# US 8,273,864 B2 · Nucleic acid molecules encoding B7-DC variants

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- Number: US 8,273,864 B2
- Document ID: US8273864B2
- Status: Granted
- Kind: B2
- Application number: 13/228,893
- Filed: 2011-09-09
- Published: 2012-01-05
- Granted: 2012-09-25
- Earliest claimed: 2002-10-04
- Assignees: Mayo Foundation for Medical Education and Research
- Inventors: Lieping Chen
- Web page: https://patentgrep.com/patent/US8273864B2

## CPC

- C07K 14/47: from mammals
- A61P 37/04: Immunostimulants
- C07K 14/70532: {B7 molecules, e.g. CD80, CD86}
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

Variant costimulatory polypeptides, nucleic acids encoding such polypeptides, and methods for using the polypeptides and nucleic acids to enhance a T cell response are provided herein.

## Claims

### Claim 1

An isolated nucleic acid molecule encoding a variant B7-DC polypeptide comprising an IgV domain, wherein the variant polypeptide is a variant of wild-type B7-DC polypeptide, has altered binding affinity for PD-1 compared to the wild-type B7-DC polypeptide, and comprises a substitution of an amino acid in the A′, B, C, C′, C″, D, E, F, or G β strand of wild-type B7-DC polypeptide according to SEQ ID NO:5 or 6.

### Claim 2 (depends on 1)

The isolated nucleic acid molecule of claim 1, wherein the variant B7-DC polypeptide is a variant of murine B7-DC.

### Claim 3 (depends on 1)

The isolated nucleic acid molecule of claim 1, wherein the variant B7-DC polypeptide is a variant of human B7-DC.

### Claim 4 (depends on 1)

The isolated nucleic acid molecule of claim 1, wherein the variant B7-DC polypeptide or fragment thereof has decreased binding affinity for PD-1 compared to wild-type B7-DC polypeptide.

### Claim 5 (depends on 4)

The isolated nucleic acid molecule of claim 4, wherein the variant B7-DC polypeptide or fragment thereof enhances an immune response in a mammal.

### Claim 6 (depends on 1)

The isolated nucleic acid molecule of claim 1, wherein the variant B7-DC polypeptide or fragment thereof has increased binding affinity for PD-1 compared to wild-type B7-DC polypeptide.

### Claim 7 (depends on 6)

The isolated nucleic acid molecule of claim 6, wherein the variant B7-DC polypeptide or fragment thereof enhances an immune response in a mammal.

### Claim 8 (depends on 1)

The isolated nucleic acid molecule of claim 1, wherein the variant polypeptide comprises a substitution of the amino acid at position 111 numbered from the initiation methionine of wild-type murine B7-DC (SEQ ID NO:6) or human B7-DC (SEQ ID NO:5).

### Claim 9 (depends on 8)

The isolated nucleic acid molecule of claim 8, wherein the substitution is with a serine residue.

### Claim 10 (depends on 1)

The isolated nucleic acid molecule of claim 1, wherein the variant polypeptide comprises a substitution of the amino acid at position 113 numbered from the initiation methionine of wild-type murine B7-DC (SEQ ID NO:6) or human B7-DC (SEQ ID NO:5).

### Claim 11 (depends on 10)

The isolated nucleic acid molecule of claim 10, wherein the substitution is with a serine residue.

### Claim 12 (depends on 1)

The isolated nucleic acid molecule of claim 1, wherein the variant polypeptide comprises a substitution of the amino acid at position 56 numbered from the initiation methionine of wild-type murine B7-DC (SEQ ID NO:6) or human B7-DC (SEQ ID NO:5).

### Claim 13 (depends on 12)

The isolated nucleic acid molecule of claim 12, wherein the substitution is with a serine residue.

### Claim 14 (depends on 1)

The isolated nucleic acid molecule of claim 1, wherein the variant polypeptide comprises a substitution of the amino acid at position 67 numbered from the initiation methionine of wild-type murine B7-DC (SEQ ID NO:6) or human B7-DC (SEQ ID NO:5).

### Claim 15 (depends on 14)

The isolated nucleic acid molecule of claim 14, wherein the substitution is with a tyrosine residue.

### Claim 16 (depends on 1)

The isolated nucleic acid molecule of claim 1, wherein the variant polypeptide comprises a substitution of the amino acid at position 71 numbered from the initiation methionine of wild-type murine B7-DC (SEQ ID NO:6) or human B7-DC (SEQ ID NO:5).

### Claim 17 (depends on 16)

The isolated nucleic acid molecule of claim 16, wherein the substitution is with a serine residue.

### Claim 18 (depends on 1)

The isolated nucleic acid molecule of claim 1, wherein the variant polypeptide comprises a substitution of the amino acid at position 101 numbered from the initiation methionine of wild-type murine B7-DC (SEQ ID NO:6) or human B7-DC (SEQ ID NO:5).

### Claim 19 (depends on 18)

The isolated nucleic acid molecule of claim 18, wherein the substitution is with a serine residue.

### Claim 20 (depends on 1)

The isolated nucleic acid molecule of claim 1, wherein the variant polypeptide comprises a substitution of the amino acid at position 105 numbered from the initiation methionine of wild-type murine B7-DC (SEQ ID NO:6) or human B7-DC (SEQ ID NO:5).

### Claim 21 (depends on 20)

The isolated nucleic acid molecule of claim 20, wherein the substitution is with an alanine residue.

### Claim 22 (depends on 1)

The isolated nucleic acid molecule of claim 1, wherein the variant polypeptide comprises a substitution of the amino acid at position 58 number from the initiation methionine of wild-type murine B7-DC (SEQ ID NO:6) or human B7-DC (SEQ ID NO:5).

### Claim 23 (depends on 22)

The isolated nucleic acid molecule of claim 22, wherein the substitution is with a tyrosine residue.

### Claim 24

An isolated nucleic acid molecule encoding a polypeptide fragment of murine B7-DC (SEQ ID NO:6) or human B7-DC (SEQ ID NO:5), the polypeptide fragment comprising amino acids 67-71, amino acids 101-105, or amino acids 111-113 of SEQ ID NO:6, wherein the polypeptide inhibits binding of endogenous B7-DC to PD-1.

### Claim 25 (depends on 24)

The isolated nucleic acid molecule of claim 24 encoding a polypeptide comprising amino acids 101-113 of murine B7-DC (SEQ ID NO:6) or human B7-DC (SEQ ID NO:5).

### Claim 26 (depends on 1)

A vector comprising the nucleic acid molecule of claim 1.

### Claim 27 (depends on 26)

The vector of claim 26, wherein the nucleic acid molecule is operably linked to an expression control sequence.

### Claim 28 (depends on 27)

An isolated host cell comprising the vector of claim 27.

### Claim 29 (depends on 27)

A method for enhancing an immune response in a mammal comprising administering to the mammal the vector of claim 27.

### Claim 30

An isolated nucleic acid molecule encoding a fusion polypeptide comprising a first fusion partner comprising a variant B7-DC polypeptide comprising an IgV domain, wherein the variant polypeptide is a variant of wild-type B7-DC polypeptide, has altered binding affinity for PD-1 compared to the wild-type B7-DC polypeptide, and comprises a substitution of an amino acid in the A′, B, C, C′, C″, D, E, F, or G β strand of wild-type B7-DC polypeptide according to SEQ ID NO:5 or 6, and a second fusion partner.

### Claim 31 (depends on 30)

The isolated nucleic acid molecule of claim 30, wherein the second fusion partner comprises one or more domains of an Ig heavy chain constant region.

### Claim 32 (depends on 30)

The isolated nucleic acid molecule of claim 30, wherein the second polypeptide comprises an amino acid sequence corresponding to the hinge, CH2 and CH3 regions of a human immunoglobulin chain.

### Claim 33 (depends on 30)

The isolated nucleic acid molecule of claim 30, wherein the first polypeptide comprises the extracellular domain of B7-DC or a fragment thereof, and wherein the second polypeptide comprises an amino acid sequence corresponding to the hinge, CH2 and CH3 regions of a human immunoglobulin chain.

### Claim 34 (depends on 30)

The isolated nucleic acid molecule of claim 30, wherein the variant B7-DC polypeptide is a variant of murine B7-DC.

### Claim 35 (depends on 30)

The isolated nucleic acid molecule of claim 30, wherein the variant B7-DC polypeptide is a variant of human B7-DC.

### Claim 36 (depends on 30)

The isolated nucleic acid molecule of claim 30, wherein the variant B7-DC polypeptide or fragment thereof has decreased binding affinity for PD-1 compared to wild-type B7-DC polypeptide.

### Claim 37 (depends on 36)

The isolated nucleic acid molecule of claim 36, wherein the variant B7-DC polypeptide or fragment thereof enhances an immune response in a mammal.

### Claim 38 (depends on 30)

The isolated nucleic acid molecule of claim 30, wherein the variant B7-DC polypeptide or fragment thereof has increased binding affinity for PD-1 compared to wild-type B7-DC polypeptide.

### Claim 39 (depends on 38)

The isolated nucleic acid molecule of claim 38, wherein the variant B7-DC polypeptide or fragment thereof enhances an immune response in a mammal.

### Claim 40

An isolated nucleic acid molecule encoding a fusion polypeptide comprising the IgV domain of B7-DC fused to a second polypeptide, wherein the IgV domain of B7-DC is from human (SEQ ID NO:5) or murine B7-DC (SEQ ID NO:6) and comprises an amino acid substitution relative to wildtype human (SEQ ID NO:5) or murine (SEQ ID NO:6) B7-DC IgV domain, wherein the substitution alters binding affinity for PD-1 compared to the wild-type B7-DC polypeptide.

### Claim 41 (depends on 40)

The isolated nucleic acid of claim 40, wherein the second polypeptide comprises an amino acid sequence corresponding to the hinge, CH2 and CH3 regions of a human immunoglobulin chain.

### Claim 42 (depends on 41)

The isolated nucleic acid molecule of claim 41, wherein the IgV domain of B7-DC comprises an amino acid substitution at position 111 numbered from the initiation methionine of wild-type murine B7-DC (SEQ ID NO:6) or human B7-DC (SEQ ID NO:5).

### Claim 43 (depends on 42)

The isolated nucleic acid molecule of claim 42, wherein the substitution is with a serine residue.

### Claim 44 (depends on 41)

The isolated nucleic acid molecule of claim 41, wherein the IgV domain of B7-DC comprises an amino acid substitution at position 113 numbered from the initiation methionine of wild-type murine B7-DC (SEQ ID NO:6) or human B7-DC (SEQ ID NO:5).

### Claim 45 (depends on 44)

The isolated nucleic acid molecule of claim 44, wherein the substitution is with a serine residue.

### Claim 46 (depends on 41)

The isolated nucleic acid molecule of claim 41, wherein the IgV domain of B7-DC comprises an amino acid substitution at position 56 numbered from the initiation methionine of wild-type murine B7-DC (SEQ ID NO:6) or human B7-DC (SEQ ID NO:5).

### Claim 47 (depends on 46)

The isolated nucleic acid molecule of claim 46, wherein the substitution is with a serine residue.

### Claim 48 (depends on 41)

The isolated nucleic acid molecule of claim 41, wherein the IgV domain of B7-DC comprises an amino acid substitution at position 67 numbered from the initiation methionine of wild-type murine B7-DC (SEQ ID NO:6) or human B7-DC (SEQ ID NO:5).

### Claim 49 (depends on 48)

The isolated nucleic acid molecule of claim 48, wherein the substitution is with a tyrosine residue.

### Claim 50 (depends on 41)

The isolated nucleic acid molecule of claim 41, wherein the IgV domain of B7-DC comprises an amino acid substitution at position 71 numbered from the initiation methionine of wild-type murine B7-DC (SEQ ID NO:6) or human B7-DC (SEQ ID NO:5).

### Claim 51 (depends on 50)

The isolated nucleic acid molecule of claim 50, wherein the substitution is with a serine residue.

### Claim 52 (depends on 41)

The isolated nucleic acid molecule of claim 41, wherein the IgV domain of B7-DC comprises an amino acid substitution at position 101 numbered from the initiation methionine of wild-type murine B7-DC (SEQ ID NO:6) or human B7-DC (SEQ ID NO:5).

### Claim 53 (depends on 52)

The isolated nucleic acid molecule of claim 52, wherein the substitution is with a serine residue.

### Claim 54 (depends on 41)

The isolated nucleic acid molecule of claim 41, wherein the IgV domain of B7-DC comprises an amino acid substitution at position 105 numbered from the initiation methionine of wild-type murine B7-DC (SEQ ID NO:6) or human B7-DC (SEQ ID NO:5).

### Claim 55 (depends on 54)

The isolated nucleic acid molecule of claim 54, wherein the substitution is with an alanine.

### Claim 56 (depends on 41)

A vector comprising the nucleic acid molecule of claim 41.

### Claim 57 (depends on 56)

The vector of claim 56, wherein the nucleic acid molecule is operably linked to an expression control sequence.

### Claim 58 (depends on 57)

An isolated host cell comprising the vector of claim 57.

### Claim 59 (depends on 57)

A method for enhancing an immune response in a mammal comprising administering to the mammal the vector of claim 57.

### Claim 60 (depends on 40)

A method for enhancing an immune response in a mammal comprising administering to the mammal a polypeptide encoded by a polynucleotide of claim 40.

### Claim 61 (depends on 58)

A method for enhancing an immune response in a mammal comprising administering to the mammal a polypeptide produced by the host cell of claim 58.