# US 10,118,971 B2 · Protein in which electrical interaction is introduced within hydrophobic interaction site and preparation method therefor

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- Number: US 10,118,971 B2
- Document ID: US10118971B2
- Status: Granted
- Kind: B2
- Application number: 14/776,045
- Filed: 2014-03-13
- Published: 2016-06-02
- Granted: 2018-11-06
- Earliest claimed: 2013-03-13
- Assignees: Ibentrus, Inc.
- Applicants: Ibentrus, Inc.
- Inventors: Hoeon Kim
- Web page: https://patentgrep.com/patent/US10118971B2

## CPC

- C07K 16/32: against translation products of oncogenes
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/108: Orthomyxoviridae (F), e.g. influenza virus
- C07K 16/241: {Tumor Necrosis Factors}
- C07K 16/244: {Interleukins [IL]}
- C07K 16/247: {IL-4}
- C07K 16/26: against hormones {; against hormone releasing or inhibiting factors}
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- C07K 16/2866: {against receptors for cytokines, lymphokines, interferons}
- C07K 16/2875: {against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154 (against NGF C07K16/22, against TNF C07K16/241)}
- C07K 16/2878: {against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95}
- C07K 16/468: {Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies}
- 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 2317/31: multispecific
- C07K 2317/515: Complete light chain, i.e. VL + CL
- C07K 2317/522: CH1 domain
- C07K 2317/526: CH3 domain
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- C07K 2319/00: Fusion polypeptide
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C07K 2319/32: fusions with soluble part of a cell surface receptor, "decoy receptors"
- 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)

## Abstract

The present invention provides a protein or antibody in which, of a pair of hydrophobic amino acids selected from within a hydrophobic interaction site of the protein, one hydrophobic amino acid is transformed into a substance having a positive electrical charge and the other hydrophobic amino acid is transformed into a substance having a negative electrical charge, and electrostatic interaction is introduced within the hydrophobic interaction site of the protein by means of the positive charge and the negative charge. The present invention also provides a method for preparing the protein or antibody, and a method for measuring the degree of coupling between a heavy chain and a light chain, using the antibody. The protein or antibody in accordance with to the present invention has a low contamination by a homodimer or a monomer, and thus a heterodimer can be obtained in high purity.

## Claims

### Claim 1

A method of preparation of a protein with increased chain selectivity, the method comprising the following steps:
(a) selecting at least two pairs of hydrophobic amino acids from the group consisting of 351 leucine and 351 leucine pair, 395 proline and 397 valine pair, and 395 proline and 395 proline pair between CH3 domains in a protein comprising two Fc domains of human IgG1 of SEQ ID NO: 1, or IgG4 of SEQ ID NO: 4;
(b) modifying one hydrophobic amino acid to a positive charged amino acid selected from the group consisting of lysine, arginine and histidine, and the other hydrophobic amino acid to a negative charged amino acid selected from the group consisting of aspartic acid and glutamic acid, in the selected pairs of hydrophobic amino acids; and
(c) binding with electrostatic interaction by contacting between the positive charged amino acid and the negative charged amino acid.

### Claim 2

A protein comprising two Fc domains of human IgG1 of SEQ ID NO: 1, or IgG4 of SEQ ID NO: 4, characterized in that at least two hydrophobic amino acid pairs selected from the group consisting of 351 leucine and 351 leucine pair, 395 proline and 397 valine pair, and 395 proline and 395 proline pair between CH3 domains of the protein are mutated by substituting one hydrophobic amino acid of each amino acid pair with a positive charged amino acid selected from the group consisting of lysine, arginine and histidine, and the other hydrophobic amino acid with a negative charged amino acid selected from the group consisting of aspartic acid and glutamic acid, whereby electrostatic interaction is introduced between the mutated amino acids in the protein.

### Claim 3

A protein comprising two Fc domains of human IgG1 of SEQ ID NO: 1, or IgG4 of SEQ ID NO: 4, wherein the protein comprises a mutation of Z4, Z6, Z7, Z8, Z10, Z11, Z12, or Z14 as defined in the following table:
Chain A
Chain B
Z4
L351K/P395K
L351D/P395D
Z6
T394K/P395K
T394D/P395D
Z7
T394K/V397K
T394D/V397D
Z8
P395K/V397K
P395D/V397D
Z10
L351K/T394K/P395K
L351D/T394D/P395D
Z11
L351K/T394K/V397K
L351D/T394D/V397D
Z12
L351K/P395K/V397K
L351D/P395D/V397D
Z14
L351K/T394K/P395K/V397K
L351D/T394D/P395D/V397D.

### Claim 4 (depends on 2)

The protein according to claim 2 wherein the protein is a fusion antibody comprising a pair of ectodomains selected from the group consisting of TNFR2 (tumor necrosis factor receptor 2), Her3 (human epidermal growth factor receptor 3), Tie2 (angiopoietin receptor 2), TGFbR1 (transforming growth factor, beta receptor 1), BMPR1b (bone morphogenetic protein receptor type 1 b), II-12R-b1 (interleukin 12 receptor subunit beta 1), IL-4Ra (interleukin 4 receptor alpha), ITGA4 (integrin subunit alpha 4), ITGA2B (integrin subunit alpha 2B), IFNAR1 (interferon-alpha receptor 1), IL-12A (interleukin 12 receptor subunit alpha), IL-4 (interleukin 4), IFN-alpha (interferon-alpha), BMP2 (bone morphogenetic protein 2), IL1 RL1 (interleukin 1 receptor-like 1) , IL-17RA (interleukin 17 receptor alpha), IL-17A (interleukin 17 alpha), Fas (tumor necrosis factor receptor superfamily member 6), FltD2 (domain 2 of fms related tyrosine kinase), Her1 (human epidermal growth factor receptor 1), Tie1 angiopoietin receptor 1), TGFbR2 (transforming growth factor beta receptor 2), IL-12R-b2 (interleukin12 receptor subunit beta 2), IL-13Ra1 (interleukin 13 receptor subunit alpha 1), ITGB1 (integrin subunit beta 1), ITGB3 (integrin subunit beta 3), IFNAR2 (interferon-alpha/beta receptor 2), IL-12B (interleukin 12 beta), IL-13 (interleukin 13), IFN-beta (interferon beta), BMP7 (bone morphogenetic protein 7), IL-1RAP (Interleukin-1 receptor accessory protein), IL-17RC (interleukin 12 receptor C) and IL-17F (interleukin 17), and each of the selected pair of ectodomains is fused with each of the two Fc domains of the protein.

### Claim 5 (depends on 4)

The protein according to claim 4 wherein the pair of ectodomains is Her3/FltD2 combination, Her1/Her3 combination, or Tie1/Tie2 combination.

### Claim 6 (depends on 2)

The protein according to claim 2, wherein
the protein is an antibody comprising two human IgG1- or IgG4-type heavy chains and two human kappa-type light chains, and further comprising a mutation selected from the group consisting of V1-V5, W1-W8, V2P, V3P, W4P, V3W4, W4V3, V3V1, and V3W1 as defined in the following table, thereby having an increased coupling between the heavy chain and the light chain:
LN (light
HP (heavy
HN (heavy
LP (light chain
chain
chain mutated
chain mutated
mutated with
mutated with
with positive
with negative
positive amino
negative
amino acid)
amino acid)
acid)
amino acid)
V1
F45K
F45D
F98K
F98D
V3
W103K
W103D
P44K
P44D
V4
V37K
V37D
F98K
F98D
V5
F45K
F45D
Y87K
Y87D
W1
L128K
L128D
F118K
F118D
W2
A141K
A141D
F116K
F116D
W3
L145K
L145D
V133K
V133D
W4
V185K
V185D
L135K
L135D
W5
L128K
L128D
V133K
V133D
W6
A141K
A141D
L135K
L135D
W7
L145K/V185K
L145D/V185D
V133K/L135K
V133D/
L135D
W8
A141K/V185K
A141D/V185D
F116K/L135K
F116D/
L135D
V3P
W103K
—
—
P44D
W2P
A141K
—
—
F116D
W4P
V185K
—
—
L135D
V3W4
W103K
V185D
L135K
P44D
W4V3
V185K
W103D
P44K
L135D
V3V1
W103K
F45D
F98K
P44D
V3W1
W103K
L128D
F118K
P44D.

### Claim 7 (depends on 6)

The protein according to claim 6, wherein the mutation is V3W1.

### Claim 8 (depends on 2)

The protein according to claim 2, wherein,
the protein is an antibody comprising two human IgG1- or IgG4-type heavy chains and two human kappa-type light chains, and
one or more hydrophobic amino acid pairs selected from the group consisting of 128 leucine and 118 phenylalanine pair, 128 leucine and 133 valine pair, 141 alanine and 116 phenylalanine pair, 141 alanine and 135 leucine pair, 145 leucine and 133 valine pair, 170 phenylalanine and 135 leucine pair, 185 valine and 118 phenylalanine pair, and 185 valine and 135 leucine pair between a CH1 domain of the heavy chain and a CL domain of the light chain are mutated by substituting one hydrophobic amino acid of each amino acid pair with a positive charged amino acid selected from the group consisting of lysine, arginine and histidine, and the other hydrophobic amino acid with a negative charged amino acid selected from the group consisting of aspartic acid and glutamic acid, thereby having an increased coupling between the heavy chain and the light chain.

### Claim 9 (depends on 3)

The protein according to claim 3, wherein,
the protein is an antibody comprising two human IgG1- or IgG4-type heavy chains and two human kappa-type light chains, and
one or more hydrophobic amino acid pairs selected from the group consisting of 128 leucine and 118 phenylalanine pair, 128 leucine and 133 valine pair, 141 alanine and 116 phenylalanine pair, 141 alanine and 135 leucine pair, 145 leucine and 133 valine pair, 170 phenylalanine and 135 leucine pair, 185 valine and 118 phenylalanine pair, and 185 valine and 135 leucine pair between a CH1 domain of the heavy chain and a CL domain of the light chain are mutated by substituting one hydrophobic amino acid of each amino acid pair with a positive charged amino acid selected from the group consisting of lysine, arginine and histidine, and the other hydrophobic amino acid with a negative charged amino acid selected from the group consisting of aspartic acid and glutamic acid, thereby having an increased coupling between the heavy chain and the light chain.