# US 6,723,705 B1 · Tumor cells modified to express B7-2 with increased immunogenicity and uses therefor

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- Number: US 6,723,705 B1
- Document ID: US6723705B1
- Status: Granted
- Kind: B1
- Application number: 09/206,132
- Filed: 1998-12-07
- Granted: 2004-04-20
- Earliest claimed: 1993-08-19
- Assignees: Gentics Institute, Inc.
- Inventors: Gordon J. Freeman; Lee M. Nadler; Gary S. Gray
- Web page: https://patentgrep.com/patent/US6723705B1

## CPC

- A01K 67/0271: Chimeric vertebrates, e.g. comprising exogenous cells
- C07K 14/70532: {B7 molecules, e.g. CD80, CD86}
- C07K 16/2827: {against B7 molecules, e.g. CD80, CD86}
- C12N 15/8509: {for producing genetically modified animals, e.g. transgenic}
- A01K 2217/05: Animals comprising random inserted nucleic acids (transgenic)
- A01K 2217/075: inducing loss of function, i.e. knock out
- A01K 2267/0331: Animal model for proliferative diseases
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K 2319/00: Fusion polypeptide
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- 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)
- 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)

## Abstract

Tumor cells modified to express one or more T cell costimulatory molecules are disclosed. Preferred costimulatory molecules are B7-2 and B7-3. The tumor cells of the invention can be modified by transfection with nucleic acid encoding B7-2 and/or B7-3, by using an agent which induces or increases expression of B7-2 and/or B7-3 on the tumor cell or by coupling B7-2 and/or B7-3 to the tumor cell. Tumor cells modified to express B7-2 and/or B7-3 can be further modified to express B7. Tumor cells further modified to express MHC class I and/or class II molecules or in which expression of an MHC associated protein, the invariant chain, is inhibited are also disclosed. The modified tumor cells of the invention can be used in methods for treating a patient with a tumor, preventing or inhibiting metastatic spread of a tumor or preventing or inhibiting recurrence of a tumor. A method for specifically inducing a CD4+ T cell response against a tumor and a method for treating a tumor by modification of tumor cells in vivo are disclosed.

## Claims

### Claim 1

A method for treating a mammalian subject having a solid tumor, comprising direct injection of a nucleic acid molecule encoding B7-2 molecule in a form suitable for expression of the B7-2 molecule, into cells of the tumor, wherein the 87-2 molecule has the ability to costimulate a T cell and the ability to bind a CD28 or CTLA4 ligands, such that the growth of the tumor is inhibited.

### Claim 2 (depends on 1)

The method of claim 1 wherein B7-2 comprises the amino acid sequence shown in SEQ ID NO:2.

### Claim 3 (depends on 1)

The method of claim 1, wherein the nucleic acid molecule encoding B7-2 comprises the nucleic sequence shown in SEQ ID No:1.

### Claim 4 (depends on 1)

The method of claim 1 wherein the solid tumor is selected from the group consisting of carcinoma, sarcoma, melanoma and neuroblastoma.

### Claim 5

A method for modifying cells of a solid tumor in vivo to express a B7-2 molecule, comprising direct injection of a nucleic acid molecule encoding a B7-2 molecule in a form suitable for expression of the B7-2 molecule, into the tumor cells, wherein the 87-2 molecule has the ability to costimulate a T cell and the ability to bind a CD28 or CTLA4 ligand, such that B7-2 is expressed by the tumor cells.

### Claim 6 (depends on 5)

The method of claim 5 wherein the B7-2 molecule comprises the amino acid sequence shown in SEQ ID NO:2.

### Claim 7 (depends on 5)

The method of claim 5 wherein the nucleic acid encoding a B7-2 molecule comprises the nucleic acid sequence shown in SEQ ID NO:1.

### Claim 8 (depends on 1, 5)

The method of claim 1 or 5 wherein the nucleic acid molecule encoding B7-2 is a viral vector.

### Claim 9 (depends on 8)

The method of claim 8 wherein the viral vector is selected from the group consisting of a retroviral vector an adenoviral vector, and an adeno-associated vector.

### Claim 10 (depends on 1, 5)

The method of claim 1 or 5 wherein the nucleic acid molecule encoding B7-2 is a plasmid expression vector.

### Claim 11 (depends on 1, 5)

The method of claim 1 or 5 wherein the tumor cells are further transfected with at least one nucleic acid molecule encoding a B7 protein.

### Claim 12 (depends on 1, 5)

The method of claim 1 or 5 wherein the tumor cells are further injected with at least one nucleic acid molecule encoding at least one MHC class II α chain protein and at least one MHC class II β chain protein in a for suitable for expression of the MHC class II α chain protein(s) and the MHC class II β chain protein(s).

### Claim 13 (depends on 1, 5)

The method of claim 1 or 5 wherein the tumor cells are further transfected with at least one nucleic acid molecule encoding at least one MHC class I α chain protein in a form suitable for expression of the MHC class I protein(s).

### Claim 14 (depends on 1, 5)

The method of claim 1 or 5 wherein the tumor cells are further transfected with a nucleic acid molecule encoding a β-2 microglobulin protein in a form suitable for expression of the β-2 microglobulin protein.

### Claim 15 (depends on 1, 5)

The method of claim 1 or 5 wherein expression of the MHC class II invariant chain is inhibited in the tumor cells by transfection of the tumor cells with a nucleic acid molecule which is antisense to a regulatory or a coding region of the invariant chain gene.

### Claim 16 (depends on 1)

The method of claim 1 wherein the solid tumor is selected from a group consisting of a carcinoma, sarcoma, melanoma and neuroblastoma.

### Claim 17

A method of increasing the immunogenicity of a cells of a solid tumor comprising, direct injection of a nucleic acid molecule encoding a B7-2 molecule in a form suitable for expression of the B7-2 molecule, into the tumor cells, wherein the B7-2 molecule has the ability to costimulate a T cell and the ability to bind a CD28 or CTLA4 ligand, such that B7-2 is expressed by the tumor cells, to thereby increase the immunogenicity of the tumor cells.

### Claim 18 (depends on 17)

The method of claim 17 wherein B7-2 comprises the amino acid sequence shown in SEQ ID NO:2.

### Claim 19 (depends on 17)

The method of claim 17 wherein the nucleic acid molecule encoding a B7-2 molecule comprises the nucleic sequence shown in SEQ ID NO:9.

### Claim 20 (depends on 17)

The method of claim 17 wherein the solid tumor is selected from a group consisting of a carcinoma, sarcoma, melanoma and neuroblastoma.

### Claim 21 (depends on 17)

The method of claim 17 wherein the rumor cells are further transfected with at least one nucleic acid molecule encoding a B7 protein.

### Claim 22 (depends on 17)

The method of claim 17 wherein the tumor cells are further injected with at least one nucleic acid molecule encoding at least one MHC class II α chain protein and at least one MHC class II β chain protein in a from suitable for expression of the MHC class II α chain protein(s) and the MHC class II β chain protein(s).

### Claim 23 (depends on 17)

The method of claim 17 wherein the tumor cells are further transfected with at least one nucleic acid molecule encoding at least one MHC class I α chain protein in a form suitable for expression of the MHC class I protein(s).

### Claim 24 (depends on 17)

The method of claim 17 wherein the tumor cells are further transfected with a nucleic acid molecule encoding a β-2 microglobulin protein in a form suitable for expression of the β-2 microglobulin protein.

### Claim 25 (depends on 17)

The method of claim 17 wherein expression of the MHC class II invariant chain is inhibited in the tumor cells by transfection of the tumor cells with a nucleic acid molecule which is antisense to a regulatory or a coding region of the invariant chain gene.