# US 10,870,701 B2 · Multispecific fab fusion proteins and use thereof

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- Number: US 10,870,701 B2
- Document ID: US10870701B2
- Status: Granted
- Kind: B2
- Application number: 16/085,542
- Filed: 2017-03-15
- Published: 2019-03-28
- Granted: 2020-12-22
- Earliest claimed: 2016-03-15
- Assignees: Generon (Shanghai) Corporation Ltd.
- Applicants: Generon (Shanghai) Corporation Ltd.
- Inventors: Yumin Cui; Zhihua Huang; Hanyang Chen; Xinfeng Zhang; Bo Qi; Xiaoqiang Yan
- Web page: https://patentgrep.com/patent/US10870701B2

## CPC

- C07K 16/2809: {against the T-cell receptor (TcR)-CD3 complex}
- A61K 31/573: substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
- A61P 35/00: Antineoplastic agents
- C07K 16/2821: {against ICAM molecules, e.g. CD50, CD54, CD102}
- C07K 16/30: from tumour cells
- C07K 16/3023: {Lung}
- C07K 16/3046: {Stomach, Intestines}
- C12N 15/62: DNA sequences coding for fusion proteins
- A61K 2039/505: {comprising antibodies}
- C07K 2317/31: multispecific
- C07K 2317/55: Fab or Fab'
- C07K 2317/62: comprising only variable region components
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2317/94: Stability, e.g. half-life, pH, temperature or enzyme-resistance
- 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)

## Abstract

The present invention provides multispecific Fab fusion proteins (MSFP) that specifically bind to CD3 and EpCAM. The present invention further provides uses of the MSFPs for the preparation of pharmaceutical compositions, methods of treating cancer, and kits comprising the MSFPs. Also provided are anti-EpCAM antibodies or antigen-binding fragments thereof.

## Claims

### Claim 1

An anti-EpCAM antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising: (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and a light chain variable region (VL) comprising: (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18.

### Claim 2 (depends on 1)

The anti-EpCAM antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises the amino acid sequence of SEQ ID NO:19; and/or wherein the VL comprises the amino acid sequence of SEQ ID NO:20.

### Claim 3 (depends on 1)

The anti-EpCAM antibody of claim 1, wherein the anti-EpCAM antibody is a multispecific antibody.

### Claim 4 (depends on 1)

The antigen-binding fragment of the anti-EpCAM antibody of claim 1, wherein the antigen-binding fragment is a single-chain FAT (scFv).

### Claim 5 (depends on 4)

The antigen-binding fragment of the anti-EpCAM antibody of claim 4, wherein the scFv comprises the amino acid sequence of SEQ ID NO:21.

### Claim 6 (depends on 1)

A multispecific Fab fusion protein comprising the anti-EpCAM antigen-binding fragment of claim 1.

### Claim 7 (depends on 6)

The multispecific Fab fusion protein of claim 6, comprising a Fab fragment that specifically binds to CD3, a first copy of the anti-EpCAM antigen-binding fragment, and a second copy of the anti-EpCAM antigen binding fragment; wherein the first copy of the anti-EpCAM antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment; and wherein the second copy of the anti-EpCAM antigen binding fragment is fused to the N-terminus of the VL of the Fab fragment.

### Claim 8 (depends on 7)

The multispecific Fab fusion protein of claim 7, wherein the Fab fragment specifically binds to the N-terminus of CD3 epsilon.

### Claim 9 (depends on 8)

The multispecific Fab fusion protein of claim 8, wherein the Fab fragment specifically binds to an epitope within amino acids 1-27 of CD3 epsilon.

### Claim 10 (depends on 9)

The multispecific Fab fusion protein of claim 9, wherein the VH of the Fab fragment comprises a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, and/or the VL of the Fab fragment comprises a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6.

### Claim 11 (depends on 10)

The multispecific Fab fusion protein of claim 10, wherein the VH of the Fab fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:7 and 39-43, and/or wherein the VL of the Fab fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:8 and 44-47.

### Claim 12 (depends on 7)

The multispecific Fab fusion protein of claim 7, wherein the Fab fragment comprises a human immunoglobulin heavy chain constant region 1 (CH1) comprising the amino acid sequence of SEQ ID NO:9.

### Claim 13 (depends on 7)

The multispecific Fab fusion protein of claim 7, wherein the Fab fragment comprises a human lambda light chain constant region (CL) comprising the amino acid sequence of SEQ ID NO:10.

### Claim 14 (depends on 7)

The multispecific Fab fusion protein of claim 7, wherein the CH1 and the CL of the Fab fragment are connected by one or more disulfide bonds.

### Claim 15 (depends on 7)

The multispecific Fab fusion protein of claim 7, comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:22, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:23.

### Claim 16 (depends on 7)

A method of treating a cancer in an individual, comprising administering to the individual an effective amount of the multispecific Fab fusion protein of claim 7.

### Claim 17 (depends on 16)

The method of claim 16, wherein the multispecific Fab fusion protein is administered intravenously.

### Claim 18 (depends on 16)

The method of claim 16, wherein the cancer is selected from the group consisting of small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, skin cancer, renal cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, bile duct cancer, and head and neck cancer.

### Claim 19 (depends on 18)

The method of claim 18, wherein the cancer is colorectal adenocarcinoma.

### Claim 20 (depends on 18)

The method of claim 18, wherein the cancer is lung adenocarcinoma.