# US 2014/0157443 A1 · Methods and compositions for detecting and modulating a novel mtor complex

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- Number: US 2014/0157443 A1
- Document ID: US20140157443A1
- Status: Published application
- Kind: A1
- Application number: 14/111,447
- Filed: 2012-04-12
- Published: 2014-06-05
- Earliest claimed: 2011-04-14
- Assignees: St. Jude Children's Research Hospital
- Inventors: Gerard C. Grosveld; Frank C. Harwood; Ramon I. Klein-Geltink
- Web page: https://patentgrep.com/patent/US20140157443A1

## CPC

- C07K 16/40: against enzymes
- A01K 67/0275: Genetically modified vertebrates, e.g. transgenic
- A61K 31/4709: Non-condensed quinolines and containing further heterocyclic rings
- A61K 31/5377: not condensed and containing further heterocyclic rings, e.g. timolol
- C07K 14/4702: {Regulators; Modulating activity}
- C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes}
- C12N 9/12: transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N 9/1205: {Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases}
- C12Q 1/485: {involving kinase}
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- G01N 33/57557: {of other specific parts of the body, e.g. brain}
- G01N 33/57595: {involving intracellular compounds}
- G01N 2333/91215: with a definite EC number (2.7.1.-)
- G01N 2500/00: Screening for compounds of potential therapeutic value
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Provided herein is a novel mTOR-comprising complex, mT-ORC3, which comprises mTOR and the Ets transcription factor TEL2. Specific mTORC3 binding agents and modulating agents are provided, along with kits and methods for the detection of mTORC3. Methods of modulating the activity of mTORC3 or modulating cell growth and/or survival are also provided. Further provided are methods for screening for mTORC3 binding agents and for mTORC3 modulating agents. Various methods of diagnosis and treatment are further provided.

## Claims

### Claim 1

An isolated mTOR complex 3 (mTORC3), wherein said mTORC3 comprises:
a) a first polypeptide comprising an mTOR polypeptide or a biologically active variant or fragment thereof; and
b) a second polypeptide comprising a TEL2 polypeptide or a biologically active variant or fragment thereof.

### Claim 2 (depends on 1)

The isolated mTORC3 of claim 1, wherein said first polypeptide comprises the mTOR polypeptide of SEQ ID NO: 2, a biologically active fragment thereof, or a biologically active variant thereof having at least 80% sequence identity to the mTOR polypeptide of SEQ ID NO: 2.

### Claim 3 (depends on 1)

The isolated mTORC3 of claim 1, wherein said second polypeptide comprises the TEL2 polypeptide of SEQ ID NO: 4, a biologically active fragment thereof, or a biologically active variant thereof having at least 80% sequence identity to the TEL2 polypeptide of SEQ ID NO: 4.

### Claim 4 (depends on 1)

The isolated mTORC3 of claim 1, wherein said mTORC3 has a molecular weight greater than 1.5 MDa.

### Claim 5 (depends on 1)

The isolated mTORC3 of claim 1, wherein said mTORC3 further comprises 4E-BP1.

### Claim 6

An antibody that specifically binds to an mTOR complex 3 (mTORC3), wherein said mTORC3 comprises:
a) a first polypeptide comprising an mTOR polypeptide or a biologically active variant or fragment thereof; and
b) a second polypeptide comprising a TEL2 polypeptide or a biologically active variant or fragment thereof.

### Claim 7 (depends on 6)

The antibody of claim 6, wherein said first polypeptide comprises the mTOR polypeptide of SEQ ID NO: 2, a biologically active fragment thereof, or a biologically active variant thereof having at least 80% sequence identity to the mTOR polypeptide of SEQ ID NO: 2.

### Claim 8 (depends on 6)

The antibody of claim 6, wherein said second polypeptide comprises the TEL2 polypeptide of SEQ ID NO: 4, a biologically active fragment thereof, or a biologically active variant thereof having at least 80% sequence identity to the TEL2 polypeptide of SEQ ID NO: 4.

### Claim 9 (depends on 6)

The antibody of claim 6, wherein said mTORC3 further comprises 4E-BP1.

### Claim 10 (depends on 6)

The antibody of claim 6, wherein said antibody is a monoclonal antibody.

### Claim 11 (depends on 6)

The antibody of claim 6, wherein said antibody is bispecific, wherein a first antigen binding domain specifically interacts with said first polypeptide and said second antigen binding domain specifically interacts with said second polypeptide.

### Claim 12 (depends on 6)

The antibody of claim 6, wherein said antibody specifically inhibits the activity of an mTOR complex 3.

### Claim 13

A mixture of a first and a second antibody comprising:
a) a first antibody having a first chemical moiety, wherein said first antibody specifically binds to a first polypeptide comprising an mTOR polypeptide or a biologically active variant or fragment thereof; and,
b) a second antibody having a second chemical moiety, wherein said second antibody specifically binds to a second polypeptide comprising a TEL2 polypeptide or a biologically active variant or fragment thereof;
wherein said first and said second chemical moiety allow for the detection of an mTOR complex 3 (mTORC3).

### Claim 14 (depends on 13)

The mixture of said first and said second antibody of claim 13, wherein said first polypeptide comprises the mTOR polypeptide of SEQ ID NO: 2, a biologically active fragment thereof, or a biologically active variant thereof having at least 80% sequence identity to the mTOR polypeptide of SEQ ID NO: 2.

### Claim 15 (depends on 13)

The mixture of said first and said second antibody of claim 13, wherein said second polypeptide comprises the TEL2 polypeptide of SEQ ID NO: 4, a biologically active fragment thereof, or a biologically active variant thereof having at least 80% sequence identity to the TEL2 polypeptide of SEQ ID NO: 4.

### Claim 16

A compound that specifically inhibits the activity of an mTOR complex 3.

### Claim 17 (depends on 16)

The compound of claim 16, wherein said compound comprises a small molecule.

### Claim 18 (depends on 6)

A pharmaceutical composition comprising the antibody of claim 6 and a pharmaceutically acceptable carrier.

### Claim 19

A kit for determining the level of expression of a polynucleotide encoding an mTOR polypeptide and a polynucleotide encoding a TEL2 polypeptide in a sample comprising:
a) a first polynucleotide or pair of polynucleotides capable of specifically detecting or specifically amplifying a polynucleotide encoding an mTOR polypeptide or a biologically active variant or fragment thereof; and
b) a second polynucleotide or pair of polynucleotides capable of specifically detecting or specifically amplifying a polynucleotide encoding a TEL2 polypeptide or a biologically active variant or fragment thereof;
wherein the encoded polypeptides are capable of associating with one another in an mTOR complex 3 (mTORC3).

### Claim 20 (depends on 19)

The kit of claim 19, wherein
a) the first polynucleotide or pair of polynucleotides is capable of specifically detecting or amplifying a polynucleotide encoding the amino acid sequence of SEQ ID NO:2 or a sequence having at least 80% sequence identity to SEQ ID NO:2; and,
b) the second polynucleotide or pair of polynucleotides is capable of specifically detecting or amplifying a polynucleotide encoding the amino acid sequence of SEQ ID NO:4 or a sequence having at least 80% sequence identity to SEQ ID NO:4.

### Claim 21 (depends on 19)

The kit of claim 19, wherein:
a) said first pair of polynucleotides comprises a first and a second primer that share sufficient sequence homology or complementarity to said polynucleotide encoding an mTOR polypeptide or biologically active variant or fragment thereof to specifically amplify said polynucleotide encoding an mTOR polypeptide or biologically active variant or fragment thereof; and
b) said second pair of polynucleotides comprises a third and a forth primer that share sufficient sequence homology or complementarity to said polynucleotide encoding an TEL2 polypeptide or biologically active variant or fragment thereof to specifically amplify said polynucleotide encoding a TEL2 polypeptide or biologically active variant or fragment thereof.

### Claim 22 (depends on 19)

The kit of claim 19, wherein said kit comprises:
a) a first polynucleotide that can specifically detect said polynucleotide encoding an mTOR polypeptide or biologically active variant or fragment thereof, wherein said first polynucleotide comprises at least one DNA molecule of a sufficient length of contiguous nucleotides identical or complementary to SEQ ID NO:1; and
b) a second polynucleotide that can specifically detect said polynucleotide encoding a TEL2 polypeptide or biologically active variant or fragment thereof, wherein said second polynucleotide comprises at least one DNA molecule of a sufficient length of contiguous nucleotides identical or complementary to SEQ ID NO:3.

### Claim 23 (depends on 19)

The kit of claim 19, wherein said kit comprises
a) a first polynucleotide that hybridizes under stringent conditions to the sequence of SEQ ID NO:1; and
b) a second polynucleotide that hybridizes under stringent conditions to the sequence of SEQ ID NO:3.

### Claim 24 (depends on 6)

A kit for detecting the presence of an mTOR complex 3 (mTORC3) in a sample comprising an antibody of claim 6.

### Claim 25

A method for detecting the level of expression of a polynucleotide encoding an mTOR polypeptide and a polynucleotide encoding a TEL2 polypeptide in a sample comprising
a) contacting said sample with
i) a first and a second primer capable of specifically amplifying a first amplicon of a polynucleotide encoding an mTOR polypeptide or a biologically active variant or fragment thereof; and,
ii) a third and a fourth primer capable of specifically amplifying a second amplicon of a polynucleotide encoding a TEL2 polypeptide or a biologically active variant or fragment thereof;
wherein the encoded polypeptides are capable of associating with one another in an mTOR complex 3 (mTORC3);
b) amplifying said first and said second amplicon; and
c) detecting said first and said second amplicon and thereby detecting the level of expression of a polynucleotide encoding an mTOR polypeptide and a polynucleotide encoding a TEL2 polypeptide in said sample.

### Claim 26 (depends on 25)

The method of claim 25, wherein said first and said second primer comprise at least 8 consecutive polynucleotides of SEQ ID NO: 1 or the complement thereof, and said third and said fourth primer comprise at least 8 consecutive polynucleotides of SEQ ID NO:3 or the complement thereof.

### Claim 27

A method for detecting the level of expression of a polynucleotide encoding an mTOR polypeptide and a polynucleotide encoding a TEL2 polypeptide in a sample, said method comprising:
a) contacting said sample with
i) a first polynucleotide capable of specifically detecting a polynucleotide encoding an mTOR polypeptide or a biologically active variant or fragment thereof; and,
ii) a second polynucleotide capable of specifically detecting a polynucleotide encoding a TEL2 polypeptide or a biologically active variant or fragment thereof;
wherein the encoded polypeptides are capable of associating with one another in an mTOR complex 3 (mTORC3); and
b) detecting said polynucleotide encoding the mTOR polypeptide or an active variant or fragment thereof and the polynucleotide encoding the TEL2 polypeptide or an active variant or fragment thereof.

### Claim 28 (depends on 6)

A method for detecting an mTOR complex 3 (mTORC3), said method comprising:
a) contacting a sample with the antibody of claim 6; and
b) detecting a complex comprising the mTORC3 and the antibody; thereby detecting said mTORC3.

### Claim 29

A method for identifying an mTOR complex 3 (mTORC3) binding agent, wherein the method comprises the steps of:
a) contacting the mTORC3 or a cell comprising the mTORC3 with a test compound; and
b) detecting a complex comprising the mTORC3 and the test compound.

### Claim 30 (depends on 29)

The method of claim 29, wherein said method further comprises assaying the kinase activity of the mTORC3 to thereby determine if said test compound modulates the activity of the mTORC3 complex.

### Claim 31 (depends on 29)

The method of claim 29, wherein said method further comprises contacting at least one of an mTORC1, an mTORC2, a cell comprising an mTORC1, and a cell comprising an mTORC2, and assaying for a complex comprising the mTORC 1 or mTORC2 and the test compound, thereby determining if said test compound specifically binds to the mTORC3 complex.

### Claim 32 (depends on 29)

The method of claim 29, wherein said method is a cell-free method.

### Claim 33

A method for screening for an mTOR complex 3 (mTORC3) antagonist, wherein said method comprises contacting mTORC3 with a test compound and assaying the kinase activity of the mTORC3 to thereby identify a compound that reduces the activity of the mTORC3.

### Claim 34 (depends on 33)

The method of claim 33, wherein said method further comprises contacting at least one of an mTORC1, an mTORC2, a cell comprising an mTORC1, and a cell comprising an mTORC2, and assaying the kinase activity of the mTORC1 or mTORC2, thereby determining if said mTORC3 antagonist specifically reduces the activity of the mTORC3 complex.

### Claim 35 (depends on 29)

The method of claim 29, wherein said test compound comprises an antibody.

### Claim 36 (depends on 29)

The method of claim 29, wherein said test compound comprises a small molecule.

### Claim 37

A method for reducing cell growth or cell survival, said method comprising contacting a cell expressing an mTOR complex 3 (mTORC3) with a specific mTORC3 antagonist.

### Claim 38 (depends on 37)

The method of claim 37, wherein said specific mTORC3 complex antagonist comprises an antibody.

### Claim 39 (depends on 37)

The method of claim 37, wherein said specific mTORC3 complex antagonist comprises a small molecule.

### Claim 40

A method for treating or preventing a cancer in a subject in need thereof, wherein said method comprises administering to the subject a therapeutically effective amount of a specific mTORC3 complex antagonist.

### Claim 41 (depends on 40)

The method of claim 40, wherein said specific mTORC3 complex antagonist comprises an antibody.

### Claim 42 (depends on 40)

The method of claim 40, wherein said specific mTORC3 complex antagonist comprises a small molecule.

### Claim 43

A method for diagnosing a cancer in a subject or determining the severity of a cancer in a subject, wherein said method comprises the steps of:
a) evaluating the level of an mTOR complex 3 (mTORC3) in a biological sample from said subject;
b) comparing the level of said mTORC3 in the biological sample of said subject to a control; and
c) diagnosing said cancer in said subject, wherein the level of said mTORC3 in the biological sample of said subject is relatively higher than the control; or determining the cancer of said subject is more severe than the control, wherein the level of mTORC3 in the biological sample of said subject is relatively higher than the control.

### Claim 44 (depends on 6, 43)

The method of claim 43, wherein said evaluating the level of mTORC3 in a sample of said subject comprises detecting the level of mTORC3 with an antibody of claim 6.

### Claim 45 (depends on 43)

The method of claim 43, wherein said method further comprises administering to the subject a therapeutically effective amount of a specific mTORC3 complex antagonist.

### Claim 46 (depends on 40)

The method of claim 40, wherein said cancer comprises a solid tumor cancer.

### Claim 47 (depends on 40)

The method of claim 40, wherein said cancer comprises a pediatric cancer.

### Claim 48 (depends on 40)

The method of claim 40, wherein said cancer is selected from the group consisting of acute lymphocytic leukemia, acute myeloid leukemia, ependymoma, Ewing's sarcoma, glioblastoma, medulloblastoma, neuroblastoma, osteosarcoma, rhabdomyosarcoma, rhabdoid cancer, nephroblastoma (Wilm's tumor), hepatocellular carcinoma, esophageal carcinoma, liposarcoma, bladder cancer, gastric cancer, myxofibrosarcoma, colon cancer, kidney cancer, histiosarcoma, ovarian cancer, endometrial carcinoma, lung cancer, and breast cancer.

### Claim 49

A method for treating or preventing a non-B cell cancer in a subject in need thereof, wherein said method comprises administering to the subject a therapeutically effective amount of a specific TEL2 antagonist, wherein said non-B cell cancer is selected from the group consisting of ependymoma, Ewing's sarcoma, glioblastoma, medulloblastoma, neuroblastoma, osteosarcoma, rhabdomyosarcoma, rhabdoid cancer, nephroblastoma (Wilm's tumor), esophageal carcinoma, liposarcoma, bladder cancer, gastric cancer, myxofibrosarcoma, colon cancer, kidney cancer, histiosarcoma, ovarian cancer, endometrial carcinoma, lung cancer, and breast cancer.

### Claim 50

A method for diagnosing a non-B cell cancer in a subject or determining the severity of a non-B cell cancer in a subject, wherein said method comprises the steps of:
a) evaluating the expression of TEL2 in a biological sample from said subject;
b) comparing the expression of TEL2 in said biological sample of said subject with a control; and
c) diagnosing said non-B cell cancer in said subject, wherein the expression level of TEL2 in the biological sample of said subject is relatively higher than the control; or determining the non-B cell cancer of said subject is more severe than the control, wherein the expression level of TEL2 in the sample of said subject is relatively higher than the control, wherein said non-B cell cancer is selected from the group consisting of ependymoma, Ewing's sarcoma, glioblastoma, medulloblastoma, neuroblastoma, osteosarcoma, rhabdomyosarcoma, rhabdoid cancer, nephroblastoma (Wilm's tumor), esophageal carcinoma, liposarcoma, bladder cancer, gastric cancer, myxofibrosarcoma, colon cancer, kidney cancer, histiosarcoma, ovarian cancer, endometrial carcinoma, lung cancer, and breast cancer.

### Claim 51 (depends on 50)

The method of claim 50, wherein said method further comprises administering to the subject a therapeutically effective amount of a specific mTORC3 complex antagonist or a specific TEL2 antagonist.

### Claim 52

A method for treating an Epstein-Barr virus infection in a subject in need thereof, wherein said method comprises administering a therapeutically effective amount of a specific mTORC3 complex antagonist.

### Claim 53

A non-human transgenic animal having stably incorporated into its genome a polynucleotide that encodes a TEL2 polypeptide or a biologically active variant or fragment thereof, wherein said polynucleotide is heterologous to the genome.

### Claim 54 (depends on 53)

The non-human transgenic animal of claim 53, wherein said polynucleotide encodes the TEL2 polypeptide of SEQ ID NO: 4, a biologically active fragment thereof, or a biologically active variant thereof having at least 80% sequence identity to the TEL2 polypeptide of SEQ ID NO: 4.

### Claim 55 (depends on 53)

The non-human transgenic animal of claim 53, wherein said non-human transgenic animal comprises a single copy of the stably incorporated polynucleotide.

### Claim 56 (depends on 53)

The non-human transgenic animal of claim 53, wherein said polynucleotide encoding the TEL2 polypeptide further comprises a TEL2 promoter.

### Claim 57 (depends on 53)

The non-human transgenic animal of claim 53, wherein said non-human transgenic animal is heterozygous for a p53 mutation that inhibits p53 activity.

### Claim 58 (depends on 53)

The non-human transgenic animal of claim 53, wherein said non-human transgenic animal is a rodent.

### Claim 59 (depends on 13)

A pharmaceutical composition comprising the mixture of a first and a second antibody of claim 13 and a pharmaceutically acceptable carrier.

### Claim 60 (depends on 16)

A pharmaceutical composition comprising the compound of claim 16 and a pharmaceutically acceptable carrier.

### Claim 61 (depends on 13, 43)

The method of claim 43, wherein evaluating the level of mTORC3 in a sample of said subject comprises detecting the level of mTORC3 with the mixture of a first and a second antibody of claim 13.

### Claim 62 (depends on 43)

The method of claim 43, wherein said cancer comprises a solid tumor cancer.

### Claim 63 (depends on 43)

The method of claim 43, wherein said cancer comprises a pediatric cancer.

### Claim 64 (depends on 43)

The method of claim 43, wherein said cancer is selected from the group consisting of acute lymphocytic leukemia, acute myeloid leukemia, ependymoma, Ewing's sarcoma, glioblastoma, medulloblastoma, neuroblastoma, osteosarcoma, rhabdomyosarcoma, rhabdoid cancer, nephroblastoma (Wilm's tumor), hepatocellular carcinoma, esophageal carcinoma, liposarcoma, bladder cancer, gastric cancer, myxofibrosarcoma, colon cancer, kidney cancer, histiosarcoma, ovarian cancer, endometrial carcinoma, lung cancer, and breast cancer.