# US 10,501,555 B2 · Humanized anti-trop-2 monoclonal antibodies and uses thereof

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- Number: US 10,501,555 B2
- Document ID: US10501555B2
- Status: Granted
- Kind: B2
- Application number: 15/532,598
- Filed: 2015-12-04
- Published: 2018-01-04
- Granted: 2019-12-10
- Earliest claimed: 2014-12-04
- Assignees: Abruzzo Theranostic S.R.L.
- Applicants: Abruzzo Theranostic S.R.L.
- Inventors: Emanuela Guerra; Saverio Alberti
- Web page: https://patentgrep.com/patent/US10501555B2

## CPC

- C07K 16/30: from tumour cells
- A61K 39/395: Antibodies (agglutinins A61K38/36 {; as drug carriers A61K47/50}); Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61P 35/00: Antineoplastic agents
- A61P 35/04: specific for metastasis
- A61K 2039/505: {comprising antibodies}
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/33: Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
- C07K 2317/732: Antibody-dependent cellular cytotoxicity [ADCC]
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- 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

The present invention refers to humanized anti-Trop-2 antibodies and their fragments, derivatives and conjugates that are able to recognise and bind with high affinity distinct regions of the Trop-2 molecule. The present invention also teaches the use of such antibodies and of pharmaceutical compositions thereof for diagnosis and therapy of human pathologies, in particular cancer.

## Claims

### Claim 1

A humanized antibody, or fragments or conjugates thereof, which recognizes and binds the same epitope or epitopes as the antibody 2G10 produced by the hybridoma deposited in the Advanced Biotechnology Center (ABC) with the number PD 08020;
(a) wherein the humanized antibody comprises (i) a variable domain framework region from a heavy chain of a human antibody or from a human consensus framework; and/or (ii) a variable domain framework region from a light chain of a human antibody or from a human consensus framework; and
(b) wherein (i) at least one amino acid in the variable domain framework region from the heavy chain is substituted with a corresponding amino acid from a heavy chain of a mouse antibody or mouse consensus framework and/or (ii) at least one amino acid in the variable domain framework region from the light chain is substituted with a corresponding amino acid from a light chain of a mouse antibody or mouse consensus framework; and
(c) wherein (i) the amino acid substitutions in the heavy chain are:
F (murine) instead of Y (human) at position 27, and/or
S (murine) instead of T (human) at position 30, and/or
L (murine) instead of V (human) at position 37, and/or
I (murine) instead of M (human) at position 48, and/or
A (murine) instead of G (human) at position 49, and/or
L (murine) instead of I (human) at position 70, and/or
V (murine) instead of R (human) at position 72; and/or
(ii) the amino acid substitution in the light chain is S (murine) instead of Y (human) at position 49; and
(d) wherein the humanized antibody comprises (i) a heavy chain complementary determining region (CDRH3) amino acid sequence having the amino acid sequence of SEQ ID NO:9, and a heavy chain complementary determining region (CDRH2) amino acid sequence having the amino acid sequence of SEQ ID NO:8, and a heavy chain complementary determining region (CDRH1) amino acid sequence having the amino acid sequence of SEQ ID NO:7; and (ii) a light chain complementary determining region (CDRL3) amino acid sequence having the amino acid sequence of SEQ ID NO: 12, and a light chain complementary determining region (CDRL2) amino acid sequence having the amino acid sequence of SEQ ID NO: 11, and a light chain complementary determining region (CDRL1) amino acid sequence having the amino acid sequence of SEQ ID NO: 10.

### Claim 2 (depends on 1)

A kit for diagnosing tumors and metastases comprising a humanized antibody or fragment or conjugate thereof according to claim 1 and means for detecting tumors and metastases.

### Claim 3 (depends on 1)

The humanized antibody, or fragments or conjugates thereof according to claim 1 wherein the amino acid substitutions of the heavy chain of the human antibody further comprise at least one of:
K (murine) instead of R (human) at position 67, and/or
A (murine) instead of V (human) at position 68.

### Claim 4 (depends on 1)

The humanized antibody, or fragments or conjugates thereof according to claim 1 wherein the amino acid substitutions of the heavy chain of the human antibody comprise:
F (murine) instead of Y (human) at position 27 and
S (murine) instead of T (human) at position 30 and
L (murine) instead of V (human) at position 37, and
I (murine) instead of M (human) at position 48, and
A (murine) instead of G (human) at position 49, and
L (murine) instead of I (human) at position 70, and
V (murine) instead of R (human) at position 72.

### Claim 5 (depends on 1)

The humanized antibody, or fragments or conjugates thereof according to claim 1 wherein the heavy chain of the human antibody (claim 1(a)(i)) comprises the sequence of SEQ ID NO: 90.

### Claim 6 (depends on 1)

The humanized antibody, or fragments or conjugates thereof according to claim 1 wherein the amino acid substitutions in the light chain further comprise at least one of:
(i) Y (murine) instead of S (human) at position 67; or
(ii) T (murine) instead of I (human) at position 2 and Y (murine) instead of S (human) at position 67; or
(iii) R (murine) instead of T (human) at position 22 and Y (murine) instead of S (human) at position 67; or
(iv) E (murine) instead of K (human) at position 42 and Y (murine) instead of S (human) at position 67; or
(v) S (murine) instead of G (human) at position 64 and Y (murine) instead of S (human) at position 67; or
(vi) Y (murine) instead of S (human) at position 67 and V (murine) instead of T (human) at position 72; or
(vii) Y (murine) instead of S (human) at position 67 , and D (murine) instead of T (human) at position 85.

### Claim 7 (depends on 1)

The humanized antibody, or fragments or conjugates thereof according to claim 1 wherein the light chain of the human antibody (claim 1(a)(ii)) has the sequence of SEQ ID NO:92.

### Claim 8 (depends on 1)

The humanized antibody, or fragments or conjugates thereof, according to claim 1, comprising a heavy chain variable region amino acid sequence having at least 80% identity to the amino acid sequence selected from the group consisting of amino acid residues 20-138 of SEQ ID NOs: 18, 32, or 34 or fragments thereof and/or a light chain variable region amino acid sequence having at least 80% identity to the amino acid sequence selected from the group consisting of amino add residues 21-127 of SEQ ID NOs: 20, 36, 38 or 40 or fragments thereof, or fragments or conjugates thereof.

### Claim 9 (depends on 8)

The humanized antibody, or fragments or conjugates thereof according to claim 8, comprising a heavy chain variable region amino acid sequence comprising amino acid residues 20-138 of SEQ ID NO:32 or SEQ ID NO:34 and a light chain variable region amino acid sequence comprising amino acid residues 21-127 of SEQ ID NO:40.

### Claim 10 (depends on 8)

The humanized antibody, or fragments or conjugates thereof, according to claim 8, comprising a heavy chain variable region amino acid sequence having at least 80% identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 32, or 34 or fragments thereof and/or a light chain variable region amino acid sequence having at least 80% identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 36, 38 or 40 or fragments thereof, or fragments or conjugates thereof.

### Claim 11 (depends on 10)

The humanized antibody, or fragments or conjugates thereof according to claim 10, comprising a heavy chain variable region amino acid sequence comprising SEQ ID NO:32 or SEQ ID NO:34 and a light chain variable region amino acid sequence comprising SEQ ID NO:40.

### Claim 12 (depends on 1)

The humanized antibody, or fragments or conjugates thereof according to claim 1 wherein the humanized antibody comprises (i) a variable domain framework region from a heavy chain of a human antibody or from a human consensus framework; and (ii) a variable domain framework region from a light chain of a human antibody or from a human consensus framework.

### Claim 13 (depends on 1)

The humanized antibody, or fragments or conjugates thereof according to claim 1 wherein (i) an amino acid in the variable domain framework region from the heavy chain of a human antibody is substituted with the corresponding amino acid from a heavy chain of a mouse antibody or mouse consensus framework and (ii) an amino acid in the variable domain framework region from the light chain of a human antibody is substituted with the corresponding amino acid from a light chain of a mouse antibody or mouse consensus framework.

### Claim 14 (depends on 1)

A nucleic acid molecule encoding a humanized antibody or fragments or conjugates thereof according to claim 1, or hybridizing with the nucleic acid, or a degenerate sequence thereof.

### Claim 15 (depends on 14)

The nucleic acid molecule according to claim 14 comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 17, 19, 31, 33, 35, 37, 39, 45, 47 and 49.

### Claim 16 (depends on 1)

An expression vector comprising a nucleic acid encoding the humanized antibody or fragments or conjugates of claim 1.

### Claim 17 (depends on 1)

A host cell that produces a humanized antibody or fragments or conjugates thereof according to claim 1.

### Claim 18 (depends on 1)

A pharmaceutical composition that comprises a humanized antibody or fragment or conjugate thereof according to claim 1 and an excipient and/or adjuvant and/or carrier and/or diluent that is pharmaceutically acceptable.