# US 5,705,151 A · Gene therapy for T cell regulation

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- Number: US 5,705,151 A
- Document ID: US5705151A
- Status: Granted
- Kind: A
- Application number: 08/446,918
- Filed: 1995-05-18
- Granted: 1998-01-06
- Earliest claimed: 1995-05-18
- Assignees: National Jewish Center for Immunology and Respiratory Medicine
- Inventors: Steve W. Dow; Robyn E. Elmslie
- Web page: https://patentgrep.com/patent/US5705151A

## CPC

- C12N 15/62: DNA sequences coding for fusion proteins
- A61K 39/39: characterised by the immunostimulating additives, e.g. chemical adjuvants
- C07K 14/31: from Staphylococcus (G)
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K 2039/53: {DNA (RNA) vaccination}
- A61K 2039/55522: {Cytokines; Lymphokines; Interferons}
- C07K 2319/55: containing a fusion with a toxin, e.g. diphteria toxin
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- 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)

## Abstract

The present invention provides a nucleic acid-based therapeutic composition to treat an animal with disease by controlling the activity of effector cells, including T cells, macrophages, monocytes and/or natural killer cells, in the animal. The present invention also relates to methods of gene therapy involving different modes of administration of a therapeutic composition to treat animals with different types of diseases. Also included in the present invention are recombinant molecules for use in a therapeutic composition and recombinant cells useful as a tumor vaccine. Therapeutic compositions of the present invention include superantigen-encoding nucleic acid molecules, either in the presence or absence of a cytokine-encoding nucleic acid molecule, depending upon the disease being treated.

## Claims

### Claim 1

A method to treat a mammal that has cancer, said method comprising administering to said mammal a therapeutic composition comprising:
(a) a liposome delivery vehicle; and
(b) a recombinant construct comprising a first isolated nucleic acid sequence encoding a superantigen, and a second isolated nucleic acid sequence encoding a cytokine, said first and second nucleic acid sequences being operatively linked to one or more transcription control sequences;
wherein said first and said second nucleic acid sequences encoding said superantigen and said cytokine, respectively, are coexpressed at or adjacent to said cancer; and
wherein coexpression of said superantigen and said cytokine produces a result selected from the group consisting of alleviation of said cancer, reduction of a tumor associated with said cancer, elimination of a tumor associated with said cancer, prevention of meta static cancer, and stimulation of effector cell immunity against said cancer.

### Claim 2

A method to treat a mammal that has cancer, said method comprising administering to said mammal a therapeutic composition comprising:
(a) a liposome delivery vehicle;
(b) a first recombinant construct comprising an isolated nucleic acid sequence encoding a superantigen operatively linked to one or more transcription control sequences; and
(c) a second recombinant construct comprising an isolated nucleic acid sequence encoding a cytokine operatively linked to one or more transcription control sequences;
wherein said nucleic acid sequences encoding said superantigen and said cytokine, respectively, are coexpressed at or adjacent to said cancer; and
wherein coexpression of said superantigen and said cytokine produces a result selected from the group consisting of alleviation of said cancer, reduction of a tumor associated with said cancer, elimination of a tumor associated with said cancer, prevention of metastatic cancer, and stimulation of effector cell immunity against said cancer.

### Claim 3

A method to treat a mammal that has cancer, said method comprising:
(a) removing cells of said mammal;
(b) transfecting said cells in vitro with a recombinant construct comprising a first isolated nucleic acid sequence encoding a superantigen, and a second isolated nucleic acid sequence encoding a cytokine, said first and second nucleic acid sequences being operatively linked to one or more transcription control sequences; and
(c) reintroducing said transfected cells to said mammal;
wherein said first and said second nucleic acid sequences encoding said superantigen and said cytokine, respectively, are coexpressed at or adjacent to said cancer; and
wherein coexpression of said superantigen and said cytokine produces a result selected from the group consisting of alleviation of said cancer, reduction of a tumor associated with said cancer, elimination of a tumor associated with said cancer, prevention of metastatic cancer, and stimulation of effector cell immunity against said cancer.

### Claim 4

A method to treat a mammal that has cancer, said method comprising:
(a) removing cells of said mammal;
( b) transletting said cells in vitro with a therapeutic composition comprising:
(i) a first recombinant construct comprising an isolated nucleic acid sequence encoding a superantigen operatively linked to one or more transcription control sequences; and
(ii) a second recombinant construct comprising an isolated nucleic acid sequence encoding a cytokine operatively linked to one or more transcription control sequences; and
(c) reintroducing said transfected cells to said mammal;
wherein said nucleic acid sequences encoding said superantigen and said cytokine, respectively, are coexpressed at or adjacent to said cancer; and
wherein coexpression of said superantigen and said cytokine produces a result selected from the group consisting of alleviation of said cancer, reduction of a tumor associated with said cancer, elimination of a tumor associated with said cancer, prevention of metastatic cancer, and stimulation of effector cell immunity against said cancer.

### Claim 5 (depends on 1, 2, 3, 4)

The method of claim 1, 2, 3 or 4, wherein said superantigen is selected from the group consisting of staphylococcal enterotoxins, retroviral antigens, streptococcal antigens, mycoplasma antigens, mycobacteria antigens, viral antigens and protozoan antigens.

### Claim 6 (depends on 1, 2, 3, 4)

The method of claim 1, 2, 3 or 4, wherein said superantigen comprises staphylococcal enterotoxins.

### Claim 7 (depends on 1, 2, 3, 4)

The method of claim 1, 2, 3 or 4, wherein said superantigen is selected from the group consisting of SEA, SEB, SEC.sub.1, SEC.sub.2, SEC.sub.3, SED, SEE and TSST.

### Claim 8 (depends on 1, 2, 3, 4)

The method of claim 1, 2, 3 or 4, wherein said superantigen is from a virus selected from the group consisting of mouse mammary tumor virus, rabies virus and herpes virus.

### Claim 9 (depends on 1, 2, 3, 4)

The method of claim 1, 2, 3 or 4, wherein said cytokine is selected from the group consisting of hematopoietic growth factors, interleukins, interferons, immunoglobulin superfamily molecules, tumor necrosis factor family molecules and chemokines.

### Claim 10 (depends on 1, 2, 3, 4)

The method of claim 1, 2, 3 or 4, wherein said cytokine is selected from the group consisting of granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor, tumor necrosis factor .alpha., interleukin-1, interleukin-6 and interleukin-12.

### Claim 11 (depends on 1, 2, 3, 4)

The method of claim 1, 2, 3 or 4, wherein said cytokine is selected from the group consisting of granulocyte macrophage colony stimulating factor and tumor necrosis factor.

### Claim 12 (depends on 1, 2, 3, 4)

The method of claim 1, 2, 3 or 4, wherein said cytokine comprises granulocyte macrophage colony stimulating factor.

### Claim 13 (depends on 1, 2, 3, 4)

The method of claim 1, 2, 3 or 4, wherein said transcription control sequences are selected from the group consisting of RSV control sequences, CMV control sequences, retroviral LTR sequences, SV-40 control sequences and .beta.-actin control sequences.

### Claim 14 (depends on 2, 4)

The method of claim 2 or 4, wherein said first recombinant construct is selected from the group consisting of PCR.sub.3 -SEB, PCR.sub.3 -SEA, PCR.sub.3 -SEB.S, PCR.sub.3 -SEA.S, PCR.sub.3 -TSST and wherein said second recombinant construct is PCR.sub.3 -GM.sub.3.

### Claim 15 (depends on 1, 2)

The method of claim 1 or 2, wherein said liposome delivery vehicle includes a compound capable of specifically delivering said liposome to said cancer.

### Claim 16 (depends on 1, 2)

The method of claim 1 or 2, wherein said therapeutic composition is administered to said mammal at or adjacent to said cancer.

### Claim 17 (depends on 1, 2, 3, 4)

The method of claim 1, 2, 3 or 4, wherein said mammal is a human.

### Claim 18 (depends on 1, 2, 3, 4)

The method of claim 1, 2, 3 or 4, wherein said mammal is selected from the from the group consisting of humans, dogs, cats, sheep, cattle, horses and pigs.

### Claim 19 (depends on 1, 2, 3, 4)

The method of claim 1, 2, 3 or 4, wherein said cancer is selected from the group consisting of melanomas, squamous cell carcinoma, breast cancers, head and neck carcinomas, thyroid carcinomas, soft tissue sarcomas, bone sarcomas, te sticular cancers, prostatic cancers, ovarian cancers, bladder cancers, skin cancers, brain cancers, angiosarcomas, hemangiosarcomas, mast cell tumors, primary hepatic cancers, lung cancers, pancreatic cancers, gastrointestinal cancers, renal cell carcinomas, and hematopoietic neoplasias.

### Claim 20 (depends on 1, 2, 3, 4)

The method of claim 1, 2, 3 or 4, wherein said cancer is selected from the group consisting of melanomas, lung cancers, thyroid carcinomas, breast cancers, renal cell carcinomas, squamous cell carcinomas, brain tumors and skin cancers.

### Claim 21 (depends on 1, 2, 3, 4)

The method of claim 1, 2, 3 or 4, wherein said cytokine is an interleukin.

### Claim 22 (depends on 21)

The method of claim 21, wherein said interleukin is selected from the group consisting of interleukin-1 (IL-1), interleukin-6 (IL-6) and interleukin-12 (IL-12).

### Claim 23 (depends on 22)

The method of claim 22, wherein said interleukin is selected from the group consisting of interleukin-1 (IL-1), interleukin-6 (IL-6) and interleukin-12 (IL-12).

### Claim 24

A method for increasing effector cell immunity against a cancer in a mammal, said method comprising administering to said mammal a therapeutic composition comprising:
(a) a liposome delivery vehicle; and
(b) a recombinant construct comprising a first isolated nucleic acid sequence encoding a superantigen; and a second isolated nucleic acid sequence encoding a cytokine, said first and second nucleic acid sequences being operatively linked to one or more transcription control sequences;
wherein said first and said second nucleic acid sequences encoding said superantigen and said cytokine, respectively, are coexpressed at or adjacent to said cancer; and
wherein coexpression of said superantigen and said cytokine results in an increase in effector immunity against said cancer.

### Claim 25

A method for increasing effector cell immunity against a cancer in a mammal, said method comprising administering to said mammal a therapeutic composition comprising:
(a) a liposome delivery vehicle;
(b) a first recombinant construct comprising an isolated nucleic acid sequence encoding a superantigen operatively linked to one or more transcription control sequences; and
(c) a second recombinant construct comprising an isolated nucleic acid sequence encoding a cytokine operatively linked to one or more transcription control sequences;
wherein said nucleic acid sequences encoding said superantigen and said cytokine, respectively, are coexpressed at or adjacent to said cancer; and
wherein coexpression of said superantigen and said cytokine results in an increase in effector immunity against said cancer.

### Claim 26

A method for increasing effector cell immunity against a cancer in a mammal, said method comprising:
(a) removing cells of said mammal;
(b) transfecting said cells in vitro with a recombinant construct comprising a first isolated nucleic acid sequence encoding a superantigen, and a second isolated nucleic acid sequence encoding a cytokine, said first and second nucleic acid sequences being operatively linked to one or more transcription control sequences; and
(c) reintroducing said transfected cells to said mammal;
wherein said first and said second nucleic acid sequences encoding said superantigen and said cytokine, respectively, are coexpressed at or adjacent to said cancer; and
wherein coexpression of said superantigen and said cytokine results in an increase in effector immunity against said cancer.

### Claim 27

A method for increasing effector cell immunity against a cancer in a mammal, said method comprising:
(a) removing cells of said mammal;
(b) transfecting said cells in vitro with a therapeutic composition comprising:
(i) a first recombinant construct comprising an isolated nucleic acid sequence encoding a superantigen operatively linked to one or more transcription control sequences; and
(ii) a second recombinant construct comprising an isolated nucleic acid sequence encoding a cytokine operatively linked to one or more transcription control sequences; and
(c) reintroducing said transfected cells to said mammal;
wherein said nucleic acid sequences encoding said superantigen and said cytokine, respectively, are coexpressed at or adjacent to said cancer; and
wherein coexpression of said superantigen and said cytokine results in an increase in effector immunity against said cancer.

### Claim 28

A composition comprising a recombinant construct comprising a first isolated nucleic acid sequence encoding a superantigen and a second isolated nucleic acid sequence encoding a cytokine, wherein said isolated nucleic acid sequences are operatively linked to one or more transcription control sequences.

### Claim 29

A composition comprising:
(a) a first recombinant construct comprising an isolated nucleic acid sequence encoding a superantigen operatively linked to one or more transcription control sequences; and
(b) a second recombinant construct comprising an isolated nucleic acid sequence encoding a cytokine operatively linked to one or more transcription control sequences.

### Claim 30 (depends on 28, 29)

The composition of claim 28 or 29, wherein said composition further comprises a pharmaceutically acceptable carrier.

### Claim 31 (depends on 30)

The composition of claim 30, wherein said pharmaceutically acceptable carrier is selected from the group consisting of an aqueous physiologically balanced solution, an artificial lipid-containing substrate, a natural lipid-containing substrate, an oil, an ester, a glycol, a virus and metal particles.

### Claim 32 (depends on 30)

The composition of claim 30, wherein said pharmaceutically acceptable carrier comprises a delivery vehicle that delivers said nucleic acid sequences to a cancer in a mammal.

### Claim 33 (depends on 32)

The composition of claim 32, wherein said delivery vehicle is selected from the group consisting of liposomes, micelles, and cells.

### Claim 34 (depends on 32)

The composition of claim 32, wherein said delivery vehicle comprises a liposome.

### Claim 35 (depends on 28, 29)

The composition of claim 28 or 29, wherein said superantigen is selected from the group consisting of staphylococcal enterotoxins, retroviral antigens, streptococcal antigens, mycoplasma antigens, mycobacteria antigens, virus antigens and protozoan antigens.

### Claim 36 (depends on 28, 29)

The composition of claim 28 or 29, wherein said superantigen comprises staphylococcal enterotoxins.

### Claim 37 (depends on 28, 29)

The composition of claim 28 or 29, wherein said superantigen is selected from the group consisting of SEA, SEB, SEC.sub.1, SEC.sub.2, SEC.sub.3, SED, SEE and TSST.

### Claim 38 (depends on 28, 29)

The composition of claim 28 or 29, wherein said superantigen is from a virus selected from the group consisting of mouse mammary tumor virus, rabies virus and herpes virus.

### Claim 39 (depends on 28, 29)

The composition of claim 28 or 29, wherein said cytokine is selected from the group consisting of hematopoietic growth factors, interleukins, interferons, immunoglobulin superfamily molecules, tumor necrosis factor family molecules and chemokines.

### Claim 40 (depends on 28, 29)

The composition of claim 28 or 29, wherein said cytokine is selected from the group consisting of granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor, tumor necrosis factor .alpha., interleukin-1, interleukin-6 and interleukin-12.

### Claim 41 (depends on 28, 29)

The composition of claim 28 or 29, wherein said cytokine is granulocyte macrophage colony stimulating factor and tumor necrosis factor .alpha..

### Claim 42 (depends on 28, 29)

The composition of claim 28 or 29, wherein said transcription control sequences are selected from the group consisting of KSV control sequences, CMV control sequences, retroviral LTR control sequences, SV-40 control sequences and .beta.-actin control sequences.

### Claim 43 (depends on 29)

The composition of claim 29, wherein said first recombinant construct is selected from the group consisting of PCR.sub.3 -SEB, PCR.sub.3 -SEA, PCR.sub.3 -SEB.S, PCR.sub.3 -SEA.S, PCR.sub.3 -TSST and wherein said second recombinant construct is PCR.sub.3 -GM.sub.3.

### Claim 44 (depends on 34)

The composition of claim 34, wherein said composition is useful for treating a cancer selected from the group consisting of melanomas, squamous cell carcinoma, breast cancers, head and neck carcinomas, thyroid carcinomas, soft tissue sarcomas, bone sarcomas, testicular cancers, prostatic cancers, ovarian cancers, bladder cancers, skin cancers, brain cancers, angiosarcomas, hemangiosarcomas, mast cell tumors, primary hepatic cancers, lung cancers, pancreatic cancers, gastrointestinal cancers, renal cell carcinomas, and hematopoietic neoplasias, such that treatment of said cancer produces a result selected from the group consisting of alleviation of said cancer, reduction of a tumor associated with said cancer, elimination of a tumor associated with said cancer, prevention of metastatic cancer, and stimulation of effector cell immunity against said cancer.

### Claim 45 (depends on 34)

The composition of claim 34, wherein said composition is useful for treating a cancer selected from the group consisting of melanomas, lung cancers, thyroid carcinomas, breast cancers, renal cell carcinomas, squamous cell carcinomas, brain tumors and skin cancers such that treatment of said cancer produces a result selected from the group consisting of alleviation of said cancer, reduction of a tumor associated with said cancer, elimination of a tumor associated with said cancer, prevention of metastatic cancer, and stimulation of effector cell immunity against said cancer.

### Claim 46 (depends on 28, 29)

The therapeutic composition of claim 28 or 29, wherein said cytokine is an interleukin.

### Claim 47

A recombinant construct comprising an isolated nucleic acid sequence encoding a superantigen and an isolated nucleic acid sequence encoding a cytokine, wherein said isolated nucleic acid sequences are operatively linked to one or more transcription control sequences.

### Claim 48 (depends on 47)

The construct of claim 47, wherein said transcription control sequence is selected from the group consisting of RSV control sequences, CMV control sequences, retroviral LTR control sequences, SV-40 control sequences and .beta.-actin control sequences.

### Claim 49 (depends on 47)

The construct of claim 47, wherein said superantigen-encoding nucleic acid sequence encodes a toxin selected from the group consisting of staphylococcal enterotoxlns, retroviral antigens, streptococcal antigens, mycoplasma antigens, mycobacteria antigens, virus antigens and protozoan antigens.

### Claim 50 (depends on 47)

The construct of claim 47, wherein said superantigen-encoding nucleic acid sequence encodes a toxin selected from the group consisting of a SEA, SEB, SEC.sub.1, SEC.sub.2, SEC.sub.3, SED, SEE and TSST gene.

### Claim 51 (depends on 47)

The construct of claim 47, wherein said superantigen-encoding nucleic acid sequence lacks a bacterial leader sequence.

### Claim 52 (depends on 32)

The composition of claim 32, wherein said delivery vehicle comprises DOTMA and DOPE.