# US 11,993,642 B2 · Trivalent, bispecific antibodies

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- Number: US 11,993,642 B2
- Document ID: US11993642B2
- Status: Granted
- Kind: B2
- Application number: 16/413,469
- Filed: 2019-05-15
- Published: 2020-02-27
- Granted: 2024-05-28
- Earliest claimed: 2009-04-07
- Assignees: Hoffmann-La Roche Inc.
- Applicants: Hoffmann-La Roche Inc.
- Inventors: Ulrich Brinkmann; Rebecca Croasdale; Eike Hoffmann; Christian Klein; Ekkehard Moessner; Juergen Michael Schanzer; Claudio Sustmann; Pablo Umana
- Web page: https://patentgrep.com/patent/US11993642B2

## CPC

- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- A61P 35/00: Antineoplastic agents
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- C07K 16/32: against translation products of oncogenes
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/31: multispecific
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2319/00: Fusion polypeptide
- 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

## Abstract

The present invention relates to trivalent, bispecific antibodies, methods for their production, pharmaceutical compositions containing the antibodies, and uses thereof.

## Claims

### Claim 1

A nucleic acid encoding a trivalent, bispecific antibody comprising
a) a full length antibody that specifically binds to a first antigen wherein the full length antibody comprises two antibody heavy chains and two antibody light chains, wherein the CH3 domain of one heavy chain and the CH3 domain of the other heavy chain each meet at an interface which comprises an alteration in the original interface between the antibody CH3 domains, wherein
i) in the CH3 domain of one heavy chain an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the interface of the CH3 domain of one heavy chain which is positionable in a cavity within the interface of the CH3 domain of the other heavy chain, and wherein
ii) in the CH3 domain of the other heavy chain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the interface of the second CH3 domain within which the protuberance within the interface of the first CH3 domain is positionable;
b) a polypeptide comprising
ba) an antibody heavy chain variable domain (VH); or
bb) an antibody heavy chain variable domain (VH) and an antibody constant domain 1 (CH1), wherein the N-terminus of the VH domain of the polypeptide is fused via a peptide connector to the C-terminus of the CH3 domain of one of the two heavy chains of the full length antibody;
c) a polypeptide comprising
ca) an antibody light chain variable domain (VL), or
cb) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL);
wherein the N-terminus of the VL domain of the polypeptide is fused via a peptide connector to the C-terminus of the CH3 domain of the other of the two heavy chains of the full length antibody;
and wherein the antibody heavy chain variable domain (VH) of the polypeptide under b) and the antibody light chain variable domain (VL) of the polypeptide under c) together form an antigen-binding site specifically binding to a second antigen; and
wherein the peptide connectors under b) and c) are peptides with a length between 5 and 50 amino acids.

### Claim 2 (depends on 1)

The nucleic acid of claim 1, wherein
i) the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W); and
ii) the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

### Claim 3 (depends on 2)

The nucleic acid of claim 2, wherein both CH3 domains are further altered by the introduction of cysteine as an amino acid in each CH3 domain such that a disulfide bridge between both CH3 domains can be formed.

### Claim 4 (depends on 3)

The nucleic acid of claim 3, wherein
the CH3 domain under i) comprises a T366W mutation; and
the CH3 domain under ii) comprises T366S, L368A, and Y407V mutations,
wherein the numbering is according to the EU index of Kabat.

### Claim 5 (depends on 4)

The nucleic acid of claim 4, wherein
the CH3 domain under i) comprises Y349C and T366W mutations; and
the CH3 domain under ii) comprises S354C, T366S, L368A, and Y407V mutations,
wherein the numbering is according to the EU index of Kabat.

### Claim 6 (depends on 1)

The nucleic acid of claim 1, wherein
the antibody heavy chain variable domain (VH) of the polypeptide under b) and the antibody light chain variable domain (VL) of the polypeptide under c) are linked and stabilized via an interchain disulfide bridge by introduction of a disulfide bond between the following positions:
i) heavy chain variable domain (VH) position 44 and light chain variable domain (VL) position 100;
ii) heavy chain variable domain (VH) position 105 and light chain variable domain (VL) position 43; or
iii) heavy chain variable domain (VH) position 101 and light chain variable domain (VL) position 100,
wherein the numbering is according to the EU index of Kabat.

### Claim 7 (depends on 6)

The nucleic acid of claim 6, wherein
the antibody heavy chain variable domain (VH) of the polypeptide under b) and the antibody light chain variable domain (VL) of the polypeptide under c) are linked and stabilized via an interchain disulfide bridge by introduction of a disulfide bond between heavy chain variable domain (VH) position 44 and light chain variable domain (VL) position 100,
wherein the numbering is according to the EU index of Kabat.

### Claim 8 (depends on 1)

The nucleic acid of claim 1, wherein the peptide connectors under b) and c) are identical peptides with a length between 25 and 50 amino acids.

### Claim 9 (depends on 1)

The nucleic acid of claim 1, wherein the peptide connectors under b) and c) comprise glycine (G) and serine (S) residues.

### Claim 10 (depends on 9)

The nucleic acid of claim 9, wherein the peptide connectors under b) and c) comprise GGGGS (SEQ ID NO: 38) repeats.