# US 10,118,951 B2 · Multimerization of recombinant protein by fusion to a sequence from lamprey

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- Number: US 10,118,951 B2
- Document ID: US10118951B2
- Status: Granted
- Kind: B2
- Application number: 15/536,726
- Filed: 2015-12-18
- Published: 2018-03-01
- Granted: 2018-11-06
- Earliest claimed: 2014-12-19
- Assignees: Sanofi Pasteur Limited
- Applicants: Sanofi Pasteur
- Inventors: Régis Sodoyer; Isabelle Legastelois
- Web page: https://patentgrep.com/patent/US10118951B2

## CPC

- C07K 14/005: from viruses
- A61P 31/16: for influenza or rhinoviruses
- A61P 37/04: Immunostimulants
- C07K 14/461: {from fish}
- C12N 15/62: DNA sequences coding for fusion proteins
- C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes}
- C07K 2319/00: Fusion polypeptide
- C07K 2319/21: containing a His-tag
- C12N 2760/16122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2760/16134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2800/22: Vectors comprising a coding region that has been codon optimised for expression in a respective host
- 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
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention relates to polymerized recombinant proteins, to recombinant nucleic acids coding for the polymerized recombinant proteins, to expression cassettes comprising the recombinant nucleic acids, to host cells transformed by the expression cassettes and to a method for multimerizing a recombinant protein. The polymerized proteins of the invention may be used in pharmaceutical or immunogenic compositions. In particular, the recombinant proteins may be antigens, antibodies or scaffolds. In particular, the polymerized recombinant protein may be an influenza haemagglutinin.

## Claims

### Claim 1

A molecule which comprises a first amino acid sequence which has at least 80% identity to SEQ ID NO: 1 and a second amino acid sequence which is heterologous to said first sequence, wherein said molecule does not comprise a leucine-rich repeat (LRR) module from a lamprey VLR-B antibody.

### Claim 2 (depends on 1)

The molecule of claim 1, wherein said molecule does not comprise a sequence selected from the group of sequences defined by SEQ ID NO: 29.

### Claim 3 (depends on 1)

The molecule of claim 1, wherein the only amino acid sequence in said molecule which is derived from a lamprey VLR-B antibody is the sequence having at least 80% identity to SEQ ID NO: 1.

### Claim 4 (depends on 1)

The molecule of claim 1, comprising cysteine residues at the positions within the molecule corresponding to positions 2, 7, 13, 19, 21, 24 and 27 of SEQ ID NO:1.

### Claim 5 (depends on 1)

The molecule of claim 1, comprising SEQ ID NO: 2.

### Claim 6 (depends on 1)

The molecule of claim 1, wherein there is a linker between the first amino acid sequence and the second heterologous amino acid sequence.

### Claim 7 (depends on 1)

The molecule of claim 1, wherein the second heterologous amino acid sequence encodes an antigen.

### Claim 8 (depends on 7)

The molecule of claim 7 wherein the antigen is selected from influenza virus, HIV, cytomegalovirus, dengue virus, yellow fever virus, tick-borne encephalitis virus, hepatitis virus, japanese encephalitis virus, human papillomavirus, coxsackievirus, herpes simplex virus, rubella virus, mumps virus, measles virus, rabies virus, polio virus, rotavirus, respiratory syncytial virus, Ebola virus, Chikungunya virus, Mycobacterium tuberculosis, Staphylococcus aureus, Staphylococcus epidermidis, E. coli, Clostridium difficile, Bordetella pertussis, Clostridium tetani, Haemophilus influenzae type b, Chlamydia pneumoniae, Chlamydia trachomatis, Porphyromonas gingivalis, Pseudomonas aeruginosa, Mycobacterium diphtherias, Shigella, Neisseria meningitidis, Streptococcus pneumoniae and Plasmodium falciparum.

### Claim 9 (depends on 8)

The molecule of claim 8, wherein the antigen is from influenza virus and is selected from a haemaglutinin (HA), a matrix 2 protein (M2), and an HAM2 fusion protein.

### Claim 10 (depends on 8)

The molecule of claim 8, wherein the antigen is from Shigella and is selected from IpaD and MxiH.

### Claim 11 (depends on 1)

The molecule of claim 1, wherein the second heterologous amino acid sequence encodes an antibody or a scaffold.

### Claim 12 (depends on 11)

The molecule of claim 11 wherein the antibody or scaffold is specific for an antigen selected from the group consisting of influenza virus, HIV, cytomegalovirus, dengue virus, yellow fever virus, tick-borne encephalitis virus, hepatitis virus, japanese encephalitis virus, human papillomavirus, coxsackievirus, herpes simplex virus, rubella virus, mumps virus, measles virus, rabies virus, polio virus, rotavirus, respiratory syncytial virus, Ebola virus, Chikungunya virus, Mycobacterium tuberculosis, Staphylococcus aureus, Staphylococcus epidermidis, E. coli, Clostridium difficile, Bordetella pertussis, Clostridium tetani, Haemophilus influenzae type b, Chlamydia pneumoniae, Chlamydia trachomatis, Porphyromonas gingivalis, Pseudomonas aeruginosa, Mycobacterium diphtherias, Shigella, Neisseria meningitidis, Streptococcus pneumoniae and Plasmodium falciparum.

### Claim 13 (depends on 11)

The molecule of claim 11 wherein the antibody is selected from a monoclonal antibody, a single domain antibody (dAb), a single-chain variable fragment (scFv), a Fab, a F(ab′)2 and a diabody (Db).

### Claim 14 (depends on 11)

The molecule of claim 11 wherein the second heterologous amino acid sequence encodes an antibody or scaffold selected from a bi-specific antibody, a multi-specific antibody, a bi-specific scaffold, and a multi-specific scaffold.

### Claim 15

A recombinant nucleic acid which comprises a first nucleic acid sequence with at least 80% identity to SEQ ID NO: 3 and a second nucleic acid sequence which is heterologous to said first sequence, wherein said recombinant nucleic acid does not encode a leucine-rich repeat (LRR) module from a lamprey VLR-B antibody.

### Claim 16 (depends on 15)

The recombinant nucleic acid of claim 15 wherein said first nucleic acid sequence encodes an amino acid sequence which comprises cysteine residues at positions within said amino acid sequence that correspond to positions 2, 7, 13, 19, 21, 24 and 27 of SEQ ID NO:1.

### Claim 17 (depends on 15)

The recombinant nucleic acid of claim 15, comprising SEQ ID NO: 4.

### Claim 18 (depends on 15)

An expression cassette comprising the recombinant nucleic acid of claim 15, wherein the recombinant nucleic acid is operably linked to a promoter.

### Claim 19 (depends on 18)

A host cell transformed with the expression cassette of claim 18.

### Claim 20 (depends on 1)

A pharmaceutical composition comprising the molecule of claim 1, and a pharmaceutically acceptable carrier or diluent.

### Claim 21 (depends on 20)

A method for inducing an immune response to an antigen in a subject comprising administering an effective amount of the molecule of claim 20 to the subject.

### Claim 22

A method for multimerizing a recombinant protein comprising:
a) fusing a nucleic acid sequence having at least 80% identity to SEQ ID NO: 3 to the nucleic acid sequence coding for said recombinant protein, with the proviso that said recombinant protein does not comprise a leucine-rich repeat (LRR) module from a lamprey VLR-B antibody,
b) expressing the fusion protein encoded by said nucleic acid sequence, under conditions which lead to the multimerization of said recombinant protein.