# US 2020/0224206 A1 · Amplicon Expression Vector Vaccines

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- Number: US 2020/0224206 A1
- Document ID: US20200224206A1
- Status: Published application
- Kind: A1
- Application number: 16/703,915
- Filed: 2019-12-05
- Published: 2020-07-16
- Earliest claimed: 2018-12-05
- Assignees: LineaRx, Inc.; Evvivax S. R. L.
- Applicants: LineaRx, Inc.; Evvivax S. R. L.
- Inventors: Michael E. Hogan; Stephen Hughes; Antonella Conforti; Luigi Aurisicchio; Fabio Palombo
- Web page: https://patentgrep.com/patent/US20200224206A1

## CPC

- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- C12N 15/64: General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
- A61K 38/1774: {Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)}
- A61K 39/0011: {Cancer antigens}
- A61K 39/001157: {Telomerase or TERT [telomerase reverse transcriptase]}
- A61K 39/00116: {Serine proteases, e.g. kallikrein}
- A61K 41/0047: {Sonopheresis, i.e. ultrasonically-enhanced transdermal delivery, electroporation of a pharmacologically active agent}
- A61P 35/00: Antineoplastic agents
- C12N 15/85: for animal cells
- A01K 2207/12: Animals modified by administration of exogenous cells
- A01K 2227/105: Murine
- A01K 2267/0331: Animal model for proliferative diseases
- A61K 2039/53: {DNA (RNA) vaccination}
- A61K 2039/54: {characterised by the route of administration}
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS

## Abstract

Provided herein are non-plasmid derived DNA vaccines comprised solely of enzymatically produced amplicon expression vectors and their method of use to elicit antigen-specific immune responses in a subject. The enzymatically produced amplicon expression vectors may be specifically utilized as a DNA based cancer vaccine to express desired antigens or other immunogenic polypeptides within a subject to induce a specific anti-cancer antigen-specific immune response. The enzymatically produced amplicon expression vectors may also be utilized to express cancer-specific neoantigens.

## Claims

### Claim 1

A method of treating cancer in a subject, said method comprising:
Identifying in a subject cancer neoantigens;
Without the use of plasmid-derived DNA, assembling an amplicon expression vector template comprising a promotor, one or more open reading frames (ORF) encoding the identified neoantigens and a terminator via gene synthesis;
amplifying the amplicon expression vector template via the polymerase chain reaction (PCR) and phosphorothioate modified PCR primers to produce a plurality of amplicon expression vectors;
purifying and concentrating the amplicon expression vectors to create an efficacious therapeutic dose for the subject; and
administering the therapeutic dose of amplicon expression vectors via electroporation in conjunction with one or more immune checkpoint inhibitors.

### Claim 2 (depends on 1)

The method of claim 1, wherein the immune checkpoint inhibitors include one or more from the group of PD-1, CTLA-4, PD-L1.

### Claim 3 (depends on 1)

The method of claim 1, wherein the amplicon expression vector template further comprises an ORF for a small ubiquitin-related modifier (SUMO).

### Claim 4 (depends on 1)

The method of claim 1, wherein the amplicon expression vector template further comprises an ORF encoding for tissue plasminogen activator (TPA).

### Claim 5 (depends on 2)

The method of claim 2, wherein the amplification via PCR is conducted using a polymerase with an error rate less than 7×10−5 error/bp.

### Claim 6 (depends on 1)

The method of claim 1, wherein the amplicon expression vector template encodes for between 2 and 20 neoantigens.

### Claim 7 (depends on 1)

The method of claim 1, wherein the amplicon expression vector template encodes for between 5 and 15 neoantigens.

### Claim 8 (depends on 2)

The method of claim 2, wherein the neoantigens are identified from the subject's circulating tumor cells (CTC).

### Claim 9 (depends on 4)

The method of claim 4, wherein the amplicon expression vector template further comprises an ORF encoding for an immunomodulator.

### Claim 10

A method of prophylactically treating cancer in a subject, said method comprising:
Without the use of plasmid-derived DNA, assembling an amplicon expression vector template comprising a promotor, an ORF encoding a consensus sequence for telomerase reverse transcriptase and terminator;
amplifying the amplicon expression vector template via the polymerase chain reaction (PCR) and phosphorothioate modified PCR primers to produce a plurality of amplicon expression vectors;
purifying and concentrating the amplicon expression vectors to create an efficacious prophylactic dose for the subject; and
administering the prophylactic dose of amplicon expression vectors via electroporation.

### Claim 11 (depends on 10)

The method of claim 10, wherein the amplification via PCR is conducted using a polymerase with an error rate less than 7×10−5 error/bp.

### Claim 12 (depends on 11)

The method of claim 11, wherein the amplicon expression vector template further comprises an ORF for a small ubiquitin-related modifier (SUMO).

### Claim 13 (depends on 10)

The method of claim 10, wherein the amplicon expression vector template further comprises an ORF encoding for tissue plasminogen activator (TPA).

### Claim 14 (depends on 11)

The method of claim 11, wherein the amplicon expression vector template further comprises an ORF encoding for an immunomodulator.

### Claim 15

A method of treating cancer in a subject, said method comprising:
Without the use of plasmid-derived DNA, assembling an amplicon expression vector template comprising a promotor, an ORF encoding a consensus sequence for telomerase reverse transcriptase and terminator;
amplifying the amplicon expression vector template via the polymerase chain reaction (PCR) and phosphorothioate modified PCR primers to produce a plurality of amplicon expression vectors;
purifying and concentrating the amplicon expression vectors to create an efficacious therapeutic dose for the subject; and
administering the therapeutic dose of amplicon expression vectors via electroporation in conjunction with one or more immune checkpoint inhibitors.

### Claim 16 (depends on 15)

The method of claim 15, wherein the immune checkpoint inhibitors include one or more from the group of PD-1, CTLA-4, PD-L1.

### Claim 17 (depends on 16)

The method of claim 16 wherein the amplicon expression vector template further comprises an ORF for a small ubiquitin-related modifier (SUMO).

### Claim 18 (depends on 15)

The method of claim 15, wherein the amplicon expression vector template further comprises an ORF encoding for tissue plasminogen activator (TPA).

### Claim 19 (depends on 15)

The method of claim 15, wherein the amplicon expression vector template further comprises an ORF encoding for an immunomodulator.

### Claim 20 (depends on 15)

The method of claim 15, wherein the amplification via PCR is conducted using a polymerase with an error rate less than 7×10−5 error/bp.