# US 12,258,408 B2 · Oncolytic virus expressing PD-1 binding protein and application of oncolytic virus

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- Number: US 12,258,408 B2
- Document ID: US12258408B2
- Status: Granted
- Kind: B2
- Application number: 17/288,006
- Filed: 2019-10-24
- Published: 2021-12-30
- Granted: 2025-03-25
- Earliest claimed: 2018-10-26
- Assignees: Shanghai Yuansong Biotechnology Co. Ltd.
- Applicants: Shanghai Yuansong Biotechnology Co. Ltd.
- Inventors: Kangjian Zhang; Xianlong Fang; Jinfa Gu; Xinyuan Liu
- Web page: https://patentgrep.com/patent/US12258408B2

## CPC

- C07K 16/2818: {against CD28 or CD152}
- A61K 35/761: Adenovirus
- A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- A61P 35/00: Antineoplastic agents
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 15/86: Viral vectors
- A61K 2039/505: {comprising antibodies}
- A61K 2039/53: {DNA (RNA) vaccination}
- A61K 2039/545: {characterised by the dose, timing or administration schedule}
- C07K 2317/52: Constant or Fc region; Isotype
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/64: comprising a combination of variable region and constant region components
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2319/02: containing a signal sequence
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C12N 2710/10021: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2710/10032: Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
- C12N 2710/10321: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2710/10343: viral genome or elements thereof as genetic vector
- 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)
- 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
- 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

Provided is an oncolytic virus expressing a PD-1 binding protein, i.e., an oncolytic adenovirus comprising a fusion protein expressing a PD-1 single-chain antibody, wherein the fusion protein has a structure of S-VL-L-VH-Fc, S is an optional signal peptide sequence, VL is the light chain variable region of the PD-1 single-chain antibody, L is a flexible linker, VH is the heavy chain variable region of the PD-1 single-chain antibody and Fc is an immunoglobulin Fc fragment.

## Claims

### Claim 1

An oncolytic virus comprising a nucleic acid encoding a PD-1 binding protein capable of inhibiting PD-1 activity, wherein the PD-1 binding protein is a fusion polypeptide comprising a PD-1 single-chain antibody and an immunoglobulin Fc fragment; and
the fusion polypeptide has a structure of S-VL-L-VH-Fc;
S is an optional signal peptide sequence, and the signal peptide sequence is MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 14), or MDMRVPAQLLGLLLLWFPGSRC (SEQ ID NO: 16);
VL is the light chain variable region of the PD-1 single-chain antibody, comprising CDR1 with sequence RAGONVONYLA (SEQ ID NO: 17), CDR2 with sequence NAQSLQT (SEQ ID NO: 18) and CDR3 with sequence QQYNSWPT (SEQ ID NO: 19);
L is a flexible linker comprising or mainly consisting of Ala (A), Thr (T), Gly (G) and/or Ser(S);
VH is the heavy chain variable region of the PD-1 single-chain antibody, comprising CDR1 with sequence GFSLSTSGT (SEQ ID NO: 20), CDR2 with sequence CWEDS (SEQ ID NO: 21) and CDR3 with sequence EDSGYFWFPY (SEQ ID NO: 22); and
Fc is an immunoglobulin Fc fragment, wherein the Fc fragment is derived from the Fc fragment of IgG, IgA, IgD, IgE or IgM.

### Claim 2 (depends on 1)

The oncolytic virus according to claim 1, wherein the virus is an adenovirus.

### Claim 3 (depends on 2)

The oncolytic virus according to claim 2, wherein the virus comprises ELA gene driven by a survivin promoter, and the endogenous promoter of the E1A gene in viral genome is replaced by the survivin promoter; and/or the activity of E1B gene is reduced or completely inactivated.

### Claim 4 (depends on 1)

The oncolytic virus according to claim 1, wherein the nucleic acid is operably linked to a promoter.

### Claim 5 (depends on 1)

The oncolytic virus according to claim 1, wherein the oncolytic virus was deposited at China Center for Type Culture Collection on Aug. 21, 2018, under the accession number CCTCC NO: V201853.

### Claim 6 (depends on 1)

A pharmaceutical composition comprising a pharmaceutically effective amount of the oncolytic virus according to claim 1, and optionally a pharmaceutically acceptable carrier.

### Claim 7 (depends on 6)

The pharmaceutical composition according to claim 6, wherein the pharmaceutical composition is formulated for oral, nebulized inhalation, intravenous, intramuscular, subcutaneous, perfusion, intralesional injection, or intratumoral administration.

### Claim 8 (depends on 6)

The pharmaceutical composition according to claim 6, comprising the oncolytic virus in an amount of about 108 vp to 1012 vp.

### Claim 9 (depends on 1)

A method of treating a proliferative tumor disease, comprising administering the oncolytic virus according to claim 1 to a subject in need thereof, wherein the tumor is selected from the group consisting of prostate cancer, breast cancer, colorectal cancer, lung cancer, liver cancer, melanoma, head and neck cancer, lymphoma, gastric cancer, esophageal cancer, ovarian cancer, head and neck squamous cell carcinoma, bladder cancer, and kidney cancer.

### Claim 10 (depends on 9)

The method according to claim 9, wherein the oncolytic virus is administered to the subject in an amount of about 108 vp to 1012 vp by oral, nebulized inhalation, intravenous, intramuscular, subcutaneous, perfusion, intralesional injection, or intratumoral administration, with an administration number of 1-6, and an administration interval of every 1, 2, 3, 4, 5, 6, 7 or more days, or 1, 2, 3, 4, 5, 6 or more times over the course of one day.

### Claim 11 (depends on 3)

The oncolytic virus according to claim 3, wherein E1B gene is knocked out from the viral genome.

### Claim 12 (depends on 1)

The oncolytic virus according to claim 1, wherein the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4 and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 3.

### Claim 13 (depends on 1)

The oncolytic virus according to claim 1, wherein L is 1-50 amino acids in length.

### Claim 14 (depends on 1)

The oncolytic virus according to claim 1, wherein L is (Gly4Ser) m, and m is a natural number between 1 and 10.

### Claim 15 (depends on 1)

The oncolytic virus according to claim 1, wherein L is (Gly4Ser)6 or L is A(EAAAK)nA, and n is a natural number between 1 and 9.

### Claim 16 (depends on 1)

The oncolytic virus according to claim 1, wherein the Fc fragment is derived from the Fc fragment of IgG.

### Claim 17 (depends on 1)

The oncolytic virus according to claim 1, wherein the sequence of the Fc fragment is shown in SEQ ID NO: 9.

### Claim 18 (depends on 4)

The oncolytic virus according to claim 4, wherein the promoter is a CMV promoter.