# US 11,999,787 B2 · Tie2-binding agents and methods of use

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- Number: US 11,999,787 B2
- Document ID: US11999787B2
- Status: Granted
- Kind: B2
- Application number: 17/208,292
- Filed: 2021-03-22
- Published: 2021-10-28
- Granted: 2024-06-04
- Earliest claimed: 2020-03-24
- Assignees: Genentech, Inc.
- Applicants: Genentech, Inc.
- Inventors: Minhong Yan; Gu Zhang; Nicholas John Agard; Danielle Marie Dicara; Philip E. Hass; Julie Q. Hang; Erin L. Christensen; Robert Paul Morse; Sarah Sanowar; Vittal Shivva
- Web page: https://patentgrep.com/patent/US11999787B2

## CPC

- C07K 16/2863: {against receptors for growth factors, growth regulators}
- A61K 9/0048: {Eye, e.g. artificial tears}
- A61K 39/3955: {against proteinaceous materials, e.g. enzymes, hormones, lymphokines}
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
- A61K 47/6849: {the antibody targeting a receptor, a cell surface antigen or a cell surface determinant}
- A61P 27/02: Ophthalmic agents
- A61K 2039/505: {comprising antibodies}
- A61K 2039/54: {characterised by the route of administration}
- C07K 2317/21: from primates, e.g. man
- 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 2317/35: Valency
- C07K 2317/52: Constant or Fc region; Isotype
- C07K 2317/55: Fab or Fab'
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/71: Decreased effector function due to an Fc-modification
- C07K 2317/75: Agonist effect on antigen
- 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 2319/61: containing an enzyme fusion for detection (lacZ, luciferase)
- 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 invention provides Tie-2 antibodies and fragments thereof and conjugates and methods of using the same.

## Claims

### Claim 1

A conjugate that binds to Tie2, wherein the conjugate comprises two, three, four, five, six, seven, or eight antibodies or antigen-binding fragments thereof, wherein each of the antibodies or fragments thereof bind Tie2 and comprises:
a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence NTDIS (SEQ ID NO:3), (b) CDR-H2 comprising the amino acid sequence RISPSDGNTYYADSVKG (SEQ ID NO:4), and (c) CDR-H3 comprising the amino acid sequence RTRWASWAFDY (SEQ ID NO:7), and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence RASQDVSTAVA (SEQ ID NO:8), (e) CDR-L2 comprising the amino acid sequence SASFLYS (SEQ ID NO:9), and (f) CDR-L3 comprising the amino acid sequence QQSYTTPPT (SEQ ID NO:10), and
wherein each of the antibodies or antigen-binding fragment thereof is linked to a multimerizing moiety.

### Claim 2 (depends on 1)

The conjugate of claim 1, wherein the multimerizing moiety comprises a polyol, wherein the polyol is a is a multi-armed polyol, selected from a dimer, a tetramer, a hexamer, and an octamer.

### Claim 3 (depends on 2)

The conjugate of claim 2, wherein the multi-armed polyol is a hexamer.

### Claim 4 (depends on 2)

The conjugate of claim 2, wherein the polyol is a polyethylene glycol (PEG).

### Claim 5 (depends on 4)

The conjugate of claim 4, wherein the PEG has a weight average molecular weight of from about 500 Daltons (Da) to about 300,000 Da.

### Claim 6 (depends on 4)

The conjugate of claim 4, wherein the PEG has the structure of general formula (Ib):
wherein each m is independently an integer from 3-250; each R1 is independently either absent or is a linking group; and each R2 is independently either hydrogen or a terminal reactive group;
wherein at least one R2 is a terminal reactive group and is covalently linked to the antibody or antigen-binding fragment thereof.

### Claim 7 (depends on 6)

The conjugate according to claim 6, wherein at least one R1 is a linking group, wherein R1 and R2 when taken together are selected from:
and combinations thereof; wherein each i is independently an integer of 0-10; j is an integer of 0-10; and R2 is a terminal reactive group selected from the group consisting of a thiol reactive group, an amino reactive group, and combinations thereof.

### Claim 8 (depends on 6)

The conjugate of claim 6, wherein each R2 is a maleamide and is covalently linked to the antibody or antigen-binding fragment thereof.

### Claim 9 (depends on 2)

The conjugate of claim 2, wherein each of the antibodies or antigen-binding fragment thereof is a Fab and the polyol is covalently linked to the Fab through a free sulfhydryl group of an engineered cysteine amino acid, wherein the engineered cysteine is selected from the group consisting of
T120C, G166C, G178C, T187C, and T209C in the heavy chain (HC); or
the engineered cysteine is selected from the group consisting of Q124C, R142C, Q155C, L201C, T206C, K107C, K126C, and K149C in the light chain (LC);
wherein the residue number is according to EU numbering.

### Claim 10 (depends on 9)

The conjugate of claim 9, wherein the engineered cysteine is T209C in the HC, wherein the residue number is according to EU numbering.

### Claim 11 (depends on 1)

A pharmaceutical composition comprising the conjugate of claim 1 and a pharmaceutically acceptable carrier.

### Claim 12 (depends on 1)

The conjugate of claim 1, wherein the VH of each of the antibodies or antigen-binding fragments thereof comprises SEQ ID NO:20 and the VL of each of the antibodies or antigen-binding fragments thereof comprises SEQ ID NO:21.

### Claim 13 (depends on 12)

The conjugate of claim 12, wherein each of the antibodies or antigen-binding fragments thereof is a Fab comprising the Fab HC sequence of SEQ ID NO: 55 and the Fab LC sequence of SEQ ID NO:25.

### Claim 14

A conjugate which binds Tie2 comprising:
a Fab comprising a heavy chain (HC) having SEQ ID NO:55 and a light chain (LC) having SEQ ID NO:25; conjugated to a polyol which has the structure of Formula 1 (b)
wherein each m is independently an integer from 10-30, wherein R1 and R2 taken together has the structure
wherein R2 is a maleamide; and
wherein the polyol is linked to the Fab HC at residue C209 (EU numbering).

### Claim 15 (depends on 14)

A pharmaceutical composition comprising the conjugate of claim 14 and a pharmaceutically acceptable carrier.

### Claim 16

A conjugate that binds to Tie2,
wherein the conjugate comprises six Fabs that bind Tie2, wherein each of the six Fabs comprises:
a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence NTDIS (SEQ ID NO:3), (b) CDR-H2 comprising the amino acid sequence RISPSDGNTYYADSVKG (SEQ ID NO:4), and (c) CDR-H3 comprising the amino acid sequence RTRWASWAFDY (SEQ ID NO:7), and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence RASQDVSTAVA (SEQ ID NO:8), (e) CDR-L2 comprising the amino acid sequence SASFLYS (SEQ ID NO:9), and (f) CDR-L3 comprising the amino acid sequence QQSYTTPPT (SEQ ID NO:10), and
wherein each of the Fabs is linked to an arm of a hexameric polyethylene glycol (PEG) molecule.

### Claim 17 (depends on 16)

The conjugate of claim 16, wherein each of the Fabs is linked to the arm of the PEG through a free sulfhydryl group of an engineered cysteine amino acid, wherein
the engineered cysteine is selected from the group consisting of T120C, G166C, G178C, T187C, and T209C in the Fab heavy chain (HC); or
the engineered cysteine is selected from the group consisting of Q124C, R142C, Q155C, L201C, T206C, K107C, K126C, and K149C in the Fab light chain (LC);
wherein the residue number is according to EU numbering.

### Claim 18 (depends on 17)

The conjugate of claim 17, wherein the engineered cysteine is T209C in the HC, wherein the residue number is according to EU numbering.

### Claim 19 (depends on 17)

The conjugate of claim 17, wherein the PEG has a weight average molecular weight of from about 500 Daltons (Da) to about 300,000 Da.

### Claim 20 (depends on 19)

The conjugate of claim 19, wherein the PEG has the structure of general formula (Ib):
wherein each m is independently an integer from 3-250; each R1 is independently either absent or is a linking group; and each R2 is independently either hydrogen or a terminal reactive group; and
wherein at least one R2 is a terminal reactive group and is covalently linked to the antibody or antigen-binding fragment thereof.

### Claim 21 (depends on 20)

The conjugate of claim 20, wherein the VH of each of the six Fabs comprises SEQ ID NO:20 and the VL of each of the six Fabs comprises SEQ ID NO:21.

### Claim 22 (depends on 21)

The conjugate of claim 21, wherein the Fab HC of each of the six Fabs comprises the sequence of SEQ ID NO:55 and the Fab LC of each of the six Fabs comprises the sequence of SEQ ID NO:25.

### Claim 23 (depends on 22)

The conjugate of claim 22, wherein the PEG has a weight average molecular weight of from about 1000 Daltons (Da) to about 10,000 Da.

### Claim 24 (depends on 23)

The conjugate of claim 23, wherein each of the six Fabs is linked to the arm of the PEG through a free sulfhydryl group of an engineered cysteine amino acid in each of the six Fabs, wherein the engineered cysteine is T209C in the Fab heavy chain (HC), wherein the residue number is according to EU numbering.

### Claim 25 (depends on 24)

The conjugate of claim 24, wherein each R2 is a terminal reactive group and is covalently linked to the Fab HC.

### Claim 26 (depends on 25)

The conjugate of claim 25, wherein each R2 is a maleimide and is covalently linked to the Fab HC.

### Claim 27 (depends on 26)

A pharmaceutical composition comprising the conjugate of claim 26 and a pharmaceutically acceptable carrier.

### Claim 28 (depends on 16)

A pharmaceutical composition comprising the conjugate of claim 16 and a pharmaceutically acceptable carrier.

### Claim 29

A conjugate that binds to Tie2,
wherein the conjugate comprises six Fabs that bind Tie2,
wherein each of the six Fabs comprises a Fab heavy chain (HC) comprising the sequence of SEQ ID NO: 55 and a Fab light chain (LC) comprising the sequence of SEQ ID NO: 25,
wherein each of the six Fabs is linked to an arm of a hexameric polyethylene glycol (PEG) molecule, wherein the hexameric PEG molecule has the structure of general formula (Ib):
wherein each m is independently an integer from 10-30, wherein R1 and R2 when taken together has the structure
wherein i and j are 2,
wherein R2 is a maleamide and is covalently linked to one of the six Fabs through a free sulfhydryl group of engineered cysteine amino acid T209C of the Fab HC (EU numbering).

### Claim 30 (depends on 29)

A pharmaceutical composition comprising the conjugate of claim 29 and a pharmaceutically acceptable carrier.