# US 11,306,156 B2 · Modified antigen binding polypeptide constructs and uses thereof

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- Number: US 11,306,156 B2
- Document ID: US11306156B2
- Status: Granted
- Kind: B2
- Application number: 15/314,496
- Filed: 2015-05-29
- Published: 2017-07-20
- Granted: 2022-04-19
- Earliest claimed: 2014-05-28
- Assignees: Zymeworks Inc.
- Applicants: Zymeworks Inc.
- Inventors: Mario Sanches; Thomas Spreter Von Kreudenstein; Dunja Urosev; Stacey A. L. Tom-Yew; Adam Louis Corper; Igor Edmondo Paolo D'Angelo; Yang-Chieh Chou; Surjit Bhimarao Dixit
- Web page: https://patentgrep.com/patent/US11306156B2

## CPC

- C07K 16/468: {Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies}
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- C07K 16/32: against translation products of oncogenes
- C07K 16/36: against blood coagulation factors
- 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 2317/51: Complete heavy chain or Fd fragment, i.e. VH + CH1
- C07K 2317/515: Complete light chain, i.e. VL + CL
- C07K 2317/52: Constant or Fc region; Isotype
- C07K 2317/522: CH1 domain
- C07K 2317/55: Fab or Fab'
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- 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
- C12N 15/1034: {Isolating an individual clone by screening libraries}
- C40B 40/08: Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)

## Abstract

The present invention provides heterodimer pairs that can comprise a first heterodimer and a second heterodimer wherein each heterodimer comprises an immunoglobulin heavy chain or fragment thereof and an immunoglobulin light chain or fragment thereof. At least one of the heterodimers can comprise one or more amino acid modifications in the CH1 and/or CL domains, one or more amino acid modifications in the VH and/or VL domains, or a combination thereof. The modified amino acid(s) can be part of the interface between the light chain and heavy chain and are typically modified to create preferential pairing between each heavy chain and a desired light chain such that when the two heavy chains and two light chains of the heterodimer pair are co-expressed in a cell, the heavy chain of the first heterodimer preferentially pairs with one of the light chains rather than the other. Likewise, the heavy chain of the second heterodimer typically preferentially pairs with the second light chain rather than first.

## Claims

### Claim 1

An antigen binding-polypeptide construct comprising at least a first heterodimer and a second heterodimer,
the first heterodimer comprising a first human or humanized immunoglobulin G (IgG) heavy chain polypeptide sequence (H1), and a first human or humanized immunoglobulin kappa light chain polypeptide sequence (L1), and binding to a first epitope; and the second heterodimer comprising a second human or humanized immunoglobulin G (IgG) heavy chain polypeptide sequence (H2), and a second human or humanized immunoglobulin kappa light chain polypeptide sequence (L2), and binding to a second epitope, wherein the H1 and L1 polypeptide sequences of the first heterodimer are different from the corresponding H2 and L2 polypeptide sequences of the second heterodimer,
wherein H1 and H2 each comprise a heavy chain variable domain (VH domain) and a heavy chain constant domain (CH1 domain);
wherein L1 and L2 each comprise a light chain variable domain (VL domain) and a light chain constant domain (CL domain);
wherein H1, H2, L1, and L2 comprise the following amino acid substitutions that promote preferential pairing of H1 with L1 as compared to L2 and of H2 with L2 as compared to L1, at positions identified according to the Kabat numbering system:
a) H1 comprises amino acid substitutions 143E, 145T, 179E, and 188L, L1 comprises amino acid substitutions 124R and 178R, H2 comprises amino acid substitution 179K, and L2 comprises amino acid substitutions 124E, 176L, and 180E,
b) H1 comprises amino acid substitutions 143E and 145T, L1 comprises amino acid substitutions 124R, 160K and 178R, H2 comprises amino acid substitution 179K, and L2 comprises amino acid substitutions 124E, 178E, and 180E,
c) H1 comprises amino acid substitutions 143E, 145T, 179E, and 188L, L1 comprises amino acid substitution 124R and 178R, H2 comprises amino acid substitution 179K, and L2 comprises amino acid substitutions 124E, 131T, 178Y, and 180E,
d) H1 comprises amino acid substitutions 143E, 145T, 179E, and 188L, L1 comprises amino acid substitution 124R and 178R, H2 comprises amino acid substitution 179K, and L2 comprises amino acid substitutions 124E, 131T, 178F, and 180E,
e) H1 comprises amino acid substitutions 143E, 145T, and 188L, L1 comprises amino acid substitutions 124R, 160K and 178R, H2 comprises amino acid substitution 179K, and L2 comprises amino acid substitutions 124E, 131T, 178Y, and 180E,
f) H1 comprises amino acid substitutions 143E, 145T, and 188L, L1 comprises amino acid substitution 124R, 160K and 178R, H2 comprises amino acid substitution 179K, and L2 comprises amino acid substitutions 124E, 131T, 178F, and 180E,
g) H1 comprises amino acid substitutions 143E, 145T, and 188L, L1 comprises amino acid substitutions 124R, 160K and 178R, H2 comprises amino acid substitution 179K, and L2 comprises amino acid substitutions 124E, 176L, and 180E,
h) H1 comprises amino acid substitutions 143E, 145T, 179E, and 188L, L1 comprises amino acid substitutions 124R and 178R, H2 comprises amino acid substitution 179R, and L2 comprises amino acid substitutions 124E, 176L, and 180E,
i) H1 comprises amino acid substitutions 143E, 145T, 179E, and 188L, L1 comprises amino acid substitutions 124R and 178R, H2 comprises amino acid substitution 179R, and L2 comprises amino acid substitutions 124E, 131T, 178Y, and 180E,
j) H1 comprises amino acid substitutions 143E, 145T, 179E, and 188L, L1 comprises amino acid substitutions 124R and 178R, H2 comprises amino acid substitution 179R, and L2 comprises amino acid substitutions 124E, 131T, 178F, and 180E,
k) H1 comprises amino acid substitutions 143E and 145T, L1 comprises amino acid substitutions 124R, 160K and 178R, H2 comprises amino acid substitution 179R, and L2 comprises amino acid substitutions 124E, 178E, and 180E,
l) H1 comprises amino acid substitutions 143E and 145T, L1 comprises amino acid substitutions 124R, 160K and 178R, H2 comprises amino acid substitution 179R, and L2 comprises amino acid substitutions 124E and 178E,
m) H1 comprises amino acid substitutions 143E, 145T, and 188L, L1 comprises amino acid substitutions 124R, 160K and 178R, H2 comprises amino acid substitution 179R, and L2 comprises amino acid substitutions 124E, 176L, and 180E,
n) H1 comprises amino acid substitutions 143E, 145T, and 188L, L1 comprises amino acid substitutions 124R, 160K and 178R, H2 comprises amino acid substitution 179R, and L2 comprises amino acid substitutions 124E, 131T, 178Y, and 180E,
o) H1 comprises amino acid substitutions 143E and 145T, L1 comprises amino acid substitutions 124R, 160K and 178R, H2 comprises amino acid substitution 186K, and L2 comprises amino acid substitutions 124E, 160E and 178E,
p) H1 comprises amino acid substitutions 143E and 145T, L1 comprises amino acid substitutions 124R, 160K and 178R, H2 comprises amino acid substitution 186K, and L2 comprises amino acid substitutions 124E and 178E,
q) H1 comprises amino acid substitutions 143E and 145T, L1 comprises amino acid substitutions 124R, 160K and 178R, H2 comprises amino acid substitution 186K, and L2 comprises amino acid substitutions 124E, 178E and 180E,
r) H1 comprises amino acid substitutions 143E, 145T, 179E and 188L, L1 comprises amino acid substitutions 124R and 178R, H2 comprises amino acid substitution 186K, and L2 comprises amino acid substitutions 124E, 131T, 178F and 180E,
s) H1 comprises amino acid substitutions 143E, 145T, 179E and 188L, L1 comprises amino acid substitutions 124R and 178R, H2 comprises amino acid substitution 186K, and L2 comprises amino acid substitutions 124E, 131T, 178Y and 180E,
t) H1 comprises amino acid substitutions 143E, 145T, 179E and 188L, L1 comprises amino acid substitutions 124R and 178R, H2 comprises amino acid substitution 186K, and L2 comprises amino acid substitutions 124E, 176L and 180E,
u) H1 comprises amino acid substitutions 143E, 145T and 188L, L1 comprises amino acid substitutions 124R, 160K and 178R, H2 comprises amino acid substitution 186K, and L2 comprises amino acid substitutions 124E, 131T, 178Y and 180E,
v) H1 comprises amino acid substitutions 143E, 145T and 188L, L1 comprises amino acid substitutions 124R, 160K and 178R, H2 comprises amino acid substitution 186K, and L2 comprises amino acid substitutions 124E, 176L and 180E,
w) H1 comprises amino acid substitutions 143E and 145T, L1 comprises amino acid substitutions 124R, 160K and 178R, H2 comprises amino acid substitution 186R, and L2 comprises amino acid substitutions 124E, 178E and 180E,
x) H1 comprises amino acid substitutions 143E, 145T, 179E and 188L, L1 comprises amino acid substitutions 124R and 178R, H2 comprises amino acid substitution 186R, and L2 comprises amino acid substitutions 124E, 131T, 178Y and 180E, or
y) H1 comprises amino acid substitutions 143E, 145T, 179E and 188L, L1 comprises amino acid substitutions 124R and 178R, H2 comprises amino acid substitution 186R, and L2 comprises amino acid substitutions 124E, 176L and 180E,
wherein when both L1 and L2 are co-expressed with at least one of H1 and H2, the pairing of H1-L1 to H1-L2 and the pairing of H2-L2 to H2-L1 is greater than the pairing of H1-L1 to H1-L2 and the pairing of H2-L2 to H2-L1 in the absence of the amino acid substitutions.

### Claim 2 (depends on 1)

The construct of claim 1, wherein the construct further comprises an Fc comprising a first CH3 sequence and a second CH3 sequence, and a first CH2 sequence and a second CH2 sequence, and wherein the Fc is coupled, with or without one or more linkers, to the first heterodimer and to the second heterodimer.

### Claim 3 (depends on 2)

The construct of claim 2, wherein the first CH2 sequence and the second CH2 sequence comprise mutations that selectively alter the affinity of the Fc for Fcgamma receptors.

### Claim 4 (depends on 2)

The construct according to claim 2, wherein the Fc is coupled to H1 and H2 by one or more linkers.

### Claim 5 (depends on 4)

The construct of claim 4, wherein the Fc is a human Fc, a human IgG1 Fc, a human IgA Fc, a human IgG Fc, a human IgD Fc, a human IgE Fc, a human IgM Fc, a human IgG2 Fc, a human IgG3 Fc, or a human IgG4 Fc.

### Claim 6 (depends on 5)

The construct of claim 5, wherein the Fc is a heterodimeric Fc.

### Claim 7 (depends on 6)

The construct of claim 6, wherein the Fc comprises one or more substitutions in at least one of the CH3 sequences that promote the formation of a heterodimeric Fc with stability comparable to a wild-type homodimeric Fc.

### Claim 8 (depends on 7)

The construct of claim 7, wherein the Fc comprises:
i) a heterodimeric IgG1 Fc having the substitutions L351Y_F405A_Y407V in the first Fc polypeptide, and the substitutions T366L_K392M_T394W in the second Fc polypeptide;
ii) a heterodimeric IgG1 Fc having the substitutions L351Y_F405A_Y407V in the first Fc polypeptide, and the substitutions T366L_K392L_T394W in the second Fc polypeptide;
iii) a heterodimeric IgG1 Fc having the substitutions T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and the substitutions T350V_T366L_K392L_T394W in the second Fc polypeptide;
iv) a heterodimeric IgG1 Fc having the substitutions T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and the substitutions T350V_T366L_K392M_T394W in the second Fc polypeptide; or
v) a heterodimeric IgG1 Fc having the substitutions T350V_L351Y_S400E_F405A_Y407V in the first Fc polypeptide, and the substitutions T350V_T366L_N390R_K392M_T394W in the second Fc polypeptide;
wherein the numbering of amino acid residues in the CH3 sequence is according to the EU numbering system.

### Claim 9 (depends on 1)

The construct according to claim 1, wherein the construct is multispecific or bispecific.

### Claim 10 (depends on 1)

The construct according to claim 1, wherein the construct is conjugated to a therapeutic agent or drug moiety.

### Claim 11 (depends on 1)

The construct according to claim 1, wherein
a) H1 comprises amino acid substitutions 143E, 145T, 179E and 188L, L1 comprises amino acid substitutions 124R and 178R, H2 comprises amino acid substitution 186K, and L2 comprises amino acid substitutions 124E, 131T, 178Y and 180E.

### Claim 12 (depends on 11)

The construct according to claim 11, wherein the construct further comprises an Fc comprising a first CH3 sequence and a second CH3 sequence and a first CH2 sequence and a second CH2 sequence, and wherein the Fc is coupled, with or without one or more linkers, to the first heterodimer and to the second heterodimer.

### Claim 13 (depends on 12)

The construct according to claim 12, wherein the Fc is coupled to H1 and H2 by one or more linkers.

### Claim 14 (depends on 12)

The construct of claim 12, wherein the first CH2 sequence and the second CH2 sequence comprise mutations that selectively alter the affinity of the Fc for Fcgamma receptors.

### Claim 15 (depends on 13)

The construct of claim 13 wherein the Fc is a human Fc, a human IgG1 Fc, a human IgA Fc, a human IgG Fc, a human IgD Fc, a human IgE Fc, a human IgM Fc, a human IgG2 Fc, a human IgG3 Fc, or a human IgG4 Fc.

### Claim 16 (depends on 15)

The construct of claim 15, wherein the Fc is a heterodimeric Fc.

### Claim 17 (depends on 16)

The construct of claim 16, wherein the Fc comprises one or more substitutions in at least one of the CH3 sequences that promote the formation of a heterodimeric Fc with stability comparable to a wild-type homodimeric Fc.

### Claim 18 (depends on 17)

The construct of claim 17, wherein the Fc comprises:
i) a heterodimeric IgG1 Fc having the substitutions L351Y_F405A_Y407V in the first Fc polypeptide, and the substitutions T366L_K392M_T394W in the second Fc polypeptide;
ii) a heterodimeric IgG1 Fc having the substitutions L351Y_F405A_Y407V in the first Fc polypeptide, and the substitutions T366L_K392L_T394W in the second Fc polypeptide;
iii) a heterodimeric IgG1 Fc having the substitutions T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and the substitutions T350V_T366L_K392L_T394W in the second Fc polypeptide;
iv) a heterodimeric IgG1 Fc having the substitutions T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and the substitutions T350V_T366L_K392M_T394W in the second Fc polypeptide; or
v) a heterodimeric IgG1 Fc having the substitutions T350V_L351Y_S400E_F405A_Y407V in the first Fc polypeptide, and the substitutions T350V_T366L_N390R_K392M_T394W in the second Fc polypeptide;
wherein the numbering of amino acid residues in the CH3 sequence is according to the EU numbering system.

### Claim 19 (depends on 12)

The construct according to claim 12, wherein the construct is multispecific or bispecific.

### Claim 20 (depends on 19)

The construct according to claim 19, wherein the construct is conjugated to a therapeutic agent or drug moiety.

### Claim 21 (depends on 1)

The construct according to claim 1, wherein H1 comprises amino acid substitutions 143E, 145T, 179E and 188L, L1 comprises amino acid substitutions 124R and 178R, H2 comprises amino acid substitution 186K, and L2 comprises amino acid substitutions 124E, 176L and 180E.

### Claim 22 (depends on 21)

The construct according to claim 21, wherein the construct further comprises an Fc comprising a first CH3 sequence and a second CH3 sequence and a first CH2 sequence and a second CH2 sequence, and wherein the Fc is coupled, with or without one or more linkers, to the first heterodimer and to the second heterodimer.

### Claim 23 (depends on 22)

The construct according to claim 22, wherein the Fc is coupled to H1 and H2 by one or more linkers.

### Claim 24 (depends on 22)

The construct of claim 22, wherein the first CH2 sequence and the second CH2 sequence comprise mutations that selectively alter the affinity of the Fc for Fcgamma receptors.

### Claim 25 (depends on 23)

The construct of claim 23 wherein the Fc is a human Fc, a human IgG1 Fc, a human IgA Fc, a human IgG Fc, a human IgD Fc, a human IgE Fc, a human IgM Fc, a human IgG2 Fc, a human IgG3 Fc, or a human IgG4 Fc.

### Claim 26 (depends on 25)

The construct of claim 25, wherein the Fc is a heterodimeric Fc.

### Claim 27 (depends on 26)

The construct of claim 26, wherein the Fc comprises one or more substitutions in at least one of the CH3 sequences that promote the formation of a heterodimeric Fc with stability comparable to a wild-type homodimeric Fc.

### Claim 28 (depends on 27)

The construct of claim 27, wherein the Fc comprises:
i) a heterodimeric IgG1 Fc having the substitutions L351Y_F405A_Y407V in the first Fc polypeptide, and the substitutions T366L_K392M_T394W in the second Fc polypeptide;
ii) a heterodimeric IgG1 Fc having the substitutions L351Y_F405A_Y407V in the first Fc polypeptide, and the substitutions T366L_K392L_T394W in the second Fc polypeptide;
iii) a heterodimeric IgG1 Fc having the substitutions T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and the substitutions T350V_T366L_K392L_T394W in the second Fc polypeptide;
iv) a heterodimeric IgG1 Fc having the substitutions T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and the substitutions T350V_T366L_K392M_T394W in the second Fc polypeptide; or
v) a heterodimeric IgG1 Fc having the substitutions T350V_L351Y_S400E_F405A_Y407V in the first Fc polypeptide, and the substitutions T350V_T366L_N390R_K392M_T394W in the second Fc polypeptide;
wherein the numbering of amino acid residues in the CH3 sequence is according to the EU numbering system.

### Claim 29 (depends on 28)

The construct according to claim 28, wherein the construct is multispecific or bispecific.

### Claim 30 (depends on 29)

The construct according to claim 29, wherein the construct is conjugated to a therapeutic agent or drug moiety.

### Claim 31 (depends on 1)

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

### Claim 32 (depends on 1)

An isolated polynucleotide or set of isolated polynucleotides comprising at least one sequence that encodes the construct according to claim 1.

### Claim 33 (depends on 32)

A vector or set of vectors comprising one or more of the polynucleotides or sets of polynucleotides according to claim 32.

### Claim 34 (depends on 32)

An isolated cell comprising a polynucleotide or set of polynucleotides according to claim 32.

### Claim 35 (depends on 1)

A method of obtaining the construct according to claim 1 from a host cell culture, the method comprising the steps of:
(a) obtaining a host cell culture comprising at least one host cell comprising one or more nucleic acid sequences encoding the construct; and
(b) recovering the construct from the host cell culture.

### Claim 36 (depends on 1)

A method of obtaining the construct according to claim 1, comprising the steps of:
(a) obtaining H1, L1, H2, and L2;
(b) allowing H1 to pair preferentially with L1 as compared to L2 and H2 to pair preferentially with L2 as compared to L1; and
(c) obtaining the construct.

### Claim 37 (depends on 1)

A method of preparing the construct according to claim 1, comprising:
a. Obtaining a polynucleotide or set of polynucleotides encoding at least one construct;
b. Determining the optimal ratios of each of the polynucleotide or set of polynucleotides for introduction into at least one host cell, wherein the optimal ratios are determined by assessing the amount of H1-L1 and H2-L2 heterodimer pairs formed upon expression of H1, L1, H2, and L2 as compared to mispaired H1-L2 and H2-L1 heterodimer pairs formed upon expression of H1, L1, H2, and L2;
c. Selecting a preferred optimal ratio, wherein transfection of at least one host cell with the preferred optimal ratio of the polynucleotide or set of polynucleotides results in expression of the construct;
d. Transfecting the at least one host cell with the optimal ratio of the polynucleotide or set of polynucleotides; and
e. Culturing the at least one host cell to express the construct.