WO2024102838A1 - Récepteurs d'interleukine-7 génétiquement modifiés et leurs utilisations - Google Patents

Récepteurs d'interleukine-7 génétiquement modifiés et leurs utilisations Download PDF

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WO2024102838A1
WO2024102838A1 PCT/US2023/079129 US2023079129W WO2024102838A1 WO 2024102838 A1 WO2024102838 A1 WO 2024102838A1 US 2023079129 W US2023079129 W US 2023079129W WO 2024102838 A1 WO2024102838 A1 WO 2024102838A1
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cell
amino acid
polypeptide
engineered
locus
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PCT/US2023/079129
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English (en)
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Andreia COSTA
Melissa Chiasson
Rupesh AMIN
Eric Eymard
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Century Therapeutics, Inc.
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Publication of WO2024102838A1 publication Critical patent/WO2024102838A1/fr

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Definitions

  • the application relates to engineered interleukin-7 (IL-7) receptors, particularly IL-7 receptor alpha chain (IL-7Ra) polypeptides comprising one or more mutations, and their uses in enhancing expansion and/or persistence of a population of cells (e.g., immune cells) and in prevention and/or treatment of a disease, including a cancer.
  • IL-7Ra IL-7 receptor alpha chain
  • Related polynucleotides, vectors, cells and pharmaceutical compositions are also provided.
  • Adoptive cell therapy typically involves isolating cells from a donor, culturing and/or manipulating cells in vitro, and then transferring the cells to a patient for the treatment of a disease. While ACT has emerged as a powerful and potentially curative therapy, one challenge that remains is its lack of efficacy due to limited expansion and/or persistence of immune cells post infusion. Lymphodepletion regimens involving, e g., chemotherapy and/or radiation have been used to enhance in vivo immune cell expansion, however, such approaches can be associated with unwanted side effects.
  • immune cells may be generated from cancer patients who have undergone multiple rounds of chemotherapy and/or drug treatments prior to their eligibility for immune therapy, and as such, quality and/or counts of the cells to be engineered may be low. There is therefore a need for means to enable T-cell expansion without such toxic lymphodepl eting agents.
  • Sources of immune cells for use in ACT include those that have been differentiated from induced pluripotent stem cells (iPSCs). These cells can be modified to be allogeneic. There remains an unmet need for therapeutically sufficient and functional engineered allogeneic iPSC-derived therapies for treating cancer. In engineering cell therapies, it is desirable to engineer the cells with multiple functionalities including means to increase and enable cell expansion.
  • iPSCs induced pluripotent stem cells
  • the present disclosure describes engineered interleukin-7 (IL-7) receptor compositions and methods for their use in enhancing expansion and/or persistence of immune cells and in immunotherapy.
  • IL-7 engineered interleukin-7
  • an engineered IL-7 receptor alpha chain (IL-7Ra) polypeptide comprising one or more amino acid mutations in the IL-7Ra cytoplasmic domain, wherein the one or more amino acid mutations promote IL-7 cytokine independent signaling.
  • the one or more amino acid mutations comprise at least one Box 2 motif insertion, at least one substitution at the IL-7Ra YXXM motif, at least one YXXM motif insertion, or a combination thereof.
  • the Box 2 motif is inserted from about 10 amino acids to about 50 amino acids away from the transmembrane domain.
  • the Box 2 motif is inserted 3' to amino acid position E301 with respect to
  • the Box 2 motif comprises the sequence of ISPLEVLERDK (SEQ ID NO: 3), or an amino acid sequence having at least 80% identity thereof.
  • the IL-7Ra polypeptide comprises the amino acid sequence of
  • the IL-7Ra YXXM motif substitution comprises an amino acid substitution at amino acid positions V450, T451, or M452, or a combination thereof, with respect to SEQ ID NO: 1.
  • the YXXM motif substitution comprises V450M and T45 IN with respect to SEQ ID NO: 1.
  • the IL-7Ra polypeptide comprises the amino acid sequence of
  • the IL-7Ra polypeptide comprises the amino acid sequence of
  • the YXXM motif insertion comprises a IL-7Ra YXXM motif or a YXXM motif comprising the sequence of YMNM (SEQ ID NO: 17), inserted 3' to amino acid position Q459 with respect to SEQ ID NO: 1.
  • the IL-7Ra polypeptide comprises the amino acid sequence of
  • the IL-7Ra YXXM motif substitution comprises M452L with respect to SEQ ID NO: 1.
  • the IL-7Ra polypeptide comprises the amino acid sequence of MTILGTTFGMVFSLLQVVSGESGYAQNGDLEDAELDDYSFSCYSQLEVNGSQHSLTC AFEDPDVNITNLEFEICGALVEVKCLNFRKLQEIYFIETKKFLLIGKSNICVKVGEKSLT CKKIDLTTIVKPEAPFDLSVVYREGANDFVVTFNTSHLQKKYVKVLMHDVAYRQEK DENKWTHVNLSSTKLTLLQRKLQPAAMYEIKVRSIPDHYFKGFWSEWSPSYYFRTPE INNSSGEMDPILLTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKP
  • the IL-7Ra is capable of inducing constitutive IL-7R activity.
  • the constitutive IL-7R activity elicits signaling via a JAK- STAT pathway or phosphoinositide-3 kinase (PI3K)-protein kinase B pathway, or a combination thereof.
  • PI3K phosphoinositide-3 kinase
  • the signaling via the JAK-STAT pathway is STAT5 signaling.
  • a polynucleotide encoding an engineered IL- 7Ra polypeptide described herein.
  • the polynucleotide sequence encoding the engineered IL- 7Ra polypeptide comprises the nucleotide sequence of SEQ ID NO: 6, 8, 10. 12. 14. or 16, or a nucleotide sequence having at least 80% sequence identity thereof.
  • the polynucleotide sequence encoding the engineered IL- 7Ra polypeptide is operably linked to a promoter.
  • the promoter is an inducible promoter.
  • the polynucleotide described herein is a DNA molecule.
  • the polynucleotide described herein is an RNA molecule.
  • a recombinant vector comprising a polynucleotide described herein.
  • the vector is a viral vector.
  • the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, an alphaviral vector, a herpes virus vector, a baculoviral vector, or a vaccinia virus vector.
  • the vector is a non-viral vector.
  • the non-viral vector is a minicircle plasmid, a Sleeping Beauty transposon, a piggyBac transposon, or a single or double stranded DNA molecule that is used as a template for homology directed repair (HDR) based gene editing.
  • HDR homology directed repair
  • an isolated host cell comprising a polynucleotide described herein or a recombinant vector described herein.
  • an isolated host cell comprising an engineered IL-7Ra encoded by a polynucleotide described herein.
  • the host cell is an iPSC or a population thereof.
  • the engineered IL-7Ra polynucleotide is integrated into a gene locus selected from the group consisting of an AAV S 1 locus, a B2M locus, a CIITA locus, a CCR5 locus, a CD70 locus, a CLYBL locus, an NKG2A locus, an NKG2D locus, a CD38 locus, a TRAC locus, a TRBC1 locus, a ROSA26 locus, an HTRP locus, a GAPDH locus, a RUNX1 locus, a TAPI locus, a TAP2 locus, a TAPBP locus, an NLRC5 locus, a RFXANK locus, a RFX5 locus, a RFXAP locus, a CISH locus, a CBLB locus, a S0CS2 locus, a PD1 locus, a CTLA4 locus
  • the engineered IL-7Ra polynucleotide is integrated into a gene locus by targeted genome editing comprising using a method selected from the group consisting of a CRISPR method, a zinc finger nuclease method, a TALEN method, a homing nuclease method, a homology recombination method, and any functional variation thereof.
  • the host cell is an immune cell.
  • the host cell is a B cell, a T cell, a natural killer (NK) cell, a mesenchymal stem cell (MSC), or a macrophage.
  • NK natural killer
  • MSC mesenchymal stem cell
  • the host cell is an B cell.
  • the host cell is a T cell.
  • the host cell is an a T-cell, a y5 T-cell, a CD8+ T-cell, a CD4+ T-cell, a cytotoxic T-cell, an invariant natural killer T (iNKT) cell, a memory T-cell, a memory stem T-cell (TSCM), a naive T-cell, an effector T-cell, a T-helper cell, or a regulatory T-cell (Treg).
  • iNKT invariant natural killer T
  • TSCM memory stem T-cell
  • Reg regulatory T-cell
  • the host cell is an NK cell.
  • the host cell is an NK cell derived from peripheral, cord blood, iPS cells, and/or a cell line.
  • the host cell further expresses one or more antigenrecognition molecules.
  • the one or more antigen-recognition molecules are selected from a[3 T cell receptors (TCRs). synthetic T cell receptors and antigen receptor (STARs), chimeric antigen receptor (CARs), T cell antigen couplers (TACs), T cell receptor fusion constructs (TruCs), or antibodies (e.g., bispecific antibodies), or a combination thereof
  • the target antigen for the antigen-recognition molecule is selected from the group consisting of 17-1 A antigen, A3, A33 antigen, AFP, B7H4, Ba 733, BCMA, BrE3 antigen.
  • p53 PAP, PDGFRA, PLGF. PSA, PSMA. ROBOL RS5, SI 00. SLAM F7. SLITRK6, TAC, TAG-72, tenascin-C, tenascin-R, tenascin-W, tenascin-X, Thomson- Friedenreich antigen, Tn antigen, TRAILR1, TRAILR2, TRAILR3, TRAILR4, VEGF, atumor necrosis antigen, an angiogenesis antigen, and an oncogene antigen.
  • a primary cell an induced pluripotent stem cell
  • iPSC chimeric antigen receptors
  • a co-stimulatory signaling domain (vi) a co-stimulatory signaling domain; and, optionally, at least one of:
  • HLA- E human leukocyte antigen E
  • HLA-G human leukocyte antigen G
  • an exogenous polynucleotide encoding a natural killer (NK) cell receptor immunoglobulin gamma Fc region receptor III (FcyRIII, cluster of differentiation 16 (CD 16)) and/or an NKG2D protein;
  • NK natural killer
  • FcyRIII cluster of differentiation 16
  • a pharmaceutical composition comprising an isolated host cell described herein, or a primary cell or an induced pluripotent stem cell (iPSC), or a derivative cell thereof, described herein, and a pharmaceutically acceptable carrier and/or excipient.
  • iPSC induced pluripotent stem cell
  • a method of enhancing expansion and/or persistence of a population of host cells comprising expressing a polynucleotide described herein or a recombinant vector described herein in a host cell.
  • a method of generating an isolated host cell described herein comprising genetically modifying the host cell with a polynucleotide described herein or a recombinant vector described herein.
  • the host cell is an iPSC or a population thereof.
  • the host cell is an immune cell.
  • the host cell is a B cell, a T cell, a natural killer (NK) cell, a mesenchymal stem cell (MSC), or a macrophage.
  • NK natural killer
  • MSC mesenchymal stem cell
  • the host cell is an B cell.
  • the host cell is a T cell.
  • the host cell is an 0,(3 T-cell, a y5 T-cell, a CD8+ T-cell, a CD4+ T-cell, a cytotoxic T-cell, an invariant natural killer T (iNKT) cell, a memory T-cell, a memory stem T-cell (TSCM), a naive T-cell, an effector T-cell, a T-helper cell, or a regulatory T-cell (Treg).
  • iNKT invariant natural killer T
  • TSCM memory stem T-cell
  • Reg regulatory T-cell
  • the host cell is an NK cell.
  • the host cell is an NK cell derived from peripheral, cord blood, iPS cells, and/or a cell line.
  • the method may further comprise genetically modifying a host cell described herein to express a chimeric antigen receptor (CAR) described herein.
  • CAR chimeric antigen receptor
  • a method for preventing or treating a cancer comprising administering to a subject in need thereof, a therapeutically effective amount of a host cell described herein, a primary cell or an induced pluripotent stem cell, or a derivative cell thereof, described herein, or a pharmaceutical composition described herein.
  • the cancer is lung cancer, pancreatic cancer, liver cancer, melanoma, bone cancer, breast cancer, colon cancer, leukemia, uterine cancer, ovarian cancer, lymphoma, brain cancer, cervical cancer, head and neck cancer, liver cancer, prostate cancer, renal cell carcinoma, bladder cancer, other solid tumor cancer, or hematologic malignancy.
  • the brain cancer is a glioblastoma.
  • the hematologic malignancy is a leukemia, a lymphoma, or a myeloma.
  • the leukemia is acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), or chronic myeloid leukemia (CML)
  • ALL acute lymphocytic leukemia
  • CLL chronic lymphocytic leukemia
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • the lymphoma is Hodgkin's lymphoma or non-Hodgkin’s lymphoma.
  • the non-Hodgkin’s lymphoma is Diffuse Large B-cell Lymphoma (DLBCL).
  • the subject is human.
  • FIGs. 1A-1B depict IL-7Ra mutant designs.
  • a schematic representation of a full- length wild-type IL-7Ra comprising an ectodomain, a transmembrane domain, and a cytoplasmic domain used as a template for the IL-7Ra mutant designs described herein is shown in Fig. 1A.
  • the wild-type IL-7Ra YXXM motif comprises the amino acid sequence YVTM (SEQ ID NO: 21).
  • IL-7Ra mutant designs included: (1) CD28 YXXM (YVTM449- 452YMNM IL-7Ra); (2) CD122 (IL2/15Rp) YXXM; (3) YXXM duplication (YXXM Dup); (4) CD122 (IL2/15RP) Box 2; (5) CD28 YXXM + Box 2; and (6) CD28 YXXM duplication (CD28 YXXM Dup) + Box 2 (Fig. IB).
  • Fig. 2 shows an exemplary workflow for testing the IL-7Ra mutants.
  • FIG. 3 depicts a graph showing secreted alkaline phosphatase (SEAP) production 24 hours post-transfection for the IL-7Ra YXXM and CD 122 Box 2 site mutants, which were tested in the presence (10 ng/mL IL-7 and 5 ng/mL IL-7) and absence (no IL-7 added) of exogenous IL-7.
  • SEAP secreted alkaline phosphatase
  • Figs. 4A-4C illustrate validation in Ba/F3 cells to confirm constitutive activity of IL- 7Ra CD 122 Box 2 insertion mutants.
  • a graph showing Ba/F3 cell counts post passage day D0- D3 for IL-7Ra mutants (CD28 YXXM + Box 2, CD28 YXXM duplication + Box 2, CD 122 Box 2, CD28 YXXM, YXXM duplication), constitutively signaling cytokine receptor (C7R), wild type (WT) IL-7Ra, and no virus (control), in the presence of 10 mg/mL recombinant human (rhu)-IL-7 (Fig. 4A) and when no exogenous cytokine was added (Fig. 4B).
  • Figs. 5A-5G demonstrate a strong positive selection effect of the IL-7Ra constructs in the absence of cytokine supplementation.
  • a graph of the percentage of positive GFP cells at D3 post transduction (p.t.) and D7 post re-plate (p. rp.) for the 10 ng/mL IL-7 group and the no cytokine group Fig. 5G).
  • the present disclosure provides engineered interleukin-7 (IL-7) receptors, and fragments and derivatives thereof, particularly IL-7 receptor alpha chain (IL-7Ra) polypeptides comprising one or more mutations, for example, in the IL-7Ra cytoplasmic domain.
  • IL-7Ra IL-7 receptor alpha chain
  • such mutation(s) are capable of promoting cytokine-independent signaling, e.g., within a cell.
  • related polynucleotides, vectors, cells and pharmaceutical compositions Methods for enhancing expansion and/or persistence of a population host cells, generating genetically modified host cells, as well as for treating subjects using the genetically modified host cells and/or pharmaceutical compositions described herein are also provided.
  • the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers and are intended to be non-exclusive or open-ended.
  • a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus.
  • “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).
  • the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term "and/or.”
  • subject means any animal, preferably a mammal, most preferably a human.
  • mammal encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably a human.
  • the subject is a patient. In some embodiments, the subject is an individual.
  • any numerical values, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.”
  • references that include a numerical parameter would include variations that, using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.), would not vary the least significant digit.
  • a numerical value typically includes ⁇ 10% of the recited value.
  • a concentration of I mg/mL includes 0.9 mg/mL to 1.1 mg/mL.
  • a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v).
  • cytokine refers to any of various cell signaling molecules that are capable of regulating an immune system, for example, in response to inflammation and/or infection, and can aid cell-to-cell communication in immune responses.
  • cytokines include, without limitation, interleukins (ILs), chemokines, interferons, lymphokines, and tumor necrosis factors.
  • IL-7 refers to IL-7 polypeptides or derivatives thereof having substantial amino acid sequence identity to a mature, wild-type mammalian IL-7.
  • IL-7 polypeptide can refer to an amino acid sequence of a recombinant or non-recombinant polypeptide having an amino acid sequence of: i) a native allelic variant of an IL-7 polypeptide, ii) a biologically active fragment of an IL-7 polypeptide, or iii) a biologically active variant or analog of an IL-7 polypeptide.
  • the IL-7 can be obtained from any mammalian species, or obtained from a recombinant expression system (e.g., yeast, bacteria, etc.).
  • the IL-7 may be glycosylated, partially glycosylated, or non-glycosylated.
  • An exemplary human wild-type IL-7 polypeptide may comprise an amino acid sequence of SEQ ID NO: 22 (see also, e.g., UniProtKB P 13232).
  • interleukin-7 is a non-hematopoietic cell-derived cytokine which, among other things, can promote survival and/or proliferation of B-cells, T cells, long term memory T cells, and various other immune cells.
  • IL-7 may be requisite for T cell development and participates in the restoration and maintenance of homeostasis of mature T cells.
  • IL-7 also can play a role in naive and memory T cell homeostasis and survival in the periphery.
  • IL-7 may be secreted by stromal cells, e.g., in the thymus and/or bone marrow (BM), and may be produced by fibroblastic reticular cells, e.g., in T cell zones of lymph nodes.
  • IL-7 can signal via binding to its heterodimeric receptor, which is typically a cell surface protein comprising two chains: the alpha (a) receptor chain (IL-7Ra, CD 127), and the common cytokine receptor y-chain (yc, CD 132) shared with receptors for IL-2, IL-4, IL-9, IL- 15, and IL-21.
  • IL-7Ra alpha receptor chain
  • yc common cytokine receptor y-chain
  • IL-7 signals through binding its IL-7Ra (CD127) and y-chain (yc) via their extracellular domains to form a ternary' complex which activates, e.g., the JAK/STAT. phosphoinositol 3-kinase (PI3K)/Akt, and/or SRC pathways.
  • IL-7Ra CD127
  • yc y-chain
  • IL-7 receptor refers to any form of IL-7R, or fragment or derivative thereof, capable of inducing IL-7R activity.
  • Non-limiting examples of IL-7R activity include eliciting signaling via a JAK-STAT pathway or a phosphoinositide-3 kinase (PI3K)-protein kinase B pathway, or a combination thereof.
  • the signaling via the JAK-STAT pathway is STAT5 signaling.
  • An exemplary IL-7R, or fragment or derivative thereof, of the present disclosure is alpha (a) receptor chain (IL-7Ra, CD 127), or a fragment or derivative thereof.
  • an IL-7Ra or fragment or derivative thereof comprises an engineered IL-7R, or fragment or derivative thereof, described herein.
  • an engineered IL-7Ra, or fragment or derivative thereof may comprise a polypeptide comprising one or more amino acid mutations at one or more locations in its amino acid sequence as compared to an amino acid sequence of a reference IL-7Ra polypeptide, e.g., a reference sequence comprising or consisting of a wild-type IL-7Ra polypeptide.
  • FIG. 1 A schematic representation of an exemplary full-length (459 aa) wild-type IL-7Ra polypeptide (see also, e.g., SEQ ID NO: 1; UniProtKB P16871) comprising an ectodomain (21-239 aa), a transmembrane domain (240-264 aa) and a cytoplasmic domain (265-459 aa) that may be used a reference sequence (i.e., template) for the engineered IL-7Ra described herein is displayed in Fig. 1.
  • fragment when referring to a protein means a protein that is shorter or has fewer amino acids than the full-length protein.
  • a fragment can be, for example, an N- terminal fragment (i.e., removal of a portion of the C-terminal end of the protein), a C-terminal fragment (i.e., removal of a portion of the N-terminal end of the protein), or an internal fragment.
  • fragment when referring to a nucleic acid means a nucleic acid that is shorter or has fewer nucleotides than the full-length nucleic acid.
  • a fragment can be, for example, a 5’ fragment (i.e., removal of a portion of the 3' end of the nucleic acid), a 3' fragment (i.e., removal of a portion of the 5' end of the protein), or an internal fragment.
  • derivative refers to a nucleic acid, or protein, or a variant, or an analog thereof comprising one or more mutations and/or chemical modifications as compared to a corresponding full-length wild-type nucleic acid, or protein.
  • Non-limiting examples of chemical modifications involving nucleic acids include, for example, modifications to the sugar moiety, phosphate moiety, base moiety, phosphate-sugar backbone, or a combination thereof.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman. Proc. Nat 'I. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates. Inc. and John Wiley & Sons, Inc. (1995 Supplement) (Ausubel)).
  • BLAST and BLAST 2.0 algorithms are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402, respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra .
  • HSPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold (Altschul et al., supra .
  • These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues; always > 0
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negativescoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlength
  • E expectation
  • BLOSUM62 scoring matrix see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity 7 between two sequences (see, e.g., Karlin & Altschul, Proc. Nat’l. Acad. Sci. USA 90:5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • a further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below 7 .
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.
  • isolated means a biological component (such as a nucleic acid, peptide, protein, or cell) has been substantially separated, produced apart from, or purified away from other biological components of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, proteins, cells, and tissues.
  • Nucleic acids, peptides, proteins, and cells that have been “isolated” thus include nucleic acids, peptides, proteins, and cells purified by standard purification methods and purification methods described herein.
  • isolated nucleic acids, peptides, proteins, and cells can be part of a composition and still be isolated if the composition is not part of the native environment of the nucleic acid, peptide, protein, or cell.
  • the term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • polynucleotide synonymously referred to as “nucleic acid molecule,” “nucleotide” or “nucleic acid,” refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA.
  • DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the term “polynucleotide” also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stabili ty or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short nucleic acid chains, often referred to as “oligonucleotides”.
  • a “construct” refers to a macromolecule or complex of molecules comprising a polynucleotide to be delivered to a host cell, either in vitro or in vivo.
  • vector expression vector
  • expression construct are used interchangeably to refer to a composition of matter which can be used to deliver a nucleic acid of interest to the interior of a cell and mediate its expression within the cell.
  • vectors are autonomously replicating plasmids and viruses (such as, e.g., adenoviral vectors, adeno- associated virus vectors (AAV), lentiviral vectors, Sindbis virus vectors, etc.).
  • an expression construct can be replicated in a living cell, or it can be made synthetically.
  • an expression vector comprises a promoter operably linked to a polynucleotide (e.g., a polynucleotide encoding an engineered IL-7Ra polypeptide, or fragment or derivative thereof, described herein) which promoter controls the initiation of transcription by RNA polymerase and expression of the polynucleotide.
  • Typical promoters for mammalian cell expression include, e.g., SV40 early promoter, CMV immediate early promoter (see, e.g.. U.S. Pat. Nos.
  • mouse mammary tumor virus LTR promoter mouse mammary tumor virus LTR promoter
  • Ad MLP adenovirus major late promoter
  • herpes simplex virus promoter murine metallothionein gene promoter
  • U6 or Hl RNA pol III promoter mouse mammary tumor virus LTR promoter
  • Ad MLP adenovirus major late promoter
  • herpes simplex virus promoter murine metallothionein gene promoter
  • U6 or Hl RNA pol III promoter U6 or Hl RNA pol III promoter
  • Non-limiting examples of promoters useful for expressing, e.g., IL-7Ra polypeptides and/or chimenc antigen receptors (CARs) described herein in the methods of the present disclosure include, e.g., MND promoter, synapsin promoter, CamKIIa promoter, ubiquitin promoter, CAG promoter, CMV promoter, and (3-actin promoter. These and other promoters can be obtained from commercially available plasmids, using techniques well known in the art. See, e.g., Sambrook et al., supra. Enhancer elements may be used in association with promoters to increase expression levels of the vectors.
  • Examples include the SV40 early gene enhancer, as described in Dijkema et al., EMBO J. (1985) 4:761, the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777, which is incorporated herein by reference in its entirety, and elements derived from human CMV, as described in Boshart et al., Cell (1985) 41:521, which is incorporated herein by reference in its entirety, such as elements included in the CMV intron A sequence.
  • LTR long terminal repeat
  • Transcription terminator/polyadenylation signals may also be present in the expression vector.
  • sequences include, but are not limited to, those derived from SV40, as described in Sambrook et al., supra, as well as a bovine growth hormone terminator sequence (see, e.g., U.S. Pat. No. 5,122,458, which is incorporated herein by reference in its entirety).
  • 5'-UTR sequences can be placed adjacent to the coding sequence in order to enhance expression of the same.
  • Such sequences include UTRs which include, e.g., an Internal Ribosome Entry Site (IRES) present in the leader sequences of picomaviruses such as the encephalomyocarditis virus (EMCV) UTR (Jang et al. J. Virol. (1989) 63: 1651-1660, which is incorporated herein by reference in its entirety).
  • IRES Internal Ribosome Entry Site
  • EMCV encephalomyocarditis virus
  • Other useful picomavirus UTR sequences include, e.g., the polio leader sequence, hepatitis A virus leader and the hepatitis C IRES.
  • the term “host cell” means any cell that contains a heterologous nucleic acid.
  • the heterologous nucleic acid can be a vector (e.g., an expression vector).
  • a host cell can be a cell from any organism that is selected, modified, transformed, grown, used or manipulated in any way. for the production of a substance by the cell, for example the expression by the cell of a gene, a DNA or RNA sequence, a protein or an enzyme.
  • An appropriate host may be determined.
  • the host cell may be selected based on the vector backbone and the desired result.
  • a plasmid or cosmid can be introduced into a prokaryote host cell for replication of several types of vectors.
  • Bacterial cells such as, but not limited to DH5a, JM109, and KCB, SURE® Competent Cells, and SOLOP ACK Gold Cells, can be used as host cells for vector replication and/or expression. Additionally, bacterial cells such as E. coli LE392 could be used as host cells for phage viruses. Eukaryotic cells that can be used as host cells include, but are not limited to yeast (e.g., YPH499, YPH500 and YPH501), insects and mammals. Examples of mammalian eukaryotic host cells for replication and/or expression of a vector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, CHO, Saos, and PC12.
  • Host cells of the present disclosure include T cells and natural killer cells (NK cells) that contain the DNA or RNA sequences encoding the polypeptide(s) disclosed herein.
  • host cells may be used for enhancing T cell activity, NK cell activity, treatment of cancer, and/or treatment of autoimmune disease.
  • operably linked refers to the operational linkage of nucleic acid sequences or amino acid sequences so that they are placed in functional relationships with each other.
  • a promoter is operably linked with a coding sequence or functional RNA when it is capable of affecting the expression of that coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter).
  • Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
  • the term “expression” as used herein, refers to the biosynthesis of a gene product.
  • the term encompasses the transcription of a gene into RNA.
  • the term also encompasses translation of RNA into one or more polypeptides, and further encompasses all naturally occurring post-transcriptional and post-translational modifications.
  • the expressed polypeptides can be within the cytoplasm of a host cell, into the extracellular milieu such as the growth medium of a cell culture or anchored to the cell membrane.
  • regulatory element refers to any cis-acting genetic element that controls some aspect of the expression of nucleic acid sequences.
  • the term “promoter” comprises essentially the minimal sequences required to initiate transcription.
  • the term “promoter” includes the sequences to start transcription, and in addition, also include sequences that can upregulate or downregulate transcription, commonly termed “enhancer elements” and “repressor elements”, respectively.
  • the term “promoter” comprises essentially the minimal sequences required to initiate transcription.
  • the term “promoter” includes the sequences to start transcription, and in addition, also include sequences that can upregulate or downregulate transcription, commonly termed “enhancer elements” and ⁇ ‘repressor elements”, respectively.
  • the terms '‘peptide,” “polypeptide,” or “protein” can refer to a molecule comprised of amino acids and can be recognized as a protein by those of skill in the art.
  • the conventional one-letter or three-letter code for amino acid residues is used herein.
  • the terms “peptide,” “polypeptide,” and “protein” can be used interchangeably herein to refer to polymers of amino acids of any length.
  • the polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation.
  • acetylation phosphorylation
  • any other manipulation or modification such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • other modifications known in the art.
  • signal peptide refers to a leader sequence at the aminoterminus (N-terminus) of a nascent polypeptide, e.g., an IL-7Ra polypeptide disclosed herein, which co-translationally or post-translationally directs the nascent protein to the endoplasmic reticulum and subsequent surface expression or secretion.
  • extracellular domain refers to the part of a molecule of the application , e.g., an IL-7Ra polypeptide or a chimeric antigen receptor (CAR) described herein, that is located outside of the cell membrane and can be capable of binding to an antigen, target or ligand.
  • CAR chimeric antigen receptor
  • transmembrane domain refers to the portion of a molecule of the application, e.g., an IL-7Ra polypeptide or a CAR described herein, that extends across the cell membrane and anchors the molecule to cell membrane.
  • intracellular domain or “cytoplasmic domain,” refers to the part of a molecule of the application, e.g., an IL-7Ra polypeptide or a CAR described herein, that is located inside of the cell membrane and can be capable of transducing an effector signal.
  • the peptide sequences described herein are written according to the usual convention whereby the N-terminal region of the peptide is on the left and the C-terminal region is on the right. Although isomeric forms of the amino acids are known, it is the L-form of the amino acid that is represented unless otherwise expressly indicated.
  • pluripotent refers to the abi 1 i ty of a cell to form all lineages of the body or soma or the embryo proper.
  • embryonic stem cells are a type of pluripotent stem cells that are able to form cells from each of the three germs layers, the ectoderm, the mesoderm, and the endoderm.
  • Pluripotency is a continuum of developmental potencies ranging from the incompletely or partially pluripotent cell (e.g., an epiblast stem cell or EpiSC), which is unable to give rise to a complete organism to the more primitive, more pluripotent cell, which is able to give rise to a complete organism (e.g., an embryonic stem cell).
  • the incompletely or partially pluripotent cell e.g., an epiblast stem cell or EpiSC
  • EpiSC epiblast stem cell
  • a complete organism e.g., an embryonic stem cell
  • induced pluripotent stem cells means that the stem cells are produced from differentiated adult, neonatal or fetal cells that have been induced or changed or reprogrammed into cells capable of differentiating into tissues of all three germ or dermal layers: mesoderm, endoderm, and ectoderm.
  • the iPSCs produced do not refer to cells as they are found in nature.
  • the induced pluripotent stem cell (iPSC) parental cell lines may be generated from peripheral blood mononuclear cells (PBMCs) or T-cells using any known method for introducing re-programming factors into non-pluripotent cells such as the episomal plasmidbased process as previously described in U.S. Pat. Nos. 8,546,140; 9,644,184; 9,328,332; and 8,765,470, the complete disclosures of which are incorporated herein by reference in their entirety for all intended purposes.
  • PBMCs peripheral blood mononuclear cells
  • T-cells any known method for introducing re-programming factors into non-pluripotent cells such as the episomal plasmidbased process as previously described in U.S. Pat. Nos. 8,546,140; 9,644,184; 9,328,332; and 8,765,470, the complete disclosures of which are incorporated herein by reference in their entirety for all intended purposes.
  • the reprogramming factors may be in a form of polynucleotides, and thus are introduced to the non-pluripotent cells by vectors such as a retrovirus, a Sendai virus, an adenovirus, an episome, and a mini-circle.
  • the one or more polynucleotides encoding at least one reprogramming factor are introduced by a lentiviral vector.
  • the one or more polynucleotides are introduced by an episomal vector.
  • the one or more polynucleotides are introduced by a Sendai viral vector.
  • the iPSCs are clonal iPSCs or are obtained from a pool of iPSCs and the genome edits are introduced by making one or more targeted integration and/or in/del at one or more selected sites.
  • the iPSCs are obtained from human T cells having antigen specificity and a reconstituted TCR gene (hereinafter, also refer to as “T-iPS” cells) as described in US Pat. Nos. 9,206,394, and 10,787,642 hereby incorporated by reference into the present application in their entirety 7 for all intended purposes.
  • immune cell' or “immune-effector cell’ refers to a cell that is involved in an immune response. Immune response includes, for example, the promotion of an immune effector response. Examples of immune cells include T cells, B cells, natural killer (NK) cells, mast cells, and myeloid-derived phagocytes.
  • NK natural killer
  • engineered immune cell or “engineered immune-effector cell” refers to an immune cell that has been genetically modified by the addition of exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell.
  • T cell and “T lymphocyte” are interchangeable and used synonymously herein.
  • T cell includes thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes.
  • a T cell can be a T helper (Th) cell, for example a T helper 1 (Thl) or a T helper 2 (Th2) cell.
  • Th T helper 1
  • Th2 T helper 2
  • the T cell can be a helper T cell (HTL; CD4+ T cell) CD4+ T cell, a cytotoxic T cell (CTL; CD8+ T cell), a tumor infiltrating cytotoxic T cell (TIL; CD8+ T cell), CD4+CD8+ T cell, or any other subset of T cells.
  • TTL helper T cell
  • CTL cytotoxic T cell
  • TIL tumor infiltrating cytotoxic T cell
  • CD4+CD8+ T cell CD4+CD8+ T cell
  • Other illustrative populations of T cells suitable for use in particular embodiments include naive T cells and memory T cells.
  • NK.T cells which refer to a specialized population of T cells that express a semi -invariant a[3 T-cell receptor, but also express a variety of molecular markers that are typically associated with NK cells, such as NK1.1.
  • NKT cells include NK1.1+ andNKl.
  • NKT cells The TCR on NKT cells is unique in that it recognizes glycolipid antigens presented by the MHC I-like molecule CD Id. NKT cells can have either protective or deleterious effects due to their abilities to produce cytokines that promote either inflammation or immune tolerance. Also included are "‘gamma-delta T cells (y5 T cells),” which refer to a specialized population that to a small subset of T cells possessing a distinct TCR on their surface, and unlike the majority of T cells in which the TCR is composed of two glycoprotein chains designated a- and P-TCR chains, the TCR in yS T cells is made up of a y-chain and a 5-chain.
  • Tregs refers to T cells that suppress an abnormal or excessive immune response and play a role in immune tolerance.
  • Tregs cells are typically transcription factor Foxp3 -positive CD4+T cells and can also include transcription factor Foxp3-negative regulatory T cells that are IL-10-producing CD4+T cells.
  • NK cell refers to a differentiated lymphocyte with a CD 16+ CD56+ and/or CD57+ TCR- phenotype. NKs are characterized by their ability' to bind to and kill cells that fail to express “self’ MHC/HLA antigens by the activation of specific cytolytic enzymes, the ability to kill tumor cells or other diseased cells that express a ligand for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response.
  • CAR chimeric antigen receptor
  • CARs refers to a recombinant polypeptide comprising at least an extracellular domain that binds specifically to an antigen or a target, a transmembrane domain and an intracellular signaling domain. Engagement of the extracellular domain of the CAR with the target antigen on the surface of a target cell can result in clustering of the CAR and deliver an activation stimulus to the CAR-containing cell.
  • CARs may redirect the specificity of immune effector cells and trigger proliferation, cytokine production, phagocytosis and/or production of molecules that can mediate cell death of the target antigen-expressing cell in a maj or histocompatibility (MHC)-independent manner.
  • MHC histocompatibility
  • signaling domain refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire signaling domain is present, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal.
  • intracellular signaling domain is thus meant to include any truncated portion of the signaling domain sufficient to transduce the effector function signal.
  • Non-limiting examples of signaling domains include, e.g., signaling domains derived from DAP 10, DAP12, Fc epsilon receptor I y chain (FCER1G), FcR (3, NKG2D, CD35, CD3e, CD3y, CD3 , CD5, CD22, CD226. CD66d, CD79A. or CD79B.
  • the term "antigen” refers to any agent (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portions thereof, or combinations thereof) molecule capable of being bound by an antibody, T-cell receptor or alternative scaffold.
  • An antigen is also able to provoke an immune response.
  • An example of an immune response may involve, without limitation, antibody production, or the activation of specific immunologically competent cells, or both.
  • an antigen need not be encoded by a “gene’’ at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample, or might be macromolecule besides a polypeptide.
  • a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a fluid with other biological components, organisms, subunits of proteins/ anti gens, killed or inactivated whole cells or lysates.
  • antigen-binding moiety refers to a target-specific binding element that may be any ligand which binds to an antigen of interest, or a polypeptide or fragment thereof, wherein the ligand can be either naturally-derived or synthetic.
  • Antigen-binding moieties include, for example, without limitation, antibodies, polypeptides derived from T-cell receptors (e.g., TCR variable domains) polypeptides derived from antibodies including, e.g., Fv fragments, single chain variable fragments (scFv), and Fab, Fab', F(ab’)2; secreted factors (e.g., cytokines, growth factors), and any ligand or receptor fragment that binds to an antigen of interest.
  • T-cell receptors e.g., TCR variable domains
  • polypeptides derived from antibodies including, e.g., Fv fragments, single chain variable fragments (scFv), and Fab, Fab', F(ab’)2
  • secreted factors e.g., cytokines, growth factors
  • antigen-recognition molecule refers to any molecule comprising an antigen-binding moiety.
  • antigen-recognition molecules include, without limitation, ot(3 T cell receptors (TCRs), synthetic T cell receptors and antigen receptors (STARs). chimeric antigen receptor (CARs), T cell antigen couplers (TACs), T cell receptor fusion constructs (TruCs), or antibodies (e.g., bispecific antibodies), or any combination thereof.
  • antibody is used in a broad sense and includes immunoglobulin or antibody molecules including human, humanized, composite and chimeric antibodies and antibody fragments that are monoclonal or polyclonal. In general, antibodies are proteins or peptide chains that exhibit binding specificity’ to a specific antigen. Antibody structures are well known. Immunoglobulins can be assigned to five major classes (i.e., IgA, IgD, IgE. IgG and IgM), depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4.
  • the antibodies of the application can be of any of the five major classes or corresponding sub-classes.
  • the antibodies of the application are IgGl, IgG2, IgG3 or IgG4.
  • Antibody light chains of vertebrate species can be assigned to one of two clearly distinct types, namely kappa and lambda, based on the ammo acid sequences of their constant domains.
  • the antibodies of the application can contain a kappa or lambda light chain constant domain.
  • the antibodies of the application include heavy and/or light chain constant regions from rat or human antibodies.
  • antibodies contain an antigen-binding region that is made up of a light chain variable region and a heavy chain variable region, each of which contains three domains (i.e., complementarity determining regions 1-3; CDR1, CDR2, and CDR3).
  • the light chain variable region domains are alternatively referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCDR2. and HCDR3.
  • an “isolated antibody” refers to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to the specific tumor antigen is substantially free of antibodies that do not bind to the tumor antigen). In addition, an isolated antibody is substantially free of other cellular material and/or chemicals.
  • the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts.
  • the monoclonal antibodies of the application can be made by the hybridoma method, phage display technology, single lymphocyte gene cloning technology, or by recombinant DNA methods.
  • the monoclonal antibodies can be produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, such as a transgenic mouse or rat, having a genome comprising a human heavy chain transgene and a light chain transgene.
  • the term “antigen-binding fragment” refers to an antibody fragment such as, for example, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv').
  • a disulfide stabilized diabody a single-chain antibody molecule (scFv), a single domain antibody (sdAb), a scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a minibody, a nanobody, a domain antibody, a bivalent domain antibody, a light chain variable domain (VL), a variable domain (VHH) of a camelid antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure.
  • An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment binds.
  • single-chain antibody refers to a conventional single-chain antibody in the field, which comprises a heavy chain variable region and a light chain variable region connected by a short peptide of about 15 to about 20 amino acids (e.g., a linker peptide).
  • single domain antibody refers to a conventional single domain antibody in the field, which comprises a heavy chain variable region and a heavy chain constant region or which comprises only a heavy chain variable region.
  • human antibody refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide.
  • humanized antibody refers to a non-human antibody that is modified to increase the sequence homology to that of a human antibody, such that the antigen-binding properties of the antibody are retained, but its antigenicity in the human body is reduced.
  • chimeric antibody refers to an antibody wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species.
  • the variable region of both the light and heavy chains often corresponds to the variable region of an antibody derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capability, while the constant regions correspond to the sequences of an antibody derived from another species of mammal (e g., human) to avoid eliciting an immune response in that species.
  • multispecific antibody refers to an antibody that comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope.
  • the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein).
  • the first and second epitopes overlap or substantially overlap.
  • the first and second epitopes do not overlap or do not substantially overlap.
  • the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein).
  • a multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain.
  • a multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.
  • bispecific antibody refers to a multispecific antibody that binds no more than two epitopes or two antigens.
  • a bispecific antibody is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.
  • the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein).
  • the first and second epitopes overlap or substantially overlap.
  • the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein).
  • a bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a second epitope.
  • a bispecific antibody comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope.
  • a bispecific antibody comprises a scFv, or fragment thereof, having binding specificity for a first epitope, and a scFv, or fragment thereof, having binding specificity for a second epitope.
  • a bispecific antibody comprises a VHH having binding specificity for a first epitope, and a VHH having binding specificity for a second epitope.
  • an antigen binding domain or antigen binding fragment that “specifically binds to a tumor antigen” refers to an antigen binding domain or antigen binding fragment that binds a tumor antigen, with a KD of 1 / 10 7 M or less, preferably l xl(T 8 M or less, more preferably 5 / 10 M or less, I / 10 9 M or less, 5/ 10 10 M or less, or 1 x 10 10 M or less.
  • KD refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and is expressed as a molar concentration (M).
  • KD values for antibodies can be determined using methods in the art in view of the present disclosure.
  • the KD of an antigen binding domain or antigen binding fragment can be determined by using surface plasmon resonance, such as by using a biosensor system, e.g., a Biacore® system, or by using bio-layer interferometry technology, such as an Octet RED96 system.
  • a biosensor system e.g., a Biacore® system
  • bio-layer interferometry technology such as an Octet RED96 system.
  • composition means a product comprising an isolated polynucleotide of the application, an isolated polypeptide of the application, and/or an isolated host cell of the application (e.g., an immune cell or population thereof) together with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier e.g., an immune cell or population thereof.
  • Polynucleotides, polypeptides, and/or host cells or populations thereof of the application and compositions comprising them can also be useful in the manufacture of a medicament for therapeutic applications mentioned herein.
  • carrier refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid containing vesicle, microsphere, liposomal encapsulation, and/or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application.
  • compositions of the disclosure refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., a human).
  • a mammal e.g., a human
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.
  • the term “therapeutically effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity (e.g., alleviation of symptoms associated with cancer or autoimmune disease) upon administration to a subject in need thereof.
  • a desired activity e.g., alleviation of symptoms associated with cancer or autoimmune disease
  • the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, the mode of administration, and the like.
  • An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.
  • therapeutically effective amount refers to an amount of an active ingredient or component that elicits the desired biological or medicinal response in a subject.
  • a therapeutically effective amount can be determined empirically and in a routine manner, in relation to the stated purpose.
  • a therapeutically effective amount refers to the amount of therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of the disease, disorder or condition to be treated or a symptom associated therewith; (ii) reduce the duration of the disease, disorder or condition to be treated, or a symptom associated therewith; (iii) prevent the progression of the disease, disorder or condition to be treated, or a symptom associated therewith; (iv) cause regression of the disease, disorder or condition to be treated, or a symptom associated therewith; (v) prevent the development or onset of the disease, disorder or condition to be treated, or a symptom associated therewith; (vi) prevent the recurrence of the disease, disorder or condition to be treated, or a symptom associated therewith; (vii) reduce hospitalization of a subject having the disease, disorder or condition to be treated, or a symptom associated therewith; (viii) reduce hospitalization length of a subject having the
  • the therapeutically effective amount or dosage can vary according to various factors, such as the disease, disorder or condition to be treated, the means of administration, the target site, the physiological state of the subject (including, e.g., age, body weight, health), whether the subject is a human or an animal, other medications administered, and whether the treatment is prophylactic or therapeutic. Treatment dosages are optimally titrated to optimize safety and efficacy.
  • the terms “treat,’’ “treating,” and “treatment” are all intended to refer to an amelioration or reversal of at least one measurable physical parameter related to a cancer, which is not necessarily discernible in the subject, but can be discernible in the subject.
  • the terms “treat,” “treating,” and “treatment,” can also refer to causing regression, preventing the progression, or at least slowing down the progression of the disease, disorder, or condition.
  • “treat,” “treating,” and “treatment” refer to an alleviation, prevention of the development or onset, or reduction in the duration of one or more symptoms associated with the disease, disorder, or condition, such as a tumor or more preferably a cancer.
  • “treat,” “treating,” and “treatment” refer to prevention of the recurrence of the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment”’ refer to an increase in the survival of a subject having the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment” refer to elimination of the disease, disorder, or condition in the subject.
  • Gene editing is a type of genetic engineering in which DNA is inserted, deleted, and/or replaced in the genome of a targeted cell.
  • Targeted genome editing (interchangeable with “targeted genomic editing” or “targeted genetic editing”) enables insertion, deletion, and/or substitution at pre-selected sites in the genome.
  • targeted genomic editing or “targeted genetic editing”
  • targeted genome editing enables insertion, deletion, and/or substitution at pre-selected sites in the genome.
  • an endogenous sequence is deleted or disrupted at the insertion site during targeted editing, an endogenous gene comprising the affected sequence can be knocked-out or knocked-down due to the sequence deletion or disruption. Therefore, targeted editing can also be used to disrupt endogenous gene expression with precision.
  • targeted integration referring to a process involving insertion of one or more exogenous sequences at pre-selected sites in the genome, with or without deletion of an endogenous sequence at the insertion site
  • the present application provides, among other things, engineered IL-7 receptor alpha chain (IL-7Ra) polypeptides, and fragments and derivatives thereof.
  • IL-7Ra engineered IL-7 receptor alpha chain
  • IL-7Ra is expressed in T cells, pre-B cells, bone marrow (BM) macrophages, early thymocytes, and other various immune cells.
  • IL-7-mediated signaling elicits downstream signaling pathways via, for example, Janus kinase 1 (JAK1), JAK3, and phosphoinositide 3-kinase (PI3K), which can further lead to activation and phosphorylation of signal transducer and activator of transcription 5 (STAT5).
  • JAK1 Janus kinase 1
  • PI3K phosphoinositide 3-kinase
  • STAT5 signal transducer and activator of transcription 5
  • B-cell lymphoma 2 (Bcl-2) family members such as reduced expression of pro-apoptotic molecules, e.g., Bad, Bax, and Bim, and enhanced expression of anti-apoptotic molecules, e.g., Bcl-2, Bcl-xl, and Mcl-1.
  • the engineered IL-7Ra polypeptides described herein are capable of inducing constitutive IL-7R activity'.
  • the constitutive IL- 7R activity can elicit signaling via a JAK-STAT pathway or a phosphoinositide-3 kinase (PI3K)-protein kinase B pathway, or a combination thereof.
  • the signaling via the JAK-STAT pathway may be STAT5 signaling.
  • the engineered IL-7Ra polypeptide described herein may comprise one or more amino acid mutations compared to its corresponding wild-type IL-7Ra polypeptide.
  • the one or more mutations in the engineered IL-7Ra polypeptide may, for example, promote cytokine-independent signaling. In some embodiments, the one or more mutations in the engineered IL-7Ra polypeptide may, for example, promote homodimerization of the IL-7Ra polypeptides.
  • the IL-7Ra polypeptides described herein can be engineered to comprise one or more mutations that facilitate signaling in the absence of binding of the cognate cytokine and thus, in regard to cytokine signaling, may be constitutively active.
  • the engineered IL-7Ra polypeptide, or the functional fragment or derivative thereof, described herein may comprise amino acid mutation(s) in one or more positions.
  • Non-limiting examples of amino acid mutations comprise amino acid substitutions, insertions, and/or deletions.
  • Amino acid substitution means that an amino acid residue is substituted for a replacement amino acid residue at the same position.
  • Inserted amino acid residues may be inserted at any position and may be inserted such that some or all of the inserted amino acid residues are immediately adjacent one another or may be inserted such that none of the inserted amino acid residues is immediately adjacent to another inserted amino acid residue.
  • the engineered IL-7Ra polypeptide described herein may comprise at least one ectodomain, at least one transmembrane domain, and/or at least one cytoplasmic domain, or any combination thereof.
  • the at least one ectodomain, at least one transmembrane domain, and/or at least one cytoplasmic domain may be derived from IL-7Ra.
  • the engineered IL-7Ra polypeptide may comprise one or more amino acid mutations in the ectodomain, transmembrane domain, and/or the cytoplasmic domain of the engineered IL-7Ra polypeptide.
  • the engineered IL-7Ra polypeptide described herein may comprise one or more amino acid mutation(s) (e.g., amino acid substitutions, insertions, and/or deletions) in the IL- 7Ra cytoplasmic domain.
  • amino acid mutation(s) e.g., amino acid substitutions, insertions, and/or deletions
  • the engineered IL-7Ra polypeptide, or the functional fragment or derivative thereof may comprise amino acid mutation(s) in one or more positions.
  • the engineered IL-7Ra polypeptide, or the functional fragment or derivative thereof may comprise amino acid mutation(s) in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
  • the engineered IL-7Ra polypeptide, or fragment or derivative thereof may comprise one or more mutations, i.e., amino acid substitutions, insertions, or deletions, or combination thereof, at one or more location in its amino acid sequence as compared to an amino acid sequence of a reference IL-7Ra polypeptide.
  • the engineered IL-7Ra polypeptide may include a substitution(s) of one or more amino acids in the amino acid sequence of a parent IL-7Ra polypeptide with a similar or homologous amino acid(s) or a dissimilar amino acid(s).
  • the engineered IL-7Ra polypeptide described herein may include any amino acid sequence having an identity of at least about 50% or more, about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more.
  • the engineered IL-7Ra polypeptide, or fragment or derivative thereof may comprise one or more amino acid mutations at one or more locations in its amino acid sequence as compared to an amino acid sequence of a reference IL-7Ra polypeptide, e.g., a reference sequence comprising or consisting of a wild-type IL-7Ra polypeptide.
  • FIG. 1 A schematic representation of an exemplary full-length (459 aa) wild-type IL-7Ra polypeptide (see also, e.g., SEQ ID NO: 1; UniProtKB P16871) comprising an ectodomain (21-239 aa), a transmembrane domain (240-264 aa) and a cytoplasmic domain (265-459 aa) that may be used a reference sequence (i.e., template) for the engineered IL-7Ra described herein is displayed in Fig. 1
  • the one or more amino acid mutations in the amino acid sequence of the engineered IL-7Ra polypeptide may be described in reference to the amino acid sequence of SEQ ID NO: 1.
  • the wild-type IL-7Ra polypeptide reference sequence (i.e., template) for the engineered IL-7Ra polypeptide comprises the amino acid sequence of SEQ ID NO: 1, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%.
  • the nucleotide sequence that encodes the wild-tj pe IL-7Ra polypeptide reference sequence for the engineered IL-7Ra polypeptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 1, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%.
  • the nucleotide sequence that encodes the wild-type IL-7Ra polypeptide reference sequence for the engineered IL-7Ra polypeptide comprises the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%. at least about 92%, at least about 93%. at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with SEQ ID NO: 2.
  • the wild-type IL-7Ra polypeptide reference sequence for the engineered IL-7Ra polypeptide comprises the amino acid sequence of SEQ ID NO: 1.
  • the nucleotide sequence that encodes the wild-type IL-7Ra polypeptide reference sequence for the engineered IL-7Ra polypeptide comprises the nucleotide sequence of SEQ ID NO: 2.
  • the wild-type IL-7Ra polypeptide reference sequence (i.e., template) for the engineered IL-7Ra polypeptide, or the fragment or derivative thereof, comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 1.
  • the wild-type IL-7Ra polypeptide reference sequence for the engineered IL-7Ra polypeptide, or the fragment or derivative thereof comprises the amino acid sequence of SEQ ID NO: 1.
  • the w ild-type IL-7Ra polypeptide reference sequence for the engineered IL-7Ra polypepride consists of the amino acid sequence of SEQ ID NO: 1.
  • the engineered IL-7Ra polypeptide may comprise a mutation at any amino acid, or combination thereof, contained therein. In some embodiments, the engineered IL-7Ra polypeptide may comprise a substitution at any amino acid, or combination thereof, contained therein. In some embodiments, the IL-7Ra polypeptide may comprise an insertion at any amino acid, or combination thereof, contained therein. In some embodiments, the IL-7Ra polypeptide may comprise a deletion at any amino acid, or combination thereof, contained therein.
  • amino acid mutation(s) to a protein or fragment of derivative thereof are those which may confer or modify physicochemical or functional properties.
  • the amino acid sequence of the IL-7Ra polypeptide disclosed herein may be mutated, for example, to enhance activity of the IL-7Ra polypeptide, i.e., enhance cytokine-dependent signaling.
  • the activity of the IL-7Ra polypeptide after the one or more mutations is enhanced by more than 2%, more than 5%, more than 10%, more than 20%, more than 40%, more than 60%, more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or 100% or more as compared to the activity before mutation.
  • the amino acid sequence of the IL-7Ra polypeptide disclosed herein may be mutated, for example, to promote cytokine-independent signaling.
  • the ammo acid sequence of the IL-7Ra polypeptide disclosed herein may be mutated, for example, to promote homodimerization.
  • the engineered IL-7Ra polypeptide described herein may include conservative modifications and/or substitutions at various positions of IL-7Ra.
  • a conservative amino acid substitution would not substantially change the structural characteristics of the parent sequence.
  • amino acids belonging to one of the following groups represent conservative changes: Group I: Pro, Gly, Gin, Asn, Ala, Ser, Thr; Group II: Thr, Cys. Ser, Tyr,; Group III: Leu, Met. Ala, Vai. He. Phe; Group IV: Arg, His, Lys; Group V : Trp, His, Phe, Tyr,; and Group VI: Glu, Asp.
  • a conservative ammo acid substitution would not substantially change the structural characteristics of the parent sequence.
  • the engineered IL-7Ra polypeptide, or the functional fragment or derivative thereof may comprise mutations(s), e.g.. substitution(s). in one or more positions selected from E301, V450, T451. M452, Q459, or a combination thereof, with respect to the amino acid sequence of SEQ ID NO: 1.
  • the amino acid substitutions(s) may comprise an alanine (A) substitution, an arginine (R) substitution, a methionine substitution (M).
  • N an asparagine (N) substitution, an aspartic acid (D) substitution, a leucine (L) substitution, a lysine (K) substitution, a phenylalanine (F) substitution, a glutamine (Q) substitution, a glutamic acid (E) substitution, a serine (S) substitution, and a threonine (T) substitution, or any combination thereof.
  • N an asparagine
  • D aspartic acid
  • L leucine
  • K a lysine
  • F a phenylalanine
  • Q glutamine
  • E glutamic acid
  • S serine
  • T threonine
  • the engineered IL-7Ra polypeptide, or fragment or derivative thereof, described herein may comprise one or more amino acid mutations comprising at least one Box 2 motif insertion, at least one substitution at the IL-7Ra YXXM motif, at least one YXXM motif insertion, or a combination thereof.
  • the Box 2 motif may be inserted in the IL-7Ra cytoplasmic domain of the engineered IL-7Ra polypeptide, or fragment or derivative thereof, described herein.
  • the Box 2 motif may be derived from CD122.
  • a non-limiting example of a Box 2 motif comprises the amino acid sequence of ISPLEVLERDK (SEQ ID NO: 3).
  • the Box 2 motif that may be used in accordance with the present disclose may comprise any of various Box 2 motifs, or fragments or derivatives thereof, as described by Murakami and colleagues (see, e.g., Murakami et al., “Critical cytoplasmic region of the interleukin 6 signal transducer gp!30 is conserved in the cytokine receptor family” ProcNatl Acad Sci U S A. 1991 Dec 15;88(24): 11349-53, the content of which is incorporated herein by reference in its entirety for all purposes).
  • Box 1 is proline rich and is located approximately 10 amino acids from the C-terminal end of the transmembrane region of a cytokine receptor
  • Box 2 is about 10-50 amino acids further downstream and is rich in hydrophobic residues. Aside from these attributes, these regions share little sequence homology' betw een different cytokine receptors.
  • the Box 2 motif of the engineered IL-7Ra described herein may be inserted in the IL-7Ra cytoplasmic domain from about 60 amino acids to about 100 amino acids, from about 50 amino acids to about 90 amino acids, from about 40 amino acids to about 80 amino acids, from about 30 amino acids to about 70 amino acids, from about 20 amino acids to about 60 amino acids, from about 10 amino acids to about 50 amino acids, from about 9 amino acids to about 45 amino acids, from about 8 amino acids to about 40 amino acids, from about 7 amino acids to about 35 amino acids, from about 6 amino acids to about 30 amino acids, from about 5 amino acids to about 25 amino acids, from about 4 amino acids to about 20 amino acids, from about 3 amino acids to about 15 amino acids, from about 2 amino acids to about 10 amino acids, or from about 1 amino acids to about 5 amino acids, away from the IL-7Ra transmembrane domain.
  • the Box 2 motif is inserted from about 10 amino acids to about 50 amino acids away from the IL-7Ra transmembrane domain.
  • the Box 2 motif may be inserted in the IL-7Ra cytoplasmic domain at an amino acid position in the range of from about position 265 to about position 459, from about position 270 to about position 455, from about position 275 to about position 450, from about position 280 to about position 445, from about position 285 to about position 440, from about position 290 to about position 435, from about position 295 to about position 430, from about position 300 to about position 425, from about position 305 to about position 420, from about position 310 to about position 415, from about position 315 to about position 410, from about position 320 to about position 405, from about position 325 to about position 400, from about position 330 to about position 395, from about position 335 to about position 390, from about position 340 to about position 385, from about position 345 to about position 380, from about position 350 to about position 375, from about position 355 to about position 370, from about position 360 to about position 365 with respect to S
  • the Box 2 motif may be inserted in the IL-7Ra cytoplasmic domain at an amino acid position in the range of from about position 250 to about 325 with respect to SEQ ID NO: 1. In some embodiments, the Box 2 motif may be inserted in the IL- 7Ra cytoplasmic domain at an amino acid position in the range of from about position 275 to about 314 with respect to SEQ ID NO: 1.
  • the Box 2 motif may be inserted in the IL-7Ra cytoplasmic domain at least about 1 amino acid away from the IL-7Ra transmembrane domain. In some embodiments, the Box 2 motif may be inserted in the IL-7Ra cytoplasmic domain at most about 50 amino acids away from the IL-7Ra transmembrane domain. In various embodiments, the IL-7Ra cytoplasmic domain may be inserted at least about 2. 3, 4, 5, 6, 7, 8, 9. 10, 11, 12, 13, 14. 15. 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31. 32. 33. 34. 35. 36. 37. 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 amino acids or more away from the IL-7Ra transmembrane domain.
  • the Box 2 motif may be inserted 3' to amino acid position E301 with respect to the amino acid sequence of SEQ ID NO: 1.
  • the Box 2 motif comprises the amino acid sequence of SEQ ID NO: 3, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%. sequence identity with SEQ ID NO: 3.
  • the nucleotide sequence that encodes the Box 2 motif comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 3, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with SEQ ID NO: 3.
  • the Box 2 motif comprises the amino acid sequence of SEQ ID NO: 3.
  • the Box 2 motif consists of the amino acid sequence of SEQ ID NO: 3.
  • the nucleotide sequence that encodes the Box 2 motif comprises the nucleotide sequence of SEQ ID NO: 4, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with SEQ ID NO: 4.
  • the nucleotide sequence that encodes the Box 2 motif comprises the nucleotide sequence of SEQ ID NO: 4.
  • the nucleotide sequence that encodes the Box 2 motif consists of the nucleotide sequence of SEQ ID NO: 4.
  • a non-limiting example of an engineered IL-7Ra polypeptide which comprises a Box 2 motif insertion, e.g., a Box 2 motif inserted 3' to amino acid position E301 with respect to the amino acid sequence of SEQ ID NO: 1, is the amino acid sequence of SEQ ID NO: 7.
  • Other examples of engineered IL-7Ra polypeptides comprising a Box 2 motif insertion include the amino acid sequences of SEQ ID NOs: 5, 7, and 13.
  • the engineered IL-7Ra polypeptide comprises the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with SEQ ID NO: 7.
  • the nucleotide sequence that encodes the engineered IL-7Ra polypeptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with SEQ ID NO: 7.
  • the nucleotide sequence that encodes the engineered IL-7Ra polypeptide comprises the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%. at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with SEQ ID NO: 8.
  • the engineered IL-7Ra polypeptide comprises the amino acid sequence of SEQ ID NO: 7.
  • the nucleotide sequence that encodes the engineered IL-7Ra polypeptide comprises the nucleotide sequence of SEQ ID NO: 8.
  • the engineered IL-7Ra polypeptide, or the fragment or derivative thereof comprises aIL-7Ra YXXM motif substitution, where "X " can be any amino acid.
  • the IL-7Ra YXXM motif substitution may comprise an amino acid substitution at amino acid positions V450, T451 , or M452, or a combination thereof, with respect to SEQ ID NO: 1.
  • the YXXM motif substitution may comprise V450M and/or T45 IN with respect to SEQ ID NO: 1.
  • the YXXM motif may be derived from CD28.
  • the YXXM motif may comprise the amino acid sequence YMNM (SEQ ID NO: 17).
  • the YXXM motif may consist of the amino acid sequence YMNM (SEQ ID NO: 17).
  • a non-limiting example of an engineered IL-7Ra polypeptide which comprises a IL-7Ra YXXM motif substitution is the amino acid sequence of SEQ ID NO: 9.
  • the engineered IL-7Ra polypeptide comprises the amino acid sequence of SEQ ID NO: 9, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with SEQ ID NO: 9.
  • the nucleotide sequence that encodes the engineered IL-7Ra polypeptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 9, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%. at least about 96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with SEQ ID NO: 9.
  • the nucleotide sequence that encodes the engineered IL-7Ra polypeptide comprises the nucleotide sequence of SEQ ID NO: 10, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with SEQ ID NO: 10.
  • the engineered IL-7Ra polypeptide comprises the amino acid sequence of SEQ ID NO: 9.
  • the nucleotide sequence that encodes the engineered IL-7Ra polypeptide comprises the nucleotide sequence of SEQ ID NO: 10.
  • the engineered IL-7Ra polypeptide, or the fragment or derivative thereof may comprise a Box 2 motif insert or a IL-7Ra YXXM motif substitution, or a combination thereof.
  • Such engineered IL-7Ra polypeptide for example, without limitation, may comprise or consist of the amino acid sequence of SEQ ID NO: 5.
  • the engineered IL-7Ra polypeptide comprises the amino acid sequence of SEQ ID NO: 5, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with SEQ ID NO: 5.
  • the nucleotide sequence that encodes the engineered IL-7Ra polypeptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 5, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%. sequence identity with SEQ ID NO: 5.
  • the nucleotide sequence that encodes the engineered IL-7Ra polypeptide comprises the nucleotide sequence of SEQ ID NO: 6, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%. at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with SEQ ID NO: 6.
  • the engineered IL-7Ra polypeptide comprises the amino acid sequence of SEQ ID NO: 5.
  • the nucleotide sequence that encodes the engineered IL-7Ra polypeptide comprises the nucleotide sequence of SEQ ID NO: 6.
  • the YXXM motif insertion described herein may comprise a IL-7Ra YXXM motif, e.g., the amino acid sequence YVTM (SEQ ID NO: 21).
  • a wild-type IL-7Ra polypeptide see, e.g., SEQ ID NO: 1
  • the fragment or derivative thereof may comprise a IL-7Ra YXXM motif, where the YXXM motif may comprise the amino acid sequence YVTM (SEQ ID NO: 21).
  • the IL- 7Ra YXXM motif comprising the sequence YVTM may correspond to amino acid positions 449-452 with respect to SEQ ID NO: 1.
  • the YXXM motif may be derived from CD28.
  • the YXXM motif insertion described herein may comprise a YXXM motif comprising the amino acid sequence YMNM (SEQ ID NO: 17).
  • YXXM motif insertions described herein may be inserted 3' to amino acid position Q459 with respect to SEQ ID NO: 1.
  • a YXXM motif insertion may comprise a IL-7Ra YXXM motif or a YXXM motif comprising the sequence of YMNM (SEQ ID NO: 17), inserted 3' to amino acid position Q459 with respect to SEQ ID NO: 1.
  • a YXXM motif insertion comprising a IL-7Ra YXXM motif or a YXXM motif comprising the sequence of YMNM (SEQ ID NO: 17) is inserted 3' to amino acid position Q459 with respect to SEQ ID NO: 1
  • the YMNM motif (SEQ ID NO: 17) is introduced into a YXXM location on the IL-7Ra chain in order to effectively recruit STAT5 and PI3K.
  • an engineered IL-7Ra polypeptide described herein may comprise at least one YXXM motif .
  • an engineered IL-7Ra polypeptide described herein may comprise two or more YXXM motifs .
  • the two or more YXXM motifs may be derived from IL-7Ra.
  • the YXXM motif comprises the amino acid sequence YVTM (SEQ ID NO: 21).
  • the engineered IL-7Ra polypeptide, or the fragment or derivative thereof may comprise a YXXM motif insertion comprising the amino acid sequence of YVTM (SEQ ID NO: 21).
  • Such engineered IL-7Ra polypeptide for example, without limitation, may comprise or consist of the amino acid sequence of SEQ ID NO: 11.
  • the engineered IL-7Ra polypeptide comprises the amino acid sequence of SEQ ID NO: 11, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with SEQ ID NO: 11.
  • the nucleotide sequence that encodes the engineered IL-7Ra polypeptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 11, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with SEQ ID NO: 11.
  • the nucleotide sequence that encodes the engineered IL-7Ra polypeptide comprises the nucleotide sequence of SEQ ID NO: 12, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%. at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with SEQ ID NO: 12.
  • the engineered IL-7Ra polypeptide comprises the amino acid sequence of SEQ ID NO: 11.
  • the nucleotide sequence that encodes the engineered IL-7Ra polypeptide comprises the nucleotide sequence of SEQ ID NO: 12.
  • the engineered IL-7Ra polypeptide described herein comprises two or more YXXM motifs
  • the YXXM motif comprises the amino acid sequence of YMNM (SEQ ID NO: 17).
  • the two or more YXXM motifs may be derived from CD28.
  • the engineered IL-7Ra polypeptide described herein comprises two or more YXXM motifs comprising the amino acid sequence of YMNM (SEQ ID NO: 17)
  • the engineered IL-7Ra polypeptide may further comprise a Box 2 insert described herein.
  • the Box 2 motif may be derived from CD 122.
  • a nonlimiting example of a Box 2 motif comprises the amino acid sequence of SEQ ID NO: 3.
  • the engineered IL-7Ra polypeptide, or the fragment or derivative thereof may comprise two YXXM motifs.
  • each of the two YXXM motifs may comprise the amino acid sequence of YMNM (SEQ ID NO: 17).
  • the engineered IL-7Ra polypeptide comprising the two YXXM motifs each comprising the amino acid sequence of YMNM (SEQ ID NO: 17) may further comprise a Box 2 insert comprising the amino acid sequence of SEQ ID NO: 3.
  • Such engineered IL-7Ra polypeptide for example, without limitation, may comprise or consist of the amino acid sequence of SEQ ID NO: 13.
  • the engineered IL-7Ra polypeptide comprises the amino acid sequence of SEQ ID NO: 13, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with SEQ ID NO: 13.
  • the nucleotide sequence that encodes the engineered IL-7Ra polypeptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 13, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%. at least about 96%, at least about 97%, at least about 98% or at least about 99%. sequence identity with SEQ ID NO: 13.
  • the nucleotide sequence that encodes the engineered IL-7Ra polypeptide comprises the nucleotide sequence of SEQ ID NO: 14, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with SEQ ID NO: 14.
  • the engineered IL-7Ra poly peptide comprises the amino acid sequence of SEQ ID NO: 13.
  • the nucleotide sequence that encodes the engineered IL-7Ra polypeptide comprises the nucleotide sequence of SEQ ID NO: 14.
  • a IL-7Ra YXXM motif substitution described herein may comprise an amino acid substitution at amino acid positions V450, T451, or M452, or a combination thereof, with respect to SEQ ID NO: 1.
  • a IL-7Ra YXXM motif substitution may comprise, e.g., V450L, T451S, and/or M452L with respect to SEQ ID NO: 1.
  • the YXXM motif when the YXXM motif comprises a substitution comprising M452L with respect to SEQ ID NO: 1, the YXXM motif may comprise a YXXL motif, where 'X " is any amino acid.
  • the YXXL motif may be derived from CD 122.
  • the YXXL motif may comprise the amino acid sequence ofYLSL (SEQ ID NO: 18).
  • the engineered IL-7Ra polypeptide, or fragment or derivative thereof comprises a IL-7Ra YXXM motif substitution comprising V450L, T451S, and/or M452L with respect to SEQ ID NO: 1.
  • the engineered IL-7Ra polypeptide, or the fragment or derivative thereof may comprise a YXXL motif which may comprise the amino acid sequence of YLSL (SEQ ID NO: 18).
  • Such engineered IL-7Ra polypeptide for example, without limitation, may comprise or consist of the amino acid sequence of SEQ ID NO: 15.
  • the engineered IL-7Ra polypeptide comprises the amino acid sequence of SEQ ID NO: 15, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with SEQ ID NO: 15.
  • the nucleotide sequence that encodes the engineered IL-7Ra polypeptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 15, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with SEQ ID NO: 15.
  • the nucleotide sequence that encodes the engineered IL-7Ra polypeptide comprises the nucleotide sequence of SEQ ID NO: 16, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with SEQ ID NO: 16.
  • the engineered IL-7Ra polypeptide comprises the amino acid sequence of SEQ ID NO: 15.
  • the nucleotide sequence that encodes the engineered IL-7Ra polypeptide comprises the nucleotide sequence of SEQ ID NO: 16.
  • the engineered IL-7Ra polypeptide, or the fragment or derivative thereof comprises a leader sequence.
  • the leader sequence may be positioned at the N-terminus of the extracellular domain.
  • the leader sequence may be optionally cleaved from the extracellular during cellular processing and localization of the IL-7Ra polypeptide to the cellular membrane. Any of various leader sequences known to one of skill in the art may be used as the leader sequence.
  • Non-limiting examples of peptides from which the leader sequence may be derived include granulocyte-macrophage colony-stimulating factor receptor (GMCSFR), FceR, human immunoglobulin (IgG) heavy chain (HC) variable region, CD8a, or any of various other proteins secreted by T cells.
  • GMCSFR granulocyte-macrophage colony-stimulating factor receptor
  • FceR human immunoglobulin (IgG) heavy chain (HC) variable region
  • CD8a or any of various other proteins secreted by T cells.
  • the leader sequence is compatible with the secretory pathw ay of a T cell.
  • the engineered IL-7Ra polypeptide described herein may be truncated, at the N-terminal and/or the C-terminal end(s), thereby resulting in the production of a truncated engineered IL-7Ra polypeptide, e.g., a truncated functional fragment, which retains the ability to impart at least one IL-7R activity'.
  • an N-terminally and/or C-terminally truncated functional fragment of an engineered IL-7Ra polypeptide described herein may comprise or consist of an amino acid sequence or a functional variant thereof having at least about 30%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%. at least about 91%, at least about 92%. at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with SEQ ID NO: 1 .
  • the truncated engineered IL-7Ra polypeptide may comprise a truncated ectodomain.
  • the ectodomain can be truncated resulting in the production of a shortened ectodomain amino acid sequence.
  • the ectodomain may be truncated so as to eliminate pairing of an IL-7Ra described herein with a common-y chain receptor.
  • an ectodomain w hich has been truncated to eliminate pairing with a common-y chain receptor may be truncated from amino acid position 1 to about 200, amino acid position 1 to about 201, amino acid position 1 to about 202, amino acid position 1 to about 203, amino acid position 1 to about 204, amino acid position 1 to about 205, amino acid position 1 to about 206, amino acid position 1 to about 207, amino acid position 1 to about 208, amino acid position 1 to about 209, amino acid position 1 to about 210, amino acid position 1 to about 211, amino acid position 1 to about 212, amino acid position 1 to about 213.
  • amino acid position 1 to about 214 amino acid position 1 to about 215, amino acid position 1 to about 216, amino acid position 1 to about 217, amino acid position 1 to about 218, amino acid position 1 to about 219, amino acid position 1 to about 220, amino acid position 1 to about 221.
  • an ectodomain of an IL-7Ra polypeptide described herein when truncated to eliminate pairing with a common-y chain receptor, ajuxtamembrane portion of the ectodomain (i. e.
  • an ectodomain of an IL-7Ra polypeptide described herein when truncated to eliminate pairing with a common -y chain receptor, ajuxtamembrane portion of the ectodomain may remain intact.
  • the ectodomain can be truncated such that the ectodomain is eliminated.
  • the signal peptide when the truncated engineered IL-7Ra polypeptide comprises a truncated ectodomain, the signal peptide is retained.
  • the signal peptide may comprise, without limitation, the amino acid sequence MTILGTTFGMVFSLLQVVSG (SEQ ID NO: 20), or a variant thereof, described herein.
  • the signal peptide may comprise, e.g., a MARS signal peptide such as that which may be derived from an IGK V region exon, which may comprise the amino acid sequence MARSPAQLLGLLLLWLSGARC (SEQ ID NO: 29), or variant thereof, described herein.
  • the signal peptide may comprise, e.g., a CD33 signal peptide, which may comprise the amino acid sequence MPLLLLLPLLWAGALA (SEQ ID NO: 30), or variant thereof, described herein.
  • the signal peptide may, for example, mediate insertion of the truncated engineered IL-7Ra polypeptide into the cellular membrane.
  • the signal peptide when the signal peptide is retained, the signal peptide may direct the truncated engineered IL-7Ra polypeptide to the membrane of the endoplasmic reticulum (ER) and initiate translocation into the ER lumen.
  • the signal peptide is not retained and is absent from the truncated engineered IL-7Ra polypeptide.
  • the signal peptide is not retained and a non-endogenous signal peptide is present instead.
  • the engineered IL-7Ra polypeptide may comprise a truncated cytoplasmic domain, e.g., a cytoplasmic domain derived from IL-7Ra described herein.
  • the cytoplasmic domain is truncated resulting in the production of a shortened cytoplasmic domain amino acid sequence.
  • an engineered IL-7Ra polypeptide described herein comprises a truncated cytoplasmic domain
  • the engineered IL-7Ra polypeptide may be truncated from amino acid position 270 to about 459, amino acid position 270 to about 458, amino acid position 270 to about 457, amino acid position 270 to about 456, amino acid position 270 to about 455, amino acid position 270 to about 454, amino acid position 270 to about 453, amino acid position 270 to about 452, amino acid position 270 to about 451, amino acid position 270 to about 450.
  • amino acid position 270 to about 449 ammo acid position 270 to about 448, amino acid position 270 to about 447, amino acid position 270 to about 446, amino acid position 270 to about 445, amino acid position 270 to about 444, amino acid position 270 to about 443, amino acid position 270 to about 442, amino acid position 270 to about 441, amino acid position 270 to about 440, amino acid position 270 to about 439, amino acid position 270 to about 438.
  • amino acid position 270 to about 407 amino acid position 270 to about 406, amino acid position 270 to about 405, amino acid position 270 to about 404, amino acid position 270 to about 403, amino acid position 270 to about 402, amino acid position 270 to about 401, amino acid position 270 to about 400.
  • amino acid position 270 to about 389 amino acid position 270 to about 388, amino acid position 270 to about 387, amino acid position 270 to about 386, amino acid position 270 to about 385, amino acid position 270 to about 384, amino acid position 270 to about 383, amino acid position 270 to about 382, amino acid position 270 to about 381, amino acid position 270 to about 380, amino acid position 270 to about 379, amino acid position 270 to about 378.
  • amino acid position 270 to about 287 amino acid position 270 to about 286, amino acid position 270 to about 285, ammo acid position 270 to about 284, amino acid position 270 to about 283, amino acid position 270 to about 282, amino acid position 270 to about 281, amino acid position 270 to about 280, amino acid position 270 to about 279, amino acid position 270 to about 278, amino acid position 270 to about 277, amino acid position 270 to about 276.
  • the cytoplasmic domain may be eliminated.
  • the engineered IL-7Ra polypeptide may comprise a truncated ectodomain and/or a truncated cytoplasmic domain.
  • the N-tenninal end of the truncated engineered IL-7Ra polypeptide may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110. 120, 130, 140, 150, 160, 170, 180, 190. 200, 210, 220. 230 or more amino acids.
  • the C-termmal end of the truncated engineered IL-7Ra polypeptide may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or more amino acids.
  • a truncated engineered IL-7Ra polypeptide described herein may retain IL-7 receptor activity (e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% of IL-7 receptor activity) relative to an IL-7Ra polypeptide which is not truncated.
  • a truncated engineered IL-7Ra polypeptide described herein may retain signaling via a JAK-STAT pathway (e.g., at least 20, 30, 40, 50, 60. 70. 80, 90, 95, or 100% of signaling via a JAK-STAT) relative to a IL-7Ra polypeptide which is not truncated.
  • the signaling via the JAK-STAT pathway is STAT5 signaling.
  • a truncated engineered IL-7Ra polypeptide described herein may retain signaling via a phosphoinositide-3 kinase (PI3K)-protein kinase B pathway (e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 95 or 100% of signaling via a PI3K-protein kinase B pathway) relative to a IL-7Ra polypeptide which is not truncated.
  • PI3K phosphoinositide-3 kinase
  • truncations may provide an advantage of reducing construct size for expression.
  • the leader sequence comprises the amino acid sequence LHRSRASEFAAT (SEQ ID NO: 19), or a variant thereof having at least about 50% or more, about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more.
  • the nucleotide sequence that encodes the leader sequence comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 19, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%. at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with SEQ ID NO: 19.
  • the leader sequence comprises the amino acid sequence of SEQ ID NO: 19.
  • the leader sequence consists of the amino acid sequence of SEQ ID NO: 19.
  • the engineered IL-7Ra polypeptide, or fragment or derivative thereof comprises a signal peptide.
  • a signal peptide of the present disclosure may comprise a signal peptide at the amino-terminus (N-terminus) of a nascent polypeptide, e.g.. an engineered IL-7Ra polypeptide disclosed herein, which co- translationally or post-translationally directs the nascent protein to the endoplasmic reticulum and subsequent surface expression or secretion.
  • the signal peptide comprises the amino acid sequence MTILGTTFGMVFSLLQVVSG (SEQ ID NO: 20), or a variant thereof having at least about 50% or more, about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more.
  • the nucleotide sequence that encodes the signal peptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with SEQ ID NO: 20.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 20.
  • the signal peptide consists of the amino acid sequence of SEQ ID NO: 20.
  • the signal peptide comprises the amino acid sequence MARSPAQLLGLLLLWLSGARC (SEQ ID NO: 29), or a variant thereof having at least about 50% or more, about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more.
  • the nucleotide sequence that encodes the signal peptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%. sequence identity with SEQ ID NO: 29.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 29.
  • the signal peptide consists of the amino acid sequence of SEQ ID NO: 29.
  • the signal peptide comprises the amino acid sequence MPLLLLLPLLWAGALA (SEQ ID NO: 30), or a variant thereof having at least about 50% or more, about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more. 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more.
  • the nucleotide sequence that encodes the signal peptide comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, sequence identity with SEQ ID NO: 30.
  • the signal peptide comprises the amino acid sequence of SEQ ID NO: 30.
  • the signal peptide consists of the amino acid sequence of SEQ ID NO: 30.
  • polypeptides e.g., engineered IL-7Ra polypeptides, or fragments of derivatives thereof, described herein.
  • the polynucleotide can comprise any type of nucleotides, including, but not limited to DNA and RNA. which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides.
  • the polynucleotide can comprise naturally-occurring or non-naturally-occurring intemucleotide linkages, or both types of linkages.
  • the polynucleotide described herein is a DNA molecule. In various embodiments, the polynucleotide described herein is an RNA molecule. [00213] In certain embodiments, the polynucleotide sequence that encodes the engineered IL-7Ra polypeptide, or fragment or derivative thereof, comprises the nucleotide sequence of SEQ ID NOs: 6, 8, 10, 12, 14, or 16, or a variant thereof comprising at least about 50% or more, about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more,
  • the polynucleotide sequence encoding the engineered IL- 7Ra polypeptide comprises the nucleotide sequence of SEQ ID NO: 6, 8, 10, 12, 14, or 16, or a nucleotide sequence having at least 80% sequence identity thereof.
  • the polynucleotide sequence that encodes the engineered IL-7Ra polypeptide, or fragment or derivative thereof consists of the nucleotide sequence of SEQ ID NOs: 6, 8, 10, 12, 14, or 16.
  • the present disclosure provides recombinant vectors comprising a polynucleotide described herein.
  • the recombinant vector comprises a polynucleotide encoding the engineered IL-7Ra polypeptide, or fragment or derivative thereof, described herein.
  • a recombinant vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses.
  • a vector can be selected from the pUC series (Fermentas Life Sciences, Glen Bumie, Md.), the pBluescript series (Stratagene, LaJolla, Calif), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif).
  • Bacteriophage vectors such as ZGTI 0.
  • XGT11 XZapII (Stratagene), EMBL4. and XNM1149
  • plant expression vectors useful in the context of the disclosure include pBIOl, pBU01.2, pBU01.3, pBI121 and pBIN19 (Clontech).
  • animal expression vectors useful in the context of the disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).
  • the recombinant vector is a viral vector.
  • Suitable viral vectors include, without limitation, retroviral vectors, lentiviral vectors, alphaviral vectors, adenoviral vectors, adeno-associated viral vectors (AAVs), herpes viral vectors, vaccinia vectors, and fowl pox viral vectors.
  • the viral vectors have a native or engineered capacity to transform a host cell (e.g., T cell).
  • the recombinant vector is a non-viral vector.
  • the viral vector may be a mini circle plasmid, a Sleeping Beauty transposon, a piggyBac transposon, or a single or double stranded DNA molecule that is used as a template for homology directed repair (HDR) based gene editing.
  • the vector is a plasmid.
  • Recombinant vectors can be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al.. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994.
  • Constructs of expression vectors, which are circular or linear can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived, e.g., from ColEl, 2p plasmid, k, SV40, bovine papilloma virus, and the like.
  • a recombinant vector can include one or more marker genes, which allow for selection of transformed or transfected hosts.
  • Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like.
  • Suitable marker genes for the recombinant expression vectors include, for instance, neomycin/G418 resistance genes, puromycin resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.
  • Vectors useful in the context of the disclosure can be "‘naked’’ nucleic acid vectors (i.e., vectors having little or no proteins, sugars, and/or lipids encapsulating them), or vectors complexed with other molecules.
  • Other molecules that can be suitably combined with the vectors include without limitation viral coats, cationic lipids, liposomes, polyamines. gold particles, and targeting moieties such as ligands, receptors, or antibodies that target cellular molecules.
  • Vector DNA can be introduced into a host cell, e.g., an immune effector cell, via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, gene gun, or electroporation.
  • the polynucleotide encoding the polypeptide described herein is operably linked to at least a regulatory element.
  • the regulatory' element can be capable of mediating expression of the polypeptide in the host cell.
  • Regulatory elements include, but are not limited to. promoters, enhancers, initiation sites, polyadenylation (poly A) tails, IRES elements, response elements, and termination signals.
  • the regulatory element regulates polypeptide expression.
  • the regulatory element increased the expression of the polypeptide (e.g., first polypeptide or second polypeptide).
  • the regulatory element increases the expression of the polypeptide (e.g., first polypeptide or second polypeptide) once the host cell is activated. In certain embodiments, the regulatory element decreases expression of the polypeptide (e.g., first polypeptide or second polypeptide). In certain embodiments, the regulatory' element decreases expression of the polypeptide (e.g., first polypeptide or second polypeptide) once the host cell is activated.
  • the polynucleotide sequence encoding the engineered IL- 7Ra polypeptide, or fragment or derivative thereof, described herein is operably linked to a promoter.
  • promoters included CMV, EFla, PGK, CAG, UBC, SV40, human beta actin, or other constitutive, inducible, temporal-, tissue-, or cell type-specific promoters.
  • the promoter is a CAG promoter.
  • the polynucleotides described herein are placed operably under the control of a Kozak consensus sequence.
  • the polynucleotides are operatively linked to a terminator/polyadenylation signal.
  • the terminator/ polyadenylation signal is a SV40 signal.
  • Other terminator sequences can also be used, examples of which include, but are not limited to BGH. hGH. and PGK.
  • one or more of the exogenous IL7R polynucleotides described are integrated at one or more loci on the chromosome of a cell such as an iPSC.
  • the integration of the exogenous polynucleotide into the gene locus is produced by way of targeted genome editing.
  • targeted genome editing include any method selected from the group consisting of a CRISPR method, a zinc finger nuclease method, a TALEN method, a homing nuclease method, a homology 7 recombination method, and any functional variation thereof.
  • Targeted editing can be achieved either through a nuclease-independent approach, or through a nuclease-dependent approach.
  • nuclease-independent targeted editing approach homologous recombination is guided by homologous sequences flanking an exogenous polynucleotide to be inserted, through the enzymatic machinery of the host cell.
  • targeted editing could be achieved with higher frequency through specific introduction of double strand breaks (DSBs) by specific rare-cutting endonucleases.
  • DSBs double strand breaks
  • Such nuclease-dependent targeted editing utilizes DNA repair mechanisms including non- homologous end joining (NHEJ), which occurs in response to DSBs. Without a donor vector containing exogenous genetic material, the NHEJ often leads to random insertions or deletions (in/dels) of a small number of endogenous nucleotides.
  • NHEJ non- homologous end joining
  • the exogenous genetic material can be introduced into the genome during homology directed repair (HDR) by homologous recombination, resulting in a "‘targeted integration.’ 7
  • HDR homology directed repair
  • DSBs available endonucleases capable of introducing specific and targeted DSBs include, but not limited to, zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), RNA-guided CRISPR (Clustered Regular Interspaced Short Palindromic Repeats) systems. Additionally, DICE (dual integrase cassette exchange) system utilizing phiC31 and Bxbl integrases is also a promising tool for targeted integration.
  • ZFN zinc-finger nucleases
  • TALEN transcription activator-like effector nucleases
  • CRISPR Clustered Regular Interspaced Short Palindromic Repeats
  • ZFNs are targeted nucleases comprising a nuclease fused to a zinc finger DNA binding domain.
  • a “zinc finger DNA binding domain” or “ZFBD” it is meant a polypeptide domain that binds DNA in a sequence-specific manner through one or more zinc fingers.
  • a zinc finger is a domain of about 30 amino acids within the zinc finger binding domain whose structure is stabilized through coordination of a zinc ion. Examples of zinc fingers include, but not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers.
  • a “designed” zinc finger domain is a domain not occurring in nature whose design/composition results principally from rational criteria, e.g., application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. See, for example, US6, 140,081; US6,453,242; and US6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.
  • a “selected” zinc finger domain is a domain not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection.
  • ZFNs are described in greater detail in US7,888,121 and US7,972,854, the complete disclosures of which are incorporated herein by reference.
  • the most recognized example of a ZFN in the art is a fusion of the Fokl nuclease with a zinc finger DNA binding domain.
  • a TALEN is a targeted nuclease comprising a nuclease fused to a TAL effector DNA binding domain.
  • transcription activator-like effector DNA binding domain By “transcription activator-like effector DNA binding domain,” “TAL effector DNA binding domain,” or “TALE DNA binding domain,” it is meant the polypeptide domain of TAL effector proteins that is responsible for binding of the TAL effector protein to DNA.
  • TAL effector proteins are secreted by plant pathogens of the genus Xanthomonas during infection. These proteins enter the nucleus of the plant cell, bind effector-specific DNA sequences via their DNA binding domain, and activate gene transcription at these sequences via their transactivation domains.
  • TAL effector DNA binding domain specificity depends on an effector-variable number of imperfect 34 amino acid repeats, which comprise polymorphisms at select repeat positions called repeat variable-diresidues (RVD).
  • RVD repeat variable-diresidues
  • TALENs are described in greater detail in US2011/0145940, which is herein incorporated by reference.
  • the most recognized example of a TALEN in the art is a fusion polypeptide of the Fokl nuclease to a TAL effector DNA binding domain.
  • a targeted nuclease that finds use in the subject methods is a targeted Spoil nuclease, a polypeptide comprising a Spoil polypeptide having nuclease activity fused to a DNA binding domain, e.g. a zinc finger DNA binding domain, a TAL effector DNA binding domain, etc. that has specificity for a DNA sequence of interest.
  • a DNA binding domain e.g. a zinc finger DNA binding domain, a TAL effector DNA binding domain, etc. that has specificity for a DNA sequence of interest.
  • targeted nucleases suitable for the present application include, but not limited to Bxbl, phiC3 1, R4, PhiBTl, and Wp/SPBc/TP901-l, whether used individually or in combination.
  • targeted nucleases include naturally occurring and recombinant nucleases; CRISPR related nucleases from families including cas, cpf, cse, csy, csn, csd, cst, csh, csa, csm, and cmr; restriction endonucleases; meganucleases; homing endonucleases, and the like.
  • CRISPR/Cas9 requires two major components: (1) a Cas9 endonuclease and (2) the crRNA-tracrRNA complex.
  • CRISPR/Cpfl comprises two major components: (1) a Cpfl endonuclease and (2) a crRNA.
  • RNP ribonucleoprotein
  • the crRNA can be combined to form a chimeric guide RNA (gRNA) to guide Cpfl to target selected sequences. These two components can then be delivered to mammalian cells via transfection or transduction.
  • gRNA chimeric guide RNA
  • MAD7 is an engineered Casl2a variant originating from the bacterium Eubacterium rectale that has a preference for 5'-TTTN-3' and 5'-CTTN-3' PAM sites and does not require a tracrRNA. See, for example, WO2018/236548, the disclosure of which is incorporated herein by reference. Additional descriptions of CRISPR-MAD7 methods can be found in, e.g., CRISPR J., April 2020, 3(2):97-108.
  • DICE mediated insertion uses a pair of recombinases, for example, phiC31 and Bxbl, to provide unidirectional integration of an exogenous DNA that is tightly restricted to each enzyme’s own small attB and attP recognition sites. Because these target att sites are not naturally present in mammalian genomes, they must be first introduced into the genome, at the desired integration site. See, for example, US2015/0140665 and Farriggo et al., Methods Mol Biol, 2017. 1642:69-85, the disclosures of which are incorporated herein by reference.
  • a construct comprising one or more exogenous polynucleotides for targeted genome integration.
  • the construct further comprises a pair of homologous arms specific to a desired integration site, and the method of targeted integration comprises introducing the construct to cells to enable site specific homologous recombination by the cell host enzymatic machinery.
  • the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, and introducing a ZFN expression cassette comprising a DNA-binding domain specific to a desired integration site to the cell to enable a ZFN-mediated insertion.
  • the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, and introducing a TALEN expression cassette comprising a DNA-binding domain specific to a desired integration site to the cell to enable a TALEN-mediated insertion.
  • the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, introducing a Cpfl expression cassette, and a gRNA comprising a guide sequence specific to a desired integration site to the cell to enable a Cpfl -mediated insertion.
  • the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, introducing a Cas9 expression cassette, and a gRNA comprising a guide sequence specific to a desired integration site to the cell to enable a Cas9- mediated insertion.
  • the method of targeted integration in a cell comprises introducing a construct comprising one or more “at” sites of a pair of DICE recombinases to a desired integration site in the cell, introducing a construct comprising one or more exogenous polynucleotides to the cell, and introducing an expression cassette for DICE recombinases, to enable DICE-mediated targeted integration.
  • Sites for targeted integration include, but are not limited to, genomic safe harbors, which are intragenic or extragenic regions of the human genome that, theoretically, are able to accommodate predictable expression of newly integrated DNA without adverse effects on the host cell or organism.
  • the genome safe harbor for the targeted integration is one or more loci of genes selected from the group consisting of AAVS1, CCR5, ROSA26, HTRP, GAPDH, TCR and RUNX1 genes.
  • a TCR gene is selected from the group consisting of a TRA gene, a TRB gene, a TRD gene, and a TRG gene.
  • the site for targeted integration is selected for deletion or reduced expression of an endogenous gene at the insertion site.
  • a deletion with respect to expression of a gene includes any genetic modification that abolishes the expression of the gene. Examples of a deletion of expression of a gene include, e.g., a removal or deletion of a DNA sequence of the gene, an insertion of an exogenous polynucleotide sequence at a locus of the gene, and one or more substitutions within the gene, which abolishes the expression of the gene.
  • Genes for target deletion include, but are not limited to, genes of major histocompatibility complex (MHC) class I and MHC class II proteins. Multiple MHC class I and class II proteins must be matched for histocompatibility in allogeneic recipients to avoid allogeneic rejection problems.
  • MHC deficient including MHC-class I deficient, or MHC-class II deficient, or both, refers to cells that either lack, or no longer maintain, or have reduced level of surface expression of a complete MHC complex comprising a MHC class I protein heterodimer and/or a MHC class II heterodimer, such that the diminished or reduced level is less than the level naturally detectable by other cells or by synthetic methods.
  • MHC class I deficiency can be achieved by functional deletion of any region of the MHC class I locus (chromosome 6p21), or deletion or reducing the expression level of one or more MHC class-I associated genes including, not being limited to, beta-2 microglobulin (B2M) gene, TAPI gene, TAP2 gene and tapasin genes.
  • B2M gene encodes a common subunit essential for cell surface expression of all MHC class I heterodimers.
  • B2M null cells are MHC-I deficient.
  • MHC class II deficiency can be achieved by functional deletion or reduction of MHC-II associated genes including, not being limited to, RFXANK, CIITA, RFX5 and RFXAP.
  • CIITA is a transcriptional coactivator, functioning through activation of the transcription factor RFX5 required for class II protein expression.
  • CIITA null cells are MHC-II deficient.
  • one or more of the exogenous polynucleotides are integrated at one or more loci of genes selected from the group consisting of B2M, TAPI, TAP2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes to thereby delete or reduce the expression of the gene(s) with the integration.
  • Other genes that may be targeted for deletion include NKG2A, CD38, CD70 and CD33.
  • the exogenous polynucleotides are integrated at one or more loci on the chromosome of the cell, preferably the one or more loci are of genes selected from the group consisting of AAVS1, CCR5, ROSA26. HTRP. GAPDH, RUNX1, B2M. TAPI.
  • TAP2 Tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRa constant region, TCRb constant region, NKG2A, NKG2D, CD38, CIS, CBL-B, S0CS2, PD1, CTLA4, LAG3, TIM3, or TIGIT genes, provided at least one of the one or more loci is of a MHC gene, such as a gene selected from the group consisting ofB2M, TAPI, TAP2, Tapasin, RFXANK. CIITA. RFX5 and RFXAP genes.
  • the one or more exogenous polynucleotides are integrated at a locus of an MHC class-I associated gene, such as a B2M gene, TAPI gene, TAP2 gene, or Tapasin gene; and at a locus of an MHC-II associated gene, such as a RFXANK, CIITA. RFX5, RFXAP, or CIITA gene; and optionally further at a locus of a safe harbor gene selected from the group consisting of AAVS1. CCR5. ROSA26, HTRP, GAPDH, TCR and RUNX1 genes.
  • a TCR gene is selected from the group consisting of a TRA gene, a TRB gene, a TRD gene, and a TRG gene.
  • the one or more of the exogenous polynucleotides are integrated at the loci of CIITA. AAVS1 and B2M genes.
  • an exogenous polynucleotide is integrated at a gene locus; (ii) a different exogenous polynucleotide is integrated at a locus of CIITA gene; and (iii) another different exogenous polynucleotide is integrated at a locus of B2M gene; wherein integrations of the exogenous polynucleotides delete or reduce expression of CIITA and B2M genes.
  • an exogenous polynucleotide is integrated at a gene locus; (ii) another exogenous polynucleotide is integrated at a locus of CIITA gene; and (iii) yet another exogenous polynucleotide is integrated at a locus of B2M gene; wherein integrations of the exogenous polynucleotides eliminate or reduce expression of CIITA and B2M genes.
  • a first exogenous polynucleotide is integrated at a safe harbor locus;
  • a second exogenous polynucleotide is integrated at a locus of CIITA gene; and
  • a third exogenous polynucleotide is integrated at a locus of B2M gene; wherein integrations of the exogenous polynucleotides eliminate or reduce expression of CIITA and B2M genes.
  • a first exogenous polynucleotide is integrated at a AAV S 1 , CCR5, ROS A26, HTRP, GAPDH, TRA, TRB, TRD, TRG or RUNX1 gene locus;
  • a second exogenous polynucleotide is integrated at a CIITA gene locus;
  • a third exogenous polynucleotide is integrated at a B2M gene locus; wherein integrations of the exogenous polynucleotides eliminate or reduce expression of CIITA and B2M genes.
  • an exogenous polynucleotide is integrated at a CD70 gene locus.
  • an exogenous polynucleotide is integrated at a gene locus;
  • a different exogenous polynucleotide is integrated at a CIITA gene locus;
  • another different exogenous polynucleotide is integrated at a B2M gene locus;
  • yet another different exogenous polynucleotide is integrated at a CD70 gene locus; wherein integrations of the exogenous polynucleotides eliminate or reduce expression of the CD70, CIITA and B2M genes.
  • a first exogenous polynucleotide is integrated at a gene locus;
  • a second exogenous polynucleotide is integrated at a CIITA gene locus;
  • a third exogenous polynucleotide is integrated at a B2M gene locus;
  • a fourth exogenous polynucleotide is integrated at a CD70 gene locus; wherein integrations of the exogenous polynucleotides eliminate or reduce expression of the CD70, CIITA and B2M genes.
  • a first exogenous polynucleotide is integrated at a safe harbor gene locus;
  • a second exogenous polynucleotide is integrated at a CIITA gene locus;
  • a third exogenous polynucleotide is integrated at a B2M gene locus;
  • a fourth exogenous polynucleotide is integrated at a CD70 gene locus; wherein integrations of the exogenous polynucleotides eliminate or reduce expression of the CD70, CIITA and B2M genes.
  • a first exogenous polynucleotide is integrated at a AAV S 1 , CCR5, ROSA26, HTRP, GAPDH, TRA, TRB, TRD, TRG or RUNX1 gene locus;
  • a second exogenous polynucleotide is integrated at a CIITA gene locus;
  • a third exogenous polynucleotide is integrated at a B2M gene locus;
  • a fourth exogenous polynucleotide is integrated at a CD70 gene locus; wherein integrations of the exogenous polynucleotides eliminate or reduce expression of the CD70, CIITA and B2M genes.
  • the present disclosure provides an isolated host cell comprising any of the various polynucleotides described herein, for example, without limitation, polynucleotides encoding an engineered IL-7Ra polypeptide, or fragment or derivative thereof, described herein.
  • an isolated host cell of the present disclosure may comprise an engineered IL-7Ra encoded by the polynucleotide described herein.
  • the present disclosure provides an isolated host cell comprising any of the various recombinant vectors disclosed herein.
  • the isolated host cell disclosed herein may comprise two or more polynucleotides or recombinant vectors described herein.
  • the host cell may be an induced pluripotent stem cell (iPSC) or a population thereof.
  • the host cell may be an immune cell, or a population thereof.
  • the immune cell, or a population thereof can be derived from an iPSC disclosed herein.
  • the isolated host cell, or population thereof may be a B cell, a T cell, a natural killer (NK) cell, a natural killer T cell (NKT cell), a mesenchymal stem cell (MSC), or a macrophage.
  • NK natural killer
  • NKT cell natural killer T cell
  • MSC mesenchymal stem cell
  • the isolated host cell, or population thereof may be a B cell.
  • the B cell can be, for example, a transitional B cell, a naive B cell, a plasma B cell, or a memory B cell.
  • the B cell is a T-independent B cell.
  • the isolated host cell, or population thereof may be a T cell.
  • T-cells may include, for example, without limitation, thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes.
  • a T-cell can be a T helper (Th) cell, for example a T helper 1 (Thl) or a T helper 2 (Th2) cell.
  • the T-cell can be a helper T-cell (HTL; CD4+ T-cell) CD4+ T-cell, a cytotoxic T-cell (CTL; CD8+ T-cell), a tumor infiltrating cytotoxic T-cell (TIL; CD8+ T-cell), CD4+ CD8+ T-cell, or any other subset of T-cells.
  • HTL helper T-cell
  • CTL cytotoxic T-cell
  • TIL tumor infiltrating cytotoxic T-cell
  • CD4+ CD8+ T-cell CD4+ CD8+ T-cell, or any other subset of T-cells.
  • Other illustrative populations of T-cells suitable for use in particular embodiments include naive T-cells memory T-cells, and NKT cells.
  • the T-cell may be an af> T-cell, a yd T-cell, a CD8+ T-cell, a CD4+ T-cell, a cytotoxic T-cell, an invariant natural killer T (iNKT) cell, a memory' T-cell, a memory' stem T-cell (TSCM), a naive T-cell, an effector T-cell, a T-helper cell, or a regulatory T-cell (Treg).
  • the T cell may be derived from peripheral, cord blood, iPS cells, and/or a cell line.
  • the T cell when the T cell is derived from an IPS cell, the T cell may be any of various T-cells described herein, e.g., an a
  • the host cell, or population thereof may be an NK cell.
  • the NK cell may be derived from peripheral, cord blood, iPS cells, and/or a cell line.
  • the IL7R polynucleotide is incorporated into an immune-effector cell differentiated from an iPSC cell or a derivative thereof.
  • iPSCs are differentiated into a cell ty pe which is then cultured and differentiated into another cell ty pe.
  • an iPSC can be differentiated into a progenitor cell such as a NK progenitor cell, which is then cultured under conditions to become a mature cell such as aNK cell.
  • a progenitor cell such as a NK progenitor cell
  • the derivative cell is a hematopoietic cell, including, but not limited to, hematopoietic stem and progenitor cells (HSCs), hematopoietic multipotent progenitor cells, T cell progenitors, natural killer (NK) cell progenitors, B cell progenitors, CD34+ hematopoietic progenitor cells, T cells, NKT cells, NK cells, B cells, antigen presenting cells (APC), monocytes and macrophages.
  • the derivative cell is an immune effector cell, such as a NK cell or a T cell.
  • the iPSC is produced from whole peripheral blood mononuclear cells. In some embodiments, the iPSC is produced from an NK cell. In some embodiments, the iPSC is produced from a T cell. In some embodiments, the iPSC is produced from a reprogrammed NK cell. In some embodiments, the iPSC is produced from a reprogrammed T cell.
  • the method includes differentiating the iPSC under conditions to promote, facilitate or generate a specific differentiated cell.
  • the differentiated cells is further cultured to produce a cell derived from the differentiated cell, e.g., a derivative cell.
  • An iPSC can be differentiated by any method known in the art. Exemplary methods are described in US10,947,502; US8,846,395; US8,945,922; US8,318,491; W02010/099539; WO20I0/14I801; WO2012/109208; W02016/010148; WO2017/070333; W02017/070337; WO2017/179720; WO2018/048828; and WO2019/157597 and WO2020/252477; the contents of which are herein incorporated by reference in their entireties.
  • the differentiation protocol may use feeder cells or may be feeder-free.
  • Feeder cells or feeders include cells of one type that are co-cultured with cells of a second type to provide an environment in which the cells of the second type can grow, expand, or differentiate, as the feeder cells provide stimulation, growth factors and nutrients for the support of the second cell type.
  • the differentiated iPSCs are NK cells which are prepared by a method of differentiating an iPSC into an NK cell.
  • the iPSCs are subjected to a differentiation protocol including the addition of recombinant human IL-12p70 (e.g., IL-12) to the culture media for the final 24 hours of culture.
  • IL-12p70 e.g., IL-12
  • cells that are primed with IL- 12 demonstrate more rapid cell killing compared to those that are differentiated in the absence of IL- 12.
  • the cells differentiated using the IL- 12 conditions demonstrate improved cancer cell growth inhibition.
  • recombinant human IL-12p70 (human IL-12) includes a IL-12 p40 subunit and/or a IL- 12 p35 subunit.
  • a IL- 12 p40 subunit is connected to a IL-12 p35 subunit by way of a linker which can be any of those described herein.
  • recombinant human IL-12p70 includes a IL- 12 p40 subunit, a Whitlow linker, and a IL-12 p35 subunit.
  • the recombinant human IL-12p70 protein has at least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. sequence identity to SEQ ID NO: 25.
  • the recombinant human IL-12p70 protein is encoded by a polynucleotide sequence having at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to SEQ ID NO: 26.
  • the differentiated iPSCs are T cells which are prepared by a method of differentiating an iPSC into a T cell.
  • Protocols for generating a T cell from an iPSC include those disclosed, for example, in W02010/099539, W02010/141801, W02017/070333, W02017/070337, WO2017/179720, WO2018/048828, WO2019/157597 and WO2020/252477, the contents of which are herein incorporated by reference in their entireties.
  • the differentiated iPSCs are T cells which are prepared by a method of differentiating an iPSC into an T cell.
  • the iPSCs are subjected to a differentiation protocol including the addition of recombinant human DLL-4 protein to the culture media.
  • the cells are cultured in medium comprising human DLL-4 protein for the final hours (e g., 12, 18, 20, or 24 hours) of culture.
  • recombinant human DLL-4 protein comprises an amino acid sequence having at least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to SEQ ID NO: 27.
  • recombinant human DLL-4 comprises an amino acid sequence having at least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to SEQ ID NO: 28.
  • DLL-4 variant protein having an amino acid sequence selected from the group consisting of SEQ ID NOS: 27-28.
  • the differentiated iPSCs are CD34-positive (CD34+) cells which are prepared by a method of differentiating an iPSC into a CD34+ cell.
  • Protocols for generating a CD34+ cell from an iPSC include those disclosed, for example, in W02010/099539, W02010/141801, W02017/070333, WO2017/070337, WO2018/048828, WO2019/157597 and WO2020/252477, the contents of which are herein incorporated by reference in their entireties.
  • iPSC cells are differentiated into hematopoietic progenitor cells (HPCs).
  • iPSC cells are in HDM-I media plus Hl 152.
  • HDM media contains IMDM medium, Ham’s F12 medium, CTS B27 minus vitamin A supplement, non-essential amino acids, ascorbic acid, Mg 2-phosphate, monothioglycerol, and heparin.
  • HDM-I media can contain HDM + CHIR99021 GSK3 inhibitor. FGF2, and VEGF.
  • the cells are further cultured in HDM-II medium comprising HDM media in addition to BMP4, FGF2, and VEGF.
  • the cells are further cultured in HDM-III medium comprising HDM in addition to BMP4, SCF, TPO, FLT3L, and IL3. The resulting HPCs can be collected.
  • HPCs are differentiated to produce NK or T cells.
  • the HPCs are cultured in retronectin/DLL-4-coated bioreactors, e.g., G-Rex bioreactors.
  • Notch signaling factors, cytokines, and growth factors can be added to culture medium to facilitate differentiation into lymphoid lineage and subsequent NK or T cell maturation and activation.
  • maturation and/or activation of NK or T cells from HPCs includes culturing the HPCs in a culture medium comprising a recombinant IL- 12 protein.
  • IL- 12 is a cytokine that stimulates the production of interferon-gamma (IFN-y) and tumor necrosis factor-alpha (TNF-a) from T cells and natural killer (NK) cells.
  • a recombinant IL-12 protein comprises human IL-12p70.
  • recombinant IL-12 comprises ahuman IL-12p70 p40 subunit and ahuman IL-12p70 p35 subunit.
  • recombinant IL-12 protein comprises a human IL-12p70 p40 subunit, a human IL-12p70 p35 subunit and a linker.
  • recombinant IL- 12 protein comprises from N- to C-terminus: a human IL-12p70 p40 subunit, a linker, and a human IL- 12p70 p35 subunit.
  • recombinant IL-12 protein comprises from N- to C-terminus: a human IL-12p70 p35 subunit, a linker, and a human IL-12p70 p40 subunit.
  • recombinant IL-12 protein comprises from N- to C-terminus: a human IL- 12p70 p40 subunit, a Whitlow linker, and a human IL-12p70 p35 subunit. In various embodiments, recombinant IL-12 protein comprises fromN- to C-terminus: ahuman IL-12p70 p35 subunit, a Whitlow linker, and a human IL-12p70 p40 subunit. In some embodiments, a recombinant IL- 12 protein comprises an amino acid sequence having at least 90%, at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to SEQ ID NO: 25.
  • a recombinant IL-12 protein is encoded by a polynucleotide having at least 90%, at least 90%, 91%, 82%. 93%. 94%. 95%. 96%. 97%, 98%, 99% or 100%, sequence identity to SEQ ID NO: 26.
  • Antigen-recognition molecules having at least 90%, at least 90%, 91%, 82%. 93%. 94%. 95%. 96%. 97%, 98%, 99% or 100%, sequence identity to SEQ ID NO: 26.
  • the host cell may further express one or more antigenrecognition molecules.
  • the antigen-recognition molecules are selected from aP T cell receptors (TCRs), synthetic T cell receptors and antigen receptor (STARs), chimeric antigen receptor (CARs), T cell antigen couplers (TACs), T cell receptor fusion constructs (TruCs), or antibodies (e.g., bispecific antibodies), or a combination thereof.
  • the antigen-recognition molecule(s) may target one or more antigens (i.e., a target antigen).
  • the target antigen targeting by the antigen-recognition molecule(s) disclosed herein may include, for example, without limitation, 17-1 A antigen, A3, A33 antigen, AFP, B7H4, Ba 733, BCMA, BrE3 antigen, CA125, CA9 (CAIX), CD1, CDla, CD3, CD5. CD15, CD16, CD19, CD20, CD21, CD22. CD22.
  • SI 00 SLAM F7, SLITRK6, TAC, TAG-72, tenascin-C, tenascin-R, tenascin-W, tenascin-X, Thomson-Friedenreich antigen, Tn antigen, TRAILR1, TRAILR2, TRAILR3, TRAILR4, VEGF, a tumor necrosis antigen, an angiogenesis antigen, and/or an oncogene antigen.
  • the host cell may further express one or more antigenrecognition molecules, and the antigen recognition molecule(s) is a CAR.
  • a CAR of the present disclosure herein may comprise a recombinant polypeptide comprising an extracellular domain that binds specifically to an antigen or a target, a transmembrane domain and an intracellular signaling domain. Engagement of the extracellular domain of the CAR with the target antigen on the surface of a target cell can result in clustering of the CAR and deliver an activation stimulus to the CAR-containing cell.
  • a CAR described herein comprises a leader sequence.
  • the leader sequence may be positioned at the N-terminus the extracellular domain.
  • the leader sequence may be optionally cleaved from the extracellular domain during cellular processing and localization of the CAR to the cellular membrane. Any of various leader sequences known to one of skill in the art may be used as the leader sequence.
  • Non-limiting examples of peptides from which the leader sequence may be derived include granulocyte-macrophage colonystimulating factor receptor (GMCSFR), FceR. human immunoglobulin (IgG) heavy chain (HC) variable region, CD8a, or any of various other proteins secreted by T cells.
  • the leader sequence is compatible with the secretory pathway of a T cell.
  • the leader sequence is derived from human immunoglobulin heavy chain (HC).
  • the CAR of the present disclosure comprises a transmembrane domain which may be fused in frame between an extracellular domain and a cytoplasmic domain.
  • the transmembrane domain may be derived from the protein contributing to the extracellular domain, the protein contributing to the signaling or co-signaling domain, or by a totally different protein.
  • the transmembrane domain can be selected or modified by amino acid substitution, deletions, or insertions to minimize interactions with other members of the CAR complex.
  • the transmembrane domain can be selected or modified by amino acid substitution, deletions, or insertions to avoid binding of proteins naturally associated with the transmembrane domain.
  • the transmembrane domain includes additional amino acids to allow for flexibility and/or optimal distance between the domains connected to the transmembrane domain.
  • the transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.
  • Non-limiting examples of transmembrane domains of particular use in this disclosure may be derived from (i.e. comprise at least the transmembrane domain(s) of) the a, (3 or chain of the T cell receptor (TCR), CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD8a, CD9.
  • TCR T cell receptor
  • CD28 CD3 epsilon
  • CD45 CD45
  • CD4 CD5
  • CD8a CD9.
  • CD16 CD22, CD33, CD37, CD40, CD64, CD80, CD86.
  • CD134 CD137, or CD 154.
  • the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. For example, a triplet of phenylalanine, tryptophan and/or valine can be found at each end of a synthetic transmembrane domain.
  • the transmembrane domain will be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
  • it will be desirable to employ the transmembrane domain of , or FceRly and -P, MB1 (Iga.), B29 or CD3- y, ⁇ , or r
  • the transmembrane domain is derived from CD8 or CD28.
  • the CAR comprises a spacer region between the extracellular domain and the transmembrane domain, wherein the domain, linker, and the transmembrane domain are in frame with each other.
  • spacer region' as used herein generally means any oligo- or polypeptide that functions to link the domain to the transmembrane domain.
  • a spacer region can be used to provide more flexibility and accessibility for the domain.
  • a spacer region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
  • a spacer region may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region.
  • the spacer region may be a synthetic sequence that corresponds to a naturally occurring spacer region sequence, or may be an entirely synthetic spacer region sequence.
  • Non-limiting examples of spacer regions which may be used in accordance with the disclosure include apart of human CD8a chain, partial extracellular domain of CD28. FcyRllla receptor, IgG, IgM, IgA, IgD, IgE, an Ig hinge, or functional fragment thereof.
  • additional linking amino acids are added to the spacer region to ensure that the antigen-binding domain is an optimal distance from the transmembrane domain.
  • the spacer when the spacer is derived from an Ig, the spacer may be mutated to prevent Fc receptor binding.
  • the spacer region comprises a hinge domain.
  • the hinge domain may be derived from CD8a, CD28, or an immunoglobulin (IgG).
  • IgG hinge may be from IgGl, IgG2, IgG3, IgG4, IgMl, IgM2, IgAl, IgA2, IgD, IgE, or a chimera thereof.
  • the hinge domain comprises an immunoglobulin IgG hinge or functional fragment thereof.
  • the IgG hinge is from IgGl, IgG2, IgG3, IgG4, IgMl, IgM2, IgAl, IgA2, IgD, IgE, or a chimera thereof.
  • the hinge domain comprises the CHI, CH2, CH3 and/or hinge region of the immunoglobulin.
  • the hinge domain comprises the core hinge region of the immunoglobulin.
  • the term ‘“core hinge” can be used interchangeably with the term “short hinge” (a.k.a “SH”).
  • the hinge domain is a fragment of the immunoglobulin hinge.
  • the hinge domain is derived from CD8 or CD28.
  • the transmembrane domain and/or hinge domain is derived from CD8 or CD28. In some embodiments, both the transmembrane domain and hinge domain are derived from CD8. In some embodiments, both the transmembrane domain and hinge domain are derived from CD28.
  • the CAR of the present disclosure comprises a cytoplasmic domain, which comprises at least one intracellular signaling domain.
  • cytoplasmic domain also comprises one or more co-stimulatory signaling domains.
  • the cytoplasmic domain is responsible for activation of at least one of the normal effector functions of the host cell (e.g., T cell) in which the CAR has been placed in.
  • effector function refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
  • signaling domain refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire signaling domain is present, in many cases it is not necessary to use the entire chain.
  • intracellular signaling domain is thus meant to include any truncated portion of the signaling domain sufficient to transduce the effector function signal.
  • Non-limiting examples of signaling domains which can be used in the CARs of the present disclosure include, e.g., signaling domains derived from DAP10, DAP12, Fc epsilon receptor I y chain (FCER1G), FcR (3, CD35, CD3s, CD3y, CD3i CD5, CD22, CD226, CD66d, CD79A, and CD79B.
  • FCER1G Fc epsilon receptor I y chain
  • FcR 3, CD35, CD3s, CD3y, CD3i CD5, CD22, CD226, CD66d, CD79A, and CD79B.
  • the cytoplasmic domain comprises a CD3 ⁇ signaling domain.
  • the cytoplasmic domain further comprises one or more costimulatory signaling domains.
  • the one or more co-stimulatory signaling domains are derived from CD28, 41BB, IL2Rb, CD40, 0X40 (CD134), CD80, CD86, CD27, ICOS, NKG2D, DAP 10, DAP 12, 2B4 (CD244), BTLA, CD30, GITR, CD226, CD79A, and HVEM.
  • the CAR of the present disclosure comprises a hinge region, a transmembrane domain and a co-stimulatory signaling domain all derived from CD28.
  • the CAR of the present disclosure comprises one costimulatory signaling domains. In some embodiments, the CAR of the present disclosure comprises two or more costimulatory signaling domains. In certain embodiments, the CAR of the present disclosure comprises two, three, four, five, six or more costimulatory signaling domains.
  • the signaling domain(s) and costimulatory signaling domain(s) can be placed in any order.
  • the signaling domain is upstream of the costimulatory signaling domains.
  • the signaling domain is downstream from the costimulatory signaling domains. In the cases where two or more costimulatory domains are included, the order of the costimulatory signaling domains could be switched.
  • the choice of antigen-binding domain may depend upon the type and number of antigens that define the surface of a target cell.
  • the antigen-binding domain may be chosen to recognize an antigen that acts as a cell surface marker on target cells associated with a particular disease state.
  • the CARs of the present disclosure can be genetically modified to target a tumor antigen of interest by way of engineering a desired antigen-binding domain that specifically binds to an antigen (e.g., on a tumor cell).
  • Nonlimiting examples of cell surface markers that may act as targets for the antigen-binding domain in the CAR of the disclosure include those associated with tumor cells or autoimmune diseases.
  • the antigen-binding domain binds to at least one tumor antigen or autoimmune antigen.
  • the antigen-binding domain binds to at least one tumor antigen. In some embodiments, the antigen-binding domain binds to two or more tumor antigens. In some embodiments, the two or more tumor antigens are associated with the same tumor. In some embodiments, the two or more tumor antigens are associated with different tumors.
  • the antigen-binding domain binds to at least one autoimmune antigen. In some embodiments, the antigen-binding domain binds to two or more autoimmune antigens. In some embodiments, the two or more autoimmune antigens are associated with the same autoimmune disease. In some embodiments, the two or more autoimmune antigens are associated with different autoimmune diseases.
  • the tumor antigen is associated with glioblastoma, ovarian cancer, cervical cancer, head and neck cancer, liver cancer, prostate cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, or hematologic malignancy.
  • tumor antigen associated with glioblastoma include HER2, EGFRvIII, EGFR, CD133, PDGFRA, FGFR1, FGFR3.
  • tumor antigens associated with ovarian cancer include FOLR1, FSHR, MUC16, MUC 1, Mesothelin, CA125, EpCAM, EGFR, PDGFRa, Nectin-4, and B7H4.
  • Non-limiting examples of the tumor antigens associated with cervical cancer or head and neck cancer include GD2, MUC1, Mesothelin, HER2, and EGFR.
  • Non-limiting examples of tumor antigen associated with liver cancer include Claudin 18.2, GPC-3, EpCAM, cMET, and AFP.
  • Non-limiting examples of tumor antigens associated with hematological malignancies include CD22, CD79, BCMA, GPRC5D, SLAM F7, CD33, CLL1, CD123, and CD70.
  • Non-limiting examples of tumor antigens associated with bladder cancer include Nectin-4 and SLITRK6.
  • antigens that may be targeted by the antigen-binding domain include, but are not limited to, alpha-fetoprotein, A3, antigen specific for A33 antibody, Ba 733, BrE3-antigen, carbonic anhydrase EX, CD1, CDla, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD123, CD 138, colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, CSAp, EGFR, EGP-I, EGP-2, Ep-CAM, EphAl , EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphAlO, EphBl, EphB2, EphB3, EphB4, EphB6, FIt-I, Flt-3, folate receptor, HLA-
  • KC4-antigen KS-1 -antigen, KS1-4, Le-Y, macrophage inhibition factor (MIF), MAGE, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, antigen specific for PAM-4 antibody, placental growth factor, p53, prostatic acid phosphatase, PSA, PSMA, RS5, SI 00, TAC, TAG-72, tenascin, TRAIL receptors, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, ED-B fibronectin, 17-1A- antigen, an angiogenesis marker, an oncogene marker or an oncogene product.
  • MIF macrophage inhibition factor
  • MAGE MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, antigen specific for PAM-4 antibody, placental growth factor, p53, prostatic acid phosphatase, PSA, PSMA, RS5, SI 00
  • the antigen targeted by the antigen-binding domain is CD 19.
  • the antigen-binding domain comprises an anti-CD19 scFv.
  • the antigen is associated with an autoimmune disease or disorder.
  • Such antigens may be derived from cell receptors and cells which produce “self directed antibodies.
  • the antigen is associated with an autoimmune disease or disorder such as Rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome, Systemic lupus erythematosus, sarcoidosis, Type 1 diabetes mellitus, insulin dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis, Myasthenia gravis, Hashimoto's thyroiditis, Graves' disease, chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, Crohn's disease or ulcerative colitis.
  • RA Rheumatoid arthritis
  • MS
  • autoimmune antigens that may be targeted by the CAR disclosed herein include but are not limited to platelet antigens, myelin protein antigen, Sm antigens in snRNPs, islet cell antigen, Rheumatoid factor, and anticitrullinated protein, citrullinated proteins and peptides such as CCP-1, CCP-2 (cyclical citrullinated peptides), fibrinogen, fibrin, vimentin, filaggrin, collagen I and II peptides, alpha-enolase, translation initiation factor 4G1, perinuclear factor, keratin, Sa (cytoskeletal protein vimentin), components of articular cartilage such as collagen II, IX, and XI, circulating serum proteins such as RFs (IgG, IgM), fibrinogen, plasminogen, ferritin, nuclear components such as RA33/hnRNP A2, Sm, eukaryotic translation elongation factor 1 alpha
  • neutrophil nuclear proteins such as lactoferrin and 25-35 kD nuclear protein
  • granular proteins such as bactericidal permeability increasing protein (BPI), elastase, cathepsin G, myeloperoxidase, proteinase 3, platelet antigens, myelin protein antigen, islet cell antigen, rheumatoid factor, histones, ribosomal P proteins, cardiolipin, vimentin, nucleic acids such as dsDNA, ssDNA, and RNA, ribonuclear particles and proteins such as Sm antigens (including but not limited to SmD's and SmB7B), U1RNP, A2/B1 hnRNP, Ro (SSA), and La (SSB) antigens.
  • BPI bactericidal permeability increasing protein
  • elastase cathepsin G
  • myeloperoxidase proteinase 3
  • platelet antigens my
  • the scFv fragment used in the CAR of the present disclosure may include a linker between the VH and VL domains.
  • the linker can be a peptide linker and may include any naturally occurring amino acid. Exemplary amino acids that may be included into the linker are Gly. Ser Pro, Thr, Glu, Lys, Arg. He, Leu, His and The.
  • the linker should have a length that is adequate to link the VH and the VL in such a way that they form the correct conformation relative to one another so that they retain the desired activity, such as binding to an antigen.
  • the linker may be about 5-50 amino acids long. In some embodiments, the linker is about 10-40 amino acids long.
  • the linker is about 10-35 amino acids long. In some embodiments, the linker is about 10-30 amino acids long. In some embodiments, the linker is about 10-25 amino acids long. In some embodiments, the linker is about 10-20 amino acids long. In some embodiments, the linker is about 15-20 amino acids long.
  • Exemplary linkers that may be used are Gly rich linkers, Gly and Ser containing linkers, Gly and Ala containing linkers, Ala and Ser containing linkers, and other flexible linkers.
  • an iPSC or a derivative cell thereof may comprise an exogenous polynucleotide encoding an artificial cell death polypeptide.
  • artificial cell death polypeptide refers to an engineered protein designed to prevent potential toxicity or otherwise adverse effects of a cell therapy.
  • the artificial cell death polypeptide could mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional genetic regulation and/or antibody-mediated depletion.
  • the artificial cell death polypeptide is activated by an exogenous molecule, e.g. an antibody, that when activated, triggers apoptosis and/or cell death of a therapeutic cell.
  • an artificial cell death polypeptide comprises an inactivated cell surface receptor that comprises an epitope specifically recognized by an antibody, particularly a monoclonal antibody, which is also referred to herein as a monoclonal antibodyspecific epitope.
  • an antibody particularly a monoclonal antibody, which is also referred to herein as a monoclonal antibodyspecific epitope.
  • the inactivated cell surface receptor When expressed by iPSCs or derivative cells thereof, the inactivated cell surface receptor is signaling inactive or significantly impaired, but can still be specifically recognized by an antibody.
  • the specific binding of the antibody to the inactivated cell surface receptor enables the elimination of the iPSCs or derivative cells thereof by ADCC and/or ADCP mechanisms, as well as, direct killing with antibody drug conjugates with toxins or radionuclides.
  • the inactivated cell surface receptor comprises an epitope that is selected from epitopes specifically recognized by an antibody, including but not limited to, ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab. gemtuzumab.
  • an antibody including but not limited to, ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, ritux
  • the inactivated cell surface receptor comprises an epitope that is specifically recognized by cetuximab. In certain embodiments, the inactivated cell surface receptor comprises an epitope that is specifically recognized by trastuzumab.
  • the inactivated cell surface receptor comprises an epitope that is specifically recognized by bevacizumab. In certain embodiments, the inactivated cell surface receptor comprises an epitope that is specifically recognized by avelumab. In certain embodiments, the inactivated cell surface receptor comprises an epitope that is specifically recognized by ipilimumab.
  • Epidermal grow th factor receptor also know n as EGFR, ErbBl and HER1, is a cellsurface receptor for members of the epidermal growth factor family of extracellular ligands.
  • truncated EGFR/ "tEGFR.
  • short EGFR” or sEGFR refers to an inactive EGFR variant that lacks the EGF-binding domains and the intracellular signaling domains of the EGFR.
  • An exemplary' tEGFR variant contains residues 322-333 of domain 2, all of domains 3 and 4 and the transmembrane domain of the native EGFR sequence containing the cetuximab binding epitope.
  • tEGFR variant on the cell surface enables cell elimination by an antibody that specifically binds to the tEGFR, such as cetuximab (Erbitux®), as needed. Due to the absence of the EGF-binding domains and intracellular signaling domains, tEGFR is inactive when expressed by iPSCs or derivative cell thereof.
  • An exemplary inactivated cell surface receptor of the application comprises a tEGFR variant.
  • expression of the inactivated cell surface receptor in an engineered immune cell expressing a chimeric antigen receptor (CAR) induces cell suicide of the engineered immune cell when the cell is contacted with an anti-EGFR antibody.
  • CAR chimeric antigen receptor
  • a subject who has previously received an engineered immune cell of the present disclosure that comprises a heterologous polynucleotide encoding an inactivated cell surface receptor comprising a tEGFR variant can be administered an anti-EGFR antibody in an amount effective to ablate in the subject the previously administered engineered immune cell.
  • the anti-EGFR antibody is cetuximab, matuzumab, necitumumab or panitumumab, preferably the anti-EGFR antibody is cetuximab.
  • the iPSC cell or a derivative cell thereof optionally comprises an exogenous polynucleotide encoding a cytokine, such as interleukin- 15 or interleukin-2.
  • Interleukin- 15 refers to a cytokine that regulates T and NK cell activation and proliferation, or a functional portion thereof.
  • a “functional portion” (“biologically active portion”) of a cytokine refers to a portion of the cytokine that retains one or more functions of full length or mature cytokine.
  • Such functions for IL- 15 include the promotion of NK cell survival, regulation of NK cell and T cell activation and proliferation as well as the support of NK cell development from hematopoietic stem cells.
  • the sequence of a variety of IL- 15 molecules are known in the art.
  • the IL- 15 is a wild-type IL-15.
  • the IL-15 is a human IL-15.
  • the IL-15 is a membrane bound form, where all or a functional portion of the IL- 15 protein is fused to all or a portion of a transmembrane protein that anchors the expressed IL-15 as a cell membrane-bound polypeptide (mbIL15)”, for example the construct described in US Patent US9629877B2, hereby incorporated by reference into the present application.
  • mbIL15 cell membrane-bound polypeptide
  • Interleukin-2 refers to a cytokine that regulates T and NK cell activation and proliferation, or a functional portion thereof.
  • the IL-2 is a wild-type IL-2.
  • an inactivated cell surface receptor comprises a monoclonal antibody-specific epitope operably linked to a cytokine, preferably by an autoprotease peptide sequence.
  • the autoprotease peptide include, but are not limited to, a peptide sequence selected from the group consisting of porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2 A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2 A (BmCPV2A), a Flacherie Virus 2 A (BmIFV2A), and a combination thereof.
  • P2A porcine teschovirus-1 2A
  • FMDV foot-and-mouth disease virus
  • E2A Equine Rhinitis A Virus
  • T2A a cytoplasmic polyhedrosis virus 2 A
  • the autoprotease peptide is an autoprotease peptide of porcine tesehovirus-1 2A (P2A).
  • an inactivated cell surface receptor comprises a truncated epithelial growth factor receptor (tEGFR) variant operably linked to an interleukin- 15 (IL- 15) or IL-2 by an autoprotease peptide sequence.
  • tEGFR truncated epithelial growth factor receptor
  • MHC I and/or MHC II knock-out and/or knock down can be incorporated in the cells for use in “allogeneic” cell therapies, in which cells are harvested from a subject, modified to knock-out or knock-down, e.g., disrupt, B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP gene expression, and then returned to a different subject.
  • knock-out or knock-down e.g., disrupt, B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP gene expression
  • Knocking out or knocking down the B2M, TAP I, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes as described herein can: (1) prevent Graft versus Host response; (2) prevent Host versus Graft response; and/or (3) improve cell safety and efficacy.
  • a presently disclosed invention comprises independently knocking out and/or knocking down one or more genes selected from the group consisting of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes in an iPSC cell.
  • a presently disclosed method comprises independently knocking out and/or knocking down two genes selected from the group consisting B2M, TAP 1, TAP 2. Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes in an 1PSC cell, in particular, B2M and CIITA to achieve class I and II HLA disruption.
  • an iPSC or derivative cell thereof of the application can be further modified by introducing an exogenous polynucleotide encoding one or more proteins related to immune evasion, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G).
  • disruption of the B2M gene eliminates surface expression of all MHC class I molecules, leaving cells vulnerable to lysis by NK cells through the “missing self’ response.
  • Exogenous HLA-E expression can lead to resistance to NK-mediated lysis (Gomalusse et al., Nat Biotechnol. 2017; 35(8): 765-772).
  • Incorporating MHC I and/or MHC II knock-out and/or knock down in the cells for use in “allogeneic” cell therapies will allow the cell product candidates to escape recognition and destruction by the host immune system.
  • the reduction in allogeneic reactivity enabled by use of this technology will allow repeat dosing of the CAR-modified cell therapies to improve their therapeutic potential.
  • the cells In combination with the extended killing capability of optimized immune cells derived from single genetically engineered cell cloning, the cells will have the capacity for repeat dosing to maximize durability of response and efficacy. Additionally, this technology 7 may permit dosing in patients with limited or no immune preconditioning regimens.
  • an iPSC or derivative cell thereof of the application can be further modified by introducing an exogenous polynucleotide encoding one or more proteins related to immune evasion, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G)
  • proteins related to immune evasion such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G)
  • the iPSC or derivative cell thereof comprises an exogenous polypeptide encoding at least one of a human leukocyte antigen E (HLA-E) and human leukocyte antigen G (HLA-G).
  • the exogenous polynucleotide encodes a polypeptide comprising a signal peptide operably linked to a mature B2M protein that is fused to an HLA-E or HLA-G via a linker.
  • the genomic editing at one or more selected sites may comprise insertions of one or more exogenous polynucleotides encoding other additional artificial cell death polypeptides, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins promoting engraftment, trafficking, homing, viability, self-renewal, persistence, and/or survival of the genome-engineered iPSCs or derivative cells thereof.
  • the exogenous polynucleotides for insertion are operatively linked to (1) one or more exogenous promoters comprising CMV, EFla, PGK, CAG.
  • UBC or other constitutive, inducible, temporal-, tissue-, or cell type-specific promoters; or (2) one or more endogenous promoters comprised in the selected sites comprising AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hll, beta-2 microglobulin, GAPDH, TCR or RUNX1, or other locus meeting the criteria of a genome safe harbor.
  • the genome- engineered iPSCs generated using the above method comprise one or more different exogenous polynucleotides encoding proteins comprising caspase, thymidine kinase, cytosine deaminase, B-cell CD20, ErbB2 or CD79b wherein when the genome-engineered iPSCs comprise two or more suicide genes, the suicide genes are integrated in different safe harbor locus comprising AAVS1, CCR5, ROSA26, collagen. HTRP. Hll, Hll, beta-2 microglobulin, GAPDH, TCR or RUNX1.
  • exogenous polynucleotides encoding proteins may include those encoding PET reporters, homeostatic cytokines, and inhibitory' checkpoint inhibitory proteins such as PD1, PD-L1, and CTLA4, as well as proteins that target the CD47/signal regulator ⁇ ' protein alpha (SIRPa) axis.
  • SIRPa CD47/signal regulator ⁇ ' protein alpha
  • the cell may comprise an exogenous polynucleotide encoding a CD 16 protein and/or an NKG2D protein, wherein the CD 16 protein and the NKG2D protein may be operably linked by an autoprotease peptide as disclosed in co-pending patent application PCT/US23/68079.
  • the cells of the present invention may comprise genetically engineered iPSCs and cells derived therefrom that exogenously express recombinant CD16 and recombinant NKG2D.
  • the CD16 protein (which is also referred to as “low affinity immunoglobulin gamma Fc region receptor III-A” or “Fc gamma receptor Illa”) is a wildtype CD 16 protein.
  • the human wildtype CD 16 protein has the amino acid sequence set forth in NCBI Ref. Seq. No. NP_000560.7 or UniProt No. P08637.
  • the coding sequence of human wildtype CD16 is set forth in NCBI Ref. No. NM_000569.8.
  • the CD 16 protein is a CD 16 variant protein.
  • the CD 16 variant protein has an amino acid sequence having at least 90%, e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity' to wildtype CD16.
  • the CD 16 variant is a high affinity CD 16 variant.
  • the CD 16 variant is a noncleav able CD 16 variant.
  • the CD 16 variant is a high affinity and noncl eavable CD 16 variant.
  • the CD16 variant comprises one or more amino acid substitutions selected from the group consisting of F158V, F176V, S197P, D205A, S219A, T220A, and any combination thereof.
  • the CD 16 variant has an F 158V substitution and one or more substitutions selected from Fl 76V, S197P, D205A, S219A, T220A, and any combination thereof.
  • the CD16 variant has an F176V substitution and one or more substitutions selected from F158V, S197P, D205A, S219A, T220A, and any combination thereof.
  • the CD 16 variant has an S197P, substitution and one or more substitutions selected from F158V, F176V, D205A, S219A, T220A, and any combination thereof.
  • the CD 16 variant has a D205A substitution and one or more substitutions selected from Fl 58V, Fl 76V, S197P, S219A, T220A, and any combination thereof.
  • the CD 16 variant has a substitution and one or more substitutions selected from F158V, F176V, S197P, D205A, S219A, T220A, and any combination thereof.
  • the CD16 variant has an S219A substitution and one or more substitutions selected from F158V, F176V, S197P, D205A, T220A, and any combination thereof. In some embodiments, the CD 16 variant has a T220A substitution and one or more substitutions selected from Fl 58V, Fl 76V, S197P, D205A, S219A, T220A, and any combination thereof.
  • the NKG2D protein (which is also referred to as NKG2-D type II integral membrane protein, CD314, killer cell lectin-like receptor subfamily KI member 1 or KLRK1) is a wildtype NKG2D protein.
  • the human wildtype NKG2D protein has the amino acid sequence set forth in NCBI Ref. Seq. Nos. NP_001186734.1 or NP_031386.2 or UniProt No. P26718.
  • the coding sequence of human wildtype NKG2D is set forth in NCBI Ref. Nos. NM_001199805.1 or NM_007360.3.
  • the NKG2D protein is a NKG2D variant protein.
  • the NKG2D variant protein has an amino acid sequence having at least 90%, e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to wildtype NKG2D.
  • constructs containing autoprotease peptide sequences including 2A peptides that can induce ribosomal skipping during translation of an polypeptide.
  • 2A peptides function to '‘cleave” an mRNA transcript by making the ribosome skip the synthesis of a peptide bond at the C -terminus, between the glycine (G) and proline (P) residues, thereby leading to separation between the end of the 2A sequence and the next peptide downstream.
  • 2A peptides include, but are not limited to, a porcine tesehovirus-1 2A (P2A) peptide, a foot-and-mouth disease virus (FMDV) 2A (F2A) peptide, an Equine Rhinitis A Virus (ERAV) 2A (E2A) peptide, a Thosea asigna virus 2A (T2A) peptide, a cytoplasmic polyhedrosis virus 2A (BmCPV2A) peptide, and a Flacherie Virus 2A (BmIFV2A) peptide.
  • P2A porcine tesehovirus-1 2A
  • FMDV foot-and-mouth disease virus
  • F2A foot-and-mouth disease virus
  • E2A Equine Rhinitis A Virus
  • T2A cytoplasmic polyhedrosis virus
  • BmCPV2A cytoplasmic polyhedrosis virus
  • BmIFV2A Flacherie Virus 2A
  • Another optional genome edit is the insertion of a polynucleotide encoding a a membrane-bound interleukin 12 (IL- 12) comprising a first polypeptide comprising an IL- 12 alpha subunit p35, a second polypeptide comprising an IL-12 beta subunit p40 and a transmembrane fused to the terminus of the first and/or second IL- 12 subunit polypeptide as disclosed in co-pending patent application PCT/US23/68105.
  • IL- 12 membrane-bound interleukin 12
  • the polynucleotide encoding the membrane bound IL-12 is fused to a polynucleotide encoding an ADAM17 protease cleavage site peptide for the activation induced release of the IL-12 through the protease ADAMI 7.
  • ADAMI 7 is expressed by activated lymphocytes and is directly involved in the liberation of other immune mediators like TNFa that are similarly presented as a membrane anchored form. When this membrane tethered IL- 12 is expressed on engineered iNK or T cells, it remains cell associated. Upon cell activation and the increased expression of ADAMI 7, the protease cleaves the membrane stalk and releases IL-12 into the extracellular space.
  • the cell of the invention may further comprise (i) an exogenous polynucleotide encoding a membrane-bound interleukin 12 (IL- 12) comprising a first polypeptide comprising an IL-12 alpha subunit p35 or a polypeptide at least 90% similar thereto, a second polypeptide comprising an IL- 12 beta subunit p40 or a polypeptide at least 90% similar thereto, and a transmembrane domain fused to the terminus of the first and/or second IL- 12 subunit polypeptide.
  • IL- 12 membrane-bound interleukin 12
  • the genome-engineered iPSCs generated using the method provided herein comprise in/del at one or more endogenous genes associated with targeting modality, receptors, signaling molecules, transcription factors, drug target candidates, immune response regulation and modulation, or proteins suppressing engraftment, trafficking, homing, viability, self-renewal, persistence, and/or survival of the iPSCs or derivative cells thereof.
  • compositions comprise polypeptides (e.g., engineered IL-7Ra polypeptides, or fragments or derivatives thereof), polynucleotides, vectors comprising same, and/or cell compositions described herein.
  • compositions of the present disclosure include, but are not limited to pharmaceutical compositions.
  • pharmaceutical composition means a product comprising an isolated polynucleotide of the disclosure, an isolated polypeptide of the disclosure, and/or an isolated host cell of the disclosure (e.g.. an immune cell or population thereof) together with a pharmaceutically acceptable carrier.
  • Polynucleotides, polypeptides, and/or isolated host cells or populations thereof of the disclosure and compositions comprising them can also be useful in the manufacture of a medicament for therapeutic applications mentioned herein
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide described herein, and a pharmaceutically accepted carrier and/or excipient.
  • a carrier described herein may comprise any excipient, diluent, fdler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid containing vesicle, microsphere, liposomal encapsulation, and/or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application.
  • a pharmaceutically acceptable carrier of the present disclosure comprise a non-toxic material that does not interfere with the effectiveness of a composition described herein or the biological activity of a composition described herein.
  • any pharmaceutically acceptable carrier suitable for use in a polynucleotide, polypeptide, and/or host cell or population cell thereof can be used.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polynucleotide or a recombinant vector described herein, and a pharmaceutically accepted carrier and/or excipient.
  • the pharmaceutical composition comprises a polynucleotide encoding engineered IL-7Ra polypeptides, or fragment or derivative thereof, and a pharmaceutically accepted carrier and/or excipient.
  • the present disclosure provides pharmaceutical composition
  • a pharmaceutically acceptable carrier and/or excipient comprising an isolated host cells or population thereof, described herein and a pharmaceutically acceptable carrier and/or excipient.
  • Examples of pharmaceutical carriers include but are not limited to sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.
  • compositions comprising isolated host cells disclosed herein may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, phosphate buffered saline and the like
  • carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins such as glucose, mannose, sucrose or dextrans, mannitol
  • compositions comprising isolated host cells disclosed herein may comprise one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the
  • the compositions are formulated for parenteral administration, e.g., intravascular (intravenous or intraarterial), intraperitoneal, intratumoral, intraventricular, intrapleural or intramuscular administration.
  • parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • An injectable pharmaceutical composition is preferably sterile.
  • the composition is reconstituted from a lyophilized preparation prior to administration.
  • the isolated host cells may be mixed with substances that adhere to or penetrate them prior to their administration, e.g., but not limited to, nanoparticles.
  • compositions of the present disclosure such as but not limited to polynucleotides (e.g., polynucleotides encoding engineered IL-7Ra polypeptides, or fragments or derivatives thereof, described herein), and/or recombinant vectors comprising same, for enhancing expansion and/or persistence of a population of host cells described herein.
  • the method comprises expressing the polynucleotides described herein and/or the recombinant vector of any in a host cell described herein.
  • compositions of the present disclosure for generating an isolated host cell (e.g., immune cell or population thereof) described herein.
  • the method comprises genetically modifying an isolated host cell described herein with a polynucleotide described herein and/or a recombinant vector described herein.
  • the host cell that may be used in accordance with the above-described methods may be any of various host cells or populations thereof of the present disclosure.
  • the host cell may be an iPSC or a population thereof.
  • the host cell may be, for example, without limitation, a B cell, a T cell, a natural killer (NK) cell, a mesenchymal stem cell (MSC), or a macrophage.
  • the isolated host cell, or population thereof may be a B cell.
  • the B cell can be, for example, a transitional B cell, a naive B cell, a plasma B cell, or a memory B cell.
  • the B cell is a T-independent B cell.
  • the isolated host cell, or population thereof may be a T cell.
  • T-cells may include, for example, without limitation, thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes.
  • a T-cell can be a T helper (Th) cell, for example a T helper 1 (Thl) or a T helper 2 (Th2) cell.
  • the T-cell can be a helper T-cell (HTL; CD4+ T-cell) CD4+ T-cell, a cytotoxic T-cell (CTL; CD8+ T-cell), a tumor infiltrating cytotoxic T-cell (TIL; CD8+ T-cell), CD4+ CD8+ T-cell, or any other subset of T-cells.
  • HTL helper T-cell
  • CTL cytotoxic T-cell
  • TIL tumor infiltrating cytotoxic T-cell
  • CD4+ CD8+ T-cell CD4+ CD8+ T-cell, or any other subset of T-cells.
  • Other illustrative populations of T-cells suitable for use in particular embodiments include naive T-cells memory T-cells, and NKT cells.
  • the T-cell may be an a(3 T-cell receptor (TCR) T-cell, a y5 T-cell, a CD8+ T-cell, a CD4+ T-cell, a cytotoxic T-cell, an invariant natural killer T (iNKT) cell, a memory T-cell, a memory stem T-cell (TSCM), a naive T-cell, an effector T-cell, a T- helper cell, or a regulatory T-cell (Treg).
  • TCR T-cell receptor
  • the host cell, or population thereof may be an NK cell.
  • the NK cell may be derived from peripheral, cord blood, iPS cells, and/or a cell line.
  • the method may further comprise genetically modifying the host cell (e.g., immune cell or population thereof) to express a chimeric antigen receptor (CAR) described herein.
  • CAR chimeric antigen receptor
  • the disclosure provides a method of preventing or treating a disease or a condition in a subject in need thereof.
  • the methods comprise administering to the subject in need thereof a therapeutically effective amount of any of the cells, e.g., host cells or population thereof, of the disclosure, and/or a composition, e.g., a pharmaceutical composition, of the disclosure.
  • the composition may be, for example, a pharmaceutical composition comprising an isolated host cell disclosed herein.
  • the disease or condition is cancer.
  • the present disclosure provides a method for preventing or treating a cancer, the method comprising administering to an individual in need thereof, a pharmaceutically effective amount of the host cell, immune-effector cell, or the population thereof disclosed herein, and/or a pharmaceutical composition disclosed herein.
  • the cancer can, for example, be a solid or a liquid cancer.
  • the cancer can, for example, be selected from the group consisting of a lung cancer, a gastric cancer, a colon cancer, a hepatocellular carcinoma, a renal cell carcinoma, a bladder urothelial carcinoma, a metastatic melanoma, a breast cancer, an ovarian cancer, a cervical cancer, a head and neck cancer, a pancreatic cancer, an endometrial cancer, a prostate cancer, a thyroid cancer, a glioma, a glioblastoma, and other solid tumors, and a non-Hodgkin's lymphoma (NHL), Hodgkin’s lymphoma/disease (HD), an acute lymphocytic leukemia (ALL), a chronic lymphocytic leukemia (CLL), a chronic myelogenous leukemia (CML), a multiple myeloma (MM), an acute myeloid leukemia (AML), and other liquid tumors.
  • NHL non-Hodg
  • the cancer may be, for example, without limitation, a lung cancer, pancreatic cancer, liver cancer, melanoma, bone cancer, breast cancer, colon cancer, leukemia, uterine cancer, ovarian cancer, lymphoma, or brain cancer.
  • the cancer may be a lung cancer, pancreatic cancer, liver cancer, melanoma, bone cancer, breast cancer, colon cancer, leukemia, uterine cancer, ovarian cancer, lymphoma, brain cancer (e.g., glioblastoma), cervical cancer, head and neck cancer, liver cancer, prostate cancer, renal cell carcinoma, bladder cancer, other solid tumor cancer, or hematologic malignancy.
  • the cancer is selected from the group consisting of leukemias, such as AML, CML, ALL and CLL, lymphomas, such as Hodgkin lymphoma, non-Hodgkin lymphoma and multiple myeloma, and solid cancers such as sarcomas, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterus cancer, ovary cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, renal cancer, adrenal cancer, stomach cancer, testicular cancer, cancer of the gall bladder and biliary tracts, thyroid cancer, thymus cancer, cancer of bone, and cerebral cancer, as well as cancer of unknown primary (CUP).
  • leukemias such as AML, CML, ALL and CLL
  • lymphomas such as Hodgkin lymphoma, non-Hodgkin lymphoma and multiple myeloma
  • solid cancers such as
  • the cancer is selected from myeloma and lymphoma (e.g., Hodgkin's or non-Hodgkin's).
  • the lymphoma is Diffuse Large B-cell Lymphoma (DLBCL).
  • Primary cancer cells can be readily distinguished from non-cancerous cells by well- established techniques, particularly histological examination.
  • the definition of a ‘cancer cell”, as used herein, includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells.
  • a “clinically detectable” tumor is one that is detectable on the basis of tumor mass; e.g., by procedures such as computed tomography (CT) scan, magnetic resonance imaging (MRI), X- ray, ultrasound, or palpation on physical examination, and/or which is detectable because of the expression of one or more cancer-specific antigens in a sample obtainable from a patient.
  • CT computed tomography
  • MRI magnetic resonance imaging
  • X- ray X-ray
  • ultrasound X-ray
  • Cancer conditions may be characterized by the abnormal proliferation of malignant cancer cells and may include leukemias, such as AML, CML, ALL and CLL, lymphomas, such as Hodgkin lymphoma, non-Hodgkin lymphoma and multiple myeloma, and solid cancers such as sarcomas, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterus cancer, ovary cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, renal cancer, adrenal cancer, stomach cancer, testicular cancer, cancer of the gall bladder and biliary tracts, thyroid cancer, thymus cancer, cancer of bone, and cerebral cancer, as well as cancer of unknown primary (CUP).
  • leukemias such as AML, CML, ALL and CLL
  • lymphomas such as Hodgkin lymphoma, non-Hodgkin lymphoma and multiple myeloma
  • Cancer cells within an individual may be immunologically distinct from normal somatic cells in the individual (i.e. the cancerous tumor may be immunogenic).
  • the cancer cells may be capable of eliciting a systemic immune response in the individual against one or more antigens expressed by the cancer cells.
  • the tumor antigens that elicit the immune response may be specific to cancer cells or may be shared by one or more normal cells in the individual.
  • the cancer cells of an individual suitable for treatment as described herein may express the antigen and/or may be of correct HLA type to bind the antigen receptor expressed by the T cells.
  • An individual suitable for treatment as described above may be a mammal.
  • the individual is a human.
  • non-human mammals especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g. murine, primate, porcine, canine, or rabbit animals) may be employed.
  • the individual may have minimal residual disease (MRD) after an initial cancer treatment. In some embodiments, the individual may have no minimal residual disease after one or more cancer treatments or repeated dosing.
  • MRD minimal residual disease
  • An individual with cancer may display at least one identifiable sign, symptom, or laboratory finding that is sufficient to make a diagnosis of cancer in accordance with clinical standards know n in the art. Examples of such clinical standards can be found in textbooks of medicine such as Harrison’s Principles of Internal Medicine, 15 th Ed., Fauci AS et al., eds., McGraw-Hill, New York. 2001.
  • a diagnosis of a cancer in an individual may include identification of a particular cell type (e.g. a cancer cell) in a sample of a body fluid or tissue obtained from the individual.
  • An anti-tumor effect is a biological effect which can be manifested by a reduction in the rate of tumor growth, decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition.
  • An “‘anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies, also T cells which may be obtained according to the methods of the present invention, as described herein in prevention of the occurrence of tumors in the first place.
  • Treatment may be any treatment and/or therapy, whether of a human or an animal (e.g. in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition or delay of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, cure or remission (whether partial or total) of the condition, preventing, delaying, abating or arresting one or more symptoms and/or signs of the condition or prolonging survival of a subject or patient beyond that expected in the absence of treatment.
  • some desired therapeutic effect is achieved, for example, the inhibition or delay of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, cure or remission (whether partial or total) of the condition, preventing, delaying, abating or arresting one or more symptoms and/or signs of the condition or prolonging survival of a subject or patient beyond that expected in the absence of treatment.
  • Treatment may also be prophylactic (i.e. prophylaxis).
  • an individual susceptible to or at risk of the occurrence or re-occurrence of cancer may be treated as described herein. Such treatment may prevent or delay the occurrence or re-occurrence of cancer in the individual.
  • treatment may include inhibiting cancer growth, including complete cancer remission, and/or inhibiting cancer metastasis.
  • Cancer growth generally refers to any one of a number of indices that indicate change within the cancer to a more developed form.
  • indices for measuring an inhibition of cancer growth include a decrease in cancer cell survival, a decrease in tumor volume or morphology (for example, as determined using computed tomographic (CT), sonography, or other imaging method), a delayed tumor growth, a destruction of tumor vasculature, improved performance in delayed hypersensitivity skin test, an increase in the activity of T cells, and a decrease in levels of tumor-specific antigens.
  • Administration of T cells modified as described herein may improve the capacity of the individual to resist cancer growth, in particular growth of a cancer already present the subject and/or decrease the propensity for cancer growth in the individual.
  • the composition comprises a therapeutically effective amount of an isolated polynucleotide, an isolated polypeptide, and/or a host cell or population thereof.
  • therapeutically effective amount refers to an amount of an active ingredient or component that elicits the desired biological or medicinal response in a subject.
  • a therapeutically effective amount can be determined empirically and in a routine manner, in relation to the stated purpose.
  • a therapeutically effective amount means an amount of the cells and/or the pharmaceutical composition that modulates an immune response in a subject in need thereof.
  • a therapeutically effective amount refers to the amount of therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of the disease, disorder or condition to be treated or a symptom associated therewith; (ii) reduce the duration of the disease, disorder or condition to be treated, or a symptom associated therewith; (iii) prevent the progression of the disease, disorder or condition to be treated, or a symptom associated therewith; (iv) cause regression of the disease, disorder or condition to be treated, or a symptom associated therewith; (v) prevent the development or onset of the disease, disorder or condition to be treated, or a symptom associated therewith; (vi) prevent the recurrence of the disease, disorder or condition to be treated, or a symptom associated therewith; (vii) reduce hospitalization of a subject having the disease, disorder or condition to be treated, or a symptom associated therewith; (viii) reduce hospitalization length of a subject having
  • the therapeutically effective amount or dosage can vary according to various factors, such as the disease, disorder or condition to be treated, the means of administration, the target site, the physiological state of the subject (including, e.g., age, body weight, health), whether the subject is a human or an animal, other medications administered, and whether the treatment is prophylactic or therapeutic. Treatment dosages are optimally titrated to optimize safety and efficacy.
  • the compositions described herein are formulated to be suitable for the intended route of administration to a subject.
  • the compositions described herein can be formulated to be suitable for intravenous, subcutaneous, or intramuscular administration.
  • the cells of the disclosure and/or the pharmaceutical compositions of the disclosure can be administered in any convenient manner known to those skilled in the art.
  • the cells of the disclosure can be administered to the subject by aerosol inhalation, injection, ingestion, transfusion, implantation, and/or transplantation.
  • the compositions comprising the cells of the disclosure can be administered transarterially, subcutaneously, intradermally, intratumorally, intranodally. intramedullary, intramuscularly, intrapleurally, by intravenous (i.v.) injection, or intraperitoneally.
  • the cells of the disclosure can be administered with or without lymphodepletion of the subject.
  • compositions comprising cells of the disclosure can be provided in sterile liquid preparations, typically isotonic aqueous solutions with cell suspensions, or optionally as emulsions, dispersions, or the like, which are typically buffered to a selected pH.
  • the compositions can comprise carriers, for example, water, saline, phosphate buffered saline, and the like, suitable for the integrity and viability of the cells, and for administration of a cell composition.
  • Sterile injectable solutions can be prepared by incorporating cells of the disclosure in a suitable amount of the appropriate solvent with various other ingredients, as desired.
  • Such compositions can include a pharmaceutically acceptable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like, that are suitable for use with a cell composition and for administration to a subject, such as a human.
  • Suitable buffers for providing a cell composition are well known in the art. Any vehicle, diluent, or additive used is compatible with preserving the integrity and viability of the cells of the disclosure.
  • the cells of the disclosure and/or the pharmaceutical compositions of the disclosure can be administered in any physiologically acceptable vehicle.
  • a cell population comprising cells of the disclosure can comprise a purified population of cells.
  • the ranges in purity in cell populations comprising genetically modified cells of the disclosure can be from about 50% to about 55%, from about 55% to about 60%, from about 60% to about 65%, from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95%, or from about 95% to about 100%. Dosages can be readily adjusted by those skilled in the art, for example, a decrease in purity could require an increase in dosage.
  • a disease or disorder such as a cancer and/or an autoimmune disease by administering any of the cells described herein.
  • the teachings of the present disclosure may be relevant to any and all cancers.
  • the cancer treated by methods of the present disclosure is a solid tumor.
  • the cancer treated by methods of the present disclosure is a hematologic malignancy.
  • NK cells described herein that have been differentiated from iPSCs and engineered (e.g., modified) to express one or more of the IL7R exogenous polynucleotide constructs provided can be administered to a patient for treating cancer and/or an autoimmune disease.
  • T cells e.g., gamma-delta T cells
  • T cells described herein that have been differentiated from iPSCs and engineered to express one or more of the exogenous IL7R polynucleotide constructs provided can be administered to a patient for treating cancer and/or an autoimmune disease.
  • CD34+ cells described herein that have been differentiated from iPSCs and engineered to express one or more of the exogenous IL7R polynucleotide constructs provided can be administered to a patient for treating cancer and/or an autoimmune disease.
  • the cells may express one or more of additional exogenous polynucleotide constructs including, but not limited to, an exogenous polynucleotide construct encoding a CD 16 protein, an NKG2D protein and a self-cleavage peptide; an exogenous polynucleotide construct encoding a CAR; an exogenous polynucleotide construct encoding an HLA-E protein; an exogenous polynucleotide construct encoding an HLA-G protein; an exogenous polynucleotide construct encoding an HLA-E protein and an HLA-G protein; an exogenous polynucleotide construct encoding an HLA-E protein, an HLA-G protein, and a self-cleavage peptide; an exogenous polynucleotide construct encoding a fusion protein containing IL-15 and IL-15Ra; and an exogenous polynucleotide construct encoding an exogenous poly
  • such cells can be administered to treat a patient with cancer, such as any type of cancer.
  • the cancer treated by methods of the present disclosure include a glioblastoma, ovarian cancer, cervical cancer, head and neck cancer, liver cancer, prostate cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, other solid tumor cancer, or hematologic malignancy.
  • the hematologic malignancy is a leukemia (e.g., acute lymphocytic (ALL), chronic lymphocytic (CLL), acute myeloid (AML), chronic myeloid (CML)), myeloma, or lymphoma (e.g., Hodgkin's or non-Hodgkin's (NHL)).
  • ALL acute lymphocytic
  • CLL chronic lymphocytic
  • AML acute myeloid
  • CML chronic myeloid
  • myeloma e.g., Hodgkin's or non-Hodgkin's (NHL)
  • the disease is an autoimmune disease or disorder.
  • the autoimmune disease or disorder is rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome, systemic lupus erythematosus, sarcoidosis, type 1 diabetes mellitus.
  • IDDM insulin dependent diabetes mellitus
  • autoimmune thyroiditis reactive arthritis
  • ankylosing spondylitis scleroderma
  • polymyositis dermatomyositis
  • psoriasis vasculitis
  • Wegener's granulomatosis Myasthenia gravis
  • Hashimoto's thyroiditis Graves' disease, chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, Crohn's disease or ulcerative colitis.
  • the methods can be used to target multiple antigens (or multiple epitopes in the same antigen) in the same disease (e.g., tumor or autoimmune disease), or multiple antigens in different diseases (e.g., tumor or autoimmune disease).
  • the methods as described can be used to target multiple antigens (or multiple epitopes in the same antigen) in the same disease (e.g., tumor or autoimmune disease), or multiple antigens in different diseases (e.g.. tumor or autoimmune disease).
  • the cells of the disclosure are generally administered as a dose based on cells per kilogram (cells/kg) of body weight of the subject to which the cells and/or pharmaceutical compositions comprising the cells are administered.
  • the cell doses are in the range of about 10 4 to about IO 10 cells/kg of body weight, for example, about 10 5 to about IO 9 , about 10 5 to about 10 8 , about 10 5 to about 10 7 , or about I O' to about 10 6 , depending on the mode and location of administration.
  • a higher dose is used than in regional administration, where the immune cells of the disclosure are administered in the region of a tumor and/or cancer.
  • Exemplary dose ranges include, but are not limited to, 1 x 10 4 to 1 x 10 8 , 2 x 10 4 to 1 x 10 8 , 3 x 10 4 to 1 x 10 8 , 4 x 10 4 to 1 x 10 8 , 5 x 10 4 to 6 x 10 8 , 7 x 10 4 to 1 x 10 8 , 8 x 10 4 to 1 x 10 8 , 9 x 10 4 to 1 x 10 8 , 1 x 10 5 to 1 x 10 8 , 1 x 10 5 to 9 x 10 7 , 1 x 10 5 to 8 x 10 7 , 1 x 10 5 to 7 x 10 7 , 1 x 10 5 to 6 x 10 7 , 1 x 10 5 to 5 x 10 7 , 1 x 10 5 to 4 x 10 7 , 1 x 10 7 , 1 x
  • the dose can be adjusted to account for whether a single dose is being administered or whether multiple doses are being administered. The precise determination of what would be considered an effective dose can be based on factors particular to each subject. [00378] As used herein, the terms “treat,”’ “treating,’” and “treatment” are all intended to refer to an amelioration or reversal of at least one measurable physical parameter related to a cancer, which is not necessarily discernible in the subject, but can be discernible in the subject.
  • treat can also refer to causing regression, preventing the progression, or at least slowing dow n the progression of the disease, disorder, or condition.
  • “treat,” “treating,” and “treatment” refer to an alleviation, prevention of the development or onset, or reduction in the duration of one or more symptoms associated with the disease, disorder, or condition, such as a tumor or more preferably a cancer.
  • “treat,” “treating,” and “treatment” refer to prevention of the recurrence of the disease, disorder, or condition.
  • “treat,” “treating,” and “treatment” refer to an increase in the survival of a subject having the disease, disorder, or condition.
  • “treat,” “treating,” and “treatment” refer to elimination of the disease, disorder, or condition in the subject.
  • the cells of the disclosure and/or the pharmaceutical compositions of the disclosure can be administered in combination with one or more additional therapeutic agents.
  • the one or more therapeutic agents are selected from the group consisting of a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA), mononuclear blood cells, feeder cells, feeder cell components or replacement factors thereof, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (ImiD).
  • ImiD immunomodulatory drug
  • the present Example relates to design and testing of IL-7Ra mutants described herein.
  • the IL-7Ra mutants were designed to test the effects of modification of wild-type (wt) IL-7Ra on cell functionality' (see, e.g., Example 2, below).
  • a schematic representation of a full-length (459 aa) wild-type IL-7Ra (see, e g., SEQ ID NO: 1 ; UniProtKB P16871) comprising an ectodomain (21-239 aa), a transmembrane domain (240-264 aa) and a cytoplasmic domain (265-459 aa) used as a template for the IL-7Ra mutant designs is displayed in Fig.
  • the cytoplasmic domain comprises a Box 1/2 site which can sen e as a landing pad for Janus kinase (JAK), e.g., JAK1 and JAK3, and a YXXM motif comprising the sequence YVTM (SEQ ID NO: 21) at amino acid positions 449-452.
  • Y449 may be critical for both signal transducer and activator of transcription 5 (STAT5) and phosphoinositide 3-kinase (PI3K) binding
  • M452 may be critical for PI3K binding.
  • Fig. IB illustrates exemplary' IL-7Ra mutants designed in this Example, including: (1) CD28 YXXM (YVTM449-452YMNM IL-7Ra) (SEQ ID NO: 9): CD28 (cluster of differentiation 28)-denved YXXM motif, YMNM (see, e.g., SEQ ID NO: 17); (2) CD122 (IL2/15RP) YXXM (see, e.g., SEQ ID NO: 15): CD122 (IL2/15R0, interleukin 2/15 receptor beta)-derived YXXM motif, YXXL motif comprising the amino acid sequence of YLSL (SEQ ID NO: 18); (3) YXXM duplication (YXXM Dup) (see, e.g., SEQ ID NO: 11): additional wt YXXM motif, YVTM (SEQ ID NO: 21) inserted at amino acid position 459; (4) CD122
  • IL-7Ra mutants were cloned into the Xhol site of a lentiviral plasmid with an MND promoter and a P2A-GFP 3’ of the mutant. Ligation reactions were transformed into chemically competent 5-alpha cells from New England Biolabs (NEB). Finished plasmids w ere purified by lysing the bacteria; precipitating out high molecular weight genomic DNA; filtering the remaining plasmid DNA via column chromatography; and, selective elution of the plasmid DNA using a nuclease-free water according to manufacturer instruction.
  • Plasmids were complexed and transfected into HEK-Blue cells.
  • Secreted alkaline phosphatase (SEAP) expression w as quantified by reading optical density at 620-655 nm using a microplate reader.
  • Lentivirus was produced from lentiviral vector transfected into Lenti-XTM 293T cells from Takara (Cat. # 632180) cells using a lentivirus packaging protocol, as described below, and validated in murine interleukin-3 dependent pro-B cell line Ba/F3.
  • the present Example was designed to test the effects of the IL-7Ra mutants described herein on cell functionality, in particular, in the presence and absence of exogenous IL-7.
  • a SEAP reporter assay was performed based on procedures described herein (below), and in accordance with the manufacturer's recommended protocol. SEAP production was measured 24 hours post-transfection for IL-7Ra YXXM and Box site mutants tested in the presence and absence of exogenous IL-7 (Fig. 3).
  • Relative absorbance units were measured for IL-7Ra YXXM and Box site mutants (CD28 YXXM, CD 122 (IL2/15RP) YXXM, YXXM duplication, CD122 (IL2/15RP) Box 2, CD28 YXXM + Box 2, CD28 YXXM duplication + Box 2) and wild type (WT) IL-7Ra tested in the presence (10 ng/mL and 5 ng/mL IL-7) and in the absence of IL-7 (no IL-7 added).
  • Controls conditions were HEK cells, HEK cells + 100 ng/mL IL- 15 (positive [+ve] control), QUANTI-Blue (QB) solution BLANK, and Pmax-GFP (green fluorescent protein). Each condition was tested in triplicate. SEAP production was limited to 24 hrs. The QB step was a 26 pL supernatant + 234 pL QB solution to account for increased volume after IL-7 addition.
  • CD28 YXXM and YXXM Dup designs responded similar to WT IL-7Ra.
  • Two IL-7Ra CD122 Box 2 insertion mutants responded at levels similar to a constitutively signaling cytokine receptor (C7R) control in the absence of IL-7 (see encircled data points. Fig. 3), exhibiting cytokine-independent functionality above control (+ Ctrl cells alone) levels (dotted line, Fig. 3).
  • This Example relates to validation experiments performed in Ba/F3 cells, a murine interleukin-3 dependent pro-B cell line, to confirm the constitutive activity of IL-7Ra CD 122 Box 2 insertion mutants.
  • Ba/F3 cells were counted via Cellometer on post passage day D0-D3 for IL-7Ra mutants (CD28 YXXM + Box 2, CD28 YXXM duplication + Box 2, CD 122 Box 2, CD28 YXXM, YXXM duplication), constitutively signaling cytokine receptor (C7R), wild ty pe (WT) IL-7Ra, and no virus (control), in the presence of 10 mg/rnL recombinant human (rhu)-IL-7 (Fig.
  • IL-7Ra CD 122 Box 2 insertion mutants (CD28 YXXM + Box 2 and CD 122 Box 2) showed enhanced cell proliferation at levels similar to the C7R group in both the presence and absence of exogenous cytokine, thereby confirming constitutive activity of the IL-7Ra CD 122 Box 2 insertion mutants.
  • Example 4 Strong positive selection effect of the IL-7Rq constructs in the absence of cytokine supplementation.
  • Findings described in this Example support a strong positive selection effect of the IL-7Ra constructs in the absence of cytokine supplementation.
  • two conditions were set up for each construct (CD28 YXXM + Box 2; CD28 YXXM Dup + Box 2; CD122 Box 2; CD28 YXXM; YXXM Dup; C7R; WT; no virus control; On-GFP): (1) a 10 ng/mL IL-7 group to test the ability of the transduced IL-7R receptors to provide required STAT5 signaling; and (2) a no cytokine group (i.e., cells alone with no supplemental cytokines added) to assess cytokine-independent functionality.
  • Flow cytometric data corresponding to CD28 YXXM + Box 2, CD28 YXXM Dup + Box 2 and GFP (+ve) positive control conditions at D3 post transduction (p.t.) are shown in Figs. 5A-5C, respectively, and at D7 post re-plate (p. rp.) in the absence of exogenous cytokine, as shown in Figs. 5D-5F, respectively.
  • Fig. 5G depicts the percentage of positive GFP cells at D3 post transduction (p.t.) and D7 post re-plate (p. rp.) for the 10 ng/mL IL-7 group and the no cytokine group.
  • Mission Lentivirus Packaging mix Sigma Aldrich, Cat. # SHP0001-1.7 mL
  • the Lentivirus Packaging Mix Powered by MISSION® Genomics is an optimized mixture of two plasmids expressing the gag, pol, rev and VSV-G genes required for higher-titer lentivirus production.
  • ViralBoost Reagent Alstem Bio Cat. # VB100
  • Tubes were prepared and labeled. For each well, one tube of DNA was prepared and one tube of OPTIMEM in which to dilute the DNA was prepared.
  • the cell supernatant was harvested, and the supernatant filtered through a 0.45 pM Poly ethersulfone (PES) filter into a 15 mL conical tube. The steps below for concentration of lentivirus were performed, otherwise the supernatant was frozen and stored at -80°C for subsequent cell transduction.
  • PES Poly ethersulfone
  • a centrifuge was chilled to 4°C.
  • Lentivirus with LentiX concentrator was spun down for 45 minutes at 4°C at 1500 x g. While spinning, a tray of dry ice was prepared and cryotubes were labeled for lentivirus storage.
  • SEAP Reporter Assay The procedure for the SEAP Reporter Assay protocol was as follows:
  • Day 1 5e3 HEK-BlueTM IL-2 cells were plated in a 96-well flat bottom plate using phenol red free media containing heat inactivated FBS and incubate at 37°C overnight.
  • Day 2 HEK-BlueTM IL-2 cells were transfected with plasmid and incubated at 37°C.
  • Remaining cells were analyzed using flow cytometry to assess expression of plasmid introduced constructs using a green fluorescent protein (GFP) readout.
  • GFP green fluorescent protein
  • SEQ ID NO: 2 wild-type (wt) IL-7Ra nucleotide sequence
  • SEQ ID NO: 20 signal peptide, wild-type (wt) IL-7Ra, amino acid sequence MTILGTTFGMVFSLLQVVSG
  • SEQ ID NO: 22 human IL-7, amino acid sequence MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSN CLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILL NCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKIL MGTKEH
  • YXXL motif amino acid sequence, where “X” is any amino acid YXXL
  • SEQ ID NO: 26 Polynucleotide sequence encoding IL- 12 protein, nucleotide sequence

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Abstract

La présente divulgation concerne des récepteurs d'interleukine-7 (IL-7) génétiquement modifiés, et des fragments et des dérivés de ceux-ci, en particulier des polypeptides de chaîne alpha du récepteur IL-7 (IL-7Rα) comprenant une ou plusieurs mutations, par exemple, dans le domaine cytoplasmique d'IL-7Rα. Dans certains aspects, de telles mutations sont capables de favoriser une signalisation indépendante de la cytokine telle qu'à l'intérieur d'une cellule. L'invention concerne en outre des polynucléotides, des vecteurs, des cellules et des compositions pharmaceutiques associés. L'invention concerne également des procédés permettant d'améliorer l'expansion et/ou la persistance d'une population de cellules hôtes, de générer des cellules hôtes génétiquement modifiées, et de traiter des sujets à l'aide des cellules hôtes génétiquement modifiées et/ou des compositions pharmaceutiques.
PCT/US2023/079129 2022-11-09 2023-11-08 Récepteurs d'interleukine-7 génétiquement modifiés et leurs utilisations WO2024102838A1 (fr)

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