# US 11,077,183 B2 · CD40 ligand fusion protein vaccine

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- Number: US 11,077,183 B2
- Document ID: US11077183B2
- Status: Granted
- Kind: B2
- Application number: 15/393,543
- Filed: 2016-12-29
- Published: 2017-06-29
- Granted: 2021-08-03
- Earliest claimed: 2005-11-07
- Assignees: MicroVAX, LLC
- Applicants: MicroVAX, LLC
- Inventors: Yucheng Tang; Albert B. Deisseroth
- Web page: https://patentgrep.com/patent/US11077183B2

## CPC

- A61K 39/145: Orthomyxoviridae, e.g. influenza virus
- A61K 39/12: Viral antigens
- A61K 39/39: characterised by the immunostimulating additives, e.g. chemical adjuvants
- A61P 31/04: Antibacterial agents
- A61P 31/12: Antivirals
- A61P 31/16: for influenza or rhinoviruses
- C07K 14/005: from viruses
- C07K 14/70575: {NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154 (NGF C07K14/48, TNF C07K14/525)}
- A61K 39/385: Haptens or antigens, bound to carriers
- A61K 2039/5256: {expressing foreign proteins}
- A61K 2039/53: {DNA (RNA) vaccination}
- A61K 2039/545: {characterised by the dose, timing or administration schedule}
- A61K 2039/55: {characterised by the host/recipient, e.g. newborn with maternal antibodies}
- A61K 2039/575: {humoral response}
- A61K 2039/585: {wherein the target is cancer}
- A61K 2039/6031: {Proteins}
- C07K 2319/02: containing a signal sequence
- C07K 2319/06: containing a lysosomal/endosomal localisation signal
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C12N 2710/10034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2710/10043: viral genome or elements thereof as genetic vector
- C12N 2710/10343: viral genome or elements thereof as genetic vector
- C12N 2710/20034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- 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
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided are methods of generating an immune response to any of various antigens including foreign antigens such as infectious agent antigens. In general, the method comprises administering an expression vector encoding a transcription unit encoding a secretable fusion protein, the fusion protein containing the foreign antigen and CD40 ligand and also administering the encoded fusion protein. In another approach, an immune response to the foreign antigen is elicited using the encoded fusion protein without administering the vector. The invention methods may be used to immunize an individual against an infectious agent such as influenza virus. Methods of obtaining an immune response in older individuals also is described.

## Claims

### Claim 1

A vaccine composition for generating in an individual an immune response against one or more influenza antigens resulting from an infectious influenza virus, comprising:
an effective amount of an expression vector,
said expression vector comprising a transcription unit encoding a secretable fusion protein comprising at least first and second influenza antigen fragments, each influenza antigen fragment from an extracellular region of the influenza virus, and each of said first and second influenza antigen fragments comprising at least first and second epitopes wherein the first and second influenza antigen fragments are linked to the amino terminus of an extracellular domain of a CD40 ligand, for generating both a cellular and antibody immune response,
said first influenza antigen fragment comprising, the first epitope that is recognized and bound by Class II MHC for eliciting antigen specific antibodies, and the second epitope that is recognized and bound by Class I MHC for eliciting antigen specific CD8+ effector T cells, and
said second influenza antigen fragment comprising the first epitope that is recognized and bound by Class II MHC for eliciting antigen specific antibodies, and the second epitope that is recognized and bound by Class I MHC for eliciting antigen specific CD8+ effector T cells,
wherein each of said epitopes in both the first and second influenza antigen fragments has a distinct amino acid sequence from each of the other epitopes.

### Claim 2 (depends on 1)

The composition of claim 1 wherein said influenza antigen fragments are from an influenza virus belonging to at least one or both of classes H3N2 or H5N1.

### Claim 3 (depends on 1)

The composition of claim 1 wherein each of said influenza antigen fragments is from a different viral influenza protein.

### Claim 4 (depends on 1)

The composition of claim 1 wherein at least one of said first and second influenza antigen fragments is from the extracellular domain of hemagglutinin or neuraminidase or Matrix protein 2.

### Claim 5 (depends on 1)

The composition of claim 1 wherein said expression vector is a viral vector or a plasmid vector.

### Claim 6 (depends on 5)

The composition of claim 5 wherein said viral vector is an adenoviral vector.

### Claim 7 (depends on 1)

The composition of claim 1 wherein said antibodies are neutralizing antibodies.

### Claim 8 (depends on 1)

The composition of claim 1 wherein the transcription unit encodes a secretory signal sequence linked to the first and second influenza antigen fragments connected to a linker connected to the extracellular domain of the CD40 ligand.

### Claim 9

A vaccine composition for increasing the immune responsiveness in an individual against one or more foreign antigens on an infectious agent, comprising:
an expression vector comprising a transcription unit encoding a secretable fusion protein comprising two or more foreign antigen fragments each from the extracellular domain of the one or more foreign antigens on the infectious agent, and an extracellular domain of a CD40L ligand, wherein the two or more foreign antigen fragments are linked to an amino terminus of the extracellular domain of the CD40L ligand,
a first one of said foreign antigen fragments comprising a first epitope that contains a binding site that is recognized and bound by Class II MHC for eliciting foreign antigen specific antibodies, and further comprising a second epitope that contains a binding site that is recognized and bound by Class I MHC for eliciting foreign antigen specific CD8+ T cells, and
a second one of said foreign antigen fragments comprising a first epitope and a second epitope, each of said first and second epitopes in the second foreign antigen fragment having a distinct amino acid sequence from each of the amino acid sequence epitopes in the first foreign antigen fragment, and said first and second epitopes of said second foreign antigen each having a binding site respectively recognized and bound by Class II MHC and Class I MHC.

### Claim 10 (depends on 9)

The composition of claim 9 wherein said infectious agent comprises one or more proteins of the same strain or different strains and said foreign antigen fragments are from said one or more proteins.

### Claim 11 (depends on 9)

The composition of claim 9 wherein said infectious agent is one of bacterial, fungal, viral or protozoan.

### Claim 12 (depends on 9)

The composition of claim 9 wherein said foreign antigen specific antibodies are neutralizing antibodies.

### Claim 13

A vaccine composition employing an expression vector comprising a transcription unit encoding a secretable fusion protein comprising first and second connected and distinct foreign antigen fragments, each fragment from a different protein of an infectious agent, and an extracellular domain of a CD40 ligand,
said first foreign antigen fragment from a first protein comprising at least first and second epitopes linked to the amino terminus of the extracellular domain of said CD40 ligand, wherein:
the first one of said epitopes that contains antigenic determinant that includes a binding site that is recognized and bound by Class II MHC for eliciting foreign antigen specific antibodies, and
the second one of said epitopes that contains antigenic determinant that includes a binding site that is recognized and bound by Class I MHC for eliciting foreign antigen specific CD8+effector T cells, and
said second foreign antigen fragment from a second protein, and comprising at least a first epitope and a second epitope, said second fragment first epitope and second epitope each containing a binding site that is respectively recognized and bound by Class II MHC and Class I MHC.

### Claim 14 (depends on 13)

The composition of claim 13 wherein said foreign antigen specific antibodies are neutralizing antibodies.

### Claim 15 (depends on 13)

The composition of claim 13 wherein the first and second foreign antigen fragments are each from proteins of the same strain or different strains.

### Claim 16 (depends on 13)

The composition of claim 13, wherein said foreign antigen fragments are one of bacterial, fungal, viral or protozoan.

### Claim 17

A composition for generating an immune response in an individual against one or more influenza antigens on an infectious agent for limiting progression of the infectious agent for multiple strains of the influenza virus, comprising,
an expression vector comprising a transcription unit encoding a secretable fusion protein comprising the extracellular domain of the CD40 ligand and an extracellular portion of the Matrix protein 2 influenza antigen SEQ ID NO 77, wherein said Matrix protein 2 is connected at the amino terminus of the CD40 ligand and functions to increase the immunogenicity of said Matrix protein 2 so as to increase both the level of Matrix protein 2 specific CD8+ effector T cells and neutralizing antibodies, against the infectious agent.