Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1998-04-30
2001-11-27
Huff, Sheela (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S391300
Reexamination Certificate
active
06323322
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to single-chain antigen-binding molecules capable of glycosylation. More specifically, the invention relates to antigen-binding proteins having Asn-linked glycosylation sites capable of attaching a carbohydrate moiety. The invention also relates to multivalent antigen-binding molecules capable of glycosylation. The invention further relates to glycosylated antigen-binding molecules capable of polyalkylene oxide conjugation. Compositions of, genetic constructions for, methods of use, and methods for producing glycosylated antigen-binding proteins capable of polyalkylene oxide conjugation are disclosed. The invention also relates to methods for producing a polypeptide having increased glycosylation and the polypeptide produced by the described methods.
2. Description of the Background Art
Antibodies are proteins generated by the immune system to provide a specific molecule capable of complexing with an invading molecule, termed an antigen. Natural antibodies have two identical antigen-binding sites, both of which are specific to a particular antigen. The antibody molecule “recognizes ” the antigen by complexing its antigen-binding sites with areas of the antigen termed epitopes. The epitopes fit into the conformational architecture of the antigen-binding sites of the antibody, enabling the antibody to bind to the antigen.
The IgG antibody, e.g., is composed of two identical heavy and two identical light polypeptide chains, held together by interchain disulfide bonds. The remainder of this discussion on antibodies will refer only to one pair of light/heavy chains, as each light/heavy pair is identical. Each individual light and heavy chain folds into regions of approximately 110 amino acids, assuming a conserved three-dimensional conformation. The light chain comprises one variable region (V
L
) and one constant region (C
L
), while the heavy chain comprises one variable region (V
H
) and three constant regions (C
H
1, C
H
2 and C
H
3). Pairs of regions associate to form discrete structures. In particular, the light and heavy chain variable regions associate to form an “Fv ” area which contains the antigen-binding site.
Recent advances in immunobiology, recombinant DNA technology, and computer science have allowed the creation of single polypeptide chain molecules that bind antigen. These single-chain antigen-binding molecules (“SCA”) or single-chain variable fragments of antibodies (“sFv”) incorporate a linker polypeptide to bridge the individual variable regions, V
L
and V
H
, into a single polypeptide chain. A description of the theory and production of single-chain antigen-binding proteins is found in Ladner et al., U.S. Pat. Nos. 4,946,778, 5,260,203, 5,455,030 and 5,518,889. The single-chain antigen-binding proteins produced under the process recited in the above U.S. patents have binding specificity and affinity substantially similar to that of the corresponding Fab fragment. A computer-assisted method for linker design is described more particularly in Ladner et al., U.S. Pat. Nos. 4,704,692 and 4,881,175, and WO 94/12520.
The in vivo properties of SCA polypeptides are different from MAbs and antibody fragments. Due to their small size, SCA polypeptides clear more rapidly from the blood and penetrate more rapidly into tissues (Milenic, D. E. et al.,
Cancer Research
51:6363-6371 (1991); Colcher et al.,
J. Natl. Cancer Inst
. 82:1191 (1990); Yokota et al.,
Cancer Research
52:3402 (1992)). Due to lack of constant regions, SCA polypeptides are not retained in tissues such as the liver and kidneys. Due to the rapid clearance and lack of constant regions, SCA polypeptides will have low immunogenicity. Thus, SCA polypeptides have applications in cancer diagnosis and therapy, where rapid tissue penetration and clearance, and ease of microbial production are advantageous.
A multivalent antigen-binding protein has more than one antigen-binding site. A multivalent antigen-binding protein comprises two or more single-chain protein molecules. Enhanced binding activity, di- and multi-specific binding, and other novel uses of multivalent antigen-binding proteins have been demonstrated. See, Whitlow, M., et al.,
Protein Engng
. 7:1017-1026 (1994); Hoogenboom, H. R.,
Nature Biotech
. 15:125-126 (1997); and WO 93/11161.
Carbohydrate modifications of proteins fall into three general categories: N-linked (or Asn-linked) modification of asparagine, O-linked modification of serine or threonine and glycosyl-phosphatidylinositol derivation of the C-terminus carboxyl group. Each of these transformations is catalyzed by one or more enzymes which demonstrate different peptide sequence requirements and reaction specificities. N-linked glycosylation is catalyzed by a single enzyme, oligosaccharyl transferase (OT), and involves the co-translational transfer of a lipid-linked tetradecasaccharide (GlcNAc
2
-Man
9
-Glc
3
) to an asparagine side chain within a nascent polypeptide (see, Imperiali, B. and Hendrickson, T. L.,
Bioorganic
&
Med. Chem
. 3:1565-1578 (1995)). The asparagine residue must reside within the tripeptide N-linked glycosylation consensus sequence Asn-Xaa-Thr/Ser (NXT/S), where Xaa can be any of the 20 natural amino acids except proline.
A natural N-linked glycosylation sequence (Asn-Val-Thr) at amino acid positions 18-20 (Kabat's numbering) was identified in the framework-1 (FR-1) region of the light chain variable domain of a murine anti-B cell lymphoma antibody, LL-2 (Leung, S.-o. et al.,
J. Immunol
. 154:5919-5926 (1995)). By a single Arg to Asn mutation, an N-linked glycosylation sequence similar to that of LL-2 was introduced in the FR-1 segment of a nonglycosylated, humanized anti-carcinoembryonic Ag (CEA) Ab, MN-14 (Leung, S.-O. et al.,
J. Immunol
. 154:5919-5926 (1995), which disclosure is incorporated herein by reference).
An sFv having a C-terminus that has cross-linking means by disulfide bonds at cysteine residues has been reported (Huston et al., U.S. Pat. No. 5,534,254). A monoclonal antibody has also been reported that is covalently bound to a diagnostic or therapeutic agent through a carbohydrate moiety at an Asn-linked glycosylation site at about amino acid position 18 of the V
L
region (Hansen et al., U.S. Pat. No. 5,443,953). Binding studies of an anti-dextran antibody that is Asn-linked glycosylated in the V
H
chain have been performed which show that slight changes in the position of the Asn-linked carbohydrate moiety in the V
H
region result in substantially different effects on antigen binding (Wright et al.,
EMBO J
. 10:2717-2723 (1991)). It has also been shown that glycosylation at position 19 within the V
H
region of an sFv enhanced expression of the overall amount of sFv intracellularly, of which approximately half was glycosylated (Greenman, J., et al.,
J. Immunol. Methods
194:169-180 (1996)), and enhanced synthesis and secretion of the glycosylated sFv over the nonglycosylated sFv (Jost, C. R., et al.,
J. Biol. Chem
. 269:26267-26273 (1994)). Co et al., U.S. Pat. No. 5,714,350, relates to increasing binding affinity of an antibody by eliminating a glycosylation site.
The covalent attachment of strands of a polyalkylene glycol or polyalkylene oxide to a polypeptide molecule is disclosed in U.S. Pat. No. 4,179,337 to Davis et al., as well as in Abuchowski and Davis “Enzymes as Drugs,” Holcenberg and Roberts, Eds., pp. 367-383, John Wiley and Sons, New York (1981), and Zalipsky et al., WO 92/16555. These references disclosed that proteins and enzymes modified with polyethylene glycols have reduced immunogenicity and antigenicity and have longer lifetimes in the bloodstream, compared to the parent compounds. The resultant beneficial properties of the chemically modified conjugates are very useful in a variety of therapeutic applications.
To effect covalent attachment of polyethylene glycol (PEG) and similar poly(alkylene oxides) to a molecule, the hydroxyl end groups of the polymer must first be converted into reactive functional groups. This
Filpula David
Lee Lihsyng S.
Shorr Robert
Wang Maoliang
Whitlow Marc
Enzon Inc.
Helms Larry R.
Huff Sheela
Sterne Kessler Goldstein & Fox P.L.L.C.
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