EP4536282A2 - Cellules de récepteur d'antigène chimérique dépendant du glycane améliorées - Google Patents

Cellules de récepteur d'antigène chimérique dépendant du glycane améliorées

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Publication number
EP4536282A2
EP4536282A2 EP23824455.2A EP23824455A EP4536282A2 EP 4536282 A2 EP4536282 A2 EP 4536282A2 EP 23824455 A EP23824455 A EP 23824455A EP 4536282 A2 EP4536282 A2 EP 4536282A2
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EP
European Patent Office
Prior art keywords
cell
car
antigen
modified cell
domain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP23824455.2A
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German (de)
English (en)
Inventor
Michael Demetriou
Raymond Wenhou Zhou
Yanni ZHU
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of California
University of California Berkeley
University of California San Diego UCSD
Original Assignee
University of California
University of California Berkeley
University of California San Diego UCSD
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Application filed by University of California, University of California Berkeley, University of California San Diego UCSD filed Critical University of California
Publication of EP4536282A2 publication Critical patent/EP4536282A2/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4256—Tumor associated carbohydrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the polypeptide N-acetylgalactosaminyltransferase is selected from the group consisting of ppGalNAc-Tl (GALNT1), ppGalNAc-T2 (GALNT2), ppGalNAc-T3 (GALNT3), ppGalNAc-T4 (GALNT4), ppGalNAc-T5 (GALNT5), ppGalNAc-T6 (GALNT6), ppGalNAc-T7 (GALNT7), ppGalNAc-T8 (GALNT8), ppGalNAc-T9 (GALNT9), ppGalNAc-TIO (GALNT10), ppGalNAc-T12 (GALNT12), ppGalNAc-T13 (GALNT13), ppGalNAc-T14 (GALNT14), ppGalNAc-T15 (GALNT1), ppGalNAc-T12 (GALNT
  • the CAR selectively targets a TACA selected from the group consisting of pi, 6 branching, pi,6GlcNAc-branched N-glycans, T antigen, Tn antigen, sialyl-T epitopes, Tn epitopes, sialyl-Tn epitopes, a2, 6 sialylation, Sialylation, sialyl-Lewisx/a, di-sialyl-Lewisx/a, sialyl 6-sulfo Lexisx, Globo H, GD2, GD3, GM3, and Fucosyl GM1.
  • the CAR selectively targets pi,6GlcNAc-branched N-glycans, GalNAc, Tn antigen, GalNAca-ser, GalNAc, or GalNAcpi.
  • the modified cell comprising a CAR of SEQ ID NO: 23-29 shows reduced tonic signalingsignaling when compared to a modified cell comprising a CAR of SEQ ID NO: 21 or 22.
  • TACA tumor-associated carbohydrate antigen
  • TACA tumor-associated carbohydrate antigen
  • an antigen binding domain comprising a deletion in the amino acid sequence set forth in SEQ ID NOs: 30-54; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 30-54; (b) a CD8 or CD28 hinge domain; (c) a CD8 or CD28 transmembrane domain; (d) a CD28 costimulatory and/or a 4- IBB costimulatory domain;
  • the expression construct further comprises a promoter.
  • the promoter is selected from an EF-la promoter, a T cell Receptor alpha (TRAC) promoter, interleukin 2 (IL-2) promoter, or cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a Moloney Murine Leukemia Virus (MoMuLV) promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, or a Rous sarcoma virus promoter.
  • the expression construct is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno- associated viral vector.
  • the expression construct is a lentiviral vector.
  • the expression construct is a self-inactivating lentiviral vector.
  • Another aspect of the present disclosure provides a method of treating a cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective composition comprising a modified cell comprising a chimeric antigen receptor that selectively binds a tumor-associated carbohydrate antigen (TACA), where the CAR comprises: (i) an antigen binding domain, and wherein the antigen binding domain comprises a deletion in the amino acid sequence set forth in SEQ ID NOs: 30-54; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 30- 54; (ii) a CD
  • FIG. IB shows a size exclusion chromatography (SEC) analysis of a GlyTRl L " PHAXCDS an j p rotejn standards, Sigma (Cat# MWGF1000-1KT). SEC analysis was conducted using a GE Superdex 200 Increase3.2/300 columns. Molecular weights were calculated from trendlines generated from retention times.
  • FIG. 2B shows chromatographs demonstrating that GlyTR2 CD30 l f3, C D 3 was predominantly made up of large multimers and that GlyTR2 slCD301(4)xCD3 w ith stiff-linkers was predominantly a monomer.
  • FIGs. 3C-E show that GlyTRl LPHA(2) or GlyTR2 slCD301(4) CAR T cells readily killed ovarian and breast cancer cells.
  • FIG. 3C shows flow cytometry analyses on day 3 and day 7 characterizing the cell size and surface expression of the GlyTR-CARs.
  • a modified cell comprising an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising: an amino acid sequence set forth in SEQ ID NOs: 23-29; or (ii) an amino acid sequence having 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%, at least 99% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 23-29.
  • CAR chimeric antigen receptor
  • Antigen or “Ag” is defined as a molecule that provokes an immune response. This immune response may involve other antibody production, or the activation of specific immunologically-competent cells, or both.
  • any macromolecule including virtually all proteins or peptides, can serve as an antigen.
  • antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein.
  • the term “Allogeneic” refers to a graft derived from a different animal of the same species.
  • the term “Antibody” refers to an immunoglobulin molecule, which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules.
  • the antibodies in the present disclosure may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies (scFv) and humanized antibodies.
  • antibody fragment refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.
  • a tumor antigen is a cell surface molecule that is overexpressed in a cancer cell in comparison to a normal cell, for instance, 1-fold over expression, 2-fold overexpression, 3 -fold overexpression or more in comparison to a normal cell.
  • a tumor antigen is a cell surface molecule that is inappropriately synthesized in the cancer cell, for instance, a molecule that contains deletions, additions, or mutations in comparison to the molecule expressed on a normal cell.
  • a tumor antigen will be expressed exclusively on the cell surface of a cancer cell, entirely or as a fragment (e.g., MHC/peptide), and not synthesized or expressed on the surface of a normal cell.
  • Co-stimulatory ligand includes a molecule on an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell, and the like) that specifically binds a cognate co-stimulatory molecule on a T cell, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like.
  • an antigen presenting cell e.g., an aAPC, dendritic cell, B cell, and the like
  • Co-stimulatory signal refers to a signal, which in combination with a primary signal, such as TCR/CD3 ligation, leads to T cell proliferation and/or upregulation or downregulation of key molecules.
  • a costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule.
  • a costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors.
  • a "Disease” refers to a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.
  • a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.
  • epitope As used herein, the term “Epitope” as used herein is defined as a small chemical molecule on an antigen that can elicit an immune response, inducing B and/or T cell responses.
  • An antigen can have one or more epitopes. Most antigens have many epitopes; i.e., they are multivalent. In general, an epitope is roughly about 10 amino acids and/or sugars in size. In certain exemplary embodiments, die epitope is about 4-18 amino acids, about 5-16 amino acids, about 6-14 amino acids, about 7-12 amino acids, or about 8-10 amino acids.
  • pression is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
  • the fusion protein further comprises a third domain comprising two polypeptide monomers, where each monomer comprises a hinge, a CH2 domain and a CH3 domain.
  • the third domain comprises in an amino to carboxyl order: hinge-CH2-CH3-linker-hinge-CH2-CH3.
  • the CH2 domain comprises an intra-domain cysteine disulfide bridge.
  • immune effector cell refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response.
  • immune effector cells include T cells (e.g., alpha/eta T cells and gamma/delta T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloic-derived phagocytes.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • a N-acetyl-galactosamine (GalNAc) sugar residue is attached to a serine or threonine of a glycoprotein (GalNAcal-O-Ser/Thr, Tn antigen) and is usually elongated by the T-synthase (core 1 P3 -galactosyltransferase) in the Golgi apparatus that attaches a galactose residue to Thomsen-Friedenreich (TF) antigen (Tn antigen).
  • T-synthase core 1 P3 -galactosyltransferase
  • Tumor -Associated Carbohydrate Antigens Defining Tumor Malignancy Basis for Development of Anti-Cancer Vaccines, in The Molecular Immunology of Complex Carbohydrates — 2. Advances in Experimental Medicine and Biology, vol 491. Springer, Boston, MA (Wu et al (eds)).
  • carbohydrate structures may be free standing and/or attached to proteins or lipids, known as glycoproteins and
  • the term ’’Overexpressed tumor antigen” or ’’’overexpression of the tumor antigen is intended to indicate an abnormal level of expression of the tumor antigen in a cell from a disease area like a solid tumor within a specific tissue or organ of the patient relative to the level of expression in a normal cell from that tissue or organ.
  • Patients having solid tumors or a hematological malignancy characterized by overexpression of the tumor antigen can be determined by standard assays known in the art.
  • parenteral administration of an immunogenic composition includes, e.g., subcutaneous (s.c), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.
  • nucleic acid As used herein, the term "Polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric "Nucleotides.” The monomelic nucleotides can be hydrolyzed into nucleosides.
  • polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCRTM, and the like, and by synthetic means.
  • recombinant means i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCRTM, and the like, and by synthetic means.
  • Specifically binds or “selectively binds,” as used herein with respect to an antibody, antigen-binding domain, a CAR, or a bi-specific fusion protein, is meant an antibody, antigen-binding domain, a CAR, or a bi-specific fusion protein which recognizes a specific antigen (e.g., a TACA), but does not substantially recognize or bind other molecules in a sample.
  • a specific antigen e.g., a TACA
  • an antibody, antigen-binding domain, a CAR, or a bi-specific fusion protein that specifically binds to an antigen (e.g., a TACA) from one species may also bind to that antigen from one or more species.
  • Specifically binds means an antibody or binding fragment thereof (e.g., scFv) which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample.
  • an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific.
  • an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.
  • Stimulatory molecule means a molecule on a T cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell.
  • Stimulatory ligand means a ligand that when present on an antigen presenting cell (e.g., an aAPC, a dendritic cell, a B-cell, and the like) can specifically bind with a cognate binding partner (referred to herein as a“ stimulatory molecule”) on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like.
  • an antigen presenting cell e.g., an aAPC, a dendritic cell, a B-cell, and the like
  • a cognate binding partner referred to herein as a“ stimulatory molecule”
  • TCRs may exist in alpha/beta and gamma/delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain is composed of two extracellular domains, a variable and constant domain.
  • the TCR may be modified on any cell comprising a TCR, including, for example, a helper T cell, a cytotoxic T cell, a memory T cell, regulatory T cell, natural killer T cell, and gamma delta T cell.
  • the terms “ameliorate T cell exhaustion,” “inhibit T cell exhaustion,” “reduce T cell exhaustion” and the like refer to a condition of restored functionality of T cells characterized by one or more of the following: decreased expression and/or level of one or more of PD-1, TIM-3, and LAG-3; increased memory cell formation and/or maintenance of memory markers (e.g., CD62L); prevention of apoptosis; increased antigen-induced cytokine (e.g., IL-2) production and/or secretion; enhanced cytotoxicity /killing capacity; increased recognition of tumor targets with low surface antigen; enhanced proliferation in response to antigen.
  • memory markers e.g., CD62L
  • antigen-induced cytokine e.g., IL-2
  • Xenogeneic refers to a graft derived from an animal of a different species.
  • TACA tumor-associated carbohydrate antigen
  • TACA tumor-associated carbohydrate antigen
  • an antigen binding domain comprising a deletion in the amino acid sequence set forth in SEQ ID NOs: 30-54; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 30-54; a CD8 a or CD28 hinge domain; a CD8 or CD28 transmembrane domain; a CD28 costimulatory and/or a 4- IBB costimulatory domain; and a CD3 zeta intracellular
  • TACA tumor-associated carbohydrate antigen
  • TACAs tumor-associated carbohydrate antigens
  • the antigen binding domain comprises multiple (e.g., more than one) TACA binding domains. In some embodiments, the antigen binding domain comprises one, two, three, four, five, six, seven, eight, nine, ten, or more TACA binding domains. In one embodiment, the antigen binding domain comprises one TACA binding domain. In one embodiment, the antigen binding domain comprises two TACA binding domains. In one embodiment, the antigen binding domain comprises three TACA binding domains. In one embodiment, the antigen binding domain comprises four TACA binding domains.
  • the TACA-binding domain is a peptide sequence derived from a lectin protein.
  • the lectin is selected from the group consisting of a mammalian lectin, human lectin, plant lectin, bacterial lectin, viral lectin, fungal lectin, and protozoan lectin.
  • the antigen binding domain comprises a TACA- binding domain derived from a lectin. In some embodiments, the antigen binding domain comprises at least two TACA binding domains from a lectin.
  • a subject CAR of the present disclosure comprises an antigen binding domain (e.g., a tumor-associated carbohydrate antigen (TACA), a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain.
  • a subject CAR of the present disclosure may optionally comprise a hinge domain.
  • a subject CAR of the present disclosure comprises an antigen binding domain (e.g., TACA binding domain), a hinge domain, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain.
  • each of the domains of a subject CAR is separated by a linker.
  • a subject CAR of the present disclosure is mutated to prevent CAR- induced T cell exhaustion caused by T cell exhaustion.
  • the antigen binding domain of the CAR is mutated to modulate the CAR signaling.
  • the antigen binding domain may be operably linked to another domain of the CAR, such as the transmembrane domain, the costimulatory signaling domain or the intracellular signaling domain, each described elsewhere herein, for expression in the cell.
  • the antigen binding domains described herein can be combined with any of the transmembrane domains, any of the costimulatory signaling domains, any of the intracellular signaling domains, or any of the other domains described herein that may be included in a CAR of the present disclosure.
  • linker and “spacer” are used interchangeably herein.
  • the linker is typically rich in glycine for flexibility, as well as serine or threonine for solubility. Multiple linker may be used to connect the more than one TACA binding domains.
  • the more than one TACA binding domains can be operably linked by a linker, such as a linker may be selected from the group consisting of a peptide linker, a non-peptide linker, a chemical unit, a hindered cross-linker, a non-hindered cross-linker.
  • the linker is a peptide linker.
  • the peptide linker can be a glycine-serine linker.
  • the peptide linker can be at least about 4, at least about 6, at least about 8, at least about 10, at least about 12, at least about 14, or at least about 15 amino acids in length.
  • the CAR of the present disclosure can be designed to comprise a transmembrane domain that connects the antigen binding domain of the CAR to the intracellular domain.
  • the transmembrane domain of the subject CAR is a region that is capable of spanning the plasma membrane of a cell (e.g., an immune cell or precursor thereof).
  • the transmembrane domain is for insertion into a cell membrane, e.g., a eukaryotic cell membrane.
  • the transmembrane domain is interposed between the antigen binding domain and the intracellular domain of a CAR
  • the transmembrane domain is naturally associated with one or more of the domains in the CAR.
  • the transmembrane domain can 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.
  • 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, e.g., a Type I transmembrane protein. Where the source is synthetic, the transmembrane domain may be any artificial sequence that facilitates insertion of the CAR into a cell membrane, e.g., an artificial hydrophobic sequence.
  • the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • transmembrane domains described herein can be combined with any of the antigen binding domains described herein, any of the costimulatory signaling domains described herein, any of the intracellular signaling domains described herein, or any of the other domains described herein that may be included in a subject CAR.
  • a subject CAR of the present disclosure also includes an intracellular domain.
  • the intracellular domain of the CAR is responsible for activation of at least one of the effector functions of the cell in which the CAR is expressed (e.g., immune cell).
  • the intracellular domain transduces the effector function signal and directs the cell (e.g., immune cell) to perform its specialized function, e.g., harming and/or destroying a target cell.
  • the intracellular domain or otherwise the cytoplasmic domain of the CAR is responsible for activation of the cell in which the CAR is expressed.
  • Examples of an intracellular domain for use in the invention include, but are not limited to, the cytoplasmic portion of a surface receptor, co-stimulatory molecule, and any molecule that acts in concert to initiate signal transduction in the T cell, as well as any derivative or variant of these elements and any synthetic sequence that has the same functional capability.
  • the intracellular domain comprises a costimulatory signaling domain and an intracellular signaling domain.
  • NKpSO B7-H6
  • DAP 12 see, e.g., Topfer et al., J. Immunol. (2015) 194(7): 3201-3212
  • NKG2D NKp44
  • NKp46 NKp46
  • DAP10 CD3z
  • a suitable intracellular signaling domain can be an IT AM motif-containing portion that is derived from a polypeptide that contains an GGAM motif.
  • a suitable intracellular signaling domain can be an IT AM motif-containing domain from any ITAM motif-containing protein.
  • a suitable intracellular signaling domain need not contain the entire sequence of the entire protein from which it is derived.
  • the intracellular signaling domain is derived from FCsRIG (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma-chain; fc-epsilon RI -gamma; fcR gamma; fceRl gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.).
  • FCsRIG also known as FCRG
  • Fc epsilon receptor I gamma chain Fc receptor gamma-chain
  • fcR gamma fceRl gamma
  • high affinity immunoglobulin epsilon receptor subunit gamma immunoglobulin E receptor, high affinity, gamma chain; etc.
  • the intracellular signaling domain is derived from T- cell surface glycoprotein CD3 delta chain (also known as CD36; CD3- DELTA; CD3 antigen, delta subunit; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T-cell receptor T3 delta chain; T-cell surface glycoprotein CD3 delta chain; etc.).
  • the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 epsilon chain (also known as CD3s, T-cell surface antigen T3/Leu-4 epsilon chain, T- cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3 epsilon, T3e, etc.).
  • the intracellular signaling domain is derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; Ig-alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein; etc.).
  • an intracellular signaling domain suitable for use in a subject CAR of the present disclosure includes a DAP10/CD28 type signaling chain.
  • an intracellular signaling domain suitable for use in a subject CAR of the present disclosure includes a ZAP70 polypeptide.
  • the costimulatory domain comprises a 4- IBB costimulatory domain or the amino acid sequence of SEQ ID NO: 58. In some embodiments, the costimulatory domain comprises a CD28 costimulatory domain or the amino acid sequence of SEQ ID NO: 88. In some embodiments, the costimulatory domain comprises a 4- IBB and a CD28 costimulatory domain.
  • Intracellular signaling domain comprises a 4- IBB costimulatory domain or the amino acid sequence of SEQ ID NO: 58. In some embodiments, the costimulatory domain comprises a CD28 costimulatory domain or the amino acid sequence of SEQ ID NO: 88. In some embodiments, the costimulatory domain comprises a 4- IBB and a CD28 costimulatory domain.
  • the intracellular domain comprises an intracellular signaling domain.
  • the isolated nucleic acid molecule encoding the chimeric antigen receptor (CAR) comprises an intracellular domain that may be from the intracellular signaling domain of a molecule selected from the group consisting of T cell receptor (TCR) zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-delta, CD3-epsilon, CD3-zeta, CD3, CD5, CD22, CD79a, CD79b, and CD66d.
  • TCR T cell receptor
  • the intracellular signaling domain comprises a CD3zeta signaling domain; or the amino acid sequence of SEQ ID NO: 59.
  • the intracellular domain comprises an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NO: 59.
  • the hinge region of the CAR is a hydrophilic region which is located between the antigen binding domain and the transmembrane domain. In some embodiments, this domain facilitates proper protein folding for the CAR.
  • the hinge region is an optional component for the CAR.
  • the chimeric antigen receptor (CAR) may further comprise a hinge domain.
  • the hinge domain is a protein selected from the group consisting of a CD8a, a CD28 hinge, an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, and an artificial spacer sequence.
  • the hinge domain is a CD8a hinge domain.
  • the hinge domain is a CD28 hinge domain.
  • the hinge domain comprises the amino acid sequence of SEQ ID NO: 77 or 86.
  • the hinge domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, SEQ ID NO: 68, 71-77, and 86.
  • the CAR of the present disclosure includes a hinge region that connects the antigen binding domain with the transmembrane domain, which, in turn, connects to the intracellular domain.
  • the hinge region is preferably capable of supporting the antigen binding domain to recognize and bind to the target antigen on the target cells (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135).
  • the hinge region is a flexible domain, thus allowing the antigen binding domain to have a structure to optimally recognize the specific structure and density of the target antigens on a cell such as tumor cell. The flexibility of the hinge region permits the hinge region to adopt many different conformations.
  • the hinge region is an immunoglobulin heavy chain hinge region.
  • the hinge region is a hinge region polypeptide derived from a receptor (e.g., a CD8 or CD28 -derived hinge region).
  • a CAR of the invention may be synthesized using 9-fluorenyl methoxycarbonyl (Fmoc) solid phase chemistry with direct incorporation of phosphothreonine as the N- fluorenylmethoxy-carbonyl-O-benzyl-L-phosphothreonine derivative.
  • Fmoc 9-fluorenyl methoxycarbonyl
  • N-terminal or C-terminal fusion proteins comprising a CAR of the present disclosure conjugated with other molecules may be prepared by fusing, through recombinant techniques, the N-terminal or C-terminal of the chimeric protein, and the sequence of a selected protein or selectable marker with a desired biological function.
  • the resultant fusion proteins contain the CAR of the disclosure fused to the selected protein or marker protein as described herein.
  • proteins, which may be used to prepare fusion proteins include immunoglobulins, glutathione-S- transferase (GST), hemagglutinin (HA), and truncated myc.
  • the fragments include peptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post-translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.
  • the CARs of the present disclosure can be post-translationally modified.
  • post-translational modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc.
  • Some modifications or processing events require introduction of additional biological machinery.
  • processing events such as signal peptide cleavage and core glycosylation, are examined by adding canine microsomal membranes or Xenopus egg extracts (U.S. Pat. No. 6,103,489) to a standard translation reaction.
  • the targeting domain can be a membrane spanning domain, a membrane binding domain, or a sequence directing the protein to associate with for example vesicles or with the nucleus.
  • the targeting domain can target a peptide to a particular cell type or tissue.
  • the targeting domain can be a cell surface ligand or an antibody against cell surface antigens of a target tissue (e.g., bone, regenerating bone, degenerating bone, cartilage).
  • a targeting domain may target the peptide of the invention to a cellular component.
  • One aspect of the present disclosure relates to an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain that selectively binds a tumor-associated carbohydrate antigen (TACA), a hinge domain, a transmembrane domain, a costimulatory signaling region, and an intracellular signaling domain.
  • CAR chimeric antigen receptor
  • One aspect of the present disclosure provides an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain that selectively binds a tumor-associated carbohydrate antigen (TACA), where the antigen binding domain comprises a TACA-binding domain derived from a lectin; and the antigen binding domain comprises one or more TACA binding domains; a transmembrane domain; a costimulatory signaling region; and an intracellular signaling domain.
  • CAR chimeric antigen receptor
  • TACA tumor-associated carbohydrate antigen
  • the antigen binding domain comprises a deletion in the TACA binding domain (TBD).
  • the deletion is in the N-terminal and/or C- terminal region of the TACA binding domain (TBD).
  • the deletion is at least about 2 amino acids, at least about 5 amino acids, at least about 10 amino acids, at least about 15 amino acids, at least about 16 amino acids, at least about 17 amino acids, at least about 18 amino acids, at least about 19 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, at least about 35 amino acids, at least about 36 amino acids, at least about 38 amino acids, at least about 40 amino acids, at least about 45 amino acids, or more.
  • the deletion is at least about 10 amino acids, at least about 18 amino acids, or at least about 36 amino acids. In one embodiment, the deletion is at least about 36 amino acids.
  • the antigen binding domain comprises a deletion that removes disulfide-bonded cysteine residues in the TACA binding domain (TBD).
  • the expression of a CAR comprising a deletion in the TACA binding domain (TBD) of the antigen-binding domain is similar to the expression of a CAR comprising the wild-type TBD.
  • the antigen binding domain comprises a deletion in the carbohydrate binding domain of the TACA binding domain comprising an amino acid sequence set forth in SEQ ID NOs: 30-54; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 30-54.
  • the antigen binding domain comprises the amino acid sequence set forth in SEQ ID NOs: 34-39; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 34-39.
  • the antigen binding domain may be operably linked to another domain of the CAR, such as the transmembrane domain, the costimulatory signaling domain or the intracellular signaling domain, each described elsewhere herein, for expression in the cell.
  • the antigen binding domains described herein can be combined with any of the transmembrane domains, any of the costimulatory signaling domains, any of the intracellular signaling domains, or any of the other domains described herein that may be included in a CAR of the present disclosure.
  • the isolated nucleic acid sequence encoding a chimeric antigen receptor of the present disclosure can be obtained using any of the many recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.
  • the isolated nucleic acid may comprise any type of nucleic acid, including, but not limited to DNA and RNA.
  • the nucleic acid molecules of the present disclosure can be modified to improve stability in serum or in growth medium for cell cultures. Modifications can be added to enhance stability, functionality, and/or specificity and to minimize immunostimulatory properties of the nucleic acid molecule of the disclosure.
  • the 3 '-residues may be stabilized against degradation, e.g., they may be selected such that they consist of purine nucleotides, particularly adenosine or guanosine nucleotides.
  • substitution of pyrimidine nucleotides by modified analogues e.g., substitution of uridine by 2'-deoxythymidine is tolerated and does not affect function of the molecule.
  • the nucleic acid molecule may contain at least one modified nucleotide analogue.
  • the ends may be stabilized by incorporating modified nucleotide analogues.
  • the 2' OH-group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or ON, wherein R is Ci-Ce alkyl, alkenyl or alkynyl and halo is F, CI, Br or I.
  • the nucleic acid molecule comprises at least one of the following chemical modifications: 2'-H, 2'-0-methyl, or 2'-OH modification of one or more nucleotides.
  • a nucleic acid molecule of the disclosure can have enhanced resistance to nucleases.
  • a nucleic acid molecule can include, for example, 2'-modified ribose units and/or phosphorothioate linkages.
  • the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents.
  • the nucleic acid molecules of the disclosure can include 2'-0-methyl, 2'-fluorine, 2'-0- methoxyethyl, 2'-0-aminopropyl, 2'- amino, and/or phosphorothioate linkages.
  • LNA locked nucleic acids
  • ENA ethylene nucleic acids
  • certain nucleobase modifications such as 2-amino-A, 2-thio (e.g., 2-thio-U), G-clamp modifications, can also increase binding affinity to a target.
  • the nucleic acid molecule of the disclosure preferably has one or more of the following properties: Nucleic acid agents discussed herein include otherwise unmodified RNA and DNA as well as RNA and DNA that have been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates.
  • Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably as occur naturally in the human body.
  • the art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al., Nucleic Acids Res., 1994, 22:2183-2196.
  • Such rare or unusual RNAs, often termed modified RNAs are typically the result of a post-transcriptional modification and are within the term unmodified RNA as used herein.
  • Modified RNA refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature, preferably different from that which occurs in the human body. While they are referred to as “modified RNAs" they will of course, because of the modification, include molecules that are not, strictly speaking, RNAs. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to be presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone. Modifications of the nucleic acid of the disclosure may be present at one or more of, a phosphate group, a sugar group, backbone, N-terminus, C-terminus, or nucleobase.
  • the vector may be provided to a cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals.
  • Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses.
  • the expression construct is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno- associated viral vector.
  • the expression construct is a lentiviral vector.
  • the expression construct is a self-inactivating lentiviral vector.
  • the expression construct comprises an isolated nucleic acid encoding a CAR described herein.
  • retroviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retroviral systems are known in the art.
  • adenovirus vectors are used.
  • a number of adenovirus vectors are known in the art.
  • lentivirus vectors are used.
  • vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells.
  • Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
  • the composition includes a vector derived from an adeno-associated virus (AAV).
  • Adeno- associated viral (AAV) vectors have become powerful gene delivery tools for the treatment of various disorders.
  • AAV vectors possess a number of features that render them ideally suited for gene therapy, including a lack of pathogenicity, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner. Expression of a particular gene contained within an AAV vector can be specifically targeted to one or more types of cells by choosing the appropriate combination of AAV serotype, promoter, and delivery method.
  • the vector also includes conventional control elements which are operably linked to the transgene in a manner which permits its transcription, translation and/or expression in a cell transfected with the plasmid vector or infected with the virus produced by the invention.
  • "Operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
  • Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • efficient RNA processing signals such as splicing and polyadenylation (poly A) signals
  • sequences that stabilize cytoplasmic mRNA sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • a great number of expression control sequences, including promoters, which are native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized.
  • Additional promoter elements e.g., enhancers, regulate the frequency of transcriptional initiation.
  • these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • tk thymidine kinase
  • the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • individual elements can function either cooperatively or independently to activate transcription.
  • constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, Moloney Murine Leukemia Virus (MoMuLV) promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters.
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HSV human immunodeficiency virus
  • LTR long terminal repeat
  • MoMuLV Moloney Murine Leukemia Virus
  • avian leukemia virus promoter an
  • inducible promoters are also contemplated as part of the invention.
  • the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired or turning off the expression when expression is not desired.
  • inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • Enhancer sequences found on a vector also regulates expression of the gene contained therein.
  • enhancers are bound with protein factors to enhance the transcription of a gene.
  • Enhancers may be located upstream or downstream of the gene it regulates. Enhancers may also be tissue-specific to enhance transcription in a specific cell or tissue type.
  • the vector of the present invention comprises one or more enhancers to boost transcription of the gene present within the vector.
  • Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).
  • Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
  • the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter.
  • Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven.
  • the terms “ameliorate T cell exhaustion,” “inhibit T cell exhaustion,” “reduce T cell exhaustion” and the like refer to a condition of restored functionality of T cells characterized by one or more of the following: decreased expression and/or level of one or more of PD-1, TIM-3, and LAG-3; increased memory cell formation and/or maintenance of memory markers (e.g., CD62L); prevention of apoptosis; increased antigen-induced cytokine (e.g., IL-2) production and/or secretion; enhanced cytotoxicity /killing capacity; increased recognition of tumor targets with low surface antigen; enhanced proliferation in response to antigen.
  • memory markers e.g., CD62L
  • antigen-induced cytokine e.g., IL-2
  • Modified cells that express CARs undergo tonic, antigen-independent signaling due to receptor clustering and replicate the fundamental biology of T cell exhaustion, as shown by high levels of PD-1, TIM-3, and LAG-3 expression, diminished antigen-induced cytokine production and excessive programmed cell death.
  • tonic signaling is highly dependent upon CAR receptor levels in CAR T cells
  • control of CAR expression levels can be used to regulate the level of tonic signaling (e.g., in vitro or in vivo). Since tonic signaling is highly dependent upon CAR receptor levels, precise control of CAR expression levels also precisely regulated levels of tonic signaling.
  • SUBSTITUTE SHEET (RULE 26) functionality including, but not limited to, cytotoxic activities against tumor cells; promotion of CAR T cell survival and function; induction of cytokine expression such as expression of interleukin-2 (IL-2) to promote T cell survival, expression of Fas Ligand (FasL) and/or tumor necrosis factor-related apoptosis inducing ligand (TRAIL) to induce tumor cell apoptosis, and/or to induce interferon (IFN)-gamma to activate innate immune responses (e.g., against cancer); and/or potentiate the induction of cell cycle arrest and/or apoptosis.
  • CAR T cells of the present disclosure sensitize cancer cells to induction of cell cycle arrest and/or apoptosis, including cells that are normally resistant to such inducing stimuli.
  • the modified cell comprises an isolated nucleic acid encoding a chimeric antigen receptor (CAR) comprising: an antigen-binding domain that selectively binds a tumor- associated carbohydrate antigen (TACA), a hinge domain, a transmembrane domain, a costimulatory signaling region, and an intracellular signaling domain.
  • CAR chimeric antigen receptor
  • TACA tumor-associated carbohydrate antigen
  • the modified cell comprises the chimeric antigen receptor that selectively binds a tumor- associated carbohydrate antigen (TACA).
  • the antigen binding domain comprises a mutation in the TACA binding domain (TBD) selected from a substitution, a deletion, or an insertion.
  • the antigen binding domain comprises a deletion in the TACA binding domain (TBD).
  • the deletion is in the N-terminal and/or C-terminal region of the TACA binding domain (TBD).
  • the deletion is at least about 2 amino acids, at least about 5 amino acids, at least about 10 amino acids, at least about 15 amino acids, at least about 16 amino acids, at least about 17 amino acids, at least about 18 amino acids, at least about 19 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, at least about 35 amino acids, at least about 36 amino acids, at least about 38 amino acids, at least about 40 amino acids, at least about 45 amino acids, or more. In some embodiments, the deletion is at least about 10 amino acids, at least about 18 amino acids, or at least about 36 amino acids. In some embodiments, the deletion is at least about 36 amino acids.
  • the antigen binding domain comprises a deletion that removes disulfide-bonded cysteine residues in the TACA binding domain (TBD).
  • the expression of a CAR comprising a deletion in the TACA binding domain (TBD) of the antigen-binding domain is similar to the expression of a CAR comprising the wild-type TBD.
  • the modified cell expressing a CAR with a deletion in the TACA binding domain (TBD) of the antigen-binding domain exhibits reduced tonic signaling when compared to a modified cell comprising a CAR comprising the wild-type TBD.
  • the modified cell expressing a CAR with a deletion in the TACA binding domain (TBD) of the antigen-binding domain is less susceptible to experience exhaustion when compared to a modified cell comprising a CAR comprising the wild-type TBD. In some embodiments, the modified cell expressing a CAR with a deletion in the TACA binding domain (TBD) of the antigen-binding domain is less susceptible to experience exhaustion induced by a tonically signaling TACA CAR when compared to a modified cell comprising a CAR comprising the wild-type TBD.
  • the antigen binding domain comprises one, two, three, four, five, six, seven, eight, nine, ten, or more TACA binding domains.
  • the TACA-binding domain is derived from a lectin.
  • the lectin is selected from a galectin, a siglec, a selectin; a C-type lectin; CD301, a polypeptide N- acetylgalactosaminyltransferase (ppGalNAc-T), L-PHA (Phaseolus vulgaris leukoagglutinin); E-PHA (Phaseolus vulgaris erythroagglutinen); tomato lectin (Lycopersicon esculentum lectin; LEA); peanut lectin (Arachis hypogaea Agglutinin; PNA); potato lectin (Solanum tuberosum lectin), pokeweed mitogen (Phytolacca American lectin), wheat germ agglutinin (Triticum Vulgaris lectin); Artocarpus polyphemus lectin (Jacalin letin); Vicia villosa Agglutinin (VVA); Helix pomatia Agglutinin (HP
  • the antigen binding domain selectively targets a TACA selected from the group consisting of pi, 6 branching, pi,6GlcNAc-branched N-glycans, T antigen, Tn antigen, sialyl-T epitopes, Thomsen-nouveau (Tn) epitopes (Tn antigen), sialyl- Tn epitopes (sialyl-Tn antigen), a2, 6 sialylation, Sialylation, sialyl-Lewisx/a, di-sialyl- Lewisx/a, sialyl 6-sulfo Lexisx, Globo H, GD2, GD3, GM3, and Fucosyl GM1.
  • TACA selected from the group consisting of pi, 6 branching, pi,6GlcNAc-branched N-glycans, T antigen, Tn antigen, sialyl-T epitopes, Thomsen-nouveau (Tn) epitopes (T
  • the antigen binding domain comprises a deletion in the TACA binding domain of the antigen binding domain comprising an amino acid sequence set forth in SEQ ID NOs: 30-54; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least
  • the antigen binding domain comprises the amino acid sequence set forth in SEQ ID NOs: 34-39; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 34-39.
  • the antigen binding comprises an amino acid sequence having at least 90% homology to SEQ ID NO: 34-39.
  • the transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 78 or SEQ ID NO: 87. In some embodiments, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In certain exemplary embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • the intracellular domain comprises a costimulatory signaling domain.
  • the chimeric antigen receptor (CAR) comprises a costimulatory domain that is a costimulatory domain of a molecule selected from the group consisting of CD27, CD28, 4-IBB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, CD8, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, DAP 10, DAP 12, Lek, Fas, and a combination thereof.
  • the intracellular domain comprises an intracellular signaling domain.
  • the isolated nucleic acid molecule encoding the chimeric antigen receptor (CAR) comprises an intracellular domain that may be from the intracellular signaling domain of a molecule selected from the group consisting of T cell receptor (TCR) zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-delta, CD3-epsilon, CD3-zeta, CD3, CD5, CD22, CD79a, CD79b, and CD66d.
  • TCR T cell receptor
  • the intracellular signaling domain comprises a CD3zeta signaling domain; or the amino acid sequence of SEQ ID NO: 59.
  • the intracellular domain comprises an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NO: 59.
  • the CAR selectively targets a TACA selected from the group consisting of pi, 6 branching, pi,6GlcNAc-branched N-glycans, T antigen, Tn antigen, sialyl-T epitopes, Tn epitopes, sialyl-Tn epitopes, a2, 6 sialylation, Sialylation, sialyl— Lewisx/a, di -si alyl -Lewi sx/a, sialyl 6-sulfo Lexisx, Globo H, GD2, GD3, GM3, and Fucosyl GM1.
  • TACA selected from the group consisting of pi, 6 branching, pi,6GlcNAc-branched N-glycans, T antigen, Tn antigen, sialyl-T epitopes, Tn epitopes, sialyl-Tn epitopes, a2, 6 sialylation, Sialylation, sialyl— Lewisx/
  • Modified cells may be produced by stably transfecting host cells with an expression vector including a nucleic acid of the present disclosure. Additional methods to generate a modified cell of the present disclosure include, without limitation, chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes and/or cationic polymers), non-chemical transformation methods (e.g., electroporation, optical transformation, gene electrotransfer and/or hydrodynamic delivery) and/or particle-based methods (e.g., impalefection, using a gene gun and/or magnetofection). Transfected cells expressing a subject CAR of the present disclosure may be expanded ex vivo.
  • chemical transformation methods e.g., using calcium phosphate, dendrimers, liposomes and/or cationic polymers
  • non-chemical transformation methods e.g., electroporation, optical transformation, gene electrotransfer and/or hydrodynamic delivery
  • particle-based methods e.g., impalefection, using a gene gun and/or
  • the cell is genetically modified by contacting the cell with an isolated nucleic acid encoding the TACA CAR as described herein.
  • the nucleic acid sequence is delivered into cells using a retroviral or lentiviral vector.
  • retroviral and lentiviral vectors expressing a peptide of the invention can be delivered into different types of eukaryotic cells as well as into tissues and whole organisms using transduced cells as carriers or cell- free local or systemic delivery of encapsulated, bound or naked vectors.
  • the method used can be for any purpose where stable expression is required or sufficient.
  • Transfected cells expressing a subject CAR or bi-specific fusion protein of the present disclosure may be expanded ex vivo.
  • the cell is genetically modified by contacting the cell with an isolated nucleic acid encoding the CAR or the bi-specific fusion protein as described herein.
  • the nucleic acid sequence is delivered into cells using a retroviral or lentiviral vector.
  • retroviral and lentiviral vectors expressing a peptide of the invention can be delivered into different types of eukaryotic cells as well as into tissues and whole organisms using transduced cells as carriers or cell- free local or systemic delivery of encapsulated, bound or naked vectors. The method used can be for any purpose where stable expression is required or sufficient.
  • the cell may be of any suitable cell type that can express the desired peptide.
  • the modified cell is used in a method where the cell is introduced into a recipient.
  • the cell is autologous, allogeneic, syngeneic or xenogeneic with respect to recipient.
  • compositions and methods can be applied to the modulation of T cell activity in basic research and therapy, in the fields of cancer, stem cells, acute and chronic infections, and autoimmune diseases, including the assessment of the ability of the genetically modified T cell to kill a target cancer cell.
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
  • Viral vectors, and especially retroviral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.
  • Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.
  • Adenovirus expression vectors contain adenovirus sequences sufficient to: (a) support packaging of the expression vector and (b) to ultimately express the subject CAR in the host cell.
  • the adenovirus genome is a 36 kb, linear, double stranded DNA, where a foreign DNA sequence (e.g., a nucleic acid encoding the TACA- CAR or bi-specific fusion protein) may be inserted to substitute large pieces of adenoviral DNA in order to make the expression vector of the present invention. See, e.g., Danthinne and Imperiale, Gene Therapy 7(20): 1707-1714(2000).
  • Lentivirus vectors are derived from lentiviruses, which are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. See, e.g., U.S. Patent Nos. 6,013,516 and 5,994, 136.
  • Some examples of lentiviruses include the human immunodeficiency viruses (HTV-1, HTV-2) and the simian immunodeficiency virus (SIV).
  • Lentivirus vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe.
  • Expression vectors including a nucleic acid of the present disclosure can be introduced into a host cell by any means known to persons skilled in the art.
  • the expression vectors may include viral sequences for transfection, if desired.
  • the expression vectors may be introduced by fusion, electroporation, biolistics, transfection, lipofection, or the like.
  • the host cell may be grown and expanded in culture before introduction of the expression vectors, followed by the appropriate treatment for introduction and integration of the vectors. The host cells are then expanded and may be screened by virtue of a marker present in the vectors.
  • Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
  • the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution.
  • Lipids suitable for use can be obtained from commercial sources.
  • DMPC dimyristyl phosphatidylcholine
  • DCP dicetyl phosphate
  • Choi cholesterol
  • DMPG dimyristyl phosphatidylglycerol
  • Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20°C.
  • the isolated nucleic acid encoding the CAR or bi-specific fusion protein of the disclosure and introduced into a cell of the present disclosure comprises an RNA.
  • the RNA is mRNA.
  • the RNA is an in vitro transcribed (IVT) RNA.
  • IVT in vitro transcribed
  • the RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template.
  • DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase.
  • the source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA.
  • the DNA to be used for PCR contains an open reading frame.
  • the DNA can be from a naturally occurring DNA sequence from the genome of an organism.
  • the DNA is a full length gene of interest of a portion of a gene.
  • the gene can include some or all of the 5' and/or 3' untranslated regions (UTRs).
  • the gene can include exons and introns.
  • the DNA to be used for PCR is a human gene.
  • the DNA to be used for PCR is a human gene including the 5' and 3' UTRs.
  • the DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism.
  • An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism.
  • Any DNA polymerase useful for PCR can be used in the methods disclosed herein.
  • the reagents and polymerase are commercially available from a number of sources. Chemical structures with the ability to promote stability and/or translation efficiency may also be used.
  • the RNA preferably has 5' and 3' UTRs. In one embodiment, the 5' UTR is between zero and 3000 nucleotides in length. The length of 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs.
  • the 5' and 3' UTRs can be the naturally occurring, endogenous 5' and 3' UTRs for the gene of interest.
  • UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template.
  • the use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3' UTR sequences can decrease the stability of mRNA. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.
  • the 5' UTR can contain the Kozak sequence of the endogenous gene.
  • a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence.
  • Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art.
  • the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells.
  • various nucleotide analogues can be used in the 3' or 5' UTR to impede exonuclease degradation of the mRNA.
  • the polyA/T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100T tail (size can be 50-5000 T), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination.
  • Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines.
  • Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E- PAP).
  • E- PAP E. coli polyA polymerase
  • increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA.
  • the attachment of different chemical groups to the 3' end can increase mRNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds.
  • ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.
  • RNAs produced by the methods disclosed herein include a 5' cap.
  • the 5' cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7: 1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun, 330:958-966 (2005)).
  • the RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence.
  • IRS internal ribosome entry site
  • RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as "gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).
  • the RNA is electroporated into the cells, such as in vitro transcribed RNA.
  • the formulations and methodology of electroporation of nucleic acid constructs into mammalian cells as taught in e.g., US 2004/0014645, US 2005/0052630A1, US 2005/0070841 Al, US 2004/0059285 Al, US 2004/0092907A1.
  • the various parameters including electric field strength required for electroporation of any known cell type are generally known in the relevant research literature as well as numerous patents and applications in the field. See e.g., U.S. Pat. No. 6,678,556, U.S. Pat. No. 7,171,264, and U.S. Pat. No. 7, 173,116.
  • Apparatus for therapeutic application of electroporation are available commercially, e.g., the MedPulserTM DNA Electroporation Therapy System (Inovio/Genetronics, San Diego, Calif), and are described in patents such as U.S. Pat. No. 6,567,694; U.S. Pat. No. 6,516,223, U.S. Pat. No. 5,993,434, U.S. Pat. No. 6, 181,964, U.S. Pat. No. 6,241,701, and U.S. Pat. No. 6,233,482; electroporation may also be used for transfection of cells in vitro as described e.g., in US20070128708A1. Electroporation may also be utilized to deliver nucleic acids into cells in vitro.
  • electroporation-mediated administration into cells of nucleic acids including expression constructs utilizing any of the many available devices and electroporation systems known to those of skill in the art presents an exciting new means for delivering an RNA of interest to a target cell.
  • the disclosed methods can be applied to the modulation of host cell activity in basic research and therapy, in the fields of cancer, stem cells, acute and chronic infections, and autoimmune diseases, including the assessment of the ability of the genetically modified host cell to kill a target cancer cell.
  • the methods also provide the ability to control the level of expression over a wide range by changing, for example, the promoter or the amount of input RNA, making it possible to individually regulate the expression level.
  • the PCR- based technique of mRNA production greatly facilitates the design of the mRNAs with different structures and combination of their domains.
  • the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.
  • the target cell is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, e.g., an iPS cell generated from a subject, manipulated to alter (e.g., induce a mutation in) or manipulate the expression of one or more target genes, and differentiated into, e.g., a T cell, e.g., a CD8 + T cell (e.g., a CD8 + naive T cell, central memory T cell, or effector memory T cell), a CD4 + T cell, a stem cell memory T cell, a lymphoid progenitor cell or a hematopoietic stem cell.
  • iPS induced pluripotent stem
  • the methods include isolating immune cells from the subject, preparing, processing, culturing, and/or engineering them
  • preparation of the engineered cells includes one or more culture and/or preparation steps.
  • the cells for engineering as described may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject.
  • the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered.
  • the subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
  • the cells in some embodiments are primary cells, e.g., primary human cells.
  • the samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with viral vector), washing, and/or incubation.
  • the biological sample can be a sample obtained directly from a biological source or a sample that is processed.
  • Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
  • the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product.
  • exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom.
  • Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
  • the cells are derived from cell lines, e.g., T cell lines.
  • the cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, and pig.
  • isolation of the cells includes one or more preparations and/or non-affinity based cell separation steps.
  • cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents.
  • cells are separated based on one or more properties, such as density, adherent properties, size, sensitivity and/or resistance to particular components.
  • a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated“flow- through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions.
  • the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca 2+ - free, Mg 2+ -free PBS, PlasmaLyte A, or another saline solution with or without buffer.
  • the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
  • one or more of tire T cell populations is enriched for or depleted of cells that are positive for (markeri-) or express high levels (markerhigh) of one or more particular markers, such as surface markers, or that are negative for (marker-) or express relatively low levels (markerlow) of one or more markers.
  • specific subpopulations of T cells such as cells positive or expressing high levels of one or more surface markers, e.g., CD28 + , CD62L + , CCR7 + , CD27 + , CD127 + , CD4 + , CD8 + , CD45RA + , and/or CD45RO + T cells, are isolated by positive or negative selection techniques.
  • cells are enriched for or depleted of cells positive or expressing high surface levels of CD122, CD95, CD25, CD27, and/or IL7-Ra (CD 127).
  • CD8 + T cells are enriched for cells positive for CD45RO (or negative for CD45RA) and for CD62L.
  • CD3 + , CD28 + T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).
  • T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14.
  • a CD4 + or CD8 + selection step is used to separate CD4 + helper and CD8 + cytotoxic T cells.
  • Such CD4 + and CD8 + populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.
  • combining TCM-enriched CD8 + T cells and CD4 + T cells further enhances efficacy.
  • memory T cells are present in both CD62L + and CD62L- subsets of CD8 + peripheral blood lymphocytes.
  • PBMC can be enriched for or depleted of CD62L-CD8 + and/or CD62L + CD8 + fractions, such as using anti-CD8 and anti- CD62L antibodies.
  • a CD4 + T cell population and/or a CD8 + T population is enriched for central memory (TCM) cells.
  • the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD8, and/or CD127.
  • the enrichment is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B.
  • isolation of a CD8 + population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD 14, CD45RA, and positive selection or enrichment for cells expressing CD62L.
  • enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD14 and CD45RA, and a positive selection based on CD62L.
  • Such selections in certain aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order.
  • the same CD4 expression- based selection step used in preparing the CD8 + cell population or subpopulation also is used to generate the CD4 + cell population or subpopulation, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.
  • CD4 + T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens.
  • CD4 + lymphocytes can be obtained by standard methods.
  • naive CD4 + T lymphocytes are CD45RO", CD45RA + , CD62L + , CD4 + T cells.
  • central memory CD4 + cells are CD62L + and CD45RO + .
  • effector CD4+ cells are CD62L- and CD45RO.
  • a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8.
  • the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection.
  • the cells are incubated and/or cultured prior to or in connection with genetic engineering.
  • the incubation steps can include culture, cultivation, stimulation, activation, and/or propagation.
  • the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.
  • the conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
  • the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex.
  • T cells are isolated from peripheral blood by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient.
  • T cells can be isolated from an umbilical cord.
  • a specific subpopulation of T cells can be further isolated by positive or negative selection techniques.
  • the cord blood mononuclear cells so isolated can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD8, CD14, CD19, and CD56. Depletion of these cells can be accomplished using an isolated antibody, a biological sample comprising an antibody, such as ascites, an antibody bound to a physical support, and a cell bound antibody.
  • T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient or by counterflow centrifugal elutriation.
  • a specific subpopulation of T cells such as CD3 + , CD28 + , CD4 + , CD8 + , CD45RA + , and CD45RO + T cells, can be further isolated by positive or negative selection techniques.
  • T cells are isolated by incubation with anti-CD3/anti-CD28 (i.e., 3x28)- conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells.
  • the time period is about 30 minutes.
  • the time period ranges from 30 minutes to 36 hours or longer and all integer values there between.
  • the time period is at least 1, 2, 3, 4, 5, or 6 hours.
  • the time period is 10 to 24 hours.
  • the incubation time period is 24 hours.
  • use of longer incubation times, such as 24 hours can increase cell yield.
  • TIL tumor infiltrating lymphocytes
  • subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points.
  • the skilled artisan would recognize that multiple rounds of selection can also be used in the context of this invention.
  • a T cell population by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • An exemplary method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
  • a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8.
  • it may be desirable to enrich for or positively select for regulatory T cells which typically express CD4 + , CD25 + , CD62L M , GITR + , and FoxP3 + .
  • cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present invention.
  • a blood sample or an apheresis product is taken from a generally healthy subject.
  • Expanding the immune cells by the methods disclosed herein can be multiplied by about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700 fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or greater, and any and all whole or partial integers therebetween.
  • the immune cells expand in the range of about 20-fold to about 50-fold.
  • the immune cells can be incubated in cell medium in a culture apparatus for a period of time or until the cells reach confluency or high cell density for optimal passage before passing the cells to another culture apparatus.
  • the culturing apparatus can be of any culture apparatus commonly used for culturing cells in vitro.
  • the level of confluence is 70% or greater before passing the cells to another culture apparatus.
  • the level of confluence is 90% or greater.
  • a period of time can be any time suitable for the culture of cells in vitro.
  • the immune cell medium may be replaced during the culture of the immune cells at any time. In certain exemplary embodiments, the immune cell medium is replaced about every 2 to 3 days.
  • the immune cells are then harvested from the culture apparatus whereupon the immune cells can be used immediately or cryopreserved to be stored for use at a later time.
  • the invention includes cryopreserving the expanded immune cells.
  • the cryopreserved immune cells are thawed prior to introducing nucleic adds into the immune cell.
  • the method comprises isolating immune cells and expanding the immune cells.
  • the invention further comprises cryopreserving the immune cells prior to expansion.
  • the cryopreserved immune cells are thawed for electroporation with the RNA encoding the chimeric membrane protein.
  • Cell culture refers generally to cells taken from a living organism and grown under controlled condition.
  • a primary cell culture is a culture of cells, tissues or organs taken directly from an organism and before the first subculture.
  • Cells are expanded in culture when they are placed in a growth medium under conditions that facilitate cell growth and/or division, resulting in a larger population of the cells.
  • the rate of cell proliferation is typically measured by the amount of time required for the cells to double in number, otherwise known as the doubling time.
  • the primary stimulatory signal and the co- stimulatory signal for the T cell may be provided by different protocols.
  • the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in "cis” formation) or to separate surfaces (i.e., in "trans” formation). Alternatively, one agent may be coupled to a surface and the other agent in solution.
  • the agent providing the co- stimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution.
  • the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents.
  • a surface such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents.
  • the two agents are immobilized on beads, either on the same bead, i.e., "cis," or to separate beads, i.e., "trans.”
  • the agent providing the primary activation signal is an anti-CD3 antibody or an antigen- binding fragment thereof and the agent providing the co-stimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof; and both agents are co-immobilized to the same bead in equivalent molecular amounts.
  • a 1 : 1 ratio of each antibody bound to the beads for CD4+ T cell expansion and T cell growth is used.
  • a ratio of anti CD3 :CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed as compared to the expansion observed using a ratio of 1 : 1. In one particular embodiment an increase of from about 1 to about 3 fold is observed as compared to the expansion observed using a ratio of 1 : 1. In one embodiment, the ratio of CD3 :CD28 antibody bound to the beads ranges from 100: 1 to 1 : 100 and all integer values there between. In one aspect of the present invention, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the ratio of CD3 :CD28 is less than one.
  • Ratios of particles to cells from 1 :500 to 500: 1 and any integer values in between may be used to stimulate T cells or other target cells.
  • the ratio of particles to cells may depend on particle size relative to the target cell. For example, small sized beads could only bind a few cells, while larger beads could bind many.
  • the ratio of cells to particles ranges from 1 : 100 to 100: 1 and any integer values in-between and in further embodiments the ratio comprises 1 :9 to 9: 1 and any integer values in between, can also be used to stimulate T cells.
  • the ratio of anti-CD3- and anti-CD28-coupled particles to T cells that result in T cell stimulation can vary as noted above, however certain preferred values include 1 : 100, 1 :50, 1 :40, 1 :30, 1 :20, 1 : 10, 1 :9, 1 :8, 1 :7, 1 :6, 1 :5, 1 :4, 1 :3, 1 :2, 1 : 1, 2: 1, 3: 1, 4:1, 5: 1, 6:1, 7: 1, 8: 1, 9: 1, 10: 1, and 15: 1 with one preferred ratio being at least 1 :1 particles per T cell. In one embodiment, a ratio of particles to cells of 1 :1 or less is used. In one particular embodiment, a preferred particle: cell ratio is 1 : 5.
  • the agent-coated beads and cells prior to culture, are not separated but are cultured together.
  • the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.
  • cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached (3x28 beads) to contact the T cells.
  • the cells for example, 104 to 109 T cells
  • beads for example, DYNABEADS® M-450 CD3/CD28 T paramagnetic beads at a ratio of 1 : 1
  • a buffer preferably PBS (without divalent cations such as, calcium and magnesium).
  • the target cell may be very rare in the sample and comprise only 0.01% of the sample or the entire sample (i.e., 100%) may comprise the target cell of interest.
  • the cells may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between.
  • Conditions appropriate for immune cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN- gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-beta, and TNF-a or any other additives for the growth of cells known to the skilled artisan.
  • serum e.g., fetal bovine or human serum
  • IL-2 interleukin-2
  • insulin IFN- gamma
  • IL-4 interleukin-7
  • GM-CSF GM-CSF
  • IL-10 interleukin-12
  • IL-15 IL-15
  • additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N- acetyl-cysteine and 2-mercaptoethanol .
  • Media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of immune cells.
  • Antibiotics e.g., penicillin and streptomycin
  • the target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% C02).
  • the medium used to culture the immune cells may include an agent that can costimulate the immune cells.
  • an agent that can stimulate CD3 is an antibody to CD3
  • an agent that can stimulate CD28 is an antibody to CD28.
  • a cell isolated by the methods disclosed herein can be expanded approximately 10-fold, 20-fold, 30-fold, 40-fold, 50- fold, 60-fold, 70-fold, 80- fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000- fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or greater.
  • the method of expanding the immune cells can further comprise isolating the expanded immune cells for further applications.
  • the method of expanding can further comprise a subsequent electroporation of the expanded immune cells followed by culturing.
  • the subsequent electroporation may include introducing a nucleic acid encoding an agent, such as a transducing the expanded immune cells, transfecting the expanded immune cells, or electroporating the expanded immune cells with a nucleic acid, into the expanded population of immune cells, wherein the agent further stimulates the immune cell.
  • the agent may stimulate the immune cells, such as by stimulating further expansion, effector function, or another immune cell function.
  • T cells that have been exposed to varied stimulation times may exhibit different characteristics.
  • typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (TH, CD4 + ) that is greater than the cytotoxic or suppressor T cell population (Tc, CD8 + ).
  • Tc cytotoxic or suppressor T cell population
  • Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of Tc cells. Accordingly, depending on the purpose of treatment, infusing a subject with a T cell population comprising predominately of TH cells may be advantageous. Similarly, if an antigen-specific subset of Tc cells has been isolated it may be beneficial to expand this subset to a greater degree.
  • the present disclosure provides a scaffold or substrate composition comprising a peptide comprising a TACA-binding domain, a nucleic acid molecule encoding a peptide comprising a TACA-binding domain, a cell modified to express a peptide comprising a TACA-binding domain, or a combination thereof.
  • a peptide comprising a TACA-binding domain, a nucleic acid molecule encoding a peptide comprising a TACA-binding domain, a cell modified to express a peptide comprising a TACA-binding domain, or a combination thereof is present within a scaffold.
  • a peptide comprising a TACA-binding domain in another embodiment, is applied to the surface of a scaffold.
  • the scaffold of the invention may be of any type known in the art. Non-limiting examples of such a scaffold includes a, hydrogel, electrospun scaffold, foam, mesh, sheet, patch, and sponge.
  • the antigen binding domain comprises a mutation in the TACA binding domain (TBD) selected from a substitution, a deletion, or an insertion.
  • the antigen binding domain comprises a deletion in the TACA binding domain (TBD).
  • the deletion is in the N-terminal and/or C-terminal region of the TACA binding domain (TBD).
  • the antigen binding domain comprises a deletion in the TACA binding domain (TBD), and the deletion is in the N-terminus of the TBD and is at least about 18 amino acids. In some embodiments, the antigen binding domain comprises a deletion in the TACA binding domain (TBD), and in the C-terminus and is at least about 10 amino acids. In some embodiments, the antigen binding domain comprises a deletion in the TACA binding domain (TBD), and in the N-terminus of the TBD and is at least about 36 amino acids. In some embodiments, the antigen binding domain comprises a deletion in the TACA binding domain (TBD), and in the N-terminus of the TBD and is at least about 18 amino acids and in the C-terminus and is at least about 10 amino acids.
  • the lectin is a siglec selected from the group consisting of siglec-1 (sialoadhesion), siglec-2 (CD22), siglec-3 (CD33), siglec-4 (myelin associated glycoprotein), siglec-5, siglec-6, siglec-7, siglec-8, siglec-9, siglec-10, siglec-11, siglec-12, siglec-13, siglec-14, siglec-15, siglec-16, siglec-17, Siglec E, Siglec F, siglec G and siglec H.
  • the composition comprises an antigen binding domain that selectively targets pi,6GlcNAc-branched N-glycans, Tn epitopes (Tn antigen), sialyl-Tn epitopes (sialyl-Tn antigen), GalNAca-Serine, GalNAca-Threonine, GalNAc, or GalNAcpi.
  • the composition comprises an antigen binding domain comprises a deletion in the TACA binding domain of the antigen binding domain comprising an amino acid sequence set forth in SEQ ID NOs: 30-54; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 30-54.
  • the transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 78 or SEQ ID NO: 87. In some embodiments, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In certain exemplary embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • the composition comprises an intracellular domain comprising a costimulatory signaling domain.
  • the chimeric antigen receptor (CAR) comprises a costimulatory domain that is a costimulatory domain of a molecule selected from the group consisting of CD27, CD28, 4-IBB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, CD8, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, DAP 10, DAP 12, Lek, Fas, and a combination thereof.
  • the costimulatory domain comprises a 4- IBB costimulatory domain or the amino acid sequence of SEQ ID NO: 58. In some embodiments, the costimulatory domain comprises a CD28 costimulatory domain or the amino acid sequence of SEQ ID NO: 88. In some embodiments, the costimulatory domain comprises a 4-IBB and a CD28 costimulatory domain.
  • the hinge domain is a protein selected from the group consisting of a CD8a, a CD28 hinge, an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, and an artificial spacer sequence.
  • the hinge domain is a CD8a hinge domain
  • n one embodiment, the hinge domain is a CD28 hinge domain.
  • the hinge domain comprises the amino acid sequence of SEQ ID NO: 77 or 86.
  • the hinge domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, SEQ ID NO: 68, 71-77, and 86.
  • compositions comprising modified cell comprising an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising: an amino acid sequence set forth in SEQ ID NOs: 23-29; or an amino acid sequence having 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%, at least 99% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 23-29.
  • the isolated nucleic acid expressed in the modified cell comprises an expression vector; and/or an in vitro transcribed RNA.
  • the composition comprises a CAR that selectively targets a TACA selected from the group consisting of pi, 6 branching, pi,6GlcNAc-branched N- glycans, T antigen, Tn antigen, sialyl-T epitopes, Tn epitopes, sialyl-Tn epitopes, a2, 6 sialylation, Sialylation, sialyl-Lewisx/a, di-sialyl-Lewisx/a, sialyl 6-sulfo Lexisx, Globo H, GD2, GD3, GM3, and Fucosyl GM1.
  • a TACA selected from the group consisting of pi, 6 branching, pi,6GlcNAc-branched N- glycans, T antigen, Tn antigen, sialyl-T epitopes, Tn epitopes, sialyl-Tn epitopes, a2, 6 sialylation, Sialylation, sia
  • the composition is a pharmaceutical composition.
  • the composition may include a pharmaceutical composition and further comprises one or more pharmaceutically or physiologically acceptably carriers, diluents, adjuvants, or excipients.
  • Such compositions 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.
  • Compositions of the present disclosure are preferably formulated for parenteral administration (e.g., intravenous administration).
  • additional ingredients include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials.
  • compositions of the disclosure are known in the art and described, for example in Genaro, ed. (1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA), which is incorporated herein by reference.
  • Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like.
  • a suitable carrier such as sterile water, physiological saline, glucose, dextrose, or the like.
  • the compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and/or colors, depending upon the route of administration and the preparation desired. Standard texts may in certain aspects be consulted to prepare suitable preparations.
  • the composition includes an anti-oxidant and a chelating agent that inhibits the degradation of one or more components of the composition.
  • Preferred antioxidants for some compounds are BHT, BHA, alpha- tocopherol and ascorbic acid in the preferred range of about 0.01% to 0.3% and more preferably BHT in the range of 0.03% to 0.1% by weight by total weight of the composition.
  • the chelating agent is present in an amount of from 0.01% to 0.5% by weight by total weight of the composition.
  • Particularly preferred chelating agents include edetate salts (e.g., disodium edetate) and citric acid in the weight range of about 0.01% to 0.20% and more preferably in the range of 0.02% to 0.10% by weight by total weight of the composition.
  • the chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are the particularly preferred antioxidant and chelating agent respectively for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.
  • Liquid suspensions may be prepared using conventional methods to achieve suspension of the peptide or other composition of the disclosure in an aqueous or oily vehicle.
  • Aqueous vehicles include, for example, water, and isotonic saline.
  • Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
  • Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents.
  • Oily suspensions may further comprise a thickening agent.
  • Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxy cetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively).
  • Known emulsifying agents include, but are not limited to, lecithin, and acacia.
  • Known preservatives include, but are not limited to, methyl, ethyl, or n- propyl-para- hydroxybenzoates, ascorbic acid, and sorbic acid.
  • compositions or formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration.
  • the modified immune cell populations are administered parenterally.
  • parenteral includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration.
  • the immune cells of the present disclosure are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
  • the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an immunotherapeutic composition comprising the modified cell described herein; the chimeric antigen receptor (CAR) described herein; or a the composition described herein.
  • an immunotherapeutic composition comprising the modified cell described herein; the chimeric antigen receptor (CAR) described herein; or a the composition described herein.
  • One aspect of the present disclosure provides a method of treating a cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective composition comprising a modified cell comprising a chimeric antigen receptor that selectively binds a tumor-associated carbohydrate antigen (TACA).
  • TACA tumor-associated carbohydrate antigen
  • T cells can be preferentially selected for or against at culture initiation or at other time points during the process.
  • subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points.
  • the skilled person would recognize that multiple rounds of selection can also be used in the context of this invention.
  • a nucleic acid encoding the TACA CAR of the present disclosure is introduced into the immune cells such that the immune cells will express, preferably stably, the CAR.
  • the modified immune cells may be applied as a dispersion injected at or near the site of interest.
  • the cells may be in a physiologically- acceptable medium
  • the treatment method is subject to many variables, such as the cellular response to the TACA CAR, the efficiency of expression of the TACA CAR by the immune cells and, as appropriate, the level of secretion, the activity of the expressed CAR, the particular need of the subject, which may vary with time and circumstances, the rate of loss of the cellular activity as a result of loss of modified immune cells or the expression activity of individual cells, and the like. Therefore, it is expected that for each individual patient, even if there were universal cells, which could be administered to the population at large, each patient would be monitored for the proper dosage for the individual, and such practices of monitoring a patient are routine in the art.
  • the isolated nucleic acid encoding the CAR comprises an expression vector; and/or an in vitro transcribed RNA.
  • the CAR selectively targets a TACA selected from the group consisting of [31, 6 branching, pi,6GlcNAc-branched N-glycans, T antigen, Tn antigen, sialyl-T epitopes, Tn epitopes, sialyl-Tn epitopes, a2, 6 sialylation, Sialylation, sialyl— Lewis x/a , di -si alyl -Lewi s x/a , sialyl 6-sulfo Lexis x , Globo H, GD2, GD3, GM3, and Fucosyl GM1.
  • the CAR selectively targets pi,6GlcNAc-branched N-glycans, GalNAc, Tn antigen,
  • the present disclosure provides a method of treating cancer.
  • the method may be used to treat any cancer, including a hematological malignancy, a solid tumor, a primary or a metastasizing tumor.
  • Cancers that may be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors.
  • the cancers may comprise nonsolid tumors (such as hematological tumors, for example, leukemias and lymphomas) or may comprise solid tumors.
  • Types of cancers to be treated with the CARs of the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas.
  • sarcomas e.g., sarcomas, carcinomas, and melanomas.
  • Adult tumor s/cancers and pediatric tumors/cancers are also included.
  • the cancer is selected from the group consisting of a hematological malignancy, a solid tumor, a primary or a metastasizing tumor, a leukemia, a carcinoma, a blastoma, a sarcoma, a leukemia, lymphoid malignancies, a melanoma and a lymphoma, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas.
  • the cancer may be tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors.
  • the cancer may comprise non-solid tumors (such as hematological tumors, for example, leukemias and lymphomas) or may comprise solid tumors.
  • he cancer is selected from the group consisting of a hematological malignancy, a solid tumor, a primary or a metastasizing tumor, a leukemia, a carcinoma, a blastoma, a sarcoma, a leukemia, lymphoid malignancies, a melanoma and a lymphoma.
  • Hematologic cancers are cancers of the blood or bone marrow.
  • leukemias include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.
  • acute leukemias such as acute lymphocytic
  • An exemplary type of cancer to be treated with the modified immune cells (e.g., modified T cells comprising a TACA CAR) or pharmaceutical compositions of the present disclosure include non-small cell lung cancer.
  • Lung cancer is a leading cause of cancer- related mortality around the world and remains a significant unmet need despite advances in therapy.
  • Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer cases in the US, with a significant proportion of the remaining 15% being small cell lung cancers (SCLC).
  • SCLC small cell lung cancers
  • Surgical resection remains the single most consistent and successful option for localized NSCLC; however, close to 70% of patients with lung cancer present with locally advanced or metastatic disease at the time of diagnosis. Molina et al, Mayo Clin Proc, 83(5):584-594 (2008). Overall, the prognosis for lung cancer patients is poor, with 5-year relative survival less than 18%. The median OS time for patients with stage IV NSCLC is 4 months, while l-and 5-year survival is less than 16% and 2%, respectively. Cetin et al. Clin Epidemiol, 3: 139-148 (2011).
  • pancreatic adenocarcinoma An exemplary type of cancer to be treated with the modified immune cells (e.g., modified T cells comprising a TACA CAR) or pharmaceutical compositions of the invention include pancreatic adenocarcinoma.
  • Pancreatic ductal adenocarcinoma is a highly lethal malignancy. It is the fourth leading cause of cancer-related death in the United States with approximately 45,000 new cases per year. Surgical resection is the only potentially curative treatment, however with tire majority of patients presenting with advanced disease only 15- 20% of patients are candidates for surgical intervention.
  • An exemplary type of cancer to be treated with the modified immune cells (e.g., modified T cells comprising a TACA CAR) or pharmaceutical compositions of the invention include epithelial ovarian cancer.
  • Epithelial ovarian cancers generally include fallopian tube malignancies as well as primary peritoneal cancers. More than 70% of women with epithelial ovarian cancer present with advanced disease at the time of first diagnosis. Although patients with advanced disease can achieve complete remission after surgical cytoreduction and platinum- and taxane-based chemotherapy, up to 80% eventually experience recurrence. Herzog et al.. Gynecol Oncol Res Pract, 4: 13 (2017).
  • a TACA-binding lectin and a lectin- binding composition e.g., T cell engineered to express an anti-lectin CAR.
  • this method can have the ability to time limit the T cell response as the half-life of the lectin is much shorter than the engineered T cell.
  • the engineered T cells may remain for years, but without the lectin, the T cells would be inactive, thereby allowing for easier targeting of solid cancers by limiting persistence of the response.
  • a population of modified immune cells are administered to the subject.
  • the population of modified immune cells comprises immune cells selected from the group consisting of natural killer (NK) cells, NKT cells, and T cells.
  • the population of modified immune cells comprises modified T cells.
  • the modified immune cells are autologous or heterologous immune cells.
  • the present disclosure provides a type of cellular therapy where T cells are genetically modified to express a peptide of the invention, and the cell is infused to a recipient in need thereof.
  • the infused cell is able to kill tumor cells in the recipient.
  • the modified cells are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control.
  • the modified cells disclosed herein can undergo robust in vivo T cell expansion and can persist for an extended amount of time.
  • the modified T cells of the invention evolve into specific memory T cells that can be reactivated to inhibit any additional tumor formation or growth.
  • modified T cells of the invention can undergo robust in vivo T cell expansion and persist at high levels for an extended amount of time in blood and bone marrow and form specific memory T cells.
  • the modified immune cells of the present disclosure to be administered may be autologous, with respect to the subject undergoing therapy or heterologous.
  • the administration of the immune cells of the present disclosure may be carried out in any convenient manner known to those of skill in the art.
  • the immune cells of the present disclosure may be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation.
  • the compositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
  • the immune cells of the present disclosure are injected directly into a site of inflammation in the subject, a local disease site in the subject, a lymph node, an organ, a tumor, and the like.
  • compositions of the present invention may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations.
  • pharmaceutical compositions of the present invention may comprise a composition as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions 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
  • proteins such as glucose, mannose
  • compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented).
  • the quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient' s disease, although appropriate dosages may be determined by clinical trials.
  • an immunologically effective amount When “an immunologically effective amount,” “an anti-tumor effective amount,” “an tumor-inhibiting effective amount,” or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).
  • compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (iv.) injection, or intraperitoneally.
  • the compositions of the present invention are administered to a patient by intradermal or subcutaneous injection.
  • the compositions of the present invention are administered by i.v. injection.
  • the compositions of be injected directly into a tumor or lymph node.
  • the composition of the invention is administered during surgical resection or debulking of a tumor or diseased tissue.
  • the composition may be administered to the site in order to further treat the tumor.
  • the method comprises administering to the subject a scaffold comprising a peptide comprising a TACA- binding domain, a nucleic acid molecule encoding a peptide comprising a TACA-binding domain, a cell modified to express a peptide comprising a TACA-binding domain, or a combination thereof.
  • compositions and pharmaceutical compositions of the invention include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.
  • mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.
  • the individual populations or subtypes are present at or near a desired output ratio (sudi as CD4 + to CD8 + ratio), e.g., within a certain tolerated difference or error of such a ratio.
  • the cells are administered at or within a tolerated difference of a desired dose of one or more of the individual populations or sub-types of cells, such as a desired dose of CD4 + cells and/or a desired dose of CD8 + cells.
  • the desired dose is a desired number of cells of the sub-type or population, or a desired number of such cells per unit of body weight of the subject to whom the cells are administered, e.g., cells/kg.
  • the desired dose is at or above a minimum number of cells of the population or subtype, or minimum number of cells of the population or sub-type per unit of body weight.
  • the dosage is based on a desired fixed dose of total cells and a desired ratio, and/or based on a desired fixed dose of one or more, e.g., each, of the individual sub-types or sub-populations.
  • the dosage is based on a desired fixed or minimum dose of T cells and a desired ratio of CD4 + to CD8 + cells, and/or is based on a desired fixed or minimum dose of CD4 + and/or CD8 + cells.
  • the modified immune cells, or individual populations of subtypes of immune cells are administered to the subject at a range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650
  • the dose of total cells and/or dose of individual subpopulations of cells is within a range of between at or about 1 x 10 5 cells/kg to about 1 x 10 11 cells/kg, 10 4 , and at or about 10 11 cells/kilograms (kg) body weight, such as between 10 5 and 10 6 cells / kg body weight, for example, at or about 1 x 10 5 cells/kg, 1.5 x 10 5 cells/kg, 2 x 10 5 cells/kg, or 1 x 10 6 cells/kg body weight.
  • the cells are administered at, or within a certain range of error of, between at or about 10 4 and at or about 10 9 T cells/kilograms (kg) body weight, such as between 10 4 and 10 6 T cells / kg body weight, for example, at or about 1 x 10 4 T cells/kg, 1.5 x 10 4 T cells/kg, 2 x 10 5 T cells/kg, or 1 x 10 6 T cells/kg body weight.
  • a suitable dosage range of modified cells for use in a method of the present disclosure includes, without limitation, from about 1 x 10 4 cells/kg to about 1 x 10 6 cells/kg, from about 1 x 10 6 cells/kg to about 1 x 10 7 cells/kg, from about 1 x 10 7 cells/kg about 1 x 10 8 cells/kg, from about 1 x 10 8 cells/kg about 1 x 10 9 cells/kg, from about 1 x 10 9 cells/kg about 1 x 10 10 cells/kg, from about 1 x 10 10 cells/kg about 1 x 10 11 cells/kg.
  • a suitable dosage for use in a method of the present disclosure is about 1 x 10s cells/kg.
  • a suitable dosage for use in a method of the present disclosure is about 1 x 10 7 cells/kg. In other embodiments, a suitable dosage is from about 1 x 10 7 total cells to about 5 x 10 7 total cells. In some embodiments, a suitable dosage is from about 1 x 10 4 total cells to about 5 x 10 4 total cells. In some embodiments, a suitable dosage is from about 1.4 x 10 7 total cells to about 1.1 x 10 9 total cells. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about 7 x 10 9 total cells. In an exemplary embodiment, a suitable dosage is from about 1 x 10 7 total cells to about 3 x 10 7 total cells.
  • the modified immune cells are administered at or within a tolerated range of a desired output ratio of multiple cell populations or sub-types, such as CD4 + and CD8 + cells or sub-types.
  • the desired ratio can be a specific ratio or can be a range of ratios, for example, in some embodiments, the desired ratio (e.g., ratio of CD4 + to CD8 + cells) is between at or about 5: 1 and at or about 5: 1 (or greater than about 1 :5 and less than about 5: 1), or between at or about 1 :3 and at or about 3 : 1 (or greater than about 1 :3 and less than about 3: 1), such as between at or about 2: 1 and at or about 1 :5 (or greater than about 1 :5 and less than about 2: 1, such as at or about 5: 1, 4.5: 1, 4: 1, 3.5: 1, 3: 1, 2.5:1, 2: 1, 1.9: 1, 1.8: 1, 1.7: 1, 1.6: 1, 1.5: 1, 1.4:
  • the tolerated difference is within about 1%, about 2%, about 3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value in between these ranges.
  • a dose of modified cells is administered to a subject in need thereof, in a single dose or multiple doses. In some embodiments, a dose of modified cells is administered in multiple doses, e.g., once a week or every 7 days, once every 2 weeks or every 14 days, once every 3 weeks or every 21 days, once every 4 weeks or every 28 days. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof by rapid intravenous infusion. In some embodiments, a dose of modified cells is administered to a subject in need thereof, in a fractionated dose or split dose.
  • the modified immune cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent.
  • the modified immune cells in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order.
  • the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa.
  • the cells are administered prior to the one or more additional therapeutic agents.
  • the cells are administered after the one or more additional therapeutic agents.
  • the one or more additional agents includes a cytokine, such as IL-2, for example, to enhance persistence.
  • the methods comprise administration of a chemotherapeutic agent.
  • the biological activity of the engineered cell populations in some embodiments is measured, e.g., by any of a number of known methods.
  • Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry.
  • the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al., J.
  • the biological activity of the cells is measured by assaying expression and/or secretion of one or more cytokines, such as CD 107a, IFNy, IL-2, and TNF.
  • the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.
  • the subject can be administered, in addition to the CAR, a secondary treatment.
  • the subject can be administered conditioning therapy prior to CAR T cell therapy.
  • the present disclosure provides a method of treatment comprising administering a conditioning therapy prior to administering CAR T therapy (e.g., modified T cells comprising a TACA CAR of the present disclosure).
  • Administration of a conditioning therapy prior to TACA CAR T cell therapy may increase the efficacy of the TACA CAR T cell therapy.
  • TACA target density in cancer cells can be about 100-1000 fold greater than typical protein antigens.
  • increasing the number of TACA binding domains in GlyTR may drive cancer cells specificity by enhancing binding avidity, distinction to antibodies, where high affinity is used to achieve specificity.
  • High avidity binding is accomplished by the combination of high-density target expression and the presence of multiple carbohydrate-binding domains.
  • the combination of high target density and multiple binding sites should lead to high specificity for high expressing over low expressing cells.
  • specificity of a multi-valent GlyTR protein for TACAs would not be determined by presence or absence of the target, but rather a threshold density of target expression specifically detected by GlyTRs with multiple TACA binding domains.
  • GlyTR2 pi,6GlcNAc-branched N- glycans
  • GlyTR2 Tn antigen
  • L-PHA is a plant lectin with high specificity for pi,6GlcNAc-branched N-glycans.
  • GlyTR l LPHAxCD3 was predominantly a dimer of ⁇ 100kDa versus 55kDa predicted (FIG. IB) and thus contained two L-PHA and two anti-CD3 binding domains. Dimer formation is not unexpected as native L-PHA is a tetramer.
  • GlyTR l LPHA(2)xCD3 GlyTR 1LPHAxLPHAxCD3
  • two L-PHA domains linked in tandem by three flexible linkers i.e. (GGGGS)3
  • FIG. 1A Size exclusion chromatography
  • SEC Size exclusion chromatography
  • dimeric GlyTRl LPHA(2)xCD3 (four L-PHA domains) bound to target cancer cells were significantly better than original dimeric GlyTRl LPHAxCD3 (two L-PHA domains), leading to a >3000 fold increase in cancer cell killing activity.
  • dimeric GlyTRl LPHA(2)xCD3 potently triggered human T cell dependent killing of many diverse liquid and solid cancer types with an ECso as low as ⁇ 100 femtomolar, including multiple myeloma, T cell leukemia, acute myeloid leukemia, (AML), pancreatic cancer, colon cancer, non-small cell lung cancer, prostate cancer, ovarian cancer and breast cancer.
  • MDA-MD- 231-Fluc triple negative breast cancer
  • SKOV3-Fluc ovarian cancer
  • MHC class I genes i.e., P2 microglobulin
  • Flow cytometry confirmed loss of HLA ABC class I at the cell surface (data not shown).
  • mice were injected intra-peritoneal (i.p.) with MDA- MB -231 -Fluc-Ml" or SKOV3 -Fluc-Ml "cells and once tumor was established after 5 days, mice were injected i.p. with purified CD8 + T cells every 3- 4 days for 2 or 3 injections, respectively, along with GlyTRl LPHAxLPHAxCD3 j p twice daily.
  • GlyTRl LPHAxLPHAxCD3 a lOug twice daily induced marked tumor regression, with many mice displaying undetectable disease after ⁇ 1 week of treatment.
  • dimeric GlyTRl LPHA(2)xCD3 treatment of humanized mice did not induce 1) “on-target, off cancer” toxicity in major organs, or 2) non- specific T cell activation.
  • the lack of “on-target, off cancer” organ toxicity was consistent with fluorescently tagged dimeric GlyTRl LPHA(2)xCD3 not s ig n if lcan ly accumulating in mouse tissues with the highest target expression, namely kidney, stomach and small intestine.
  • Lack of kidney toxicity was also consistent with the molecular weight of dimeric GlyTRl LPHA(2)xCD3 (about 182 kDa) being well above the glomerular filtration cut-off of about 70kDa.
  • Tn antigen Although not found on the cell surface of normal human tissue, Tn antigens are expressed in -90% of human carcinomas and many hematopoietic cancers. Indeed, Tn antigens are one of the most specific human cancer associated structures known and promote cell motility, invasiveness and metastasis.
  • the Tn antigen is a single N-acetyl- galactosamine (GalNAc) a-O-linked to serine/threonine in proteins like mucins.
  • GalNAc N-acetyl- galactosamine
  • a Tn is a biosynthetic precursor of O-glycans that is normally extended with al, 3 linked galactose.
  • the chaperone protein COSMC a protein required by T-synthase to add galactose to GalNAc, is frequently altered in cancer. Mis- localization of enzymes within the ER/Golgi may also lead to abnormal Tn antigen expression in human cancer.
  • the Tn antigen can be abnormally extended with Sialic Acid to make the sTn antigen; which is also not typically expressed in normal tissue.
  • CD301 (CLEC10 A, macrophage galactose lectin) was utilized.
  • CD301 (CLEC10) is a transmembrane lectin expressed in macrophages and dendritic cells that functions as a pattern recognition receptor for non-self antigens, and binds to Tn + cancers. See e.g., Nollau et al., J. histochemistry and cytochemistry, 61 : 199-205 (2013); Lenos et al., Oncotarget 6: 26278-26290 (2015).
  • CD301 also strongly binds to three other well-known cancer specific glycan antigens containing 3- and 4- hydroxyl exposed GalNAc, namely sTn and the gangliosides GD2 and GM236. These three glycan antigens are the only TACAs that have reached Phase III immunotherapy clinical trials, with an anti-GD2 monoclonal antibody being FDA approved for neuroblastoma.
  • CD301 also binds the invertebrate glycan LacdiNAc (GalNAc i,4GlcNAc). Mammalian cells generally do not express LacdiNAc, but expression is often induced in many human cancers. The blood group A glycan antigen has a terminal GalNAc residue, however CD301 is expressed in blood group A individuals without inducing toxicity. Indeed, CD301 failed to bind blood group A positive RBC or blood vessels on a tissue microarray (data not shown). Finally, a fully human protein CD301 should be poorly immunogenic. As such, a human CD301 provides high specificity for Tn antigen and three other well-known TACAs.
  • GalNAc i,4GlcNAc invertebrate glycan LacdiNAc
  • the extracellular domain of human CD301 was combined with a scFv domain specific to CD3. See e.g., International Application NO. PCT/US2016/030113. However, this protein was unable to be expressed in CHO cells, presumably because of protein mis-folding.
  • the CD301 extracellular domain consists of a neck region and a single TACA binding domain (TBD). The neck region promotes trimerization of CD301. See e.g., Jegouzo et al., Glycobiology 23:853-864 (2013); Napoletano et al., Eur. J. Immunol. 42:936-945 (2012).
  • deletion of the neck region should avoid multimerization and may promote folding of GlyTR2 proteins.
  • a GlyTR2 CD301xCD3 containing a single CD301 TACA binding domain without most of the neck region was readily expressed and bound Tn hlgh Jurkat- TCR -/- leukemic T cells (FIGs. 2A).
  • Jurkat-TCRP ⁇ " leukemic T cells express maximal levels of Tn antigen due to mutation of the chaperone protein COSMC, a protein required by T-synthase to extend GalNAc with galactose and produce mature O-glycans.
  • Soluble Tn antigen (GalNAca-Ser) and GalNAc but not related sugars galactose and GlcNAc blocked binding of GlyTR2 CD301xCD3 to Tn 1 ⁇ 11 Jurkat-TCRP' /_ leukemic T cells, confirming specificity of GlyTR2 CD301(3)xCD3 for Tn antigen.
  • Adding a fourth CD301 domain i.e. GlyTR2 CD301(4)xCD3 ) further improved binding relative to GlyTR2 CD301(3)xCD3 with three binding domains.
  • GlyTR2 slCD301(4)xCD3 (stiff-linkers, four CD301 domains) bound to Tn hlgh Jurkat-TCRP' /_ leukemic T cells similar to GlyTR2 CD301(3)xCD3 (flexible linkers, three CD301 domains), but bound significantly better to a wide diversity of lower Tn expressing tumor cell lines.
  • GalNAc but not the related sugar GlcNAc readily blocked binding of GlyTR2 slCD301(4)xCD3 to Tn + MM1R multiple myeloma cells, confirming specificity of binding to Tn antigen. Given these data, GlyTR2 slCD301(4)xCD3 was selected for further characterization.
  • GlyTR2 slCD301(4)xCD3 In vitro and in vivo cancer killing by Qiypp2 slCD301(4)xCD3 GlyTR2 slCD301(4)xCD3 dose- dependently triggered T cell mediated killing of diverse Tn + liquid and solid cancers with ECso in the high pM to low nM range, including multiple myeloma, T cell leukemia, AML, pancreatic cancer, colon cancer, non-small cell lung cancer, prostate cancer, ovarian cancer and breast cancer. There was little killing without PBMCs/T cells, confirming killing by GlyTR2 slCD301(4)xCD3 requires T cells.
  • GlyTR2 slCD301(4)xCD3 To first assess GlyTR2 slCD301(4)xCD3 activity in vivo, we maximized Tn antigen expression in MDA-MB-231-Fluc-Ml- breast cancer cells by deleting the gene COSMC (i.e., MDA-MB-231-luc + MI' / 'C' / ' cells). GlyTR2 slCD301(4)xCD3 readily induced killing of these cells by purified CD8 + T cells in vitro. In mice with established breast cancer tumors, 15 days of GlyTR2 slCD301(4)xCD3 treatment dose dependently induced tumor regression in NSG mice humanized with CD8 + T cells compared to with CD8 + T cells compared to control mice.
  • SKOV3 ovarian cancer cells knocked out for MHC class I were utilized.
  • GlyTR2 slCD301(4)xCD3 treatment induced marked ovarian tumor regression in NSG mice humanized with CD8 + T cells relative to control mice (.
  • Injection of fluorescently labelled GlyTR2 slCD301(4)xCD3 into NSG mice with or without metastatic MDA-MB-23 I -Fluc-MI ⁇ 'C' 7 ' cells demonstrated accumulation of GlyTR2 slCD301(4)xCD3 in lungs with but not without cancer, indicating specificity for cancer cells in vivo.
  • tonic signaling was evidenced by: 1) continued blasting on day 7 despite resting for 4 days (i.e. significantly larger cells compared to nontransduced based on side vs forward scatter (SSC/FSC)) (FIG. 3C); 2) increased cell death on day 7 compared to non-transduced (i.e., 58.3% vs 87.4% live cells on SSC/FSC) (FIG. 3 C); and 3) IFNy production in the absence of cancer cells (FIG. 4A).
  • SSC/FSC side vs forward scatter
  • GlyTR2 slCD301(4) CAR T cells also expressed elevated IFNy production in the absence of cancer cells relative to non-transduced (FIG. 4B). Moreover, high but not low expression of GlyTR2 slCD301(4) CAR triggered cell surface CAR clustering and induction of the 4- IBB activation marker (FIG. 4 C), further indicating tonic signaling.
  • Tonic signaling by CARs is a common design issue and may help cancer cell killing but may also increase risk of toxicity from excessive T cell activation. Both intracellular and extracellular domains can drive tonic signaling. Long et al., Nat. Med. 21 :581-590 (2015). In the case of GlyTR CAR T cells, tonic signaling is likely driven by both the extracellular and intracellular domains. For example, the two L-PHA domains in GlyTRl LPHA(2)xCD3 bi-specific protein induce dimerization; thus the two L-PHA domains in the GlyTRl-CAR are likely similarly dimerizing at the cell surface to drive tonic signaling.
  • mice with established breast cancer tumors 15 days of GlyTR2 slCD301(4)xCD3 treatment dose dependently induced tumor regression in NSG mice humanized with CD8 + T cells compared to with CD8 + T cells compared to control mice (FIGs. 3F-G).
  • MDA-MB-231 triple negative breast cancer cells or SK0V3 ovarian cancer cells knocked out for MHC class I were utilized.
  • GlyTR2 slCD301(4)xCD3 treatment induced marked ovarian tumor regression in NSG mice humanized with CD8 + T cells relative to control mice (FIGS. 3F-G).
  • linker peptides that separate domains in fusion proteins play important roles in their expression, stability and function50.
  • Chen et al. Advanced drug delivery reviews 65: 1357-1369 (2013).
  • Three repeats of the flexible (GGGGS)n linker separates the L-PHA domains in both the dimeric GlyTRl LPHA(2)xCD3 bispecific protein and GlyTRl-CAR.
  • the alpha helix forming linker A(EAAAK)nA is a rigid linker that has been used successfully in fusion proteins to maintain distance between two different domains including in the GlyTR2 bi-specific proteins. See Example 2; Chen et al. , Advanced drug delivery reviews 65: 1357-1369 (2013).
  • deleting the first five amino acids in one or both of the two L-PHA domains in the GlyTRl-CAR is expected to reduce CAR dimerization/tonic signaling. Maximizing binding avidity of the GlyTRl-CAR via increased valency for > 1 ,6G!cN Ac- branched N- glycans.
  • the GlyTR2 slCD301(4) CAR contained four CD301 domains, which may promote multimerization and associated tonic signaling at the cell surface. However, reducing the GlyTR2 CAR to a single CD301 domain (i.e., GlyTR2 CD301 CAR) did not significantly impact tonic signaling as evidenced by continued clustering of the CAR and induction of the 4-1BB activation marker in high > low CAR expressing cells (FIG. 5B).
  • variant 181-316 markedly reduced tonic signaling as evidenced by reduced 4-1BB induction and IFNy production in the absence of cancer cells, yet was just as potent as the parental GlyTR2CD301 CAR at killing cancer cells (FIGs. 5F-G). Additional deletion of the 10 C-terminal amino acids (i.e. variant 181-306) also displayed reduced tonic signaling but was less potent than variant 181-316 in killing cancer cells across three different donors (FIG. 5A, C-G).
  • a range includes each individual member.
  • a group having 1-3 cells refers to groups having 1, 2, or 3 cells.
  • a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

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Abstract

L'invention concerne des compositions et des méthodes de traitement de maladies associées à une glycosylation aberrante de molécules de surface cellulaire et à l'expression d'antigènes glucidiques associés à une tumeur (TACA). L'invention concerne également des récepteurs antigéniques chimériques (CAR) spécifiques d'antigènes glucidiques associés à une tumeur (TACA-CAR), des vecteurs codant les TACA-CAR, et des cellules recombinées comprenant les CAR TACA.
EP23824455.2A 2022-06-13 2023-06-09 Cellules de récepteur d'antigène chimérique dépendant du glycane améliorées Pending EP4536282A2 (fr)

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