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  Non-human primate fc receptors and methods of useUSPTO Application #: 20070106069Title: 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. (end of abstract)
Agent: Merchant & Gould PC - Minneapolis, MN, US Inventors: Leonard G. Presta, Angela K. Namenuk USPTO Applicaton #: 20070106069 - Class: 536023500 (USPTO) Related Patent Categories: Organic Compounds -- Part Of The Class 532-570 Series, Azo Compounds Containing Formaldehyde Reaction Product As The Coupling Component, Carbohydrates Or Derivatives, Nitrogen Containing, Dna Or Rna Fragments Or Modified Forms Thereof (e.g., Genes, Etc.), , Encodes An Animal Polypeptide The Patent Description & Claims data below is from USPTO Patent Application 20070106069. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [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. BACKGROUND OF THE INVENTION [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 amplification of the immune response. As such, FcRs play a central role in development of antibody specificity and effector cell function. Deo et al., 1997, Immunology Today 18:127-135. [0003] FcRs are defined by their specificity for immunoglobulin isotypes; Fc receptors for IgG antibodies are referred to as Fc.gamma.R, for IgE as Fc.epsilon.R, for IgA as Fc.alpha.R and so on. FcRn is a special class of Fc receptor found on neonatal cells and is responsible for, among other things, transporting maternal IgG from milk across the infants intestinal epithelial cells. Three subclasses of human gamma receptors have been identified: Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16). Because each human Fc.gamma.R subclass is encoded by two or three genes, and alternative RNA spicing leads to multiple transcripts, a broad diversity in Fc.gamma. isoforms exists. The three genes encoding the human Fc.gamma.RI subclass (Fc.gamma.RIA, Fc.gamma.RIB and Fc.gamma.RIC) are clustered in region 1q21.1 of the long arm of chromosome 1; the genes encoding Fc.gamma.RII isoforms (Fc.gamma.RIIA, Fc.gamma.RIIB and Fc.gamma.RIIC) and the two genes encoding Fc.gamma.RIII (Fc.gamma.RIIIA and Fc.gamma.RIIIB) are all clustered in region 1q22. FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J Lab. Clin. Med. 126:330-41 (1995). [0004] Human Fc.gamma.RI is a heteroligomeric complex composed of an .alpha.-chain and .gamma.-chain. The .alpha.-chain is a 70-72 kDa glycoprotein having 3 extracellular C-2 Ig like domains, a 21 amino acid membrane domain and a charged cytoplasmic tail of 61 amino acids. van de Winkel et al., 1993, Immunology Today 14:215-221. The .gamma.-chain is a homodimer that is involved in cell surface assembly and cell signaling into the interior of the cell. Each chain of .gamma. homodimer includes a motif involved in cellular activation designated the ITAM motif. Human Fc.gamma.RI binds monomeric IgG with high affinity (10.sup.-7-10.sup.-9M) through the action of the third extracellular C-2 domain. [0005] Fc.gamma.RII is a 40 kDa glycoprotein having two C2 set Ig-like extracellular domains, a 27-29 amino acid transmembrane domain, and a cytoplasmic domain having variable length, from 44 to 76 amino acids. There are six known isoforms of the human Fc.gamma.RII, differing for the most part in their heterogeneous cytoplasmic domains. Human Fc.gamma.RIIA includes an ITAM motif in the cytoplasmic region of the molecule, and upon crosslinking of the receptor this motif is associated with cellular activation. In contrast, human Fc.gamma.RIIB includes an inhibitory motif in its cytoplasmic region designated ITIM. When the Fc.gamma.RIIB is crosslinked, cellular activation is inhibited. In general, Fc.gamma.RII binds monomeric IgG poorly (>10.sup.7 M.sup.-1), but has high affinity for complexed IgG. [0006] Human Fc.gamma.RIII has two major isoforms, Fc.gamma.RIIIA and Fc.gamma.RIIIB, both isoforms are between 50 to 80 kDa, having two C2 Ig-like extracellular domains. The Fc.gamma.RIIIA .alpha.-chain is anchored to the membrane by a 25 amino acid transmembrane domain, while Fc.gamma.RIIIB is linked to the membrane via a glycosyl phosphatidyl-inositol (GPI) anchor. Human Fc.gamma.RIIIA is a heteroligomeric complex with the .alpha.-chain complexed with a heterodimeric .gamma.-67 (gamma-delta) chain or .gamma.-.gamma. chain. The .gamma.-chain includes a cytoplasmic tail with an ITAM motif. The .alpha.-chain is homologous to the .alpha.-chain and is also involved in cell signaling and cell surface assembly. The .gamma.-.delta. (gamma-delta) chain also includes an ITAM motif in its cytoplasmic region. In both cases, the Fc.gamma.RIII binds monomeric IgG with low affinity, and binds complexed IgG with high affinity. [0007] Human FcRn is a heterodimer composed of a .beta.-2 microglobulin chain and a .alpha. chain. The .beta.-2 microglobulin chain is approximately 15 kDa and is similar to the .beta.-2 microglobulin chain present in MHC class I heterodimers. The presence of a .beta.-2 microglobulin chain in FcRn makes it the only known Fc receptor to fall within the MHC class I family of proteins. Ghetie et al., 1997 Immunology Today 18(12):592-598. The .alpha. chain is a 37-40 kDa integral membrane glycoprotein having a single glycosylation site. Evidence suggests that FcRn is involved in transferring maternal IgG across the neonatal gut and in regulating serum IgG levels. FcRn is also found in adults on many tissues. [0008] As discussed above, human Fc.gamma.Rs, with the exception of Fc.gamma.RIIB, contain a cytoplasmic .about.26 amino acid immunoreceptor tyrosine-based activation motif (ITAM). It is believed that this motif is involved in cell signaling and effector cell function. Crosslinking of Fc.gamma.Rs may lead to the phosphorylation of tyrosine residues within the ITAM motif by src-family tyrosine kinases (PTKs), followed by association and activation of the phosphorylated ITAM motif with syk-family PTKs. Deo et al., 1997, Immunology Today 18:127-135. Once activated, a poorly understood signaling cascade is translated into biological responses. [0009] Human Fc.gamma.RIIB members contain a distinct 13 amino acid immuno-receptor tyrosine-based inhibitory motif (TIM) in their cytoplasmic domain. Human Fc.gamma.RIIB is expressed on B lymphocytes and binds to IgG complexes. However, rather than activating cells, crosslinking of the IIB receptor results in a signal inhibiting B cell activation and antibody secretion. (Camigorea et al., 1992, Cytoplasmic Domain Heterogeneity and Function of IgG Receptors in B Lymphocytes, Science 256:1808.) [0010] Because of the central role of Fc.gamma.R as a trigger molecule in numerous immune responses, it has become a target for developing potential therapeutics. For example, several ongoing clinical trials are based on activating a cancer patient's effector cells by treating the patient with tumor-specific monoclonal antibodies (Mabs). These studies have shown that the tumor-specific antibodies mediate their effects in part through Fc.gamma.R binding, and subsequent effector cell activity. Adams et al., 1984, Proc. Natl. Acad. Sci. 81:3506-3510; Takahashi et al., 1995, Gastroenterology 108:172-182; Riethmeuller et al., 1994, Lancet 343:1177-1183, Clynes, R. A., Towers, T. L., Presta, L. G., and Ravetch, J. V., 2000, Nature Med. 6:443-446. Further, a novel series of bispecific molecule antibodies (BSMs), molecules engineered to have one arm specific for a tumor cell and the other arm specific for a target Fc.gamma.R, are in clinical trials to specifically target a tumor for Fc.gamma.R mediated, effector cell destruction of the tumor cells. Valone et al., 1995, J. Clin. Oncol. 13:2281-2292; Repp et al., 1995, Hematother 4:415-421. In addition, Fc.gamma.Rs can be used as therapeutic targets in a number of infectious diseases, and for that matter, a number of autoimmune disorders. With regard to infectious diseases, BSMs are being developed to target any number of microorganisms to a patient's Fc.gamma.R expressing effector cells (Deo et al., 1997, Immunology Today 18:127-135), while soluble Fc.gamma.Rs have been used to inhibit the Arthus reaction, and Fc.gamma.R blocking agents have been used to reduce the severity of several autoimmune disorders. Ierino et al., 1993, J. Exp. Med. 178:1617-1628; Debre et al., 1993, Lancet 342:945-949. [0011] As antibodies have become increasingly used as therapeutic agents, there is a need to develop animal models for evaluating the toxicity, efficacy and pharmacokinetics of such therapeutic agents. In addition to rodent models for evaluating efficacy of antibody therapeutics, primate models have been used for evaluation of therapeutic antibody pharmacokinetics, toxicity, and efficacy (Anderson, D. R., Grillo-Lopez, A., Varns, C., Chambers, K. S., and Hanna, N. (1997) Biochem. Soc. Trans. 25, 705-708). However, there is only sparse information available regarding the interaction of human antibodies with primate Fey receptors and the effects of this interaction on interpretation of pharmacokinetic, toxicity, and efficacy studies in primates. [0012] Although many advances have been made in elucidating Fc.gamma.R activity and identifying and engineering Fc.gamma.R ligands, there still remains a need in the art to identify other Fc.gamma.Rs and to identify and engineer other Fc.gamma.R ligands, both activating and inhibiting. These new receptors and receptor ligands possess potential therapeutic value in a number of disease states, including, the destruction of tumor cells and infectious material, as well as in blocking portions of the immune response involved in several autoimmune disorders. As antibodies and other Fc.gamma.R ligands are used as therapeutic agents, there is also a need to develop models to test the efficacy, toxicity, and pharmacokinetics of these therapeutic agents, especially in vivo. SUMMARY OF INVENTION [0013] The invention is based upon, among other things, the isolation and sequencing of polynucleotides encoding Fc receptor polypeptides from non-human primates, such as cynomolgus monkeys and chimps. The cynomolgus monkey or chimp FcR polynucleotides and polypeptides of the invention are useful, inter alia, for evaluation of binding of antibodies of any subclass (especially antibodies with prospective therapeutic utility) to cynomolgus or chimpanzee FcR polypeptides prior to in vivo evaluation in a primate. [0014] The invention provides polynucleotide molecules encoding non-human primate Fc receptor polypeptides. The polynucleotides of the invention encode non-human primate Fc receptor polypeptides with an amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO. 29, SEQ ID NO. 64 or fragments thereof. Fc receptor polynucleotide molecules of the invention include those molecules having a nucleic acid sequence as shown in SEQ ID NOs: 1, 3, 5, 7, 13, 22, and 27, as well as polynucleotides having substantial nucleic acid identity with the nucleic acid sequences of SEQ ID NOs 1, 3, 5, 7, 13, 22, and 27. .beta.-2 microglobulin polynucleotide molecules of the invention also include molecules having a nucleic acid sequence as shown in SEQ ID NO: 23, as well as polynucleotides having substantial nucleic acid identity with the nucleic acid sequences of SEQ ID NO: 23. [0015] The present invention also provides non-human primate Fc.gamma. receptors and non-human primate .beta.-2 microglobulin. Fc.gamma. polypeptides of the invention include those having an amino acid sequence shown in SEQ ID NOs: 9, 11, 15, 17, 18, 20, 29, and 64 as well as polypeptides having substantial amino acid sequence identity to the amino acid sequences of SEQ ID NOs 9, 11, 15, 17, 18, 20, 29, and 64 and useful fragments thereof. .beta.-2 microglobulin polypeptides of the invention include those having an amino acid sequence shown in SEQ ID NO: 25, as well as polypeptides having substantial amino acid sequence identity to the amino acid sequence of SEQ ID NO: 25 and useful fragments thereof. [0016] In another aspect the invention provides polynucleotide molecules encoding mature non-human primate Fc receptor polypeptides. The polynucleotides of the invention encode mature non-human primate Fc receptor polypeptides with an amino acid sequence of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO. 71, SEQ ID NO. 72 or fragments thereof. Fc receptor polynucleotide molecules of the invention include those molecules having a nucleic acid sequence as shown in SEQ ID NOs: 1, 3, 5, 7, 13, 22, 23 and 27, as well as polynucleotides having substantial nucleic acid identity with the nucleic acid sequences of SEQ ID NOs 1, 3, 5, 7, 13, 22, 23, and 27. [0017] In another aspect of the invention, a method of obtaining a nucleic acid encoding a nonhuman primate Fc receptor is provided. The method comprises amplifying a nucleic acid from a nonhuman primate cell with a primer set comprising a forward and a reverse primer, wherein the primer sets are selected from the group consisting of SEQ ID NO:31 and SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46, SEQ ID NO:47 and SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50, SEQ ID NO:51 and SEQ ID NO:52, and SEQ ID NO:53 and SEQ ID NO:54; and isolating the amplified nucleic acid. The nonhuman primate cell is a preferably a cynomologus spleen cell or a chimp spleen cell. [0018] The invention includes variants, derivatives, and fusion proteins of the non-human primate Fc.gamma. receptor polypeptides and .beta.-2 microglobulin. For example, the fusion proteins of the invention include the non-human primate Fc.gamma. receptor polypeptides fused to heterologous protein or peptide that confers a desired function, i.e., purification, stability, or secretion. The fusion proteins of the invention can be produced, for example, from an expression construct containing a polynucleotide molecule encoding one of the polypeptides of the invention in frame with a polynucleotide molecule encoding the heterologous protein. [0019] The invention also provides vectors, plasmids, expression systems, host cells, and the like, containing the polynucleotides of the invention. Several recombinant methods for the production of the polypeptides of the invention include expression of the polynucleotide molecules in cell free expression systems, in cellular hosts, in tissues, and in animal models, according to known methods. [0020] The non-human primate Fc.gamma. receptors are useful in animal models for the evaluation of the therapeutic safety, efficacy and pharmacokenetics of agents, especially agents having a Fc region. A method of the invention involves contacting an agent with Fc receptor binding domain with a non-human primate Fc receptor polypeptide, preferably a mature soluble polypeptide, and determining the effect of contact on at least biological property of the Fc region containing molecule. A method of the invention involves contacting a cell expressing at least one non-human primate Fc.gamma. receptor polypeptide with an agent having a Fc region and determining whether the agent alters biological activity of the cell or is toxic to the cell. The invention also includes a method for screening variants of agents including an Fc region for the ability of such variants to bind to and activate FcRs. An example of such variants include antibodies that have amino acid substitutions at specific residues that may alter binding affinity for one or more Fc receptor classes. [0021] Another example, of screening for agents with FcR binding domains includes identifying agents that have an altered affinity for a Fc.gamma. receptor having an ITAM region compared to a Fc.gamma. receptor having an ITIM region. In addition, the invention provides reagents, compositions, and methods that are useful identifying an agent that has an altered affinity for a Fc.gamma. receptor having an ITIM region, or for a method for identifying an agent with increased binding affinity for a Fc.gamma. receptor having an ITAM region. Continue reading... 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