Non-human primate Fc receptors and methods of use

Abstract

The invention provides isolated non-human primate Fc receptor polypeptides, the nucleic acid molecules encoding the Fc receptor polypeptides, and the processes for production of recombinant forms of the Fc receptor polypeptides, including fusions, variants, and derivatives thereof. The invention also provides methods for evaluating the safety, efficacy and biological properties of Fc region containing molecules using the non-human primate Fc receptor polypeptides.

Description

[0001] The invention generally relates to purified and isolated non-human primate Fc receptor polypeptides, the nucleic acid molecules encoding the FcR polypeptides, and the processes for production of non-human primate Fc receptor polypeptides as well as to methods for evaluating the safety, efficacy and biological properties of therapeutic agents.[0002] Fc receptors (FcRs) are membrane receptors expressed on a number of immune effector cells. Upon interaction with target immunoglobulins, FcRs mediate a number of cellular responses, including, activation of cell mediated killing, induction of mediator release from the cell, uptake and destruction of antibody coated particles, and transport of immunoglobulins. Deo et al., 1997, Immunology Today 18:127-135. Further, it has been shown that antigen-presenting cells, e.g., macrophages and dendritic cells, undergo FcR mediated internalization of antigen-antibody complexes, allowing for antigen presentation and the consequent amplificatio...

AI Technical Summary

Benefits of technology

[0049] The term "fusion protein" is a polypeptide having two portions combined where each of the portions is a polypeptide having a different property. This property may be a biological property, such as activity in vitro or in vivo. The property may also be a simple chemical or physical property, such as binding to a target molecule, catalysis of a reaction etc. The two portions may be linked directly by a single peptide bond or through a peptide linker containing one or more amino acid residues. The fused polypeptide may be used, among other things, to determine the location of the fusion protein in a cell, enhance the stability of the fusion protein, facilitate the oligomerization of the protein, or facilitate the purification of the fusion protein. Examples of such fusion proteins include proteins expressed as fusion with a portion of an immunoglobulin molecule, proteins expressed as fusion proteins with a leucine zipper moiety, Fc receptors polypeptides fused to glutathione S-transferase, and Fc receptor polypeptides fused with one or more amino acids that serve to allow detection or purification of the receptor such as Gly6-His tag.

[0070] For example, in one aspect of the invention, a method is provided for identifying agents that selectively activate ITAM motifs in target Fc receptors while failing to activate ITIM motifs in other Fc receptors. Preferably these agents are antibodies and more preferably these agents are monoclonal antibodies. These identified agents may have uses in designing therapeutic antibodies which preferentially bind to and activate only ITAM-containing Fc.gamma.R (i.e. not simultaneously engaging the inhibitory ITIM-containing receptors) which could thereby improve the cytotoxicity or phagocytosis ability of the therapeutic antibody or the ability of the therapeutic antibody to be internalized by antigen-presenting cells for increased immune system response against the target antigen.

[0071] Finally, the cynomolgus Fc.gamma.R polynucleotides and polypeptides of the invention permit a more detailed analysis of Fc.gamma.R-mediated molecular interactions. The amino acids in human IgG1 which interact with human Fc.gamma.R have been mapped (Shields, R. L., Namenuk, A. K., Hong, K., Meng, Y. G., Rae, J., Briggs, J., Xie, D., Lai, J., Stadlen, A., Li, B., Fox, J. A., and Presta, L. G. (2001) J. Biol. Chem. 276, 6591-6604). Testing the binding of these same human IgG1 variants against cynomolgus Fc.gamma.R can aid in mapping the interaction of specific amino acids in the human IgG1 with amino acids in the Fc.gamma.R.

[0093] The invention also provides cynomolgus and chimp Fc.gamma.R polypeptides, cynomolgus FcRn polypeptide, .beta.-2 microglobulin nucleic acid molecules, or fragments and variants thereof, ligated to heterologous polynucleotides to encode fusion proteins. The heterologous polynucleotides can be ligated to the 3' or 5' end of the nucleic acid molecules of the invention, for example SEQ ID NOs: 1, 3, 5, 7, 13, 22, 25 or 27, to avoid interfering with the in-frame expression of the resultant cynomolgus and chimp Fc.gamma.R, cynomolgus FcRn, and .beta.-2 microglobulin polypeptides. Alternatively, the heterologous polynucleotide can be ligated within the coding region of the nucleic acid molecule of the invention. Heterologous polynucleotides can encode a single amino acid, peptide, or polypeptides that provide for secretion, improved stability, or facilitate purification of the cynomolgus and chimp encoded polypeptides of the invention.

[0117] However, the binding patterns of human IgG subclasses to other cynomolgus FcRs, especially Fc.gamma.RI, indicate that the non-human primates can be used as effective models to evaluate the safety, efficacy and pharmokenetics of Fc region binding molecules.

[0141] A method of the invention involves evaluating the binding of a Fc region containing polypeptide or agent to cynomolgus or chimp Fc receptor polypeptide by contacting the Fc region containing molecule with a cynomolgus or chimp Fc receptor polypeptide. The cynomolgus or chimp Fc receptor polypeptide can be soluble or can be expressed as a membrane bound protein on transiently infected cells. Binding of the Fc region containing molecule to the cynomolgus or chimp Fc receptor polypeptide indicates that the Fc region containing molecule or polypeptide is suitable for in vivo evaluation in a primate. Binding to cynomolgus FcRn molecules provides an indication that Fc region containing molecule or polypeptide will have a longer half-life in vivo.

Problems solved by technology

However, there is only sparse information available regarding the interaction of human antibodies with primate Fc.gamma. receptors and the effects of this interaction on interpretation of pharmacokinetic, toxicity, and efficacy studies in primates.

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