BACKGROUND OF THE INVENTION

1. Field of Art

The present invention relates to the biochemical arts, particularly to conjugates with immunoglobulin Fc domains.

2. Discussion of Related Art

The immunoglobulin Fc domain has found widespread use as a carrier protein for a variety of therapeutic and diagnostic molecules. Antibodies comprise two functionally independent parts, a variable domain known as “Fab”, which binds antigen, and a constant domain known as “Fc”, which links to such effector functions as complement activation and attack by phagocytic cells. An Fc has a long serum half-life, whereas a Fab is short-lived. (Capon et al. (1989),Nature337: 525-31). When constructed together with a therapeutic protein or peptide, an Fc domain can provide longer half-life, or can incorporate such functions as Fc receptor binding, protein A binding, complement fixation and perhaps even placental transfer. Id.

Numerous fusions of proteins and peptides have been engineered at either the amino- or carboxy-termini. Also, a variety of enzymes and synthetic reporter molecules have been chemically conjugated to the side chains of non-terminal amino acids as well as the derivatized carbohydrate moieties of the Fc domain. Further, several polymers, such as polyethylene glycol (PEG) have been conjugated to the Fc domain for the purpose of improved half-life in vivo and reduced immunogenicity.

The success of the drug Enbrel® (etanercept) brought to fruition the promise of therapeutic agents modified with the constant domain of an antibody. Table 1 summarizes several examples of the use of Fc fusion proteins known in the art.

TABLE 1Fc fusion with therapeutic proteinsTherapeuticForm of FcFusion partnerimplicationsReferenceIgG1N-terminus ofHodgkin's disease;U.S. Pat. No.CD30-Lanaplastic5,480,981lymphoma; T-cellleukemiaMurineIL-10anti-inflammatory;Zheng et al. (1995),Fcγ2atransplant rejectionJ. Immunol.154:5590-600IgG1TNF receptorseptic shockFisher et al. (1996),N. Engl. J. Med.334: 1697-1702;Van Zee, K. et al.(1996),J. Immunol.156: 2221-30IgG, IgA,TNF receptorinflammation,U.S. Pat. No.IgM, or IgEautoimmune5,808,029, issued(excludingdisordersSep. 15, 1998the firstdomain)IgG1CD4 receptorAIDSCapon et al. (1989),Nature337: 525-31IgG1, IgG3N-terminus ofanti-cancer,Harvill et al. (1995),IL-2antiviralImmunotech.1:95-105IgG1C-terminus ofosteoarthritis;WO 97/23614,OPGbone densitypublishedJul. 3, 1997IgG1N-terminus ofAnti-obesityWO 98/28427, filedleptinDec. 11, 1997Human IgCTLA-4autoimmuneLinsley (1991),C□1disordersJ. Exp. Med.174:561-9

A more recent development is fusion of randomly generated peptides with the Fc domain. See U.S. Pat. No. 6,660,843, issued Dec. 9, 2003 to Feige et al. (incorporated by reference in its entirety). Such molecules have come to be known as “peptibodies.” They include one or more peptides linked to the N-terminus, C-terminus, amino acid side chains, or to more than one of these sites. Peptibody technology enables design of therapeutic agents that incorporate peptides that target one or more ligands or receptors, tumor-homing peptides, membrane-transporting peptides, and the like. Peptibody technology has proven useful in design of a number of such molecules, including linear and disulfide-constrained peptides, “tandem peptide multimers” (i.e., more than one peptide on a single chain of an Fc domain). See, for example, U.S. Pat. No. 6,660,843; U.S. Pat. App. No. 2003/0195156 A1, published Oct. 16, 2003 (corresponding to WO 02/092620, published Nov. 21, 2002); U.S. Pat. App. No. 2003/0176352, published Sep. 18, 2003 (corresponding to WO 03/031589, published Apr. 17, 2003); U.S. Ser. No. 09/422,838, filed Oct. 22, 1999 (corresponding to WO 00/24770, published May 4, 2000); U.S. Pat. App. No. 2003/0229023, published Dec. 11, 2003; WO 03/057134, published Jul. 17, 2003; U.S. Pat. App. No. 2003/0236193, published Dec. 25, 2003 (corresponding to PCT/US04/010989, filed Apr. 8, 2004); U.S. Ser. No. 10/666,480, filed Sep. 18, 2003 (corresponding to WO 04/026329, published Apr. 1, 2004), U.S. Patent App. No. 2006/0140934, published Jun. 29, 2006 (corresponding to WO 2006/036834, published Apr. 4, 2006), each of which is hereby incorporated by reference in its entirety. The art would benefit from further technology enabling such rational design of polypeptide therapeutic agents.

Conventional approaches for chemical conjugation to the immunoglobulin Fc domain include random coupling to naturally occurring primary amines such as lysine and the amino-terminus or carboxylic acids such as glutamic acid, aspartic acid and the carboxy terminus. Alternatively, semi-selective site-specific coupling may be achieved through N-terminal conjugation under appropriate conditions, or derivatized carbohydrates as found on Fc proteins isolated from eukaryotic sources, or by partial reduction and coupling of native cysteine residues. (E.g., Kim et al., A pharmaceutical composition comprising an immunoglobulin Fc region as a carrier, WO 2005/047337). While each of these approaches has been applied successfully, they typically suffer from varying degrees of conjugate heterogeneity, relatively low yields and sometimes, significant losses in functional activity are also observed. The art would benefit from a process for selective, site-specific conjugation to the immunoglobulin Fc domain without significant loss in functional activity.

SUMMARY OF THE INVENTION

The present invention concerns compositions of matter and a process for making them. The inventive composition of matter, which is a pharmacologically active compound, comprises a monomeric or multimeric Fc domain having at least one additional functional moiety that is covalently bound (or conjugated), either directly or through a linker, to one or more specifically selected conjugation site(s) in the Fc domain through the side chain of an amino acid residue at the conjugation site(s). Such an internal conjugation site may be already present in a native Fc domain sequence or can be added by insertion (i.e., between amino acids in the native Fc domain) or by replacement (i.e., removing amino acid residue(s) and substituting different canonical and/or non-canonical amino acid residue(s)) in the native Fc domain sequence in order to create or “engineer” the conjugation site. In the latter case, the number of amino acid residues added need not correspond to the number of amino acid residues removed from the previously existing Fc domain sequence.

This inventive process of preparing a pharmacologically active compound comprising an Fc domain includes:

a. selecting at least one internal conjugation site of an Fc domain sequence, said conjugation site being amenable to conjugation of an additional moiety by a defined coupling chemistry through the side chain of an amino acid residue at the conjugation site; and

b. conjugating a predetermined functional moiety to the selected conjugation site by employing the defined conjugation chemistry.

In some embodiments, the functional moiety is a half-life extending moiety and/or a pharmacologically active moiety, which can be, for example, a polypeptide, a peptide, a peptidomimetic, or a non-peptide organic moiety. In other embodiments the additional functional moiety is a moiety detectably labeled with a radioisotope, an enzyme (e.g., a peroxidase or a kinase), a biotinyl moiety, a fluorophore, or a chromophore. Alternatively, the additional functional moiety is an immobilized substrate, such as but not limited to, a plate surface, a bead, a particle, a microparticle, a nanoparticle, a chip, a liposome, a matrix, or the like, provided that in a chain of additional functional moieties, the immobilized substrate is the additional moiety most distal from the Fc domain, and there can be no more than one immobilized substrate in the chain.

The inventive process can be employed to modify an Fc domain that is already linked through an N- or C-terminus or side chain to a polypeptide (e.g., a soluble fragment of TNF-R2, as in etanercept) or to a peptide (e.g., as described in U.S. Pat. App. Nos. 2003/0195156 A1, 2003/0176352, 2003/0229023, and 2003/0236193; WO 00/24770; WO 04/026329). The process described throughout can also be employed to modify an Fc domain that is part of an antibody (e.g., adalimumab, epratuzumab, infliximab, Herceptin®, and the like). In this way, different molecules can be produced that have additional functionalities, such as a binding domain to a different epitope, an additional binding domain to the precursor molecule's existing epitope, or an additional half-life extending moiety.

The compounds of this invention may be prepared by standard synthetic methods, recombinant DNA techniques, or any other methods of preparing peptides and fusion proteins with reference to the disclosure of this specification.

The compounds of this invention may be used for therapeutic or prophylactic purposes by formulating them by methods known for other proteinacious molecules and administering an effective amount to a patient, such as a human (or other mammal) in need thereof. Other related aspects are also included in the current invention.

Numerous additional aspects and advantages of the present invention will become apparent upon consideration of the figures and detailed description of the invention.

