# US 2019/0169278 A1 · Anti-Polyubiquitin Multispecific Antibodies

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- Number: US 2019/0169278 A1
- Document ID: US20190169278A1
- Status: Published application
- Kind: A1
- Application number: 16/211,669
- Filed: 2018-12-06
- Published: 2019-06-06
- Earliest claimed: 2016-06-24
- Assignees: Genentech, Inc.
- Applicants: Genentech, Inc.
- Inventors: Vishva Dixit; Marissa Matsumoto; Erick Castellanos
- Web page: https://patentgrep.com/patent/US20190169278A1

## 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 having greater avidity for a mixed-topology polyubiquitin than a single-topology polyubiquitin and multispecific anti-polyubiquitin antibodies, and methods of using the same.

## Claims

### Claim 1

An antibody with a greater avidity for a mixed-topology polyubiquitin than a single-topology polyubiquitin, wherein the mixed-topology polyubiquitin comprises a first linkage and a second linkage, wherein the first linkage and the second linkage differ from one another.

### Claim 2

A multispecific antibody that binds a mixed-topology polyubiquitin comprising a first linkage and a second linkage, the antibody comprising a first antigen recognition site specific for the first linkage, and a second antigen recognition site specific for the second linkage, wherein the first linkage and the second linkage differ from one another.

### Claim 3 (depends on 2)

The antibody of claim 2, comprising a first VH/VL unit specific for the first linkage, and a second VH/VL unit specific for the second linkage.

### Claim 4 (depends on 2)

The antibody of claim 2, which 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 5 (depends on 2)

The antibody of claim 2, wherein the first linkage or the second linkage is a K11 linkage, a K48 linkage, a K63 linkage, or a C-terminal to N-terminal-linkage.

### Claim at position 6

6-8. (canceled)

### Claim 9 (depends on 2)

The antibody of claim 2, wherein the first linkage and the second linkage are:
a) a K11 linkage and a K48 linkage;
b) a K11 linkage and a K63 linkage;
c) a K11 linkage and a C- to N-terminal linkage;
d) a K48 linkage and a K63 linkage;
e) a K48 linkage and a C- to N-terminal linkage; or
f) a K63 linkage and a C- to N-terminal linkage.

### Claim 10 (depends on 2)

The antibody of claim 2, comprising a first half antibody that comprises:
a) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9,
(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10,
(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11,
(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12,
(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and
(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14;
b) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 23,
(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 24,
(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 25,
(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 26,
(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 27, and
(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28;
c) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37,
(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38,
(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39,
(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 40,
(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and
(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42; or
d) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 51,
(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 52,
(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 53,
(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 54,
(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 55, and
(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 56.

### Claim 11 (depends on 10)

The antibody of claim 10, comprising a second half antibody that comprises:
a) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9,
(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10,
(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11,
(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12,
(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and
(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14;
b) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 23,
(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 24,
(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 25,
(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 26,
(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 27, and
(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28;
c) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37,
(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38,
(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39,
(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 40,
(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and
(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42; or
d) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 51,
(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 52,
(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 53,
(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 54,
(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 55, and
(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 56,
wherein the HVRs of the second half antibody are not identical to the HVRs of the first half antibody.

### Claim 12 (depends on 2)

The antibody of claim 2, comprising first and second half antibodies, wherein:
a) one of the first and second half antibodies comprises
(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9,
(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10,
(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11,
(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12,
(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and
(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14, and the other of the first and second half antibodies comprises
(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 23,
(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 24,
(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 25,
(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 26,
(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 27, and
(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28;
b) one of the first and second half antibodies comprises
(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9,
(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10,
(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11,
(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12,
(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and
(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14, and the other of the first and second half antibodies comprises
(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37,
(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38,
(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39,
(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 40,
(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and
(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42;
c) one of the first and second half antibodies comprises
(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9,
(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10,
(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11,
(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12,
(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and
(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14, and the other of the first and second half antibodies comprises
(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 51,
(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 52,
(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 53,
(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 54,
(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 55, and
(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 56;
d) one of the first and second half antibodies comprises
(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 23,
(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 24,
(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 25,
(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 26,
(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 27, and
(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28, and the other of the first and second half antibodies comprises
(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37,
(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38,
(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39,
(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 40,
(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and
(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42;
e) one of the first and second half antibodies comprises
(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 23,
(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 24,
(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 25,
(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 26,
(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 27, and
(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28, and the other of the first and second half antibodies comprises
(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 51,
(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 52,
(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 53,
(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 54,
(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 55, and
(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 56; or
f) one of the first and second half antibodies comprises
(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37,
(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38,
(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39,
(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 40,
(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and
(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42, and the other of the first and second half antibodies comprises
(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 51,
(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 52,
(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 53,
(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 54,
(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 55, and
(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 56.

### Claim at position 11

13-15. (canceled)

### Claim 16 (depends on 2)

The antibody of claim 2, comprising 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;
b) a VL sequence of SEQ ID NO: 21 and a VH sequence of SEQ ID NO: 22;
c) a VL sequence of SEQ ID NO: 35 and a VH sequence of SEQ ID NO: 36; or
d) a VL sequence of SEQ ID NO: 49 and a VH sequence of SEQ ID NO: 50.

### Claim 17 (depends on 16)

The antibody of claim 16, comprising 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;
b) a VL sequence of SEQ ID NO: 21 and a VH sequence of SEQ ID NO: 22;
c) a VL sequence of SEQ ID NO: 35 and a VH sequence of SEQ ID NO: 36; or
d) a VL sequence of SEQ ID NO: 49 and a VH sequence of SEQ ID NO: 50;
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 18 (depends on 2)

The antibody of claim 2, comprising first and second half antibodies, wherein:
a) 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;
b) 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: 35 and a VH sequence of SEQ ID NO: 36;
c) 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: 49 and a VH sequence of SEQ ID NO: 50;
d) one 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,
and the other of the first and second half antibodies comprises a VL sequence of SEQ ID NO: 35 and a VH sequence of SEQ ID NO: 36;
e) one 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,
and the other of the first and second half antibodies comprises a VL sequence of SEQ ID NO: 49 and a VH sequence of SEQ ID NO: 50; or
f) one of the first and second half antibodies comprises a VL sequence of SEQ ID NO: 35 and a VH sequence of SEQ ID NO: 36,
and the other of the first and second half antibodies comprises a VL sequence of SEQ ID NO: 49 and a VH sequence of SEQ ID NO: 50.

### Claim 19 (depends on 2)

The antibody of claim 2, which is a monoclonal antibody.

### Claim 20 (depends on 2)

The antibody of claim 2, which is a mouse, rabbit, human, humanized, or chimeric antibody.

### Claim 21 (depends on 2)

The antibody of claim 2, wherein the antibody is an IgG antibody.

### Claim at position 18

22-23. (canceled)

### Claim 24 (depends on 2)

The antibody of claim 2, comprising 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 25 (depends on 24)

The antibody of claim 24, wherein the antibody is an IgG1 or IgG4 antibody and wherein (i) the knob mutation comprises a T366W mutation and/or (ii) the hole mutation comprises T366S, L368A, and Y407V mutations.

### Claim at position 21

26-41. (canceled)

### Claim 42 (depends on 12)

The antibody of claim 12, comprising 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;
b) a light chain sequence of SEQ ID NO: 16 and a heavy chain sequence of SEQ ID NO: 18;
c) a light chain sequence of SEQ ID NO: 30 and a heavy chain sequence of SEQ ID NO: 32; or
d) a light chain sequence of SEQ ID NO: 44 and a heavy chain sequence of SEQ ID NO: 46,
optionally wherein a C-terminal lysine is missing from one or more heavy chains.

### Claim 43

(canceled)

### Claim 44 (depends on 12)

The antibody of claim 12, comprising 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;
b) a light chain sequence of SEQ ID NO: 16 and a heavy chain sequence of SEQ ID NO: 20;
c) a light chain sequence of SEQ ID NO: 30 and a heavy chain sequence of SEQ ID NO: 34; or
d) a light chain sequence of SEQ ID NO: 44 and a heavy chain sequence of SEQ ID NO: 48,
optionally wherein a C-terminal lysine is missing from one or more heavy chains.

### Claim at position 25

45-49. (canceled)

### Claim 50 (depends on 2)

The antibody of claim 2, comprising 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;
b) 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: 30 and a heavy chain sequence of SEQ ID NO: 34;
c) 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: 44 and a heavy chain sequence of SEQ ID NO: 48;
d) 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;
e) 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: 30 and a heavy chain sequence of SEQ ID NO: 32;
f) 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: 44 and a heavy chain sequence of SEQ ID NO: 46;
g) the first half antibody comprises a light chain sequence of SEQ ID NO: 16 and a heavy chain sequence of SEQ ID NO: 18,
and the second half antibody comprises a light chain sequence of SEQ ID NO: 30 and a heavy chain sequence of SEQ ID NO: 34;
h) the first half antibody comprises a light chain sequence of SEQ ID NO: 16 and a heavy chain sequence of SEQ ID NO: 18,
and the second half antibody comprises a light chain sequence of SEQ ID NO: 44 and a heavy chain sequence of SEQ ID NO: 48;
i) the first half antibody comprises a light chain sequence of SEQ ID NO: 30 and a heavy chain sequence of SEQ ID NO: 32,
and the second half antibody comprises a light chain sequence of SEQ ID NO: 44 and a heavy chain sequence of SEQ ID NO: 48;
j) the first half antibody comprises a light chain sequence of SEQ ID NO: 16 and a heavy chain sequence of SEQ ID NO: 20,
and the second half antibody comprises a light chain sequence of SEQ ID NO: 30 and a heavy chain sequence of SEQ ID NO: 32;
k) the first half antibody comprises a light chain sequence of SEQ ID NO: 16 and a heavy chain sequence of SEQ ID NO: 20,
and the second half antibody comprises a light chain sequence of SEQ ID NO: 44 and a heavy chain sequence of SEQ ID NO: 46; or
l) the first half antibody comprises a light chain sequence of SEQ ID NO: 30 and a heavy chain sequence of SEQ ID NO: 34,
and the second half antibody comprises a light chain sequence of SEQ ID NO: 44 and a heavy chain sequence of SEQ ID NO: 46;
optionally wherein a C-terminal lysine is missing from one or more heavy chains.

### Claim at position 27

51-66. (canceled)

### Claim 67 (depends on 2)

The antibody of claim 2, conjugated to a label.

### Claim at position 29

68-69. (canceled)

### Claim 70 (depends on 2)

A composition comprising the antibody of claim 2, wherein the composition is substantially free of monospecific antibodies, unassembled half antibodies, or both monospecific antibodies and unassembled half antibodies.

### Claim 71

(canceled)

### Claim 72 (depends on 1)

A pharmaceutical formulation comprising a pharmaceutically acceptable carrier and at least one of:
a) the antibody of claim 1; or
b) an immunoconjugate comprising the antibody of (a) and a cytotoxic agent;
optionally wherein the composition is substantially free of monospecific antibodies, unassembled half antibodies, or both monospecific antibodies and unassembled half antibodies.

### Claim 73 (depends on 2)

Isolated nucleic acid encoding:
the antibody of claim 2.

### Claim 74 (depends on 73)

A host cell comprising the nucleic acid of claim 73.

### Claim 75 (depends on 73)

A method of producing an antibody comprising culturing the host cell of claim 73 so that the antibody is produced.

### Claim 76 (depends on 2)

A method of making the antibody of claim 2, comprising forming the antibody from a first half antibody and a second half antibody.

### Claim 77 (depends on 2)

A method of detecting a mixed-topology polyubiquitin in a biological sample comprising contacting the biological sample with the antibody of claim 2 under conditions permissive for binding of the antibody to the mixed-topology polyubiquitin, and detecting whether a complex is formed between the antibody and mixed-topology polyubiquitin in the biological sample.

### Claim at position 38

78-86. (canceled)

### Claim 87 (depends on 2)

A method of detecting a polyubiquitinated protein in a biological sample, the polyubiquitinated protein being polyubiquitinated at two or more positions with at least first and second polyubiquitins, with the first and second polyubiquitins having different linkages, comprising contacting the biological sample with the antibody of claim 2 under conditions permissive for binding of the antibody to the polyubiquitinated protein, and detecting whether a complex is formed between the antibody and polyubiquitinated protein in the biological sample.

### Claim 88

(canceled)