Methods of treating cancer using antibodies that immunospecifically bind to TRAIL receptors

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) based on U.S. Provisional Application Ser. No. 60/608,362 filed Sep. 10, 2004. This application is also a continuation-in-part and claims benefit of priority under 35 U.S.C. § 120 of International Application No. PCT/US03/25457, filed Aug. 15, 2003, which claims benefit under 35 U.S.C. § 119(e) based on U.S. Provisional Application Nos. 60/403,382, filed Aug. 15, 2002; 60/425,730, filed Nov. 13, 2002; and 60/468,050, filed May 6, 2003. This application is also a continuation-in-part and claims benefit of priority under 35 U.S.C. § 120 of Non-Provisional application Ser. No. 10/139,785, filed May 7, 2002, now U.S. Pat. No. 7,064,189, issued Jun. 20, 2006, which claims benefit under 35 U.S.C. § 119(e) based on U.S. Provisional Application Nos. 60/293,473, filed May 25, 2001; 60/294,981, filed Jun. 4, 2001; 60/309,176, filed Aug. 2, 2001; 60/323,807, filed Sep. 21, 2001; 60/327,364, filed Oct. 9, 2001; 60/331,044, filed Nov. 7, 2001; 60/331,310, filed Nov. 14, 2001; 60/341,237, filed Dec. 20, 2001; and 60/369,860, filed Apr. 5, 2002. Each of the above-identified priority applications is hereby incorporated by reference in its entirety.

[0002] The present invention relates to antibodies and related molecules that immunospecifically bind to TRAIL receptor, TR4. Such antibodies have uses, for example, in the prevention and treatment of cancers and other proliferative disorders. The invention also relates to nucleic acid molecules encoding anti-TR4 antibodies, vectors and host cells containing these nucleic acids, and methods for producing the same. The present invention relates to methods and compositions for preventing, detecting, diagnosing, treating or ameliorating a disease or disorder, especially cancer and other hyperproliferative disorders, comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that immunospecifically bind to TR4.

[0003] Many biological actions, for instance, response to certain stimuli and natural biological processes, are controlled by factors, such as cytokines. Many cytokines act through receptors by engaging the receptor and producing an intra-cellular response.

[0004] For example, tumor necrosis factors (TNF) alpha and beta are cytokines which act through TNF receptors to regulate numerous biological processes, including protection against infection and induction of shock and inflammatory disease. The TNF molecules belong to the “TNF-ligand” superfamily, and act together with their receptors or counter-ligands, the “TNF-receptor” superfamily. So far, at least eighteen members of the TNF ligand superfamily have been identified and at least nineteen members of the TNF-receptor superfamily have been characterized (See, e.g., Locksley et el., Cell (2001) 104:487-501).

[0005] Among the ligands there are included TNF-α, lymphotoxin-α (LT-α, also known as TNF-β), LT-β (found in complex heterotrimer LT-α2-β), FasL, CD40L, CD27L, CD30L, 4-1BBL, OX40L and nerve growth factor (NGF). The superfamily of TNF receptors includes the p55TNF receptor, p75TNF receptor, TNF receptor-related protein, FAS antigen or APO-1, CD40, CD27, CD30, 4-1BB, OX40, low affinity p75 and NGF-receptor (Meager, A., Biologicals, 22:291-295 (1994)).

[0006] Many members of the TNF-ligand superfamily are expressed by activated T-cells, implying that they are necessary for T-cell interactions with other cell types which underlie cell ontogeny and functions. (Meager, A., supra).

[0007] Considerable insight into the essential functions of several members of the TNF receptor family has been gained from the identification and creation of mutants that abolish the expression of these proteins. For example, naturally occurring mutations in the FAS antigen and its ligand cause lymphoproliferative disease (Watanabe-Fukunaga, R., et al., Nature 356:314 (1992)), perhaps reflecting a failure of programmed cell death. Mutations of the CD40 ligand cause an X-linked immunodeficiency state characterized by high levels of immunoglobulin M and low levels of immunoglobulin G in plasma, indicating faulty T-cell-dependent B-cell activation (Allen, R. C. et al., Science 259:990 (1993)). Targeted mutations of the low affinity nerve growth factor receptor cause a disorder characterized by faulty sensory innovation of peripheral structures (Lee, K. F. et al., Cell 69:737 (1992)).

[0008] TNF and LT-α are capable of binding to two TNF receptors (the 55- and 75-kd TNF receptors). A large number of biological effects elicited by TNF and LT-α, acting through their receptors, include hemorrhagic necrosis of transplanted tumors, cytotoxicity, a role in endotoxic shock, inflammation, immunoregulation, proliferation and anti-viral responses, as well as protection against the deleterious effects of ionizing radiation. TNF and LT-α are involved in the pathogenesis of a wide range of diseases, including endotoxic shock, cerebral malaria, tumors, autoimmune disease, AIDS and graft-host rejection (Beutler, B. and Von Huffel, C., Science 264:667-668 (1994)). Mutations in the p55 Receptor cause increased susceptibility to microbial infection.

[0009] Moreover, an about 80 amino acid domain near the C-terminus of TNFR1 (p55) and Fas was reported as the “death domain,” which is responsible for transducing signals for programmed cell death (Tartaglia et al., Cell 74:845 (1993)).

[0010] Apoptosis, or programmed cell death, is a physiologic process essential to the normal development and homeostasis of multicellular organisms (H. Steller, Science 267, 1445-1449 (1995)). Derangements of apoptosis contribute to the pathogenesis of several human diseases including cancer, neurodegenerative disorders, and acquired immune deficiency syndrome (C. B. Thompson, Science 267, 1456-1462 (1995)). Recently, much attention has focused on the signal transduction and biological function of two cell surface death receptors, Fas/APO-1 and TNFR-1 (J. L. Cleveland, et al., Cell 81, 479-482 (1995); A. Fraser, et al., Cell 85, 781-784 (1996); S. Nagata, et al., Science 267, 1449-56 (1995)). Both are members of the TNF receptor family which also include TNFR-2, low affinity NGFR, CD40, and CD30, among others (C. A. Smith, et al., Science 248, 1019-23 (1990); M. Tewari, et al., in Modular Texts in Molecular and Cell Biology M. Purton, Heldin, Carl, Ed. (Chapman and Hall, London, 1995). While family members are defined by the presence of cysteine-rich repeats in their extracellular domains, Fas/APO-1 and TNFR-1 also share a region of intracellular homology, appropriately designated the “death domain”, which is distantly related to the Drosophila suicide gene, reaper (P. Golstein, et al., Cell 81, 185-6 (1995); K. White et al., Science 264, 677-83 (1994)). This shared death domain suggests that both receptors interact with a related set of signal transducing molecules that, until recently, remained unidentified. Activation of Fas/APO-1 recruits the death domain-containing adapter molecule FADD/MORT1 (A. M. Chinnaiyan, et al., Cell 81, 505-12 (1995); M. P. Boldin, et al., J. Biol Chem 270, 7795-8 (1995); F. C. Kischkel, et al., EMBO 14, 5579-5588 (1995)), which in turn binds and presumably activates FLICE/MACH1, a member of the ICE/CED-3 family of pro-apoptotic proteases (M. Muzio et al., Cell 85, 817-827 (1996); M. P. Boldin, et al, Cell 85, 803-815 (1996)). While the central role of Fas/APO-1 is to trigger cell death, TNFR-1 can signal an array of diverse biological activities-many of which, stem from its ability to activate NF-kB (L. A. Tartaglia, et al., Immunol Today 13, 151-3 (1992)). Accordingly, TNFR-1 recruits the multivalent adapter molecule TRADD, which like FADD, also contains a death domain (H. Hsu, et al., Cell 81, 495-504 (1995); H. Hsu, et al., Cell 84, 299-308 (1996)). Through its associations with a number of signaling molecules including FADD, TRAF2, and RIP, TRADD can signal both apoptosis and NF-kB activation (H. Hsu, et al., Cell 84, 299-308 (1996); H. Hsu, et al., Immunity 4, 387-396 (1996)).

[0011] One TNF-related apoptosis inducing ligand has been reported by several groups and has been ascribed the name Apoptosis Inducing Molecule I (AIM-I) (Intenation Application No. WO 97/33899) and TNF-related apoptosis-inducing ligand or (TRAIL) (Wiley, S. R. et al., Immunity 3:673-682 (1995)). Pitti, R. M. et al., refer to the new molecule as Apo-2 ligand or (“Apo-2L”). For convenience, it will be referred to herein as TRAIL. The amino acid sequence of TRAIL is given in SEQ ID NO:66.

[0012] Unlike FAS ligand whose transcripts appear to be largely restricted to stimulated T-cells, significant levels of TRAIL are seen in many tissues, and it is constitutively transcribed by some cell lines. It has been shown that TRAIL acts independently from FAS ligand (Wiley, S. R., et al. (1995)), supra). Studies by Marsters, S. A. et al., have indicated that TRAIL activates apoptosis rapidly, within a time frame that is similar to death signalling by FAS/Apo-1L but much faster than TNF-induced apoptosis (Current Biology, 6:750-752 (1996)).

[0013] As many as five TRAIL receptors have been identified, including TR4 (also known as TRAIL receptor 1 (TRAIL-R1) and death receptor 4 (DR4), Pan et al., Science 276:111-3 (1997), International Patent Application Nos. WO98/32856, WO00/67793, WO99/37684, WO2000/34355, WO99/02653, SEQ ID NO:1); TR7 (also referred to as TRAIL receptor 2 (TRAIL-R2), DR5, and KILLER, Pan et al., Science 277:815-8 (1997), Sheridan et al., Science 277:818-21 (1997), Chaudhury et al., Immunity 7:821-30 (1997), International Patent Application Nos. WO98/46643, WO99/09165, WO99/11791, WO98/41629, WO00/66156, and WO98/35986, SEQ ID NO:3); TR1 (also referred to as Osteoprotegrin (OPG) osteoclastogenesis inhibitory factor (OCIF), TNFRSF11B, and FTHMA-090 (International Patent Application Nos. WO98/12344, WO2000/54651, WO2001/04137, WO66/26217, WO98/07840, WO2000/21554, WO99/53942, and WO2001/03719, SEQ ID NO:5); TR5 (also referred to as TRAIL receptor 3 (TRAIL-R3), decoy receptor 1 (DcR1) and TRID) (Degli-Esposti et al., J. Exp. Med. 186:1165-70 (1997), International Patent Application Nos. WO98/30693, WO00/71150, WO99/00423, EP867509, WO98/58062, SEQ ID NO:2); and TR10 (also referred to as TRAIL Receptor 4 (TRAIL-R4), DcR2, and TRUNDD, Pan et al., FEBS Lett. 424:41-5 (1998), Degli-Eposti et al., Immunity 7:813-20 (1997), International Patent Application Nos. WO98/54202, WO00/73321, WO2000/08155, WO99/03992, WO 2000/34355 and WO9910484, SEQ ID NO:4). TR4 and TR7 contain death domains in their cytoplasmic tails and the triggering of these receptors results in apoptosis. On the other hand, TR1, TR5 and TR10 can inhibit apoptosis induced by the cytotoxic ligand TRAIL in part because of their absent or truncated cytoplasmic death domains, respectively. Each of the publications and patents cited above is hereby incorporated by reference in their entireties, particularly with respect to the nucleotide and amino acid sequences of the TRAIL receptors disclosed therein.

[0014] The effects of TNF family ligands and TNF family receptors are varied and influence numerous functions, both normal and abnormal, in the biological processes of the mammalian system. There is a clear need, therefore, for identification and characterization of compositions, such as antibodies, that influence the biological activity of TNF receptors, both normally and in disease states. In particular, there is a need to isolate and characterize antibodies that modulate the biological activities of TRAIL receptors.

[0015] The present invention encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a TR4 polypeptide or polypeptide fragment or variant of TR4. In particular, the invention encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or polypeptide fragment or variant of human TR4 such as that of SEQ ID NO:1. In some embodiments, an antibody of the invention that immunospecifically bind to a TR4 polypeptide, also bind TR7 (e.g., SEQ ID NO:3), but not other proteins, including (TR1, TR5, and TR10 (SEQ ID NOS:5, 2 and 4.)

[0016] The present invention relates to methods and compositions for preventing, treating or ameliorating a disease or disorder comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that immunospecifically bind to TR4 or a fragment or variant thereof. In specific embodiments, the present invention relates to methods and compositions for preventing, treating or ameliorating a disease or disorder associated with TR4 function or TR4 ligand function or aberrant TR4 or TR4 ligand expression, comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that immunospecifically bind to a TR4 or a fragment or variant thereof. In highly preferred embodiments, the present invention relates to antibody-based methods and compositions for preventing, treating or ameliorating cancers and other hyperproliferative disorders (e.g., leukemia, carcinoma, and lymphoma). Other diseases and disorders which can be treated, prevented or ameliorated with the antibodies of the invention include, but are not limited to, neurodegenerative disorders (e.g., Parkinson's disease, Alzheimer's disease, and Huntington's disease), immune disorders (e.g., lupus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Hashimoto's disease, and immunodeficiency syndrome), inflammatory disorders (e.g., asthma, allergic disorders, and rheumatoid arthritis), infectious diseases (e.g., AIDS, herpes viral infections, and other viral infections) and proliferative disorders.

[0017] In highly preferred embodiments, antibodies of the present invention are used in methods and compositions for preventing, diagnosing, prognosing, treating or ameliorating the following types of cancer: breast cancer, lung cancer, (including non-small cell lung cancer), colon cancer, cancer of the urinary tract, bladder cancer, kidney cancer, pancreatic cancer, liver cancer, stomach cancer, prostate cancer, leukemia, Non-Hodgkin's lymphoma, esophageal cancer, brain cancer, leukemia, ovarian cancer, testicular cancer, melanoma, uterine cancer, cervical cancer, cancer of the larynx, rectal cancer, and cancers of the oral cavity. In specific embodiments, antibodies of the invention are administered in combination with chemotherapeutics such as paclitaxel (Taxol), irinotecan (Camptosar, CPT-11), irinotecan analogs, and gemcitabine (GEMZAR™)) or other therapeutic agents useful in the treatment of cancers.

[0018] The present invention also encompasses methods and compositions for detecting, diagnosing, or prognosing diseases or disorders comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that immunospecifically bind to TR4 or a fragment or variant thereof. In specific embodiments, the present invention also encompasses methods and compositions for detecting, diagnosing, or prognosing diseases or disorders associated with TR4 function or TR4 ligand function or aberrant TR4 or TR4 ligand expression, comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that immunospecifically bind to TR4 or a fragment or variant thereof. In highly preferred embodiments, the present invention relates to antibody-based methods and compositions for detecting, diagnosing, or prognosing cancers and other hyperproliferative disorders (e.g., leukemia, carcinoma, and lymphoma). Other diseases and disorders which can be detected, diagnosed or prognosed with the antibodies of the invention include, but are not limited to, neurodegenerative disorders (e.g., Parkinson's disease, Alzheimer's disease, and Huntington's disease), immune disorders (e.g., lupus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Hashimoto's disease, and immunodeficiency syndrome), inflammatory disorders (e.g., asthma, allergic disorders, and rheumatoid arthritis), infectious diseases (e.g., AIDS, herpes virus infections, and other viral infections), and proliferative disorders.

[0019] Another embodiment of the present invention includes the use of the antibodies of the invention as a diagnostic tool to monitor the expression of TR4 expression on cells.

[0020] The present inventors have generated single chain Fv's (scFvs) that immunospecifically bind TR4 polypeptides (e.g., SEQ ID NOs:1). Thus, the invention encompases these scFvs, listed in Table 1. In addition, the invention encomasses cell lines engineered to express antibodies corresponding to these scFvs which are deposited with the American Type Culture Collection (“ATCC”) as of the dates listed in Table 1 and given the ATCC Deposit Numbers identified in Table 1 The ATCC is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure.

[0021] Further, the present invention encompasses the polynucleotides encoding the scFvs, as well as the amino acid sequences encoding the scFvs. Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs (e.g., VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of any one of the scFvs referred to in Table 1), that immunospecifically bind to TR4 or fragments or variants thereof are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies and/or molecules. In highly preferred embodiments, the present invention encompasses antibodies, or fragments or variants thereof, that bind to the extracellular regions/domains of TR4 or fragments and variants thereof.

[0022] The present invention also provides antibodies that bind TR4 polypeptides which are coupled to a detectable label, such as an enzyme, a fluorescent label, a luminescent label, or a bioluminescent label. The present invention also provides antibodies that bind TR4 polypeptides which are coupled to a therapeutic or cytotoxic agent. The present invention also provides antibodies that bind TR4 polypeptides which are coupled to a radioactive material.

[0023] The present invention also provides antibodies that bind TR4 polypeptides that act as either TR4 agonists or TR4 antagonists. In specific embodiments, the antibodies of the invention stimulate apoptosis of TR4 expressing cells. In other specific embodiments, the antibodies of the invention inhibit TRAIL binding to TR4. In other specific embodiments, the antibodies of the invention upregulate TR4 expression.

[0024] The present invention also provides antibodies that inhibit apoptosis of TR4 expressing cells. In other specific embodiments, the antibodies of the invention downregulate TR4 expression.

[0025] In further embodiments, the antibodies of the invention have a dissociation constant (K_D) of 10⁻⁷ M or less. In preferred embodiments, the antibodies of the invention have a dissociation constant (K_D) of 10⁻⁹ M or less.

[0026] The present invention further provides antibodies that stimulate apoptosis of TR4 expressing cells better than an equal concentration of TRAIL polypeptide stimulates apoptosis of TR4 expressing cells.

[0027] The present invention further provides antibodies that stimulate apoptosis of TR4 expressing cells equally well in the presence or absence of antibody cross-linking reagents; and/or stimulate apoptosis with equal or greater potency as an equal concentration of TRAIL in the absence of a cross-linking antibody or other cross-linking agent.

[0028] In further embodiments, antibodies of the invention have an off rate (k_off) of 10⁻³/sec or less. In preferred embodiments, antibodies of the invention have an off rate (k_off) of 10⁻⁴/sec or less. In other preferred embodiments, antibodies of the invention have an off rate (k_off) of 10⁻⁵/sec or less.

[0029] The present invention also provides for antibodies that preferentially bind TR4 and/or TR7 relative to their ability to bind other proteins (including TR1, TR5 and TR10).

[0030] In certain embodiments, properties of the antibodies of the present invention, as detailed in the Examples below, make the antibodies better therapeutic agents than previously described TR4 binding antibodies.

[0031] The present invention also provides panels of antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants) wherein the panel members correspond to one, two, three, four, five, ten, fifteen, twenty, or more different antibodies of the invention (e.g., whole antibodies, Fabs, F(ab′)₂ fragments, Fd fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and scFvs). The present invention further provides mixtures of antibodies, wherein the mixture corresponds to one, two, three, four, five, ten, fifteen, twenty, or more different antibodies of the invention (e.g., whole antibodies, Fabs, F(ab′)₂ fragments, Fd fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and scFvs)). The present invention also provides for compositions comprising, or alternatively consisting of, one, two, three, four, five, ten, fifteen, twenty, or more antibodies of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof). A composition of the invention may comprise, or alternatively consist of, one, two, three, four, five, ten, fifteen, twenty, or more amino acid sequences of one or more antibodies or fragments or variants thereof. Alternatively, a composition of the invention may comprise, or alternatively consist of, nucleic acid molecules encoding one or more antibodies of the invention.

[0032] The present invention also provides for fusion proteins comprising an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) of the invention, and a heterologous polypeptide (i.e., a polypeptide unrelated to an antibody or antibody domain). Nucleic acid molecules encoding these fusion proteins are also encompassed by the invention. A composition of the present invention may comprise, or alternatively consist of, one, two, three, four, five, ten, fifteen, twenty or more fusion proteins of the invention. Alternatively, a composition of the invention may comprise, or alternatively consist of, nucleic acid molecules encoding one, two, three, four, five, ten, fifteen, twenty or more fusion proteins of the invention.

[0033] The present invention also provides for a nucleic acid molecule(s), generally isolated, encoding an antibody (including molecules, such as scFvs, VH domains, or VL domains, that comprise, or alternatively consist of, an antibody fragment or variant thereof) of the invention. The present invention also provides a host cell transformed with a nucleic acid molecule of the invention and progeny thereof. The present invention also provides a method for the production of an antibody (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof) of the invention. The present invention further provides a method of expressing an antibody (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof) of the invention from a nucleic acid molecule. These and other aspects of the invention are described in further detail below.

Definitions

[0037] The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody multimers and antibody fragments as well as variants (including derivatives) of antibodies, antibody multimers and antibody fragments. Examples of molecules which are described by the term “antibody” herein include, but are not limited to: single chain Fvs (scFvs), Fab fragments, Fab′ fragments, F(ab′)₂, disulfide linked Fvs (sdFvs), Fvs, and fragments comprising or alternatively consisting of, either a VL or a VH domain. The term “single chain Fv” or “scFv” as used herein refers to a polypeptide comprising a VL domain of antibody linked to a VH domain of an antibody. Antibodies that immunospecifically bind to TR4 may have cross-reactivity with other antigens, e.g., another TRAIL Receptor. Preferably, antibodies that immunospecifically bind to TR4 do not cross-react with other antigens (e.g., other TRAIL receptors or other members of the Tumor Necrosis Factor Receptor superfamily). Antibodies that immunospecifically bind to TR4 can be identified, for example, by immunoassays or other techniques known to those of skill in the art, e.g., the immunoassays described in the Examples below.

[0038] Antibodies of the invention include, but are not limited to, monoclonal, multispecific, human or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), intracellularly-made antibodies (i.e., intrabodies), and epitope-binding fragments of any of the above. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂) or subclass of immunoglobulin molecule. Preferably, an antibody of the invention comprises, or alternatively consists of, a VH domain, VH CDR, VL domain, or VL CDR having an amino acid sequence of any one of those referred to in Table 1, or a fragment or variant thereof. In a preferred embodiment, the immunoglobulin is an IgG1 isotype. In another preferred embodiment, the immunoglobulin is an IgG4 isotype. Immunoglobulins may have both a heavy and light chain. An array of IgG, IgE, IgM, IgD, IgA, and IgY heavy chains may be paired with a light chain of the kappa or lambda forms.

[0039] Antibodies of the invention may also include multimeric forms of antibodies. For example, antibodies of the invention may take the form of antibody dimers, trimers, or higher-order multimers of monomeric immunoglobulin molecules. Dimers of whole immunoglobulin molecules or of F(ab′)₂ fragments are tetravalent, whereas dimers of Fab fragments or scFv molecules are bivalent. Individual monomers withon an antibody multimer may be identical or different, i.e., they may be heteromeric or homomeric antibody multimers. For example, individual antibodies within a multimer may have the same or different binding specificities.

[0040] Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers, and other higher-order antibody multimers. Alternatively, antibody homodimers may be formed through chemical linkage techniques known in the art. For example, heterobifunctional crosslinking agents including, but not limited to, SMCC [succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate] and SATA [N-succinimidyl S-acethylthio-acetate] (available, for example, from Pierce Biotechnology, Inc. (Rockford, Ill.)) can be used to form antibody multimers. An exemplary protocol for the formation of antibody homodimers is given in Ghetie et al., Proceedings of the National Academy of Sciences USA (1997) 94:7509-7514, which is hereby incorporated by reference in its entirety. Antibody homodimers can be converted to Fab′2 homodimers through digestion with pepsin. Another way to form antibody homodimers is through the use of the autophilic T15 peptide described in Zhao and Kohler, The Journal of Immunology (2002) 25:396-404, which is hereby incorporated by reference in its entirety.

[0041] Alternatively, antibodies can be made to multimerize through recombinant DNA techniques. IgM and IgA naturally form antibody multimers through the interaction with the mature J chain polypeptide (e.g., SEQ ID NO:67). Non-IgA or non-IgM molecules, such as IgG molecules, can be engineered to contain the J chain interaction domain of IgA or IgM, thereby conferring the ability to form higher order multimers on the non-IgA or non-IgM molecules. (see, for example, Chintalacharuvu et al., (2001) Clinical Immunology 101:21-31. and Frigerio et al., (2000) Plant Physiology 123:1483-94., both of which are hereby incorporated by reference in their entireties.) IgA dimers are naturally secreted into the lumen of mucosa-lined organs. This secretion is mediated through interaction of the J chain with the polymeric IgA receptor (pIgR) on epithelial cells. If secretion of an IgA form of an antibody (or of an antibody engineered to to contain a J chain interaction domain) is not desired, it can be greatly reduced by expressing the antibody molecule in association with a mutant J chain that does not interact well with pIgR (e.g., SEQ ID NOS:68-70; Johansen et al., The Journal of Immunology (2001) 167:5185-5192 which is hereby incorporated by reference in its entirety). SEQ ID NO:68 is a mutant form of a human mature J chain with C134S mutation compared to the mature form of human J chain (SEQ ID NO:67). SEQ ID NO:69 is a mutant form of a human mature J chain with amino acids 113-137 deleted compared to the mature form of human J chain (SEQ ID NO:67). SEQ ID NO:70 shows a mutant form of human mature J chain with C109S and C134S mutation compared to the mature form of human J chain (SEQ ID NO:67). Expression of an antibody with one of these mutant J chains will reduce its ability to bind to the polymeric IgA receptor on epithelial cells, thereby reducing transport of the antibody across the epithelial cell and its resultant secretion into the lumen of mucosa lined organs. ScFv dimers can also be formed through recombinant techniques known in the art; an example of the construction of scFv dimers is given in Goel et al., (2000) Cancer Research 60:6964-6971 which is hereby incorporated by reference in its entirety. Antibody multimers may be purified using any suitable method known in the art, including, but not limited to, size exclusion chromatography.

[0042] Unless otherwise defined in the specification, specific binding or immunospecifc binding by an anti-TR4 antibody means that the anti-TR4 antibody binds TR4 but does not significantly bind to (i.e., cross react with) proteins other than TR4, such as other proteins in the same family of proteins). An antibody that binds TR4 protein and does not cross-react with other proteins is not necessarily an antibody that does not bind said other proteins in all conditions; rather, the TR4-specific antibody of the invention preferentially binds TR4 compared to its ability to bind said other proteins such that it will be suitable for use in at least one type of assay or treatment, i.e., give low background levels or result in no unreasonable adverse effects in treatment. It is well known that the portion of a protein bound by an antibody is known as the epitope. An epitope may either be linear (i.e., comprised of sequential amino acids residues in a protein sequences) or conformational (i.e., comprised of one or more amino acid residues that are not contiguous in the primary structure of the protein but that are brought together by the secondary, tertiary or quaternary structure of a protein). Given that TR4-specific antibodies bind to epitopes of TR4, an antibody that specifically binds TR4 may or may not bind fragments of TR4 and/or variants of TR4 (e.g., proteins that are at least 90% identical to TR4) depending on the presence or absence of the epitope bound by a given TR4-specific antibody in the TR4 fragment or variant. Likewise, TR4-specific antibodies of the invention may bind species orthologues of TR4 (including fragments thereof) depending on the presence or absence of the epitope recognized by the antibody in the orthologue. Additionally, TR4-specific antibodies of the invention may bind modified forms of TR4, for example, TR4 fusion proteins. In such a case when antibodies of the invention bind TR4 fusion proteins, the antibody must make binding contact with the TR4 moiety of the fusion protein in order for the binding to be specific. Antibodies that specifically bind to TR4 can be identified, for example, by immunoassays or other techniques known to those of skill in the art, e.g., the immunoassays described in the Examples below.

[0043] In some embodiments the present invention encompasses antibodies that immunospecifically or specifically bind both TR4 and TR7. Specific binding or immunospecifc binding by an antibody that immunospecifically binds TR4 and TR7 means that the antibody binds TR4 and TR7 but does not significantly bind to (i.e., cross react with) proteins other than TR4 or TR7, such as other proteins in the same family of proteins). An antibody that binds TR4 and TR7 proteins and does not cross-react with other proteins is not necessarily an antibody that does not bind said other proteins in all conditions; rather, the antibody that immunospcifically or specifically binds both TR4 and TR7 preferentially binds TR4 and TR7 compared to its ability to bind said other proteins such that it will be suitable for use in at least one type of assay or treatment, i.e., give low background levels or result in no unreasonable adverse effects in treatment. It is well known that the portion of a protein bound by an antibody is known as the epitope. An epitope may either be linear (i.e., comprised of sequential amino acids residues in a protein sequences) or conformational (i.e., comprised of one or more amino acid residues that are not contiguous in the primary structure of the protein but that are brought together by the secondary, tertiary or quaternary structure of a protein). Given that antibodies that bind TR4 and TR7 bind to epitopes common to TR4 and TR7, an antibody that specifically binds TR4 and TR7 may or may not bind fragments of TR4, TR7 and/or variants of TR4 or TR7 (e.g., proteins that are at least 90% identical to TR4 or TR7, respectively) depending on the presence or absence of the epitope bound by a given antibody in the TR4 or TR7 fragment or variant. Likewise, antibodies of the invention that immunospecifically bind TR4 and TR7 may bind species orthologues of TR4 and/or TR7 (including fragments thereof) depending on the presence or absence of the epitope recognized by the antibody in the orthologues. Additionally, antibodies of the invention that immunospecifically bind TR4 and TR7 may bind modified forms of TR4 or TR7, for example, TR4 or TR7 fusion proteins. In such a case when antibodies of the invention bind fusion proteins, the antibody must make binding contact with the TR4 or TR7 moiety of the fusion protein in order for the binding to be specific. Antibodies that specifically bind to TR4 or TR7 can be identified, for example, by immunoassays or other techniques known to those of skill in the art, e.g., the immunoassays described in the Examples below.

[0044] The term “variant” as used herein refers to a polypeptide that possesses a similar or identical function as a TR4 polypeptide, a fragment of a TR4 polypeptide, an anti-TR4 antibody or antibody fragment thereof, but does not necessarily comprise a similar or identical amino acid sequence of a TR4 polypeptide, a fragment of a TR4 polypeptide, an anti-TR4 antibody or antibody fragment thereof, or possess a similar or identical structure of a TR4 polypeptide, a fragment of a TR4 polypeptide, an anti-TR4 antibody or antibody fragment thereof, respectively. A variant having a similar amino acid sequence refers to a polypeptide that satisfies at least one of the following: (a) a polypeptide comprising, or alternatively consisting of, an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence of TR4 polypeptide (SEQ ID NO:1), a fragment of, an anti-TR4 antibody or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence of any one or more scFvs referred to in Table 1) described herein; (b) a polypeptide encoded by a nucleotide sequence, the complementary sequence of which hybridizes under stringent conditions to a nucleotide sequence encoding TR4 (SEQ ID NO:1), a fragment of a TR4 polypeptide, an anti-TR4 antibody or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence of any one of those referred to in Table 1), described herein, of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, or at least 150 amino acid residues; and (c) a polypeptide encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%, identical to the nucleotide sequence encoding a TR4 polypeptide, a fragment of a TR4 polypeptide, an anti-TR4 antibody or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence of any one or more scFvs referred to in Table 1), described herein. A polypeptide with similar structure to a TR4 polypeptide, a fragment of a TR4 polypeptide, an anti-TR4 antibody or antibody fragment thereof, described herein refers to a polypeptide that has a similar secondary, tertiary or quaternary structure of a TR4 polypeptide, a fragment of a TR4 polypeptide, an anti-TR4 antibody, or antibody fragment thereof, described herein. The structure of a polypeptide can determined by methods known to those skilled in the art, including but not limited to, X-ray crystallography, nuclear magnetic resonance, and crystallographic electron microscopy.

[0045] To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions/total number of positions×100%). In one embodiment, the two sequences are the same length.

[0046] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-2268(1990), modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-5877(1993). The BLASTn and BLASTx programs of Altschul, et al. J. Mol. Biol. 215:403-410(1990) have incorporated such an alogrithm. BLAST nucleotide searches can be performed with the BLASTn program (score=100, wordlength=12) to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention. BLAST protein searches can be performed with the BLASTx program (score=50, wordlength=3) to obtain amino acid sequences homologous to a protein molecule of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. Nucleic Acids Res. 25:3589-3402(1997). Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. (See world wide web at ncbi.nlm.nih.gov).

[0047] Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0) which is part of the GCG sequence alignment software package has incorporated such an alogrithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti Comput. Appl. Biosci., 10:3-5(1994); and FASTA described in Pearson and Lipman Proc. Natl. Acad. Sci 85:2444-8(1988). Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.

[0048] The term “derivative” as used herein, refers to a variant polypeptide of the invention that comprises, or alternatively consists of, an amino acid sequence of a TR4 polypeptide, a fragment of a TR4 polypeptide, or an antibody of the invention that immunospecifically binds to a TR4 polypeptide, which has been altered by the introduction of amino acid residue substitutions, deletions or additions. The term “derivative” as used herein also refers to a TR4 polypeptide, a fragment of a TR4 polypeptide, an antibody that immunospecifically binds to a TR4 polypeptide which has been modified, e.g., by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, a TR4 polypeptide, a fragment of a TR4 polypeptide, or an anti-TR4 antibody, may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A derivative of a TR4 polypeptide, a fragment of a TR4 polypeptide, or an anti-TR4 antibody, may be modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Further, a derivative of a TR4 polypeptide, a fragment of a TR4 polypeptide, or an anti-TR4 antibody, may contain one or more non-classical amino acids. A polypeptide derivative possesses a similar or identical function as a TR4 polypeptide, a fragment of a TR4 polypeptide, or an anti-TR4 antibody, described herein.

[0049] The term “epitopes” as used herein refers to portions of TR4 having antigenic or immunogenic activity in an animal, preferably a mammal. An epitope having immunogenic activity is a portion of TR4 that elicits an antibody response in an animal. An epitope having antigenic activity is a portion of TR4 to which an antibody immunospecifically binds as determined by any method known in the art, for example, by the immunoassays described herein. Antigenic epitopes need not necessarily be immunogenic.

[0050] The term “fragment” as used herein refers to a polypeptide comprising an amino acid sequence of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 35 amino acid residues, at least 40 amino acid residues, at least 45 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, at least 150 amino acid residues, at least 175 amino acid residues, at least 200 amino acid residues, or at least 250 amino acid residues, of the amino acid sequence of TR4, or an anti-TR4 antibody (including molecules such as scFv's, that comprise, or alternatively consist of, antibody fragments or variants thereof).

[0051] The term “fusion protein” as used herein refers to a polypeptide that comprises, or alternatively consists of, an amino acid sequence of an anti-TR4 antibody of the invention and an amino acid sequence of a heterologous polypeptide (i.e., a polypeptide unrelated to an antibody or antibody domain).

[0052] The term “host cell” as used herein refers to the particular subject cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

[0053] Antibodies of the present invention are preferably provided in an isolated form, and preferably are substantially purified. By “isolated” is intended an antibody removed from its native environment. Thus, for eaxample, a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention.

[0054] By “isolated antibody” is intended an antibody removed from its native environment. Thus, an antibody produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention.

Antibody Structure

[0055] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light/heavy chain pair form the antibody binding site.

[0056] Thus, an intact IgG antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same.

[0057] The chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs. The CDRs from the heavy and the ligt chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J Mol. Biol. 196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989).

[0058] A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab′ fragments. See, e.g., Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny et al. J. Immunol. 148:1547 1553 (1992). In addition, bispecific antibodies may be formed as “diabodies” (Holliger et al. “‘Diabodies’: small bivalent and bispecific antibody fragments” PNAS USA 90:6444-6448 (1993)) or “Janusins” (Traunecker et al. “Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on HIV infected cells” EMBO J 10:3655-3659 (1991) and Traunecker et al. “Janusin: new molecular design for bispecific reagents” Int J Cancer Suppl 7:51-52 (1992)).

[0059] Production of bispecific antibodies can be a relatively labor intensive process compared with production of conventional antibodies and yields and degree of purity are generally lower for bispecific antibodies. Bispecific antibodies do not exist in the form of fragments having a single binding site (e.g., Fab, Fab′, and Fv).

Anti-TR4 Antibodies

[0060] Using phage display technology, the present inventors have identified single chain antibody molecules (“scFvs”) that immunospecifically bind to TR4 (or fragments or variants thereof). Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs (e.g., including VH domains, VH CDRS, VL domains, or VL CDRs having an amino acid sequence of any one of those referred to in Table 1), that immunospecifically bind to TR4 (or fragments or variants thereof) are also encompassed by the invention, as are nucleic acid molecules that encode these scFvs, and/or molecules.

[0061] In particular, the invention relates to scFvs comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 42-53, preferably SEQ ID NOs:42 and 43 as referred to in Table 1 below. Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs (e.g., including VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of any one of those referred to in Table 1), that immunospecifically bind to TR4 are also encompassed by the invention, as are nucleic acid molecules that encode these scFvs, and/or molecules (e.g., SEQ ID NOs:54-65).

[0062] ScFvs corresponding to SEQ ID NOS:42-53 were selected for their ability bind TR4 polypeptide.

[0063] The present invention provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or a polypeptide fragment of TR4. In particular, the invention provides antibodies corresponding to the scFvs referred to in Table 1. Such scFvs may routinely be “converted” to immunoglobulin molecules by inserting, for example, the nucleotide sequences encoding the VH and/or VL domains of the scFv into an expression vector containing the constant domain sequences and engineered to direct the expression of the immunoglobulin molecule, as described in more detail in Example 5 below.

[0064] NS0 cell lines that express IgG1 antibodies that comprise the VH and VL domains of scFvs of the invention have been deposited with the American Type Culture Collection (“ATCC”) on the dates listed in Table 1 and given the ATCC Deposit Numbers identified in Table 1. The ATCC is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure. Accordingly, in one embodiment, the invention provides antibodies that comprise the VH and VL domains of scFvs of the invention.

[0065] In a preferred embodiment, an antibody of the invention is the antibody expressed by cell line NSO αTRAIL 1985 BU #81 P:15 Jun. 21, 2001 (See Table 1).

[0066] In a preferred embodiment, an antibody of the invention is the antibody expressed by cell line TRAIL (NSO) 14G03 #39 P:14 Jul. 2, 2001 (See Table 1).

[0067] In a preferred embodiment, an antibody of the invention is the antibody expressed by cell line NSO anti-TRAIL 14F08 #28 P:11 (See Table 1).

[0068]

TABLE 1
scFvs that Immunospecifically bind to TRAIL Receptors
	scFv	scFv									Cell
	protein	DNA									Line
	SEQ	SEQ	AAs of	AAs of	AAs of	AAs of	AAs of	AAs of	AAs of	AAs of	Express-	ATCC	ATCC
	ID	ID	VH	VH	VH	VH	VL	VL	VL	VL	ing	Deposit	Deposit
scFv	NO:	NO:	Domain	CDR1	CDR2	CDR3	Domain	CDR1	CDR2	CDR3	antibody	Number	Date
T1014A04	42	54	1-118	26-35	50-66	99-107	135-245	157-170	186-192	225-234	NSO	PTA-	Jul. 30,
											αTRAIL	3571	2001
											1985
											BU #81
											P: 15
											Jun. 21,
											2001
T1014G03	43	55	1-118	26-35	50-66	99-107	135-245	157-170	186-192	225-234	TRAIL	PTA-	Jul. 30,
											(NSO)	3570	2001
											14G03
											#39
											P: 14
											Jul. 2,
											2001
T1014A02	44	56	1-116	26-35	50-65	98-105	134-244	156-168	184-190	223-233
T1014A12	45	57	1-118	26-35	50-66	99-107	135-245	157-170	186-192	225-234
T1014B01	46	58	1-118	26-35	50-66	99-107	135-245	157-170	186-192	225-234
T1014B11	47	59	1-118	26-35	50-66	99-107	135-245	157-170	186-192	225-234
T1014F08	48	60	1-118	26-35	50-66	99-107	135-245	157-170	186-192	225-234	NSO	PTA-	Aug. 29,
											anti-	3675	2001
											TRAIL
											14F08
											#28
											P: 11
T1014G04	49	61	1-118	26-35	50-66	99-107	135-245	157-170	186-192	225-234
T1015A02	50	62	1-123	26-37	52-67	100-112	140-250	162-174	190-196	229-239
T1015A07	51	63	1-118	26-35	50-66	99-107	135-245	157-170	186-192	225-234
T1015E01	52	64	1-118	26-35	50-66	99-107	135-245	157-170	186-192	225-234
T1006F07	53	65	1-125	26-35	50-66	99-114	142-249	164-174	190-196	229-238

[0069] The present invention encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a TR4 polypeptide or a fragment, variant, or fusion protein thereof. A TR4 polypeptide includes, but is not limited to, TR4 (SEQ ID NO:1) or the polypeptide encoded by the cDNA in clone HCUDS60 contained in ATCC Deposit 97853 deposited Jan. 21, 1997. In some embodiments, antibodies of the present invention may immunospecifically bind to both TR4 as described above and to TR7 (SEQ ID NO:3) or the polypeptide encoded by the cDNA in clone HLYBX88 contained in ATCC Deposit 97920 deposited Mar. 7, 1997. TRAIL receptors may be produced through recombinant expression of nucleic acids encoding the polypeptides of SEQ ID NOS:1-5, (TR4, TR5, TR7, TR10, and TR1; e.g., the cDNAs in the ATCC Deposit Numbers 97853, (TR4) 97798 (TR5, deposited Nov. 20, 1996), 97920 (TR7), or 209040 (TR10, deposited May 15, 1997).

[0070] In one embodiment, the antibodies of the invention preferentially bind TR4 (SEQ ID NO:1), or fragments, variants, or fusion proteins thereof (e.g., the extracellular region of TR4 fused to an Fc domain) relative to their ability to bind other proteins including TR1, TR5, TR7, or TR10 (SEQ ID NOS:5, 2, 3, and 4) or fragments, variants, or fusion proteins thereof. In other preferred embodiments, the antibodies of the invention preferentially bind to TR4 and TR7 (SEQ ID NOS:1 and 3), or fragments or variants thereof relative to their ability to bind other proteins including TR1, TR5 or TR10 (SEQ ID NOS:5, 2 and 4) or fragments, variants, or fusion proteins thereof. In other preferred embodiments, the antibodies of the invention bind TR1 TR4, TR5, TR7 and TR10 (SEQ ID NOS:5, 1, 2, 3, and 4). An antibody's ability to preferentially bind one antigen compared to another antigen may be determined using any method known in the art.

TR4 Polypeptides

[0071] In certain embodiments of the present invention, the antibodies of the present invention bind TR4 polypeptide, or fragments or variants thereof. The following section describes the TR4 polypeptides, fragments and variants that may be bound by the antibodies of the invention in more detail. The TR4 polypeptides, fragments and variants which may be bound by the antibodies of the invention are also described in International Publication Numbers, for example, WO98/32856 and WO00/67793 which are herein incorporated by reference in their entireties.

[0072] In certain embodiments, the antibodies of the present invention immunospecifically bind TR4 polypeptide. An antibody that immunospecifically binds TR4 may, in some embodiments, bind fragments, variants (including species orthologs of TR4), multimers or modified forms of TR4. For example, an antibody immunospecific for TR4 may bind the TR4 moiety of a fusion protein comprising all or a portion of TR4.

[0073] TR4 proteins may be found as monomers or multimers (i.e., dimers, trimers, tetramers, and higher multimers). Accordingly, the present invention relates to antibodies that bind TR4 proteins found as monomers or as part of multimers. In specific embodiments, antibodies of the invention bind TR4 monomers, dimers, trimers or tetramers. In additional embodiments, antibodies of the invention bind at least dimers, at least trimers, or at least tetramers containing one or more TR4 polypeptides.

[0074] Antibodies of the invention may bind TR4 homomers or heteromers. As used herein, the term homomer, refers to a multimer containing only TR4 proteins of the invention (including TR4 fragments, variants, and fusion proteins, as described herein). These homomers may contain TR4 proteins having identical or different polypeptide sequences. In a specific embodiment, a homomer of the invention is a multimer containing only TR4 proteins having an identical polypeptide sequence. In another specific embodiment, antibodies of the invention bind TR4 homomers containing TR4 proteins having different polypeptide sequences. In specific embodiments, antibodies of the invention bind a TR4 homodimer (e.g., containing TR4 proteins having identical or different polypeptide sequences). In additional embodiments, antibodies of the invention bind at least a homodimer, at least a homotrimer, or at least a homotetramer of TR4.

[0075] In specific embodiments antibodies of the presnt invention bind TR4 homotrimers (e.g., containing TR4 proteins having identical or different polypeptide sequences).

[0076] As used herein, the term heteromer refers to a multimer containing heterologous proteins (i.e., proteins containing polypeptide sequences that do not correspond to a polypeptide sequences encoded by the TR4 gene) in addition to the TR4 proteins of the invention. In a specific embodiment, antibodies of the invention bind a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the antibodies of the invention bind at least a homodimer, at least a homotrimer, or at least a homotetramer containing one or more TR4 polypeptides.

[0077] In specific embodiments antibodies of the presnt invention bind a TR4 heterotrimer (e.g., containing 1 or 2 TR4 proteins and 2 or 1, respectively, TR7 proteins).

[0078] Multimers bound by one or more antibodies of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation. Thus, in one embodiment, multimers bound by one or more antibodies of the invention, such as, for example, homodimers or homotrimers, are formed when TR4 proteins contact one another in solution. In another embodiment, heteromultimers bound by one or more antibodies of the invention, such as, for example, heterotrimers or heterotetramers, are formed when proteins of the invention contact antibodies to the TR4 polypeptides (including antibodies to the heterologous polypeptide sequence in a fusion protein) in solution. In other embodiments, multimers bound by one or more antibodies of the invention are formed by covalent associations with and/or between the TR4 proteins of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence of the protein (e.g., the polypeptide sequence recited in SEQ ID NO:1 or the polypeptide encoded by the deposited cDNA clone of ATCC Deposit 97853). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences of the proteins which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a TR4 fusion protein. In one example, covalent associations are between the heterologous sequence contained in a fusion protein (see, e.g., U.S. Pat. No. 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in a TR4-Fc fusion protein (as described herein). In another specific example, covalent associations of fusion proteins are between heterologous polypeptide sequences from another TNF family ligand/receptor member that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication No. WO 98/49305, the contents of which are herein incorporated by reference in its entirety).

[0079] The multimers that may be bound by one or more antibodies of the invention may be generated using chemical techniques known in the art. For example, proteins desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimers that may be bound by one or more antibodies of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the polypeptide sequence of the proteins desired to be contained in the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Further, proteins that may be bound by one or more antibodies of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide sequence of the protein and techniques known in the art may be applied to generate multimers containing one or more of these modified proteins (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the protein components desired to be contained in the multimer that may be bound by one or more antibodies of the invention (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).

[0080] Alternatively, multimers that may be bound by one or more antibodies of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, proteins contained in multimers that may be bound by one or more antibodies of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In a specific embodiment, polynucleotides coding for a homodimer that may be bound by one or more antibodies of the invention are generated by ligating a polynucleotide sequence encoding a TR4 polypeptide to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant TR4 polypeptides which contain a transmembrane domain and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, two or more TR4 polypeptides are joined through synthetic linkers (e.g., peptide, carbohydrate or soluble polymer linkers). Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple TR4 polypeptides separated by peptide linkers may be produced using conventional recombinant DNA technology. In specific embodiments, antibodies of the invention bind proteins comprising multiple TR4 polypeptides separated by peptide linkers.

[0081] Another method for preparing multimer TR4 polypeptides involves use of TR4 polypeptides fused to a leucine zipper or isoleucine polypeptide sequence. Leucine zipper domains and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric TR4 proteins are those described in PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a soluble TR4 polypeptide fused to a peptide that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric TR4 is recovered from the culture supernatant using techniques known in the art. In specific embodiments, antibodies of the invention bind TR4-leucine zipper fusion protein monomers and/or TR4-leucine zipper fusion protein multimers.

[0082] Certain members of the TNF family of proteins are believed to exist in trimeric form (Beutler and Huffel, Science 264:667, 1994; Banner et al., Cell 73:431, 1993). Thus, trimeric TR4 may offer the advantage of enhanced biological activity. Preferred leucine zipper moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, now U.S. Pat. No. 5,716,805, hereby incorporated by reference. In specific embodiments, antibodies of the invention bind TR4-leucine zipper fusion protein trimers.

[0083] Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric TR4. In specific embodiments, antibodies of the invention bind TR4-fusion protein monomers and/or TR4 fusion protein trimers.

[0084] Antibodies of the invention that bind TR4 receptor polypeptides may bind them as isolated polypeptides or in their naturally occurring state. By “isolated polypeptide” is intended a polypeptide removed from its native environment. Thus, a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention. Also, intended as an “isolated polypeptide” are polypeptides that have been purified, partially or substantially, from a recombinant host cell. For example, a recombinantly produced version of the TR4 polypeptide is substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). Thus, antibodies of the present invention may bind recombinantly produced TR4 receptor polypeptides. In a specific embodiment, antibodies of the present invention bind a TR4 receptor expressed on the surface of a cell, wherein said TR4 polypeptide is encoded by a polynucleotide encoding amino acids 1 to 468 of SEQ ID NO:1 operably associated with a regulatory sequence that controls expression of said polynucleotide.

[0085] Antibodies of the present invention may bind TR4 polypeptide fragments comprising or alternatively, consisting of, an amino acid sequence contained in SEQ ID NO:1, encoded by the cDNA contained in ATCC deposit Number 97853, or encoded by nucleic acids which hybridize (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in ATCC deposit Number 97853, or the complementary strand thereto. Protein fragments may be “free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Antibodies of the present invention may bind polypeptide fragments, including, for example, fragments that comprise or alternatively, consist of from about amino acid residues: 1 to 23, 24 to 43, 44 to 63, 64 to 83, 84 to 103, 104 to 123, 124 to 143, 144 to 163, 164 to 183, 184 to 203, 204 to 223, 224 to 238, 239 to 264, 265 to 284, 285 to 304, 305 to 324, 325 to 345, 346 to 366, 367 to 387, 388 to 418, 419 to 439, and/or 440 to 468 of SEQ ID NO:1. In this context “about” includes the particularly recited value, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Moreover, polypeptide fragments bound by the antibodies of the invention can be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175 or 200 amino acids in length. In this context “about” includes the particularly recited value, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.

[0086] Preferably, antibodies of the present invention bind polypeptide fragments selected from the group: a polypeptide comprising or alternatively, consisting of, the TR4 receptor extracellular domain (predicted to constitute amino acid residues from about 24 to about 238 in SEQ ID NO:1); a polypeptide comprising or alternatively, consisting of, both TR4 cysteine rich domains (both of which may be found in the protein fragment consisting of amino acid residues from about 131 to about 229 in SEQ ID NO:1); a polypeptide comprising or alternatively, consisting of, the TR4 cysteine rich domain consisting of amino acid residues from about 131 to about 183 in SEQ ID NO:1); a polypeptide comprising or alternatively, consisting of, the TR4 cysteine rich domain consisting of amino acid residues from about 184 to about 229 in SEQ ID NO:1); a polypeptide comprising or alternatively, consisting of, the TR4 receptor transmembrane domain (predicted to constitute amino acid residues from about 239 to about 264 in SEQ ID NO:1); a polypeptide comprising or alternatively, consisting of, fragment of the predicted mature TR4 polypeptide, wherein the fragment has a TR4 functional activity (e.g., antigenic activity or biological acitivity); a polypeptide comprising or alternatively, consisting of, the TR4 receptor intracellular domain (predicted to constitute amino acid residues from about 265 to about 468 in SEQ ID NO:1); a polypeptide comprising or alternatively, consisting of, the TR4 receptor extracellular and intracellular domains with all or part of the transmembrane domain deleted; a polypeptide comprising, or alternatively consisting of, the TR4 receptor death domain (predicted to constitute amino acid residues from about 379 to about 422 in SEQ ID NO:1); and a polypeptide comprising, or alternatively, consisting of, one, two, three, four or more, epitope bearing portions of the TR4 receptor protein. In additional embodiments, the polypeptide fragments of the invention comprise, or alternatively, consist of, any combination of 1, 2, 3, 4, 5, 6, 7, or all 8 of the above members. The amino acid residues constituting the TR4 receptor extracellular, transmembrane and intracellular domains have been predicted by computer analysis. Thus, as one of ordinary skill would appreciate, the amino acid residues constituting these domains may vary slightly (e.g., by about 1 to about 15 amino acid residues) depending on the criteria used to define each domain. Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0087] It is believed that one or both of the extracellular cysteine rich motifs of TR4 is important for interactions between TR4 and its ligands (e.g., TRAIL). Accordingly, in highly preferred embodiments, antibodies of the present invention bind TR4 polypeptide fragments comprising, or alternatively consisting of amino acid residues 131 to 183, and/or 184 to 229 of SEQ ID NO:1. In another highly preferred embodiment, antibodies of the present invention bind TR4 polypeptides comprising, or alternatively consisting of both of the extracellular cysteine rich motifs (amino acid residues 131 to 229 of SEQ ID NO:1.) In another preferred embodiment, antibodies of the present invention bind TR4 polypeptides comprising, or alternatively consisting the extracellular soluble domain of TR4 (amino acid residues 24-238 of SEQ ID NO:1.) In highly preferred embodiments, the antibodies of the invention that bind all or a portion of the extracellular soluble domain of TR4 (e.g., one or both cysteine rich domains) prevent TRAIL ligand from binding to TR4. In other highly preferred embodiments, the antibodies of the invention that bind all or a portion of the extracellular soluble domain of TR4 (e.g., one or both cysteine rich domains) agonize the TR4 receptor. In other highly preferred embodiments, the antibodies of the invention that bind all or a portion of the extracellular soluble domain of TR4 (e.g., one or both cysteine rich domains) induce cell death of the cell expressing the TR4 receptor.

[0088] Antibodies of the invention may also bind fragments comprising, or alternatively, consisting of structural or functional attributes of TR4. Such fragments include amino acid residues that comprise alpha-helix and alpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheet-forming regions (“beta-regions”), turn and turn-forming regions (“turn-regions”), coil and coil-forming regions (“coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, surface forming regions, and high antigenic index regions (i.e., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1.5, as identified using the default parameters of the Jameson-Wolf program) of complete (i.e., full-length) TR4. Certain preferred regions are those set out in Table 2 and include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence depicted in (SEQ ID NO:1), such preferred regions include; Garnier-Robson predicted alpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasman predicted alpha-regions, beta-regions, and turn-regions; Kyte-Doolittle predicted hydrophilic regions; Eisenberg alpha and beta amphipathic regions; Emini surface-forming regions; and Jameson-Wolf high antigenic index regions, as predicted using the default parameters of these computer programs.

[0089] The data representing the structural or functional attributes of TR4 set forth in Table 2, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. Column I represents the results of a Garnier-Robson analysis of alpha helical regions; Column II represents the results of a Chou-Fasman analysis of alpha helical regions; Column III represents the results of a Garnier Robson analysis of beta sheet regions; Column IV represents the results of a Chou-Fasman analysis of beta sheet regions; Column V represents the results of a Garnier Robson analysis of turn regions; Column VI represents the results of a Chou-Fasman analysis of turn regions; Column VII represents the results of a Garnier Robson analysis of coil regions; Column VIII represents a Kyte-Doolittle hydrophilicity plot; Column; Column IX represents the results of an Eisenberg analysis of alpha amphipathic regions; Column X represents the results of an Eisenberg analysis of beta amphipathic regions; Column XI represents the results of a Karplus-Schultz analysis of flexible regions; Column XII represents the Jameson-Wolf antigenic index score; and Column XIII represents the Emini surface probability plot.

[0090] In a preferred embodiment, the data presented in columns VIII, XII, and XIII of Table 2 can be used to determine regions of TR4 which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, XII, and/or XIII by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.

[0091] The above-mentioned preferred regions set out in Table 2 include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in SEQ ID NO:1. As set out in Table 2, such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions. Among preferred polypeptide fragments bound by one or more antibodies of the invention are those that comprise regions of TR4 that combine several structural features, such as several (e.g., 1, 2, 3, or 4) of the same or different region features set out above and in Table 2.

[0092]

TABLE 2
Res	Position	I	II	III	IV	V	VI	VII	VIII	IX	X	XI	XII	XIII
Met	1	.	.	B	.	.	.	.	0.12	.	.	.	−0.10	0.90
Ala	2	.	.	.	.	.	.	C	−0.08	*	*	.	0.25	1.08
Pro	3	.	.	.	.	.	.	C	0.42	*	*	.	0.10	0.86
Pro	4	.	.	.	.	.	T	C	−0.04	*	*	.	1.05	1.69
Pro	5	A	.	.	.	.	T	.	0.31	.	*	F	1.00	1.24
Ala	6	A	.	.	.	.	T	.	0.10	.	*	F	1.00	1.10
Arg	7	A	.	.	.	.	T	.	0.34	.	*	.	0.10	0.58
Val	8	.	.	B	B	.	.	.	−0.03	.	*	.	−0.30	0.37
His	9	.	.	B	B	.	.	.	−0.52	.	*	.	−0.30	0.37
Leu	10	.	.	B	B	.	.	.	−1.12	.	*	.	−0.60	0.17
Gly	11	.	.	B	B	.	.	.	−1.12	.	*	.	−0.60	0.18
Ala	12	.	.	B	B	.	.	.	−2.09	.	*	.	−0.60	0.14
Phe	13	.	.	B	B	.	.	.	−1.54	.	*	.	−0.60	0.12
Leu	14	.	.	B	B	.	.	.	−1.72	.	.	.	−0.60	0.18
Ala	15	.	.	B	B	.	.	.	−0.91	.	.	.	−0.60	0.27
Val	16	.	.	B	B	.	.	.	−0.78	.	.	.	−0.60	0.51
Thr	17	.	.	B	B	.	.	.	−0.53	.	.	F	−0.45	0.95
Pro	18	.	.	.	B	.	.	C	−0.13	.	.	F	0.05	0.93
Asn	19	.	.	.	.	.	T	C	0.09	.	.	F	0.60	1.69
Pro	20	.	.	.	.	.	T	C	0.09	.	.	F	0.60	1.18
Gly	21	.	.	.	.	T	T	.	0.64	.	.	F	0.65	0.77
Ser	22	.	.	.	.	.	T	C	0.61	.	.	F	0.45	0.64
Ala	23	.	.	.	.	.	.	C	0.51	.	.	F	0.25	0.41
Ala	24	.	.	.	.	.	T	C	0.51	.	.	F	0.45	0.60
Ser	25	.	.	B	.	.	T	.	0.13	.	.	F	0.85	0.78
Gly	26	A	.	.	.	.	T	.	−0.11	.	.	F	0.85	0.78
Thr	27	A	.	.	.	.	T	.	−0.40	.	.	F	0.85	0.78
Glu	28	A	A	.	.	.	.	.	−0.40	.	.	F	0.45	0.58
Ala	29	A	A	.	.	.	.	.	−0.12	.	.	.	0.30	0.60
Ala	30	A	A	.	.	.	.	.	−0.03	.	.	.	0.30	0.60
Ala	31	A	A	.	.	.	.	.	0.01	.	.	.	0.30	0.53
Ala	32	A	A	.	.	.	.	.	0.37	.	.	.	−0.30	0.71
Thr	33	A	.	.	.	.	T	.	−0.49	*	.	F	1.00	1.40
Pro	34	A	.	.	.	.	T	.	−0.19	.	.	F	1.00	1.03
Ser	35	.	.	B	.	.	T	.	0.06	.	.	F	0.40	1.07
Lys	36	.	.	B	.	.	T	.	0.34	.	.	F	0.25	0.73
Val	37	.	.	B	B	.	.	.	0.63	.	.	F	−0.15	0.64
Trp	38	.	.	B	B	.	.	.	0.36	.	.	F	−0.15	0.64
Gly	39	.	.	B	B	.	.	.	0.22	*	*	F	−0.15	0.32
Ser	40	.	.	.	.	.	.	C	0.63	*	*	F	−0.05	0.43
Ser	41	.	.	.	.	.	T	C	−0.30	*	*	F	0.45	0.80
Ala	42	.	.	.	.	.	T	C	0.56	*	*	F	1.05	0.57
Gly	43	.	.	.	.	.	T	C	0.63	*	*	F	1.35	0.73
Arg	44	.	.	B	.	.	T	.	1.09	*	*	F	1.49	0.84
Ile	45	.	.	B	.	.	.	.	1.04	*	*	F	1.78	1.63
Glu	46	.	.	B	.	.	.	.	1.00	*	*	F	2.12	1.63
Pro	47	.	.	B	.	.	T	.	1.24	*	*	F	2.51	0.83
Arg	48	.	.	.	.	T	T	.	1.70	*	*	F	3.40	1.17
Gly	49	.	.	.	.	T	T	.	1.24	*	*	F	3.06	1.32
Gly	50	.	.	.	.	T	T	.	1.54	*	*	F	2.57	0.84
Gly	51	.	.	.	.	.	T	C	0.73	*	*	F	2.03	0.44
Arg	52	.	.	.	.	.	T	C	0.73	*	*	F	1.39	0.36
Gly	53	.	.	B	.	.	T	.	0.31	*	*	F	0.85	0.57
Ala	54	.	.	B	.	.	T	.	0.36	.	*	F	0.85	0.83
Leu	55	.	.	B	.	.	.	.	0.10	.	*	F	0.65	0.57
Pro	56	.	.	B	.	.	.	.	0.10	.	*	F	−0.25	0.57
Thr	57	.	.	B	.	.	.	.	−0.01	.	*	F	−0.25	0.55
Ser	58	.	.	B	.	.	T	.	0.30	.	.	F	0.10	1.16
Met	59	.	.	B	.	.	T	.	0.54	.	.	F	0.40	1.02
Gly	60	.	.	B	.	.	T	.	1.14	.	.	F	0.25	0.70
Gln	61	.	.	.	.	T	T	.	1.06	.	.	F	0.65	0.81
His	62	.	.	.	.	.	.	C	0.78	.	*	F	0.40	1.10
Gly	63	.	.	.	.	.	T	C	1.19	.	*	F	0.60	1.12
Pro	64	.	.	.	.	.	T	C	1.20	.	*	F	1.20	1.27
Ser	65	.	.	.	.	.	T	C	1.66	.	*	F	1.05	0.94
Ala	66	.	.	B	.	.	T	.	1.07	.	*	F	1.30	1.86
Arg	67	.	.	B	.	.	.	.	0.76	*	*	.	1.29	1.22
Ala	68	.	.	B	.	.	.	.	1.21	*	*	.	1.48	0.90
Arg	69	.	.	B	.	.	T	.	0.83	.	*	.	2.17	1.74
Ala	70	.	.	B	.	.	T	.	0.92	.	*	F	2.51	0.90
Gly	71	.	.	.	.	T	T	.	1.17	.	*	F	3.40	1.37
Arg	72	.	.	.	.	.	T	C	0.84	.	*	F	2.71	0.69
Ala	73	.	.	.	.	.	T	C	1.54	*	.	F	2.48	1.06
Pro	74	.	.	.	.	.	T	C	1.22	*	.	F	2.70	2.10
Gly	75	.	.	.	.	.	T	C	1.22	*	.	F	2.62	1.66
Pro	76	.	.	.	.	.	T	C	1.68	*	*	F	2.24	1.66
Arg	77	.	.	.	.	.	.	C	1.57	*	.	F	2.60	2.10
Pro	78	.	A	B	.	.	.	.	1.57	*	.	F	1.94	3.68
Ala	79	.	A	B	.	.	.	.	1.48	*	.	F	1.68	2.40
Arg	80	.	A	B	.	.	.	.	1.61	*	*	F	1.42	1.64
Glu	81	.	A	B	.	.	.	.	1.93	*	*	F	1.16	1.64
Ala	82	A	A	.	.	.	.	.	1.01	*	*	F	0.90	3.19
Ser	83	A	.	.	.	.	T	.	1.33	*	*	F	1.30	1.34
Pro	84	A	.	.	.	.	T	.	1.07	*	*	F	1.30	1.52
Arg	85	A	.	.	.	.	T	.	0.92	*	*	F	1.00	1.12
Leu	86	A	.	.	.	.	T	.	0.97	.	*	.	0.85	1.13
Arg	87	A	.	.	B	.	.	.	1.24	.	*	.	0.75	1.46
Val	88	A	.	.	B	.	.	.	0.84	*	*	.	0.75	1.08
His	89	A	.	.	B	.	.	.	1.10	.	*	.	−0.15	1.13
Lys	90	A	.	.	B	.	.	.	0.29	*	*	F	0.90	1.16
Thr	91	.	.	B	B	.	.	.	0.24	*	*	F	0.00	1.35
Phe	92	.	.	B	B	.	.	.	−0.72	*	*	.	−0.30	0.74
Lys	93	.	.	B	B	.	.	.	−0.72	*	*	.	−0.30	0.27
Phe	94	.	.	B	B	.	.	.	−1.03	*	.	.	−0.60	0.14
Val	95	.	.	B	B	.	.	.	−1.93	*	.	.	−0.60	0.16
Val	96	.	.	B	B	.	.	.	−2.43	.	*	.	−0.60	0.06
Val	97	.	.	B	B	.	.	.	−2.54	.	*	.	−0.60	0.06
Gly	98	.	.	B	B	.	.	.	−2.59	.	*	.	−0.60	0.06
Val	99	.	.	B	B	.	.	.	−2.74	.	.	.	−0.60	0.15
Leu	100	.	.	B	B	.	.	.	−2.74	*	.	.	−0.60	0.15
Leu	101	.	.	B	B	.	.	.	−2.10	*	.	.	−0.60	0.11
Gln	102	.	.	B	B	.	.	.	−1.54	*	.	.	−0.60	0.23
Val	103	.	.	B	B	.	.	.	−1.50	.	.	.	−0.60	0.37
Val	104	.	.	B	.	.	T	.	−1.23	.	.	.	−0.20	0.61
Pro	105	.	.	B	.	.	T	.	−1.01	*	.	F	0.25	0.35
Ser	106	A	.	.	.	.	T	.	−0.51	*	.	F	−0.05	0.48
Ser	107	A	.	.	.	.	T	.	−1.40	*	*	F	0.25	0.94
Ala	108	A	.	.	.	.	.	.	−0.50	.	*	F	0.05	0.43
Ala	109	A	.	.	.	.	.	.	−0.46	.	*	.	0.50	0.63
Thr	110	A	.	.	.	.	.	.	−0.28	.	*	.	−0.10	0.39
Ile	111	A	.	.	.	.	.	.	0.02	.	*	.	−0.10	0.53
Lys	112	.	.	B	.	.	.	.	0.32	.	*	.	0.50	0.87
Leu	113	.	.	B	.	.	.	.	0.61	.	*	F	1.05	1.04
His	114	.	.	B	.	.	.	.	0.31	.	*	F	1.30	1.99
Asp	115	.	.	.	.	.	T	C	0.28	*	*	F	1.80	0.70
Gln	116	.	.	.	.	T	T	.	0.86	.	*	F	1.65	0.84
Ser	117	.	.	.	.	T	T	.	0.81	.	.	F	2.50	0.89
Ile	118	.	.	.	.	T	T	.	1.62	.	.	F	2.25	0.92
Gly	119	.	.	.	.	.	.	C	1.37	.	.	F	1.00	0.92
Thr	120	.	.	.	.	.	.	C	1.37	.	.	F	0.45	0.72
Gln	121	.	.	B	.	.	.	C	1.33	.	.	F	0.65	1.79
Gln	122	.	.	B	.	.	.	.	1.33	.	.	F	0.20	2.46
Trp	123	.	.	B	.	.	.	.	2.01	.	.	.	0.05	2.28
Glu	124	.	.	.	.	.	.	C	1.54	.	.	.	0.25	2.04
His	125	.	.	.	.	.	.	C	1.51	.	.	.	0.10	0.97
Ser	126	.	.	.	.	.	T	C	1.51	.	.	F	0.45	0.91
Pro	127	.	.	.	.	T	T	.	0.70	.	.	F	1.55	0.91
Leu	128	.	.	.	.	T	T	.	0.32	.	.	F	0.65	0.55
Gly	129	.	.	.	.	T	T	.	0.11	.	.	F	0.65	0.22
Glu	130	.	.	.	.	T	.	.	−0.07	.	.	F	0.45	0.22
Leu	131	.	.	B	.	.	.	.	−0.11	*	.	.	0.18	0.42
Cys	132	.	.	B	.	.	.	.	−0.20	*	.	F	1.21	0.42
Pro	133	.	.	B	.	.	T	.	0.58	*	*	F	1.69	0.32
Pro	134	.	.	.	.	T	T	.	1.03	.	*	F	1.47	0.53
Gly	135	.	.	.	.	T	T	.	0.73	.	*	F	2.80	1.94
Ser	136	.	.	.	.	.	T	C	1.54	*	.	F	2.32	1.68
His	137	.	.	.	.	.	.	C	2.32	*	.	F	2.48	1.88
Arg	138	.	.	B	.	.	.	.	2.32	*	.	F	2.34	3.72
Ser	139	.	.	B	.	.	.	.	2.19	*	.	F	2.40	4.29
Glu	140	.	.	.	.	T	.	.	1.94	*	.	F	2.86	3.12
Arg	141	.	.	.	.	T	T	.	1.58	*	.	F	3.40	1.61
Pro	142	.	.	.	.	T	T	.	1.61	.	*	F	2.91	0.64
Gly	143	.	.	.	.	T	T	.	1.61	.	*	F	2.57	0.60
Ala	144	.	.	.	.	T	T	.	1.24	.	*	.	2.08	0.60
Cys	145	.	.	.	.	T	.	.	0.93	.	*	.	1.41	0.21
Asn	146	.	.	B	.	.	.	.	0.82	.	*	.	0.84	0.30
Arg	147	.	.	B	.	.	.	.	0.69	*	.	.	1.01	0.52
Cys	148	.	.	B	.	.	T	.	0.18	*	.	F	1.83	0.96
Thr	149	.	.	B	.	.	T	.	0.42	*	.	F	1.70	0.44
Glu	150	.	.	B	.	.	T	.	0.84	*	.	F	1.53	0.22
Gly	151	.	.	B	.	.	T	.	0.53	*	.	F	0.76	0.65
Val	152	.	.	B	B	.	.	.	0.42	.	*	F	0.19	0.65
Gly	153	.	.	B	B	.	.	.	0.50	.	.	.	−0.13	0.61
Tyr	154	.	.	B	B	.	.	.	0.51	.	.	.	−0.60	0.62
Thr	155	.	.	B	B	.	.	.	0.51	.	.	F	−0.30	1.12
Asn	156	.	.	.	B	.	.	C	0.86	.	.	F	0.20	1.81
Ala	157	.	.	.	.	T	T	.	0.90	.	.	F	0.80	1.86
Ser	158	.	.	.	.	T	T	.	0.54	.	.	F	0.80	1.06
Asn	159	.	.	.	.	T	T	.	0.20	.	.	F	0.35	0.57
Asn	160	.	.	.	.	T	T	.	−0.16	*	.	F	0.35	0.57
Leu	161	.	A	B	.	.	.	.	−0.97	*	.	.	−0.60	0.23
Phe	162	.	A	B	.	.	.	.	−0.59	.	.	.	−0.60	0.12
Ala	163	.	A	B	.	.	.	.	−0.96	.	.	.	−0.60	0.11
Cys	164	.	A	B	.	.	.	.	−1.27	*	.	.	−0.60	0.07
Leu	165	.	.	B	.	.	T	.	−1.86	.	.	.	−0.20	0.12
Pro	166	.	.	B	.	.	T	.	−1.71	*	.	.	−0.20	0.12
Cys	167	.	.	.	.	T	T	.	−0.97	*	.	.	0.20	0.12
Thr	168	A	.	.	.	.	T	.	−0.68	.	.	.	0.10	0.30
Ala	169	A	.	.	.	.	.	.	−0.01	.	.	.	0.50	0.26
Cys	170	A	.	.	.	.	T	.	0.80	.	.	.	0.70	0.80
Lys	171	A	.	.	.	.	T	.	1.01	.	.	F	1.15	0.96
Ser	172	A	.	.	.	.	T	.	1.68	.	*	F	1.30	1.65
Asp	173	A	.	.	.	.	T	.	2.10	.	*	F	1.30	5.33
Glu	174	A	A	.	.	.	.	.	2.39	.	*	F	0.90	5.22
Glu	175	A	A	.	.	.	.	.	2.84	.	*	F	1.24	5.22
Glu	176	A	A	.	.	.	.	.	2.13	.	*	F	1.58	4.83
Arg	177	.	A	.	.	T	.	.	2.12	.	.	F	2.32	1.50
Ser	178	.	.	.	.	.	T	C	1.81	.	.	F	2.86	1.25
Pro	179	.	.	.	.	T	T	.	1.50	*	.	F	3.40	1.04
Cys	180	.	.	.	.	T	T	.	1.61	*	.	F	2.61	0.77
Thr	181	.	.	.	.	T	T	.	1.61	*	.	F	2.67	1.12
Thr	182	.	.	.	.	T	.	.	1.19	*	*	F	2.38	1.16
Thr	183	.	.	.	.	T	T	.	0.90	.	.	F	2.49	3.13
Arg	184	.	.	.	.	T	T	.	0.44	.	.	F	2.40	2.19
Asn	185	.	.	.	.	T	T	.	1.11	.	.	F	2.50	0.81
Thr	186	.	.	.	.	T	T	.	0.76	*	.	F	2.25	0.98
Ala	187	.	.	.	.	T	.	.	1.11	*	.	.	1.65	0.27
Cys	188	.	.	.	.	T	.	.	1.21	*	.	.	1.40	0.33
Gln	189	.	.	B	.	.	.	.	0.76	*	.	.	0.75	0.36
Cys	190	.	.	B	.	.	.	.	0.44	.	.	.	0.50	0.35
Lys	191	.	.	B	.	.	T	.	0.06	.	*	F	0.85	0.94
Pro	192	.	.	.	.	T	T	.	0.76	.	.	F	0.65	0.47
Gly	193	.	.	.	.	T	T	.	1.42	.	*	F	1.74	1.72
Thr	194	.	.	B	.	.	T	.	1.42	.	*	F	1.68	1.38
Phe	195	.	.	B	.	.	.	.	2.09	.	*	F	1.82	1.49
Arg	196	.	.	.	.	T	.	.	1.74	.	*	F	2.56	2.42
Asn	197	.	.	.	.	T	T	.	1.37	.	*	F	3.40	2.25
Asp	198	.	.	.	.	T	T	.	1.71	.	*	F	3.06	2.63
Asn	199	.	.	.	.	.	T	C	1.42	.	*	F	2.52	2.32
Ser	200	A	.	.	.	.	T	.	1.46	.	*	F	1.98	1.43
Ala	201	A	.	.	.	.	.	.	1.46	.	*	.	1.14	0.46
Glu	202	A	.	.	.	.	.	.	1.50	*	.	.	0.80	0.56
Met	203	A	.	.	.	.	.	.	0.83	*	.	.	1.11	0.83
Cys	204	A	.	.	.	.	T	.	0.53	*	.	.	1.62	0.44
Arg	205	.	.	.	.	T	T	.	0.52	*	.	.	2.33	0.34
Lys	206	.	.	.	.	T	T	.	0.77	*	.	F	2.49	0.50
Cys	207	.	.	.	.	T	T	.	0.10	*	.	F	3.10	0.92
Ser	208	.	.	.	.	T	.	.	0.49	*	*	F	2.59	0.25
Thr	209	.	.	.	.	T	.	.	1.27	*	*	F	1.98	0.19
Gly	210	.	.	.	.	T	.	.	0.81	*	.	F	1.67	0.71
Cys	211	.	.	B	.	.	T	.	0.17	*	*	F	1.16	0.53
Pro	212	.	.	.	.	T	T	.	−0.02	*	*	F	1.25	0.36
Arg	213	.	.	.	.	T	T	.	0.32	*	*	F	0.65	0.27
Gly	214	.	.	B	.	.	T	.	−0.22	*	*	.	0.85	1.01
Met	215	.	.	B	B	.	.	.	0.17	*	*	.	0.30	0.48
Val	216	.	.	B	B	.	.	.	0.83	*	*	.	0.79	0.49
Lys	217	.	.	B	B	.	.	.	0.38	*	*	.	0.98	0.83
Val	218	.	.	B	B	.	.	.	−0.04	*	*	F	1.32	0.45
Lys	219	.	.	B	B	.	.	.	0.09	.	*	F	1.51	0.88
Asp	220	.	.	B	.	.	.	.	0.40	.	*	F	1.90	0.68
Cys	221	.	.	B	.	.	.	.	0.96	.	*	F	0.81	0.96
Thr	222	.	.	.	.	.	T	C	0.91	.	*	F	1.62	0.65
Pro	223	.	.	.	.	T	T	.	0.88	.	*	F	1.63	0.65
Trp	224	.	.	.	.	T	T	.	0.83	.	*	F	0.54	0.84
Ser	225	A	.	.	.	.	T	.	0.17	.	.	F	1.00	1.01
Asp	226	A	A	.	.	.	.	.	−0.02	.	.	F	0.45	0.35
Ile	227	A	A	.	.	.	.	.	0.26	*	.	.	−0.30	0.25
Glu	228	A	A	.	.	.	.	.	0.51	*	.	.	0.30	0.25
Cys	229	.	A	B	.	.	.	.	0.80	*	.	.	0.60	0.30
Val	230	A	A	.	.	.	.	.	0.80	*	*	.	0.60	0.74
His	231	A	A	.	.	.	.	.	0.46	*	*	.	0.60	0.58
Lys	232	A	A	.	.	.	.	.	1.34	*	.	F	0.60	1.06
Glu	233	.	A	.	.	T	.	.	1.00	*	.	F	1.30	2.30
Ser	234	.	.	.	.	T	T	.	1.63	*	.	F	1.70	1.68
Gly	235	.	.	.	.	T	T	.	2.49	*	.	F	1.70	1.14
Asn	236	.	.	.	.	T	T	.	1.63	*	.	F	1.40	1.06
Gly	237	.	.	.	.	.	T	C	1.30	*	.	F	0.45	0.55
His	238	.	.	.	B	.	.	C	0.44	.	.	.	−0.40	0.59
Asn	239	.	.	.	B	.	.	C	−0.14	.	.	.	−0.40	0.27
Ile	240	.	.	B	B	.	.	.	−0.61	.	.	.	−0.60	0.19
Trp	241	.	.	B	B	.	.	.	−1.47	.	.	.	−0.60	0.12
Val	242	.	.	B	B	.	.	.	−1.98	.	.	.	−0.60	0.05
Ile	243	.	.	B	B	.	.	.	−2.26	.	.	.	−0.60	0.06
Leu	244	.	.	B	B	.	.	.	−3.07	.	.	.	−0.60	0.08
Val	245	.	.	B	B	.	.	.	−3.03	.	.	.	−0.60	0.09
Val	246	.	.	B	B	.	.	.	−3.60	.	.	.	−0.60	0.09
Thr	247	.	.	B	B	.	.	.	−2.96	.	.	.	−0.60	0.08
Leu	248	.	.	B	B	.	.	.	−2.88	.	.	.	−0.60	0.17
Val	249	.	.	B	B	.	.	.	−2.88	.	*	.	−0.60	0.19
Val	250	.	.	B	B	.	.	.	−2.83	.	.	.	−0.60	0.11
Pro	251	.	.	B	B	.	.	.	−2.83	.	.	.	−0.60	0.11
Leu	252	.	.	B	B	.	.	.	−3.11	.	.	.	−0.60	0.11
Leu	253	A	.	.	B	.	.	.	−3.16	.	.	.	−0.60	0.15
Leu	254	A	.	.	B	.	.	.	−3.11	.	.	.	−0.60	0.07
Val	255	A	.	.	B	.	.	.	−3.14	.	.	.	−0.60	0.07
Ala	256	A	.	.	B	.	.	.	−3.79	.	.	.	−0.60	0.06
Val	257	.	.	B	B	.	.	.	−3.64	.	.	.	−0.60	0.05
Leu	258	.	.	B	B	.	.	.	−3.50	.	.	.	−0.60	0.04
Ile	259	.	.	B	B	.	.	.	−3.36	.	.	.	−0.60	0.02
Val	260	.	.	B	B	.	.	.	−3.39	.	.	.	−0.60	0.02
Cys	261	.	.	B	B	.	.	.	−3.14	.	.	.	−0.60	0.01
Cys	262	.	.	B	B	.	.	.	−2.59	.	.	.	−0.60	0.02
Cys	263	.	.	B	B	.	.	.	−2.12	.	.	.	−0.60	0.03
Ile	264	.	.	B	B	.	.	.	−1.90	.	.	.	−0.60	0.06
Gly	265	.	.	.	.	T	T	.	−1.39	.	.	F	0.35	0.06
Ser	266	.	.	.	.	T	T	.	−1.07	.	.	F	0.35	0.11
Gly	267	.	.	.	.	T	T	.	−0.40	.	.	F	0.65	0.16
Cys	268	.	.	.	.	T	T	.	0.06	.	.	F	1.25	0.27
Gly	269	.	.	.	.	T	.	.	0.99	.	*	F	1.39	0.31
Gly	270	.	.	.	.	T	.	.	0.67	.	.	F	2.03	0.62
Asp	271	.	.	.	.	.	T	C	0.37	.	.	F	2.37	0.62
Pro	272	.	.	.	.	T	T	.	0.71	*	*	F	2.91	0.62
Lys	273	.	.	.	.	T	T	.	1.49	*	*	F	3.40	1.05
Cys	274	.	.	B	.	.	T	.	0.98	*	*	.	2.51	1.23
Met	275	.	.	B	B	.	.	.	0.66	*	*	.	1.62	0.59
Asp	276	.	.	B	B	.	.	.	−0.04	*	*	.	1.28	0.16
Arg	277	.	.	B	B	.	.	.	−0.12	.	*	.	0.04	0.26
Val	278	.	.	B	B	.	.	.	−0.06	.	*	.	−0.60	0.27
Cys	279	.	.	B	B	.	.	.	−0.20	.	.	.	0.30	0.32
Phe	280	.	.	B	B	.	.	.	0.06	.	*	.	−0.60	0.13
Trp	281	.	.	B	B	.	.	.	−0.76	.	.	.	−0.60	0.18
Arg	282	.	.	B	B	.	.	.	−1.68	.	.	.	−0.60	0.28
Leu	283	.	.	B	B	.	.	.	−0.71	.	.	.	−0.60	0.26
Gly	284	.	.	.	B	T	.	.	−0.39	.	*	.	−0.20	0.49
Leu	285	.	.	.	B	.	.	C	0.10	.	*	.	0.50	0.25
Leu	286	.	.	.	B	.	.	C	0.04	.	*	.	0.20	0.46
Arg	287	.	.	.	B	.	.	C	−0.66	.	.	F	0.65	0.46
Gly	288	.	.	.	.	.	T	C	0.16	.	.	F	1.35	0.57
Pro	289	.	.	.	.	.	T	C	0.50	.	*	F	2.70	1.19
Gly	290	.	.	.	.	.	T	C	1.31	*	*	F	3.00	1.01
Ala	291	A	.	.	.	.	T	.	1.53	.	*	F	2.50	1.65
Glu	292	A	.	.	.	.	.	.	1.39	.	.	F	2.00	1.08
Asp	293	A	.	.	.	.	.	.	1.73	.	.	F	1.70	1.48
Asn	294	A	.	.	.	.	T	.	1.94	.	*	.	1.45	2.36
Ala	295	A	.	.	.	.	T	.	1.40	.	.	.	1.15	2.36
His	296	A	.	.	.	.	T	.	1.18	*	.	.	1.00	0.99
Asn	297	A	.	.	.	.	T	.	0.88	.	.	.	0.10	0.51
Glu	298	A	.	.	.	.	.	.	0.88	*	.	.	−0.10	0.67
Ile	299	A	.	.	.	.	.	.	0.29	*	*	.	−0.10	0.80
Leu	300	A	.	.	.	.	.	.	0.88	*	*	.	−0.10	0.50
Ser	301	A	.	.	.	.	.	.	0.61	*	.	F	0.65	0.48
Asn	302	A	.	.	.	.	T	.	−0.20	*	.	F	0.25	0.92
Ala	303	A	.	.	.	.	T	.	−0.50	*	.	F	0.25	0.92
Asp	304	A	.	.	.	.	T	.	0.08	*	.	F	0.85	0.92
Ser	305	.	.	.	.	.	T	C	0.19	*	.	F	1.05	0.83
Leu	306	.	.	.	B	.	.	C	−0.37	*	.	F	0.05	0.71
Ser	307	.	.	B	B	.	.	.	−0.67	*	.	F	−0.15	0.31
Thr	308	.	.	B	B	.	.	.	−0.08	*	.	.	−0.60	0.31
Phe	309	.	.	B	B	.	.	.	−0.08	*	.	.	−0.30	0.66
Val	310	A	.	.	B	.	.	.	0.22	.	.	F	−0.15	0.85
Ser	311	A	A	.	.	.	.	.	0.43	.	.	F	0.00	1.03
Glu	312	A	A	.	.	.	.	.	0.73	.	.	F	0.00	1.17
Gln	313	A	A	.	.	.	.	.	0.74	.	.	F	0.90	2.73
Gln	314	A	A	.	.	.	.	.	1.44	.	.	F	0.90	2.73
Met	315	A	A	.	.	.	.	.	2.30	.	.	F	0.90	2.73
Glu	316	A	A	.	.	.	.	.	2.39	.	.	F	0.90	2.73
Ser	317	A	A	.	.	.	.	.	1.80	.	*	F	0.90	2.44
Gln	318	A	A	.	.	.	.	.	1.80	.	*	F	0.90	2.49
Glu	319	A	A	.	.	.	.	.	0.99	.	*	F	0.90	2.40
Pro	320	A	A	.	.	.	.	.	1.28	.	*	F	0.90	1.48
Ala	321	A	A	.	.	.	.	.	0.93	.	.	F	0.60	1.23
Asp	322	A	A	.	B	.	.	.	0.38	.	.	F	0.45	0.70
Leu	323	A	A	.	B	.	.	.	0.07	.	.	F	−0.15	0.34
Thr	324	.	A	B	B	.	.	.	−0.79	.	.	F	−0.15	0.48
Gly	325	.	A	B	B	.	.	.	−0.58	.	.	.	−0.30	0.21
Val	326	.	.	B	B	.	.	.	−0.29	.	.	.	−0.60	0.45
Thr	327	.	.	B	B	.	.	.	−0.50	.	.	.	−0.60	0.42
Val	328	.	.	B	B	.	.	.	−0.03	.	*	F	−0.17	0.65
Gln	329	.	.	B	B	.	.	.	0.28	.	*	F	0.11	0.87
Ser	330	.	.	.	.	.	T	C	0.03	.	*	F	2.04	1.05
Pro	331	.	.	.	.	.	T	C	0.89	.	*	F	2.32	1.42
Gly	332	.	.	.	.	T	T	.	0.53	.	*	F	2.80	1.42
Glu	333	A	.	.	.	.	T	.	0.58	.	*	F	1.97	0.57
Ala	334	.	.	B	.	.	.	.	−0.23	.	*	.	0.74	0.30
Gln	335	.	.	B	.	.	.	.	−0.28	.	.	.	0.46	0.25
Cys	336	.	.	B	.	.	.	.	−0.28	.	.	.	0.18	0.14
Leu	337	.	.	B	.	.	.	.	−0.52	.	*	.	−0.40	0.22
Leu	338	.	.	B	.	.	.	.	−0.52	.	*	.	−0.40	0.13
Gly	339	.	A	.	.	.	.	C	−0.52	.	*	F	0.05	0.42
Pro	340	A	A	.	.	.	.	.	−0.52	.	*	F	−0.15	0.51
Ala	341	A	A	.	.	.	.	.	−0.20	.	*	F	0.60	1.07
Glu	342	A	A	.	.	.	.	.	0.31	.	*	F	0.90	1.07
Ala	343	A	A	.	.	.	.	.	1.12	*	*	F	0.75	0.93
Glu	344	A	A	.	.	.	.	.	1.58	.	*	F	0.90	1.60
Gly	345	A	A	.	.	.	.	.	1.90	.	*	F	0.90	1.80
Ser	346	A	.	.	.	.	T	.	2.60	.	*	F	1.30	3.50
Gln	347	A	.	.	.	.	T	.	1.79	.	*	F	1.30	3.96
Arg	348	A	.	.	.	.	T	.	1.57	.	*	F	1.30	3.30
Arg	349	.	.	B	.	.	T	.	0.71	.	*	F	1.30	2.03
Arg	350	.	.	B	B	.	.	.	0.84	.	*	F	0.75	0.87
Leu	351	.	.	B	B	.	.	.	0.56	.	*	.	0.60	0.69
Leu	352	.	.	B	B	.	.	.	0.56	.	*	.	0.30	0.35
Val	353	.	.	B	B	.	.	.	0.10	*	*	.	−0.30	0.29
Pro	354	.	.	B	.	.	T	.	−0.60	*	.	.	−0.20	0.35
Ala	355	.	.	.	.	T	T	.	−0.71	.	*	.	0.50	0.43
Asn	356	.	.	.	.	.	T	C	−0.11	.	.	F	1.65	0.96
Gly	357	.	.	.	.	.	T	C	0.39	.	.	F	1.95	0.96
Ala	358	.	.	.	.	.	.	C	1.24	.	.	F	2.20	1.37
Asp	359	.	.	.	.	.	T	C	1.14	.	.	F	3.00	1.48
Pro	360	A	.	.	.	.	T	.	0.92	*	.	F	2.50	2.16
Thr	361	A	.	.	.	.	T	.	0.32	.	.	F	1.90	1.76
Glu	362	A	.	.	.	.	T	.	−0.14	.	.	F	1.60	1.04
Thr	363	A	.	.	B	.	.	.	−0.26	.	.	F	0.15	0.56
Leu	364	A	.	.	B	.	.	.	−0.96	*	.	.	−0.60	0.33
Met	365	A	.	.	B	.	.	.	−0.74	*	.	.	−0.60	0.17
Leu	366	A	.	.	B	.	.	.	−0.39	*	.	.	−0.60	0.19
Phe	367	A	.	.	B	.	.	.	−1.09	*	.	.	−0.60	0.47
Phe	368	A	.	.	B	.	.	.	−1.37	*	.	.	−0.60	0.41
Asp	369	A	.	.	B	.	.	.	−0.56	*	.	.	−0.60	0.50
Lys	370	A	A	.	.	.	.	.	−0.84	*	.	.	−0.30	0.93
Phe	371	A	A	.	B	.	.	.	−0.89	*	.	.	−0.30	0.75
Ala	372	A	A	.	B	.	.	.	−0.40	*	.	.	−0.30	0.34
Asn	373	.	A	B	B	.	.	.	−0.40	*	.	.	−0.60	0.26
Ile	374	.	A	B	B	.	.	.	−0.40	*	.	.	−0.60	0.26
Val	375	.	A	B	B	.	.	.	−0.74	.	.	.	−0.60	0.43
Pro	376	.	A	.	B	.	.	C	−0.33	.	.	.	−0.10	0.36
Phe	377	.	.	.	.	T	T	.	0.26	.	.	.	0.20	0.54
Asp	378	.	.	.	.	T	T	.	0.26	.	.	F	0.80	1.21
Ser	379	.	.	.	.	T	T	.	0.33	.	.	F	1.40	1.35
Trp	380	A	.	.	.	.	T	.	0.59	*	*	F	0.40	1.29
Asp	381	A	A	.	.	.	.	.	0.91	*	.	F	−0.15	0.76
Gln	382	A	A	.	.	.	.	.	1.61	*	.	.	−0.15	1.11
Leu	383	A	A	.	.	.	.	.	0.80	*	.	.	−0.15	1.84
Met	384	A	A	.	.	.	.	.	1.10	*	.	.	0.30	0.91
Arg	385	A	A	.	.	.	.	.	0.58	*	.	.	0.30	0.87
Gln	386	A	A	.	.	.	.	.	0.27	*	.	.	−0.30	0.87
Leu	387	A	A	.	.	.	.	.	0.31	*	.	.	0.45	1.27
Asp	388	A	A	.	.	.	.	.	1.12	*	.	.	0.75	1.30
Leu	389	A	A	.	.	.	.	.	1.72	*	.	F	0.60	1.21
Thr	390	A	.	.	.	.	T	.	0.72	*	.	F	1.30	2.54
Lys	391	A	.	.	.	.	T	.	0.72	.	*	F	1.30	1.07
Asn	392	A	.	.	.	.	T	.	0.68	*	*	F	1.30	2.16
Glu	393	A	.	.	.	.	T	.	−0.18	*	.	F	1.30	1.11
Ile	394	.	.	B	B	.	.	.	0.74	*	.	F	0.75	0.41
Asp	395	.	.	B	B	.	.	.	0.47	*	*	.	0.60	0.50
Val	396	.	.	B	B	.	.	.	0.08	*	*	.	0.60	0.29
Val	397	.	.	B	B	.	.	.	−0.23	.	.	.	0.51	0.41
Arg	398	.	.	B	.	.	T	.	−0.82	*	.	.	1.12	0.36
Ala	399	.	.	B	.	.	T	.	−0.28	*	.	.	0.73	0.49
Gly	400	.	.	.	.	T	T	.	−0.49	*	.	F	2.09	0.65
Thr	401	.	.	.	.	.	T	C	0.02	*	*	F	2.10	0.51
Ala	402	.	.	.	.	.	.	C	0.88	*	*	F	1.09	0.50
Gly	403	.	.	.	.	.	T	C	0.18	*	*	F	1.68	0.85
Pro	404	.	.	.	.	.	T	C	−0.04	.	.	F	1.47	0.59
Gly	405	.	.	.	.	.	T	C	0.06	.	.	F	1.26	0.48
Asp	406	A	.	.	.	.	T	.	−0.22	.	.	F	0.25	0.76
Ala	407	A	A	.	.	.	.	.	−0.23	.	.	.	−0.30	0.50
Leu	408	A	A	.	.	.	.	.	−0.70	.	.	.	−0.60	0.50
Tyr	409	A	A	.	.	.	.	.	−1.09	*	.	.	−0.60	0.25
Ala	410	A	A	.	.	.	.	.	−0.70	*	.	.	−0.60	0.24
Met	411	A	A	.	.	.	.	.	−0.99	*	.	.	−0.60	0.59
Leu	412	A	A	.	.	.	.	.	−1.26	*	.	.	−0.60	0.39
Met	413	A	A	.	.	.	.	.	−0.44	*	.	.	−0.60	0.29
Lys	414	A	A	.	B	.	.	.	−0.16	*	.	.	−0.60	0.47
Trp	415	A	A	.	B	.	.	.	0.12	*	.	.	0.15	1.14
Val	416	A	A	.	B	.	.	.	0.38	*	*	.	0.45	1.66
Asn	417	A	.	.	.	.	T	.	1.30	*	.	F	1.75	0.82
Lys	418	A	.	.	.	.	T	.	1.90	*	.	F	2.20	1.53
Thr	419	.	.	.	.	.	T	C	1.27	*	.	F	3.00	3.32
Gly	420	.	.	.	.	.	T	C	1.26	*	.	F	2.70	2.08
Arg	421	.	.	.	.	T	.	.	1.22	*	.	F	2.40	1.40
Asn	422	.	.	.	.	.	T	C	1.19	*	.	F	1.65	0.68
Ala	423	.	.	B	.	.	T	.	0.83	.	.	.	1.00	0.93
Ser	424	.	.	B	.	.	T	.	0.33	.	.	.	0.70	0.69
Ile	425	.	.	B	.	.	T	.	−0.13	.	*	.	−0.20	0.35
His	426	.	A	B	.	.	.	.	−0.24	.	*	.	−0.60	0.29
Thr	427	.	A	B	.	.	.	.	−0.83	*	*	.	−0.60	0.36
Leu	428	A	A	.	.	.	.	.	−1.06	*	*	.	−0.60	0.52
Leu	429	A	A	.	.	.	.	.	−0.76	*	*	.	−0.60	0.31
Asp	430	A	A	.	.	.	.	.	0.24	*	*	.	−0.30	0.38
Ala	431	A	A	.	.	.	.	.	−0.32	*	*	.	0.30	0.89
Leu	432	A	A	.	.	.	.	.	−0.01	*	*	.	0.75	1.07
Glu	433	A	A	.	.	.	.	.	0.80	*	*	.	0.75	1.11
Arg	434	A	A	.	.	.	.	.	1.72	*	*	F	0.90	1.90
Met	435	A	A	.	.	.	.	.	1.69	*	*	F	0.90	4.52
Glu	436	A	A	.	.	.	.	.	1.69	*	*	F	0.90	3.55
Glu	437	A	A	.	.	.	.	.	2.54	*	.	F	0.90	1.83
Arg	438	A	A	.	.	.	.	.	2.54	*	*	F	0.90	3.70
His	439	A	A	.	.	.	.	.	2.48	*	*	F	0.90	3.70
Ala	440	A	A	.	.	.	.	.	2.19	*	*	F	0.90	4.28
Lys	441	A	A	.	.	.	.	.	2.19	*	*	F	0.90	1.53
Glu	442	A	A	.	.	.	.	.	2.19	*	.	F	0.90	1.95
Lys	443	A	A	.	.	.	.	.	1.27	*	*	F	0.90	3.22
Ile	444	A	A	.	.	.	.	.	0.49	*	*	F	0.90	1.33
Gln	445	A	A	.	.	.	.	.	0.22	*	*	F	0.75	0.63
Asp	446	A	A	.	.	.	.	.	0.18	*	*	F	−0.15	0.23
Leu	447	A	A	.	.	.	.	.	−0.12	*	.	.	−0.30	0.56
Leu	448	A	A	.	.	.	.	.	−0.51	*	.	.	0.55	0.43
Val	449	A	A	.	.	.	.	.	0.42	*	.	F	0.95	0.26
Asp	450	A	.	.	.	.	T	.	−0.28	*	.	F	1.60	0.62
Ser	451	.	.	.	.	T	T	.	−1.17	*	.	F	2.25	0.65
Gly	452	.	.	.	.	T	T	.	−0.60	*	.	F	2.50	0.62
Lys	453	.	.	B	.	.	T	.	−0.60	.	.	F	1.25	0.58
Phe	454	.	A	B	.	.	.	.	0.26	.	.	.	0.15	0.36
Ile	455	.	A	B	.	.	.	.	0.26	.	.	.	0.20	0.62
Tyr	456	.	A	B	.	.	.	.	0.21	.	.	.	0.55	0.52
Leu	457	.	A	B	.	.	.	.	0.24	.	.	.	−0.03	0.59
Glu	458	.	A	B	.	.	.	.	−0.14	.	.	F	0.54	1.22
Asp	459	.	A	.	.	T	.	.	0.26	.	.	F	1.66	0.77
Gly	460	.	.	.	.	T	T	.	0.56	.	.	F	2.78	1.26
Thr	461	.	.	.	.	.	T	C	−0.06	*	.	F	2.70	0.73
Gly	462	.	.	.	.	.	T	C	0.46	*	.	F	2.13	0.33
Ser	463	.	.	.	.	.	T	C	−0.36	.	.	F	1.26	0.44
Ala	464	A	.	.	.	.	.	.	−0.36	.	.	.	0.14	0.25
Val	465	.	.	B	.	.	.	.	−0.40	.	.	.	0.17	0.44
Ser	466	.	.	B	.	.	.	.	−0.48	.	.	.	−0.10	0.42
Leu	467	.	.	B	.	.	.	.	−0.52	.	.	.	−0.10	0.53
Glu	468	A	.	.	.	.	.	.	−0.61	.	.	.	0.50	0.92