# US 11,028,182 B2 · Antigen-binding constructs targeting HER2

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- Number: US 11,028,182 B2
- Document ID: US11028182B2
- Status: Granted
- Kind: B2
- Application number: 15/572,364
- Filed: 2016-05-13
- Published: 2018-09-27
- Granted: 2021-06-08
- Earliest claimed: 2015-05-13
- Assignees: Zymeworks Inc.
- Applicants: Zymeworks Inc.
- Inventors: Eric Escobar-Cabrera; Leonard G. Presta
- Web page: https://patentgrep.com/patent/US11028182B2

## CPC

- C07K 16/32: against translation products of oncogenes
- A61P 35/00: Antineoplastic agents
- G01N 33/57515: of the breast
- G01N 33/5753: of the stomach or small intestine
- G01N 33/5759: {involving compounds localised on the membrane of tumour or cancer cells}
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/31: multispecific
- C07K 2317/35: Valency
- C07K 2317/526: CH3 domain
- C07K 2317/55: Fab or Fab'
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/567: Framework region [FR]
- C07K 2317/64: comprising a combination of variable region and constant region components
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2317/77: Internalization into the cell
- 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
- G01N 2333/71: for growth factors; for growth regulators
- 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
- 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

Described herein are high affinity antigen binding constructs, e.g., antibodies, directed to the ECD2 domain of HER2. The antigen-binding constructs comprise at least one antigen-binding polypeptide construct that binds to ECD2 of HER2 (HER2 ECD2) with increased affinity compared to a wild-type 2C4 antibody. Such antigen-binding polypeptide constructs comprise one or more amino acid modifications in the framework region and/or CDRs compared to the amino acid sequence of a wild-type 2C4 antibody that increase affinity of the antigen-binding polypeptide construct for ECD2 by 2-fold or greater. The antigen-binding constructs can inhibit the growth of HER2-expressing breast cancer cells and gastric cancer cells. Antigen-binding constructs in biparatopic format are internalized in HER2-expressing cells.

## Claims

### Claim 1

An antigen-binding construct comprising a variant first antigen-binding polypeptide construct which monovalently binds a first HER2 ECD2 (human epidermal growth factor receptor 2 extracellular domain 2) antigen, the variant first antigen-binding polypeptide construct comprising a heavy chain variable (VH) domain as set forth in SEQ ID NO:2 and a light chain variable (VL) domain as set forth in SEQ ID NO:11 and comprising an amino acid modification at position 75 in the VH domain (Kabat numbering) wherein the lysine at position 75 has been substituted with a tryptophan (H_K75W), glutamic acid (H_K75E), or tyrosine (H_K75Y),
optionally wherein the variant first antigen-binding polypeptide construct comprises H_K75W and further comprises the following substitutions or set of substitutions, numbering according to Kabat numbering system:
H_T30Q; or
H_T30Y; or
H_G56Y; or
H_S99W; or
L_Y49W; or
L_Y96G; or
H_S99W and L_Y49W; or
L_Y49W and L_Y96G; or
H_T30Q and L_Y49W; or
H_T30Q and H_S99W; or
H_T30Q and L_Y96G; or
H_T30Y and L_Y49W; or
H_S99W and L_Y49W and L_Y96G; or
H_T3000 and H_S99W and L_Y96G or
H_T3000 and H_S99W and L_Y49W; or
H_T3000 and L_Y49W and L_Y96G; or
H_T30Y and L_Y49W and L_Y96G, and
optionally wherein the variant first antigen-binding polypeptide construct comprises H_K75E and further comprises the following substitutions or set of substitutions, numbering according to Kabat numbering system
L_Y49W; or
H_T30Q; or
H_S74W; or
H_S99W.

### Claim 2 (depends on 1)

The antigen-binding construct of claim 1, wherein the variant first antigen-binding polypeptide construct comprises H_K75W.

### Claim 3 (depends on 1)

The antigen-binding construct of claim 1, wherein the variant first antigen-binding polypeptide construct comprises H_K75W and further comprises the following substitutions or set of substitutions, numbering according to Kabat numbering system:
H_T30Q; or
H_T30Y; or
H_G56Y; or
H_S99W; or
L_Y49W; or
L_Y96G; or
H_S99W and L_Y49W; or
L_Y49W and L_Y96G; or
H_T30Q and L_Y49W; or
H_T30Q and H_S99W; or
H_T30Q and L_Y96G; or
H_T30Y and L_Y49W; or
H_S99W and L_Y49W and L_Y96G; or
H_T30Q and H_S99W and L_Y96G or
H_T30Q and H_S99W and L_Y49W; or
H_T30Q and L_Y49W and L_Y96G; or
H_T30Y and L_Y49W and L_Y96G.

### Claim 4 (depends on 1)

The antigen-binding construct of claim 1, wherein the variant first antigen-binding polypeptide construct comprises H_K75E.

### Claim 5 (depends on 1)

The antigen-binding construct of claim 1, wherein the variant first antigen-binding polypeptide construct comprises H_K75E and further comprises the following substitutions or set of substitutions, numbering according to Kabat numbering system
L_Y49W; or
H_T30Q; or
H_S74W; or
H_S99W.

### Claim 6 (depends on 1)

The antigen-binding construct of claim 1, wherein the variant first antigen-binding polypeptide construct comprises H_K75Y.

### Claim 7 (depends on 1)

The antigen-binding construct of claim 1, wherein the variant first antigen-binding polypeptide construct is a Fab.

### Claim 8 (depends on 1)

The antigen-binding construct of claim 1, wherein the antigen-binding construct is one-armed.

### Claim 9 (depends on 7)

The antigen binding construct of claim 7, further comprising a first linker polypeptide operably linked to the variant first antigen-binding polypeptide construct.

### Claim 10 (depends on 1)

The antigen-binding construct of claim 1, wherein the antigen-binding construct comprises a second antigen-binding polypeptide construct that binds to a second antigen.

### Claim 11 (depends on 10)

The antigen-binding construct of claim 10, further comprising a first linker polypeptide operably linked to the variant first antigen-binding polypeptide construct, and a second linker polypeptide operably linked to the second antigen-binding polypeptide construct.

### Claim 12 (depends on 11)

The antigen-binding construct of claim 11, wherein the second antigen is a HER2 ECD2 antigen and the second antigen-binding polypeptide construct is identical to the variant first antigen-binding polypeptide construct.

### Claim 13 (depends on 11)

The antigen-binding construct of claim 11, wherein the second antigen is a HER2 ECD4 antigen and the second antigen-binding polypeptide construct is an scFv comprising the VH and VL domain of trastuzumab and a glycine-serine linker.

### Claim 14 (depends on 9)

The antigen-binding construct according to claim 9, wherein the first linker polypeptide is operably linked to a heterodimeric human IgG1 Fc comprising a first Fc polypeptide and a second Fc polypeptide each comprising a different CH3 sequence.

### Claim 15 (depends on 11)

The antigen-binding construct according to claim 11, wherein the first and second linker polypeptides are operably linked to a heterodimeric human IgG1 Fc comprising a first Fc polypeptide and a second Fc polypeptide each comprising a different CH3 sequence.

### Claim 16 (depends on 14)

The antigen-binding construct of claim 14, wherein the CH3 sequence of each Fc polypeptide comprises one or more modifications that promote the formation of a heterodimeric Fc with stability comparable to a wild-type homodimeric Fc, the heterodimeric IgG1 Fc having
a) the modifications L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T366L_K392M_T394W in the second polypeptide; or
b) the modifications L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T366L_K392L_T394W in the second Fc polypeptide; or
c) the modifications T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T350V_T366L_K392L_T394W in the second Fc polypeptide; or
d) the modifications T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T350V_T366L_K392M_T394W in the second Fc polypeptide; or
e) the modifications T350V_L351Y_S400E_F405A_Y407V in the first Fc polypeptide, and the modifications T350V_T366L_N390R_K392M_T394W in the second Fc polypeptide; or
f) the modifications T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T366I_N390R_K392M_T394W in the second Fc polypeptide; or
g) the modifications L351Y_S400E_F405A_Y407V in the first Fc polypeptide, and the modifications T350V_T366L_K392L_T394W in the second Fc polypeptide, wherein the numbering of amino acid residues in the Fc is according to the EU numbering system.

### Claim 17 (depends on 15)

The antigen-binding construct of claim 15, wherein the CH3 sequence of each Fc polypeptide comprises one or more modifications that promote the formation of a heterodimeric Fc with stability comparable to a wild-type homodimeric Fc, the heterodimeric IgG1 Fc having
a) the modifications L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T366L_K392M_T394W in the second polypeptide; or
b) the modifications L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T366L_K392L_T394W in the second Fc polypeptide; or
c) the modifications T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T350V_T366L_K392L_T394W in the second Fc polypeptide; or
d) the modifications T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T350V_T366L_K392M_T394W in the second Fc polypeptide; or
e) the modifications T350V_L351Y_S400E_F405A_Y407V in the first Fc polypeptide, and the modifications T350V_T366L_N390R_K392M_T394W in the second Fc polypeptide; or
f) the modifications T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T366I_N390R_K392M_T394W in the second Fc polypeptide; or
g) the modifications L351Y_S400E_F405A_Y407V in the first Fc polypeptide, and the modifications T350V_T366L_K392L_T394W in the second Fc polypeptide, wherein the numbering of amino acid residues in the Fc is according to the EU numbering system.

### Claim 18 (depends on 1)

The antigen-binding construct of claim 1, wherein the VH consists of SEQ ID NO: 515 and the VL consists of SEQ ID NO: 815; or the VH consists of SEQ ID NO: 125 and the VL consists of SEQ ID NO: 915; or the VH consists of SEQ ID NO: 515 and the VL consists of SEQ ID NO: 95; or the VH consists of SEQ ID NO: 35 and the VL consists of SEQ ID NO: 815; or the VH consists of SEQ ID NO: 45 and the VL consists of SEQ ID NO: 95.

### Claim 19 (depends on 1)

A pharmaceutical composition comprising the antigen-binding construct of claim 1, and a pharmaceutical carrier, optionally selected from a buffer, an antioxidant, a low molecular weight molecule, a drug, a protein, an amino acid, a carbohydrate, a lipid, a chelating agent, a stabilizer, or an excipient.

### Claim 20 (depends on 1)

An isolated polynucleotide or set of isolated polynucleotides comprising at least one nucleic acid sequence that encodes the antigen-binding construct of claim 1, optionally wherein said polynucleotide or set of polynucleotides is cDNA.

### Claim 21 (depends on 20)

A vector or set of vectors comprising one or more of the polynucleotides or sets of polynucleotides according to claim 20, optionally selected from the group consisting of a plasmid, a viral vector, a non-episomal mammalian vector, an expression vector, and a recombinant expression vector.

### Claim 22 (depends on 20)

An isolated cell comprising a polynucleotide or set of polynucleotides according to claim 20, the isolated cell optionally selected from a hybridoma, a Chinese Hamster Ovary (CHO) cell, or a HEK293 cell.

### Claim 23 (depends on 1, 20)

A method of producing the antigen-binding construct according to claim 1, comprising culturing a host cell under conditions suitable for expressing the antigen-binding construct wherein the host cell comprises a polynucleotide or set of polynucleotides according to claim 20, and purifying the construct.

### Claim 24 (depends on 1)

A method of inhibiting cell growth of a HER2-expressing (HER2+) cell, the method comprising contacting the cell with the antigen-binding construct of claim 1.

### Claim 25 (depends on 24)

The method of claim 24, wherein the cell is a breast cancer cell.

### Claim 26 (depends on 24)

The method of claim 24, wherein the cell is a gastric cancer cell.

### Claim 27 (depends on 24)

The method of claim 24, wherein the method is performed in vitro.

### Claim 28 (depends on 1)

A method of treating a subject having a HER2-expressing (HER2+) cancer, optionally wherein the cancer is breast cancer or gastric cancer, the method comprising administering to the subject the antigen-binding construct of claim 1.