# US 12,215,148 B2 · Antigen binding molecule formats

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- Number: US 12,215,148 B2
- Document ID: US12215148B2
- Status: Granted
- Kind: B2
- Application number: 16/987,487
- Filed: 2020-08-07
- Published: 2021-02-11
- Granted: 2025-02-04
- Earliest claimed: 2019-08-08
- Assignees: Regeneron Pharmaceuticals, Inc.
- Applicants: Regeneron Pharmaceuticals, Inc.
- Inventors: Tong Zhang; Erica Pyles; Michael Rosconi; Nina Liu; Supriya Patel; Eric Smith; Andrew Murphy; Chiayang Lin; Samuel Davis
- Web page: https://patentgrep.com/patent/US12215148B2

## CPC

- C07K 16/244: {Interleukins [IL]}
- A61K 39/395: Antibodies (agglutinins A61K38/36 {; as drug carriers A61K47/50}); Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K 47/6845: {the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon}
- A61K 47/6849: {the antibody targeting a receptor, a cell surface antigen or a cell surface determinant}
- A61P 35/00: Antineoplastic agents
- C07K 16/2809: {against the T-cell receptor (TcR)-CD3 complex}
- C07K 16/30: from tumour cells
- C07K 16/46: Hybrid immunoglobulins (hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00)
- A61K 2039/505: {comprising antibodies}
- C07K 2317/31: multispecific
- C07K 2317/35: Valency
- C07K 2317/52: Constant or Fc region; Isotype
- C07K 2317/522: CH1 domain
- C07K 2317/53: Hinge
- C07K 2317/55: Fab or Fab'
- C07K 2317/64: comprising a combination of variable region and constant region components
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- 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

## Abstract

Antigen binding molecules (ABMs) comprising Fab domains in non-native configurations, ABM conjugates comprising the ABMs and cytotoxic or cytostatic agents, pharmaceutical compositions containing the ABMs and ABM conjugates, methods of using the ABMs, ABM conjugates and pharmaceutical compositions for treating cancer, nucleic acids encoding the ABMs, cells engineered to express the ABMs, and methods of producing ABMs.

## Claims

### Claim 1

A method, comprising contacting a target molecule having a molecular weight of less than 100 kDa exclusive of post-translational modifications with an antigen-binding molecule comprising:
(a) a first half antibody comprising in an N- to C-terminal orientation:
(i) a first Fc domain which lacks a Fab domain at its N-terminus; and
(ii) a first Fab domain that binds to the target molecule, the first Fab domain comprising a first heavy chain variable region (VH) associated with a first light chain variable region (VL) on a separate polypeptide chain; and
(b) a second half antibody comprising in an N- to C-terminal orientation:
(i) a second Fc domain which lacks a Fab domain at its N-terminus, the second Fc domain associated with the first Fc domain to form an Fc heterodimer; and
(ii) a second Fab domain that binds to the target molecule, the second Fab domain comprising a second VH associated with a second VL on a separate polypeptide chain.

### Claim 2 (depends on 1)

The method of claim 1, wherein the first half antibody comprises a first hinge domain N-terminal to the first Fc domain and the second half antibody comprises a second hinge domain N-terminal to the second Fc domain.

### Claim 3 (depends on 1)

The method of claim 1, wherein the antigen-binding molecule has one hinge region.

### Claim 4 (depends on 3)

The method of claim 3, wherein the hinge region comprises a chimeric hinge sequence.

### Claim 5 (depends on 4)

The method of claim 4, wherein the chimeric hinge sequence comprises SEQ ID NO:22 or SEQ ID NO:23.

### Claim 6 (depends on 1)

The method of claim 1, wherein the antigen-binding molecule has two hinge regions.

### Claim 7 (depends on 1)

The method of claim 1, wherein the first Fab domain and the second Fab domain are not in the form of single chain Fabs.

### Claim 8 (depends on 1)

The method of claim 1, wherein the antigen-binding molecule is bispecific.

### Claim 9 (depends on 1)

The method of claim 1, wherein the first Fc domain and the second Fc domain comprise knob-in-hole mutations as compared to a wild-type Fc domain.

### Claim 10 (depends on 1)

The method of claim 1, wherein the antigen-binding molecule is bivalent.

### Claim 11 (depends on 1)

The method of claim 1, wherein the target molecule has a molecular weight of less than 75 kDa exclusive of post-translational modifications.

### Claim 12 (depends on 1)

The method of claim 1, wherein the target molecule has a molecular weight of less than 60 kDa exclusive of post-translational modifications.

### Claim 13 (depends on 1)

The method of claim 1, wherein the target molecule has a molecular weight of less than 45 kDa exclusive of post-translational modifications.

### Claim 14 (depends on 1)

The method of claim 1, wherein the target molecule has a molecular weight ranging from 5 kDa to 30 kDa exclusive of post-translational modifications.

### Claim 15 (depends on 1)

The method of claim 1, wherein contacting the target molecule is performed in vitro.

### Claim 16 (depends on 1)

The method of claim 1, wherein contacting the target molecule is performed in vivo.

### Claim 17 (depends on 1)

The method of claim 1, wherein,
(a) the first half antibody comprises:
(i) a first polypeptide comprising, in an N- to C-terminal orientation:
(1) the first Fc domain;
(2) a first linker;
(3) the first VH; and
(4) a first CH1 domain; and
(ii) a second polypeptide associated with the first polypeptide comprising, in an N-to-C terminal orientation, the first VL and a first CL domain, wherein the first VL and the first CL domain are associated with the first VH and the first CH1 domain to form the first Fab domain; and
(b) the second half antibody comprises:
(i) a third polypeptide comprising, in an N- to C-terminal orientation:
(1) the second Fc domain;
(2) a second linker;
(3) the second VH; and
(4) a second CH1 domain; and
(ii) a fourth polypeptide associated with the third polypeptide comprising, in an N-to-C terminal orientation, the second VL and a second CL domain, wherein the second VL and the second CL domain are associated with the second VH and the second CH1 domain to form the second Fab domain.

### Claim 18 (depends on 17)

The method of claim 17, wherein the first polypeptide comprises a first hinge sequence between the first Fc domain and the first linker, and the third polypeptide comprises a second hinge sequence between the second Fc domain and the second linker.

### Claim 19 (depends on 18)

The method of claim 18, wherein (1) the first hinge sequence comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, and (2) the second hinge sequence comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.

### Claim 20 (depends on 19)

The method of claim 19, wherein (1) the first hinge sequence comprises the amino acid sequence of SEQ ID NO:2, and (2) the second hinge sequence comprises the amino acid sequence of SEQ ID NO:2.

### Claim 21 (depends on 17)

The method of claim 17, wherein the first Fc domain is an IgG4 Fc domain.

### Claim 22 (depends on 17)

The method of claim 17, wherein the first Fc domain is an IgG1 Fc domain.

### Claim 23 (depends on 17)

The method of claim 17, wherein the second Fc domain is an IgG4 Fc domain.

### Claim 24 (depends on 17)

The method of claim 17, wherein the second Fc domain is an IgG1 Fc domain.

### Claim 25 (depends on 16)

The method of claim 16, wherein the antigen-binding molecule is provided in the form of a pharmaceutical composition comprising the antigen-binding molecule and an excipient.