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

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] Fe.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.-7), 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.-.delta. (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 P-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 a 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 (ITIM) 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 Fc.gamma. 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: 1, 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 proteinor 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.

[0022] These and various other features as well as advantages which characterize the invention will be apparent from a reading of the following detailed description and a review of the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

[0023] FIG. 1A: FIG. 1A illustrates monomeric IgG subclass binding to human Fc.gamma.RI.

[0024] FIG. 1B: FIG. 1B illustrates monomeric IgG subclass binding to cynomolgus Fc.gamma.RI.

[0025] FIG. 2: FIG. 2 illustrates hexameric immune complex binding to cynomolgus Fc.gamma.RIIA.

[0026] FIG. 3A: FIG. 3A illustrates hexameric immune complex binding to human Fc.gamma.RIIB.

[0027] FIG. 3B: FIG. 3B illustrates hexameric immune complex binding to cynomolgus Fc.gamma.RIIB.

[0028] FIG. 4A: FIG. 4A illustrates hexameric immune complex binding to human Fc.gamma.RIIIA-F158.

[0029] FIG. 4B: FIG. 4B illustrates hexameric immune complex binding to human Fc.gamma.RIIIA-V158.

[0030] FIG. 4C: FIG. 4C illustrates hexameric immune complex binding to cynomolgus Fc.gamma.RIIIA.

[0031] FIG. 5: FIG. 5 illustrates hexameric immune complex binding of human IgG 1 variants to cynomolgus Fc.gamma.RIIA.

[0032] FIG. 6: FIG. 6 illustrates hexameric immune complex binding of human IgG variants to cynomolgus Fc.gamma.RIIB.

[0033] FIG. 7: FIG. 7 illustrates hexameric immune complex binding of human IgG variants to cynomolgus Fc.gamma.RIIIA.

[0034] FIG. 8: FIG. 8 illustrates concentration dependent monomeric IgG subclass binding to human FcRn.

[0035] FIG. 9: FIG. 9 illustrates concentration dependent monomeric IgG subclass binding to cynomolgus FcRn (S3).

[0036] FIG. 10: FIG. 10 illustrates concentration dependent monomeric IgG subclass binding to cynomolgus FcRn (N3).

IDENTIFICATION OF SEQUENCES AND SEQUENCE IDENTIFIERS

[0037]

1 SEQ ID ACCESSION NO. DESCRIPTION LOCATION NO. 1 Cynomolgus DNA for a Fc.gamma.RI .alpha.-chain Table 3 -- 2 Human DNA for a Fc.gamma.RI .alpha.-chain Table 3 GenBank L03418 3 Cynomolgus DNA for a Fc.gamma.RIIA Table 5 -- 4 Human DNA for a Fc.gamma.RIIA Table 5 GenBank M28697 5 Cynomolgus DNA for a Fc.gamma.RIIB Table 6 -- 6 Human DNA for a Fc.gamma.RIIB Table 6 GenBank X52473 7 Cynomolgus DNA for a Fc.gamma.RIIIA .alpha.-chain Table 7 -- 8 Human DNA for a Fc.gamma.RIIIA .alpha.-chain Table 7 GenBank X52645 9 Amino acid sequence of a cynomolgus Fc.gamma.RI .alpha.-chain Table 10 -- 10 Amino acid sequence of a human Fc.gamma.RI .alpha.-chain Table 10 GenBank P12314 11 Amino acid sequence of a cynomolgus Fc.gamma.RI/III gamma chain Table 12 -- 12 Amino acid sequence of a human Fc.gamma.RI/III gamma chain Table 12 GenBank P30273 13 DNA sequence for a cynomolgus gamma chain DNA Table 4 -- 14 DNA sequence for a human gamma chain DNA Table 4 GenBank M33195 15 Amino acid sequence of a cynomolgus Fc.gamma.RIIA Table 11 -- 16 Amino acid sequence of a human Fc.gamma.RIIA Table 11 GenBank P12318 17 Amino acid sequence of a chimp Fc.gamma.RIIA Table 11 -- 18 Amino acid sequence of a cynomolgus Fc.gamma.RIIB Table 11 -- 19 Amino acid sequence of a human Fc.gamma.RIIB Table 11 GenBank X52473 20 Amino acid sequence of a cynomolgus Fc.gamma.RIIIA .alpha.-chain Table 11 -- 21 Amino acid sequence of a human Fc.gamma.RIIIA .alpha.-chain Table 11 GenBank P08637 22 DNA sequence for a chimp Fc.gamma.RIIA Table 5 -- 23 Cynomolgus B-2 microglobulin DNA Table 8 24 Human B-2 microglobulin DNA Table 8 AB 021288 25 Amino acid sequence of cynomolgus B-2 microglobulin Table 13 -- 26 Amino acid sequence of human .beta.-2 microglobulin Table 13 P01884 27 Cynomolgus FcRn .alpha. -chain DNA Table 9 -- 28 Human FcRn .alpha. -chain DNA Table 9 U12255 29 Amino acid sequence of cynomolgus FcRn .alpha. -chain (S3) Table 14 -- 30 Amino acid sequence of human FcRn .alpha. -chain Table 14 U12255 31 Cynomolgus Fc.gamma.RI full-length forward primer Table 1 32 Cynomolgus Fc.gamma.RI full-length reverse primer Table 1 33 Cynomolgus Fc.gamma.RI-H6-GST forward primer Table 1 34 Cynomolgus Fc.gamma.RI-H6-GST reverse primer Table 1 35 Cynomolgus Fc.gamma.RIIB full-length forward primer Table 1 36 Cynomolgus Fc.gamma.RIIB full-length reverse primer Table 1 37 Cynomolgus Fc.gamma.RIIB-H6-GST forward primer Table 1 38 Cynomolgus Fc.gamma.RIIB-H6-GST reverse primer Table 1 39 Cynomolgus Fc.gamma.RIIIA full-length forward primer Table 1 40 Cynomolgus Fc.gamma.RIIIA full-length reverse primer Table 1 41 Cynomolgus Fc.gamma.RIIIA-H6-GST forward primer Table 1 42 Cynomolgus Fc.gamma.RIIIA-H6-GST reverse primer Table 1 43 Cynomolgus Fc gamma chain forward primer Table 1 44 Cynomolgus Fc gamma chain reverse primer Table 1 45 Cynomolgus .beta.-2 Microglobulin forward primer Table 1 46 Cynomolgus .beta.-2 Microglobulin reverse primer Table 1 47 Cynomolgus Fc.gamma.RIIA full-length forward primer Table 1 48 Cynomolgus Fc.gamma.RIIA full-length reverse primer Table 1 49 Cynomolgus Fc.gamma.RIIA-H6-GST forward primer Table 1 50 Cynomolgus Fc.gamma.RIIA-H6-GST reverse primer Table 1 51 Cynomolgus FcRn full-length forward primer Table 1 52 Cynomolgus FcRn full-length reverse primer Table 1 53 Cynomolgus FcRn-H6 forward primer Table 1 54 Cynomolgus FcRn-H6 reverse primer Table 1 55 PCR primer 0F1 Table 2 56 PCR primer 0R1 Table 2 57 PCR primer 0F2 Table 2 58 PCR primer 0F3 Table 2 59 PCR primer 0R2 Table 2 60 PCR primer 0F4 Table 2 61 PCR primer 0R3 Table 2 62 PCR primer 0F5 Table 2 63 PCR primer 0R4 Table 2 64 Amino acid sequence of cynomologus FcRn .alpha.-chain (N3) Table 14 65 Amino acid sequence of a mature cynomolgus Fc.gamma.RI .alpha.-chain Table 10 66 Amino acid sequence of a mature cynomolgus Fc.gamma.RIIA Table 11 Table 21 67 Amino acid sequence of a mature chimp Fc.gamma.RIIA Table 11 68 Amino acid sequence of a mature cynomolgus Fc.gamma.RIIB Table 11 Table 22 69 Amino acid sequence of a mature cynomolgus Fc.gamma.RIIIA .alpha.-chain Table 11 Table 23 70 Amino acid sequence of a mature cynomolgus .beta.-2 microglobulin Table 13 71 Amino acid sequence of a mature cynomolgus Fc.gamma.Rn .alpha.-chain (S3) Table 14 72 Amino acid sequence of a mature cynomolgus FcRn .alpha.-chain (N3) Table 14

DETAILED DESCRIPTION OF THE INVENTION

[0038] The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0039] Throughout the present specification and claims, the numbering of the residues in an IgG heavy chain is that of the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), expressly incorporated herein by reference. The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0040] The term "amino acids" refers to any of the twenty naturally occurring amino acids as well as any modified amino acid sequences. Modifications may include natural processes such as posttranslational processing, or may include chemical modifications which are known in the art. Modifications include but are not limited to: phosphorylation, ubiquitination, acetylation, amidation, glycosylation, covalent attachment of flavin, ADP-ribosylation, cross linking, iodination, methylation, and alike.

[0041] The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, fully synthetic antibodies, and antibody fragments so long as they exhibit the desired biological activity.

[0042] The term "antisense" refers to polynucleotide sequences that are complementary to a target "sense" polynucleotide sequence.

[0043] The term "complementary" or "complementarity" refers to the ability of a polynucleotide in a polynucleotide molecule to form a base pair with another polynucleotide in a second polynucleotide molecule. For example, the sequence A-G-T is complementary to the sequence T-C-A. Complementarity may be partial, in which only some of the polynucleotides match according to base pairing, or complete, where all the polynucleotides match according to base pairing.

[0044] The term "expression" refers to transcription and translation occurring within a host cell. The level of expression of a DNA molecule in a host cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of DNA molecule encoded protein produced by the host cell (Sambrook et al., 1989, Molecular cloning: A Laboratory Manual, 18.1-18.88).

[0045] The term "Fc region" is used to define a C-terminal region of an immunoglobulin heavy chain. Although the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region stretches from amino acid residue at position Cys226 to the carboxyl-terminus. The term "Fc region-containing molecule" refers to an molecule, such as an antibody or immunoadhesin, which comprises an Fc region. The Fc region of an IgG comprises two constant domains, CH2 and CH3. The "CH2" domain of a human IgG Fc region (also referred to as "C.gamma.2" domain) usually extends from amino acid 231 to amino acid 340. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. Burton, Molec. Immunol.22:161-206 (1985).

[0046] The term "Fc receptor" refers to a receptor that binds to the Fc region of an antibody or Fc region containing molecule. The preferred Fc receptor is a receptor which binds an IgG antibody (Fc.gamma.R) and includes receptors of the Fc.gamma.RI, Fc.gamma.RII, Fc.gamma.RIII, and FcRn subclasses, including allelic variants and alternatively spliced forms of these receptors. The term "FcR polypeptide" is used to describe a polypeptide that forms a receptor that binds to the Fc region of an antibody or Fc region containing molecule. The term "Fc receptor polypeptide" also includes both the mature polypeptide and the polypeptide with the signal sequence. The term "Fc.gamma.R polypeptide" is used to describe a polypeptide that forms a receptor that binds to the Fc region of an IgG antibody or IgG Fe region containing molecule. For example, Fc.gamma.RI and Fc.gamma.RIII receptors each include a Fc receptor polypeptide .alpha.-chain and a Fc receptor polypeptide homo or hetereodimer of a .gamma.-chain. FcRn receptors include an Fc receptor polypeptide alpha chain and a .beta.-2 microglobulin. Typically, the .alpha.-chains have the extracellular regions that bind to the Fc-region containing agent. 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). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein.

[0047] The term "fragment" is used to describe a portion of an Fc receptor polypeptide or a nucleic acid encoding a portion of an Fc receptor polypeptide. The fragment is preferably capable of binding to a Fc region containing molecule. The structure of human Fc.gamma. .alpha.-chain of Fc.gamma.RI/III and Fc.gamma.RIIA or B has been characterized and includes a signal sequence, 2 or 3 extracellular C-2 Ig like domains; a transmembrane domain; and an intracellular cytoplasmic tail. Fragments of an Fc receptor .alpha.-chain or Fc.gamma.RIIA or B include, but are not limited to, soluble Fc receptor polypeptides with one or more of the extracellular C-2 Ig like domains, the transmembrane domain, or intracellular domain of the Fc receptor polypeptides.

[0048] The term "binding domain" refers to the region of a polypeptide that binds to another molecule. In the case of an Fc receptor polypeptide or FcR, the binding domain can comprise a portion of a polypeptide chain thereof (e.g. the .alpha.-chain thereof) which is responsible for binding an Fc region of an immunoglobulin or other Fc region containing molecule. One useful binding domain is the extracellular domain of an Fc receptor .alpha.-chain polypeptide.

[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.

[0050] The term "homology" refers to a degree of complementarity or sequence identity between polynucleotides.

[0051] The term "host cell" or "host cells" refers to cells established in ex vivo culture. It is a characteristic of host cells discussed in the present disclosure that they be capable of expressing Fc receptors. Examples of suitable host cells useful for aspects of the present invention include, but are not limited to, insect and mammalian cells. Specific examples of such cells include SF9 insect cells (Summers and Smith, 1987, Texas Agriculture Experiment Station Bulletin, 1555), human embryonic kidney cells (293 cells), Chinese hamster ovary (CHO) cells (Puck et al., 1958, Proc. Natl. Acad. Sci. USA 60, 1275-1281), human cervical carcinoma cells (HELA) (ATCC CCL 2), human liver cells (Hep G2) (ATCC HB8065), human breast cancer cells (MCF-7) (ATCC HTB22), and human colon carcinoma cells (DLD-1) (ATCC CCL 221), Daudi cells (ATCC CRL-213), and the like.

[0052] The term "hybridization" refers to the pairing of complementary polynucleotides during an annealing period. The strength of hybridization between two polynucleotide molecules is impacted by the homology between the two molecules, stringency of the conditions involved, the melting temperature of the formed hybrid and the G:C ratio within the polynucleotides.

[0053] As used herein, the term "immunoadhesin" designates antibody-like molecules which combine the "binding domain" of a heterologous "adhesin" protein (e.g. a receptor, ligand or enzyme) with one or more immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of the adhesin amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site (antigen combining site) of an antibody (i.e. is "heterologous") and an immunoglobulin constant domain sequence. The immunoglobulin constant domain sequence is preferably the Fc portion of an immunoglobulin.

[0054] "Immune complex" refers to the relatively stable structure which forms when at least one target molecule and at least one Fc region-containing polypeptide bind to one another forming a larger molecular weight complex. Examples of immune complexes are antigen-antibody aggregates and target molecule-immunoadhesin aggregates. Immune complex can be administered to a mammal, e.g. to evaluate clearance of the immune complex in the mammal or can be used to evaluate the binding properties of FcR or Fc receptor polypeptides.

[0055] The term "isolated" refers to a polynucleotide or polypeptide that has been separated or recovered from at least one contaminant of its natural environment. Contaminants of one natural environment are materials, which would interfere with using the polynucleotide or polypeptide therapeutically or in assays. Ordinarily, isolated polypeptides or polynucleotides are prepared by at least one purification step.

[0056] A "native sequence" polypeptide refers to a polypeptide having the same amino acid sequence as the corresponding polypeptide derived from nature. The term specifically encompasses naturally occurring truncated or secreted forms of the polypeptide, naturally occurring variant forms (e.g. alternatively spliced forms) and naturally occurring allelic variants. A "mature polypeptide" refers to a polypeptide that does not contain a signal peptide.

[0057] The term "nucleic acid sequence" refers to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along a polypeptide chain. The deoxyribonucleotide sequence thus codes for the amino acid sequence.

[0058] The term "polynucleotide" refers to a linear sequence of nucleotides. The nucleotides are either a linear sequence of polyribonucleotides or polydeoxyribonucleotides, or a mixture of both. Examples of polynucleotides in the context of the present invention include--single and double stranded DNA, single and double stranded RNA, and hybrid molecules that have both mixtures of single and double stranded DNA and RNA. Further, the polynucleotides of the present invention may have one or more modified nucleotides.

[0059] The terms, "protein," "peptide," and "polypeptide" are used interchangeably to denote an amino acid polymer or a set of two or more interacting or bound amino acid polymers.

[0060] The term "purify," or "purified" refers to a target protein that is free from at least 5-10% of the contaminating proteins. Purification of a protein from contaminating proteins can be accomplished through any number of well known techniques, including, ammonium sulfate or ethanol precipitation, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Various protein purification techniques are illustrated in Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly updates).

[0061] The term "Percent (%) nucleic acid or amino acid sequence identity" describes the percentage of nucleic acid sequence or amino acid residues that are identical with amino acids in a reference polypeptide, after aligning the sequence and introducing gaps, if necessary to achieve the maximum sequence identity, and not considering any conservative substitutions as part of the sequence identity. For purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

[0062] where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Preferably, % sequence identity can be determined by aligning the sequences manually and again multiplying 100 times the fraction X/Y, where X is the number of amino acids scored as identical matches by manual comparison and Y is the total number of amino acids in B. Further, the above described methods can also be used for purposes of determining % nucleic acid sequence identity. Alternatively, computer programs commonly employed for these purposes, such as the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), that uses the algorithm of Smith and Waterman, 1981, Adv. Appl. Math, 2: 482-489 can be used.

[0063] Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained by manual alignment. However, the ALIGN-2 sequence comparison computer program can be used as described in WO 00/15796.

[0064] The term "stringency" refers to the conditions (temperature, ionic strength, solvents, etc) under which hybridization between polynucleotides occurs. A hybridization reaction conducted under high stringency conditions is one that will only occur between polynucleotide molecules that have a high degree of complementary base pairing (about 85% to 100% of sequence identity). Conditions for high stringency hybridization, for example, may include an overnight incubation at about 42.degree. C. for about 2.5 hours in 6.times. SSC/0.1% SDS, followed by washing of the filters in 1.0.times. SSC at 65.degree. C., 0.1% SDS. A hybridization reaction conducted under moderate stringency conditions is one that will occur between polynucleotide molecules that have an intermediate degree of complementary base pairing (about 50% to 84% identity).

[0065] As used herein the term "variant" means a polynucleotide or polypeptide with a sequence that differs from a native polynucleotide or polypeptide. Variants can include changes that result in amino acid substitutions, additions, and deletions in the resulting variant polypeptide when compared to a full length native sequence or a mature polypeptide sequence.

[0066] The term "vector," "extra-chromosomal vector" or "expression vector" refers to a first piece of DNA, usually double-stranded, which may have inserted into it a second piece of DNA, for example a piece of heterologous DNA like the cDNA of cynomolgus Fc.gamma.RI. Heterologous DNA is DNA that may or may not be naturally found in the host cell and includes additional copies of nucleic acid sequences naturally present in the host genome. The vector transports the heterologous DNA into a suitable host cell. Once in the host cell the vector may be capable of integrating into the host cell chromosomes. The vector may also contain the necessary elements to select cells containing the integrated DNA as well as elements to promote transcription of mRNA from the transfected DNA. Examples of vectors within the scope of the present invention include, but are not limited to, plasmids, bacteriophages, cosmids, retroviruses, and artificial chromosomes.

Modes of Carrying Out the Invention

[0067] The invention is based upon, among other things, the isolation and sequencing of nucleic acids encoding Fc receptor polypeptides from non-human primates, such as cynomolgus monkeys and chimps. In particular, the invention provides isolated polynucleotides encoding FcR polypeptides with an amino acid sequence of SEQ ID NO: 9, 11, 15, 17, 18, 20, 29, 64 or fragments thereof. The invention also provides isolated polynucleotides encoding mature FcR polypeptides with an amino acid sequence of SEQ ID NO: 65, 66, 67, 68, 69, 71 or 72, or fragments thereof. The invention also provides an isolated polynucleotide encoding .beta.-2 microglobulin having an amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 70.

[0068] The cynomolgus monkey or chimp Fc receptor polynucleotides and polypeptides of the invention are useful 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. Evaluation could include testing binding to primate FcRs or Fc receptor polypeptides in an ELISA-format assay or to transiently- or stably-transfected human or primate cells (e.g. CHO, COS). Evaluation of the ability of a human antibody to bind to cynomolgus or other primate FcRs or Fc receptor polypeptides (either in an ELISA- or transfected cell format) could be used as a preliminary test prior to evaluation of pharmacokinetics/pharmacodynamics in vivo. Binding of antibodies or antibody variants to cynomolgus FcRn or FcRn polypeptides would be useful to identify antibodies or antibody variants that could have a longer half life in vivo. Binding of antibodies to FcRn correlates with a longer half life in vivo.

[0069] The primate FcRs or Fc receptor polypeptides could also be used to screen for variants (e.g. protein-sequence or carbohydrate) of primate or human IgG which exhibit either improved or reduced binding to these receptors or receptor polypeptides; such variants could then be evaluated in vivo in a primate model for altered efficacy of the antibody, e.g. augmentation or abrogation of IgG effector functions. In addition, soluble cynomolgus or chimpanzee Fc receptor polypeptides could be evaluated as therapeutics in primate models.

[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.

[0072] Within the application, unless otherwise stated, the techniques utilized may be found in any of several well-known references, such as: Molecular Cloning: A Laboratory Manual (Sambrook et al. (1989) Molecular cloning: A Laboratory Manual), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991 Academic Press, San Diego, Calif.), "Guide to Protein Purification" in Methods in Enzymology (M. P. Deutshcer, 3d., (1990) Academic Press, Inc.), PCR Protocols: A Guide to Methods and Applications (Innis et al. (1990) Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual of Basic Technique, 2.sup.nd ed. (R. I. Freshney (1987) Liss, Inc., New York, N.Y.), and Gene Transfer and Expression Protocols, pp 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.).

Polynucleotide Sequences

[0073] One aspect of the invention provides isolated nucleic acid molecules encoding Fc receptor polypeptides from cynomolgus monkeys and chimps. Due to the degeneracy of the genetic code, two DNA sequences may differ and yet encode identical amino acid sequences. The present invention thus provides isolated nucleic acid molecules comprising a polynucleotide sequence encoding cynomolgus FcR polypeptides, wherein the polynucleotide sequences encode a polypeptide with an amino acid sequence of SEQ ID NO: 9, or SEQ ID NO: 11, or SEQ ID NO: 15, or SEQ ID NO: 18, or SEQ ID NO: 20, or SEQ ID NO: 29, or SEQ ID NO: 64, or fragments thereof. The present invention also provides isolated nucleic acid molecules comprising a polynucleotide sequence encoding a chimp Fc.gamma.R polypeptide of the invention, wherein the polynucleotide sequence encodes a polypeptide with an amino acid sequence of SEQ ID NO: 17 or fragments thereof. The invention also provides for isolated nucleic acid molecules comprising a polynucleotide sequence encoding cynomolgus .beta.-2 microglobulin with an amino acid sequence of SEQ ID NO: 25.

[0074] The present invention also provides isolated nucleic acid molecules comprising a polynucleotide sequence encoding mature nonprimate FcR polypeptides, wherein the polynucleotide sequences encode a polypeptide with an amino acid sequence of SEQ ID NO: 65, 66, 68, 67, 69, 70, 71, or 72.

[0075] The nucleotide sequences shown in the tables, in most instances, begin at the coding sequence for the signal sequence of the Fc receptor polypeptide.

[0076] Nucleotide sequences of the non-human primate receptors have been aligned with human sequences for FcR polypeptides or .beta.-2 microglobulin to determine % sequence identity. Nucleotide sequences of primate and human proteins are aligned manually and differences in nucleotide or protein sequence noted. Percent identity is calculated as number of identical residues/number of total residues. When the sequences differ in the total number of residues, two values for percent identity are provided, using the two different numbers for total residues. Some nucleic acid sequences for human FcR are known to those of skill in the art and are identified by GenBank accession numbers.

[0077] In one embodiment, the invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a cynomolgus Fc.gamma.RI .alpha.-chain. One example of a cynomolgus Fc.gamma.RI .alpha.-chain has an amino acid sequence including the signal sequence as shown in Table 10 (SEQ. ID. NO: 9). The mature cynomolgus Fc.gamma.RI .alpha.-chain has an amino acid sequence shown in Table 10 (SEQ ID NO: 65). An example of an isolated nucleic acid encoding a cynomolgus Fc.gamma.RI .alpha.-chain is shown in Table 3 (SEQ ID NO: 1). A nucleic acid sequence encoding a cynomolgus Fc.gamma.RI .alpha.-chain has about 91% or 96% sequence identity when aligned with a human nucleic acid sequence (SEQ ID NO: 2) encoding a Fc.gamma.RI .alpha.-chain as shown in Table 3 (GenBank Accession No. L03418).

[0078] In another embodiment, the invention provides an isolated nucleic acid comprising a polynucleotide sequence encoding a cynomolgus gamma chain of Fc.gamma.RI/III. An example of such a nucleic acid sequence is shown in Table 4 (SEQ ID NO: 13). An example of a cynomolgus gamma chain polypeptide is shown in Table 12 (SEQ ID NO: 11). A nucleic acid encoding a cynomolgus gamma chain has about 99% sequence identity when aligned with a human nucleic acid sequence (SEQ ID NO: 14) encoding a FcR gamma chain as shown in Table 4 (GenBank Accession No. M33195).

[0079] In another embodiment, the invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a cynomolgus Fc.gamma.RIIA. One example of cynomolgus Fc.gamma.RIIA has an amino acid sequence including the signal sequence as shown in Table 11 (SEQ. ID. NO: 15). The mature cynomolgus Fc.gamma.RIIA has an amino acid sequence as shown in Table 21 (SEQ ID NO: 66). An example of an isolated nucleic acid encoding a cynomolgus Fc.gamma.RIIA is shown in Table 5 (SEQ ID NO: 3). A nucleic acid sequence encoding a cynomolgus Fc.gamma.RIIA .alpha.-chain has about 94% sequence identity when aligned with a human nucleic acid sequence (SEQ ID NO: 4) encoding a Fc.gamma.RIIA as shown in Table 5 (Genbank Accession No. M28697).

[0080] The invention also provides for isolated nucleic acids comprising a polynucleotide encoding Fc.gamma.R from chimps such as an isolated nucleic acid comprising a polynucleotide encoding a Fc.gamma.RIIA receptor. One example of a chimp Fc.gamma.RIIA has an amino acid sequence including the signal sequence as shown in Table 11 (SEQ. ID. NO: 17). The mature chimp Fc.gamma.RIIA has an amino acid sequence as shown in Table 11 (SEQ ID NO: 67). An example of an isolated nucleic acid encoding a chimp Fc.gamma.RIIA is shown in Table 5 (SEQ ID NO: 22). A nucleic acid sequence having a sequence of SEQ ID NO: 22 has about 99% sequence identity when aligned with a human nucleic acid sequence (SEQ ID NO: 4) encoding a Fc.gamma.RIIA as shown in Table 5 (GenBank Accession No. M28697).

[0081] In another embodiment, the invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a cynomolgus Fc.gamma.RIIB. One example of a cynomolgus Fc.gamma.RIIB has an amino acid sequence as shown in Table 11 (SEQ. ID. NO: 18). The mature cynomolgus Fc.gamma.RIIB has an amino acid sequence as shown in Table 22 (SEQ ID NO: 68). An example of an isolated nucleic acid encoding a cynomolgus Fc.gamma.RIIB is shown in Table 6 (SEQ ID NO: 5). A nucleic acid sequence encoding a cynomolgus Fc.gamma.RIIB has about 94% sequence identity when aligned with a human nucleic acid sequence (SEQ ID NO: 6) encoding a Fc.gamma.RIIB as shown in Table 6 (GenBank Accession No.X52473).

[0082] In another embodiment, the invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a cynomolgus Fc.gamma.RIIIA .alpha.-chain. One example of a cynomolgus Fc.gamma.RIIIA has an amino acid sequence as shown in Table 11 (SEQ. ID. NO: 20). The mature cynomolgus Fc.gamma.RIIIA has an amino acid sequence as shown in Table 23 (SEQ ID NO: 69). An example of an isolated nucleic acid encoding a cynomolgus Fc.gamma.RIIIA .alpha.-chain is shown in Table 7 (SEQ ID NO: 7). A nucleic acid sequence cynomolgus Fc.gamma.RIIIA .alpha.-chain has about 96% sequence identity when aligned with a human nucleic acid sequence (SEQ ID NO: 8) encoding a Fc.gamma.RIIIA .alpha.-chain as shown in Table 7 (GenBank Accession No.X52645).

[0083] The invention also provides isolated nucleic acid molecules having a polynucleotide sequence encoding a cynomolgus Fc receptor (FcRn) .alpha.-chain. One example of a cynomolgus Fc receptor .alpha.-chain (S3) has an amino acid sequence of SEQ ID NO. 29 as shown in Table 14. An allele has been identified encoding a polypeptide with an amino acid sequence which differs from that of SEQ ID NO: 29 by a substitution of an asparagine for a serine at the third residue in the mature polypeptide. This polypeptide sequence has been designated SEQ ID NO: 64. The mature polypeptides of FcRn .alpha.-chain (S3) and FcRn .alpha.-chain (N3) have the amino acid sequences of SEQ ID NO: 71 and 72, respectively. An example of an isolated nucleic acid encoding a cynomolgus FcRn .alpha.-chain is SEQ ID NO: 27 shown in Table 9. A nucleic acid encoding a cynomolgus FcRn has about 97% sequence identity when aligned with a human sequence (SEQ ID NO: 28) encoding a human FcRn .alpha.-chain as shown in Table 9 (GenBank Accession No. U12255).

[0084] In another embodiment, the invention provides isolated nucleic acid molecules comprising a polynucleotide sequence encoding cynomolgus .beta.-2 microglobulin. One example of a cynomolgus .beta.-2 microglobulin has an amino acid sequence as shown in Table 13 (SEQ ID NO: 25). The mature P-2 microglobulin has a sequence as shown in Table 13 (SEQ ID NO: 70). An example of an isolated nucleic acid encoding a cynomolgus .beta.-2 microglobulin is shown in Table 8 (SEQ ID NO: 23). A nucleic acid cynomolgus .beta.-2 microglobulin has about 95% sequence identity when aligned with a human sequence (SEQ ID NO: 24) encoding .beta.-2 microglobulin as shown in Table 8 (GenBank Accession No. AB021288).

[0085] The non-human primate nucleic acids of the invention include cDNA, chemically synthesized DNA, DNA isolated by PCR, and combinations thereof. RNA transcribed from cynomolgus or chimp cDNA is also encompassed by the invention. The cynomolgus DNA can be obtained using standard methods from tissues such as the spleen or liver and as described in the Examples below. The chimp Fc.gamma.R DNA can be obtained using standard methods from tissues such as spleen or liver and as described in the Examples below.

[0086] 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. Some of the primer sets provide for amplification of an extracellular fragment of the Fc receptor polypeptides fused to GlyHis-GST.

[0087] Fragments of the cynomolgus and chimp Fc.gamma.R-encoding nucleic acid molecules described herein, as well as polynucleotides capable of hybridizing to such nucleic acid molecules, may be used in a number of ways including as a probe or as primers in a polymerase chain reaction (PCR). Such probes may be used, e.g., to detect the presence of Fc.gamma.R polynucleotides in in vitro assays, as well as in Southern and Northern blots. Cell types expressing the Fc.gamma.R may also be identified by the use of such probes. Such procedures are well known, and the skilled artisan will be able to choose a probe of a length suitable to the particular application. For PCR, 5' and 3' primers corresponding to the termini of the nucleic acid molecules are employed to isolate and amplify that sequence using conventional techniques. Fragments useful as probes are typically oligonucleotides about 18 to 20 nucleotides, including up to the full length of the polynucleotides encoding the Fc.gamma.R. Fragments useful as PCR primers typically are oligonucleotides of 20 to 50 nucleotides.

[0088] Other useful fragments of the different cynomolgus Fc.gamma.R polynucleotides are antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence capable of binding to a target Fc.gamma.R mRNA (using a sense strand), or DNA (using an antisense strand) sequence.

[0089] Other useful fragments include polynucleotides that encode domains of a FC.gamma. receptor polypeptide. The fragments are preferably capable of binding to a Fc region containing molecule. One embodiment of a polynucleotide fragment is a fragment that encodes extracellular domains of a Fc.gamma. receptor polypeptide in which the transmembrane and cytoplasmic domains have been deleted. Other domains of Fc.gamma. receptors are identified in, for example, Table 10 and Table 11. Nucleic acid fragments encoding one or more polypeptide domains are included within the scope of the invention.

[0090] The invention also provides variant cynomolgus and chimp Fc.gamma.R nucleic acid molecules as well as variant cynomolgus .beta.-2 microglobulin nucleic acid molecules. Variant polynucleotides can include changes to the nucleic acid sequence that result in amino acid substitutions, additions, and deletions in the resultant variant polypeptide when compared to a native polypeptide, for instance SEQ ID NOs: 9, 11, 15, 17, 18, 20, 25, 29, or 64. The changes to the variant nucleic acid sequences can include changes to the nucleic acid sequence that result in replacement of an amino acid by a residue having similar physiochemical properties, such as substituting one aliphatic residue (Ile, Val, Leu, or Ala) for another, or substitutions between basic residues Lys and Arg, acidic residues Glu and Asp, amide residues Gln and Asn, hydroxyl residues Ser and Tyr, or aromatic residues Phe and Tyr. Variant polynucleotide sequences of the present invention are preferably at least about 95% identical, more preferably at least about 96% identical, more preferably at least about 97% or 98% identical, and most preferably at least about 99% identical, to a nucleic acid sequence encoding the full length native sequence, a polypeptide lacking a signal sequence, an extracellular domain of the polypeptide, or a nucleic acid encoding a fragment of the Fc.gamma. receptor polypeptide or .beta.-2 microglobulin of sequences of SEQ ID NOs: 1, 3, 5, 7, 23 or 27.

[0091] The percentage of sequence identity between the sequences and a variant sequence as discussed above may also be determined, for example, by comparing the variant sequence with a reference sequence using any of the computer programs commonly employed for this purpose, such as ALIGN 2 or by using manual alignment. Percent identity is calculated as [number of identical residues]/[number of total residues]. When the sequences differed in the total number of residues, two values for percent identity are provided, using the two different numbers for total residues.

[0092] Alterations of the cynomolgus monkey and chimp Fc.gamma.R polypeptides, and cynomolgus monkey .beta.-2 microglobulin, nucleic acid and amino acid sequences may be accomplished by any of a number of known techniques. For example, mutations may be introduced at particular locations by procedures well known to the skilled artisan, such as oligonucleotide-directed mutagenesis, which is described by Walder et al., 1986, Gene, 42:133; Bauer et al., 1985, Gene 37:73; Craik, 1985, BioTechniques, 12-19; Smith et al., 1981, Genetic Engineering: Principles and Methods, Plenum Press; and U.S. Pat. No. 4,518,584 and U.S. Pat. No. 4,737,462.

[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.

[0094] A preferred embodiment is a nucleic acid sequence encoding an extracellular domain of the .alpha.-chain of Fc.gamma.RI, Fc.gamma.R or FcRn fused to Gly(His).sub.6-gst tag or Fc.gamma.RIIA or IIB fused to Gly(His).sub.6-gst tag obtained as described in Example 1. The Gly(His).sub.6-gst tag provides for ease of purification of polypeptides encoded by the nucleic acid.

[0095] The cynomolgus and chimp Fc.gamma.R polypeptide and .beta.-2 microglobulin nucleic acid molecules of the invention can be cloned into prokaryotic or eukaryotic host cells to express the resultant polypeptides of the invention. Any recombinant DNA or RNA method can be use to create the host cell that expresses the target polypeptides of the invention, including, but not limited to, transfection, transformation or transduction. Methods and vectors for genetically engineering host cells with the polynucleotides of the present invention, including fragments and variants thereof, are well known in the art, and can be found in Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1988, and updates). Vectors and host cells for use with the present invention are described in the Examples provided herein.

[0096] The invention also provides isolated nucleic acids comprising a polynucleotide encoding the mature Fc receptor polypeptide. The isolated nucleic acids can further comprise a nucleic acid sequence encoding a heterologous signal sequence. A heterologous signal sequence is one obtained from a polynucleotide encoding a polypeptide different than the native sequence non-human primate Fc receptor polypeptides of the invention. Heterologous signal sequences include signal sequences from human Fc receptor polypeptides as well as from polypeptides like tissue plasminogen activator.

Polypeptide Sequences

[0097] Another aspect of the invention is directed to FcR polypeptides from non-human primates such as cynomolgus monkeys and chimps. The Fc.gamma.R polypeptides include Fc.gamma.RI .alpha.-chain, Fc.gamma.RIIA, Fc.gamma.RIIB, Fc.gamma.RIIIA .alpha.-chain, FcRn .alpha.-chain, FcR.gamma.I/III .gamma.-chain, and .beta.-2 microglobulin. The polypeptides bind IgG antibody or other molecules having a Fc region. Some of the receptors are low affinity receptors which preferably bind to IgG antibody complexes. FcR polypeptides also mediate effector cell functions such as antibody dependent cellular cytotoxicity, induction of mediator release from the cell, uptake and destruction of antibody coated particles, and transport of immunoglobulins.

[0098] Amino acid sequences of the Fc.gamma.R polypeptides derived from cynomolgus monkeys and chimps are aligned with the amino acid sequences encoding human Fc.gamma.R polypeptides to determine the % of sequence identity with the human sequences. Amino acid sequences of primate and human proteins are aligned manually and differences in nucleotide or protein sequence noted. Percent identity is calculated as number of identical residues/number of total residues. When the sequences differ in the total number of residues, two values for percent identity are provided, using the two different numbers for total residues. Some amino acid sequences encoding human Fc.gamma.R polypeptides are known to those skill in the art and are identified by GenBank Accession numbers.

[0099] The polypeptide sequences shown in the tables are numbered starting from the signal sequence or from the first amino acid of the mature protein. When the amino acid residues of the polypeptide are numbered starting from the signal sequence the numbers are identified by the number of the residue and a line. When the amino acid residues of the polypeptide are also numbered from the first amino acid of the mature human protein, the amino acid is designated by the number and A symbol. In Table 11, the first N terminal residue of the cynomologus sequences is designated with an asterisk, but the numbering is still that corresponding to the mature human protein. The numbering of the amino acid residues of the FcR polypeptides is sequential.

[0100] The non-human primate receptors were also analyzed to compare the binding of the non-human primate Fc receptor polypeptides to various subclasses of human IgG and IgG variants to human Fc receptors. The binding to the subclasses also included binding to IgG4b. IgG4b is a form of IgG4, but has a change in the hinge region at amino acid residue 228 from serine to a proline. This change results in a molecule that is more stable than the native IgG4 due to increase formation of interchain disulfide bonds as described in Angal, S., King, D. J., Bodmer, M. W., Turner, A., Lawson, D. G., Robert, G., Pedley B. and Adair, J. R (1993) A single amino acid substitution abolishes heterogeneity of chimeric--mouse/human (IgG4) antibody. Molec. Immunology 30:105-108.

[0101] One embodiment of the invention is a cynomolgus Fc.gamma.RI polypeptide. A cynomolgus Fc.gamma.RI binds to IgG and other molecules having an Fc region, preferably human monomeric IgG. One example of an .alpha.-chain of a cynomolgus Fc.gamma.RI is a polypeptide having a sequence of SEQ ID NO: 9. Based on the alignment with the human sequence, the mature cynomolgus Fc.gamma.RI has a sequence of SEQ ID NO: 65. An extracellular fragment obtained as described in example 1 has an amino acid sequence of .DELTA.1 to .DELTA.269 as shown in table 10.

[0102] An alignment of the amino acid sequence .alpha.-chain of the Fc.gamma.RI from human and cynomolgus monkeys is also shown in Table 10. The amino acid numbers shown below the amino acids with the symbol .DELTA. are numbered from the start of the mature polypeptide not including the signal sequence. The numbers above the amino acid residues represent the numbering of the residues starting at the signal sequence. Each of the domains of the Fc.gamma.RI .alpha.-chain are shown including signal sequence, extracellular domain 1, extracellular domain 2, extracellular domain 3, and the transmembrane and intracellular sequence. The alignment of a human sequence of SEQ ID NO: 10 (GenBank Accession No. P12314) with a cynomolgus Fc.gamma.RI .alpha.-chain sequence starting from the signal sequence shows about a 90% or 94% sequence identity with the human sequence depending on whether the 3' extension present on the human sequence was used in the calculation.

[0103] This alignment of the cynomolgus sequence with the human sequence shows that the cynomolgus Fc.gamma.RI .alpha.-chain has the same number of amino acids in the signal sequence, the three extracellular domains, and transmembrane domain as found in the human Fc.gamma.RI sequence (Table 10). In contrast, the cynomolgus Fc.gamma.RI .alpha.-chain intracellular domain is shorter than that of the human Fc.gamma.RI .alpha.-chain by seventeen amino acids (Table 10). A cynomolgus Fc.gamma.RI .alpha.-chain binds to human monomeric subclasses as follows: IgG3.gtoreq.IgG1>IgG4b>>>IgG2, which is similar to that of the human Fc.gamma.RI.

[0104] Fc receptors of the I and IIIA subclass are complex molecules including an .alpha.-chain complexed to either a homo or hetero dimer of a .gamma.-chain. The invention also includes a cynomolgus FcR gamma chain. One example of a gamma chain polypeptide has an amino acid sequence of SEQ ID NO: 11 as shown in Table 12. When the cynomolgus gamma chain amino acid sequence is aligned with a human sequence for the gamma chain of SEQ ID NO: 12 (GenBank Accession No. P30273) it has about 99% sequence identity with the human sequence. The ITAM motif of the cynomolgus gamma chain is identical to that of the human gamma chain.

[0105] Another embodiment of the invention is a cynomolgus Fc.gamma.RIIA. A cynomolgus Fc.gamma.RIIA binds to immunoglobulins and other molecules having an Fc region, preferably immunoglobulins complexed to an antigen or each other. More preferably, the receptor binds a dimeric or hexameric immune complex of human Ig. One example of a cynomolgus Fc.gamma.RIIA has an amino acid sequence of SEQ ID NO: 15. The mature cynomolgus Fc.gamma.RIIA has an amino acid sequence of SEQ ID NO: 66 (Table 21). an extracellular fragment obtained with the primers of example 1 has an amino acid sequence of .DELTA.1 to .DELTA.182 as shown in Table 21.

[0106] The cynomolgus Fc.gamma.RIIA sequence was aligned with a human amino acid sequence of Fc.gamma.RIIA as shown in Table 11 (SEQ ID NO: 16) (Accession No. P12318). In table 11, the amino acid numbers shown below the amino acids with the symbol A are numbered from the start of the mature human polypeptide not including the signal sequence. The numbers above the amino acid residues represent the numbering of the residues starting at the signal sequence. When the cynomolgus sequence is aligned with the human sequence it has about 87% or 89% sequence identity with the human sequence depending on whether the alignment starts with the MAMETQ sequence. This alignment shows that the cynomolgus Fc.gamma.RIIA has fewer amino acids in the signal peptide sequence than found in the human Fc.gamma.RIIA (Table 11). Cynomolgus Fc.gamma.RIIA has about the same number of amino acids in the two extracellular domains, transmembrane domain, and intracellular domain as found in the human Fc.gamma.RIIA sequence (Table 11). Notably, the cynomolgus Fc.gamma.RIIA contains the identical two ITAM motifs as found in the human receptor (Table 11).

[0107] The cynomolgus Fc.gamma.RIIA binds to hexameric complexes of subclasses IgG with the following binding pattern: IgG3=IgG2>IgG1>IgG4b, IgG4. A human Fc.gamma.RIIA isoform with an arginine at the amino acid corresponding to the amino acid 131 (R131) binds hexameric IgG subclasses as follows: IgG3>IgG1>>>IgG2&g- t;IgG4. A human Fc.gamma.RIIA isoform with a histidine at the amino acid corresponding to the amino acid 131 (H1131) binds hexameric IgG subclasses as follows: IgG3.gtoreq.IgG1=IgG2>>>IgG4. Cynomolgus Fc.gamma.RIIA with an amino acid sequence of SEQ ID NO: 15 has H 131 and binds to human subclasses of IgG in a similar manner to those human Fc receptors with the H131 isoform variant. However, the cynomolgus Fc receptor binds IgG2 as efficiently as it binds IgG3.

[0108] Another embodiment of the invention is a chimp Fc.gamma.RIIA. A chimp Fc.gamma.RIIA binds to immunoglobulins and other molecules having an Fc region, preferably immunoglobulins complexed to an antigen or each other. Preferably the receptor binds a dimeric or hexameric immune complex of human Ig. One example of a chimp Fc.gamma.RIIIA has an amino acid sequence of SEQ ID NO: 17. Based on the alignment with the human sequence, the mature chimp Fc.gamma.RIIA has an amino acid sequence of SEQ ID NO: 67.

[0109] The chimp Fc.gamma.RIIA amino acid sequence was aligned starting with the signal sequence with a human sequence for Fc.gamma.RIIA of SEQ ID NO: 16 as shown in Table 11 (Accession No. P12318). The alignment shows that when compared to the human sequence, the chimp sequence has about 97% sequence identity. This alignment also shows that the chimpanzee Fc.gamma.RIIA has one less amino acid in the signal peptide sequence than found in the human Fc.gamma.RIIA .alpha.-chain (Table 11). Chimpanzee Fc.gamma.RIIA has the same number of amino acids in the two extracellular domains, transmembrane domain, and intracellular domain as found in the human Fc.gamma.RIIA sequence (Table 11). Notably, the chimpanzee Fc.gamma.RIIA contains the identical two ITAM motifs as found in the human and cynomolgus receptors (Table 11).

[0110] Another embodiment of the invention is a cynomolgus Fc.gamma.RIIB. A cynomolgus Fc.gamma.RIIB binds to immunoglobulins and other molecules having an Fc region, preferably immunoglobulins complexed to an antigen or each other. More preferably, the receptor binds a dimeric or hexameric immune complex of human Ig. One example of a cynomolgus Fc.gamma.RIIB has an amino acid sequence of SEQ ID NO: 18. The mature cynomolgus Fc.gamma.RIIB has an amino acid sequence of SEQ ID NO: 68 (Table 22). an extracellular fragment obtained with the primers of example 1 has an amino acid sequence of .DELTA.1 to .DELTA.184 as ahown in table 22.

[0111] The cynomolgus Fc.gamma.RIIB has about 92% sequence identity with a human amino acid sequence of Fc.gamma.RIIB as shown in Table 11 (SEQ ID NO: 19) (Accession No. X52473). An alignment of the cynomolgus sequence with the human sequence shows that the cynomolgus Fc.gamma.RIIB has about the same number of amino acids in the signal peptide, two extracellular domains, and transmembrane domain as found in the human Fc.gamma.RIIB sequence (Table 11). The cynomolgus Fc.gamma.RIIB has three amino acids inserted in the N-terminal portion of the intracellular domain (compared to human Fc.gamma.RIIB) (Table 11). Notably, the cynomolgus Fc.gamma.RIIB intracellular domain contains the identical ITIM motif as found in the human receptor (Table 11).

[0112] The cynomolgus Fc.gamma.RIIB binds to hexameric complexes of subclasses IgG with the following binding pattern: IgG2.gtoreq.IgG3>IgG1>IgG4b, IgG4. A human Fc.gamma.RIIB binds hexameric IgG subclasses as follows: IgG3.gtoreq.IgG1>IgG2>IgG4. The cynomolgus Fc.gamma.RIIB binds IgG2 much more efficiently than the human Fc.gamma.RIIB.

[0113] Another embodiment of the invention is a cynomolgus Fc.gamma.RIIIA. A cynomolgus receptor Fc.gamma.RIIIA binds to immunoglobulins and other molecules having an Fc region, preferably immunoglobulins complexed. Preferably, the receptor binds a dimeric or hexameric immune complex of human Ig. One example of an amino acid sequence of the .alpha.-chain of Fc.gamma.RIIIA is SEQ ID NO: 20. The mature cynomolgus Fc.gamma.RIIIA .alpha.-chain has a sequence of SEQ ID NO: 69 (Table 23). An extracellular fragment obtained using the primer as described in example 1 has an amino acid sequence of .DELTA.1 to .DELTA.187 as shown in Table 23.

[0114] The cynomolgus Fc.gamma.RIIIA .alpha.-chain sequence was aligned with a human amino acid sequence of Fc.gamma.RIIIA as shown in Table 11 (SEQ ID NO: 21) (Accession No. P08637). In table 11, the amino acid numbers shown below the amino acids with the symbol A are numbered from the start of the mature human polypeptide not including the signal sequence. The numbers above the amino acid residues represent the numbering of the residues starting at the signal sequence. The alignment with the human and cynomolgus Fc.gamma.RIIIA sequence shows the sequence has about 91% sequence identity to the human sequence. This alignment of the cynomolgus sequence with the human sequence shows that the cynomolgus Fc.gamma.RIIIA .alpha.-chain has about the same number of amino acids in the signal peptide, the two extracellular domains, the transmembrane domain, and intracellular domain as found in the human Fc.gamma.RIIIA sequence (Table 11). Neither the cynomolgus nor human intracellular domains contain an ITAM motif; the activating ITAM motif for human Fc.gamma.RIIIA is supplied by the associated .gamma.-chain and the same situation most likely occurs in cynomolgus monkeys.

[0115] The cynomolgus Fc.gamma.RIIIA .alpha.-chain binds to hexameric complexes of subclasses IgG with the following binding pattern: IgG1>IgG3>>IgG2>IgG4b, IgG4. A human Fc.gamma.RIIIA isoform with a phenylalanine at the amino acid corresponding to the amino acid 158 (F158) binds hexameric IgG subclasses as follows: IgG3=IgG1>>>IgG2, IgG4. A human Fc.gamma.RIIA isoform with a valine at the amino acid corresponding to the amino acid 158 (V158) binds hexameric IgG subclasses as follows: IgG1>IgG3>>>IgG2A, IgG4. Cynomolgus Fc.gamma.RIIIA with an amino acid sequence of SEQ ID NO: 20 has an isoleucine at amino acid position corresponding to amino acid 158 and binds human Ig subclasses similar to human Fc.gamma.RIIIA VI 58.

[0116] Human IgG1 binds to human Fc.gamma.RIIIA-V158 better than it does to human Fc.gamma.RIIIA-F158 (Koene, H. R., Kleijer, M., Algra, J., Roos, D., von dem Borne, E. G. K., and de Hass, M. (1997) Blood 90, 1109-1114; Wu, J., Edberg, J. C., Redecha, P. B., Bansal, V., Guyre, P. M., Coleman, K., Salmon, J. E., and Kimberly, R. P. (1997) J. Clin. Invest. 100, 1059-1070; 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). In humans, the Fc.gamma.RIIIA-F158 allele predominates with approximately 90% of humans having at least one Fc.gamma.RIIIA-F158 allele (Lehrnbecher, T., Foster, C. B., Zhu, S., Leitman, S. F., Goldin, L. R., Huppi, K., and Chanock, S. J. (1999) Blood 94, 4220-4232). In addition, recent studies have begun to correlate specific disease states with the Fc.gamma.RIIIA polymorphic status of individuals (Wu, J., Edberg, J. C., Redecha, P. B., Bansal, V., Guyre, P. M., Coleman, K., Salmon, J. E., and Kimberly, R. P. (1997) J. Clin. Invest. 100, 1059-1070; Lehrnbecher, T., Foster, C. B., Zhu, S., Venzon, D., Steinberg, S. M., Wyvill, K., Metcalf, J. A., Cohen, S. S., Kovacs, J., Yarchoan, R., Blauvelt, A., and Chanock, S. J. (2000) Blood 95, 2386-2390; Nieto, A., Caliz, R., Pascual, M., Mataran, L., Garcia, S., and Martin, J. (2000) Arthritis & Rheumatism 43, 735-739). Notably, the chimpanzee and cynomolgus Fc.gamma.RIIIA have valine and isoleucine, respectively, at position 158. The similarity of binding of the four human subclasses of IgG to cynomolgus Fc.gamma.RIIIA and human Fc.gamma.RIIIA-V158 (as opposed to human Fc.gamma.RIIIA-F158) suggests that evaluation of human antibodies in primate models should account for the primate model reflecting only a minority of humans with respect to binding to Fc.gamma.RIIIA receptors, i.e. Fc.gamma.RIIIA-V158/V158 homozygotes. For example, since human Fc.gamma.RIIIA-V158 exhibits superior antibody-dependent cellular cytotoxicity (ADCC) compared to human Fc.gamma.RIIIA-F158 (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), primate models may overestimate the efficacy of human antibody effector functions associated with Fc.gamma.RIIIA.

[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.

[0118] The invention also provides for Fc receptor polypeptides identified as FcRn. Amino acid sequences of cynomolgus FcRn are shown in Table 14. In Table 14, the numbers shown below the amino acids and designated with the signal .DELTA. are numbered from the start of the mature polypeptide. Two alleles were identified and are shown in Table 14. A cynomologus FcRn .alpha.-chain has an amino acid sequence of SEQ ID NO: 29 with a serine at residue 3 of the mature polypeptide. A cynomolgus FcRn .alpha.-chain has a sequence of SEQ ID NO: 64 and has an asparagine at residue 3 of the mature polypeptide. The mature polypeptides of FcRn .alpha.-chain S3 and FcRn .alpha.-chain N3 have a sequence of SEQ ID NO: 71 and 72, respectively. A extracellular fragment of a FcRn as obtained using the primers as described in example 1 has an amino acid sequence of .DELTA.1 to .DELTA.274 as shown in table 14.

[0119] A sequence alignment of cynomolgus FcRn .alpha.-chain sequences to human FcRn .alpha.-chain (SEQ ID NO: 20) (GenBank Accession No. U12255) shows that the cynomolgus sequence is about 97% identical to the human sequence. Cynomolgus FcRn (S3) and FcRn (N3) .alpha.-chains bind to subclasses of IgG with the following binding pattern: IgG3>>IgG4>IgG2>IgG1, which is similar to that of the human FcRn .alpha.-chain.

[0120] The invention also includes cynomolgus .beta.-2 microglobulin polypeptides. A cynomolgus .beta.-2 microglobulin polypeptide has a sequence of SEQ ID NO: 25, Table 13. The mature .beta.-2 microglobulin polypeptide has a sequence of SEQ ID NO: 70. When the cynomolgus .beta.-2 microglobulin sequence is aligned with a human sequence for .beta.-2 microglobulin (SEQ ID NO: 26; GenBank Accession No. P01884), it shows that the cynomolgus sequence has about 92% sequence identity to human .beta.-2 microglobulin.

[0121] Variants, derivatives, fusion proteins, and fragments of the different cynomolgus and chimp Fc.gamma.R polypeptides that retain any of the biological activities of the FcRs, are also within the scope of the present invention. Note that one of ordinary skill in the art will readily be able to determine whether a variant, derivative, or fragment of a Fc.gamma.R polypeptide displays activity by subjecting the variant, derivative, or fragment to a immunoglobulin binding assay as described below in Example 3.

[0122] Derivatives of the different cynomolgus and chimp Fc.gamma.Rs can be polypeptides modified by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, polyethylene glycol (PEG) groups, lipids, phosphate, acetyl groups and the like.

[0123] In another embodiment, the polypeptides of the invention include fragments of the polypeptides that lack a portion or all of the transmembrane and intracellular domains: e.g. amino acid residues of the mature polypeptide as follows: Fc.gamma.RI .alpha.-chain amino acid residues 270-336 of SEQ ID NO: 65; Fc.gamma.RIIA amino acid residues 183 to 282 of SEQ ID NO: 66; chimp Fc.gamma.RIIA amino acid residues 172 to 281 of SEQ ID NO: 67; Fc.gamma.RIIB amino acid residues 185 to 252 of SEQ ID NO: 68, Fc.gamma.RIIIA .alpha.-chain amino acid residues 188 to 234 of SEQ ID NO: 69; or FcRn amino acid residues 275 to 342 of SEQ ID NO: 71 or SEQ ID NO: 72. A soluble Fc.gamma.R polypeptide may include a portion of the transmembrane domain and intracellular, as long as the polypeptide is secreted from the cell in which it is produced. Preferably, the fragments are capable of binding to an Fc region containing molecule.

[0124] Fragments of polypeptides also include one or more domain of the polypeptide identified in Table 10 or Table 11, including signal peptide, domain 1, domain 2, domain 3, transmembrane/intracellular, or a cytoplasmic domain including the ITAM or ITIM motif. Exemplary fragments of the polypeptides also include soluble polypeptides having only domain 1, domain 2 and domain 3 amino acid sequences of the corresponding mature Fc.gamma.R polypeptides: e.g., amino acid residues .DELTA.1 to .DELTA.269 of cynomolgus Fc.gamma.RI (Table 10), amino acid residues .DELTA.1 to .DELTA.182 of cynomolgus Fc.gamma.RIIA (Table 21), amino acid residues .DELTA.1 to .DELTA.184 of cynomolgus Fc.gamma.RIIB (Table 22), amino acid residues .DELTA.1 to .DELTA.187 of cynomolgus Fc.gamma.RIIIA (Table 23), and amino acids .DELTA.1 to .DELTA.274 of cynomolgus FcRn (Table 14).

[0125] Cynomolgus or chimp Fc.gamma.R variants within the scope of the invention may comprise conservatively substituted sequences, meaning that one or more amino acid residues of each polypeptide may be replaced by different residues that do not alter the secondary and/or tertiary structure of the polypeptide. Such substitutions may include the replacement of an amino acid by a residue having similar physicochemical properties, such as substituting one aliphatic residue (Ile, Val, Leu or Ala) for another, or substitution between basic residues Lys and Arg, acidic residues Glu and Asp, amide residues Gln and Asn, hydroxyl residues Ser and Tyr, or aromatic residues Phe and Tyr. Further information regarding making phenotypically silent amino acid exchanges may be found in Bowie et al., Science 247:1306-1310 (1990). Other variants which might retain substantially the biological activities of the proteins are those where amino acid substitutions have been made in areas outside functional regions of the protein.

[0126] The invention also provides variant cynomolgus and chimp FcR polypeptides. Variant polypeptide can include changes to the polypeptide sequence that result in the amino acid substitutions, additions, and deletions in the resultant variant polypeptide when compared to the native polypeptide, for instance SEQ ID NOs: 9, 15, 17, 18, 20, 25, 29, or 64. The changes to the variant polypeptide sequences can include changes to the nucleic acid sequence that result in replacement of an amino acid by a residue having similar physiochemical properties, such as substituting one aliphatic residue (Ile, Val, Leu, or Ala) for another, or substitutions between basic residues Lys and Arg, acidic residues Glu and Asp, amide residues Gln and Asn, hydroxyl residues Ser and Tyr, or aromatic residues Phe and Tyr. Variant polypeptide sequences of the present invention are preferably at least about 90% identical, more preferably at least about 91% identical, more preferably at least 92% or 93% identical, more preferably 94% identical, more preferably 95% or 96% identical, more preferably 97% or 98% identical, and most preferably at least about 99% identical, to a full length native sequence, a polypeptide lacking a signal sequence, an extracellular domain of the polypeptide, or a fragment of the Fc.gamma. receptor or .beta.-2 microglobulin of sequences of SEQ ID NOs: 9, 15, 17, 18, 20, 25, 29, or 64.

[0127] Another embodiment of the present invention are polypeptides of the invention fused to heterologous amino acids, peptides, or polypeptides. Such amino acids, peptides, or polypeptides, preferably facilitate purification of the polypeptide. Many of the available peptides used for such a function allow selective binding of the fusion protein to a binding partner. For example, the cynomolgus Fc.gamma.RI polypeptide, having a sequence as shown in SEQ ID NO:9, may be modified to comprise a peptide to form a fusion protein which specifically binds to a binding partner, or peptide tag. Non-limiting examples of such peptide tags include the 6-His tag, Gly/His.sub.6/GST tag, thioredoxin tag, hemaglutinin tag, Glylh156 tag, and OmpA signal sequence tag. Full length, variable and truncated polypeptides of the present invention may be fused to such heterologous amino acids, peptides, or polypeptides. For example, the transmembrane and intracellular domains of cynomolgus Fc.gamma.RIA can be replaced by DNA encoding the Gly/His.sub.6/GST tag fused as His271. As will be understood by one of skill in the art, the binding partner which recognizes and binds to the peptide may be any molecule or compound including metal ions (e.g., metal affinity columns), antibodies, or fragments thereof, and any protein or peptide which binds the peptide, such as the FLAG tag. The polypeptides of the present invention can also be fused to the immunoglobulin constant domain of an antibody to form immunoadhesin molecules.

[0128] The polypeptides of the present invention are preferably provided in an isolated form, and preferably are purified. The polypeptides may be recovered and purified from recombinant cell cultures by well-known methods, including ammonium sulfate or ethanol precipitation, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. In a preferred embodiment, high performance liquid chromatography (HPLC) is employed for purification.

Vectors and Host Cells

[0129] The present invention also relates to vectors comprising the polynucleotide molecules of the invention, as well as host cell transformed with such vectors. Any of the polynucleotide molecules of the invention may be joined to a vector, which generally includes a selectable marker and an origin of replication, for propagation in a host. Host cells are genetically engineered to express the polypeptides of the present invention. The vectors include DNA encoding any of the polypeptides described above or below, operably linked to suitable transcriptional or translational regulatory sequences, such as those derived from a mammalian, microbial, viral, or insect gene. Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences which control transcription and translation. Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA encoding the target protein. Thus, a promoter nucleotide sequence is operably linked to a cynomolgus monkey or chimp Fc.gamma.R DNA sequence, FcRn .alpha.-chain DNA sequence, or .beta.-2 microglobulin DNA sequence if the promoter nucleotide sequence directs the transcription of the Fc.gamma.R sequence.

[0130] Expression of non-human primate receptors of the invention can also be accomplished by removing the native nucleic acid encoding the signal sequence or replacing the native nucleic acid signal sequence with a heterologous signal sequence. Heterologous signal sequences include those from human Fc receptor polypeptides or other polypeptides, such as tissue plasminogen activator. Nucleic acids encoding signal sequences from heterologous sources are known to those of skill in the art.

[0131] Selection of suitable vectors to be used for the cloning of polynucleotide molecules encoding the target polypeptides of this invention will depend upon the host cell in which the vector will be transformed, and, where applicable, the host cell from which the target polypeptide is to be expressed. Suitable host cells for expression of the polypeptides of the invention include prokaryotes, yeast, and higher eukaryotic cells, each of which is discussed below.

[0132] Expression of functional cynomolgus monkey or chimp Fc.gamma.R polypeptides of the invention may require the genetic engineering of a host cell to contemporaneously express two or more polypeptide molecules. As was discussed previously, most Fc.gamma.Rs are complex molecules requiring the expression of both a IgG binding and a signal transducing polypeptide chain. The complex of two or more polypeptide chains forms the functional receptor. As such, for example, a host cell may be co-transfected with a first vector expressing the Fc.gamma.RI .alpha.-chain, having a first selection marker, and a second vector expressing the Fc.gamma.RI .gamma.-chain, having a second selection marker. Only host cells that have acquired both vectors and are expressing both polypeptides would survive and express functional Fc.gamma.RI. Other methods are envisioned for the co-transfection of multiple polypeptide chains into target host cells, including the linked expression of target polypeptides from the same vector.

[0133] The cynomolgus monkey or chimp Fc.gamma.R, FcRn, or .beta.-2 microglobulin polypeptides to be expressed in such host cells may also be fusion proteins which include regions from heterologous proteins. Such regions may be included to allow, e.g., secretion, improved stability, or facilitated purification of the polypeptide. For example, a sequence encoding an appropriate signal peptide can be incorporated into expression vectors. A DNA sequence for a signal peptide (secretory leader) may be fused in-frame to the target sequence so that target protein is translated as a fusion protein comprising the signal peptide. The DNA sequence for a signal peptide can replace the native nucleic acid encoding a signal peptide or in addition to the nucleic acid sequence encoding the native sequence signal peptide. A signal peptide that is functional in the intended host cell promotes extracellular secretion of the polypeptide. Preferably, the signal sequence will be cleaved from the target polypeptide upon secretion from the cell. Non-limiting examples of signal sequences that can be used in practicing the invention include the yeast I-factor and the honeybee melatin leader in Sf9 insect cells.

[0134] Suitable host cells for expression of target polypeptides of the invention include prokaryotes, yeast, and higher eukaryotic cells. Suitable prokaryotic hosts to be used for the expression of these polypeptides include bacteria of the genera Escherichia, Bacillus, and Salmonella, as well as members of the genera Pseudomonas, Streptomyces, and Staphylococcus. For expression in, e.g., E. coli, a target polypeptide may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in a prokaryotic host. The N-terminal Met may optionally then be cleaved from the expressed polypeptide.

[0135] Expression vectors for use in prokaryotic hosts generally comprise one or more phenotypic selectable marker genes. Such genes generally encode, e.g., a protein that confers antibiotic resistance or that supplies an auxotrophic requirement. A wide variety of such vectors are readily available from commercial sources. Examples include pSPORT vectors, pGEM vectors (Promega), pPROEX vectors (LTI, Bethesda, Md.), Bluescript vectors (Stratagene), and pQE vectors (Qiagen).

[0136] The cynomolgus monkey or chimp Fc.gamma.R, FcRn, or P-2 microglobulin, may also be expressed in yeast host cells from genera including Saccharomyces, Pichia, and Kluveromyces. Preferred yeast hosts are S. cerevisiae and P. pastoris. Yeast vectors will often contain an origin of replication sequence from a 2T yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Vectors replicable in both yeast and E. coli (termed shuttle vectors) may also be used. In addition to the above-mentioned features of yeast vectors, a shuttle vector will also include sequences for replication and selection in E. coli. Direct secretion of the target polypeptides expressed in yeast hosts may be accomplished by the inclusion of nucleotide sequence encoding the yeast I-factor leader sequence at the 5' end of the cynomolgus Fc.gamma.R-encoding nucleotide sequence.

[0137] Insect host cell culture systems may also be used for the expression of the polypeptides of the invention. In a preferred embodiment, the target polypeptides of the invention are expressed using a baculovirus expression system. Further information regarding the use of baculovirus systems for the expression of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988).

[0138] In another preferred embodiment, the cynomolgus Fc.gamma.R polypeptides are individually expressed in mammalian host cells. Non-limiting examples of suitable mammalian cell lines include the COS-7 line of monkey kidney cells (Gluzman et al., Cell 23:175 (1981)), Chinese hamster ovary (CHO) cells (Puck et al., Proc. Natl. Acad. Sci. USA, 60:1275-1281 (1958), CV-1 and human cervical carcinoma cells (HELA) (ATCC CCL 2). Preferably, HEK293 cells are used for expression of the target proteins of this invention.

[0139] The choice of a suitable expression vector for expression of the target polypeptides of the invention will of course depend upon the specific mammalian host cell to be used, and is within the skill of the ordinary artisan. Examples of suitable expression vectors include pcDNA3.1/Hygro (Invitrogen), 409, and pSVL (Pharmacia Biotech). A preferred vector for expression of the cynomolgus Fc.gamma.R polypeptides is pRK. Eaton, D. L., Wood, W. I., Eaton, D., Hass, P. E., Hollingshead, P., Wion, K., Mather, J., Lawn, R. M., Vehar, G. A., and Gorman, C. (1986) Biochemistry 25:8343-47. Expression vectors for use in mammalian host cells may include transcriptional and translational control sequences derived from viral genomes. Commonly used promoter sequences and enhancer sequences which may be used in the present invention include, but are not limited to, those derived from human cytomegalovirus (CMV), Adenovirus 2, Polyoma virus, and Simian virus 40 (SV40). Methods for the construction of mammalian expression vectors are disclosed, for example, in Okayama and Berg (Mol. Cell. Biol. 3:280 (1983)); Cosman et al. (Mol. Immunol. 23:935 (1986)) and Cosman et al. (Nature 312:768 (1984)).

Method of Evaluating Biological Properties, Safety and Efficacy of Fc Region Containing Molecules

[0140] One aspect of the invention includes a method for the evaluation of the pharmacokinetics/pharmacodynamics of FcR binding molecules such as humanized antibodies with cynomolgus monkey or chimp Fc receptors prior to an in vivo evaluation in a primate. This aspect of the invention is based on the finding that cynomolgus and chimp FcR polypeptides have a high degree of sequence identity with human Fc receptor polypeptides and bind to IgG subclasses in a similar manner. Evaluations can include testing, for example, humanized antibodies of any subclass (especially antibodies with prospective therapeutic utility) on target Fc receptors of the invention in an ELISA-format assay or to transiently expressing cells.

[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.

[0142] The invention also provides for screening variants of Fc region containing molecules such as antibody variants for their biological properties, safety, efficacy and pharmcokenetics. Antibody variants are typically altered at one or more residues and then the variants are analyzed for alteration in biological activities including altered binding affinity for Fc receptors. Screening for alterations in biological activities by variants may be tested both in vivo and in vitro. For example, receptor polypeptides of the present invention can be used in an ELISA-format assay or transiently infected cells. Antibody variants which bind to cynomolgus and/or chimp FcR polypeptides, such as the .alpha.-chain of Fc.gamma.RII, Fc.gamma.RIII or FcRn or Fc.gamma.RIIA or Fc.gamma.RIIB, are variants that are suitable for in vivo evaluation in primates as a therapeutic agent.

[0143] Direct binding and binding affinity determination between the different Fc region containing molecules is preferably performed against soluble extracellular domains of cynomolgus Fc.gamma.R polypeptides. For example, the transmembrane domain and intracellular domain of a target Fc.gamma.R can be replaced by DNA encoding a Gly-His.sub.6 tag or glutathione S-transferase (GST) (see Example 3). The Gly-His.sub.6 tag or GST provide a convenient method for immobilizing the Fc binding region of the receptor to a solid support for identification and/or determination of binding affinities between the receptor and target antibody variant. Potential assays include ELISA-format assays, co-precipitation format assays, and column chromatographic format assays. Identified Fc region containing molecules should directly interact with the soluble cynomolgus Fc.gamma.R and have equivalent or greater binding affinities for the cynomolgus Fc.gamma.R, as compared to corresponding human Fc.gamma.R.

[0144] Another aspect of the invention provides methods of identifying agents that have altered binding to a cynomolgus Fc.gamma.R comprising an ITAM and/or ITIM region. A method of the invention involves identifying an agent that has increased binding affinity for an FcR comprising an ITAM region and a decreased affinity for a FcR comprising an ITIM region.

[0145] Target agents include molecules that have a Fc region, preferably an antibody and more preferably an IgG antibody. If the target agent is an antibody it may be a variant antibody with an altered amino acids sequence compared to the native sequence of the antibody. Preferably variant antibodies have had amino acid substitutions in regions of the antibody that are involved in binding to Fc.gamma. receptor, including amino acids corresponding to amino acids 226 to 436 in a human IgG. Variant antibodies can be prepared using standard methods such as site specific oligonucleotide or PCR mediated methods as described previously. Examples of variant antibodies includes alanine variants of human IgG1, anti IgE E27 prepared as described in Shields et al., J. Biol. Chem. 276:6591 (2001).

[0146] Binding affinities of antibodies and/or variant antibodies are determined using standard methods as described in Shields et al., J. Biol. Chem. 276:6591 (2001) and in Examples 3-7 below. Binding affinities are preferably determined by binding to cells that express a Fc.gamma. receptor of the type being analyzed. However, binding affinities of antibodies or Fc region containing molecules can also be determined using soluble Fc.gamma. receptors or Fey receptors expressed on or secreted from a host cell.

[0147] A variant antibody that has an increased affinity for a cynomolgus Fc.gamma.RIIA compared with a human Fc.gamma.RIIA is an antibody that has a change in amino acid sequence at the position corresponding to amino acid 298 of human IgG1. One such variant has a change at that position from serine to alanine and is designated as S298A. Another variant antibody with a change at that position is designated as S298A/E333A/K334 which is a variant antibody with alanine in positions corresponding to amino acid 298, 333 and 334 of native sequence IgG1. These variants have increased binding affinity to a cynomolgus Fc.gamma.RIIA compared to a human Fc.gamma.RIIA.

[0148] In another method of the invention, target agents with altered binding affinity to a cynomolgus Fc.gamma.RIIB as compared to human Fc.gamma.RIIB are identified. The agents are preferably variants of native sequence antibodies. Binding affinities are determined as described above and as shown in the Examples below. Agents with enhanced binding to a Fc.gamma.RIIB may preferentially stimulate ITIM inhibitory functions. Agents with decreased affinity for a cynomolgus Fc.gamma.RIIB may have decreased stimulation of inhibitory function.

[0149] Variant antibodies that have decreased affinity for a cynomolgus Fc.gamma.RIIB compared to a human Fc.gamma.RIIB are: R255A, E258A, S37A, D280A and R301M.

[0150] Another embodiment of the invention involves the use of variant antibodies S298A or S298A/E333A/K334 to identify agents that can activate Fc.gamma. receptors comprising an ITAM while not engaging Fey receptors comprising an ITIM region.

[0151] Variant antibodies with S298A, and S292A/E333A/K334, have increased binding affinity to a cynomolgus Fc.gamma.RIIA, and decreased binding affinity to a cynomolgus Fc.gamma.RIIB. Such methods can be conducted in vivo or in vitro.

[0152] These methods are also useful for identifying the location of amino acid in native sequence antibodies that can be modified to increase binding of the antibody to FcR polypeptides, preferably human and cynomolgus Fc.gamma.R, comprising an ITAM region and/or to decrease binding affinity to Fc.gamma.R comprising an ITIM region. Modifications to the amino acid sequence at the identified locations can be prepared by standard methods.

[0153] Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.

EXAMPLES

Example 1

Molecular Cloning of Cynomolgus and Chimp Fc Receptor DNA and .beta.-2 Microglobulins

[0154] Materials and Methods:

Cloning of Cynomolgus Monkey Fc.gamma.R

[0155] Since cynomolgus monkey DNA shares approximately 90% homology to human DNA, a series of PCR primers for each Fc.gamma.R was designed based on the sequence of the corresponding human receptor. Each sense primer starts at a site immediately 5' of the coding region or at the start of the coding region. The antisense primers were designed in the same way, i.e. immediately 3' of the C terminal stop codon or at the C terminal stop codon. Primers incorporated endonuclease restriction sites used to subclone PCR product into a pRK vector (Eaton et al.). The sequences of the primers are shown in Table 1.

2TABLE 1 Restriction sites are underlined. Receptor Cyno Fc.gamma.RI Full-Length Forward CAGGTCAATCTCTAGACTCCCACCAGCTTGGAG Primer (SEQ ID NO: 31) Reverse GGTCAACTATAAGCTTGGACGGTCCAGATCGAT Primer (SEQ ID NO: 32) Restriction XbaI/HindIII Sites Receptor Cyno Fc.gamma.RI-H6-GST Forward CAGGTCAATCATCGATATGTGGTTCTTGACAGCT Primer (SEQ ID NO: 33) Reverse GGTCAACTATGCTAGCATGGTGATGATGGTGGTGCCAG Primer ACAGGAGTTGGTA (SEQ ID NO: 34) Restriction ClaI/NheI Sites Receptor Cyno Fc.gamma.RIIB Full-Length Forward CAGGTCAATCTCTAGAATGGGAATCCTGTCATTCTT Primer (SEQ ID NO: 35) Reverse GGTCAACTATAAGCTTCTAAATACGGTTCTGGTC Primer (SEQ ID NO: 36) Restriction Xbal/HindIII Sites Receptor Cyno Fc.gamma.RIIB-H6-GST Forward CAGGTCAATCATCGATATGCTTCTGTGGACAGC Primer (SEQ ID NO: 37) Reverse GGTCAACTATGGTGACCTATCGGTGAAGAGCTGC Primer (SEQ ID NO: 38) Restriction ClaI/BstEII Sites Receptor Cyno Fc.gamma.RIIIA Full-Length Forward CAGGTCAATCTCTAGAATGTGGCAGCTGCTCCT Primer (SEQ ID NO: 39) Reverse TCAACTATAAGCTTATGTTCAGAGATGCTGCTG Primer (SEQ ID NO: 40) Restriction XbaI/HindIII Sites Receptor Cyno Fc.gamma.RIIIA-H6-GST Forward CAGGTCAATCTCTAGAATGTGGCAGCTGCTCCT Primer (SEQ ID NO: 41) Reverse GGTCAACTATGGTCACCTTGGTACCCAGGTGGAAA Primer (SEQ ID NO: 42) Restriction XbaI/BstEII Sites Receptor Cyno Fc .gamma. Chain Forward CAGGTCAATCATCGATGAATTCCCACCATGATTCCAGC Primer AGTGGTC (SEQ ID NO: 43) Reverse GGTCAACTATAAGCTTCTACTGTGGTGGTTTCTCA Primer (SEQ ID NO: 44) Restriction EcoRI/HindIII Sites Receptor Cyno .beta.-2 Microglobulin Forward CAGGTCAATCATCGATTCGGGCCGAGATGTCT Primer (SEQ ID NO: 45) Reverse GGTCAACTATTCTAGATTACATGTCTCGATCCCA Primer (SEQ ID NO: 46) Restriction ClaI/XbaI Sites Receptor Cyno Fc.gamma.RIIA Full-Length Forward CAGGTCAATCTCTAGAATGTCTCAGAATGTATGTC Primer (SEQ ID NO: 47) Reverse GGTCAACTATAAGCTTTTAGTTATTACTGTTGTCATA Primer (SEQ ID NO: 48) Restriction XbaI/HindIII Sites Receptor Cyno Fc.gamma.RIIA-H6-GST Forward CAGGTCAATCATCGATATGTCTCAGAATGTATGTC Primer (SEQ ID NO: 49) Reverse GGTCAACTATGGTGACCCATCGGTGAAGAGCTGC Primer (SEQ ID NO: 50) Restriction ClaI/BstEII Sites Receptor Cyno FcRn Full-Length Forward CAGGTCAATCATCGATAGGTCGTCCTCTCAGC Primer (SEQ ID NO: 51) Reverse GGTCAACTATGAATTCTCGGAATGGCGGATGG Primer (SEQ ID NO: 52) Restriction ClaI/EcoRI Sites Receptor Cyno FcRn-H6 Forward CAGGTCAATCATCGATAGGTCGTCCTC- TCAGC Primer (SEQ ID NO: 53) Reverse GGTCAACTATGAATTCATGGTGATGATGGTGGTGCGAG Primer GACTTGGCTGGAGTTTC (SEQ ID NO: 54) Restriction ClaI/EcoRI Sites

[0156] The cDNA for FcRs was isolated by reverse transcriptase-PCR (GeneAmp, PerkinElmer Life Sciences) of oligo(dT)-primed RNA from cynomologus spleen cells using primers as shown in Table 1. The cDNA was subcloned into previously described pRK mammalian cell expression vectors, as described in Eaton et al., 1986, Biochemistry, 25:8343-8347. PCR reactions were set up using 200 ng of cDNA vector library from cynomolgus spleen and ExTaq Premix (Panvera, Madison, Wis.) according to the manufacturers instructions. After denaturation at 90.degree. C. for 30 s, 25 cycles were run with annealing at 55.degree. C. for 1 min, elongation at 72.degree. C. for 3 min, and denaturation at 98.degree. C. for 30 s. DNA bands migrating at the expected size (Fc.gamma.RI, Fc.gamma.RIIIA, FcRn, 1100 base pairs; Fc.gamma.RIIA, Fc.gamma.RIIB, 1000 base pairs; Fc.gamma. chain, 300 base pairs; .beta.-2 microglobulin, 400 base pairs) were isolated, cloned into pRK vectors, then transformed into Escherichia coli XL 1-Blue (Stratagene, San Diego, Calif.). Individual clones were selected and double-stranded DNA for each was purified using Qiagen mini-prep DNA kits (cat. # 27106; Qiagen). DNA sequencing was performed on an Applied Biosystems model 377 sequencer using Big-Dye Terminator Cycle Sequencing kits (Applied Biosystems, Foster City, Calif.).

[0157] Initial PCR reactions for Fc.gamma.RIIA did not reveal a PCR product. To determine whether or not Fc.gamma.RIIA was present in cynomolgus monkeys, a sense primer was designed in a region conserved between human Fc.gamma.RIIA, human Fc.gamma.RIIB, and cynomolgus Fc.gamma.RIIB (OF1, Table 2). An antisense primer was designed based on the consensus sequence in the region encoding the ITAM of human Fc.gamma.RIIA (OR1, Table 2). Using these two PCR primers (OF1, OR1) and the PCR protocol described above, a PCR product of approximately 700 base pairs was obtained. The PCR band was isolated and subcloned into a pRK vector, individual clones were isolated and sequenced as described above. Sequence analysis revealed that the fragment had 90% identity to human Fc.gamma.RIIA.

[0158] In order to determine the DNA sequence at the 5' end of the receptor, a nested PCR reaction was utilized. For the first step of the nested PCR reaction, a sense PCR primer (OF2, Table 2) was designed to lay down on the pRK vector 5' of the vector cloning site. This primer was used in conjunction with reverse primer OR1. The PCR reaction was performed on the cDNA library as described above, the product was diluted 1:500 and 1 .mu.L was used as a template for the second step of the nested PCR reaction. Due to the fact that primer OF2 would lay down on all members of the cDNA library (all members being cloned into separate pRK vectors), only a small quantity of PCR fragment was obtained and hence this was used as a template for amplification in the second step. The sense primer (OF3, Table 2) for the second step was designed to lay down on the pRK vector sequence 3' of OF2 and the reverse primer (OR2, Table 2) was based on partial sequence of Fc.gamma.RIIA determined above. The second step of the nested PCR reaction revealed a band of approximately 600 base pairs. The band was isolated and individual clones were prepared and sequenced as described above.

[0159] The DNA sequence at the 3' end of the receptor was determined in a similar manner. An initial PCR reaction on the cDNA library was performed using the forward primer OF4, designed from the sequence of the Fc.gamma.RIIA fragment, and the reverse primer OR3, designed to lay down in the pRK vector 3' from the end of the Fc.gamma.RIIA. The resultant fragment was used as template for the second step of the nested PCR reaction. The second step used the forward primer OF5, designed from the sequence of the Fc.gamma.RIIA fragment, and the reverse primer OR4, designed to lay down in the pRK vector 5' from primer OR3. The second step of the nested PCR reaction revealed a band of approximately 800 base pairs. The band was isolated and individual clones were sequenced as described above. PCR primers for the full length Fc.gamma.RIIA were designed based on the information acquired from the nested PCR reactions. Full length Fc.gamma.RIIA was cloned using the method described for all other receptors. The sequences of the primers described above are shown in Table 2.

3TABLE 2 (SEQ ID NO: 55) OF1 CAGGTCAATCTCTAGACAGTGGTTCCACAATGG (SEQ ID NO: 56) OR1 GGTCAACTATAAGCTTAAGAGTCAGGTAGATGTTT (SEQ ID NO: 57) OF2 CAGGTCAATC TCTAGA ATACATAACCTTATGTATCAT (SEQ ID NO: 58) OF3 CAGGTCAATC TCTAGA TATAGAATAACATCCACTTTG (SEQ ID NO: 59) OR2 GGTCAACTAT AAGCTT CAGAGTCATGTAGCCG (SEQ ID NO: 60) OF4 CAGGTCAATC TCTAGA ATTCCACTGATCCTGTGAA (SEQ ID NO: 61) OR3 GGTCAACTAT AAGCTT GCTTTATTTGTGAAATTTGTG (SEQ ID NO: 62) OF5 CAGGTCAATC TCTAGA ACTTGGACGTCAAACGATT (SEQ ID NO: 63) OR4 GGTCAACTAT AAGCTT CTGCAATAAACAAGTTGGG

Example 2

Alignment of Nucleotide and Amino Acid Sequences of Cynomolgus, Chimp and Human Fc.gamma.R

[0160] Nucleotide and amino acid sequences for FcR polypeptides from human, cynomolgus and chimps were aligned and % sequence identity calculated.

[0161] Nucleotide and amino acid sequences of primate and human proteins were aligned manually and differences in nucleotide or protein sequence noted. Percent identity was calculated as [number of identical residues]/[number of total residues]. When the sequences differed in the total number of residues, two values for percent identity are provided, using the two different numbers for total residues. Nucleotide sequences begin at the coding sequence for the signal sequence.

[0162] The alignment of nucleic acid sequences for human (SEQ ID NO: 2) and cynomolgus Fc.gamma.RI .alpha.-chain (SEQ ID NO: 1) as shown in Table 3 below. The dots indicate locations of nucleotide sequence differences. An analysis of the % sequence identity shows that the human and cynomolgus nucleotide sequences encoding Fc.gamma.RI .alpha.-chain have about 91% or 96% sequence identity depending on whether the nucleotides of 3' extensions are included in the calculation.

4TABLE 3 Alignment of Human and Cynomolgus High-Affinity Fc.gamma.RI DNA 1030 matches in an overlap of 1074: 95.9% identity 1030 matches in an overlap of 1128: 91.3% identity 10 20 30 40 50 Human ATGTGGTTCTTGACAACTCTGCTCCTTTGGGTTCCAGTTGATGGGCA- AGT .cndot. Cyno ATGTGGTTCTTGACAGCTCTGCTCCTTTGGGTTCCAGTTGATGGGCAAGT 60 70 80 90 100 Human GGACACCACAAAGGCAGTGATCACTTTGCAGCCTCCATGGGTCAGCGTGT .cndot. Cyno GGATACCACAAAGGCAGTGATCACTTTGCAGCCTCCATGGGTCA- GCGTGT 110 120 130 140 150 Human TCCAAGAGGAAACCGTAACCTTGCACTGTGAGGTGCTCCATCTGCCTGGG .cndot. .cndot. .cndot. .cndot. .cndot. Cyno TCCAAGAGGAAACTGTAACCTTACAGTGTGAGGTG- CCCCGTCTGCCTGGG 160 170 180 190 200 Human AGCAGCTCTACACAGTGGTTTCTCAATGGCACAGCCACTCAG- ACCTCGAC .cndot. Cyno AGCAGCTCCACACAGTGGTTTCTCAATGGCACAGCCACTCAGACCTCGAC 210 220 230 240 250 Human CCCCAGCTACAGAATCACCTCTGCCAGTGTCAATGACAGTGGTGAATACA .cndot. .cndot. Cyno TCCCAGCTACAGAATCACCTCTGCCAGTGTCAAGGACAGTGGTGAATACA 260 270 280 290 300 Human GGTGCCAGAGAGGTCTCTCAGGGCGAAGTGACCCCATACAGCTGGAAATC .cndot. Cyno GGTGCCAGAGAGGTCCCTCAGGGCGAAGTGACCCC- ATACAGCTGGAAATC 310 320 330 340 350 Human CACAGAGGCTGGCTACTACTGCAGGTCTCCAGCAGAGTCTTC- ACGGAAGG .cndot. .cndot. .cndot. Cyno CACAGAGACTGGCTACTACTGCAGGTATCCAGCAGAG- TCTTCACAGAAGG 360 370 380 390 400 Human AGAACCTCTGGCCTTGAGGTGTCATGCGTGGAAGGATAAGCTGG- TGTACA .cndot. Cyno AGAACCTCTGGCCTTGAGGTGTCATGCATGGAAGGATAAGCTGGTGTACA 410 420 430 440 450 Human ATGTGCTTTACTATCGAAATGGCAAAGCCTTTAAGTTTTTCCACTGGAAT .cndot. .cndot. .cndot. Cyno ATGTGCTTTACTATCAAAATGGCAAAGCCTTTAAGTTTTTCTACCGGAAT 460 470 480 490 500 Human TCTAACCTCACCATTCTGAAAACCAACATAAGTCACAATGGCACCTACCA .cndot. .cndot. .cndot. .cndot. Cyno TCTCAACTCACCATTCTGAAAACCAACATAAGTCACAACGGCGCCTACCA 510 520 530 540 550 Human TTGCTCAGGCATGGGAAAGCATCGCTACACATCAGCAGGAATATCTGTCA .cndot. .cndot. Cyno CTGCTCAGGCATGGGAAAGCATCGCTACACATCAGCAGGAGTATCTGTCA 560 570 580 590 600 Human CTGTGAAAGAGCTATTTCCAGCTCCAGTGCTGAATGCATCTGTGACATCC .cndot. Cyno CTGTGAAAGAGCTATTTCCAGCTCCAGTGCTGAATGCATCCGTGACATCC 610 620 630 640 650 Human CCACTCCTGGAGGGGAATCTGGTCACCCTGAGCTGTGAAACAAAGTTGCT .cndot. Cyno CCGCTCCTGGAGGGGAATCTGGTCACCCTGAGCTGTGAAACAAA- GTTGCT 660 670 680 690 700 Human CTTGCAGAGGCCTGGTTTGCAGCTTTACTTCTCCTTCTACATGGGCAGCA .cndot..cndot. Cyno TCTGCAGAGGCCTGGTTTGCAGCTTTACTTCTCCTTCTACATGGGCAGCA 710 720 730 740 750 Human AGACCCTGCGAGGCAGGAACACATCCTCTGAATACCAAATACTAACTGCT .cndot. Cyno AGACCCTGCGAGGCAGGAACACGTCCTC- TGAATACCAAATACTAACTGCT 760 770 780 790 800 Human AGAAGAGAAGACTCTGGGTTATACTGGTGCGAGGC- TGCCACAGAGGATGG .cndot. .cndot..cndot. .cndot. .cndot. Cyno AGAAGAGAAGACTCTGGGTTTTACTGGTGCGAGGCCACCACAGAAGACGG 810 820 830 840 850 Human AAATGTCCTTAAGCGCAGCCCTGAGTTGGAGCTTCAAGTGCTTGGCCTCC Cyno AAATGTCCTTAAGCGCAGCCCTGAGTTGGAGCTTCAAGTGCTTGGCCTCC 860 870 880 890 900 Human AGTTACCAACTCCTGTCTGGTTTCATGTCCTTTTCTATCTGGCAGTGGGA .cndot. .cndot. Cyno AGTTACCAACTCCTGTCTGGCTTCATGTCCTTTTCTATCTGGTAGTGGGA 910 920 930 940 950 Human ATAATGTTTTTAGTGAACACTGTTCTCTGGGTGACAATACGTAAAGAACT Cyno ATAATGTTTTTAGTGAACACTGTTCTCTGGGTGACAATACGTAAAGAACT 960 970 980 990 1000 Human GAAAAGAAAGAAAAAGTGGGATTTAGAAATCTCTTTGGATTCTGGTCATG .cndot. .cndot. .cndot. Cyno GAAAAGAAAGAAAAAGTGGAATTTAGAAATATCTTTGGATTCTGCTCATG 1010 1020 1030 1040 1050 Human AGAAGAAGGTAATTTCCAGCCTTCAAGAAGACAGACATTTAGAAGAAGAG .cndot. Cyno AGAAGAAGGTAACTTCCAGCCTTCAAGAAGACAGACAT- TTAGAAGAAGAG 1060 1070 1080 1090 1100 Human CTGAAATGTCAGGAACAAAAAGAAGAACAGCTGCAGGAAGGGGTG- CACCG .cndot..cndot. .cndot. .cndot. Cyno CTGAAGAGTCAGGAACAAGAATAA 1110 1120 Human GAAGGAGCCCCAGGGGGCCACGTAGCAG 3' extension

[0163] The Human DNA sequence shown in Table 3 has GenBank Accession No. L03418. Porges, A. J. Redecha, P. B., Doebele, R., Pan, L. C., Salmon, J. E. and Kimberly, R. P., Novel Fc gamma receptor I family gene products in human mononuclear cells, J. Clin Invest. 90, 2102-2109 (1992).

[0164] An alignment of nucleic acid sequences encoding human (SEQ ID NO: 14) and cynomolgus (SEQ ID NO: 13) gamma chain is shown in Table 4.

[0165] Analysis of the % sequence identity shows that the nucleic acid sequences encoding human and cynomolgus Fc.gamma.RI/III gamma chain have about 99% identity.

5TABLE 4 Alignment of Human and Cynomolgus Gamma-Chain DNA 258 matches in an overlap of 261: 98.9% identity 10 20 30 40 50 Human ATGATTCCAGCAGTGGTCTTGCTCTTACTCCTTTTGGTTGAACAAGC- AGC Cyno ATGATTCCAGCAGTGGTCTTGCTCTTACTCCTTTTGGTTGAACAAGCAG- C 60 70 80 90 100 Human GGCCCTGGGAGAGCCTCAGCTCTGCTATATCCTGGATGCCATCCTGTTTC Cyno GGCCCTGGGAGAGCCTCAGCTCTGCTATATCCTGGATGCCATCCTGTTTC 110 120 130 140 150 Human TGTATGGAATTGTCCTCACCCTCCTCTACTGTCGACTGAAGATCCAAGTG Cyno TGTATGGAATTGTCCTCACCCTCCTCTACTGTCGACTGAAGATCCAAGTG 160 170 180 190 200 Human CGAAAGGCAGCTATAACCAGCTATGAGAAATCAGATGGTGTTTACACGGG .cndot. Cyno CGAAAGGCAGCTATAGCCAGCTATGAGAAATCAGA- TGGTGTTTACACGGG 210 220 230 240 250 Human CCTGAGCACCAGGAACCAGGAGACTTACGAGACTCTGAAGCA- TGAGAAAC .cndot. .cndot. Cyno CCTGAGCACCAGGAACCAGGAAACTTATGAGACTCTGAAGCATGAGAAAC 260 Human CACCACAGTAG Cyno CACCACAGTAG

[0166] The DNA sequence for the human gamma chain as GenBank Accession No. M33195 J05285. Kuester, H., Thompson, H. and Kinet, J.-P., Characterization and expression of the gene for the human receptor gamma subunit: Definition of a new gene family, J. Biol. Chem. 265, 6448-6452 (1990).

[0167] An alignment of the human (SEQ ID NO: 4), chimp (SEQ ID NO: 22) and cynomolgus (SEQ ID NO: 3) nucleic acid sequence encoding Fc.gamma.RIIA is shown in Table 5. An analysis of the % sequence identity shows that the human and cynomolgus sequences encoding Fc.gamma.RIIA have about 94% sequence identity. A comparison of chimp and human sequences encoding Fc.gamma.RIIA have about 99% sequence identity.

6TABLE 5 Alignment of Human, Cynomolgus and Chimp Low-Affinity Fc.gamma.RIIA DNA Human/Cyno 878 matches in an overlap of 933: 94.1% identity without one gap of three nucleotides 878 matches in an overlap of 936: 93.8% identity with one gap of three nucleotides Human/Chimp 924 matches in an overlap of 933: 99.0% identity without one gap of three nucleotides 924 matches in an overlap of 936: 98.7% identity with one gap of three nucleotides 10 20 30 40 50 Chimp ATGTCTCAGAATGTATGTCCCAGAAACCTGTGGCTGCTTCAACCATTGAC Human ATGTCTCAGAATGTATGTCCCAGAAACCTGTGGCTGCTTCAACCATTGAC .cndot. .cndot. Cyno ATGTCTCAGAATGTATGTCCCGGCAACCTGTGGCTGCTTCAACCATTGAC 60 70 80 90 100 Chimp AGTTTTGCTGCTGCTGGCTTCTGCAGACAGTCAAGCT---GCTCCCCCAA .cndot..cndot..cndot. Human AGTTTTGCTGCTGCTGGCTTCTGCAGACAGTCAAGCTGCAGCTCCCCCAA .cndot. .cndot..cndot..cndot. .cndot. Cyno AGTTTTGCTGCTGCTGGCTTCTGCAGACAGTCAAACT---GCTC- CCCCGA 110 120 130 140 150 Chimp AGGCTGTGCTGAAACTTGAGCCCCCGTGGATCAACGTGCTCCAGGAGGAC Human AGGCTGTGCTGAAACTTGAGCCCCCGTGGATCAACGTGCTCCAGGAGGAC .cndot. .cndot. Cyno AGGCTGTGCTGAAACTCGAGCCCCCGTGGATCAACGTGCTCCGGGAGGAC 160 170 180 190 200 Chimp TCTGTGACTCTGACATGCCGGGGGGCTCGCAGCCCTGAGAGCGACTCCAT .cndot. Human TCTGTGACTCTGACATGCCAGGGGGCTCGCAGCCCTGAGAGCGACTCCAT .cndot. .cndot..cndot. .cndot. .cndot. .cndot. .cndot. Cyno TCTGTGACTCTGACGTGCGGGGGCGCTCACAGCCCTGAC- AGCGACTCCAC 210 220 230 240 250 Chimp TCAGTGGTTCCACAATGGGAATCTCATCCCCACCCACACGCAGCCC- AGCT .cndot. Human TCAGTGGTTCCACAATGGGAATCTCATTCCCACCCACACGCAGCCCAGCT .cndot. .cndot. .cndot. Cyno TCAGTGGTTCCACAATGGGAATCGCATCCCCACCCACACACAGCCCAGCT 260 270 280 290 300 Chimp ACAGGTTCAAGGCCAACAACAATGACAGCGGGGAGTACACGTGCCAGACT Human ACAGGTTCAAGGCCAACAACAATGACAGCGGGGAGTACACGTGCCAGACT .cndot. .cndot. Cyno ACAGGTTCAAGGCCAACAACAATGATAGCGGGGAGTACAGGTGCCAGACT 310 320 330 340 350 Chimp GGCCAGACCAGCCTCAGCGACCCTGTGCATCTGACTGTGCTTTCCGAATG Human GGCCAGACCAGCCTCAGCGACCCTGTGCATCTGACTGTGCTTTCCGAATG .cndot. .cndot. .cndot. .cndot. Cyno GGCCGGACCAGCCTCAGCGACCCTGTTCATCTGACTGTGCTTTCTGAGTG 360 370 380 390 400 Chimp GCTGGTGCTCCAGACCCCTCACCTGGAGTTCCAGGAGGGAGAAACCATCG .cndot. Human GCTGGTGCTCCAGACCCCTCACCTGGAGTTCCAGGAGGGAGAAACCATCA .cndot. .cndot. .cndot. Cyno GCTGGCGCTTCAGACCCCTCACCTGGAGTTCCGGGAGGGAGAAACCATCA 410 420 430 440 450 Chimp TGCTGAGGTGCCACAGCTGGAAGGACAAGCCTCTGGTCAAGGTCACATTC Human TGCTGAGGTGCCACAGCTGGAAGGACAAGCCTCTGGTCAAGGTCACATTC .cndot. Cyno TGCTGAGGTGCCACAGCTGGAAGGACAAGCCTCTGATCAAGGTCACATTC 460 470 480 490 500 Chimp TTCCAGAATGGAAAATCCCAGAAATTCTCCCATTTGGATCCCAACCTCTC .cndot. .cndot. .cndot. Human TTCCAGAATGGAAAATCCCAGAAATTCTCCCGTTTGGATCCCACCTTCTC .cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot..cndot. Cyno TTCCAGAATGGAATAGCCAAGAAATTTTCCC- ATATGGATCCCAATTTCTC 510 520 530 540 550 Chimp CATCCCACAAGCAAACCACAGTCACAGTGGTGATTAC- CACTGCACAGGAA Human CATCCCACAAGCAAACCACAGTCACAGTGGTGATTACC- ACTGCACAGGAA Cyno CATCCCACAAGCAAACCACAGTCACAGTGGTGATTACCAC- TGCACAGGAA 560 570 580 590 600 Chimp ACATAGGCTACACGCTGTTCTCATCCAAGCCTGTGACCATCACTGTC- CAA Human ACATAGGCTACACGCTGTTCTCATCCAAGCCTGTGACCATCACTGTCC- AA .cndot. .cndot..cndot. .cndot. .cndot. Cyno ACATAGGCTACACACCATACTCATCCAAACCTGTGACCATCACT- GTCCAA 610 620 630 640 650 Chimp GCGCCCAGCGTGGGCAGCTCTTCACCAGTGGGGATCATTGTGGCTGTGGT .cndot. .cndot. .cndot. Human GTGCCCAGCATGGGCAGCTCTTCACCAATGGGGATCATTGTGGCTGTGGT .cndot. .cndot. Cyno GTGCCCAGCGTGGGCAGCTCTTCACCGATGGGGATCATTGTGGCTGTGGT 660 670 680 690 700 Chimp CATTGCGACTGCTGTAGCAGCCATTGTTGCTGCTGTAGTGGCCTTGATCT Human CATTGCGACTGCTGTAGCAGCCATTGTTGCTGCTGTAGTGGCCTTGATCT .cndot. .cndot. .cndot. .cndot. Cyno CACTGGGATTGCTGTAGCGGCCATTGTTGCTGCTGTAGTGGCCTTGATCT 710 720 730 740 750 Chimp ACTGCAGGAAAAAGCGGATTTCAGCCAATTCCACTGATCCTGTGAAGGCT Human ACTGCAGGAAAAAGCGGATTTCAGCCAATTCCACTGATCCTGTGAAGGCT Cyno ACTGCAGGAAAAAGCGGATTTCAGCCAATTCCACTGATCCTGTGAAGGCT 760 770 780 790 800 Chimp GCCCAATTTGAGCCACCTGGACGTCAAATGATTGCCATCAGAAAGAGACA Human GCCCAATTTGAGCCACCTGGACGTCAAATGATTGCCATCAGAAAGAGACA .cndot. .cndot. .cndot. .cndot. Cyno GCCCGATTTGAGCCACTTGGACGTCAAACGATTGCCCTCAGAAAGAGACA 810 820 830 840 850 Chimp ACTTGAAGAAACCAACAATGACTATGAAACAGCTGACGGCGGCTACATGA Human ACTTGAAGAAACCAACAATGACTATGAAACAGCTGACGGCGGCTACATGA .cndot. Cyno ACTTGAAGAAACCAACAATGACTATGAAACAGCCGACGGCGGCTACATGA 860 870 880 890 900 Chimp CTCTGAACCCCAGGGCACCTACTGACGATGATAAAAACATCTACCTGACT Human CTCTGAACCCCAGGGCACCTACTGACGATGATAAAAACATCTACCTGACT .cndot. .cndot. Cyno CTCTGAACCCCAGGGCACCTACTGATGATGATAGAAACATCTACCTGACT 910 920 930 Chimp CTTCCTCCCAACGACCATGTCAACAG- TAATAACTAA Human CTTCCTCCCAACGACCATGTCAACAGTAATAACTAA .cndot. .cndot. .cndot. Cyno CTTTCTCCCAACGACTATGACAACAGTAATAACTAA

[0168] The sequence for the human Fc.gamma.RIIA receptor has GenBank Accession No. M28697. Seki, T., Identification of multiple isoforms of the low-affinity human IgG Fc receptor, Immunogenetics 30, 5-12 (1989).

[0169] Alignment of the nucleic acid sequences encoding human (SEQ ID NO: 6) and cynomolgus (SEQ ID NO: 5) Fc.gamma.RIIB is shown in Table 6.

[0170] Analysis of the % sequence identity shows that the human and cynomolgus sequences encoding Fc.gamma.RIIB have about 94% identity.

7TABLE 6 Alignment of Human and Cynomolgus Low-Affinity Fc.gamma.RIIB DNA 837 matches out of 885: 94.6% identity (without gap) 837 matches out of 894: 93.6% identity (with gap) 10 20 30 40 50 Human ATGGGAATCCTGTCATTCTTACCTGTCCTTGCCACTG- AGAGTGACTGGGC .cndot. Cyno ATGGGAATCCTGTCATTCTTACCTGTCCTTGCTACTGAGAGTGACTGGGC 60 70 80 90 100 Human TGACTGCAAGTCCCCCCAGCCTTGGGGTCATATGCTTCTGTGGACAGCTG .cndot. .cndot. .cndot. Cyno TGACTGCAAGTCCTCCCAGCCTTGGGGCCACATGCTTCTGTGGACAGCTG 110 120 130 140 150 Human TGCTATTCCTGGCTCCTGTTGCTGGGACACCTGCAGCTCCCCCAAAGGCT .cndot. Cyno TGCTATTCCTGGCTCCTGTTGCTGGGACACCTGCAGCTCCCCCGAAGGCT 160 170 180 190 200 Human GTGCTGAAACTCGAGCCCCAGTGGATCAACGTGCTCCAGGAGGACTCTGT .cndot. .cndot. Cyno GTGCTGAAACTCGAGCCCCCGTGGATCAACGTGCTCCGGGAGGACTCTGT 210 220 230 240 250 Human GACTCTGACATGCCGGGGGACTCACAGCCCTGAGAGCGACTCCATTCAGT .cndot. .cndot. .cndot..cndot. .cndot. .cndot. Cyno GACTCTGACGTGCGGGGGCGCTCACAGCCCTGACAGCGACTCCA- CTCAGT 260 270 280 290 300 Human GGTTCCACAATGGGAATCTCATTCCCACCCACACGCAGCCCAGCTACAGG .cndot. Cyno GGTTCCACAATGGGAATCTCATCCCCACCCACACGCAGCCCAGCTACAGG 310 320 330 340 350 Human TTCAAGGCCAACAACAATGACAGCGGGGAGTACACGTGCCAGACTGGCCA .cndot. .cndot. .cndot. Cyno TTCAAGGCCAACAACAATGATAGCGGGGAGTACAGGTGCCAGACTGGCCG 360 370 380 390 400 Human GACCAGCCTCAGCGACCCTGTGCATCTGACTGTGCTTTCTGAGTGGCTGG .cndot. Cyno GACCAGCCTCAGCGACCCTGTTC- ATCTGACTGTGCTTTCTGAGTGGCTGG 410 420 430 440 450 Human TGCTCCAGACCCCTCACCTGGAGTTCC- AGGAGGGAGAAACCATCGTGCTG .cndot. .cndot. .cndot. Cyno CGCTCCAGACCCCTCACCTGGAGTTCCGGGAGGGAGAAACCATCTTGCTG 460 470 480 490 500 Human AGGTGCCACAGCTGGAAGGACAAGCCTCTGGTCAAGGTCACATTCTTCCA .cndot. Cyno AGGTGCCACAGCTGGAAGGACAAGCCTCTGATCAAGGTCACATTCTTCCA 510 520 530 540 550 Human GAATGGAAAATCCAAGAAATTTTCCCGTTCGGATCCCAACTTCTCCATCC .cndot. .cndot. .cndot..cndot. .cndot. Cyno GAATGGAATATCCAAGAAATTTTCCCATATGAATCCCAACTTCTCCATCC 560 570 580 590 600 Human CACAAGCAAACCACAGTCACAGTGGTGATTACCACTGCACAGGAAACATA Cyno CACAAGCAAACCACAGTCACAGTGGTGATTACCACTGCACAGGAAACATA 610 620 630 640 650 Human GGCTACACGCTGTACTCATCCAAGCCTGTGACCATCACTGTCCAAGCTCC .cndot. .cndot..cndot. .cndot. .cndot..cndot. Cyno GGCTACACACCATACTCATCCAAACCTGTGACCATCA- CTGTCCAAGTGCC 660 670 680 690 700 Human ---------CAGCTCTTCACCGATGGGGATCATTGTGGCTGTGG- TCACTG .cndot..cndot..cndot..cndot..cndot..cndot..cndot..- cndot..cndot. .cndot. Cyno CAGCATGGGCAGCTCTTCACCGATAGGGATCATTGTGGCTGTGGTCACTG 710 720 730 740 750 Human GGATTGCTGTAGCGGCCATTGTTGCTGCTGTAGTGGCCTTGATCTACTGC Cyno GGATTGCTGTAGCGGCCATTGTTGCTGCTGTAGTGGCCTTGATCTACTGC 760 770 780 790 800 Human AGGAAAAAGCGGATTTCAGCCAATCCCACTAATCCTGATGAGGCTGACAA .cndot. Cyno AGGAAAAAGCGGATTTCAGCCAATCCCACTAATCCTGACGAGGCTGACAA 810 820 830 840 850 Human AGTTGGGGCTGAGAACACAATCACCTATTCACTTCTCATGCACCCGGATG .cndot. .cndot. Cyno AGTTGGGGCTGAGAACACAATCACCTATTCACTTCTCATGCATCCGGACG 860 870 880 Human CTCTGGAAGAGCCTGATGACCAGAACCGTATTTAG .cndot. .cndot. Cyno CTCTGGAAGAGCCTGATGACCAAAACCGNG- TTTAG

[0171] The human sequence for Fc.gamma.RIIB has GenBank Accession No. X52473. Engelhardt, W., Geerds, C. and Frey, J., Distribution, inducibility and biological function of the cloned and expressed human beta Fc receptor II, Eur. J. Immunol. 20 (6), 1367-1377 (1990)

[0172] Alignment of the nucleic acid sequences encoding a human (SEQ ID NO: 8) and cynomolgus (SEQ ID NO: 7) Fc.gamma.RIIIA is shown in Table 7.

[0173] Analysis of the % sequence identity shows that the human and cynomolgus nucleic acid sequences encoding Fc.gamma.RIIIA have about 96% identity.

8TABLE 7 Alignment of Human and Cynomolgus Low-Affinity Fc.gamma.RIIIA DNA 733 matches in an overlap of 765: 95.8% identity 10 20 30 40 50 Human ATGTGGCAGCTGCTCCTCCCAACTGCTCTGCTAC- TTCTAGTTTCAGCTGG Cyno ATGTGGCAGCTGCTCCTCCCAACTGCTCTGCTACTT- CTAGTTTCAGCTGG 60 70 80 90 100 Human CATGCGGACTGAAGATCTCCCAAAGGCTGTGGTGTTCCTGGAG- CCTCAAT .cndot. Cyno CATGCGGGCTGAAGATCTCCCAAAGGCTGTGGTGTTCCTGGAGCCTCAAT 110 120 130 140 150 Human GGTACAGGGTGCTCGAGAAGGACAGTGTGACTCTGAAGTGCCAGGGAGCC .cndot. Cyno GGTACAGGGTGCTCGAGAAGGACCGTG- TGACTCTGAAGTGCCAGGGAGCC 160 170 180 190 200 Human TACTCCCCTGAGGACAATTCCACACAGTGGTTTC- ACAATGAGAGCCTCAT .cndot. Cyno TACTCCCCTGAGGACAATTCCACACGGTGGTTTCACAATGAGAGCCTCAT 210 220 230 240 250 Human CTCAAGCCAGGCCTCGAGCTACTTCATTGACGCTGCCACAGTCGACGACA .cndot. .cndot..cndot. .cndot. .cndot. .cndot. Cyno CTCAAGCCAGACCTCGAGCTACTTCATTGCTGCTG- CCAGAGTCAACAACA 260 270 280 290 300 Human GTGGAGAGTACAGGTGCCAGACAAACCTCTCCACCCTCAGTG- ACCCGGTG .cndot. .cndot. Cyno GTGGAGAGTACAGGTGCCAGACAAGCCTCTCCACACTCAGTGACCCGGTG 310 320 330 340 350 Human CAGCTAGAAGTCCATATCGGCTGGCTGTTGCTCCAGGCCCCTCGGTGGGT .cndot. .cndot. Cyno CAGCTGGAAGTCCATATCGGCTGGCTATTGCTCCAGGCCCCTCGGTGGGT 360 370 380 390 400 Human GTTCAAGGAGGAAGACCCTATTCACCTGAGGTGTCACAGCTGGAAGAACA .cndot..cndot. Cyno GTTCAAGGAGGAAGAATCTATTCACCTG- AGGTGTCACAGCTGGAAGAACA 410 420 430 440 450 Human CTGCTCTGCATAAGGTCACATATTTACAGAATGGC- AAAGGCAGGAAGTAT .cndot..cndot. .cndot. Cyno CTCTTCTGCATAAGGTCACGTATTTACAGAATGGCAAAGGCAGGAAGTAT 460 470 480 490 500 Human TTTCATCATAATTCTGACTTCTACATTCCAAAAGCCACACTCAAAGACAG .cndot. Cyno TTTCATCAGAATTCTGACTTCTACATTCCAA- AAGCCACACTCAAAGACAG 510 520 530 540 550 Human CGGCTCCTACTTCTGCAGGGGGCTTTTTGGGAGTAAA- AATGTGTCTTCAG .cndot. .cndot. .cndot. Cyno CGGCTCCTACTTCTGCAGGGGACTTATTGGGAGT- AAAAATGTATCTTCAG 560 570 580 590 600 Human AGACTGTGAACATCACCATCACTCAAGGTTTGGCAGTGTCA- ACCATCTCA .cndot. .cndot. Cyno AGACTGTGAACATCACCATCACTCAAGATTTGGCAGTGTCATCC- ATCTCA 610 620 630 640 650 Human TCATTCTTTCCACCTGGGTACCAAGTCTCTTTCTGCTTGGTGATGGTACT .cndot. Cyno TCATTCTTTCCACCTGGGTACCAAGTCTCTTTCTGCCTGGTGATGGTACT 660 670 680 690 700 Human CCTTTTTGCAGTGGACACAGGACTATATTTCTCTGTGAAGACAAACATTC .cndot. .cndot. .cndot. Cyno CCTTTTTGCAGTGGACACAGGACTATATTTCTCTATGAAGAAAAGCATTC 710 720 730 740 750 Human GAAGCTCAACAAGAGACTGGAAGGACCATAAATTTAAATGGAGAAAGGAC .cndot. .cndot. .cndot. .cndot. Cyno CAAGCTCAACAAGGGACTGGGAGGACCATAAATTTAAATGGAGCAAGGAC 760 Human CCTCAAGACAAATGA Cyno CCTCAAGACAAATGA

[0174] The human sequence for Fc.gamma.III has GenBank Accession No. X52645 M31937). Ravetch, J. V. and Perussia, B., Alternative membrane forms of Fc gamma RIII(CD16) on human natural killer cells and neutrophils. Cell type-specific expression of two genes that differ in single nucleotide substitutions, J. Exp. Med. 170 (2), 481-497 (1989).

[0175] Alignment of the nucleic acid sequences encoding a human (SEQ ID NO: 24) and cynomolgus (SEQ ID NO: 23) .beta.-2 microglobulin is shown in Table 8.

[0176] Analysis of the % sequence identity shows that the human and cynomolgus nucleic acid sequences encoding .beta.-2 microglobulin have about 95% identity.

9TABLE 8 Alignment of Human and Cynomolgus .beta.2-Microglobulin DNA 341/360 = 94.7% identity 10 20 30 40 50 Human ATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGG .cndot. .cndot. .cndot. .cndot. Cyno ATGTCTCCCTCAGTGGCCTTAGCCGTGCTGGCGCTACTCTCTCTTTCTGG 60 70 80 90 100 Human CCTGGAGGCTATCCAGCGTACTCCAAAGATTCAGGTTTACTCACGTCATC .cndot. Cyno CCTGGAGGCTATCCAGCGTACTCCAAAGATTCAGGTTTACTCACGCCATC 110 120 130 140 150 Human CAGCAGAGAATGGAAAGTCAAATTTCCTGAATTGCTATGTGTCTGGGTTT .cndot. .cndot. .cndot. Cyno CACCAGAGAATGGAAAGCCAAATTTCCTGAATTGCTATGTGTCTGGATTT 160 170 180 190 200 Human CATCCATCCGACATTGAAGTTGACTTACTGAAGAATGGAGAGAGAATTGA .cndot. .cndot. .cndot. .cndot. .cndot. Cyno CATCCATCTGATATTGAAGTTGACTTACTGAAGAATGGAGAGAA- AATGGG 210 220 230 240 250 Human AAAAGTGGAGCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTTTCTATC .cndot. Cyno AAAAGTGGAGCATTCAGACTTGTCTTTCAGCAAAGACTGGTCTTTCTATC 260 270 280 290 300 Human TCTTGTACTACACTGAATTCACCCCCACTGAAAAAGATGAGTATGCCTGC .cndot. Cyno TCTTGTACTACACTGAATTCACC- CCCAATGAAAAAGATGAGTATGCCTGC 310 320 330 340 350 Human CGTGTGAACCATGTGACTTTGTCACAG- CCCAAGATAGTTAAGTGGGATCG .cndot..cndot. .cndot. .cndot. Cyno CGTGTGAACCATGTGACTTTGTCAGGGCCCAGGACAGTTAAGTGGGATCG 360 Human AGACATGTAA Cyno AGACATGTAA

[0177] The DNA sequence for the human .beta.-2 microglobulin has GenBank Accession No. ABO21288. Matsumoto, K., Minamitani, T., Human mRNA for beta 2-microglobulin, DDBJ/EMBL/GenBank databases (1998).

[0178] Alignment of the nucleic acid sequences encoding a human (SEQ ID NO: 28) and cynomolgus (SEQ ID NO: 27) FcRn .alpha.-chain is shown in Table 9.

[0179] Analysis of the % sequence identity shows that the human and cynomolgus nucleic acid sequences encoding FcRn .alpha.-chain have about 97% identity.

10TABLE 9 Alignment of Human and Cynomolgus FcRn .alpha.-Chain DNA 1062/1098 = 96.7% identity 10 20 30 40 50 Human ATGGGGGTCCCGCGGCCTCAGCCCTGGGCGCTGGGGCTCCTGCTCTTTCT .cndot. Cyno ATGAGGGTCCCGCGGCCTCAGCCCTGGGCGCTGGGGCTCCT- GCTCTTTCT 60 70 80 90 100 Human CCTTCCTGGGAGCCTGGGCGCAGAAAGCCACCTCTCCCTCCTGTACC- ACC .cndot. .cndot. Cyno CCTGCCCGGGAGCCTGGGCGCAGAAAGCCACCTCTCCCTCCTGTACCACC 110 120 130 140 150 Human TTACCGCGGTGTCCTCGCCTGCCCCGGGGACTCCTGCCTTCTGGGTGTCC .cndot. .cndot. .cndot. Cyno TCACCGCGGTGTCCTCGCCCGCCCCGGGGACGCCTGCCTTCTGGGTGTCC 160 170 180 190 200 Human GGCTGGCTGGGCCCGCAGCAGTACCTGAGCTACAATAGCCTGCGGGGCGA .cndot. .cndot. .cndot. .cndot. Cyno GGCTGGCTGGGCCCGCAGCAGTACCTGAGCTACGACAGCCTGAGGGGCCA 210 220 230 240 250 Human GGCGGAGCCCTGTGGAGCTTGGGTCTGGGAAAACCAGGTGTCCTGGTATT .cndot. Cyno GGCGGAGCCCTGTGGAGCTTGGGTCTGGGAAAACCAAGTGTCCTGGTATT 260 270 280 290 300 Human GGGAGAAAGAGACCACAGATCTGAGGATCAAGGAGAAGCTCTTTCTGGAA Cyno GGGAGAAAGAGACCACAGATCTGAGGATCAAGGAGAAGCTCTTTCTGGAA 310 320 330 340 350 Human GCTTTCAAAGCTTTGGGGGGAAAAGGTCCCTACACTCTGCAGGGCCTGCT .cndot. Cyno GCTTTCAAAGCTTTGGGGGGAAAA- GGCCCCTACACTCTGCAGGGCCTGCT 360 370 380 390 400 Human GGGCTGTGAACTGGGCCCTGACAACACCTCG- GTGCCCACCGCCAAGTTCG .cndot. Cyno GGGCTGTGAACTGAGCCCTGACAACACCTCGGTGCCCACCGCCAAGTTCG 410 420 430 440 450 Human CCCTGAACGGCGAGGAGTTCATGAATTTCGACCTCAAGCAGGGCACCTGG Cyno CCCTGAACGGCGAGGAGTTCATGAATTTCGACCTCAAGCAGGGCACCTGG 460 470 480 490 500 Human GGTGGGGACTGGCCCGAGGCCCTGGCTATCAGTCAGCGGTGGCAGCAGCA Cyno GGTGGGGACTGGCCCGAGGCCCTGGCTATCAGTCAGCGGTGGCAGCAGCA 510 520 530 540 550 Human GGACAAGGCGGCCAACAAGGAGCTCACCTTCCTGCTATTCTCCTGCCCGC .cndot. Cyno GGACAAGGCGGCCAACAAGGAGCTCACCTTCCTGCTATTCTCCTGCCCAC 560 570 580 590 600 Human ACCGCCTGCGGGAGCACCTGGAGAGGGGCCGCGGAAACCTGGAGTGGAAG .cndot. .cndot. Cyno ACCGGCTGCGGGAGCACCTGGAGAGGGGCCGTGGAAACCTGGAGTGGAAG 610 620 630 640 650 Human GAGCCCCCCTCCATGCGCCTGAAGGCCCGACCCAGCAGCCCTGGCTTTTC .cndot. .cndot. Cyno GAGCCCCCCTCCATGCGCCTGAAGGCCCGACCCGGCAACCCTGGCTTTTC 660 670 680 690 700 Human CGTGCTTACCTGCAGCGCCTTCTCCTTCTACCCTCCGGAGCTGCAACTTC .cndot. .cndot. Cyno CGTGCTTACCTGCAGCGCCTTCTCCTTCTACCCTCCGGAACTGCAACTGC 710 720 730 740 750 Human GGTTCCTGCGGAATGGGCTGGCCGCTGGCACCGGCCAGGGTGACTTCGGC .cndot. .cndot. .cndot. Cyno GGTTCCTGCGGAATGGGATGGCCGCTGGCACCGGACAGGGCGACTTCGGC 760 770 780 790 800 Human CCCAACAGTGACGGATCCTTCCACGCCTCGTCGTCACTAACAGTCAAAAG .cndot. Cyno CCCAACAGTGACGGCTCCTTCCACGCCTCGTCGTCA- CTAACAGTCAAAAG 810 820 830 840 850 Human TGGCGATGAGCACCACTACTGCTGCATTGTGCAGCACGCGGGG- CTGGCGC .cndot. Cyno TGGCGATGAGCACCACTACTGCTGCATCGTGCAGCACGCGGGGCTGGCGC 860 870 880 890 900 Human AGCCCCTCAGGGTGGAGCTGGAATCTCCAGCCAAGTCCTCCGTGCTCGTG .cndot. .cndot. Cyno AGCCCCTCAGGGTGGAGCTGGAAACTCCAGCCAAGTCCTCGGTGCTCGTG 910 920 930 940 950 Human GTGGGAATCGTCATCGGTGTCTTGCTACTCACGGCAGCGGCTGTAGGAGG Cyno GTGGGAATCGTCATCGGTGTCTTGCTACTCACGGCAGCGGCTGTAGGAGG 960 970 980 990 1000 Human AGCTCTGTTGTGGAGAAGGATGAGGAGTGGGCTGCCAGCCCCTTGGATCT Cyno AGCTCTGTTGTGGAGAAGGATGAGGAGTGGGCTGCCAGCCCCTTGGATCT 1010 1020 1030 1040 1050 Human CCCTTCGTGGAGACGACACCGGGGTCCTCCTGCCCACCCCAGGGGAGGCC .cndot. .cndot. .cndot..cndot. .cndot. Cyno CCCTCCGTGGAGATGACACCGGGTCCCTCCTGCCCACCCCGGGGGAGGCC 1060 1070 1080 1090 Human CAGGATGCTGATTTGAAGGATGTAAATGTGATTCCAGCCACCGCCTGA .cndot. .cndot. .cndot. .cndot. Cyno CAGGATGCTGATTCGAAGGATATAAATGTGATCCCAGCCACTGCCTGA

[0180] The DNA sequence for the human FcRn .alpha.-chain has GenBank Accession No. U12255. Story, C. M., Mikulska, J., and Simister, N. E., A major histocompatibility complex class I-like Fc receptor cloned from human placenta: Possible role in transfer of immunoglobulin G from mother to fetus, J. Exp. Med. 180, 2377-2381 (1994).

[0181] An alignment of the amino acid sequences for human (SEQ ID NO: 10) and cynomologus (SEQ ID NO: 9) Fc.gamma.RI .alpha.-chain is shown in Table 10. As described previously, the .alpha.-chain of Fc.gamma.RI has various domains, including a signal peptide, three extracellular C-2 Ig like domains, a transmembrane domain and an intracellular domain. The amino acid numbers shown below the amino acids with the symbol .DELTA. are numbered from the start of the mature polypeptide not including the signal sequence. Based on the alignment with the human sequence, the mature cynomolgus Fc.gamma.RI has an amino acid sequences of residues .DELTA.1 to .DELTA.336 (SEQ ID NO: 65). The n-terminal sequence of cynomologus sequences Fc.gamma.RI may vary from that shown below. It would be within the skill in the art to express the nucleic acid sequence encoding the cynomologus Fc.gamma.RI sequence and identify the n-terminal sequence. An extracellular fragment of cynolomolgus Fc.gamma.RI obtained using the primers of example 1 has an amino acid sequence of .DELTA.1 to .DELTA.269. Any numbers above the amino acid residues represent the numbering of the residues starting at the signal sequence.

[0182] Analysis of the % sequence identity shows that the amino acid sequences for human and cynomolgus Fc.gamma.RI have about 90% identity when the 3' extension is taken into account and about 94% when the 3' extension is not included.

11TABLE 10 Alignment of Human and Cynomolgus High-Affinity Fc.gamma.RI Human MWFLTTLLLWVPVDGQVDTTK .cndot. Cyno MWFLTALLLWVPVDGQVDTTK Domain 1 Human AVISLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTAT .cndot. .cndot. .cndot..cndot. Cyno AVITLQPPWVSVFQEETVTLQCEVPRLPGSSSTQWFLNGTAT .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. 1 10 20 30 40 70 80 90 100 .vertline. .vertline. .vertline. .vertline. Human QTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHR .cndot. .cndot. Cyno QTSTPSYRITSASVKDSGEYRCQRGPS- GRSDPIQLEIHR .DELTA. .DELTA. .DELTA. .DELTA. 50 60 70 80 Domain 2 Human GWLLLQVSSRVFTEGEPLALRCHAWKDK- LVYNVLYYRNGKAFKF .cndot. .cndot. Cyno DWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYQNGKAF- KF .DELTA. .DELTA. .DELTA. .DELTA. 90 100 110 120 150 160 170 180 190 .vertline. .vertline. .vertline. .vertline. .vertline. Human FHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGIS- VTVKELFP .cndot..cndot. .cndot. .cndot. .cndot. Cyno FYRNSQLTILKTNISHNGAYHCSGMGKHRYTSAGV- SVTVKELFP .DELTA. .DELTA. .DELTA. .DELTA. 130 140 150 160 Domain 3 Human APVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMG- SKTLRG Cyno APVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKT- LRG .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. 170 180 190 200 210 Human RNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPE- LELQVLGLQLP .cndot. .cndot. Cyno RNTSSEYQILTARREDSGFYWCEATTEDGNVLKRSPELELQVLGLQLP .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. 220 230 240 250 260 transmembrane/intracellular Human TPVWFHVLFYLAVGIMFLVNTVLWVTIRKELKRKKKWDLEISLDSGHE .cndot. .cndot. .cndot. .cndot. Cyno TPVWLHVLFYLVVGIMFLVNTVLWVTIRKELKRKKKWNLEISLDSAHE .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. 270 280 290 300 310 Human KKVTSSLQEDRHLEEELKCQEQKEEQLQEGVHRKEPQGAT .cndot. .cndot. Cyno KKVTSSLQEDRHLEEELKSQEQE .DELTA. .DELTA. .DELTA. .DELTA. 320 330 340 350 Human vs Cyno 335/357 = 93.8% identity without human 3' extension 335/374 = 89.6% identity with human 3' extension

[0183] The amino acid sequence for human Fc.gamma.RI has Accession Nos.: P112314; P12315; EMBL; X14356; CAA32537.1. EMBL; X14355; CAA32536.1. PIR; S03018. PIR; S03019. PIR; A41357. PIR; B41357. HSSP; P12319; 1ALT. MIM; 146760; -. InterPro; IPR003006; -. Pfam; PF00047; Allen J. M., Seed B., Nucleic Acids Res. 16, 11824-11824, 1988, Nucleotide sequence of three cDNAs for the human high affinity Fc receptor (FcRI); Allen J. M., Seed B., Science 243, 378-381, 1989, Isolation and expression of functional high-affinity Fc receptor complementary DNAs.

[0184] An alignment of amino acid sequences for human, cynomolgus, and chimp sequences for Fc.gamma.RIIA (cynomolgus/SEQ ID NO: 15; human/SEQ ID NO: 16; chimp/SEQ ID NO. 17), Fc.gamma.RIIB (cynomolgus/SEQ ID NO: 18; human/SEQ ID NO: 19), and Fc.gamma.RIIIA (cynomolgus/SEQ ID NO: 20; human/SEQ ID NO: 21) is shown in Table 11.

[0185] The sequence is divided into domains as described previously: signal peptide, 3 extracellular C-2 like domains, and a transmembrane intracellular domain. In Table 11, the amino acid numbers shown below the amino acids with the symbol A are numbered from the start of the mature human polypeptide not including the signal sequence. The mature polypeptides for cynomolgus and chimp Fc.gamma.RIIA, cynomolgous Fc.gamma.RIIB, and cynomolgus Fc.gamma.RIIIA start at the amino acid identified with the asterisk in Table 11 and are separately shown in Tables 21, 22, and 23, and are as follows:

[0186] 1) cynomolgus Fc.gamma.RIIA amino acids .DELTA.1 to .DELTA.282 (SEQ ID NO: 66), N terminal sequence TAPPKA (Table 21);

[0187] 2) chimp Fc.gamma.RIIA amino .DELTA.1 to .DELTA.249 (SEQ ID NO: 67)(based on alignment with the human sequence);

[0188] 3) cynomolgus Fc.gamma.RIIB amino acids .DELTA.1 to .DELTA.252 (SEQ ID NO: 68), N terminal sequence TPAAPP (table 22); and

[0189] 4) cynomolgus Fc.gamma.RIIIA amino acids .DELTA.1 to .DELTA.234 (SEQ ID NO: 69), N terminal sequence EDLPKA (table 23).

[0190] In table 11, any numbers above the amino acid residues represent the numbering of the residues starting at the signal sequence. The asterisks in the table indicate the start of the n-terminal sequence for cynomologus Fc.gamma.RIIA, Fc.gamma.RIIB, and Fc.gamma.RIIIA.

[0191] Extracellular fragments of the Fc receptor polypeptides were obtained using the primers described in example 1. An extracellular fragment of Fc.gamma.RIIA obtained using the primers of example 1 has an amino acid sequence of .DELTA.1 to .DELTA.182, as shown in table 21. An extracellular fragment of Fc.gamma.RIIB obtained using the primers of example 1 has an amino acid sequence of .DELTA.1 to .DELTA.184, as shown in Table 22. An extracellular fragment of Fc.gamma.RIIIA obtained using the primers of example 1 has an amino acid sequence of .DELTA.1 to .DELTA.187, as shown in Table 23.

[0192] Analysis of the % sequence identity shows the following:

[0193] 1) Chimp and human amino acid sequences for Fc.gamma.RIIA have about 97% identity;

[0194] 2) Cynomolgus and human amino acid sequences for Fc.gamma.RIIA have about 87% identity with MAMETQ (possible portion of signal peptide) and 89% identity without MAMETQ in the alignment;

[0195] 3) Cynomolgus and chimp amino acid sequences for Fc.gamma.RIIA have about 87% identity including MAMETQ in the alignment and 89% without MAMETQ in the alignment;

[0196] 4) Cynomolgus and human amino acid sequences for Fc.gamma.RIIB have about 92% identity; and

[0197] 5) Cynomolgus and human amino acid sequences for Fc.gamma.RIIIA have about 91% identity.

12TABLE 11 Alignment of Human, Cynomolgus and Chimp Low-Affinity Fc.gamma.RIIA, Fc.gamma.RIIB, Fc.gamma.RIIIA signal peptide .cndot..cndot..cndot..cnd- ot..cndot..cndot. .cndot. .cndot..cndot. IIA-human ---------MAMETQMSQNVCPRNLWLLQPLTVLLLLASADSQAA IIA- chimp ---------MAMETQMSQNVCPRNLWLLQPLTVLLLLASADSQA- IIA-cyno ---------------MSQNVCPGNLWLLQPLTVLLLLASADSQT- * .cndot. IIB-human MGILSFLPVLATESDWADCKSPQPW- GHMLLWTAVLFLAPVAGTPA IIB-cyno MGILSFLPVLATESDWADCKSSQPWGHM- LLWTAVLFLAPVAGTPA * .cndot. IIIA-human MWQLLLPTALLLLVSAGMRTE IIIA-cyno MWQLLLPTALLLLVSAGMRAE .DELTA. * 1 Domain 1 .cndot. .cndot. .cndot. .cndot. .cndot. IIA-human APPKAVLKLEPPWINVLQEDSVTLTCQGA- RSPESDSIQWFHN IIA-chimp APPKAVLKLEPPWINVLQEDSVTLTCRGARSPES- DSIQWFHN IIA-cyno APPKAVLKLEPPWINVLREDSVTLTCGGAHSPDSDSTQWF- HN .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. 1 10 20 30 40 .cndot. .cndot. .cndot. .cndot. .cndot. .cndot. IIB-human APPKAVLKLEPQWINVLQEDSVTLTCRGT- HSPESDSIQWFHN IIB-cyno APPKAVLKLEPPWINVLREDSVTLTCGGAHSPDSD- STQWFHN .cndot. .cndot. IIIA-human DLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQ- WFHN IIIA-cyno DLPKAVVFLEPQWYRVLEKDRVTLKCQGAYSPEDNSTRWFHN .DELTA. .DELTA. .DELTA. .DELTA. 10 20 30 40 .cndot. .cndot. .cndot. IIA-human GNLIPTHTQPSYRFKANNNDSGEYTCQTGQTSLSDPVHLTVLSE IIA-chimp GNLIPTHTQPSYRFKANNNDSGEYTCQTGQTSLSDPVHLTVLSE IIA-cyno GNRIPTHTQPSYRFKANNNDSGEYRCQTGRTSLSDPVHLTVLSE .DELTA. .DELTA. .DELTA. .DELTA. 50 60 70 80 .cndot. .cndot. IIB-human GNLIPTHTQPSYRFKANNNDSGEYTCQTGQTSLSDPVHLTVLSE IIB-cyno GNLIPTHTQPSYRFKANNNDSGEYRCQTGRTSLSDPVHLTVLSE .cndot. .cndot. .cndot. .cndot..cndot. .cndot. IIIA-human ESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIG IIIA-cyno ESLISSQTSSYFIAAARVNNSGEYRCQTSLSTLSDPVQLEVHIG .DELTA. .DELTA. .DELTA. .DELTA. 50 60 70 80 Domain 2 .cndot. .cndot. .cndot. .cndot. .cndot..cndot..cndot. IIA-human WLVLQTPHLEFQEGETIMLRCHSWK- DKPLVKVTFFQNGKSQKFS IIA-chimp WLVLQTPHLEFQEGETIVLRCHSWKDKP- LVKVTFFQNGKSQKFS IIA-cyno WLALQTPHLEFREGETIMLRCHSWKDKPLIKV- TFFQNGIAKKFS .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. 90 100 110 120 130 .cndot. .cndot. .cndot. .cndot. .cndot. IIB-human WLVLQTPHLEFQEGETIVLRCHSWKDKPLVKVTFFQNGKSKKFS IIB-cyno WLALQTPHLEFREGETILLRCHSWKDKPLIKVTFFQNGISKKFS .cndot..cndot. .cndot. IIIA-human WLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYF IIIA-cyno WLLLQAPRWVFKEEESIHLRCHSWKNTLLHKVTYLQNGKGRKYF .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. 90 100 110 120 130 .cndot..cndot. .cndot..cndot. .cndot..cndot. .cndot. IIA-human RLDPTFSIPQANHSHSGDYHCTGNIGYTLFSSKPVTI- TVQV IIA-chimp HLDPNLSIPQANHSHSGDYHCTGNIGYTLFSSKPVTITVQA IIA-cyno HMDPNFSIPQANHSHSGDYHCTGNIGYTPYSSKPVTITVQV .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. 131 140 150 160 170 .cndot..cndot..cndot. .cndot. .cndot. IIB-human RSDPNFSIPQANHSHSGDYHCTGNIGYTLYSSKPVTITV- QA IIB-cyno HMNPHFSIPQANHSHSGDYHCTGNIGYTPYSSKPVTITVQV .cndot. .cndot. IIIA-human HHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQ IIIA-cyno HQNSDFYIPKATLKDSGSYFCRGLIGSKNVSSETVNITITQ .DELTA. .DELTA. .DELTA. .DELTA. 140 150 158 170 transmembrane/intracellular .cndot. .cndot. .cndot..cndot..cndot. IIA-human PSMGSSSPMGIIVAVVIATAVAAI- VAAVVALIYCRKKRISANSTD IIA-chimp PSVGSSSPVGIIVAVVIATAVAAIVA- AVVALIYCRKKRISANSTD IIA-cyno PSVGSSSPMGIIVAVVTGIAVAAIVAAVV- ALIYCRKKRISANSTD .DELTA. .DELTA. .DELTA. .DELTA. 180 190 200 210 .cndot..cndot..cndot. .cndot. IIB-human P---SSSPMGIIVAVVTGIAVAAIVAAVVALIYCRKKRISANPTN IIB-cyno PSMGSSSPIGIIVAVVTGIAVAAIVAAVVALIYCRKKRISANPTN .cndot. .cndot. .cndot. .cndot..cndot. .cndot. IIIA-human GLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVK- TNIRSST IIIA-cyno DLAVSSISSFFPPGYQVSFCLVMVLLFAVDTGLYFSMKKS- IPSST .DELTA. .DELTA. .DELTA. .DELTA. 180 190 200 210 .cndot. .cndot. .cndot. .cndot. ITAM motif IIA-human PVKAAQFEPPGRQMIAIRKRQLEETNNDYETADGGYMTLNP- RAPT IIA-chimp PVKAAQFEPPGRQMIAIRKRQLEETNNDYETADGGYMTLNPRA- PT IIA-cyno PVKAARFEPLGRQTIALRKRQLEETNNDYETADGGYMTLNPRAPT .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. 220 230 240 250 260 .cndot. IIB-human PDEADKVGAENTITYSLLMHPDALEEPDDQNRI IIB-cyno PDEADKVGAENTITYSLLMHPDALEEPDDQNRV ITIM motif .cndot. .cndot. IIIA-human RDWKDHKFKWRKDPQDK IIIA-cyno RDWEDHKFKWSKDPQDK .DELTA. .DELTA. 220 230 ITAM motif .cndot. .cndot. .cndot..cndot. IIA-human DDDKNIYLTLPPNDHVNSNN IIA-chimp DDDKNIYLTLPPNDHVNSNN IIA-cyno DDDRNIYLTLSPNDYDNSNN .DELTA. .DELTA. 270 280 IIA chimp/human 308/317 = 97.2% identity cyno/human 277/317 = 87.4% identity (+MAMETQ) 277/311 = 89.1% identity (-MAMETQ) cyno/chimp 276/316 = 87.3% identity (+MAMETQ) 276/310 = 89.0% identity (-MAMETQ) IIB cyno/human 270/294 = 91.8% identity IIIA cyno/human 232/254 = 91.3% identity

[0198] The human amino acid sequence for FcRIIA has the following Accession Nos.: P12318; EMBL; M31932; AAA35827.1. EMBL; Y00644; CAA68672.1. EMBL; J03619; AAA35932.1. EMBL; A21604; CAA01563.1. PIR; A31932. PIR; JL0118. PIR; S02297. PIR; S00477. PIR; S06946. HSSP; P12319; 1ALT. MIM; 146790; -. InterPro; IPR003006; -. Pfam; PF00047. Brooks D. G., Qiu W. Q., Luster A. D., Ravetch J. V., J. Exp. Med. 170, 1369-1385, 1989, Structure and expression of human IgG FcRII(CD32). Functional heterogeneity is encoded by the alternatively spliced products of multiple genes; Stuart S. G., Trounstine M. L., Vaux D. J. T., Koch T., Martens C. L., Moore K. W., J. Exp. Med. 166, 1668-1684, 1987, Isolation and expression of cDNA clones encoding a human receptor for IgG (Fc gamma RII); Hibbs M. L., Bonadonna L., Scott B. M., Mckenzie I. F. C., Hogarth P. M., Proc. Natl. Acad. Sci. U.S.A. 85, 2240-2244, 1988, Molecular cloning of a human immunoglobulin G Fc receptor; Stengelin S., Stamenkovic I., Seed B., EMBO J. 7, 1053-1059, 1988, Isolation of cDNAs for two distinct human Fc receptors by ligand affinity cloning; Salmon J. E., Millard S., Schachter L. A., Arnett F. C., Ginzler E. M., Gourley M. F., Ramsey-Goldman R., Peterson M. G. E., Kimberly R. P., J. Clin. Invest. 97, 1348-1354, 1996, Fc gamma RIIA alleles are heritable risk factors for lupus nephritis in African Americans.

[0199] The human sequence for Fc.gamma.RIIB has Accession No. X52473. Engelhardt, W., Geerds, C. and Frey, J., Distribution, inducibility and biological function of the cloned and expressed human beta Fc receptor II, Eur. J. Immunol. 20 (6), 1367-1377 (1990).

[0200] The human amino acid sequence for Fc.gamma.RIIIA has Accession Nos.: P08637; EMBL; X52645; CAA36870.1. EMBL; Z46222; CAA86295.1. PIR; JL0107. MIM; 146740; -. InterPro; IPR003006; -. Pfam; PF00047; Ravetch J. V., Perussia B., J. Exp. Med. 170, 481497, 1989, Alternative membrane forms of Fc gamma RIII(CD16) on human natural killer cells and neutrophils. Cell type-specific expression of two genes that differ in single nucleotide substitutions; Gessner J. E., Grussenmeyer T., Kolanus W., Schmidt R. E., J. Biol. Chem. 270, 1350-1361, 1995, The human low affinity immunoglobulin G Fc receptor III-A and III-B genes: Molecular characterization of the promoter regions; de Haas M., Koene H. R., Kleijer M., de Vries E., Simsek S., van Tol M. J. D., Roos D., von dem Borne A. E. G. K., J. Immunol. 156, 3948-3955, 1996, A triallelic Fc gamma receptor type IIIA polymorphism influences the binding of human IgG by NK cell Fc gamma RIIIa; Koene H. R., Kleijer M., Algra J., Roos D., von dem Borne A. E. G. K., de Haas M., Blood 90, 1109-1114, 1997, Fc gammaRIIIa-158V/F polymorphism influences the binding of IgG by natural killer cell Fc gammaRIIIa, independently of the Fc gammaRIIIa-48L/R/H phenotype; Wu J., Edberg J. C., Redecha P. B., Bansal V., Guyre P. M., Coleman K., Salmon J. E., Kimberly R. P., J. Clin. Invest. 100, 1059-1070, 1997, A novel polymorphism of FcgammaRIIIa (CD16) alters receptor function and predisposes to autoimmune disease.

13TABLE 21 Sequence of Mature FcRIIA Domain 1 TAPPKAVLKLEPPWINVLREDSVTLTCGGAHSPDSDSTQWFHN .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. 1 10 20 30 40 GNRIPTHTQPSYRFKANNNDSGEYRCQTGRTSLSDPVHLTVLSE .DELTA. .DELTA. .DELTA. .DELTA. 50 60 70 80 Domain 2 WLALQTPHLEFREGETIMLRCHSWKDKPLIKVTFFQNGIAKKFS .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. 90 100 110 120 130 HMDPNFSIPQANHSHSGDYHCTGNIGYTPYSSKPVTITVQV .DELTA. .DELTA. .DELTA. .DELTA. 140 150 160 170 Intracellular/transmembrane domain PSVGSSSPMGIIVAVVTGIAVAAIVAAVVA- LIYCRKKRISANSTD .DELTA. .DELTA. .DELTA. .DELTA. 180 190 200 210 ITAM PVKAARFEPLGRQTIALRKRQLEETNNDYETADGGYMTLNPRAPT .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. 220 230 240 250 260 ITAM DDDRNIYLTLSPNDYDNSNN .DELTA. .DELTA. 270 280

[0201]

14TABLE 22 Sequence of Mature Fc.gamma.RIIB Domain 1 TPAAPPKAVLKLEPPWINVLREDSVTLTCGGAHSPDSDS- TQWFHN .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. 1 10 20 30 40 GNLIPTHTQPSYRFKANNNDSGEYRCQTGRTSLSDPVHLTVLSE .DELTA. .DELTA. .DELTA. .DELTA. 50 60 70 80 Domain 2 WLALQTPHLEFREGETILLRCHSWKDKPLIKVTFFQNGISKKFS .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. 90 100 110 120 130 HMNPNFSIPQANHSHSGDYHCTGNIGYTPYSSKPVTITVQV .DELTA. .DELTA. .DELTA. .DELTA. 140 150 160 170 Transmembrane/intracellular PSMGSSSPIGIIVAVVTGIAVAAIVAAVVALIYCRKK- RISANPTN .DELTA. .DELTA. .DELTA. .DELTA. 180 190 200 210 ITIM motif PDEADKVGAENTITYSLLMHPDALEEPDDQNRV .DELTA. .DELTA. .DELTA. .DELTA. 220 230 240 250

[0202]

15TABLE 23 Sequence for Mature Fc.gamma.RIIIA Domain 1 EDLPKAVVFLEPQWYRVLEKDRVTLKCQGAYSPEDNS- TRWFHN .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. 1 10 20 30 40 ESLISSQTSSYFIAAARVNNSGEYRCQTSLSTLSDPVQLEVHIG .DELTA. .DELTA. .DELTA. .DELTA. 50 60 70 80 Domain 2 WLLLQAPRWVFKEEESIHLRCHSWKNTLLHKVTYLQNGKGRKYF .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. 90 100 110 120 130 HQNSDFYIPKATLKDSGSYFCRGLIGSKNVSSETVNITITQ .DELTA. .DELTA. .DELTA. .DELTA. 140 150 160 170 Transmembrane/intracellular DLAVSSISSFFPPGYQVSFCLVMVLLFAVDTGLYFSM- KKSIPSST .DELTA. .DELTA. .DELTA. .DELTA. 180 190 200 210 RDWEDHKFKWSKDPQDK .DELTA. .DELTA. 220 230

[0203] An alignment of the nucleic acid sequence encoding the human (SEQ ID NO: 12) and cynomolgus (SEQ ID NO: 11) gamma chain of Fc.gamma.RI/III is shown in Table 12.

[0204] Analysis of % sequence identity shows that the nucleic acid sequences encoding human and cynomolgus gamma chain Fc.gamma.RI/III have about 99% identity.

16TABLE 12 Alignment of Human and Cynomolgus Fc.gamma.RI/III Gamma-Chain 1 10 .vertline. .vertline. Human MIPAVVLLLLLLVEQAAA Cyno MIPAVVLLLLLLVEQAAA 20 30 40 50 .vertline. .vertline. .vertline. .vertline. Human LGEPQLCYILDAILFLYGIVLTLLYCRLKIQV Cyno LGEPQLCYILDAILFLYGIVLTLLYCRLKIQV 60 70 80 .vertline. .vertline. .vertline. Human RKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ .cndot. Cyno RKAAIASYEKSDGVYTGLSTRNQETYETLKHEKPPQ ITAM motif ITAM motif Cyno vs Human = 85/86 = 98.8% identity

[0205] An amino acid sequence for human gamma chain has Accession Nos.: P30273; EMBL; M33195; AAA35828.1. EMBL; M33196; -. PIR; A35241. MIM; 147139; -. Kuester H., Thompson H., Kinet J.-P., J. Biol. Chem. 265, 6448-6452, 1990, Characterization and expression of the gene for the human Fc receptor gamma subunit. Definition of a new gene family.

[0206] An alignment of the amino acid sequences for human (SEQ ID NO: 26) and cynomolgus (SEQ ID NO: 25) .beta.-2 microglobulin is shown in Table 13. The mature .beta.-2 microglobulin has an amino acid sequence of amino acids .DELTA.1 to .DELTA.99 (SEQ ID NO: 70).

[0207] Analysis of the % sequence identity shows that the amino acid sequences for human and cynomolgus .beta.-2 microglobulin have about 92% identity with no deletions or insertions.

17TABLE 13 Alignment of Human and Cynomolgus .beta.2-Microglobulin Human MSRSVALAVLALLSLSGLEA .cndot. Cyno MSPSVALAVLALLSLSGLEA Human IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSD .cndot. .cndot. .cndot..cndot..cndot. Cyno IQRTPKIQVYSRHPPENGKPNFLNCYVSGF- HPSDIEVDLLKNGEKMGKVEHSD .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. 1 10 20 30 40 50 Human LSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM .cndot. .cndot. .cndot..cndot. Cyno LSFSKDWSFYLLYYTEFTPNEKDEYACRVNHVTLSGPRTVKWDRDM .DELTA. .DELTA. .DELTA. .DELTA. 60 70 80 90

[0208] Cyno vs Human 109/119=91.6% identity

[0209] The human amino acid sequence for .beta.-2 microglobulin has Accession Nos.: P01884; EMBL; M17987; AAA51811.1. EMBL; M17986; AAA51811.1. EMBL; AB021288; BAA35182.1. EMBL; AF072097; AAD48083.1. EMBL; V00567; CAA23830.1. EMBL; M30683; AAA87972.1. EMBL; M30684; AAA88008.1. PIR; A02179. PIR; A28579. PDB; 1HLA. Guessow D., Rein R., Ginjaar I., Hochstenbach F., Seemann G., Kottman A., Ploegh H. L., The human beta 2-microglobulin gene. Primary structure and definition of the transcriptional unit, J. Immunol. 139, 3132-3138 (1987); Matsumoto K., Minamitani T., Human mRNA for beta 2-microglobulin, Medline: Embl/genbank/ddbj database (1998); Zhao Z., Huang X., Li N., Zhu X., Cao X., A novel gene from human dendritic cell, Embl/genbank/ddbj databases (1998); Rosa F., Berissi H., Weissenbach J., Maroteaux L., Fellous M., Revel M., The beta-2-microglobulin mRNA in human Daudi cells has a mutated initiation codon but is still inducible by interferon, EMBO J. 2, 239-243 (1983); Suggs S. V., Wallace R. B., Hirose T., Kawashima E. H., Itakura K., Use of synthetic oligonucleotides as hybridization probes: isolation of cloned cDNA sequences for human beta 2-microglobulin, Proc. Natl. Acad. Sci. USA 78, 6613-6617 (1981); Cunningham B. A., Wang J. L., Berggard I., Peterson P. A., The complete amino acid sequence of beta 2-microglobulin, Biochem. 12, 4811-4822 (1973); Lawlor D. A., Warren E., Ward F. E., Parham P., Comparison of class I MHC alleles in human and apes, Immunol. Rev. 113, 147-185 (1990); Bjorkman P. J., Saper M. A., Samraoui B., Bennett W. S., Strominger J. L., Wiley D. C., Structure of the human class I histocompatibility antigen, HLA-A2, Nature 329, 506-512 (1987); Saper M. A., Bjorkman P. J., Wiley D. C., Refined structure of the human histocompatibility antigen HLA-A2 at 2.6A resolution, J. Mol. Biol. 219, 277-319 (1991); Collins E. J., Garboczi D. N., Karpusas M. N., Wiley D. C., The three-dimentional structure of a class I major histocompatibility complex molecule missing the alpha 3 domain of the heavy chain, Proc. Natl. Acad. Sci USA 92, 1218-1221 (1995).

[0210] An alignment of the amino acid sequences for human (SEQ ID NO: 30) and cynomolgus FcRn .alpha.-chain (SEQ ID NO: 29) is shown in Table 14. Two alleles of cynomolgus FcRn were identified. One sequence is that of SEQ ID NO: 29 and has a serine at position 3 (S3) of the mature polypeptide. Another sequence is SEQ ID NO: 64 has an asparagine at position 3 (N3) in the mature polypeptide. The mature polypeptide of FcRnS3 .alpha.-chain has a sequence of amino acids .DELTA.1 to .DELTA.342 (SEQ ID NO: 71). The mature polypeptide of FcRnN3 .alpha.-chain has a sequence of .DELTA.1 to .DELTA.342 (SEQ ID NO: 72). An extracellular fragment of the FcRn prepared by the method of example 1, has an amino acid sequence of .DELTA.1 to .DELTA.274.

[0211] Analysis of the % sequence identity shows that the amino acid sequences for human and cynomolgus FcRn have about 97% identity with no deletions or insertions.

18TABLE 14 Alignment of Human and Cynomolgus FcRn .alpha.-Chain 354/365 = 97% identity Signal Cyno MRVPRPQPWALGLLLFLLPGSLG .cndot. Human MGVPRPQPWALGLLLFLLPGSLG Extracellular Domain Cyno AESHLSLLYHLTAVSSPAPGTPAFWVSGWLGPQQYLSYDSLRGQAEPCGA CynoN3 N .cndot. .cndot. Human AESHLSLLYHLTAVSSPAPGTPAFWV- SGWLGPQQYLSYNSLRGEAEPCGA .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. 10 20 30 40 50 Cyno WVWENQVSWYWEKETTDLRIKEKLFLEAFKALGGKGPYTLQGLLGCELSP .cndot. Human WVWENQVSWYWEKETTDLRIKEKLFLEAFKALGGKGPYTLQGLLGCELGP .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. 60 70 80 90 100 Cyno DNTSVPTAKFALNGEEFMNFDLKQGTWGGDWPEALAISQRWQQQDKAA- NK Human DNTSVPTAKFALNGEEFMNFDLKQGTWGGDWPEALAISQRWQQQDKAAN- K .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. 110 120 130 140 150 Cyno ELTFLLFSCPHRLREHLERGRGNLEWKEPPSMR- LKARPGNPGFSVLTCSA .cndot..cndot. Human ELTFLLFSCPHRLREHLERGRGNLEWKEPPSMRLKA- RPSSPGFSVLTCSA .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. 160 170 180 190 200 Cyno FSFYPPELQLRFLRNGMAAGTGQGDFGPNSDGSFHASSSLTVKSGDEHHY .cndot. Human FSFYPPELQLRFLRNGLAAGTGQGDFGPNSDGS- FHASSSLTVKSGDEHHY .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. 210 220 230 240 250 Cyno CCIVQHAGLAQPLRVELETPAKSS .cndot. Human CCIVQHAGLAQPLRVELESPAKSS .DELTA. .DELTA. 260 270 Transmembrane/Intracellular Cyno VLVVGIVIGVLLLTAAAVGGALLWRRMR- SGLPAPWISLRGDDTGSLLPTP 0 Human VLVVGIVIGVLLLTAAAVGGALLWRRMRSGLPAPWISLR- GDDTGVLLPTP .DELTA. .DELTA. .DELTA. .DELTA. .DELTA. 280 290 300 310 320 Cyno GEAQDADSKDINVIPATA .cndot. .cndot. Human GEAQDADLKDVNVIPATA .DELTA. .DELTA. 330 340

[0212] The human amino acid sequence for FcRn has Accession No.: U12255. Story C. M., Mikulska J., Simister N. E., A major histocompatibility complex class I-like Fc receptor cloned from human placenta: Possible role in transfer of immunoglobulin G from mother to fetus, J. Exp. Med. 180, 2377-2381 (1994).

Example 3

Cynomolgus Fe.gamma.RI And Human Fc.gamma.RI Bind Human IgG Subclasses Equivalently

[0213] Materials and Methods:

[0214] Human IgG2, IgG3, and IgG4 isotypes of E27 (IgG 1) were constructed by subcloning the appropriate heavy chain Fc cDNA from a human spleen cDNA library into a pRK vector containing the E27 variable heavy domain. All IgG subclasses and variants were expressed using the same E27 .kappa. light chain as described in 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 or U.S. Pat. No. 6,194,551.

[0215] Following cotransfection of heavy and light chain plasmids into 293 cells, IgG1, IgG2, IgG4 and variants were purified by protein A chromatography. IgG3 was purified using protein G chromatography. All protein preparations were analyzed using a combination of SDS-polyacrylamide gel electrophoresis, ELISA, and spectroscopy.

[0216] The cDNA for Human Fc.gamma.RI was isolated by reverse transcriptase-PCR (GeneAmp, PerkinElmer Life Sciences) of oligo(dT)-primed RNA from U937 cells using primers that generated a fragment encoding the .alpha.-chain extra-cellular domain. Human Fc.gamma.R extracellular domains bound to Gly/6-His/GST fusions were prepared as described in 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 or U.S. Pat. No. 6,194,551. The cDNA was subcloned into previously described pRK mammalian cell expression vectors, as described in Eaton et al., 1986, Biochemistry, 25:8343-8347. The cDNA for cynomolgus Fc.gamma.RI was isolated as described in Example 1.

[0217] To facilitate the purification of the expressed human and cynomologus Fc.gamma.RI, the transmembrane domain and intracellular domain of each were replaced by DNA encoding a Gly-His.sub.6 tag and human glutathione S-transferase (GST). The GST sequence was obtained by PCR from the pGEX4T2 plasmid (Amersham Pharmacia Biotech) with NheI and XbaI restriction sites at the 5' and 3' ends, respectively. The expressed Fc.gamma.RI contained the extracellular domains of the .alpha.-chain fused at His271 to Gly/His.sub.6/GST. Primers used to subclone the extracellular portion of the cynomolgus Fc.gamma.RI .alpha.-chain are shown in Table 1.

[0218] The cynomolgus and human Fc.gamma.RI plasmids were transfected into human embryonic kidney 293 cells by calcium phosphate precipitation (Gorman, C. M., Gies, D. R., and McCray, G. (1990) DNA Prot. Engineer. Tech. 2, 3-10). Supernatants were collected 72 hours after conversion to serum-free PSO.sub.4 medium supplemented with 10 mg/liter recombinant bovine insulin, 1 mg/liter human transferrin, and trace elements. Proteins were purified by nickel-nitrilotriacetic acid chromatography (Qiagen, Valencia, Calif.). Purified protein was analyzed through a combination of 4-20% SDS-polyacrylamide gel electrophoresis, ELISA, and amino acid analysis.

[0219] Standard enzyme-linked immunoabsorbent assays (ELISA) were performed in order to detect and quantify interactions between cynomologus Fc.gamma.RI or human Fc.gamma.RI and human IgG1, IgG2, IgG3, or IgG4 (Table 15). ELISA plates (Nunc) were coated with 150 ng/well by adding 100 .mu.L of 1.5 .mu.g/ml stock solution cynomologus Fc.gamma.RI or human Fc.gamma.RI in PBS for 48 hours at 4.degree. C. After washing plates five times with wash buffer, (PBS, pH 7.4 containing 0.5% Tween-20), plates were blocked with 250 .mu.L of assay buffer (50 mM Tris-buffered saline, 0.05% Tween-20, 0.5% RIA-grade bovine serum albumin, 2 mM EDTA, pH 7.4) at 25.degree. C. for 1 hours. Plates were washed five times with wash buffer.

[0220] Serial 3-fold dilutions of monomeric antibody (10.0-0.0045 .mu.g/ml) were added to plates and incubated for 2 hours. After washing plates five times with assay buffer, the detection reagent was added. Several different horseradish peroxidase (HRP)-conjugated reagents were used to detect the IgG-Fc.gamma.RI interaction, including: HRP-Protein G (Bio-Rad), goat HRP-anti-human IgG (Boehringer-Mannheim, Indianapolis, Ind.), and murine HRP-anti-human Kappa light chain. After incubation with detecting reagent at 25.degree. C. for 90 minutes, plates were washed five times with wash buffer and 100 .mu.l of 0.4 mg/ml o-phenylenediamine dihydrochloride (Sigma, St. Louis, Mo.) was added. Absorbance at 490 nm was read using a Vmax plate reader (Molecular Devices, Mountain View, Calif.). Note that values reported in Table 15 are the mean+deviation relative to binding of human IgG1 at an IgG1 concentration of 0.370 .mu.g/ml. Titration plots for human IgG using murine HRP-anti-human Kappa light chain as detecting reagent are shown for cynomolgus Fc.gamma.RI (FIG. 1B) and human Fc.gamma.RI (FIG. 1A).

[0221] Results and Discussion:

[0222] As illustrated in Table 15, the pattern of binding of cynomolgus Fc.gamma.RI and human Fc.gamma.RI to the four human IgG subclasses was similar, regardless of the detection reagent. In each case, human or cynomolgus showed the highest level of binding to IgG3 and the lowest level of binding to IgG2. In particular, the pattern for both human and cynomolgus receptor-IgG interaction was IgG3.gtoreq.IgG1>IgG4>>&- gt;IgG2. Note that the data from the human Fc.gamma.RI-IgG binding interactions corresponds to data previously reported. Gessner et al, 1998, Ann. Hematol. 76:231-248; Deo et al., 1997, Immunology Today 18:127-135; Van de Winkel, 1993, Immunology Today 14:215-221.

19TABLE 15 Binding of monomeric human IgG subclasses to cynomolgus and human Fc.gamma.RI.sup.a Cynomolgus Fc.gamma.RI Human Fc.gamma.RI Subclass ProtG.sup.b anti-huIgG anti-kappa ProtG E27IgG1 1.00 1.00 1.00 1.00 E27IgG2 0.13 .+-. 0.04 0.04, 0.04 0.11, 0.14 0.08, 0.08 E27IgG3 1.01 .+-. 0.06 1.22, 1.15 1.32, 1.37 1.14, 1.03 E27IgG4 0.52 .+-. 0.04 0.44, 0.45 0.60, 0.63 0.27, 0.27 .sup.aDetection reagents were HRP-conjugated Protein G (ProtG), HRP-conjugated murine anti-human IgG, heavy chain specific (anti-huIgG), or HRP-conjugated murine anti-human kappa light chain (anti-kappa). Values are the ratio of OD.sub.490 nm (E27IgG subclass) to OD.sub.490 nm (E27IgG1) at 0.37 .mu.g/ml. .sup.bMean .+-. S.D., n = 4.

[0223] As illustrated in FIGS. 1A and 1B, binding affinity of the human and cynomolgus Fc.gamma.RI is similar for each of the tested IgG subclasses. In both cases, human and cynomolgus receptors showed a markedly higher affinity for IgG3 and IgG1 as compared to the IgG4 and IgG2. FIGS. 1A and 1B also shows that the IgG subclass binding to Fc.gamma.RI is concentration-dependent and saturable.

[0224] This data illustrates that cynomolgus Fc.gamma.RI can replace human Fc.gamma.RI in the detection of IgG subclasses as human and cynomolgus reveal similar binding patterns of interaction with similar affinities for each IgG subclass.

Example 4

Cynomolgus Fc.gamma.RIIA Binds Human IgG2

[0225] Materials and Methods

[0226] ELISA assays analyzing human IgG subclass binding to cynomolgus Fc.gamma.RIIA were performed using essentially the methods as described in Example 3. However, because Fc.gamma.RIIA is a low-affinity Fc.gamma.R, hexameric complexes of each human IgG subclass was formed prior to addition to the Fc receptor. Hexameric complexes were formed by mixing the human IgG subclass with a human IgG at a 1:1 molar ratio. Liu, J., Lester, P., Builder, S., and Shire, S. J. (1995) Biochemistry 34:10474-10482. Preparation of the hexameric complexes and their use in Fc.gamma.RII and Fc.gamma.RIII assays were as described in 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. A plasmid encoding human Fc.gamma.RIIA(R131) can be readily prepared using the sequence information as described in GenBank or other published sources and see Warmerdam et al., 1991 J. of Immunology 147:1338-1343 and Clark et al., 1991 J of Immunology 21:1911-1916.

[0227] Results and Discussion:

[0228] As illustrated by Table 16, the pattern of cynomolgus Fc.gamma.RIIA binding to hexameric complexes of the human IgG subclasses was IgG3=IgG2>IgG1>IgG4. Previous analysis of human IgG subclass binding to the two polymorphic human Fc.gamma.RIIA forms showed the pattern: human Fc.gamma.RIIA(R131)-IgG3.gtoreq.IgG1>>>IgG2.gtore- q.IgG4 and Fc.gamma.RIIA(H 131)-IgG3.gtoreq.IgG1=IgG2>>>IgG4. Gessner et al, 1998, Ann. Hematol. 76:231-248; Deo et al., 1997, Immunology Today 18:127-135; Van de Winkel, 1993, Immunology Today 14:215-221. These binding patterns show that cynomolgus Fc.gamma.RIIA, which has a histidine at amino acid 131, is comparable to the human Fc.gamma.RIIA(H131), both of which bind human IgG2. In contrast, human Fc.gamma.RIIA(R131) has been reported to bind human IgG2 poorly. Note also that cynomolgus Fc.gamma.RIIA binds human IgG2 as efficiently as it binds human IgG3, a difference from the human Fc.gamma.RIIA(H 131) receptor.

20TABLE 16 Binding of hexameric complexes of human IgG subclasses to cynomolgus and human Fc.gamma.RIIA.sup.a Subclass ProtG anti-huIgG anti-kappa Cynomolgus Fc.gamma.RIIA E27IgG1 1.00 1.00 1.00 E27IgG2 2.11 1.27 2.20 .+-. 0.93.sup.b E27IgG3 1.10 1.56 2.44 .+-. 0.47 E27IgG4 0.12 0.12 0.42 .+-. 0.18 Human Fc.gamma.RIIA(H131) E27IgG1 1.00 1.00 1.00 E27IgG2 0.95 0.83 0.84 E27IgG3 0.78 1.03 0.98 E27IgG4 0.25 0.47 0.19 Human Fc.gamma.RIIA(R131) E27IgG1 1.00 1.00 1.00 E27IgG2 0.63 0.40 0.47 E27IgG3 1.17 1.14 0.85 E27IgG4 0.59 0.44 0.27 .sup.aDetection reagents were HRP-conjugated Protein G (ProtG), HRP-conjugated murine anti-human IgG, heavy chain specific (anti-huIgG), or HRP-conjugated murine anti-human kappa light chain (anti-kappa). Values are the ratio of OD.sub.490 nm (E27IgG subclass) to OD.sub.490 nm (E27IgG1) at 0.123 .mu.g/ml. .sup.bMean .+-. SD, n = 3.

[0229] The binding of cynomolgus Fc.gamma.RIIA to each IgG subclass generally increased as the concentration of each antibody subclass increased (FIG. 2).

[0230] The data from table 16 and FIG. 2 illustrates that cynomolgus Fc.gamma.RIIA binds human IgG2 and IgG3 with high efficiency and may be a preferable agent for use in detecting these human subclasses to either of the two human polymorphic forms of Fc.gamma.RIIA.

Example 5

Cynomolgus Fc.gamma.RIIB Binds Human IgG2

[0231] Materials and Methods

[0232] The methods used to detect Fc.gamma.RIIB binding to human IgG subclasses was essentially as shown in Examples 3 and 4. Plasmid encoding human Fc.gamma.RIIB is known and readily obtainable by those of skill in the art and see Kurucz et al., 2000, Immunol Lett 75(1):33-40. Data reported in Table 17-represent the mean.+-.deviation relative to binding of human IgG1 at an IgG1 concentration of 0.370 .mu.g/ml.

[0233] Results and Discussion:

[0234] Table 17 illustrates the binding of hexameric complexes of the human IgG subclasses to human and cynomolgus Fc.gamma.RIIB. The binding pattern between the IgG subclasses and human Fc.gamma.RIIB is IgG3.gtoreq.IgG1>IgG2>IgG4 and between the IgG subclasses and cynomolgus Fc.gamma.RIIB is IgG2.gtoreq.IgG3>IgG1>IgG4. This binding pattern was the same for both human (FIG. 3A) and cynomolgus (FIG. 3B) over a range of IgG concentrations.

[0235] This data illustrates that cynomolgus Fc.gamma.RIIB has a stronger binding affinity for IgG2 than does human Fc.gamma.RIIB.

21TABLE 17 Binding of Hexameric Complexes of Human IgG Subclasses to Cynomolgus and Human Fc.gamma.RIIB Cynomolgus Fc.gamma.RIIB Human Fc.gamma.RIIB Subclass ProtG.sup.b anti-huIgG.sup.c anti-kappa.sup.d ProtG.sup.d E27IgG1 1.00 1.00 1.00 1.00 E27IgG2 1.89 .+-. 0.37 1.26 .+-. 0.15 2.73 .+-. 1.00 0.43 .+-. 0.10 E27IgG3 1.25 .+-. 0.17 1.69 .+-. 0.20 2.99 .+-. 1.26 1.03 .+-. 0.13 E27IgG4 0.48 .+-. 0.11 0.58 .+-. 0.16 0.64 .+-. 0.21 0.23 .+-. 0.08 .sup.aDetection reagents were HRP-conjugated Protein G (ProtG), HRP-conjugated murine anti-human IgG, heavy chain specific (anti-huIgG), or HRP-conjugated murine anti-human kappa light chain (anti-kappa). Values are the ratio of OD.sub.490 nm (E27IgG subclass) to OD.sub.490 nm (E27IgG1) at 0.37 .mu.g/ml. .sup.bMean .+-. SD, n = 8. .sup.cMean .+-. SD, n = 5. .sup.dMean .+-. SD, n = 3.

Example 6

Cynomolgus Fc.gamma.RIIIA And Human Fc.gamma.RIIIA-V158 Exhibit Equivalent Binding To Human IgG Subclasses

[0236] Materials and Methods:

[0237] The methods used to detect Fc.gamma.RIIIA binding to human IgG subclasses was essentially as shown in Examples 3 and 4. As described previously, a human DNA sequence for Fc.gamma.RIIA .alpha.-chain is known and readily obtainable by those of skill in the art. Data reported in Table 18 represents the mean.+-.deviation relative to binding of human IgG1 at an IgG1 concentration of 0.370 .mu.g/ml.

[0238] Results and Discussion:

[0239] As illustrated in Table 18, cynomolgus Fc.gamma.RIIIA and human Fc.gamma.RIIIA-V 158 both bind human IgG subclasses with essentially the same pattern, IgG1>IgG3>>IgG2.gtoreq.IgG4, as compared to human Fc.gamma.RIIIA-F158, which binds with the pattern, IgG3=IgG1>>>IgG2=IgG4. The human Fc.gamma.RIIIA-F158-human IgG subclass binding data is in agreement with previous reports. Gessner et al, 1998, Ann. Hematol. 76:231-248; Deo et al., 1997, Immunology Today 18:127-135; Van de Winkel, 1993, Immunology Today 14:215-221. FIGS. 4A, 4B, and 4C illustrate the binding pattern for human Fc.gamma.RIIIA-F158, human Fc.gamma.RIIIA-V158, and cynomolgus Fc.gamma.RIIIA, respectively, for increasing concentrations of each IgG subclass and indicate that the binding interactions are specific and concentration dependent and saturable.

[0240] The data illustrates that cynomolgus Fc.gamma.RIIIA and human Fc.gamma.RIIIA-V158 have equivalent binding interactions with the human IgG subclasses, and in particular that cynomolgus Fc.gamma.RIIIA has preferred binding to the IgG2 subclass as compared to the human Fc.gamma.RIIIA.

22TABLE 18 Binding of Hexameric Complexes of Human IgG Subclasses to Cynomolgus and Human Fc.gamma.RIIIA Subclass Cynomolgus.sup.b Human(F158).sup.c Human(V158).sup.c E27IgG1 1.00 1.00 1.00 E27IgG2 0.11 .+-. 0.02 0.06, 0.13 0.06, 0.03 E27IgG3 0.82 .+-. 0.08 0.75, 0.82 0.79, 0.82 E27IgG4 0.15 .+-. 0.04 0.06, 0.11 0.06, 0.04 .sup.aDetection reagent was HRP-conjugated Protein G. Values are the ratio of OD.sub.490 nm (E27IgG subclass) to OD.sub.490 nm (E27IgG1) at 0.37 .mu.g/ml for cynomolgus Fc.gamma.RIIIA and human Fc.gamma.RIIIA(V158) and 1.11 .mu.g/ml for human Fc.gamma.RIIIA(F158). .sup.bMean .+-. SD, n = 4. .sup.cHuman(F158) and Human(V158) are polymorphic forms of human Fc.gamma.RIIIA with phenylalanine or valine at receptor position 158.

Example 7

Cynomolgus Fc.gamma.RIIA Binds Human IgG1 Variants S298A and S298A/E333A/K334A

[0241] Materials and Methods:

[0242] Site-directed mutagenesis on E27 IgG1 was essentially as described in Shields et al., 2001, J. Biol. Chem., 276:6591-6604. Briefly, site-directed mutagenesis was used to generate IgG1 variants in which a number of solvent-exposed residues in the CH2 and CH3 domains were individually altered to alanine. The alanine variants were D265A, S298A, S37A, R292A, D280A and S298A/E333A.

[0243] ELISA reactions were essentially as described in Examples 3-6, where IgG variants were incubated with the Fc receptors, rather than native IgG protein. Note that for the values provided in Table 19, human receptors are (Absorbance Variant/Absorbance Native IgG1) at 1 .mu.g/ml and for cynomolgus receptors, values are (Absorbance Variant/Absorbance Native IgG1) at 0.370 .mu.g/ml.

[0244] Results and Discussion:

[0245] As illustrated by Table 19 and FIGS. 5-7, the binding pattern of all IgG variants to cynomolgus Fc.gamma.RI was similar to that for human Fc.gamma.RI. With regard to IgG variant binding to cynomolgus Fc.gamma.RIIA, the pattern generally followed the same pattern for human polymorph Fc.gamma.RIIA(H131). (FIG. 5). As above, this likely reflects the fact that the cynomolgus Fc.gamma.RIIA has a histidine as residue 131. Note, however, that there were two notable exceptions, variant S298A and variant S298A/E333A/K334A had improved binding to the cynomolgus Fc.gamma.RIIA as compared to native human IgG1, and these same variants bound poorly to human Fc.gamma.RIIA.

[0246] Referring to Table 19 and FIG. 6, the pattern of variant IgG binding to cynomolgus Fc.gamma.RIIB exhibited several differences from the binding pattern for human Fc.gamma.RIIB. In particular, variants R255A, E255A, E258A, S37A, D280A, and R301A bound the cynomolgus Fc.gamma.RIIB equivalently as they had native human IgG, whereas these same variants all exhibited improved binding to the human Fc.gamma.RIIB when compared to native human IgG.

[0247] Referring to Table 19 and FIG. 7, the binding pattern of the variant IgG to cynomolgus Fc.gamma.RIIIA followed the binding pattern established for human polymorph Fc.gamma.IIIA-V 158, as compared to the binding pattern for human polymorph Fc.gamma.IIIA-F 158. This likely reflects the fact that the cynomolgus Fc.gamma.RIIIA has a similar amino acid residue, isoleucine, at position 158 as does human Fc.gamma.RIIIA-V158 (compared to the phenylalanine located in Fc.gamma.RIIIA-F158).

[0248] Blocking the inhibitory signals (e.g., ITIM-containing Fc.gamma.RIIB) mediated by Fc receptors, which counterbalance the activating signals (e.g., ITAM-containing Fc.gamma.RI, Fc.gamma.RIIA, and Fc.gamma.RIIIA) mediated by Fc receptors, may provide for improved therapeutic efficacy of antibodies. An unexpected result shown in Table 19 is that variants having S298A showed improved binding to cynomolgus Fc.gamma.RIIA, maintained native-like binding to cynomolgus Fc.gamma.RI and Fc.gamma.RIIIA, and showed significantly decreased binding to cynomolgus Fc.gamma.RIIB. Two variants in particular, S298A and S298A/E333A/K334A may be used to selectively engage the activating ITAM-containing Fc receptors, while simultaneously not engaging the inhibitory ITIM-containing Fc.gamma.RIIB.

23TABLE 19 Binding of Human E27 IgG1 Variants to Human and Cynomolgus Fc.gamma.R Variant Fc.gamma.RI Fc.gamma.RIIA Fc.gamma.RIIB Fc.gamma.RIIIA S239A Human 0.81 .+-. 0.09 0.73 .+-. 0.25 0.76 .+-. 0.36 0.26 .+-. 0.08 Cynomolgus N/A 0.68 .+-. 0.04 N/A N/A R255A Human 0.99 .+-. 0.12 1.30 .+-. 0.20 1.59 .+-. 0.42 0.98 + 0.18 Cynomolgus 0.85 .+-. 0.15 1.09 .+-. 0.07 0.80 .+-. 0.06 0.91 .+-. 0.08 E258A Human 1.18 .+-. 0.13 1.33 .+-. 0.22 1.65 .+-. 0.38 1.12 .+-. 0.12 Cynomolgus 0.91 .+-. 0.08 0.88 .+-. 0.05 0.99 .+-. 0.07 0.93 .+-. 0.11 D265A Human 0.16 .+-. 0.05 0.07 .+-. 0.01 0.13 .+-. 0.05 0.09 .+-. 0.06 Cynomolgus N/A 0.05 .+-. 0.02 0.05 0.04 .+-. 0.01 S37A Human 1.09 .+-. 0.08 1.52 .+-. .22(R) 1.84 .+-. 0.43 1.05 .+-. 0.24 1.10 .+-. .12(H) Cynomolgus 1.02 .+-. 0.09 1.23 .+-. 0.34 1.04 .+-. 0.30 0.88 .+-. 0.11 H268A Human 1.10 .+-. 0.11 1.21 .+-. .14(R) 1.44 .+-. 0.22 0.54 .+-. 0.12 0.97 .+-. .15(H) Cynomolgus 1.02 .+-. 0.09 0.99 .+-. 0.07 1.20 0.86 .+-. 0.07 D280A Human 1.04 .+-. 0.08 1.34 .+-. 0.14 1.60 .+-. 0.31 1.09 .+-. 0.20 Cynomolgus 0.97 .+-. 0.08 1.45 .+-. 0.18 1.20 .+-. 0.11 0.99 .+-. 0.04 R292A Human 0.95 .+-. 0.05 0.27 .+-. 0.13 0.17 .+-. 0.07 0.89 .+-. 0.17 Cynomolgus 0.87 .+-. 0.08 0.80 .+-. 0.23 0.63 .+-. 0.06 0.90 .+-. 0.09 E293A Human 1.11 .+-. 0.07 1.08 .+-. 0.19 1.07 .+-. 0.20 0.31 .+-. 0.13 Cynomolgus N/A 0.92 .+-. 0.07 N/A N/A S298A Human 1.11 .+-. 0.03 0.40 .+-. .15(R) 0.23 .+-. 0.13 1.34 .+-. 0.20(F) 0.24 .+-. .08(H) 1.07 .+-. .07(V) Cynomolgus 1.06 .+-. 0.09 2.07 .+-. 0.30 0.20 .+-. 0.09 0.98 .+-. 0.13 R301M Human 1.06 .+-. 0.12 1.29 .+-. 0.17 1.56 .+-. 0.12 0.48 .+-. 0.21 Cynomolgus 1.00 .+-. 0.09 1.62 .+-. 0.30 1.27 .+-. 0.20 0.85 .+-. 0.08 P329A Human 0.48 .+-. 0.10 0.08 .+-. 0.02 0.12 .+-. 0.08 0.21 .+-. 0.03 Cynomolgus N/A 0.21 .+-. 0.06 N/A N/A E333A Human 0.98 .+-. 0.15 0.92 .+-. 0.12 0.76 .+-. 0.11 1.27 .+-. 0.17 Cynomolgus N/A 0.67 .+-. 0.09 N/A N/A K334A Human 1.06 .+-. 0.07 1.01 .+-. 0.15 0.90 .+-. 0.12 1.39 .+-. 0.19(F) 1.10 .+-. .07(V) Cynomolgus 1.08 .+-. 0.09 0.92 .+-. 0.15 0.66 .+-. 0.14 1.00 .+-. 0.15 A339T Human 1.06 .+-. 0.04 1.09 .+-. 0.03 1.20 .+-. 0.03 1.34 .+-. 0.09 Cynomolgus N/A 1.05 .+-. 0.02 N/A N/A S298A/E333A/K334A Human N/A 0.35 .+-. 0.13 0.18 .+-. 0.08 1.51 .+-. 0.31(F) 1.11 .+-. .08(V) Cynomolgus 1.19 .+-. 0.08 1.99 .+-. 0.24 0.12 .+-. 0.04 1.08 .+-. 0.15

Example 8

Cynomolgus FcRn And Human FcRn Bind Human IgG Subclasses Equivalently

[0249] Materials and Methods:

[0250] Human IgG2, IgG3, and IgG4 isotypes of E27 (IgG1) were constructed by subcloning the appropriate heavy chain Fc cDNA from a human spleen cDNA library into a pRK vector containing the E27 variable heavy domain. All IgG subclasses and variants were expressed using the same E27 .kappa. light chain.

[0251] Following cotransfection of heavy and light chain plasmids into 293 cells, IGGI, IgG2, IgG4 and variants were purified by protein A chromatography. IgG3 was purified using protein G chromatography. All protein preparations were analyzed using a combination of SDS-polyacrylamide gel electrophoresis, ELISA, and spectroscopy.

[0252] Herceptin.TM. IgG1 was essentially constructed as described in Coussens et al., 1985, Science, 230:1132-39. Herceptin.TM. IgG1 is a recombinant DNA-derived monoclonal antibody having an IgG1 .kappa. chain that contains a consensus amino acid framework with complementary-determining regions of a murine antibody (4D5) that binds HER2.

[0253] The cDNA for cynomologus FcRn was isolated by reverse transcriptase-PCR (GeneAmp, PerkinElmer Life Sciences) of oligo(dT)-primed RNA from cynomologus spleen cells using primers that generated a fragment encoding the .alpha.-chain extra-cellular domain as described in Example 1. The cDNA was subcloned into previously described pRK mammalian cell expression vectors, as described in Eaton et al., 1986, Biochemistry, 25:8343-8347. Two DNA sequences were identified and confirmed that differed at base 77, one sequence had base G, giving Ser 3 in the mature polypeptide, and the other had base A giving Aspargine 3 in the mature polypeptide. The cDNA for cynomolgus FcRn (S3) and FcRn (N3) were isolated essentially as described in Example 1.

[0254] The cynomolgus and human FcRn plasmids were transfected into human embryonic kidney cells by calcium phosphate precipitation (Gorman, C. M., Gies, D. R., and McCray, G, 1990, DNA Prot. Engineer. Tech., 2:3-10). Supernatants were collected 72 hours after conversion to serum-free PSO.sub.4 medium supplemented with 10 mg/liter recombinant bovine insulin, 1 mg/liter human transferrin, and trace elements. Proteins were purified using nickel nitrothiacetic acid chromatography (Qiagen, Valencia, Calif.). Purified protein was analyzed through a combination of 4-20% SDS-polyacrylamide gel electrophoresis, ELISA, and amino acid analysis.

[0255] Standard enzyme-linked immunoabsorbent assays (ELISA) were performed in order to detect and quantify interactions between cynomolgus FcRn (S3), FcRn (N3) or human FcRn and human IgG1 (including herceptin IgG1), IgG2, IgG3, or IgG4 (table 20). ELISA plates (Nunc) were coated with 2 .mu.g/ml streptavidin (Zymed Laboratories Inc., South San Francisco, Calif.) in 50 mM carbonate buffer, pH 9.6, at 4.degree. C. overnight. Plates were blocked with PBS, 0.5% BSA, 10 ppm Proclin 300 (Supelco, Bellefonte, Pa.), pH 7.2 at 25.degree. C. for 1 h. FcRn-Gly-His.sub.6 was biotynylated using a standard protocol with biotin-X--NHS (Research Organics, Cleveland, Ohio) and bound to streptavidin coated plates at 2 .mu.g/ml in PBS, 0.5 BSA, 0.05% polysorbate-20 (sample buffer), pH 7.2 at 25.degree. C. for 1 h. Plates were then rinsed with sample buffer, pH 6.0. Eight serial 2-fold dilutions of E27 standard or variants in sample buffer at pH 6.0 were incubated for 2 h. Plates were rinsed with sample buffer pH 6.0 and bound IgG was detected with peroxidase-conjugated goat F(ab').sub.2 anti-human IgG F(ab').sub.2 (Jackson ImmunoResearch) in pH 6.0 sample buffer using 3,3',5,5'-tetramethlbenzidine (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) as substrate. Absorbance at 450 nm was read on a V.sub.max plate reader (Molecular Devices).

[0256] The data shown in Table 20 was plotted as saturation binding curves.

[0257] Results and Discussion:

[0258] As illustrated in Table 20 and corresponding FIGS. 8-10, the pattern of binding of cynomolgus FcRn (S3), FcRn (N3) and human FcRn to the four human IgG subclasses was similar. In each case, human and cynomolgus FcRns showed the highest level of binding to IgG3 and the lowest level of binding to IgG1. In particular, the pattern for both human and cynomolgus receptor-IgG interaction was IgG3>>IgG4>IgG- 2>IgG1. Note that the data from the human FcRn-IgG binding interactions corresponds to data previously reported. AP West Jr. arid P. J. Bjorkman Biochemistry 39:9698 (2000).

[0259] In addition, the data illustrates that the binding affinity of the human and cynomolgus FcRns is similar for IgG1, IgG2, and IgG3, and is slightly stronger for IgG4, as compared to the human FcRn for IgG4. As illustrated graphically in FIGS. 8-10, binding of the human and cynomolgus FcRns to the human IgG subclasses is concentration-dependent and saturable.

24TABLE 20 Binding of Human IgG Subclasses to Human FcRn Subclass Cyno S3.sup.a Cyno N3.sup.a Human.sup.b Human.sup.c E27IgG1 1.00, 1.00 1.00, 1.00 1.00 1.00 E27IgG2 1.30, 1.15 1.49, 1.39 1.06 .+-. 0.10 0.93 .+-. 0.16 E27IgG3 3.82, 3.59 4.34, 3.97 5.60 .+-. 1.31 1.55 .+-. 0.45 E27IgG4 1.52, 1.44 1.59, 1.62 1.06 .+-. 0.23 0.95 .+-. 0.14 .sup.aAssay with NeutrAvidin coated on plate followed by FcRn-biotin, then sample and detection with HRP-conjugated goat anti-human F(ab').sub.2. Values are the ratio of OD.sub.490 nm (E27IgG subclass) to OD.sub.490 nm (E27IgG1) at [mAb] = 50 ng/ml for two assays. Cyno S3 and N3 differ only in the amino acid at position 3. .sup.bAssay with NeutrAvidin coated on plate followed by FcRn-biotin, then sample and detection with HRP-conjugated goat anti-human F(ab').sub.2. Values are the ratio of OD.sub.490 nm (E27IgG subclass) to OD.sub.490 nm (E27IgG1) at [mAb] = 50 ng/ml for five assays. A second, separate lot of E27IgG1 showed a ratio of 0.81 .+-. 0.03 (mean .+-. S.D., n = 3) compared to the E27IgG1 used as standard. .sup.cAssay with human IgE coated on the plate followed by sample, then FcRn-biotin and detection with HRP-conjugated streptavidin. Values are the ratio of OD.sub.490 nm (E27IgG subclass) to OD.sub.490 nm (E27IgG1) at [mAb] = 50 ng/ml for four assays. A second, separate lot of E27IgG1 showed ratios of 0.92 and 0.88 compared to the E27IgG1 used as standard.

[0260] This data illustrates that cynomolgus FcRn can replace human FcRn in the detection of human IgG subclasses as human and cynomolgus FcRn reveal similar binding patterns of interaction with similar affinities for each IgG subclass.

[0261] It will be clear that the invention is well adapted to attain the ends and advantages mentioned as well as those inherent therein. While a presently preferred embodiment has been described for purposes of this disclosure, various changes and modifications may be made which are well within the scope of the invention. Numerous other changes may be made which will readily suggest themselves to those skilled in the art and which are encompassed in the spirit of the invention disclosed herein and as defined in the appended claims.

[0262] All publications cited herein are hereby incorporated by reference.

Sequence CWU 1

1

72 1 1074 DNA Cynomolgus misc_feature (1)..(1074) FcgammaRI alpha-chain 1 atgtggttct tgacagctct gctcctttgg gttccagttg atgggcaagt ggataccaca 60 aaggcagtga tcactttgca gcctccatgg gtcagcgtgt tccaagagga aactgtaacc 120 ttacagtgtg aggtgccccg tctgcctggg agcagctcca cacagtggtt tctcaatggc 180 acagccactc agacctcgac tcccagctac agaatcacct ctgccagtgt caaggacagt 240 ggtgaataca ggtgccagag aggtccctca gggcgaagtg accccataca gctggaaatc 300 cacagagact ggctactact gcaggtatcc agcagagtct tcacagaagg agaacctctg 360 gccttgaggt gtcatgcatg gaaggataag ctggtgtaca atgtgcttta ctatcaaaat 420 ggcaaagcct ttaagttttt ctaccggaat tctcaactca ccattctgaa aaccaacata 480 agtcacaacg gcgcctacca ctgctcaggc atgggaaagc atcgctacac atcagcagga 540 gtatctgtca ctgtgaaaga gctatttcca gctccagtgc tgaatgcatc cgtgacatcc 600 ccgctcctgg aggggaatct ggtcaccctg agctgtgaaa caaagttgct tctgcagagg 660 cctggtttgc agctttactt ctccttctac atgggcagca agaccctgcg aggcaggaac 720 acgtcctctg aataccaaat actaactgct agaagagaag actctgggtt ttactggtgc 780 gaggccacca cagaagacgg aaatgtcctt aagcgcagcc ctgagttgga gcttcaagtg 840 cttggcctcc agttaccaac tcctgtctgg cttcatgtcc ttttctatct ggtagtggga 900 ataatgtttt tagtgaacac tgttctctgg gtgacaatac gtaaagaact gaaaagaaag 960 aaaaagtgga atttagaaat atctttggat tctgctcatg agaagaaggt aacttccagc 1020 cttcaagaag acagacattt agaagaagag ctgaagagtc aggaacaaga ataa 1074 2 1128 DNA Homo sapiens misc_feature (1)..(1128) FcgammaRI alpha-chain 2 atgtggttct tgacaactct gctcctttgg gttccagttg atgggcaagt ggacaccaca 60 aaggcagtga tcactttgca gcctccatgg gtcagcgtgt tccaagagga aaccgtaacc 120 ttgcactgtg aggtgctcca tctgcctggg agcagctcta cacagtggtt tctcaatggc 180 acagccactc agacctcgac ccccagctac agaatcacct ctgccagtgt caatgacagt 240 ggtgaataca ggtgccagag aggtctctca gggcgaagtg accccataca gctggaaatc 300 cacagaggct ggctactact gcaggtctcc agcagagtct tcacggaagg agaacctctg 360 gccttgaggt gtcatgcgtg gaaggataag ctggtgtaca atgtgcttta ctatcgaaat 420 ggcaaagcct ttaagttttt ccactggaat tctaacctca ccattctgaa aaccaacata 480 agtcacaatg gcacctacca ttgctcaggc atgggaaagc atcgctacac atcagcagga 540 atatctgtca ctgtgaaaga gctatttcca gctccagtgc tgaatgcatc tgtgacatcc 600 ccactcctgg aggggaatct ggtcaccctg agctgtgaaa caaagttgct cttgcagagg 660 cctggtttgc agctttactt ctccttctac atgggcagca agaccctgcg aggcaggaac 720 acatcctctg aataccaaat actaactgct agaagagaag actctgggtt atactggtgc 780 gaggctgcca cagaggatgg aaatgtcctt aagcgcagcc ctgagttgga gcttcaagtg 840 cttggcctcc agttaccaac tcctgtctgg tttcatgtcc ttttctatct ggcagtggga 900 ataatgtttt tagtgaacac tgttctctgg gtgacaatac gtaaagaact gaaaagaaag 960 aaaaagtggg atttagaaat ctctttggat tctggtcatg agaagaaggt aatttccagc 1020 cttcaagaag acagacattt agaagaagag ctgaaatgtc aggaacaaaa agaagaacag 1080 ctgcaggaag gggtgcaccg gaaggagccc cagggggcca cgtagcag 1128 3 933 DNA Cynomolgus misc_feature (1)..(933) FcgammaRIIA 3 atgtctcaga atgtatgtcc cggcaacctg tggctgcttc aaccattgac agttttgctg 60 ctgctggctt ctgcagacag tcaaactgct cccccgaagg ctgtgctgaa actcgagccc 120 ccgtggatca acgtgctccg ggaggactct gtgactctga cgtgcggggg cgctcacagc 180 cctgacagcg actccactca gtggttccac aatgggaatc gcatccccac ccacacacag 240 cccagctaca ggttcaaggc caacaacaat gatagcgggg agtacaggtg ccagactggc 300 cggaccagcc tcagcgaccc tgttcatctg actgtgcttt ctgagtggct ggcgcttcag 360 acccctcacc tggagttccg ggagggagaa accatcatgc tgaggtgcca cagctggaag 420 gacaagcctc tgatcaaggt cacattcttc cagaatggaa tagccaagaa attttcccat 480 atggatccca atttctccat cccacaagca aaccacagtc acagtggtga ttaccactgc 540 acaggaaaca taggctacac accatactca tccaaacctg tgaccatcac tgtccaagtg 600 cccagcgtgg gcagctcttc accgatgggg atcattgtgg ctgtggtcac tgggattgct 660 gtagcggcca ttgttgctgc tgtagtggcc ttgatctact gcaggaaaaa gcggatttca 720 gccaattcca ctgatcctgt gaaggctgcc cgatttgagc cacttggacg tcaaacgatt 780 gccctcagaa agagacaact tgaagaaacc aacaatgact atgaaacagc cgacggcggc 840 tacatgactc tgaaccccag ggcacctact gatgatgata gaaacatcta cctgactctt 900 tctcccaacg actatgacaa cagtaataac taa 933 4 936 DNA Homo sapiens misc_feature (1)..(936) FcgammaRIIA 4 atgtctcaga atgtatgtcc cagaaacctg tggctgcttc aaccattgac agttttgctg 60 ctgctggctt ctgcagacag tcaagctgca gctcccccaa aggctgtgct gaaacttgag 120 cccccgtgga tcaacgtgct ccaggaggac tctgtgactc tgacatgcca gggggctcgc 180 agccctgaga gcgactccat tcagtggttc cacaatggga atctcattcc cacccacacg 240 cagcccagct acaggttcaa ggccaacaac aatgacagcg gggagtacac gtgccagact 300 ggccagacca gcctcagcga ccctgtgcat ctgactgtgc tttccgaatg gctggtgctc 360 cagacccctc acctggagtt ccaggaggga gaaaccatca tgctgaggtg ccacagctgg 420 aaggacaagc ctctggtcaa ggtcacattc ttccagaatg gaaaatccca gaaattctcc 480 cgtttggatc ccaccttctc catcccacaa gcaaaccaca gtcacagtgg tgattaccac 540 tgcacaggaa acataggcta cacgctgttc tcatccaagc ctgtgaccat cactgtccaa 600 gtgcccagca tgggcagctc ttcaccaatg gggatcattg tggctgtggt cattgcgact 660 gctgtagcag ccattgttgc tgctgtagtg gccttgatct actgcaggaa aaagcggatt 720 tcagccaatt ccactgatcc tgtgaaggct gcccaatttg agccacctgg acgtcaaatg 780 attgccatca gaaagagaca acttgaagaa accaacaatg actatgaaac agctgacggc 840 ggctacatga ctctgaaccc cagggcacct actgacgatg ataaaaacat ctacctgact 900 cttcctccca acgaccatgt caacagtaat aactaa 936 5 885 DNA Cynomolgus misc_feature (1)..(885) FcgammaRIIB 5 atgggaatcc tgtcattctt acctgtcctt gctactgaga gtgactgggc tgactgcaag 60 tcctcccagc cttggggcca catgcttctg tggacagctg tgctattcct ggctcctgtt 120 gctgggacac ctgcagctcc cccgaaggct gtgctgaaac tcgagccccc gtggatcaac 180 gtgctccggg aggactctgt gactctgacg tgcgggggcg ctcacagccc tgacagcgac 240 tccactcagt ggttccacaa tgggaatctc atccccaccc acacgcagcc cagctacagg 300 ttcaaggcca acaacaatga tagcggggag tacaggtgcc agactggccg gaccagcctc 360 agcgaccctg ttcatctgac tgtgctttct gagtggctgg cgctccagac ccctcacctg 420 gagttccggg agggagaaac catcttgctg aggtgccaca gctggaagga caagcctctg 480 atcaaggtca cattcttcca gaatggaata tccaagaaat tttcccatat gaatcccaac 540 ttctccatcc cacaagcaaa ccacagtcac agtggtgatt accactgcac aggaaacata 600 ggctacacac catactcatc caaacctgtg accatcactg tccaagtgcc cagcatgggc 660 agctcttcac cgatagggat cattgtggct gtggtcactg ggattgctgt agcggccatt 720 gttgctgctg tagtggcctt gatctactgc aggaaaaagc ggatttcagc caatcccact 780 aatcctgacg aggctgacaa agttggggct gagaacacaa tcacctattc acttctcatg 840 catccggacg ctctggaaga gcctgatgac caaaaccgng tttag 885 6 876 DNA Homo sapiens misc_feature (1)..(876) FcgammaRIIB 6 atgggaatcc tgtcattctt acctgtcctt gccactgaga gtgactgggc tgactgcaag 60 tccccccagc cttggggtca tatgcttctg tggacagctg tgctattcct ggctcctgtt 120 gctgggacac ctgcagctcc cccaaaggct gtgctgaaac tcgagcccca gtggatcaac 180 gtgctccagg aggactctgt gactctgaca tgccggggga ctcacagccc tgagagcgac 240 tccattcagt ggttccacaa tgggaatctc attcccaccc acacgcagcc cagctacagg 300 ttcaaggcca acaacaatga cagcggggag tacacgtgcc agactggcca gaccagcctc 360 agcgaccctg tgcatctgac tgtgctttct gagtggctgg tgctccagac ccctcacctg 420 gagttccagg agggagaaac catcgtgctg aggtgccaca gctggaagga caagcctctg 480 gtcaaggtca cattcttcca gaatggaaaa tccaagaaat tttcccgttc ggatcccaac 540 ttctccatcc cacaagcaaa ccacagtcac agtggtgatt accactgcac aggaaacata 600 ggctacacgc tgtactcatc caagcctgtg accatcactg tccaagctcc cagctcttca 660 ccgatgggga tcattgtggc tgtggtcact gggattgctg tagcggccat tgttgctgct 720 gtagtggcct tgatctactg caggaaaaag cggatttcag ccaatcccac taatcctgat 780 gaggctgaca aagttggggc tgagaacaca atcacctatt cacttctcat gcacccggat 840 gctctggaag agcctgatga ccagaaccgt atttag 876 7 765 DNA Cynomolgus misc_feature (1)..(765) FcgammaRIIIA alpha-chain 7 atgtggcagc tgctcctccc aactgctctg ctacttctag tttcagctgg catgcgggct 60 gaagatctcc caaaggctgt ggtgttcctg gagcctcaat ggtacagggt gctcgagaag 120 gaccgtgtga ctctgaagtg ccagggagcc tactcccctg aggacaattc cacacggtgg 180 tttcacaatg agagcctcat ctcaagccag acctcgagct acttcattgc tgctgccaga 240 gtcaacaaca gtggagagta caggtgccag acaagcctct ccacactcag tgacccggtg 300 cagctggaag tccatatcgg ctggctattg ctccaggccc ctcggtgggt gttcaaggag 360 gaagaatcta ttcacctgag gtgtcacagc tggaagaaca ctcttctgca taaggtcacg 420 tatttacaga atggcaaagg caggaagtat tttcatcaga attctgactt ctacattcca 480 aaagccacac tcaaagacag cggctcctac ttctgcaggg gacttattgg gagtaaaaat 540 gtatcttcag agactgtgaa catcaccatc actcaagatt tggcagtgtc atccatctca 600 tcattctttc cacctgggta ccaagtctct ttctgcctgg tgatggtact cctttttgca 660 gtggacacag gactatattt ctctatgaag aaaagcattc caagctcaac aagggactgg 720 gaggaccata aatttaaatg gagcaaggac cctcaagaca aatga 765 8 765 DNA Homo sapiens misc_feature (1)..(765) FcgammaRIIIA alpha-chain 8 atgtggcagc tgctcctccc aactgctctg ctacttctag tttcagctgg catgcggact 60 gaagatctcc caaaggctgt ggtgttcctg gagcctcaat ggtacagggt gctcgagaag 120 gacagtgtga ctctgaagtg ccagggagcc tactcccctg aggacaattc cacacagtgg 180 tttcacaatg agagcctcat ctcaagccag gcctcgagct acttcattga cgctgccaca 240 gtcgacgaca gtggagagta caggtgccag acaaacctct ccaccctcag tgacccggtg 300 cagctagaag tccatatcgg ctggctgttg ctccaggccc ctcggtgggt gttcaaggag 360 gaagacccta ttcacctgag gtgtcacagc tggaagaaca ctgctctgca taaggtcaca 420 tatttacaga atggcaaagg caggaagtat tttcatcata attctgactt ctacattcca 480 aaagccacac tcaaagacag cggctcctac ttctgcaggg ggctttttgg gagtaaaaat 540 gtgtcttcag agactgtgaa catcaccatc actcaaggtt tggcagtgtc aaccatctca 600 tcattctttc cacctgggta ccaagtctct ttctgcttgg tgatggtact cctttttgca 660 gtggacacag gactatattt ctctgtgaag acaaacattc gaagctcaac aagagactgg 720 aaggaccata aatttaaatg gagaaaggac cctcaagaca aatga 765 9 357 PRT Cynomolgus MISC_FEATURE (1)..(357) FcgammaRI <chain 9 Met Trp Phe Leu Thr Ala Leu Leu Leu Trp Val Pro Val Asp Gly Gln 1 5 10 15 Val Asp Thr Thr Lys Ala Val Ile Thr Leu Gln Pro Pro Trp Val Ser 20 25 30 Val Phe Gln Glu Glu Thr Val Thr Leu Gln Cys Glu Val Pro Arg Leu 35 40 45 Pro Gly Ser Ser Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55 60 Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val Lys Asp Ser 65 70 75 80 Gly Glu Tyr Arg Cys Gln Arg Gly Pro Ser Gly Arg Ser Asp Pro Ile 85 90 95 Gln Leu Glu Ile His Arg Asp Trp Leu Leu Leu Gln Val Ser Ser Arg 100 105 110 Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys 115 120 125 Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Gln Asn Gly Lys Ala Phe 130 135 140 Lys Phe Phe Tyr Arg Asn Ser Gln Leu Thr Ile Leu Lys Thr Asn Ile 145 150 155 160 Ser His Asn Gly Ala Tyr His Cys Ser Gly Met Gly Lys His Arg Tyr 165 170 175 Thr Ser Ala Gly Val Ser Val Thr Val Lys Glu Leu Phe Pro Ala Pro 180 185 190 Val Leu Asn Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val 195 200 205 Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln 210 215 220 Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn 225 230 235 240 Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser Gly 245 250 255 Phe Tyr Trp Cys Glu Ala Thr Thr Glu Asp Gly Asn Val Leu Lys Arg 260 265 270 Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Leu Gln Leu Pro Thr Pro 275 280 285 Val Trp Leu His Val Leu Phe Tyr Leu Val Val Gly Ile Met Phe Leu 290 295 300 Val Asn Thr Val Leu Trp Val Thr Ile Arg Lys Glu Leu Lys Arg Lys 305 310 315 320 Lys Lys Trp Asn Leu Glu Ile Ser Leu Asp Ser Ala His Glu Lys Lys 325 330 335 Val Thr Ser Ser Leu Gln Glu Asp Arg His Leu Glu Glu Glu Leu Lys 340 345 350 Ser Gln Glu Gln Glu 355 10 374 PRT Homo sapiens MISC_FEATURE (1)..(374) FcgammaRI alpha-chain 10 Met Trp Phe Leu Thr Thr Leu Leu Leu Trp Val Pro Val Asp Gly Gln 1 5 10 15 Val Asp Thr Thr Lys Ala Val Ile Ser Leu Gln Pro Pro Trp Val Ser 20 25 30 Val Phe Gln Glu Glu Thr Val Thr Leu His Cys Glu Val Leu His Leu 35 40 45 Pro Gly Ser Ser Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55 60 Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser 65 70 75 80 Gly Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile 85 90 95 Gln Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg 100 105 110 Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys 115 120 125 Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys Ala Phe 130 135 140 Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu Lys Thr Asn Ile 145 150 155 160 Ser His Asn Gly Thr Tyr His Cys Ser Gly Met Gly Lys His Arg Tyr 165 170 175 Thr Ser Ala Gly Ile Ser Val Thr Val Lys Glu Leu Phe Pro Ala Pro 180 185 190 Val Leu Asn Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val 195 200 205 Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln 210 215 220 Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn 225 230 235 240 Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser Gly 245 250 255 Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val Leu Lys Arg 260 265 270 Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Leu Gln Leu Pro Thr Pro 275 280 285 Val Trp Phe His Val Leu Phe Tyr Leu Ala Val Gly Ile Met Phe Leu 290 295 300 Val Asn Thr Val Leu Trp Val Thr Ile Arg Lys Glu Leu Lys Arg Lys 305 310 315 320 Lys Lys Trp Asp Leu Glu Ile Ser Leu Asp Ser Gly His Glu Lys Lys 325 330 335 Val Thr Ser Ser Leu Gln Glu Asp Arg His Leu Glu Glu Glu Leu Lys 340 345 350 Cys Gln Glu Gln Lys Glu Glu Gln Leu Gln Glu Gly Val His Arg Lys 355 360 365 Glu Pro Gln Gly Ala Thr 370 11 86 PRT Cynomolgus MISC_FEATURE (1)..(86) FcgammaRI/III gamma-chain 11 Met Ile Pro Ala Val Val Leu Leu Leu Leu Leu Leu Val Glu Gln Ala 1 5 10 15 Ala Ala Leu Gly Glu Pro Gln Leu Cys Tyr Ile Leu Asp Ala Ile Leu 20 25 30 Phe Leu Tyr Gly Ile Val Leu Thr Leu Leu Tyr Cys Arg Leu Lys Ile 35 40 45 Gln Val Arg Lys Ala Ala Ile Ala Ser Tyr Glu Lys Ser Asp Gly Val 50 55 60 Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys 65 70 75 80 His Glu Lys Pro Pro Gln 85 12 86 PRT Homo sapiens MISC_FEATURE (1)..(86) FcgammaRI/III gamma-chain 12 Met Ile Pro Ala Val Val Leu Leu Leu Leu Leu Leu Val Glu Gln Ala 1 5 10 15 Ala Ala Leu Gly Glu Pro Gln Leu Cys Tyr Ile Leu Asp Ala Ile Leu 20 25 30 Phe Leu Tyr Gly Ile Val Leu Thr Leu Leu Tyr Cys Arg Leu Lys Ile 35 40 45 Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val 50 55 60 Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys 65 70 75 80 His Glu Lys Pro Pro Gln 85 13 261 DNA Cynomolgus misc_feature (1)..(261) gamma chain 13 atgattccag cagtggtctt gctcttactc cttttggttg aacaagcagc ggccctggga 60 gagcctcagc tctgctatat cctggatgcc atcctgtttc tgtatggaat tgtcctcacc 120 ctcctctact gtcgactgaa gatccaagtg cgaaaggcag ctatagccag ctatgagaaa 180 tcagatggtg tttacacggg cctgagcacc aggaaccagg aaacttatga gactctgaag 240 catgagaaac caccacagta g 261 14 261 DNA Homo sapiens misc_feature (1)..(261) gamma chain 14 atgattccag cagtggtctt gctcttactc cttttggttg aacaagcagc ggccctggga 60 gagcctcagc tctgctatat cctggatgcc atcctgtttc tgtatggaat tgtcctcacc 120 ctcctctact gtcgactgaa gatccaagtg cgaaaggcag ctataaccag ctatgagaaa 180 tcagatggtg tttacacggg cctgagcacc aggaaccagg agacttacga gactctgaag 240 catgagaaac caccacagta g 261 15 310 PRT Cynomolgus MISC_FEATURE (1)..(310) FcgammaRIIA 15 Met Ser Gln Asn Val Cys Pro Gly Asn Leu Trp Leu Leu Gln Pro Leu 1 5 10 15 Thr Val Leu Leu Leu Leu Ala Ser Ala Asp Ser Gln Thr Ala Pro Pro 20 25 30 Lys Ala Val Leu Lys Leu Glu Pro Pro Trp Ile Asn Val Leu Arg Glu 35 40 45 Asp Ser Val Thr Leu Thr Cys Gly Gly Ala His Ser Pro Asp Ser Asp 50 55 60 Ser Thr Gln Trp Phe His Asn Gly Asn Arg Ile Pro Thr His Thr Gln 65 70 75 80 Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn Asp Ser Gly Glu Tyr Arg

85 90 95 Cys Gln Thr Gly Arg Thr Ser Leu Ser Asp Pro Val His Leu Thr Val 100 105 110 Leu Ser Glu Trp Leu Ala Leu Gln Thr Pro His Leu Glu Phe Arg Glu 115 120 125 Gly Glu Thr Ile Met Leu Arg Cys His Ser Trp Lys Asp Lys Pro Leu 130 135 140 Ile Lys Val Thr Phe Phe Gln Asn Gly Ile Ala Lys Lys Phe Ser His 145 150 155 160 Met Asp Pro Asn Phe Ser Ile Pro Gln Ala Asn His Ser His Ser Gly 165 170 175 Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr Thr Pro Tyr Ser Ser Lys 180 185 190 Pro Val Thr Ile Thr Val Gln Val Pro Ser Val Gly Ser Ser Ser Pro 195 200 205 Met Gly Ile Ile Val Ala Val Val Thr Gly Ile Ala Val Ala Ala Ile 210 215 220 Val Ala Ala Val Val Ala Leu Ile Tyr Cys Arg Lys Lys Arg Ile Ser 225 230 235 240 Ala Asn Ser Thr Asp Pro Val Lys Ala Ala Arg Phe Glu Pro Leu Gly 245 250 255 Arg Gln Thr Ile Ala Leu Arg Lys Arg Gln Leu Glu Glu Thr Asn Asn 260 265 270 Asp Tyr Glu Thr Ala Asp Gly Gly Tyr Met Thr Leu Asn Pro Arg Ala 275 280 285 Pro Thr Asp Asp Asp Arg Asn Ile Tyr Leu Thr Leu Ser Pro Asn Asp 290 295 300 Tyr Asp Asn Ser Asn Asn 305 310 16 317 PRT Homo sapiens MISC_FEATURE (1)..(317) FcgammaRIIA 16 Met Ala Met Glu Thr Gln Met Ser Gln Asn Val Cys Pro Arg Asn Leu 1 5 10 15 Trp Leu Leu Gln Pro Leu Thr Val Leu Leu Leu Leu Ala Ser Ala Asp 20 25 30 Ser Gln Ala Ala Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Pro 35 40 45 Trp Ile Asn Val Leu Gln Glu Asp Ser Val Thr Leu Thr Cys Gln Gly 50 55 60 Ala Arg Ser Pro Glu Ser Asp Ser Ile Gln Trp Phe His Asn Gly Asn 65 70 75 80 Leu Ile Pro Thr His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn 85 90 95 Asn Asp Ser Gly Glu Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser 100 105 110 Asp Pro Val His Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr 115 120 125 Pro His Leu Glu Phe Gln Glu Gly Glu Thr Ile Met Leu Arg Cys His 130 135 140 Ser Trp Lys Asp Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly 145 150 155 160 Lys Ser Gln Lys Phe Ser Arg Leu Asp Pro Thr Phe Ser Ile Pro Gln 165 170 175 Ala Asn His Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly 180 185 190 Tyr Thr Leu Phe Ser Ser Lys Pro Val Thr Ile Thr Val Gln Val Pro 195 200 205 Ser Met Gly Ser Ser Ser Pro Met Gly Ile Ile Val Ala Val Val Ile 210 215 220 Ala Thr Ala Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr 225 230 235 240 Cys Arg Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala 245 250 255 Ala Gln Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg 260 265 270 Gln Leu Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr 275 280 285 Met Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr 290 295 300 Leu Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn 305 310 315 17 316 PRT Chimp MISC_FEATURE (1)..(316) FcgammaRIIA 17 Met Ala Met Glu Thr Gln Met Ser Gln Asn Val Cys Pro Arg Asn Leu 1 5 10 15 Trp Leu Leu Gln Pro Leu Thr Val Leu Leu Leu Leu Ala Ser Ala Asp 20 25 30 Ser Gln Ala Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Pro Trp 35 40 45 Ile Asn Val Leu Gln Glu Asp Ser Val Thr Leu Thr Cys Arg Gly Ala 50 55 60 Arg Ser Pro Glu Ser Asp Ser Ile Gln Trp Phe His Asn Gly Asn Leu 65 70 75 80 Ile Pro Thr His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn 85 90 95 Asp Ser Gly Glu Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp 100 105 110 Pro Val His Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr Pro 115 120 125 His Leu Glu Phe Gln Glu Gly Glu Thr Ile Val Leu Arg Cys His Ser 130 135 140 Trp Lys Asp Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly Lys 145 150 155 160 Ser Gln Lys Phe Ser His Leu Asp Pro Asn Leu Ser Ile Pro Gln Ala 165 170 175 Asn His Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr 180 185 190 Thr Leu Phe Ser Ser Lys Pro Val Thr Ile Thr Val Gln Ala Pro Ser 195 200 205 Val Gly Ser Ser Ser Pro Val Gly Ile Ile Val Ala Val Val Ile Ala 210 215 220 Thr Ala Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr Cys 225 230 235 240 Arg Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala Ala 245 250 255 Gln Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg Gln 260 265 270 Leu Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr Met 275 280 285 Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr Leu 290 295 300 Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn 305 310 315 18 294 PRT Cynomolgus MISC_FEATURE (1)..(294) FcgammaRIIB 18 Met Gly Ile Leu Ser Phe Leu Pro Val Leu Ala Thr Glu Ser Asp Trp 1 5 10 15 Ala Asp Cys Lys Ser Ser Gln Pro Trp Gly His Met Leu Leu Trp Thr 20 25 30 Ala Val Leu Phe Leu Ala Pro Val Ala Gly Thr Pro Ala Ala Pro Pro 35 40 45 Lys Ala Val Leu Lys Leu Glu Pro Pro Trp Ile Asn Val Leu Arg Glu 50 55 60 Asp Ser Val Thr Leu Thr Cys Gly Gly Ala His Ser Pro Asp Ser Asp 65 70 75 80 Ser Thr Gln Trp Phe His Asn Gly Asn Leu Ile Pro Thr His Thr Gln 85 90 95 Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn Asp Ser Gly Glu Tyr Arg 100 105 110 Cys Gln Thr Gly Arg Thr Ser Leu Ser Asp Pro Val His Leu Thr Val 115 120 125 Leu Ser Glu Trp Leu Ala Leu Gln Thr Pro His Leu Glu Phe Arg Glu 130 135 140 Gly Glu Thr Ile Leu Leu Arg Cys His Ser Trp Lys Asp Lys Pro Leu 145 150 155 160 Ile Lys Val Thr Phe Phe Gln Asn Gly Ile Ser Lys Lys Phe Ser His 165 170 175 Met Asn Pro Asn Phe Ser Ile Pro Gln Ala Asn His Ser His Ser Gly 180 185 190 Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr Thr Pro Tyr Ser Ser Lys 195 200 205 Pro Val Thr Ile Thr Val Gln Val Pro Ser Met Gly Ser Ser Ser Pro 210 215 220 Ile Gly Ile Ile Val Ala Val Val Thr Gly Ile Ala Val Ala Ala Ile 225 230 235 240 Val Ala Ala Val Val Ala Leu Ile Tyr Cys Arg Lys Lys Arg Ile Ser 245 250 255 Ala Asn Pro Thr Asn Pro Asp Glu Ala Asp Lys Val Gly Ala Glu Asn 260 265 270 Thr Ile Thr Tyr Ser Leu Leu Met His Pro Asp Ala Leu Glu Glu Pro 275 280 285 Asp Asp Gln Asn Arg Val 290 19 291 PRT Homo sapiens MISC_FEATURE (1)..(291) FcgammaRIIB 19 Met Gly Ile Leu Ser Phe Leu Pro Val Leu Ala Thr Glu Ser Asp Trp 1 5 10 15 Ala Asp Cys Lys Ser Pro Gln Pro Trp Gly His Met Leu Leu Trp Thr 20 25 30 Ala Val Leu Phe Leu Ala Pro Val Ala Gly Thr Pro Ala Ala Pro Pro 35 40 45 Lys Ala Val Leu Lys Leu Glu Pro Gln Trp Ile Asn Val Leu Gln Glu 50 55 60 Asp Ser Val Thr Leu Thr Cys Arg Gly Thr His Ser Pro Glu Ser Asp 65 70 75 80 Ser Ile Gln Trp Phe His Asn Gly Asn Leu Ile Pro Thr His Thr Gln 85 90 95 Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn Asp Ser Gly Glu Tyr Thr 100 105 110 Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp Pro Val His Leu Thr Val 115 120 125 Leu Ser Glu Trp Leu Val Leu Gln Thr Pro His Leu Glu Phe Gln Glu 130 135 140 Gly Glu Thr Ile Val Leu Arg Cys His Ser Trp Lys Asp Lys Pro Leu 145 150 155 160 Val Lys Val Thr Phe Phe Gln Asn Gly Lys Ser Lys Lys Phe Ser Arg 165 170 175 Ser Asp Pro Asn Phe Ser Ile Pro Gln Ala Asn His Ser His Ser Gly 180 185 190 Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr Thr Leu Tyr Ser Ser Lys 195 200 205 Pro Val Thr Ile Thr Val Gln Ala Pro Ser Ser Ser Pro Met Gly Ile 210 215 220 Ile Val Ala Val Val Thr Gly Ile Ala Val Ala Ala Ile Val Ala Ala 225 230 235 240 Val Val Ala Leu Ile Tyr Cys Arg Lys Lys Arg Ile Ser Ala Asn Pro 245 250 255 Thr Asn Pro Asp Glu Ala Asp Lys Val Gly Ala Glu Asn Thr Ile Thr 260 265 270 Tyr Ser Leu Leu Met His Pro Asp Ala Leu Glu Glu Pro Asp Asp Gln 275 280 285 Asn Arg Ile 290 20 254 PRT Cynomolgus MISC_FEATURE (1)..(254) FcgammaRIIIA 20 Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala 1 5 10 15 Gly Met Arg Ala Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30 Gln Trp Tyr Arg Val Leu Glu Lys Asp Arg Val Thr Leu Lys Cys Gln 35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Arg Trp Phe His Asn Glu 50 55 60 Ser Leu Ile Ser Ser Gln Thr Ser Ser Tyr Phe Ile Ala Ala Ala Arg 65 70 75 80 Val Asn Asn Ser Gly Glu Tyr Arg Cys Gln Thr Ser Leu Ser Thr Leu 85 90 95 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110 Ala Pro Arg Trp Val Phe Lys Glu Glu Glu Ser Ile His Leu Arg Cys 115 120 125 His Ser Trp Lys Asn Thr Leu Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140 Gly Lys Gly Arg Lys Tyr Phe His Gln Asn Ser Asp Phe Tyr Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Ile 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190 Asp Leu Ala Val Ser Ser Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln 195 200 205 Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 210 215 220 Leu Tyr Phe Ser Met Lys Lys Ser Ile Pro Ser Ser Thr Arg Asp Trp 225 230 235 240 Glu Asp His Lys Phe Lys Trp Ser Lys Asp Pro Gln Asp Lys 245 250 21 254 PRT Homo sapiens MISC_FEATURE (1)..(254) FcgammaRIIIA 21 Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala 1 5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30 Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60 Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr 65 70 75 80 Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140 Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190 Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln 195 200 205 Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 210 215 220 Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp 225 230 235 240 Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 245 250 22 933 DNA Chimp misc_feature (1)..(933) FcgammaRIIA 22 atgtctcaga atgtatgtcc cagaaacctg tggctgcttc aaccattgac agttttgctg 60 ctgctggctt ctgcagacag tcaagctgct cccccaaagg ctgtgctgaa acttgagccc 120 ccgtggatca acgtgctcca ggaggactct gtgactctga catgccgggg ggctcgcagc 180 cctgagagcg actccattca gtggttccac aatgggaatc tcatccccac ccacacgcag 240 cccagctaca ggttcaaggc caacaacaat gacagcgggg agtacacgtg ccagactggc 300 cagaccagcc tcagcgaccc tgtgcatctg actgtgcttt ccgaatggct ggtgctccag 360 acccctcacc tggagttcca ggagggagaa accatcgtgc tgaggtgcca cagctggaag 420 gacaagcctc tggtcaaggt cacattcttc cagaatggaa aatcccagaa attctcccat 480 ttggatccca acctctccat cccacaagca aaccacagtc acagtggtga ttaccactgc 540 acaggaaaca taggctacac gctgttctca tccaagcctg tgaccatcac tgtccaagcg 600 cccagcgtgg gcagctcttc accagtgggg atcattgtgg ctgtggtcat tgcgactgct 660 gtagcagcca ttgttgctgc tgtagtggcc ttgatctact gcaggaaaaa gcggatttca 720 gccaattcca ctgatcctgt gaaggctgcc caatttgagc cacctggacg tcaaatgatt 780 gccatcagaa agagacaact tgaagaaacc aacaatgact atgaaacagc tgacggcggc 840 tacatgactc tgaaccccag ggcacctact gacgatgata aaaacatcta cctgactctt 900 cctcccaacg accatgtcaa cagtaataac taa 933 23 360 DNA Cynomolgus misc_feature (1)..(360) B-2 microglobulin 23 atgtctccct cagtggcctt agccgtgctg gcgctactct ctctttctgg cctggaggct 60 atccagcgta ctccaaagat tcaggtttac tcacgccatc caccagagaa tggaaagcca 120 aatttcctga attgctatgt gtctggattt catccatctg atattgaagt tgacttactg 180 aagaatggag agaaaatggg aaaagtggag cattcagact tgtctttcag caaagactgg 240 tctttctatc tcttgtacta cactgaattc acccccaatg aaaaagatga gtatgcctgc 300 cgtgtgaacc atgtgacttt gtcagggccc aggacagtta agtgggatcg agacatgtaa 360 24 360 DNA Homo sapiens misc_feature (1)..(360) B-2 microglobulin 24 atgtctcgct ccgtggcctt agctgtgctc gcgctactct ctctttctgg cctggaggct 60 atccagcgta ctccaaagat tcaggtttac tcacgtcatc cagcagagaa tggaaagtca 120 aatttcctga attgctatgt gtctgggttt catccatccg acattgaagt tgacttactg 180 aagaatggag agagaattga aaaagtggag cattcagact tgtctttcag caaggactgg 240 tctttctatc tcttgtacta cactgaattc acccccactg aaaaagatga gtatgcctgc 300 cgtgtgaacc atgtgacttt gtcacagccc aagatagtta agtgggatcg agacatgtaa 360 25 119 PRT Cynomolgus MISC_FEATURE (1)..(119) Beta-2 microglobulin 25 Met Ser Pro Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser 1 5 10 15 Gly Leu Glu Ala Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg 20 25 30 His Pro Pro Glu Asn Gly Lys Pro Asn Phe Leu Asn Cys Tyr Val Ser 35 40 45 Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu 50 55 60 Lys Met Gly Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp 65 70 75 80 Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Asn Glu Lys Asp 85 90 95 Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gly Pro Arg Thr 100 105 110 Val Lys Trp Asp Arg Asp Met 115 26 119 PRT Homo sapiens MISC_FEATURE (1)..(119) Beta-2 microglobulin 26 Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser 1 5 10 15 Gly Leu Glu Ala Ile

Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg 20 25 30 His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser 35 40 45 Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu 50 55 60 Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp 65 70 75 80 Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp 85 90 95 Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile 100 105 110 Val Lys Trp Asp Arg Asp Met 115 27 1098 DNA Cynomolgus misc_feature (1)..(1098) FcRn alpha-chain 27 atgagggtcc cgcggcctca gccctgggcg ctggggctcc tgctctttct cctgcccggg 60 agcctgggcg cagaaagcca cctctccctc ctgtaccacc tcaccgcggt gtcctcgccc 120 gccccgggga cgcctgcctt ctgggtgtcc ggctggctgg gcccgcagca gtacctgagc 180 tacgacagcc tgaggggcca ggcggagccc tgtggagctt gggtctggga aaaccaagtg 240 tcctggtatt gggagaaaga gaccacagat ctgaggatca aggagaagct ctttctggaa 300 gctttcaaag ctttgggggg aaaaggcccc tacactctgc agggcctgct gggctgtgaa 360 ctgagccctg acaacacctc ggtgcccacc gccaagttcg ccctgaacgg cgaggagttc 420 atgaatttcg acctcaagca gggcacctgg ggtggggact ggcccgaggc cctggctatc 480 agtcagcggt ggcagcagca ggacaaggcg gccaacaagg agctcacctt cctgctattc 540 tcctgcccac accggctgcg ggagcacctg gagaggggcc gtggaaacct ggagtggaag 600 gagcccccct ccatgcgcct gaaggcccga cccggcaacc ctggcttttc cgtgcttacc 660 tgcagcgcct tctccttcta ccctccggaa ctgcaactgc ggttcctgcg gaatgggatg 720 gccgctggca ccggacaggg cgacttcggc cccaacagtg acggctcctt ccacgcctcg 780 tcgtcactaa cagtcaaaag tggcgatgag caccactact gctgcatcgt gcagcacgcg 840 gggctggcgc agcccctcag ggtggagctg gaaactccag ccaagtcctc ggtgctcgtg 900 gtgggaatcg tcatcggtgt cttgctactc acggcagcgg ctgtaggagg agctctgttg 960 tggagaagga tgaggagtgg gctgccagcc ccttggatct ccctccgtgg agatgacacc 1020 gggtccctcc tgcccacccc gggggaggcc caggatgctg attcgaagga tataaatgtg 1080 atcccagcca ctgcctga 1098 28 1098 DNA Homo sapiens misc_feature (1)..(1098) FcRn alpha-chain 28 atgggggtcc cgcggcctca gccctgggcg ctggggctcc tgctctttct ccttcctggg 60 agcctgggcg cagaaagcca cctctccctc ctgtaccacc ttaccgcggt gtcctcgcct 120 gccccgggga ctcctgcctt ctgggtgtcc ggctggctgg gcccgcagca gtacctgagc 180 tacaatagcc tgcggggcga ggcggagccc tgtggagctt gggtctggga aaaccaggtg 240 tcctggtatt gggagaaaga gaccacagat ctgaggatca aggagaagct ctttctggaa 300 gctttcaaag ctttgggggg aaaaggtccc tacactctgc agggcctgct gggctgtgaa 360 ctgggccctg acaacacctc ggtgcccacc gccaagttcg ccctgaacgg cgaggagttc 420 atgaatttcg acctcaagca gggcacctgg ggtggggact ggcccgaggc cctggctatc 480 agtcagcggt ggcagcagca ggacaaggcg gccaacaagg agctcacctt cctgctattc 540 tcctgcccgc accgcctgcg ggagcacctg gagaggggcc gcggaaacct ggagtggaag 600 gagcccccct ccatgcgcct gaaggcccga cccagcagcc ctggcttttc cgtgcttacc 660 tgcagcgcct tctccttcta ccctccggag ctgcaacttc ggttcctgcg gaatgggctg 720 gccgctggca ccggccaggg tgacttcggc cccaacagtg acggatcctt ccacgcctcg 780 tcgtcactaa cagtcaaaag tggcgatgag caccactact gctgcattgt gcagcacgcg 840 gggctggcgc agcccctcag ggtggagctg gaatctccag ccaagtcctc cgtgctcgtg 900 gtgggaatcg tcatcggtgt cttgctactc acggcagcgg ctgtaggagg agctctgttg 960 tggagaagga tgaggagtgg gctgccagcc ccttggatct cccttcgtgg agacgacacc 1020 ggggtcctcc tgcccacccc aggggaggcc caggatgctg atttgaagga tgtaaatgtg 1080 attccagcca ccgcctga 1098 29 365 PRT Cynomolgus MISC_FEATURE (1)..(365) FcRn (S3) 29 Met Arg Val Pro Arg Pro Gln Pro Trp Ala Leu Gly Leu Leu Leu Phe 1 5 10 15 Leu Leu Pro Gly Ser Leu Gly Ala Glu Ser His Leu Ser Leu Leu Tyr 20 25 30 His Leu Thr Ala Val Ser Ser Pro Ala Pro Gly Thr Pro Ala Phe Trp 35 40 45 Val Ser Gly Trp Leu Gly Pro Gln Gln Tyr Leu Ser Tyr Asp Ser Leu 50 55 60 Arg Gly Gln Ala Glu Pro Cys Gly Ala Trp Val Trp Glu Asn Gln Val 65 70 75 80 Ser Trp Tyr Trp Glu Lys Glu Thr Thr Asp Leu Arg Ile Lys Glu Lys 85 90 95 Leu Phe Leu Glu Ala Phe Lys Ala Leu Gly Gly Lys Gly Pro Tyr Thr 100 105 110 Leu Gln Gly Leu Leu Gly Cys Glu Leu Ser Pro Asp Asn Thr Ser Val 115 120 125 Pro Thr Ala Lys Phe Ala Leu Asn Gly Glu Glu Phe Met Asn Phe Asp 130 135 140 Leu Lys Gln Gly Thr Trp Gly Gly Asp Trp Pro Glu Ala Leu Ala Ile 145 150 155 160 Ser Gln Arg Trp Gln Gln Gln Asp Lys Ala Ala Asn Lys Glu Leu Thr 165 170 175 Phe Leu Leu Phe Ser Cys Pro His Arg Leu Arg Glu His Leu Glu Arg 180 185 190 Gly Arg Gly Asn Leu Glu Trp Lys Glu Pro Pro Ser Met Arg Leu Lys 195 200 205 Ala Arg Pro Gly Asn Pro Gly Phe Ser Val Leu Thr Cys Ser Ala Phe 210 215 220 Ser Phe Tyr Pro Pro Glu Leu Gln Leu Arg Phe Leu Arg Asn Gly Met 225 230 235 240 Ala Ala Gly Thr Gly Gln Gly Asp Phe Gly Pro Asn Ser Asp Gly Ser 245 250 255 Phe His Ala Ser Ser Ser Leu Thr Val Lys Ser Gly Asp Glu His His 260 265 270 Tyr Cys Cys Ile Val Gln His Ala Gly Leu Ala Gln Pro Leu Arg Val 275 280 285 Glu Leu Glu Thr Pro Ala Lys Ser Ser Val Leu Val Val Gly Ile Val 290 295 300 Ile Gly Val Leu Leu Leu Thr Ala Ala Ala Val Gly Gly Ala Leu Leu 305 310 315 320 Trp Arg Arg Met Arg Ser Gly Leu Pro Ala Pro Trp Ile Ser Leu Arg 325 330 335 Gly Asp Asp Thr Gly Ser Leu Leu Pro Thr Pro Gly Glu Ala Gln Asp 340 345 350 Ala Asp Ser Lys Asp Ile Asn Val Ile Pro Ala Thr Ala 355 360 365 30 365 PRT Homo sapiens MISC_FEATURE (1)..(365) FcRn alpha-chain 30 Met Gly Val Pro Arg Pro Gln Pro Trp Ala Leu Gly Leu Leu Leu Phe 1 5 10 15 Leu Leu Pro Gly Ser Leu Gly Ala Glu Ser His Leu Ser Leu Leu Tyr 20 25 30 His Leu Thr Ala Val Ser Ser Pro Ala Pro Gly Thr Pro Ala Phe Trp 35 40 45 Val Ser Gly Trp Leu Gly Pro Gln Gln Tyr Leu Ser Tyr Asn Ser Leu 50 55 60 Arg Gly Glu Ala Glu Pro Cys Gly Ala Trp Val Trp Glu Asn Gln Val 65 70 75 80 Ser Trp Tyr Trp Glu Lys Glu Thr Thr Asp Leu Arg Ile Lys Glu Lys 85 90 95 Leu Phe Leu Glu Ala Phe Lys Ala Leu Gly Gly Lys Gly Pro Tyr Thr 100 105 110 Leu Gln Gly Leu Leu Gly Cys Glu Leu Gly Pro Asp Asn Thr Ser Val 115 120 125 Pro Thr Ala Lys Phe Ala Leu Asn Gly Glu Glu Phe Met Asn Phe Asp 130 135 140 Leu Lys Gln Gly Thr Trp Gly Gly Asp Trp Pro Glu Ala Leu Ala Ile 145 150 155 160 Ser Gln Arg Trp Gln Gln Gln Asp Lys Ala Ala Asn Lys Glu Leu Thr 165 170 175 Phe Leu Leu Phe Ser Cys Pro His Arg Leu Arg Glu His Leu Glu Arg 180 185 190 Gly Arg Gly Asn Leu Glu Trp Lys Glu Pro Pro Ser Met Arg Leu Lys 195 200 205 Ala Arg Pro Ser Ser Pro Gly Phe Ser Val Leu Thr Cys Ser Ala Phe 210 215 220 Ser Phe Tyr Pro Pro Glu Leu Gln Leu Arg Phe Leu Arg Asn Gly Leu 225 230 235 240 Ala Ala Gly Thr Gly Gln Gly Asp Phe Gly Pro Asn Ser Asp Gly Ser 245 250 255 Phe His Ala Ser Ser Ser Leu Thr Val Lys Ser Gly Asp Glu His His 260 265 270 Tyr Cys Cys Ile Val Gln His Ala Gly Leu Ala Gln Pro Leu Arg Val 275 280 285 Glu Leu Glu Ser Pro Ala Lys Ser Ser Val Leu Val Val Gly Ile Val 290 295 300 Ile Gly Val Leu Leu Leu Thr Ala Ala Ala Val Gly Gly Ala Leu Leu 305 310 315 320 Trp Arg Arg Met Arg Ser Gly Leu Pro Ala Pro Trp Ile Ser Leu Arg 325 330 335 Gly Asp Asp Thr Gly Val Leu Leu Pro Thr Pro Gly Glu Ala Gln Asp 340 345 350 Ala Asp Leu Lys Asp Val Asn Val Ile Pro Ala Thr Ala 355 360 365 31 33 DNA Cynomolgus misc_feature (1)..(33) FcgammaRI - forward primer 31 caggtcaatc tctagactcc caccagcttg gag 33 32 33 DNA Cynomolgus misc_feature (1)..(33) FcgammaRI - reverse primer 32 ggtcaactat aagcttggac ggtccagatc gat 33 33 34 DNA Cynomolgus misc_feature (1)..(34) FcgammaRI-H6-GST - forward primer 33 caggtcaatc atcgatatgt ggttcttgac agct 34 34 51 DNA Cynomolgus misc_feature (1)..(51) FcgammaRI-H6-GST - reverse primer 34 ggtcaactat gctagcatgg tgatgatggt ggtgccagac aggagttggt a 51 35 36 DNA Cynomolgus misc_feature (1)..(36) FcgammaRIIB - forward primer 35 caggtcaatc tctagaatgg gaatcctgtc attctt 36 36 34 DNA Cynomolgus misc_feature (1)..(34) FcgammaRIIB - reverse primer 36 ggtcaactat aagcttctaa atacggttct ggtc 34 37 33 DNA Cynomolgus misc_feature (1)..(33) FcgammaRIIB-H6-GST - forward primer 37 caggtcaatc atcgatatgc ttctgtggac agc 33 38 34 DNA Cynomolgus misc_feature (1)..(34) FcgammaRIIB-H6-GST - reverse primer 38 ggtcaactat ggtgacctat cggtgaagag ctgc 34 39 33 DNA Cynomolgus misc_feature (1)..(33) FcgammaRIIIA - forward primer 39 caggtcaatc tctagaatgt ggcagctgct cct 33 40 33 DNA Cynomolgus misc_feature (1)..(33) FcgammaRIIIA - reverse primer 40 tcaactataa gcttatgttc agagatgctg ctg 33 41 33 DNA Cynomolgus misc_feature (1)..(33) FcgammaRIIIA-H6-GST - forward primer 41 caggtcaatc tctagaatgt ggcagctgct cct 33 42 35 DNA Cynomolgus misc_feature (1)..(35) FcgammaRIIIA-H6-GST - reverse primer 42 ggtcaactat ggtcaccttg gtacccaggt ggaaa 35 43 45 DNA Cynomolgus misc_feature (1)..(45) Fc gamma - forward primer 43 caggtcaatc atcgatgaat tcccaccatg attccagcag tggtc 45 44 35 DNA Cynomolgus misc_feature (1)..(35) Fc gamma - reverse primer 44 ggtcaactat aagcttctac tgtggtggtt tctca 35 45 32 DNA Cynomolgus misc_feature (1)..(32) B-2 microglobulin - forward primer 45 caggtcaatc atcgattcgg gccgagatgt ct 32 46 34 DNA Cynomolgus misc_feature (1)..(34) B-2 microglobulin - reverse primer 46 ggtcaactat tctagattac atgtctcgat ccca 34 47 35 DNA Cynomolgus misc_feature (1)..(35) FcgammaRIIA - forward primer 47 caggtcaatc tctagaatgt ctcagaatgt atgtc 35 48 37 DNA Cynomolgus misc_feature (1)..(37) FcgammaRIIA - reverse primer 48 ggtcaactat aagcttttag ttattactgt tgtcata 37 49 35 DNA Cynomolgus misc_feature (1)..(35) FcgammaRIIA-H6-GST - forward primer 49 caggtcaatc atcgatatgt ctcagaatgt atgtc 35 50 34 DNA Cynomolgus misc_feature (1)..(34) FcgammaRIIA-H6-GST - reverse primer 50 ggtcaactat ggtgacccat cggtgaagag ctgc 34 51 32 DNA Cynomolgus misc_feature (1)..(32) FcRn - forward primer 51 caggtcaatc atcgataggt cgtcctctca gc 32 52 32 DNA Cynomolgus misc_feature (1)..(32) FcRn - reverse primer 52 ggtcaactat gaattctcgg aatggcggat gg 32 53 32 DNA Cynomolgus misc_feature (1)..(32) FcRn-H6 - forward primer 53 caggtcaatc atcgataggt cgtcctctca gc 32 54 55 DNA Cynomolgus misc_feature (1)..(55) FcRn-H6 - reverse primer 54 ggtcaactat gaattcatgg tgatgatggt ggtgcgagga cttggctgga gtttc 55 55 33 DNA Artificial Sequence PCR primer OF1 55 caggtcaatc tctagacagt ggttccacaa tgg 33 56 35 DNA artificial sequence PCR primer OR1 56 ggtcaactat aagcttaaga gtcaggtaga tgttt 35 57 37 DNA artificial sequence PCR primer OF2 57 caggtcaatc tctagaatac ataaccttat gtatcat 37 58 37 DNA artificial sequence PCR primer OF3 58 caggtcaatc tctagatata gaataacatc cactttg 37 59 32 DNA artificial sequence PCR primer OR2 59 ggtcaactat aagcttcaga gtcatgtagc cg 32 60 35 DNA artificial sequence PCR primer OF4 60 caggtcaatc tctagaattc cactgatcct gtgaa 35 61 37 DNA artificial sequence PCT primer OR3 61 ggtcaactat aagcttgctt tatttgtgaa atttgtg 37 62 35 DNA artificial sequence PCR primer OF5 62 caggtcaatc tctagaactt ggacgtcaaa cgatt 35 63 35 DNA artificial sequence PCR primer OR4 63 ggtcaactat aagcttctgc aataaacaag ttggg 35 64 365 PRT Cynomolgus MISC_FEATURE (1)..(365) FcRn (N3) 64 Met Arg Val Pro Arg Pro Gln Pro Trp Ala Leu Gly Leu Leu Leu Phe 1 5 10 15 Leu Leu Pro Gly Ser Leu Gly Ala Glu Asn His Leu Ser Leu Leu Tyr 20 25 30 His Leu Thr Ala Val Ser Ser Pro Ala Pro Gly Thr Pro Ala Phe Trp 35 40 45 Val Ser Gly Trp Leu Gly Pro Gln Gln Tyr Leu Ser Tyr Asp Ser Leu 50 55 60 Arg Gly Gln Ala Glu Pro Cys Gly Ala Trp Val Trp Glu Asn Gln Val 65 70 75 80 Ser Trp Tyr Trp Glu Lys Glu Thr Thr Asp Leu Arg Ile Lys Glu Lys 85 90 95 Leu Phe Leu Glu Ala Phe Lys Ala Leu Gly Gly Lys Gly Pro Tyr Thr 100 105 110 Leu Gln Gly Leu Leu Gly Cys Glu Leu Ser Pro Asp Asn Thr Ser Val 115 120 125 Pro Thr Ala Lys Phe Ala Leu Asn Gly Glu Glu Phe Met Asn Phe Asp 130 135 140 Leu Lys Gln Gly Thr Trp Gly Gly Asp Trp Pro Glu Ala Leu Ala Ile 145 150 155 160 Ser Gln Arg Trp Gln Gln Gln Asp Lys Ala Ala Asn Lys Glu Leu Thr 165 170 175 Phe Leu Leu Phe Ser Cys Pro His Arg Leu Arg Glu His Leu Glu Arg 180 185 190 Gly Arg Gly Asn Leu Glu Trp Lys Glu Pro Pro Ser Met Arg Leu Lys 195 200 205 Ala Arg Pro Gly Asn Pro Gly Phe Ser Val Leu Thr Cys Ser Ala Phe 210 215 220 Ser Phe Tyr Pro Pro Glu Leu Gln Leu Arg Phe Leu Arg Asn Gly Met 225 230 235 240 Ala Ala Gly Thr Gly Gln Gly Asp Phe Gly Pro Asn Ser Asp Gly Ser 245 250 255 Phe His Ala Ser Ser Ser Leu Thr Val Lys Ser Gly Asp Glu His His 260 265 270 Tyr Cys Cys Ile Val Gln His Ala Gly Leu Ala Gln Pro Leu Arg Val 275 280 285 Glu Leu Glu Thr Pro Ala Lys Ser Ser Val Leu Val Val Gly Ile Val 290 295 300 Ile Gly Val Leu Leu Leu Thr Ala Ala Ala Val Gly Gly Ala Leu Leu 305 310 315 320 Trp Arg Arg Met Arg Ser Gly Leu Pro Ala Pro Trp Ile Ser Leu Arg 325 330 335 Gly Asp Asp Thr Gly Ser Leu Leu Pro Thr Pro Gly Glu Ala Gln Asp 340 345 350 Ala Asp Ser Lys Asp Ile Asn Val Ile Pro Ala Thr Ala 355 360 365 65 336 PRT Cynomolgus MISC_FEATURE (1)..(336) FcgammaRI alpha-chain 65 Ala Val Ile Thr Leu Gln Pro Pro Trp Val Ser Val Phe Gln Glu Glu 1 5 10 15 Thr Val Thr Leu Gln Cys Glu Val Pro Arg Leu Pro Gly Ser Ser Ser 20 25 30 Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln Thr Ser Thr Pro Ser 35 40 45 Tyr Arg Ile Thr Ser Ala Ser Val Lys Asp Ser Gly Glu Tyr Arg Cys 50 55 60 Gln Arg Gly Pro Ser Gly Arg Ser Asp Pro Ile Gln Leu Glu Ile His 65 70 75 80 Arg Asp Trp Leu Leu Leu Gln Val Ser Ser Arg Val Phe Thr Glu Gly 85 90 95 Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys Asp Lys Leu Val Tyr 100 105 110 Asn Val Leu Tyr Tyr Gln Asn Gly Lys Ala Phe Lys Phe Phe Tyr Arg 115 120 125 Asn Ser Gln Leu Thr Ile Leu Lys Thr Asn Ile Ser His Asn Gly Ala 130 135 140 Tyr His Cys Ser Gly Met Gly Lys His Arg Tyr Thr Ser Ala Gly Val 145 150 155 160 Ser Val Thr Val Lys Glu Leu Phe Pro Ala Pro Val Leu Asn Ala Ser 165 170 175 Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val Thr Leu Ser Cys Glu

180 185 190 Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln Leu Tyr Phe Ser Phe 195 200 205 Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn Thr Ser Ser Glu Tyr 210 215 220 Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser Gly Phe Tyr Trp Cys Glu 225 230 235 240 Ala Thr Thr Glu Asp Gly Asn Val Leu Lys Arg Ser Pro Glu Leu Glu 245 250 255 Leu Gln Val Leu Gly Leu Gln Leu Pro Thr Pro Val Trp Leu His Val 260 265 270 Leu Phe Tyr Leu Val Val Gly Ile Met Phe Leu Val Asn Thr Val Leu 275 280 285 Trp Val Thr Ile Arg Lys Glu Leu Lys Arg Lys Lys Lys Trp Asn Leu 290 295 300 Glu Ile Ser Leu Asp Ser Ala His Glu Lys Lys Val Thr Ser Ser Leu 305 310 315 320 Gln Glu Asp Arg His Leu Glu Glu Glu Leu Lys Ser Gln Glu Gln Glu 325 330 335 66 282 PRT Cynomolgus MISC_FEATURE (1)..(282) FcgammaRIIA 66 Thr Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Pro Trp Ile Asn 1 5 10 15 Val Leu Arg Glu Asp Ser Val Thr Leu Thr Cys Gly Gly Ala His Ser 20 25 30 Pro Asp Ser Asp Ser Thr Gln Trp Phe His Asn Gly Asn Arg Ile Pro 35 40 45 Thr His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn Asp Ser 50 55 60 Gly Glu Tyr Arg Cys Gln Thr Gly Arg Thr Ser Leu Ser Asp Pro Val 65 70 75 80 His Leu Thr Val Leu Ser Glu Trp Leu Ala Leu Gln Thr Pro His Leu 85 90 95 Glu Phe Arg Glu Gly Glu Thr Ile Met Leu Arg Cys His Ser Trp Lys 100 105 110 Asp Lys Pro Leu Ile Lys Val Thr Phe Phe Gln Asn Gly Ile Ala Lys 115 120 125 Lys Phe Ser His Met Asp Pro Asn Phe Ser Ile Pro Gln Ala Asn His 130 135 140 Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr Thr Pro 145 150 155 160 Tyr Ser Ser Lys Pro Val Thr Ile Thr Val Gln Val Pro Ser Val Gly 165 170 175 Ser Ser Ser Pro Met Gly Ile Ile Val Ala Val Val Thr Gly Ile Ala 180 185 190 Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr Cys Arg Lys 195 200 205 Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala Ala Arg Phe 210 215 220 Glu Pro Leu Gly Arg Gln Thr Ile Ala Leu Arg Lys Arg Gln Leu Glu 225 230 235 240 Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr Met Thr Leu 245 250 255 Asn Pro Arg Ala Pro Thr Asp Asp Asp Arg Asn Ile Tyr Leu Thr Leu 260 265 270 Ser Pro Asn Asp Tyr Asp Asn Ser Asn Asn 275 280 67 281 PRT Chimp MISC_FEATURE (1)..(281) FcgammaRIIA 67 Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Pro Trp Ile Asn Val 1 5 10 15 Leu Gln Glu Asp Ser Val Thr Leu Thr Cys Arg Gly Ala Arg Ser Pro 20 25 30 Glu Ser Asp Ser Ile Gln Trp Phe His Asn Gly Asn Leu Ile Pro Thr 35 40 45 His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn Asp Ser Gly 50 55 60 Glu Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp Pro Val His 65 70 75 80 Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr Pro His Leu Glu 85 90 95 Phe Gln Glu Gly Glu Thr Ile Val Leu Arg Cys His Ser Trp Lys Asp 100 105 110 Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly Lys Ser Gln Lys 115 120 125 Phe Ser His Leu Asp Pro Asn Leu Ser Ile Pro Gln Ala Asn His Ser 130 135 140 His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr Thr Leu Phe 145 150 155 160 Ser Ser Lys Pro Val Thr Ile Thr Val Gln Ala Pro Ser Val Gly Ser 165 170 175 Ser Ser Pro Val Gly Ile Ile Val Ala Val Val Ile Ala Thr Ala Val 180 185 190 Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr Cys Arg Lys Lys 195 200 205 Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala Ala Gln Phe Glu 210 215 220 Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg Gln Leu Glu Glu 225 230 235 240 Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr Met Thr Leu Asn 245 250 255 Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr Leu Thr Leu Pro 260 265 270 Pro Asn Asp His Val Asn Ser Asn Asn 275 280 68 252 PRT Cynomolgus MISC_FEATURE (1)..(252) FcgammaaRIIB 68 Thr Pro Ala Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Pro Trp 1 5 10 15 Ile Asn Val Leu Arg Glu Asp Ser Val Thr Leu Thr Cys Gly Gly Ala 20 25 30 His Ser Pro Asp Ser Asp Ser Thr Gln Trp Phe His Asn Gly Asn Leu 35 40 45 Ile Pro Thr His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn 50 55 60 Asp Ser Gly Glu Tyr Arg Cys Gln Thr Gly Arg Thr Ser Leu Ser Asp 65 70 75 80 Pro Val His Leu Thr Val Leu Ser Glu Trp Leu Ala Leu Gln Thr Pro 85 90 95 His Leu Glu Phe Arg Glu Gly Glu Thr Ile Leu Leu Arg Cys His Ser 100 105 110 Trp Lys Asp Lys Pro Leu Ile Lys Val Thr Phe Phe Gln Asn Gly Ile 115 120 125 Ser Lys Lys Phe Ser His Met Asn Pro Asn Phe Ser Ile Pro Gln Ala 130 135 140 Asn His Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr 145 150 155 160 Thr Pro Tyr Ser Ser Lys Pro Val Thr Ile Thr Val Gln Val Pro Ser 165 170 175 Met Gly Ser Ser Ser Pro Ile Gly Ile Ile Val Ala Val Val Thr Gly 180 185 190 Ile Ala Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr Cys 195 200 205 Arg Lys Lys Arg Ile Ser Ala Asn Pro Thr Asn Pro Asp Glu Ala Asp 210 215 220 Lys Val Gly Ala Glu Asn Thr Ile Thr Tyr Ser Leu Leu Met His Pro 225 230 235 240 Asp Ala Leu Glu Glu Pro Asp Asp Gln Asn Arg Val 245 250 69 234 PRT Cynomolgus MISC_FEATURE (1)..(234) FcgammaRIIIA - Alpha chain 69 Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro Gln Trp Tyr Arg 1 5 10 15 Val Leu Glu Lys Asp Arg Val Thr Leu Lys Cys Gln Gly Ala Tyr Ser 20 25 30 Pro Glu Asp Asn Ser Thr Arg Trp Phe His Asn Glu Ser Leu Ile Ser 35 40 45 Ser Gln Thr Ser Ser Tyr Phe Ile Ala Ala Ala Arg Val Asn Asn Ser 50 55 60 Gly Glu Tyr Arg Cys Gln Thr Ser Leu Ser Thr Leu Ser Asp Pro Val 65 70 75 80 Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln Ala Pro Arg Trp 85 90 95 Val Phe Lys Glu Glu Glu Ser Ile His Leu Arg Cys His Ser Trp Lys 100 105 110 Asn Thr Leu Leu His Lys Val Thr Tyr Leu Gln Asn Gly Lys Gly Arg 115 120 125 Lys Tyr Phe His Gln Asn Ser Asp Phe Tyr Ile Pro Lys Ala Thr Leu 130 135 140 Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Ile Gly Ser Lys Asn 145 150 155 160 Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln Asp Leu Ala Val 165 170 175 Ser Ser Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln Val Ser Phe Cys 180 185 190 Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly Leu Tyr Phe Ser 195 200 205 Met Lys Lys Ser Ile Pro Ser Ser Thr Arg Asp Trp Glu Asp His Lys 210 215 220 Phe Lys Trp Ser Lys Asp Pro Gln Asp Lys 225 230 70 99 PRT Cynomolgus MISC_FEATURE (1)..(99) Beta-2 microglobulin 70 Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg His Pro Pro Glu 1 5 10 15 Asn Gly Lys Pro Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro 20 25 30 Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu Lys Met Gly Lys 35 40 45 Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu 50 55 60 Leu Tyr Tyr Thr Glu Phe Thr Pro Asn Glu Lys Asp Glu Tyr Ala Cys 65 70 75 80 Arg Val Asn His Val Thr Leu Ser Gly Pro Arg Thr Val Lys Trp Asp 85 90 95 Arg Asp Met 71 342 PRT Cynomolgus MISC_FEATURE (1)..(342) FcgammaRn alpha-chain (S3) 71 Ala Glu Ser His Leu Ser Leu Leu Tyr His Leu Thr Ala Val Ser Ser 1 5 10 15 Pro Ala Pro Gly Thr Pro Ala Phe Trp Val Ser Gly Trp Leu Gly Pro 20 25 30 Gln Gln Tyr Leu Ser Tyr Asp Ser Leu Arg Gly Gln Ala Glu Pro Cys 35 40 45 Gly Ala Trp Val Trp Glu Asn Gln Val Ser Trp Tyr Trp Glu Lys Glu 50 55 60 Thr Thr Asp Leu Arg Ile Lys Glu Lys Leu Phe Leu Glu Ala Phe Lys 65 70 75 80 Ala Leu Gly Gly Lys Gly Pro Tyr Thr Leu Gln Gly Leu Leu Gly Cys 85 90 95 Glu Leu Ser Pro Asp Asn Thr Ser Val Pro Thr Ala Lys Phe Ala Leu 100 105 110 Asn Gly Glu Glu Phe Met Asn Phe Asp Leu Lys Gln Gly Thr Trp Gly 115 120 125 Gly Asp Trp Pro Glu Ala Leu Ala Ile Ser Gln Arg Trp Gln Gln Gln 130 135 140 Asp Lys Ala Ala Asn Lys Glu Leu Thr Phe Leu Leu Phe Ser Cys Pro 145 150 155 160 His Arg Leu Arg Glu His Leu Glu Arg Gly Arg Gly Asn Leu Glu Trp 165 170 175 Lys Glu Pro Pro Ser Met Arg Leu Lys Ala Arg Pro Gly Asn Pro Gly 180 185 190 Phe Ser Val Leu Thr Cys Ser Ala Phe Ser Phe Tyr Pro Pro Glu Leu 195 200 205 Gln Leu Arg Phe Leu Arg Asn Gly Met Ala Ala Gly Thr Gly Gln Gly 210 215 220 Asp Phe Gly Pro Asn Ser Asp Gly Ser Phe His Ala Ser Ser Ser Leu 225 230 235 240 Thr Val Lys Ser Gly Asp Glu His His Tyr Cys Cys Ile Val Gln His 245 250 255 Ala Gly Leu Ala Gln Pro Leu Arg Val Glu Leu Glu Thr Pro Ala Lys 260 265 270 Ser Ser Val Leu Val Val Gly Ile Val Ile Gly Val Leu Leu Leu Thr 275 280 285 Ala Ala Ala Val Gly Gly Ala Leu Leu Trp Arg Arg Met Arg Ser Gly 290 295 300 Leu Pro Ala Pro Trp Ile Ser Leu Arg Gly Asp Asp Thr Gly Ser Leu 305 310 315 320 Leu Pro Thr Pro Gly Glu Ala Gln Asp Ala Asp Ser Lys Asp Ile Asn 325 330 335 Val Ile Pro Ala Thr Ala 340 72 342 PRT Cynomolgus MISC_FEATURE (1)..(342) FcgammaRn alpha-chain (N3) 72 Ala Glu Asn His Leu Ser Leu Leu Tyr His Leu Thr Ala Val Ser Ser 1 5 10 15 Pro Ala Pro Gly Thr Pro Ala Phe Trp Val Ser Gly Trp Leu Gly Pro 20 25 30 Gln Gln Tyr Leu Ser Tyr Asp Ser Leu Arg Gly Gln Ala Glu Pro Cys 35 40 45 Gly Ala Trp Val Trp Glu Asn Gln Val Ser Trp Tyr Trp Glu Lys Glu 50 55 60 Thr Thr Asp Leu Arg Ile Lys Glu Lys Leu Phe Leu Glu Ala Phe Lys 65 70 75 80 Ala Leu Gly Gly Lys Gly Pro Tyr Thr Leu Gln Gly Leu Leu Gly Cys 85 90 95 Glu Leu Ser Pro Asp Asn Thr Ser Val Pro Thr Ala Lys Phe Ala Leu 100 105 110 Asn Gly Glu Glu Phe Met Asn Phe Asp Leu Lys Gln Gly Thr Trp Gly 115 120 125 Gly Asp Trp Pro Glu Ala Leu Ala Ile Ser Gln Arg Trp Gln Gln Gln 130 135 140 Asp Lys Ala Ala Asn Lys Glu Leu Thr Phe Leu Leu Phe Ser Cys Pro 145 150 155 160 His Arg Leu Arg Glu His Leu Glu Arg Gly Arg Gly Asn Leu Glu Trp 165 170 175 Lys Glu Pro Pro Ser Met Arg Leu Lys Ala Arg Pro Gly Asn Pro Gly 180 185 190 Phe Ser Val Leu Thr Cys Ser Ala Phe Ser Phe Tyr Pro Pro Glu Leu 195 200 205 Gln Leu Arg Phe Leu Arg Asn Gly Met Ala Ala Gly Thr Gly Gln Gly 210 215 220 Asp Phe Gly Pro Asn Ser Asp Gly Ser Phe His Ala Ser Ser Ser Leu 225 230 235 240 Thr Val Lys Ser Gly Asp Glu His His Tyr Cys Cys Ile Val Gln His 245 250 255 Ala Gly Leu Ala Gln Pro Leu Arg Val Glu Leu Glu Thr Pro Ala Lys 260 265 270 Ser Ser Val Leu Val Val Gly Ile Val Ile Gly Val Leu Leu Leu Thr 275 280 285 Ala Ala Ala Val Gly Gly Ala Leu Leu Trp Arg Arg Met Arg Ser Gly 290 295 300 Leu Pro Ala Pro Trp Ile Ser Leu Arg Gly Asp Asp Thr Gly Ser Leu 305 310 315 320 Leu Pro Thr Pro Gly Glu Ala Gln Asp Ala Asp Ser Lys Asp Ile Asn 325 330 335 Val Ile Pro Ala Thr Ala 340

Claims

1-43. (cancelled)

44. A method for evaluating at least one biological property of an Fc region containing molecule comprising: a) contacting an isolated non-human primate Fc receptor polypeptide with an Fc region containing molecule; and b) determining the effect of the contact on at least one biological property of the Fc region containing molecule.

45. A method according to claim 44, wherein the Fc region containing molecule is an antibody.

46. A method according to claim 45, wherein the antibody is a humanized antibody.

47. A method according to claim 46, wherein the antibody is an antibody variant.

48. A method according to claim 47, wherein the non-human primate Fc receptor polypeptide is a soluble receptor.

49. A method according to claim 48, wherein the non-human primate receptor polypeptide is selected from the group consisting of Fc.gamma.RI .alpha.-chain, Fc.gamma.RIIA, Fc.gamma.RIIB, Fc.gamma.RIIA .alpha.-chain, FcRn .alpha.-chain and mixtures thereof.

50. A method according to claim 44, wherein the non-human primate receptor polypeptide is expressed on a cell.

51. A method according to claim 44, wherein the biological property is the binding affinity of the Fc region containing molecule for the non-human primate receptor polypeptide.

52. A method according to claim 44, wherein the biological property is the toxicity of the Fc region containing molecule.

53. A method according to claim 44, wherein the isolated non-human primate Fc receptor polypeptide is a FcRn .alpha.-chain and the biological property is the half-life of the Fc region containing molecule.

54. A method according to claim 44, wherein the non-human primate Fc receptor polypeptide comprises an amino sequence of 1 to 265 of SEQ ID NO: 65.

55. A method according to claim 44, wherein the non-human primate Fc receptor polypeptide comprises an amino acid sequence of 1 to 172 of SEQ ID NO: 66.

56. A method according to claim 44, wherein the non-human primate Fc receptor polypeptide comprises an amino acid sequence of 1 to 174 of SEQ ID NO: 68.

57. A method according to claim 47, wherein the non-human primate receptor polypeptide comprises an amino acid sequence of amino acids 1 to 172 of SEQ ID NO: 69.

58. A method according to claim 44, wherein the non-human primate Fc receptor polypeptide comprises an amino acid sequence of amino acids 1 to 171 of SEQ ID NO: 67.

59. A method for evaluating at least one biological property of an Fc region containing molecule comprising: a) contacting a Fc region containing molecule with a cell transformed with an isolated nucleic acid encoding a nonhuman primate Fc receptor polypeptide; and b) determining the effect of the contact on at least one biological property of the Fc region containing molecule.

60. A method according to claim 59, wherein the Fc region containing molecule is an antibody or antibody variant.

61. A method according to claim 59, wherein the biological property is the binding affinity of the Fc region containing molecule for the non-human primate Fc receptor polypeptide.

62. A method according to claim 59, wherein the cell is transformed with at least two nucleic acids according to claim 1.

63. A method according to claim 62, wherein the nucleic acids comprise a nucleic acid that encodes a cynomolgus Fc.gamma.RI .alpha.-chain of SEQ ID NO: 9 and a nucleic acid that encodes a cynomolgus Fc.gamma.R gamma chain of SEQ ID NO: 11.

64. A method according to claim 62, wherein the nucleic acids comprise a nucleic acid that encodes a cynomolgus Fc.gamma.RIII .alpha.-chain of SEQ ID NO: 20 and a nucleic acid that encodes a cynomolgus Fc.gamma.R gamma chain of SEQ ID NO: 11.

65. A method according to claim 62, wherein the nucleic acids comprise a nucleic acid that encodes a cynomolgus Fc.gamma.R .alpha.-chain of SEQ ID NO: 29 and a nucleic acid sequence that encodes a cynomolgus .beta.-2 microglobulin of SEQ ID NO:25.

66. A method for identifying an agent that has an increased affinity for at least one cynomolgus Fc receptor polypeptide with an ITAM region compared to human Fc receptor polypeptide comprising: a) determining the binding affinity of the agent to at least one cynomolgus Fc receptor polypeptide associated a polypeptide with an ITAM region; b) determining the binding affinity of the agent to the corresponding human Fc receptor polypeptide; and c) selecting agents that have an increased affinity for the cynomolgus Fc.gamma. receptor polypeptide associated with a polypeptide with an ITAM region compared to the corresponding human Fc receptor.

67. A method according to claim 66, wherein the agent is an antibody.

68. A method according to claim 67, wherein the agent is an IgG antibody.

69. A method according to claim 67, wherein the Fc receptor polypeptide is selected from the group consisting of Fc.gamma.R1 .alpha.-chain, Fc.gamma.RIIA, Fc.gamma.RIIIA .alpha.-chain and mixtures thereof.

70. A method for identifying an agent that has an altered affinity for a cynomolgus Fc receptor polypeptide with an ITIM region compared to corresponding human Fc receptor polypeptide comprising: a) determining a binding affinity for the agent to be at least one cynomolgus Fc.gamma.RIIB receptor polypeptide; b) determining a binding affinity of the agent to corresponding human Fc.gamma.RIIB receptor polypeptide; and c) selecting agents with altered affinity for a cynomolgus Fc.gamma.RIIB receptor polypeptide with an ITIM region compared to corresponding human Fc.gamma.RIIB polypeptide.

71. A method according to claim 70, wherein the agent is an antibody.

72. A method for identifying an agent with increased binding affinity for a cynomolgus Fc receptor polypeptide with an ITAM region and decreased affinity for a cynomolgus Fc receptor polypeptide with an ITIM region comprising: a) determining a binding affinity of the agent for at least one cynomolgus Fc receptor polypeptide associated with an ITAM region and a binding affinity of the agent to the corresponding human Fc receptor polypeptide; b) determining the binding affinity of the agent for at least one cynomolgus Fc receptor polypeptide with an ITIM region and a binding affinity of the agent for the corresponding human Fc receptor polypeptide; and c) selecting an agent with enhanced binding for a cynomolgus Fc receptor polypeptide with an ITAM region and a decreased affinity for a cynomolgus Fc receptor polypeptide with an ITIM region compared to the corresponding human Fc receptor polypeptides.

73. A method according to claim 72, wherein the Fc.gamma. receptor with an ITAM region is an Fc.gamma. receptor IIA and the Fc.gamma. receptor with an ITIM region is a Fc.gamma. receptor IIB.

74. A method according to claim 73, wherein the agent is an antibody.

75-90. (cancelled)