# US 12,110,332 B2 · Bifunctional fusion protein targeting CD47 and PD-L1

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- Number: US 12,110,332 B2
- Document ID: US12110332B2
- Status: Granted
- Kind: B2
- Application number: 16/765,008
- Filed: 2017-11-20
- Published: 2020-11-12
- Granted: 2024-10-08
- Earliest claimed: 2017-11-20
- Assignees: Taizhou Mabtech Pharmaceutical Co., Ltd.
- Applicants: Taizhou Mabtech Pharmaceutical Co., Ltd.
- Inventors: Yajun Guo
- Web page: https://patentgrep.com/patent/US12110332B2

## CPC

- C07K 16/2827: {against B7 molecules, e.g. CD80, CD86}
- A61P 35/00: Antineoplastic agents
- A61P 37/02: Immunomodulators
- C07K 14/70503: {Immunoglobulin superfamily}
- C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere}
- C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes}
- C12N 15/62: DNA sequences coding for fusion proteins
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- A61K 2039/505: {comprising antibodies}
- C07K 16/2803: {against the immunoglobulin superfamily}
- C07K 2317/31: multispecific
- C07K 2317/51: Complete heavy chain or Fd fragment, i.e. VH + CH1
- C07K 2317/515: Complete light chain, i.e. VL + CL
- C07K 2317/52: Constant or Fc region; Isotype
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2317/732: Antibody-dependent cellular cytotoxicity [ADCC]
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2319/00: Fusion polypeptide
- C07K 2319/70: containing domain for protein-protein interaction
- C07K 2319/74: containing a fusion for binding to a cell surface receptor
- 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)
- 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)

## Abstract

A bifunctional fusion protein targeting CD47 and PD-L1, which belongs to the field of biomedicine and solves the problems that anti-PD-1/PD-L1 treatment has poor effect on low immunogenic tumors and anti-CD47 treatment has poor targeting properties. The fusion protein consists of a CD47-binding moiety and a PD-L1-binding moiety linked by means of a disulfide bond, can block both the binding of CD47 to SIRPa and the binding of PD-L1 to PD-1, not only activating macrophage phagocytosis of tumor cells and promoting antigen presentation in innate immunity, but also promoting tumor-specific T cell activation in acquired immunity, and has lower hematological toxicity. The fusion protein has better anti-tumor efficacy and hematological safety than anti-PD-L1 or anti-CD47 therapy alone, and animals from which a tumor has disappeared after the treatment of the fusion protein produce immunity against re-inoculation of the same tumor cell.

## Claims

### Claim 1

A bifunctional protein, characterized in that the bifunctional protein consists of two moieties linked by disulfide bonds,
wherein one moiety is a CD47 binding moiety formed by linking SIRPα mutant to an antibody Fc, and the other moiety is a Programmed death-ligand 1 (PD-L1) binding moiety formed by linking a light chain and a heavy chain of anti-PD-L1 antibody by disulfide bond, the PD-L1 binding moiety having a Fc segment;
wherein the bifunctional protein has a Knob-in-Hole structure formed by the CD47 binding moiety and the PD-L1 binding moiety, the Fc of the CD47 binding moiety has four amino acid mutations of y349c, t366s, 1368a and y407v, and the Fc segment of the PD-L1 binding moiety has two amino acid mutations of t366w and s354c; and
wherein the CD47 binding moiety comprises SEQ ID NO: 1, the light chain of the PD-L1 binding moiety comprises SEQ ID NO: 2, and the heavy chain comprises in SEQ ID NO: 3.

### Claim 2 (depends on 1)

A method for treating a tumor comprising: administrating the bifunctional protein of claim 1 to a subject with the tumor.