US-12624124-B2 - Heterodimeric proteins and methods for producing and purifying them

US12624124B2US 12624124 B2US12624124 B2US 12624124B2US-12624124-B2

Abstract

The present invention relates to engineered heteromultimeric proteins, and more specifically, to methods for producing and purifying heterodimeric proteins, such as bispecific antibodies and other heterodimeric proteins comprising immunoglubulin-like hinge sequences. Methods for producing and purifying such engineered heterodimeric proteins and their use in diagnostics and therapeutics are also provided.

Inventors

Weihsien HO
Jaume Pons
Arvind Rajpal
Pavel Strop

Assignees

RINAT NEUROSCIENCE CORP.

Dates

Publication Date: 20260512
Application Date: 20220622

Claims (7)

1 . A method of purifying a heterodimeric protein, wherein the purification comprises a chromatography step, wherein the heterodimeric protein comprises: a hinge region comprising a first immunoglobulin-like hinge polypeptide and a second immunoglobulin-like hinge polypeptide which interact together to form a dimeric hinge interface, wherein electrostatic interactions between one or more charged amino acids within the hinge interface favor interaction between the first and second hinge polypeptides over interaction between two first hinge polypeptides or two second hinge polypeptides, thereby promoting heterodimer formation over homodimer formation, wherein the hinge region is a human IgG2 hinge region, wherein each of the first hinge polypeptide and the second hinge polypeptide comprises at least two amino acid modifications relative to a wild-type IgG2 hinge region at a position of Cys223, Glu225 or Pro228, according to EU numbering scheme, wherein the wild-type amino acid in the first hinge polypeptide is replaced with an amino acid having an opposite charge to the corresponding amino acid in the second hinge polypeptide, and the heterodimeric protein further comprising an immunoglobulin-like CH3 region comprising a first CH3 polypeptide fused to the first hinge polypeptide and a second CH3 polypeptide fused to the second hinge polypeptide, wherein the first CH3 polypeptide and the second CH3 polypeptide comprise at least one amino acid modification relative to a wild-type IgG2 CH3 region sequence at a position of Leu368 or Lys409, according to EU numbering scheme.
2 . The method of claim 1 , wherein the chromatography step is ion exchange chromatography.
3 . The method of claim 1 , wherein the heterodimeric protein is a bispecific IgG2 antibody.
4 . The method of claim 3 , wherein the bispecific antibody comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein the first heavy chain comprises the first hinge polypeptide and the first CH3 polypeptide, and wherein the second heavy chain comprises the second hinge polypeptide and the second CH3 polypeptide.
5 . The method of claim 4 , wherein the first heavy chain and second heavy chain each comprise an amino aid modification of Ala330Ser and Pro331Ser, according to the EU numbering scheme.
6 . The method of claim 5 , wherein a first antibody variable domain of the bispecific antibody binds to a T cell receptor and the second and a second antibody variable domain of the bispecific antibody binds to a tumor antigen.
7 . The method of claim 6 , wherein the first antibody variable domain binds to CD3.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a divisional application of U.S. application Ser. No. 16/157,951 filed Oct. 11, 2018, which is a divisional application of U.S. application Ser. No. 15/351,275, filed Nov. 14, 2016, now U.S. Pat. No. 10,138,303, which is a divisional of U.S. application Ser. No. 13/697,683, filed on Jan. 17, 2013, now U.S. Pat. No. 9,527,926, which is a National Stage Application under 35 U.S.C. § 371 of PCT/US2011/036419, filed on May 13, 2011, which claims the benefits of U.S. Provisional Application No. 61/345,047 filed May 14, 2010, and U.S. Provisional Application No. 61/485,097 filed May 11, 2011, which are both hereby incorporated by reference in their entireties. REFERENCE TO SEQUENCE LISTING This application is being filed electronically via EFS-Web and includes an electronically submitted sequence listing in.txt format. The txt file contains a sequence listing entitled “PC71659D_SequenceListing_ST25.txt” created on Jun. 21, 2022, and having a size of 18 KB. The sequence listing contained in this txt file is part of the specification and is incorporated herein by reference in its entirety. FIELD The present invention relates to engineered heteromultimeric proteins, and more specifically, to methods for producing and purifying heterodimeric proteins, such as bispecific antibodies and other heterodimeric proteins comprising immunoglobulin-like hinge sequences. Methods for producing and purifying such engineered heterodimeric proteins and their use in diagnostics and therapeutics are also provided. BACKGROUND Antibodies possess a variety of properties which make them useful as therapeutic molecules. In addition to their ability to bind with high affinity to a molecular target inside or outside of cells with high specificity and selectivity, antibodies render their targeted binding partners susceptible to Fc-receptor cell-mediated phagocytosis and killing through effector functions, such as complement induced pathways and ADCC (antibody-dependent cell-mediated cytotoxicity) related activities. Further, antibodies may be engineered in a variety of ways to further increase their therapeutic utility. Antibodies having extended in vivo half-lives, for example, may be produced by engineering Fc fusion molecules, by treatment with biocompatible polymers such as polyethylene glycol (PEG), or “pegylation” and by other engineering methods well known in the art. Antibodies have binding specificities for at least two different antigens, called bispecific antibodies (BsAbs), have also been engineered. See Nolan, O. and R. O'Kennedy (1990) Biochim Biophvs Acta 1040(1): 1-1 1.; de Leij, L. et al., Adv Drug Deliv Rev 31(1-2): 105-129 (1998); and Carter, P. J Immunol Methods 248(1-2): 7-15 (2001)). While classical antibodies have identical sequences in each of the two arms (containing the antigen binding sites of Fab region) of the Y-shaped molecule, bispecific antibodies have different sequences in each of the two Fab regions so that each arm of the Y-shaped molecule binds to a different antigen or epitope. By being able to bind two different antigenic molecules or different epitopes, BsAbs offer a wide variety of clinical applications as targeting agents for in vitro and in vivo diagnostics and immunotherapies. In diagnostic areas, BsAbs have been used, e.g., to study functional properties of cell surface molecules, different Fc receptors and their ability to mediate cytotoxicity (Fanger et al., Crit. Rev. Immunol. 12:101-124 (1992); Nolan et al., Biochem. Biophys. Acta. 1040:1-11 (1990); and to immobilize enzymes and other agents to produce immunodiagnostic and immunoassay reagents and methods. Bispecific antibodies can also be used for in vitro or in vivo diagnoses of various disease states, including cancer (Songsivilai et al., Clin. Exp. Immunol. 79:315 (1990)). For example, one arm of the BsAb can be engineered to bind a tumor-associated antigen and the other arm to bind a detectable marker. (See, e.g., Le Doussal et al., J. Nucl. Med. 34:1662-1671 (1993), in which a BsAb having one arm which bound a carcinoembryonic antigen (CEA) and another arm which bound DPTA was used for radioimmunodetection of colorectal and thyroid carcinomas. See also Stickney et al., Cancer Res. 51:6650-6655 (1991), describing a strategy for detecting colorectal cancers expressing CEA by radioimmunodetection. The use of bispecific antibodies for immunotherapy of cancer has been reviewed (see e.g., Nolan and O'Kennedy 1990, supra; de Leij et al. (1998) supra; and Carter, P. (2001) supra.) BsAbs can be used to direct a patient's cellular immune defense mechanisms specifically to a tumor cell or an infectious agent (e.g., virally infected cells such as HIV or influenza virus; protozoa such as Toxoplasma gondii). In particular, one can redirect immune modulated cytotoxicity by engineering one arm of the BsAb to bind to a desired target (e.g. tumor cell or pathogen) and the other arm of the BsAb to