# US 11,440,972 B2 · Bispecific antibodies and uses thereof

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- Number: US 11,440,972 B2
- Document ID: US11440972B2
- Status: Granted
- Kind: B2
- Application number: 17/398,853
- Filed: 2021-08-10
- Published: 2022-01-27
- Granted: 2022-09-13
- Earliest claimed: 2017-08-01
- Assignees: Ab Studio Inc.
- Applicants: AB Studio Inc.
- Inventors: Yue Liu; Wenyan Cai; Jiadong Shi
- Web page: https://patentgrep.com/patent/US11440972B2

## CPC

- C07K 16/2887: {against CD20}
- C07K 16/468: {Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies}
- A61P 35/00: Antineoplastic agents
- A61P 35/02: specific for leukemia
- C07K 16/2809: {against the T-cell receptor (TcR)-CD3 complex}
- C07K 16/2827: {against B7 molecules, e.g. CD80, CD86}
- C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere}
- A61K 2039/505: {comprising antibodies}
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/31: multispecific
- C07K 2317/515: Complete light chain, i.e. VL + CL
- C07K 2317/524: CH2 domain
- C07K 2317/526: CH3 domain
- C07K 2317/567: Framework region [FR]
- C07K 2317/71: Decreased effector function due to an Fc-modification
- C07K 2317/72: Increased effector function due to an Fc-modification
- C07K 2317/732: Antibody-dependent cellular cytotoxicity [ADCC]
- C07K 2317/734: Complement-dependent cytotoxicity [CDC]
- C07K 2317/77: Internalization into the cell
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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

This disclosure relates to bispecific antibodies or antigen-binding fragments thereof, wherein the bispecific antibodies or antigen-binding fragments thereof specifically bind to two different antigens with different binding affinities.

## Claims

### Claim 1

A method of making a bispecific antibody or antigen-binding fragment thereof, the method comprising:
(a) selecting a first antigen and a second antigen, and identifying a first antibody or antigen-binding fragment thereof that binds to the first antigen and a second antibody or antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or the antigen-binding fragment thereof comprises a first heavy chain variable region (VHa) and a first light chain variable region (VLa), and the second antibody or the antigen-binding fragment thereof comprises a second heavy chain variable region (VHb) and a second light chain variable region (VLb);
(b) determining the amino acid sequence of VHa, VLa, VHb, and VLb;
(c) aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is greater than 80%;
(d) designing a common light chain variable region (VLc), wherein the VLc, when it associates with VHa, maintains the affinity to the first antigen;
(e) redesigning the VHa and VHb sequences, thereby obtaining VHa′ and VHb′ to increase the difference of biochemical or biophysical characteristics between a first protein comprising two polypeptides each comprising VHa′ and two polypeptides each comprising VLc, and a second protein comprising two polypeptides each comprising VHb′ and two polypeptides each comprising VLc; and
(f) producing a bispecific antibody or antigen-binding fragment thereof that has two light chain variable regions and two heavy chain variable regions, wherein the two light chain variable regions each comprises VLc, and the two heavy chain variable regions comprise VHa′ and VHb′ respectively, wherein VHa′ and VLc associate with each other, forming a first antigen-binding region that specifically binds to the first antigen, and VHb′ and VLc associate with each other, forming a second antigen-binding region that specifically binds to the second antigen, wherein the binding affinity of the first antigen-binding region when it binds to the first antigen is at least 10 times greater than the binding affinity of the second antigen binding region when it binds to the second antigen.

### Claim 2 (depends on 1)

The method of claim 1, wherein in step (d), the binding affinity of the VLc-VHb to the second antigen can decrease.

### Claim 3 (depends on 1)

The method of claim 1, wherein the method further comprises
(g) developing a buffer system to purify the bispecific antibody or the antigen-binding fragment thereof.

### Claim 4 (depends on 1)

The method of claim 1, wherein the first antigen-binding region specifically binds to the first antigen with a binding affinity greater than 108 M−1, wherein the second antigen-binding region specifically binds to the second antigen with a binding affinity less than 106M−1.

### Claim 5 (depends on 1)

The method of claim 1, wherein the binding affinity of the first antigen-binding region when it binds to the first antigen is at least 1000 times greater than the binding affinity of the second antigen-binding region when it binds to the second antigen.

### Claim 6 (depends on 1)

The method of claim 1, wherein the first antigen is a cancer-specific antigen, and the second antigen is CD3.

### Claim 7 (depends on 1)

The method of claim 1, wherein the first antigen is CD20, and the second antigen is CD3.

### Claim 8 (depends on 1)

The method of claim 1, wherein the first antigen is a cancer-specific antigen, and the second antigen is a cancer-associated antigen.

### Claim 9 (depends on 1)

The method of claim 1, wherein the first antigen is PD-L1 and the second antigen is CD55.

### Claim 10 (depends on 2)

The method of claim 2, wherein in step (d), if an amino acid in the light chain variable region is identified to be important for binding to the second antigen, but not important in the binding with the first antigen, then the amino acid in VLa can be changed to the corresponding amino acid in VLb.

### Claim 11 (depends on 2)

The method of claim 2, wherein step (d) comprises the following steps:
(i) analyzing interaction between the VLa and the VHa and interaction between the VLb and the VHb and introducing one or more mutations on VLa;
(ii) if an amino acid in the light chain variable region is identified to be important for binding to the second antigen, but not important in the binding with the first antigen, then the amino acid in VLa can be changed to the corresponding amino acid in VLb, thereby creating a modified VLa sequence; and
(iii) analyzing interaction between the modified VLa sequence and the VHa and interaction between the modified VLa sequence and the VHb, and repeating step (ii) until obtaining the VLc.