# US 12,215,143 B2 · Anti-polyubiquitin multispecific antibodies

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- Number: US 12,215,143 B2
- Document ID: US12215143B2
- Status: Granted
- Kind: B2
- Application number: 17/576,344
- Filed: 2022-01-14
- Published: 2022-08-04
- Granted: 2025-02-04
- Earliest claimed: 2016-06-24
- Assignees: Genentech, Inc.
- Applicants: Genentech, Inc.
- Inventors: Vishva Dixit; Marissa Matsumoto; Erick Castellanos
- Web page: https://patentgrep.com/patent/US12215143B2

## CPC

- C07K 16/18: against material from animals or humans
- A61P 35/00: Antineoplastic agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 15/62: DNA sequences coding for fusion proteins
- G01N 33/68: involving proteins, peptides or amino acids {(involving lipoproteins G01N33/92)}
- G01N 33/6857: {Antibody fragments}
- C07K 2317/31: multispecific
- C07K 2317/41: Glycosylation, sialylation, or fucosylation
- C07K 2317/52: Constant or Fc region; Isotype
- C07K 2317/526: CH3 domain
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/626: Diabody or triabody
- G01N 2440/36: addition of addition of other proteins or peptides, e.g. SUMOylation, ubiquitination
- 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)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The invention provides antibodies that bind mixed-topology polyubiquitin and multispecific anti-polyubiquitin antibodies, and methods of using the same.

## Claims

### Claim 1

A nucleic acid encoding a multispecific antibody that binds a mixed-topology polyubiquitin comprising a first linkage and a second linkage, the antibody comprising a first VH/VL unit specific for the first linkage, and a second VH/VL unit specific for the second linkage, wherein the first linkage and the second linkage are a K11 linkage, and a K48 linkage, or a K48 linkage and a K11 linkage, respectively, wherein the multispecific-antibody comprises first and second half antibodies, wherein,
a) one of the first and second half antibodies comprises
(i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10,
(iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11,
(iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12,
(v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and
(vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and
the other of the first and second half antibodies comprises
(i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24,
(iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 25,
(iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26,
(v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 27, and
(vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 28;
b) one of the first and second half antibodies comprises
(i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10,
(iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11,
(iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12,
(v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and
(vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and
the other of the first and second half antibodies comprises
(i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 37,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 38,
(iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39,
(iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40,
(v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41, and
(vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42;
c) one of the first and second half antibodies comprises
(i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10,
(iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11,
(iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12,
(v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and
(vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and
the other of the first and second half antibodies comprises
(i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 51,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 52,
(iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 53,
(iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 54,
(v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 55, and
(vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56;
d) one of the first and second half antibodies comprises
(i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24,
(iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 25,
(iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26,
(v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 27, and
(vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 28, and
the other of the first and second half antibodies comprises
(i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 37,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 38,
(iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39,
(iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40,
(v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41, and
(vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42;
e) one of the first and second half antibodies comprises
(i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24,
(iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 25,
(iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26,
(v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 27, and
(vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 28, and
the other of the first and second half antibodies comprises
(i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 51,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 52,
(iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 53,
(iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 54,
(v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 55, and
(vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56; or
f) one of the first and second half antibodies comprises
(i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 37,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 38,
(iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39,
(iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40,
(v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41, and
(vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42, and
the other of the first and second half antibodies comprises
(i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 51,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 52,
(iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 53,
(iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 54,
(v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 55, and
(vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56.

### Claim 2 (depends on 1)

The nucleic acid encoding a multispecific antibody of claim 1, wherein the multispecific antibody is: a knob-in-hole bispecific antibody; a bispecific antibody comprising a leucine zipper; a cross-linked pair of antibodies; an antibody Fc-heterodimeric molecule; a diabody; a triabody; a tetrabody; a single-chain Fv dimer; a trispecific antibody; an octopus antibody; or a dual acting FAb.

### Claim 3 (depends on 1)

The nucleic acid encoding a multispecific antibody of claim 1, wherein the first linkage and the second linkage are:
a K11 linkage and a K48 linkage.

### Claim 4 (depends on 1)

The nucleic acid encoding a multispecific antibody of claim 1, wherein the multispecific antibody comprises first and second half antibodies, wherein the first half antibody comprises:
a) a VL sequence of SEQ ID NO: 7 and a VH sequence of SEQ ID NO: 8; or
b) a VL sequence of SEQ ID NO: 21 and a VH sequence of SEQ ID NO: 22.

### Claim 5 (depends on 1)

The nucleic acid encoding a multispecific antibody of claim 1, wherein the multispecific antibody comprises first and second half antibodies, wherein the second half antibody comprises:
a) a VL sequence of SEQ ID NO: 7 and a VH sequence of SEQ ID NO: 8; or
b) a VL sequence of SEQ ID NO: 21 and a VH sequence of SEQ ID NO: 22;
wherein the VL and VH sequences of the second half antibody are not identical to the VL and VH sequences of the first half antibody.

### Claim 6 (depends on 1)

The nucleic acid encoding a multispecific antibody of claim 1, wherein the multispecific antibody comprises first and second half antibodies, wherein:
one of the first and second half antibodies comprises a VL sequence of SEQ ID NO: 7 and a VH sequence of SEQ ID NO: 8;
and the other of the first and second half antibodies comprises a VL sequence of SEQ ID NO: 21 and a VH sequence of SEQ ID NO: 22.

### Claim 7 (depends on 1)

The nucleic acid encoding a multispecific antibody of claim 1, wherein the multispecific antibody is a monoclonal antibody.

### Claim 8 (depends on 1)

The nucleic acid encoding a multispecific antibody of claim 1, wherein the multispecific antibody is an IgG antibody.

### Claim 9 (depends on 1)

The nucleic acid encoding a multispecific antibody of claim 1, wherein the bispecific antibody comprises first and second half antibodies, wherein the first half antibody comprises a first heavy chain constant region comprising a knob mutation and the second half antibody comprises a second heavy chain constant region comprising a hole mutation, or wherein the first half antibody comprises a first heavy chain constant region comprising a hole mutation and the second half antibody comprises a second heavy chain constant region comprising a knob mutation.

### Claim 10 (depends on 9)

The nucleic acid encoding a multispecific antibody of claim 9, wherein the multispecific antibody is an IgG1 antibody and wherein the knob mutation comprises a T366W mutation.

### Claim 11 (depends on 1)

The nucleic acid encoding a multispecific antibody of claim 1, wherein the multispecific antibody comprises first and second half antibodies, wherein the first half antibody comprises:
a) a light chain sequence of SEQ ID NO: 2 and a heavy chain sequence of SEQ ID NO: 4; or
b) a light chain sequence of SEQ ID NO: 16 and a heavy chain sequence of SEQ ID NO: 18;
optionally wherein the multispecific antibody comprises one or more heavy chains and a C-terminal lysine is missing from the one or more heavy chains.

### Claim 12 (depends on 1)

The nucleic acid encoding a multispecific antibody of claim 1, wherein the multispecific antibody comprises first and second half antibodies, wherein the first half antibody comprises:
a) a light chain sequence of SEQ ID NO: 2 and a heavy chain sequence of SEQ ID NO: 6; or
b) a light chain sequence of SEQ ID NO: 16 and a heavy chain sequence of SEQ ID NO: 20;
optionally wherein the multispecific antibody comprises one or more heavy chains and a C-terminal lysine is missing from the one or more heavy chains.

### Claim 13 (depends on 1)

The nucleic acid encoding a multispecific antibody of claim 1, wherein the multispecific antibody comprises first and second half antibodies, wherein:
a) the first half antibody comprises a light chain sequence of SEQ ID NO: 2 and a heavy chain sequence of SEQ ID NO: 4,
and the second half antibody comprises a light chain sequence of SEQ ID NO: 16 and a heavy chain sequence of SEQ ID NO: 20; or
b) the first half antibody comprises a light chain sequence of SEQ ID NO: 2 and a heavy chain sequence of SEQ ID NO: 6,
and the second half antibody comprises a light chain sequence of SEQ ID NO: 16 and a heavy chain sequence of SEQ ID NO: 18;
optionally wherein the multispecific antibody comprises one or more heavy chains and a C-terminal lysine is missing from the one or more heavy chains.

### Claim 14 (depends on 1)

A host cell comprising the nucleic acid of claim 1.

### Claim 15 (depends on 14)

A method of producing an antibody or half antibody comprising culturing the host cell of claim 14 under conditions suitable for producing the antibody or half antibody, thereby producing the antibody or half antibody.