Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-01-15
2004-11-09
Helms, Larry R. (Department: 1642)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100, C435S325000, C435S326000, C530S387300
Reexamination Certificate
active
06815540
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to the modification of immunoglobuling superfamily (IgSF) domains and derivatives thereof so as to increase their solubility, and hence the yield, and ease of handling.
Small antibody fragments show exciting promise for use as therapeutic agents, diagnostic reagents, and for biochemical research. Thus, they are needed in large amounts, and the expression of antibody fragments, e.g. Fv, single-chain-Fv (scFv), or Fab in the periplasm of
E. coli
(Skerra & Plückthun 1988; Better et al., 1988) is now used routinely in many laboratories. Expression yields vary widely, however, especially in the case of scFvs. While some fragments yield up to several mg of functional, soluble protein per litre and OD of culture broth in shake flask 25 culture (Carter et al., 1992, Plückthun et al. 1996), other fragments may almost exclusively lead to insoluble material, often found in so-called inclusion bodies. Functional protein may be obtained from the latter in modest yields by a laborious and time-consuming refolding process. The factors influencing antibody expression levels are still only poorly understood.
Folding efficiency and stability of the antibody fragments, protease lability and toxicity of the expressed proteins to the host cells often severely limit actual production levels, and several attempts have been tried to increase expression yields. For example, Knappik & Plückthun (1995) have identified key residues in the antibody framework which influence expression yields dramatically. Similarly, Ullrich et al. (1995) found that point mutations in the CDRs can increase the yields in periplasmic antibody fragment expression. Nevertheless, these strategies are only applicable to a few antibodies.
The observations by Knappik & Plückthun (1995) indicate that optimizing those parts of the antibody fragment which are not directly involved in antigen recognition can significantly improve folding properties and production yields of recombinant Fv and scFv constructs. The causes for the improved expression behavior lie in the decreased aggregation behavior of these molecules. For other molecules, fragment stability and protease resistance may also be affected. The understanding of how specific sequence modifications change these properties is still very limited and currently under active investigation.
Difficulties in expressing and manipulating protein domains may arise because amino acids which are normally buried within the protein structure become exposed when only a portion of the whole molecule is expressed. Aggregation may occur through interaction of newly solvent-exposed hydrophobic residues originally forming the contact regions between adjacent domains. Leistler and Perham (1994) could show that a certain domain of glutathione reductase may be expressed separately from its neighboring domains, but the protein showed non-specific association in vitro forming multimeric protein species. The introduction of hydrophilic residues instead of exposed hydrophobic amino acids could decrease this aggregation tendency and thus stabilize this isolated domain. Both wild type and modified domains were exclusively found in inclusion bodies and had to be refolded. Although in vitro experiments contributed a lot to define various intermolecular interactions, which drive folding processes, they are only of limited value in predicting the folding behaviour of different polypeptide chains in vivo (Gething & Sambrook, 1992). Thus, Leistler and Perham do not teach or suggest how to increase expression yields of soluble protein domains.
In the case of antibodies, two chains comprising several domains dimerize, each domain consisting of a b-barrel whose two b-sheets are held together by a disulphide bond, forming the so-called immunoglobulin fold. Two domains, one variable domain (VL) and one constant domain (CL) are adjacent along the longitudinal axis in the light chain (VL-CL), and four domains, one variable domain (VH) and three constant domain (CH1 to CH3) are adjacent along the longitudinal axis in the heavy chain (VH-CH1-CH2-CH3). In the dimer formed by chains a and b, two such domains associate laterally: VLa with VHa, CLa with CH1a, VLb with VHb, CLb with CH1b CH2a with CH2b and CH3a with CH3b. In WO 92/01787 (Johnson et al., 1992), it is taught that isolated single domains, e.g. VH, can be modified in the former VL/VH interface region by exchanging hydrophobic residues by hydrophilic ones without changing the specificity of the parent domain. The rationale for WO 92/01787 was the assumption that exposed hydrophobic residues might lead to non-specific binding, interaction with surfaces and decreased stability. Data for increase in binding specificity was given, but increase in expression level was not shown. Furthermore, WO 92/01787 would not be applicable to any antibody fragment containing the complete antigen binding site, as it must contain VL and VH. In the case of T cell receptors, two chains (a and b) dimerize, each consisting of a variable (V) and a constant (C) domain with the immunoglobulin fold, and one transmembrane domain. In each chain, the variable and constant domains are adjacent along the longitudinal axis in the chains (Va-Ca; Vb-Cb) and associate laterally with the corresponding domains of the second chain (Va-Vb; Ca-Cb).
Various other molecules of the immunoglobulin superfamily, such as CD2, CD4, CD16, CD22, comprise only one chain, wherein two or more domains (variable and/or constant) with the immunoglobulin fold are adjacent along the longitudinal axis in the chains.
SUMMARY OF THE INVENTION
The present inventors have found that expression problems are largely associated with a part of the molecule that has hitherto not been regarded relevant for expression studies and which comprises the interface between adjacent domains within an immunoglobulin chain. This surprising finding forms the basis of the present invention, which provides a general solution to the problems associated with production of domains or fragments of the immunoglobulin superfamily (IgSF), especially antibody fragments, which exhibit poor solubility or reduced levels of expression.
In addition to lateral interactions between domains of different chains described above, there are well documented contacts between adjacent domains within individual chains along the longitudinal axis. For example, in the case of an antibody (Lesk & Chothia, 1988), the “bottom” of VL makes contact with the “top” of CL, and, in a similar manner there are contacts between VH and CH1. The contacts at these inter-domain interfaces are probably essential for the compact arrangement of the Fab fragment, and, as is typical for such contacts, are at least partially hydrophobic in nature (Lesk & Chothia, 1988).
The basis of the present invention is the surprising finding that the solubility (and hence the yield) of antibody fragments comprising at least one domain can be dramatically increased by decreasing the hydrophobicity of former interfaces at the “end” of said domain, where it would normally adjoin a second domain within a chain in a larger antibody fragment or full antibody. This is surprising and could not have been predicted from the prior art (WO 92/01787), because the size of the longitudinal interface, for example, in a scFv fragment, is much smaller than that between VH and VL, and therefore, the amino acids which make up the interfaces between VH and CH1 or between VL and CL in a Fab fragment represent a much smaller proportion of the total surface area of the scFv molecule, and would accordingly be expected to play less of a role in determining the physical properties of the molecule.
The present invention has the additional advantage that because the alterations effected in the molecules that lead to said decreased hydrophobicity of former interfaces are located at the most distant part of the domain from the CDRs, applying the invention is unlikely to have a deleterious effect on the binding properties of the molecule. This is not the case in WO 92/01787, wh
Honegger Annemarie
Nieba Lars
Plückthun Andreas
Heller Ehrman White and McAuliffe LLP
Helms Larry R.
University of Zurich
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