Drug – bio-affecting and body treating compositions – In vivo diagnosis or in vivo testing
Reexamination Certificate
2001-08-20
2004-11-23
Saunders, David (Department: 1644)
Drug, bio-affecting and body treating compositions
In vivo diagnosis or in vivo testing
C424S009200, C424S133100, C435S007210, C530S387300
Reexamination Certificate
active
06821505
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of the transport of serum proteins and antibodies mediated by the Fc receptor, FcRn, and further to the effect on serum half life of agents that interact with the Fc receptor in a pH dependent way.
2. Description of Related Art
IgGs constitute the most prevalent immunoglobin class in the serum of man and other mammals and are maintained at remarkably constant levels. Recent studies indicate that the major histocompatibility complex (MHC)-class I related receptor, FcRn, is involved in the homeostasis of serum IgGs (Ghetie et al., 1996; Junghans and Anderson, 1996; Israel et al., 1996). This receptor most likely acts as a salvage receptor, and this would be consistent with its known ability to transcytose IgGs in intact form across the neonatal gut (Wallace and Rees, 1980; Rodewald and Kraehenbuhl, 1984) and yolk sac (Roberts et al., 1990; Israel et al., 1995) or placenta (Kristoffersen and Matre, 1996; Simister et al., 1996; Leach et al., 1996). The interaction site of FcRn on mouse IgG1(mIgG1) has been mapped using site-directed mutagenesis of recombinant Fc-hinge fragments, followed by analysis of these fragments both in vivo and in vitro(Kim et al., 1994b; Medesan et al., 1996; 1997). From these studies, I253 (EU numbering (Edelman et al., 1969)), H310, H435 and to a lesser extent, H436 play a central role in this interaction. These amino acids are located at the CH2-CH3 domain interface (Deisenhofer, 1981), and the mapping of the functional site to these residues is consistent with the X-ray crystallographic structure of rat FcRn complexed with rat Fc (Burmeister et al., 1994b).
The FcRn interaction site encompasses three spatially close loops comprised of sequences that are distal in the primary amino acid sequence. The central role of Fc histidines in building this site accounts for the marked pH dependence (binding at pH 6.0, release at pH 7.4) of the Fc-FcRn interaction (Rodewald and Kraehenbuhl, 1984; Raghavan et al., 1995; Popov et al., 1996), as the pKa of one of the imidazole protons lies in this pH range. I253, H310, H435 and to a lesser degree, H436, are highly conserved in IgGs of both human and rodent IgGs (Kabat et al., 1991). This, taken together with the isolation of a human homolog of FcRn (Story et al., 1994), indicate that the molecular mechanisms involved in IgG homeostasis are common to both mouse and man and this has implications for the modulation of the pharmacokinetics of IgGs for use in therapy.
To date, in studies to identify the FcRn interaction site on Fc, mutations of Fc-hinge fragments have been made that reduce the serum half lives of the corresponding Fc-hinge fragments (Medesan et al., 1997; Kim et al., 1994a). The correlation between serum half life and binding affinity for FcRn is excellent for these mutated Fc-hinge fragments (Kim et al., 1994b; Popov et al., 1996), suggesting that if the affinity of the FcRn-Fc interaction could be increased, whilst still retaining pH dependence, this would result in an Fc fragment with prolonged serum persistence. Production of such a fragment would be a significant advance in the engineering of a new generation of therapeutic IgGs with improved pharmacokinetics such as increased persistence in the circulation. But to date, no such fragments have been produced.
Immunoglobulin Fc domains are also of great interest for purposes of studying the mechanisms of antibody stabilization, catabolism and antibody interactions with further molecules of the immune system. These include, depending on the class of antibody, interactions with complement, and binding to specific receptors on other cells, including macrophages, neutrophils and mast cells. More detailed knowledge of the biology of Fe regions is important in understanding various molecular processes of the immune system, such as phagocytosis, antibody-dependent cell-mediated cytotoxicity and allergic reactions.
The production of a longer-lived Fc fragment that has increased binding to FcRn would be attractive, since such a fragment could be used to tag therapeutic reagents. Chimeric proteins produced in this manner would have the advantage of high in vivo stability which would allow fewer doses of the agent to be used in therapy and possibly even allow lower doses of the agent to be used through its increased persistence in the bloodstream. Unfortunately,methodology for generating proteins, such as antibody fragments, with increased serum persistence has not yet been developed.
SUMMARY OF THE INVENTION
The present invention seeks to overcome deficiencies in the art by providing functional proteins, antibodies or other agents that have an increased serum half-life through the interaction with Fc receptor (FcRn). These functional agents include any molecule that binds to FcRn in a pH dependent way such that binding affinity is strong at about pH 6 to about pH 6.5 relative to binding at pH 7.4. Physiologically, this allows the agent to be salvaged by FcRn at lower pH and released into the essentially neutral pH environment of the serum. The present disclosure includes protein and peptide compositions having altered serum half-lives relative to IgG, methods of making such proteins or peptides, either starting with a known sequence or by screening random sequences, and methods of screening unknown candidate agents for pH dependent FcRn binding. In addition, disclosed herein are methods of making an agent with altered serum half-life by conjugating or otherwise binding of that agent to a moiety identified as having an increased serum half-life through its interaction with FcRn. Such agents would include, but are not limited to antibodies, fragments of antibodies, hormones, receptor ligands, immunotoxins, therapeutic drugs of any kind, T-cell receptor binding antigens and any other agent that may be bound to the increased serum half life moieties of the present invention.
Also disclosed are methods of increasing the FcRn binding affinity of an FcRn binding protein or peptide so that the protein or peptide will have an increased serum half-life. These methods include identifying amino acids that directly interact with FcRn. These amino acids may be identified by their being highly conserved over a range of species, or by any other method. Other methods would include, for example, mutation or blocking of the amino acid and screening for reduced binding to FcRn, or by a study of three dimensional structure of the interaction, or by other methods known in the art. When those residues are identified that directly interact, then secondary amino acids are identified whose side chains are in the spatial vicinity of the direct interaction. In the case of antibodies, these secondary amino acids often occur in loops so that they are exposed to the solvent. In this way, mutation of these amino acids is not expected to disrupt the native protein structure. These identified secondary amino acids are then randomly mutated and the mutated proteins or peptides are then screened for increased binding affinity for FcRn at about pH 6 relative to the non-mutated protein or peptide. This method is applicable to any protein or peptide that binds FcRn in a pH dependent way and all such proteins or peptides would be encompassed by the present claimed invention. It is also understood that random mutation, in and of itself, does not constitute the invention, and that the secondary amino acids may be specifically mutated or modified or derivatized in any way known in the art and then screened for the effect on FcRn binding.
In certain broad aspects, the invention encompasses the design and production of recombinant antibody or antibody Fc-hinge domains engineered to have increased in vivo, or serum half lives. The Fc-hinge domain mutants with increased serum half lives of the present invention are generally defined as mutants in which one or more of the natural residues at the CH2-CH3 domain interface of the Fc-hinge fragment have been exchanged for alternate amino acids. Such Fc-hing
Board of Regents , The University of Texas System
Fulbright & Jaworski
Saunders David
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