro-3-[hydroxymethyl]butyl)guanine (FHBG), Glu-urea-Lys-(Ahx)-[(HBED-CC)](PSMA-11), PSMA-617, 2-(phosphonomethyl) 1,3-Dicarboxypropyl)ureido]glutaric acid (DUPA), PSMAN, N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid, MIP-1072, MIP-1095, MIP-1404, MIP-1405, and N -[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-L-glutamate (ZJ43), (S)-2-(4-iodobenzylphosphonylmethyl)-glutaric acid (GPI-18431), 2-[(3-{4-[(2-amino-4-hydroxy-pteridin-6-ylmethyl)-amino]-benzoylamino}-3-carboxy-propyl)-hydroxy-phosphorylmethyl]-glutaric acid (MPE), (2S,3'S)-{[(3'-amino-3'-carboxy-propyl)-hydroxyphosphoryl]methyl}-glutaric acid (EPE) and (2S)-2-{[(2-carboxy-ethyl)-hydroxy-phosphoryl]methyl}-glutaric acid (SPE).In some embodiments, the PSMA-targeting molecule is or includes 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid (DCFPyL) or N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine ​​(DCFBC).

[0063] In some embodiments, the PSMA targeting molecule is or comprises an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof optionally has a binding site that specifically binds to PSMA. In some embodiments, the antibody or antigen-binding fragment thereof is selected from J591, DFO-J591, CYT-356, J415, 3 / A12, 3 / F11, 3 / E7, D2B, 107-1A4, YPSMA-1, YPSMA-2, 3E6, 2G7, 24.4E6, GCP-02, GCP-04, GCP-05, J533, E99, 1G9, 3C6, 4.40, 026, D7-Fc, D7-CH3, 4D4, A5, and antigen-binding fragments and derivatives thereof, or comprises CDR3, V H and / or V L , and / or compete for binding to PSMA or bind to the same PSMA epitope as any of the foregoing.

[0064] In some embodiments, the PSMA-targeting molecule is or includes an aptamer or a conjugate thereof. In some embodiments, the aptamer includes A9, A10, A10g, A10-3.2, or SZT101, or a conjugate thereof.

[0065] In some embodiments, the encoded recombinant receptor is or includes a chimeric receptor and / or a recombinant antigen receptor.

[0066] In some embodiments, the encoded recombinant receptor is capable of binding to a target antigen that is associated with, specific for, and / or expressed on cells or tissues of a disease or disorder. In some embodiments, the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer. In some embodiments, the target antigen is a tumor antigen.

[0067] In some embodiments, the target antigen is selected from αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), cancer-testis antigen, cancer / testis antigen 1B (CTAG,Also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclins, cyclin A2, CC motif chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), truncated epidermal growth factor protein (tEGFR), epidermal growth factor receptor type III mutant (EGFRvIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor-like protein 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), glypican (GPC3), G protein-coupled receptor 5D (GPCR5D), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimer, human high score melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22Rα), IL-13 receptor alpha 2 (IL-13Rα2), kinase insert domain receptor (KDR), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, leucine-rich repeat-containing protein 8 family member A (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer cell group 2 member D (NKG2D) ligand, melanin A (MART-1), neural cell adhesion molecule (NCAM), carcinoembryonic antigen, preferentially expressed melanoma antigen (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein (TPBG, also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-related protein 1 (TRP1, also known as TYRP1 or gp75), tyrosinase-related protein 2 (TRP2,Also known as dopachrome tautomerase, dopachrome delta isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor 1 (WT-1), pathogen-specific or pathogen-expressed antigens, or antigens associated with a universal tag, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens. In some embodiments, the target antigen is selected from ROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2 / neu (receptor tyrosine kinase erbB2), L1-CAM, CD19, CD20, CD22, mesothelin, CEA and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, erb-B2, erb-B3, erb-B4, erbB dimer, EGFR vIII, folate binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, GD2, GD3, G protein-coupled receptor 5D (GPCR5D), HMW-MAA, IL-22R-α, IL-13R-α2, kinase insert domain receptor (KDR), kappa light chain, Lewis Y, L1 cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, preferentially expressed melanoma antigen (PRAME), survivin, TAG72, B7-H6, IL-13 receptor α2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250 / CAIX, HLA-A1, MAGE A1, HLA-A2, NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7 / 8, avb6 integrin, 8H9, NCAM, VEGF receptor, 5T4, fetal AchR, NKG2D ligand, CD44v6, dual antigen, cancer-testis antigen, mesothelin, murine CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp 100, carcinoembryonic antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), Her2 / neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms tumor 1 (WT-1), cyclins, cyclin A2, CCL-1, CD138, pathogen-specific antigens, and antigens associated with universal tags.

[0068] In some embodiments, the encoded recombinant receptor is or includes a functional non-TCR antigen receptor or TCR or an antigen-binding fragment thereof. In some embodiments, the encoded recombinant receptor is a chimeric antigen receptor (CAR).

[0069] In some embodiments, the encoded recombinant receptor includes an extracellular domain comprising an antigen binding domain.

[0070] In some embodiments, the antigen binding domain is or comprises an antibody or an antibody fragment thereof, wherein the antibody fragment is optionally a single chain fragment. In some embodiments, the fragment comprises an antibody variable region connected by a flexible linker. In some embodiments, the fragment comprises an scFv.

[0071] In some embodiments, the encoded recombinant receptor further comprises a spacer and / or a hinge region.

[0072] In some embodiments, the encoded recombinant receptor includes an intracellular signaling region. In some embodiments, the intracellular signaling region includes an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or includes a primary signaling domain, a signaling domain capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain containing an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling domain is or includes the intracellular signaling domain of the CD3 chain, optionally the CD3-zeta (CD3ζ) chain, or a signaling portion thereof.

[0073] In some embodiments, the recombinant receptor further comprises a transmembrane domain disposed between the extracellular domain and the intracellular signaling region.

[0074] In some embodiments, the intracellular signaling region further comprises a costimulatory signaling region. In some embodiments, the costimulatory signaling region comprises an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof. In some embodiments, the costimulatory signaling region comprises an intracellular signaling domain of CD28, 4-1BB or ICOS or a signaling portion thereof.

[0075] In some embodiments, the costimulatory signaling region is located between the transmembrane domain and the intracellular signaling region.

[0076] In some embodiments, the polynucleotide comprises, in 5' to 3' order: i) a nucleic acid encoding a signal peptide; ii) a nucleic acid encoding a CAR comprising an scFv; a spacer; a transmembrane domain; an intracellular region comprising a costimulatory signaling region, and an intracellular signaling domain of the CD3-zeta (CD3ζ) chain, or a signaling portion thereof; iii) a nucleic acid sequence encoding a self-cleaving peptide or a peptide that causes ribosome skipping (which is optionally T2A, P2A, E2A, or F2A); and iv) a nucleic acid encoding a PSMA or a modified form thereof, optionally comprising the amino acid sequence set forth in SEQ ID NO: 52, or a fragment thereof; or an amino acid sequence that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 52, or a fragment thereof, and comprising a deletion of said one or more N-terminal amino acid residues.

[0077] Also provided are vectors, such as vectors containing any of the polynucleotides described herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector. In some embodiments, the vector is a lentiviral vector or a gamma-retroviral vector.

[0078] Also provided is a vector set comprising a first vector and a second vector. In some embodiments, the first vector comprises any first polynucleotide described herein, and the second vector comprises a second polynucleotide described herein. Also provided is a composition comprising such a vector set.

[0079] Also provided are methods for producing engineered cells. In some embodiments, the methods comprise introducing into a cell any of the polynucleotides, any of the polynucleotides in the set, or any of the compositions comprising the polynucleotide sets described herein, or any of the vectors or vectors in the set, or any of the compositions comprising the vector sets described herein. Also provided are engineered cells produced using such production methods.

[0080] Also provided are engineered cells containing any of the polynucleotides, polynucleotides in the set, compositions containing the set of polynucleotides described herein, any of the vectors or vectors in the set, or compositions containing the set of vectors described herein.

[0081] Also provided are compositions containing any of the engineered cells described herein. In some embodiments, the compositions further contain a pharmaceutically acceptable excipient. In some embodiments, the compositions further contain a PSMA-targeting molecule, such as any of the PSMA-binding molecules described herein.

[0082] Also provided are methods of treatment. In some embodiments, the methods of treatment involve administering to a subject any of the engineered cells or compositions described herein. In some embodiments, the methods further involve administering to a subject a PSMA-targeting molecule or a composition comprising a PSMA-targeting molecule. In some embodiments, the PSMA-targeting molecule is or includes a therapeutic agent, or further includes a therapeutic agent.

[0083] Also provided are therapeutic methods involving administering to a subject any of the engineered cells or compositions described herein and a PSMA-targeting molecule that is, includes, or also includes a therapeutic agent, or a composition containing a PSMA-targeting molecule that is, includes, or also includes a therapeutic agent.

[0084] Also provided are methods of treatment involving administering to a subject who has been administered any of the engineered cells or compositions described herein: a PSMA-targeting molecule that is, includes, or also includes a therapeutic agent; or a composition containing a PSMA-targeting molecule that is, includes, or also includes a therapeutic agent. In some embodiments of any of the provided embodiments, the recombinant receptor is a chimeric receptor and / or a recombinant antigen receptor, such as a chimeric antigen receptor (CAR).

[0085] In some embodiments, the administration of a PSMA-targeting molecule or a composition containing a PSMA-targeting molecule is performed simultaneously with the administration of an engineered cell or a composition containing an engineered cell, or sequentially in any order. In some embodiments, the administration of a PSMA-targeting molecule or a composition containing a PSMA-targeting molecule is performed simultaneously with the administration of an engineered cell or a composition containing an engineered cell, optionally in the same or different compositions. In some embodiments, the administration of a PSMA-targeting molecule or a composition containing a PSMA-targeting molecule is performed sequentially with the administration of an engineered cell or a composition containing an engineered cell, in any order.

[0086] In some embodiments, the PSMA-targeting molecule or portion thereof: is capable of binding to PSMA and / or a modified form thereof, and / or is capable of binding to the active site of PSMA and / or the active site of a modified form of PSMA, and / or is capable of being cleaved by PSMA and / or by a modified form of PSMA; and / or is an antagonist, selective antagonist, inverse agonist, selective inverse agonist, agonist, selective agonist, inhibitor, and / or selective inhibitor of PSMA and / or its modified forms.

[0087] In some embodiments, PSMA or a modified form thereof is expressed on one or more engineered cells.

[0088] In some embodiments, the subject has a disease, disorder or condition, optionally a cancer, a tumor, an autoimmune disease, disorder or condition, or an infectious disease.

[0089] In some embodiments, the PSMA-targeting molecule is or includes a small molecule, a ligand, an antibody or antigen-binding fragment thereof, an aptamer, a peptide, or a conjugate thereof.

[0090] In some embodiments, the PSMA targeting molecule is or includes a small molecule. In some embodiments, the small molecule is selected from 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid (DCFPyL), N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine ​​(DCFBC), (aminostyryl)pyridinium (ASP) dye, 2-(3-[1-carboxy-5-[(5-iodo-pyridine-3-carbonyl)-amino]-pentyl]-ureido)-glutaric acid (YC-VI-11), 2-[3-[1-carboxy-5-(4-iodo-benzoylamino)-pentyl]-ureido]-glutaric acid (DCIBzL), or YC-7), 1-(3-carboxy-4-(3-hydroxy-6-oxo-6H-xanthen-9-yl)phenylamino)-9,16,24-trioxo-1-thioxo-2,8,17,23,25-pentaazaoctacosane-7,22,26,28-tetracarboxylic acid (YC-36), Glu-NH-CO-NH-Lys(Ahx)-HBED-CC(PSMA-HBED-CC), 9-(4-fluoro-3-[hydroxymethyl]butyl)guanine (FHBG), Glu-urea-Lys-(Ahx)-[(HBED-CC)](PSMA-11), PSMA-617, 2-(phosphonomethyl) 1,3-Dicarboxypropyl)ureido]glutaric acid (DUPA), PSMAN, N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid, MIP-1072, MIP-1095, MIP-1404, MIP-1405, and N -[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-L-glutamate (ZJ43), (S)-2-(4-iodobenzylphosphonylmethyl)-glutaric acid (GPI-18431), 2-[(3-{4-[(2-amino-4-hydroxy-pteridin-6-ylmethyl)-amino]-benzoylamino}-3-carboxy-propyl)-hydroxy-phosphorylmethyl]-glutaric acid (MPE), (2S,3'S)-{[(3'-amino-3'-carboxy-propyl)-hydroxyphosphoryl]methyl}-glutaric acid (EPE) and (2S)-2-{[(2-carboxy-ethyl)-hydroxy-phosphoryl]methyl}-glutaric acid (SPE).In some embodiments, the PSMA-targeting molecule is or includes 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid (DCFPyL) or N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine ​​(DCFBC).

[0091] In some embodiments, the PSMA targeting molecule is or comprises an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof optionally has a binding site that specifically binds to PSMA. In some embodiments, the antibody or antigen-binding fragment thereof is selected from J591, DFO-J591, CYT-356, J415, 3 / A12, 3 / F11, 3 / E7, D2B, 107-1A4, YPSMA-1, YPSMA-2, 3E6, 2G7, 24.4E6, GCP-02, GCP-04, GCP-05, J533, E99, 1G9, 3C6, 4.40, 026, D7-Fc, D7-CH3, 4D4, A5, and antigen-binding fragments and derivatives thereof, or comprises CDR3, V H and / or V L , and / or compete for binding to PSMA or bind to the same PSMA epitope as any of the foregoing.

[0092] In some embodiments, the PSMA-targeting molecule is or includes an aptamer or a conjugate thereof. In some embodiments, the aptamer includes A9, A10, A10g, A10-3.2, or SZT101, or a conjugate thereof.

[0093] In some embodiments, the therapeutic agent is capable of modulating the tumor microenvironment (TME) or is cytotoxic to the tumor.

[0094] In some embodiments, the therapeutic agent is an immunomodulatory agent, a cytotoxic agent, an anticancer agent, or a radiotherapeutic agent.

[0095] In some embodiments, the PSMA-targeting molecule is or comprises a prodrug that is or comprises a therapeutic agent or is capable of converting to or exposing a therapeutic agent, and / or the PSMA-targeting molecule is capable of being cleaved after binding to PSMA or a modified form thereof, wherein the cleavage produces at least one cleavage product comprising the therapeutic agent. In some embodiments, the PSMA-targeting molecule is or comprises Mipsagargin (G-202 (8-O-(12-aminododecanoyl)-8-O-debutyrylthapsigargin)-Asp-γ-Glu-γ-Glu-γ-GluGluOH).

[0096] In some embodiments, the PSMA-targeting molecule is an antibody-drug conjugate (ADC).

[0097] In some embodiments, the PSMA-targeting molecule further comprises a therapeutic agent, and the therapeutic agent is directly or indirectly (optionally via a linker) attached to a portion of the PSMA-targeting molecule that is capable of binding to PSMA or a modified form thereof.

[0098] In some embodiments, the linker is a peptide or polypeptide, or a chemical linker. In some embodiments, the linker is a releasable linker or a cleavable linker. In some embodiments, the linker is capable of being cleaved after PSMA or its modified form is bound by a PSMA targeting molecule, wherein the cleavage produces at least one cleavage product comprising a therapeutic agent. In some embodiments, the releasable linker or cleavable linker is released or cleaved in the presence of one or more conditions or factors present in the tumor microenvironment (TME), wherein the cleavage produces at least one cleavage product comprising a therapeutic agent. In some embodiments, the one or more conditions or factors present in the tumor microenvironment (TME) include matrix metalloproteinases (MMPs), hypoxic conditions, or acidic conditions.

[0099] In some embodiments, the PSMA-targeting molecule induces killing or destruction of one or more engineered cells and / or cells or tissues present in the subject that are specifically recognized by the recombinant receptor.

[0100] In some embodiments, the therapeutic agent comprises a cytotoxic agent. In some embodiments, the cytotoxic agent is or comprises a toxin. In some embodiments, the toxin is a peptide toxin, ricin A chain toxin, abrin A chain, diphtheria toxin (DT) A chain, Pseudomonas exotoxin, Shiga toxin A chain, gelonin, momordin, pokeweed antiviral protein, saporin, trichosanthin, proaerolysin, or barley toxin.

[0101] In some embodiments, the therapeutic agent comprises a photosensitizer. In some embodiments, the photosensitizer comprises pyropheophorbide-a (Ppa) or YC-9.

[0102] In some embodiments, administration of a PSMA-targeting molecule does not or does not substantially induce killing or destruction of healthy tissues or healthy cells, cells or tissues that do not contain engineered cells and / or do not express the antigen.

[0103] In some embodiments, the therapeutic agent is an immunomodulatory agent. In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor or modulator or a cytokine.

[0104] In some embodiments, the immunomodulator is an immune checkpoint inhibitor that can inhibit or block the function of an immune checkpoint molecule or a signaling pathway involving an immune checkpoint molecule. In some embodiments, the immune checkpoint molecule is selected from PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM3, VISTA, an adenosine receptor or extracellular adenosine, optionally adenosine 2A receptor (A2AR) or adenosine 2B receptor (A2BR), or an adenosine or pathway involving any of the above.

[0105] In some embodiments, the method further comprises detecting cells expressing PSMA or a modified form thereof, and / or detecting binding of a PSMA-targeting molecule to PSMA or a modified form thereof and / or the presence of a PSMA-targeting molecule. In some embodiments, the detecting is performed in vivo and / or the detecting is performed via in vivo imaging.

[0106] Also provided are methods for detecting engineered cells. In some embodiments, the methods comprise contacting any of the engineered cells or compositions provided herein with a PSMA-targeting molecule; and detecting binding of the PSMA-targeting molecule to or with PSMA or a modified form thereof and / or the engineered cells and / or the presence of the PSMA-targeting molecule. In some embodiments, the contacting comprises administering the PSMA-targeting molecule to a subject who has already been administered the engineered cells.

[0107] Also provided are methods of detecting the presence or absence of engineered cells in a subject that express a chimeric receptor and / or a recombinant antigen receptor and PSMA or a modified form thereof, the subject having previously been administered any of the provided engineered cells or any of the provided compositions, the methods comprising: (a) administering a PSMA-targeting molecule to a subject that has previously been administered any of the engineered cells provided herein or any of the compositions provided herein, wherein the engineered cells express a chimeric receptor and / or a recombinant antigen receptor and PSMA or a modified form thereof in the subject; and (b) detecting binding of the PSMA-targeting molecule to PSMA or a modified form thereof and / or to the engineered cells and / or the presence of the PSMA-targeting molecule in the subject.

[0108] Also provided are methods for detecting the presence or absence of engineered cells expressing a recombinant receptor and PSMA or a modified form thereof in a subject that has previously been administered any engineered cell or composition provided herein. In some embodiments, the method comprises administering a PSMA-targeting molecule to the subject; and detecting the binding of the PSMA-targeting molecule to PSMA or a modified form thereof and / or to the engineered cells and / or the presence of the PSMA-targeting molecule in the subject. In some embodiments, the detection is performed via in vivo imaging. In some of the embodiments provided, the recombinant receptor is a chimeric receptor and / or a recombinant antigen receptor, such as a chimeric antigen receptor (CAR).

[0109] In some embodiments, the PSMA-targeting molecule or portion thereof: is capable of binding to PSMA and / or its modified forms, and / or is capable of binding to the active site of PSMA and / or the active site of a modified form of PSMA, and / or is capable of being cleaved by PSMA and / or by a modified form of PSMA; and / or is an antagonist, selective antagonist, inverse agonist, selective inverse agonist, agonist, selective agonist, inhibitor, and / or selective inhibitor of PSMA and / or its modified forms. In some embodiments, the PSMA-targeting molecule is or comprises a small molecule, a ligand, an antibody or antigen-binding fragment thereof, an aptamer, a peptide, or a conjugate thereof.

[0110] In some embodiments, the PSMA targeting molecule is or includes a small molecule. In some embodiments, the small molecule is selected from 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid (DCFPyL), N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine ​​(DCFBC), (aminostyryl)pyridinium (ASP) dye, 2-(3-[1-carboxy-5-[(5-iodo-pyridine-3-carbonyl)-amino]-pentyl]-ureido)-glutaric acid (YC-VI-11), 2-[3-[1-carboxy-5-(4-iodo-benzoylamino)-pentyl]-ureido]-glutaric acid (DCIBzL), or YC-7), 1-(3-carboxy-4-(3-hydroxy-6-oxo-6H-xanthen-9-yl)phenylamino)-9,16,24-trioxo-1-thioxo-2,8,17,23,25-pentaazaoctacosane-7,22,26,28-tetracarboxylic acid (YC-36), Glu-NH-CO-NH-Lys(Ahx)-HBED-CC(PSMA-HBED-CC), 9-(4-fluoro-3-[hydroxymethyl]butyl)guanine (FHBG), Glu-urea-Lys-(Ahx)-[(HBED-CC)](PSMA-11), PSMA-617, 2-(phosphonomethyl) 1,3-Dicarboxypropyl)ureido]glutaric acid (DUPA), PSMAN, N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid, MIP-1072, MIP-1095, MIP-1404, MIP-1405, and N -[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-L-glutamate (ZJ43), (S)-2-(4-iodobenzylphosphonylmethyl)-glutaric acid (GPI-18431), 2-[(3-{4-[(2-amino-4-hydroxy-pteridin-6-ylmethyl)-amino]-benzoylamino}-3-carboxy-propyl)-hydroxy-phosphorylmethyl]-glutaric acid (MPE), (2S,3'S)-{[(3'-amino-3'-carboxy-propyl)-hydroxyphosphoryl]methyl}-glutaric acid (EPE) and (2S)-2-{[(2-carboxy-ethyl)-hydroxy-phosphoryl]methyl}-glutaric acid (SPE).In some embodiments, the PSMA-targeting molecule is or includes 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid (DCFPyL) or N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine ​​(DCFBC).

[0111] In some embodiments, the PSMA targeting molecule is or comprises an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof optionally has a binding site that specifically binds to PSMA. In some embodiments, the antibody or antigen-binding fragment thereof is selected from J591, DFO-J591, CYT-356, J415, 3 / A12, 3 / F11, 3 / E7, D2B, 107-1A4, YPSMA-1, YPSMA-2, 3E6, 2G7, 24.4E6, GCP-02, GCP-04, GCP-05, J533, E99, 1G9, 3C6, 4.40, 026, D7-Fc, D7-CH3, 4D4, A5, and antigen-binding fragments and derivatives thereof, or comprises CDR3, V H and / or V L , and / or compete for binding to PSMA or bind to the same PSMA epitope as any of the foregoing.

[0112] In some embodiments, the PSMA-targeting molecule is or includes an aptamer or a conjugate thereof. In some embodiments, the aptamer includes A9, A10, A10g, A10-3.2, or SZT101, or a conjugate thereof.

[0113] In some embodiments, the PSMA-targeting molecule provides a signal or induces a detectable signal or is capable of binding to a moiety that provides a signal or induces a detectable signal; and / or the PSMA-targeting molecule is or comprises a moiety that provides a signal or induces a detectable signal. In some embodiments, wherein the detecting comprises identifying a signal in a subject or a sample from a subject, wherein: the PSMA-targeting molecule provides the signal or induces the detectable signal or is capable of binding to a moiety that provides the signal or induces the detectable signal; and / or the PSMA-targeting molecule is or comprises a moiety that provides the signal or induces the detectable signal.

[0114] In some embodiments, the moiety comprises a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a chromophoric compound, a quantum dot, a nanoparticle, a metal chelate, or an enzyme.

[0115] In some embodiments, the PSMA targeting molecule is or includes an imaging probe or detection agent, which is optionally a radioligand.

[0116] In some embodiments, the PSMA targeting molecule is or comprises the moiety, and / or the PSMA targeting molecule is capable of being cleaved after binding to PSMA or a modified form thereof, wherein the cleavage produces at least one cleavage product comprising the moiety and / or is fluorescent and / or radioactive.

[0117] In some embodiments, the PSMA-targeting molecule further comprises a moiety that provides a signal or induces a detectable signal, and said moiety is directly or indirectly (optionally via a linker) attached to a portion of the PSMA-targeting molecule capable of binding to PSMA or a modified form thereof.

[0118] In some embodiments, the linker is a releasable linker or a cleavable linker. In some embodiments, the linker is capable of being cleaved after binding to PSMA or a modified form thereof, wherein the cleavage produces at least one cleavage product comprising the moiety and / or is fluorescent and / or radioactive. In some embodiments, the releasable linker or cleavable linker is released or cleaved in the presence of one or more conditions or factors present in the tumor microenvironment (TME), wherein the cleavage produces at least one cleavage product comprising the moiety and / or is fluorescent and / or radioactive. In some embodiments, the one or more conditions or factors present in the tumor microenvironment (TME) include matrix metalloproteinases (MMPs), hypoxic conditions, or acidic conditions.

[0119] In some embodiments, the radiotherapeutic agent, radioisotope, radioligand, or radioactive cleavage product comprises 11 C. 18 F. 64 Cu, 68 Ga, 68 Ge, 86 Y. 89 Zr, 90 Y. 99m Tc, 111 In, 123 I. 125 I. 177 Lu and / or 213 Bi.

[0120] In some embodiments, the PSMA targeting molecule is 2-(3-{1-carboxy-5-[(6-[ 18 F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid ( 18 F-DCFPyL) or N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-[ 18 F]Fluorobenzyl-L-cysteine ​​( 18 F-DCFBC).

[0121] In some embodiments, said contacting and / or said detecting is performed in vivo and / or said detecting is performed via in vivo imaging.

[0122] In some embodiments, the in vivo imaging is selected from magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT), computed tomography (CT), computed axial tomography (CAT), electron beam computed tomography (EBCT), high resolution computed tomography (HRCT), hypocycloidal tomography, positron emission tomography (PET), scintigraphy, gamma cameras, beta+ detectors, gamma detectors, fluorescence imaging, low light imaging, X-rays, bioluminescence imaging, and near infrared (NIR) optical tomography. In some embodiments, the in vivo imaging is positron emission tomography (PET), optionally coupled with computed tomography (CT).

[0123] In some embodiments, the methods are capable of detecting as few as, or as few as, approximately 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 cells, or as few as, approximately 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 cells present in a specified volume. In some embodiments, the specified volume is the volume of a liquid, a sample, and / or an organ or tissue and / or is between about 10 μL and about 100 μL.

[0124] In some embodiments, the contacting and / or the detecting are performed in vitro or ex vivo. In some embodiments, the contacting and / or the detecting comprise immunohistochemistry (IHC), immunocytochemistry, or flow cytometry. In some embodiments, the method is capable of detecting as little as or as little as, or as little as or as little as about 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 cells, or as little as or as little as about 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 cells present in a specified volume. In some embodiments, the specified volume is the volume of a liquid, a sample, and / or an organ or tissue and / or is between about 10 μL and about 100 μL.

[0125] In some embodiments, the method further comprises determining the number or concentration of engineered cells administered to the subject. In some embodiments, determining comprises comparing the signal to a standard curve. In some embodiments, the standard curve is generated by detecting signals from a plurality of control samples containing a defined number of cells expressing PSMA or a modified form thereof, the plurality of control samples having been contacted with a PSMA-targeting molecule.

[0126] In some embodiments, the in vivo imaging is positron emission tomography (PET), optionally coupled with computed tomography (CT), and the PSMA targeting molecule is 2-(3-{1-carboxy-5-[(6-[18F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid (18F-DCFPyL).

[0127] Also provided herein are methods for selecting, isolating, or separating cells expressing PSMA or a modified form thereof. In some embodiments, the method comprises contacting a plurality of cells containing any of the engineered cells described herein with a PSMA targeting molecule; and selecting, isolating, or separating cells that are recognized or bound by the PSMA targeting molecule. In some of any of the provided embodiments, the recombinant receptor is a chimeric receptor and / or a recombinant antigen receptor, such as a chimeric antigen receptor (CAR).

[0128] Also provided herein are methods for selecting, isolating, or separating cells expressing PSMA or a modified form thereof. In some embodiments, the method comprises selecting, isolating, or separating cells recognized or bound by a PSMA targeting molecule from a plurality of cells containing any engineered cell herein that has been contacted with a PSMA targeting molecule. In some embodiments of any of the provided embodiments, the recombinant receptor is a chimeric receptor and / or a recombinant antigen receptor, such as a chimeric antigen receptor (CAR).

[0129] In some embodiments, the PSMA-targeting molecule or portion thereof: is capable of binding to PSMA and / or a modified form thereof, and / or is capable of binding to the active site of PSMA and / or the active site of a modified form of PSMA, and / or is capable of being cleaved by PSMA and / or by a modified form of PSMA; and / or is an antagonist, selective antagonist, inverse agonist, selective inverse agonist, agonist, selective agonist, inhibitor, and / or selective inhibitor of PSMA and / or its modified forms.

[0130] In some embodiments, the plurality of cells includes engineered cells containing any of a polynucleotide, set of polynucleotides, or composition containing a set of polynucleotides described herein, or a vector, set of vectors, or composition containing a set of vectors described herein.

[0131] In some embodiments, the plurality of cells comprising engineered cells comprises peripheral blood leukocytes from a subject that has been administered any of the engineered cells or compositions described herein.

[0132] In some embodiments, the PSMA-targeting molecule is or includes a small molecule, a ligand, an antibody or antigen-binding fragment thereof, an aptamer, a peptide, or a conjugate thereof.

[0133] In some embodiments, the PSMA targeting molecule is or includes a small molecule. In some embodiments, the small molecule is selected from 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid (DCFPyL), N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine ​​(DCFBC), (aminostyryl)pyridinium (ASP) dye, 2-(3-[1-carboxy-5-[(5-iodo-pyridine-3-carbonyl)-amino]-pentyl]-ureido)-glutaric acid (YC-VI-11), 2-[3-[1-carboxy-5-(4-iodo-benzoylamino)-pentyl]-ureido]-glutaric acid (DCIBzL), or YC-7), 1-(3-carboxy-4-(3-hydroxy-6-oxo-6H-xanthen-9-yl)phenylamino)-9,16,24-trioxo-1-thioxo-2,8,17,23,25-pentaazaoctacosane-7,22,26,28-tetracarboxylic acid (YC-36), Glu-NH-CO-NH-Lys(Ahx)-HBED-CC(PSMA-HBED-CC), 9-(4-fluoro-3-[hydroxymethyl]butyl)guanine (FHBG), Glu-urea-Lys-(Ahx)-[(HBED-CC)](PSMA-11), PSMA-617, 2-(phosphonomethyl) 1,3-Dicarboxypropyl)ureido]glutaric acid (DUPA), PSMAN, N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid, MIP-1072, MIP-1095, MIP-1404, MIP-1405, and N -[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-L-glutamate (ZJ43), (S)-2-(4-iodobenzylphosphonylmethyl)-glutaric acid (GPI-18431), 2-[(3-{4-[(2-amino-4-hydroxy-pteridin-6-ylmethyl)-amino]-benzoylamino}-3-carboxy-propyl)-hydroxy-phosphorylmethyl]-glutaric acid (MPE), (2S,3'S)-{[(3'-amino-3'-carboxy-propyl)-hydroxyphosphoryl]methyl}-glutaric acid (EPE) and (2S)-2-{[(2-carboxy-ethyl)-hydroxy-phosphoryl]methyl}-glutaric acid (SPE).In some embodiments, the PSMA-targeting molecule is or includes 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid (DCFPyL) or N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine ​​(DCFBC).

[0134] In some embodiments, the PSMA targeting molecule is or comprises an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof optionally has a binding site that specifically binds to PSMA. In some embodiments, the antibody or antigen-binding fragment thereof is selected from J591, DFO-J591, CYT-356, J415, 3 / A12, 3 / F11, 3 / E7, D2B, 107-1A4, YPSMA-1, YPSMA-2, 3E6, 2G7, 24.4E6, GCP-02, GCP-04, GCP-05, J533, E99, 1G9, 3C6, 4.40, 026, D7-Fc, D7-CH3, 4D4, A5, and antigen-binding fragments and derivatives thereof, or comprises CDR3, V H and / or V L , and / or compete for binding to PSMA or bind to the same PSMA epitope as any of the foregoing.

[0135] In some embodiments, the PSMA-targeting molecule is or includes an aptamer or a conjugate thereof. In some embodiments, the aptamer includes A9, A10, A10g, A10-3.2, or SZT101, or a conjugate thereof.

[0136] In some embodiments, the PSMA-targeting molecule is contained in a matrix or immobilized on a solid support. In some embodiments, the solid support comprises magnetic particles.

[0137] Also provided herein are kits. In some embodiments, the kit comprises a composition containing a therapeutically effective amount of any engineered cell provided herein; and a composition containing a PSMA targeting molecule. In some embodiments, the PSMA targeting molecule is or includes or further includes a therapeutic agent and / or the PSMA targeting molecule provides a signal or induces a detectable signal or is capable of binding to a portion that provides a signal or induces a detectable signal. In some of any of the provided embodiments, the recombinant receptor is a chimeric receptor and / or a recombinant antigen receptor, such as a chimeric antigen receptor (CAR). In some embodiments, wherein the detection comprises identifying a signal in a subject or a signal from a sample of a subject, wherein: the PSMA targeting molecule provides the signal or induces the detectable signal or is capable of binding to a portion that provides the signal or induces the detectable signal; and / or the PSMA targeting molecule is or includes a portion that provides the signal or induces the detectable signal.

[0138] In some embodiments, the kit further comprises instructions for administering the engineered cells and the PSMA-targeting molecule as a combination therapy to a subject for treating a disease or condition. In some embodiments, the kit further comprises instructions for administering the PSMA-targeting molecule to a subject receiving or having been administered the engineered cells for detecting the engineered cells.

[0139] Also provided herein are kits comprising a composition comprising a therapeutically effective amount of any of the engineered cells provided herein; and instructions for administering a PSMA-targeting molecule to a subject who is receiving or has been administered the engineered cells to detect the engineered cells.

[0140] Also provided herein are kits comprising a composition comprising a PSMA-targeting molecule; and instructions for administering a PSMA-targeting molecule to a subject receiving or having been administered a therapeutically effective amount of any of the engineered cells provided herein to detect the engineered cells. In some embodiments of any of the provided embodiments, the recombinant receptor is a chimeric receptor and / or a recombinant antigen receptor, such as a chimeric antigen receptor (CAR).

[0141] In some embodiments, the instructions further specify determining the number or concentration of engineered cells administered to the subject. In some embodiments, the instructions specify that the determining comprises comparing the signal to a standard curve. In some embodiments, the instructions further specify that the standard curve is generated by detecting the signal from a plurality of control samples containing a defined number of cells expressing PSMA or a modified form thereof, the plurality of control samples having been contacted with a PSMA-targeting molecule. In some embodiments, the instructions specify that the detection is performed via positron emission tomography (PET) (optionally coupled to computed tomography (CT)), and the PSMA-targeting molecule is 2-(3-{1-carboxy-5-[(6-[ 18 F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid ( 18 F-DCFPyL).

[0142] Also provided herein are kits comprising a composition comprising a therapeutically effective amount of any engineered cell provided herein; and instructions for administering the engineered cells to a subject for treatment of a disease or condition in combination therapy with a PSMA-targeting molecule, the PSMA-targeting molecule being or including or further comprising a therapeutic agent for treating the disease or condition. In some any of the provided embodiments, the recombinant receptor is a chimeric receptor and / or a recombinant antigen receptor, such as a chimeric antigen receptor (CAR).

[0143] In some embodiments, the PSMA-targeting molecule or therapeutic agent is capable of modulating the tumor microenvironment (TME) or is cytotoxic to the tumor. In some embodiments, the therapeutic agent is an immunomodulatory agent, a cytotoxic agent, an anticancer agent, or a radiotherapeutic agent.

[0144] Also provided herein are kits comprising a composition comprising a PSMA-targeting molecule and instructions for administering the PSMA-targeting molecule to a subject for treatment of a disease or condition in combination therapy with a therapeutically effective amount of any of the engineered cells provided herein to treat the disease or condition. In some embodiments of any of the provided embodiments, the recombinant receptor is a chimeric receptor and / or a recombinant antigen receptor, such as a chimeric antigen receptor (CAR).

[0145] In some embodiments, the PSMA-targeting molecule is or includes a small molecule, a ligand, an antibody or antigen-binding fragment thereof, an aptamer, a peptide, or a conjugate thereof.

[0146] In some embodiments, the PSMA targeting molecule is or includes a small molecule. In some embodiments, the small molecule is selected from 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid (DCFPyL), N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine ​​(DCFBC), (aminostyryl)pyridinium (ASP) dye, 2-(3-[1-carboxy-5-[(5-iodo-pyridine-3-carbonyl)-amino]-pentyl]-ureido)-glutaric acid (YC-VI-11), 2-[3-[1-carboxy-5-(4-iodo-benzoylamino)-pentyl]-ureido]-glutaric acid (DCIBzL), or YC-7), 1-(3-carboxy-4-(3-hydroxy-6-oxo-6H-xanthen-9-yl)phenylamino)-9,16,24-trioxo-1-thioxo-2,8,17,23,25-pentaazaoctacosane-7,22,26,28-tetracarboxylic acid (YC-36), Glu-NH-CO-NH-Lys(Ahx)-HBED-CC(PSMA-HBED-CC), 9-(4-fluoro-3-[hydroxymethyl]butyl)guanine (FHBG), Glu-urea-Lys-(Ahx)-[(HBED-CC)](PSMA-11), PSMA-617, 2-(phosphonomethyl) 1,3-Dicarboxypropyl)ureido]glutaric acid (DUPA), PSMAN, N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid, MIP-1072, MIP-1095, MIP-1404, MIP-1405, and N -[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-L-glutamate (ZJ43), (S)-2-(4-iodobenzylphosphonylmethyl)-glutaric acid (GPI-18431), 2-[(3-{4-[(2-amino-4-hydroxy-pteridin-6-ylmethyl)-amino]-benzoylamino}-3-carboxy-propyl)-hydroxy-phosphorylmethyl]-glutaric acid (MPE), (2S,3'S)-{[(3'-amino-3'-carboxy-propyl)-hydroxyphosphoryl]methyl}-glutaric acid (EPE) and (2S)-2-{[(2-carboxy-ethyl)-hydroxy-phosphoryl]methyl}-glutaric acid (SPE).In some embodiments, the PSMA-targeting molecule is or includes 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid (DCFPyL) or N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine ​​(DCFBC).

[0147] In some embodiments, the PSMA targeting molecule is or comprises an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof optionally has a binding site that specifically binds to PSMA. In some embodiments, the antibody or antigen-binding fragment thereof is selected from J591, DFO-J591, CYT-356, J415, 3 / A12, 3 / F11, 3 / E7, D2B, 107-1A4, YPSMA-1, YPSMA-2, 3E6, 2G7, 24.4E6, GCP-02, GCP-04, GCP-05, J533, E99, 1G9, 3C6, 4.40, 026, D7-Fc, D7-CH3, 4D4, A5, and antigen-binding fragments and derivatives thereof, or comprises CDR3, V H and / or V L , and / or compete for binding to PSMA or bind to the same PSMA epitope as any of the foregoing.

[0148] In some embodiments, the PSMA-targeting molecule is or includes an aptamer or a conjugate thereof. In some embodiments, the aptamer includes A9, A10, A10g, A10-3.2, or SZT101, or a conjugate thereof.

[0149] Also provided herein are PSMA targeting molecules. In some embodiments, the PSMA targeting molecule includes a portion capable of binding to PSMA or a modified form thereof, wherein the PSMA targeting molecule is or further includes an immunomodulator. In some embodiments, the immunomodulator is capable of regulating, optionally increasing the activity or immune response of immune cells and / or is capable of regulating the tumor microenvironment (TME). In some of any of the provided embodiments, the recombinant receptor is a chimeric receptor and / or a recombinant antigen receptor, such as a chimeric antigen receptor (CAR).

[0150] In some embodiments, the PSMA-targeting molecule is or comprises a prodrug that is or comprises an immunomodulatory agent or is capable of converting to or revealing an immunomodulatory agent, and / or the PSMA-targeting molecule is capable of being cleaved after binding to PSMA or a modified form thereof, wherein the cleavage produces at least one cleavage product comprising the immunomodulatory agent.

[0151] In some embodiments, the immunomodulator is directly or indirectly (optionally via a linker) attached to a portion of a PSMA targeting molecule that is capable of binding to PSMA or a modified form thereof. In some embodiments, the linker is a peptide or polypeptide, or a chemical linker. In some embodiments, the linker is a releasable linker or a cleavable linker. In some embodiments, the linker is capable of being cleaved after PSMA or a modified form thereof is bound by the binding molecule, wherein the cleavage produces at least one cleavage product comprising the immunomodulator. In some embodiments, the releasable linker or cleavable linker is released or cleaved in the presence of one or more conditions or factors present in the tumor microenvironment (TME), wherein the cleavage produces at least one cleavage product comprising the immunomodulator. In some embodiments, the one or more conditions or factors present in the tumor microenvironment (TME) include matrix metalloproteinases (MMPs), hypoxic conditions, or acidic conditions.

[0152] In some embodiments, the immunomodulator is an immune checkpoint inhibitor or a modulator or a cytokine. In some embodiments, the immunomodulator is an immune checkpoint inhibitor that can inhibit or block the function of an immune checkpoint molecule or a signaling pathway involving an immune checkpoint molecule. In some embodiments, the immune checkpoint molecule is selected from PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM3, VISTA, adenosine receptors or extracellular adenosine, optionally adenosine 2A receptor (A2AR) or adenosine 2B receptor (A2BR), or an adenosine or pathway involving any of the above.

[0153] Also provided herein are PSMA targeting molecules. In some embodiments, the PSMA targeting molecule includes a portion capable of binding to PSMA or a modified form thereof, wherein the PSMA targeting molecule is or further includes a therapeutic agent capable of regulating the tumor microenvironment (TME), wherein the therapeutic agent is connected to the portion of the PSMA targeting molecule by a releasable or cleavable linker that responds to one or more conditions or factors present in the TME. In some embodiments, one or more conditions or factors present in the tumor microenvironment (TME) include matrix metalloproteinases (MMPs), hypoxic conditions, or acidic conditions. In some embodiments, the therapeutic agent is an immunomodulator, a cytotoxic agent, an anticancer agent, or a radiotherapeutic agent. In some of the embodiments provided, the recombinant receptor is a chimeric receptor and / or a recombinant antigen receptor, such as a chimeric antigen receptor (CAR).

[0154] In some embodiments, the PSMA-targeting molecule is an antibody-drug conjugate (ADC).

[0155] In some embodiments, the PSMA-targeting molecule or portion thereof: is capable of binding to PSMA and / or a modified form thereof, and / or is capable of binding to the active site of PSMA and / or the active site of a modified form of PSMA, and / or is capable of being cleaved by PSMA and / or by a modified form of PSMA; and / or is an antagonist, selective antagonist, inverse agonist, selective inverse agonist, agonist, selective agonist, inhibitor, and / or selective inhibitor of PSMA and / or its modified forms.

[0156] In some embodiments, the PSMA-targeting molecule is or includes a small molecule, a ligand, an antibody or antigen-binding fragment thereof, an aptamer, a peptide, or a conjugate thereof.

[0157] In some embodiments, the PSMA targeting molecule is or includes a small molecule. In some embodiments, the small molecule is selected from 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid (DCFPyL), N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine ​​(DCFBC), (aminostyryl)pyridinium (ASP) dye, 2-(3-[1-carboxy-5-[(5-iodo-pyridine-3-carbonyl)-amino]-pentyl]-ureido)-glutaric acid (YC-VI-11), 2-[3-[1-carboxy-5-(4-iodo-benzoylamino)-pentyl]-ureido]-glutaric acid (DCIBzL), or YC-7), 1-(3-carboxy-4-(3-hydroxy-6-oxo-6H-xanthen-9-yl)phenylamino)-9,16,24-trioxo-1-thioxo-2,8,17,23,25-pentaazaoctacosane-7,22,26,28-tetracarboxylic acid (YC-36), Glu-NH-CO-NH-Lys(Ahx)-HBED-CC(PSMA-HBED-CC), 9-(4-fluoro-3-[hydroxymethyl]butyl)guanine (FHBG), Glu-urea-Lys-(Ahx)-[(HBED-CC)](PSMA-11), PSMA-617, 2-(phosphonomethyl) 1,3-Dicarboxypropyl)ureido]glutaric acid (DUPA), PSMAN, N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid, MIP-1072, MIP-1095, MIP-1404, MIP-1405, and N -[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-L-glutamate (ZJ43), (S)-2-(4-iodobenzylphosphonylmethyl)-glutaric acid (GPI-18431), 2-[(3-{4-[(2-amino-4-hydroxy-pteridin-6-ylmethyl)-amino]-benzoylamino}-3-carboxy-propyl)-hydroxy-phosphorylmethyl]-glutaric acid (MPE), (2S,3'S)-{[(3'-amino-3'-carboxy-propyl)-hydroxyphosphoryl]methyl}-glutaric acid (EPE) and (2S)-2-{[(2-carboxy-ethyl)-hydroxy-phosphoryl]methyl}-glutaric acid (SPE).In some embodiments, the PSMA-targeting molecule is or includes 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid (DCFPyL) or N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine ​​(DCFBC).

[0158] In some embodiments, the PSMA targeting molecule is or comprises an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof optionally has a binding site that specifically binds to PSMA. In some embodiments, the antibody or antigen-binding fragment thereof is selected from J591, DFO-J591, CYT-356, J415, 3 / A12, 3 / F11, 3 / E7, D2B, 107-1A4, YPSMA-1, YPSMA-2, 3E6, 2G7, 24.4E6, GCP-02, GCP-04, GCP-05, J533, E99, 1G9, 3C6, 4.40, 026, D7-Fc, D7-CH3, 4D4, A5, and antigen-binding fragments and derivatives thereof, or comprises CDR3, V H and / or V L , and / or compete for binding to PSMA or bind to the same PSMA epitope as any of the foregoing.

[0159] In some embodiments, the PSMA-targeting molecule is or includes an aptamer or a conjugate thereof. In some embodiments, the aptamer includes A9, A10, A10g, A10-3.2, or SZT101, or a conjugate thereof.

[0160] Also provided herein are methods of treatment comprising administering to a subject any of the PSMA-targeting molecules described herein. In some of any provided embodiments, the recombinant receptor is a chimeric receptor and / or a recombinant antigen receptor, such as a chimeric antigen receptor (CAR).

[0161] Also provided herein are articles of manufacture. In some embodiments, the articles of manufacture include any of the engineered cells, compositions, polynucleotides, sets of polynucleotides, compositions comprising sets of polynucleotides, vectors, sets of vectors, compositions comprising sets of vectors, kits, or PSMA-targeting molecules provided herein. In some of any of the provided embodiments, the recombinant receptor is a chimeric receptor and / or a recombinant antigen receptor, such as a chimeric antigen receptor (CAR). BRIEF DESCRIPTION OF THE DRAWINGS

[0162] Figures 1A-1BDepicted are exemplary FACS plots showing the detection level of binding of anti-PSMA antibody (PSMA (mAb)) and CD4 in a sample enriched for CAR+ T cells. Cells were engineered to express anti-CD19 CAR and full-length wild-type PSMA (WT PSMA), carrying an amino acid substitution at position 2 (PSMA (W2G) ) or a 9-amino acid N-terminal deletion (PSMA (N9del) ) or, as a control, cells were transduced with a vector that did not encode CAR and / or did not encode PSMA (mock). Figure 1C Shown are the geometric mean fluorescence intensity (gMFI) of PSMA (or N-terminally modified variants) expression on CD4+ and CD8+ T cells, as determined by binding of anti-PSMA antibodies. Figure 1D Shown are the gMFI of PSMA (or N-terminally modified variants) expression on CD4+ or CD8+ cells transduced to express the respective PSMA or one of the variants and on T cells expressing truncated EGFR (EGFRt) or mock transduced. Figures 1E-1F Depicted are exemplary FACS graphs showing the detection level of binding of YC-36-FITC (a FITC-conjugated analog of DCFPyL) and CD8 in samples enriched for CAR+ T cells expressing anti-CD19 CAR and PSMA (or N-terminally modified variants). Figure 1G Depicted are exemplary FACS plots showing modified PSMA (N9del) Co-expression on the surface of CAR-expressing cells as determined by anti-idiotypic antibodies specific for the binding domain of the CAR. Figures 1H-1L Depicted are exemplary FACS plots showing that cells have been treated with WT PSMA or modified variants (PSMA (W2G) and PSMA (N9del) ) expression of PSMA and anti-CD19 CAR in CD4+ or CD8+ cells transduced with WT cells. Figure 1M The results show that the expression of anti-CD19 CAR in cells co-expressing a truncated PSMA variant (CD19-tPSMA; PSMA (N9del) Figure 5 gMFI of CAR surface expression by flow cytometry in cells expressing either the truncated receptor or a surrogate marker (CD19-t receptor) as a control.

[0163] Figure 2A Shown are the cytolytic activity of CAR+ cells expressing PSMA (or N-terminally modified variants) or CAR+ cells expressing an alternative EGFRt marker, as assessed by measuring the loss of viable NLR-labeled CD19-expressing target cells (NLR+ cells) at an effector cell to target cell ratio of 4:1. Figure 2BShown is a comparison of the results for different killing indices at E:T ratios (4:1, 2:1, 1:1 and 1:2). Figure 2C Shown is the expression of anti-CD19 CAR / PSMA in the presence of the reagent 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid (DCFPyL) that binds to the catalytic domain of PSMA at an E:T ratio of 4:1 (N9del) Cytolytic activity of T cells. Figure 2D Shown is the expression of anti-CD19 CAR / PSMA in the presence of 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid (DCFPyL) at an E:T ratio of 4:1 or 1:1 (N9del) The killing index of T cells.

[0164] Figure 3A (IFN-γ), Figure 3B (TNF-α) and Figure 3C (IL-2) depicts the expression of anti-CD19 CAR / WTPSMA, anti-CD19 CAR / PSMA (W2G) , anti-CD19 CAR / PSMA (N9del) The amount of cytokine production by T cells or mock-transduced cells (mock) in response to incubation with target cells expressing CD19. Figure 3D Shown are the effects of anti-CD19 CAR expressing cells co-expressing a truncated PSMA variant (CD19-tPSMA; PSMA (N9del) ) or IFN-γ production in cells expressing a truncated receptor as a control surrogate marker (CD19-t receptor). Figure 3E Figure 3 shows cells expressing anti-CD19 CAR / PSMA cultured in the presence of different concentrations of DCFPyL at E:T ratios of 4:1 and 1:1. (N9del) The amount of IFN-γ produced in co-cultures of T cells and K562-CD19 target cells.

[0165] Figure 4A shows that after receiving a certain dose of anti-CD19 CAR / PSMA (N9del) Figure 3. Tumor growth over time in mice bearing tumors that were transduced with either engineered T cells expressing anti-CD19 CAR / EGFRt or mock-transduced cells (mock). Mean radiance (p / s / cm2) as determined by bioluminescence imaging of mouse models bearing tumors expressing bioluminescent proteins. 2 Figure 4B shows the survival curves of mice in each group.

[0166] Figure 5AFigure 2 shows mice injected with T cells containing 50% CAR+ cells and 50% non-CAR+ T cells (such that mice were injected with 500 (0.5k), 2,500 (2.5k), 5,000 (5k), 25,000 (25k), 50,000 (50k), 250,000 (250k), 500,000 (0.5M), or 2,500,000 (2.5M) cells expressing anti-CD19 CAR / PSMA (Ndel) An exemplary positron emission tomography-computed tomography (PET / CT) scan of the engineered cells was also performed with 400 μCi of 2-(3-{1-carboxy-5-[(6-[ 18 F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid ([ 18 F]DCFPyL) injected mice. Shown are mice that have received anti-CD19 CAR / PSMA expression (N9del) and 400 μCi[ 18 F] Cross-sectional view of an exemplary animal with 2.5M engineered cells of DCFPyL. Figure 5B Shown is a sample from another study in which NOD / Scid / gc- / - (NSG) mice were injected with 50 μL of Example positron emission tomography-computed tomography (PET / CT) of 2,000 (2k), 4,000 (4k), 20,000 (20k), 40,000 (40k), 200,000 (200k), 400,000 (400k), or 2,000,000 (2M) anti-CD19 CAR+ T cells expressing PSMA-N9del. PET results are expressed as percentage of injected dose per cubic centimeter of tissue imaged (%ID / cc).

[0167] Figure 6 Shown are bioluminescent imaging and PET / CT scans of mice with disseminated tumors that had received a dose of anti-CD19 CAR / PSMA expressing (N9del) , anti-CD19 CAR / EGFRt engineered T cells, analog transduced cells (analogs) or no T cells were received. The top graph shows bioluminescent imaging of tumors and spontaneous metastases that occurred in the exemplary mice of each test group. The bottom graph shows corresponding PET / CT scans of CAR+ cells expressing modified PSMA detected in each mouse shown in the corresponding portion of the top graph using a PSMA targeting agent.

[0168] Figure 7 The use of PSMA-targeted radiolabeled agents ([ 18F]DCFPyL) and PET imaging to detect different amounts of engineered anti-CD19 CAR / PSMA in vitro (N9del) The left image depicts CAR+ / PSMA expressing T cells in 20 μL PBS. (N9del) The right panel depicts the PET imaging results.

[0169] Figure 8 Shown are survival curves representing the survival of mice with disseminated tumor xenograft models over time that had received doses of anti-CD19 CAR / PSMA expressing (N9del) engineered T cells or mimic-transduced cells (mock), or received no treatment (no treatment).

[0170] Figures 9A-9D Describes the effect of anti-CD19 CAR / PSMA on the (N9del) Figure 1 shows exemplary bioluminescence imaging results on days 0 and 11 after CAR+ T cell injection and PET / CT imaging results on days 5 and 12 after CAR+ T cell injection in mice with disseminated tumors that were injected with engineered T cells containing CAR+ T cells. The first and third images show the bioluminescence imaging results on days 0 and 11, respectively, and the second and fourth images show the PET / CT scan results of detecting CAR+ cells expressing modified PSMA using a PSMA targeting agent on days 5 and 12, respectively. Figure 9E Depicted are the bioluminescence imaging results of untreated mice (Untreated) and the mock study group (Mock Treated) on day 0 and the PET / CT imaging results on day 9. Arrows indicate the location of the tumor.

[0171] Figure 10 Describes the determination of the expression of anti-CD19 CAR / PSMA in patients who have received a certain dose (N9del) Immunohistochemistry results for the presence of tumor cells and PSMA-expressing CAR+ cells in mice with disseminated tumors engineered with T cells. Anti-GFP antibodies were used to detect Nalm6-GFP-ffluc tumor cells or anti-CD19 CAR / PSMA antibodies were used to detect (N9del) Tumor tissue sections were stained with anti-PSMA antibodies for T cells.

[0172] Figure 11A The use of PSMA-specific PET ligands [ 18F]DCFPyL was used to determine the standard curve for the number of PSMA-expressing cells by PET / CT imaging. The standard curve was determined by plotting the total voxels (units of graphical information in three-dimensional space) of PET images from in vitro phantom imaging experiments (n=8) against the corresponding cell number. The resulting linear regression equation was y=5×10 -6 x+0.0122(R 2 =0.9989). Error bars show standard deviation. Figure 11B and 11C Anti-CD19 CAR / PSMA was given on days 4, 5, 6, 7, 8, 9, 10, 11 and 12 after the initial BLI on day 0. (N9del) Exemplary bioluminescence imaging and PET / CT imaging results of tumor model mice expressing cells. The total number of CAR+ cells in the tumor was extrapolated based on the standard curve and is listed in Table 1.

[0173] Figure 12 The results show the difference between the 6th and 13th days after administration in the cells that had been given anti-CD19 CAR / PSMA, as determined by counting the number of PSMA+ cells stained with anti-PSMA antibody. (N9del) The density of administered CAR+ T cells present in tumor biopsies obtained from mice with tumor models expressing CAR+ T cells, as well as the density of CAR+ T cells in human tumor biopsies after administration of CAR+ T cells.

[0174] Figures 13A-13B Depicts the determination of the presence of anti-CD19 CAR / PSMA in patients who had received (N9del) Example results of immunohistochemistry for the presence of tumor cells and PSMA-expressing CAR+ cells in mice with disseminated tumors using engineered T cells. Anti-GFP antibodies were used to detect Nalm6-GFP-ffluc tumor cells or anti-CD19 CAR / PSMA antibodies were used to detect (N9del) Tumor tissue sections were stained with anti-PSMA antibodies for T cells.

[0175] Figures 14A-14B Anti-CD19 CAR / PSMA was given on days 10, 11 and 12 after the initial BLI on day 0. (N9del) Exemplary bioluminescence imaging and PET / CT imaging results of tumor model mice expressing cells. Figures 14C-14D The total number of CAR+ cells in the tumors extrapolated based on the standard curve was plotted relative to the Figures 14A-14BGraph depicting the percentage of CAR+ T cells in peripheral blood (PPB) samples or in live cells within bone marrow (BM) samples in each mouse as determined using flow cytometry by staining with anti-PSMA antibody. DETAILED DESCRIPTION

[0176] Provided herein are engineered cells that express prostate-specific membrane antigen (PSMA) or a modified form thereof (e.g., variant PSMA) and a recombinant molecule (such as a recombinant receptor, e.g., a chimeric antigen receptor (CAR)). Also provided are related methods and compositions including treatment methods and compositions. In some embodiments, the engineered cells also express a recombinant receptor, such as a recombinant antigen receptor and / or a chimeric receptor. In some embodiments, the engineered cells co-express PSMA (typically a modified PSMA) and a recombinant receptor (e.g., CAR). Also provided are methods involving the use of PSMA targeting agents or PSMA targeting molecules to detect and / or target such cells. The methods include detection and imaging methods and methods for, for example, combination therapies of engineered cells and one or more additional therapeutic agents.

[0177] The methods provided include those in which PSMA or modified PSMA expressed by cells can be used, for example, for therapeutic, diagnostic, monitoring and / or regulatory results or methods. In some embodiments, PSMA or modified PSMA is used to detect (e.g., imaging) cells in vivo in a subject to which cells are or have been given. In some embodiments, PSMA or modified PSMA is used to target or deliver therapeutic agents to antigens, cells, or tissues, for example, recognized by cells or recombinant receptors. In some embodiments, PSMA or modified PSMA is targeted for treating a disease or condition and / or for detecting, imaging, identifying, selecting, separating, separating, engineering cells, processing cells, and / or removing cells; including combining genetically engineered cells, for example, cells engineered with recombinant receptors (e.g., CAR). In some embodiments, PSMA or its modified form is used as a marker, for example, for confirming the engineering of a cell (e.g., transduction of a cell) and / or for confirming the presence or quantity of an engineered cell. Also provided are polynucleotides and vectors encoding PSMA and / or recombinant receptors, methods for engineering such cells, and methods of utilizing such engineered cells, including methods for treating, detecting, selecting, isolating, or separating such cells. Also provided are PSMA-targeting molecules that can target or bind to PSMA, such as PSMA or a modified form thereof expressed on the surface of an engineered cell. In some embodiments, the PSMA-targeting molecule includes a therapeutic agent and / or moiety, such as a moiety that provides a signal or induces a detectable signal. In some embodiments, the PSMA-targeting molecule can be combined with an engineered cell for use in the methods provided herein.

[0178] A variety of strategies can be used to produce and give engineered cells for adoptive therapy. Typically, cells are engineered by introducing one or more genetically engineered nucleic acids or their products. Nucleic acids and their products include genetically engineered antigen receptors, including engineered T cell receptors (TCR) and functional non-TCR antigen receptors (such as chimeric antigen receptors (CAR), including activated, stimulated and costimulatory CARs), and combinations thereof. For example, strategies can be used to engineer cells (such as T cells) expressing chimeric receptors (such as CAR), and administer compositions containing such engineered cells to subjects.

[0179] In the whole process of producing engineered cells, it may be beneficial to be able to identify, detect, locate and / or select cells transduced and / or cells expressing desired recombinant molecules (such as recombinant receptors). Similarly, after or substantially simultaneously with the administration of engineered cells for adoptive therapy, it may also be necessary or desirable to detect, monitor, observe, locate or identify the adoptively transferred cells. For example, it may be desirable to determine or assess the presence or absence or degree of amplification, persistence, immunogenicity of the adoptively transferred cells, or to determine the biodistribution and / or pharmacokinetics of the adoptively transferred cells, and / or to provide a mechanism to exhaust or reduce the number of adoptively transferred cells in the subject.

[0180] In some cases, certain available methods for determining the biodistribution and / or pharmacokinetics of administered cells (e.g., adoptively transferred cells) may not be entirely satisfactory. For example, it may be difficult to determine the systemic spatial distribution of adoptively transferred cells, the specific location of cells, such as within or around the site or location of a disease or disorder (e.g., a tumor), the persistence of cells in vivo, and / or the occurrence of adverse effects (e.g., toxicity).

[0181] The method for determining the pharmacokinetics of adoptive transfer cells can include extracting peripheral blood from a subject who has been given engineered cells, and determining the number or ratio of engineered cells in peripheral blood. Approaches for selecting and / or separating cells can include using chimeric antigen receptor (CAR) specific antibodies (e.g., Brentjens et al., Sci. Transl. Med. 2013 March; 5 (177): 177ra38), protein L (Zheng et al., J. Transl. Med. 2012 February; 10: 29), epitope tags such as Strep- tag sequences (Liu et al. (2016) Nature Biotechnology, 34: 430) for directly introducing the specific sites in CAR (the binding reagents of Strep- tags are used to directly assess CAR); International Patent Application Publication No. WO 2015095895) and monoclonal antibodies specifically bound to CAR polypeptides (see International Patent Application Publication No. WO 2014190273). In some cases, extrinsic marker genes can be used in conjunction with engineered cell therapies to allow detection or selection of cells, and in some cases can also be used to promote cell suicide. In some cases, a truncated epidermal growth factor receptor (EGFRt) can be co-expressed with a transgene of interest (e.g., encoding a CAR or TCR) in a transduced cell (see, e.g., U.S. Patent No. 8,802,374). EGFRt can contain a marker gene that is expressed by the antibody cetuximab. or other therapeutic anti-EGFR antibodies or binding molecules that can be used to identify or select cells that have been engineered with an EGFRt construct and another recombinant receptor (e.g., a chimeric antigen receptor (CAR)), and / or to eliminate or isolate cells that express the receptor. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. 2016 Apr; 34(4): 430-434).

[0182] In some embodiments, the advantages of the compositions and methods provided are that they allow for detection or imaging of adoptively transferred cells with a high degree of sensitivity, which in some respects can provide the ability to directly assess circulating engineered cells (e.g., CAR T cells) in vivo after infusion. In some respects, the advantages of the embodiments provided are their applicability for in vivo use, for example, by providing detection of cells with reagents (such as PSMA targeting molecules) in a manner of targeting, binding and / or detecting administered cells (such as CAR T cells) without affecting one or more functions of the engineered cells and / or the ability to detect or separate during cell production. In some respects, the advantages of the embodiments provided are also that they allow in vivo detection without changing the sequence or function of the engineered recombinant receptor. In some embodiments, the embodiments provided are based on the following observations: without changing the function of the engineered cells, prostate-specific membrane antigen (PSMA) or its modified form is effectively expressed on the cell surface of the engineered cells for detection, for example, as shown by assessing in vitro cytotoxicity and / or in vivo anti-tumor activity. In some embodiments, the embodiments provided can also be used to detect the presence, biodistribution and transport of administered cells engineered to express recombinant receptors at primary and metastatic tumor sites in vivo.

[0183] In some embodiments, the provided agents, compositions, products, combinations and methods are useful and / or advantageous in determining the biodistribution and / or pharmacokinetics of administered cell compositions. In some aspects, the methods are advantageous in that they provide safe, highly sensitive, minimally invasive, real-time, relatively non-immunogenic and / or do not affect the function of the engineered cells. In some aspects, the provided embodiments include cell surface markers that can assist in the production, monitoring and / or post-administration stages involving the transduced cell product. For example, in some aspects, methods are provided for effectively selecting and isolating transgene-positive cells and for monitoring transgene-expressing cells in vivo and ex vivo. In some embodiments, the provided engineered cells, nucleic acids, vectors, compositions, PSMA-targeting molecules and methods provide one or more advantages over existing labeling or selection strategies for incorporating engineered cells.

[0184] In some embodiments, the prostate-specific membrane antigen (PSMA) or a modified form thereof that is engineered to be expressed on the engineered cells provided herein is human PSMA. In some embodiments, cells are typically engineered by introducing one or more genetically engineered nucleic acids or their products (e.g., those encoding recombinant receptors and / or PSMA or modified PSMA). It is advantageous to engineer cells to express PSMA or its modified form (particularly human PSMA, to administer engineered cells to human subjects) because PSMA is not immunogenic. PSMA is an antigen that is expressed in the human body in a highly tissue-specific manner. Therefore, the expression of PSMA or its modified form allows the use of PSMA-targeting molecules that specifically bind to and / or target PSMA to detect and target specific cells without causing an immune response or having immunogenic properties.

[0185] In some embodiments, engineered cells expressing PSMA or a modified form thereof can be used to target PSMA-targeting molecules, which can include a portion capable of binding to PSMA and a detectable portion or agent. In some aspects, PSMA-targeting molecules can be used in conjunction with the engineered cells provided herein to allow for delivery or targeting of therapeutic agents; detection, identification, or imaging of cells; selection, isolation, or separation of cells; and to promote cell suicide or cell removal. Various known agents that can specifically target PSMA can be used in conjunction with the embodiments for therapeutic and / or detection purposes.

[0186] In some aspects, targeting PSMA or its modified forms can facilitate detection of detectable moieties or agents, which can allow for detection of engineered cells in subjects to whom the engineered cells have been administered. In particular, in some embodiments, PSMA-targeting molecules and systems can be used for in vivo imaging of PSMA-expressing cells. The use of PSMA-targeting molecules for in vivo imaging in some aspects allows for minimally invasive, real-time, accurate, and / or rapid determination, assessment, and / or confirmation, or monitoring of the biodistribution of engineered cells.

[0187] In some embodiments, PSMA or its modified form as a marker makes the engineered cells compatible with detection using minimally invasive, rapid, accurate and / or sensitive methods. For example, the provided PSMA or its modified form can be recognized and / or specifically bound by a ligand, antibody or its antigen-binding fragment or other PSMA-targeting molecule, which is used for real-time or in vivo imaging of cells, such as positron emission tomography (PET), computed tomography (CT) and single photon emission computed tomography (SPECT) using a radionuclide-labeled ligand. PET is a minimally invasive, rapid, accurate and sensitive method for detecting specific cells in the body and provides whole-body spatial resolution. In some aspects, the provided embodiments provide the advantage of accurately and quickly providing a quantitative assessment of the administered cells (e.g., the biodistribution and / or pharmacokinetics of the administered cells) without the use of invasive methods. In some aspects, the provided embodiments provide advantages over existing methods for assessing biodistribution or pharmacokinetics, such as peripheral blood draws, which do not provide any spatial information or specific information about the tumor site; or biopsies, which are invasive and only provide information about the tumor site. Thus, in some aspects, engineering cells to express PSMA or a modified form thereof (e.g., one that can be recognized, targeted, or bound by a PSMA-targeting molecule) can provide certain advantages. In addition, provided engineered cells, nucleic acids, vectors, compositions, PSMA-targeting molecules, and methods provide cell surface molecular targets and reagents to facilitate the processing, production, and / or function of cells, and / or to facilitate the selection, isolation, killing, and / or removal of cells.

[0188] In some embodiments, engineered cells expressing PSMA or a modified form thereof and a PSMA-targeting molecule can be used to target therapeutic agents (including immunomodulatory agents or cytotoxic agents) to specific sites, locations, microenvironments, and / or specific types of targeted cells. For example, PSMA or a modified form thereof can be targeted to deliver a PSMA-targeting molecule (which can include a moiety capable of binding to PSMA or a modified form thereof and a therapeutic agent) to a site, location, and / or microenvironment containing the engineered cells, such as a tumor microenvironment (TME).

[0189] Also provided are methods for using cells expressing PSMA or a modified form thereof. Methods for cell isolation and genetic engineering are provided. Nucleic acids (e.g., constructs, e.g., viral vectors) encoding PSMA or a modified form thereof and / or nucleic acids and / or proteins encoding PSMA or a modified form thereof, as well as methods for introducing such nucleic acids into cells, e.g., by transduction, are provided. Also provided are PSMA-targeting molecules comprising a moiety capable of binding to PSMA or a modified form thereof and an immunomodulatory agent. Also provided are compositions containing the engineered cells, as well as methods, kits, and articles of manufacture for administering and monitoring cells and compositions to a subject (e.g., for adoptive cell therapy).

[0190] All publications (including patent documents, scientific articles, and databases) mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If definitions set forth herein are contrary to or otherwise inconsistent with definitions set forth in patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth herein take precedence over those incorporated herein by reference.

[0191] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0192] I. Engineered cells expressing prostate-specific membrane antigen (PSMA) and recombinant receptors

[0193] Provided herein are engineered cells expressing prostate-specific membrane antigen (PSMA) or a modified form thereof (e.g., a variant of PSMA), and related compositions, articles, combinations, methods, and uses, including those involving administration to cells and detection, binding, or targeting of cells using PSMA targeting agents or molecules. Engineered cells also typically express recombinant molecules, such as recombinant receptors, e.g., chimeric antigen receptors (CARs). In some embodiments, methods, compositions, and uses are related to provided engineered cells.

[0194] A. Prostate-specific membrane antigen (PSMA)

[0195] In some embodiments, prostate-specific membrane antigen (PSMA; also known as glutamate carboxypeptidase 2 or folate hydrolase 1) or a modified form thereof is expressed in an engineered cell, e.g., a primary cell, such as an immune cell, e.g., a T cell, including cells further engineered to have a recombinant receptor (e.g., CAR). In some embodiments, PSMA is human PSMA.

[0196] PSMA is a type II transmembrane protein containing a short cytoplasmic amino terminus, a single transmembrane domain, and a large extracellular domain. PSMA contains an amino acid sequence that exhibits similarity to proteins of the peptidase family M28, including cocatalytic metallopeptidases. Wild-type, full-length human PSMA is a 750-amino acid protein comprising an intracellular portion of 19 amino acid residues, a transmembrane portion of 24 amino acid residues, and an extracellular portion of 707 amino acid residues. In humans, PSMA is encoded by the FOLH1 gene (e.g., as described in GenBank Accession No. DD461260 (as shown in SEQ ID NO: 26)) and its isoforms and variants. An exemplary human PSMA amino acid sequence is shown, for example, in UniProt Accession No. Q04609 (as shown in SEQ ID NO: 23).

[0197] In some aspects, the intracellular (N-terminal) portion of PSMA contains amino acid residues that participate in cellular internalization (e.g., clathrin-dependent endocytic internalization of the molecule). In some aspects, cellular internalization is mediated by the following N-terminal amino acids: for example, the amino acid residues at positions 1-5 of the exemplary human PSMA amino acid sequence shown in SEQ ID NO: 23 (see, e.g., Rajasekaran et al. (2003) Mol. Biol. Cell. 14: 4835-4845).

[0198] In some cases, the extracellular portion of PSMA folds into three distinct structural and functional domains: a protease domain (residues 56-116 and 352-590), an apical domain (residues 117-351), and a C-terminal helical domain (residues 592-750), the positions of which are referenced to the wild-type human PSMA sequence (e.g., the amino acid sequence set forth in SEQ ID NO: 23) (see, e.g., Davis et al., (2005) Proc. Natl. Acad. Sci. 102(17):5981-5986; Mesters et al., (2006) EMBO Journal 25:1375-1384). PSMA typically contains a binuclear zinc site and can act as a glutamate carboxypeptidase or folate hydrolase, catalyzing the hydrolytic cleavage of glutamate from poly-γ-glutamylated folate. PSMA also has N-acetylated α-linked acid dipeptidase (NAALADase) activity and dipeptidyl peptidase type IV activity. The enzymatic site contains two zinc ions and is composed of two pockets, namely the glutamate-sensitive pocket (S1' pocket) and the non-pharmacophore pocket (S1 pocket). Amino acid residues from the three domains are generally involved in substrate recognition, binding and / or catalytic activity. In some cases, with reference to the positions of the wild-type human PSMA sequence (e.g., the amino acid sequence set forth in SEQ ID NO: 23), active site residues and / or residues involved in substrate binding and / or catalytic activity in PSMA include amino acid residues at positions 210, 257, 269, 272, 377, 387, 387, 424, 424, 425, 433, 436, 453, 517, 518, 519, 552, 553, 534, 535, 536, 552, 553, 628, 666, 689, 699 and / or 700. In some cases, the active site residues include one or more residues to coordinate the active zinc ion, such as one or more residues corresponding to positions His377, Asp387, Glu425, Asp453, and / or His553 with reference to the wild-type human PSMA sequence (e.g., the amino acid sequence set forth in SEQ ID NO: 23). In some embodiments, the N-acetylated alpha-linked acid dipeptidase (NAALADase) domain of PSMA can also be defined as including amino acid residues 274-587, which are positions with reference to the wild-type human PSMA sequence (e.g., the amino acid sequence set forth in SEQ ID NO: 23) (Speno et al., (1999) Molecular Pharmacology 55: 179-185).

[0199] In some embodiments, any PSMA or modified forms thereof associated with the provided disclosure is a recombinant PSMA. In some embodiments, any PSMA or modified forms thereof associated with the provided disclosure is expressed by a heterologous, recombinant, exogenous, and / or transgenic nucleic acid molecule (i.e., a nucleic acid not normally present in a cell or a sample obtained from a cell, such as a nucleic acid obtained from another organism or cell, e.g., not normally found in the engineered cell and / or the organism from which the cell was derived).

[0200] In some embodiments, PSMA or a modified form thereof (e.g., recombinant PSMA or a modified form thereof) associated with the provided disclosure contains one or more active site residues and / or residues involved in PSMA substrate binding and / or PSMA catalytic activity, such as one or more amino acid residues corresponding to the amino acid residues at positions 210, 257, 269, 272, 377, 387, 387, 424, 424, 425, 433, 436, 453, 517, 518, 519, 552, 553, 534, 535, 536, 552, 553, 628, 666, 689, 699, and / or 700 of the wild-type human PSMA sequence (e.g., the amino acid sequence set forth in SEQ ID NO: 23). In some embodiments, PSMA or a modified form thereof (e.g., recombinant PSMA or a modified form thereof) associated with the provided disclosure exhibits the same or substantially the same substrate recognition as wild-type human PSMA (e.g., the amino acid sequence set forth in SEQ ID NO: 23) or an allelic or other variant thereof, and / or a sequence that exhibits at least or about at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 23.

[0201] In some embodiments, PSMA or a modified form thereof (e.g., recombinant PSMA or a modified form thereof) associated with the provided disclosure does not bind to or recognize a substrate or ligand of wild-type human PSMA (e.g., the amino acid sequence set forth in SEQ ID NO: 23), and / or exhibits reduced binding and / or recognition thereof, for example, by greater than or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some embodiments, PSMA or a modified form thereof (e.g., recombinant PSMA or a modified form thereof) is modified, for example, by amino acid replacement, insertion, or substitution of one or more residues involved in active site residues and / or involved in PSMA substrate binding and / or PSMA catalytic activity, for example, to a wild-type human PSMA sequence (e.g., the amino acid sequence set forth in SEQ ID NO: 23) or an allelic or other variant thereof, and / or exhibits binding to and / or recognition of a wild-type human PSMA sequence (e.g., the amino acid sequence set forth in SEQ ID NO: 23). or a sequence having at least or about at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to NO: 23 or one or more amino acid residues corresponding to the amino acid residue at position 210, 257, 269, 272, 377, 387, 387, 424, 424, 425, 433, 436, 453, 517, 518, 519, 552, 553, 534, 535, 536, 552, 553, 628, 666, 689, 699 and / or 700.

[0202] In some embodiments, PSMA or a modified form thereof (e.g., recombinant PSMA or a modified form thereof) related to the provided disclosure contains a protease domain or portion thereof and / or exhibits catalytic activity (e.g., activity that catalyzes the hydrolytic cleavage of glutamic acid from poly-γ-glutamic acid folate). In some embodiments, PSMA or a modified form thereof (e.g., recombinant PSMA or a modified form thereof) related to the provided disclosure contains one or more residues associated with zinc binding and / or residues for catalytic activity, such as one or more residues His377, Asp387, Glu425, Asp453, and / or His553. In some embodiments, PSMA or a modified form thereof (e.g., recombinant PSMA or a modified form thereof) associated with the provided disclosure contains a NAALADase domain and / or includes amino acid residues 274-587, 274-700, or 247-750, with reference to positions in a wild-type human PSMA sequence, such as the amino acid sequence set forth in SEQ ID NO: 23, or an allelic or other variant thereof and / or a sequence that exhibits at least or about at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 23.

[0203] In some embodiments, PSMA or a modified form thereof (e.g., recombinant PSMA or a modified form thereof) has no catalytic activity, or exhibits reduced catalytic activity, e.g., reduced by greater than or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, e.g., exhibits no and / or exhibits reduced catalytic activity in catalyzing the hydrolytic cleavage of glutamate from poly-gamma-glutamic acid folate. In some embodiments, PSMA or a modified form thereof (e.g., recombinant PSMA or a modified form thereof) is modified, for example, by amino acid replacement, insertion, or substitution of one or more residues associated with zinc binding and / or catalytic activity, such as one or more residues His377, Asp387, Glu425, Asp453, and / or His553, with reference to the wild-type human PSMA sequence (e.g., the amino acid sequence set forth in SEQ ID NO: 23), or an allelic or other variant thereof, and / or a sequence that exhibits at least or about at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 23.

[0204] PSMA also exists as a monomer on the membrane surface, but only has enzymatic activity as a dimer. The C-terminal domain participates in the formation of a dimerization interface with the protease domain and the apical domain of another PSMA monomer (see, for example, Davis et al., (2005) Proc. Natl. Acad. Sci. 102(17):5981-5986; Mesters et al., (2006) EMBO Journal 25:1375-1384). In some embodiments, PSMA or its modified form related to the provided disclosure includes one or more residues involved in dimerization and / or is capable of forming dimers. In some embodiments, PSMA or its modified form is modified by amino acid replacement, insertion, or substitution in one or more amino acid residues involved in dimerization and / or is unable to form dimers.

[0205] In some cases, PSMA or its modified forms related to the present disclosure are capable of being recognized and / or specifically bound by a ligand, an antibody or its antigen-binding fragment, or other PSMA-targeting molecule. In some cases, PSMA or its modified forms related to the present disclosure contain an epitope, such as an epitope that can be recognized by an antibody or its antigen-binding fragment. In some aspects, the epitope is a linear epitope. In other aspects, the epitope is a conformational epitope. In some embodiments, exemplary epitopes that can be recognized by PSMA-targeting antibodies or their antigen-binding fragments include amino acid residues 57-134, 91-108, 100-104, 118-135, 271-288, 469-486, 638-657, 640-657, or 716-723, with reference to the wild-type human PSMA sequence (e.g., the amino acid sequence set forth in SEQ ID NO: 23).

[0206] In some embodiments, the engineered cells provided contain or express PSMA or a modified form thereof. In some embodiments, the PSMA is a mammalian PSMA or a modified form thereof. In some embodiments, the PSMA is a human PSMA or a modified form thereof. In some embodiments, the PSMA provided in conjunction with the present disclosure is a wild-type PSMA (optionally a wild-type human PSMA) or an allelic variant or other variant thereof, such as an alternative isoform or fragment thereof. An exemplary sequence encoding human PSMA is shown in SEQ ID NO: 26, which encodes the human PSMA shown in SEQ ID NO: 23. In some embodiments, the PSMA is encoded by a modified nucleic acid sequence (e.g., a nucleic acid sequence modified to be CpG-free and / or codon-optimized). In some embodiments, the modified nucleic acid sequence is codon-optimized for expression in human cells. In some aspects, codon optimization involves balancing the percentage of selected codons with the abundance of the disclosed human transfer RNA so that neither is overloaded or limited. In some embodiments, the CpG-free nucleic acid sequence encoding PSMA is or includes a modified cDNA sequence that does not contain a CpG sequence. In some aspects, the CpG-free nucleic acid and / or codon-optimized sequence does not alter the protein sequence compared to wild-type or unmodified PSMA. An exemplary CpG-free nucleic acid sequence encoding PSMA is set forth in SEQ ID NO: 27. In some aspects, the PSMA encoded by the CpG-free PSMA has a significant percent identity to the protein sequence set forth in SEQ ID NO: 23.

[0207] In some embodiments, PSMA or a modified form thereof (e.g., recombinant PSMA or a modified form thereof) is encoded by the nucleic acid sequence set forth in SEQ ID NO: 26 or 27, or a nucleic acid sequence that exhibits at least or about 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NO: 26 or 27, or a fragment thereof. In some embodiments, PSMA or a modified form thereof (e.g., recombinant PSMA or a modified form thereof) is encoded by a nucleic acid sequence that encodes a fragment containing an extracellular domain and / or a transmembrane domain encoded by SEQ ID NO: 26 or 27, or an amino acid sequence that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 26 or 27.

[0208] In some embodiments, PSMA or a modified form thereof (e.g., recombinant PSMA or a modified form thereof) is encoded by the nucleic acid sequence set forth in SEQ ID NO: 53, or a nucleic acid sequence that exhibits at least or about 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NO: 53 or a fragment thereof. In some embodiments, PSMA or a modified form thereof (e.g., recombinant PSMA or a modified form thereof) is encoded by a nucleic acid sequence that encodes a fragment containing an extracellular domain and / or a transmembrane domain encoded by SEQ ID NO: 53, or an amino acid sequence that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 53.

[0209] In some embodiments, the wild-type or unmodified PSMA is human PSMA and / or comprises the amino acid sequence set forth in SEQ ID NO: 23, an amino acid sequence exhibiting at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or a fragment thereof. In some embodiments, the fragment comprises the extracellular domain and / or transmembrane domain of the amino acid sequence set forth in SEQ ID NO: 23, or an amino acid sequence exhibiting at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0210] In some embodiments, PSMA or a modified form thereof (e.g., recombinant PSMA or a modified form thereof) comprises the amino acid sequence set forth in SEQ ID NO: 23, or a fragment thereof, or the extracellular and / or transmembrane domain of SEQ ID NO: 23, or an amino acid sequence that exhibits at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NO: 23, or a fragment thereof, or the extracellular and / or transmembrane domain of SEQ ID NO: 23. In some embodiments, PSMA or a modified form thereof (e.g., recombinant PSMA or a modified form thereof) is full-length PSMA. In some embodiments, PSMA is wild-type PSMA or unmodified PSMA. In some embodiments, PSMA is wild-type human PSMA. In some embodiments, PSMA comprises or consists essentially of the sequence set forth in SEQ ID NO: 23.

[0211] In some embodiments, the modified PSMA is a recombinant modified PSMA. In some embodiments, the modified PSMA associated with the provided disclosure is expressed by a heterologous, recombinant, exogenous, and / or transgenic nucleic acid molecule (i.e., a nucleic acid not normally present in a cell or a sample obtained from a cell, such as a nucleic acid obtained from another organism or cell, e.g., not normally found in the engineered cell and / or the organism from which the cell was derived).

[0212] In some embodiments, PSMA or its modified form is a modified PSMA (e.g., a recombinant modified PSMA) comprising one or more amino acid modifications compared to wild-type or unmodified PSMA. In some embodiments, the one or more amino acid modifications comprise one or more amino acid substitutions, deletions, and / or insertions. In some embodiments, the one or more amino acid modifications comprise, for example, deletions or truncations of one or more consecutive amino acid residues at, from, or near the N-terminus or C-terminus of wild-type or unmodified PSMA. In some embodiments, the modified PSMA exhibits altered PSMA enzymatic activity and / or ligand binding ability and / or cellular internalization. In some embodiments, the modified PSMA (i) exhibits reduced endogenous signaling; (ii) exhibits increased cell surface expression; and / or (iii) exhibits reduced cellular internalization compared to wild-type or unmodified PSMA.

[0213] In some embodiments, PSMA or a modified form thereof (e.g., recombinant PSMA or a modified form thereof) contains one or more amino acid substitutions. In some embodiments, the modified PSMA comprises at least one amino acid substitution, for example, at the second amino acid residue, tryptophan is substituted with glycine (corresponding to W2G), with reference to the position in the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, the modified PSMA comprises at least one amino acid substitution corresponding to W2G, or does not comprise W2, or does not comprise any residue at position 2, with reference to the position in the amino acid sequence set forth in SEQ ID NO: 23. For example, in some embodiments, the modified PSMA comprises the amino acid sequence set forth in SEQ ID NO: 24, or a fragment thereof, or an amino acid sequence that exhibits at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 24, or a fragment thereof, and comprises at least one amino acid substitution.

[0214] In some embodiments, the modified PSMA comprises an amino acid substitution (to alanine) at one or more amino acid residues at position 2, 3, 4, 5, 6, 7, 8, 9, 10, or 14, with reference to the position in the PSMA sequence set forth in SEQ ID NO: 23.

[0215] In some embodiments, the PSMA is a modified PSMA comprising a deletion of one or more N-terminal amino acid residues in the intracellular portion compared to wild-type or unmodified PSMA. In some embodiments, the PSMA is a modified PSMA comprising a deletion of one or more consecutive amino acid residues in the intracellular portion compared to wild-type or unmodified PSMA. Wild-type full-length human PSMA is a 750 amino acid protein comprising an intracellular portion of 19 amino acid residues, a transmembrane portion of 24 amino acid residues, and an extracellular portion of 707 amino acid residues. In some embodiments, the modified PSMA contains a deletion at or near the N-terminus (the N-terminus corresponds to the 5' end of the coding sequence in the nucleic acid sequence encoding PSMA or its modified form), wherein the deletion is in the intracellular portion of the PSMA. In some aspects, a modified PSMA containing one or more deletions (optionally consecutive amino acid residues) in the intracellular portion is also referred to as a truncated form of PSMA, a truncated PSMA, or tPSMA.

[0216] In some aspects, the truncated PSMA or tPSMA contains a deletion or truncation of one or more amino acid residues (optionally consecutive amino acid residues) at or near the N-terminus of wild-type or unmodified PSMA. In some aspects, the modified PSMA contains a deletion or truncation of one or more amino acid residues (e.g., one or more consecutive amino acid residues) within the intracellular portion or domain of PSMA. In some embodiments, the PSMA protein containing the deletion of the N-terminal amino acid allows the N-terminally modified PSMA to be successfully localized to the cell membrane and centrosome, and / or (i) exhibits reduced endogenous signaling compared to wild-type or unmodified PSMA; (ii) exhibits increased cell surface expression; and / or (iii) exhibits reduced cellular internalization. In some embodiments, the modified PSMA exhibits reduced endogenous signaling or reduced cellular internalization, for example, a reduction of greater than or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some embodiments, the modified PSMA exhibits increased cell surface expression or increased localization to the cell membrane and centrosome, for example, an increase of greater than or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some aspects, cell surface expression and / or cellular internalization can be assessed using cell imaging techniques (such as confocal microscopy) using labeled binding molecules (e.g., antibodies) that specifically bind to PSMA or a variant thereof.

[0217] In some embodiments, the modified PSMA (e.g., tPSMA) contains or retains a methionine as the first residue, which in some cases is required for translation. In some embodiments, the PSMA is a modified PSMA that comprises a deletion of one or more N-terminal amino acid residues (optionally contiguous amino acid residues) in the intracellular portion compared to wild-type or unmodified PSMA, but does not include a deletion of the initial methionine that is required for translation.

[0218] In some embodiments, the modified PSMA (e.g., tPSMA) contains a deletion of up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 N-terminal amino acid residues compared to wild-type or unmodified PSMA. In some embodiments, the modified PSMA contains a deletion of up to 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 N-terminal amino acid residues compared to wild-type or unmodified PSMA. In some aspects, the modified PSMA is a truncated PSMA (tPSMA) containing a deletion of up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 N-terminal amino acid residues compared to wild-type or unmodified PSMA in which the first N-terminal methionine is retained or not deleted.

[0219] In some embodiments, with reference to positions in the PSMA sequence set forth in SEQ ID NO: 23, the modified PSMA contains a deletion of a contiguous amino acid sequence at the N-terminus starting at residue 2, 3, 4, or 5 and through residue 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21. In some embodiments, with reference to positions in the PSMA sequence set forth in SEQ ID NO: 23, the modified PSMA contains a deletion of residues 2-22, 2-21, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3.

[0220] In some cases, the modified PSMA can contain a deletion of the first N-terminal residue (e.g., methionine). In some embodiments, with reference to the positions in the PSMA sequence set forth in SEQ ID NO: 23, the modified PSMA contains a deletion of residues 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2.

[0221] In some aspects, the nucleic acid sequence encoding PSMA or a modified form thereof is contained within a polynucleotide that also contains a nucleic acid sequence encoding another protein (e.g., a different protein), for example, in an expression construct. In some aspects, the nucleic acid sequence encoding PSMA or a modified form thereof is contained within a single open reading frame in which the nucleic acid sequence encodes a different molecule (e.g., a recombinant receptor). In some embodiments, the nucleic acid sequence encoding PSMA or a modified form thereof is separated from the nucleic acid sequence encoding the different molecule (e.g., a recombinant receptor) by a sequence encoding a self-cleaving peptide (e.g., a 2A sequence) or a protease recognition site (e.g., furin). Thus, the ORF encodes a single polypeptide that is processed into a single protein during translation (in the case of 2A) or thereafter. In some embodiments of such polynucleotides, the nucleic acid encoding PSMA may or may not encode an initial methionine. In some aspects, if the sequence encoding PSMA is located further downstream (e.g., closer to the 3' end of the coding sequence) relative to the sequence encoding the other protein, for example, if PSMA is translated after the other protein, then it may not be necessary to retain the initial methionine. In some aspects, retention of the initial methionine may be desirable if the sequence encoding PSMA is located further upstream (e.g., closer to the 5' end of the coding sequence) or occurs before the sequence encoding another protein, e.g., if PSMA is translated first.

[0222] In some aspects, the modified PSMA is a truncated PSMA (tPSMA) that contains a deletion of the nine N-terminal amino acid residues (excluding the N-terminal methionine required for translation) compared to wild-type or unmodified PSMA, e.g., PSMA (N9del) In some embodiments, the modified PSMA is a truncated PSMA (tPSMA) containing a deletion of residues 2-10 of the sequence set forth in SEQ ID NO: 23, or a fragment thereof, or an amino acid sequence that exhibits at least or about 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the sequence set forth in SEQ ID NO: 23, or a fragment thereof. In some embodiments, the modified PSMA comprises the amino acid sequence set forth in SEQ ID NO: 52, or a fragment thereof, or an amino acid sequence that exhibits at least or about 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the sequence set forth in SEQ ID NO: 52, or a fragment thereof.

[0223] In some aspects, the modified PSMA is a truncated PSMA (tPSMA) containing a deletion of 9 N-terminal amino acid residues compared to wild-type or unmodified PSMA. In some embodiments, the modified PSMA is a truncated PSMA (tPSMA) containing a deletion of residues 1-9 of the sequence set forth in SEQ ID NO: 23, or a fragment thereof, or an amino acid sequence that exhibits at least or about 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence set forth in SEQ ID NO: 23, or a fragment thereof. In some embodiments, the modified PSMA comprises the amino acid sequence set forth in SEQ ID NO: 25, or a fragment thereof, or an amino acid sequence that exhibits at least or about 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 25, or a fragment thereof.

[0224] In some embodiments, PSMA or a modified form thereof comprises the amino acid sequence set forth in SEQ ID NO: 25 or 52, or a fragment thereof, or an amino acid sequence that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 25 or 52, or a fragment thereof, and comprises a deletion of one or more N-terminal amino acid residues.

[0225] In some embodiments, the modified PSMA is encoded by the nucleic acid sequence set forth in SEQ ID NO: 53, or a nucleic acid sequence that exhibits at least or about 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 53, or a fragment thereof, such as a fragment containing an extracellular domain and / or a transmembrane domain. In some embodiments, the modified PSMA is encoded by a nucleic acid sequence that does not include a deletion of a methionine start codon required for translation (e.g., encoding a polypeptide containing an initial methionine). In some embodiments, the modified PSMA is encoded by a modified nucleic acid sequence (e.g., a nucleic acid sequence that has been modified to be CpG-free and / or codon-optimized).

[0226] In some embodiments, the modified PSMA comprises all or substantially all of the transmembrane domain of wild-type or unmodified PSMA; or the modified PSMA comprises a transmembrane domain having the same or at least the same number of amino acids as the transmembrane domain of wild-type or unmodified PSMA.

[0227] In some embodiments, PSMA or its modified form comprises an amino acid sequence that is bound by or recognized by a PSMA targeting molecule (e.g., an antibody or its antigen-binding fragment). In some embodiments, PSMA or its modified form comprises an epitope recognized by an antibody or its antigen-binding fragment. In some embodiments, with reference to the amino acid sequence set forth in SEQ ID NO: 23 or a position in an amino acid sequence that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, PSMA or its modified form comprises a sequence having amino acids at residues 57-134, 91-108, 100-104, 118-135, 271-288, 469-486, 638-657, 640-657, or 716-723.

[0228] In some embodiments, PSMA or its modified form comprises at least one or more extracellular domains of PSMA or a portion thereof. In some embodiments, PSMA or its modified form comprises at least one or more extracellular domains, with reference to the position of the wild-type human PSMA sequence (e.g., the amino acid sequence set forth in SEQ ID NO: 23), wherein the at least one or more extracellular domains are selected from the protease domain (residues 56-116 and 352-590), the apical domain (residues 117-351), and the C-terminal helical domain (residues 592-750). In some embodiments, the modified PSMA contains at least two extracellular domains of PSMA.

[0229] In some embodiments, PSMA or its modified forms comprise an extracellular catalytic domain or an enzymatically active domain. In some embodiments, PSMA or its modified forms comprise an N-acetylated α-linked acid dipeptidase (NAALADase) domain. In some embodiments, PSMA or its modified forms comprise a sequence having amino acids at residues 274-587, 247-700, or 247-750, with reference to the amino acid sequence set forth in SEQ ID NO: 23 or positions in an amino acid sequence exhibiting at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, PSMA or its modified forms comprise active site residues and / or residues involved in substrate binding and / or catalytic activity. In some embodiments, PSMA or a modified form thereof comprises residues at positions 210, 257, 269, 272, 377, 387, 387, 424, 424, 425, 433, 436, 453, 517, 518, 519, 552, 553, 534, 535, 536, 552, 553, 628, 666, 689, 699, and / or 700, e.g., conserved residues, with reference to positions in the amino acid sequence set forth in SEQ ID NO: 23.

[0230] In some embodiments, PSMA or its modified form is a modified PSMA comprising one or more deletions of a domain or region of the extracellular portion. For example, in some embodiments, with reference to the position in the amino acid sequence set forth in SEQ ID NO: 23, the domain or region deletion of the extracellular portion comprises a deletion of the amino acid sequence at positions 44-273 or 588-750. In some embodiments, the modified PSMA comprises a deletion of one or more amino acid residues (optionally one or more consecutive amino acid residues) in the extracellular portion that is not bound or recognized by a PSMA targeting molecule (e.g., a ligand or antibody or its antigen-binding fragment). In some embodiments, PSMA or its modified form comprises a deletion of one or more C-terminal amino acid residues (optionally one or more consecutive amino acid residues). In some embodiments, with reference to the position in the PSMA set forth in SEQ ID NO: 23, PSMA or its modified form comprises a deletion of amino acid residues 103-750, 626-750, 721-747, or 736-750. In some embodiments, PSMA or a modified form thereof comprises a deletion of 15 C-terminal amino acid residues with reference to the position in PSMA set forth in SEQ ID NO:23.

[0231] In some embodiments, the modified PSMA includes a PSMA described in, for example, International PCT Publication No. WO 2015143029, Rajasekaran et al. (2003) Mol. Biol. Cell. 14:4835-4845, Rajasekaran et al. (2008) Mol Cancer Ther. (2008) 7(7):2142-2151, Barinka et al. (2004) Eur. J. Biochem. 271:2782-2790, and Davis et al. (2005) Proc. Natl. Acad. Sci. 102(17)-5981-5986.

[0232] In some embodiments, PSMA or a modified form thereof, optionally an extracellular portion, is capable of being recognized by a PSMA targeting molecule or portion thereof, such as any PSMA targeting molecule or portion thereof described herein.

[0233] B. Recombinant receptors

[0234] In some embodiments, engineered cells expressing recombinant receptors and PSMA or modified PSMA are provided, such as immune cells, such as T cells. Receptors include antigen receptors and receptors containing one or more components thereof. Recombinant receptors may include chimeric receptors (such as those containing a ligand binding domain or its binding fragment and an intracellular signaling domain), functional non-TCR antigen receptors, chimeric antigen receptors (CARs), and T cell receptors (TCRs) (such as transgenic TCRs), and any of the aforementioned components. In some embodiments, the engineered cells provided express recombinant receptors and PSMA or its modified form (typically recombinant PSMA or modified PSMA). Chimeric receptors (such as CARs) typically include (in some aspects via a linker and / or one or more transmembrane domains) an extracellular antigen (or ligand) binding domain connected to one or more intracellular signaling components.

[0235] 1. Chimeric Antigen Receptor (CAR)

[0236] In some embodiments, there is provided an engineered cell (such as a T cell) that expresses a CAR specific for a specific antigen (or marker or ligand), such as an antigen expressed on the surface of a specific cell type. In some embodiments, the antigen is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, compared to normal or non-targeted cells or tissues, the antigen is selectively expressed or overexpressed on cells of a disease or illness, such as tumor cells or pathogenic cells. In other embodiments, the antigen is expressed on normal cells and / or expressed on engineered cells.

[0237] In certain embodiments, a recombinant receptor (e.g., a chimeric receptor) contains an intracellular signaling region comprising a cytoplasmic signaling domain (also interchangeably referred to as an intracellular signaling domain), e.g., a cytoplasmic (intracellular) region capable of inducing a primary activation signal in a T cell, e.g., a cytoplasmic signaling domain of a T cell receptor (TCR) component (e.g., a cytoplasmic signaling domain of the zeta chain of the CD3-zeta (CD3ζ) chain, or a functional variant or signaling portion thereof); and / or the intracellular signaling region comprises an immunoreceptor tyrosine-based activation motif (ITAM).

[0238] In some embodiments, the chimeric receptor also contains an extracellular ligand binding domain that specifically binds to a ligand (e.g., antigen) antigen. In some embodiments, the chimeric receptor is a CAR that contains an extracellular antigen recognition domain that specifically binds to an antigen. In some embodiments, the ligand (e.g., antigen) is a protein expressed on the surface of a cell. In some embodiments, CAR is a TCR-like CAR, and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, which is recognized on the surface of a cell in the context of a major histocompatibility complex (MHC) molecule, as is TCR.

[0239] Exemplary antigen receptors (including CARs) and methods of engineering and introducing such receptors into cells include, for example, those described in International Patent Application Publication Nos. WO 200014257, WO 2013126726, WO 2012 / 129514, WO 2014031687, WO 2013 / 166321, WO 2013 / 071154, WO 2013 / 123061, U.S. Patent Application Publication Nos. US 2002131960, US 2013287748, US 20130149337, U.S. Patent Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European Patent Application No. EP 2537416, and / or those described in Sadelain et al. Cancer Discov. 2013 Apr;3(4):388-398; Davila et al. (2013) PLoS ONE 8(4):e61338;Turtle et al., Curr. Opin. Immunol., 2012 Oct;24(5):633-39;Wu et al., Cancer, 2012 Mar 18(2):160-75. In some aspects, the antigen receptor includes a CAR as described in U.S. Patent No. 7,446,190, and those described in International Patent Application Publication No. WO / 2014055668A1. Examples of CARs include CARs as disclosed in any of the above-mentioned publications, such as WO 2014031687, US 8,339,645, US 7,446,179, US 2013 / 0149337, U.S. Patent No. 7,446,190, U.S. Patent No. 8,389,282; Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO 2014031687, US 8,339,645, US 7,446,179, US 2013 / 0149337, US Patent No.: 7,446,190, and US Patent No.: 8,389,282.

[0240] In some embodiments, CAR is constructed to have specificity for a specific antigen (or marker or ligand), such as an antigen expressed in a specific cell type targeted by adoptive therapy (e.g., a cancer marker) and / or an antigen intended to induce an attenuation response (e.g., an antigen expressed on a normal or non-diseased cell type). Therefore, CAR typically includes one or more antigen binding molecules, such as one or more antigen binding fragments, domains or portions, or one or more antibody variable domains, and / or antibody molecules in its extracellular portion. In some embodiments, CAR includes one or more antigen binding portions of an antibody molecule, such as a variable heavy chain (V) derived from a monoclonal antibody (mAb). H ) and variable light chain (V L ) of a single-chain antibody fragment (scFv).

[0241] In some embodiments, antibody or its antigen binding portion thereof is expressed on the cell as part of a recombinant receptor (such as an antigen receptor).The antigen receptor includes functional non-TCR antigen receptors, such as chimeric antigen receptors (CAR).Generally, the CAR containing antibodies or antigen binding fragments showing TCR-like specificity for peptide-MHC complexes may also be referred to as TCR-like CARs.In some embodiments, in some aspects, the MHC-peptide complex of TCR-like CAR has specific extracellular antigen binding domains connected to one or more intracellular signaling components by a joint and / or one or more transmembrane domains.In some embodiments, such molecules can generally simulate or approach signals by natural antigen receptors (such as TCR), and optionally simulate or approach signals by combining such receptors with costimulatory receptors.

[0242] In some embodiments, recombinant receptors (e.g., chimeric receptors, e.g., CARs) include ligand binding domains that bind to (e.g., specifically bind to) an antigen (or ligand). The antigen targeted by the chimeric receptor is included in antigens expressed in the case of a disease, disorder, or cell type targeted by adoptive cell therapy. The diseases and disorders include proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including blood cancers, immune system cancers, such as lymphomas, leukemias, and / or myeloma, such as B leukemia, T leukemia, and myeloid leukemia, lymphomas, and multiple myeloma.

[0243] In some embodiments, the antigen (or ligand) is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen (or ligand) is selectively expressed or overexpressed on cells (e.g., tumor or pathogenic cells) of a disease or condition, as compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or expressed on engineered cells.

[0244] In some embodiments, the CAR contains an antibody or antigen-binding fragment (e.g., scFv) that specifically recognizes an antigen (e.g., an intact antigen) expressed on the surface of a cell.

[0245] In some embodiments, the antigen (or ligand) is a tumor antigen or a cancer marker. In some embodiments, the antigen (or ligand) is or includes orphan tyrosine kinase receptor ROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2 / neu (receptor tyrosine kinase erbB2), L1-CAM, CD19, CD20, CD22, mesothelin, CEA, hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin receptor A2 (EPHa2), Her2 / neu (receptor tyrosine kinase erbB2), Her3 (erb-B3), Her4 (erb-B4), erbB dimer, epidermal growth factor receptor type III mutant (EGFR vIII), folate binding protein (FBP), FCRL5, Fc receptor-like protein 5 (FCRL5;Also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor, ganglioside GD2, ganglioside GD3, G protein-coupled receptor 5D (GPCR5D), HMW-MAA, IL-22R-α, IL-13R-α2, kinase insert domain receptor (KDR), kappa light chain, leucine-rich repeat-containing protein 8 family member A (LRRC8A), Lewis Y, L1 cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, preferentially expressed melanoma antigen (PRAME), survivin, TAG72, B7-H6, IL-13 receptor alpha 2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250 / CAIX, human leukocyte antigen A (HLA-A1), MAGE A1, HLA-A2, NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7 / 8, αvβ6 integrin (avb6 integrin), 8H9, NCAM, VEGF receptor, 5T4, fetal AchR, natural killer cell group 2 member D (NKG2D) ligand, CD44v6, dual antigen, cancer-testis antigen, mesothelin, murine CMV, mucin 1 (MUC1), MUC16, prostate stem cell antigen (PSCA), NKG2D, cancer-testis antigen (cancer / testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2)), MART-1, glycoprotein 100 ( gp100), carcinoembryonic antigen, ROR1, trophoblast glycoprotein (TPBG also known as 5T4), TAG72, VEGF-R2, carcinoembryonic antigen (CEA), Her2 / neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms tumor 1 (WT-1), cyclins, cyclin A2, C-C motif chemokine ligand 1 (CCL-1), CD138, pathogen-specific antigens and antigens associated with universal tags, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens.

[0246] In some embodiments, the antigen or ligand is a tumor antigen or cancer marker. In some embodiments, the antigen or ligand antigen is or includes αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), cancer-testis antigen, cancer / testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, CC motif chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), truncated epidermal growth factor protein (tEGFR), epidermal growth factor receptor type III mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor-like protein 5 (FCRL5;Also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), glypican (GPC3), G protein-coupled receptor 5D (GPCR5D), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimer, human high molecular weight melanoma-associated antigen (HM W-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22Rα), IL-13 receptor alpha 2 (IL-13Rα2), kinase insert domain receptor (KDR), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, leucine-rich repeat-containing protein 8 family member A (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A 10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer cell group 2 member D (NKG2D) ligand, melanin A (MART-1), neural cell adhesion molecule (NCAM), carcinoembryonic antigen, preferentially expressed melanoma antigen (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein (TPBG, also known as 5T 4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-related protein 1 (TRP1, also known as TYRP1 or gp75), tyrosinase-related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor 1 (WT-1), pathogen-specific or pathogen-expressed antigens, or antigens associated with universal tags, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens.;

[0247] In some embodiments, the antigen targeted by the receptor includes an antigen associated with a B cell malignancy, such as any of a number of known B cell markers. In some embodiments, the antigen targeted by the receptor is CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igκ, Igλ, CD79a, CD79b, or CD30.

[0248] In some embodiments, the antigen is a pathogen-specific antigen or an antigen expressed by a pathogen. In some embodiments, the antigen is a viral antigen (e.g., a viral antigen from HIV, HCV, HBV, etc.), a bacterial antigen, and / or a parasite antigen.

[0249] In some embodiments, the antigen is or includes a pathogen-specific or pathogen-expressed antigen, such as a viral antigen (eg, a viral antigen from HIV, HCV, HBV), a bacterial antigen, and / or a parasitic antigen.

[0250] In some embodiments, an antibody or antigen-binding fragment (e.g., scFv or V H domain) specifically recognizes an antigen, such as CD 19. In some embodiments, the antibody or antigen-binding fragment is derived from an antibody or antigen-binding fragment that specifically binds to CD 19, or is a variant of an antibody or antigen-binding fragment that specifically binds to CD 19.

[0251] In some embodiments, scFv and / or V H The domain is derived from FMC63. FMC63 generally refers to a mouse monoclonal IgG1 antibody produced against Nalm-1 and -16 cells expressing human CD19 (Ling, NR, et al. (1987). Leucocyte typing III.302). In some embodiments, the scFv and / or V H The domain is derived from SJ25C1. SJ25C1 refers to a mouse monoclonal IgG1 antibody raised against Nalm-1 and -16 cells expressing human CD19 (Ling, NR, et al. (1987). Leucocyte typing III. 302).

[0252] In some aspects, CAR contains a ligand-(for example, antigen-) binding domain that binds or identifies (for example, specifically binds) a universal tag or a universal epitope. In some aspects, the binding domain can be bound to molecules, labels, polypeptides and / or epitopes, and the molecules, labels, polypeptides and / or epitopes can be connected to different binding molecules (for example, antibodies or Fab) that identify the antigens related to a disease or disorder. Exemplary labels or epitopes include dyes (for example, fluorescein isothiocyanate) or biotin. In some aspects, binding molecules (for example, antibodies or Fab) are connected to labels, and the label identifies the antigens (for example, tumor antigens) related to the disease or disorder of the engineered cells with expression of the CAR specific to the label, to achieve the cytotoxicity or other effector functions of the engineered cells. In some aspects, the specificity of CAR to the antigens related to a disease or disorder is provided by the binding molecules (for example, antibodies) of labels, and the binding molecules of different labels can be used for targeting different antigens. Exemplary CARs specific for a universal tag or universal epitope include, for example, those described in US 9,233,125, WO 2016 / 030414, Urbanska et al., (2012) Cancer Res 72: 1844-1852, and Tamada et al., (2012). Clin Cancer Res 18: 6436-6445.

[0253] In some embodiments, CAR contains TCR-like antibodies, such as antibodies or antigen-binding fragments (e.g., scFv), which specifically recognize intracellular antigens (e.g., tumor-associated antigens) present on the cell surface as MHC-peptide complexes. In some embodiments, antibodies or antigen-binding portions thereof that recognize MHC-peptide complexes can be expressed on cells as part of a recombinant receptor (e.g., antigen receptor). The antigen receptor includes a functional non-TCR antigen receptor, such as a chimeric antigen receptor (CAR). Typically, CARs containing antibodies or antigen-binding fragments that exhibit TCR-like specificity for peptide-MHC complexes can also be referred to as TCR-like CARs.

[0254] Reference to a "major histocompatibility complex" (MHC) refers to a protein, typically a glycoprotein, containing a polymorphic peptide binding site or binding groove, which in some cases can be complexed with peptide antigens of a polypeptide (including peptide antigens processed by cellular machinery). In some cases, MHC molecules can be displayed or expressed on the surface of a cell, including as a complex with a peptide, i.e., an MHC-peptide complex, for presenting an antigen in a conformation recognizable by an antigen receptor on a T cell (e.g., a TCR or TCR-like antibody). Typically, MHC class I molecules are heterodimers having a membrane spanning α chain, in some cases three α domains, and non-covalently associated β2 microglobulin. Typically, MHC class II molecules are composed of two transmembrane glycoproteins, α and β, both of which typically span the membrane. MHC molecules can include an effective portion of the MHC, which contains an antigen binding site or a site for binding to a peptide and a sequence required for recognition by an appropriate antigen receptor. In some embodiments, MHC class I molecules deliver peptides derived from the cytosol to the cell surface, where the MHC-peptide complex is expressed by a T cell (e.g., typically CD8 + In some embodiments, MHC class II molecules deliver peptides derived from vesicle systems to the cell surface, where the peptides are typically expressed by CD4 T cells. + T cell recognition. Typically, MHC molecules are encoded by a set of linked loci, collectively referred to as H-2 in mice and human leukocyte antigens (HLA). Therefore, human MHC is also commonly referred to as human leukocyte antigens (HLA).

[0255] The term "MHC-peptide complex" or "peptide-MHC complex" or variants thereof refers to a complex or associate of a peptide antigen and an MHC molecule, for example, typically formed by non-covalent interactions of the peptide in the binding groove or cleft of the MHC molecule. In some embodiments, the MHC-peptide complex is present or displayed on the surface of a cell. In some embodiments, the MHC-peptide complex can be specifically recognized by an antigen receptor (e.g., a TCR, a TCR-like CAR, or an antigen-binding portion thereof).

[0256] In some embodiments, the peptide (e.g., peptide antigen or epitope) of a polypeptide can be associated with an MHC molecule, for example, for identification by an antigen receptor. Typically, the peptide is derived from or based on a fragment of a longer biomolecule (e.g., a polypeptide or protein). In some embodiments, the length of the peptide is typically about 8 to about 24 amino acids. In some embodiments, the length of the peptide is or is about 9 to 22 amino acids, for identification in MHC class II complexes. In some embodiments, the length of the peptide is or is about 8 to 13 amino acids, for identification in MHC class I complexes. In some embodiments, after identifying the peptide in the background of an MHC molecule (e.g., MHC-peptide complex), an antigen receptor (e.g., TCR or TCR-like CAR) produces or triggers an activation signal to a T cell, inducing a T cell response, such as T cell proliferation, cytokine production, cytotoxic T cell response or other responses.

[0257] In some embodiments, TCR-like antibodies or antigen-binding portions are known or can be produced by known methods (see, e.g., U.S. Published Application Nos. US 2002 / 0150914; US 2003 / 0223994; US 2004 / 0191260; US 2006 / 0034850; US 2007 / 00992530; US 20090226474; US 20090304679; and International PCT Publication No. WO 03 / 068201).

[0258] In some embodiments, antibodies or antigen-binding portions thereof that specifically bind to MHC-peptide complexes can be produced by immunizing a host with an effective amount of an immunogen containing a specific MHC-peptide complex. In some cases, the peptide of the MHC-peptide complex is an epitope of an antigen that is capable of binding to MHC, such as a tumor antigen, such as a universal tumor antigen, a myeloma antigen, or other antigens as described below. In some embodiments, an effective amount of an immunogen is then administered to the host for eliciting an immune response, wherein the immunogen maintains its three-dimensional form for a period of time sufficient to induce an immune response to the three-dimensional presentation of the peptide in the binding groove of the MHC molecule. Serum collected from the host is then assayed to determine whether the desired antibodies that recognize the three-dimensional presentation of the peptide in the binding groove of the MHC molecule have been produced. In some embodiments, the antibodies produced can be evaluated to confirm that the antibodies can distinguish between MHC-peptide complexes and separate MHC molecules, separate target peptides, and complexes of MHC and unrelated peptides. The desired antibodies can then be isolated.

[0259] In some embodiments, antibodies or antigen-binding portions thereof that specifically bind to an MHC-peptide complex can be produced using an antibody library display method (e.g., a phage antibody library). In some embodiments, a phage display library of mutant Fab, scFv, or other antibody formats can be produced, for example, wherein members of the library are mutated at one or more residues of one or more CDRs. See, for example, U.S. Published Application Nos. US20020150914, US2014 / 0294841; and Cohen CJ. et al. (2003) J Mol. Recogn. 16:324-332.

[0260] The term "antibody" as used herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including whole antibodies and functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy chains (V H ) region, single-chain antibody fragments (including single-chain variable fragments (scFv)) and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term covers genetically engineered and / or otherwise modified forms of immunoglobulins, such as intracellular antibodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugate antibodies (heteroconjugateantibody), multispecific (e.g., bispecific) antibodies, double antibodies, three antibodies and four antibodies, tandem two-scFv, tandem three-scFv. Unless otherwise indicated, the term "antibody" should be understood to cover its functional antibody fragments. The term also covers complete or full-length antibodies, including antibodies of any class or subclass (including IgG and its subclasses, IgM, IgE, IgA and IgD).

[0261] In some embodiments, antigen-binding proteins, antibodies, and antigen-binding fragments thereof specifically recognize the antigen of a full-length antibody. In some embodiments, the heavy and light chains of the antibody can be full-length or can be antigen-binding portions (Fab, F(ab')2, Fv, or single-chain Fv fragments (scFv)). In other embodiments, the antibody heavy chain constant region is selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, particularly selected from, for example, IgG1, IgG2, IgG3, and IgG4, more particularly IgG1 (for example, human IgG1). In another embodiment, the antibody light chain constant region is selected from, for example, κ or λ, particularly κ.

[0262] The antibodies provided include antibody fragments. An "antibody fragment" is a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; variable heavy chain (V H ) region, single-chain antibody molecules (such as scFv) and single domain V H Single antibodies; and multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single-chain antibody fragment comprising a variable heavy chain region and / or a variable light chain region, such as scFv.

[0263] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding the antibody to an antigen. H and V L ) generally have a similar structure, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, for example, Kindt et al. Kuby Immunology, 6th edition, WH Freeman and Co., page 91 (2007)). A single V H or V L The domain may be sufficient to confer antigen binding specificity. In addition, a V domain from an antibody that binds to the antigen may be used. H or V L The domains are separated to separate antibodies that bind to the specific antigen to screen for complementary V L or V H Libraries of domains. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0264] Single domain antibodies are antibody fragments comprising all or part of the heavy chain variable domains or all or part of the light chain variable domains of an antibody. In certain embodiments, single domain antibodies are human single domain antibodies. In some embodiments, CAR comprises an antibody heavy chain domain that specifically binds to an antigen, such as a cancer marker or a cell surface antigen of a cell or disease (e.g., a tumor cell or cancer cell) to be targeted, such as any target antigen described herein or known.

[0265] Antibody fragments can be prepared by various techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, such as a fragment comprising an arrangement that does not exist in nature (such as two or more antibody regions or chains connected by a synthetic linker (e.g., a peptide linker), and / or a fragment that can be produced without enzymatic digestion of naturally occurring intact antibodies. In some embodiments, the antibody fragment is a scFv.

[0266] "Humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDR and all or substantially all FR amino acid residues are derived from people FR. Humanized antibodies optionally can include at least a portion of an antibody constant region derived from a human antibody. "Humanized form" of a non-human antibody refers to a variant of the non-human antibody, which has undergone humanization to generally reduce immunogenicity to people while retaining the specificity and avidity of the parent non-human antibody. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody derived from CDR residues), for example, to restore or improve antibody specificity or avidity.

[0267] Therefore, in some embodiments, chimeric antigen receptors (including TCR-like CARs) include an extracellular portion containing an antibody or antibody fragment. In some embodiments, the antibody or fragment includes scFv. In some aspects, chimeric antigen receptors include an extracellular portion containing an antibody or fragment and an intracellular signaling region. In some embodiments, the intracellular signaling region includes an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or includes a primary signaling domain, a signaling domain capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM).

[0268] In some embodiments, the recombinant receptor (e.g., CAR, such as the antibody portion thereof) further comprises a spacer, which can be or include at least a portion of an immunoglobulin constant region or a variant or modified form thereof, such as a hinge region (e.g., an IgG4 hinge region) and / or a C H 1 / C LIn some embodiments, the recombinant receptor further comprises a spacer and / or hinge region. In some embodiments, the constant region or portion is human IgG such as IgG4 or IgG1. In some aspects, the portion of the constant region is used as a spacer region between an antigen recognition component (e.g., scFv) and a transmembrane domain. Compared to the absence of a spacer, the length of the spacer can provide enhanced cellular reactivity after antigen binding. In some examples, the length of the spacer is or is about 12 amino acids or is no more than 12 amino acids in length. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids (and including any integer between the endpoints of any listed ranges). In some embodiments, the spacer region has about 12 or fewer amino acids, about 119 or fewer amino acids, or about 229 or fewer amino acids. Exemplary spacers include an IgG4 hinge alone, an IgG4 hinge connected to a CH2 and CH3 domain, or an IgG4 hinge connected to a CH3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, Hudecek et al. (2015) Cancer Immunol Res. 3(2):125-135; or International Patent Application Publication No. WO 2014031687. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 1 and is encoded by the sequence shown in SEQ ID NO: 2. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 3. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 4.

[0269] In some embodiments, the constant region or portion is of IgD. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 5. In some embodiments, the spacer has an amino acid sequence that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 1, 3, 4, and 5.

[0270] In some aspects, the spacer is a polypeptide spacer, such as one or more selected from the group consisting of: (a) comprising or consisting of all or part of an immunoglobulin hinge or a modified form thereof or comprising about 15 amino acids or less and not comprising the CD28 extracellular region or the CD8 extracellular region, (b) comprising or consisting of all or part of an immunoglobulin hinge (optionally an IgG4 hinge) or a modified form thereof and / or comprising about 15 amino acids or less and not comprising the CD28 extracellular region or the CD8 extracellular region, or (c) being or being about 12 amino acids in length and / or comprising or consisting of all or part of an immunoglobulin hinge (optionally an IgG4 hinge) or a modified form thereof; or (d) comprising or consisting of SEQ ID NO:1, the amino acid sequence shown in 3-5 or any variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity, or (e) comprises the formula X1PPX2P (SEQ ID NO:66) (wherein X1 is glycine, cysteine ​​or arginine and X2 is cysteine ​​or threonine) or consists of it. In some embodiments, the spacer comprises a sequence from an immunoglobulin. In some embodiments, the spacer comprises all or part of an immunoglobulin hinge (optionally an IgG4 hinge and / or an IgG1 hinge), or consists of it. In some embodiments, the spacer comprises an immunoglobulin heavy chain constant region (optionally C H 2 and / or C H Part of Zone 3).

[0271] Antigen recognition domains are typically connected to one or more intracellular signaling components (such as by antigen receptor complexes (such as TCR complexes) (in the case of CAR) simulation activation and / or via another cell surface receptor simulation signal signaling components). Therefore, in some embodiments, antigen binding components (e.g., antibodies) are connected to one or more transmembrane and intracellular signaling regions. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain that is naturally associated with a domain in the receptor (e.g., CAR) is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid such domains from being combined with the transmembrane domain of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0272] In some embodiments, membrane spaning domain is derived from natural or synthetic source.When source is natural, in some aspects, described domain can be derived from any membrane-bound protein or transmembrane protein.Membrane spaning region includes those membrane spaning regions (that is, including at least one or more membrane spaning regions) derived from the following items: α, β or ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, in some embodiments, membrane spaning domain is synthetic. In some aspects, synthetic membrane spaning domain mainly comprises hydrophobic residues, such as leucine and valine. In some aspects, the triplet of phenylalanine, tryptophan and valine will be found at each end of synthetic membrane spaning domain. In some embodiments, connection is by joint, spacer and / or one or more membrane spaning domains.

[0273] Intracellular signaling regions include those that simulate or approach signals by natural antigen receptors, simulate or approach signals by such receptors in combination with costimulatory receptors and / or simulate or approach signals only by costimulatory receptors. In some embodiments, a short oligopeptide or polypeptide linker (e.g., a linker of 2 to 10 amino acids in length, such as a linker containing glycine and serine, e.g., a glycine-serine doublet) is present and forms a connection between the transmembrane domain of the CAR and the cytoplasmic signaling domain.

[0274] Receptor such as CAR generally includes at least one intracellular signaling component or multiple intracellular signaling components. In some embodiments, the receptor includes the intracellular component of the TCR complex, such as the TCR CD3 chain such as CD3 ζ chain that mediates T cell activation and cytotoxicity. Therefore, in some aspects, the antigen binding domain is connected to one or more cell signaling modules. In some embodiments, the cell signaling module includes CD3 transmembrane domain, CD3 intracellular signaling domain and / or other CD transmembrane domain. In some embodiments, the receptor (such as CAR) also includes a part of one or more other molecules (such as Fc receptor gamma, CD8, CD4, CD25 or CD16). For example, in some aspects, CAR includes a chimeric molecule between CD3-zeta (CD3-ζ) or Fc receptor gamma and CD8, CD4, CD25 or CD16.

[0275] In some embodiments, after connecting the CAR, the cytoplasmic domain or intracellular signaling region of the CAR activates at least one of the normal effector functions or responses of immune cells (e.g., engineered to express the T cells of the CAR). For example, in some situations, CAR induces the function of T cells, such as cytolytic activity or T helper activity, such as the secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling region of an antigen receptor component or a costimulatory molecule (e.g., if its transduction effector function signal) is used instead of a complete immunostimulatory chain. In some embodiments, the intracellular signaling region (e.g., comprising one or more intracellular signaling domains) includes the cytoplasmic sequence of a T cell receptor (TCR), and in some aspects also includes a co-receptor (which acts in parallel with this receptor in a natural context to start signal transduction after antigen receptor engagement) and / or any derivative or variant of such molecules, and / or any synthetic sequence with the same functional capabilities.

[0276] In the case of natural TCR, full activation generally requires not only signal conduction by TCR, but also costimulatory signals. Therefore, in some embodiments, in order to promote full activation, components for generating secondary or costimulatory signals are also included in the CAR. In other embodiments, CAR does not include components for generating costimulatory signals. In some aspects, other CARs are expressed in the same cell and are provided for generating components of secondary or costimulatory signals.

[0277] In some aspects, T cell activation is described as being mediated by two categories of cytoplasmic signaling sequences: those sequences that initiate antigen-dependent primary activation by TCR (primary cytoplasmic signaling sequences) and those sequences that act in an antigen-independent manner to provide stimulation or costimulatory signals (secondary cytoplasmic signaling sequences). In some aspects, CAR includes one or both of such signaling components.

[0278] In some aspects, CAR includes a primary cytoplasmic signaling sequence for regulating the primary activation of the TCR complex. The primary cytoplasmic signaling sequence that works in a stimulating manner can contain a signaling motif (which is referred to as an activation motif or ITAM based on immunoreceptor tyrosine). The example of ITAM containing a primary cytoplasmic signaling sequence includes those derived from TCR or CD3ζ, FcRγ or FcRβ. In some embodiments, the cytoplasmic signaling molecule in CAR contains a cytoplasmic signaling domain, a portion thereof or a sequence derived from CD3ζ.

[0279] In some embodiments, CAR includes signaling regions and / or transmembrane portions of costimulatory receptors such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some aspects, the same CAR includes signaling regions and costimulatory components.

[0280] In some embodiments, the signaling region is included in one CAR, and the costimulatory component is provided by another CAR that recognizes another antigen. In some embodiments, CAR includes activation or stimulation CAR and costimulatory CAR expressed on the same cell (see WO 2014 / 055668).

[0281] In certain embodiments, the intracellular signaling region comprises a CD28 transmembrane and signaling domain connected to a CD3 (e.g., CD3-ζ) intracellular domain. In some embodiments, the intracellular signaling region comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9) costimulatory domain connected to a CD3 ζ intracellular domain.

[0282] In some embodiments, CAR includes one or more, such as two or more costimulatory domains and activation domains, such as primary activation domains, in the cytoplasmic portion.Exemplary CARs include the intracellular components of CD3-ζ, CD28, and 4-1BB.

[0283] In some cases, CAR is referred to as first generation, second generation and / or third generation CAR.In some aspects, first generation CAR is the CAR that provides only the signal induced by CD3 chain after antigen binding; In some aspects, second generation CAR is the CAR that provides this signal and costimulatory signal, for example, including the CAR of the intracellular signaling domain from costimulatory receptor (such as CD28 or CD137); In some aspects, third generation CAR is the CAR of multiple costimulatory domains including different costimulatory receptors in some aspects.

[0284] In some embodiments, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or antibody fragment described herein. In some aspects, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or fragment described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment comprises an scFv or single domain V H Antibodies, and the intracellular domain contains an ITAM. In some aspects, the intracellular signaling domain comprises the signaling domain of the zeta chain of the CD3-zeta (CD3ζ) chain. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain disposed between the extracellular domain and the intracellular signaling region.

[0285] In some aspects, the membrane-spanning domain contains the membrane-spanning portion of CD28. Extracellular domain and membrane-spanning can be connected directly or indirectly. In some embodiments, the extracellular domain and membrane-spanning are connected by a spacer (any spacer as described herein). In some embodiments, chimeric antigen receptor contains the intracellular domain of a T cell costimulatory molecule, such as between a membrane-spanning domain and an intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB.

[0286] In some embodiments, CAR contains an antibody (e.g., an antibody fragment), a transmembrane domain (which is or contains the transmembrane portion of CD28 or a functional variant thereof), and an intracellular signaling domain containing a signaling portion of CD28 or a functional variant thereof and a signaling portion of CD3ζ or a functional variant thereof. In some embodiments, CAR contains an antibody, such as an antibody fragment, a transmembrane domain (which is or contains the transmembrane portion of CD28 or a functional variant thereof), and an intracellular signaling domain containing a signaling portion of 4-1BB or a functional variant thereof and a signaling portion of CD3ζ or a functional variant thereof. In some such embodiments, the receptor further comprises a spacer containing a portion of an Ig molecule (e.g., a human Ig molecule, such as an Ig hinge, e.g., an IgG4 hinge), such as a spacer containing only a hinge.

[0287] In some embodiments, the transmembrane domain of the receptor (e.g., CAR) is the transmembrane domain of human CD28 or a variant thereof, such as the 27 amino acid transmembrane domain of human CD28 (Accession No.: P10747.1), or a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence that exhibits at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 8; in some embodiments, the transmembrane domain of the portion containing the recombinant receptor comprises the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence that exhibits at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 9. NO:9 has an amino acid sequence that is at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical.

[0288] In some embodiments, the chimeric antigen receptor contains the intracellular domain of a T cell co-stimulatory molecule. In some aspects, the T cell co-stimulatory molecule is CD28 or 4-1BB.

[0289] In some embodiments, the intracellular signaling region comprises the intracellular co-stimulatory signaling domain of human CD28, or a functional variant or portion thereof, such as a 41 amino acid domain thereof, and / or such a domain having an LL to GG substitution at positions 186-187 of the native CD28 protein. In some embodiments, the intracellular signaling region and / or domain can comprise the amino acid sequence set forth in SEQ ID NO: 10 or 11, or an amino acid sequence that exhibits at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10 or 11. In some embodiments, the intracellular region comprises the intracellular co-stimulatory signaling domain of 4-1BB or a functional variant or portion thereof, such as the 42 amino acid cytoplasmic domain of human 4-1BB (Accession No. Q07011.1) or a functional variant or portion thereof, such as the amino acid sequence shown in SEQ ID NO: 12, or an amino acid sequence that exhibits at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12. In some embodiments, the intracellular region comprises the intracellular co-stimulatory signaling domain of human 4-1BB or a functional variant or portion thereof.

[0290] In some embodiments, the intracellular signaling region comprises a human CD3 chain, optionally a CD3 zeta stimulatory signaling domain, or a functional variant thereof, such as the 112 AA cytoplasmic domain of isoform 3 of human CD3 zeta (Accession No.: P20963.2), or a CD3 zeta signaling domain as described in U.S. Patent No. 7,446,190 or U.S. Patent No. 8,911,993. In some embodiments, the intracellular signaling region comprises the amino acid sequence shown in SEQ ID NO: 13, 14, or 15, or an amino acid sequence that exhibits at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13, 14, or 15.

[0291] In some aspects, the spacer contains only the hinge region of IgG, such as only the hinge of IgG4 or IgG1, such as the hinge-only spacer shown in SEQ ID NO: 1. In other embodiments, the spacer is H 2 and / or C H In some embodiments, the spacer is an Ig hinge connected to the C3 domain, for example, an IgG4 hinge. H2 and C H 3 domains, such as an IgG4 hinge, as shown in SEQ ID NO: 3. In some embodiments, the spacer is only attached to the C H The spacer is an Ig hinge connected to the 3 domains, such as an IgG4 hinge, as shown in SEQ ID NO: 4. In some embodiments, the spacer is or comprises a glycine-serine-rich sequence or other flexible linker, such as a known flexible linker.

[0292] In some embodiments, the CAR comprises:

[0293] (i) an scFv specific for an antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule which is optionally or optionally comprises 4-1BB, and a cytoplasmic signaling domain derived from a molecule containing a primary signaling ITAM which is optionally or comprises a CD3 zeta signaling domain and optionally further comprises a spacer between the transmembrane domain and the scFv, in order;

[0294] (ii) in order, a scFv specific for an antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule which is or optionally comprises a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from a molecule containing a primary signaling ITAM, which is optionally a CD3 zeta signaling domain; or

[0295] (iii) in order, a scFv specific for an antigen, a spacer, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule which is optionally a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from a molecule containing a primary signaling ITAM (which is optionally or optionally comprises a CD3 zeta signaling domain); and:

[0296] The spacer is optionally a polypeptide spacer that (a) comprises or consists of all or part of an immunoglobulin hinge, or a modified form thereof, or comprises about 15 amino acids or less and does not comprise the extracellular region of CD28 or CD8, (b) comprises or consists of all or part of an immunoglobulin hinge (optionally IgG4), or a modified form thereof, and / or comprises about 15 amino acids or less and does not comprise the extracellular region of CD28 or CD8, or (c) is 12 amino acids or about 12 amino acids in length and / or comprises or consists of all or part of an immunoglobulin hinge (optionally IgG4), or a modified form thereof; or (d) has or consists of the sequence of SEQ ID NO: 1, which is a sequence encoded by SEQ ID NO: 2, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 NO:58 or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of the foregoing, or (e) comprises or consists of the formula X1PPX2P (SEQ ID NO:66), wherein X1 is glycine, cysteine ​​or arginine and X2 is cysteine ​​or threonine; and / or

[0297] the costimulatory domain comprises SEQ ID NO: 12 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12; and / or

[0298] The primary signaling domain comprises SEQ ID NO: 13 or 14 or 15 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto; and / or

[0299] The scFv comprises the CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 59), the CDRL2 sequence of SRLHSGV (SEQ ID NO: 60) and / or the CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 61) and / or the CDRH1 sequence of DYGVS (SEQ ID NO: 62), the CDRH2 sequence of VIWGSETTYYNSALKS (SEQ ID NO: 63) and / or the CDRH3 sequence of YAMDYWG (SEQ ID NO: 64). NO:64), or wherein the scFv comprises the variable heavy chain region of FMC63 and the variable light chain region of FMC63 and / or the CDRL1 sequence of FMC63, the CDRL2 sequence of FMC63, the CDRL3 sequence of FMC63, the CDRH1 sequence of FMC63, the CDRH2 sequence of FMC63 and the CDRH3 sequence of FMC63, or binds to the same epitope as any one of the foregoing or competes with the binding of any one of the foregoing, and optionally wherein the scFv comprises the variable heavy chain region of FMC63 and the variable light chain region of FMC63 and / or the CDRL1 sequence of FMC63, the CDRL2 sequence of FMC63, the CDRL3 sequence of FMC63, the CDRH1 sequence of FMC63, the CDRH2 sequence of FMC63 and the CDRH3 sequence of FMC63 H , optionally comprising a linker of SEQ ID NO: 65 and V L , and / or the scFv comprises a flexible linker and / or comprises the amino acid sequence shown in SEQ ID NO: 65.

[0300] 2. T cell receptor (TCR)

[0301] In some embodiments, engineered cells (e.g., T cells) are provided that express a T cell receptor (TCR) or an antigen binding portion thereof that recognizes a peptide epitope or T cell epitope of a target polypeptide (e.g., an antigen of a tumor, virus, or autoimmune protein).

[0302] In some embodiments, a "T cell receptor" or "TCR" is a molecule containing variable α and β chains (also referred to as TCRα and TCRβ, respectively) or variable γ and δ chains (also referred to as TCRα and TCRβ, respectively), or an antigen binding portion thereof, and is capable of specifically binding to a peptide bound to an MHC molecule. In some embodiments, the TCR is in the form of αβ. Typically, TCRs in the form of αβ and γδ are generally similar in structure, but the T cells expressing them may have different anatomical locations or functions. The TCR can be found on the surface of a cell or in a soluble form. Typically, the TCR is found on the surface of a T cell (or T lymphocyte), where it is typically responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.

[0303] Unless otherwise indicated, the term "TCR" should be understood to encompass complete TCRs and their antigen-binding portions or antigen-binding fragments. In some embodiments, TCR is a complete or full-length TCR, including a TCR in αβ form or γδ form. In some embodiments, TCR is an antigen-binding portion that is less than the full-length TCR but binds to a specific peptide bound in an MHC molecule (such as bound to an MHC-peptide complex). In some cases, the antigen-binding portion or fragment of TCR may contain only a portion of the structural domain of the full-length or complete TCR, but is still capable of binding to a peptide epitope (such as an MHC-peptide complex) bound to the complete TCR. In some cases, the antigen-binding portion contains the variable domains of TCR (such as the variable α chain and variable β chain of TCR), which are sufficient to form a binding site for binding to a specific MHC-peptide complex. Typically, the variable chain of TCR contains complementary determining regions that participate in the recognition of peptides, MHC and / or MHC-peptide complexes.

[0304] In some embodiments, the variable domains of TCR contain hypervariable loops or complementary determining regions (CDRs), which are typically the main contributors to antigen recognition and binding ability and specificity. In some embodiments, the CDRs of TCR or their combinations form all or substantially all of the antigen binding sites of a given TCR molecule. Each CDR in the variable region of a TCR chain is typically separated by a framework region (FR), which, compared to CDR, typically displays lower variability between TCR molecules (see, for example, Jores et al., Proc. Nat'l Acad. Sci. USA 87: 9138, 1990; Chothia et al., EMBO J. 7: 3745, 1988; See also Lefranc et al., Dev. Comp. Immunol. 27: 55, 2003). In some embodiments, CDR3 is the main CDR responsible for antigen binding or specificity, or the antigen recognition for the processed peptide portion of the peptide-MHC complex in a given TCR variable region and / or the most important CDR for interacting with it in three CDRs. In some cases, the CDR1 of the α chain may interact with the N-terminal portion of some antigenic peptides. In some cases, the CDR1 of the β chain may interact with the C-terminal portion of the peptide. In some cases, the CDR2 may have the strongest effect or be the primary responsible CDR for interaction or recognition with the MHC portion of the MHC-peptide complex. In some embodiments, the variable region of the β chain may contain an additional hypervariable region (CDR4 or HVR4), which is typically involved in superantigen binding rather than antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).

[0305] In some embodiments, the TCR may also contain a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd ed., Current Biology Publications, p. 4:33, 1997). In some aspects, each chain of the TCR may have an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, the TCR associates with an invariant protein of the CD3 complex that mediates signal transduction.

[0306] In some embodiments, the TCR chain contains one or more constant domains. For example, the extracellular portion of a given TCR chain (e.g., an α chain or a β chain) can contain two immunoglobulin-like domains adjacent to the cell membrane, such as a variable domain (e.g., Vα or Vβ; typically based on amino acids 1 to 116 of Kabat numbering, Kabat et al., "Sequences of Proteins of Immunological Interest", US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th edition) and a constant domain (e.g., an α chain constant domain or Cα, typically based on positions 117 to 259 of Kabat numbering, or a β chain constant domain or Cβ). β , typically based on positions 117 to 295 of the chains of Kabat. For example, in some cases, the extracellular portion of a TCR formed from two chains contains two membrane-proximal constant domains and two membrane-distal variable domains, wherein each variable domain contains a CDR. The constant domain of a TCR may contain a short linker sequence in which cysteine ​​residues form a disulfide bond, thereby connecting the two chains of the TCR. In some embodiments, a TCR may have additional cysteine ​​residues in each α and β chain, such that the TCR contains two disulfide bonds in the constant domain.

[0307] In some embodiments, the TCR chain contains a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain contains a cytoplasmic tail. In some cases, the structure allows TCR to associate with other molecules (such as CD3 and its subunits). For example, the TCR containing a constant domain and a transmembrane region can anchor the protein in the cell membrane and associate with the constant subunits of the CD3 signal transduction device or complex. The intracellular tail of the CD3 signal transduction subunit (for example, CD3γ, CD3δ, CD3ε and CD3ζ chain) contains one or more immunoreceptor tyrosine-based activation motifs or ITAMs involved in the signal transduction ability of the TCR complex.

[0308] In some embodiments, the TCR can be a heterodimer of two chains α and β (or optionally γ and δ), or it can be a single-chain TCR construct. In some embodiments, the TCR is a heterodimer containing two separate chains (α and β chains or γ and δ chains) that are connected, for example, by one or more disulfide bonds.

[0309] In some embodiments, TCR can be produced from one or more known TCR sequences (such as Vα, β chain sequences), and the substantially full-length coding sequences of the one or more known TCR sequences are easily obtained. Methods for obtaining full-length TCR sequences (including V chain sequences) from cell sources are well known. In some embodiments, nucleic acids encoding TCRs can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of nucleic acids encoding TCRs in one or more given cells or isolated from the one or more given cells, or by synthesis of publicly available TCR DNA sequences.

[0310] In some embodiments, the TCR is obtained from a biological source, such as from a cell (such as from a T cell, such as a cytotoxic T cell), a T cell hybridoma, or other publicly available sources. In some embodiments, the T cell can be obtained from cells isolated in vivo. In some embodiments, the TCR is a thymus-selected TCR. In some embodiments, the TCR is a new epitope-restricted TCR. In some embodiments, the T cell can be a cultured T cell hybridoma or clone. In some embodiments, the TCR or its antigen-binding portion or its antigen-binding fragment can be produced synthetically based on the knowledge of the TCR sequence.

[0311] In some embodiments, TCR is produced by screening candidate TCR libraries for target polypeptide antigens or their target T cell epitopes and identifying or selecting TCR. TCR libraries can be produced by amplifying Vα and Vβ libraries from T cells, which are isolated from subjects, including cells present in PBMC, spleen or other lymphoid organs. In some cases, T cells can be amplified from tumor infiltrating lymphocytes (TIL). In some embodiments, TCR libraries can be produced from CD4+ or CD8+ cells. In some embodiments, TCR can be amplified from the T cell sources of normal or healthy subjects, i.e., normal TCR libraries. In some embodiments, TCR can be amplified from the T cell sources of diseased subjects, i.e., diseased TCR libraries. In some embodiments, degenerate primers are used to amplify the gene pool of Vα and Vβ, such as by performing RT-PCR in samples (such as T cells) obtained from people. In some embodiments, scTv libraries can be assembled from original Vα and Vβ libraries, wherein the amplified products are cloned or assembled to be separated by joints. Depending on the source of the subject and the cell, the library can be HLA allele-specific. Alternatively, in some embodiments, TCR library can be produced by mutagenesis or diversification of parent or scaffold TCR molecules. In some aspects, TCR is subjected to directed evolution, such as by mutagenesis such as described α or β chain. In some aspects, the specific residues in the CDR of TCR are changed. In some embodiments, the TCR selected can be modified by affinity maturation. In some embodiments, antigen-specific T cells can be selected, such as by screening to assess the CTL activity for described peptide. In some aspects, the TCR for example present on antigen-specific T cells can be selected, such as by binding activity, such as specific affinity or avidity to described antigen.

[0312] In some embodiments, the antigen receptor of genetic engineering includes recombinant T cell receptor (TCR) and / or TCR cloned from naturally occurring T cells. In some embodiments, the high-affinity T cell clone of target antigen (for example, cancer antigen) is identified and separated from the patient, and introduced into the cell. In some embodiments, TCR clones for target antigen have been produced in transgenic mice engineered with human immune system genes (for example, human leukocyte antigen system or HLA). See, for example, tumor antigens (see, for example, Parkhurst et al. (2009) Clin Cancer Res.15:169-180 and Cohen et al. (2005) J Immunol.175:5799-5808. In some embodiments, phage display is used to separate TCR for target antigen (see, for example, Varela-Rohena et al. (2008) Nat Med.14:1390-1395 and Li (2005) Nat Biotechnol.23:349-354.

[0313] In some embodiments, TCR or its antigen-binding portion thereof is modified or engineered. In some embodiments, directed evolution methods are used to produce a TCR with a changed characteristic such as a higher affinity for a specific MHC-peptide complex. In some embodiments, directed evolution is achieved by display methods, including but not limited to yeast display (Holler et al., (2003) Nat Immunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci USA, 97, 5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54) or T cell display (Chervin et al. (2008) J Immunol Methods, 339, 175-84). In some embodiments, display approach relates to engineering or modification of known parents or reference TCR. For example, in some cases, a wild-type TCR can be used as a template to generate a mutagenized TCR in which one or more residues of a CDR are mutated, and mutants are selected that have the desired altered property (e.g., higher affinity for the desired target antigen).

[0314] In some embodiments, the peptides for producing or generating the target polypeptide of the target TCR are known or can be easily identified by a skilled person. In some embodiments, peptides suitable for producing TCR or antigen binding portion can be determined based on the presence of HLA restriction motifs in the target polypeptide (target polypeptide as described below). In some embodiments, available computer prediction models are used to identify peptides. In some embodiments, for predicting MHC class I binding sites, such models include but are not limited to ProPred1 (Singh and Raghava (2001) Bioinformatics 17 (12): 1236-1237) and SYFPEITHI (see Schuler et al. (2007) Immunoinformatics Methods in Molecular Biology, 409 (1): 75-93 2007). In some embodiments, the MHC restricted epitope is HLA-A0201, which is expressed in approximately 39%-46% of all Caucasians and therefore represents a suitable choice for preparing the MHC antigens of TCR or other MHC-peptide binding molecules.

[0315] The use of computer prediction models for HLA-A0201 binding motifs and cleavage sites for the proteasome and immunoproteasome is known. Such models for predicting MHC class I binding sites include, but are not limited to, ProPred1 (described in more detail in Singh and Raghava, ProPred: prediction of HLA-DR binding sites. BIOINFORMATICS 17(12): 1236-1237 2001) and SYFPEITHI (see Schuler et al. SYFPEITHI, Database for Searching and T-Cell Epitope Prediction., Immunoinformatics Methods in Molecular Biology, Vol. 409(1): 75-93 2007).

[0316] In some embodiments, the TCR or its antigen-binding portion can be a recombinantly produced natural protein or a mutant form thereof (in which one or more properties (such as binding characteristics) have been altered). In some embodiments, the TCR can be derived from one of various animal species, such as humans, mice, rats, or other mammals. The TCR can be cell-bound or in a soluble form. In some embodiments, for the purposes of the provided methods, the TCR is in a cell-bound form expressed on the surface of a cell.

[0317] In some embodiments, the TCR is a full-length TCR. In some embodiments, the TCR is an antigen-binding portion. In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the TCR is a single-chain TCR (sc-TCR). In some embodiments, the dTCR or scTCR has a structure as described in WO 03 / 020763, WO 04 / 033685, or WO 2011 / 044186.

[0318] In some embodiments, the TCR contains a sequence corresponding to a transmembrane sequence. In some embodiments, the TCR does contain a sequence corresponding to a cytoplasmic sequence. In some embodiments, the TCR is capable of forming a TCR complex with CD3. In some embodiments, any TCR (including dTCR or scTCR) can be connected to a signaling domain that produces an active TCR on the surface of a T cell. In some embodiments, the TCR is expressed on the surface of a cell.

[0319] In some embodiments, dTCR contains a first polypeptide (wherein the sequence corresponding to the TCR α chain variable region sequence is fused to the N-terminus of the sequence corresponding to the TCR α chain constant region extracellular sequence) and a second polypeptide (wherein the sequence corresponding to the TCR β chain variable region sequence is fused to the N-terminus of the sequence corresponding to the TCR β chain constant region extracellular sequence), and the first polypeptide and the second polypeptide are connected by a disulfide bond. In some embodiments, the bond may correspond to the natural interchain disulfide bond present in the natural dimeric α β TCR. In some embodiments, the interchain disulfide bond is not present in the natural TCR. For example, in some embodiments, one or more cysteines can be incorporated into the constant region extracellular sequence of the dTCR polypeptide pair. In some cases, natural and non-natural disulfide bonds may be required. In some embodiments, TCR contains a transmembrane sequence to anchor to the membrane.

[0320] In some embodiments, the dTCR contains a TCR α chain (comprising a variable α domain, a constant α domain, and a first dimerization motif attached to the C-terminus of the constant α domain) and a TCR β chain (comprising a variable β domain, a constant β domain, and a first dimerization motif attached to the C-terminus of the constant β domain), wherein the first and second dimerization motifs are susceptible to interacting to form a covalent bond between amino acids of the first dimerization motif and amino acids of the second dimerization motif, thereby linking the TCR α chain and the TCR β chain together.

[0321] In some embodiments, the TCR is a scTCR. Generally, scTCRs can be generated using known methods, see, for example, Soo Hoo, WF et al. PNAS (USA) 89, 4759 (1992); Wülfing, C. and Plückthun, A., J. Mol. Biol. 242, 655 (1994); Kurucz, I. et al. PNAS (USA) 90 3830 (1993); International Publication Nos. WO96 / 13593, WO 96 / 18105, WO99 / 60120, WO99 / 18129, WO 03 / 020763, WO2011 / 044186; and Schlueter, CJ et al. J. Mol. Biol. 256, 859 (1996). In some embodiments, the scTCR contains an introduced non-native interchain disulfide bond to promote association of the TCR chains (see, e.g., International Publication No. WO 03 / 020763). In some embodiments, the scTCR is a non-disulfide-linked truncated TCR in which a heterologous leucine zipper fused to its C-terminus promotes chain association (see, e.g., International Publication No. WO 99 / 60120). In some embodiments, the scTCR contains a TCR alpha variable domain covalently linked to a TCR beta variable domain via a peptide linker (see, e.g., International Publication No. WO 99 / 18129).

[0322] In some embodiments, the scTCR contains a first segment composed of an amino acid sequence corresponding to the variable region of the TCR α chain, a second segment composed of an amino acid sequence corresponding to the variable region sequence of the TCR β chain (fused to the N-terminus of an amino acid sequence corresponding to the extracellular sequence of the TCR β chain constant domain), and a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.

[0323] In some embodiments, the scTCR contains a first segment consisting of an α chain variable region sequence fused to the N-terminus of the α chain extracellular constant domain sequence and a second segment consisting of a β chain variable region sequence fused to the N-terminus of the β chain extracellular constant and transmembrane sequences and, optionally, a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.

[0324] In some embodiments, the scTCR contains a first segment composed of the TCR β chain variable region sequence fused to the N-terminus of the β chain extracellular constant domain sequence and a second segment composed of the α chain variable region sequence fused to the N-terminus of the α chain extracellular constant and transmembrane sequences and, optionally, a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.

[0325] In some embodiments, the linker connecting the first and second TCR segments of the scTCR can be any linker capable of forming a single polypeptide chain while retaining the TCR binding specificity. In some embodiments, the linker sequence may, for example, have the formula -P-AA-P-, where P is proline and AA represents an amino acid sequence, wherein the amino acids are glycine and serine. In some embodiments, the first segment and the second segment are paired so that their variable region sequences are oriented for such binding. Thus, in some cases, the linker has a length sufficient to span the distance between the C-terminus of the first segment and the N-terminus of the second segment, or vice versa, but cannot be too long to block or reduce the binding of the scTCR to the target ligand. In some embodiments, the linker may contain from or about 10 to 45 amino acids, such as 10 to 30 amino acids or 26 to 41 amino acid residues, for example 29, 30, 31 or 32 amino acids. In some embodiments, the linker has the formula -PGGG-(SGGGG)5-P-, wherein P is proline, G is glycine and S is serine (SEQ ID NO: 28). In some embodiments, the linker has the sequence GSADDAKKDAAKKDGKS (SEQ ID NO: 29)

[0326] In some embodiments, the scTCR contains covalent disulfide bonds linking residues from the immunoglobulin region of the constant domain of the α chain to residues from the immunoglobulin region of the constant domain of the β chain. In some embodiments, interchain disulfide bonds are not present in native TCRs. For example, in some embodiments, one or more cysteines can be incorporated into the constant region extracellular sequences of the first and second segments of the scTCR polypeptide. In some cases, both native and non-native disulfide bonds may be desired.

[0327] In some embodiments of dTCRs or scTCRs containing introduced interchain disulfide bonds, no native disulfide bonds are present. In some embodiments, one or more native cysteines that form native interchain disulfide bonds are substituted for another residue, such as a serine or alanine. In some embodiments, the introduced disulfide bonds can be formed by mutating non-cysteine ​​residues on the first and second segments to cysteine. Exemplary non-native disulfide bonds for TCRs are described in published International PCT No. WO 2006 / 000830.

[0328] In some embodiments, the TCR or antigen-binding fragment thereof exhibits affinity for the target antigen with an equilibrium binding constant of at or between about 10<-5 > and 10<-12 > M and all individual values ​​and ranges therein. In some embodiments, the target antigen is an MHC-peptide complex or ligand.

[0329] In some embodiments, one or more nucleic acids encoding TCRs (such as α and β chains) can be amplified by PCR, cloning, or other suitable methods, and cloned into a suitable expression vector. The expression vector can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and amplification or for expression or for both, such as plasmids and viruses.

[0330] In some embodiments, the vector can be a vector of the following series: pUC series (Fermentas LifeSciences), pBluescript series (Stratagene, La Jolla, California), pET series (Novagen, Madison, Wisconsin), pGEX series (Pharmacia Biotech, Uppsala, Sweden) or pEX series (Clontech, Palo Alto, California). In some cases, phage vectors such as λG10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 can also be used. In some embodiments, plant expression vectors can be used and include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). In some embodiments, animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). In some embodiments, viral vectors are used, such as retroviral vectors.

[0331] In some embodiments, standard recombinant DNA techniques can be used to prepare recombinant expression vectors. In some embodiments, the vector may contain regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacteria, fungi, plants or animals) into which the vector is introduced, and whether the vector is based on DNA or RNA, as appropriate and in consideration of the fact that the vector is based on RNA. In some embodiments, the vector may contain a non-natural promoter, which is operably linked to a nucleotide sequence encoding a TCR or antigen binding portion (or other MHC-peptide binding molecules). In some embodiments, the promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, a SV40 promoter, a RSV promoter, and a promoter found in the long terminal repeat sequence of a mouse stem cell virus. Other known promoters are also contemplated.

[0332] In some embodiments, after obtaining T cell clones, TCR α and β chains are separated and cloned into gene expression vectors. In some embodiments, TCR α and β genes are connected by picornavirus 2A ribosomal jumping peptides so that two chains are co-expressed. In some embodiments, the genetic transfer of TCR is completed by retrovirus or lentiviral vector or by transposon (see, for example, Baum et al. (2006) Molecular Therapy:The Journal of the American Society of Gene Therapy.13:1050-1063; Frecha et al. (2010) Molecular Therapy:The Journal of the American Society of Gene Therapy.18:1748-1757; and Hackett et al. (2010) Molecular Therapy:The Journal of the American Society of Gene Therapy.18:674-683.

[0333] In some embodiments, to generate a vector encoding a TCR, the total cDNA of the α and β chains isolated from a T cell clone expressing the target TCR is PCR amplified and cloned into an expression vector. In some embodiments, the α and β chains are cloned into the same vector. In some embodiments, the α and β chains are cloned into different vectors. In some embodiments, the generated α and β chains are incorporated into a retroviral (e.g., lentiviral) vector.

[0334] 3. Chimeric Autoantibody Receptor (CAAR)

[0335] In some embodiments, the recombinant receptor is a chimeric autoantibody receptor (CAAR). In some embodiments, CAAR is specific for autoantibodies. In some embodiments, cells expressing CAAR (e.g., T cells engineered to express CAAR) can be used to specifically bind to and kill cells expressing autoantibodies, rather than cells expressing normal antibodies. In some embodiments, cells expressing CAAR can be used to treat autoimmune diseases associated with the expression of autoantigens, such as autoimmune diseases. In some embodiments, cells expressing CAAR can target B cells that ultimately produce autoantibodies and display the autoantibodies on their cell surfaces, marking these B cells as disease-specific targets for therapeutic intervention. In some embodiments, CAAR-expressing cells can be used to effectively target and kill pathogenic B cells in autoimmune diseases by targeting disease-causing B cells using antigen-specific chimeric autoantibody receptors. In some embodiments, the recombinant receptor is a CAAR, such as any one described in U.S. Patent Application Publication No. US 2017 / 0051035.

[0336] In some embodiments, the CAAR comprises an autoantibody binding domain, a transmembrane domain, and an intracellular signaling region. In some embodiments, the intracellular signaling region comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling region comprises a secondary or costimulatory signaling region (secondary intracellular signaling region).

[0337] In some embodiments, the autoantibody binding domain comprises an autoantigen or fragment thereof. The choice of autoantigen can depend on the type of autoantibody being targeted. For example, the autoantigen may be selected because it recognizes autoantibodies on target cells (e.g., B cells) associated with a particular disease state (e.g., an autoimmune disease, such as an autoantibody-mediated autoimmune disease). In some embodiments, the autoimmune disease includes pemphigus vulgaris (PV). Exemplary autoantigens include desmoglein 1 (Dsg1) and Dsg3.

[0338] 4. Multi-targeting

[0339] In some embodiments, cell and method include multi-targeting strategy, for example, two or more genetically engineered receptors are expressed on a cell, each receptor recognizes the same or different antigens, and generally each includes different intracellular signaling components. Such multi-targeting strategy is described in, for example, the following documents: International Patent Application Publication No. WO 2014055668A1 (description of the combination of activation and costimulation CAR, for example, targeting is present alone on off-target (such as normal cells), but only present together on two different antigens on the cell of the disease to be treated or illness) and Fedorov et al., Sci.Transl.Medicine, 5 (215) (December 2013) (describing expression of activation and inhibitory CAR cells, for example, wherein activation CAR is bound to a kind of antigen expressed on the cell of normal or non-diseased cells and disease to be treated or illness simultaneously, and inhibitory CAR is bound to another kind of antigen expressed only on normal cells or cells not wishing to treat).

[0340] For example, in some embodiments, the cell includes a receptor expressing a first genetically engineered antigen receptor (for example, CAR or TCR), which is typically after specifically binding to an antigen (such as a first antigen) identified by a first receptor, can induce activation or stimulation signals to the cell. In some embodiments, the cell also includes a second genetically engineered antigen receptor (for example, CAR or TCR), such as a chimeric costimulatory receptor, which is typically after specifically binding to a second antigen identified by a second receptor, can induce costimulatory signals to immune cells. In some embodiments, the first antigen is identical to the second antigen. In some embodiments, the first antigen is different from the second antigen.

[0341] In some embodiments, the first and / or second genetically engineered antigen receptor (such as CAR or TCR) can induce activation or stimulation signal to the cell.In some embodiments, receptor includes intracellular signaling components containing ITAM or ITAM-like motifs.In some embodiments, the activation induced by the first receptor is related to the change of protein expression in signal transduction or cells, resulting in starting immune response (such as ITAM phosphorylation) and / or starting the signal transduction cascade mediated by ITAM, forming immune synapse and / or the clustering of molecules near the bound receptor (such as CD4 or CD8, etc.), activating one or more transcription factors (such as NF-κB and / or AP-1) and / or inducing factors (such as cytokines) gene expression, proliferation and / or survival.

[0342] In some embodiments, the first receptor and / or the second receptor include an intracellular signaling domain or region of a costimulatory receptor, such as CD28, CD137 (4-1BB), OX40, and / or ICOS. In some embodiments, the first receptor and the second receptor include intracellular signaling domains of different costimulatory receptors. In one embodiment, the first receptor contains a CD28 costimulatory signaling region, and the second receptor contains a 4-1BB costimulatory signaling region, or vice versa.

[0343] In some embodiments, the first receptor and / or the second receptor comprises an intracellular signaling domain containing an ITAM or ITAM-like motif and an intracellular signaling domain of a co-stimulatory receptor.

[0344] In some embodiments, the first receptor contains an intracellular signaling domain comprising an ITAM or ITAM-like motif, and the second receptor contains an intracellular signaling domain of a co-stimulatory receptor. The co-stimulatory signal combined with the activation or stimulatory signal induced in the same cell is a co-stimulatory signal that results in an immune response, such as a robust and sustained immune response, such as increased gene expression, secretion of cytokines and other factors, and T cell-mediated effector functions (such as cell killing).

[0345] In some embodiments, the connection of the independent first receptor and the connection of the independent second receptor will not induce a robust immune response. In some aspects, if only one receptor is connected, the cell becomes tolerant to the antigen or is unresponsive to the antigen, or is suppressed, and / or is not induced to proliferate or secrete factors or realize effector functions. However, in some such embodiments, when multiple receptors are connected, as when encountering cells expressing the first and second antigens, desired responses are achieved, such as complete immune activation or stimulation, such as indicated by secretion, proliferation, persistence and / or execution of immune effector functions (such as cytotoxic killing of target cells) by one or more cytokines.

[0346] In some embodiments, the two receptors induce activation and inhibitory signals to the cell, respectively, such that binding of one receptor to its antigen activates the cell or induces a response, but binding of the second inhibitory receptor to its antigen induces a signal that inhibits or attenuates the response. An example is a combination of an activating CAR with an inhibitory CAR or iCAR. For example, a strategy can be used in which the activating CAR binds to an antigen that is expressed in a disease or disorder but is also expressed on normal cells, and the inhibitory receptor binds to a separate antigen that is expressed on normal cells but not on cells of the disease or disorder.

[0347] In some embodiments, multi-targeting strategies are used in situations where an antigen associated with a particular disease or condition is expressed on non-diseased cells and / or on the engineered cells themselves, either transiently (e.g., after stimulation associated with genetic engineering) or permanently. In such situations, specificity, selectivity, and / or efficacy may be improved by requiring the connection of two separate and individually specific antigen receptors.

[0348] In some embodiments, multiple antigens (e.g., the first and second antigens) are expressed on targeted cells, tissues, or diseases or conditions (e.g., on cancer cells). In some aspects, cells, tissues, diseases, or conditions are multiple myeloma or multiple myeloma cells. In some embodiments, one or more of the multiple antigens are typically also expressed on cells (e.g., normal or non-diseased cells or tissues, and / or engineered cells themselves) that do not need to be targeted with cell therapy. In such embodiments, specificity and / or efficacy are achieved by needing to connect multiple receptors to achieve the response of the cell.

[0349] C. Nucleic Acids and Vectors

[0350] Also provided are one or more polynucleotides (e.g., nucleic acid molecules) encoding PSMA or a modified form thereof and / or a recombinant receptor, vectors for genetically engineering cells to express such PSMA or a modified form thereof and receptor, and methods for producing engineered cells.

[0351] In some embodiments, polynucleotides encoding any PSMA or modified forms thereof described herein are provided. In some aspects, the polynucleotide contains a single coding sequence, such as a coding sequence encoding only PSMA or a modified form thereof. In other cases, the polynucleotide contains at least two different coding sequences, such as a first nucleic acid sequence encoding PSMA or a modified form thereof and a second nucleic acid sequence encoding a recombinant receptor. In some aspects, the recombinant receptor is or contains a chimeric antigen receptor (CAR). In some aspects, the recombinant receptor is or contains a T cell receptor (TCR), such as a transgenic TCR. In some embodiments, the polynucleotide and vector are used to co-express PSMA or a modified form thereof and the recombinant receptor in a cell.

[0352] Also provided are groups or combinations of polynucleotides. In some embodiments, the group or combination comprises a first polynucleotide comprising a nucleic acid encoding prostate-specific membrane antigen (PSMA) or a modified form thereof, and a second polynucleotide comprising a nucleic acid encoding a recombinant receptor. Compositions containing such groups or combinations of polynucleotides are also provided. In some embodiments, the group or combination of polynucleotides is used together to engineer a cell. In some embodiments, the first and second polynucleotides in the group are introduced simultaneously or sequentially in any order into the cell being engineered.

[0353] In some embodiments, provided polynucleotides containing nucleic acids encoding PSMA or a modified form thereof include the nucleic acid sequence set forth in SEQ ID NO: 26, 27, or 53, or a nucleic acid sequence that exhibits at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NO: 26, 27, or 53, or a fragment thereof.

[0354] In some cases, the nucleic acid sequence encoding PSMA or its modified form contains a signal sequence encoding a signal peptide. In some aspects, the signal sequence can encode a signal peptide derived from a natural polypeptide. In other aspects, the signal sequence can encode a heterologous or non-natural signal peptide, such as an exemplary signal peptide of the GMCSFRα chain shown in SEQ ID NO: 31 and encoded by the nucleotide sequence shown in SEQ ID NO: 30. In some cases, the nucleic acid sequence encoding a recombinant receptor (e.g., a chimeric antigen receptor (CAR)) contains a signal sequence encoding a signal peptide. Non-limiting exemplary examples of signal peptides include, for example, a GMCSFRα chain signal peptide shown in SEQ ID NO: 31 and encoded by the nucleotide sequence shown in SEQ ID NO: 30, or a CD8α signal peptide shown in SEQ ID NO: 32.

[0355] In some embodiments, the polynucleotide encoding PSMA or a modified form thereof and / or a recombinant receptor contains at least one promoter operably linked to control the expression of PSMA or a modified form thereof and / or a recombinant receptor. In some examples, the polynucleotide contains two, three, or more promoters operably linked to control the expression of PSMA or a modified form thereof and / or a recombinant receptor.

[0356] In certain cases where a nucleic acid molecule encodes two or more different polypeptide chains, each of the polypeptide chains can be encoded by a separate nucleic acid molecule. For example, two separate nucleic acids are provided, and each can be transferred or introduced into a cell separately for expression in the cell.

[0357] In some embodiments, the nucleic acid encoding the recombinant receptor and the nucleic acid encoding PSMA or a modified form thereof are operably linked to the same promoter and are optionally separated by an internal ribosome entry site (IRES) or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping (which is optionally T2A, P2A, E2A, or F2A). In some embodiments, the nucleic acid encoding PSMA or a modified form thereof and the nucleic acid encoding the recombinant receptor are operably linked to two different promoters. In some embodiments, the nucleic acid encoding PSMA or a modified form thereof and the nucleic acid encoding the recombinant receptor are present or inserted at different locations within the cellular genome. In some embodiments, the polynucleotide encoding the recombinant receptor is introduced into a composition containing cultured cells, for example, by retroviral transduction, transfection, or transformation.

[0358] In some embodiments, such as those in which the polynucleotide comprises a first and a second nucleic acid sequence, the coding sequence encoding each different polypeptide chain can be operably linked to a promoter, which can be the same or different. In some embodiments, the nucleic acid molecule can contain a promoter that drives expression of two or more different polypeptide chains. In some embodiments, such nucleic acid molecules can be polycistronic (bicistronic or tricistronic, see, for example, U.S. Patent No. 6,060,273). In some embodiments, the transcription unit can be engineered as a bicistronic unit containing an IRES (internal ribosome entry site), which allows for co-expression of gene products (e.g., encoding PSMA or a modified form thereof and encoding a recombinant receptor) from a single promoter. Alternatively, in some cases, a single promoter can direct the expression of RNA containing two or three genes (e.g., encoding PSMA or a modified form thereof and encoding a recombinant receptor) in a single open reading frame (ORF), the genes separated from each other by a sequence encoding a self-cleaving peptide (e.g., a 2A sequence) or a protease recognition site (e.g., furin). Thus, the ORF encodes a single polypeptide that is processed into a single protein during translation (in the case of 2A) or thereafter. In some cases, peptides (such as T2A) can cause the ribosome to skip the synthesis of the peptide bond at the C-terminus of the 2A element (ribosome skipping), resulting in a separation between the end of the 2A sequence and the adjacent downstream peptide (see, e.g., de Felipe, Genetic Vaccines and Ther. 2:13 (2004) and de Felipe et al. Traffic 5:616-626 (2004)). Various 2A elements are known. Examples of 2A sequences that can be used in the methods and systems disclosed herein include, but are not limited to, 2A sequences from the following viruses: foot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 22), equine rhinitis A virus (E2A, e.g., SEQ ID NO: 21), Tetrasomy tadalafil betavirus (T2A, e.g., SEQ ID NO: 6 or 17), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO: 19 or 20), as described in U.S. Patent Publication No. 20070116690.

[0359] In some embodiments, the nucleic acid encoding PSMA or a modified form thereof and the nucleic acid encoding a recombinant receptor are operably linked to the same promoter and are optionally separated by an internal ribosome entry site (IRES) or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping (which is optionally T2A, P2A, E2A, or F2A). In some embodiments, the nucleic acid encoding PSMA or a modified form thereof and the nucleic acid encoding a recombinant receptor are operably linked to two different promoters. In some embodiments, the nucleic acid encoding PSMA or a modified form thereof and the nucleic acid encoding a recombinant receptor are present or inserted at different locations within the genome of the cell.

[0360] In some embodiments, polynucleotides are also provided, comprising a nucleic acid sequence encoding any PSMA or a modified form thereof and a nucleic acid sequence encoding any chimeric receptor and / or recombinant antigen receptor (e.g., a CAR provided herein). In some aspects, the nucleic acid encoding PSMA or a modified form thereof and the nucleic acid encoding CAR are contained within one polynucleotide. In some embodiments, the polynucleotides provided comprise, in 5' to 3' order: i) a nucleic acid encoding a signal peptide; ii) a nucleic acid encoding a CAR comprising an scFv; a spacer; a transmembrane domain; an intracellular region comprising a costimulatory signaling region, and an intracellular signaling domain of the CD3-zeta (CD3ζ) chain, or a signaling portion thereof; iii) a nucleic acid sequence encoding a self-cleaving peptide or a peptide that causes ribosome skipping (which is optionally T2A, P2A, E2A, or F2A); and iv) a nucleic acid encoding a PSMA or a modified form thereof, optionally comprising the amino acid sequence set forth in SEQ ID NO: 52, or a fragment thereof; or an amino acid sequence that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 52, or a fragment thereof, and comprising a deletion of said one or more N-terminal amino acid residues.

[0361] In some embodiments, the encoded CAR comprises:

[0362] (i) an scFv specific for an antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule which is optionally or optionally comprises 4-1BB, and a cytoplasmic signaling domain derived from a molecule containing a primary signaling ITAM which is optionally or comprises a CD3 zeta signaling domain and optionally further comprises a spacer between the transmembrane domain and the scFv, in order;

[0363] (ii) in order, a scFv specific for an antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule which is or optionally comprises a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from a molecule containing a primary signaling ITAM, which is optionally a CD3 zeta signaling domain; or

[0364] (iii) in order, a scFv specific for an antigen, a spacer, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule which is optionally a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from a molecule containing a primary signaling ITAM (which is optionally or optionally comprises a CD3 zeta signaling domain); and:

[0365] The spacer is optionally a polypeptide spacer that (a) comprises or consists of all or part of an immunoglobulin hinge, or a modified form thereof, or comprises about 15 amino acids or less and does not comprise the extracellular region of CD28 or CD8, (b) comprises or consists of all or part of an immunoglobulin hinge (optionally IgG4), or a modified form thereof, and / or comprises about 15 amino acids or less and does not comprise the extracellular region of CD28 or CD8, or (c) is 12 amino acids or about 12 amino acids in length and / or comprises or consists of all or part of an immunoglobulin hinge (optionally IgG4), or a modified form thereof; or (d) has or consists of the sequence of SEQ ID NO: 1, which is a sequence encoded by SEQ ID NO: 2, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 NO:58 or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of the foregoing, or (e) comprises or consists of the formula X1PPX2P (SEQ ID NO:66), wherein X1 is glycine, cysteine ​​or arginine and X2 is cysteine ​​or threonine; and / or

[0366] the costimulatory domain comprises SEQ ID NO: 12 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12; and / or

[0367] The primary signaling domain comprises SEQ ID NO: 13 or 14 or 15 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto; and / or

[0368] The scFv comprises the CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 59), the CDRL2 sequence of SRLHSGV (SEQ ID NO: 60) and / or the CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 61) and / or the CDRH1 sequence of DYGVS (SEQ ID NO: 62), the CDRH2 sequence of VIWGSETTYYNSALKS (SEQ ID NO: 63) and / or the CDRH3 sequence of YAMDYWG (SEQ ID NO: 64). NO:64), or wherein the scFv comprises the variable heavy chain region of FMC63 and the variable light chain region of FMC63 and / or the CDRL1 sequence of FMC63, the CDRL2 sequence of FMC63, the CDRL3 sequence of FMC63, the CDRH1 sequence of FMC63, the CDRH2 sequence of FMC63 and the CDRH3 sequence of FMC63, or binds to the same epitope as any one of the foregoing or competes with the binding of any one of the foregoing, and optionally wherein the scFv comprises the variable heavy chain region of FMC63 and the variable light chain region of FMC63 and / or the CDRL1 sequence of FMC63, the CDRL2 sequence of FMC63, the CDRL3 sequence of FMC63, the CDRH1 sequence of FMC63, the CDRH2 sequence of FMC63 and the CDRH3 sequence of FMC63 H , optionally comprising a linker of SEQ ID NO: 65 and V L , and / or the scFv comprises a flexible linker and / or comprises the amino acid sequence shown in SEQ ID NO: 65.

[0369] In some embodiments, a polynucleotide encoding PSMA or a modified form thereof and / or a recombinant receptor is introduced into a composition containing cultured cells, for example, by retroviral transduction, transfection, or transformation.

[0370] Also provided are vectors or constructs containing such nucleic acids and / or polynucleotides. In some embodiments, the vector or construct contains one or more promoters that are operably linked to a nucleic acid encoding PSMA or a modified form thereof and / or a recombinant receptor to drive its expression. In some embodiments, the promoter is operably linked to one or more nucleic acid molecules or polynucleotides. Thus, vectors are also provided, such as those containing any polynucleotide provided herein. In some embodiments, the vector includes a first polynucleotide encoding PSMA or a modified form thereof and a second polynucleotide encoding a recombinant receptor (e.g., CAR).

[0371] In some cases, the vector is a viral vector, such as a retroviral vector, for example, a lentiviral vector or a γ-retroviral vector. A group or combination of vectors is also provided. In some embodiments, the group or combination of vectors comprises a first vector and a second vector, wherein the first vector comprises a first polynucleotide (e.g., a first polynucleotide encoding PSMA or a modified form thereof), and the second vector comprises a second polynucleotide encoding a recombinant receptor (e.g., CAR). A composition containing such a group or combination of vectors is also provided. In some embodiments, the group or combination of vectors is used together for the engineering of cells. In some embodiments, the first vector and the second vector in the group are introduced into the cells for engineering simultaneously or in any order.

[0372] In some embodiments, the vector includes a viral vector, such as a retrovirus or lentivirus, a non-viral vector or a transposon, such as the Sleeping Beauty transposon system; a vector derived from Simian Virus 40 (SV40), an adenovirus, an adeno-associated virus (AAV); a lentiviral vector or a retroviral vector, such as a γ-retroviral vector, a retroviral vector derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV) or adeno-associated virus (AAV).

[0373] Any PSMA or its modified form and / or recombinant receptor described herein can be encoded by a polynucleotide containing one or more nucleic acid sequences encoding PSMA or its modified form and / or recombinant receptor in any combination or arrangement. For example, one, two, three or more polynucleotides can encode one, two, three or more different polypeptides, such as PSMA or its modified form and / or recombinant receptor. In some embodiments, one vector or construct contains a nucleic acid sequence encoding PSMA or its modified form, and a separate vector or construct contains a nucleic acid sequence encoding a recombinant receptor (e.g., CAR). In some embodiments, the nucleic acid encoding PSMA or its modified form and the nucleic acid encoding the recombinant receptor are operably linked to two different promoters. In some embodiments, the nucleic acid encoding the recombinant receptor is present downstream of the nucleic acid encoding PSMA or its modified form.

[0374] D. Cells and Cell Preparation for Engineering

[0375] Provided herein are cells, such as engineered cells containing PSMA or a modified form thereof and / or a recombinant receptor. Also provided are populations of such cells, compositions containing such cells and / or enriched for such cells, for example, wherein cells expressing PSMA or a modified form thereof and / or a recombinant receptor (e.g., a chimeric receptor) constitute at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the total cells or a certain type of cell (e.g., T cells or CD8+ or CD4+ cells) in the composition. Compositions include pharmaceutical compositions and formulations for administration (e.g., for adoptive cell therapy). Also provided are methods for engineering, producing or generating such cells, methods for administering cells and compositions to subjects (e.g., patients), and methods for detecting, selecting, isolating or separating such cells.

[0376] Therefore, there is provided a genetically engineered cell expressing PSMA or its modified form and / or recombinant receptor (such as CAR).Cells are typically eukaryotic cells (such as mammalian cells), and are typically human cells.In some embodiments, the cell is derived from blood, bone marrow, lymph or lymphoid organs, and is a cell of the immune system, such as a cell of innate immunity or adaptive immunity, such as bone marrow or lymphocytes, including lymphocytes, typically T cells and / or NK cells.Other exemplary cells include stem cells, such as pluripotent stem cells and multipotent stem cells, including induced pluripotent stem cells (iPSC).Cells are typically primary cells, such as those directly separated from a subject and / or separated and frozen from a subject.In some embodiments, cells include one or more subgroups of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells and subgroups thereof, such as those defined by the following: function, activation state, maturity, the possibility of differentiation, amplification, recycling, positioning and / or lasting ability, antigen specificity, antigen receptor type, presence in a particular organ or compartment, markers or cytokine secretion characteristics and / or degree of differentiation. With respect to the subject to be treated, the cells may be allogeneic and / or autologous. The methods include existing methods. In some aspects, as with the prior art, the cells are pluripotent and / or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods comprise isolating cells from a subject, preparing, processing, culturing, and / or engineering them as described herein, and reintroducing them into the same patient before or after cryopreservation. In some embodiments, the engineered cells are not cells that typically or normally express PSMA. In some embodiments, the engineered cells are not cells whose expression of PSMA increases during a disease state. In some embodiments, the cells are not prostate cells. In some embodiments, the cells do not express PSMA prior to being engineered by introducing a recombinant or heterologous nucleic acid sequence encoding PSMA or a variant thereof.

[0377] Among the subtypes and subsets of T cells and / or CD4+ and / or CD8+ T cells, there are naive T (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes (such as stem cell memory T (T SCM ), central memory T(T CM ), effect memory T(T EM) or terminally differentiated effector memory T cells), tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells (such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper cell T cells), α / β T cells and δ / γ T cells. In some embodiments, the cell is a regulatory T cell (Treg). In some embodiments, the cell further comprises recombinant FOXP3 or a variant thereof.

[0378] In some embodiments, the cell is a natural killer (NK) cell.In some embodiments, the cell is a monocyte or granulocyte, such as a bone marrow cell, a macrophage, a neutrophil, a dendritic cell, a mast cell, an eosinophil and / or a basophil.

[0379] In some embodiments, the cell includes one or more nucleic acids introduced via genetic engineering, thereby expressing a recombinant or genetically engineered product of such nucleic acid. In some embodiments, the nucleic acid is heterologous, i.e., not normally present in the cell or a sample obtained from the cell, such as a nucleic acid obtained from another organism or cell, for example, the nucleic acid is not normally found in the engineered cell and / or the organism from which such cell is derived. In some embodiments, the nucleic acid is not naturally occurring, such as a nucleic acid not found in nature, including nucleic acids comprising chimeric combinations of nucleic acids encoding various domains from a variety of different cell types.

[0380] In some embodiments, the preparation of engineered cells includes one or more cultivation and / or preparation steps. The cell for engineering can be separated from a sample (such as a biological sample, for example, obtained from or derived from a subject). In some embodiments, the subject is isolated from a cell and is suffering from a disease or illness or needs a cell therapy or will be given a cell therapy. In some embodiments, the subject is a person who needs a specific therapeutic intervention (such as adoptive cell therapy, in which cells are separated, processed and / or engineered).

[0381] Therefore, in some embodiments, the cell is a primary cell, such as a primary human cell. Samples include tissues, fluids and other samples taken directly from the subject, as well as samples from one or more processing steps (such as separation, centrifugation, genetic engineering (such as transduction with a viral vector), washing and / or incubation). Biological samples can be samples obtained directly from a biological source or processed samples. Biological samples include, but are not limited to, body fluids (such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat), tissue and organ samples, including processed samples derived therefrom.

[0382] In some aspects, the sample from which the cell is derived or separated is a sample derived from blood or blood, or is or is derived from apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMC), leukocytes, bone marrow, thymus, biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix uteri, testis, ovary, tonsil or other organs and / or cells derived therefrom. In the context of cell therapy (such as adoptive cell therapy), sample includes samples from autologous and allogeneic sources.

[0383] In some embodiments, the cells are derived from a cell line, such as a T cell line. In some embodiments, the cells are obtained from a xenogeneic source, such as from a mouse, rat, non-human primate, or pig.

[0384] In some embodiments, the separation of cells includes one or more preparation and / or non-affinity-based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example to remove unwanted components, enrich for desired components, crack, or remove cells that are sensitive to specific reagents. In some examples, cells are separated based on one or more characteristics (such as density, adhesion properties, size, sensitivity to specific components, and / or resistance).

[0385] In some instances, cells from the subject's circulating blood are obtained, for example, by apheresis or leukapheresis. In some aspects, the sample contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and / or platelets, and in some aspects contains cells other than red blood cells and platelets.

[0386] In some embodiments, the blood cells collected from the experimenter are washed, for example, to remove the plasma fraction and the cells are placed in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the washing solution lacks calcium and / or magnesium and / or many or all divalent cations. In some aspects, according to the manufacturer's instructions, washing steps are completed by a semi-automatic " flow-through " centrifuge (for example, Cobe 2991 cell processor, Baxter). In some aspects, according to the manufacturer's instructions, washing steps are completed by tangential flow filtration (TFF). In some embodiments, after washing, the cells are resuspended in a variety of biocompatible buffers (for example, as a precipitant containing Ca). ++ / Mg ++ In certain embodiments, the components of the blood cell sample are removed and the cells are resuspended directly in culture medium.

[0387] In some embodiments, the methods include density-based cell separation methods, such as preparing white blood cells from peripheral blood by lysing red blood cells and centrifuging through Percoll or Ficoll gradients.

[0388] In some embodiments, separation method comprises the expression or existence of one or more specific molecules (such as surface markers, for example surface proteins, intracellular markers or nucleic acids) based on the cell to separate different cell types.In some embodiments, any known method for separating based on this type of mark can be used.In some embodiments, separation is the separation based on affinity or immunoaffinity.For example, in some respects, separation comprises the expression or expression level of one or more marks (generally cell surface markers) based on the cell to separate cells and cell mass, for example, by and with the antibody or binding partner specifically bound to this type of mark, then typically washing steps and from those cells not bound to the antibody or binding partner, separation of the cells in conjunction with the antibody or binding partner.

[0389] Such separation steps can be based on positive selection (where cells that have bound the reagent are retained for further use) and / or negative selection (where cells that have not bound the antibody or binding partner are retained). In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful in cases where there are no antibodies available to specifically identify cell types in a heterogeneous population, making it best to perform separation based on markers expressed by cells other than the desired population.

[0390] Separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell (such as those expressing a marker) refers to increasing the number or percentage of such cells, but need not result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal or depletion of a particular type of cell (such as those expressing a marker) refers to reducing the number or percentage of such cells, but need not result in the complete removal of all such cells.

[0391] In some examples, multiple rounds of separation steps are performed, wherein the positive or negatively selected fractions from one step are subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can simultaneously deplete cells expressing multiple markers, such as by incubating the cells with multiple antibodies or binding partners, each specific for the marker being targeted for negative selection. Similarly, multiple cell types can be positively selected simultaneously by incubating the cells with multiple antibodies or binding partners expressed on various cell types.

[0392] For example, in some aspects, a specific subpopulation of T cells, such as cells that are positive for or express high levels of one or more surface markers (e.g., CD28 + 、CD62L + 、CCR7 + 、CD27 + 、CD127 + 、CD4 + 、CD8 + 、CD45RA + and / or CD45RO + T cells) are isolated by positive or negative selection techniques.

[0393] For example, anti-CD3 / anti-CD28 conjugated magnetic beads (e.g., M-450 CD3 / CD28 T Cell Expander) positively selects CD3 + ,CD28 + T cells.

[0394] In some embodiments, isolation is performed by enriching for a particular cell population by positive selection or depleting for a particular cell population by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agents that bind to a marker expressed (marked) on the positively or negatively selected cells, respectively. + ) or expressed at relatively high levels (marker 高 ) specifically binds to one or more surface markers of the cell.

[0395] In some embodiments, T cells are separated from the PBMC sample by negative selection for markers expressed on non-T cells (e.g., B cells, monocytes, or other leukocytes, such as CD14). In some aspects, CD4 + or CD8 + Selection step for isolation of CD4 + Helper T cells and CD8 + Cytotoxic T cells. Such CD4 T cells can be isolated by positive or negative selection for markers expressed or expressed at relatively high levels on one or more naive, memory and / or effector T cell subsets. + and CD8 + The populations were further classified into subpopulations.

[0396] In some embodiments, CD8 + The cells are further enriched or depleted for naive, central memory, effector memory and / or central memory stem cells. In some embodiments, the cells are further enriched or depleted for central memory T cells (TCM ) cells to increase efficacy, such as to improve long-term survival, expansion and / or engraftment after administration, which in some aspects is particularly robust in such subpopulations. See Terakura et al. (2012) Blood. 1: 72-82; Wang et al. (2012) J Immunother. 35(9): 689-701. In some embodiments, the combination is enriched for T CM CD8 + T cells and CD4 + T cells further enhance efficacy.

[0397] In one embodiment, memory T cells are present in CD8 + CD62L on peripheral blood lymphocytes + and CD62L - PBMCs can be targeted against CD62L, for example, using anti-CD8 and anti-CD62L antibodies. - CD8 + and / or CD62L + CD8 + Fractions are enriched or depleted.

[0398] In some embodiments, the central memory T (T CM In some aspects, enrichment of T cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD 127; in some aspects, it is based on negative selection of cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, enrichment of T cells is performed by depletion of cells expressing CD4, CD14, CD45RA and positive selection or enrichment of cells expressing CD62L. CM CD8 + In one aspect, the central memory T (T CM ) cells are enriched starting with a negative cell fraction selected based on CD4 expression, negatively selected based on the expression of CD14 and CD45RA and positively selected based on CD62L. In some aspects, this selection is performed simultaneously, and in other aspects, sequentially in any order. In some aspects, the preparation of CD8 + The same selection steps based on CD4 expression of cell populations or subpopulations are also used to generate CD4 + Cell populations or subpopulations are prepared such that positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the method, optionally after one or more further positive or negative selection steps.

[0399] In certain examples, a PBMC sample or other leukocyte sample is subjected to selection for CD4+ cells, wherein negative and positive fractions are retained. The negative fraction is then subjected to negative selection based on expression of CD14 and CD45RA or CD19, and positive selection based on markers characteristic of central memory T cells (such as CD62L or CCR7), wherein the positive and negative selections are performed in any order.

[0400] CD4+ T helper cells are classified into naive, central memory, and effector cells by identifying cell populations that display cell surface antigens. + Lymphocytes can be obtained by standard methods. In some embodiments, naive CD4 + T lymphocytes are CD45RO-, CD45RA + 、CD62L + 、CD4 + In some embodiments, central memory CD4 + cells are CD62L + CD45RO + In some embodiments, effector CD4 + cells are CD62L - CD45RO - .

[0401] In one example, to enrich for CD4 + Cells, monoclonal antibody cocktails typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibodies or binding partners are bound to a solid support or matrix (such as magnetic beads or paramagnetic beads) to allow cell separation for positive and / or negative selection. For example, in some embodiments, immunomagnetic (or affinity magnetic) separation techniques are used to separate or isolate cells and cell populations (reviewed in Methods in Molecular Medicine, Vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In vitro and In vivo, pp. 17-25, SA Brooks and U. Schumacher, eds. Humana Press Inc., Totowa, New Jersey).

[0402] In some aspects, a sample or composition of cells to be separated is incubated with a small magnetizable or magnetically responsive material, such as a magnetically responsive particle or microparticle, such as paramagnetic beads (e.g., such as Dynal beads or MACS beads). The magnetically responsive material (e.g., particle) is typically attached directly or indirectly to a binding partner (e.g., an antibody) that specifically binds to a molecule (e.g., a surface marker) present on a cell, a plurality of cells, or a population of cells that one wishes to separate (e.g., for which one wishes to negatively or positively select).

[0403] In some embodiments, the magnetic particles or beads include magnetically responsive materials that are combined with specific binding members (such as antibodies or other binding partners). There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles are included in those described in Molday, U.S. Patent No. 4,452,773 and European Patent Specification EP 452342 B (which are hereby incorporated by reference). Particles of colloid size, such as those described in Owen U.S. Patent No. 4,795,698 and Liberti et al., U.S. Patent No. 5,200,084 are other examples.

[0404] The incubation is typically performed under conditions such that the antibody or binding partner, or a molecule that specifically binds to such an antibody or binding partner attached to the magnetic particles or beads (such as a secondary antibody or other reagent), specifically binds to the cell surface molecule, if present on cells within the sample.

[0405] In some aspects, the sample is placed in a magnetic field, and those cells with magnetic responses or magnetizable particles attached thereto are attracted to the magnet and separated from the unlabeled cells. For positive selection, cells attracted by the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selections is performed during the same selection step, wherein the positive and negative fractions are retained and further processed or subjected to another separation step.

[0406] In certain embodiments, magnetic responsive particles are coated in a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme or streptavidin. In certain embodiments, magnetic particles are attached to cells by coating with a primary antibody specific for one or more markers. In certain embodiments, the cells are labeled with a primary antibody or a binding partner instead of beads, and then magnetic particles coated with a cell type specific secondary antibody or other binding partner (e.g., streptavidin) are added. In certain embodiments, streptavidin-coated magnetic particles are used in combination with a biotinylated primary antibody or a secondary antibody.

[0407] In some embodiments, the magnetically responsive particles remain attached to the cells, which are subsequently incubated, cultured, and / or engineered; in some aspects, the particles remain attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of competing unlabeled antibodies, magnetizable particles, or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0408] In some embodiments, affinity-based selection is via magnetic activated cell sorting (MACS) (Miltenyi Biotec, Auburn, California). Magnetic activated cell sorting (MACS) systems can select cells attached to magnetized particles in high purity. In certain embodiments, MACS operates in such a mode that non-target and target species are eluted sequentially after applying an external magnetic field. That is, the cells attached to the magnetized particles remain in place, and the unattached species are eluted. Then, after completing the first elution step, the species that are trapped in the magnetic field and prevented from eluting are released in a certain manner so that they can be eluted and recovered. In some aspects, the non-target cells are labeled and exhausted from a heterogeneous cell population.

[0409] In certain embodiments, use such system, device or equipment to separate or separate, described system, device or equipment carries out one or more of separation, cell preparation, separation, processing, hatching, cultivation and / or preparation step of described method.In some aspects, described system is used for carrying out each in these steps in closed or sterile environment, for example, to minimize error, user operation and / or pollution.In one example, described system is the system as described in PCT publication number WO 2009 / 072003 or US 20110003380A1.

[0410] In some embodiments, the system or device performs one or more (e.g., all) of the separation, processing, engineering, and formulation steps in an integrated or standalone system and / or in an automatic or programmable manner. In some aspects, the system or device includes a computer and / or computer program in communication with the system or device that allows a user to program, control, evaluate results of, and / or adjust various aspects of the processing, separation, engineering, and formulation steps.

[0411] In some respects, CliniMACS system (Miltenyi Biotec) is used to carry out described separation and / or other steps, for example, for being used for in closed and aseptic system on clinical scale level automatic separation of cells.Parts can comprise integrated microcomputer, magnetic separation unit, peristaltic pump and various pinch valves.In some respects, all parts of described instrument of integrated computer control and instruct described system to carry out repetitive program with standardized sequence.In some respects, magnetic separation unit comprises removable permanent magnet and the support for selecting post.Peristaltic pump controls the flow velocity of whole tube group, and guarantees together with pinch valve the controlled flow of buffer by described system and the continuous suspension of cell.

[0412] In some respects, the CliniMACS system uses the magnetizable particles of antibody coupling, which are provided in a sterile, pyrogen-free solution. In some embodiments, after labeling cells with magnetic particles, the cells are washed to remove excessive particles. The cell preparation bag is then connected to the tube set, which is in turn connected to the bag and cell collection bag containing buffer. The tube set is composed of pre-assembled sterile tubes (comprising pre-column and separation column) and is only for disposable use. After starting the separation procedure, the system automatically applies the cell sample to the separation column. The labeled cells remain in the column, and unlabeled cells are removed by a series of washing steps. In some embodiments, the cell mass used for use with the method described herein is unlabeled and is not retained in the column. In some embodiments, the cell mass used for use with the method described herein is labeled and retained in the column. In some embodiments, the cell mass used for use with the method described herein is eluted from the column after removing the magnetic field, and is collected in the cell collection bag.

[0413] In certain embodiments, use CliniMACS Prodigy system (Miltenyi Biotec) to separate and / or other steps. In some aspects, the CliniMACS Prodigy system is equipped with a cell processing consortium, which allows washing and fractionation of cells by centrifugal automation. The CliniMACS Prodigy system can also include an onboard camera and image recognition software, which determines best cell fractionation terminal by distinguishing the macroscopic layer of the source cell product. For example, peripheral blood can be automatically separated into red blood cells, white blood cells and plasma layers. The CliniMACS Prodigy system can also include an integrated cell cultivation chamber, which realizes cell culture protocols such as cell differentiation and amplification, antigen loading and long-term cell culture. The input port can allow aseptic removal and supplementary culture medium, and can use an integrated microscope to monitor cells. See, eg, Klebanoff et al. (2012) J Immunother. 35(9):651-660, Terakura et al. (2012) Blood. 1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.

[0414] In some embodiments, cell mass as described herein is collected and enriched (or exhausted) by flow cytometry, wherein the cell dyed for multiple cell surface markers is carried in fluid stream. In some embodiments, cell mass as described herein is collected and enriched (or exhausted) by preparation scale (FACS) classification. In certain embodiments, by using microelectromechanical system (MEMS) chip in conjunction with FACS-based detection system to collect and enriched (or exhausted) cell mass as described herein (see, for example, WO 2010 / 033140, Cho et al. (2010) Lab Chip 10:1567-1573; With Godin et al. (2008) J Biophoton.1 (5): 355-376). In both cases, cells can be marked with multiple markers, allowing to separate well-defined T cell subsets with high purity.

[0415] In some embodiments, antibody or binding partner is labeled with one or more detectable labels to promote separation for positive and / or negative selection.For example, separation can be based on the combination with fluorescently labeled antibodies.In some examples, separation cells are carried in fluid stream based on the combination of antibodies or other binding partners specific to one or more cell surface markers, such as by fluorescence activated cell sorting (FACS), including preparation scale (FACS) and / or microelectromechanical systems (MEMS) chips, for example, in combination with flow cytometry systems.Such methods allow positive and negative selection to be carried out simultaneously based on multiple markers.

[0416] In some embodiments, the preparation method includes the step of freezing (for example, cryopreservation) cells before or after separation, incubation and / or engineering. In some embodiments, freezing and subsequent thawing steps remove the granulocytes in the cell mass, and remove monocytes to a certain extent. In some embodiments, the cells are suspended in a freezing solution to remove plasma and platelets, for example after a washing step. In some aspects, any of a variety of known freezing solutions and parameters can be used. An example involves using PBS containing 20% ​​DMSO and 8% human serum albumin (HSA), or other suitable cell freezing culture media. It is then diluted with culture medium 1: 1 so that the final concentrations of DMSO and HSA are 10% and 4% respectively. The cells are then frozen to -80 ° C at a rate of 1 ° / minute and stored in the gas phase of a liquid nitrogen storage tank.

[0417] In some embodiments, provided methods include cultivating, incubating, culturing and / or genetic engineering steps.For example, in some embodiments, provided methods are for incubating exhausted cell populations and culturing starting compositions and / or engineering them.

[0418] Thus, in some embodiments, the cell population is incubated in a culture initiation composition. Incubation and / or engineering can be performed in a culture vessel, such as a unit, chamber, well, column, tube, tube set, valve, vial, petri dish, bag, or other container for culturing or growing cells.

[0419] In some embodiments, cells are incubated and / or cultured prior to or in connection with genetic engineering. The incubation step may include culturing, cultivating, stimulating, activating and / or propagating. In some embodiments, the composition or cells are incubated in the presence of stimulating conditions or stimulating agents. These conditions include those designed for the following: conditions for inducing proliferation, amplification, activation and / or survival of cells in a population, for simulating antigen exposure and / or for initiating cells to be genetically engineered, such as to introduce PSMA or a modified form thereof and a recombinant receptor (e.g., CAR).

[0420] Conditions may include one or more of the following: specific culture medium, temperature, oxygen content, carbon dioxide content, time, agents (e.g., nutrients, amino acids, antibiotics, ions and / or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors and any other agents intended to activate cells)).

[0421] In some embodiments, stimulation conditions or agents include one or more agents (e.g., ligands) that can activate the intracellular signaling region of the TCR complex. In some aspects, the agent opens or starts the TCR / CD3 intracellular signaling cascade in T cells. Such agents may include antibodies such as antibodies specific for TCR, such as anti-CD3. In some embodiments, stimulation conditions include one or more agents (e.g., ligands) that can stimulate co-stimulatory receptors (e.g., anti-CD28). In some embodiments, such agents and / or ligands can be bound to solid supports such as beads and / or one or more cytokines. Optionally, amplification methods can also include adding anti-CD3 and / or anti-CD28 antibodies (e.g., with a concentration of at least about 0.5 ng / ml) to the culture medium. In some embodiments, stimulants include IL-2, IL-15, and / or IL-7. In some aspects, IL-2 concentration is at least about 10 units / mL.

[0422] In some aspects, incubation is performed according to techniques such as those described in U.S. Pat. No. 6,040,177 to Riddell et al.; Klebanoff et al. (2012) J Immunother. 35(9):651-660, Terakura et al. (2012) Blood. 1:72-82; and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0423] In some embodiments, T cells are expanded by the following method: feeder cells (such as non-dividing peripheral blood mononuclear cells (PBMC)) are added to the culture starting composition (for example, so that the resulting cell population contains at least about 5, 10, 20 or 40 or more PBMC feeder cells, so that each T lymphocyte in the initial population is expanded); and incubating the culture (for example, for a time sufficient to expand the number of T cells). In some aspects, the non-dividing feeder cells can include γ-irradiated PBMC feeder cells. In some embodiments, the PBMC is irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to the culture medium before adding the T cell population.

[0424] In some embodiments, the stimulating conditions include a temperature suitable for human T lymphocyte growth, for example, at least about 25 degrees Celsius, typically at least about 30 degrees Celsius, and typically at or about 37 degrees Celsius. Optionally, hatching can also include adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rad. In some aspects, the LCL feeder cells are provided with any suitable amount (such as the ratio of LCL feeder cells to initial T lymphocytes is at least about 10: 1).

[0425] In some embodiments, antigen-specific T cells, such as antigen-specific CD4+ and / or CD8+ T cells, are obtained by stimulating naive or antigen-specific T lymphocytes with an antigen. For example, antigen-specific T cell lines or clones can be generated against cytomegalovirus antigens by isolating T cells from an infected subject and stimulating the cells in vitro with the same antigen.

[0426] E. Methods for genetic engineering

[0427] Various methods for introducing genetically engineered components such as PSMA or its modified forms and recombinant receptors (e.g., CAR or TCR) are well known and can be used with the methods and compositions provided. Exemplary methods include those for transferring nucleic acids encoding polypeptides or receptors, including via viral vectors, such as retroviruses or lentiviruses, non-viral vectors, or transposons (e.g., Sleeping Beauty transposon systems). Gene transfer methods can include transduction, electroporation, or other methods that result in gene transfer into cells.

[0428] In some embodiments, gene transfer is accomplished by first stimulating the cells, such as by combining it with a stimulus that induces a response (such as proliferation, survival, and / or activation, e.g., as measured by expression of cytokines or activation markers), and then transducing the activated cells and expanding them in culture to numbers sufficient for clinical use.

[0429] In some situations, it may be necessary to prevent the possibility that overexpression of stimulatory factors (e.g., lymphokines or cytokines) may potentially lead to undesirable results or lower efficacy in the subject (e.g., factors associated with toxicity in the subject). Therefore, in some situations, engineered cells include gene segments that cause the cells to be susceptible to negative selection in vivo (e.g., when given in adoptive immunotherapy). For example, in some aspects, cells are engineered so that they can be eliminated due to changes in the in vivo conditions of the patient to whom they are given. A negative selective phenotype can be produced by the insertion of a gene that confers sensitivity to the administered agent (e.g., compound). Negatively selectable genes include the herpes simplex virus type 1 thymidine kinase (HSV-I TK) gene (Wigler et al., Cell 11:223, 1977), which confers sensitivity to ganciclovir; the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene; the cellular adenine phosphoribosyltransferase (APRT) gene; and bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

[0430] In some embodiments, the recombinant nucleic acid is transferred into cells using recombinant infectious viral particles, such as, for example, vectors derived from simian virus 40 (SV40), adenovirus, or adeno-associated virus (AAV). In some embodiments, the recombinant nucleic acid is transferred into T cells using a recombinant lentiviral vector or a retroviral vector, such as a gamma-retroviral vector (see, for example, Koste et al. (2014) Gene Therapy 2014 Apr 3. doi: 10.1038 / gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 Nov 29(11): 550-557).

[0431] In some embodiments, the retroviral vector has long terminal repeats (LTRs), such as those derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include those derived from any avian or mammalian cell source. Retroviruses are typically amphotropic, meaning that they are able to infect host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol, and / or env sequences. Many illustrative retroviral systems have been described (e.g., U.S. Patent Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, AD (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109).

[0432] Methods for lentiviral transduction are known. Exemplary methods are described, for example, in Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101: 1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.

[0433] In some embodiments, the recombinant nucleic acid is transferred into T cells via electroporation (see, e.g., Chicaybam et al. (2013) PLoS ONE 8(3):e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16):1431-1437). In some embodiments, the recombinant nucleic acid is transferred into T cells by transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4):427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506:115-126). Other methods for introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-promoted microparticle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)).

[0434] Other methods and vectors for transferring nucleic acids encoding recombinant products are, for example, those described in International Patent Application Publication No. WO 2014055668 and US Patent No. 7,446,190.

[0435] In some embodiments, cells (e.g., T cells) can be transfected, for example, with nucleic acids encoding PSMA or a modified form thereof and / or a recombinant receptor (e.g., a T cell receptor (TCR) or a chimeric antigen receptor (CAR)) during or after expansion. For example, such transfection of genes for introducing the desired polypeptide or receptor can be performed with any suitable retroviral vector. The genetically modified cell population can then be freed from the initial stimulator (e.g., a CD3 / CD28 stimulator) and subsequently stimulated with a second type of stimulator, for example, via a de novo introduced receptor. The second type of stimulator can include an antigenic stimulator in the form of a peptide / MHC molecule, a cognate (cross-linked) ligand of a genetically introduced receptor (e.g., a natural ligand of a CAR), or any ligand (e.g., an antibody) that directly binds within the framework of the new receptor (e.g., by recognizing a constant region within the receptor). See, e.g., Cheadle et al., “Chimeric antigen receptors for T-cell based therapy” Methods Mol Biol. 2012; 907: 645-66 or Barrett et al., Chimeric Antigen Receptor Therapy for Cancer Annual Review of Medicine Vol. 65: 333-347 (2014).

[0436] Other nucleic acids (e.g., genes for introduction) include those for improving therapeutic efficacy, such as by promoting the viability and / or function of transferred cells; genes for providing genetic markers for selecting and / or evaluating cells, such as to assess survival or positioning in vivo; genes for improving safety, such as by making cells susceptible to negative selection in vivo, such as Lupton SD et al., Mol. and Cell Biol., 11: 6 (1991) and Riddell et al., Human Gene Therapy 3: 319-338 (1992) as described; see also PCT / US91 / 08442 and PCT / US94 / 05601 of Lupton et al., which describe the use of a dual-function selective fusion gene obtained by fusing a dominant positive selective marker to a negative selective marker. See, for example, Riddell et al., U.S. Patent No. 6,040,177, columns 14-17.

[0437] As described above, in some embodiments, cells are incubated and / or cultured prior to or in conjunction with genetic engineering. The incubation step can include culturing, growing, stimulating, activating, propagating, and / or freezing for preservation (e.g., cryopreservation).

[0438] II. PSMA-Targeting Molecules

[0439] In some embodiments, the PSMA targeting molecule is used in combination with an engineered cell provided herein (e.g., an engineered cell expressing PSMA or a modified form thereof and / or a recombinant receptor (e.g., CAR)) and / or used in the methods provided herein. In some embodiments, the PSMA targeting molecule is capable of binding to PSMA or a modified form thereof, or comprises a portion capable of binding to PSMA or a modified form thereof. In some embodiments, the PSMA targeting molecule is or further comprises one or more therapeutic agents and / or one or more portions that provide a signal or induce a detectable signal (e.g., a detectable portion). In some embodiments, the PSMA targeting molecule comprises a portion that is capable of binding to PSMA or a modified form thereof and a therapeutic agent and / or a portion that provides a signal or induces a detectable signal (e.g., a detectable portion).

[0440] In some embodiments, the PSMA-targeting molecule or portion thereof is capable of binding to PSMA and / or a modified form thereof, and / or is capable of binding to the active site of PSMA and / or the active site of a modified form of PSMA, and / or is capable of being cleaved by PSMA and / or by a modified form of PSMA; and / or is an antagonist, selective antagonist, inverse agonist, selective inverse agonist, agonist, selective agonist, inhibitor, and / or selective inhibitor of PSMA and / or a modified form thereof.

[0441] In some embodiments, the PSMA-targeting molecule is capable of binding a moiety that provides a signal or induces a detectable signal. In some embodiments, the PSMA-targeting molecule contains a therapeutic moiety that is activated, cleaved, and / or released upon binding to PSMA or a modified form thereof, or is activated, cleaved, and / or released under specific conditions (e.g., conditions proximate to the site, location, or microenvironment of a disease or disorder), such as in a tumor microenvironment (TME).

[0442] In some embodiments, the PSMA-targeting molecule comprises a moiety capable of binding to PSMA or a modified form thereof. In some embodiments, the moiety capable of binding to PSMA or a modified form thereof is or comprises a small molecule, a ligand, an antibody or antigen-binding fragment thereof, an aptamer, a peptide, a nanoparticle, or a conjugate thereof. In some embodiments, the PSMA-targeting molecule is or further comprises a therapeutic agent and / or moiety that provides a signal or induces a detectable signal. In some embodiments, the PSMA-targeting molecule is or comprises a moiety capable of binding to PSMA or a modified form thereof that is linked to or conjugated to a therapeutic agent and / or moiety (e.g., a detectable moiety).

[0443] In some embodiments, the PSMA-targeting molecule is a known and useful agent for detecting and / or binding PSMA (e.g., human PSMA). PSMA has been used as a target for prostate cancer imaging based on low background and tissue-specific expression, increased expression in more advanced prostate cancers, a large extracellular domain target, human biocompatibility, the diversity of available probes, and proven clinical utility and internalization and endosomal recycling. Several PSMA-binding ligands have been developed to deliver imaging and therapeutic agents for prostate cancer, including low molecular weight nuclear, fluorescent, and multimodal imaging probes (Chen et al., Biochem. Biophys. Res. Comm. 2009, 390(3):624-629; Banerjee et al., Oncotarget 2011; 2(12):1244-1253; see also Maurer et al. (2016) Nature Reviews Urology 13:226-235; Rowe et al. (2016) Prostate Cancer Prostatic Dis. 19(3):223-230; Mease et al. (2013) Curr Top Med Chem. 13(8):951-962). Exemplary other PSMA-targeting molecules are described below.

[0444] In some embodiments, the PSMA-targeting molecule provides a signal or induces a detectable signal or is capable of binding to a moiety that provides a signal or induces a detectable signal; and / or the PSMA-targeting molecule is or comprises a moiety that provides a signal or induces a detectable signal. In some embodiments, the PSMA-targeting molecule includes those that are known or available that bind to PSMA, such as those used to treat and / or diagnose prostate cancer.

[0445] In some embodiments, a PSMA-targeting molecule is also provided, comprising a portion capable of binding to PSMA or a modified form thereof and an immunomodulatory agent. In some embodiments, the immunomodulatory agent is capable of modulating, optionally increasing, the activity of immune cells or an immune response and / or modulating the tumor microenvironment (TME).

[0446] A. PSMA binding part

[0447] 1. Small molecules

[0448] In some embodiments, the PSMA-targeting molecule is or comprises a ligand and / or a small molecule. In some embodiments, the PSMA-targeting molecule comprises a moiety capable of binding to PSMA or a modified form thereof, which is or comprises a ligand and / or a small molecule. In some embodiments, the PSMA-targeting molecule is or comprises a small molecule capable of binding to the active site or substrate binding site of PSMA.

[0449] In some embodiments, the PSMA targeting molecule is or comprises an antagonist, selective antagonist, inverse agonist, selective inverse agonist, agonist, selective agonist, inhibitor, and / or selective inhibitor of PSMA and / or its modified forms. In some embodiments, the PSMA targeting molecule is or comprises an inhibitor of PSMA. In some embodiments, the PSMA targeting molecule is or comprises a small molecule, and / or a low molecular weight molecule, and / or a low molecular weight inhibitor. In some embodiments, the PSMA targeting molecule is or comprises a moiety capable of binding to PSMA or its modified forms, which moiety is a small molecule, a therapeutic agent, and / or a detectable moiety.

[0450] As described above, PSMA possesses an enzymatic site in its extracellular domain that cleaves endogenous substrates such as N-acetylaspartylglutamate (NAAG), tri-α-glutamic acid peptides, and poly-γ-glutamylfolate, and the enzymatic site contains two zinc ions and is composed of two pockets: a glutamate-sensitive pocket (S1' pocket) and a non-pharmacophore pocket (S1 pocket). Some PSMA inhibitors contain a zinc-binding moiety and glutamate or a glutamate isostere located in the S1' pocket. The non-pharmacophore pocket contains an arginine-rich region and can accommodate a medium-sized lipophilic moiety.

[0451] In some cases, small molecule PSMA inhibitors are typically zinc-binding compounds attached to glutamic acid or a glutamic acid isostere and are classified into three categories of scaffolds: (1) phosphonate-, phosphate-, and phosphoramidate; (2) thiol; and (3) urea. In some embodiments, the scaffold is a hydroxamate. In some embodiments, the small molecule scaffolds share common features including: a) glutaric acid that fits within the S1' binding pocket of the PSMA active site as a glutamate mimetic; and b) a zinc-binding group that interacts with the catalytic zinc atom in the PSMA active site. Due to their high binding affinity and ease of synthesis, urea-based small molecules (e.g., urea-based PSMA inhibitors) have been developed for bioimaging, such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) using radionuclides.

[0452] In some embodiments, the PSMA targeting molecule or portion thereof includes any of those described in, for example, WO 2015143029; WO 2016 / 065142; US 201013257499; US 2012 / 0067162; US201213566849; US 201214008715; US 201214126296; U...

Claims

1. An engineered cell comprising: Modified prostate-specific membrane antigen; and Recombinant antigen receptors; The modified prostate-specific membrane antigen comprises one or more amino acid modifications compared to the wild-type prostate-specific membrane antigen comprising the sequence shown in SEQ ID NO: 23, wherein the modified prostate specific membrane antigen comprises at least 95% sequence identity to SEQ ID NO: 23, wherein the modified prostate specific membrane antigen is a modified human prostate specific membrane antigen, wherein the one or more amino acid modifications comprise one or more amino acid substitutions, deletions and / or insertions; wherein the modified prostate-specific membrane antigen comprises at least one amino acid modification corresponding to a W2G substitution, a W2 substitution, or a W2 deletion and / or a deletion of up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 N-terminal amino acid residues, and wherein compared to the wild-type prostate-specific membrane antigen, the modified prostate-specific membrane antigen (i) exhibits reduced endogenous signaling; (ii) exhibits increased cell surface expression; and / or (iii) exhibits reduced cellular internalization.

2. An engineered cell comprising: encoding a modified prostate-specific membrane antigen; and a nucleic acid encoding a recombinant antigen receptor; The modified prostate-specific membrane antigen comprises one or more amino acid modifications compared to the wild-type prostate-specific membrane antigen comprising the sequence shown in SEQ ID NO: 23, wherein the modified prostate specific membrane antigen comprises at least 95% sequence identity to SEQ ID NO: 23, wherein the modified prostate specific membrane antigen is a modified human prostate specific membrane antigen, wherein the one or more amino acid modifications comprise one or more amino acid substitutions, deletions and / or insertions; wherein the modified prostate specific membrane antigen comprises at least one amino acid modification corresponding to a W2G substitution, a substitution of W2, or a deletion of W2 and / or a deletion of up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 N-terminal amino acid residues, and wherein compared to the wild-type prostate specific membrane antigen, the modified prostate specific membrane antigen (i) exhibits reduced endogenous signaling; (ii) exhibits increased cell surface expression; and / or (iii) exhibits reduced cellular internalization.

3. The engineered cell of claim 1, wherein the modified prostate-specific membrane antigen is expressed on the surface of the cell.

4. The engineered cell of claim 1, wherein the modified prostate-specific membrane antigen comprises an extracellular portion and a transmembrane domain.

5. The engineered cell according to claim 1, wherein the modified prostate-specific membrane antigen can be recognized by a prostate-specific membrane antigen targeting molecule or a portion thereof.

6. The engineered cell according to claim 5, wherein the prostate-specific membrane antigen targeting molecule or a portion thereof: capable of binding to prostate-specific membrane antigen and / or the modified prostate-specific membrane antigen, and / or Capable of binding to the active site of prostate-specific membrane antigen and / or the active site of the modified prostate-specific membrane antigen, and / or capable of being cleaved by prostate-specific membrane antigen and / or cleaved by said modified prostate-specific membrane antigen; and / or It is an antagonist, inverse agonist, agonist, and / or inhibitor of prostate-specific membrane antigen and / or the modified prostate-specific membrane antigen.

7. An engineered cell according to claim 1, wherein the modified prostate-specific membrane antigen comprises an N-acetylated α-linked acid dipeptidase domain, and / or comprises one or more active site residues and / or residues involved in prostate-specific membrane antigen substrate binding and / or prostate-specific membrane antigen catalytic activity.

8. The engineered cell according to claim 1, wherein the modified prostate-specific membrane antigen comprises an amino acid modification at position 2 with reference to the position in the amino acid sequence shown in SEQ ID NO:

23.

9. An engineered cell according to claim 8, wherein the modified prostate-specific membrane antigen comprises the amino acid sequence shown in SEQ ID NO: 24 or a fragment thereof, or an amino acid sequence that exhibits at least 95% sequence identity with SEQ ID NO: 24 or a fragment thereof and comprises at least one amino acid substitution compared to SEQ ID NO:

24.

10. The engineered cell of claim 1, wherein the modified prostate specific membrane antigen comprises a deletion of one or more of the 18 N-terminal amino acid residues compared to wild-type prostate specific membrane antigen.

11. The engineered cell of claim 10, wherein the modified prostate specific membrane antigen comprises a deletion of at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 N-terminal amino acid residues compared to the wild-type prostate specific membrane antigen.

12. The engineered cell of claim 1, wherein the modified prostate-specific membrane antigen comprises a deletion of a continuous amino acid sequence starting from an amino acid residue at position 2, 3, 4 or 5 and up to the N-terminus at position 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 compared to the wild-type prostate-specific membrane antigen.

13. The engineered cell of claim 1, wherein the modified prostate-specific membrane antigen comprises the amino acid sequence shown in SEQ ID NO: 52 or a fragment thereof, or an amino acid sequence that exhibits at least 95% sequence identity to SEQ ID NO: 52 or a fragment thereof and comprises a deletion of one or more N-terminal amino acid residues and contains a methionine start codon.

14. The engineered cell of claim 1, wherein the modified prostate-specific membrane antigen is encoded by: a nucleic acid sequence as set forth in SEQ ID NO: 53 or a fragment thereof; or a nucleic acid sequence exhibiting at least 95% sequence identity to SEQ ID NO: 53 or a fragment thereof and containing a nucleic acid encoding a methionine start codon.

15. The engineered cell according to claim 1, wherein the modified prostate specific membrane antigen or a modified form thereof is encoded by a nucleic acid sequence modified to be CpG-free and / or codon-optimized.

16. The engineered cell of claim 15, wherein the modified prostate-specific membrane antigen is encoded by: a nucleic acid sequence as set forth in SEQ ID NO: 27 or a fragment thereof; or a nucleic acid sequence exhibiting at least 95% sequence identity to SEQ ID NO: 27 or a fragment thereof and containing a nucleic acid encoding a methionine start codon.

17. The engineered cell of claim 2, wherein the modified prostate-specific membrane antigen comprises an N-acetylated α-linked acid dipeptidase domain, and / or comprises one or more active site residues and / or residues involved in prostate-specific membrane antigen substrate binding and / or prostate-specific membrane antigen catalytic activity.

18. An engineered cell according to claim 2, wherein the modified prostate-specific membrane antigen comprises the amino acid sequence shown in SEQ ID NO: 23 or its extracellular and / or transmembrane domain, or an amino acid sequence that exhibits at least 95% sequence identity with SEQ ID NO: 23 or its extracellular and / or transmembrane domain.

19. The engineered cell of claim 2, wherein the modified prostate-specific membrane antigen comprises an amino acid modification at position 2 with reference to the position in the amino acid sequence shown in SEQ ID NO:

23.

20. The engineered cell of claim 19, wherein the modified prostate-specific membrane antigen comprises the amino acid sequence shown in SEQ ID NO: 24 or a fragment thereof, or an amino acid sequence that exhibits at least 95% sequence identity to SEQ ID NO: 24 or a fragment thereof and comprises at least one amino acid substitution compared to SEQ ID NO:

24.

21. The engineered cell of claim 2, wherein the modified prostate specific membrane antigen comprises a deletion of one or more of the 18 N-terminal amino acid residues compared to wild-type prostate specific membrane antigen.

22. The engineered cell of claim 21, wherein the modified prostate specific membrane antigen comprises a deletion of at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 N-terminal amino acid residues compared to the wild-type prostate specific membrane antigen.

23. The engineered cell of claim 2, wherein the modified prostate-specific membrane antigen comprises a deletion of a continuous amino acid sequence starting from an amino acid residue at position 2, 3, 4 or 5 and up to the N-terminus at position 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 compared to the wild-type prostate-specific membrane antigen.

24. The engineered cell of claim 2, wherein the modified prostate-specific membrane antigen comprises the amino acid sequence shown in SEQ ID NO: 52 or a fragment thereof, or an amino acid sequence that exhibits at least 95% sequence identity to SEQ ID NO: 52 or a fragment thereof and comprises a deletion of one or more N-terminal amino acid residues and contains a methionine start codon.

25. The engineered cell of claim 2, wherein the nucleic acid encoding the modified prostate-specific membrane antigen comprises the sequence shown in SEQ ID NO: 53 or a fragment thereof; or a nucleic acid sequence that exhibits at least 95% sequence identity to SEQ ID NO: 53 or a fragment thereof and contains a methionine start codon.

26. The engineered cell of claim 2, wherein the nucleic acid encoding the modified prostate specific membrane antigen is modified to be CpG-free and / or codon-optimized.

27. The engineered cell of claim 26, wherein the nucleic acid encoding the modified prostate-specific membrane antigen comprises the sequence shown in SEQ ID NO: 27 or a fragment thereof; or a nucleic acid sequence that exhibits at least 95% sequence identity to SEQ ID NO: 27 or a fragment thereof and contains a methionine start codon.

28. The engineered cell according to any one of claims 1-27, wherein: The modified prostate-specific membrane antigen comprises all or substantially all of the transmembrane domain of the wild-type prostate-specific membrane antigen; or The modified prostate-specific membrane antigen comprises a transmembrane domain having the same or at least the same number of amino acids as the transmembrane domain of the wild-type prostate-specific membrane antigen.

29. The engineered cell of claim 5 or 6, wherein the prostate-specific membrane antigen targeting molecule comprises a small molecule, a ligand, an antibody or an antigen-binding fragment thereof, an aptamer, a peptide or a conjugate thereof.

30. The engineered cell of claim 5 or 6, wherein the prostate specific membrane antigen targeting molecule comprises a small molecule.

31. The engineered cell of claim 30, wherein the small molecule is selected from the group consisting of: 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid, N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine, (aminostyryl)pyridinium dye, 2-(3-[1-carboxy-5-[(5-iodo-pyridine-3-carbonyl)-amino]-pentyl]-ureido)-glutaric acid, 2-[3-[1-carboxy-5-[(4-iodo-benzoyl)carbamoyl]-4-fluorobenzyl-L-cysteine, [(4-(3-(4-fluoro-3-[hydroxymethyl]butyl)guanine]-glutaric acid, 1-(3-carboxy-4-(3-hydroxy-6-oxo-6H-xanthen-9-yl)phenylamino)-9,16,24-trioxo-1-thioxo-2,8,17,23,25-pentaazaoctacosane-7,22,26,28-tetracarboxylic acid, Glu-NH-CO-NH-Lys(Ahx)-HBED-CC, 9-(4-fluoro-3-[hydroxymethyl]butyl)guanine, Glu-urea-Lys-(Ahx)-[(HBED-CC)], PSMA-617, 2-(phosphonomethyl) 2-(2-(4-(1,3-dicarboxypropyl)ureido)pentanedioic acid, 2-PMPA, fluorobenzylphosphoramidate, (2S,4S)-2-fluoro-4-(phosphonomethyl)pentanedioic acid, N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-S-methyl-L-cysteine, EuK-Subkff-DOTAGA, 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid, PSMAN, N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid, MIP-1072, MIP-1095, MIP-1404, MIP-14 05 and N-[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-L-glutamate, (S)-2-(4-iodobenzylphosphonylmethyl)-glutaric acid, 2-[(3-{4-[(2-amino-4-hydroxy-pteridin-6-ylmethyl)-amino]-benzoylamino}-3-carboxy-propyl)-hydroxy-phosphorylmethyl]-glutaric acid, (2S,3'S)-{[(3'-amino-3'-carboxy-propyl)-hydroxyphosphoryl]methyl}-glutaric acid and (2S)-2-{[(2-carboxy-ethyl)-hydroxy-phosphoryl]methyl}-glutaric acid.

32. The engineered cell of claim 31, wherein the prostate-specific membrane antigen targeting molecule comprises 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid or N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine.

33. The engineered cell according to claim 5 or 6, wherein the prostate-specific membrane antigen targeting molecule comprises an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof has a binding site that specifically binds to the prostate-specific membrane antigen.

34. The engineered cell according to claim 5 or 6, wherein the prostate-specific membrane antigen targeting molecule comprises an aptamer or a conjugate thereof.

35. The engineered cell of any one of claims 1-27, wherein the recombinant antigen receptor is capable of binding to a target antigen that is associated with, specific to, and / or expressed on cells or tissues of a tumor.

36. The engineered cell of claim 35, wherein the target antigen is a tumor antigen.

37. The engineered cell of claim 35, wherein the target antigen is selected from the group consisting of: αvβ6 integrin, B cell maturation antigen, B7-H3, B7-H6, carbonic anhydrase 9, cancer-testis antigen, cancer / testis antigen 1B, carcinoembryonic antigen, cyclin, cyclin A2, CC motif chemokine ligand 1, CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD1 33, CD138, CD171, chondroitin sulfate proteoglycan 4, epidermal growth factor protein, truncated epidermal growth factor protein, epidermal growth factor receptor type III mutation, epithelial glycoprotein 2, epithelial glycoprotein 40, ephrin B2, ephrin receptor A2, estrogen receptor, Fc receptor-like protein 5, fetal acetylcholine receptor, folate binding protein, folate receptor alpha, ganglioside GD2, O-acetylated GD2, ganglioside GD3, glycoprotein 100, phosphatidylinositol proteoglycan, G protein-coupled receptor 5D, Her2 / neu , Her3, Her4, erbB dimer, human high molecular weight melanoma-associated antigen, hepatitis B surface antigen, human leukocyte antigen A1, human leukocyte antigen A2, IL-22 receptor alpha, IL-13 receptor alpha 2, kinase insert domain receptor, L1 cell adhesion molecule, leucine-rich repeat-containing protein 8 family member A, Lewis Y, melanoma-associated antigen A1, melanoma-associated antigen A3, melanoma-associated antigen A6, melanoma-associated antigen A10, mesothelin, c-Met, murine cytomegalovirus, mucin 1, M UC16, natural killer cell family 2 member D ligand, melanin A, neural cell adhesion molecule, carcinoembryonic antigen, preferentially expressed melanoma antigen, progesterone receptor, prostate specific antigen, prostate stem cell antigen, prostate specific membrane antigen, receptor tyrosine kinase-like orphan receptor 1, survivin, trophoblast glycoprotein, tumor-associated glycoprotein 72, tyrosinase-related protein 1, tyrosinase-related protein 2, vascular endothelial growth factor receptor, vascular endothelial growth factor receptor 2, Wilms tumor 1, and / or molecules expressed by HIV, HCV, or HBV.

38. The engineered cell of any one of claims 1-27, wherein the recombinant antigen receptor comprises a T cell receptor or an antigen binding fragment thereof.

39. The engineered cell of any one of claims 1-27, wherein the recombinant antigen receptor is a chimeric antigen receptor.

40. The engineered cell of claim 39, wherein the recombinant antigen receptor comprises an extracellular domain comprising an antigen binding domain.

41. The engineered cell of any one of claims 1-27, wherein the recombinant antigen receptor comprises an extracellular domain comprising an antigen binding domain.

42. The engineered cell of claim 41, wherein the antigen binding domain comprises an antibody or an antibody fragment thereof.

43. The engineered cell of claim 42, wherein the fragment comprises an antibody variable region connected by a flexible linker.

44. The engineered cell of claim 42, wherein the fragment comprises a scFv.

45. The engineered cell of claim 40, wherein the recombinant antigen receptor further comprises a spacer and / or a hinge region.

46. ​​The engineered cell of claim 45, wherein the recombinant antigen receptor comprises an intracellular signaling domain.

47. The engineered cell of any one of claims 1-27, wherein the recombinant antigen receptor comprises an intracellular signaling domain.

48. The engineered cell of claim 46, wherein the intracellular signaling region comprises an intracellular signaling domain.

49. The engineered cell of claim 48, wherein the intracellular signaling domain comprises a primary signaling domain, a signaling domain capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor component, and / or a signaling domain comprising an immunoreceptor tyrosine-based activation motif.

50. The engineered cell of claim 49, wherein the intracellular signaling domain comprises the intracellular signaling domain of the CD3 chain or a signaling portion thereof.

51. The engineered cell of claim 46, wherein the recombinant antigen receptor further comprises a transmembrane domain disposed between the extracellular domain and the intracellular signaling region.

52. The engineered cell of claim 51, wherein the intracellular signaling region further comprises a co-stimulatory signaling region.

53. The engineered cell of claim 52, wherein the co-stimulatory signaling region comprises an intracellular signaling domain of a T cell co-stimulatory molecule or a signaling portion thereof.

54. The engineered cell of claim 52, wherein the co-stimulatory signaling region comprises an intracellular signaling domain of CD28, 4-1BB or ICOS, or a signaling portion thereof.

55. The engineered cell of claim 52, wherein the co-stimulatory signaling region is located between the transmembrane domain and the intracellular signaling region.

56. The engineered cell of any one of claims 2 and 17-27, wherein the nucleic acid encoding the modified prostate specific membrane antigen and the nucleic acid encoding the recombinant antigen receptor are contained within one or more polynucleotides contained by the cell.

57. The engineered cell of claim 56, wherein the one or more polynucleotides is one polynucleotide and the nucleic acid encoding the modified prostate-specific membrane antigen and the nucleic acid encoding the recombinant antigen receptor are operably linked to the same promoter.

58. The engineered cell of claim 57, wherein the recombinant antigen receptor is a chimeric antigen receptor, and the nucleic acid encoding the modified prostate specific membrane antigen and the nucleic acid encoding the chimeric antigen receptor are contained within one polynucleotide contained by the cell, the polynucleotide comprising, in 5' to 3' order: i) a nucleic acid encoding a signal peptide; ii) a nucleic acid encoding the recombinant antigen receptor, wherein the recombinant antigen receptor is a chimeric antigen receptor comprising an scFv; a spacer; a transmembrane domain; an intracellular region comprising a co-stimulatory signaling region, and an intracellular signaling domain of a CD3-zeta chain or a signaling portion thereof; iii) a nucleic acid sequence encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and iv) a nucleic acid encoding the modified prostate-specific membrane antigen, wherein the prostate-specific membrane antigen or a modified form thereof comprises the amino acid sequence shown in SEQ ID NO: 52 or a fragment thereof; or an amino acid sequence that exhibits at least 95% sequence identity with SEQ ID NO: 52 or a fragment thereof and comprises a deletion of the one or more N-terminal amino acid residues.

59. The engineered cell of claim 56, wherein the nucleic acid encoding the modified prostate specific membrane antigen and the nucleic acid encoding the recombinant antigen receptor are operably linked to two different promoters.

60. The engineered cell of claim 59, wherein the nucleic acid encoding the recombinant antigen receptor is present downstream of the nucleic acid encoding the modified prostate specific membrane antigen.

61. An engineered cell according to claim 56, wherein the one or more polynucleotides comprise two different polynucleotides, the nucleic acid encoding the modified prostate-specific membrane antigen and the nucleic acid encoding the recombinant antigen receptor are operably linked to two different promoters, and / or the nucleic acid encoding the modified prostate-specific membrane antigen and the nucleic acid encoding the recombinant antigen receptor are present in or inserted into different locations within the genome of the cell.

62. The engineered cell of any one of claims 1-27, wherein: The cell is an immune cell; The cell is a T cell; The cells are NK cells; and / or The cells are derived from pluripotent or multipotent cells.

63. The engineered cell of claim 62, wherein: The cell is a T cell selected from the group consisting of a central memory T cell, an effector memory T cell, a naive T cell, a stem cell central memory T cell, an effector T cell and a regulatory T cell; and / or The cells comprise a plurality of cells comprising at least 50% of a population of cells selected from the group consisting of CD4+ T cells, CD8+ T cells, central memory T cells, effector memory T cells, naive T cells, stem cell central memory T cells, effector T cells, and regulatory T cells.

64. The engineered cell of claim 63, wherein the cell is a regulatory T cell.

65. The engineered cell of claim 64, further comprising recombinant FOXP3 or a variant thereof.

66. A polynucleotide comprising a first nucleic acid encoding a modified prostate-specific membrane antigen and a second nucleic acid encoding a recombinant antigen receptor, wherein the modified prostate-specific membrane antigen comprises one or more amino acid modifications compared to a wild-type prostate-specific membrane antigen comprising the sequence shown in SEQ ID NO: 23, wherein the modified prostate-specific membrane antigen comprises at least 95% or more sequence identity to SEQ ID NO: 23, wherein the modified prostate-specific membrane antigen is a modified human prostate-specific membrane antigen, wherein the modified prostate-specific membrane antigen comprises at least one amino acid modification corresponding to a W2G substitution, a substitution of W2, or a deletion of W2 and / or a deletion of up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 N-terminal amino acid residues, and wherein the encoded modified prostate-specific membrane antigen (i) exhibits reduced endogenous signaling; (ii) exhibits increased cell surface expression; and / or (iii) exhibits reduced cellular internalization compared to a wild-type prostate-specific membrane antigen.

67. The polynucleotide of claim 66, wherein the nucleic acid encoding the modified prostate-specific membrane antigen and the nucleic acid encoding the recombinant antigen receptor are operably linked to the same promoter.

68. The polynucleotide of claim 66, wherein the nucleic acid encoding the modified prostate-specific membrane antigen and the nucleic acid encoding the recombinant antigen receptor are each operably linked to different promoters.

69. The polynucleotide of claim 66, wherein the nucleic acid encoding the recombinant antigen receptor is present downstream of the nucleic acid encoding the modified prostate-specific membrane antigen.

70. A polynucleotide set comprising a first polynucleotide comprising a nucleic acid encoding a modified prostate-specific membrane antigen and a second polynucleotide comprising a nucleic acid encoding a recombinant antigen receptor, wherein the modified prostate-specific membrane antigen comprises one or more amino acid modifications compared to a wild-type prostate-specific membrane antigen comprising the sequence shown in SEQ ID NO: 23, wherein the modified prostate-specific membrane antigen comprises a sequence different from that of SEQ ID NO: 23 at least 95% or more sequence identity, wherein the modified prostate specific membrane antigen is a modified human prostate specific membrane antigen, wherein the modified prostate specific membrane antigen comprises at least one amino acid modification corresponding to a W2G substitution, a substitution of W2, or a deletion of W2 and / or a deletion of up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 N-terminal amino acid residues, and wherein compared to the wild-type prostate specific membrane antigen, the encoded modified prostate specific membrane antigen (i) exhibits reduced endogenous signaling; (ii) exhibits increased cell surface expression; and / or (iii) exhibits reduced cellular internalization.

71. A composition comprising the polynucleotide set according to claim 70.

72. A polynucleotide according to any one of claims 66-69, a polynucleotide set according to claim 70, or a composition according to claim 71, wherein the nucleic acid encoding the modified prostate-specific membrane antigen and the nucleic acid encoding the recombinant antigen receptor are each independently operably linked to a promoter.

73. The polynucleotide of any one of claims 66-69, the set of polynucleotides of claim 70, or the composition of claim 71, wherein the encoded modified prostate specific membrane antigen is capable of being expressed on the surface of a cell.

74. The polynucleotide of any one of claims 66-69, the set of polynucleotides of claim 70, or the composition of claim 71, wherein the encoded modified prostate-specific membrane antigen comprises an extracellular portion and a transmembrane domain.

75. The polynucleotide of any one of claims 66-69, the set of polynucleotides of claim 70, or the composition of claim 71, wherein the modified prostate-specific membrane antigen is recognizable by a prostate-specific membrane antigen targeting molecule or a portion thereof.

76. The polynucleotide, set of polynucleotides or composition of claim 75, wherein the prostate-specific membrane antigen targeting molecule or portion thereof: capable of binding to prostate-specific membrane antigen and / or the modified prostate-specific membrane antigen, and / or Capable of binding to the active site of prostate-specific membrane antigen and / or the active site of the modified prostate-specific membrane antigen, and / or capable of being cleaved by prostate-specific membrane antigen and / or cleaved by said modified prostate-specific membrane antigen; and / or It is an antagonist, inverse agonist, agonist, and / or inhibitor of prostate-specific membrane antigen and / or the modified prostate-specific membrane antigen.

77. A polynucleotide according to any one of claims 66-69, a polynucleotide set according to claim 70, or a composition according to claim 71, wherein the encoded modified prostate-specific membrane antigen comprises an N-acetylated α-linked acid dipeptidase domain, and / or comprises one or more active site residues and / or residues involved in prostate-specific membrane antigen substrate binding and / or prostate-specific membrane antigen catalytic activity.

78. The polynucleotide of any one of claims 66-69, the set of polynucleotides of claim 70, or the composition of claim 71, wherein the encoded modified prostate specific membrane antigen is a modified human prostate specific membrane antigen.

79. A polynucleotide according to any one of claims 66-69, a polynucleotide set according to claim 70, or a composition according to claim 71, wherein the encoded modified prostate-specific membrane antigen comprises the amino acid sequence shown in SEQ ID NO: 23 or its extracellular and / or transmembrane domain, or an amino acid sequence that exhibits at least 95% sequence identity with SEQ ID NO: 23 or its extracellular and / or transmembrane domain.

80. The polynucleotide of any one of claims 66-69, the set of polynucleotides of claim 70, or the composition of claim 71, wherein the encoded modified prostate-specific membrane antigen comprises one or more amino acid modifications compared to wild-type or unmodified prostate-specific membrane antigen.

81. A polynucleotide, a polynucleotide group or a composition according to claim 80, wherein the wild-type or unmodified prostate-specific membrane antigen is human prostate-specific membrane antigen, and / or comprises the amino acid sequence shown in SEQ ID NO: 23 or an amino acid sequence that exhibits at least 95% sequence identity thereto.

82. The polynucleotide, set of polynucleotides or composition of claim 80, wherein the one or more amino acid modifications comprise one or more amino acid substitutions, deletions and / or insertions.

83. A polynucleotide according to any one of claims 66-69, a polynucleotide set according to claim 70, or a composition according to claim 71, wherein the encoded modified prostate-specific membrane antigen comprises an amino acid modification at position 2, with reference to the position in the amino acid sequence shown in SEQ ID NO:

23.

84. A polynucleotide, a polynucleotide group or a composition according to claim 83, wherein the encoded modified prostate-specific membrane antigen comprises the amino acid sequence shown in SEQ ID NO: 24 or a fragment thereof, or an amino acid sequence that exhibits at least 95% sequence identity with SEQ ID NO: 24 or a fragment thereof and comprises at least one amino acid substitution compared to SEQ ID NO:

24.

85. A polynucleotide according to any one of claims 66-69, a polynucleotide set according to claim 70, or a composition according to claim 71, wherein the encoded modified prostate-specific membrane antigen comprises a deletion of one or more of the 18 N-terminal amino acid residues compared to wild-type prostate-specific membrane antigen.

86. A polynucleotide, a set of polynucleotides or a composition according to claim 85, wherein the encoded modified prostate specific membrane antigen comprises a deletion of at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 N-terminal amino acid residues compared to the wild-type prostate specific membrane antigen.

87. A polynucleotide according to any one of claims 66-69, a polynucleotide set according to claim 70, or a composition according to claim 71, wherein the encoded modified prostate-specific membrane antigen comprises a deletion of a continuous amino acid sequence starting from an amino acid residue at position 2, 3, 4 or 5 and up to the N-terminus at position 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18, with reference to the position in the amino acid sequence shown in SEQ ID NO:

23.

88. A polynucleotide according to any one of claims 66-69, a polynucleotide set according to claim 70, or a composition according to claim 71, wherein the encoded modified prostate-specific membrane antigen comprises the amino acid sequence shown in SEQ ID NO: 52 or a fragment thereof; or an amino acid sequence that exhibits at least 95% sequence identity with SEQ ID NO: 52 or a fragment thereof and comprises a deletion of one or more N-terminal amino acid residues and contains a methionine start codon.

89. A polynucleotide according to any one of claims 66-69, a polynucleotide set according to claim 70, or a composition according to claim 71, wherein the nucleic acid encoding the modified prostate-specific membrane antigen comprises the sequence shown in SEQ ID NO: 53 or a fragment thereof, or a nucleic acid sequence that exhibits at least 95% sequence identity with SEQ ID NO: 53 or a fragment thereof and contains a nucleic acid encoding a methionine start codon.

90. The polynucleotide of any one of claims 66-69, the set of polynucleotides of claim 70, or the composition of claim 71, wherein the modified prostate-specific membrane antigen is encoded by a nucleic acid sequence modified to be CpG-free and / or codon-optimized.

91. A polynucleotide, a polynucleotide group or a composition according to claim 90, wherein the nucleic acid encoding the modified prostate-specific membrane antigen comprises the sequence shown in SEQ ID NO: 27 or a fragment thereof, or a nucleic acid sequence that exhibits at least 95% sequence identity with SEQ ID NO: 27 or a fragment thereof and contains a nucleic acid encoding a methionine start codon.

92. The polynucleotide of any one of claims 66-69, the set of polynucleotides of claim 70, or the composition of claim 71, wherein: The encoded modified prostate-specific membrane antigen comprises all or substantially all of the transmembrane domain of the wild-type or unmodified prostate-specific membrane antigen; or The encoded modified prostate specific membrane antigen comprises a transmembrane domain having the same or at least the same number of amino acids as the transmembrane domain of the wild-type or unmodified prostate specific membrane antigen.

93. The polynucleotide, set of polynucleotides or composition of claim 75, wherein the prostate-specific membrane antigen targeting molecule comprises a small molecule, a ligand, an antibody or an antigen-binding fragment thereof, an aptamer, a peptide or a conjugate thereof.

94. The polynucleotide, set of polynucleotides or composition of claim 93, wherein the prostate specific membrane antigen targeting molecule comprises a small molecule.

95. The polynucleotide, set of polynucleotides or composition of claim 94, wherein the small molecule is selected from the group consisting of 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid, N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine, (aminostyryl)pyridinium dye, 2-(3-[1-carboxy-5-[(5-iodo-pyridine-3-carbonyl)-amino]-pentyl]-ureido)-glutaric acid, 2-[3-[1-carboxy-5-(4 [-iodo-benzoylamino)-pentyl]-ureido]-pentanedioic acid, 1-(3-carboxy-4-(3-hydroxy-6-oxo-6H-xanthen-9-yl)phenylamino)-9,16,24-trioxo-1-thioxo-2,8,17,23,25-pentaazaoctacosane-7,22,26,28-tetracarboxylic acid, Glu-NH-CO-NH-Lys(Ahx)-HBED-CC, 9-(4-fluoro-3-[hydroxymethyl]butyl)guanine, Glu-urea-Lys-(Ahx)-[(HBED-CC)], PSMA-617, 2- (Phosphonomethyl) glutaric acid, 2-PMPA, fluorobenzyl phosphoramidate, (2S,4S)-2-fluoro-4-(phosphonomethyl) glutaric acid, N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-S-methyl-L-cysteine, EuK-Subkff-DOTAGA, 2-[3-(1,3-dicarboxypropyl)ureido] glutaric acid, PSMAN, N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid, MIP-1072, MIP-1095, MIP-1404, MIP- 1405 and N-[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-L-glutamate, (S)-2-(4-iodobenzylphosphonylmethyl)-glutaric acid, 2-[(3-{4-[(2-amino-4-hydroxy-pteridin-6-ylmethyl)-amino]-benzoylamino}-3-carboxy-propyl)-hydroxy-phosphorylmethyl]-glutaric acid, (2S,3'S)-{[(3'-amino-3'-carboxy-propyl)-hydroxyphosphoryl]methyl}-glutaric acid and (2S)-2-{[(2-carboxy-ethyl)-hydroxy-phosphoryl]methyl}-glutaric acid.

96. A polynucleotide, a set of polynucleotides or a composition according to claim 95, wherein the prostate-specific membrane antigen targeting molecule comprises 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid or N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine.

97. The polynucleotide, group of polynucleotides or composition of claim 93, wherein the prostate-specific membrane antigen targeting molecule comprises an antibody or an antigen-binding fragment thereof having a binding site that specifically binds to the prostate-specific membrane antigen.

98. The polynucleotide, set of polynucleotides or composition of claim 93, wherein the prostate-specific membrane antigen targeting molecule comprises an aptamer or a conjugate thereof.

99. A polynucleotide according to any one of claims 66-69, a polynucleotide set according to claim 70, or a composition according to claim 71, wherein the encoded recombinant antigen receptor is capable of binding to a target antigen that is associated with, specific to, and / or expressed on cells or tissues of a tumor.

100. The polynucleotide, set of polynucleotides or composition of claim 99, wherein the target antigen is a tumor antigen.

101. The polynucleotide, set of polynucleotides or composition of claim 99, wherein the target antigen is selected from the group consisting of: αvβ6 integrin, B cell maturation antigen, B7-H3, B7-H6, carbonic anhydrase 9, cancer-testis antigen, cancer / testis antigen 1B, carcinoembryonic antigen, cyclin, cyclin A2, CC motif chemokine ligand 1, CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, C D123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4, epidermal growth factor protein, truncated epidermal growth factor protein, epidermal growth factor receptor type III mutation, epithelial glycoprotein 2, epithelial glycoprotein 40, ephrin B2, ephrin receptor A2, estrogen receptor, Fc receptor-like protein 5, fetal acetylcholine receptor, folate binding protein, folate receptor alpha, ganglioside GD2, O-acetylated GD2, ganglioside GD3, glycoprotein 100, phosphatidylinositol proteoglycan, G protein-coupled receptor 5D, Her 2 / neu, Her3, Her4, erbB dimer, human high molecular weight melanoma-associated antigen, hepatitis B surface antigen, human leukocyte antigen A1, human leukocyte antigen A2, IL-22 receptor alpha, IL-13 receptor alpha 2, kinase insert domain receptor, L1 cell adhesion molecule, leucine-rich repeat-containing protein 8 family member A, Lewis Y, melanoma-associated antigen A1, melanoma-associated antigen A3, melanoma-associated antigen A6, melanoma-associated antigen A10, mesothelin, c-Met, murine cytomegalovirus, mucin 1 , MUC16, natural killer cell family 2 member D ligand, melanin A, neural cell adhesion molecule, carcinoembryonic antigen, preferentially expressed melanoma antigen, progesterone receptor, prostate specific antigen, prostate stem cell antigen, prostate specific membrane antigen, receptor tyrosine kinase-like orphan receptor 1, survivin, trophoblast glycoprotein, tumor-associated glycoprotein 72, tyrosinase-related protein 1, tyrosinase-related protein 2, vascular endothelial growth factor receptor, vascular endothelial growth factor receptor 2, Wilms tumor 1, and / or molecules expressed by HIV, HCV, or HBV.

102. The polynucleotide of any one of claims 66-69, the set of polynucleotides of claim 70, or the composition of claim 71, wherein the encoded recombinant antigen receptor comprises a T cell receptor or an antigen binding fragment thereof.

103. The polynucleotide of any one of claims 66-69, the set of polynucleotides of claim 70, or the composition of claim 71, wherein the encoded recombinant antigen receptor is a chimeric antigen receptor.

104. The polynucleotide, set of polynucleotides or composition of claim 103, wherein the encoded recombinant antigen receptor comprises an extracellular domain comprising an antigen binding domain.

105. The polynucleotide of any one of claims 66-69, the set of polynucleotides of claim 70, or the composition of claim 71, wherein the encoded recombinant antigen receptor comprises an extracellular domain comprising an antigen binding domain.

106. The polynucleotide, set of polynucleotides or composition of claim 104, wherein the antigen binding domain comprises an antibody or an antibody fragment thereof.

107. The polynucleotide, set of polynucleotides or composition of claim 106, wherein the fragment comprises an antibody variable region connected by a flexible linker.

108. The polynucleotide, set of polynucleotides or composition of claim 106, wherein the fragment comprises a scFv.

109. The polynucleotide, set of polynucleotides or composition of claim 104, wherein the encoded recombinant antigen receptor further comprises a spacer and / or a hinge region.

110. The polynucleotide, set of polynucleotides or composition of claim 109, wherein the encoded recombinant antigen receptor comprises an intracellular signaling domain.

111. The polynucleotide of any one of claims 66-69, the set of polynucleotides of claim 70, or the composition of claim 71, wherein the encoded recombinant antigen receptor comprises an intracellular signaling domain.

112. The polynucleotide, set of polynucleotides or composition of claim 110, wherein the intracellular signaling region comprises an intracellular signaling domain.

113. The polynucleotide, set of polynucleotides or composition cell of claim 112, wherein the intracellular signaling domain comprises a primary signaling domain, a signaling domain capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor component, and / or a signaling domain comprising an immunoreceptor tyrosine-based activation motif.

114. The polynucleotide, set of polynucleotides or composition of claim 113, wherein the intracellular signaling domain comprises the intracellular signaling domain of the CD3 chain or a signaling portion thereof.

115. The polynucleotide, set of polynucleotides or composition of claim 110, wherein the recombinant antigen receptor further comprises a transmembrane domain disposed between the extracellular domain and the intracellular signaling region.

116. The polynucleotide, set of polynucleotides or composition of claim 115, wherein the intracellular signaling region further comprises a co-stimulatory signaling region.

117. The polynucleotide, set of polynucleotides or composition of claim 116, wherein the co-stimulatory signaling region comprises an intracellular signaling domain of a T cell co-stimulatory molecule or a signaling portion thereof.

118. The polynucleotide, set of polynucleotides or composition of claim 116, wherein the co-stimulatory signaling region comprises an intracellular signaling domain of CD28, 4-1BB or ICOS, or a signaling portion thereof.

119. The polynucleotide, set of polynucleotides or composition of claim 116, wherein the co-stimulatory signaling region is located between the transmembrane domain and the intracellular signaling region.

120. The polynucleotide, set of polynucleotides or composition of claim 119, wherein the polynucleotide comprises, in 5' to 3' order: i) a nucleic acid encoding a signal peptide; ii) a nucleic acid encoding the recombinant antigen receptor, wherein the recombinant antigen receptor is a chimeric antigen receptor comprising an scFv; a spacer; a transmembrane domain; an intracellular region comprising a co-stimulatory signaling region, and an intracellular signaling domain of a CD3-zeta chain or a signaling portion thereof; iii) a nucleic acid sequence encoding a self-cleaving peptide or a peptide that causes ribosome skipping; and iv) a nucleic acid encoding the modified prostate-specific membrane antigen, wherein the prostate-specific membrane antigen or a modified form thereof comprises the amino acid sequence shown in SEQ ID NO: 52 or a fragment thereof; or an amino acid sequence that exhibits at least 95% sequence identity with SEQ ID NO: 52 or a fragment thereof and comprises a deletion of the one or more N-terminal amino acid residues.

121. A vector comprising the polynucleotide of any one of claims 66-69 and 72-120.

122. The vector according to claim 121, which is a viral vector.

123. The vector according to claim 122, which is a retroviral vector.

124. The vector of claim 123, which is a lentiviral vector or a gammaretroviral vector.

125. A vector set comprising a first vector and a second vector, wherein the first vector comprises a first polynucleotide according to any one of claims 70-120, and the second vector comprises a second polynucleotide according to any one of claims 70-120.

126. A composition comprising the vector set according to claim 125.

127. A method for producing an engineered cell, the method comprising introducing into the cell a polynucleotide according to any one of claims 66-69 and 72-120, or a polynucleotide in the group of polynucleotides according to any one of claims 70 and 72-120, or a polynucleotide in the composition according to any one of claims 71-120, or a vector according to any one of claims 121-124, or a vector in the group of vectors according to claim 125, or a vector in the composition according to claim 126.

128. An engineered cell produced by the method of claim 127.

129. An engineered cell comprising a polynucleotide according to any one of claims 66-69 and 72-120, or a polynucleotide in the polynucleotide group according to any one of claims 70 and 72-120, or a polynucleotide in the composition according to any one of claims 71-120, or a vector according to any one of claims 121-124, or a vector in the vector group according to claim 125, or a vector in the composition according to claim 126.

130. The engineered cell of claim 128, wherein: The cell is an immune cell; The cell is a T cell; The cells are NK cells; and / or The cells are derived from pluripotent or multipotent cells.

131. The engineered cell of claim 129, wherein: The cell is an immune cell; The cell is a T cell; The cells are NK cells; and / or The cells are derived from pluripotent or multipotent cells.

132. A composition comprising the engineered cells of any one of claims 1-65 and 128-131.

133. The composition of claim 132, further comprising a pharmaceutically acceptable excipient.

134. The composition of claim 132, further comprising a prostate specific membrane antigen targeting molecule.

135. Use of the engineered cell of any one of claims 1-65 and 128-131 or the composition of any one of claims 132-134 in the preparation of a medicament for treating a tumor in a subject, the treatment comprising administering the engineered cell or the composition to the subject.

136. The use according to claim 135, wherein the treatment further comprises: A prostate-specific membrane antigen targeting molecule or a composition comprising a prostate-specific membrane antigen targeting molecule is administered to the subject.

137. The use of claim 136, wherein the prostate-specific membrane antigen targeting molecule comprises a therapeutic agent.

138. The use according to claim 136 or 137, wherein the administration of the prostate-specific membrane antigen targeting molecule or a composition comprising the prostate-specific membrane antigen targeting molecule is performed simultaneously with or sequentially in any order with the administration of the engineered cells or a composition comprising the engineered cells.

139. The use according to claim 136 or 137, wherein the administration of the prostate-specific membrane antigen targeting molecule or a composition comprising the prostate-specific membrane antigen targeting molecule is carried out simultaneously with the administration of the engineered cells or a composition comprising the engineered cells.

140. The use according to claim 136 or 137, wherein the administration of the prostate-specific membrane antigen targeting molecule or the composition comprising the prostate-specific membrane antigen targeting molecule and the administration of the engineered cells or the composition comprising the engineered cells are performed sequentially in any order.

141. The use according to claim 136 or 137, wherein the prostate specific membrane antigen targeting molecule or a portion thereof: capable of binding to prostate-specific membrane antigen and / or the modified prostate-specific membrane antigen, and / or Capable of binding to the active site of prostate-specific membrane antigen and / or the active site of the modified prostate-specific membrane antigen, and / or capable of being cleaved by prostate-specific membrane antigen and / or cleaved by said modified prostate-specific membrane antigen; and / or It is an antagonist, inverse agonist, agonist, and / or inhibitor of prostate-specific membrane antigen and / or the modified prostate-specific membrane antigen.

142. The use according to claim 136 or 137, wherein the modified prostate specific membrane antigen is expressed on one or more of the engineered cells.

143. The use according to claim 136 or 137, wherein the prostate-specific membrane antigen targeting molecule comprises a small molecule, a ligand, an antibody or an antigen-binding fragment thereof, an aptamer, a peptide or a conjugate thereof.

144. The use according to claim 136 or 137, wherein the prostate-specific membrane antigen targeting molecule comprises a small molecule.

145. The use according to claim 144, wherein the small molecule is selected from the group consisting of: 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid, N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine, (aminostyryl)pyridinium dye, 2-(3-[1-carboxy-5-[(5-iodo-pyridine-3-carbonyl)-amino]-pentyl]-ureido)-glutaric acid, 2-[3-[1-carboxy-5-[(4-iodo-benzoylamino]- )-pentyl]-ureido]-pentanedioic acid, 1-(3-carboxy-4-(3-hydroxy-6-oxo-6H-xanthen-9-yl)phenylamino)-9,16,24-trioxo-1-thioxo-2,8,17,23,25-pentaazaoctacosane-7,22,26,28-tetracarboxylic acid, Glu-NH-CO-NH-Lys(Ahx)-HBED-CC, 9-(4-fluoro-3-[hydroxymethyl]butyl)guanine, Glu-urea-Lys-(Ahx)-[(HBED-CC)], PSMA-617, 2-(phosphonomethyl) 2-(2-(4-(1,3-dicarboxypropyl)ureido)pentanedioic acid, 2-PMPA, fluorobenzylphosphoramidate, (2S,4S)-2-fluoro-4-(phosphonomethyl)pentanedioic acid, N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-S-methyl-L-cysteine, EuK-Subkff-DOTAGA, 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid, PSMAN, N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid, MIP-1072, MIP-1095, MIP-1404, MIP-14 05 and N-[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-L-glutamate, (S)-2-(4-iodobenzylphosphonylmethyl)-glutaric acid, 2-[(3-{4-[(2-amino-4-hydroxy-pteridin-6-ylmethyl)-amino]-benzoylamino}-3-carboxy-propyl)-hydroxy-phosphorylmethyl]-glutaric acid, (2S,3'S)-{[(3'-amino-3'-carboxy-propyl)-hydroxyphosphoryl]methyl}-glutaric acid and (2S)-2-{[(2-carboxy-ethyl)-hydroxy-phosphoryl]methyl}-glutaric acid.

146. The use according to claim 145, wherein the prostate-specific membrane antigen targeting molecule comprises 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid or N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine.

147. The use according to claim 136 or 137, wherein the prostate-specific membrane antigen targeting molecule comprises an antibody or an antigen-binding fragment thereof, and the antibody or the antigen-binding fragment thereof has a binding site that specifically binds to the prostate-specific membrane antigen.

148. The use according to claim 136 or 137, wherein the prostate-specific membrane antigen targeting molecule comprises an aptamer or a conjugate thereof.

149. The use of claim 137, wherein the therapeutic agent is capable of modulating the tumor microenvironment or is cytotoxic to the tumor.

150. The use of claim 137, wherein the therapeutic agent is an immunomodulatory agent, a cytotoxic agent, an anticancer agent, or a radiotherapeutic agent.

151. according to the described purposes of claim 137, wherein said prostate-specific membrane antigen targeting molecule comprises a prodrug, and said prodrug comprises said therapeutic agent or can be converted into or expose said therapeutic agent, and / or said prostate-specific membrane antigen targeting molecule can be cleaved after combining with said modified prostate-specific membrane antigen, wherein cleavage produces at least one cleavage product comprising said therapeutic agent.

152. The use according to claim 151, wherein the prostate-specific membrane antigen targeting molecule comprises misagar (G-202 (8- O -(12-aminododecanoyl)-8- O -Debutyrylthapsigargin)-Asp- γ -Glu- γ -Glu- γ -GluGluOH).

153. The use according to claim 136 or 137, wherein the prostate specific membrane antigen targeting molecule is an antibody-drug conjugate.

154. according to the use described in claim 136 or 137, wherein said prostate-specific membrane antigen targeting molecule also comprises a therapeutic agent, and said therapeutic agent is directly or indirectly connected to the part of said prostate-specific membrane antigen targeting molecule capable of binding to said modified prostate-specific membrane antigen via a linker.

155. The use according to claim 154, wherein the linker is a peptide or polypeptide or a chemical linker.

156. The use according to claim 155, wherein the linker is a releasable linker or a cleavable linker.

157. The use according to claim 156, wherein the linker is capable of being cleaved after the modified prostate-specific membrane antigen is bound by the prostate-specific membrane antigen targeting molecule, wherein the cleavage produces at least one cleavage product comprising the therapeutic agent.

158. The use of claim 156, wherein the releasable linker or the cleavable linker is released or cleaved in the presence of one or more conditions or factors present in a tumor microenvironment, wherein the cleavage produces at least one cleavage product comprising the therapeutic agent.

159. The use of claim 158, wherein the one or more conditions or factors present in the tumor microenvironment comprise matrix metalloproteinases, hypoxic conditions, or acidic conditions.

160. The use according to claim 136 or 137, wherein the prostate-specific membrane antigen targeting molecule induces killing or destruction of one or more of the engineered cells and / or cells or tissues present in the subject that are specifically recognized by the recombinant antigen receptor.

161. The use of claim 137, wherein the therapeutic agent comprises a cytotoxic agent.

162. The use of claim 161, wherein the cytotoxic agent comprises a toxin.

163. The use according to claim 162, wherein the toxin is a peptide toxin, ricin A chain toxin, abrin A chain, diphtheria A chain, Pseudomonas exotoxin, Shiga A chain, gelonin, momordica charantia toxin protein, pokeweed antiviral protein, saporin, trichosanthin, aeromonas lysogen or barley toxin.

164. The use of claim 137, wherein the therapeutic agent comprises a photosensitizer.

165. The use according to claim 136 or 137, wherein the administration of the prostate-specific membrane antigen targeting molecule does not or substantially does not induce killing or destruction of healthy tissues or healthy cells, cells or tissues that do not contain the engineered cells and / or do not express the antigen.

166. The use of claim 137, wherein the therapeutic agent is an immunomodulator.

167. The use of claim 166, wherein the immunomodulator is an immune checkpoint inhibitor or modulator or cytokine.

168. The use according to claim 167, wherein the immunomodulator is an immune checkpoint inhibitor, which can inhibit or block the function of immune checkpoint molecules or signal transduction pathways involving immune checkpoint molecules.

169. The use according to claim 168, wherein the immune checkpoint molecule is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM3, VISTA, adenosine receptor or extracellular adenosine.

170. According to the use described in any one of claims 135-137, the treatment also includes detecting cells expressing the modified prostate-specific membrane antigen, and / or detecting the binding of the prostate-specific membrane antigen targeting molecule to the modified prostate-specific membrane antigen and / or the presence of the prostate-specific membrane antigen targeting molecule.

171. The use according to claim 170, wherein the detecting is performed in vivo and / or the detecting is performed via in vivo imaging.

172. Use of a prostate-specific membrane antigen targeting molecule in preparing a kit for detecting engineered cells, comprising: (a) contacting the engineered cell according to any one of claims 1-66 and 128-131 or the composition according to any one of claims 132-134 with the prostate-specific membrane antigen targeting molecule; and (b) detecting the binding of the prostate-specific membrane antigen targeting molecule to or with the modified prostate-specific membrane antigen and / or the engineered cells and / or the presence of the prostate-specific membrane antigen targeting molecule.

173. The use of claim 172, wherein the contacting comprises administering the prostate-specific membrane antigen targeting molecule to a subject to which the engineered cells have been administered.

174. Use of a prostate-specific membrane antigen targeting molecule in the preparation of a kit for detecting the presence or absence of engineered cells in a subject, comprising: (a) administering the prostate-specific membrane antigen targeting molecule to a subject who has previously been administered an engineered cell according to any one of claims 1-65 and 128-131 or a composition according to any one of claims 132-134, wherein the engineered cell expresses a recombinant antigen receptor and a modified prostate-specific membrane antigen in the subject; and (b) detecting the binding of the prostate-specific membrane antigen targeting molecule to the modified prostate-specific membrane antigen and / or to the engineered cells and / or the presence of the prostate-specific membrane antigen targeting molecule in the subject.

175. The use according to claim 174, wherein the detecting is performed via in vivo imaging.

176. The use according to any one of claims 172-175, wherein the prostate-specific membrane antigen targeting molecule or a portion thereof: capable of binding to prostate-specific membrane antigen and / or the modified prostate-specific membrane antigen, and / or Capable of binding to the active site of prostate-specific membrane antigen and / or the active site of the modified prostate-specific membrane antigen, and / or capable of being cleaved by prostate-specific membrane antigen and / or cleaved by said modified prostate-specific membrane antigen; and / or It is an antagonist, inverse agonist, agonist, and / or inhibitor of prostate-specific membrane antigen and / or the modified prostate-specific membrane antigen.

177. The use according to any one of claims 172-175, wherein the prostate-specific membrane antigen targeting molecule comprises a small molecule, a ligand, an antibody or an antigen-binding fragment thereof, an aptamer, a peptide or a conjugate thereof.

178. The use according to any one of claims 172-175, wherein the prostate-specific membrane antigen targeting molecule comprises a small molecule.

179. The use according to claim 178, wherein the small molecule is selected from the group consisting of: 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid, N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine, (aminostyryl)pyridinium dye, 2-(3-[1-carboxy-5-[(5-iodo-pyridine-3-carbonyl)-amino]-pentyl]-ureido)-glutaric acid, 2-[3-[1-carboxy-5-[(4-iodo-benzoylamino]- )-pentyl]-ureido]-pentanedioic acid, 1-(3-carboxy-4-(3-hydroxy-6-oxo-6H-xanthen-9-yl)phenylamino)-9,16,24-trioxo-1-thioxo-2,8,17,23,25-pentaazaoctacosane-7,22,26,28-tetracarboxylic acid, Glu-NH-CO-NH-Lys(Ahx)-HBED-CC, 9-(4-fluoro-3-[hydroxymethyl]butyl)guanine, Glu-urea-Lys-(Ahx)-[(HBED-CC)], PSMA-617, 2-(phosphonomethyl) 2-(2-(4-(1,3-dicarboxypropyl)ureido)pentanedioic acid, 2-PMPA, fluorobenzylphosphoramidate, (2S,4S)-2-fluoro-4-(phosphonomethyl)pentanedioic acid, N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-S-methyl-L-cysteine, EuK-Subkff-DOTAGA, 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid, PSMAN, N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid, MIP-1072, MIP-1095, MIP-1404, MIP-14 05 and N-[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-L-glutamate, (S)-2-(4-iodobenzylphosphonylmethyl)-glutaric acid, 2-[(3-{4-[(2-amino-4-hydroxy-pteridin-6-ylmethyl)-amino]-benzoylamino}-3-carboxy-propyl)-hydroxy-phosphorylmethyl]-glutaric acid, (2S,3'S)-{[(3'-amino-3'-carboxy-propyl)-hydroxyphosphoryl]methyl}-glutaric acid and (2S)-2-{[(2-carboxy-ethyl)-hydroxy-phosphoryl]methyl}-glutaric acid.

180. The use according to claim 179, wherein the prostate-specific membrane antigen targeting molecule comprises 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid or N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine.

181. The use according to any one of claims 172-175, wherein the prostate-specific membrane antigen targeting molecule comprises an antibody or an antigen-binding fragment thereof.

182. The use according to any one of claims 172-175, wherein the prostate-specific membrane antigen targeting molecule comprises an aptamer or a conjugate thereof.

183. The use of any one of claims 173-175, wherein the detecting comprises identifying a signal in the subject or a signal in a sample from the subject, wherein: The prostate-specific membrane antigen targeting molecule provides the signal or induces the detectable signal or is capable of binding to a portion that provides the signal or induces the detectable signal; and / or The prostate specific membrane antigen targeting molecule comprises a moiety that provides the signal or induces a detectable signal.

184. The use of claim 183, wherein the moiety comprises a radioactive isotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a chromogenic compound, a quantum dot, a nanoparticle, a metal chelate, or an enzyme.

185. The use according to claim 183, wherein the prostate-specific membrane antigen targeting molecule comprises an imaging probe or a detection agent.

186. according to the use described in claim 183, wherein said prostate-specific membrane antigen targeting molecule comprises said part, and / or said prostate-specific membrane antigen targeting molecule can be cut after binding to said modified prostate-specific membrane antigen, wherein cutting produces at least one cleavage product comprising said part and / or is fluorescent and / or radioactive.

187. according to the described purposes of claim 183, wherein said prostate-specific membrane antigen targeting molecule also comprises the part that provides signal or induces detectable signal, and said part is directly or indirectly connected to the part of said prostate-specific membrane antigen targeting molecule that can combine with said prostate-specific membrane antigen via a joint.

188. The use according to claim 187, wherein the linker is a releasable linker or a cleavable linker.

189. The use according to claim 188, wherein the linker is capable of being cleaved upon binding to the modified prostate-specific membrane antigen, wherein the cleavage produces at least one cleavage product comprising the portion and the cleavage product is fluorescent and / or radioactive.

190. The use of claim 189, wherein the releasable linker or the cleavable linker is released or cleaved in the presence of one or more conditions or factors present in the tumor microenvironment, wherein the cleavage produces at least one cleavage product comprising the portion and the cleavage product is fluorescent and / or radioactive.

191. The use of claim 190, wherein the one or more conditions or factors present in the tumor microenvironment comprise matrix metalloproteinases, hypoxic conditions, or acidic conditions.

192. The use according to claim 189, wherein the radioactive cleavage product comprises 11 C. 18 F. 64 Cu, 68 Ga, 68 Ge, 86 Y. 89 Zr, 90 Y. 99m Tc, 111 In, 123 I. 125 I. 177 Lu and / or 213 Bi.

193. according to the described purposes of claim 185, wherein said prostate specific membrane antigen targeting molecule is 2-(3-{1-carboxyl-5-[(6-[ 18 F] fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid or N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-[ 18 F] Fluorobenzyl-L-cysteine.

194. The use according to any one of claims 172-175, wherein said contacting and / or said detecting is performed in vivo and / or said detecting is performed via in vivo imaging.

195. The use according to claim 194, wherein the in vivo imaging is selected from the group consisting of: magnetic resonance imaging, single photon emission computed tomography, computed tomography, computed axial tomography, electron beam computed tomography, high resolution computed tomography, hypocycloidal tomography, positron emission tomography, scintigraphy, gamma camera, beta+ detector, gamma detector, fluorescence imaging, low light imaging, X-ray, bioluminescence imaging and near infrared optical tomography.

196. The use according to claim 194, wherein the in vivo imaging is positron emission tomography.

197. The use of claim 196, wherein the kit is capable of detecting as low as or as few as, or as low as or as few as approximately 4,000, 5,000, 6,000, 7,000, 8,000, 9,000 or 10,000 cells, or as low as or as few as approximately 4,000, 5,000, 6,000, 7,000, 8,000, 9,000 or 10,000 cells present in a specified volume.

198. The use according to claim 197, wherein the specified volume is of a liquid and / or the volume of an organ or tissue and / or is between 10 μL and 100 μL.

199. The use according to any one of claims 172-175, wherein the contacting and / or the detecting is performed in vitro or ex vivo.

200. The use according to any one of claims 172-175, wherein said contacting and / or said detecting comprises immunohistochemistry, immunocytochemistry or flow cytometry.

201. The use of claim 200, wherein the kit is capable of detecting as low as or as few as, or as low as or as few as approximately 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000 or 10,000 cells, or as low as or as few as approximately 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000 or 10,000 cells present in a specified volume.

202. The use according to claim 201, wherein the specified volume is of a liquid and / or the volume of an organ or tissue and / or is between 10 μL and 100 μL.

203. The use of claim 183, wherein the detecting further comprises determining the number or concentration of the engineered cells administered in the subject.

204. The use of claim 203, wherein determining comprises comparing the signal to a standard curve.

205. The use according to claim 204, wherein the standard curve is generated by detecting the signal from a plurality of control samples containing a defined number of cells expressing the modified prostate-specific membrane antigen, the plurality of control samples having been contacted with the prostate-specific membrane antigen targeting molecule.

206. The use according to claim 194, wherein the in vivo imaging is positron emission tomography and the prostate-specific membrane antigen targeting molecule is 2-(3-{1-carboxyl-5-[(6-[ 18 F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid.

207. A method for selecting, isolating or separating cells expressing a modified prostate specific membrane antigen, the method comprising: (a) contacting a plurality of cells comprising the engineered cells according to any one of claims 1-65 and 128-131 with a prostate specific membrane antigen targeting molecule; and (b) selecting, isolating or separating cells recognized or bound by the prostate-specific membrane antigen targeting molecule.

208. The method of claim 207, wherein the prostate-specific membrane antigen targeting molecule or a portion thereof: capable of binding to prostate-specific membrane antigen and / or the modified prostate-specific membrane antigen, and / or Capable of binding to the active site of prostate-specific membrane antigen and / or the active site of the modified prostate-specific membrane antigen, and / or capable of being cleaved by prostate-specific membrane antigen and / or cleaved by said modified prostate-specific membrane antigen; and / or It is an antagonist, inverse agonist, agonist, and / or inhibitor of prostate-specific membrane antigen and / or the modified prostate-specific membrane antigen.

209. The method of claim 207, wherein the plurality of cells comprises engineered cells comprising a polynucleotide according to any one of claims 66-69 and 72-120, a polynucleotide set according to any one of claims 70 and 72-120, or a composition according to any one of claims 71-120, or a vector according to any one of claims 121-124, a vector set according to claim 125, or a composition according to claim 126.

210. The method of claim 207, wherein the plurality of cells comprising the engineered cells comprises peripheral blood leukocytes from a subject who has been administered the engineered cells of any one of claims 1-65 and 128-131 or the composition of any one of claims 132-134.

211. The method of claim 207, wherein the prostate-specific membrane antigen targeting molecule comprises a small molecule, a ligand, an antibody or an antigen-binding fragment thereof, an aptamer, a peptide or a conjugate thereof.

212. The method of claim 207, wherein the prostate-specific membrane antigen targeting molecule comprises a small molecule.

213. The method of claim 212, wherein the small molecule is selected from the group consisting of: 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid, N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine, (aminostyryl)pyridinium dye, 2-(3-[1-carboxy-5-[(5-iodo-pyridine-3-carbonyl)-amino]-pentyl]-ureido)-glutaric acid, 2-[3-[1-carboxy-5-(4-iodo-benzoylamino)- )-pentyl]-ureido]-pentanedioic acid, 1-(3-carboxy-4-(3-hydroxy-6-oxo-6H-xanthen-9-yl)phenylamino)-9,16,24-trioxo-1-thioxo-2,8,17,23,25-pentaazaoctacosane-7,22,26,28-tetracarboxylic acid, Glu-NH-CO-NH-Lys(Ahx)-HBED-CC, 9-(4-fluoro-3-[hydroxymethyl]butyl)guanine, Glu-urea-Lys-(Ahx)-[(HBED-CC)], PSMA-617, 2-(phosphonomethyl) 2-(2-(4-(1,3-dicarboxypropyl)ureido)pentanedioic acid, 2-PMPA, fluorobenzylphosphoramidate, (2S,4S)-2-fluoro-4-(phosphonomethyl)pentanedioic acid, N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-S-methyl-L-cysteine, EuK-Subkff-DOTAGA, 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid, PSMAN, N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid, MIP-1072, MIP-1095, MIP-1404, MIP-14 05 and N-[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-L-glutamate, (S)-2-(4-iodobenzylphosphonylmethyl)-glutaric acid, 2-[(3-{4-[(2-amino-4-hydroxy-pteridin-6-ylmethyl)-amino]-benzoylamino}-3-carboxy-propyl)-hydroxy-phosphorylmethyl]-glutaric acid, (2S,3'S)-{[(3'-amino-3'-carboxy-propyl)-hydroxyphosphoryl]methyl}-glutaric acid and (2S)-2-{[(2-carboxy-ethyl)-hydroxy-phosphoryl]methyl}-glutaric acid.

214. The method of claim 213, wherein the prostate-specific membrane antigen targeting molecule comprises 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid or N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine.

215. The method of claim 207, wherein the prostate-specific membrane antigen targeting molecule comprises an antibody or an antigen-binding fragment thereof.

216. The method of claim 207, wherein the prostate-specific membrane antigen targeting molecule comprises an aptamer or a conjugate thereof.

217. The method of claim 207, wherein the prostate-specific membrane antigen targeting molecule is contained in a matrix or is immobilized on a solid support.

218. The method of claim 217, wherein the solid support comprises magnetic particles.

219. A kit comprising: (a) a composition comprising a therapeutically effective amount of an engineered cell according to any one of claims 1-65 and 128-131; and (b) A composition comprising a prostate-specific membrane antigen targeting molecule.

220. The kit of claim 219 for identifying a signal in a subject or a signal in a sample from a subject, wherein: The prostate-specific membrane antigen targeting molecule provides the signal or induces the detectable signal or is capable of binding to a portion that provides the signal or induces the detectable signal; and / or The prostate specific membrane antigen targeting molecule comprises a moiety that provides the signal or induces a detectable signal.

221. The kit of claim 219, further comprising instructions for administering the engineered cells and the prostate-specific membrane antigen targeting molecule in combination therapy to a subject being treated for a disease or condition to treat the disease or condition.

222. The kit of claim 219, further comprising instructions for administering the prostate-specific membrane antigen targeting molecule to a subject receiving or having been administered the engineered cells to detect the engineered cells.

223. The kit of claim 222, wherein the instructions further specify determining the number or concentration of engineered cells administered to the subject.

224. The kit of claim 223, wherein the instructions further specify that determining comprises comparing the signal to a standard curve.

225. The kit of claim 222, wherein the instructions specify that detection is performed via positron emission tomography and the prostate-specific membrane antigen targeting molecule is 2-(3-{1-carboxy-5-[(6-[ 18 F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid.

226. A kit comprising: (a) a composition comprising a therapeutically effective amount of an engineered cell according to any one of claims 1-65 and 128-131; and (b) instructions for administering the engineered cells to a subject for treatment of a disease or condition in combination therapy with a prostate-specific membrane antigen targeting molecule that comprises or also comprises a therapeutic agent for treating the disease or condition.

227. The kit of claim 226, wherein the prostate-specific membrane antigen targeting molecule or the therapeutic agent is capable of modulating the tumor microenvironment or is cytotoxic to the tumor.

228. The kit of claim 226, wherein the therapeutic agent is an immunomodulatory agent, a cytotoxic agent, an anticancer agent, or a radiotherapeutic agent.

229. The kit according to any one of claims 219-228, wherein the prostate-specific membrane antigen targeting molecule comprises a small molecule, a ligand, an antibody or an antigen-binding fragment thereof, an aptamer, a peptide or a conjugate thereof.

230. The kit of any one of claims 219-228, wherein the prostate-specific membrane antigen targeting molecule comprises a small molecule.

231. The kit of claim 230, wherein the small molecule is selected from the group consisting of: 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid, N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine, (aminostyryl)pyridinium dye, 2-(3-[1-carboxy-5-[(5-iodo-pyridine-3-carbonyl)-amino]-pentyl]-ureido)-glutaric acid, 2-[3-[1-carboxy-5-[(4-iodo-benzoyl)carbamoyl]-4-fluorobenzyl-L-cysteine, [(4-(3-(4-fluoro-3-[hydroxymethyl]butyl)guanine]-glutaric acid, 1-(3-carboxy-4-(3-hydroxy-6-oxo-6H-xanthen-9-yl)phenylamino)-9,16,24-trioxo-1-thioxo-2,8,17,23,25-pentaazaoctacosane-7,22,26,28-tetracarboxylic acid, Glu-NH-CO-NH-Lys(Ahx)-HBED-CC, 9-(4-fluoro-3-[hydroxymethyl]butyl)guanine, Glu-urea-Lys-(Ahx)-[(HBED-CC)], PSMA-617, 2-(phosphonomethyl) 2-(2-(4-(1,3-dicarboxypropyl)ureido)pentanedioic acid, 2-PMPA, fluorobenzylphosphoramidate, (2S,4S)-2-fluoro-4-(phosphonomethyl)pentanedioic acid, N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-S-methyl-L-cysteine, EuK-Subkff-DOTAGA, 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid, PSMAN, N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid, MIP-1072, MIP-1095, MIP-1404, MIP-14 05 and N-[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-L-glutamate, (S)-2-(4-iodobenzylphosphonylmethyl)-glutaric acid, 2-[(3-{4-[(2-amino-4-hydroxy-pteridin-6-ylmethyl)-amino]-benzoylamino}-3-carboxy-propyl)-hydroxy-phosphorylmethyl]-glutaric acid, (2S,3'S)-{[(3'-amino-3'-carboxy-propyl)-hydroxyphosphoryl]methyl}-glutaric acid and (2S)-2-{[(2-carboxy-ethyl)-hydroxy-phosphoryl]methyl}-glutaric acid.

232. The kit of claim 231, wherein the prostate-specific membrane antigen targeting molecule comprises 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid or N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine.

233. A kit according to any one of claims 219-228, wherein the prostate-specific membrane antigen targeting molecule comprises an antibody or an antigen-binding fragment thereof, which optionally has a binding site that specifically binds to a prostate-specific membrane antigen.

234. The kit according to any one of claims 219-228, wherein the prostate-specific membrane antigen targeting molecule comprises an aptamer or a conjugate thereof.

235. A prostate-specific membrane antigen targeting molecule comprising a portion capable of binding to a modified prostate-specific membrane antigen, wherein the modified prostate-specific membrane antigen comprises one or more amino acid modifications compared to a wild-type prostate-specific membrane antigen comprising a sequence shown in SEQ ID NO: 23, wherein the modified prostate-specific membrane antigen comprises a portion capable of binding to a modified prostate-specific membrane antigen. 23 at least 95% sequence identity, wherein the modified prostate specific membrane antigen is a modified human prostate specific membrane antigen, wherein the one or more amino acid modifications comprise one or more amino acid substitutions, deletions and / or insertions; wherein the modified prostate specific membrane antigen comprises at least one amino acid modification corresponding to a W2G substitution, a W2 substitution, or a W2 deletion and / or a deletion of up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 N-terminal amino acid residues, and wherein compared to wild-type prostate specific membrane antigen, the modified prostate specific membrane antigen (i) exhibits reduced endogenous signaling; (ii) exhibits increased cell surface expression; and / or (iii) exhibits reduced cellular internalization, and wherein the prostate specific membrane antigen targeting molecule is or further comprises an immunomodulator.

236. The prostate-specific membrane antigen targeting molecule according to claim 235, wherein the immunomodulator is capable of regulating the activity or immune response of immune cells and / or is capable of regulating the tumor microenvironment.

237. A prostate-specific membrane antigen targeting molecule according to claim 235, wherein the prostate-specific membrane antigen targeting molecule comprises a prodrug, the prodrug comprises the immunomodulator or can be converted into or expose the immunomodulator, and / or the prostate-specific membrane antigen targeting molecule can be cleaved after binding to the modified prostate-specific membrane antigen, wherein the cleavage produces at least one cleavage product comprising the immunomodulator.

238. The prostate-specific membrane antigen targeting molecule according to claim 235, wherein the immunomodulator is directly or indirectly linked via a linker to a portion of the prostate-specific membrane antigen targeting molecule that is capable of binding to the modified prostate-specific membrane antigen.

239. The prostate-specific membrane antigen targeting molecule according to claim 238, wherein the linker is a peptide or polypeptide or a chemical linker.

240. The prostate-specific membrane antigen targeting molecule of claim 238, wherein the linker is a releasable linker or a cleavable linker.

241. A prostate-specific membrane antigen targeting molecule according to claim 240, wherein the linker is capable of being cleaved after the modified prostate-specific membrane antigen is bound by a binding molecule, wherein the cleavage produces at least one cleavage product comprising the immunomodulator.

242. A prostate-specific membrane antigen targeting molecule according to claim 240, wherein the releasable linker or the cleavable linker is released or cleaved in the presence of one or more conditions or factors present in the tumor microenvironment, wherein the cleavage produces at least one cleavage product comprising the immunomodulator.

243. The prostate-specific membrane antigen targeting molecule of claim 242, wherein the one or more conditions or factors present in the tumor microenvironment comprise matrix metalloproteinases, hypoxic conditions, or acidic conditions.

244. The prostate-specific membrane antigen targeting molecule of claim 235, wherein the immunomodulator is an immune checkpoint inhibitor or regulator or a cytokine.

245. The prostate-specific membrane antigen targeting molecule according to claim 235, wherein the immunomodulator is an immune checkpoint inhibitor, which can inhibit or block the function of immune checkpoint molecules or signal transduction pathways involving immune checkpoint molecules.

246. A prostate-specific membrane antigen targeting molecule according to claim 245, wherein the immune checkpoint molecule is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM3, VISTA, adenosine receptor or extracellular adenosine.

247. A prostate-specific membrane antigen targeting molecule comprising a portion capable of binding to a modified prostate-specific membrane antigen, wherein the modified prostate-specific membrane antigen comprises one or more amino acid modifications compared to a wild-type prostate-specific membrane antigen comprising a sequence shown in SEQ ID NO: 23, wherein the modified prostate-specific membrane antigen comprises a portion capable of binding to a modified prostate-specific membrane antigen. 23 at least 95% sequence identity, wherein the modified prostate specific membrane antigen is a modified human prostate specific membrane antigen, wherein the one or more amino acid modifications comprise one or more amino acid substitutions, deletions and / or insertions; wherein the modified prostate specific membrane antigen comprises at least one amino acid modification corresponding to a W2G substitution, a W2 substitution, or a W2 deletion and / or a deletion of up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 N-terminal amino acid residues, and wherein compared to wild-type prostate specific membrane antigen, the modified prostate specific membrane antigen (i) exhibits reduced endogenous signaling; (ii) exhibits increased cell surface expression; and / or (iii) exhibits reduced cellular internalization, and wherein the prostate specific membrane antigen targeting molecule is or further comprises a therapeutic agent capable of modulating a tumor microenvironment, wherein the therapeutic agent is linked to the portion of the prostate specific membrane antigen targeting molecule via a releasable or cleavable linker that is responsive to one or more conditions or factors present in the tumor microenvironment.

248. The prostate-specific membrane antigen targeting molecule of claim 247, wherein the one or more conditions or factors present in the tumor microenvironment comprise matrix metalloproteinases, hypoxic conditions, or acidic conditions.

249. The prostate-specific membrane antigen targeting molecule of claim 247, wherein the therapeutic agent is an immunomodulator, a cytotoxic agent, an anticancer agent, or a radiotherapeutic agent.

250. The prostate specific membrane antigen targeting molecule according to claim 247, which is an antibody-drug conjugate.

251. The prostate-specific membrane antigen targeting molecule according to any one of claims 235-250, wherein the prostate-specific membrane antigen targeting molecule or a portion thereof: capable of binding to prostate-specific membrane antigen and / or the modified prostate-specific membrane antigen, and / or capable of binding to the active site of prostate-specific membrane antigen and / or the modified form of the modified prostate-specific membrane antigen, and / or capable of being cleaved by prostate-specific membrane antigen and / or cleaved by said modified prostate-specific membrane antigen; and / or It is an antagonist, inverse agonist, agonist, and / or inhibitor of prostate-specific membrane antigen and / or the modified prostate-specific membrane antigen.

252. The prostate-specific membrane antigen targeting molecule according to any one of claims 235-250, wherein the prostate-specific membrane antigen targeting molecule comprises a small molecule, a ligand, an antibody or an antigen-binding fragment thereof, an aptamer, a peptide or a conjugate thereof.

253. The prostate-specific membrane antigen targeting molecule according to any one of claims 235-250, wherein the prostate-specific membrane antigen targeting molecule comprises a small molecule and the small molecule is selected from the group consisting of: 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid, N-[N-[(S)-1,3-dicarboxypropyl]amino]-pentyl}-ureido)-glutaric acid, acyl]-4-fluorobenzyl-L-cysteine, (aminostyryl) pyridinium dye, 2-(3-[1-carboxy-5-[(5-iodo-pyridine-3-carbonyl)-amino]-pentyl]-ureido)-glutaric acid, 2-[3-[1-carboxy-5-(4-iodo-benzoylamino)-pentyl]-ureido]-glutaric acid, 1-(3-carboxy-4-(3-hydroxy-6-oxo-6H-xanthen-9-yl)benzene 9-(4-fluoro-3-[hydroxymethyl]butyl)guanine, Glu-urea-Lys-(Ahx)-[(HBED-CC)], PSMA- 617, 2-(Phosphonomethyl)glutaric acid, 2-PMPA, fluorobenzamide phosphoroester, (2S,4S)-2-fluoro-4-(phosphonomethyl)glutaric acid, N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-S-methyl-L-cysteine, EuK-Subkff-DOTAGA, 2-[3-(1,3-dicarboxypropyl)ureido]glutaric acid, PSMAN, N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid, MIP-1072, MIP-1095, MIP-1404, MIP-1405 and N-[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-L-glutamate, (S)-2-(4-iodobenzylphosphonylmethyl)-glutaric acid, 2-[(3-{4-[(2-amino-4-hydroxy-pteridin-6-ylmethyl)-amino]-benzoylamino}-3-carboxy-propyl)-hydroxy-phosphorylmethyl]-glutaric acid, (2S,3'S)-{[(3'-amino-3'-carboxy-propyl)-hydroxyphosphoryl]methyl}-glutaric acid and (2S)-2-{[(2-carboxy-ethyl)-hydroxy-phosphoryl]methyl}-glutaric acid.

254. The prostate-specific membrane antigen targeting molecule of claim 253, wherein the prostate-specific membrane antigen targeting molecule comprises 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-glutaric acid or N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine.

255. The prostate-specific membrane antigen targeting molecule according to any one of claims 235-250, wherein the prostate-specific membrane antigen targeting molecule comprises an antibody or an antigen-binding fragment thereof.

256. The prostate-specific membrane antigen targeting molecule according to any one of claims 235-250, wherein the prostate-specific membrane antigen targeting molecule comprises an aptamer or a conjugate thereof.

257. Use of a prostate-specific membrane antigen targeting molecule according to any one of claims 235-256 in the preparation of a medicament for treating a tumor in a subject, the treatment comprising administering the prostate-specific membrane antigen targeting molecule to the subject.

258. A preparation comprising an engineered cell according to any one of claims 1-65 and 128-131, a composition according to any one of claims 132-134, a polynucleotide according to any one of claims 66-69 and 72-120, a polynucleotide set according to any one of claims 70 and 72-120, or a composition according to any one of claims 71-120, or a vector according to any one of claims 121-124, a vector set according to claim 125, or a composition according to claim 126, a kit according to any one of claims 219-234, or a prostate-specific membrane antigen targeting molecule according to any one of claims 235-256.

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