Variant ch3 domains engineered for preferential ch3 heterodimerization, multi-specific antibodies comprising the same, and methods of making thereof

Patent metadata

Field	Value
Publication number	US-2025215082-A1
Application number	US-202318727720-A
Country	US
Kind code	A1
Filing date	Jan 11, 2023
Priority date	Jan 11, 2022
Publication date	Jul 3, 2025
Grant date	—

Abstract

Variant CH3 domain polypeptides are provided that preferentially form CH3-CH3 heterodimers over CH3-CH3 homodimers. Such variant CH3 domains can be used to promote desired Fc pairing, thus providing for efficient development of bispecific and multispecific antibodies as well as Fc fusions of different formats. Methods of producing bispecific antibodies using such variant CH3 domains and for producing libraries containing such variant CH3 domains are also provided.

First claim

What is claimed is:

1 . A method of producing a heteromeric molecule, wherein the heteromeric molecule comprises:
(A) a first polypeptide comprising a first variant CH3 domain polypeptide, the first variant CH3 domain polypeptide comprising a T366V substitution, according to EU numbering; and (B) a second polypeptide comprising a second variant CH3 domain polypeptide, the second variant CH3 domain polypeptide comprising a Y407V substitution according to EU numbering;
wherein the first polypeptide and the second polypeptide are bound to or paired with each other optionally via at least one disulfide bond,
the method comprising:
(i) incubating in a reducing environment (i-1) a first parent molecule comprising at least two of the first polypeptides bound to or paired with each other optionally via at least one disulfide bond and (i-2) a second parent molecule comprising at least two of the second polypeptides bound to or paired with each other optionally via at least one disulfide bond;
(ii) placing the incubation product of step (i) in a less reducing or non-reducing environment, thereby forming the heteromeric molecule;

optionally wherein:
(a) the first variant CH3 domain polypeptide is derived from a CH3 domain of a human IgG and/or the second variant CH3 domain polypeptide is derived from a CH3 domain of a human IgG, optionally wherein the T366V substitution is relative to a CH3 domain of a human IgG and/or the Y407V substitution is relative to a CH3 domain of a human IgG;
(b) the first variant CH3 domain polypeptide is derived from a CH3 domain of a human IgG1 and/or the second variant CH3 domain polypeptide is derived from a CH3 domain of a human IgG1, optionally wherein the T366V substitution is relative to SEQ ID NO: 1, 2, 3, or 4 and/or the Y407V substitution is relative to SEQ ID NO: 1, 2, 3, or 4;
(c) the first variant CH3 domain polypeptide is derived from a CH3 domain of a human IgG2 and/or the second variant CH3 domain polypeptide is derived from a CH3 domain of a human IgG2, optionally wherein the T366V substitution is relative to SEQ ID NO: 722 and/or the Y407V substitution is relative to SEQ ID NO: 722;
(d) the first variant CH3 domain polypeptide is derived from a CH3 domain of a human IgG3 and/or the second variant CH3 domain polypeptide is derived from a CH3 domain of a human IgG3, optionally wherein the T366V substitution is relative to SEQ ID NO: 723 and/or the Y407V substitution is relative to SEQ ID NO: 723; and/or
(e) the first variant CH3 domain polypeptide is derived from a CH3 domain of a human IgG4 and/or the second variant CH3 domain polypeptide is derived from a CH3 domain of a human IgG4, optionally wherein the T366V substitution is relative to SEQ ID NO: 724 and/or the Y407V substitution is relative to SEQ ID NO: 724.

and optionally wherein the heteromeric molecule comprises one or more of the following features:
(A) the first polypeptide further comprises a first antigen-binding domain;
(B) the second polypeptide further comprises a second antigen-binding domain;
(C) the heteromeric molecule further comprises a third polypeptide optionally comprising a third antigen-binding domain, optionally wherein the third polypeptide is bound to or paired with the first polypeptide; and/or
(D) the heteromeric molecule further comprises a fourth polypeptide optionally comprising a fourth antigen-binding domain, optionally wherein the fourth polypeptide is bound to or paired with the second polypeptide,

further optionally wherein the heteromeric molecule is a multi-specific antibody or antigen-binding antibody fragment and optionally comprises a structure depicted in any one of FIGS. 2 - 8 ; optionally wherein the heteromeric molecule comprises (a) an IgG or (b) an IgG and one or more scFvs conjugated to the IgG, further optionally comprising IgG1, IgG2, IgG3 or IgG4 constant regions.

2 . The method of claim 1 , which comprises one or more of the following features:
(I) (I-1-i) the first polypeptide comprises a first antigen-binding domain which forms a first antigen-binding site specific for a first epitope and/or (I-1-ii) the heteromeric molecule comprises a third polypeptide comprising a third antigen-binding domain which forms a third antigen-binding site specific for a third epitope, optionally wherein the first epitope is the same as or different from the third epitope; or
(I-2) the first polypeptide comprises a first antigen-binding domain and the heteromeric molecule comprises a third polypeptide comprising a third antigen-binding domain, wherein the first antigen-binding domain and the third antigen-binding domain form a first antigen-binding site specific for a first epitope; and/or
(II) (II-1-i) the second polypeptide comprises a second antigen-binding domain which forms a second antigen-binding site specific for a second epitope and/or (I-1-ii) the heteromeric molecule comprises a fourth polypeptide comprising a fourth antigen-binding domain which forms a fourth antigen-binding site specific for a fourth epitope, optionally wherein the second epitope is the same as or different from the fourth epitope; or
(II-2) the second polypeptide comprises a second antigen-binding domain and the heteromeric molecule comprises a fourth polypeptide comprising a fourth antigen-binding domain, wherein the second antigen-binding domain and the fourth antigen-binding domain form a second antigen-binding site specific for a second epitope.

3 . The method of claim 1 , wherein the step (i) comprises one or more of the following features:
(a) the incubating is performed at a temperature between about 15° C. and about 40° C., between about 20° C. and about 40° C., between about 25° C. and about 35° C., between about 28° C. and about 32° C., or between about 29° C. and about 31° C., or at about 30° C.; (b) the incubating is performed for about 30 minutes to about 20 hours, for about 1 hour to about 15 hours, for about 2 hours to about 10 hours, for about 3 hours to about 7 hours, or for about 4 hours to about 6 hours, or for about 5 hours; (c) the incubating is performed at about 30° C. for about 5 hours; (d) the reducing environment comprises at least one reducing agent, optionally at least one mildly reducing agent; (e) the reducing environment comprises at least one reducing agent selected from 2-mercaptoethylamine (2-MEA), b-mercapto-ethanol (BME), L-cysteine, dithiothreitol (DTT), or dithionite; (f) the reducing environment comprises at least one reducing agent selected from:
about 25 to about 125 mM, about 50 mM to about 100 mM, about 70 to about 80 mM, or about 75 mM of 2-MEA,
about 20 to about 500 μM, about 40 to about 250 μM, about 80 to about 150 μM, about 90 to about 120 μM, or about 100 μM of BME,
about 20 to about 500 μM, about 40 to about 250 μM, about 80 to about 150 μM,
about 90 to about 120 μM, or about 100 μM of L-cysteine,
about 15 to about 400 μM, about 20 to about 200 μM, about 25 to about 100 μM,
about 30 to about 70 μM, or about 50 μM of DTT, or
about 20 to about 500 μM, about 40 to about 250 μM, about 80 to about 150 μM,
about 90 to about 120 μM, or about 100 μM of dithionite;
(g) the reducing environment comprises at least 2-MEA, optionally at about 75 mM; (h) the at least two of the first polypeptides are bound to or paired with each other via at least one disulfide bond and/or the at least two of the second polypeptides are bound to or paired with each other via at least one disulfide bond; (i) the first antibody and/or the second antibody is/are produced in a mammalian cell, a yeast cell, an insect cell, a plant cell, or a bacterial cell; and/or (j) the first antibody and/or the second antibody is/are produced in a Chinese hamster ovary (CHO) cell or a Human embryonic kidney (HEK) cell.

4 . The method of claim 1

Assignees

• Adimab Llc

Inventors

• Stein Caitlin
• Pejchal Robert
• Mccreary Julia
• Barlow Kyle
• Sivasubramanian Arvind
• Battles Michael Benjamin
• Sharkey Beth H

Classifications

• C07K2317/94

  Stability, e.g. half-life, pH, temperature or enzyme-resistance · CPC title

• C07K2317/569

  Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody® · CPC title

• C07K2317/526

  CH3 domain · CPC title

• C07K2317/31

  multispecific · CPC title

• C07K16/32

  against translation products of oncogenes · CPC title