# US 11,896,655 B2 · Circumsporozoite proteins with increased expression in mammalian cells

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- Number: US 11,896,655 B2
- Document ID: US11896655B2
- Status: Granted
- Kind: B2
- Application number: 17/440,597
- Filed: 2020-03-18
- Published: 2022-09-08
- Granted: 2024-02-13
- Earliest claimed: 2019-03-18
- Assignees: Fred Hutchinson Cancer Center
- Applicants: Fred Hutchinson Cancer Center
- Inventors: Marie Pancera; Connor Weidle
- Web page: https://patentgrep.com/patent/US11896655B2

## CPC

- A61K 39/015: Hemosporidia antigens, e.g. Plasmodium antigens
- A61K 31/355: Tocopherols, e.g. vitamin E
- A61P 33/06: Antimalarials
- C07K 14/445: Plasmodium
- A61K 2039/52: {Bacterial cells; Fungal cells; Protozoal cells}
- A61K 2039/627: {characterised by the linker}
- C07K 2319/40: containing a tag for immunodetection, or an epitope for immunisation
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Mutated and/or truncated malarial circumsporozoite proteins (CSP) and associated nucleic acids that are more stable and highly expressed in mammalian cells are described. The mutated and/or truncated CSP and associated nucleic acids can be expressed to produce malaria vaccine antigens.

## Claims

### Claim 1

A mutant N-terminus domain (NTD) of a circumsporozoite protein (CSP) comprising
i) a KKNSR (SEQ ID NO: 1) to SSNSS (SEQ ID NO: 2) mutation directly upstream of a SLGENDD (SEQ ID NO: 3) cleavage site of a wild-type Plasmodium falciparum CSP; or
ii) a KKNSR (SEQ ID NO: 1) to SSNSA (SEQ ID NO: 4) mutation directly upstream of a SLGENDD (SEQ ID NO: 3) cleavage site of a wild-type Plasmodium falciparum CSP;
wherein the wild-type Plasmodium falciparum CSP comprises a sequence as set forth in SEQ ID NOs: 7, 10, or 19.

### Claim 2 (depends on 1)

The mutant NTD of claim 1, further comprising a cysteine deletion.

### Claim 3 (depends on 2)

The mutant NTD of claim 2, wherein the cysteine is residue 5 of the wild-type Plasmodium falciparum CSP protein.

### Claim 4 (depends on 1)

The mutant NTD of claim 1, further comprising a cysteine to serine substitution.

### Claim 5 (depends on 1)

The mutant NTD of claim 1, expressed as a fusion protein with a secondary malaria vaccine epitope.

### Claim 6 (depends on 5)

The mutant NTD of claim 5, wherein the secondary malaria vaccine epitope is selected from RTS,S; NR2C, R2C, the junctional epitope, the minor epitope, or a junctional epitope linked to the minor epitope.

### Claim 7 (depends on 5)

The mutant NTD of claim 5, wherein the fusion protein comprises the sequence as set forth in SEQ ID NO: 12 (FL C5S-SAmut), SEQ ID NO: 13 (C5S-SAmut-23/4), SEQ ID NO: 14 (C5S-SAmut-19/3), or SEQ ID NO: 15 (C5S-SAmut-5/3).

### Claim 8

A circumsporozoite protein (CSP) comprising
i) a KKNSR (SEQ ID NO: 1) to SSNSS (SEQ ID NO: 2) mutation directly upstream of a SLGENDD (SEQ ID NO: 3) cleavage site within the N-terminal domain of a wild-type Plasmodium falciparum CSP; or
ii) a KKNSR (SEQ ID NO: 1) to SSNSA (SEQ ID NO: 4) mutation directly upstream of a SLGENDD (SEQ ID NO: 3) cleavage site within the N-terminal domain of a wild-type Plasmodium falciparum CSP;
wherein the wild-type Plasmodium falciparum CSP comprises a sequence as set forth in SEQ ID NOs: 7, 10, or 19.

### Claim 9 (depends on 8)

The CSP of claim 8, further comprising a cysteine deletion within the N-terminus of the CSP.

### Claim 10 (depends on 9)

The CSP of claim 9, wherein the cysteine is residue 5 of the wild-type Plasmodium falciparum CSP protein.

### Claim 11 (depends on 8)

The CSP of claim 8, further comprising a cysteine to serine substitution within the N-terminus of the CSP.

### Claim 12 (depends on 8)

The CSP of claim 8, having the sequence SLSSNSSSLGENDD (SEQ ID NO: 8) or SLSSNSASLGENDD (SEQ ID NO: 76) in place of the wild-type SLKKNSRSLGENDD (SEQ ID NO: 7) sequence.

### Claim 13 (depends on 8)

The CSP of claim 8, having the sequence QEYQCYGSSSNTRVLNELNYDNAGTNLYNELEMNYYGKQENWYSLSSNSSSLGENDD (SEQ ID NO: 9) or QEYQSYGSSSNTRVLNELNYDNAGTNLYNELEMNYYGKQENWYSLSSNSASLGENDD (SEQ ID NO: 11) in place of the wild-type QEYQCYGSSSNTRVLNELNYDNAGTNLYNELEMNYYGKQENWYSLKKNSRSLGEN DD (SEQ ID NO: 10) sequence.

### Claim 14

A method of stimulating an anti-malarial immune response in a subject comprising administering a therapeutically effective amount of a circumsporozoite protein (CSP) to the subject, wherein the CSP protein comprises
ii) a KKNSR (SEQ ID NO: 1) to SSNSS (SEQ ID NO: 2) mutation directly upstream of a SLGENDD (SEQ ID NO: 3) cleavage site in the N-terminal domain (NTD) of a wild-type Plasmodium falciparum CSP; or
iii) a KKNSR (SEQ ID NO: 1) to SSNSA (SEQ ID NO: 4) mutation directly upstream of a SLGENDD (SEQ ID NO: 3) cleavage site in the NTD of a wild-type Plasmodium falciparum CSP;
wherein the wild-type Plasmodium falciparum CSP comprises a sequence as set forth in SEQ ID NOs: 7, 10, or 19, thereby stimulating the anti-malarial immune response in the subject.

### Claim 15 (depends on 14)

The method of claim 14, wherein the CSP protein comprises a cysteine deletion within the N-terminus of the wild-type Plasmodium falciparum CSP.

### Claim 16 (depends on 15)

The method of claim 15, wherein the deleted cysteine was at residue 5 of the wild-type Plasmodium falciparum CSP protein.

### Claim 17 (depends on 14)

The method of claim 14, wherein the CSP protein comprises a cysteine to serine substitution.

### Claim 18 (depends on 14)

The method of claim 14, wherein the CSP comprises a malaria vaccine epitope.

### Claim 19 (depends on 18)

The method of claim 18, wherein the malaria vaccine epitope is selected from RTS,S; NR2C, R2C, the junctional epitope, the minor epitope, or a junctional epitope linked to the minor epitope.

### Claim 20 (depends on 14)

The method of claim 14, wherein the CSP comprises the sequence as set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.