WO2021252635A1 - Compositions et méthodes de traitement de cancers - Google Patents

Compositions et méthodes de traitement de cancers Download PDF

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Publication number
WO2021252635A1
WO2021252635A1 PCT/US2021/036627 US2021036627W WO2021252635A1 WO 2021252635 A1 WO2021252635 A1 WO 2021252635A1 US 2021036627 W US2021036627 W US 2021036627W WO 2021252635 A1 WO2021252635 A1 WO 2021252635A1
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seq
cell
receptor
antigen
tcr
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PCT/US2021/036627
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Xueyin Wang
Carl Alexander Kamb
Han Xu
Mark L. SANDBERG
Dora Toledo WARSHAVIAK
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A2 Biotherapeutics, Inc.
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Publication of WO2021252635A1 publication Critical patent/WO2021252635A1/fr

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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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46448—Cancer antigens from embryonic or fetal origin
    • A61K39/464482—Carcinoembryonic antigen [CEA]
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • CARs chimeric antigen receptors
  • TCRs T cell Receptors
  • the disclosure provides compositions and methods for increasing the specificity of immune cells used in adoptive cell therapy.
  • the disclosure provides immune cells comprising a two-receptor system that increases the specificity of the immune cells for target cells expressing a target antigen.
  • the immune cells comprise a first, activator receptor that activates the immune cells in response to binding of the first receptor by the target antigen.
  • the immune cells further comprise a second, inhibitory receptor specific to a non-target antigen. This second receptor inhibits activation of the immune cells when the second receptor is bound by the non-target antigen, even when the first receptor is bound by the target antigen.
  • the disclosure provides an immune cell responsive to loss of heterozygosity in a cancer cell, comprising: (a) first receptor, optionally a chimeric antigen receptor (CAR) or T cell receptor (TCR), comprising an extracellular ligand binding domain specific to a target antigen selected from: (i) a cancer cell-specific antigen, or a peptide antigen thereof in a complex with a major histocompatibility complex class I (MHC-I); or (ii) CEA cell adhesion molecule 5 (CEA), or a peptide antigen thereof in a complex with a major histocompatibility complex class I (MHC-I); and (b) a second receptor, optionally an inhibitory chimeric antigen receptor (iCAR), comprising an extracellular ligand binding domain specific to a non-target antigen selected from TNFRSF11 A, ACHRB, ITGAE, TRPVl, and SREC, or an antigen peptide thereof in a complex with a major histocompat
  • the target antigen is a cancer cell-specific antigen.
  • the target antigen is a peptide antigen of a cancer cell-specific antigen in a complex with a major histocompatibility complex class I (MHC-I).
  • MHC-I major histocompatibility complex class I
  • the cancer cell is a colorectal cancer cell.
  • the cancer cell is a pancreatic cancer cell, esophageal cancer cell, gastric cancer cell, lung adenocarcinoma cell, head-and-neck cancer cell, diffuse large B cell cancer cell, or acute myeloid leukemia cancer cell.
  • the target antigen is CEA.
  • the target antigen is a peptide antigen of CEA in a complex with a major histocompatibility complex class I (MHC-I).
  • MHC-I major histocompatibility complex class I
  • the CEA positive cancer cells comprise colorectal cancer cells, pancreatic cancer cells, esophageal cancer cells, gastric cancer cells, lung adenocarcinoma cells, head and neck cancer cells, diffuse large B cell cancer cells or acute myeloid leukemia cancer cells.
  • the target antigen is expressed by a target cell.
  • the non-target antigen is not expressed by the target cell.
  • the non-target antigen is expressed by healthy cells.
  • the first receptor and the second receptor together specifically activate the immune cell in the presence of the target cell.
  • the immune cell is a T cell. In some embodiments, the immune cell is a CD8+ CD4- T cell.
  • the CEA comprises a sequence that shares at least 95% identity to SEQ ID NO: 1.
  • the peptide antigen of CEA is IMIGVLVGV (SEQ ID NO: 2).
  • the MHC-I comprises a human leukocyte antigen A*02 allele (HLA-A*02).
  • the first receptor is a T cell receptor (TCR).
  • the first receptor is a chimeric antigen receptor (CAR).
  • the extracellular ligand binding domain of the first receptor comprises an antibody fragment, a single chain Fv antibody fragment (ScFv), or a b chain variable domain (nb).
  • the extracellular ligand binding domain of the first receptor comprises a TCR a chain variable domain and a TCR b chain variable domain.
  • the extracellular ligand binding domain of the first receptor comprises complement determining regions (CDRs) selected from SEQ ID NOs: 3-12.
  • the TCR a chain variable domain comprises a CDR-1 of TSITA (SEQ ID NO: 3), a CDR-2 of IRSNER (SEQ ID NO: 4) and a CDR-3 comprising ATDLTSGGNYK (SEQ ID NO: 5), ATDFTSGGNYK (SEQ ID NO: 6), ATDLTT GGNYK (SEQ ID NO: 7) or ATDFTTGGNYK (SEQ ID NO: 8); and (b) the TCR b chain variable domain comprises a CDR-1 of KGHPV (SEQ ID NO: 9), a CDR-2 of FQNQEV (SEQ ID NO: 10), and a CDR-3 of ASSLGLGDYEQ (SEQ ID NO: 11) or ASSLGTGDYEQ (SEQ ID NO: 12).
  • the TCR a chain variable domain comprises a CDR-1 of SEQ ID NO: 9, a CDR-2 of SEQ ID NO:
  • the TCR b chain variable domain comprises a CDR-1 of SEQ ID NO: 3, a CDR-2 of SEQ ID NO: 4 and a CDR-3 comprising SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.
  • the non-target antigen is a TNFRSFl 1 A antigen that shares at least 95% identity to SEQ ID NO: 13 and the polymorphism is selected from: (a) A or V at position 192 of SEQ ID NO: 13, or (b) H or Y at position 141 of SEQ ID NO: 13.
  • the non-target antigen is an ITGAE antigen that shares at least 95% identity to SEQ ID NO: 14 and the polymorphism is selected from (a) R or W at position 950 of SEQ ID NO: 14; or (b) V,
  • compositions comprising a therapeutically effective amount of the immune cells of the disclosure.
  • the pharmaceutical compositions further comprise a pharmaceutically acceptable carrier, diluent or excipient.
  • compositions comprising the immune cells of the disclosure, for use as a medicament in the treatment of cancer.
  • the disclosure provides a polynucleotide system, comprising one or more polynucleotides comprising polynucleotide sequences encoding: (a) a first receptor, optionally a chimeric antigen receptor (CAR) or T cell receptor (TCR), comprising an extracellular ligand binding domain specific to a target antigen selected from: (i) a cancer cell-specific antigen, or a peptide antigen thereof in a complex with a major histocompatibility complex class I (MHC-I); or (ii) CEA cell adhesion molecule 5 (CEA), or a peptide antigen thereof in a complex with a major histocompatibility complex class I (MHC-I); and (b) a second receptor, optionally an inhibitory chimeric antigen receptor (iCAR), comprising an extracellular ligand binding domain specific to a non-target antigen selected from TNFRSF11A, ACHRB, ITGAE, TRPVl, and S
  • the disclosure provides methods of killing a plurality of cancer cell and/or treating cancer in a subject, comprising administering to the subject an effective amount of the immune cells or the pharmaceutical composition of the disclosure.
  • a plurality cancer cells express the target antigen.
  • a plurality of cancer cells do not express the non-target antigen.
  • the plurality of cancer cells have lost the non-target antigen due to loss of heterozygosity (LOH).
  • LHO heterozygosity
  • the disclosure provides methods of treating a cancer in a subject comprising: (a) determining the genotype of wild type cells and a plurality of cancer cells of the subject at a polymorphic locus selected from the group consisting of rsl716 (ITGAE R950W), rs2976230 (ITGAE V1019A/V1019G), rsl805034 (TNFRSF 11 A V 192 A) and rs35211496 (TNFRSF11 A H141 Y); (b) determining the expression of CEACAM5 in a plurality of cancer cells; and (c) administering a plurality of immune cells to the subject if the wild type cells are heterozygous for the polymorphic locus and the plurality of cancer cells are hemizygous for the polymorphic locus, and the plurality of cancer cells are CEA positive, wherein the plurality of immune cells comprise: (i) a first receptor, optionally a chimeric antigen receptor (CAR) or T cell receptor (CAR)
  • the disclosure provides methods of making a plurality of immune cells, comprising: (a) providing a plurality of immune cells, and (b) transforming the plurality of immune cells with the polynucleotide system of the disclosure.
  • kits comprising the immune cells or pharmaceutical compositions of the disclosure.
  • the kits further comprise instructions for use.
  • FIG. 1 is a crystal structure of TNFRSF11A (RANK) bound to TNFRS11 (RANKL), showing that the variant TNFRSF11 A epitopes are on the protein surface, and presumably accessible to an antibody.
  • RANK TNFRSF11A
  • RNKL TNFRS11
  • FIG. 2 shows an alignment of human Integrin alpha-E (ITGAE) with human Integrin alpha-X (ITGAX, P20702, IT AX HUM AN) .
  • ITGAE human Integrin alpha-E
  • ITGAX human Integrin alpha-X
  • P20702 human Integrin alpha-X
  • IT AX HUM AN IT AX HUM AN
  • FIG. 3 is a crystal structure of the inactive conformation of ITGAX, which has 27% identity to ITGAE. The positions of the ITGAE SNPs are indicated as labeled.
  • FIG. 4 is a table showing that the addressable colorectal cancer (CRC) patient population that can be treated with a CEA TCR in combination with a RANK blocker receptor is estimated at 2,000 to 5,000 patients, depending on which RANK variant is used.
  • the subtotal above of treatable patients is 5-11 thousand, and include the percentage of high CEA+ patients, as noted.
  • FIG. 5 shows the expression of CEA (CEACAM5) in normal tissues.
  • FIG. 6 shows the expression of TNFRSF11A (RANK) in normal tissues.
  • FIG. 7 shows the expression of CEA across all TCGA cancers (with tumor and normal samples.
  • FIG. 8 shows the expression of TNFGSF11 A across TCGA cancers (with tumors and normal samples).
  • FIG. 9 is a table showing estimated deaths in the U.S. by cancer site, statistics taken from the American Cancer Society.
  • compositions and methods for treating cancers using immune cells comprising a two receptor system responsive to loss of heterozygosity in the cancer cells.
  • the two-receptor system is expressed in immune cells, for example immune cells used in adoptive cell therapy, and targets activity of these immune cells to cancer cells exhibiting loss of heterozygosity.
  • the first receptor an activator, sometimes referred to herein as an A module
  • the second receptor a blocker, or inhibitor, sometimes referred to herein as a B module
  • Each receptor contains a ligand-binding domain (LBD) that binds a specific ligand.
  • LBD ligand-binding domain
  • Signals from the two receptors upon ligand binding are integrated by the immune cell in a cellular integrator system.
  • Differential expression of ligands for the first and second receptors in cancer and wild type cells for example through loss of heterozygosity of the locus encoding the inhibitory ligand in cancer cells, mediates activation of immune cells by target cancer cells that express the first activator ligand but not the second inhibitory ligand.
  • immune cells comprising the two receptor system described herein are used to treat CEA cell adhesion molecule 5 (CEA) positive cancers.
  • CEA cell adhesion molecule 5
  • the target antigen of the activator receptor is CEA, or a peptide antigen thereof, in a complex with a major histocompatibility complex class I (MHC-I).
  • MHC-I major histocompatibility complex class I
  • CEA is predominantly expressed in normal adult in GI tissues as a surface protein that can be cleaved from the membrane and released in soluble form.
  • CEA positive cancer cells could be specifically targeted with an appropriate therapeutic.
  • normal CEA expression in non-cancer (non-target) cells has prevented the effective use of CEA for targeted therapies such as adoptive cell therapies.
  • DLT dose-limiting toxicity
  • the ligand for the activator is a CEA peptide complexed with MHC class I, for example an MHC complex comprising an HLA-A*02.
  • this CEA targeted activator receptor is paired with an inhibitory receptor, which increases the safety window of the activator by blocking its cytolytic effect on normal CEA-positive tissues. Without wishing to be bound by theory, these tissues are thought to be mostly in the gastrointestinal tract. However, the activator receptor still directs the targeted killing of tumor cells by immune cells comprising the two-receptor system, as the tumor cells do not express the ligand for the inhibitor, or blocker, receptor.
  • the target for the second, inhibitory receptor is expressed by gastrointestinal (GI) tissues but not in cancer cells, and the inhibitory receptor recognizes this target as an inhibitory stimulus.
  • An exemplary target for the second inhibitory receptor is expressed on the surface of normal GI epithelial cells, and is lost from GI tumor cells through loss of heterozygosity (LOH), leaving a single allelic form in cancer cells that can be distinguished from other alleles via an allele-specific ligand binding domain on the inhibitory receptor.
  • LHO heterozygosity
  • Exemplary targets of the inhibitory receptor include, but are not limited to, TNF receptor superfamily member 11a (TNFRSF11 A, also called RANK), integrin subunit alpha E (ITGAE), cholinergic receptor nicotinic beta 1 subunit (ACHRB, or CHRNB), transient receptor potential cation channel subfamily V member 1 (TRPVl), and scavenger receptor class F member 1 (SREC, or SCARF).
  • TNF receptor superfamily member 11a TNFRSF11 A, also called RANK
  • IGAE integrin subunit alpha E
  • ACHRB cholinergic receptor nicotinic beta 1 subunit
  • TRPVl transient receptor potential cation channel subfamily V member 1
  • SREC scavenger receptor class F member 1
  • Each of these has a common nonsynonymous variant form, with the amino-acid alteration in its extracellular domain accessible to antibodies, which can be used as a B module target for a cellular integrator designed to safely treat GI cancer patients with engineered T cells activated by an activator receptor such as a CEA or CEA pMHC responsive activator receptor.
  • an activator receptor such as a CEA or CEA pMHC responsive activator receptor.
  • the compositions and methods of the disclosure can reduce or eliminate DLT caused by expression of CEA on normal GI tissue.
  • expression of CEA while limited, is sufficiently high in the GI tract to induce adverse events of a severity that has prevented further advancement of CEA as a target for adoptive cell therapy or immunotherapy in the clinic.
  • the disclosure provides methods of targeting CEA in cancer cells to treat CEA positive cancers using adoptive cell therapies by adding a second inhibitory receptor that blocks activation of the adoptive immune cells in the presence of a second ligand (a ligand other than CEA).
  • tumor cells that express CEA are attacked by the adoptive immune cells expressing the two receptors because these tumor cells express only the activator ligand, CEA.
  • normal cells that express CEA plus the blocking ligand are protected from the adoptive immune cells.
  • the inhibitory receptor prevents activation of immune cells by the CEA-targeted activator receptor.
  • the disclosure provides methods and compositions that allow the use of potent CEA CAR and TCRs that induce on-target toxicity, and renders these CEA targeted receptors useful as a therapeutic by mitigating their toxicity. None of the existing therapeutics that have been tested in the clinic, including cell and large-molecule therapies, provide a mechanism to protect normal CEA-positive tissues.
  • the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ⁇ 15%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • isolated means material that is substantially or essentially free from components that normally accompany it in its native state.
  • obtained or “derived” is used synonymously with isolated.
  • subject refers to a vertebrate, preferably a mammal, more preferably a human. Tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
  • treatment includes any beneficial or desirable effect, and may include even minimal improvement in symptoms. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.
  • prevention As used herein, “prevented,” “preventing” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of a symptom of disease. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease. As used herein, “prevention” and similar words also includes reducing the intensity, effect, symptoms and/or burden of disease prior to onset or recurrence. [0044] As used herein, the term “amount” refers to “an amount effective” or “an effective amount” of a virus to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results.
  • a “prophylactically effective amount” refers to an amount of a virus effective to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount is less than the therapeutically effective amount.
  • a “therapeutically effective amount” of a virus or cell may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the virus or cell to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or cell are outweighed by the therapeutically beneficial effects.
  • the term “therapeutically effective amount” includes an amount that is effective to “treat” a subject (e.g., a patient).
  • An “increased” or “enhanced” amount of a physiological response is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the level of activity in an untreated cell.
  • a “decrease” or “reduced” amount of a physiological response is typically a “statistically significant” amount, and may include an decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the level of activity in an untreated cell.
  • maintain or “preserve,” or “maintenance,” or “no change,” or “no substantial change,” or “no substantial decrease” refers generally to a physiological response that is comparable to a response caused by either vehicle, or a control molecule/composition.
  • a comparable response is one that is not significantly different or measurable different from the reference response.
  • sequence identity or “sequence homology” refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively.
  • techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence.
  • Two or more sequences can be compared by determining their “percent identity.”
  • the percent identity of two sequences, whether nucleic acid or amino acid sequences is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschul, et al., J. Mol. Biol.
  • the BLAST program defines identity as the number of identical aligned symbols (generally nucleotides or amino acids), divided by the total number of symbols in the shorter of the two sequences. The program may be used to determine percent identity over the entire length of the proteins being compared. Default parameters are provided to optimize searches with short query sequences in, for example, with the blastp program.
  • the program also allows use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). Ranges of desired degrees of sequence identity are approximately 80% to 100% and integer values therebetween. Typically, the percent identities between a disclosed sequence and a claimed sequence are at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.
  • exogenous is used herein to refer to any molecule, including nucleic acids, protein or peptides, small molecular compounds, and the like that originate from outside the organism.
  • endogenous refers to any molecule that originates from inside the organism (i.e., naturally produced by the organism).
  • MOI multiplicity of infection
  • multiplicity of infection which is the ratio of agents (e.g. viral particles) to infection targets (e.g. cells).
  • agents e.g. viral particles
  • infection targets e.g. cells
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • the term “about”, when immediately preceding a number or numeral, means that the number or numeral ranges plus or minus 10%.
  • a “target cell” refers to cell that is targeted by an adoptive cell therapy.
  • a target cell can be cancer cell, which can be killed by the transplanted T cells of the adoptive cell therapy.
  • Target cells of the disclosure express a target antigen, as described herein, and do not express a non-target antigen.
  • a “non-target cell” refers to cell that is not targeted by an adoptive cell therapy.
  • normal, healthy, non- cancerous cells are non-target cells.
  • Some, or all, non-target cells in a subject may express both the target antigen and the non-target antigen.
  • Non-target cells in a subject may express the non-target antigen irrespective of whether or not these cells also express the target antigen.
  • a “target antigen” refers to an antigen expressed by a target cell, such as a cancer cell. Expression of target antigen is not limited to target cells. Target antigens may be expressed by both cancer cells and normal, non-cancer cells in a subject.
  • a “non-target antigen” refers to an antigen that is expressed by normal, non-cancer cells and is not expressed in cancer cells. This difference in expression allows the inhibitory receptor to inhibit immune cell activation in the presence of non-target cells, but not in the presence of target cells.
  • Polymorphism refers to the presence of two or more variants of a nucleotide sequence in a population.
  • a polymorphism may comprise one or more base changes, an insertion, a repeat, or a deletion.
  • a polymorphism includes e.g. a simple sequence repeat (SSR) and a single nucleotide polymorphism (SNP), which is a variation, occurring when a single nucleotide of adenine (A), thymine (T), cytosine (C) or guanine (G) is altered.
  • SSR simple sequence repeat
  • SNP single nucleotide polymorphism
  • the disclosure provides a first receptor, comprising a first extracellular ligand binding domain specific to a target antigen comprising a cancer cell-specific antigen, or a peptide antigen thereof in a complex with a major histocompatibility complex class I (MHC-I).
  • the first receptor is an activator receptor, and mediates activation of an immune cell expressing the first receptor upon binding of the target antigen by the extracellular ligand binding domain of the first receptor.
  • the first receptor is a chimeric antigen receptor (CAR).
  • the first receptor is a T cell receptor (TCR).
  • the first receptor is humanized.
  • “humanized” refers to the replacement of a sequence or a subsequence in a transgene that has been isolated or derived from a non-human species with a homologous, or functionally equivalent, human sequence.
  • a humanized antibody can be created by grafting mouse CDRs into human framework sequences, followed by back substitution of certain human framework residues for the corresponding mouse residues from the source antibody.
  • the target antigen for the first receptor is a cancer cell specific antigen.
  • Any cell surface molecule expressed by the target cancer cells may be a suitable target antigen for the first receptor ligand binding domain.
  • a cell adhesion molecule, a cell-cell signaling molecule, an extracellular domain, a molecule involved in chemotaxis, a glycoprotein, a G protein-coupled receptor, a transmembrane, a receptor for a neurotransmitter or a voltage gated ion channel can be used as a target antigen.
  • the target antigen is a peptide antigen of a cancer cell- specific antigen in a complex with a major histocompatibility complex class I (MHC-I).
  • MHC-I major histocompatibility complex class I
  • Any molecule expressed by the target cancer cells and presented by the major histocompatibility complex class I (MHC-I) on the cancer cell surface as a peptide antigen (pMHC) may be a suitable target antigen for the first receptor extracellular ligand binding domain.
  • the cancer cell-specific antigen is CEA cell adhesion molecule 5 (CEA), or a peptide antigen thereof in a complex with a major histocompatibility complex class I (MHC-I).
  • CEA CEA cell adhesion molecule 5
  • MHC-I major histocompatibility complex class I
  • MHC-I The major histocompatibility complex class I
  • HLAs Human Leukocyte Antigens
  • cancer cell-specific pMHC antigens comprising any of HLA-A, HLA-B or HLA- C are envisaged as within the scope of the disclosure.
  • the cancer cell-specific antigen comprises HLA-A.
  • HLA-A receptors are heterodimers comprising a heavy a chain and smaller b chain. The a chain is encoded by a variant of HLA-A, while the b chain ⁇ 2-microglobulin) is an invariant.
  • the MHC-I comprises a human leukocyte antigen A*02 allele (HLA-A*02).
  • the cancer cell-specific antigen comprises HLA-B.
  • HLA-B Hundreds of versions (alleles) of the HLA-B gene are known, each of which is given a particular number (such as HLA-B27).
  • the cancer cell-specific antigen comprises HLA-C.
  • HLA-C belongs to the HLA class I heavy chain paralogues. This class I molecule is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). Over one hundred HLA-C alleles are known in the art.
  • the cancer cell-specific antigen is a colorectal cancer antigen.
  • the colorectal cancer antigen comprises CEA, tumor specific M2 isoform of pyruvate kinase (M2-PK), tissue inhibitor of matrix metalloproteinase 1 (TIMP1), MMP7, PTGS2, TP53, MYBL2, CK19, CK20, Ezrin, epidermal growth factor receptor (EGFR), vascular endothelial growth factor A (VEGF), epithelial glycoprotein 40 (Ep-CAM), or guanylyl cyclase 2C (GUCY2C).
  • the colorectal cancer antigen comprises CEA, or a peptide antigen thereof in a complex with a major histocompatibility complex class I (MHC-I). In some embodiments, the colorectal cancer antigen comprises EGFR, or a peptide antigen thereof in a complex with a major histocompatibility complex class I (MHC-I).
  • the cancer cell-specific antigen is CEA cell adhesion molecule 5 (CEA), or a peptide antigen thereof in a complex with a major histocompatibility complex class I (MHC-I).
  • CEA is a 180-kE)a glycoprotein tumor- associated protein expressed by a variety of cancer cells. These cancers include adenocarcinomas, colorectal cancers and selected other epithelial cancers, including colorectal adenocarcinomas.
  • CEA is also expressed in a variety of normal epithelial cells throughout the gastrointestinal tract, for example in the highly differentiated epithelial cells in the upper third of colonic crypts (see FIG. 7 for CEA expression).
  • CEA comprises an amino acid sequence of:
  • CEA comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1.
  • CEA isoform 2 is described in NCBI record number NP 001295327.1, the contents of which are incorporated by reference herein.
  • CEA comprises an amino acid sequence of:
  • CEA comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 15.
  • the cancer cell-specific antigen is a peptide antigen derived from CEA.
  • the peptide antigen is comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a subsequence of SEQ ID NO: 1.
  • the peptide antigen comprises a sequence identical to a subsequence of SEQ ID NO: 1.
  • Exemplary CEA peptide antigens include amino acids 691-699 of SEQ ID NO: 1, amino acids 605-613 of SEQ ID NO: 1, and amino acids 694-702 of SEQ ID NO: 1.
  • the CEA peptide antigen comprises, or consists essentially of, amino acids 691-699 of SEQ ID NO: 1.
  • the peptide antigen is comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a subsequence of SEQ ID NO: 15.
  • the peptide antigen comprises a sequence identical to a subsequence of SEQ ID NO: 15.
  • the CEA peptide antigen is complexed with MHC-I.
  • the MHC-I comprises a human leukocyte antigen A*02 allele (HLA-A*02).
  • the disclosure provides a first receptor, comprising a first extracellular ligand binding domain specific to a target antigen.
  • the target antigen comprises a cancer cell-specific antigen.
  • the cancer cell-specific antigen is CEA or a CEA-derived peptide antigen complexed with MHC-I, and the ligand binding domain of the first receptor recognizes and binds to the CEA antigen.
  • the ligand binding domain is an antigen binding domain.
  • antigen binding domains include, inter alia , ScFv, SdAb, nb-only domains, and TCR antigen binding domains derived from the TCR a and b chain variable domains.
  • the first extracellular ligand binding domain may be part of a contiguous polypeptide chain including, for example, a nb-only domain, a single domain antibody fragment (sdAb) or heavy chain antibodies HCAb, a single chain antibody (scFv) derived from a murine, humanized or human antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci.
  • sdAb single domain antibody fragment
  • HCAb heavy chain antibodies
  • scFv single chain antibody
  • the first extracellular ligand binding domain comprises an antigen binding domain that comprises an antibody fragment. In further aspects, the first extracellular ligand binding domain comprises an antibody fragment that comprises a scFv or an sdAb.
  • antibody refers to a protein, or polypeptide sequences derived from an immunoglobulin molecule, which specifically binds to an antigen.
  • Antibodies can be intact immunoglobulins of polyclonal or monoclonal origin, or fragments thereof and can be derived from natural or from recombinant sources.
  • antibody fragment or “antibody binding domain” refer to at least one portion of an antibody, or recombinant variants thereof, that contains the antigen binding domain, /. e. , an antigenic determining variable region of an intact antibody, that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen and its defined epitope.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, single-chain (sc)Fv (“scFv”) antibody fragments, linear antibodies, single domain antibodies (abbreviated “sdAb”) (either VL or VH), camelid VHH domains, and multi-specific antibodies formed from antibody fragments.
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • “Heavy chain variable region” or “VH” refers to the fragment of the heavy chain that contains three CDRs interposed between flanking stretches known as framework regions, these framework regions are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.
  • a scFv may have the VL and VH variable regions in either order, e.g. , with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.
  • antibody light chain refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations.
  • Kappa (“K”) and lambda (“l”) light chains refer to the two major antibody light chain isotypes.
  • recombinant antibody refers to an antibody that is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system.
  • the term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art.
  • nb domain refers to an antigen binding domain that consists essentially of a single T Cell Receptor (TCR) beta variable domain that specifically binds to an antigen in the absence of a second TCR variable domain.
  • TCR T Cell Receptor
  • the nb-only domain engages antigen using complementarity-determining regions (CDRs).
  • CDRs complementarity-determining regions
  • Each nb-only domain contains three complement determining regions (CDR1, CDR2, and CDR3). Additional elements may be combined provided that the nb domain is configured to bind the epitope in the absence of a second TCR variable domain.
  • the extracellular ligand binding domain of the first receptor comprises an antibody fragment, a single chain Fv antibody fragment (ScFv), or a b chain variable domain (nb).
  • the extracellular ligand binding domain of the first receptor comprises a TCR a chain variable domain and a TCR b chain variable domain.
  • the first extracellular ligand binding domain comprises a TCR ligand binding domain that binds to a CEA antigen.
  • the CEA antigen is complexed with MHC-I, and the MHC-I comprises an HLA-A*02 allele.
  • Exemplary TCR antigen binding domains that bind to and recognize CEA MHC-I HLA- A*02 antigens are described in Parkhurst et al. Molecular Therapy 2011 19(3): P620-626, the contents of which are incorporated herein by reference.
  • MHC-I complexed with HLA-A*02 MHC-I comprises a TCR alpha domain of TRAV8-1*01 and TRAJ6*01, and a TCR beta domain of TRBV26*01, TRBD1*01, TRBJ2- 7*01 and TRBC2.
  • the first extracellular ligand binding domain comprises complement determining regions (CDRs) selected from SEQ ID NOs: 3-12 or sequences having at least 85% or at least 95% identity thereto.
  • CDRs complement determining regions
  • the ligand binding domain of the first receptor comprises a TCR ligand binding domain.
  • the TCR a chain variable domain comprises a CDR-1 of TSITA (SEQ ID NO: 3), a CDR-2 of IRSNER (SEQ ID NO: 4) and a CDR-3 comprising ATDLTSGGNYK (SEQ ID NO: 5), ATDFTSGGNYK (SEQ ID NO: 6), ATDLTT GGNYK (SEQ ID NO: 7) or ATDFTTGGNYK (SEQ ID NO: 8); and the TCR b chain variable domain comprises a CDR-1 of KGHPV (SEQ ID NO: 9), a CDR-2 of FQNQEV (SEQ ID NO: 10), and a CDR-3 of ASSLGLGDYEQ (SEQ ID NO:
  • the TCR a chain variable domain comprises a CDR-1 of SEQ ID NO: 9, a CDR-2 of SEQ ID NO: 10 and a CDR-3 of SEQ ID NO: 11 or SEQ ID NO: 12; and the TCR b chain variable domain comprises a CDR-1 of SEQ ID NO: 3, a CDR-2 of SEQ ID NO: 4 and a CDR-3 comprising SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, or sequences having at least 85% or at least 95% identity thereto.
  • TCR alpha and beta chains comprising the CDRs from Table 1 are shown in Table 2 below. CDRs are underlined in the sequences in Table 2. In Table 2, the TCR alpha and TCR beta chains are separated by a P2A self-cleaving peptide ATNF SLLKQ AGD VEENPGP (SEQ ID NO: 52) and a GSG linker.
  • the first receptor comprises a sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical to a sequence or subsequence of any one of SEQ ID NOS: 16-31 or 36-51. In some embodiments, the first receptor comprises a sequence or subsequence of any one of SEQ ID NOS: 16-31 or 36-51.
  • the first receptor comprises a TCR alpha chain comprising or consisting essentially of amino acids 1-270 of any one of SEQ ID NOS: 16-31, or a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical thereto.
  • the first receptor comprises a TCR alpha chain comprising or consisting essentially of amino acids 1-270 of any one of SEQ ID NOS: 16-31.
  • the first receptor comprises a TCR beta chain comprising or consisting essentially of amino acids 293-598 of any one of SEQ ID NOS: 16-31, or a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical thereto.
  • the first receptor comprises a TCR beta chain comprising or consisting essentially of amino acids 293-598 of any one of SEQ ID NOS: 16-31.
  • the first receptor comprises a TCR alpha chain comprising amino acids 1-270 of any one of SEQ ID NOS: 16-31, and a TCR beta chain comprising amino acids 293-598 of any one of SEQ ID NOS: 16-31.
  • the first receptor comprises a TCR alpha chain comprising or consisting essentially of amino acids 1-268 of any one of SEQ ID NOS: 36-51, or a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical thereto.
  • the first receptor comprises a TCR alpha chain comprising or consisting essentially of amino acids 1-268 of any one of SEQ ID NOS: 36-51.
  • the first receptor comprises a TCR beta chain comprising or consisting essentially of amino acids 291-596 of any one of SEQ ID NOS: 36-51, or a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical thereto.
  • the first receptor comprises a TCR beta chain comprising or consisting essentially of amino acids 291-596 of any one of SEQ ID NOS: 36-51.
  • the first receptor comprises a TCR alpha chain comprising amino acids 1-268 of any one of SEQ ID NOS: 36-51, and a TCR beta chain comprising amino acids 291-596 of any one of SEQ ID NOS: 36-51.
  • the extracellular ligand binding domain of the first receptor is an ScFv.
  • the ScFv domain binds to CEA.
  • the ScFv is the ligand binding domain of a CAR.
  • Exemplary CAR sequences comprising CEA targeting ScFv domains are shown in Table 3 below. In Table 3, CDR sequences are underlined.
  • a CEA ScFv comprises a CDR-H1 of EFGMN (SEQ ID NO: 91), a CDR-H2 of WINTKTGEAT YVEEFKG (SEQ ID NO: 92), a CDR-H3 of WDFAYYVEAMDY (SEQ ID NO: 93) or WDFAHYFQTMDY (SEQ ID NO: 94), a CDR-L1 of KASQNVGTNVA (SEQ ID NO: 95) or K A S A A V GT Y V A (SEQ ID NO: 96), a CDR-L2 of SASYRYS (SEQ ID NO: 97) or SASYRKR (SEQ ID NO: 98), and a CDR-L3 of HQYYTYPLFT (SEQ ID NO: 99) or sequences having at least 85% or at least 95% identity thereto.
  • a CEA ScFv comprises a CDR-H1 of EFGMN (SEQ ID NO: 91), a CDR-H2 of WINTKTGEAT YVEEFKG (SEQ ID NO: 92), a CDR-H3 of WDFAYYVEAMDY (SEQ ID NO: 93) or WDFAHYFQTMDY (SEQ ID NO: 94), a CDR-L1 of KASQNVGTNVA (SEQ ID NO: 95) or K A S A A V GT Y V A (SEQ ID NO: 96), a CDR-L2 of SASYRYS (SEQ ID NO: 97) or SASYRKR (SEQ ID NO: 98) and a CDR-L3 of HQYYTYPLFT (SEQ ID NO: 99).
  • a CEA ScFv comprises a CDR-H1 of EFGMN (SEQ ID NO: 91), a CDR-H2 of WINTKT GE AT YVEEFKG (SEQ ID NO: 92), a CDR-H3 of WDFAYYVEAMDY (SEQ ID NO: 93), a CDR-L1 of KASQNVGTNVA (SEQ ID NO: 95), a CDR-L2 of SASYRYS (SEQ ID NO: 97) and a CDR-L3 of HQYYTYPLFT (SEQ ID NO: 99).
  • a CEA ScFv comprises a CDR-H1 of EFGMN (SEQ ID NO: 91), a CDR-H2 of WINTKTGEAT YVEEFKG (SEQ ID NO: 92), a CDR-H3 of WDFAYYVEAMDY (SEQ ID NO: 93), a CDR-L1 of KASAAVGTYVA (SEQ ID NO: 96), a CDR-L2 of SASYRKR (SEQ ID NO: 98), and a CDR-L3 of HQYYTYPLFT (SEQ ID NO: 99).
  • a CEA ScFv comprises a CDR-H1 of EFGMN (SEQ ID NO: 91), a CDR-H2 of WINTKT GE AT YVEEFKG (SEQ ID NO: 92), a CDR- H3 of WDFAHYFQTMDY (SEQ ID NO: 94), a CDR-L1 of KASAAVGTYVA (SEQ ID NO: 96), a CDR-L2 of SASYRKR (SEQ ID NO: 98), and a CDR-L3 of HQ YYTYPLFT (SEQ ID NO: 99).
  • the disclosure provides a first, activator receptor and immune cells comprising same.
  • the first receptor is a chimeric antigen receptor.
  • CARs chimeric antigen receptors
  • CARs may refer to artificial receptors derived from T-cell receptors and encompasses engineered receptors that graft an artificial specificity onto a particular immune effector cell.
  • CARs may be employed to impart the specificity of a monoclonal antibody onto a T cell, thereby allowing a large number of specific T cells to be generated, for example, for use in adoptive cell therapy.
  • CARs direct specificity of the cell to a tumor associated antigen, for example.
  • Exemplary CARs comprise an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumor associated antigen binding region.
  • CARs further comprise a hinge domain.
  • CARs comprise fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to a CD3 transmembrane domain and endodomain.
  • the specificity of other CAR designs may be derived from ligands of receptors (e.g., peptides).
  • CARs comprise domains for additional co-stimulatory signaling, such as CD3, 4-1BB, FcR, CD27, CD28, CD137, DAP 10, and/or 0X40.
  • molecules can be co-expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging, gene products that conditionally ablate the T cells upon addition of a pro-drug, homing receptors, cytokines, and cytokine receptors.
  • the extracellular ligand binding domain of the first receptor is fused to the extracellular domain of a CAR.
  • the CARs of the present disclosure comprise an extracellular hinge region. Incorporation of a hinge region can affect cytokine production from CAR-T cells and improve expansion of CAR-T cells in vivo.
  • Exemplary hinges can be isolated or derived from IgD and CD8 domains, for example IgGl. In some embodiments, the hinge is isolated or derived from CD8a or CD28.
  • the CARs of the present disclosure can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the CAR.
  • the transmembrane domain that naturally is associated with one of the domains in the CAR is used.
  • a CAR comprising a CD28 co-stimulatory domain might also use a CD28 transmembrane domain.
  • 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. Transmembrane regions may be isolated or derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9,
  • 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.
  • a short oligo- or polypeptide linker preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
  • a glycine-serine doublet provides a particularly suitable linker.
  • the cytoplasmic domain or otherwise the intracellular signaling domain of the CARs of the instant invention is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed.
  • effector function refers to a specialized function of a cell.
  • intracellular signaling domain refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire domain.
  • intracellular signaling domain is thus meant to include any truncated portion of one or more intracellular signaling domains sufficient to transduce the effector function signal.
  • intracellular signaling domains for use in the CARs of the instant disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.
  • TCR T cell receptor
  • the intracellular domain of CARs of the instant disclosure comprises at least one cytoplasmic activation domain.
  • the intracellular activation domain ensures that there is T-cell receptor (TCR) signaling necessary to activate the effector functions of the CAR T-cell.
  • the at least one cytoplasmic activation is a CD247 molecule (0" ⁇ 3z) activation domain, a stimulatory killer immunoglobulin-like receptor (KIR) KIR2DS2 activation domain, or a DNAX- activating protein of 12 kDa (DAP12) activation domain.
  • T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen- independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).
  • primary cytoplasmic signaling sequences those that initiate antigen-dependent primary activation through the TCR
  • secondary cytoplasmic signaling sequences those that act in an antigen- independent manner to provide a secondary or co-stimulatory signal
  • Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way.
  • Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs.
  • ITAM contains a tyrosine separated from a leucine or an isoleucine by any two other amino acids (YxxL).
  • the cytoplasmic domain contains 1, 2, 3, 4 or 5 ITAMs.
  • An exemplary IT AM containing cytoplasmic domain is the O ⁇ 3z activation domain.
  • IT AM containing primary cytoplasmic signaling sequences that can be used in the CARs of the instant disclosure include those derived from TOIz, FcRy, FcRp, CD3y, CD35, CD3e, O ⁇ 3z, CD5, CD22, CD79a, CD79b, and CD66d.
  • the cytoplasmic domain of the CAR can be designed to comprise the O ⁇ 3z signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the instant disclosure.
  • the cytoplasmic domain of the CAR can comprise a CD3z chain portion and a co-stimulatory domain.
  • the co-stimulatory domain refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule.
  • a costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen.
  • Examples of such molecules include the co-stimulatory domain is selected from the group consisting of IL- 2Rp, Fc Receptor gamma (FcRy), Fc Receptor beta (FcRP), CD3g molecule gamma (CD3y), CD35, CD3e, CD5 molecule (CD5), CD22 molecule (CD22), CD79a molecule (CD79a), CD79b molecule (CD79b), carcinoembryonic antigen related cell adhesion molecule 3 (CD66d), CD27 molecule (CD27), CD28 molecule (CD28), TNF receptor superfamily member 9 (4- IBB), TNF receptor superfamily member 4 (0X40), TNF receptor superfamily member 8 (CD30), CD40 molecule (CD40), programmed cell death 1 (PD-1), inducible T cell costimulatory (ICOS), lymphocyte function-associated antigen-1 (LFA-1), CD2 molecule (CD2), CD7 molecule (CD7), TNF superfamily member 14 (LIGHT), killer cell lect
  • the cytoplasmic domains within the cytoplasmic signaling portion of the CARs of the instant disclosure may be linked to each other in a random or specified order.
  • a short oligo- or polypeptide linker for example between 2 and 10 amino acids in length may form the linkage.
  • a glycine-serine doublet provides an example of a suitable linker.
  • TCRs T Cell Receptors
  • the disclosure provides a first, activator receptor and immune cells comprising same.
  • the first receptor is a T cell receptor (TCR).
  • a “TCR”, sometimes also called a “TCR complex” or “TCR/CD3 complex” refers to a protein complex comprising a TCR alpha chain, a TCR beta chain, and one or more of the invariant CD3 chains (zeta, gamma, delta and epsilon), sometimes referred to as subunits.
  • the TCR alpha and beta chains can be disulfide-linked to function as a heterodimer to bind to peptide-MHC complexes.
  • TCR alpha/beta heterodimer engages peptide-MHC, conformational changes in the TCR complex in the associated invariant CD3 subunits are induced, which leads to their phosphorylation and association with downstream proteins, thereby transducing a primary stimulatory signal.
  • the TCR alpha and TCR beta polypeptides form a heterodimer
  • CD3 epsilon and CD3 delta form a heterodimer
  • two CD3 zeta form a homodimer.
  • any suitable ligand binding domain may be fused to an extracellular domain, hinge domain or transmembrane of the TCRs described herein.
  • the ligand binding domain can be an antigen binding domain of an antibody or TCR, or comprise an antibody fragment, a nb only domain, a linear antibody, a single-chain variable fragment (scFv), or a single domain antibody (sdAb).
  • the ligand binding domain is fused to one or more extracellular domains or transmembrane domains of one or more TCR subunits.
  • the TCR subunit can be TCR alpha, TCR beta, CD3 delta, CD3 epsilon, CD3 gamma or CD3 zeta.
  • the ligand binding domain can be fused to TCR alpha, or TCR beta, or portions of the ligand binding can be fused to two subunits, for example portions of the ligand binding domain can be fused to both TCR alpha and TCR beta.
  • TCR subunits include TCR alpha, TCR beta, CD3 zeta, CD3 delta, CD3 gamma and CD3 epsilon. Any one or more of TCR alpha, TCR beta chain, CD3 gamma, CD3 delta, CD3 epsilon, or CD3 zeta, or fragments or derivative thereof, can be fused to one or more domains capable of providing a stimulatory signal of the disclosure, thereby enhancing TCR function and activity.
  • TCR transmembrane domains isolated or derived from any source are envisaged as within the scope of the disclosure.
  • the transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.
  • the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the TCR complex has bound to a target.
  • a transmembrane domain of particular use in this invention may include at least the transmembrane region(s) of e.g ., the alpha, beta or zeta chain of the TCR, CD3 delta, CD3 epsilon or CD3 gamma, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
  • the transmembrane domain can be attached to the extracellular region of a polypeptide of the TCR, e.g. , the antigen binding domain of the TCR alpha or beta chain, via a hinge, e.g. , a hinge from a human protein.
  • a hinge e.g. , a hinge from a human protein.
  • the hinge can be a human immunoglobulin (Ig) hinge, e.g. , an IgG4 hinge, or a CD8a hinge.
  • the hinge is isolated or derived from CD8a or CD28.
  • the extracellular ligand binding domain is attached to one or more transmembrane domains of the TCR.
  • the transmembrane domain comprises a TCR alpha transmembrane domain, a TCR beta transmembrane domain, or both.
  • the transmembrane comprises a CD3 zeta transmembrane domain.
  • a transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 15 amino acids of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 15 amino acids of the intracellular region).
  • one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 15 amino acids of the extracellular region
  • additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 15 amino acids of the intracellular region
  • 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, e.g., to minimize interactions with other members of the receptor complex.
  • the transmembrane domain may be a natural TCR transmembrane domain, a natural transmembrane domain from a heterologous membrane protein, or an artificial transmembrane domain.
  • the transmembrane domain may be a membrane anchor domain.
  • a natural or artificial transmembrane domain may comprise a hydrophobic a-helix of about 20 amino acids, often with positive charges flanking the transmembrane segment.
  • the transmembrane domain may have one transmembrane segment or more than one transmembrane segment. Prediction of transmembrane domains/segments may be made using publicly available prediction tools (e.g. TMHMM, Krogh et al.
  • Non-limiting examples of membrane anchor systems include platelet derived growth factor receptor (PDGFR) transmembrane domain, glycosylphosphatidylinositol (GPI) anchor (added post- translationally to a signal sequence) and the like.
  • PDGFR platelet derived growth factor receptor
  • GPI glycosylphosphatidylinositol
  • TCRs of the disclosure can comprise one or more intracellular domains.
  • the intracellular domain comprises one or more domains capable of providing a stimulatory signal to a transmembrane domain.
  • the intracellular domain comprises a first intracellular domain capable of providing a stimulatory signal and a second intracellular domain capable of providing a stimulatory signal.
  • the intracellular domain comprises a first, second and third intracellular domain capable of providing a stimulatory signal.
  • the intracellular domains capable of providing a stimulatory signal are selected from the group consisting of a CD28 molecule (CD28) domain, a LCK proto-oncogene, Src family tyrosine kinase (Lck) domain, a TNF receptor superfamily member 9 (4- IBB) domain, a TNF receptor superfamily member 18 (GITR) domain, a CD4 molecule (CD4) domain, a CD8a molecule (CD8a) domain, a FYN proto-oncogene, Src family tyrosine kinase (Fyn) domain, a zeta chain of T cell receptor associated protein kinase 70 (ZAP70) domain, a linker for activation of T cells (LAT) domain, lymphocyte cytosolic protein 2 (SLP76) domain, (TCR) alpha, TCR beta, CD3 delta, CD3 gamma and CD3 epsilon intracellular domains.
  • CD28 CD28
  • LCK
  • an intracellular domain comprises at least one intracellular signaling domain.
  • An intracellular signaling domain generates a signal that promotes a function a cell, for example an immune effector function of a TCR containing cell, e.g., a TCR-expressing T-cell.
  • the intracellular domain of the first receptor of the disclosure includes at least one intracellular signaling domain.
  • the intracellular domains of CD3 gamma, delta or epsilon comprise signaling domains.
  • the extracellular domain, transmembrane domain and intracellular domain are isolated or derived from the same protein, for example T-cell receptor (TCR) alpha, TCR beta, CD3 delta, CD3 gamma, CD3 epsilon or CD3 zeta.
  • TCR T-cell receptor
  • Examples of intracellular domains for use in activator receptors of the disclosure include the cytoplasmic sequences of the TCR alpha, TCR beta, CD3 zeta, and 4- IBB, and the intracellular signaling co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.
  • the intracellular signaling domain comprises a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the proteins responsible for primary stimulation, or antigen dependent stimulation.
  • the intracellular domain comprises a CD3 delta intracellular domain, a CD3 epsilon intracellular domain, a CD3 gamma intracellular domain, a CD3 zeta intracellular domain, a TCR alpha intracellular domain or a TCR beta intracellular domain.
  • the intracellular signaling domain comprises at least one stimulatory intracellular domain.
  • the intracellular signaling domain comprises a primary intracellular signaling domain, such as a CD3 delta, CD3 gamma and CD3 epsilon intracellular domain, and one additional stimulatory intracellular domain, for example a co-stimulatory domain.
  • the intracellular signaling domain comprises a primary intracellular signaling domain, such as a CD3 delta, CD3 gamma and CD3 epsilon intracellular domain, and two additional stimulatory intracellular domains.
  • Exemplary co-stimulatory intracellular signaling domains include those derived from proteins responsible for co-stimulatory signals, or antigen independent stimulation. Co-stimulatory molecules include, but are not limited to an MHC class I molecule,
  • BTLA a Toll ligand receptor
  • DAP 10 DAP 12
  • CD30 LIGHT
  • 0X40 CD2
  • CD27 CDS
  • ICAM-1 CDS
  • LFA-1 CDlla/CD18 4-1BB
  • CD137 TNF receptor superfamily member 9
  • CD28 molecule CD28.
  • a co-stimulatory protein 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.
  • SLAM proteins signaling lymphocytic activation molecules
  • Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, a ligand that specifically binds with CD83, CD4, and the like.
  • the co-stimulatory domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof.
  • the stimulatory domain comprises a co-stimulatory domain.
  • the co-stimulatory domain comprises a CD28 or 4-1BB co-stimulatory domain.
  • CD28 and 4- IBB are well characterized co-stimulatory molecules required for full T cell activation and known to enhance T cell effector function.
  • CD28 and 4- IBB have been utilized in chimeric antigen receptors (CARs) to boost cytokine release, cytolytic function, and persistence over the first-generation CAR containing only the CD3 zeta signaling domain.
  • CARs chimeric antigen receptors
  • co-stimulatory domains for example CD28 and 4-1BB domains
  • inclusion of co-stimulatory domains, for example CD28 and 4-1BB domains, in TCRs can increase T cell effector function and specifically allow co-stimulation in the absence of co-stimulatory ligand, which is typically down-regulated on the surface of tumor cells.
  • the stimulatory domain comprises a CD28 intracellular domain or a 4-1BB intracellular domain.
  • the disclosure provides a second receptor, comprising an extracellular ligand binding domain specific to a non-target antigen selected from TNFRSF11 A, ACHRB, ITGAE, TRPV1, and SREC, or an antigen peptide thereof in a complex with a major histocompatibility complex class I (MHC-I), wherein the non-target antigen comprises a polymorphism, and immune cells comprising same.
  • the second receptor is an inhibitory chimeric antigen receptor (iCAR).
  • the second receptor is humanized.
  • the disclosure provides a second receptor, which is an inhibitory receptor, comprising an extracellular ligand binding that can discriminate between single amino- acid variant alleles of a non-target antigen.
  • This ability to discriminate between allelic variants of a non-target antigen allows the second receptor to inhibit activation of immune cells comprising the second receptor in the presence of non-target cells that express that the allele recognized by the ligand binding domain.
  • activation of immune cells is not inhibited in the presence of target cells that have lost the allele, for example cancer cells that have lost one allele of a gene through loss of heterozygosity.
  • the non-target antigen is not expressed by the target cells, and is expressed by non-target cells.
  • the non-target antigen is expressed by healthy cells, i.e. cells that are not cancer cells.
  • the target cells are a plurality of cancer cells that have lost expression of the non-target antigen through loss of heterozygosity (LOH).
  • LHO heterozygosity
  • Any cell surface molecule expressed by the non-target cells that is not expressed by target cells may be a suitable non-target antigen for the second receptor extracellular ligand binding domain.
  • a cell adhesion molecule, a cell-cell signaling molecule, an extracellular domain, a molecule involved in chemotaxis, a glycoprotein, a G protein-coupled receptor, a transmembrane, a receptor for a neurotransmitter or a voltage gated ion channel can be used as a non-target antigen.
  • the non-target antigen is selected from the group consisting of a polymorphic variant of TNFRSF11A, ACHRB, ITGAE, TRPVl, and SREC.
  • the non-target antigen is an antigen peptide comprising a polymorphic residue of TNFRSF11A, ACHRB, ITGAE, TRPVl, or SREC, in a complex with a major histocompatibility complex class I (MHC-I).
  • MHC-I major histocompatibility complex class I
  • the target antigen is a peptide antigen of a cancer cell- specific antigen in a complex with a major histocompatibility complex class I (MHC-I).
  • Non-target MHC-1 (pMHC) antigens comprising any of HLA-A, HLA-B or HLA-C are envisaged as within the scope of the disclosure.
  • the non-target antigen comprises HLA-A.
  • the non-target antigen comprises a human leukocyte antigen A*02 allele (HLA-A*02).
  • the non-target antigen comprises HLA-B.
  • the non-target antigen comprises HLA-C.
  • the non-target antigen is selected from the group consisting of TNFRSF11A, ACHRB, ITGAE, TRPV1, and SREC.
  • CEA and TNFRSF11 A are low/absent in T cells, thus avoiding the in cis challenges of other ligands.
  • LOH frequencies for the TNFRSF11 A locus are extremely high (-90% in rectal cancer).
  • the non-target antigen comprises TNFRSFl 1 A or an antigen peptide thereof in a complex with MHC-I.
  • Human TNFRSFl 1 A is located on Chrl8q: 35,237,593 - 37,208,541 and is frequently lost through LOH in colorectal cancer cells.
  • TNFRSFl 1 A comprises an amino acid sequence of:
  • TNFRSFl 1 A comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 13. Polymorphic residues of TNFRSFl 1 A are marked as bold and underlined in SEQ ID NO: 13.
  • the non-target antigen comprises a polymorphism of TNFRSFl 1 A.
  • the non-target antigen comprises a peptide derived from TNFRSF11A comprising a polymorphic residue of TNFRSF11A.
  • Polymorphic residues of TNFRSF11A include amino acid residues 141 and 192 of SEQ ID NO: 13.
  • the non-target antigen comprises a peptide of TNFRSF11A comprising amino acid 141 (rs35211496, H141Y) or 192 (rsl805034, V192A) of SEQ ID NO: 13.
  • the polymorphism of TNFRSFl 1A comprises an H141/A192V allele of TNFRSFl 1A.
  • the polymorphism of TNFRSFl 1 A comprises a sequence of:
  • the polymorphism of TNFRSFl 1 A comprises an H141Y/A192 allele of TNFRSFl 1A. In some embodiments, the polymorphism of TNFRSFl 1 A comprises a sequence of:
  • the polymorphism of TNFRSFl 1 A comprises an H141Y/A192V allele of TNFRSFl 1A. In some embodiments, the polymorphism of TNFRSFl 1 A comprises a sequence of:
  • the non-target antigen comprises a TNFRSF11 A polymorphism with an A at position 192 of SEQ ID NO: 13
  • the second receptor comprises a ligand binding domain with a higher affinity for a TNFRSF11 A ligand with an A at position 192 of SEQ ID NO: 13 than for a TNFRSF11 A ligand with a V at position 192 of SEQ ID NO: 13.
  • the non-target antigen comprises a TNFRSF11 A polymorphism with a V at position 192 of SEQ ID NO: 13
  • the second receptor comprises a ligand binding domain with a higher affinity for a TNFRSF11 A ligand with an V at position 192 of SEQ ID NO: 13 than for a TNFRSF11 A ligand with an A at position 192 of SEQ ID NO: 13.
  • the non target antigen comprises a TNFRSF11 A polymorphism with an H at position 141 of SEQ ID NO: 13
  • the second receptor comprises a ligand binding domain with a higher affinity for a TNFRSF11 A ligand with an H at position 141 of SEQ ID NO: 13 than for a TNFRSF11 A ligand with a Y at position 141 of SEQ ID NO: 13.
  • the non-target antigen comprises a TNFRSF11 A polymorphism with a Y at position 141 of SEQ ID NO: 13
  • the second receptor comprises a ligand binding domain with a higher affinity for a TNFRSF11 A ligand with a Y at position 141 of SEQ ID NO: 13 than for a TNFRSF11 A ligand with an H at position 141 of SEQ ID NO: 13.
  • TNFRSF11 A isoform 1 is described in NCBI record number AH19185.1, the contents of which are incorporated by reference in their entirety.
  • TNFRSF11 A comprises an amino acid sequence of:
  • TNFRSFl 1 A comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 32.
  • Polymorphic residues of TNFRSF11A are marked as bold and underlined in SEQ ID NO: 32.
  • the non-target antigen comprises a polymorphism of TNFRSFl 1A.
  • Polymorphic residues of TNFRSF11A include 142 and 193 of SEQ ID NO: 32.
  • the non-target antigen comprises a peptide of TNFRSFl 1 A comprising amino acid 142 or 193 of SEQ ID NO: 32.
  • the non-target antigen comprises integrin Alpha-E (ITGAE) or an antigen peptide thereof in a complex with MHC-I.
  • ITGAE comprises two polymorphisms in the extracellular domain: R950W (rsl716) with a minor allele frequency (MAF) of 0.2654 and V1019A/V1019G (rs2976230) with an MAF of 0.282.
  • R950W rsl716
  • MAF minor allele frequency
  • V1019A/V1019G rs2976230
  • MAF MAF of 0.282.
  • Human ITGAE (R950/V10109) is described in NCBI record number NP 002199.3 the contents of which are incorporated by reference herein in their entirety.
  • ITGAE comprises an amino acid sequence of:
  • ITGAE comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 14. Polymorphic residues of ITGAE are marked as bold and underlined in SEQ ID NO: 14.
  • the polymorphism of ITGAE comprises an R950W/V1019 allele of ITGAE. In some embodiments, the polymorphism of ITGAE comprises a sequence of:
  • the polymorphism of ITGAE comprises an R950/V1019A allele of ITGAE. In some embodiments, the polymorphism of ITGAE comprises a sequence of:
  • the polymorphism of ITGAE comprises an R950/V1019G allele of ITGAE. In some embodiments, the polymorphism of ITGAE comprises a sequence of:
  • the polymorphism of ITGAE comprises an R950W/V1019 allele of ITGAE. In some embodiments, the polymorphism of ITGAE comprises a sequence of:
  • the polymorphism of ITGAE comprises an R950W/V1019A allele of ITGAE. In some embodiments, the polymorphism of ITGAE comprises a sequence of:
  • the polymorphism of ITGAE comprises an R950W/V1019G allele of ITGAE. In some embodiments, the polymorphism of ITGAE comprises a sequence of:
  • the non-target antigen comprises a polymorphism of ITGAE.
  • the non-target antigen comprises a peptide derived from ITGAE comprising a polymorphic residue of ITGAE.
  • Polymorphic residues of ITGAE include amino acids 950 and 1019 of SEQ ID NO: 14.
  • the non-target antigen comprises a peptide of ITGAE comprising amino acid 950 or 1019 of SEQ ID NO: 14.
  • the non-target antigen comprises a ITGAE polymorphism with a R at position 950 of SEQ ID NO: 14
  • the second receptor comprises a ligand binding domain with a higher affinity for an ITGAE ligand with an R at position 950 of SEQ ID NO: 14 than for an ITGAE ligand with a W at position 950 of SEQ ID NO: 14.
  • the non-target antigen comprises a ITGAE polymorphism with a W at position 950 of SEQ ID NO: 14
  • the second receptor comprises a ligand binding domain with a higher affinity for an ITGAE ligand with an W at position 950 of SEQ ID NO: 14 than for an ITGAE ligand with an R at position 950 of SEQ ID NO: 14.
  • the non-target antigen comprises a ITGAE polymorphism with a V at position 1019 of SEQ ID NO: 14
  • the second receptor comprises a ligand binding domain with a higher affinity for an ITGAE ligand with a V at position 1019 of SEQ ID NO: 14 than for an ITGAE ligand with an A or G at position 1019 of SEQ ID NO: 14.
  • the non-target antigen comprises a ITGAE polymorphism with an A at position 1019 of SEQ ID NO: 14
  • the second receptor comprises a ligand binding domain with a higher affinity for an ITGAE ligand with an A at position 1019 of SEQ ID NO: 14 than for an ITGAE ligand with a V or G at position 1019 of SEQ ID NO: 14.
  • the non-target antigen comprises an ITGAE polymorphism with a G at position 1019 of SEQ ID NO: 14, and the second receptor comprises a ligand binding domain with a higher affinity for an ITGAE ligand with a G at position 1019 of SEQ ID NO: 14 than for an ITGAE ligand with a V or A at position 1019 of SEQ ID NO: 14.
  • the non-target antigen comprises ACHRB (also called CHRNB, or CHRNB1) or an antigen peptide thereof in a complex with MHC-I. Human ACHRB is described in NCBI record number NP 000738.2 the contents of which are incorporated by reference herein in their entirety.
  • ACHRB comprises an amino acid sequence of:
  • ACHRB comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 33.
  • Polymorphic residues of ACHRB are marked as bold and underlined in SEQ ID NO: 33.
  • the non-target antigen comprises a polymorphism of ACHRB.
  • the non-target antigen comprises a peptide derived from ACHRB comprising a polymorphic residue of ACHRB.
  • Polymorphic residues of ACHRB include 32 of SEQ ID NO: 33.
  • the non-target antigen comprises a peptide of ACHRB comprising amino acid 32 of SEQ ID NO: 33.
  • the non-target antigen comprises a peptide of ACHRB comprising an E at amino acid 32 of SEQ ID NO: 33.
  • the non-target antigen comprises a peptide of ACHRB comprising a G at amino acid 32 of SEQ ID NO: 33.
  • the non-target antigen comprises TRPVl or an antigen peptide thereof in a complex with MHC-I.
  • Human TRPVl is described in NCBI record number NP 542435.2, the contents of which are incorporated by reference herein in their entirety.
  • TRPVl comprises an amino acid sequence of:
  • TRPV1 comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 94%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 34. Polymorphic residues of TRPV1 are marked as bold and underlined in SEQ ID NO: 34.
  • the non-target antigen comprises a polymorphism of TRPV1.
  • the non-target antigen comprises a peptide derived from TRPV1 comprising a polymorphic residue of TRPV1.
  • Polymorphic residues of TRPV1 include positions 585, 459 and 469 of SEQ ID NO: 34.
  • the non-target antigen comprises a peptide of TRPV1 comprising amino acid 585, 459 or 469 of SEQ ID NO: 34.
  • the non-target antigen comprises a peptide of TRPV1 comprising an I at amino acid 585 of SEQ ID NO: 34.
  • the non target antigen comprises a peptide of TRPV1 comprising a V at amino acid 585 of SEQ ID NO: 34.
  • the non-target antigen comprises SREC or an antigen peptide thereof in a complex with MHC-I.
  • Human SREC isoform 1 is described in NCBI record number NP 003684.2, the contents of which are incorporated by reference herein in their entirety.
  • SREC comprises an amino acid sequence of:
  • SREC comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 94%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 35. Polymorphic residues of SREC are marked as bold and underlined in SEQ ID NO: 35.
  • the non-target antigen comprises a polymorphism of SREC.
  • the non-target antigen comprises a peptide derived from SREC comprising a polymorphic residue of SREC.
  • Polymorphic residues of SREC include positions 339 and 425 of SEQ ID NO: 35.
  • the non-target antigen comprises a peptide of SREC comprising amino acid 339 or 425 of SEQ ID NO: 35. In some embodiments, the non-target antigen comprises a peptide of SREC comprising an A at amino acid 425 of SEQ ID NO: 35. In some embodiments, the non-target antigen comprises a peptide of SREC comprising a V at amino acid 425 of SEQ ID NO: 35.
  • ICRs Inhibitory Chimeric Antigen Receptors
  • the disclosure provides a second receptor that is an inhibitory chimeric antigen receptor (iCAR).
  • the inhibitory CAR may comprise an extracellular ligand binding domain that binds to and recognizes the non-target antigen or a peptide derivative thereof in a MHC-I complex.
  • inhibitory chimeric antigen receptor refers to an antigen-binding domain that is fused to an intracellular signaling domain capable of transducing an inhibitory signal that inhibits or suppresses the immune activity of an immune cell.
  • iCARs have immune cell inhibitory potential, and are distinct and distinguishable from CARs, which are receptors with immune cell activating potential.
  • CARs are activating receptors as they include intracellular stimulatory and/or co-stimulatory domains.
  • iCARs are inhibiting receptors that contain intracellular inhibitory domains.
  • inhibitory signal refers to signal transduction or changes in protein expression in an immune cell resulting in suppression of an immune response (e.g., decrease in cytokine production). Inhibition or suppression of an immune cell can selective and/or reversible, or not selective and/or reversible.
  • iCARs of the disclosure may comprise an extracellular ligand binding domain. Any type of ligand binding domain that can regulate the activity of a receptor in a ligand dependent manner is envisaged as within the scope of the instant disclosure.
  • the ligand binding domain is an antigen binding domain.
  • antigen binding domains include, inter alia , ScFv, SdAb, nb-only domains, and TCR antigen binding domains derived from the TCR a and b chain variable domains.
  • Any type of antigen binding domain is envisaged as within the scope of the instant disclosure.
  • the extracellular ligand binding domain of the second receptor binds to and recognizes a polymorphic variant of TNFRSF11, ACHRB, ITGAE, TRPV1, SREC, or an antigen peptide thereof in a complex with a major histocompatibility complex class I (MHC-I).
  • MHC-I major histocompatibility complex class I
  • the extracellular ligand binding domain of the second receptor is an ScFv.
  • the extracellular ligand binding domain of the second receptor is fused to the extracellular domain of an iCAR.
  • the iCARs of the present disclosure comprise an extracellular hinge region.
  • Exemplary hinges can be isolated or derived from IgD and CD8 domains, for example IgGl.
  • the hinge is isolated or derived from CD 8 a or CD28.
  • the iCARs of the present disclosure can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the iCAR.
  • 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. Transmembrane regions may be isolated or derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9,
  • 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.
  • a short oligo- or polypeptide linker preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the intracellular domain of the iCAR.
  • a glycine-serine doublet provides a particularly suitable linker.
  • the disclosure provides an iCAR comprising an intracellular domain.
  • the intracellular domain of the iCARs of the instant invention is responsible for inhibiting activation of the immune cells comprising the iCAR, which would otherwise be activated in response to activation signals by the first receptor.
  • the inhibitory intracellular domain comprises an immunoreceptor tyrosine-based inhibitory motif (ITIM).
  • the inhibitory intracellular domain comprising an ITIM can be isolated or derived from an immune checkpoint inhibitor such as CTLA-4 and PD-1.
  • CTLA-4 and PD-1 are immune inhibitory receptors expressed on the surface of T cells, and play a pivotal role in attenuating or terminating T cell responses.
  • an inhibitory intracellular domain is isolated from human tumor necrosis factor related apoptosis inducing ligand (TRAIL) receptor and CD200 receptor 1.
  • TRAIL tumor necrosis factor related apoptosis inducing ligand
  • CD200 receptor 1 CD200 receptor 1.
  • the TRAIL receptor comprises TR10A, TR10B or TR10D.
  • an inhibitory intracellular domain is isolated from phosphoprotein membrane anchor with glycosphingolipid microdomains 1 (PAG1). In some embodiments, an inhibitory intracellular domain is isolated from leukocyte immunoglobulin like receptor B1 (LILRBl).
  • the inhibitory domain is isolated or derived from a human protein, for example a human TRAIL receptor, CTLA-4, PD-1, PAG1 or LILRBl protein.
  • the inhibitory domain comprises an intracellular domain, a transmembrane or a combination thereof. In some embodiments, the inhibitory domain comprises an intracellular domain, a transmembrane domain, a hinge region or a combination thereof.
  • the inhibitory domain is isolated or derived from killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 2 (KIR3DL2), killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 3 (KIR3DL3), leukocyte immunoglobulin like receptor B1 (LIRl, also called LIR-1 and LILRBl), programmed cell death 1 (PD-1), Fc gamma receptor IIB (FcgRIIB), killer cell lectin like receptor K1 (NKG2D), CTLA-4, a domain containing a synthetic consensus ITIM, a ZAP70 SH2 domain (e.g., one or both of the N and C terminal SH2 domains), or ZAP70 KI K369A (kinase inactive ZAP70).
  • KIR3DL2 three Ig domains and long cytoplasmic tail 2
  • KIR3DL3DL3 three Ig domains and long cytoplasmic tail 3
  • LIRl leukocyte immunoglobul
  • the inhibitory domain is isolated or derived from a human protein.
  • the second, inhibitory receptor comprises an inhibitory domain. In some embodiments, the second, inhibitory receptor comprises an inhibitory intracellular domain and/or an inhibitory transmembrane domain. In some embodiments, the inhibitory intracellular domain is fused to an intracellular domain of an iCAR. In some embodiments, the inhibitory intracellular domain is fused to the transmembrane domain of an iCAR.
  • the disclosure provides polynucleotides encoding the sequence(s) of the first and second receptors of the disclosure.
  • the disclosure provides immune cells comprising the polynucleotides and vectors described herein.
  • the sequence of the first and/or second receptor is operably linked to a promoter.
  • the sequence encoding the first receptor is operably linked to a first promoter
  • the sequence encoding the second receptor is operably linked to a second promoter.
  • the disclosure provides vectors comprising the polynucleotides described herein.
  • the first receptor is encoded by a first vector and the second receptor is encoded by second vector.
  • both receptors are encoded by a single vector.
  • the first and second receptors are encoded by a single vector.
  • Methods of encoding multiple polypeptides using a single vector will be known to persons of ordinary skill in the art, and include, inter alia , encoding multiple polypeptides under control of different promoters, or, if a single promoter is used to control transcription of multiple polypeptides, use of sequences encoding internal ribosome entry sites (IRES) and/or self-cleaving peptides.
  • IRS internal ribosome entry sites
  • Exemplary self-cleaving peptides include T2A, P2A, E2A and F2A self-cleaving peptides.
  • the T2A self- cleaving peptide comprises a sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO:
  • the P2A self-cleaving peptide comprises a sequence of ATNF SLLKQ AGD VEENPGP (SEQ ID NO: 52).
  • the E2A self cleaving peptide comprises a sequence of QCTNYALLKLAGDVESNPGP (SEQ ID NO:
  • the F2A self-cleaving peptide comprises a sequence of VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 111).
  • the vector is an expression vector, i.e. for the expression of the first and/or second receptor in a suitable cell.
  • 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 expression of natural or synthetic nucleic acids encoding receptors is typically achieved by operably linking a nucleic acid encoding the receptor or portions thereof to a promoter, and incorporating the construct into an expression vector.
  • the vectors can be suitable for replication and integration eukaryotes.
  • Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.
  • the polynucleotides encoding the receptors can be cloned into a number of types of vectors.
  • the polynucleotides can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • the expression vector may be provided to cells, such as immune cells, 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. (2001, 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.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).
  • 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.
  • Additional promoter elements e.g., enhancers, regulate the frequency of transcriptional initiation.
  • these are located in the region 30-110 basepairs (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.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • Another example of a suitable promoter is Elongation Growth Factor-la (EF-la).
  • 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, 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.
  • 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.
  • the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
  • Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.
  • Reporter genes are used for identifying potentially transfected or transduced 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 ah, 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 transcription.
  • the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art.
  • a host cell e.g., mammalian, bacterial, yeast, or insect cell
  • the expression vector can be transferred into a host cell by physical, chemical, or biological means.
  • Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). One method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.
  • 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.
  • 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).
  • assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.
  • “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR
  • biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.
  • Immune Cells include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and
  • the disclosure provides immune cells comprising the receptors, vectors and polynucleotides described herein.
  • the immune cells comprise: (a) first receptor, comprising a first extracellular ligand binding domain specific to a target antigen selected from: (i) a cancer cell-specific antigen, or a peptide antigen thereof in a complex with a major histocompatibility complex class I (MHC-I); or (ii) CEA cell adhesion molecule 5 (CEA), or a peptide antigen thereof in a complex with a major histocompatibility complex class I (MHC-I); and (b) a second receptor, comprising a second extracellular ligand binding specific to a non-target antigen selected from TNFRSF11, ACHRB, ITGAE, TRPV1, and SREC, or an antigen peptide thereof in a complex with a major histocompatibility complex class I (MHC-I), wherein the non-target antigen comprises a polymorphism.
  • the first receptor is a CAR or TCR.
  • immune cell refers to a cell involved in the innate or adaptive (acquired) immune systems.
  • exemplary innate immune cells include phagocytic cells such as neutrophils, monocytes and macrophages, Natural Killer (NK) cells, polymophonuclear leukocytes such as neutrophils eosinophils and basophils and mononuclear cells such as monocytes, macrophages and mast cells.
  • innate immune cells include phagocytic cells such as neutrophils, monocytes and macrophages, Natural Killer (NK) cells, polymophonuclear leukocytes such as neutrophils eosinophils and basophils and mononuclear cells such as monocytes, macrophages and mast cells.
  • NK Natural Killer
  • Immune cells with roles in acquired immunity include lymphocytes such as T-cells and B-cells.
  • T-cell refers to a type of lymphocyte that originates from a bone marrow precursor that develops in the thymus gland.
  • T-cells which develop upon migration to the thymus, which include, helper CD4+ T- cells, cytotoxic CD8+ T cells, memory T cells, regulatory CD4+ T-cells and stem memory T-cells.
  • helper CD4+ T- cells include, helper CD4+ T- cells, cytotoxic CD8+ T cells, memory T cells, regulatory CD4+ T-cells and stem memory T-cells.
  • cytotoxic CD8+ T cells include CD4+ T-cells which develop upon migration to the thymus.
  • memory T cells include, regulatory CD4+ T-cells and stem memory T-cells.
  • stem memory T-cells Different types of T-cells can be distinguished by the ordinarily skilled artisan based on their expression of markers. Methods of distinguishing between T-cell types will be readily apparent to the ordinarily skilled artisan.
  • the first receptor and the second receptor together specifically activate the immune cell in the presence of the target cell.
  • the immune cell is selected form the group consisting of T cells, B cells and Natural Killer (NK) cells.
  • the immune cell is a T cell, for example a CD8+ CD4- T cell.
  • the immune cell is non-natural. In some embodiments, the immune cell is isolated.
  • CD3+ T cells can be isolated from PBMCs using a CD3+ T cell negative isolation kit (Miltenyi), according to manufacturer’s instructions.
  • T cells can be cultured at a density of 1 x 10 L 6 cells/mL in X-Vivo 15 media supplemented with 5% human A/B serum and 1% Pen/strep in the presence of CD3/28 Dynabeads (1:1 cell to bead ratio) and 300 Units/mL of IL-2 (Miltenyi).
  • T cells can be transduced with viral vectors, such as lentiviral vectors using methods known in the art.
  • the viral vector is transduced at a multiplicity of infection (MOI) of 5.
  • Cells can then be cultured in IL-2 or other cytokines such as combinations of IL-7/15/21 for an additional 5 days prior to enrichment.
  • cytokines such as combinations of IL-7/15/21
  • Methods of isolating and culturing other populations of immune cells, such as B cells, or other populations of T cells, will be readily apparent to the person of ordinary skill in the art. Although this method outlines a potential approach it should be noted that these methodologies are rapidly evolving. For example excellent viral transduction of peripheral blood mononuclear cells can be achieved after 5 days of growth to generate a >99% CD3+ highly transduced cell population.
  • the T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041, 10040846; and U.S. Pat. Appl. Pub. No. 2006/0121005.
  • T cells of the instant disclosure are expanded and activated in vitro.
  • the T cells of the instant disclosure are expanded in vitro by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells.
  • T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody.
  • a ligand that binds the accessory molecule is used for co-stimulation of an accessory molecule on the surface of the T cells.
  • a population of T cells can be contacted with an anti-CD3 antibody and an anti- CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells.
  • an anti-CD3 antibody and an anti-CD28 antibody can be used.
  • an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besantjon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328, 1999; Garland et al., J. Immunol Meth. 227(1- 2):53-63, 1999).
  • 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.
  • 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. In certain embodiments of the invention, the ratio of anti CD28 antibody to anti CD3 antibody bound to the beads is greater than 2:1. [0222] 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. As those of ordinary skill in the art can readily appreciate, the ratio of particles to cells may depend on particle size relative to the target cell.
  • 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.
  • a ratio of 1 : 1 cells to beads is used.
  • ratios will vary depending on particle size and on cell size and type.
  • the cells such as T cells
  • the beads and the cells are subsequently separated, and then the cells are cultured.
  • 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 to contact the T cells.
  • the cells for example, CD4+ T cells
  • beads for example, DYNABEADS CD3/CD28 T paramagnetic beads at a ratio of 1 : 1
  • any cell concentration may be used.
  • it may be desirable to significantly decrease the volume in which particles and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and particles. For example, in one embodiment, a concentration of about 2 billion cells/ml is used.
  • greater than 100 million cells/ml is used.
  • a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
  • a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used.
  • concentrations of 125 or 150 million cells/ml can be used.
  • cells that are cultured at a density of lxlO 6 cells/mL are used.
  • the mixture may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between.
  • the beads and T cells are cultured together for 2-3 days.
  • Conditions appropriate for T 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-g, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFp, 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-g, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFp, and TNF-a or any other
  • 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 T cells.
  • the media comprises X-VIVO- 15 media supplemented with 5% human A/B serum, 1% penicillin/streptomycin (pen/strep) and 300 Units/ml of IL-2 (Miltenyi).
  • the T 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 T cells comprising TCRs, CARs and iCARS of the disclosure are autologous. Prior to expansion and genetic modification, a source of T cells is obtained from a subject.
  • Immune cells such as T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, any number of T cell lines available in the art, may be used. In certain embodiments of the present invention, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FicollTM separation. [0228] In some embodiments, cells from the circulating blood of an individual are obtained by apheresis.
  • the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations.
  • 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.
  • a semi-automated “flow-through” centrifuge for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5
  • the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca2+-free, Mg2+-free PBS, PlasmaLyte A, or other saline solution with or without buffer.
  • the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
  • immune cells such as 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.
  • Specific subpopulations of immune cells, such as T cells, B cells, or CD4+ T cells can be further isolated by positive or negative selection techniques.
  • T cells are isolated by incubation with anti-CD4 -conjugated beads, for a time period sufficient for positive selection of the desired T cells.
  • Enrichment of an immune cell population, such as a T cell population, by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • One method is cell sorting and/or selection via negative magnetic immune-adherence 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 CD 14, CD20, CD lib, CD 16, HLA- DR, and CD8.
  • the concentration of cells and surface can be varied.
  • it may be desirable to significantly decrease the volume in which beads and cells are mixed together i.e., increase the concentration of cells, to ensure maximum contact of cells and beads.
  • the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10° C or at room temperature.
  • T cells for stimulation can also be frozen after a washing step.
  • the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population.
  • the cells may be suspended in a freezing solution.
  • one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80° C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20° C or in liquid nitrogen.
  • Pharmaceutical Compositions may be used as well as uncontrolled freezing immediately at -20° C or in liquid nitrogen.
  • compositions comprising immune cells comprising the first and second receptors of the disclosure and a pharmaceutically acceptable diluent, carrier or excipient.
  • 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; and preservatives.
  • 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 such as glycine
  • chelating agents such as EDTA or glutathione
  • kits for killing a plurality of cancer cells, or treating cancer, in a subject comprising administering to the subject a therapeutically effective amount of a composition comprising immune cells comprising the first and second receptors of the disclosure.
  • the immune cells express both receptors in the same cell.
  • the plurality of cancer cells express the target antigen.
  • the plurality cancer cells of the subject express CEA.
  • CEA positive cancers include colorectal cancer, pancreatic cancer, esophageal cancer, gastric cancer, lung adenocarcinoma, head and neck cancer, diffuse large B cell cancer or acute myeloid leukemia cancer.
  • a plurality of cancer cells do not express a polymorphic allele of TNFRSF11, ACHRB, ITGAE, TRPV1, or SREC.
  • the cancer cells have lost an allele of TNFRSF11, ACHRB, ITGAE, TRPV1, or SREC through loss of heterozygosity at that locus.
  • the disclosure provides methods of treating a cancer in a subject comprising: (a) determining the genotype of wild type cells and a plurality of cancer cells of the subject at a polymorphic locus selected from the group consisting of rsl716 (ITGAE R950W), rs2976230 (ITGAE V1019A/V1019G), rsl805034 (TNFRSF 11 A V 192 A) and rs35211496 (TNFRSF11 A H141 Y); (b) determining the expression of CEA in a plurality of cancer cells; and (c) administering a plurality of immune cells to the subject if the wild type cells are heterozygous for the polymorphic locus and the plurality of cancer cells are hemizygous for the polymorphic locus, and the plurality of cancer cells are CEA positive, wherein the plurality of immune cells comprise: (i) a first receptor, optionally a chimeric antigen receptor (CAR) or T cell receptor (TCR
  • SNP genotyping methods include, inter alia , PCR based methods such as dual-probe TaqMan assays, array based hybridization methods and sequencing.
  • Methods of measuring the expression of the target antigen in cancer or wild type cells from a subject will be readily apparent to persons of ordinary skill in the art. These include, inter alia , methods of measuring RNA expression such as RNA sequencing and reverse transcription polymerase chain reaction (RT-PCR), as well as methods of measuring protein expression such as immunohistochemistry based methods.
  • the first ligand comprises IMIGVLVGV (SEQ ID NO: 2). In some embodiments, the first ligand is complexed with a major histocompatibility complex comprising a human leukocyte antigen A*02 allele (HLA-A*02).
  • the plurality of cancer cells comprises a TNFRSF11 A 192A allele at rsl805034, and the ligand binding domain of the second receptor has a higher affinity for a TNFRSF11 A ligand with an V at position 192 of SEQ ID NO: 13 than for a TNFRSFl 1 A ligand with an A at position 192 of SEQ ID NO: 13.
  • the plurality of cancer cells comprises a TNFRSFl 1 A 192V allele at rsl805034, and the ligand binding domain of the second receptor has a higher affinity for a TNFRSFl 1 A ligand with an A at position 192 of SEQ ID NO: 13 than for a TNFRSFl 1 A ligand with an V at position 192 of SEQ ID NO: 13.
  • the plurality of cancer cells comprises a TNFRSFl 1 A 141H allele at rs35211496, and the ligand binding domain of the second receptor has a higher affinity for a TNFRSFl 1 A ligand with an Y at position 141 of SEQ ID NO: 13 than for a TNFRSF11 A ligand with a H at position 141 of SEQ ID NO: 13.
  • the plurality of cancer cells comprises a TNFRSF11 A 141 Y allele at rs35211496, and wherein the ligand binding domain of the second receptor has a higher affinity for a TNFRSFl 1 A ligand with a H at position 141 of SEQ ID NO:
  • the plurality of cancer cells comprises an ITGAE 950R allele at rsl716, and the ligand binding domain of the second receptor has a higher affinity for an ITGAE ligand with a W at position 950 of SEQ ID NO: 14 than for an ITGAE ligand with an R at position 950 of SEQ ID NO: 14.
  • the plurality of cancer cells comprises an ITGAE 950W at rsl716, and the ligand binding domain of the second receptor has a higher affinity for an ITGAE ligand with an R at position 950 of SEQ ID NO: 14 than for an ITGAE ligand with a W at position 950 of SEQ ID NO: 14.
  • the plurality of cancer cells comprises an ITGAE 1019V allele at rs2976230, and the ligand binding domain of the second receptor has a higher affinity for an ITGAE ligand with an A or G at position 1019 of SEQ ID NO: 14 than for an ITGAE ligand with an W at position 1019 of SEQ ID NO: 14.
  • the plurality of cancer cells comprises an ITGAE 1019A allele at rs2976230, and the ligand binding domain of the second receptor has a higher affinity for an ITGAE ligand with an V or G at position 1019 of SEQ ID NO: 14 than for an ITGAE ligand with an A at position 1019 of SEQ ID NO: 14.
  • the plurality of cancer cells comprises an ITGAE 1019G allele at rs2976230, and the ligand binding domain of the second receptor has a higher affinity for an ITGAE ligand with a V or A at position 1019 of SEQ ID NO: 14 than for an ITGAE ligand with a G at position 1019 of SEQ ID NO: 14.
  • the immune cells are T cells.
  • the immune cells are allogeneic or autologous.
  • the second receptor increases the specificity of the immune cells for the CEA positive cancer cells compared to immune cells that express the first receptor but do not express the second receptor. In some embodiments, the immune cells have reduced side effects compared to immune cells that express the first receptor but do not express the second receptor.
  • kits and articles of manufacture comprising the polynucleotides and vectors encoding the receptors described herein, and immune cells comprising the receptors described herein.
  • the kit comprises articles such as vials, syringes and instructions for use.
  • the kit comprises a polynucleotide or vector comprising a sequence encoding one or more receptors of the disclosure.
  • the kit comprises a plurality of immune cells comprising the first and second receptors as described herein.
  • the plurality of immune cells comprises a plurality of T cells.
  • the kit further comprises instructions for use.
  • Example 1 Identification ofTNFRSHA as a Blocker
  • Chrl8q 35,237,593 - 37,208,54 was identified as the regions that was most frequently lost due to loss of heterozygosity in colorectal cancers.
  • Surface proteins encoded on Chr. 18q were filtered for those expressed by normal colon cells.
  • MAF minor allele frequency
  • VEP Variants with MAF ⁇ 0.1 were removed VEP (Variant Effect Predictor) was run, and only missense variants that were in protein coding regions were kept The following genes were removed: o genes without transmembrane domains o genes located in Golgi, ER, mitochondria, endosome, nucleus membrane o genes that are not highly expressed in colon (GTEx expression level ⁇ 5 TPM) o genes that are amplified as opposed to deleted loss of heterozygosity of candidate genes was checked in the TCGA Copy Number Portal
  • Candidate genes were checked for other variants in Ensembl Genome Browser o If there are variants, the location of the variation was checked (is it in the extracellular domain?)
  • TPM Transcripts per Kilobase Million (The Genotype-Tissue Expression, GTEx project, gtexportal . org/home)
  • Results in Table 7 are from the TCGA Copy Number Portal.
  • TNFRS11 A (RANK) was identified as a target for a blocker receptor to pair with a CEA TCR or CAR activator.
  • the TNFRSF11 A (RANK) receptor is expressed in a wide range of normal tissues, including the gut. Gut expression includes expression in the colon, wherein the median normal TNFRSF11 A colon expression is 23 transcripts/cell. Maximum CRC CEA expression in the colon is 8,780 transcripts/cell.
  • TNFRSF11 A is also expressed in the esophagus. The median normal esophagus TNFRSF11 A expression is 2 transcripts/cell.
  • TNFRSF11 A encodes a 616-residue protein that binds RANKL (the target of denosumab). It includes a 28 amino acid signal peptide, a 184 amino acid extracellular domain, a 21 amino acid transmembrane domain and a 383 amino acid intracellular domain.
  • TNFRSFl 1 A contains two common nonsynonymous variants, rsl805034 (V192A) which has an MAF of 0.4, and rs35211496 (H141 Y) which has MAF of about 0.2.

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Abstract

L'invention concerne des cellules immunitaires comprenant un premier récepteur activateur et un second récepteur inhibiteur, et des méthodes de préparation et d'utilisation de celles-ci pour le traitement du cancer.
PCT/US2021/036627 2020-06-11 2021-06-09 Compositions et méthodes de traitement de cancers WO2021252635A1 (fr)

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US11433100B2 (en) 2020-08-20 2022-09-06 A2 Biotherapeutics, Inc. Compositions and methods for treating ceacam positive cancers
US11602543B2 (en) 2020-08-20 2023-03-14 A2 Biotherapeutics, Inc. Compositions and methods for treating mesothelin positive cancers

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11433100B2 (en) 2020-08-20 2022-09-06 A2 Biotherapeutics, Inc. Compositions and methods for treating ceacam positive cancers
US11602543B2 (en) 2020-08-20 2023-03-14 A2 Biotherapeutics, Inc. Compositions and methods for treating mesothelin positive cancers

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