[go: up one dir, main page]

WO2025212519A1 - Protéines de liaison au dll3 et leurs utilisations - Google Patents

Protéines de liaison au dll3 et leurs utilisations

Info

Publication number
WO2025212519A1
WO2025212519A1 PCT/US2025/022337 US2025022337W WO2025212519A1 WO 2025212519 A1 WO2025212519 A1 WO 2025212519A1 US 2025022337 W US2025022337 W US 2025022337W WO 2025212519 A1 WO2025212519 A1 WO 2025212519A1
Authority
WO
WIPO (PCT)
Prior art keywords
dll3
amino acid
seq
acid sequence
car
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/022337
Other languages
English (en)
Inventor
Ting-Lan ALBERSHARDT
Amanda Balboni INIGUEZ
Jordan JARJOUR
Cassandra Eve BURNETT
Julie Marie GARCIA
Jay Carroll Daniels
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Moonlight Bio Inc
Original Assignee
Moonlight Bio Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Moonlight Bio Inc filed Critical Moonlight Bio Inc
Publication of WO2025212519A1 publication Critical patent/WO2025212519A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/11—Antigen recognition domain
    • A61K2239/13—Antibody-based
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • the present disclosure relates to molecules binding to delta- like ligand 3 (DLL3), such as anti-DLL3 chimeric antigen receptors (CARs), anti-DLL3 antibodies including antigen binding domains, bispecific antibodies, antibody conjugates, anti-DLL3 immune cell engagers, and other fusion proteins comprising anti-DLL3 antigen binding domains, and the like, polynucleotides and vectors encoding DLL3 binding molecules, and engineered cells expressing DLL3 binding proteins, and methods of making and using the DLL3 binding proteins and engineered cells expressing the DLL3 binding proteins.
  • DLL3 delta- like ligand 3
  • Delta-like ligand 3 is a Notch inhibitory ligand, which is overexpressed on the surface of some cancer cells, such as small cell lung cancer (SCLC) cells, neuroendocrine carcinoma cells, and others. In contrast, DLL3 has limited expression or no expression on normal cells. (See Rudin et al., J. Hematol. Oncol. 16(article 66) (2023).) DLL3 is also expressed, for example, in pulmonary neuroendocrine carcinoma, large cell neuroendocrine carcinoma, gastric cancer, pancreatic cancer, such as gastroenteropancreatic cancer, bladder cancer, prostate cancer, and cervical cancer, such as gastric, pancreatic, gladder, prostate, and cervical neuroendocrine carcinomas.
  • SCLC small cell lung cancer
  • cervical cancer such as gastric, pancreatic, gladder, prostate, and cervical neuroendocrine carcinomas.
  • DLL3 binding proteins include anti-DLL3 chimeric antigen receptors, anti- DLL3 immune cell engagers, anti-DLL3 antibodies, anti-DLL3 antigen binding domains, and fusion proteins comprising anti-DLL3 antigen binding domains.
  • DARIC dimerizing agent regulated immune-receptor complex
  • ATOMIC antibody tethered orthogonal multiplexing compatible
  • SNIPR synthetic intramembrane proteolysis receptor
  • rapamycin inducible Fc receptor a multi-chain DAP-CAR
  • TREM1/DAP12 CAR a TREM1/DAP12 CAR
  • DAP12/TREM1 CAR DAP12/TREM1 CAR
  • the DLL3 binding protein or anti-DLL3 CAR of embodiment 20 or 21, wherein the hinge domain comprises the amino acid sequence of any one of SEQ ID NOs: 18 or 100-106.
  • the transmembrane domain comprises a transmembrane region domain of TCR alpha chain, TCR beta chain, TCR zeta chain, TCRgamma, TCRdelta, CD3gamma, CD3delta
  • the DLL3 binding protein or anti-DLL3 CAR of embodiment 23, wherein the transmembrane domain comprises the amino acid sequence of any one of SEQ ID NOs: 20 or 107-109.
  • the intracellular signaling domain comprises an intracellular signaling domain of CD28, 41BB (CD137), OX-40 (CD134), CD2, CD7, CD27, CD30, CD40, PD-1, ICOS, LFA-1 (CDIla/CD18), CD3gamma, CD3delta, CD3epsilon, CD3zeta, LxxCD3zeta, CD3zeta Q14K, CD247, CD276 (B7-H3), LIGHT, NKG2C, Ig alpha (CD79a), DAP- 10, an Fc gamma receptor, MHC class I molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, an integrin, a Signaling Lymphocytic Activation Molecule (SLAM), an activating NK cell receptor, BTLA, a Toll ligand receptor, B7-H3, CDS, ICAM-1,
  • the intracellular signaling domain comprises an intracellular signaling domain of CD3zeta, lxxCD3zeta, CD3zeta Q14K, CD28, 4- 1BB, or OX-40, or any combination thereof.
  • CD3zeta such as comprising the amino acid sequence of SEQ ID NO: 24, 62, or 63, or an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 24, 62, or 63.
  • CD3zeta such as comprising the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 24.
  • the intracellular signaling domain comprises an intracellular signaling domain of 4- IBB and/or comprises the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 22.
  • the intracellular signaling domain comprises (a) an intracellular signaling domain of CD3zeta, and/or comprises the amino acid sequence of SEQ ID NO: 24, 62, or 63 or an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 24, 62, or 63, and further comprises (b) an intracellular signaling domain of 4 IBB, and/or comprises the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 22.
  • the signal sequence comprises a CD8alpha signal sequence, an IgK signal sequence, or a GMCSFR-alpha signal sequence.
  • DLL3 binding protein or anti-DLL3 CAR of embodiment 33 wherein the signal sequence comprises the amino acid sequence of any one of SEQ ID NOs: 12, 74, or 75.
  • DLL3 binding protein or anti-DLL3 CAR of embodiment 37 or 38 wherein the epitope recognized by the monoclonal antibody comprises any one of SEQ ID NOs: 64-73; or wherein the epitope recognized by the monoclonal antibody is a CCR4 epitope; or wherein the DLL3 binding protein or anti-DLL3 CAR comprises the amino acid sequence of SEQ ID NO: 162 or is encoded by the nucleotide sequence of SEQ ID NO: 163.
  • the DLL3 binding protein or anti-DLL3 CAR of embodiment 40 or 41 wherein the second target protein is or comprises CD1, CDla, CDlb, CDlc, CDld, CDle, CD2, CD3d, CD3e, CD3g, CD3s, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD44v6, CD45, CD46, CD47 CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD97, CD123, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD171, CD178, CD179, CD179a, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD
  • SCLC small cell lung carcinoma
  • VH comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3; and a VL comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 4, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 6,
  • VH comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3; and a VL comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 4, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 6,
  • VH comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 149, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 150, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 151 ; and a VL comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 152, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 153, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 154, or
  • VH comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 155, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 156, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 157; and a VL comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 158, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 159, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 160;
  • nucleic acid construct of embodiment 52 wherein the promoter is a constitutive promoter or an inducible promoter.
  • the constitutive promoter is or comprises a CD4 promoter, CD8a promoter, CD8b promoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter, CD3e promoter, CD3z promoter, CARD9 promoter, CARDIO promoter, CARD11 promoter, CARD14 promoter, or PIK3R3 promoter.
  • nucleic acid construct of embodiment 51 wherein the construct comprises a polynucleotide sequence encoding the DLL3 binding protein or anti-DLL3 CAR that is not operably linked to a promoter.
  • nucleic acid construct of embodiment 61, wherein the one or more nucleic acid element sequences is or comprises ribosomal binding sites, enhancer elements, activator elements, translational start sequences, translational termination sequences, transcription start sequences, transcription termination sequences, polyadenylation signal sequences, a 70 bp poly(A) tract, a 100 bp poly(A) tract, a 172 bp poly(A) tract, a 200 bp poly(A) tract, a 300 bp poly(A) tract, a 325 bp poly(A) tract, replication elements, RNA processing and export elements, transposon sequences, transposase sequences, insulator sequences, internal ribosome entry sites (IRES), 5’UTRs, 3’UTRs, mRNA 3’ end processing sequences, boundary elements, locus control regions (LCR), matrix attachment regions (MAR), recombination or cassette exchange sequences, linker sequences, cleavable linker sequence
  • nucleic acid construct of embodiment 63, wherein the non-coding RNA comprises a shRNA or a microRNA.
  • the potency enhancement polypeptide is or comprises a patient derived CARD11-PIK3R3 fusion, an engineered CARD11- PIK3R3 fusion (such as comprising the amino acid sequence of any one of SEQ ID NOs: 114,
  • a dominant negative form of an inhibitor of a cell-mediated immune response of the immune cell e.g., TGF R2 DNR
  • a cell-mediated immune response of the immune cell e.g., TGF R2 DNR
  • c-Jun CCL19, CCL21, IL2R, IL7, IL7Ralpha, IL15, IL15RA, IL18, decoy-resistant IL18 (DR-18), MyD88/CD40, PD1-CD28 switch receptor, PD1- 41BB switch receptor, CD40L-CD28 switch receptor, CTBR12 switch receptor, CD8alpha/beta, a combination thereof, or variants thereof.
  • IRS internal ribozyme entry site
  • RNA construct is an mRNA construct.
  • nucleic acid construct of embodiment 78, wherein the one or more modified nucleotides are or comprise pseudouridine, N1 -methylpseudouridine, 4 ’-thiouridine, 5- methylcytosine, 2-thio-l-methyl- 1 -deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5- aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4- methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl- pseudouridine, 4- thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5 -methyluridine, 5- methoxyuridine, 2’-O-methyl uridine, or any combination thereof.
  • nucleic acid construct of embodiment 79 wherein one or more modified nucleotides are selected from the group consisting of pseudouridine, N1 -methylpseudouridine, 5- methylcytosine, 5- methoxyuridine, and a combination thereof.
  • nucleic acid construct of embodiment 80 wherein one or more modified nucleotides comprise Nl- methylpseudouridine.
  • nucleic acid construct of any one of embodiments 51-81 comprising a first homology arm before the 5’ end of the nucleic acid sequence encoding the recombinant polypeptide and a second homology arm after the 3 ’ end of the nucleic acid sequence encoding the recombinant polypeptide; optionally, wherein the lengths of the two homology arms are between about 150 bp and about 1500 bp; optionally wherein the length of at least one of the first or second homology arms is about 200bp, 225 bp, about 250 bp, about 275 bp, about 300 bp, about 325 bp, about 350 bp, about 375 bp, about 400 bp, about 425 bp, about 450 bp, about 475 bp, or about 500 bp; optionally, wherein the lengths of both of the homology arms are about 225 bp, about 250 bp, about 275 bp, about 300 b
  • nucleic acid construct of any one of embodiments 51-82 wherein the nucleic acid construct is less than 10 kb, less than 9 kb, less than 8 kb, less than 7 kb, less than 6 kb, less than 5 kb, less than 4 kb, or less than 3 kb in length, optionally wherein the nucleic acid construct is less than about 5.0 kb in length, and optionally wherein the nucleic acid construct is less than about 4.7 kb in length.
  • a nucleic acid construct comprising, from 5’ to 3’:
  • a nucleic acid construct comprising, from 5’ to 3’:
  • (6) a polynucleotide encoding an additional potency enhancement polypeptide, a cytokine, a chemokine, or a growth factor.
  • cleavable linker encoding sequence a polynucleotide encoding an additional potency enhancement polypeptide, a cytokine, a chemokine, or a growth factor.
  • a nucleic acid construct comprising, from 5’ to 3’:
  • a nucleic acid construct comprising, from 5’ to 3’:
  • a nucleic acid construct comprising, from 5’ to 3’:
  • a nucleic acid construct comprising, from 5’ to 3’:
  • a nucleic acid construct comprising, from 5’ to 3’:
  • a nucleic acid construct comprising, from 5’ to 3’:
  • a nucleic acid construct comprising, from 5’ to 3’:
  • a nucleic acid construct comprising, from 5’ to 3’:
  • a nucleic acid construct comprising, from 5’ to 3’:
  • a nucleic acid construct comprising, from 5’ to 3’:
  • a nucleic acid construct comprising, from 5’ to 3’ :
  • a particle comprising the nucleic acid construct of any one of embodiments 51- 102 or the composition of any one of embodiments 103-105.
  • the particle of embodiment 126, wherein the particle is a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • the particle of embodiment 126, wherein the particle is a selective organ targeting (SORT) LNP.
  • SORT selective organ targeting
  • an ionizable lipid e.g., an amino lipid
  • a sterol or other structural lipid e.g., a sterol or other structural lipid
  • a non-cationic helper lipid or phospholipid e.g., a PEG-modified lipid
  • An engineered cell that expresses the DLL3 binding protein or anti-DLL3 CAR of any one of embodiments 1-50 or the nucleic acid construct of any one of embodiments 51-102, wherein in the DLL3 binding protein or anti-DLL3 CAR or the nucleic acid construct is under the control of an endogenous promoter.
  • the endogenous promoter is a TCRa promoter, a TCRb promoter, a CD3d promoter, a CD3g promoter, a CD3e promoter, a CD3z promoter, a CARD9 promoter, a CARDIO promoter, a CARD11 promoter, a CARD14 promoter, or a PIK3R3 promoter.
  • An engineered cell that expresses the DLL3 binding protein or anti-DLL3 CAR of any one of embodiments 1-50 or the nucleic acid construct of any one of embodiments 51-102, wherein in the DLL3 binding protein or anti-DLL3 CAR or the nucleic acid construct is under the control of an exogenous promoter.
  • exogenous promoter is or comprises a minimal TATA promoter, pGK promoter, actin promoter, CD4 promoter, CD8a promoter, CD8b promoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter, CD3e promoter, CD3z promoter, CARD9 promoter, CARDIO promoter, CARD11 promoter, CARD 14 promoter, PIK3R3 promoter, CD25 promoter, IL-2 promoter, IL7 promoter, IL 15 promoter, KLRG-1 promoter, HLA-DR promoter, CD38 promoter, CD69 promoter, Ki-67 promoter, CD1 la promoter, CD58 promoter, CD99 promoter, CD62L promoter, CD103 promoter, CCR4 promoter, CCR5 promoter, CCR6 promoter, CCR9 promoter, CCR10 promoter, CXCR3 promoter, CXCR4 promoter, CLA promoter,
  • TCR T-cell receptor
  • CD3 CD3 locus
  • B2M B2 microglobulin
  • CIITA class II transactivator
  • TCR locus is or comprises a TRAJ locus, a TRAC locus, a TRBC1 locus, or a TRBC2 locus, optionally wherein the TRAJ locus is or comprises a TRAJ intron splice acceptor locus, optionally wherein the cell does not express a functional gene product of an endogenous TRAJ locus.
  • CD3 locus is or comprises: a CD3d locus, a CD3g locus, a CD3e locus, or CD3z locus.
  • T cell is a regulatory T cell (Treg), a gamma delta T cell, a CD8+ T cell, an invariant iNKT cell, a MAIT cell, a CAR T cell, a tumor-infiltrating lymphocyte, or an engineered T cell comprising a transcriptional receptor.
  • Reg regulatory T cell
  • gamma delta T cell a CD8+ T cell
  • an invariant iNKT cell a MAIT cell
  • CAR T cell a tumor-infiltrating lymphocyte
  • tumor-infiltrating lymphocyte or an engineered T cell comprising a transcriptional receptor.
  • the engineered cell of embodiment 157 wherein the engineered cell comprises a nucleic acid construct encoding the second CAR or recombinant TCR; optionally, wherein the nucleic acid construct is an RNA construct.
  • the second target antigen is or comprises CD1, CDla, CDlb, CDlc, CDld, CDle, CD2, CD3d, CD3e, CD3g, CD3s, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD44v6, CD45, CD46, CD47 CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD97, CD123, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD171, CD178, CD179, CD179a, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD2
  • the engineered cell of embodiment 162, wherein the second target antigen is or comprises the EGF1 domain of DLL3, the EGF2 domain of DLL3, the EGF3 domain of DLL3, the EGF4 domain of DLL3, the EGF5 domain of DLL3, or the EGF6 domain of DLL3.
  • non-coding RNA comprises a shRNA or a microRNA.
  • the potency enhancement polypeptide is or comprises a patient derived CARD11-PIK3R3 fusion (such as comprising the amino acid sequence of any one of SEQ ID NOs: 114 or 116), an engineered CARD11-PIK3R3 fusion (such as comprising the amino acid sequence of any one of SEQ ID NOs: 118 or 120), a dominant negative form of an inhibitor of a cell-mediated immune response of the immune cell (e.g., TGF0R2 DNR), c-Jun, CCL19, CCL21, IL2R, IL7, IL7Ralpha, IL15, IL15RA, IL18, decoy-resistant IL18 (DR-18), MyD88/CD40, PD1-CD28 switch receptor, PD1-41BB switch receptor, CD40L-CD28 switch receptor, CTBR12 switch receptor, CD8alpha/beta, or a combination thereof.
  • a cell-mediated immune response of the immune cell e.g., TGF0
  • the depletion tag comprises an epitope recognized by a monoclonal antibody, optionally wherein the epitope recognized by the monoclonal antibody comprises a CD20 epitope, a RSV protein F epitope, a EGF receptor epitope, a PD-1 epitope, a CD52 epitope, a CCR4 epitope, or a QB END- 10 epitope, or a combination thereof.
  • the engineered cell of embodiment 169, wherein the epitope recognized by the monoclonal antibody comprises any one of SEQ ID NOs: 64-73, or wherein the epitope recognized by the monoclonal antibody is a CCR4 epitope.
  • the safety switch is or comprises a herpesvirus thymidine kinase (HSV-tk) gene, an Escherichia coli cytosine deaminase (EC-CD) gene, or an inducible Caspase9 (iCasp9) protein.
  • HSV-tk herpesvirus thymidine kinase
  • EC-CD Escherichia coli cytosine deaminase
  • iCasp9 inducible Caspase9
  • cancer is sarcoma, carcinoma, neuroendocrine neoplasm, neuroendocrine carcinoma, bladder cancer, brain cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, kidney cancer, renal pelvis cancer, leukemia, lung cancer, small cell lung cancer, non-small cell lung cancer, melanoma, lymphoma, pancreatic cancer, prostate cancer, neuroendocrine prostate cancer, ovarian cancer, fibrosarcoma, glioma, glioblastoma, glioblastoma multiforme, neuroblastoma, renal clear cell carcinoma, adrenocortical carcinoma, bladder urothelial carcinoma, diffuse large B-cell lymphoma, lung adenocarcinoma, pancreatic adenocarcinoma, renal cell cancer, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, indolent B cell lymphoma
  • embodiment 248 or 249 wherein the cancer is small cell lung cancer, neuroendocrine neoplasm (NEN), neuroendocrine carcinoma (NEC), glioma, glioblastoma, neuroblastoma, melanoma, Wilms & rhabdoid tumors, or neuroendocrine prostate cancer.
  • NNN neuroendocrine neoplasm
  • NEC neuroendocrine carcinoma
  • glioma glioblastoma
  • neuroblastoma neuroblastoma
  • melanoma Wilms & rhabdoid tumors
  • neuroendocrine prostate cancer or neuroendocrine prostate cancer.
  • embodiment 247-255 wherein the medicament is not for administration within 7 days after administration of cyclophosphamide, fludarabine, or bendamustine.
  • SCLC small cell lung carcinoma
  • the one or more additional therapies comprise chemotherapy, immunotherapy, surgery, radiotherapy, anti-angiogenic agent, anti- DNA repair agent, anti-inflammatory agent, an anti-neoplastic agent, a growth inhibitory agent, a cytotoxic agent, a hormonal agent, or any combination thereof, wherein for example, the one or more additional therapies may comprise an inhibitor, agonist, small molecule, peptide, protein, fusion protein, antibody, nucleic acid, virus, antibody drug conjugate, PROTAC (proteolysis targeting chimera), cell therapy, or natural substance.
  • the one or more additional therapies may comprise an inhibitor, agonist, small molecule, peptide, protein, fusion protein, antibody, nucleic acid, virus, antibody drug conjugate, PROTAC (proteolysis targeting chimera), cell therapy, or natural substance.
  • embodiment 260 wherein the one or more additional therapies comprise a second DLL3 binding protein, a second DLL3 CAR, an anti-DLL3 antibody, an anti- DLL3 immune cell engager, or tarlatamab.
  • an anti-DLL3 antibody wherein the anti-DLL3 antibody comprises: (i) a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (CDRH1) comprising the amino acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3; and/or wherein the anti-DLL3 antibody comprises a light chain variable region (VL) comprising a light chain CDR1 (CDRLI) comprising the amino acid sequence of SEQ ID NO: 4, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 6; (ii) a VH comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 149, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 150, and a CDRH3 comprising the amino acid sequence
  • anti-DLL3 antibody of any one of embodiments 263-266, wherein the antibody is an antigen binding domain, such as a Fab, F(ab)2, Fv, or scFv.
  • BiTE bispecific T cell engager antibody
  • SMITE simultaneous multiple interaction T cell engager antibody
  • TRiKE trispecific killer engager
  • embodiment 287 or 288, wherein the cancer is small cell lung cancer, neuroendocrine neoplasm (NEN), neuroendocrine carcinoma (NEC), glioma, glioblastoma, neuroblastoma, melanoma, Wilms & rhabdoid tumors, or neuroendocrine prostate cancer.
  • NNN neuroendocrine neoplasm
  • NEC neuroendocrine carcinoma
  • glioma glioblastoma
  • neuroblastoma neuroblastoma
  • melanoma Wilms & rhabdoid tumors
  • Wilms & rhabdoid tumors or neuroendocrine prostate cancer.
  • the one or more additional therapies comprise chemotherapy, immunotherapy, surgery, radiotherapy, anti- angiogenic agent, anti-DNA repair agent, anti-inflammatory agent, an anti-neoplastic agent, a growth inhibitory agent, a cytotoxic agent, a hormonal agent, or any combination thereof
  • the one or more additional therapies may comprise an inhibitor, agonist, small molecule, peptide, protein, fusion protein, antibody, nucleic acid, virus, antibody drug conjugate, PROTAC (proteolysis targeting chimera), cell therapy, or natural substance.
  • the one or more additional therapies comprise a second DLL3 binding protein, a second DLL3 CAR, an anti-DLL3 antibody, an anti-DLL3 immune cell engager, or tarlatamab.
  • Fig. 1 shows affinity and cross-reactivity data for a humanized rabbit monoclonal antibody (RabmAb), and shows that the antibody binds to the EGF6 domain of human DLL3, and that the antibody also binds to the cynomolgus DLL3, has some cross-reactivity with murine DLL3, and does not bind to human DLL1 or to human DLL4.
  • RabmAb rabbit monoclonal antibody
  • Figs. 2A-2C show the architecture of the ML73 CAR.
  • Figs. 2B-2C show that the ML73 CAR and comparative constructs showed robust expression in primary T cells as measured by percent CAR positivity (Fig. 2B) and CAR MFI (Fig. 2C).
  • Figs. 3A-3B show that the CAR T cells expressing ML73 produced significantly more cytokines IFN-gamma (Fig. 3A) or IL-2 (Fig. 3B) in response to antigen compared to comparator CARs or CARs made with alternative rabbit monoclonal antibodies.
  • Figs. 6A-6F show IL-2 (Figs. 6A-6C) and IFN-gamma (Figs. 6D-6F) secretion levels from CAR T cells co-cultured with a cell line expressing a low level of DLL3 (Fig. 6A and 6D), a middle level of DLL3 (Fig. 6B and 6E), and a high level of DLL3 (Fig. 6C and 6F), in each case showing that CAR T cells expressing ML73 CAR produced significantly higher levels of IL-2 and IFN-gamma.
  • Figs. 7A-7B show tumor progression (Fig. 7A) and percent survival (Fig. 7B) following administration of ML73 CAR T cells to mice engrafted with small cell lung cancer (SCLC) tumor cells.
  • SCLC small cell lung cancer
  • Figs. 8A-8D show results from delivery of ML73 CAR to the TRAJ intron splice acceptor locus of primary human T cells via an AAV6 delivery mechanism.
  • Fig. 8A shows CAR positivity.
  • Fig 8B shows CAR MFI, comparing the AAV delivery mechanism to a that of a lentivirus mechanism and an untransduced control, comparing the AAV delivery mechanism to a that of a lentivirus mechanism and an untransduced control.
  • Figs. 8A-8D show results from delivery of ML73 CAR to the TRAJ intron splice acceptor locus of primary human T cells via an AAV6 delivery mechanism.
  • Fig. 8A shows CAR positivity.
  • Fig 8B shows CAR MFI, comparing the AAV delivery mechanism to a that of a lentivirus mechanism and an untransduced control, comparing the AAV delivery mechanism to a that of a lentivirus mechanism and an untransduced control.
  • FIGS. 8C and 8D show IFN-gamma and IL-2 secretion, respectively, comparing the AAV delivery mechanism to a that of a lentivirus mechanism and an untransduced control, and when T cells are co-cultured with three different cell lines, A375, SHP-77, and DMS-273, the latter two being DLL3 antigen positive tumor cell lines.
  • Figs. 9A-9C show results from an in vivo experiment assessing the potency of ML73 CAR inserted into the TRAJ intron splice acceptor locus of primary human T cells via an AAV6 delivery mechanism.
  • Fig. 9A shows tumor growth for mice administered Untransduced T cells population
  • Fig. 9B and Fig. 9C show tumor growth for mice administered le6 CAR+T cells and 0.2e6 CAR+ T cells respectively.
  • a cell includes one or more cells, including mixtures thereof.
  • a and/or B is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B.”
  • a range includes each individual member.
  • a group having 1-3 articles refers to groups having 1 , 2, or 3 articles.
  • a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.
  • polypeptide and “protein” are used interchangeably to refer to a polymer of amino acids linked by peptide bonds.
  • the term is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, diabodies, etc., as well as Immune cell engagers such as bispecific T cell engager antibodies (BiTE), simultaneous multiple interaction T cell engager antibodies (SMITE), or trispecific killer engagers (TRiKE) and the like), full length antibodies, single-chain antibodies, antibody conjugates, and antibody fragments, so long as they exhibit the desired binding activity.
  • BiTE bispecific T cell engager antibodies
  • SMITE simultaneous multiple interaction T cell engager antibodies
  • TRiKE trispecific killer engagers
  • McCallum CDRs also called “Contact CDRs”
  • Other schemes include the IMGT scheme (LeFranc et al., Dev. Comp. Immunol. 27(1): 55-77 (2003), the “Aho” numbering scheme (Honegger and Pliickthun, J. Mol. Biol.
  • CDR or individual, specified CDR (e.g., CDR Hl, or CDR L2 or the like) should be understood to encompass a (or the specific) CDR as defined by any of the above schemes, or other known numbering schemes.
  • Exemplary Kabat CDR sequences for an anti-DLL3 antibody are included in the sequence table herein (e.g., SEQ ID NOs: 1-6), exemplary Martin “enhanced Chothia” CDR sequences for an anti-DLL3 antibody are included in the sequence table herein (e.g., SEQ ID NOs: 149-154), and exemplary structural modeling minimal CDR sequences for an anti-DLL3 antibody are included in the sequence table herein (e.g., SEQ ID NOs: 155-160).
  • CDRs of such an antibody may be defined by those sequences, or by sequences identified using any of the above schemes, or other known schemes.
  • “Framework” or “FR” refers to the residues of the variable region residues that are not part of the complementary determining regions (CDRs).
  • the FR of a variable region generally consists of four FRs: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR sequences generally appear in the following sequence in VH (or VL): FR1-CDR-H1(CDR-L1)-FR2- CDR- H2(CDR-L2)-FR3- CDR-H3(CDR-L3)-FR4.
  • acceptor human framework for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below.
  • An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some aspects, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.
  • the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.
  • variable region or “variable domain” interchangeably refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
  • the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementary determining regions (CDRs). See, e.g., Kindt et al. Kuby Immunology, 6 th ed., W.H. Freeman and Co., page 91 (2007).
  • a variable domain may comprise heavy chain (HC) CDR1-FR2-CDR2-FR3-CDR3 with or without all or a portion of FR1 and/or FR4; and light chain (LC) CDR1-FR2-CDR2-FR3-CDR3 with or without all or a portion of FR1 and/or FR4. That is, a variable domain may lack a portion of FR1 and/or FR4 so long as it retains antigen-binding activity.
  • a single VH or VL domain may be sufficient to confer antigen-binding specificity.
  • antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
  • each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three constant heavy domains (CHI, CH2, and CH3).
  • VH variable domain
  • CHI variable heavy domain
  • CH2 constant heavy domain
  • VL variable domain
  • CL constant light
  • Fc region herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
  • antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain.
  • an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain.
  • This may be the case where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to EU index). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447), of the Fc region may or may not be present.
  • a “full-length IgGl” for example, includes an IgGl with Gly446 and Lys447, or without Lys447, or without both Gly446 and Lys447.
  • Amino acid sequences of heavy chains including an Fc region are denoted herein without C-terminal glycine-lysine dipeptide if not indicated otherwise.
  • a heavy chain including an Fc region as specified herein, comprised in an antibody according to the invention may comprise Gly446 and Lys447 (numbering according to EU index).
  • a heavy chain including an Fc region as specified herein, comprised in an antibody according to the invention may comprise Gly446 (numbering according to EU index).
  • EU numbering system also called the EU index
  • EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • Effective functions refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.
  • the “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
  • the antibody is of the human IgGi IgG , IgGa, or IgG 4 isotype.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 6, e, y, and p, respectively.
  • the light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (A,), based on the amino acid sequence of its constant domain.
  • antibody fragment or “antigen binding domain” herein refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen (i.e. DLL3 or a domain of DLL3) to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv, and scFab); single domain antibodies (dAbs); tandem single domain antibodies (sdAbs); and multispecific antibodies formed from antibody fragments.
  • full length antibody “intact antibody”, and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or, in the case of an IgG antibody, having heavy chains that contain an Fc region as defined herein.
  • multispecific refers to a molecule that can bind to more than one different target or antigen, such as to two or three or more different targets or antigens.
  • bispecific refers to a molecule such as a binding protein or antibody that is able to specifically bind to two different targets or antigens.
  • a “multispecific” or “bispecific” antibody herein may include the appropriate full length heavy and light chains for binding to two different antigens, or it may include appropriate antibody fragments for binding to two different antigens.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • a “cell surface receptor” or “receptor” generally refers to a protein molecule that is expressed on or associated with the surface of a cell and that can bind or be recognized by molecules external to the cell, such as in the extracellular space or on the surface of other cells.
  • a “chimeric antigen receptor” or “CAR” refers to a fusion protein comprising an extracellular domain comprising an antigen binding domain of an antibody.
  • a CAR comprises an extracellular domain comprising an antigen binding domain of an antibody as well as a transmembrane domain.
  • a CAR is intended for expression on the cell surface, for example, to act as a cell surface receptor.
  • the CAR further comprises one or more intracellular signaling domains.
  • the CAR comprises a hinge between the antigen binding domain and the transmembrane domain.
  • the CAR comprises a signal peptide, which may be cleaved to form a mature protein.
  • a CAR comprise two or more intracellular signaling domains, such as CD3 (CD3zeta) (including 1XX CD3zeta and CD3zeta with a Q to K modification at position 14), as well as one or more co-stimulatory domains such as 41BB, OX-40, and/or CD28.
  • the antigen binding domain is a single chain antigen binding domain such as an scFv.
  • the CAR can be combined with a safety switch.
  • the safety switch comprises a depletion tag (or epitope tag) that is bound by a monoclonal antibody.
  • an “immune cell engager” refers to a fusion protein, such as an anti-DLL3 fusion protein, that is intended to direct immune cells to tumor cells, for example, to aid in killing of tumor cells.
  • an immune cell engager may comprise antibodies or antigen binding domains that bind to molecules on the surface of both a tumor cell and an immune cell, thus linking the two types of cells.
  • an immune cell engager may be soluble, i.e., is secreted from the cell that expresses it.
  • a “hinge” refers to a relatively flexible polypeptide sequence that may be placed between two domains of a protein to allow for greater flexibility.
  • a “depletion tag” herein refers to a substance or protein, such as in some cases a membrane-expressed protein or fragment thereof, which allows for cell depletion after administration of an antibody that recognizes that substance or protein.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA) including e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules.
  • DNA deoxyribonucleic acid
  • cDNA complementary DNA
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • the nucleic acid molecule may be linear or circular.
  • nucleic acid molecule includes both, sense and antisense strands, as well as single stranded and double stranded forms.
  • the herein described nucleic acid molecule can contain naturally occurring or non-naturally occurring nucleotides.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.
  • a “vector” is comprised within a “particle” herein, which particle may contain in some embodiments non-nucleic acid elements such as lipids or polymers. Examples of particles include, for instance, lipid nanoparticles (LNPs) or virus-like particles (VLPs) and the like, among others described herein.
  • LNPs lipid nanoparticles
  • VLPs virus-like particles
  • viral vector refers either to a nucleic acid molecule (e.g., a transfer plasmid) that includes virus-derived nucleic acid elements that generally facilitate transfer of the nucleic acid molecule or integration into the genome of a cell or to a viral particle that mediates nucleic acid transfer.
  • Viral particles will generally include various viral components and sometimes also host cell components in addition to nucleic acid(s).
  • the term viral vector may refer either to a virus or viral particle capable of transferring a nucleic acid into a cell or to the transferred nucleic acid itself.
  • Viral vectors and transfer plasmids contain structural and/or functional genetic elements that are primarily derived from a virus.
  • Viral vectors that can be used in the disclosure include, for example, retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors, lentivirus vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).
  • a recombinant polypeptide as disclosed herein can be produced in a eukaryotic host, such as a mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells).
  • the term “retroviral vector” refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus.
  • the retroviral vector can be a lentiviral vector.
  • the term “lentiviral vector” refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, including LTRs that are primarily derived from a lentivirus, which is a genus of retrovirus.
  • an engineered cell herein generally refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such cells.
  • the terms “host cell”, “host cell line”, and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells, and which are used to express the product of the introduced nucleic acid, such as a nucleic acid or protein product. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. References to “a cell” herein include reference to a population of such cells, including their progeny.
  • percent identity refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (e.g., about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection.
  • sequences are then the to be “substantially identical.”
  • This definition also refers to, or may be applied to, the complement of a test sequence.
  • This definition also includes sequences that have deletions and/or additions, as well as those that have substitutions.
  • sequence identity can exist over a region that is at least about 20 amino acids or nucleotides in length, or over a region that is 10-100 amino acids or nucleotides in length, or over the entire length of a given sequence
  • sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al, Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990). Sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with the default parameters thereof.
  • the term “mutation” refers to a point mutation, a gene fusion, a substitution, a gain-of-function mutation, a stop-gain mutation, an insertion mutation, a deletion mutation, a duplication mutation and/or a translocation.
  • the mutation may be in one or more genes.
  • the mutation may be naturally occurring. Alternatively, the mutation may be induced or engineered.
  • the term “vector” refers to a recombinant polynucleotide construct designed for transfer between host cells, and that may be used for the purpose of transformation, e.g., the introduction of heterologous DNA into a host cell.
  • the vector can be a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment.
  • the expression vector can be an integrating vector.
  • reduce or inhibit is meant the ability to cause an overall decrease of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or greater.
  • reduce or inhibit can refer to a relative reduction compared to a reference (e.g., reference level of biological activity or binding).
  • increase is meant the ability to cause an overall increase of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or greater.
  • increase can refer to a relative reduction compared to a reference (e.g., reference level of biological activity or binding).
  • reduce or increase may refer to reduction of the “level” (i.e. the amount or concentration) of a cell surface protein on a cell, for example.
  • pharmaceutical composition or “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.
  • the term “pharmaceutically acceptable carrier” means any suitable carriers, diluents or excipients. These include all aqueous and non-aqueous isotonic sterile injection solutions, which may contain anti-oxidants, buffers and solutes, which render the composition isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents, dispersion media, antifungal and antibacterial agents, isotonic and absorption agents and the like. It will be understood that compositions of the present disclosure may also include other supplementary physiologically active agents.
  • the carrier must be pharmaceutically “acceptable” in the sense of being compatible with the other ingredients of the composition and not injurious to the subject.
  • administering refers to the delivery of a bioactive composition or formulation by an administration route including, but not limited to, oral, intravenous, intra-arterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, and topical administration, or combinations thereof.
  • administration route including, but not limited to, oral, intravenous, intra-arterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, and topical administration, or combinations thereof.
  • the term includes, but is not limited to, administering by a medical professional and self-administering.
  • injection includes intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion.
  • cancer generally refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features.
  • Cancer cells can be in the form of a tumor, but such cells can exist alone within an animal subject, or can be a non-tumorigenic cancer cell, such as a leukemia cell. These terms include a solid tumor, a soft tissue tumor, or a metastatic lesion.
  • cancer includes premalignant, as well as malignant cancers.
  • the cancer is a solid tumor, a soft tissue tumor, or a metastatic lesion.
  • a “therapeutically effective” or “pharmaceutically effective” amount or number of a subject construct, nucleic acid, cell, or composition of the disclosure generally refer to an amount or number sufficient for a construct, nucleic acid, cell, or composition to accomplish a stated purpose relative to the absence of the composition, e.g., to provide a therapeutic benefit in the treatment or management of the cancer, or to delay or minimize one or more symptoms associated with the cancer.
  • a therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapeutic agents, which provides a therapeutic benefit in the treatment or management of the cancer.
  • terapéuticaally effective amount can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the cancer, or enhances the therapeutic efficacy of another therapeutic agent.
  • An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.”
  • a “reduction” of a symptom means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s).
  • the exact amount of a composition including a “therapeutically effective amount” will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols.
  • Embodiments herein include, for example, a D1L3 binding protein, which comprises an anti-DLL3 antigen binding domain.
  • the anti-DLL3 antigen binding domain binds to the EGF6 domain of DLL3.
  • the anti-DLL3 antigen binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (CDRH1) comprising the amino acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3; and/or wherein the anti-DLL3 antigen binding domain further comprises a light chain variable region (VL) comprising a light chain CDR1 (CDRL1) comprising the amino acid sequence of SEQ ID NO: 4, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 6.
  • VH heavy chain variable region
  • CDRL1 light
  • the anti-DLL3 antigen binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (CDRH1) comprising the amino acid sequence of SEQ ID NO: 149, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 150, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 151; and/or wherein the anti-DLL3 antigen binding domain further comprises a light chain variable region (VL) comprising a light chain CDR1 (CDRL1) comprising the amino acid sequence of SEQ ID NO: 152, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 153, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 154.
  • VH heavy chain variable region
  • CDRL1 light chain CDR1
  • the anti-DLL3 antigen binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (CDRH1) comprising the amino acid sequence of SEQ ID NO: 155, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 156, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 157; and/or wherein the anti-DLL3 antigen binding domain further comprises a light chain variable region (VL) comprising a light chain CDR1 (CDRL1) comprising the amino acid sequence of SEQ ID NO: 158, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 159, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 160.
  • VH heavy chain variable region
  • CDRL1 light chain CDR1
  • the anti-DLL3 antigen binding domain comprises a VH comprising an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 7, and/or a VL comprising an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 8 or 148.
  • the anti-DLL3 antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 7 and/or a VL comprising the amino acid sequence of SEQ ID NO: 8 or 148. In some embodiments, the anti-DLL3 antigen binding domain comprises a VH with an amino acid consisting of the sequence of SEQ ID NO: 7 and/or a VL with an amino acid sequence consisting of SEQ ID NO: 8 or 148. In some cases, the anti- DLL3 binding domain comprises a signal sequence. In other cases, it does not comprise a signal sequence.
  • the antigen binding domain is a single domain antibody (sdAb), a fragment antigen binding moiety (Fab), an Fv, a single chain Fv (scFv), a tandem scFv (bivalent and/or bispecific), tandem single domain antibody (sdAb), or a bi-specific Fab.
  • a single chain antigen binding domain is a camelid single-domain antibody, such as a VHH or nanobody.
  • the single chain antigen binding domain is an scFv
  • an anti-DLL3 antigen binding domain may be comprised within a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the present disclosure encompasses CARs comprising an extracellular domain comprising an anti-DLL3 antigen binding domain, such as an anti-DLL3 antigen binding domain as described above.
  • the CAR comprises an anti- DLL3 antibody fused to a CAR scaffold.
  • the anti-DLL3 CAR binds to the EGF6 domain of DLL3.
  • the anti-DLL3 CAR comprises an antigen binding domain as described above, and further comprises one or more of a linker sequence, e.g., as described above, a hinge domain, a transmembrane domain, one or more intracellular signaling domains, or a combination thereof.
  • the anti-DLL3 CAR comprises each of an antigen binding domain as described above, and further comprises one or more of a linker sequence, e.g., as described above, a hinge domain, a transmembrane domain, one or more intracellular signaling domains.
  • the CAR may also comprise a signal sequence.
  • the disclosed anti-DLL3 CARs further comprise a linker sequence or a functional fragment thereof.
  • the linker sequence comprises a Whitlow linker, a (G4S)n linker, or SG4S.
  • the disclosed anti-DLL3 CARs further comprise a hinge domain, or a functional fragment thereof.
  • the hinge domain comprises a CD8a hinge domain, a CD28 hinge domain, an IgG4 hinge domain, an IgG4 hinge-CH2-CH3 domain, or a variant thereof.
  • the disclosed anti-DLL3 CARs further comprise a transmembrane domain, or a functional fragment thereof.
  • the transmembrane domain is a CD8, CD8a, CD8P, 4- 1BB/CD137, CD28, CD34, CD4, FcaRIy, CD16, OX40/CD134, CD3 ⁇ , CD3e, CD3y, CD33, TCRa, TCRP, TCRy, TCR5, TCRiJ, CD32, CD64, CD5, CD9, CD22, CD33, CD37, CD38, CD40, CD45, CD64, CD80, CD86, CD137, CD154, LFA-1 T cell co-receptor, CD2 T cell co- receptor/adhesion molecule, CD4OL/CD154, VEGFR2, FAS, FcsRI, or FGFR2B transmembrane domain, or a variant thereof.
  • an anti-DLL3 CAR may be intended to be expressed on the cell surface, and thus,
  • the disclosed anti-DLL3 CARs may further comprise an intracellular domain comprising one or more of an activation domain and/or a costimulatory signaling domain (or a costimulatory domain).
  • the intracellular domain comprises a sequence encoding an activation domain.
  • the intracellular domain comprises a costimulatory signaling domain.
  • the intracellular domain comprises an activation domain and a costimulatory signaling domain.
  • the intracellular domain comprises an intracellular signaling domain of CD28, 41BB (CD137), OX-40 (CD134), CD2, CD7, CD27, CD30, CD40, PD-1, ICOS, LFA-1 (CDIla/CD18), CD3gamma, CD3delta, CD3epsilon, CD3zeta, l.xxCD3zeta, CD247, CD276 (B7-H3), LIGHT, NKG2C, Ig alpha (CD79a), DAP- 10, an Fc gamma receptor, MHC class I molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, an integrin, a Signaling Lymphocytic Activation Molecule (SLAM), an activating NK cell receptor, BTLA, a Toll ligand receptor, B7-H3, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), KIRDS2, SLAMF7,
  • SLAM
  • the linker sequence comprises any naturally occurring amino acid.
  • Certain exemplary linker sequences that may be included in a CAR herein are provided at SEQ ID Nos: 16, 38, or 40-60.
  • a linker is included between VH and VL domains of an scFv.
  • a linker is included between a transmembrane domain and an intracellular domain, as described below.
  • the linker comprises a G4S linker with 3 repeats (G4S)3.
  • the linker comprises the sequence of SEQ ID NO: 16.
  • the anti-DLL3 CAR described herein further comprises a hinge domain.
  • the hinge domain in some cases is located between the antigen-binding region and the transmembrane domain, if present, or between the antigen binding domain and a subsequent domain.
  • a hinge domain in some embodiments permits the movement of one or both of the antigen-binding region and transmembrane domain (or alternatively a subsequent domain) relative to each other.
  • the hinge domain comprises from about 10 to about 100 amino acids, e.g., from about 15 to about 75 amino acids, from about 20 to about 50 amino acids, or from about 30 to about 60 amino acids.
  • the hinge domain is a hinge domain of a naturally occurring protein.
  • the hinge domain may comprise a CD8a hinge domain, a CD28 hinge domain, an IgG4 hinge domain, an IgG4 hinge-CH2-CH3 domain, including a variant thereof.
  • the hinge domain comprises an amino acid sequence of any one of SEQ ID Nos: 18 or 101-106.
  • the hinge domain is a CD8a hinge domain.
  • the hinge domain comprises the amino acid sequence of SEQ ID NO: 18.
  • the anti-DLL3 CAR described herein further comprises a transmembrane domain.
  • the transmembrane domain is located after the antigen binding domain, in some cases wherein a hinge domain is located between the antigen binding domain and the transmembrane domain.
  • a transmembrane domain can include one or more additional amino acids adjacent to the transmembrane section, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane domain was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region).
  • one or more amino acid associated with the extracellular region of the protein from which the transmembrane domain was derived e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region
  • additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region
  • the transmembrane domain may be recombinant, in which case it can in some embodiments comprise predominantly hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and valine can be found at each end of a recombinant transmembrane domain.
  • the intracellular signaling domain comprises an intracellular signaling domain of CD3zeta, lxxCD3zeta, CD3zeta Q14K, CD28, 4-1BB, or OX-40, or any combination thereof, including variants thereof.
  • the intracellular signaling domain comprises an intracellular signaling domain of CD3zeta, such as comprising the amino acid sequence of SEQ ID NO: 24, 62, or 63, or an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 24, 62, or 6.
  • the intracellular signaling domain comprises an intracellular signaling domain of 4- IBB and/or comprises the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 22.
  • the intracellular signaling domain comprises (a) an intracellular signaling domain of CD3zeta, and/or comprises the amino acid sequence of SEQ ID NO: 24, 62, or 63 or an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 24, 62, or 63, and further comprises (b) an intracellular signaling domain of 41BB, and/or comprises the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 22.
  • the anti-DLL3 CARs comprise an intracellular signaling domain, which is an activation domain.
  • the activation domain is generally responsible for activation of at least one of the normal effector functions of a cell.
  • effector function describes a specialized function of a cell.
  • the effector function of a T-cell or an NK cell includes a cytolytic activity or helper activity.
  • Activation domain describes the portion of a protein which transduces the effector function signal and directs the cell to perform its specialized function. While the entire activation domain can be employed, in many cases it is not necessary to use an entire chain or domain. To the extent that a truncated portion of the activation domain is used, such truncated portion may be used in place of the intact domain as long as it transduces the effector function signal.
  • An activation domain can, in some embodiments, promote the activation of a T cell receptor (TCR) complex.
  • TCR T cell receptor
  • Such activation domains may comprise immunoreceptor tyrosinebased activation motifs (ITAMs).
  • ITAMs immunoreceptor tyrosinebased activation motifs
  • Activation domains containing ITAMs include the intracellular signaling domains of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
  • an activation domain comprises the intracellular signaling domain of CD3zeta, lxxCD3zeta, CD3zeta Q14K, or CD28.
  • the disclosed anti-DLL3 CARs comprise a costimulatory domain.
  • costimulatory domains for use in the chimeric receptors are cytoplasmic signaling domains of costimulatory proteins such as B7/CD28 family (B7-1/CD80, B7-2/CD86, B7- H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA/CD272, CD28, CTLA-4, G124/VISTA/B7-H5, ICOS/CD278, PD-1, PD-L2/B7-DC, and PDCD6); members of the TNF superfamily (4-1BB/TNFSF9/CD137, 4-1BB ligand/TNFSF9, BAFF/BlyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF7, CD27 ligand/TNFSF7, CD30/TNFRSF8, CD30 ligand/TNFSFSF
  • the costimulatory domain comprises an intracellular signaling domain of an activating receptor protein such as a4pi integrin, 02 integrins (CDl la-CD18, CDllb-CD18, CDl lb-CD18), CD226, CRTAM, CD27, NKp46, CD16, NKp30, NKp44, NKp80, CARD11, NKG2D, KIR-S, CD100, CD94/NKG2C, CD94/NKG2E, NKG2D, PEN5, CEACAM1, BY55, CRACC, Ly9, CD84, NTBA, 2B4, SAP, DAP10, DAP12, EAT2, FcRy, CD3 ⁇ , or ERT.
  • an activating receptor protein such as a4pi integrin, 02 integrins (CDl la-CD18, CDllb-CD18, CDl lb-CD18), CD226, CRTAM, CD27, NKp46, CD16, NKp30,
  • the costimulatory domain comprises an intracellular signaling domain of an inhibitory receptor protein such as KIR-L, LILRB1, CD94/NKG2A, KLRG-1, NKR-P1A, TIGIT, CEACAM, SIGLEC 3, SIGLEC 7, SIGLEC9, or LAIR-1.
  • the costimulatory domain comprises an intracellular signaling domain of CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, or a ligand that specifically binds with CD83.
  • the costimulatory domain comprises an intracellular signaling domain of 41BB (CD137).
  • the anti-DLL3 CAR comprises a signal sequence, for example, to direct the protein to the cellular membrane.
  • the signal sequence comprises a CD8, CD8a, CD28, CD16, an IgK signal sequence, or a GMCSFR-alpha signal sequence, including variants thereof.
  • the signal sequence comprises a CD8alpha signal sequence, an IgK signal sequence, or a GMCSFR-alpha signal sequence.
  • the signal sequence comprises the amino acid sequence of any one of SEQ ID Nos: 12, 74, or 75.
  • the signal sequence comprises a CD8a signal sequence, such as that comprising SEQ ID NO: 12.
  • the anti-DLL3 CAR may further comprise a depletion tag.
  • the depletion tag comprises an epitope recognized by an antibody.
  • the depletion tag comprises an epitope recognized by a monoclonal antibody.
  • the epitope recognized by the monoclonal antibody comprises a CD20 epitope, an RSV protein F epitope, an EGF receptor epitope, a PD-1 epitope, a CD52 epitope, a CCR4 epitope, or a QB END- 10 epitope, a combination thereof, including variants thereof.
  • the epitope recognized by the monoclonal antibody comprises any one of SEQ ID Nos: 64-73.
  • the anti-DLL3 CAR comprising an epitope recognized by a monoclonal antibody is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 162, or is encoded by a nucleic acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 163.
  • the anti-DLL3 CAR may have any one of a number of different CAR architectures, such as a single-chain CAR, a multi-chain CAR, a single-targeted CAR, a multi-targeted CAR, a bivalent tandem CAR, a bivalent loop CAR, a multicistronic CAR, a bicistronic CAR, a synNotch-CAR circuit, a synthetic intramembrane proteolysis receptor (SNIPR), a multi-chain DAP-CAR, a TREM1/DAP12 CAR, a DAP12/TREM1 CAR, or a dimerizing agent regulated immune-receptor complex (DARIC).
  • CAR architectures such as a single-chain CAR, a multi-chain CAR, a single-targeted CAR, a multi-targeted CAR, a bivalent tandem CAR, a bivalent loop CAR, a multicistronic CAR, a bicistronic CAR
  • An anti-DLL3 CAR may also have one of a number of different T cell receptor (TCR) architectures or other cell surface receptor architectures, such as a T cell receptor fusion construct (TruC), an HLA-independent T cell (HIT) receptor, a synthetic T cell receptor and antigen receptor (STAR), a rapamycin inducible TCR, a rapamycin inducible Fc receptor, or a constitutive TCR- like receptor.
  • TCR T cell receptor
  • TCR T cell receptor fusion construct
  • HIT HLA-independent T cell
  • STAR synthetic T cell receptor and antigen receptor
  • rapamycin inducible TCR a rapamycin inducible Fc receptor
  • constitutive TCR- like receptor such as a constitutive TCR- like receptor.
  • the CAR is a dimerizing agent regulated immune-receptor complex (DARIC), an antibody tethered orthogonal multiplexing compatible (ATOMIC), a synNotch-CAR circuit, a synthetic intramembrane proteolysis receptor (SNIPR), a rapamycin inducible Fc receptor, a multi-chain DAP-CAR, a TREM1/DAP12 CAR, or a DAP12/TREM1 CAR.
  • DARIC dimerizing agent regulated immune-receptor complex
  • ATOMIC antibody tethered orthogonal multiplexing compatible
  • SNIPR synthetic intramembrane proteolysis receptor
  • rapamycin inducible Fc receptor a multi-chain DAP-CAR
  • TREM1/DAP12 CAR a TREM1/DAP12 CAR
  • DAP12/TREM1 CAR DAP12/TREM1 CAR
  • the CAR is a bispecific CAR or tandem CAR. In some embodiments, the CAR is a bispecific CAR or tandem CAR comprising an antigen binding domain that binds a second target protein.
  • a bispecific CAR may comprise, from N-terminus to C-terminus, a signal sequence, a first antigen binding domain (e.g., an scFv), a linker, a second antigen binding domain e.g., an scFv), a hinge domain, a transmembrane domain, a costimulatory domain, and an activation domain.
  • the first antigen binding domain is an anti-DLL3 antigen binding domain.
  • the second antigen binding domain is an anti- DLL3 antigen binding domain.
  • the second target protein is or comprises CD1, CDla, CDlb, CDlc, CDld, CDle, CD2, CD3d, CD3e, CD3g, CD3s, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD44v6, CD45, CD46, CD47 CD48, CD52, CD59, CD66, CD70, CD71 , CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD97, CD123, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD171, CD178, CD179, CD179a, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD213A2, CD246, CD252, CD253, CD261,
  • the second target protein is or comprises TROP2, SSTR2, GD2, EGFR, CEA, CEACAMs, B7H3, PSMA, CA9, EPC AM, or FN-EDB. In some cases, the second target protein is or comprises TROP2, SSTR2, or GD2.
  • Trispecific Killer Engagers (TRIKEs)
  • the promoter is recognized by RNA polymerase II (e.g., a CMV, SV40 early region or adenovirus major late promoter). In another embodiment, the promoter is recognized by RNA polymerase III (e.g., a U6 or Hl promoter). In some embodiments, the promoter can be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus. Other known promoters also are contemplated.
  • CMV cytomegalovirus
  • the promoter is a regulated promoter (e.g., inducible promoter).
  • the promoter is an inducible promoter or a repressible promoter.
  • the promoter comprises a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence or a doxycycline operator sequence, or is an analog thereof or is capable of being bound by or recognized by a Lac repressor or a tetracycline repressor, or an analog thereof.
  • the polynucleotide does not include a regulatory element, e.g. promoter.
  • the nucleic acid sequence encoding the polynucleotide encoding the DLL3 binding protein contains a signal sequence that encodes a signal peptide.
  • the signal sequence may encode a signal peptide derived from a native polypeptide.
  • the signal sequence may encode a heterologous or non-native signal peptide, such as the exemplary signal peptide of a GMCSFR alpha chain, CD8, or IgK.
  • the one or more modified nucleotides are selected from the group consisting of pseudouridine, N1 -methylpseudouridine, 5- methylcytosine, 5- methoxyuridine, and a combination thereof. In some cases, the one or more modified nucleotides comprise N 1 - methylpseudouridine.
  • nucleic acid sequences introduced include nucleic acid sequences that encode a second chimeric antigen receptor or heterologous TCR, nucleic acid sequences that can improve the efficacy of therapy, such as by promoting viability and/or function of transferred cells; nucleic acid sequences to provide a genetic marker for selection and/or evaluation of the cells, such as to assess in vivo survival or localization; nucleic acid sequences to improve safety, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., Mol.
  • polynucleotides can be engineered as a bicistronic unit containing an IRES, which allows coexpression of gene products (e.g. encoding the recombinant receptor and the additional polypeptide) by a message from a single promoter.
  • a single promoter may direct expression of an RNA that contains, in a single open reading frame (ORF), two or three genes (e.g. encoding the marker and encoding the recombinant receptor) separated from one another by sequences encoding a self-cleavage peptide (e.g., 2A sequences) or a protease recognition site (e.g., furin).
  • ORF open reading frame
  • the ORF thus encodes a single polypeptide, which, either during (in the case of 2A) or after translation, is processed into the individual proteins.
  • the peptide such as a T2A
  • Various 2A elements are known.
  • the nucleic acid construct further comprises a polynucleotide sequence encoding one or more additional polypeptides and/or one or more non-coding RNA.
  • the non-coding RNA comprises a shRNA or a microRNA.
  • the nucleic acid construct further comprises a polynucleotide sequence encoding one or more cleavable linkers, such as a P2A, E2A, F2A, or T2A self-cleaving peptide.
  • a cleavable linker may allow the translated polypeptide species to separate after being produced from one long open reading frame.
  • the polynucleotide sequence encoding the recombinant polypeptide and the polynucleotide sequence encoding the one or more additional polypeptides are separated by an internal ribozyme entry site (IRES).
  • IRS internal ribozyme entry site
  • the second CAR or recombinant TCR binds to TR0P2, SSTR2, GD2, EGFR, CEA, CEACAMs, B7H3, PSMA, CA9, EPCAM, or FN-EDB. In some cases, the second CAR or recombinant TCR binds to TR0P2, SSTR2, or GD2. In some cases, the second CAR or recombinant TCR binds to DLL3. In some such cases, the second CAR or recombinant TCR binds to the EGF1 domain of DLL3, the EGF2 domain of DLL3, the EGF3 domain of DLL3, the EGF4 domain of DLL3, the EGF5 domain of DLL3, or the EGF6 domain of DLL3.
  • the potency enhancement polypeptide is or comprises a patient derived CARD11-PIK3R3 fusion, an engineered CARD11-PIK3R3 fusion (e.g., comprising the amino acid sequence of any one of SEQ ID NO: 1 14, 116, 118, or 120), a dominant negative form of an inhibitor of a cell-mediated immune response of the immune cell (e.g., TGF0R2 DNR), c-Jun, CCL19, CCL21, IL2R, IL7, IL7Ralpha, IL15, IL15RA, IL18, decoy-resistant IL18 (DR-18), MyD88/CD40, PD1-CD28 switch receptor, PD1-41BB switch receptor, CD40L-CD28 switch receptor, CTBR12 switch receptor, CD8alpha/beta, a combination thereof, or variants thereof.
  • a cell-mediated immune response of the immune cell e.g., TGF0R2 DNR
  • c-Jun C
  • a nucleic acid construct may be organized from 5’ to 3’ according to one of the following designs:
  • a promoter (1) a promoter, and (2) a polynucleotide encoding the DLL3 binding protein (i.e., an anti- DLL3 antigen binding domain or antibody, an anti-DLL3 CAR, or an anti-DLL3 T cell enhancer);
  • a promoter (2) a polynucleotide encoding the DLL3 binding protein, (3) a cleavable linker encoding sequence, and (4) a polynucleotide encoding one or more additional polypeptides;
  • a promoter (2) a polynucleotide encoding one or more additional polypeptides, (3) a cleavable linker encoding sequence, and (4) a polynucleotide encoding the DLL3 binding protein;
  • a promoter (2) a polynucleotide encoding one or more additional polypeptides, (3) a cleavable linker encoding sequence, (4) a polynucleotide encoding an additional potency enhancement polypeptide, a cytokine, a chemokine, or a growth factor, (5) a cleavable linker encoding sequence, and (6) a polynucleotide encoding the DLL3 binding protein;
  • a promoter (2) a polynucleotide encoding an additional potency enhancement polypeptide, a cytokine, a chemokine, or a growth factor, (3) a cleavable linker encoding sequence, (4) a polynucleotide encoding one or more additional polypeptides (5) a cleavable linker encoding sequence, and (6) a polynucleotide encoding the DLL3 binding protein:
  • a cleavable linker encoding sequence (2) a polynucleotide encoding one or more additional polypeptides, (3) a cleavable linker encoding sequence, (4) a polynucleotide encoding the DLL3 binding protein, and (5) a cleavable linker encoding sequence;
  • a cleavable linker encoding sequence (2) a polynucleotide encoding an additional potency enhancement polypeptide, a cytokine, a chemokine, or a growth factor, (3) a cleavable linker encoding sequence, (4) a polynucleotide encoding the DLL3 binding protein, (5) a cleavable linker encoding sequence, (6) a polynucleotide encoding one or more additional polypeptides, and (7) a cleavable linker encoding sequence; (1) a cleavable linker encoding sequence, (2) a polynucleotide encoding an additional potency enhancement polypeptide, a cytokine, a chemokine, or a growth factor, (3) a cleavable linker encoding sequence, (4) a polynucleotide encoding one or more additional polypeptides, (5) a cleavable linker
  • nucleic acid constructs may be used to express different polypeptides or nucleic acids.
  • examples include compositions comprising: (1) a first polynucleotide encoding the DLL3 binding protein, and (2) a second polynucleotide encoding one or more additional polypeptides, optionally wherein the first and the second polynucleotides are both RNA constructs;
  • a first polynucleotide encoding the DLL3 binding protein (2) a second polynucleotide sequence encoding one or more additional polypeptides, and (3) a third polynucleotide encoding an additional potency enhancement polypeptide, a cytokine, a chemokine, or a growth factor, optionally wherein one or more of the polynucleotides is an RNA construct; or
  • a polynucleotide encoding the DLL3 binding protein (2) a second nucleic acid construct encoding one or more additional polypeptides, and (3) a third nucleic acid construct encoding a polynucleotide encoding an additional potency enhancement polypeptide, a cytokine, a chemokine, or a growth factor, and optionally wherein the first, second, and/or third polynucleotides are RNA constructs.
  • one or more nucleic acid constructs herein may be comprised within one or more vectors.
  • a vector may further be comprised within a particle.
  • the vector can include, but is not limited to, viral vectors, plasmid DNA, and closed-end DNA.
  • Viral vectors can include, but are not limited to, adenoviral vectors, lentiviral vectors, retroviral vectors, adeno-associated viral vectors, vaccinia viruses, poxviruses, and herpes simplex viruses, or any of the viruses described elsewhere herein.
  • expression vectors contain selection markers such as ampicillin-resistance, hygromycin-resi stance, tetracycline resistance, kanamycin resistance, or neomycin resistance to permit detection of those cells transformed with the desired DNA sequences.
  • the polycistronic construct may be in the form of a vector.
  • Any type of vector suitable for introduction of nucleotide sequences into a host cell can be used, including, for example, plasmids, adenoviral vectors, adenoviral-associated vectors, retroviral vectors, lentiviral vectors, phages, and homology-directed repair (HDR)-based donor vectors.
  • HDR homology-directed repair
  • a retrovirus typically replicates by reverse transcription of its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome.
  • the structure of a wild-type retrovirus genome often comprises a 5' long terminal repeat (LTR) and a 3' LTR, between or within which are located a packaging signal to enable the genome to be packaged, a primer binding site, integration sites to enable integration into a host cell genome and gag, pol and env genes encoding the packaging components which promote the assembly of viral particles.
  • LTR 5' long terminal repeat
  • 3' LTR between or within which are located a packaging signal to enable the genome to be packaged
  • a primer binding site to enable integration into a host cell genome
  • gag, pol and env genes encoding the packaging components which promote the assembly of viral particles.
  • More complex retroviruses have additional features, such as rev and RRE sequences in HIV, which enable the efficient export of RNA transcripts of the integrated provirus from the nucleus to the
  • the LTRs themselves are typically similar (e.g., identical) sequences that can be divided into three elements, which are called U3, R and U5.
  • U3 is derived from the sequence unique to the 3' end of the RNA.
  • R is derived from a sequence repeated at both ends of the RNA and
  • U5 is derived from the sequence unique to the 5' end of the RNA.
  • the sizes of the three elements can vary considerably among different retroviruses.
  • the site of transcription initiation is typically at the boundary between U3 and R in one LTR and the site of poly (A) addition (termination) is at the boundary between R and U5 in the other LTR.
  • U3 contains most of the transcriptional control elements of the pro virus, which include the promoter and multiple enhancer sequences responsive to cellular and in some cases, viral transcriptional activator proteins.
  • Some retroviruses comprise any one or more of the following genes that code for proteins that are involved in the regulation of gene expression: tot, rev, tax and rex.
  • gag encodes the internal structural protein of the virus.
  • Gag protein is proteolytically processed into the mature proteins MA (matrix), CA (capsid) and NC (nucleocapsid).
  • the pol gene encodes the reverse transcriptase (RT), which contains DNA polymerase, associated RNase H and integrase (IN), which mediate replication of the genome.
  • the env gene encodes the surface (SU) glycoprotein and the transmembrane (TM) protein of the virion, which form a complex that interacts specifically with cellular receptor proteins. This interaction promotes infection, e.g., by fusion of the viral membrane with the cell membrane.
  • Retroviruses may also contain additional genes which code for proteins other than gag, pol and env. Examples of additional genes include (in HIV), one or more of vif, vpr, vpx, vpu, tat, rev and nef. EIAV has (amongst others) the additional gene S2.
  • Illustrative retroviruses suitable for use in particular embodiments include, but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV) and human immunodeficiency virus (HIV).
  • M-MuLV Moloney murine leukemia virus
  • MoMSV Moloney murine sarcoma virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • GaLV gibbon ape leukemia virus
  • FLV feline leukemia virus
  • RSV Rous Sarcoma Virus
  • the retrovirus is a Gammretrovirus. In some embodiments the retrovirus is an Epsilonretrovirus. In some embodiments the retrovirus is an Alpharetrovirus. In some embodiments the retrovirus is a Betaretrovirus. In some embodiments the retrovirus is a Deltaretrovirus. In some embodiments the retrovirus is a Spumaretro virus. In some embodiments the retrovirus is an endogenous retrovirus. In some embodiments the retrovirus is a lentivirus.
  • a retroviral or lentivirus vector further comprises one or more insulator elements, e.g., an insulator element described in Browning et al., “Insulators to Improve the Safety of Retroviral Vectors for HIV Gene Therapy,” Biomedicines, 4(1):4 (2016).
  • the vectors comprise a promoter operably linked to a polynucleotide encoding an exogenous agent.
  • the vectors may have one or more LTRs, wherein either LTR comprises one or more modifications, such as one or more nucleotide substitutions, additions, or deletions.
  • the vectors may further comprise one of more accessory elements to increase transduction efficiency (e.g., a cPPT/FLAP), viral packaging (e.g., a Psi (Y) packaging signal, RRE), and/or other elements that increase exogenous gene expression (e.g., poly (A) sequences), and may optionally comprise a WPRE or HPRE.
  • accessory elements to increase transduction efficiency e.g., a cPPT/FLAP
  • viral packaging e.g., a Psi (Y) packaging signal, RRE
  • other elements that increase exogenous gene expression e.g., poly (A) sequences
  • the nucleic acid molecules can be encapsulated in a viral capsid or a lipid nanoparticle, or can be delivered by viral or non-viral delivery means and methods known in the art, such as electroporation.
  • introduction of nucleic acids into cells may be achieved by viral transduction.
  • adeno-associated virus AAV is engineered to deliver nucleic acids to target cells via viral transduction.
  • AAV serotypes have been described, and all of the known serotypes can infect cells from multiple diverse tissue types. AAV is capable of transducing a wide range of species and tissues in vivo with no evidence of toxicity, and it generates relatively mild innate and adaptive immune responses.
  • a lentiviral vector can be used for delivery.
  • Lentiviral-derived vector systems are useful for nucleic acid delivery and gene therapy via viral transduction.
  • Lentiviral vectors offer several attractive properties as gene-delivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) the capability of infecting both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy-target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or introncontaining sequences; (vi) a potentially safer integration site profile; and (vii) a relatively easy system for vector manipulation and production.
  • the LNP comprises: (i) an ionizable lipid (e.g., an amino lipid), (ii) a sterol or other structural lipid, (iii) a non-cationic helper lipid or phospholipid, and (iv) a PEG-lipid (e.g., a PEG-modified lipid).
  • the nucleic acid is delivered with a polymer nanoparticle.
  • the nucleic acid is delivered with a protein nanoparticle.
  • the delivery used in the context of this disclosure are described in W02024006960A1; WO2019152557A1; Raguram, Aditya, Samagya Banskota, and David R. Liu. "Therapeutic in vivo delivery of gene editing agents." Cell (2022); Rurik, Joel G., et al. "CAR T cells produced in vivo to treat cardiac injury.” Science 375.6576 (2022): 91-96.
  • the one or more agent(s) capable of inducing a genetic disruption is introduced into the cell as a ribonucleoprotein (RNP) complex.
  • RNP complexes include a sequence of ribonucleotides, such as an RNA or a gRNA molecule, and a protein, such as a Cas9 protein or variant thereof.
  • the Cas9 protein is delivered as RNP complex that comprises a Cas9 protein and a gRNA molecule targeting the target sequence, e.g., using electroporation or other physical delivery method.
  • the RNP is delivered into the cell via electroporation or other physical means, e.g., particle gun, Calcium Phosphate transfection, cell compression or squeezing.
  • the RNP can cross the plasma membrane of a cell without the need for additional delivery agents (e.g., small molecule agents, lipids, etc.).
  • delivery of the one or more agent(s) capable of inducing genetic disruption, e.g., CRISPR/Cas9, as an RNP offers an advantage that the targeted disruption occurs transiently, e.g., in cells to which the RNP is introduced, without propagation of the agent to cell progenies.
  • delivery by RNP minimizes the agent from being inherited to its progenies, thereby reducing the chance of off-target genetic disruption in the progenies.
  • the genetic disruption and the integration of transgene can be inherited by the progeny cells, but without the agent itself, which may further introduce off-target genetic disruptions, being passed on to the progeny cells.
  • the DNA may typically but not necessarily include a control region, e.g., comprising a promoter, to effect expression.
  • exemplary promoters for CRISPR/Cas nuclease sequences include, e.g., CMV, EFla, EFS, MSCV, PGK, or CAG promoters.
  • Useful promoters for gRNAs include, e.g., Hl, EF-la, tRNA or U6 promoters. Promoters with similar or dissimilar strengths can be selected to tune the expression of components.
  • the polynucleotide encoding the engineered proteins of the disclosure can be delivered into cells by known methods or as described herein. For example, by vectors (e.g., viral or non-viral vectors), non- vector based methods (e.g., using naked DNA or DNA complexes), or a combination thereof.
  • the polynucleotide containing the agent(s) and/or components thereof is delivered by a vector (e.g., viral vector/virus or plasmid).
  • the vector may be any described herein.
  • the polynucleotide encoding the engineered proteins of the disclosure is delivered by a non-vector based method (e.g., using naked DNA or DNA complexes).
  • a non-vector based method e.g., using naked DNA or DNA complexes.
  • the DNA or RNA or proteins or combination thereof, e.g., ribonucleoprotein (RNP) complexes can be delivered, e.g., by organically modified silica or silicate (Ormosil), electroporation, transient cell compression or squeezing (such as described in Lee, et al.
  • delivery via electroporation comprises mixing the cells with the polynucleotide encoding the engineered proteins of the disclosure or RNP complex in a cartridge, chamber or cuvette and applying one or more electrical impulses of defined duration and amplitude.
  • delivery via electroporation is performed using a system in which cells are mixed with the DNA in a vessel connected to a device (e.g., a pump) which feeds the mixture into a cartridge, chamber or cuvette wherein one or more electrical impulses of defined duration and amplitude are applied, after which the cells are delivered to a second vessel.
  • a device e.g., a pump
  • the delivery vehicle is a non- viral vector.
  • the non-viral vector is an inorganic nanoparticle.
  • Exemplary inorganic nanoparticles include, e.g., magnetic nanoparticles (e.g., FesMnCh) and silica.
  • the outer surface of the nanoparticle can be conjugated with a positively charged polymer (e.g., polyethylenimine, polylysine, polyserine) which allows for attachment (e.g., conjugation or entrapment) of payload.
  • the non-viral vector is an organic nanoparticle.
  • Exemplary organic nanoparticles include, e.g., SNALP liposomes that contain cationic lipids together with neutral helper lipids which are coated with polyethylene glycol (PEG), and protamine-nucleic acid complexes coated with lipid.
  • Exemplary lipids and polymers for gene transfer include those described in, for example, WO 2019/195492 and WO 2020/223535.
  • the delivery vehicle is a biological non-viral delivery vehicle.
  • the vehicle is an attenuated bacterium (e.g., naturally or artificially engineered to be invasive but attenuated to prevent pathogenesis and expressing the transgene (e.g., Listeria monocytogenes, certain Salmonella strains, Bifidobacterium longum, and modified Escherichia coli), bacteria having nutritional and tissue- specific tropism to target specific cells, bacteria having modified surface proteins to alter target cell specificity).
  • the transgene e.g., Listeria monocytogenes, certain Salmonella strains, Bifidobacterium longum, and modified Escherichia coli
  • the vehicle is a genetically modified bacteriophage (e.g., engineered phages having large packaging capacity, less immunogenicity, containing mammalian plasmid maintenance sequences and having incorporated targeting ligands).
  • the vehicle is a mammalian virus-like particle.
  • modified viral particles can be generated (e.g., by purification of the “empty” particles followed by ex vivo assembly of the virus with the desired cargo).
  • the vehicle can also be engineered to incorporate targeting ligands to alter target tissue specificity.
  • the vehicle is a biological liposome.
  • the biological liposome is a phospholipid-based particle derived from human cells (e.g., erythrocyte ghosts, which are red blood cells broken down into spherical structures derived from the subject (e.g., tissue targeting can be achieved by attachment of various tissue or cell-specific ligands), or secretory exosomes -subject-derived membrane-bound nanovescicles (30 -100 nm) of endocytic origin (e.g., can be produced from various cell types and can therefore be taken up by cells without the need for targeting ligands).
  • human cells e.g., erythrocyte ghosts, which are red blood cells broken down into spherical structures derived from the subject (e.g., tissue targeting can be achieved by attachment of various tissue or cell-specific ligands), or secretory exosomes -subject-derived membrane-bound nanovescicles (30 -100 nm) of endocytic origin (e.g., can be produced from
  • an RNA polynucleotide encoding the engineered proteins of the disclosure can be delivered into cells, e.g., target cells described herein, by known methods or as described herein.
  • RNA can be delivered, e.g., by microinjection, electroporation, transient cell compression or squeezing (such as described in Lee, et al. (2012) Nano Let 12: 6322-27), lipid-mediated transfection, peptide-mediated delivery, e.g., cell-penetrating peptides, or a combination thereof.
  • delivery via electroporation comprises mixing the cells with the RNA in a cartridge, chamber or cuvette and applying one or more electrical impulses of defined duration and amplitude.
  • delivery via electroporation is performed using a system in which cells are mixed with the RNA encoding CRISPR/Cas nuclease molecules and/or gRNA molecules in a vessel connected to a device (e.g., a pump) which feeds the mixture into a cartridge, chamber or cuvette wherein one or more electrical impulses of defined duration and amplitude are applied, after which the cells are delivered to a second vessel.
  • a device e.g., a pump
  • engineered polypeptide molecules can be delivered into cells by known methods or as described herein.
  • protein molecules can be delivered, e.g., by microinjection, electroporation, transient cell compression or squeezing (such as described in Lee, et al. (2012) Nano Let 12: 6322-27), lipid-mediated transfection, peptide-mediated delivery, or a combination thereof. Delivery can be accompanied by DNA encoding a polypeptide (e.g., an engineered polypeptide, gRNA, CAR, TCR, etc.).
  • a polypeptide e.g., an engineered polypeptide, gRNA, CAR, TCR, etc.
  • the disclosure also encompasses an engineered cell that expresses a DLL3 binding protein, such as an anti-DLL3 CAR, anti-DLL3 antigen binding domain or antibody, or anti-DLL3 immune cell engager protein, optionally under the control of an endogenous promoter or optionally under the control of an exogenous promoter.
  • the engineered cell is a prokaryotic cell or a eukaryotic cell.
  • the cell is in vivo.
  • the cell is ex vivo.
  • the cell is in vitro.
  • the engineered cell is a eukaryotic cell.
  • the engineered cell is an animal cell.
  • the cell is a primary cell.
  • Primary cells are isolated directly from human or animal tissue using enzymatic or mechanical methods. Once isolated, they are placed in an artificial environment in plastic or glass containers supported with specialized medium containing essential nutrients and growth factors to support proliferation.
  • Primary cells could be of two types: adherent or suspension.
  • Adherent cells require attachment for growth and are said to be anchorage-dependent cells.
  • Adherent cells are usually derived from tissues of organs. Suspension cells do not require attachment for growth and are said to be anchorage independent cells. Most suspension cells are isolated from the blood system, but some tissue- derived cells can also be used in suspension, such as hepatocytes or intestinal cells.
  • primary cells usually have a limited lifespan, they offer a number of advantages compared to cell lines.
  • Primary cell culture enables researchers to study donors and not just cells. Several factors such as age, medical history, race, and sex can be considered when building an experimental model. With a growing trend towards personalized medicine, such donor variability and tissue complexity can be achieved with use of primary cells, but are difficult to replicate with cell lines that are more systematic and uniform in nature and do not capture the true diversity of a living tissue.
  • the starting material is otherwise modified or engineered to have altered expression of one or more genes.
  • the cells that are engineered as provided herein comprise cells derived from primary cells obtained or isolated from one or more individual subjects or donors.
  • the cells are derived from a pool of isolated primary cells obtained from a subject (e.g., the patient administered the cells).
  • the cells are derived from a pool of isolated primary cells obtained from one or more (e.g., two or more, three or more, four or more, five or more, ten or more, twenty or more, fifty or more, or one hundred or more) different donor subjects.
  • the primary cells isolated or obtained from the plurality of different donor subjects e.g., two or more, three or more, four or more, five or more, ten or more, twenty or more, fifty or more, or one hundred or more
  • the primary cells isolated or obtained from the plurality of different donor subjects are pooled together in a batch and are engineered in accord with the provided methods.
  • hiPSCs are human induced pluripotent stem cells.
  • the cell is a mesenchymal stem cell (MSCs).
  • the cell is a differentiated cell.
  • the cell is differentiated from a pluripotent stem cell. Differentiated cells are cells that have undergone differentiation. They are mature cells that perform a specialized function. Some examples of differentiated cells are T cells, NK cells, etc. Generally, these cells have a unique morphology, metabolic activity, membrane potential, and responsiveness to signals facilitating their function in a body tissue or organ.
  • the engineered cells described are derived from an ESC or iPSC or a progeny thereof.
  • the term “derived from an ESC or iPSC or a progeny thereof’ encompasses the initial ESC or iPSC that is generated and any subsequent progeny thereof.
  • the term “progeny” encompasses, e.g., a first-generation progeny, i.e., the progeny is directly derived from, obtained from, obtainable from or derivable from the initial ESC or iPSC by, e.g., traditional propagation methods.
  • progeny also encompasses further generations such as second, third, fourth, fifth, sixth, seventh, or more generations, i.e., generations of cells which are derived from, obtained from, obtainable from or derivable from the former generation by, e.g., traditional propagation methods.
  • progeny also encompasses modified cells that result from the modification or alteration of the initial ESC or iPSC or a progeny thereof.
  • the cell or stem cell, or a cell derived from such a stem cell has or is believed to have therapeutic value, such that the cell or stem cell or a cell derived or differentiated from such stem cell may be used to treat a disease, disorder, defect or injury in a subject in need of treatment for same.
  • the cell is a stem cell or progenitor cell (e.g., iPSC, ESC, hematopoietic stem cell, mesenchymal stem cell, endothelial stem cell, epithelial stem cell, adipose stem or progenitor cells, germline stem cells, lung stem or progenitor cells, mammary stem cells, olfactory adult stem cells, hair follicle stem cells, multipotent stem cells, amniotic stem cells, cord blood stem cells, or neural stem or progenitor cells).
  • the stem cells are adult stem cells (e.g., somatic stem cells or tissue specific stem cells).
  • the stem or progenitor cell is capable of being differentiated (e.g., the stem cell is totipotent, pluripotent, or multipotent).
  • the cell is isolated from embryonic or neonatal tissue.
  • the cell is a fibroblast, monocytic precursor, B cell, exocrine cell, pancreatic progenitor, endocrine progenitor, hepatoblast, myoblast, preadipocyte, progenitor cell, hepatocyte, chondrocyte, smooth muscle cell, K562 human erythroid leukemia cell line, bone cell, synovial cell, tendon cell, ligament cell, meniscus cell, adipose cell, dendritic cells, or natural killer cell.
  • the cell is manipulated (e.g., converted or differentiated) into a muscle cell, erythroid-megakaryocytic cell, eosinophil, iPS cell, macrophage, T cell, islet beta-cell, neuron, cardiomyocyte, blood cell, endocrine progenitor, exocrine progenitor, ductal cell, acinar cell, alpha cell, beta cell, delta cell, PP cell, hepatocyte, cholangiocyte, or brown adipocyte.
  • the cell is a muscle cell (e.g., skeletal, smooth, or cardiac muscle cell), erythroid-megakaryocytic cell, eosinophil, iPS cell, macrophage, T cell, islet beta-cell, neuron, cardiomyocyte, blood cell (e.g., red blood cell, white blood cell, or platelet), endocrine progenitor, exocrine progenitor, ductal cell, acinar cell, alpha cell, beta cell, delta cell, PP cell, hepatocyte, cholangiocyte, or white or brown adipocyte.
  • muscle cell e.g., skeletal, smooth, or cardiac muscle cell
  • erythroid-megakaryocytic cell eosinophil
  • iPS cell eosinophil
  • macrophage macrophage
  • T cell islet beta-cell
  • neuron e.g., cardiomyocyte
  • blood cell e.g., red blood cell, white blood cell,
  • the cell is a hormone-secreting cell (e.g., a cell that secretes insulin, oxytocin, endorphin, vasopressin, serotonin, somatostatin, gastrin, secretin, glucagon, thyroid hormone, bombesin, cholecystokinin, testosterone, estrogen, or progesterone, renin, ghrelin, amylin, or pancreatic polypeptide), an epidermal keratinocyte, an epithelial cell (e.g., an exocrine secretory epithelial cell, a thyroid epithelial cell, a keratinizing epithelial cell, a gall bladder epithelial cell, or a surface epithelial cell of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, or vagina), a kidney cell, a germ cell, a skeletal joint synovium cell, a periostea cell,
  • the cell is a T cell or NK cell that has been differentiated from an engineered ESC or iPSC. In some embodiments, the cell is an engineered cell that has been modified from a primary cell. In some embodiments, the cell comprises increased expression of the DLL3 binding protein disclosed herein.
  • ESCs and iPCSs there are a variety of different methods for the generation of ESCs and iPCSs.
  • the original induction of iPSCs was done from mouse embryonic or adult fibroblasts using the viral introduction of four transcription factors, Oct3/4, Sox2, c-Myc and Klf4; see Takahashi and Yamanaka Cell 126:663-676 (2006), hereby incorporated by reference in its entirety and specifically for the techniques outlined therein. Since then, a number of methods have been developed; see Seki et al, World J.
  • iPSCs are generated by the transient expression of one or more reprogramming factors" in the host cell, usually introduced using episomal vectors. Under these conditions, small amounts of the cells are induced to become iPSCs (in general, the efficiency of this step is low, as no selection markers are used). Once the cells are "reprogrammed", and become pluripotent, they lose the episomal vector(s) and produce the factors using the endogeneous genes.
  • any of the pluripotent stem cells described herein can be differentiated into any cells of an organism and tissue.
  • engineered cells that are differentiated into different cell types from ESCs or iPSCs for subsequent transplantation into recipient subjects. Differentiation can be assayed as is known in the art, generally by evaluating the presence of cell-specific markers.
  • the differentiated engineered pluripotent cell derivatives can be transplanted using techniques known in the art that depends on both the cell type and the ultimate use of these cells.
  • the iPSCs are differentiated into cell types selected from T cells, NK cells.
  • the cell is a T cell.
  • the T cell comprises a polynucleotide encoding the DLL3 binding protein of the present disclosure.
  • T cells of the disclosure or precursors thereof can be immune cells of the lymphoid lineage. Relevant information concerning T cells as referred to in the context of the present disclosure is known in the art, including certain information regarding desired features of T cells when used for cell therapy. It will be understood that embodiments concerning T cells described herein may be readily and appropriately combined. It will be understood that embodiments concerning T cells described herein may be readily and appropriately combined with other embodiments described herein (for example, but not limited to, the DLL3 binding protein).
  • T cell is a type of lymphocyte.
  • T cells are one of the white blood cells of the immune system and play a central role in the adaptive immune response.
  • T cells can be derived from T cells in a patient's own blood (autologous) or derived from the T cells of another healthy donor (allogeneic).
  • T cells can be obtained from a number of sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • the T cells as provided herein are primary T cells.
  • the primary T cells are isolated or obtained from and individual subject or one or more donors, such as one or more healthy donors (e.g., a subject that is not known or suspected of, e.g., not exhibiting clinical signs of, a disease or infection).
  • the T cells are populations of primary T cells.
  • the T cells are subpopulations or sub-types or subsets of primary T cells. Subpopulations and sub-types and subsets of primary T cells are herein described in further detail below.
  • methods of isolating or obtaining T lymphocytes from an individual can be achieved using known techniques.
  • engineered primary T lymphocytes that contain modifications (e.g., genetic modifications) described herein for subsequent transplantation or engraftment into subjects (e.g., recipients).
  • primary T cells are obtained (e.g., harvested, extracted, removed, or taken) from a subject or an individual.
  • primary T cells are produced from a pool of T cells such that the T cells are from one or more subjects (e.g., one or more human including one or more healthy humans).
  • the pool of primary T cells is from 1-100, 1-50, 1-20, 1-10, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more subjects.
  • the donor subject is different from the patient (e.g., the recipient that is administered the therapeutic cells).
  • the pool of T cells does not include cells from the patient.
  • one or more of the donor subjects from which the pool of T cells is obtained are different from the patient.
  • primary T cells are primary T cells of a T cell subset or a sub-type or subpopulation.
  • T cell subsets may be found in US2018/0319862A1 and/or WO20 16/090190A1, the entire contents of which are hereby incorporated by reference.
  • the T cell subset such as a CD62L+ T cell subset, that is increased in subjects upon administration of the genetically engineered cells are or include or share phenotypic characteristics with memory T cells or particular subsets thereof, such as long-lived memory T cells.
  • such memory T cells are central memory T cells (Tcm) or T memory stem cells (Tsc) cells.
  • the memory T cells are Tsc cells.
  • Tsc cells may be described as having one or more phenotypic differences or functional features compared to other memory T cell subsets or compared to naive T cells, such as being less differentiated or more naive (see e.g., Ahlers and Belyakov (2010) Blood, 115: 1678); Cieri et al (2015) Blood, 125:2865; Flynn et al. (2014) Clinical & Translational Immunology, 3, e20; Gattinoni et al. (2012) Nat. Med., 17: 1290-1297; Gattinoni et al. (2012) Nat .Reviews, 12:671; Li et al.
  • Tsc cells are thought to be the only memory T cells able to generate effector T cells and all three subsets of memory T cells (Tsc, Tcm, and Tern). In some aspects, Tsc cells have the highest survival and proliferation response to antigenic or homeostatic stimuli of all the memory T cell subsets, and the least attrition absent cognate antigen.
  • the less - differentiated Tsc cells may exhibit greater expansion, long-term viability, and target cell destruction following adoptive transfer than other memory T cells, and thus may be able to mediate more effective treatment with fewer transferred cells than would be possible for either Tcm or Tern cells.
  • the cells as provided herein are T lymphocytes differentiated from engineered pluripotent cells that contain modifications (e.g., genetic modifications) described herein and that are differentiated into T lymphocyte.
  • modifications e.g., genetic modifications
  • the methods for differentiation depend on the desired cell type using known techniques.
  • the cells differentiated into a T lymphocyte may be used for subsequent transplantation or engraftment into subjects (e.g., recipients).
  • the cell is an iPSC-derived T cell that is engineered to contain modifications (e.g., genetic modifications) described herein.
  • the cell is a primary T cell that is engineered to contain modifications (e.g., genetic modifications) described herein.
  • the cell comprises overexpression or increased expression of one or more complement inhibitor.
  • Cells disclosed herein may be T cells, for example a population of T cells. It will be understood that any reference to “a cell” e.g. “a T cell” below also applies to “a population of cells” e.g. “a population of T cells” as described in the present application.
  • the NK cells are primary NK cells.
  • NK cells including primary NK cells isolated from an individual subject or one or more donors (e.g., healthy donors) or NK cells differentiated from iPSCs derived from one or more donors (e.g., healthy donors) express CD56 (e.g., CDSb l 11 or CD56bright) and lack CD3 (e.g., CD3neg).
  • CD56 e.g., CDSb l 11 or CD56bright
  • CD3 e.g., CD3neg
  • NK cells as described herein may also express the low-affinity Fey receptor CD16, which, without wishing to be bound by theory, mediate ADCC.
  • the NK cells also express one or more natural killer cell receptors NKG2A and NKG2D or one or more natural cytotoxicity receptors NKp46, NKp44, NKp30.
  • the primary cells may be isolated from a starting source of NK cells, such as a sample containing peripheral blood mononuclear cells (PBMCs), by depletion of cells positive for CD3, CD14, and/or CD19.
  • PBMCs peripheral blood mononuclear cells
  • the cells may be subject to depletion using immunomagnetic beads having attached thereto antibodies to CD3, CD 14, and/or CD 19, respectively), thereby producing an enriched population of NK cells.
  • the NK cells can be immature NK cells and can be CD56+ and CD16-. In some embodiments, the NK cells can be mature NK cells and can be CD56- and CD16+, or CD561o and CD16+ (as described in WO 2011/068896A1 (the contents of which are incorporated herein by reference in their entirety)).
  • a primary NK cell may express CD16 and/or CD56.
  • an NK cell does not express CD3.
  • an "NK cell” is preferably defined as a cell that is CD56+ and CD3-.
  • an "NK cell” is defined as a cell that is CD16+ and CD3-.
  • NK cells are lymphocytes of the innate immune system that kill virally infected or transformed cells. Like T cells, NK cells are cytotoxic lymphocytes. Unlike T cells, NK cells do not require antigen recognition, and require integration of signals from many activating and inhibitory receptors to perform their function. Despite their similarities to T cells, NK cells behave differently under stimulation conditions and do not tolerate electroporation in the same way as T cells (as described in WO2017214569A1 (the contents of which are incorporated herein by reference in their entirety)).
  • Cells disclosed herein may be natural killer (NK) cells, for example a population of NK cells. It will be understood that any reference to “a cell” e.g. “a NK cell” below also applies to “a population of cells” e.g. “a population of NK cells” as described in the present application.
  • NK natural killer
  • “recombination” refers to a process of exchange of genetic information between two polynucleotides.
  • “homologous recombination (HR)” refers to the specialized form of such exchange that takes place, for example, during repair of double-strand breaks in cells via homology-directed repair mechanisms.
  • such transfer can involve mismatch correction of heteroduplex DNA that forms between the broken target and the template polynucleotide, and/or “synthesis-dependent strand annealing,” in which the template polynucleotide is used to resynthesize genetic information that will become part of the target, and/or related processes.
  • Such specialized HR often results in an alteration of the sequence of the target molecule such that part or all of the sequence of the template polynucleotide is incorporated into the target polynucleotide.
  • the genetic disruption of the target site or target position can be created by any mechanisms, such as ZFNs, TALENs, CRISPR/Cas9 system, or TtAgo nucleases.
  • the lengths of the two homology arms are between about 150 bp and about 1500 bp; optionally wherein the length of at least one of the first or second homology arms is about 200bp, 225 bp, about 250 bp, about 275 bp, about 300 bp, about 325 bp, about 350 bp, about 375 bp, about 400 bp, about 425 bp, about 450 bp, about 475 bp, or about 500 bp.
  • ZFNs are fusion proteins comprising an array of site-specific DNA binding domains adapted from zinc finger-containing transcription factors attached to the endonuclease domain of the bacterial FokI restriction enzyme.
  • a ZFN may have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) DNA binding domains or zinc finger domains. See, e.g., Carroll et al., Genetics Society of America (2011) 188:773-782; Kim et al., Proc. Natl. Acad. Sci. USA (1996) 93: 1156-1160.
  • Zinc fingers can be engineered to bind a predetermined nucleic acid sequence. Criteria to engineer a zinc finger to bind to a predetermined nucleic acid sequence are known in the art. See, e.g., Sera et al., Biochemistry (2002) 41 :7074-7081; Liu et al., Bioinformatics (2008) 24: 1850-1857.
  • ZFNs containing FokI nuclease domains or other dimeric nuclease domains function as a dimer.
  • a pair of ZFNs are required to target non-palindromic DNA sites.
  • the two individual ZFNs must bind opposite strands of the DNA with their nucleases properly spaced apart. See Bitinaite ⁇ / al., Proc. Natl. Acad. Sci. USA (1998) 95: 10570-10575.
  • a pair of ZFNs are designed to recognize two sequences flanking the site, one on the forward strand and the other on the reverse strand.
  • the nuclease domains dimerize and cleave the DNA at the site, generating a DSB with 5’ overhangs.
  • HDR can then be utilized to introduce a specific mutation, with the help of a repair template containing the desired mutation flanked by homology arms.
  • the repair template is usually an exogenous double-stranded DNA vector introduced to the cell. See Miller et al., Nat. Biotechnol. (2011) 29: 143-148; Hockemeyer et al., Nat. Biotechnol. (2011) 29:731- 734.
  • TALENS TALENs are another example of an artificial nuclease which can be used to edit a target gene.
  • a "TALE-nuclease” (TALEN) is a fusion protein consisting of a nucleic acidbinding domain typically derived from a Transcription Activator Like Effector (TALE) and one nuclease catalytic domain to cleave a nucleic acid target sequence.
  • the catalytic domain is preferably a nuclease domain and more preferably a domain having endonuclease activity, for instance I- TevI, ColE7, NucA and Fok-I.
  • the TALE domain can be fused to a meganuclease, for instance LCrel and I-Onul or functional variant thereof.
  • said nuclease is a monomeric TALE-Nuclease.
  • a monomeric TALE- Nuclease is a TALE-Nuclease that does not require dimerization for specific recognition and cleavage, such as the fusions of engineered TAL repeats with the catalytic domain of I-TevI described in WO2012138927.
  • Transcription Activator like Effector are proteins from the bacterial species Xanthomonas and comprise a plurality of repeated sequences. Each repeat is 33 to 35 amino acids in length, with two adjacent amino acids (termed the repeat-variable di-residue, or RVD) in position 12 and 13 that are specific to each nucleotide base of the nucleic acid targeted sequence. Binding domains with similar modular base-per-base nucleic acid binding properties (MBBBD) can also be derived from new modular proteins recently discovered in a different bacterial species. The new modular proteins have the advantage of displaying more sequence variability than TAL repeats.
  • RVDs associated with recognition of the different nucleotides are HD for recognizing C, NG for recognizing T, NI for recognizing A, NN for recognizing G or A, NS for recognizing A, C, G or T, HG for recognizing T, IG for recognizing T, NK for recognizing G, HA for recognizing C, ND for recognizing C, HI for recognizing C, HN for recognizing G, NA for recognizing G, SN for recognizing G or A and YG for recognizing T, TL for recognizing A, VT for recognizing A or G and SW for recognizing A.
  • critical amino acids 12 and 13 can be mutated towards other amino acid residues in order to modulate their specificity towards nucleotides A, T, C and G and in particular to enhance this specificity.
  • TALEN kits are sold commercially.
  • TALENs are produced artificially by fusing one or more TALE DNA binding domains (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) to a nuclease domain, for example, a FokI endonuclease domain.
  • TALE DNA binding domains e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
  • a nuclease domain for example, a FokI endonuclease domain.
  • a site-specific nuclease can be produced specific to any desired DNA sequence.
  • TALENs can be introduced into a cell to generate DSBs at a desired target site in the genome, and so can be used to knock out genes or knock in mutations in similar, HDR-mediated pathways. See Boch, Nature Biotech. (2011) 29: 135-136; Boch et al., Science (2009) 326: 1509-1512; Moscou et al., Science (2009) 326:3501.
  • Meganucleases are sequence- specific enzymes in the endonuclease family which are characterized by their capacity to recognize and cut large DNA sequences (from 14 to 40 base pairs). (Chevalier, B. S. and B. L. Stoddard, Nucleic Acids Res., 2001, 29, 3757-3774). They can cleave unique sites in living cells, thereby enhancing gene targeting by 1000-fold or more in the vicinity of the cleavage site (Puchta et al., Nucleic Acids Res., 1993, 21, 5034-5040; Rouet et al., Mol. Cell. Biol., 1994, 14, 8096-8106; Choulika et al., Mol. Cell.
  • Meganucleases are grouped into families based on their structural motifs which affect nuclease activity and/or DNA recognition.
  • the most widespread and best known meganucleases are the proteins in the LAGLID ADG family, which owe their name to a conserved amino acid sequence.
  • the GIY-YIG family members have a GIY-YIG module, which is 70- 100 residues long and includes four or five conserved sequence motifs with four invariant residues, two of which are required for activity. See Van Roey et al., Nature Struct. Biol. (2002) 9:806-811.
  • the His- Cys family meganucleases are characterized by a highly conserved series of histidines and cysteines over a region encompassing several hundred amino acid residues. See Chevalier et al., Nucleic Acids Res. (2001) 29(18):3757-3774. Members of the NHN family are defined by motifs containing two pairs of conserved histidines surrounded by asparagine residues. See Chevalier et al, Nucleic Acids Res. (2001) 29(18):3757-3774.
  • Cas proteins adapted for gene editing applications include, but are not limited to, Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpf 1), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmr5, Csel, Cse2, Csfl, Csm2, Csn2, CsxlO, Csxl 1, Csyl, Csy2, Csy3, Mad7, LEG14.
  • the CRISPR/Cas system includes a Cas protein and at least one to two ribonucleic acids that are capable of directing the Cas protein to and hybridizing to a target motif of a target polynucleotide sequence.
  • the Cas protein can comprise an alternative amino acid (e.g., D-amino acids, beta-amino acids, homocysteine, phosphoserine, etc.).
  • a Cas protein can comprise a modification to include a moiety (e.g., PEGylation, glycosylation, lipidation, acetylation, end-capping, etc.).
  • a Cas protein comprises a core Cas protein, isoform thereof, or any Cas-like protein with similar function or activity of any Cas protein or isoform thereof.
  • a Cas protein comprises a core Cas protein.
  • Exemplary Cas core proteins include, but are not limited to Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8 and Cas9.
  • a Cas protein comprises type V Cas protein.
  • a Cas protein comprises a Cas protein of an E. coli subtype (also known as CASS2). Exemplary Cas proteins of the E.
  • a Cas protein comprises a Cas protein of the Dvulg subtype (also known as CASS1).
  • Exemplary Cas proteins of the Dvulg subtype include Csdl, Csd2, and Cas5d.
  • a Cas protein comprises a Cas protein of the Tneap subtype (also known as CASS7).
  • Exemplary Cas proteins of the Tneap subtype include, but are not limited to, Cstl, Cst2, Cas5t.
  • a Cas protein comprises a Cas protein of the Hmari subtype.
  • Exemplary Cas proteins of the Hmari subtype include, but are not limited to Cshl, Csh2, and Cas5h.
  • a Cas protein comprises a Cas protein of the Apem subtype (also known as CASS5).
  • Exemplary Cas proteins of the Apem subtype include, but are not limited to Csal, Csa2, Csa3, Csa4, Csa5, and Cas5a.
  • a Cas protein comprises a Cas protein of the Mtube subtype (also known as CASS6).
  • Exemplary Cas proteins of the Mtube subtype include, but are not limited to Csml, Csm2, Csm3, Csm4, and Csm5.
  • a Cas protein comprises a RAMP module Cas protein.
  • Exemplary RAMP module Cas proteins include, but are not limited to, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6. See, e.g., Klompe et al., Nature 571, 219-225 (2019); Strecker et al., Science 365, 48- 53 (2019).
  • Examples of Cas proteins include, but are not limited to: Cas3, Cas8a, Cas5, Cas8b, Cas8c, CaslOd, Csel, Cse2, Csyl, Csy2, Csy3, and/or GSU0054.
  • a Cas protein comprises Cas3, Cas8a, Cas5, Cas8b, Cas8c, CaslOd, Csel, Cse2, Csyl, Csy2, Csy3, and/or GSU0054.
  • Cas proteins include, but are not limited to: Cas9, Csn2, and/or Cas4.
  • a Cas protein comprises Cas9, Csn2, and/or Cas4.
  • Examples of Cas proteins include, but are not limited to: CaslO, Csm2, Cmr5, CaslO, Csxl 1, and/or CsxlO.
  • a Cas protein comprises a CaslO, Csm2, Cmr5, CaslO, Csxl 1 , and/or CsxlO.
  • examples of Cas proteins include, but are not limited to: Csfl.
  • a Cas protein comprises Csfl.
  • examples of Cas proteins include, but are not limited to: Casl2a, Casl2b, Casl2c, C2c4, C2c8, C2c5, C2cl0, and C2c9; as well as CasX (Casl2e) and CasY (Casl2d).
  • the CRISPR/Cas system comprises a Cas effector protein selected from the group consisting of: a) Cas3, Cas8a, Cas5, Cas8b, Cas8c, CaslOd, Csel, Cse2, Csyl, Csy2, Csy3, and GSU0054; b) Cas9, Csn2, and Cas4; c) CaslO, Csm2, Cmr5, CaslO, Csxl 1, and CsxlO; d) Csfl; e) Casl2a, Casl2b, Casl2c, C2c4, C2c8, C2c5, C2cl0, C2c9, CasX (Casl2e), and CasY (Casl2d); and f) Casl3, Casl3a, C2c2, Casl3b, Casl3c, and Casl3d.
  • Cas effector protein selected from the
  • a Cas protein comprises any one of the Cas proteins described herein or a functional portion thereof.
  • “functional portion” refers to a portion of a peptide which retains its ability to complex with at least one ribonucleic acid (e.g., guide RNA (gRNA)) and cleave a target polynucleotide sequence.
  • the functional portion comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain.
  • the functional portion comprises a combination of operably linked Casl2a (also known as Cpfl) protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain.
  • the functional domains form a complex.
  • a functional portion of the Cas9 protein comprises a functional portion of a RuvC-like domain.
  • a functional portion of the Cas9 protein comprises a functional portion of the HNH nuclease domain.
  • a functional portion of the Casl2a protein comprises a functional portion of a RuvC-like domain.
  • the Cas protein is complexed with one to two ribonucleic acids. In some embodiments, the Cas protein is complexed with two ribonucleic acids. In some embodiments, the Cas protein is complexed with one ribonucleic acid. In some embodiments, the Cas protein is encoded by a modified nucleic acid, as described herein (e.g., a synthetic, modified mRNA).
  • At least one of the ribonucleic acids comprises a guide RNA that directs the Cas protein to and hybridizes to a target motif of the target polynucleotide sequence in a cell.
  • both of the one to two ribonucleic acids comprise a guide RNA that directs the Cas protein to and hybridizes to a target motif of the target polynucleotide sequence in a cell.
  • the ribonucleic acids of the present disclosure can be selected to hybridize to a variety of different target motifs, depending on the particular CRISPR/Cas system employed, and the sequence of the target polynucleotide, as will be appreciated by those skilled in the art.
  • the one to two ribonucleic acids can also be selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence.
  • the one to two ribonucleic acids hybridize to a target motif that contains at least two mismatches when compared with all other genomic nucleotide sequences in the cell.
  • the one to two ribonucleic acids hybridize to a target motif that contains at least one mismatch when compared with all other genomic nucleotide sequences in the cell.
  • the one to two ribonucleic acids are designed to hybridize to a target motif immediately adjacent to a deoxyribonucleic acid motif recognized by the Cas protein.
  • each of the one to two ribonucleic acids are designed to hybridize to target motifs immediately adjacent to deoxyribonucleic acid motifs recognized by the Cas protein which flank a mutant allele located between the target motifs.
  • each of the one to two ribonucleic acids comprises guide RNAs that directs the Cas protein to and hybridizes to a target motif of the target polynucleotide sequence in a cell.
  • one or two ribonucleic acids are complementary to and/or hybridize to sequences on the same strand of a target polynucleotide sequence. In some embodiments, one or two ribonucleic acids (e.g., guide RNAs) are complementary to and/or hybridize to sequences on the opposite strands of a target polynucleotide sequence. In some embodiments, the one or two ribonucleic acids (e.g., guide RNAs) are not complementary to and/or do not hybridize to sequences on the opposite strands of a target polynucleotide sequence.
  • the one or two ribonucleic acids are complementary to and/or hybridize to overlapping target motifs of a target polynucleotide sequence. In some embodiments, the one or two ribonucleic acids (e.g, guide RNAs) are complementary to and/or hybridize to offset target motifs of a target polynucleotide sequence.
  • CRISPR systems of the present disclosure comprise TnpB polypeptides.
  • TnpB polypeptides may comprise a Ruv-C-like domain.
  • the RuvC domain may be a split RuvC domain comprising RuvC-I, RuvC-II, and RuvC-III subdomains.
  • a TnpB may further comprise one or more of a HTH domain, a bridge helix domain and a zinc finger domain.
  • TnpB polypeptides do not comprise an HNH domain.
  • a TnpB protein comprises, starting at the N-terminus: a HTH domain, a RuvC-I subdomain, a bridge helix domain, a RuvC-II sub-domain, a zinger finger domain, and a RuvC-III sub-domain.
  • a RuvC-III sub-domain forms the C- terminus of a TnpB polypeptide.
  • a TnpB polypeptide is from Epsilonproteobacteria bacterium, Actinoplanes lobatus strain DSM 43150, Actinomadura celluolosilytica strain DSM 45823, Actinomadura namibiensis strain DSM 44197, Alicyclobacillus macrosprangiidus strain DSM 17980, Lipingzhangella hal ophila strain DSM 102030, or Ktedonobacter recemifer.
  • a TnpB polypeptide is from Ktedonobacter racemifer, or comprises a conserved RNA region with similarity to the 5 ’ ITR of K. racemifer TnpB loci.
  • a TnpB may comprise a Fanzor protein, a TnpB homolog found in eukaryotic genomes.
  • a CRISPR system comprising a TnpB polypeptide binds a target adjacent motif (TAM) sequence 5’ of a target polynucleotide.
  • TAM is a transposon-associated motif.
  • a TAM sequence comprises TCA.
  • a TAM sequence comprises TTCAN.
  • a TAM sequence comprises TTGAT.
  • a TAM sequence comprises ATAAA.
  • the base editing technology may be used to introduce single nucleotide variants (SNVs) into DNA or RNA in living cells.
  • SNVs single nucleotide variants
  • Base editing is a CRISPR-Cas9- based genome editing technology that allows the introduction of point mutations in RNAs or DNAs without generating DSBs.
  • CBEs cytidine base editors
  • ABEs adenine base editors
  • Base editors are composed by a catalytically dead Cas9 (dCas9) or a nickase Cas9 (nCas9) fused to a deaminase and guided by a sgRNA to the locus of interest.
  • a base editor is a nucleobase editor containing a first DNA binding protein domain that is catalytically inactive, a domain having base editing activity, and a second DNA binding protein domain having nickase activity, where the DNA binding protein domains are expressed on a single fusion protein or are expressed separately (e.g., on separate expression vectors).
  • the specific site for insertion within a gene locus may be located within any suitable region of the gene, including but not limited to a gene coding region (also known as a coding sequence or “CDS”), an exon, an intron, a sequence spanning a portion of an exon and a portion of an adjacent intron, or a regulatory region (e.g., promoter, enhancer).
  • a gene coding region also known as a coding sequence or “CDS”
  • CDS coding sequence
  • the insertion occurs in one allele of the specific genomic locus.
  • the insertion occurs in both alleles of the specific genomic locus.
  • the orientation of the transgene inserted into the target genomic locus can be either the same or the reverse of the direction of the endogenous gene in that locus.
  • polynucleotides of the present disclosure are inserted into a TRAJ (T cell receptor alpha joining) splice acceptor site locus.
  • the human TRAJ gene resides on chromosome 14 at 22,545,037-22,545,098, forward strand.
  • the TRAJ genomic sequence is set forth in Ensembl ID ENSG00000211888.
  • polynucleotides of the present disclosure are inserted into a beta-2- microglobulin (B2M) locus.
  • B2M gene encodes the B2M component of class I major histocompatibility complex (MHC I).
  • MHC I major histocompatibility complex
  • the B2M gene resides on chromosome 15: 45,003,675- 45,011,075, forward strand, and its genomic sequence is set forth in Ensembl ID ENSG00000166710.
  • polynucleotides of the present disclosure are inserted into a class II, major histocompatibility complex, trans activator (CUT A) locus.
  • the CIITA gene encodes the CIITA protein that is a positive regulator of class II major histocompatibility complex (MHC II) gene transcription.
  • Hie CIITA gene resides on chromosome 16: 10,866,222-10,943,021, forward strand, and its genomic sequence is set forth in Ensembl ID ENSG00000179583.
  • polynucleotides of the present disclosure are inserted into a caspase recruitment domain family member 11 (CARD11) locus.
  • CARD11 caspase recruitment domain family member 11
  • polynucleotides encoding the PIK3R3 protein, or a portion thereof, of the present disclosure are inserted into a CARD11 locus.
  • the CARD11 gene encodes the CARD11 protein which plays a role in various immune responses including, among others, lymphocyte activation, proliferation, and differentiation, and plays a role as a positive regulator of NF-kB activation.
  • the CARD 11 gene resides on chromosome 7: 2,906,142-3,044,228, reverse strand, and its genomic sequence is set forth in Ensembl ID ENSG00000198286.
  • polynucleotides of the present disclosure are inserted into a phosphoinositide-3 -kinase regulatory subunit 3 (PIK3R3) locus.
  • polynucleotides encoding the CARD11 protein, or a portion thereof, of the present disclosure are inserted into a PIK3R3 locus.
  • the PIK3R3 gene encodes the PIK3R3 protein which plays a role in inhibiting cell senescence and enhancing cell proliferation.
  • the PIK3R3 gene resides on chromosome 1 : 46,040,140-46,133,036, reverse strand, and its genomic sequence is set forth in Ensembl ID ENSG00000117461.
  • gRNAs for use in site-directed insertion of a polynucleotides according to various embodiments provided herein, especially in association with the CRISPR/Cas system.
  • the gRNAs comprise a crRNA sequence, which in turn comprises a complementary region (also called a spacer) that recognizes and binds a complementary target DNA of interest.
  • the length of the spacer or complementary region is generally between 15 and 30 nucleotides, usually about 20 nucleotides in length, although will vary based on the requirements of the specific CRISPR/Cas system.
  • the spacer or complementary region is fully complementary to the target DNA sequence.
  • the spacer is partially complementary to the target DNA sequence, for example at least 80%, 85%, 90%, 95%, 98%, or 99% complementary.
  • the gRNAs provided herein further comprise a tracrRNA sequence, which comprises a scaffold region for binding to a nuclease.
  • the length and/or sequence of the tracrRNA may vary depending on the specific nuclease being used for editing.
  • nuclease binding by the gRNA does not require a tracrRNA sequence.
  • the crRNA sequence may further comprise a repeat region for hybridization with complementary sequences of the tracrRNA.
  • the gRNAs provided herein comprise two or more gRNA molecules, for example, a crRNA and a tracrRNA, as two separate molecules.
  • the gRNAs are single guide RNAs (sgRNAs), including sgRNAs comprising a crRNA and a tracrRNA on a single RNA molecule.
  • the crRNA and tracrRNA are linked by an intervening tetraloop.
  • the gRNA comprises a complementary region specific to a target gene locus of interest, for example, a TRAJ locus, a TRAC locus, a TRBC1 locus, a TRBC2 locus, a CD3zeta locus, a B2M locus, a CIITA locus, a CARD11 locus, a PIK3R3 locus, or a safe harbor locus selected from the group consisting of an AAVS1 locus, an ABO locus, a CCR5 locus, a CLYBL locus, a CXCR4 locus, a F3 locus, a FUT1 locus, a HMGB 1 locus, a KDM5D locus, a LRP1 locus, a MICA locus, a MICB locus, a RHD locus, a ROSA26, locus and a SHS231 locus.
  • a target gene locus of interest for example, a TRAJ locus,
  • the activity, stability, and/or other characteristics of gRNAs can be altered through the incorporation of chemical and/or sequential modifications.
  • transiently expressed or delivered nucleic acids can be prone to degradation by, e.g., cellular nucleases.
  • the gRNAs described herein can contain one or more modified nucleosides or nucleotides which introduce stability toward nucleases. While not being bound by a particular theory, it is believed that certain modified gRNAs described herein can exhibit a reduced innate immune response when introduced into a population of cells, particularly the cells of the present technology.
  • the term “innate immune response” includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, generally of viral or bacterial origin, which involves the induction of cytokine expression and release, e.g., the interferons, and cell death.
  • Other common chemical modifications of gRNAs to improve stabilities, increase nuclease resistance, and/or reduce immune response include 2’-O-methyl modification, 2’ -fluoro modification, 2’-O-methyl phosphorothioate linkage modification, and 2’- O-methyl 3’ thioPACE modification.
  • poly(A) tract comprising one or more (and typically 5-200) adenine (A) residues.
  • the poly(A) tract can be contained in the nucleic acid sequence encoding the gRNA or can be added to the gRNA during chemical synthesis or following in vitro transcription using a polyadenosine polymerase (e.g., E. coll poly(A) polymerase).
  • polyadenosine polymerase e.g., E. coll poly(A) polymerase
  • poly(A) tracts can be added to sequences transcribed from DNA vectors through the use of polyadenylation signals. Examples of such signals are provided in Maeder.
  • Other suitable gRNA modifications include, without limitations, those described in U.S. Patent Application No. US 2017/0073674 Al and International Publication No. WO 2017/165862 Al, the entire contents of each of which are incorporated by reference herein.
  • the second CAR has a specificity for a target antigen selected from CD1, CDla, CDlb, CDlc, CDld, CDle, CD2, CD3d, CD3e, CD3g, CD3s, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD44v6, CD45, CD46, CD47 CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1 ), CD86 (B7.2), CD94, CD95, CD97, CD123, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD171, CD178, CD179, CD179a, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD213A2, CD24
  • the polypeptides or recombinant nucleic acid constructs and/or recombinant nucleic acids can alter T cell signaling through one or more T cell signaling pathways.
  • Signaling pathways can be, without limitation, the NFAT pathway, NF-KB pathway, AP-1 pathway, JAK/STAT pathway, RAS/MEK/ERK, and/or phospholipase gamma signaling, or described elsewhere herein.
  • the further polypeptide, recombinant nucleic acid construct and/or recombinant nucleic acid relates to a CARD11-PIK3R3 fusion polypeptide, such as including a CARD domain (i.e., containing protein or functional fragment thereof), a CARD inhibitory domain (ID), a coiled-coil domain, and an SH2 domain from PIK3R3.
  • the CARD11-PIK3R3 fusion polypeptide includes a functional fragment of a CARD containing protein derived from CARD1 Idomain, a coiled-coil domain, and an SH2 domain from PIK3R3.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente divulgation concerne des molécules se liant au ligand de type delta 3 (DLL3), telles que des récepteurs antigéniques chimériques (CAR) anti-DLL3, des anticorps anti-DLL3 comprenant des domaines de liaison à l'antigène, des anticorps bispécifiques, des conjugués d'anticorps, des anticorps engageant des cellules immunitaires anti-DLL3, et d'autres protéines de fusion comprenant des domaines de liaison à l'antigène anti-DLL3, et analogues, des polynucléotides et des vecteurs codant pour des molécules de liaison au DLL3, et des cellules modifiées exprimant des protéines de liaison au DLL3, et des procédés de fabrication et d'utilisation des protéines de liaison au DLL3.
PCT/US2025/022337 2024-04-01 2025-03-31 Protéines de liaison au dll3 et leurs utilisations Pending WO2025212519A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202463572743P 2024-04-01 2024-04-01
US63/572,743 2024-04-01
US202463693844P 2024-09-12 2024-09-12
US63/693,844 2024-09-12

Publications (1)

Publication Number Publication Date
WO2025212519A1 true WO2025212519A1 (fr) 2025-10-09

Family

ID=95399441

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/022337 Pending WO2025212519A1 (fr) 2024-04-01 2025-03-31 Protéines de liaison au dll3 et leurs utilisations

Country Status (1)

Country Link
WO (1) WO2025212519A1 (fr)

Citations (56)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
WO1992008796A1 (fr) 1990-11-13 1992-05-29 Immunex Corporation Genes de fusion selectionnables bifonctionnels
WO1994028143A1 (fr) 1993-05-21 1994-12-08 Targeted Genetics Corporation Genes de fusion selectables et bifonctionnels se basant sur le gene de cytosine-deaminase (cd)
WO1998017815A1 (fr) 1996-10-17 1998-04-30 Oxford Biomedica (Uk) Limited Vecteurs retroviraux
WO1999015683A1 (fr) 1997-09-25 1999-04-01 Oxford Biomedica (Uk) Limited Vecteurs retroviraux comprenant un site donneur d'epissage fonctionnel et un site accepteur d'epissage fonctionnel
US6040177A (en) 1994-08-31 2000-03-21 Fred Hutchinson Cancer Research Center High efficiency transduction of T lymphocytes using rapid expansion methods ("REM")
WO2001083692A2 (fr) 2000-04-28 2001-11-08 The Trustees Of The University Of Pennsylvania Vecteurs aav recombinants dotes de capsides aav5 et vecteurs aav5 pseudotypes dans des capsides heterologues
US20050100543A1 (en) 2003-07-01 2005-05-12 Immunomedics, Inc. Multivalent carriers of bi-specific antibodies
US20050175606A1 (en) 2001-04-11 2005-08-11 Hua-Liang Huang Cyclic single-chain trispecific antibody
WO2006020258A2 (fr) 2004-07-17 2006-02-23 Imclone Systems Incorporated Nouveau anticorps bispecifique tetravalent
US20070014794A1 (en) 1995-03-01 2007-01-18 Genentech, Inc. Method for making heteromultimeric polypeptides
WO2007024715A2 (fr) 2005-08-19 2007-03-01 Abbott Laboratories Immunoglobuline a deux domaines variables et utilisations de celle-ci
US20070116690A1 (en) 2001-12-10 2007-05-24 Lili Yang Method for the generation of antigen-specific lymphocytes
US7446190B2 (en) 2002-05-28 2008-11-04 Sloan-Kettering Institute For Cancer Research Nucleic acids encoding chimeric T cell receptors
WO2011068896A1 (fr) 2009-12-04 2011-06-09 Stem Cell & Regenerative Medicine International, Inc. Procédé de génération de cellules tueuses naturelles et de cellules dendritiques à partir d'hémangioblastes dérivés de cellules souches humaines
WO2012138927A2 (fr) 2011-04-05 2012-10-11 Philippe Duchateau Procédé de génération de nucléases tale compactes et leurs utilisations
WO2014039044A1 (fr) 2012-09-06 2014-03-13 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Procédés de production de populations de cellules souches t mémoires
WO2014055668A1 (fr) 2012-10-02 2014-04-10 Memorial Sloan-Kettering Cancer Center Compositions et procédés d'immunothérapie
US20150056705A1 (en) 2013-05-15 2015-02-26 Sangamo Biosciences, Inc. Methods and compositions for treatment of a genetic condition
WO2015161276A2 (fr) 2014-04-18 2015-10-22 Editas Medicine, Inc. Méthodes, compositions et constituants associés à crispr/cas pour l'immunothérapie du cancer
WO2016090190A1 (fr) 2014-12-03 2016-06-09 Juno Therapeutics, Inc. Procédés et compositions pour thérapie cellulaire adoptive
US20160272999A1 (en) 2013-05-29 2016-09-22 Cellectis Methods for engineering t cells for immunotherapy by using rna-guided cas nuclease system
EP2101823B1 (fr) 2007-01-09 2016-11-23 CureVac AG Anticorps code par un arn
US20170016027A1 (en) 2015-07-13 2017-01-19 Sangamo Biosciences, Inc. Delivery methods and compositions for nuclease-mediated genome engineering
US20170073674A1 (en) 2014-03-05 2017-03-16 Editas Medicine, Inc. Crispr/cas-related methods and compositions for treating usher syndrome and retinitis pigmentosa
US20170211075A1 (en) 2015-12-18 2017-07-27 Sangamo Biosciences, Inc. Targeted disruption of the t cell receptor
WO2017165862A1 (fr) 2016-03-25 2017-09-28 Editas Medicine, Inc. Systèmes et procédés pour traiter une déficience en alpha 1-antitrypsine (a1at)
WO2017214569A1 (fr) 2016-06-09 2017-12-14 Regents Of The University Of Minnesota Cellule nk à édition génomique et ses procédés de fabrication et d'utilisation
US20180044415A1 (en) * 2015-02-23 2018-02-15 Abbvie Stemcentrx Llc Anti-dll3 chimeric antigen receptors and methods of use
US20180319862A1 (en) 2015-11-05 2018-11-08 Juno Therapeutics, Inc. Chimeric receptors containing traf-inducing domains and related compositions and methods
WO2019126709A1 (fr) 2017-12-22 2019-06-27 The Broad Institute, Inc. Systèmes cas12b, procédés et compositions pour l'édition de base d'adn ciblée
WO2019152557A1 (fr) 2018-01-30 2019-08-08 Modernatx, Inc. Compositions et procédés destinés à l'administration d'agents à des cellules immunitaires
WO2019195492A1 (fr) 2018-04-05 2019-10-10 Juno Therapeutics, Inc. Procédés de production de cellules exprimant un récepteur recombinant et compositions associées
US10626373B2 (en) 2016-07-08 2020-04-21 Seoul National University R&Db Foundation Culture scaffold for enhancing differentiation of osteoblast using pattern
US20200248169A1 (en) 2017-06-26 2020-08-06 The Broad Institute, Inc. Crispr/cas-cytidine deaminase based compositions, systems, and methods for targeted nucleic acid editing
WO2020181195A1 (fr) 2019-03-06 2020-09-10 The Broad Institute, Inc. Édition de base t : a à a : t par excision d'adénine
WO2020181193A1 (fr) 2019-03-06 2020-09-10 The Broad Institute, Inc. Édition de bases t:a à a:t par méthylation de l'adénosine
WO2020181178A1 (fr) 2019-03-06 2020-09-10 The Broad Institute, Inc. Édition de base t:a à a:t par alkylation de thymine
WO2020181202A1 (fr) 2019-03-06 2020-09-10 The Broad Institute, Inc. Édition de base a:t en t:a par déamination et oxydation d'adénine
WO2020191248A1 (fr) 2019-03-19 2020-09-24 The Broad Institute, Inc. Procédés et compositions pour l'édition de séquences nucléotidiques
WO2020214842A1 (fr) 2019-04-17 2020-10-22 The Broad Institute, Inc. Éditeurs de base d'adénine présentant des effets hors cible réduits
WO2020223535A1 (fr) 2019-05-01 2020-11-05 Juno Therapeutics, Inc. Cellules exprimant un récepteur recombinant à base d'un locus modifié du tgfbr2, et polynucléotides et méthodes associés
US20210071163A1 (en) 2017-12-22 2021-03-11 The Broad Institute, Inc. Cas12b systems, methods, and compositions for targeted rna base editing
US20210079366A1 (en) 2017-12-22 2021-03-18 The Broad Institute, Inc. Cas12a systems, methods, and compositions for targeted rna base editing
US20210093667A1 (en) 2017-06-26 2021-04-01 The Broad Institute, Inc. Crispr/cas-adenine deaminase based compositions, systems, and methods for targeted nucleic acid editing
WO2021158921A2 (fr) 2020-02-05 2021-08-12 The Broad Institute, Inc. Éditeurs de base d'adénine et leurs utilisations
WO2021226558A1 (fr) 2020-05-08 2021-11-11 The Broad Institute, Inc. Méthodes et compositions d'édition simultanée des deux brins d'une séquence nucléotidique double brin cible
WO2022067130A2 (fr) 2020-09-24 2022-03-31 The Broad Institute, Inc. Arn guides d'édition primaire, leurs compositions et leurs méthodes d'utilisation
US20220127622A1 (en) 2018-09-07 2022-04-28 Beam Therapeutics Inc. Compositions and Methods for Improving Base Editing
US20220184127A1 (en) * 2019-04-08 2022-06-16 Phanes Therapeutics, Inc. Humanized anti-dll3 chimeric antigen receptors and uses thereof
WO2022164935A1 (fr) 2021-01-27 2022-08-04 Umoja Biopharma, Inc. Lentivirus permettant de générer des cellules exprimant un récepteur antigénique chimérique anti-cd19
US20220249563A1 (en) * 2019-07-17 2022-08-11 Nanjing Legend Biotech Co., Ltd. Anti-dll3 chimeric antigen receptors and uses thereof
US11673953B2 (en) * 2019-03-01 2023-06-13 Allogene Therapeutics, Inc. DLL3 targeting chimeric antigen receptors and binding agents
WO2023193015A1 (fr) 2022-04-01 2023-10-05 Sana Biotechnology, Inc. Polythérapies d'agoniste de récepteur de cytokine et de vecteur viral
WO2024006960A1 (fr) 2022-06-29 2024-01-04 Juno Therapeutics, Inc. Nanoparticules lipidiques pour l'administration d'acides nucléiques
US20240025992A1 (en) * 2020-10-22 2024-01-25 Janssen Biotech, Inc. Proteins comprising delta-like ligand 3 (dll3) antigen binding domains and their uses

Patent Citations (56)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
WO1992008796A1 (fr) 1990-11-13 1992-05-29 Immunex Corporation Genes de fusion selectionnables bifonctionnels
WO1994028143A1 (fr) 1993-05-21 1994-12-08 Targeted Genetics Corporation Genes de fusion selectables et bifonctionnels se basant sur le gene de cytosine-deaminase (cd)
US6040177A (en) 1994-08-31 2000-03-21 Fred Hutchinson Cancer Research Center High efficiency transduction of T lymphocytes using rapid expansion methods ("REM")
US20070014794A1 (en) 1995-03-01 2007-01-18 Genentech, Inc. Method for making heteromultimeric polypeptides
WO1998017815A1 (fr) 1996-10-17 1998-04-30 Oxford Biomedica (Uk) Limited Vecteurs retroviraux
WO1999015683A1 (fr) 1997-09-25 1999-04-01 Oxford Biomedica (Uk) Limited Vecteurs retroviraux comprenant un site donneur d'epissage fonctionnel et un site accepteur d'epissage fonctionnel
WO2001083692A2 (fr) 2000-04-28 2001-11-08 The Trustees Of The University Of Pennsylvania Vecteurs aav recombinants dotes de capsides aav5 et vecteurs aav5 pseudotypes dans des capsides heterologues
US20050175606A1 (en) 2001-04-11 2005-08-11 Hua-Liang Huang Cyclic single-chain trispecific antibody
US20070116690A1 (en) 2001-12-10 2007-05-24 Lili Yang Method for the generation of antigen-specific lymphocytes
US7446190B2 (en) 2002-05-28 2008-11-04 Sloan-Kettering Institute For Cancer Research Nucleic acids encoding chimeric T cell receptors
US20050100543A1 (en) 2003-07-01 2005-05-12 Immunomedics, Inc. Multivalent carriers of bi-specific antibodies
WO2006020258A2 (fr) 2004-07-17 2006-02-23 Imclone Systems Incorporated Nouveau anticorps bispecifique tetravalent
WO2007024715A2 (fr) 2005-08-19 2007-03-01 Abbott Laboratories Immunoglobuline a deux domaines variables et utilisations de celle-ci
EP2101823B1 (fr) 2007-01-09 2016-11-23 CureVac AG Anticorps code par un arn
WO2011068896A1 (fr) 2009-12-04 2011-06-09 Stem Cell & Regenerative Medicine International, Inc. Procédé de génération de cellules tueuses naturelles et de cellules dendritiques à partir d'hémangioblastes dérivés de cellules souches humaines
WO2012138927A2 (fr) 2011-04-05 2012-10-11 Philippe Duchateau Procédé de génération de nucléases tale compactes et leurs utilisations
WO2014039044A1 (fr) 2012-09-06 2014-03-13 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Procédés de production de populations de cellules souches t mémoires
WO2014055668A1 (fr) 2012-10-02 2014-04-10 Memorial Sloan-Kettering Cancer Center Compositions et procédés d'immunothérapie
US20150056705A1 (en) 2013-05-15 2015-02-26 Sangamo Biosciences, Inc. Methods and compositions for treatment of a genetic condition
US20160272999A1 (en) 2013-05-29 2016-09-22 Cellectis Methods for engineering t cells for immunotherapy by using rna-guided cas nuclease system
US20170073674A1 (en) 2014-03-05 2017-03-16 Editas Medicine, Inc. Crispr/cas-related methods and compositions for treating usher syndrome and retinitis pigmentosa
WO2015161276A2 (fr) 2014-04-18 2015-10-22 Editas Medicine, Inc. Méthodes, compositions et constituants associés à crispr/cas pour l'immunothérapie du cancer
WO2016090190A1 (fr) 2014-12-03 2016-06-09 Juno Therapeutics, Inc. Procédés et compositions pour thérapie cellulaire adoptive
US20180044415A1 (en) * 2015-02-23 2018-02-15 Abbvie Stemcentrx Llc Anti-dll3 chimeric antigen receptors and methods of use
US20170016027A1 (en) 2015-07-13 2017-01-19 Sangamo Biosciences, Inc. Delivery methods and compositions for nuclease-mediated genome engineering
US20180319862A1 (en) 2015-11-05 2018-11-08 Juno Therapeutics, Inc. Chimeric receptors containing traf-inducing domains and related compositions and methods
US20170211075A1 (en) 2015-12-18 2017-07-27 Sangamo Biosciences, Inc. Targeted disruption of the t cell receptor
WO2017165862A1 (fr) 2016-03-25 2017-09-28 Editas Medicine, Inc. Systèmes et procédés pour traiter une déficience en alpha 1-antitrypsine (a1at)
WO2017214569A1 (fr) 2016-06-09 2017-12-14 Regents Of The University Of Minnesota Cellule nk à édition génomique et ses procédés de fabrication et d'utilisation
US10626373B2 (en) 2016-07-08 2020-04-21 Seoul National University R&Db Foundation Culture scaffold for enhancing differentiation of osteoblast using pattern
US20200248169A1 (en) 2017-06-26 2020-08-06 The Broad Institute, Inc. Crispr/cas-cytidine deaminase based compositions, systems, and methods for targeted nucleic acid editing
US20210093667A1 (en) 2017-06-26 2021-04-01 The Broad Institute, Inc. Crispr/cas-adenine deaminase based compositions, systems, and methods for targeted nucleic acid editing
US20210071163A1 (en) 2017-12-22 2021-03-11 The Broad Institute, Inc. Cas12b systems, methods, and compositions for targeted rna base editing
WO2019126709A1 (fr) 2017-12-22 2019-06-27 The Broad Institute, Inc. Systèmes cas12b, procédés et compositions pour l'édition de base d'adn ciblée
US20210079366A1 (en) 2017-12-22 2021-03-18 The Broad Institute, Inc. Cas12a systems, methods, and compositions for targeted rna base editing
WO2019152557A1 (fr) 2018-01-30 2019-08-08 Modernatx, Inc. Compositions et procédés destinés à l'administration d'agents à des cellules immunitaires
WO2019195492A1 (fr) 2018-04-05 2019-10-10 Juno Therapeutics, Inc. Procédés de production de cellules exprimant un récepteur recombinant et compositions associées
US20220127622A1 (en) 2018-09-07 2022-04-28 Beam Therapeutics Inc. Compositions and Methods for Improving Base Editing
US11673953B2 (en) * 2019-03-01 2023-06-13 Allogene Therapeutics, Inc. DLL3 targeting chimeric antigen receptors and binding agents
WO2020181195A1 (fr) 2019-03-06 2020-09-10 The Broad Institute, Inc. Édition de base t : a à a : t par excision d'adénine
WO2020181202A1 (fr) 2019-03-06 2020-09-10 The Broad Institute, Inc. Édition de base a:t en t:a par déamination et oxydation d'adénine
WO2020181178A1 (fr) 2019-03-06 2020-09-10 The Broad Institute, Inc. Édition de base t:a à a:t par alkylation de thymine
WO2020181193A1 (fr) 2019-03-06 2020-09-10 The Broad Institute, Inc. Édition de bases t:a à a:t par méthylation de l'adénosine
WO2020191248A1 (fr) 2019-03-19 2020-09-24 The Broad Institute, Inc. Procédés et compositions pour l'édition de séquences nucléotidiques
US20220184127A1 (en) * 2019-04-08 2022-06-16 Phanes Therapeutics, Inc. Humanized anti-dll3 chimeric antigen receptors and uses thereof
WO2020214842A1 (fr) 2019-04-17 2020-10-22 The Broad Institute, Inc. Éditeurs de base d'adénine présentant des effets hors cible réduits
WO2020223535A1 (fr) 2019-05-01 2020-11-05 Juno Therapeutics, Inc. Cellules exprimant un récepteur recombinant à base d'un locus modifié du tgfbr2, et polynucléotides et méthodes associés
US20220249563A1 (en) * 2019-07-17 2022-08-11 Nanjing Legend Biotech Co., Ltd. Anti-dll3 chimeric antigen receptors and uses thereof
WO2021158921A2 (fr) 2020-02-05 2021-08-12 The Broad Institute, Inc. Éditeurs de base d'adénine et leurs utilisations
WO2021226558A1 (fr) 2020-05-08 2021-11-11 The Broad Institute, Inc. Méthodes et compositions d'édition simultanée des deux brins d'une séquence nucléotidique double brin cible
WO2022067130A2 (fr) 2020-09-24 2022-03-31 The Broad Institute, Inc. Arn guides d'édition primaire, leurs compositions et leurs méthodes d'utilisation
US20240025992A1 (en) * 2020-10-22 2024-01-25 Janssen Biotech, Inc. Proteins comprising delta-like ligand 3 (dll3) antigen binding domains and their uses
WO2022164935A1 (fr) 2021-01-27 2022-08-04 Umoja Biopharma, Inc. Lentivirus permettant de générer des cellules exprimant un récepteur antigénique chimérique anti-cd19
WO2023193015A1 (fr) 2022-04-01 2023-10-05 Sana Biotechnology, Inc. Polythérapies d'agoniste de récepteur de cytokine et de vecteur viral
WO2024006960A1 (fr) 2022-06-29 2024-01-04 Juno Therapeutics, Inc. Nanoparticules lipidiques pour l'administration d'acides nucléiques

Non-Patent Citations (134)

* Cited by examiner, † Cited by third party
Title
"Methods and Protocols", 2013, SPRINGER, article "Methods in Molecular Biology: Pluripotent Stem Cells"
"NCBI", Database accession no. NP_001311210.1
"Uniprot", Database accession no. E2QRCO
ABHINANDANMARTIN, MOL. IMMUNOL., vol. 45, no. 14, 2008, pages 3832 - 9
AHLERSBELYAKOV, BLOOD, vol. 115, 2010, pages 1678
ARNOVAD ET AL., JMOL BIOL, vol. 355, 2006, pages 443 - 458
ATSCHUL ET AL., J. MOLECULAR BIOL, vol. 215, 1990, pages 403
BAUM ET AL., MOLECULAR THERAPY: THE JOURNAL OF THE AMERICAN SOCIETY OF GENE THERAPY, vol. 13, 2006, pages 1050 - 1063
BELLIS REBECCA Y. ET AL: "DLL3-targeted CAR T-cell therapy in pre-clinical models for small cell lung cancer: safety, efficacy, and challenges", TRANSLATIONAL LUNG CANCER RESEARCH, vol. 13, no. 3, 1 March 2024 (2024-03-01), Hong Kong, pages 694 - 698, XP093285790, ISSN: 2218-6751, DOI: 10.21037/tlcr-23-820 *
BERGSHI, SCIENCE, vol. 271, 1996, pages 1081 - 1085
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423 - 426
BITINAITE, PROC. NATL. ACAD. SCI., vol. 95, 1998, pages 10570 - 10575
BLUESTONE ET AL., SCI. TRANSL. MED., vol. 7, 2015, pages 315
BRASH ET AL., MOL. CELL BIOL., vol. 7, 1987, pages 2031 - 2034
BROWNING ET AL.: "Insulators to Improve the Safety of Retroviral Vectors for HIV Gene Therapy", BIOMEDICINES, vol. 4, no. 1, 2016, pages 4
CARROLL ET AL., GENETICS SOCIETY OF AMERICA, vol. 188, 2011, pages 773 - 782
CAVALIERI ET AL., BLOOD, vol. 102, no. 2, 2003, pages 1637 - 1644
CERMAK ET AL., NUCL. ACIDS RES., vol. 39, 2011, pages 82
CHALLITA ET AL., J. VIROL., vol. 69, no. 2, 1995, pages 748 - 755
CHEN ET AL., J. IMMUNOL., vol. 183, 2009, pages 4094 - 4102
CHEN ET AL., PROTEIN ENG DES SEI, vol. 22, 2009, pages 249 - 256
CHEVALIER ET AL., MOL. CELL., vol. 10, 2002, pages 895 - 905
CHEVALIER, B. S.B. L. STODDARD, NUCLEIC ACIDS RES, vol. 29, no. 18, 2001, pages 3757 - 3774
CHICAYBAM ET AL., PLOS ONE, vol. 8, no. 3, 2013, pages 60298
CHOTHIALESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
CHOULIKA ET AL., MOL. CELL. BIOL., vol. 15, 1995, pages 1968 - 1973
CIERI ET AL., BLOOD, vol. 125, 2015, pages 2865
CLARKSON ET AL., NATURE, vol. 352, 1991, pages 624 - 628
COHEN-TANNOUDJI ET AL., MOL. CELL. BIOL., vol. 18, 1998, pages 1444 - 1448
CRONICAN ET AL., ACS CHEM BIOL., vol. 5, no. 8, 2010, pages 747 - 52
DANG ET AL., GENOME BIOLOGY, vol. 16, 2015, pages 280
DE FELIPE ET AL., TRAFFIC, vol. 5, 2004, pages 616 - 626
DE GRAAF ET AL., METHODS MOL. BIOL, vol. 178, 2002, pages 379 - 87
DE LIBERO: "Methods in Molecular Biology", vol. 514, 2009, HUMANA PRESS, article "T Cell Protocols"
DEVEREUX ET AL., NUCLEIC ACIDS RES, vol. 12, 1984, pages 387
DONOHO ET AL., MOL. CELL. BIOL, vol. 18, 1998, pages 4070 - 4078
DOYON ET AL., J AM CHEM SOC, vol. 128, 2006, pages 2477 - 2484
DOYON ET AL., NATURE METHODS, vol. 8, 2010, pages 74 - 79
EPINAT ET AL., NUCLEIC ACIDS RES, vol. 31, 2003, pages 2952 - 2962
EUCHNER ET AL., FRONTIERS IN IMMUNOLOGY, vol. 12, 2021
FELIPE, GENETIC VACCINES AND THER, vol. 2, 2004, pages 13
FLATMAN ET AL., J. CHROMATOGR. B, vol. 848, 2007, pages 79 - 87
FLYNN ET AL., CLINICAL & TRANSLATIONAL IMMUNOLOGY, vol. 3, 2014, pages 20
GARCIA ET AL.: "Naturally occurring T cell mutations enhance engineered T cell therapies", NATURE, vol. 626, 2024, pages 626 - 634
GATTINONI ET AL., NAT .REVIEWS, vol. 12, 2012, pages 671
GATTINONI ET AL., NAT. MED., vol. 17, 2012, pages 1290 - 1297
GUO ET AL., J. MOL. BIOL., vol. 200, 2010, pages 96
HACKETT ET AL., MOLECULAR THERAPY, vol. 18, 2010, pages 1748 - 1757
HAMILTONJENNIFER R. ET AL.: "In vivo human T cell engineering with enveloped delivery vehicles", NATURE BIOTECHNOLOGY, 2024, pages 1 - 9
HOCKEMEYER ET AL., NAT. BIOTECHNOL., vol. 29, 2011, pages 731 - 734
HOCKEMEYER ET AL., NATURE BIOTECH, vol. 29, 2011, pages 135 - 136
HOLLIGERHUDSON, NATURE BIOTECHNOLOGY, vol. 23, 2005, pages 1126 - 1136
HOLLINGER ET AL., PROC NATL ACAD. SCI., vol. 90, 1993, pages 6444 - 6448
HONEGGERPLIICKTHUN, J. MOL. BIOL., vol. 309, no. 3, 2001, pages 657 - 70
HUANGFU ET AL., NATURE BIOTECHNOL, vol. 26, no. 7, 2008, pages 795
HUSTON ET AL., PROC. NATL. ACAD. SCI., vol. 85, 1988, pages 5879 - 5883
IRIGUCHI ET AL., NATURE COMMUNICATIONS, vol. 12, 2021, pages 430
J. MANSILLA-SOTO ET AL., NATURE MEDICINE, vol. 28, no. 2, 2022, pages 345 - 351
J. WANG ET AL., AM. J. HEMATOL., vol. 97, no. 8, 2022, pages 992 - 1004
JINEK ET AL., SCIENCE, vol. 337, no. 6096, 2012, pages 816 - 821
JOHNSTON, NATURE, vol. 346, 1990, pages 776 - 777
KARDA ET AL., GENE THER, vol. 26, 2019, pages 86 - 92
KEARSE: "T Cell Protocols: Development and Activation", 2000, HUMANA PRESS
KLOMPE ET AL., NATURE, vol. 571, 2019, pages 219 - 225
KOLLMANNSPERGER ET AL., NAT COMM, vol. 7, 2016, pages 10372
KOONIN ET AL.: "Diversity, classification and evolution of CRISPR- Cas systems", CURR OPIN MICROBIOL, vol. 37, 2017, pages 67 - 78, XP055968642, DOI: 10.1016/j.mib.2017.05.008
KORTT ET AL., PROT. ENG., vol. 10, 1997, pages 423
KRIANGKUM ET AL., BIOMOL. ENG., vol. 18, 2001, pages 31 - 108
LEE ET AL., CANCER RES, vol. 71, 2011, pages 2871 - 2881
LEE ET AL., CLIN CANCER RES, vol. 18, no. 10, 2012, pages 2780 - 90
LEE ET AL., NANO LET, vol. 12, 2012, pages 6322 - 27
LEE ET AL., NANO LETT, vol. 12, 2012, pages 6322 - 27
LEFRANC ET AL., DEV. COMP. IMMUNOL., vol. 27, no. 1, 2003, pages 55 - 77
LI ET AL., PLOS ONE, vol. 8, 2013, pages 67401
LIEBERMAN, PHARMACEUTICAL DOSAGE FORMS, vol. 1-3, 1992
LIU ET AL., BIOINFORMATICS, vol. 24, 2008, pages 1850 - 1857
LLOYD, THE ART, SCIENCE AND TECHNOLOGY OF PHARMACEUTICAL COMPOUNDING, 1999
LU ET AL.: "The CBM-opathies-A Rapidly Expanding Spectrum of Human Inborn Errors of Immunity Caused by Mutations in the CARD11-BCL10-MALT1 Complex", FRONT IMMUNOL, vol. 9, 19 September 2018 (2018-09-19), pages 2078
LUNDSTROMKENNETH: "Self-replicating RNA viruses for RNA therapeutics", MOLECULES, vol. 23, no. 12, 2018, pages 3310, XP055682865, DOI: 10.3390/molecules23123310
LUPTON S. D. ET AL., MOL. AND CELL BIOL, vol. 11, no. 6, 1991
MACCALLUM ET AL., J. MOL. BIOL., vol. 262, 1996, pages 732 - 745
MACDONALD ET AL., J CLIN. INVEST, vol. 126, 2016, pages 1413 - 1424
MALI ET AL., NAT BIOTECH, vol. 31, no. 9, 2013, pages 833 - 838
MALI ET AL., NATURE METHODS, vol. 10, 2013, pages 957 - 963
MALI ET AL., SCIENCE, vol. 339, no. 6121, 2013, pages 823 - 826
MANURI ET AL., HUM GENE THER, vol. 21, no. 4, 2010, pages 427 - 437
MARTIN ET AL., PNAS, vol. 86, no. 23, 1989, pages 9268 - 9272
MIYARA ET AL., NAT. REV. RHEUMATOL., vol. 10, 2014, pages 543 - 551
MOSCOU ET AL., SCIENCE, vol. 326, 2009, pages 3501 - 1512
NAGORSENBAUERLE, EXP CELL RES, vol. 317, 2011, pages 1255 - 1260
PICKAR, DOSAGE CALCULATIONS, 1999
PORTOLANO ET AL., J. IMMUNOL., vol. 150, 1993, pages 880 - 887
PUCHTA ET AL., NUCLEIC ACIDS RES, vol. 21, 1993, pages 5034 - 5040
PUCHTA ET AL., PROC. NATL. ACAD. SCI., vol. 93, 1996, pages 5055 - 5060
PUTNAM ET AL., AM. TRANPLANT., vol. 13, 2013, pages 3010 - 3020
PUTNAM ET AL., DIABETES, vol. 58, 2009, pages 652 - 662
RAGURAMADITYASAMAGYA BANSKOTADAVID R. LIU: "Therapeutic in vivo delivery of gene editing agents", CELL, 2022
RAN ET AL., CELL, vol. 155, no. 2, 2013, pages 479 - 480
RAN ET AL., NATURE, vol. 520, 2015, pages 186 - 191
RIDDELL ET AL., HUMAN GENE THERAPY, vol. 3, 1992, pages 319 - 338
ROSENBERG ET AL., NEW ENGL J MED, vol. 319, 1988, pages 1676
ROUET ET AL., MOL. CELL. BIOL., vol. 14, 1994, pages 8096 - 8106
ROWLAND-JONES: "Lymphocytes: A Practical Approach", 1999, OXFORD UNIVERSITY PRESS
RUDIN ET AL., HEMATOL. ONCOL., vol. 16, 2023
RURIKJOEL G. ET AL.: "CAR T cells produced in vivo to treat cardiac injury", SCIENCE, vol. 375, no. 6576, 2022, pages 91 - 96
SARGENT ET AL., MOL. CELL. BIOL., vol. 17, 1997, pages 267 - 77
SEDDIKI ET AL., J. EXP. MED., vol. 203, 2006, pages 1693 - 1700
SEKI ET AL., WORLD J. STEM CELLS, vol. 7, no. 1, 2015, pages 116 - 125
SELIGMAN ET AL., NUCLEIC ACIDS RES, vol. 30, 2002, pages 3870 - 3879
SERA ET AL., BIOCHEMISTRY, vol. 41, 2002, pages 7074 - 7081
SHANKAR ET AL., STEM CELL RES THER, vol. 11, 2020, pages 234
SHARMA ET AL., MOLEC THER NUCL ACIDS, vol. 2, 2013, pages 74
SILVA ET AL., CURRENT GENE THERAPY, vol. 11, 2011, pages 11 - 27
SILVA ET AL., J MOL. BIOL., vol. 361, 2006, pages 744 - 754
SMITH ET AL., NUCLEIC ACIDS RES, vol. 363, no. 2, 2006, pages 283 - 294
STADLER ET AL., NATURE MEDICINE, 12 June 2017 (2017-06-12)
STRECKER ET AL., NATURE COMM, 2019
STRECKER ET AL., SCIENCE, vol. 365, 2019, pages 48 - 53
SU ET AL., METHODS MOL. BIOL, vol. 806, 2012, pages 287 - 299
SUSSMAN ET AL., J MOL BIOL, vol. 342, 2004, pages 31 - 41
SZCZEPEK ET AL., NATURE BIOTECH, vol. 25, 2007, pages 786 - 793
TAKAHASHIYAMANAKA, CELL, vol. 126, 2006, pages 663 - 676
THEMELI ET AL., NATURE BIOTECHNOLOGY, vol. 31, 2013, pages 928 - 933
UKENA ET AL., EXP. HEMATOL., vol. 39, 2011, pages 1152 - 1160
VAN ROEY ET AL., NATURE STRUCT. BIOL, vol. 9, 2002, pages 806 - 811
VAN TEDELOO ET AL., GENE THERAPY, vol. 7, no. 16, 2000, pages 1431 - 1437
VERHOEYEN ET AL., METHODS MOL BIOL, vol. 506, 2009, pages 115 - 114
WANG ET AL., J. IMMUNOTHER., vol. 35, no. 9, 2012, pages 689 - 701
WANG ET AL.: "Genomic profiling of Sézary syndrome identifies alterations of key T cell signaling and differentiation genes", NATURE GENETICS, vol. 47, 2015, pages 1426 - 1434, XP055844005, DOI: 10.1038/ng.3444
WARD ET AL., NATURE, vol. 334, 1989, pages 544
WOLTJEN ET AL., NATURE, vol. 458, no. 7239, 2009, pages 766 - 770
WOOD ET AL., SCIENCE, vol. 333, 2011, pages 307
ZHOU ET AL., CELL STEM CELL, vol. 8, 2009, pages 381 - 384
ZHOU ET AL., STEM CELLS, vol. 27, no. 11, 2009, pages 2667 - 74

Similar Documents

Publication Publication Date Title
US12404316B2 (en) Transgenic T cell and chimeric antigen receptor T cell compositions and related methods
US12325728B2 (en) Methods and compositions for genetically modifying lymphocytes to express polypeptides comprising the intracellular domain of CD79A and CD79B
JP6681837B2 (ja) 同種移植に適合するｔ細胞を作製するための方法
EP3380620B1 (fr) Vecteurs de transfert lentiviral optimisés et utilisations associées
US20210128617A1 (en) SYNTHETIC CARS TO TREAT IL13R-alpha-2 POSITIVE HUMAN AND CANINE TUMORS
JP2019047801A (ja) Ｒｎａガイドｃａｓヌクレアーゼ系を用いることによって免疫療法のためにｔ細胞を操作するための方法
JP2016525888A (ja) Ｒｎａガイドｃａｓヌクレアーゼ系を用いることによって免疫療法のためにｔ細胞を操作するための方法
CN116234558A (zh) 条件性地表达重组受体的工程化t细胞、相关多核苷酸和方法
JP2022547552A (ja) 前立腺幹細胞抗原（ｐｓｃａ）キメラ抗原受容体（ｃａｒ）を含む組成物および方法
JP2024534772A (ja) 同種異系細胞療法用の遺伝子改変細胞
US20240010988A1 (en) Genetically modified primary cells for allogeneic cell therapy
KR20250089526A (ko) Il18과 조합된 신규한 cd19 결합제를 포함하는 car-t 작제물 및 이의 사용 방법
WO2023193003A2 (fr) Constructions d'anticorps spécifiques de cd4 et compositions et utilisations associées
CN117165532A (zh) 具有增强的迁移能力的修饰的细胞
US20230398148A1 (en) Cells expressing a chimeric receptor from a modified invariant cd3 immunoglobulin superfamily chain locus and related polynucleotides and methods
US20250152709A1 (en) Cd3-targeted lentiviral vectors and uses thereof
WO2025212519A1 (fr) Protéines de liaison au dll3 et leurs utilisations
TW202304961A (zh) 用於將治療劑遞送至受體細胞之組合物及方法
WO2025212536A1 (fr) Cellules car-t marquées par un épitope d'anticorps thérapeutique pour un contrôle et une sécurité améliorés
WO2025171085A1 (fr) Compositions et procédés d'amélioration des agents thérapeutiques adoptifs par lymphocytes t
US20250339558A1 (en) Recombinant Viral Particle for Gene and/or Cellular Therapy
WO2024062138A1 (fr) Cellules immunitaires comprenant un gène suv39h1 modifié
WO2025235851A1 (fr) Lymphocytes t génétiquement modifiés exprimant un récepteur antigénique chimérique (car) cd19 et leurs utilisations pour une thérapie cellulaire allogénique
WO2025054202A1 (fr) Procédé de criblage d'un échantillon contenant un transgène à l'aide d'un code à barres unique
JP2025532948A (ja) 抗ｒｏｒ１キメラ抗原受容体（ｃａｒ）、ｃａｒを発現する細胞、及び関連する方法

Legal Events

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

Ref document number: 25719258

Country of ref document: EP

Kind code of ref document: A1