HUMAN ALZHEIMER'S DISEASE AND TRAUMATIC BRAIN INJURY ASSOCIATED TAU VARIANTS AS BIOMARKERS AND METHODS OF USE THEREOF

Abstract

The present invention provides detection reagents and method for determining risk of traumatic brain injury (TBI) and/or susceptibility to neurodegenerative disease in a subject.

Description

RELATED APPLICATION

[0001] This application claims the benefit of priority of U.S. Provisional Application Ser. No. 62/266,461 filed on Dec. 11, 2015, which application is incorporated by reference herein.

BACKGROUND

[0003] Numerous studies have implicated small soluble oligomeric aggregates of A.beta. as toxic species in Alzheimer's disease (AD), and increasing evidence also implicates oligomeric forms of tau as having a direct role in disease pathogenesis of AD and other tauopathies such as Frontotemporal Dementia (FTD). As the focus of A.beta. studies has slowly shifted toward soluble A.beta. species and mechanisms, new reagents were needed that could specifically identify the variety of different aggregate species present. Indeed, many contradictory studies on the role of A.beta. aggregation in AD were reported and progress impeded because suitably selective reagents were not available to characterize the aggregate species present. Increasing evidence from cell and animal models indicate that oligomeric rather than fibrillar forms of tau are toxic and correlate with neuronal degeneration, therefore well characterized reagents that can specifically recognize the diversity of tau morphologies present in the human brain are critically needed to facilitate studies to identify the most promising tau species for use as biomarkers of disease and to study toxic mechanisms.

[0004] The microtubule associating protein tau is a major component of the neurofibrillary tangles associated with AD and tauopathies that are characterized by hyperphosphorylation and aggregation of tau. Tau plays an important role in assembly and stabilization of microtubules. Tau is a natively unfolded protein, and similar to a number of other natively unfolded proteins, it can aberrantly fold into various aggregate morphologies including .beta.-sheet rich fibrillar forms. The different types of post-translational modifications of tau in AD include phosphorylation, glycosylation, glycation, prolyl-isomerization, cleavage or truncation, nitration, polyamination, ubiquitination, sumoylation, oxidation and aggregation. Tau has 85 putative phosphorylation sites, and excess phosphorylation can interfere with microtubule assembly. Tau can be modified by phosphorylation or by reactive nitrogen and oxygen species among others. Elevated total tau concentration in CSF has been correlated with AD, as has the presence of various phosphorylated tau forms, and the ratio of tau to A.beta.42. Reactive nitrogen and oxygen can modify tau facilitating formation of aggregate forms including oligomeric species. Levels of oligomeric tau have also been implicated as a potential early diagnostic for AD. Therefore, while determination of total tau and phosphorylated tau levels has demonstrated value for diagnosis of AD and other tauopathies, reagents that can selectively recognize the tau species that are most selectively involved in AD would have particular value in diagnostics for neurodegenerative diseases including tauopathies and AD.

[0005] Tau is an intrinsically unstructured protein due to its very low hydrophobic content containing a projection domain, a basic proline-rich region, and an assembly domain. Hexapeptide motifs in repeat regions of tau give the protein a propensity to form .beta.-sheet structures which facilitate interaction with tubulin to form microtubules as well as self-interaction to form pathological aggregates such as paired helical filaments (PHF). Hyperphosphorylation of tau, particularly in the assembly domain, decreases the affinity of tau to the microtubules and impairs its ability to regulate microtubule dynamics and axonal transport. In addition, parts of the basic proline-rich domain and the pseudo-repeat also stabilize microtubules by interacting with its negatively charged surface. Alternative splicing of the second, third and tenth exons of tau results in six tau isoforms of varying length in the CNS. The assembly domain in the carboxyl-terminal portion of the protein contains either three or four repeats (3R or 4R) of a conserved tubulin-binding motif depending on alternative splicing of exon 10. Tau 4R isoforms have greater microtubule binding and stabilizing ability than the 3R isoforms. Human adult brains have similar levels of 3R and 4R isoforms, while only 3R tau is expressed at the fetal stage. Mutations altering splicing of tau transcript and the ratio of 3R to 4R tau isoforms are sufficient to cause neurodegenerative disease. Therefore tau in human brain tissue can exist in a variety of different lengths and morphologies and with multiple post-translational modifications.

[0006] Tau plays a critical role in the pathogenesis of AD and studies show that reduction of tau levels in AD animal models reverses disease phenotypes and that tau is necessary for the development of cognitive deficits in AD models caused by over-expression of A.beta.. While NFTs have been implicated in mediating neurodegeneration in AD and tauopathies, animal models of tauopathy have shown that memory impairment and neuron loss do not associate well with accumulation of NFT. Animal studies showed improvement in memory and reduction in neuron loss despite the accumulation of NFTs, a regional dissociation of neuron loss and NFT pathology, and hippocampal synapse loss and dysfunction and microglial activation months before the accumulation of filamentous tau inclusions. The pathological structures of tau most closely associated with AD progression are tau oligomers. All these studies suggest that tau tangles are not acutely neurotoxic, but rather that pretangle oligomeric tau species are responsible for the neurodegenerative phenotype, similar to toxic role of oligomeric A.beta. species.

[0007] Numerous studies suggest that extracellular tau species contribute to neurotoxicity through an "infectious" model of disease progression. For example, tau pathology spreads contiguously throughout the brain from early to late stage disease, extracellular tau aggregates can propagate tau misfolding from the outside to the inside of a cell, brain extract from a transgenic mouse with aggregated mutant human tau transmits tau pathology throughout the brain in mice expressing normal human tau, induction of pro-aggregation human tau induces formation of tau aggregates and tangles composed of both human and normal murine tau (co-aggregation), and levels of tau rise in CSF in AD, whereas A.beta. levels decrease. A receptor-mediated mechanism for the spread of tau pathology by extracellular tau has been described.

[0008] Collectively, these studies all indicate that a variety of different tau forms including splice variants, post-translational modifications and different aggregated forms, both intracellular and extracellular, are vitally important in AD and other tauopathies. In order to more clearly define the role of individual tau forms in disease, there is a critical need to develop a series of well-defined reagents that selectively recognize individual tau species, and to use these reagents to identify which tau forms are the best biomarkers for AD, which forms are involved in toxicity, and which forms can distinguish between healthy and AD patients in brain tissue and CSF samples.

SUMMARY

[0009] Methods have been developed that enable generation of reagents that selectively bind disease related protein variants. The inventors have developed methods and reagents to assess neuronal damage following traumatic brain injury (TBI). The inventors have also developed methods and reagents to assess the staging of Alzheimer's Disease (AD). Phage display antibody libraries are used as a source to isolate the protein variant specific reagents.

[0010] The present invention discloses an antibody or antibody fragment that preferentially recognizes human traumatic brain injury (TBI)-associated tau and other antibody or antibody fragments that preferentially recognize different stages of AD. As used herein, the phrase "preferentially recognizes" indicates that it does not bind to or recognize non-TBI associated forms of tau or non-specific proteins. As used herein, the term "antibody" includes scFv (also called a "nanobody"), humanized, fully human or chimeric antibodies, single-chain antibodies, diabodies, and antigen-binding fragments of antibodies (e.g., Fab fragments).

[0011] In certain embodiments, the antibody is an antibody fragment that does not contain the constant domain region of an antibody.

[0012] In certain embodiments, the antibody fragment is less than 500 amino acids in length, such as between 200-450 amino acids in length, or less than 400 amino acids in length. In certain embodiments, the antibody has an amino acid sequence having at least 80% sequence identity of any one of SEQ ID NO: 42, 44, 46, or 48. In certain embodiments, the amino acid sequence has at least 90% sequence identity of any one of SEQ ID NO: 42, 44, 46, or 48. In certain embodiments, the amino acid sequence has at least 95% sequence identity of any one of SEQ ID NO: 42, 44, 46, or 48. In certain embodiments, the amino acid sequence has 100% sequence identity of any one of SEQ ID NO: 42, 44, 46, or 48. In certain embodiments, the present invention discloses a nucleic acid that encodes an antibody that preferentially recognizes human traumatic brain injury (TBI)-associated tau. In certain embodiments, the present invention provides a nucleic acid encoding an antibody that preferentially recognizes TBI-associated tau, wherein the nucleic acid has at least 80% sequence identity of any one of SEQ ID NO: 41, 43, 45, or 47. In certain embodiments, the nucleic acid sequence has at least 90% sequence identity of any one of SEQ ID NO: 41, 43, 45, or 47. In certain embodiments, the nucleic acid sequence has at least 95% sequence identity of any one of SEQ ID NO: 41, 43, 45, or 47. In certain embodiments, the nucleic acid sequence has 100% sequence identity of any one of SEQ ID NO: 41, 43, 45, or 47.

[0013] In certain embodiments, the present invention provides an antibody that preferentially recognizes a human Alzheimer's Disease (AD)-associated Tau.

[0014] In certain embodiments, the antibody is an antibody fragment that does not contain the constant domain region of an antibody.

[0015] In certain embodiments, the antibody fragment is less than 500 amino acids in length, such as between 200-450 amino acids in length, or less than 400 amino acids in length. In certain embodiments, the antibody has an amino acid sequence having at least 80% sequence identity of any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. In certain embodiments, the amino acid sequence has at least 90% sequence identity of any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. In certain embodiments, the amino acid sequence has at least 95% sequence identity of any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. In certain embodiments, the amino acid sequence has 100% sequence identity of any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.

[0016] In certain embodiments, the present invention discloses a nucleic acid that encodes an antibody that preferentially recognizes human AD-associated tau. In certain embodiments, the present invention provides a nucleic acid encoding an antibody that preferentially recognizes a human AD-associated tau, wherein the nucleic acid has at least 80% sequence identity of any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, or 39. In certain embodiments, the nucleic acid has at least 90% sequence identity of any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, or 39. In certain embodiments, the nucleic acid has at least 95% sequence identity of any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, or 39. In certain embodiments, the nucleic acid has 100% sequence identity of any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, or 39.

[0017] In certain embodiments, the present invention provides a vector comprising a nucleic acid described above.

[0018] In certain embodiments, the present invention provides a phage comprising the vector described above.

[0019] In certain embodiments, the present invention provides a method for determining risk of traumatic brain injury (TBI), assessment of the amount of neuronal damage, and/or susceptibility to neurodegenerative disease in a human, comprising the steps of:

[0020] (A) providing a sample obtained from a subject post-injury;

[0021] (B) detecting levels of human TBI-associated tau in the sample;

[0022] (C) comparing the TBI-associated tau protein level in the sample with TBI-associated tau protein level in a normal control; and

[0023] (D) determining whether the human has a risk of TBI in accordance with the result of step (C);

[0024] wherein a subject having elevated TBI-associated tau protein has a high risk of TBI.

[0025] In certain embodiments, the present invention provides a method for determining the stage of Alzheimer's disease (AD) in a human, comprising the steps of:

[0026] (A) providing a sample obtained from a human;

[0027] (B) detecting levels of stage-specific human AD-associated tau in the sample;

[0028] (C) comparing the AD-associated tau protein level in the sample with AD-associated tau protein level in a normal control; and

[0029] (D) determining whether the subject has a risk of AD in accordance with the result of step (C);

[0030] wherein a subject having elevated AD-associated tau protein has a high risk of AD.

[0031] In certain embodiments, the samples and the normal control are blood product samples or cerebrospinal fluid (CSF) samples.

[0032] In certain embodiments, the blood product is serum.

[0033] In certain embodiments, the detecting in step (B) is by means of a ligand specific for the protein.

[0034] In certain embodiments, the ligand is an antibody.

[0035] In certain embodiments, the ligand is a scFv.

[0036] In certain embodiments, the protein levels are detected by means of ELISA.

BRIEF DESCRIPTION OF THE FIGURES

[0037] FIG. 1: Immunohistochemistry stain of human AD and control brain tissue slices showing increased presence of phosphorylated tau fibrils in the human AD tissue compared to the age matched cognitively normal sample. Tau was stained using the commercially available anti-phospho-tau antibody AT8.

[0038] FIG. 2: Dotblot assay confirming the presence of immunoprecipitated tau from TBI and control CSF samples in the IP elutes following immunoprecipitation protocol.

[0039] FIGS. 3a and 3b. FIG. 3a: Western blot of human AD and control brain tissue homogenates staining with polyclonal tau antibody shows presence of increased molecular weight tau species in AD patient sample compared to ND sample. Staining with the anti-phosphorylated tau antibody, AT8 shows presence of high molecular weight phosphorylated tau species in the AD sample with no phosphorylated tau species in the ND sample. FIG. 3b: Western blot of human AD and control brain tissue homogenates after immunoprecipitation with polyclonal anti-tau antibody. Staining with anti-phosphorylated tau antibody AT8, shows presence of high molecular weight phosphorylated tau species in AD sample with its absence in the ND sample.

[0040] FIG. 4: Negative panning against bovine serum albumin.

[0041] FIG. 5: Negative panning against aggregated .alpha.-synuclein.

[0042] FIG. 6: Negative panning against AD Braak stage I tissue.

[0043] FIG. 7: Negative panning against AD Braak stage I tau IP (Black arrow indicates phage).

[0044] FIG. 8: Flow diagram indicating steps in the panning protocol. Negative selection to remove non-specific clones was followed by positive selection with tau immunoprecipitated from AD Braak stage III and Braak stage V.

[0045] FIG. 9: Indirect ELISA assay testing different AD tau phage clones with pooled AD brain tissue homogenates. X axis represents the various clones and Y axis represents luminescence signal ratio to ND controls. All the clones have high levels of binding to AD tissue (Braak stage III and V) compared to the controls.

[0046] FIG. 10: Indirect ELISA of clone 51A with individual AD brain tissue homogenates. 7 samples (Braak V), 2 samples (Braak III) and 2 samples (Braak I) were tested. This clone binds tau morphologies present in AD Braak stage III.

[0047] FIG. 11: Indirect ELISA of clone 51F with individual AD brain tissue homogenates. 7 samples (Braak V), 2 (Braak III) and 2 (Braak I) were tested. This clone binds tau morphologies in AD Braak stage V.

[0048] FIG. 12: Sandwich ELISA with AD clones 51A and M58C with 5 AD sera samples. X-Axis represents individual sera samples and Y-Axis represents ratio to healthy control. Both the clones selectively bind to tau morphologies present in AD sera over healthy control.

[0049] FIG. 13: Indirect ELISA of TBI clones with pooled TBI and control CSF. X axis represents the TBI clones and Y axis represents ratio to no sample control. Most of the clones had high levels of binding to TBI compared to control.

[0050] FIG. 14: Indirect ELISA of TBI clones with individual TBI (4 samples) and pooled control CSF. X axis represents the TBI clones and Y axis represents ratio to control CSF. Most of the clones have high levels of binding to TBI samples.

[0051] FIG. 15: Sandwich ELISA of clone T2B with 18 TBI sera samples and 3 healthy aged controls. X-Axis represents individual sera samples and Y-Axis represents ratio to no sample control. T2B selectively binds to tau species that possibly circulates in TBI individuals several years after their head trauma when compared to healthy controls.

DETAILED DESCRIPTION

[0052] A vast number of studies have correlated protein aggregation with neurodegenerative diseases including AD, Parkinson's and Dementia with Lewy Bodies. Numerous recent studies suggest that specific protein variants including selected oligomeric forms of these proteins are involved in neuronal toxicity and can interfere with important functions including long term potentiation. Various soluble oligomeric species of A.beta. and a-syn have been shown to occur early during the course of AD and PD, and increasing evidence implicates oligomeric forms of tau in AD and other tauopathies.

[0053] A novel biopanning technology has been developed that combines the imaging capability of Atomic Force Microscopy (AFM) with the diversity of antibody libraries. This unique combination of antibody diversity and imaging capability allows for the isolation of single chain antibody variable domain fragment (scFv or nanobody) reagents to an array of morphologies of key proteins involved in neurodegenerative diseases including A.beta. and alpha-synuclein (a-syn). Nanobodies have been isolated that specifically recognize monomeric, fibrillar, and two different oligomeric a-syn morphologies. The anti-oligomeric a-syn nanobodies do not cross react with oligomeric A.beta., and specifically label PD brain tissue but not AD or healthy tissue. In addition, nanobodies were isolated to different regions of full length A.beta. and to three distinct naturally occurring oligomeric A.beta. morphologies. One, A4, specifically recognizes a larger oligomeric A.beta. species, inhibits aggregation and extracellular toxicity of A.beta., does not cross react with oligomeric a-syn, and specifically labels A.beta. aggregates in human AD brain samples, but not PD or healthy brain tissue. A second nanobody, E1, recognizes a smaller trimeric or tetrameric A.beta. species, and similar to A4 inhibits aggregation and extracellular toxicity of A.beta., does not cross react with oligomeric a-syn, and labels A.beta. aggregates in human AD but not healthy brain tissue. Utilizing an AD brain derived oligomeric A.beta. preparation, a third nanobody, C6, was isolated that specifically recognizes oligomeric A.beta. species derived from human AD brain tissue, but does not recognize A.beta. aggregates generated in vitro. The different specificities of each nanobody can be readily observed when each nanobody is expressed on the surface of a filamentous bacteriophage and antibody/antigen complexes are imaged by AFM. Therefore, the combination of antibody libraries and AFM imaging technologies enables the isolation and characterization of reagents that recognize specific protein variants including a variety of different naturally occurring aggregated forms of both a-syn and A.beta..

[0054] Another powerful advantage of this AFM palming protocol is that not only is it possible to isolate and characterize reagents to specific protein morphologies, but it is possible to do so using only picograms or less of material. In addition the sample does not need to be purified, and the protein does not need to be chemically modified in any way. It is possible to actually isolate nanobodies against a single molecule of the target antigen. This unique ability to generate and characterize reagents that specifically recognize individual protein variants provides the means to generate reagents that selectively recognize an array of different tau variants present in human AD brain.

[0055] While several reagents already exist that can recognize monomeric and phosphorylated tau, these reagents cannot distinguish between different aggregated states of tau. Reagents that can detect specific forms of tau can provide very powerful tools to facilitate diagnosis of AD and other tauopathies and to follow progression of these diseases or to evaluate therapeutic strategies. While many neurodegenerative diseases have overlapping clinical symptoms and cellular and biochemical mechanisms such as an increase in inflammatory markers, and aggregation of similar proteins, the reagents presently developed have well defined specificities and selectivities for selected tau forms and facilitate specific diagnoses of AD and other tauopathies. In combination with other protein and morphology specific reagents against A.beta. and a-syn species, these reagents can be used to detect the presence of biomarkers which can readily detect and distinguish many related neurodegenerative diseases including AD, PD, FTD and LBD.

[0056] In addition to the unique reagents and ELISA protocol, other advantages of this proposal over previous studies include the use of postmortem tissue and CSF from cases with neuropathologically confirmed AD; the use of control subjects who have had standardized neuromotor assessment and postmortem neuropathologic examination, ensuring that they are not in preclinical stages of AD or other neurodegenerative disease, and the use of a significant number of cases, compensating for individual variation as well as allowing stratification for possible significant influences on disease severity.

[0057] Traumatic Brain Injury

[0058] It is well established that chronic stress and especially traumatic brain injury (TBI) can disrupt cognitive functioning. The brain is very sensitive to stress and injury and responds by expressing a variety of neuromorphological and neurochemical changes. Stress induces increases in expression levels in the hippocampus of the Amyloid Precursor Protein (APP) and BACE-1, a protease which cleaves APP. These increases are of particular relevance for soldiers suffering TBI since similar increases in hippocampal expression of APP and BACE-1 are strongly linked with the onset and progression of Alzheimer's disease (AD). BACE-1 cleavage of APP results in generation of the beta-amyloid (A.beta.) protein, the primary component of the hallmark amyloid plaques associated with AD. Numerous studies have indicated that patients suffering brain trauma are at greater risk of developing AD and at an earlier age. The brain experiences very high sheer forces and mechanical deformation following TBI, and neuronal axons, particularly in the white matter are very susceptible to injury. Resulting damage to the neuronal axons can impair protein transport leading to accumulation of proteins and swelling causing the typical axon pathology observed with TBI. Various forms of stress induce memory deficits in mice and rats, with accompanying increases in APP, BACE-1 and A.beta. levels.

[0059] Increased expression of APP and BACE-1 results in increased production of A.beta., which in turn can promote aggregation of this natively unstructured protein into a variety of soluble aggregate species some of which are potent neurotoxins that inhibit long term potentiation and other neuronal functions. A.beta. can also self-assemble into much larger aggregates which eventually form the distinctive insoluble amyloid fibrils which are a hallmark of AD brain tissue. A vast amount of literature implicates A.beta. accumulation as being central to the progression of AD, leading to formation of the A.beta. hypothesis. The major weakness of the A.beta. hypothesis however, is that the presence of amyloid plaques does not correlate well with the progression of AD. While A.beta. can form amyloid plaques, it also forms a number of soluble intermediate or metastable structures which may contribute to toxicity. Cortical levels of soluble A.beta. correlated well with the cognitive impairment and loss of synaptic function. Small, soluble aggregates of A.beta. termed A.beta.-derived diffusible ligands and spherical or annular aggregates termed protofibrils are neurotoxic. Oligomeric forms of A.beta., created in vitro or derived from cell cultures inhibit long term potentiation. The concentration of oligomeric forms of A.beta. is also elevated in transgenic mouse models of AD and in human AD brain and CSF samples. Disruption of neural connections near A.beta. plaques was also attributed to oligomeric A.beta. species. A halo of oligomeric A.beta. surrounds A.beta. plaques causing synapse loss, and oligomeric A.beta. was shown to disrupt cognitive function in transgenic animal models of AD. Different size oligomers of A.beta. have been correlated with AD, including a 56 kD aggregate and smaller trimeric and tetrameric species. Therefore, the presence of oligomeric A.beta. is strongly correlated with neuronal dysfunction and memory deficits following neuronal damage and plays a critical role in progression of AD.

[0060] Given the critical role of APP and BACE-1 in cognitive deficits associated with AD, it is likely that similar increases in APP and BACE-1 levels induced by stress and injury to the brain also lead to elevated A.beta. levels, promoting formation of neurotoxic aggregate species, and subsequent memory loss and neuronal dysfunction. Following induced trauma to the brain, substantial deposition of non-fibrillar A.beta. aggregates has been observed throughout the brain, even after only a single event. Significantly, when TBI is induced in animal models of AD, there is a substantial increase in neuronal death, memory disorders, and A.beta. accumulation, but no corresponding increase in A.beta. plaque deposition, there was even a decrease in observed plaques. A preponderance of studies now indicate that various soluble oligomeric A.beta. aggregates play a very critical role in neuronal dysfunction rather than the hallmark fibrillar A.beta. plaques that have long been associated with AD. An observed increase in A.beta. levels in CSF samples from TBI patients suggests that detection of specific A.beta. species in CSF and serum represents a promising route for early detection of AD like brain injury in soldiers suffering TBI.

[0061] Since TBI also induces axonal injury and damage to protein transport mechanisms, neurofilament proteins may also play a role in TBI and AD. Neurofilament proteins accumulate in axons following TBI, and several studies have implicated the neurofilament protein tau in this process. The second major pathological feature of AD brains is the presence of neurofibrillary tangles that contain aggregates of the microtubule associated protein, tau. Tau is also a natively unfolded protein similar to A.beta., and can aberrantly fold into various aggregate morphologies including .beta.-sheet containing fibrillar forms and different oligomeric species. Tau plays an important role in assembly and stabilization of microtubules and can undergo numerous post-translational modifications including phosphorylation, glycosylation, glycation, prolyl-isomerization, cleavage or truncation, nitration, polyamination, ubiquitination, sumoylation, oxidation and aggregation. Tau has 85 putative phosphorylation sites, and excess phosphorylation can interfere with microtubule assembly. Elevated total tau concentration in CSF has been correlated with AD, as has the presence of various phosphorylated tau forms and the ratio of tau to A.beta.42. In addition to phosphorylation, tau can be modified by reactive nitrogen and oxygen species, leading to modified tau forms that are prone to assemble into aggregate species including different oligomeric forms. Levels of oligomeric tau have also been implicated as a potential early diagnostic for AD. Therefore, determination of total tau, phosphorylated tau and oligomeric tau concentrations all have potential value as diagnostics for neurodegenerative disorders including tauopathies, AD and TBI.

[0062] Tau is a very complex protein in vivo as alternative splicing of the second, third and tenth exons of tau result in generation of six tau isoforms of varying length in the CNS. The assembly domain in the carboxyl-terminal portion of the protein contains either three or four repeats (3R or 4R) of a conserved tubulin-binding motif depending on alternative splicing of exon 10. Tau 4R isoforms have greater microtubule binding and stabilizing ability than the 3R isoforms. Human adult brains have similar levels of 3R and 4R isoforms, whereas only 3R tau is expressed at the fetal stage. In tauopathies, mutations altering the splicing of tau transcript and the ratio of 3R to 4R tau isoforms are sufficient to cause neurodegenerative disease. Therefore tau in human brain tissue can exist in a variety of different lengths and morphologies and with multiple post-translational modifications.

[0063] Tau plays a critical role in the pathogenesis of AD and studies show that reduction of tau levels in AD animal models reverses disease phenotypes and that tau is necessary for the development of cognitive deficits in AD models caused by over-expression of A.beta.. While NFTs have been implicated in mediating neurodegeneration in AD and tauopathies, animal models of tauopathy have shown that memory impairment and neuron loss do not associate well with accumulation of NFT. In animal models expressing human tau, neurodegeneration-related phenotypes including behavioral impairments, neuronal loss, and synapse lesions correlate better with the presence of soluble tau oligomers and pre-filament species than with fibrillar NFT levels. Neuronal loss also precedes NFT formation suggesting involvement of other species such as oligomeric tau variants. In addition, animal studies showed that hippocampal synapse loss and dysfunction and microglial activation occurred months before the accumulation of filamentous tau inclusions. Both brain derived and recombinant oligomeric tau aggregate species disrupt intracellular calcium levels and are toxic to cultured human neuronal cells when added extracellularly. The pathological structures of tau most closely associated with AD progression were shown to be tau oligomers. In postmortem human brains, high oligomeric tau levels were detected in the frontal lobe cortex at early stages of AD before the presence of NFTs. Oligomeric tau may also be responsible for transmission of pathology with a prion-like mechanism as NFT tau pathology spreads from brain regions seeded with oligomeric tau into other regions resulting in aggregation of endogenous tau. It has been previously shown using recombinant human tau (rhTau) that extracellular trimeric, but not monomeric or dimeric species are toxic to human neuronal cells.

[0064] All these studies suggest that tau tangles are not acutely neurotoxic, but rather that pretangle oligomeric tau species are responsible for the neurodegenerative phenotype, similar to toxic role of oligomeric A.beta. species. Therefore both toxic oligomeric A.beta. and tau species in CSF and serum have promise as early biomarkers for AD and for AD like damage in TBI patients.

[0065] Similar to the role of A.beta. and tau in AD, aggregation of alpha-synuclein (a-syn) plays a critical role in PD and synucleinopathies. A-syn is a major component of Lewy bodies and neurites. Wild-type a-syn along with the three mutant forms, A30P, E46K and A53T can assemble into Lewy body like fibrils in vitro. Since all of the mutations increase the total rate of oligomerization compared to the wild-type form of a-syn, it has been postulated that the intermediate oligomeric morphologies of a-syn are the toxic structures in PD rather than fibrils. A partially folded intermediate of a-syn helps to promote fibril formation in vitro and a protofibrillar form of a-syn is stabilized by formation of a dopamine adduct complex, suggesting a possible connection between this morphology of a-syn and dopaminergic cell death. The different morphologies of a-syn also have different affinities for various membranes, and both the oligomeric forms and fibrillar forms have been shown to disrupt membrane permeability and integrity. Aggregated forms of a-syn were shown to induce toxicity in dopaminergic neurons in vivo and several different oligomeric morphologies were shown to each have different toxic mechanisms and effects on cells. We have shown that oligomeric but not fibrillar forms of a-syn are toxic to neuronal cells. Toxic oligomeric a-syn forms were identified in living cells, in human plasma from PD patients, and in human PD brain tissue indicating that oligomeric a-syn is also a good biomarker for neuronal damage.

[0066] Clearly protein misfolding and aggregation is critically important in many devastating neurodegenerative diseases. Therefore, determining how concentration profiles of selected key forms and morphologies of A.beta., tau and a-syn vary in AD, TBI and cognitively normal patients will facilitate development of an effective diagnostic assay for these diseases. In order to assess the value of these protein aggregates as biomarkers in neuronal disease, highly specific reagents are needed that can selectively identify the different toxic protein species. Our lab has developed unique technology that enables us to isolate reagents that bind specific morphologies of a target protein. We have combined the imaging capabilities of AFM with the binding diversity of phage display antibody technology to allow us to identify the presence of specific protein morphologies and then isolate reagents that bind a target morphology. These morphology specific reagents have promise for assessing whether the specific toxic aggregate species in human samples such as serum, plasma or CSF are useful biomarkers for neuronal damage. CSF levels of A.beta. and tau have been useful to predict AD, however biomarker studies of TBI patients have been less successful, where S100B has been the only marker to consistently predict TBI and outcome. S100B is a calcium binding protein that has been implicated in various diseases including AD, diabetes, melanoma and epilepsy, so its use in predicting TBI may be limited. We have developed a series of morphology specific nanobodies that have great promise for distinguishing between different neurodegenerative diseases. These nanobodies selectively recognize toxic protein aggregate biomarkers that are associated with specific diseases, therefore these nanobodies are recognizing biomarkers that are associated with the onset and progression of specific diseases, rather than recognizing a more generic secondary effect such as inflammatory signals, microglial activation or apoptotic markers. We have shown that three different nanobodies against different oligomeric A.beta. species all selectively distinguish between AD and PD or healthy samples in post-mortem human tissue and CSF samples. We have similarly shown that two different nanobodies against different toxic oligomeric a-syn species both selectively distinguish between PD and AD or healthy post-mortem human tissue and CSF samples. When we assayed post-mortem tissue, CSF and serum samples from AD, PD and cognitively normal patients with our anti-oligomeric A.beta., a-syn and tau nanobodies, we can not only readily distinguish AD, PD and normal samples but we can also stage progression of these different diseases. Also in the preliminary data section and of direct relevance to this proposal, we show that we can not only detect the presence of toxic oligomeric morphologies of A.beta., tau and a-syn in ante-mortem human serum samples, but that there is a very distinct spike in oligomeric A.beta. species in serum many years prior to diagnosis of AD, and even several years prior to diagnosis of mild-cognitive impairment (MCI) suggesting that we can presymptomatically diagnose AD by analysis of serum samples many years before symptoms of AD occur. Since early, even presymptomatic diagnosis of AD is critical so that preventative and treatment therapies can begin before extensive neuronal damage has occurred, the studies proposed here have very high potential impact. The morphology specific nanobodies we have developed and other nanobodies that have been developed are powerful tools to characterize human tissue, CSF and serum samples and to distinguish between different neurodegenerative diseases. Since the nanobodies recognize toxic species that should be present at early stages of disease progression, these nanobodies should be useful as early presymptomatic biomarkers for different neurodegenerative diseases, and to identify soldiers who are susceptible to AD following TBI.

[0067] The following human AD-associated Tau clone sequences were identified:

TABLE-US-00001 Clone 32B: (SEQ ID NO: 1) GATTACNGCCAAGCTTGCATGCAAATTNTATTTCAAGGAGNCAGTCATAATG AAATACNTATTGCCTNCGNCAGCCGCTGGATTGTTATTACTCGCGGCCCAGCCGGCC ATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTC CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTG GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTCTTCTAATGG TGATGATACAGCTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACA ATTCCAAGGACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCC GTATATTACTGTGCGAAAGCTAATAATTCTTTTGACTACTGGGGCCAGGGAACCCTG GTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCG GGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAG ACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGG TATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAATGCATCCACTTT GCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTC TCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGGATA GTGCTACTCCTTATACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGCGGCC GCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGA TCTGAATGGGGCCGC 32B AA sequence: (SEQ ID NO: 2) MKYXLPXXAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSS YAMSWVRQAPGKGLEWVSSISSNGDDTAYADSVKGRFTISRDNSKDTLYLQMNSLRA EDTAVYYCAKANNSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSAS VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYNASTLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQDSATPYTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLNGA Clone 51A: (SEQ ID NO: 3) TATGANCCATGATTACGCCAAGCTNNCATGCAANNTNTATTTTCAAGGAGAC AGTCATAATGAAATACCTATTGCNTACGNCAGCCGCTNNGATTGTTATTACTCGCGG CCNCAGCCGGCCATGGCCGAGGTGCAGCTGTNGGAGTCTGGGGGAGGCTTGGTACA GCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGNTA TGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATAGA TTTAGCAGTCGGGTCCGGTTACATCTTACGCAGACTCCGTGAAGGGCCGGTTCACCA TCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCC GAGGACACGGCCGTATATTACTGTGCGAAACGTCAGTTGATGTTTGACTACTGGGG CCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCA GCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCT GCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAG CTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATG CTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGG ACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTAC TGTCAACAGAGTTACAGTACCCCTAATACGTTCGGCCAAGGGACCAAGGTGGAAAT CAAACGGGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCA TCTCAGAAGAGGATCTGAATGGGCCGCATAG 51A AA sequence: (SEQ ID NO: 4) MAEVQLXESGGGLVQPGGSLRLSCAASGFTFSXYAMSWVRQAPGKGLEWVSIQ SGPVTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRQLMFDYWGQGT LVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQ QKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPNTF GQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLNGPH Clone 51F: (SEQ ID NO: 5) ATTNCGCCAAGCTNNCATGCAAAATTTNTATTTNAANGGAGACAGTCATAAT GAAATACCTATTGCNTACNNNNNNNCGCTGGATTGTTATTACTCGCGGNNCAGCCG GCCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGG GTCCCTGAGACTNTCCTGTGCAGCNTCTGGATTCACCTTTAGCAGCTATGCCATGAN NTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGNNNNNNGTNTCATCTATTACGTAGA CGGGTTCGTAGACACAGTACGCAGACTCCGTGAAGGGCAGGTTCACCATCTCCAGA GACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACAC GGCCGTATATTACTGTGCGAAACAGCATGATGATTTTGACTACTGGGGCCAGGGAA CCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGT GGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTA GGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAA TTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATACTGCATCCA ATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTC ACTCTCACCATCAGCAGTNTGCAACCTGAAGATTTTGCAACTTANTACTGTCAACAG CTGGATGTGTNTCCTTNGACGTTCGGNCAANNNACCAAGGTGGAAATCAA 51F AA sequence: (SEQ ID NO: 6) MAEVQLLESGGGLVQPGGSLRXSCAXSGFTFSSYAMXWVRQAPGKGLXXXSSI TTGSTQYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQHDDFDYWGQGT LVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQ QKPGKAPKLLIYTASNLQSGVPSRFSGSGSGTDFTLTISSXQPEDFATXYCQQLDVXPXT FXQXTKVEI Clone 52H: (SEQ ID NO: 7) GAGACAGTCATAGCTAGCATGAAAAAGANTTGGCTGGCGCTGGCTGGTTTAG TTTTagCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCCGGCCATGGACCTGG GTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTCAGGACGACGAAGTTCGTATCCTG ATGGCTAACGGTGCTGACGTTAACGCTGACGACTACGAAGGTTGGACTCCGCTGCA CCTGGCTGCTATGGTTGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGTACGGTGC TGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACA ACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCACATCATCATCACCAT CACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCAT AGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCT GGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAAT GCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTT CCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGANGGTGGCGGTTCT GAGGGTGGCGGTTCTGANGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACC TATTCCGGGCTATACTTATATCAACCCTCTCGACNGCACTTATCCGCCTGGTACTGA GCAAAACCCCGCTAATCCTAATCCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTT TCATGTTTCANAATAATANNTTCCGAAATNNNCNNGGTGCATTAACTGTTTATACNG GCACTGTTACTCNANNNACTGACCCCCGTTTAAAACTTATTACCAGTACACTCCNTG NNATCAT 52H AA sequence: (SEQ ID NO: 8) MKKXWLALAGLVLAFSASADYKEAQPAMDLGKKLLEAARAGQDDEVRILMA NGADVNADDYEGWTPLHLAAMVGHLEIVEVLLKYGADVNAQDKFGKTAFDISIDNGN EDLAEILQAAAHHHHHHGAAEQKLISEEDLNGAA Clone M32B: (SEQ ID NO: 9) TTCAGGAGANAGTCNTAATGAAATACCTATTGCCTACGGCAGCCGCTGGAtT GTTATTACTCGCGGNCCAGCCGGCCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGG GAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCA CCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAG TGGGTCTCAGGTATTTCTAATAATGGTAGTAATACAACTTACGCAGACTCCGTGAAG GGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA CAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGCTTCTTATACTTT TGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAG GCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCA TCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAG AGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCT CCTGATCTATAGTGCATCCTCTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAG TGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGC AACTTACTACTGTCAACAGTATTCTGGTTCTCCTGCTACGTTCGGCCNAGGGACCAA GGTGGAAATCANACGGGCGGCCGCACNTCATCATNNCCATCACGGGGCCGCAGAA NNAAAACTCATCTCAGAAGAGGANNTGAATGGGGCCGCATAGACTGTT M32B AA sequence: (SEQ ID NO: 10) MKYLLPTAAAGLLLLAXQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSY AMSWVRQAPGKGLEWVSGISNNGSNTTYADSVKGRFTISRDNSKNTLYLQMNSLRAE DTAVYYCAKASYTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYSASSLQSGVPSRFSGSGSGTDFTLTIS SLQPEDFATYYCQQYSGSPATFGXGTKVEIXRAAAXHHXHHGAAEXKLISEEX Clone M32E: (SEQ ID NO: 11) TTCAGGANANAGTCATAATGAANTACCTATTGCCTACGGCAGCCGCTGGANT NNTATTACTCGCGGCCCAgCCGGCCATGGCCCANGTGCAGCTGGTGGAGTCTGGGG GAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGNCTCCGGATTC ACCTTTANCAGCTATGACATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGA GTGGGTCTCAAGTATTAGTGGTAGTGGTCCTACCATGAACTACGCANACTCTGTGAA GGGCCGATTCACCGTCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGG ACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGGGGTACGGA CTTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGGNGGAGGCGGTT

CANGCGGAGGTGGCTCTGGCGGTGGCGGATCGTCTGAGCTGACTCAGGACCCTGCT GTGTCTGTGGCCTTGGGACAGACAGTCANCATCACATGCCAAGGANACAGCCTCNN AACCTATTATGCAAGCTGGTACCANCANAAGCCAGGACAGGCCCCTGTACTTGTCA TCTATGGNAAAAACAACCGGCCCTCANGGATCNCAGACCGATTCTCTGGCTCCAGC TCANGAAACACAGCTTCCTTGACCATCACTGNGGCTCAGGCGGAAGATGAGGCTGA CTATTACTGNAACTCCCGGGACAGCAGTGNNAACCATCTNANGAGTGTTCGGCGGA GGGANCNNGCTGACCGNCNTANGTGCGGCCGCAGNANCNNNNNCTNCNNNTCAGA ANANGATCTGAATGGGGCNNCATANACTGTTGNAAANNNGNTTANCAA M32E AA sequence: (SEQ ID NO: 12) MXYLLPTAAAGXXLLAAQPAMAXVQLVESGGGVVQPGRSLRLSCAXSGFTFXS YDMGWVRQAPGKGLEWVSSISGSGPTMNYAXSVKGRFTVSRDNSKNTLYLQMDSLRA EDTAVYYCAKGGTDFDYWGQGTLVTVSSXGGGSXGGGSGGGGSSELTQDPAVSVALG QTVXITCQGXSLXTYYASWYXXKPGQAPVLVIYXKNNRPSXIXDRFSGSSSXNTASLTI TXAQAEDEADYYXNSRDSSXNHXXSVRRR Clone M33F: (SEQ ID NO: 13) TTCAGGAGANAGTCNTAATGAAATACCTATTGCCTACGGCAGCCGCTGGATT GTTATTACTCGCGGCCCAGCCGGCCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGG GAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCA CCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAG TGGGTCTCAGCTATTACTAATGATGGTGCTGGTACAACTTACGCAGACTCCGTGAAG GGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA CAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATCTTATACTGGTTT TGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAG GCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAATCTCCA TCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAG AGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCT CCTGATCTATACTGCATCCACTTTGCAAAGTGGGNTCCCATTAAGGTTCAGTGGCAG TGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGC AACTTACTACTGTCAACAGANNTATGCTANTCCTANNACGTTCGGNCNANGGGACC NNNGNNNNAAATCANNCGGGCGGCCGCACNNCATNATNNNNNATNCNCGNNNNCG CAGAACAAAACTC M33F AA sequence: (SEQ ID NO: 14) MKYLLPTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSY AMSWVRQAPGKGLEWVSAITNDGAGTTYADSVKGRFTISRDNSKNTLYLQMNSLRAE DTAVYYCAKSYTGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYTASTLQSGXPLRFSGSGSGTDFTLTIS SLQPEDFATYYCQQXYAXPXTF Clone M34C: (SEQ ID NO: 15) CTANGCGNCCNNTTNAGATCCTCTTCTGAGANGAGTTTTTGTTCTGCGGCCCC GTGATGGTGATGATGATGTGCGGCCGCCCGTTTGATTTCCACCTTGGTCCCTTGGCC GAACGTCGCAGGAGTCTGATGAGTCTGTTGACAGTAGTAAGTTGCAAAATCTTCAG GTTGCAGACTGCTGATGGTGAGAGTGAAATCTGTCCCAGATCCACTGCCACTGAAC CTTGATGGGACCCCACTTTGCAACTGGGATGCCGGATAGATCAGGAGCTTAGGGGC TTTCCCTGGTTTCTGCTGATACCAATTTAAATAGCTGCTAATGCTCTGACTTGCCCGG CAAGTGATGGTGACTCTGTCTCCTACAGATGCAGACAGGGAGGATGGAGACTGGGT CATCTGGATGTCCGTCGACCCGCCACCGCCGCTGCCACCTCCGCCTGAACCGCCTCC ACCGCTCGAGACGGTGACCAGGGTTCCCTGGCCCCAGTAGTCAAAAGACCAAAACT GTTTCGCACAGTAATATACGGCCGTGTCCTCGGCTCTCAGGCTGTTCATTTGCAGAT ACAGCGTGTTCTTGGAATTGTCTCTGGAGATGGTGAACCGGCCCTTCACGGAGTCTG CGTACGTTGTCGGCGGACCCTGCTTCGCAATATCTGAGACCCACTCCAGCCCCTTCC CTGGAGCCTGGCGGACCCAGCTCATGGCATAGCTGCTAAAGGTGAATCCAGAGGCT GCACAGGAGAGTCTCANGGACCCCCCAGGCTGTACCAAGCCTCCCCCAGACTCCAA CAGCTGCACCTCGGCCATGGCCGGCTGGGCCGCGAGTAATAACAATCCAGCGGCTG CCGTANGCAATANGTATTTCATTATGACTGTCTCCTTGAAATAGAATTTGCATGCAA GCTTGGNNTANNATGGNCATAGCTGTTTNCTGTGTGAAATNGNTATNCNNTCNCAA TTCCNCACAANATAC M34C AA sequence: (SEQ ID NO: 16) MKYXLXTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSXRLSCAASGFTFSS YAMSWVRQAPGKGLEWVSDIAKQGPPTTYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCAKQFWSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSA SVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYPASQLQSGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQTHQTPATFGQGTKVEIKRAAAHHHHHHGAAEQKLXSEED Clone M34F: (SEQ ID NO: 17) CATTCNGATCCTCTTCTGAGANGAGTTTTTGTTCTGCGGCCCCGTGATGGTGA TGATGATGTGCGGCCGCCCGTTTGATTTCCACCTTGGTCCCTTGGCCGAACGTAATA GGAGACGGATGCGACTGTTGACAGTAGTAAGTTGCAAAATCTTCAGGTTGCAGACT GCTGATGGTGAGAGTGAAATCTGTCCCAGATCCACTGCCACTGAACCTTGATGGGA CCCCACTTTGCAAATTGGATGCCCTATAGATCAGGAGCTTAGGGGCTTTCCCTGGTT TCTGCTGATACCAATTTAAATAGCTGCTAATGCTCTGACTTGCCCGGCAAGTGATGG TGACTCTGTCTCCTACAGATGCAGACAGGGAGGATGGAGACTGGGTCATCTGGATG TCCGTCGACCCGCCACCGCCGCTGCCACCTCCGCCTGAACCGCCTCCACCGCTCGAG ACGGTGACCAGGGTTCCCTGGCCCCAGTAGTCAAACGCCGTCCAACGTTTCGCACA GTAATATACGGCCGTGTCCTCGGCTCTCAGGCTGTTCATTTGCAGATACAGCGTGTT CTTGGAATTGTCTCTGGAGATGGTGAACCGGCCCTTCACGGAGTCTGCGTAAATTGT CGGACTACCACCCCCAGCAATCGATGAGACCCACTCCAGCCCCTTCCCTGGAGCCT GGCGGACCCAGCTCATGGCATAGCTGCTAAAGGTGAATCCAGAGGCTGCACAGGAG AGTCTCANGGACCCCCCAGGCTGTACCAAGCCTCCCCCAGACTCCAACAGCTGCAC CTCGGCCATGGCCGGCTGGGCCGCGAGTAATAACAATCCAGCGGCTGCCGTANGCA ATAGGTATTTCATTATGACTGTCTCCTTGAAATAGANTTTGCATGCAAGCTTGGCGT AANTCATGGNCATAGCTGTTTCCTGTGTGAAATTGTTATCCNCTCACAANTTCCNCN CAANCATACGAANCCCGGAANGC M34F AA sequence: (SEQ ID NO: 18) MXMXYAKLACKXYFKETVIMKYLLXTAAAGLLLLAAQPAMAEVQLLESGGGL VQPGGSXRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIAGGGSPTIYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKRWTAFDYWGQGTLVTVSSGGGGSGGGGS GGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYRASNL QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHPSPITFGQGTKVEIKRAAAHHHH HHGAAEQKLXSEED Clone M34G: (SEQ ID NO: 19) CTATGCGNNNNATTCAGATCCTCTTCTGAGATGAGTTTTTGTTCTGCGGCCCC GTGATGGTGATGATGATGTGCGGCCGCCCGTTTGATTTCCACCTTGGTCCCTTGGCC GAACGTAGGAGGCGAAGTCTGAACCTGTTGACAGTAGTAAGTTGCAAAATCTTCAG GTTGCAGACTGCTGATGGTGAGAGTGAAATCTGTCCCAGATCCACTGCCACTGAAC CTTGATGGGACCCCACTTTGCAACAGGGATGCACGATAGATCAGGAGCTTAGGGGC TTTCCCTGGTTTCTGCTGATACCAATTTAAATAGCTGCTAATGCTCTGGCTTGCCCGG CAAGTGATGGTGACTCTGTCTCCTACAGATGCAGACAGGGAGGATGGAGACTGGGT CATCTGGATGTCCGTCGACCCGCCACCGCCGCTGCCACCTCCGCCTGAACCGCCTCC ACCGCTCGAGACGGTGACCAGGGTTCCCTGGCCCCAGTAGTCAAACTGCTTACCAC GTTTCGCACAGTAATATACGGCCGTGTCCTCGGCTCTCAGGCTGTTCATTTGCAGAT ACAGCGTGTTCTTGGAATTGTCTCTGGAGATGGTGAACCGGCCCTTCACGGAGTCTG CGTAATGTGTCACAGTACCATCCGGCCAAATACCTGAGACCCACTCCAGCCCCTTCC CTGGAGCCTGGCGGACCCAGCTCATGGCATAGCTGCTAAAGGTGAATCCAGAGGCT GCACAGGAGAGTCTCAGGGACCCCCCAGGCTGTACCAAGCCTCCCCCAGACTCCAA CAGCTGCACCTCGGCCATGGCCGGCTGGGCCGCGAGTAATAACAATCCAGCGGCTG CCGTANGCAATAGGTATTTCATTATGACTGTCTCCTTGAAATAGAATTTGCATGCAA GCTTGGCGTANTCATGGTCATAGCTGTTTCCTGTGNGAAATTGTTATCCGCTCACNN TTCCACNCAACATACGANCCGG M34G AA sequence: (SEQ ID NO: 20) MKYLLXTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSS YAMSWVRQAPGKGLEWVSGIWPDGTVTHYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCAKRGKQFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSA SVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYRASLLQSGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQVQTSPPTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLNX XHR Clone M35A: (SEQ ID NO: 21) CTANGCGNNNNNNTCAGATCCTCTTCTGAGATGAGTTTTTGTTCTGCGGCCCC GTGATGGTGATGATGATGTGCGGCCGCCCGTTTGATTTCCACCTTGGTCCCTTGGCC GAACGTAGAAGGATTATCATCATTCTGTTGACAGTAGTAAGTTGCAAAATCTTCAG GTTGCAGACTGCTGATGGTGAGAGTGAAATCTGTCCCAGATCCACTGCCACTGAAC CTTGATGGGACCCCACTTTGCAAAGTGGATGCATCATAAATCAGGAGCTTAGGGGC TTTCCCTGGTTTCTGCTGATACCAATTTAAATAGCTGCTAATGCTCTGACTTGCCCGG CAAGTGATGGTGACTCTGTCTCCTACAGATGCAGACAGGGAGGATGGAGACTGGGT CATCTGGATGTCCGTCGACCCGCCACCGCCGCTGCCACCTCCGCCTGAACCGCCTCC ACCGCTCGAGACGGTGACCAGGGTTCCCTGGCCCCAGTAGTCAAAACCATTAGAAG TTTTCGCACAGTAATATACGGCCGTGTCCTCGGCTCTCAGGCTGTTCATTTGCAGAT ACAGCGTGTTCTTGGAATTGTCTCTGGAGATGGTGAACCGGCCCTTCACGGAGTCTG

CGTAATATGTAGTACTACCAGTAGCATCAATAGTTGAGACCCACTCCAGCCCCTTCC CTGGAGCCTGGCGGACCCAGCTCATGGCATAGCTGCTAAAGGTGAATCCAGAGGCT GCACAGGAGAGTCTCAGGGACCCCCCAGGCTGTACCAAGCCTCCCCCAGACTCCAA CAGCTGCACCTCGGCCATGGCCGGCTGGGCCGCGAGTAATAACAATCCAGCGGCTG CCGTNNNAATANGTATTTCATTATGACTGTCTCCTTGAAATAGAATTTGCATGCAAG CTNGGNNTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAAT TCCACACAACATACG M35A AA sequence: (SEQ ID NO: 22) MQILFQGDSHNEIXIXTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSC AASGFTFSSYAMSWVRQAPGKGLEWVSTIDATGSTTYYADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCAKTSNGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMT QSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASTLQSGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQNDDNPSTFGQGTKVEIKRAAAHHHHHHGAAEQK LISEEDL Clone M35F: (SEQ ID NO: 23) TTCAGATCCTCTTCTGAGANGAGTTTTTGTTCTGCGGCCCCGTGATGGTGATG ANGATGTGCGGCCGCCCGTTTGATTTCCACCTTGGTCCCTTGGCCGAACGTAGTAGG ACTAGCATAACTCTGTTGACAGTAGTAAGTTGCAAAATCTTCAGGTTGCAGACCGCT GATGGTGAGAGTGAAATCTGTCCCAGATCCACTGCCACTGAACCTTGATGGGACCC CACTTTGCAAAGAGGATGCACCATAGATCAGGAGCTTAGGGGCTTTCCCTGGTTTCT GCTGATACCAATTTAAATAGCTGCTAATGCTCTGACTTGCCCGGCAAGTGATGGTGA CTCTGTCTCCTACAGATGCAGACAGGGAGGATGGAGACTGGGTCATCTGGATGTCC GTCGACCCGCCACCGCCGCTGCCACCTCCGCCTGAACCGCCTCCACCGCTCGAGAC GGTGACCAGGGTTCCCTGGCCCCAGTAGTCAAAAGCAGTAGCAGTTTTCGCACAGT AATATACGGCCGTGTCCTCGGCTCTCAGGCTGTTCATTTGCAGATACAGCGTGTTCT TGGAATTGTCTCTGGAGATGGTGAACCGGCCCTTCACGGAGTCTGCGTAACTTGTAG CATCACCATTAGAATAAATAGATGAGACCCACTCCAGCCCCTTCCCTGGAGCCTGG CGGACCCAGCTCATGGCATAGCTGCTAAAGGTGAATCCAGAGGCTGCACAGGAGAG TCTCAGGGACCCCCCAGGCTGTACCAAGCCTCCCCCAGACTCCAACAGCTGCACCTC GGCCATGGCCGGCTGGGCCGCGAGTAATAACAATCCAGCGGCTGCCGTNNCAATAG GTATTTCATTATGACTGTCTCCTTGAAATANAATTTGCATGCAAGCTTGGNGTAATC ATGGNCATAGCTGTTTCCTGNGTGAAATTGTTATCCGCTCACNATTCCNCACNACAT A M35F AA sequence: (SEQ ID NO: 24) MQIXFQGDSHNEIPIXTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSC AASGFTFSSYAMSWVRQAPGKGLEWVSSIYSNGDATSYADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCAKTATAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQM TQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGS GSGTDFTLTISGLQPEDFATYYCQQSYASPTTFGQGTKVEIKRAAAHXHHHHGAAEQKL XSEEDLK Clone M35G: (SEQ ID NO: 25) CTATGCGNCCCCATTCAGATCCTCTTCTGAGANGAGTTTTTGTTCTGCGGCCC CGTGATGGTGATGATGATGTGCGGCCGCCCGTTTGATTTCCACCTTGGTCCCTTGGC CGAACGTAGGAGGCGAAGTCTGAACCTGTTGACAGTAGTAAGTTGCAAAATCTTCA GGTTGCAGACTGCTGATGGTGAGAGTGAAATCTGTCCCAGATCCACTGCCACTGAA CCTTGATGGGACCCCACTTTGCAACAGGGATGCACGATAGATCAGGAGCTTAGGGG CTTTCCCTGGTTTCTGCTGATACCAATTTAAATAGCTGCTAATGCTCTGGCTTGCCCG GCAAGTGATGGTGACTCTGTCTCCTACAGATGCAGACAGGGAGGATGGAGACTGGG TCATCTGGATGTCCGTCGACCCGCCACCGCCGCTGCCACCTCCGCCTGAACCGCCTC CACCGCTCGAGACGGTGACCAGGGTTCCCTGGCCCCAGTAGTCAAACTGCTTACCA CGTTTCGCACAGTAATATACGGCCGTGTCCTCGGCTCTCAGGCTGTTCATTTGCAGA TACAGCGTGTTCTTGGAATTGTCTCTGGAGATGGTGAACCGGCCCTTCACGGAGTCT GCGTAATGTGTCACAGTACCATCCGGCCAAATACCTGAGACCCACTCCAGCCCCTTC CCTGGAGCCTGGCGGACCCAGCTCATGGCATAGCTGCTAAAGGTGAATCCAGAGGC TGCACAGGAGAGTCTCAGGGACCCCCCAGGCTGTACCAAGCCTCCCCCAGACTCCA ACAGCTGCACCTCGGCCATGGCCGGCTGGGCCGCGAGTAATAACAATCCAGCGGCT GCCGTANGCAATAGGTATTTCATTATGACTGTCTCCTTGAAATAGAATTTGCATGCA AGCTTGGCGTAANCATGGTCATAGCTGTTTCCTGTGNGAAATTGTTATCCNGCTCAC AATTCCNNCACAA M35G AA sequence: (SEQ ID NO: 26) MKYLLXTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSS YAMSWVRQAPGKGLEWVSGIWPDGTVTHYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCAKRGKQFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSA SVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYRASLLQSGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQVQTSPPTFGQGTKVEIKRAAAHHHHHHGAAEQKLXSEEDLNG XA Clone M58A: (SEQ ID NO: 27) TATGCGNNNATTCNGATCCTCTTCTGAGANGAGTTTTTGTTCTGCGGCCCCGT GATGGTGATGATGNNNTGCGGCCGCCCGTTTGATTTCCACCTTGGTCCCTTGGCCGA ACGTATTAGGACAATCAGTAGTCTGTTGACAGTAGTAAGTTGCAAAATCTTCAGGTT GCAGACTGCTGATGGTGAGAGTGAAATCTGTCCCAGATCCACTGCCACTGAACCTT GATGGGACCCCACTTTGCAAAGTGGATGCATTATAGATCAGGAGCTTAGGGGCTTT CCCTGGTTTCTGCTGATACCAATTTAAATAGCTGCTAATGCTCTGACTTGCCCGGCA AGTGATGGTGACTCTGTCTCCTACAGATGCAGACAGGGAGGATGGAGACTGGGTCA TCTGGATGTCCGTCGACCCGCCACCGCCGCTGCCACCTCCGCCTGAACCGCCTCCAC CGCTCGAGACGGTGACCAGGGTTCCCTGGCCCCAGTAGTCAAAATTAGCACCAGAT TTCGCACAGTAATATACGGCCGTGTCCTCGGCTCTCAGGCTGTTCATTTGCAGATAC AGCGTGTTCTTGGAATTGTCTCTGGAGATGGTGAACCTGCCCTTCACGGAGTCTGCG TAAGATGTAGCATAACCACTAGCAGTAATACCTGAGACCCACTCCAGCCCCTTCCCT GGAGCCTGGCGGACCCAGCTCATGGCATAGCTGCTAAAGGTGAATCCAGAGGCTGC ACAGGAGAGTCTCANGGACCCCCCAGGCTGTACCAAGCCTCCCCCAGACTCCAACA GCTGCACCTCGGCCATGGCCGGCTGGGCCGCGAGTAATAACAATCCAGCGGCTGCC GTANGCAATANGTATTTCATTATGACTGTCTCCTTGAAATAGAANTTTGCATGCAAG CTTGGNNTAATCATGGNNATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAAT TNCNCAC M58A AA sequence: (SEQ ID NO: 28) MIXPSLHAXFYFKETVIMKYXLXTAAAGLLLLAAQPAMAEVQLLESGGGLVQP GGSXRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGITASGYATSYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCAKSGANFDYWGQGTLVTVSSGGGGSGGGGSGG GGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYNASTLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTTDCPNTFGQGTKVEIKRAAAXHHH HHGAAEQKLXSEEDXNXRI Clone M58C: (SEQ ID NO: 29) GCGGCNNNTTCNGANCCTCTTCTGAGANGAGTTTTTGTTCTGCGGCCCCGTG NNGGTGATGNNNNNGTGCGGCCGCCCGTTTGATTTCCACCTTGGTCCCTTGGCCGAA CGTATTAGGGGTACTGTAACTCTGTTGACAGTAGTAAGTTGCAAAATCTTCAGGTTG CAGACTGCTGATGGTGAGAGTGAAATCTGTCCCAGATCCACTGCCACTGAACCTTG ATGGGACCCCACTTTGCAAACTGGATGCAGCATAGATCAGGAGCTTAGGGGCTTTC CCTGGTTTCTGCTGATACCAATTTAAATAGCTGCTAATGCTCTGACTTGCCCGGCAA GTGATGGTGACTCTGTCTCCTACAGATGCAGACAGGGAGGATGGAGACTGGGTCAT CTGGATGTCCGTCGACCCGCCACCGCCGCTGCCACCTCCGCCTGAACCGCCTCCACC GCTCGAGACGGTGACCAGGGTTCCCTGGCCCCAGTAGTCAAACGCCGGATGATATT TCGCACAGTAATATACGGCCGTGTCCTCGGCTCTCAGGCTGTTCATTTGCAGATACA GCGTGTTCTTGGAATTGTCTCTGGAGATGGTGAACCGGCCCTTCACGGNGTCTGCGT ACTCTGTCGGCAGACCCTGCGGCGCAATCGATGAGACCCACTCCAGCCCCTTCCCTG GAGCCTGGCGGACCCAGCTCATGGCATAGCTGCTAAAGGTGAATCCAGAGGCTGCA CAGGAGAGTCTCAGGGACCCCCCAGGCTGTACCAAGCCTCCCCCAGACTCCAACAG CTGCACCTCGGCCATGGCCGGCTGGGCCGCGAGTAATAACAATCCAGCGGCNGNCG TANNCAATAGGTATTTCATTATGACTGTCTCCTTGAAATANNATTTGCATGCAAGCT TGGNGTANTCATGGNCATAGCTGTTTNCTGNGTGNAAATTGTTATCCGCTCNNNAAT TTCCAC M58C AA sequence: (SEQ ID NO: 30) MKYLLXTXAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSS YAMSWVRQAPGKGLEWVSSIAPQGLPTEYADXVKGRFTISRDNSKNTLYLQMNSLRAE DTAVYYCAKYHPAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSAS VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQSYSTPNTFGQGTKVEIKRAAAXXHHXHGAAEQKLXSEED Clone M59F: (SEQ ID NO: 31) GCGNCCNNTTCAGATCCTCTTCTGAGATGAGTTTTTGTTCTGCGGCCCCGTGA TGGTGATGANNNNNTGCGGCCGCCCGTTTGATTTCCACCTTGGTCCCTTGGCCGAAC GTAGAAGGAGAATTACCAGTCTGTTGACAGTAGTAAGTTGCAAAATCTTCAGGTTG CAGACTGCTGATGGTGAGAGTGAAATCTGTCCCAGATCCACTGCCACTGAACCTTG ATGGGACCCCACTTTGCAAAGCGGATGCAGTATAGATCAGGAGCTTAGGGGCTTTC CCTGGTTTCTGCTGATACCAATTTAAATAGCTGCTAATGCTCTGACTTGCCCGGCAA GTGATGGTGACTCTGTCTCCTACAGATGCAGACAGGGAGGATGGAGACTGGGTCAT

CTGGATGTCCGTCGACCCGCCACCGCCGCTGCCACCTCCGCCTGAACCGCCTCCACC GCTCGAGACGGTGACCAGGGTTCCCTGGCCCCAGTAGTCAAAAGTACTATAAGATT TCGCACAGTAATATACGGCCGTGTCCTCGGCTCTCAGGCTGTTCATTTGCAGATACA GCGTGTTCTTGGAATTGTCTCTGGAGATGGTGAACCGGCCCTTCACGGAGTCTGCGT AAGCTGTACTAGCACTACTAGCAGCAATACCTGAGACCCACTCCAGCCCCTTCCCTG GAGCCTGGCGGANCCAGCTCATGGCATAGCTGCTAAAGGTGAATCCAGAGGCTGCA CAGGAGAGTCTCAGGGACCCCCCAGGCTGTACCAAGCCTCCCCCGGACTCCAACAG CTGCACCTCGGCCATGGCCGGCTGGGCCGCGAGTAATAACAATCCAGCGGCTGCCG TANGCAATANGTATTTCATTATGACTGTCTCCTTGAAATAGAATTTGCATGCAAGCT TGGCGTANTCATGGNCATAGCTGNTTCCTGTGTGAAATTGNTNATCCGCTCAC M59F AA sequence: (SEQ ID NO: 32) MKYXLXTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSS YAMSWXRQAPGKGLEWVSGIAASSASTAYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCAKSYSTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSAS VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYTASALQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQTGNSPSTFGQGTKVEIKRAAAXXHHHHGAAEQKLISEEDL Clone 4E1: (SEQ ID NO: 33) ATGGCCGAGGTGCAGCTGTCGGAGTCTGGGGGAGGCTTGGTACAGCCTGGG GGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATG AGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTAATAG TAATGGTACTTCTACATCTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAG AGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACA CGGCCGTATATTACTGTGCGAAATCTGCTTCTGATTTTGACTACTGGGGCCAGGGAA CCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGT GGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTA GGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAA TTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAATGCATCCA CTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTC ACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAG AATACTTATAGTCCTACTACGTTC 4E1 AA sequence: (SEQ ID NO: 34) MKYLLPTNAAGLLLLAANPAMAEVQLSESGGGLVQPGGSLRLSCAASGFTFSSY AMSWVRQAPGKGLEWVSGINSNGTSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKSASDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYNASTLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQNTYSPTTFGNNNKVEIKRAA Clone 4H3: (SEQ ID NO: 35) ATGGCCGAGATGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGG GGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATG AGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATATATTACTGC TAATGGTGATAGTACAACTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCA GAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGAC ACGGCCGTATATTACTGTGCGAAAAGTACTACTGATTTTGACTACTGGGGCCAGGG AACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCG GTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTG TAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTA AATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGTGCATC CAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATT TCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAAC AGACTTCTTATAGTCCTTCTACGTTCGGCCAAGGGNCCAAGGTGGAAATCAAACGG GCGGCC 4H3 AA Sequence: (SEQ ID NO: 36) MAEMQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSYI TANGDSTTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTTDFDYWGQ GTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNW YQQKPGKAPKLLIYSASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTSYSPS TFGQG?KVEIKRAAA Clone 1A5: (SEQ ID NO: 37) ATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGG GGNTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATG AGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTAATGC TAGTGGTGGTAGTACAGGTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCA GAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGAC ACGGCCGTATATTACTGTGCGAAAGCTGATGCTTATTTTGACTACTGGGGCCAGGGA ACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGG TGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGT AGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAA ATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCTGCATCCT CGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTC ACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAG GATGCTAGTGGTCCTTCTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGC GGCCGCA 1A5 AA Sequence: (SEQ ID NO: 38) MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTI NASGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKADAYFDYWGQ GTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNW YQQKPGKAPKLLIYSASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDASGPS TFGQGTKVEIKRAA Clone 3D3: (SEQ ID NO: 39) ATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGG GGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATG AGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATATATTGCTGA TGATGGTGCTAATACAGCTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCA GAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGAC ACGGCCGTATATTACTGTGCGAAAAATAATGATGGTTTTGACTACTGGGGCCAGGG AACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCG GTGGCGGGTCGACGAACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTG TAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTA AATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCTGCATC CACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATT TCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAAC AGGCTGCTACTAGTCCTTCTACGTTCGGCCAAGGGNCCAAGGTGGAAATCAAACGG GCGGNCGCAC 3D3 AA Sequence: (SEQ ID NO: 40) MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSYI ADDGANTAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNNDGFDYWG QGTLVTVSSGGGGSGGGGSGGGGSTNIQMTQSPSSLSASVGDRVTITCRASQSISSYLN WYQQKPGKAPKLLIYSASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAATS PSTFGQG?KVEIKRA?A

[0068] The following TBI Clone Sequences were identified:

TABLE-US-00002 Clone T2B: (SEQ ID NO: 41) TTCAAGGAGACAGTCATAATGAAATANCCTATTGCNTACGGCANNCGCTGGA TTGTTATTACTCGCGGCCCAGCCNGNCCATGGCCGAGGTGCAGCTGTTGGAGTCTGG GGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATT CACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGG AGTGGGTCTCAAATATTAGTTCTGATGGTGATTCTACAGCTTACGCAGACTCCGTGA AGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATG AACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGCTTCTAGTAA TTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTT CAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTC TCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAG TCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTA AGCTCCTGATCTATGCTGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTG GCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATT TTGCAACTTACTACTGTCAACAGTCTAATTCTGATCCTACTACGTTCGGCCAAGGGA CCAAGGTAATCAAACGGGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAA CAAAAACTCNTCTCAGAAGAGGATCTGAATGGNNCCGCATAGNC T2B AA sequence: (SEQ ID NO: 42) MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNI SSDGDSTAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKASSNFDYWGQ GTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNW YQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNSDP TTFGQGTKVIKRAAAHHHHHHGAAEQKLXSEEDLNXXA Clone T1H: (SEQ ID NO: 43) CAGGGGGGGCGGNGCCTATGNAAAAAACGCCAGCAACGCGGCCTTTTACGG TTCCTGGCCCTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTC TGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAAC GACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAA CCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCC GACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTA GGCACCCCAGGCTTTACACTTTATGCTCCCGGCTCGTATGTTGTGTGGAATTGTGAG CGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTGCAT GCAAATTCTATTTCAAGGAGACAGTCATAGCTAGCATGAAAAAGATTTGGCTGGCG CTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCC GGCCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTCAGGACGACG AAGTTCGTATCCTGATGGCTAACGGTGCTGACGTTAACGCTCATGACGAACAGGGT ACTACTCCGCTGCACCTGGCTGCTAAAGAAGGTCACCTGGAAATCGTTGAAGTTCTG CTGAAGTACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGA CATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCAC ATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTG AATGGCCGCNTA T1H AA sequence: (SEQ ID NO: 44) MLPARMLCGIVSGQFHTGNSYDHDYAKLACKFYFKETVIASMKKIWLALAGLV LAFSASADYKEAQPAMDLGKKLLEAARAGQDDEVRILMANGADVNAHDEQGTTPLHL AAKEGHLEIVEVLLKYGADVNAQDKFGKTAFDISIDNGNEDLAEILQAAAHHHHHHGA AEQKLISEEDLNGRX Clone T3F: (SEQ ID NO: 45) GAGCTATGAGANNNNNNCCACGCTTCCCCNAAGGGAGAAAGGCGGACAGGT ATCCCGGTAAGCNGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGNNTNCAGGG GGAAACGCCTGGTATCTTTATAGTCCTGTCGGNNTTTCGCCACCTCTGACTTGAGCG TCGATTTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAAC GCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTG CGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCG CTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGA GCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTG GCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGA GTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTCCCGGCTCGTATGTT GTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATT ACGCCAAGCTTGCATGCAAATTCTATTTCAAGGAGACAGTCATAGCTAGCATGAAA AAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGACTAC AAAGAGGCCCAGCCGGCCATGGTAGGAAGACCTGACGTTAACGCTCAGGACAAATT CGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAA TCCTGCAAGCGGCCGCACatCaTCATCACCATCACGGGGCCGCAGAACAAAAACTCN TCTCAGAAGAGGATCNGAANNNNNCGCNTAGA T3F AA sequence: (SEQ ID NO: 46) MFFPALSPDSVDNRITAFEADTARRSRTTERSESVSEEAEERPIRKPPLPARWPIHC SWHDRFPDWKAGSERNAINVSLTHAPQALHEMLPARMLCGIVSGQFHTGNSYDHDYA KLACKFYFKETVIASMKKIWLALAGLVLAFSASADYKEAQPAMVGRPDVNAQDKFGK TAFDISIDNGNEDLAEILQAAAHHHHHHGAAEQKLXSEED Clone T3G: (SEQ ID NO: 47) TCGTCAGGGGGGNCGGNAGCCTATGGAAAAAACGNNAGCAACGNGNCNTTT TTACGGNNNNTGGCCTTTTGCTGGCNTTTGCTCACATGTTCTTTCCTGCGTTNNCCCC TGATTCTGTGGATANCCGTATTACCGCCTTTNGAGTGAGCTGATACCGNTCGCCGCA GCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAAT ACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACA GGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTC ACTCATTAGGCACCCCAGGCTTTACACTTTATGCTCCCGGCTCGTATGTTGTGTGGA ATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAA GCTTGCATGCAAATTCTATTTCAAGGAGACAGTCATAGCTAGCATGAAAAAGATTT GGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAG GCCCAGCCGGCCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTCA GGACGACGAAGTTCGTATCCTGATGGCTAACGGTGCTGACGTTAACGCTTGGGACA TGACTGGTCATACTCCGCTGCACCTGGCTGCTCAGTTCGGTCACCTGGAAATCGTTG AAGTTCTGCTGAAGCACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACC GCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGC GGCCGCACatcatCATCACCATCACGGGGcCGCAGAACAAAAAcTCaTcTCAGAAGAGG ATNNGAANGNNNCCGCA T3G AA sequence: (SEQ ID NO: 48) MLPARMLCGIVSGQFHTGNSYDHDYAKLACKFYFKETVIASMKKIWLALAGLV LAFSASADYKEAQPAMDLGKKLLEAARAGQDDEVRILMANGADVNAWDMTGHTPLH LAAQFGHLEIVEVLLKHGADVNAQDKFGKTAFDISIDNGNEDLAEILQAAAHHHHHHG AAEQKLISEED Clone T1A: (SEQ ID NO: 49) TTTATAGTNCNTGTCGGGTTTCNCCACNTNTGACNTGAGCNTCGATNTTTNNN NTGCTCNNCAGGGGGGCGGAGCCTATGGAAAAACGNCAGCAACGCGNCNTTTNTN CGGTTNNTGNCNTTTTGCTGGCCTTTTGCTCACATGTTCTTTCNTGCGTTATCCCNTG ATTNTGTGGATANCCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCANNC GAACGACCGAGCGCAGNGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACG CAAACCGCCTCTCNCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTT TNNNGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTC ATTAGGCACCCCAGGCTTTACACTTTATGCTCCCGGCTCGTATGTTGTGTGGAATTG TGAGCGGATAACAATTTCACACAGGAAACAGNTATGACCATGATTACGCCAAGCTT GCATGCAAATTCTATTTCAAGGAGACAGTCATAGCTAGCATGAAAAAGATTTGGCT GGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGANTACAAAGAGGCCC AGCCGGCCATGGGCGGAACCAGCAGTTTNTTACCCAGGTCCATGGACCTGGGTCAC CTGGAAATCGTTGAAGTTCTGCTGAAGTACGGTGNTGACGTTAACGNTCAGGACAA ATTCGGTAAGACCGCTTTCGACATNTCCATCGACAACGGTAACGAGGACCTGGCTG AAATCCTGCAAGCGGCCGCACATCATCATCACCATCATCGGGCTCGCAGAACAAAA ATCATCTC T1A AA sequence: (SEQ ID NO: 50) MFFXALSXDXVDXRITAFEADTARRXRTTERXESVSEEAEERPIRKPPLXARWPI HCSWHDRFXDWKAGSERNAINVSLTHAPQALHFMLPARMLCGIVSGQFHTGNXYDHD YAKLACKFYFKETVIASMKKIWLALAGLVLAFSASAXYKEAQPAMGGTSSXLPRSMDL GHLEIVEVLLKYGXDVNXQDKFGKTAFDXSIDNGNEDLAEILQAAAHHHHHHRARRT KII Clone T1D: (SEQ ID NO: 51) GAGCCTATGGAAAAAACGCCCAGCAACGCGGCNTTTTTACGGTTCCTGGCCT TTTGCTNGNCNTTTTGNTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATA ACCGTATTACCGCCTTTGAGTGNNNNGATACCGCTCGCCGCAGCCGAACGACCGAG CGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTC TCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGA AAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCC CAGGCTTTACACTTTATGCTCCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAA CAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTGCATGCAAATT CTATTTCAAGGAGACAGTCATAGCTAGCATGAAAAAGATTTGGCTGGCGCTGGCTG

GTTTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCCGGCCATG GACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTCAGGACGACGAAGTTCG TATCCTGATGGCTAACGGTGCTGACGTTAACGCTGACGACTTCTCTGGTACTACTCC GCTGCACCTGGCTGCTCATCATGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGTA CGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCAT CGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCANNNNNNCAT CACCATCACGGGGCCGCAGAACAAAAACTCNNNCAGAAGAGGATNNGAANNNNCG CATA T1D AA sequence: (SEQ ID NO: 52) MFFPALSPDSVDNRITAFEXXDTARRSRTTERSESVSEEAEERPIRKPPLPARWPI HCSWHDRFPDWKAGSERNAINVSLTHAPQALHFMLPARMLCGIVSGQFHTGNSYDHD YAKLACKFYFKETVIASMKKIWLALAGLVLAFSASADYKEAQPAMDLGKKLLEAARA GQDDEVRILMANGADVNADDFSGTTPLHLAAHHGHLEIVEVLLKYGADVNAQDKFGK TAFDISIDNGNEDLAEILQAAAXXHHHHGAAEQKLX Clone T2C: (SEQ ID NO: 53) GANGNTCNNCAGGGGGGGCGGAGCCTATNGAAAAAACGCCAGCAACGCGG CNTTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTT ATCCCCTGATTCTGTGGATANCCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCG CCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGC CCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCAC GACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTA GCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTCCCGGCTCGTATGTTGTGT GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGC CAAGCTTGCATGCAAATTCTATTTCAAGGAGACAGTCATAGCTAGCATGAAAAAGA TTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAG AGGCCCAGCCGGCCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGT CAGGACGACGAAGTTCGTATCCTGATGGCTAACGGTGCTGACGTTAACGCTCTGGA CGAAGTTGGTTCTACTCCGCTGCACCTGGCTGCTATGGCTGGTCACCTGGAAATCGT TGAAGTGCTGAAGCACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCG CTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCG GCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGA GGATCTGAATGNNNCGCNTAG T2C AA sequence: (SEQ ID NO: 54) MLPARMLCGIVSGQFHTGNSYDHDYAKLACKFYFKETVIASMKKIWLALAGLV LAFSASADYKEAQPAMDLGKKLLEAARAGQDDEVRILMANGADVNALDEVGSTPLHL AAMAGHLEIVEVLKHGADVNAQDKFGKTAFDISIDNGNEDLAEILQAAAHHHHHHGA AEQKLISEED Clone T2F: (SEQ ID NO: 55) GGACAGGNTATCCGGTAAAGCGGCAGGGTCGGANCANNAGAGCGCACGAG GGAGCTTNNCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGNTTTCGCCCAC CTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGGCGGAGCCTATGGAA AAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCA CATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAG TGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGA GGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTC ATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAAC GCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTC CCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAG CTATGACCATGATTACGCCAAGCTTGCATGCAAATTCTATTTCAAGGAGACAGTCAT AGCTAGCATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGC ATCGGCGGACTACAAAGAGGCCCAGCCGGCCATGGACCTGGCTGCTCATGTTGGTC ACCTGGAAATCGTTGAAGTTCTGCTGAAGTACGGTGCTGACGTTAACGCTCAGGAC AAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGC TGAAATCCTGCAAGCGGCcgCACatCaTCATCACCATCACGGGGCCGCAGAACAAAAA CTCaTCTCAGAAGAGGATCTGANNNNNCGCNTAG T2F AA sequence: (SEQ ID NO: 56) MFFPALSPDSVDNRITAFEADTARRSRTTERSESVSEEAEERPIRKPPLPARWPIHC SWHDRFPDWKAGSERNAINVSLTHAPQALHFMLPARMLCGIVSGQFHTGNSYDHDYA KLACKFYFKETVIASMKKIWLALAGLVLAFSASADYKEAQPAMDLAAHVGHLEIVEVL LKYGADVNAQDKFGKTAFDISIDNGNEDLAEILQAAAHHHHHHGAAEQKLISEEDL

[0069] A "variant" of an amino acid sequence described herein, or a nucleic acid sequence encoding such an amino acid sequence, is a sequence that is substantially similar to SEQ ID NO:1-56. Variant amino acid and nucleic acid sequences include synthetically derived amino acid and nucleic acid sequences, or recombinantly derived amino acid or nucleic acid sequences. Generally, amino acid or nucleic acid sequence variants of the invention will have at least 40, 50, 60, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequence identity to SEQ ID NO: 1-56. The present invention includes variants of the amino acid sequences of the antibodies and antibody fragments described herein, as well as variants of the nucleic acid sequences encoding such amino acid sequences (i.e., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 or SEQ ID NO:56).

[0070] "Variants" are intended to include sequences derived by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end, and/or addition of one or more bases to the 5' or 3' end of the nucleic acid sequence; deletion or addition of one or more amino acids/nucleic acids at one or more sites in the sequence; or substitution of one or more amino acids/nucleic acids at one or more sites in the sequence. The amino acids described herein may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. The substitution may be a conserved substitution. A "conserved substitution" is a substitution of an amino acid with another amino acid having a similar side chain. A conserved substitution would be a substitution with an amino acid that makes the smallest change possible in the charge of the amino acid or size of the side chain of the amino acid (alternatively, in the size, charge or kind of chemical group within the side chain) such that the overall protein retains its spatial conformation but does not alter its biological activity. For example, common conserved changes might be Asp to Glu, Asn or Gln; His to Lys, Arg or Phe; Asn to Gln, Asp or Glu and Ser to Cys, Thr or Gly. Alanine is commonly used to substitute for other amino acids. The 20 essential amino acids can be grouped as follows: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and methionine having nonpolar side chains; glycine, serine, threonine, cystine, tyrosine, asparagine and glutamine having uncharged polar side chains; aspartate and glutamate having acidic side chains; and lysine, arginine, and histidine having basic side chains.

[0071] As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences makes reference to a specified percentage of residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).

[0072] Nucleic Acids and Vectors

[0073] In certain embodiments, the present invention provides a nucleic acid encoding the amino acids described herein.

[0074] In certain embodiments, the present invention provides a vector comprising the nucleic acid described herein.

[0075] In certain embodiments, the present invention provides a phage comprising the vector described herein.

[0076] As used herein, the term "nucleic acid" and "polynucleotide" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base that is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues.

[0077] A "nucleic acid fragment" is a portion of a given nucleic acid molecule. Deoxyribonucleic acid (DNA) in the majority of organisms is the genetic material while ribonucleic acid (RNA) is involved in the transfer of information contained within DNA into proteins. The term "nucleotide sequence" refers to a polymer of DNA or RNA which can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers.

[0078] The terms "nucleic acid," "nucleic acid molecule," "nucleic acid fragment," "nucleic acid sequence or segment," or "polynucleotide" may also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene, e.g., genomic DNA, and even synthetic DNA sequences. The term also includes sequences that include any of the known base analogs of DNA and RNA.

[0079] By "fragment" or "portion" is meant a full length or less than full length of the nucleotide sequence.

[0080] A "variant" of a molecule is a sequence that is substantially similar to the sequence of the native molecule. For nucleotide sequences, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis that encode the native protein, as well as those that encode a polypeptide having amino acid substitutions. Generally, nucleotide sequence variants of the invention will have in at least one embodiment 40%, 50%, 60%, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequence identity to the native (endogenous) nucleotide sequence.

[0081] As used herein, "sequence identity" or "identity" in the context of two nucleic acid sequences makes reference to a specified percentage of residues in the two sequences that are the same when aligned by sequence comparison algorithms or by visual inspection.

[0082] As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences, wherein the portion of the polynucleotide sequence may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

[0083] The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%; at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%; at least 90%, 91%, 92%, 93%, or 94%; or even at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters.

[0084] Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions (see below). Generally, stringent conditions are selected to be about 5.degree. C. lower than the thermal melting point (T.sub.m) for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1.degree. C. to about 20.degree. C., depending upon the desired degree of stringency as otherwise qualified herein. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.

[0085] As noted above, another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase "hybridizing preferentially to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. "Bind(s) substantially" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.

[0086] "Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. The T.sub.m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched nucleic acid. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the T.sub.m can be approximated from the equation of Meinkoth and Wahl: T.sub.m 81.5.degree. C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L. M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. T.sub.m is reduced by about 1.degree. C. for each 1% of mismatching; thus, T.sub.m, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the T.sub.m can be decreased 10.degree. C. Generally, stringent conditions are selected to be about 5.degree. C. lower than the thermal melting point (T.sub.m) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4.degree. C. lower than the thermal melting point (T.sub.m); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10.degree. C. lower than the thermal melting point (T.sub.m); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20.degree. C. lower than the thermal melting point (T.sub.m). Using the equation, hybridization and wash compositions, and desired T, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T of less than 45.degree. C. (aqueous solution) or 32.degree. C. (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. Generally, highly stringent hybridization and wash conditions are selected to be about 5.degree. C. lower than the thermal melting point (T.sub.m) for the specific sequence at a defined ionic strength and pH.

[0087] An example of highly stringent wash conditions is 0.15 M NaCl at 72.degree. C. for about 15 minutes. An example of stringent wash conditions is a 0.2.times.SSC wash at 65.degree. C. for 15 minutes. Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1.times.SSC at 45.degree. C. for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6.times.SSC at 40.degree. C. for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.5 M, more preferably about 0.01 to 1.0 M, Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30.degree. C. and at least about 60.degree. C. for long probes (e.g., >50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2.times. (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.

[0088] Very stringent conditions are selected to be equal to the T.sub.m for a particular probe. An example of stringent conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide, e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.1.times.SSC at 60 to 65.degree. C. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1M NaCl, 1% SDS (sodium dodecyl sulphate) at 37.degree. C., and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.5.times. to 1.times.SSC at 55 to 60.degree. C.

[0089] "Operably-linked" nucleic acids refers to the association of nucleic acid sequences on single nucleic acid fragment so that the function of one is affected by the other, e.g., an arrangement of elements wherein the components so described are configured so as to perform their usual function. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. Control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter and the coding sequence and the promoter can still be considered "operably linked" to the coding sequence.

[0090] The terms "isolated and/or purified" refer to in vitro isolation of a nucleic acid, e.g., a DNA or RNA molecule from its natural cellular environment, and from association with other components of the cell or test solution (e.g. RNA pool), such as nucleic acid or polypeptide, so that it can be sequenced, replicated, and/or expressed. For example, "isolated nucleic acid" may be a DNA molecule containing less than 31 sequential nucleotides that is transcribed into an RNAi molecule. Such an isolated RNAi molecule may, for example, form a hairpin structure with a duplex 21 base pairs in length that is complementary or hybridizes to a sequence in a gene of interest, and remains stably bound under stringent conditions (as defined by methods well known in the art, e.g., in Sambrook and Russell, 2001). Thus, the RNA or DNA is "isolated" in that it is free from at least one contaminating nucleic acid with which it is normally associated in the natural source of the RNA or DNA and is preferably substantially free of any other mammalian RNA or DNA. The phrase "free from at least one contaminating source nucleic acid with which it is normally associated" includes the case where the nucleic acid is reintroduced into the source or natural cell but is in a different chromosomal location or is otherwise flanked by nucleic acid sequences not normally found in the source cell, e.g., in a vector or plasmid.

[0091] Nucleic acid molecules having base substitutions (i.e., variants) are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the nucleic acid molecule.

[0092] "Operably-linked" refers to the association of nucleic acid sequences on single nucleic acid fragment so that the function of one of the sequences is affected by another. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation.

[0093] As used herein, the term "derived" or "directed to" with respect to a nucleotide molecule means that the molecule has complementary sequence identity to a particular molecule of interest.

[0094] In certain embodiments, the expression cassette further contains a promoter. In certain embodiments, the promoter is a regulatable promoter. In certain embodiments, the promoter is a constitutive promoter. In certain embodiments, the promoter is a PGK, CMV or RSV promoter.

[0095] The present invention provides a vector containing the expression cassette described above. Expression vectors include, but are not limited to, viruses, plasmids, and other vehicles for delivering heterologous genetic material to cells. Accordingly, the term "expression vector" as used herein refers to a vehicle for delivering heterologous genetic material to a cell. In particular, the expression vector is a recombinant adenoviral, adeno-associated virus, or lentivirus or retrovirus vector. In certain embodiments, the viral vector is an adenoviral, lentiviral, adeno-associated viral (AAV), poliovirus, HSV, or murine Maloney-based viral vector.

[0096] "Expression cassette" as used herein means a nucleic acid sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, which may include a promoter operably linked to the nucleotide sequence of interest that may be operably linked to termination signals. It also may include sequences required for proper translation of the nucleotide sequence. The coding region usually codes for a protein of interest. The expression cassette including the nucleotide sequence of interest may be chimeric. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of a regulatable promoter that initiates transcription only when the host cell is exposed to some particular stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development.

[0097] Binding Molecules

[0098] As used herein, the term "binding molecule" includes antibodies, which includes scFvs (also called a "nanobodies"), humanized, fully human or chimeric antibodies, single-chain antibodies, diabodies, and antigen-binding fragments of antibodies (e.g., Fab fragments).

[0099] In certain embodiments, the binding molecule does not contain the constant domain region of an antibody.

[0100] In certain embodiments, the binding molecule is less than 500 amino acids in length, such as between 200-450 amino acids in length, or less than 400 amino acids in length.

[0101] In certain embodiments, the binding molecule preferentially recognizes a particular stage of human AD Tau (e.g., AD Braak Stage I, compared to other AD stages).

[0102] In certain embodiments, the binding molecule preferentially recognizes tau associated with TBI.

[0103] In certain embodiments, the binding molecule binds to AD Tau. In certain embodiments, the binding molecule comprises an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. In certain embodiments, the binding molecule comprises an amino acid sequence encoded by a nucleic acid, wherein the nucleic acid has at least 80% identity to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, or 39.

[0104] In certain embodiments, the binding molecule binds to TBI Tau. In certain embodiments, the binding molecule comprises an amino acid sequence of SEQ ID NO: 42, 44, 46, or 48, 50, 52, 54, 56. In certain embodiments, the binding molecule comprises an amino acid sequence encoded by a nucleic acid, wherein the nucleic acid has at least 80% identity to SEQ ID NO:41, 43, 45, or 47, 49, 51, 53, 55.

[0105] Detection Reagents and Assays

[0106] For purposes of the diagnostic methods of the invention, the compositions or ligand of the invention (e.g., binding molecule such as an antibody or antibody fragment) may be conjugated to a detecting reagent that facilitates detection of the ligand. For example, example, the detecting reagent may be a direct label or an indirect label. The labels can be directly attached to or incorporated into the detection reagent by chemical or recombinant methods.

[0107] In one embodiment, a label is coupled to the ligand through a chemical linker. Linker domains are typically polypeptide sequences, such as poly gly sequences of between about 5 and 200 amino acids. In some embodiments, proline residues are incorporated into the linker to prevent the formation of significant secondary structural elements by the linker. In certain embodiments, linkers are flexible amino acid subsequences that are synthesized as part of a recombinant fusion protein comprising the RNA recognition domain. In one embodiment, the flexible linker is an amino acid subsequence that includes a proline, such as Gly(x)-Pro-Gly(x) where x is a number between about 3 and about 100. In other embodiments, a chemical linker is used to connect synthetically or recombinantly produced recognition and labeling domain subsequences. Such flexible linkers are known to persons of skill in the art. For example, poly(ethylene glycol) linkers are available from Shearwater Polymers, Inc. Huntsville, Ala. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.

[0108] The detectable labels can be used in the assays of the present invention to diagnose TBI, these labels are attached to the ligand of the invention, can be primary labels (where the label comprises an element that is detected directly or that produces a directly detectable element) or secondary labels (where the detected label binds to a primary label, e.g., as is common in immunological labeling). An introduction to labels, labeling procedures and detection of labels is found in Polak and Van Noorden (1997) Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, N.Y. and in Haugland (1996) Handbook of Fluorescent Probes and Research Chemicals, a combined handbook and catalogue Published by Molecular Probes, Inc., Eugene, Oreg. Patents that described the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.

[0109] Primary and secondary labels can include undetected elements as well as detected elements. Useful primary and secondary labels in the present invention can include spectral labels such as green fluorescent protein, fluorescent dyes (e.g., fluorescein and derivatives such as fluorescein isothiocyanate (FITC) and Oregon Green.TM., rhodamine and derivatives (e.g., Texas red, tetrarhodimine isothiocynate (TRITC), etc.), digoxigenin, biotin, phycoerythrin, AMCA, CyDyes.TM., and the like), radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S, .sup.14C, .sup.32P, .sup.33P, etc.), enzymes (e.g., horse radish peroxidase, alkaline phosphatase etc.), spectral calorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads. The label can be coupled directly or indirectly to a component of the detection assay (e.g., the detection reagent) according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.

[0110] Exemplary labels that can be used include those that use: 1) chemiluminescence (using horseradish peroxidase and/or alkaline phosphatase with substrates that produce photons as breakdown products as described above) with kits being available, e.g., from Molecular Probes, Amersham, Boehringer-Mannheim, and Life Technologies/Gibco BRL; 2) color production (using both horseradish peroxidase and/or alkaline phosphatase with substrates that produce a colored precipitate (kits available from Life Technologies/Gibco BRL, and Boehringer-Mannheim)); 3) fluorescence using, e.g., an enzyme such as alkaline phosphatase, together with the substrate AttoPhos (Amersham) or other substrates that produce fluorescent products, 4) fluorescence (e.g., using Cy-5 (Amersham), fluorescein, and other fluorescent tags); 5) radioactivity. Other methods for labeling and detection will be readily apparent to one skilled in the art.

[0111] Where the ligand-based compositions of the invention are contemplated to be used in a clinical setting, the labels are preferably non-radioactive and readily detected without the necessity of sophisticated instrumentation. In certain embodiments, detection of the labels will yield a visible signal that is immediately discernable upon visual inspection. One example of detectable secondary labeling strategies uses an antibody that recognizes oligomers in which the antibody is linked to an enzyme (typically by recombinant or covalent chemical bonding). The antibody is detected when the enzyme reacts with its substrate, producing a detectable product. In certain embodiments, enzymes that can be conjugated to detection reagents of the invention include, e.g., .beta.-galactosidase, luciferase, horse radish peroxidase, and alkaline phosphatase. The chemiluminescent substrate for luciferase is luciferin. One embodiment of a fluorescent substrate for .beta.-galactosidase is 4-methylumbelliferyl-.beta.-D-galactoside. Embodiments of alkaline phosphatase substrates include p-nitrophenyl phosphate (pNPP), which is detected with a spectrophotometer; 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) and fast red/napthol AS-TR phosphate, which are detected visually; and 4-methoxy-4-(3-phosphonophenyl) spiro[1,2-dioxetane-3,2'-adamantane], which is detected with a luminometer. Embodiments of horse radish peroxidase substrates include 2,2'azino-bis(3-ethylbenzthiazoline-6 sulfonic acid) (ABTS), 5-aminosalicylic acid (5AS), o-dianisidine, and o-phenylenediamine (OPD), which are detected with a spectrophotometer, and 3,3,5,5'-tetramethylbenzidine (TMB), 3,3' diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC), and 4-chloro-1-naphthol (4C1N), which are detected visually. Other suitable substrates are known to those skilled in the art. The enzyme-substrate reaction and product detection are performed according to standard procedures known to those skilled in the art and kits for performing enzyme immunoassays are available as described above.

[0112] The presence of a label can be detected by inspection, or a detector which monitors a particular probe or probe combination is used to detect the detection reagent label. Typical detectors include spectrophotometers, phototubes and photodiodes, microscopes, scintillation counters, cameras, film and the like, as well as combinations thereof. Examples of suitable detectors are widely available from a variety of commercial sources known to persons of skill. Commonly, an optical image of a substrate comprising bound labeling moieties is digitized for subsequent computer analysis.

[0113] The ligand compositions of the invention can be used in any diagnostic assay format to determine the presence of human CSF/brain-associated tau variants. A variety of immunodetection methods are contemplated for this embodiment. Such immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot, though several others are well known to those of ordinary skill. The steps of various useful immunodetection methods have been described in the scientific literature.

[0114] In general, the binding methods include obtaining a sample suspected of containing a protein, polypeptide and/or peptide (e.g., human CSF/brain-associated Tau variants), and contacting the sample with a first antibody, monoclonal or polyclonal, in accordance with the present invention, as the case may be, under conditions effective to allow the formation of complexes.

[0115] The binding methods include methods for detecting and quantifying the amount of the target oligomer component in a sample and the detection and quantification of any complexes formed during the binding process. Here, one would obtain a sample suspected of containing target oligomers, and contact the sample with an antibody fragment of the invention, and then detect and quantify the amount of complexes formed under the specific conditions.

[0116] Contacting the chosen biological sample with the antibody under effective conditions and for a period of time sufficient to allow the formation of complexes (primary complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form complexes with, i.e., to bind to, any antigens present. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or western blot, will generally be washed to remove any non-preferentially bound antibody species, allowing only those scFv molecules preferentially bound within the primary complexes to be detected.

[0117] In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological and enzymatic tags. U.S. patents concerning the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated herein by reference. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody and/or a biotin/avidin ligand binding arrangement, as is known in the art.

[0118] As noted above, a ligand of the invention may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary complexes in the composition to be determined. Alternatively, a first antibody that becomes bound within the primary complexes may be detected by means of a second binding ligand that has binding affinity for the complex. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a "secondary" ligand. The primary complexes are contacted with the labeled, secondary binding ligand or antibody under effective conditions and for a period of time sufficient to allow the formation of secondary complexes. The secondary complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary complexes is then detected.

[0119] Further methods include the detection of primary complexes by a two-step approach. A second binding ligand, such as an antibody, that has binding affinity for the scFv is used to form secondary complexes, as described above. After washing, the secondary complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of complexes (tertiary complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary complexes thus formed. This system may provide for signal amplification if this is desired.

[0120] One method of immunodetection designed by Charles Cantor uses two different antibodies. A first step biotinylated, monoclonal or polyclonal antibody or antibody fragment (in the present example a scFv) is used to detect the target antigen(s), and a second step antibody is then used to detect the biotin attached to the complex. In this method the sample to be tested is first incubated in a solution containing the first step ligand. If the target antigen is present, some of the ligand binds to the antigen to form a biotinylated ligand/antigen complex. The ligand/antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and/or complementary biotinylated DNA, with each step adding additional biotin sites to the ligand/antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin. This second step antibody is labeled, as for example with an enzyme that can be used to detect the presence of the antibody/antigen complex by histoenzymology using a chromogen substrate. With suitable amplification, a conjugate can be produced which is macroscopically visible.

[0121] Another known method of detection takes advantage of the immuno-PCR (Polymerase Chain Reaction) methodology. The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA/biotin/streptavidin/antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls. At least in theory, the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule.

[0122] As detailed above, the assays in their most simple and/or direct sense are binding assays. Certain preferred assays are the various types of enzyme linked immunosorbent assays (ELISAs) and/or radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and/or western blotting, dot blotting, FACS analyses, and/or the like may also be used.

[0123] The diagnostic assay format that may be used in the present invention could take any conventional format such as ELISA or other platforms such as luminex or biosensors. The present invention provides various ligands (e.g., scFv). These ligands can readily be modified to facilitate diagnostic assays, for example a tag (such as GFP) can be added to these ligands to increase sensitivity. In one exemplary ELISA, ligands (e.g., scFvs) are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing a target oligomer, such as a clinical sample (e.g., a biological sample obtained from the subject), is added to the wells. After binding and/or washing to remove non-specifically bound complexes, the bound antigen may be detected. Detection is generally achieved by the addition of an antibody that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA." Detection may also be achieved by the addition of a second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

[0124] In another exemplary ELISA, the samples suspected of containing the antigen are immobilized onto the well surface and/or then contacted with binding agents. After binding and/or washing to remove non-specifically bound complexes, the bound anti-binding agents are detected. Where the initial binding agents are linked to a detectable label, the complexes may be detected directly. Again, the complexes may be detected using a second antibody that has binding affinity for the first binding agents, with the second antibody being linked to a detectable label.

[0125] Another ELISA in which the antigens are immobilized, involves the use of antibody competition in the detection. In this ELISA, labeled antibodies (or nanobodies) against an antigen are added to the wells, allowed to bind, and/or detected by means of their label. The amount of an antigen in an unknown sample is then determined by mixing the sample with the labeled antibodies against the antigen during incubation with coated wells. The presence of an antigen in the sample acts to reduce the amount of antibody against the antigen available for binding to the well and thus reduces the ultimate signal. This is also appropriate for detecting antibodies against an antigen in an unknown sample, where the unlabeled antibodies bind to the antigen-coated wells and also reduces the amount of antigen available to bind the labeled antibodies.

[0126] Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non-specifically bound species, and detecting the bound immune complexes.

[0127] In coating a plate with either target oligomers or a ligand (e.g., antibody) of the invention, one will generally incubate the wells of the plate with a solution of the antigen or ligand, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein or solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

[0128] In ELISAs, it is probably more customary to use a secondary or tertiary detection means rather than a direct procedure. Thus, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or a third binding ligand.

[0129] "Under conditions effective to allow immune complex (antigen/antibody) formation" means that the conditions preferably include diluting the tau oligomers and/or scFv composition with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.

[0130] The "suitable" conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25.degree. C. to 27.degree. C., or may be overnight at about 4.degree. C. or so.

[0131] Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. An example of a washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.

[0132] To provide a detecting means, the second or third antibody will have an associated label to allow detection. This may be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact or incubate the first and second immune complex with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).

[0133] After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS), or H.sub.2O.sub.2, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generated, e.g., using a visible spectra spectrophotometer.

[0134] Diagnostic Methods

[0135] In certain embodiments, the present invention provides a method for determining risk of traumatic brain injury (TBI) and/or susceptibility to neurodegenerative disease in a subject by measuring levels of particular tau oligomeric proteins preferentially associated with different stages of AD or with TBI.

[0136] In certain embodiments, the present invention provides a method for determining risk of traumatic brain injury (TBI), assessment of the amount of neuronal damage, and/or susceptibility to neurodegenerative disease in a subject, comprising the steps of:

[0137] (A) providing a sample obtained from a subject post-injury;

[0138] (B) assessing levels of TBI-associated tau in the sample;

[0139] (C) comparing the TBI-associated tau protein level in the sample with TBI-associated tau protein level in a normal control; and

[0140] (D) determining whether the subject has a risk of TBI in accordance with the result of step (C);

[0141] wherein a subject having elevated TBI-associated tau protein has a high risk of TBI.

[0142] In certain embodiments, the sample and the normal control are blood product samples or cerebrospinal fluid (CSF) samples. In certain embodiments, the blood product is serum.

[0143] In certain embodiments, the detecting in step (B) is by means of a ligand specific for the protein.

[0144] In certain embodiments, the ligand is an antibody.

[0145] In certain embodiments, the ligand is a scFv specific for TBI-associated tau.

[0146] In certain embodiments, the protein levels are detected by means of ELISA.

[0147] The invention will now be illustrated by the following non-limiting Example.

Example 1

Isolation of Nanobodies Selective for Tau Species in TBI but Not ND CSF Samples

[0148] Morphology-specific nanobodies are used to identify the set of serum biomarkers that are diagnostic for AD, and determine if these also show up in a subset of TBI patients. Soldiers suffering TBI who show reactivity similar to the AD biomarker set, have suffered damage similar to that shown in AD brain, and should be much more susceptible to AD. The goal is to rapidly and accurately detect and quantify a selected set of toxic protein variants of A13, tau and a-syn that are characteristic of AD.

[0149] Nanobodies specific to selected tau species unique to TBI were isolated. Tau is present in human tissue in a variety of different forms since it is generated through multiple splicing events and can have a variety of different post-translational modifications. Because of the diversity of tau species, there are selected species that are indicative of particular neuronal conditions such as AD or other tauopathies. Nanobodies were generated that selectively recognize tau species present in TBI patients.

[0150] Immunoprecipitate Total Tau from Two Different Regions of Age Matched Post-Mortem Human AD and Cognitively Normal Brain Tissue

[0151] Brain Tissue and CSF: AD and normal brain tissue samples were obtained from Banner/Sun Health Brain Bank (BSHBB). Samples were obtained from two different brain regions of AD cases confirmed to have abundant tangles, the superior frontal gyrus and middle temporal gyrus. Ten different AD and 10 different control patient samples were received for a total of 20 AD and 20 control samples. All subjects had a PMI less than 5 hours. Detailed clinical and neuropathological data were available on these subjects, including MMSE, CERAD neuritic plaque density scores, Braak tangle stage and regional Lewy-typealpha-syncleinopathy density scores. Post-mortem samples were de-identified for all personal patient information. CSF from four different patients who had sustained head injury and control CSF from 15 normal/healthy individuals were obtained from Banner Sun Health. These samples were used to identify potential markers in traumatic brain injury compared to control.

[0152] Brain tissue lysis: Briefly, frozen samples were homogenized by supersonication in cold lysis buffer: 25 mM HEPESNaOH (pH 7.9), 150 mM NaCl, 1.5 mM MgCl.sub.2, 0.2 mM EDTA, 0.5% Triton-X-100, 1 mM dithiothreitol, proteaseinhibitor cocktail. The homogenized sample was centrifuged and the supernatant was frozen in -80.degree. C. The presence of fibrillar tau was verified by immunohistochemistry using slices from one AD and one ND sample and staining with an antibody against phosphorylated tau. The AD sample showed significantly more tau tangle pathology than the control sample (FIG. 1).

[0153] Immunoprecipitation of tau protein: Two polyclonal tau antibody preparations were used to immunoprecipitate tau from the AD brain homogenates, TBI CSF and corresponding controls: PA1 against amino-acids 240-450 of tau and PAS against amino-acid 1-286 to cover all isoforms. Antibody conjugates were captured using the Pierce Crosslink IP Kit A following the manufacturers' protocols. With the crosslink kit, the antibody first binds the protein A/G agarose and then is chemically crosslinked to the resin preventing the antibody from eluting off the column. Tau was eluted from the column using a low pH elution buffer. To preserve integrity of the tau aggregates, the elution buffer was neutralized with 1M Tris, pH 9.5 as recommended by the manufacturer. The brain slices (FIG. 1) were analyzed and tau eluted from TBI CSF using dot blots (FIG. 2), and tau eluted from the brain tissue by western blot (FIGS. 3a and 3b).

[0154] Biopanning--AD tau specific morphologies: Aliquots of the Sheets, Tomlinson I and J and DARPin scFv libraries were grown and combined to generate scFv library stock with titers of around 10e13. Negative panning steps were performed to remove phage clones that bind to non-target sticky protein samples including bovine serum album (FIG. 4). Several rounds of negative panning was performed against a-synuclein aggregates to remove all antibody fragments that bind generic forms of aggregated protein morphologies. Additional negative panning steps against monomeric tau, healthy tissue samples and healthy tau samples were performed to remove all antibody fragments that bind to generic forms of tau found in healthy individuals. Atomic force microscopy (AFM) imaging was performed after every negative panning step to ensure removal of non-specific antibody fragments. Final positive selection was performed using for AD braak stage III and V specific tau variant morphologies.

[0155] TBI tau specific morphologies: A clone that could bind to all forms of tau is essential and is used as a secondary detection reagent in sandwich ELISA. Hence, phage obtained after negative panning with a-synuclein (FIG. 5) was used for positive panning against monomeric tau. For panning against tau isolated from TBI, eight rounds of negative panning against control CSF samples (10 .mu.g/mL) were first performed. Cleaved mica surface was used to conserve sample. This step was used to remove all phage binding to proteins and other components present in normal CSF including any tau variants present in healthy individuals. Phage remaining after negative panning against BSA, .alpha.-synuclein and control CSF was used to carry out positive panning with tau immunoprecipitated from TBI CSF samples (FIGS. 6, 7). Positive binding clones were eluted using either Trypsin or TEA and recovered by infecting TG1 cells.

[0156] AD tau clones: After two rounds of positive panning with AD Braak stage III, unbound phage clones were used for a positive round of selection against AD Braak stage V and vice versa (FIG. 8). Each of the micas was eluted using trypin and TEA and grown on LB-Amp plates overnight at 37.degree. C. About 60 clones were obtained before negative panning with AD Braak stage III and V. Typically 50-100 clones are obtained in this step and the results obtained are encouraging. After further rounds of negative panning with AD Braak stage V and III, 15 and 20 clones were obtained against AD Braak stage III and Braak stage V respectively. The number of clones after the final rounds of positive panning was as anticipated. To ensure these clones are capable of making full length antibody fragment, their sequences were checked for any mutations and stop codons. Phage was produced for clone sequences free of any errors. Dotblots spotted with human AD homogenized brain tissue and corresponding controls were used to verify each of the phage clones.

[0157] TBI tau clone: Approximately 24 clones were recovered from the panning against tau variants present in the TBI CSF samples. Typically it is expected to recover around 20-50 clones in the positive panning step so this was an encouraging result. These clones were further sequenced to check for any stop codons or mutations. This step ensures that these clones are capable of making full length antibody fragments. Several clones isolated against TBI had complete sequences free of any stop codons, mutations or errors. Phage was produced for such clones which were verified using dotblots using TBI CSF and corresponding CSF controls. The blot was visualized using a chemiluminescent substrate and the blot was developed using film. The dotblots showed high binding to TBI and relatively lower binding to controls. Further characterization using ELISA assays were performed.

[0158] Indirect AD ELISA: Indirect ELISA was performed to check the specificity of each of the phage clones to tau variants in AD brain tissue. The assay parameters and wash steps were optimized to yield a high signal to noise ratio. Human brain homogenates (mix of individual samples classified by their Braak stage) was used to coat the plates and 2% milk was used subsequently to block the wells. Each of the phage clones were tested with pooled ND controls, AD Braak stage III and AD Braak stage V homogenates. Secondary anti-M13 and chemiluminescent substrate was used for detection. The luminescence was measured using a spectrophotometer and represented as a ratio with respect to ND control.

[0159] The clones were initially screened with pooled samples to check if they selected AD over control samples. From this initial assay it can be noted that several clones preferentially bound to tau morphologies in AD Braak stage III (51A, M58C and M59F) and there were a few clones (51F, 52H and M34G) that preferentially bound to tau forms present in AD Braak stage V (FIG. 9). This indicates that unique tau species exist during different AD Braak stages. These clones serve as a tool in tracking the progression of AD. Other clones that preferentially bound to both AD Braak stage III and V (M34F) indicate overlap of tau species common to Braak stages III and V. These clones serve as a potential secondary reagent for detection in a sandwich ELISA.

[0160] 51A and 51F clones had high binding ratios to AD Braak stage III and V respectively in the initial ELISA assay. These clones were further tested with individual AD brain tissue homogenates (FIGS. 10 through 12). They selectively bind to individual samples classified under Braak stage III and V respectively. Both these clones have relatively very low binding to ND control indicating that the negative panning steps against the ND controls was successful.

[0161] Indirect TBI ELISA: Indirect ELISA was performed to check the specificity of each of the phage clones to tau variants in TBI CSF. The assay parameters and wash steps were optimized to yield a high signal to noise ratio. Pooled TBI and control CSF samples were used to coat the plates and milk was used subsequently to block the wells. Each of the phage clones were tested with AD I, ADIII and ADV homogenates. Secondary anti-M13 and chemiluminescent substrate was used for detection. The luminescence was measured using a spectrophotometer and represented as a ratio with respect to no sample control.

[0162] Several clones preferentially bound to tau morphologies in TBI CSF over control (FIG. 13). This indicates that unique tau species circulate in the CSF of traumatic brain injured individuals compared to controls. These clones can serve as a tool in differentiating TBI and healthy individuals.

[0163] Clones that gave a high binding ratio to pooled TBI CSF samples were further tested with individual TBI and control CSF samples. As can be seen from FIGS. 14-15, each of the TBI clones were able to selectively pick out all four of the TBI CSF samples over pooled control CSF sample. Almost all the clones had high binding ratio to one of the TBI samples. This could possibly be due to the presence of high levels of TBI specific tau morphologies in this sample which were recognized by the individual clones. Each clone's binding ratio to the individual TBI samples are different indicating that each clone might not necessarily be binding to the same type of tau species. These clones can serve as a tool in recognizing specific tau forms in TBI.

[0164] All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.

[0165] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0166] Embodiments of this invention are described herein. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein.

[0167] Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Sequence CWU 1

1

571912DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(7)..(7)a, c, t, g, unknown or othermodified_base(28)..(28)a, c, t, g, unknown or othermodified_base(41)..(41)a, c, t, g, unknown or othermodified_base(59)..(59)a, c, t, g, unknown or othermodified_base(68)..(68)a, c, t, g, unknown or othermodified_base(71)..(71)a, c, t, g, unknown or other 1gattacngcc aagcttgcat gcaaattnta tttcaaggag ncagtcataa tgaaatacnt 60attgcctncg ncagccgctg gattgttatt actcgcggcc cagccggcca tggccgaggt 120gcagctgttg gagtctgggg gaggcttggt acagcctggg gggtccctga gactctcctg 180tgcagcctct ggattcacct ttagcagcta tgccatgagc tgggtccgcc aggctccagg 240gaaggggctg gagtgggtct catctatttc ttctaatggt gatgatacag cttacgcaga 300ctccgtgaag ggccggttca ccatctccag agacaattcc aaggacacgc tgtatctgca 360aatgaacagc ctgagagccg aggacacggc cgtatattac tgtgcgaaag ctaataattc 420ttttgactac tggggccagg gaaccctggt caccgtctcg agcggtggag gcggttcagg 480cggaggtggc agcggcggtg gcgggtcgac ggacatccag atgacccagt ctccatcctc 540cctgtctgca tctgtaggag acagagtcac catcacttgc cgggcaagtc agagcattag 600cagctattta aattggtatc agcagaaacc agggaaagcc cctaagctcc tgatctataa 660tgcatccact ttgcaaagtg gggtcccatc aaggttcagt ggcagtggat ctgggacaga 720tttcactctc accatcagca gtctgcaacc tgaagatttt gcaacttact actgtcaaca 780ggatagtgct actccttata cgttcggcca agggaccaag gtggaaatca aacgggcggc 840cgcacatcat catcaccatc acggggccgc agaacaaaaa ctcatctcag aagaggatct 900gaatggggcc gc 9122287PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(4)..(4)Any amino acidMOD_RES(7)..(8)Any amino acid 2Met Lys Tyr Xaa Leu Pro Xaa Xaa Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Ser Asn Gly Asp Asp Thr 65 70 75 80 Ala Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asp Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Lys Ala Asn Asn Ser Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Ile Gln Met Thr Gln 145 150 155 160 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 165 170 175 Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln 180 185 190 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asn Ala Ser Thr Leu 195 200 205 Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 210 215 220 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 225 230 235 240 Tyr Cys Gln Gln Asp Ser Ala Thr Pro Tyr Thr Phe Gly Gln Gly Thr 245 250 255 Lys Val Glu Ile Lys Arg Ala Ala Ala His His His His His His Gly 260 265 270 Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala 275 280 285 3 927DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(6)..(6)a, c, t, g, unknown or othermodified_base(25)..(26)a, c, t, g, unknown or othermodified_base(34)..(35)a, c, t, g, unknown or othermodified_base(37)..(37)a, c, t, g, unknown or othermodified_base(76)..(76)a, c, t, g, unknown or othermodified_base(81)..(81)a, c, t, g, unknown or othermodified_base(90)..(91)a, c, t, g, unknown or othermodified_base(112)..(112)a, c, t, g, unknown or othermodified_base(141)..(141)a, c, t, g, unknown or othermodified_base(220)..(220)a, c, t, g, unknown or other 3tatganccat gattacgcca agctnncatg caanntntat tttcaaggag acagtcataa 60tgaaatacct attgcntacg ncagccgctn ngattgttat tactcgcggc cncagccggc 120catggccgag gtgcagctgt nggagtctgg gggaggcttg gtacagcctg gggggtccct 180gagactctcc tgtgcagcct ctggattcac ctttagcagn tatgccatga gctgggtccg 240ccaggctcca gggaaggggc tggagtgggt ctcatagatt tagcagtcgg gtccggttac 300atcttacgca gactccgtga agggccggtt caccatctcc agagacaatt ccaagaacac 360gctgtatctg caaatgaaca gcctgagagc cgaggacacg gccgtatatt actgtgcgaa 420acgtcagttg atgtttgact actggggcca gggaaccctg gtcaccgtct cgagcggtgg 480aggcggttca ggcggaggtg gcagcggcgg tggcgggtcg acggacatcc agatgaccca 540gtctccatcc tccctgtctg catctgtagg agacagagtc accatcactt gccgggcaag 600tcagagcatt agcagctatt taaattggta tcagcagaaa ccagggaaag cccctaagct 660cctgatctat gctgcatcca gtttgcaaag tggggtccca tcaaggttca gtggcagtgg 720atctgggaca gatttcactc tcaccatcag cagtctgcaa cctgaagatt ttgcaactta 780ctactgtcaa cagagttaca gtacccctaa tacgttcggc caagggacca aggtggaaat 840caaacgggcg gccgcacatc atcatcacca tcacggggcc gcagaacaaa aactcatctc 900agaagaggat ctgaatgggc cgcatag 9274266PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(7)..(7)Any amino acidMOD_RES(33)..(33)Any amino acid 4Met Ala Glu Val Gln Leu Xaa Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Xaa Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Ile Gln Ser Gly Pro Val Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Gln Leu Met Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Thr Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 130 135 140 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser 145 150 155 160 Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 165 170 175 Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser 180 185 190 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 195 200 205 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr 210 215 220 Ser Thr Pro Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 225 230 235 240 Ala Ala Ala His His His His His His Gly Ala Ala Glu Gln Lys Leu 245 250 255 Ile Ser Glu Glu Asp Leu Asn Gly Pro His 260 265 5 833DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(4)..(4)a, c, t, g, unknown or othermodified_base(14)..(15)a, c, t, g, unknown or othermodified_base(28)..(28)a, c, t, g, unknown or othermodified_base(34)..(34)a, c, t, g, unknown or othermodified_base(37)..(37)a, c, t, g, unknown or othermodified_base(67)..(67)a, c, t, g, unknown or othermodified_base(71)..(77)a, c, t, g, unknown or othermodified_base(101)..(102)a, c, t, g, unknown or othermodified_base(177)..(177)a, c, t, g, unknown or othermodified_base(189)..(189)a, c, t, g, unknown or othermodified_base(221)..(222)a, c, t, g, unknown or othermodified_base(253)..(258)a, c, t, g, unknown or othermodified_base(261)..(261)a, c, t, g, unknown or othermodified_base(745)..(745)a, c, t, g, unknown or othermodified_base(771)..(771)a, c, t, g, unknown or othermodified_base(794)..(794)a, c, t, g, unknown or othermodified_base(800)..(800)a, c, t, g, unknown or othermodified_base(810)..(810)a, c, t, g, unknown or othermodified_base(814)..(816)a, c, t, g, unknown or other 5attncgccaa gctnncatgc aaaatttnta tttnaangga gacagtcata atgaaatacc 60tattgcntac nnnnnnncgc tggattgtta ttactcgcgg nncagccggc catggccgag 120gtgcagctgt tggagtctgg gggaggcttg gtacagcctg gggggtccct gagactntcc 180tgtgcagcnt ctggattcac ctttagcagc tatgccatga nntgggtccg ccaggctcca 240gggaaggggc tgnnnnnngt ntcatctatt acgtagacgg gttcgtagac acagtacgca 300gactccgtga agggcaggtt caccatctcc agagacaatt ccaagaacac gctgtatctg 360caaatgaaca gcctgagagc cgaggacacg gccgtatatt actgtgcgaa acagcatgat 420gattttgact actggggcca gggaaccctg gtcaccgtct cgagcggtgg aggcggttca 480ggcggaggtg gcagcggcgg tggcgggtcg acggacatcc agatgaccca gtctccatcc 540tccctgtctg catctgtagg agacagagtc accatcactt gccgggcaag tcagagcatt 600agcagctatt taaattggta tcagcagaaa ccagggaaag cccctaagct cctgatctat 660actgcatcca atttgcaaag tggggtccca tcaaggttca gtggcagtgg atctgggaca 720gatttcactc tcaccatcag cagtntgcaa cctgaagatt ttgcaactta ntactgtcaa 780cagctggatg tgtntccttn gacgttcggn caannnacca aggtggaaat caa 8336238PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(22)..(22)Any amino acidMOD_RES(26)..(26)Any amino acidMOD_RES(37)..(37)Any amino acidMOD_RES(48)..(50)Any amino acidMOD_RES(210)..(210)Any amino acidMOD_RES(218)..(218)Any amino acidMOD_RES(226)..(226)Any amino acidMOD_RES(228)..(228)Any amino acidMOD_RES(231)..(231)Any amino acidMOD_RES(233)..(233)Any amino acid 6Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Xaa Ser Cys Ala Xaa Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Ala Met Xaa Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Xaa 35 40 45 Xaa Xaa Ser Ser Ile Thr Thr Gly Ser Thr Gln Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gln His Asp Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Thr Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 130 135 140 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser 145 150 155 160 Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 165 170 175 Lys Leu Leu Ile Tyr Thr Ala Ser Asn Leu Gln Ser Gly Val Pro Ser 180 185 190 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 195 200 205 Ser Xaa Gln Pro Glu Asp Phe Ala Thr Xaa Tyr Cys Gln Gln Leu Asp 210 215 220 Val Xaa Pro Xaa Thr Phe Xaa Gln Xaa Thr Lys Val Glu Ile 225 230 235 7963DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(29)..(29)a, c, t, g, unknown or othermodified_base(660)..(660)a, c, t, g, unknown or othermodified_base(690)..(690)a, c, t, g, unknown or othermodified_base(763)..(763)a, c, t, g, unknown or othermodified_base(853)..(853)a, c, t, g, unknown or othermodified_base(861)..(862)a, c, t, g, unknown or othermodified_base(872)..(874)a, c, t, g, unknown or othermodified_base(876)..(877)a, c, t, g, unknown or othermodified_base(898)..(898)a, c, t, g, unknown or othermodified_base(912)..(912)a, c, t, g, unknown or othermodified_base(914)..(916)a, c, t, g, unknown or othermodified_base(954)..(954)a, c, t, g, unknown or othermodified_base(957)..(958)a, c, t, g, unknown or other 7gagacagtca tagctagcat gaaaaagant tggctggcgc tggctggttt agttttagcg 60tttagcgcat cggcggacta caaagaggcc cagccggcca tggacctggg taagaaactg 120ctggaagctg ctcgtgctgg tcaggacgac gaagttcgta tcctgatggc taacggtgct 180gacgttaacg ctgacgacta cgaaggttgg actccgctgc acctggctgc tatggttggt 240cacctggaaa tcgttgaagt tctgctgaag tacggtgctg acgttaacgc tcaggacaaa 300ttcggtaaga ccgctttcga catctccatc gacaacggta acgaggacct ggctgaaatc 360ctgcaagcgg ccgcacatca tcatcaccat cacggggccg cagaacaaaa actcatctca 420gaagaggatc tgaatggggc cgcatagact gttgaaagtt gtttagcaaa acctcataca 480gaaaattcat ttactaacgt ctggaaagac gacaaaactt tagatcgtta cgctaactat 540gagggctgtc tgtggaatgc tacaggcgtt gtggtttgta ctggtgacga aactcagtgt 600tacggtacat gggttcctat tgggcttgct atccctgaaa atgagggtgg tggctctgan 660ggtggcggtt ctgagggtgg cggttctgan ggtggcggta ctaaacctcc tgagtacggt 720gatacaccta ttccgggcta tacttatatc aaccctctcg acngcactta tccgcctggt 780actgagcaaa accccgctaa tcctaatccc ttctcttgag gagtctcagc ctcttaatac 840tttcatgttt canaataata nnttccgaaa tnnncnnggt gcattaactg tttatacngg 900cactgttact cnannnactg acccccgttt aaaacttatt accagtacac tccntgnnat 960cat 9638142PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(4)..(4)Any amino acid 8Met Lys Lys Xaa Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser 1 5 10 15 Ala Ser Ala Asp Tyr Lys Glu Ala Gln Pro Ala Met Asp Leu Gly Lys 20 25 30 Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile 35 40 45 Leu Met Ala Asn Gly Ala Asp Val Asn Ala Asp Asp Tyr Glu Gly Trp 50 55 60 Thr Pro Leu His Leu Ala Ala Met Val Gly His Leu Glu Ile Val Glu 65 70 75 80 Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly 85 90 95 Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala 100 105 110 Glu Ile Leu Gln Ala Ala Ala His His His His His His Gly Ala Ala 115 120 125 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala Ala 130 135 140 9890DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(10)..(10)a, c, t, g, unknown or othermodified_base(15)..(15)a, c, t, g, unknown or othermodified_base(67)..(67)a, c, t, g, unknown or othermodified_base(778)..(778)a, c, t, g, unknown or othermodified_base(799)..(799)a, c, t, g, unknown or othermodified_base(814)..(814)a, c, t, g, unknown or othermodified_base(822)..(823)a, c, t, g, unknown or othermodified_base(843)..(844)a, c, t, g, unknown or othermodified_base(866)..(867)a, c, t, g, unknown or other 9ttcaggagan agtcntaatg aaatacctat tgcctacggc agccgctgga ttgttattac 60tcgcggncca gccggccatg gccgaggtgc agctgttgga gtctggggga ggcttggtac 120agcctggggg gtccctgaga ctctcctgtg cagcctctgg attcaccttt agcagctatg 180ccatgagctg ggtccgccag gctccaggga aggggctgga gtgggtctca ggtatttcta 240ataatggtag taatacaact tacgcagact ccgtgaaggg ccggttcacc atctccagag 300acaattccaa gaacacgctg tatctgcaaa tgaacagcct gagagccgag gacacggccg 360tatattactg tgcgaaagct tcttatactt ttgactactg gggccaggga accctggtca 420ccgtctcgag cggtggaggc ggttcaggcg gaggtggcag cggcggtggc gggtcgacgg 480acatccagat gacccagtct ccatcctccc tgtctgcatc tgtaggagac agagtcacca 540tcacttgccg ggcaagtcag agcattagca gctatttaaa ttggtatcag cagaaaccag 600ggaaagcccc taagctcctg atctatagtg catcctcttt gcaaagtggg gtcccatcaa 660ggttcagtgg cagtggatct gggacagatt tcactctcac catcagcagt ctgcaacctg 720aagattttgc aacttactac tgtcaacagt attctggttc tcctgctacg ttcggccnag 780ggaccaaggt

ggaaatcana cgggcggccg cacntcatca tnnccatcac ggggccgcag 840aannaaaact catctcagaa gagganntga atggggccgc atagactgtt 89010283PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(17)..(17)Any amino acidMOD_RES(254)..(254)Any amino acidMOD_RES(261)..(261)Any amino acidMOD_RES(266)..(266)Any amino acidMOD_RES(269)..(269)Any amino acidMOD_RES(276)..(276)Any amino acidMOD_RES(283)..(283)Any amino acid 10Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Xaa Gln Pro Ala Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ser Gly Ile Ser Asn Asn Gly Ser Asn Thr 65 70 75 80 Thr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Lys Ala Ser Tyr Thr Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Ile Gln Met Thr Gln 145 150 155 160 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 165 170 175 Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln 180 185 190 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser Leu 195 200 205 Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 210 215 220 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 225 230 235 240 Tyr Cys Gln Gln Tyr Ser Gly Ser Pro Ala Thr Phe Gly Xaa Gly Thr 245 250 255 Lys Val Glu Ile Xaa Arg Ala Ala Ala Xaa His His Xaa His His Gly 260 265 270 Ala Ala Glu Xaa Lys Leu Ile Ser Glu Glu Xaa 275 280 11883DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(8)..(8)a, c, t, g, unknown or othermodified_base(10)..(10)a, c, t, g, unknown or othermodified_base(23)..(23)a, c, t, g, unknown or othermodified_base(51)..(51)a, c, t, g, unknown or othermodified_base(53)..(54)a, c, t, g, unknown or othermodified_base(86)..(86)a, c, t, g, unknown or othermodified_base(154)..(154)a, c, t, g, unknown or othermodified_base(172)..(172)a, c, t, g, unknown or othermodified_base(267)..(267)a, c, t, g, unknown or othermodified_base(434)..(434)a, c, t, g, unknown or othermodified_base(447)..(447)a, c, t, g, unknown or othermodified_base(529)..(529)a, c, t, g, unknown or othermodified_base(546)..(546)a, c, t, g, unknown or othermodified_base(555)..(556)a, c, t, g, unknown or othermodified_base(581)..(581)a, c, t, g, unknown or othermodified_base(584)..(584)a, c, t, g, unknown or othermodified_base(620)..(620)a, c, t, g, unknown or othermodified_base(639)..(639)a, c, t, g, unknown or othermodified_base(645)..(645)a, c, t, g, unknown or othermodified_base(672)..(672)a, c, t, g, unknown or othermodified_base(700)..(700)a, c, t, g, unknown or othermodified_base(734)..(734)a, c, t, g, unknown or othermodified_base(754)..(755)a, c, t, g, unknown or othermodified_base(764)..(764)a, c, t, g, unknown or othermodified_base(766)..(766)a, c, t, g, unknown or othermodified_base(785)..(785)a, c, t, g, unknown or othermodified_base(787)..(788)a, c, t, g, unknown or othermodified_base(797)..(797)a, c, t, g, unknown or othermodified_base(799)..(799)a, c, t, g, unknown or othermodified_base(802)..(802)a, c, t, g, unknown or othermodified_base(815)..(815)a, c, t, g, unknown or othermodified_base(817)..(817)a, c, t, g, unknown or othermodified_base(819)..(823)a, c, t, g, unknown or othermodified_base(826)..(826)a, c, t, g, unknown or othermodified_base(828)..(830)a, c, t, g, unknown or othermodified_base(837)..(837)a, c, t, g, unknown or othermodified_base(839)..(839)a, c, t, g, unknown or othermodified_base(854)..(855)a, c, t, g, unknown or othermodified_base(860)..(860)a, c, t, g, unknown or othermodified_base(868)..(868)a, c, t, g, unknown or othermodified_base(872)..(874)a, c, t, g, unknown or othermodified_base(876)..(876)a, c, t, g, unknown or othermodified_base(880)..(880)a, c, t, g, unknown or other 11ttcagganan agtcataatg aantacctat tgcctacggc agccgctgga ntnntattac 60tcgcggccca gccggccatg gcccangtgc agctggtgga gtctggggga ggcgtggtcc 120agcctgggag gtccctgaga ctctcctgtg cagnctccgg attcaccttt ancagctatg 180acatgggctg ggtccgccag gctccaggga aggggctgga gtgggtctca agtattagtg 240gtagtggtcc taccatgaac tacgcanact ctgtgaaggg ccgattcacc gtctccagag 300acaattccaa gaacacgctg tatctgcaaa tggacagcct gagagccgag gacacggccg 360tatattactg tgcgaaaggg ggtacggact ttgactactg gggccagggc accctggtca 420ccgtctcctc aggnggaggc ggttcangcg gaggtggctc tggcggtggc ggatcgtctg 480agctgactca ggaccctgct gtgtctgtgg ccttgggaca gacagtcanc atcacatgcc 540aagganacag cctcnnaacc tattatgcaa gctggtacca ncanaagcca ggacaggccc 600ctgtacttgt catctatggn aaaaacaacc ggccctcang gatcncagac cgattctctg 660gctccagctc angaaacaca gcttccttga ccatcactgn ggctcaggcg gaagatgagg 720ctgactatta ctgnaactcc cgggacagca gtgnnaacca tctnangagt gttcggcgga 780gggancnngc tgaccgncnt angtgcggcc gcagnancnn nnnctncnnn tcagaanang 840atctgaatgg ggcnncatan actgttgnaa annngnttan caa 88312255PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(2)..(2)Any amino acidMOD_RES(12)..(13)Any amino acidMOD_RES(23)..(23)Any amino acidMOD_RES(46)..(46)Any amino acidMOD_RES(52)..(52)Any amino acidMOD_RES(84)..(84)Any amino acidMOD_RES(139)..(139)Any amino acidMOD_RES(144)..(144)Any amino acidMOD_RES(171)..(171)Any amino acidMOD_RES(177)..(177)Any amino acidMOD_RES(180)..(180)Any amino acidMOD_RES(188)..(189)Any amino acidMOD_RES(201)..(201)Any amino acidMOD_RES(208)..(208)Any amino acidMOD_RES(210)..(210)Any amino acidMOD_RES(219)..(219)Any amino acidMOD_RES(228)..(228)Any amino acidMOD_RES(239)..(239)Any amino acidMOD_RES(246)..(246)Any amino acidMOD_RES(249)..(250)Any amino acid 12Met Xaa Tyr Leu Leu Pro Thr Ala Ala Ala Gly Xaa Xaa Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala Xaa Val Gln Leu Val Glu Ser Gly Gly Gly 20 25 30 Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Xaa Ser Gly 35 40 45 Phe Thr Phe Xaa Ser Tyr Asp Met Gly Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Pro Thr Met 65 70 75 80 Asn Tyr Ala Xaa Ser Val Lys Gly Arg Phe Thr Val Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asp Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Lys Gly Gly Thr Asp Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Xaa Gly Gly Gly Ser Xaa 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Glu Leu Thr Gln Asp Pro 145 150 155 160 Ala Val Ser Val Ala Leu Gly Gln Thr Val Xaa Ile Thr Cys Gln Gly 165 170 175 Xaa Ser Leu Xaa Thr Tyr Tyr Ala Ser Trp Tyr Xaa Xaa Lys Pro Gly 180 185 190 Gln Ala Pro Val Leu Val Ile Tyr Xaa Lys Asn Asn Arg Pro Ser Xaa 195 200 205 Ile Xaa Asp Arg Phe Ser Gly Ser Ser Ser Xaa Asn Thr Ala Ser Leu 210 215 220 Thr Ile Thr Xaa Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Xaa Asn 225 230 235 240 Ser Arg Asp Ser Ser Xaa Asn His Xaa Xaa Ser Val Arg Arg Arg 245 250 255 13854DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(10)..(10)a, c, t, g, unknown or othermodified_base(15)..(15)a, c, t, g, unknown or othermodified_base(651)..(651)a, c, t, g, unknown or othermodified_base(751)..(752)a, c, t, g, unknown or othermodified_base(760)..(760)a, c, t, g, unknown or othermodified_base(766)..(767)a, c, t, g, unknown or othermodified_base(776)..(776)a, c, t, g, unknown or othermodified_base(778)..(778)a, c, t, g, unknown or othermodified_base(780)..(780)a, c, t, g, unknown or othermodified_base(787)..(789)a, c, t, g, unknown or othermodified_base(791)..(794)a, c, t, g, unknown or othermodified_base(801)..(802)a, c, t, g, unknown or othermodified_base(816)..(817)a, c, t, g, unknown or othermodified_base(821)..(821)a, c, t, g, unknown or othermodified_base(824)..(828)a, c, t, g, unknown or othermodified_base(831)..(831)a, c, t, g, unknown or othermodified_base(833)..(833)a, c, t, g, unknown or othermodified_base(836)..(839)a, c, t, g, unknown or other 13ttcaggagan agtcntaatg aaatacctat tgcctacggc agccgctgga ttgttattac 60tcgcggccca gccggccatg gccgaggtgc agctgttgga gtctggggga ggcttggtac 120agcctggggg gtccctgaga ctctcctgtg cagcctctgg attcaccttt agcagctatg 180ccatgagctg ggtccgccag gctccaggga aggggctgga gtgggtctca gctattacta 240atgatggtgc tggtacaact tacgcagact ccgtgaaggg ccggttcacc atctccagag 300acaattccaa gaacacgctg tatctgcaaa tgaacagcct gagagccgag gacacggccg 360tatattactg tgcgaaatct tatactggtt ttgactactg gggccaggga accctggtca 420ccgtctcgag cggtggaggc ggttcaggcg gaggtggcag cggcggtggc gggtcgacgg 480acatccagat gacccaatct ccatcctccc tgtctgcatc tgtaggagac agagtcacca 540tcacttgccg ggcaagtcag agcattagca gctatttaaa ttggtatcag cagaaaccag 600ggaaagcccc taagctcctg atctatactg catccacttt gcaaagtggg ntcccattaa 660ggttcagtgg cagtggatct gggacagatt tcactctcac catcagcagt ctgcaacctg 720aagattttgc aacttactac tgtcaacaga nntatgctan tcctannacg ttcggncnan 780gggaccnnng nnnnaaatca nncgggcggc cgcacnncat natnnnnnat ncncgnnnnc 840gcagaacaaa actc 85414252PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(212)..(212)Any amino acidMOD_RES(245)..(245)Any amino acidMOD_RES(248)..(248)Any amino acidMOD_RES(250)..(250)Any amino acid 14Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ser Ala Ile Thr Asn Asp Gly Ala Gly Thr 65 70 75 80 Thr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Lys Ser Tyr Thr Gly Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Ile Gln Met Thr Gln 145 150 155 160 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 165 170 175 Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln 180 185 190 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Thr Ala Ser Thr Leu 195 200 205 Gln Ser Gly Xaa Pro Leu Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 210 215 220 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 225 230 235 240 Tyr Cys Gln Gln Xaa Tyr Ala Xaa Pro Xaa Thr Phe 245 250 15972DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(4)..(4)a, c, t, g, unknown or othermodified_base(8)..(8)a, c, t, g, unknown or othermodified_base(11)..(12)a, c, t, g, unknown or othermodified_base(15)..(15)a, c, t, g, unknown or othermodified_base(32)..(32)a, c, t, g, unknown or othermodified_base(749)..(749)a, c, t, g, unknown or othermodified_base(850)..(850)a, c, t, g, unknown or othermodified_base(857)..(857)a, c, t, g, unknown or othermodified_base(908)..(909)a, c, t, g, unknown or othermodified_base(912)..(913)a, c, t, g, unknown or othermodified_base(918)..(918)a, c, t, g, unknown or othermodified_base(930)..(930)a, c, t, g, unknown or othermodified_base(942)..(942)a, c, t, g, unknown or othermodified_base(944)..(944)a, c, t, g, unknown or othermodified_base(948)..(948)a, c, t, g, unknown or othermodified_base(950)..(951)a, c, t, g, unknown or othermodified_base(954)..(954)a, c, t, g, unknown or othermodified_base(962)..(962)a, c, t, g, unknown or othermodified_base(968)..(968)a, c, t, g, unknown or other 15ctangcgncc nnttnagatc ctcttctgag angagttttt gttctgcggc cccgtgatgg 60tgatgatgat gtgcggccgc ccgtttgatt tccaccttgg tcccttggcc gaacgtcgca 120ggagtctgat gagtctgttg acagtagtaa gttgcaaaat cttcaggttg cagactgctg 180atggtgagag tgaaatctgt cccagatcca ctgccactga accttgatgg gaccccactt 240tgcaactggg atgccggata gatcaggagc ttaggggctt tccctggttt ctgctgatac 300caatttaaat agctgctaat gctctgactt gcccggcaag tgatggtgac tctgtctcct 360acagatgcag acagggagga tggagactgg gtcatctgga tgtccgtcga cccgccaccg 420ccgctgccac ctccgcctga accgcctcca ccgctcgaga cggtgaccag ggttccctgg 480ccccagtagt caaaagacca aaactgtttc gcacagtaat atacggccgt gtcctcggct 540ctcaggctgt tcatttgcag atacagcgtg ttcttggaat tgtctctgga gatggtgaac 600cggcccttca cggagtctgc gtacgttgtc ggcggaccct gcttcgcaat atctgagacc 660cactccagcc ccttccctgg agcctggcgg acccagctca tggcatagct gctaaaggtg 720aatccagagg ctgcacagga gagtctcang gaccccccag gctgtaccaa gcctccccca 780gactccaaca gctgcacctc ggccatggcc ggctgggccg cgagtaataa caatccagcg 840gctgccgtan gcaatangta tttcattatg actgtctcct tgaaatagaa tttgcatgca 900agcttggnnt annatggnca tagctgtttn ctgtgtgaaa tngntatncn ntcncaattc 960cncacaanat ac 97216283PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(4)..(4)Any amino acidMOD_RES(6)..(6)Any amino acidMOD_RES(40)..(40)Any amino acidMOD_RES(279)..(279)Any amino acid 16Met Lys Tyr Xaa Leu Xaa Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Xaa Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ser Asp Ile Ala Lys Gln Gly Pro Pro Thr 65 70 75 80 Thr Tyr Ala Asp

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Lys Gln Phe Trp Ser Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Ile Gln Met Thr Gln 145 150 155 160 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 165 170 175 Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln 180 185 190 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Pro Ala Ser Gln Leu 195 200 205 Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 210 215 220 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 225 230 235 240 Tyr Cys Gln Gln Thr His Gln Thr Pro Ala Thr Phe Gly Gln Gly Thr 245 250 255 Lys Val Glu Ile Lys Arg Ala Ala Ala His His His His His His Gly 260 265 270 Ala Ala Glu Gln Lys Leu Xaa Ser Glu Glu Asp 275 280 17980DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(6)..(6)a, c, t, g, unknown or othermodified_base(22)..(22)a, c, t, g, unknown or othermodified_base(739)..(739)a, c, t, g, unknown or othermodified_base(840)..(840)a, c, t, g, unknown or othermodified_base(880)..(880)a, c, t, g, unknown or othermodified_base(903)..(903)a, c, t, g, unknown or othermodified_base(910)..(910)a, c, t, g, unknown or othermodified_base(942)..(942)a, c, t, g, unknown or othermodified_base(950)..(950)a, c, t, g, unknown or othermodified_base(955)..(955)a, c, t, g, unknown or othermodified_base(957)..(957)a, c, t, g, unknown or othermodified_base(961)..(961)a, c, t, g, unknown or othermodified_base(970)..(970)a, c, t, g, unknown or othermodified_base(978)..(978)a, c, t, g, unknown or other 17cattcngatc ctcttctgag angagttttt gttctgcggc cccgtgatgg tgatgatgat 60gtgcggccgc ccgtttgatt tccaccttgg tcccttggcc gaacgtaata ggagacggat 120gcgactgttg acagtagtaa gttgcaaaat cttcaggttg cagactgctg atggtgagag 180tgaaatctgt cccagatcca ctgccactga accttgatgg gaccccactt tgcaaattgg 240atgccctata gatcaggagc ttaggggctt tccctggttt ctgctgatac caatttaaat 300agctgctaat gctctgactt gcccggcaag tgatggtgac tctgtctcct acagatgcag 360acagggagga tggagactgg gtcatctgga tgtccgtcga cccgccaccg ccgctgccac 420ctccgcctga accgcctcca ccgctcgaga cggtgaccag ggttccctgg ccccagtagt 480caaacgccgt ccaacgtttc gcacagtaat atacggccgt gtcctcggct ctcaggctgt 540tcatttgcag atacagcgtg ttcttggaat tgtctctgga gatggtgaac cggcccttca 600cggagtctgc gtaaattgtc ggactaccac ccccagcaat cgatgagacc cactccagcc 660ccttccctgg agcctggcgg acccagctca tggcatagct gctaaaggtg aatccagagg 720ctgcacagga gagtctcang gaccccccag gctgtaccaa gcctccccca gactccaaca 780gctgcacctc ggccatggcc ggctgggccg cgagtaataa caatccagcg gctgccgtan 840gcaataggta tttcattatg actgtctcct tgaaatagan tttgcatgca agcttggcgt 900aantcatggn catagctgtt tcctgtgtga aattgttatc cnctcacaan ttccncncaa 960ncatacgaan cccggaangc 98018302PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(2)..(2)Any amino acidMOD_RES(4)..(4)Any amino acidMOD_RES(12)..(12)Any amino acidMOD_RES(25)..(25)Any amino acidMOD_RES(59)..(59)Any amino acidMOD_RES(298)..(298)Any amino acid 18Met Xaa Met Xaa Tyr Ala Lys Leu Ala Cys Lys Xaa Tyr Phe Lys Glu 1 5 10 15 Thr Val Ile Met Lys Tyr Leu Leu Xaa Thr Ala Ala Ala Gly Leu Leu 20 25 30 Leu Leu Ala Ala Gln Pro Ala Met Ala Glu Val Gln Leu Leu Glu Ser 35 40 45 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Xaa Arg Leu Ser Cys Ala 50 55 60 Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln 65 70 75 80 Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ala Gly Gly Gly 85 90 95 Ser Pro Thr Ile Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 100 105 110 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 115 120 125 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Arg Trp Thr Ala Phe 130 135 140 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 145 150 155 160 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Ile Gln 165 170 175 Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val 180 185 190 Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp 195 200 205 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Arg Ala 210 215 220 Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 225 230 235 240 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 245 250 255 Ala Thr Tyr Tyr Cys Gln Gln Ser His Pro Ser Pro Ile Thr Phe Gly 260 265 270 Gln Gly Thr Lys Val Glu Ile Lys Arg Ala Ala Ala His His His His 275 280 285 His His Gly Ala Ala Glu Gln Lys Leu Xaa Ser Glu Glu Asp 290 295 300 19980DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(8)..(11)a, c, t, g, unknown or othermodified_base(850)..(850)a, c, t, g, unknown or othermodified_base(912)..(912)a, c, t, g, unknown or othermodified_base(937)..(937)a, c, t, g, unknown or othermodified_base(957)..(958)a, c, t, g, unknown or othermodified_base(965)..(965)a, c, t, g, unknown or othermodified_base(976)..(976)a, c, t, g, unknown or other 19ctatgcgnnn nattcagatc ctcttctgag atgagttttt gttctgcggc cccgtgatgg 60tgatgatgat gtgcggccgc ccgtttgatt tccaccttgg tcccttggcc gaacgtagga 120ggcgaagtct gaacctgttg acagtagtaa gttgcaaaat cttcaggttg cagactgctg 180atggtgagag tgaaatctgt cccagatcca ctgccactga accttgatgg gaccccactt 240tgcaacaggg atgcacgata gatcaggagc ttaggggctt tccctggttt ctgctgatac 300caatttaaat agctgctaat gctctggctt gcccggcaag tgatggtgac tctgtctcct 360acagatgcag acagggagga tggagactgg gtcatctgga tgtccgtcga cccgccaccg 420ccgctgccac ctccgcctga accgcctcca ccgctcgaga cggtgaccag ggttccctgg 480ccccagtagt caaactgctt accacgtttc gcacagtaat atacggccgt gtcctcggct 540ctcaggctgt tcatttgcag atacagcgtg ttcttggaat tgtctctgga gatggtgaac 600cggcccttca cggagtctgc gtaatgtgtc acagtaccat ccggccaaat acctgagacc 660cactccagcc ccttccctgg agcctggcgg acccagctca tggcatagct gctaaaggtg 720aatccagagg ctgcacagga gagtctcagg gaccccccag gctgtaccaa gcctccccca 780gactccaaca gctgcacctc ggccatggcc ggctgggccg cgagtaataa caatccagcg 840gctgccgtan gcaataggta tttcattatg actgtctcct tgaaatagaa tttgcatgca 900agcttggcgt antcatggtc atagctgttt cctgtgngaa attgttatcc gctcacnntt 960ccacncaaca tacganccgg 98020289PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(6)..(6)Any amino acidMOD_RES(286)..(287)Any amino acid 20Met Lys Tyr Leu Leu Xaa Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ser Gly Ile Trp Pro Asp Gly Thr Val Thr 65 70 75 80 His Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Lys Arg Gly Lys Gln Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Ile Gln Met Thr Gln 145 150 155 160 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 165 170 175 Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln 180 185 190 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Arg Ala Ser Leu Leu 195 200 205 Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 210 215 220 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 225 230 235 240 Tyr Cys Gln Gln Val Gln Thr Ser Pro Pro Thr Phe Gly Gln Gly Thr 245 250 255 Lys Val Glu Ile Lys Arg Ala Ala Ala His His His His His His Gly 260 265 270 Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Xaa Xaa His 275 280 285 Arg 21973DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(4)..(4)a, c, t, g, unknown or othermodified_base(8)..(13)a, c, t, g, unknown or othermodified_base(849)..(851)a, c, t, g, unknown or othermodified_base(856)..(856)a, c, t, g, unknown or othermodified_base(904)..(904)a, c, t, g, unknown or othermodified_base(907)..(908)a, c, t, g, unknown or other 21ctangcgnnn nnntcagatc ctcttctgag atgagttttt gttctgcggc cccgtgatgg 60tgatgatgat gtgcggccgc ccgtttgatt tccaccttgg tcccttggcc gaacgtagaa 120ggattatcat cattctgttg acagtagtaa gttgcaaaat cttcaggttg cagactgctg 180atggtgagag tgaaatctgt cccagatcca ctgccactga accttgatgg gaccccactt 240tgcaaagtgg atgcatcata aatcaggagc ttaggggctt tccctggttt ctgctgatac 300caatttaaat agctgctaat gctctgactt gcccggcaag tgatggtgac tctgtctcct 360acagatgcag acagggagga tggagactgg gtcatctgga tgtccgtcga cccgccaccg 420ccgctgccac ctccgcctga accgcctcca ccgctcgaga cggtgaccag ggttccctgg 480ccccagtagt caaaaccatt agaagttttc gcacagtaat atacggccgt gtcctcggct 540ctcaggctgt tcatttgcag atacagcgtg ttcttggaat tgtctctgga gatggtgaac 600cggcccttca cggagtctgc gtaatatgta gtactaccag tagcatcaat agttgagacc 660cactccagcc ccttccctgg agcctggcgg acccagctca tggcatagct gctaaaggtg 720aatccagagg ctgcacagga gagtctcagg gaccccccag gctgtaccaa gcctccccca 780gactccaaca gctgcacctc ggccatggcc ggctgggccg cgagtaataa caatccagcg 840gctgccgtnn naatangtat ttcattatga ctgtctcctt gaaatagaat ttgcatgcaa 900gctnggnnta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 960cacacaacat acg 97322294PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(14)..(14)Any amino acidMOD_RES(16)..(16)Any amino acid 22Met Gln Ile Leu Phe Gln Gly Asp Ser His Asn Glu Ile Xaa Ile Xaa 1 5 10 15 Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala Ala Gln Pro Ala Met Ala 20 25 30 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 35 40 45 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 50 55 60 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 65 70 75 80 Ser Thr Ile Asp Ala Thr Gly Ser Thr Thr Tyr Tyr Ala Asp Ser Val 85 90 95 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 100 105 110 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 115 120 125 Ala Lys Thr Ser Asn Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 130 135 140 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 145 150 155 160 Gly Gly Ser Thr Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 165 170 175 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser 180 185 190 Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 195 200 205 Lys Leu Leu Ile Tyr Asp Ala Ser Thr Leu Gln Ser Gly Val Pro Ser 210 215 220 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 225 230 235 240 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asp 245 250 255 Asp Asn Pro Ser Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 260 265 270 Ala Ala Ala His His His His His His Gly Ala Ala Glu Gln Lys Leu 275 280 285 Ile Ser Glu Glu Asp Leu 290 23959DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(20)..(20)a, c, t, g, unknown or othermodified_base(55)..(55)a, c, t, g, unknown or othermodified_base(837)..(838)a, c, t, g, unknown or othermodified_base(875)..(875)a, c, t, g, unknown or othermodified_base(895)..(895)a, c, t, g, unknown or othermodified_base(906)..(906)a, c, t, g, unknown or othermodified_base(922)..(922)a, c, t, g, unknown or othermodified_base(944)..(944)a, c, t, g, unknown or othermodified_base(950)..(950)a, c, t, g, unknown or othermodified_base(954)..(954)a, c, t, g, unknown or other 23ttcagatcct cttctgagan gagtttttgt tctgcggccc cgtgatggtg atgangatgt 60gcggccgccc gtttgatttc caccttggtc ccttggccga acgtagtagg actagcataa 120ctctgttgac agtagtaagt tgcaaaatct tcaggttgca gaccgctgat ggtgagagtg 180aaatctgtcc cagatccact gccactgaac cttgatggga ccccactttg caaagaggat 240gcaccataga tcaggagctt aggggctttc cctggtttct gctgatacca atttaaatag 300ctgctaatgc tctgacttgc ccggcaagtg atggtgactc tgtctcctac agatgcagac 360agggaggatg gagactgggt catctggatg tccgtcgacc cgccaccgcc gctgccacct 420ccgcctgaac cgcctccacc gctcgagacg gtgaccaggg ttccctggcc ccagtagtca 480aaagcagtag cagttttcgc acagtaatat acggccgtgt cctcggctct caggctgttc 540atttgcagat acagcgtgtt cttggaattg tctctggaga tggtgaaccg gcccttcacg 600gagtctgcgt aacttgtagc atcaccatta gaataaatag atgagaccca ctccagcccc 660ttccctggag cctggcggac ccagctcatg gcatagctgc taaaggtgaa tccagaggct 720gcacaggaga gtctcaggga ccccccaggc tgtaccaagc ctcccccaga ctccaacagc 780tgcacctcgg ccatggccgg ctgggccgcg agtaataaca atccagcggc tgccgtnnca 840ataggtattt cattatgact gtctccttga aatanaattt gcatgcaagc ttggngtaat 900catggncata gctgtttcct gngtgaaatt gttatccgct cacnattccn cacnacata 95924295PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(4)..(4)Any amino acidMOD_RES(16)..(16)Any amino acidMOD_RES(277)..(277)Any amino acidMOD_RES(289)..(289)Any amino acid 24Met Gln Ile Xaa Phe Gln Gly Asp Ser His Asn Glu Ile Pro Ile Xaa 1 5 10 15 Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala Ala Gln Pro Ala Met Ala 20 25 30 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 35 40 45 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 50 55 60 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 65 70 75 80 Ser Ser Ile Tyr Ser Asn Gly Asp Ala Thr Ser Tyr Ala Asp Ser Val 85 90 95 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

100 105 110 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 115 120 125 Ala Lys Thr Ala Thr Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 130 135 140 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 145 150 155 160 Gly Gly Ser Thr Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 165 170 175 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser 180 185 190 Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 195 200 205 Lys Leu Leu Ile Tyr Gly Ala Ser Ser Leu Gln Ser Gly Val Pro Ser 210 215 220 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 225 230 235 240 Gly Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr 245 250 255 Ala Ser Pro Thr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 260 265 270 Ala Ala Ala His Xaa His His His His Gly Ala Ala Glu Gln Lys Leu 275 280 285 Xaa Ser Glu Glu Asp Leu Lys 290 295 25971DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(8)..(8)a, c, t, g, unknown or othermodified_base(33)..(33)a, c, t, g, unknown or othermodified_base(851)..(851)a, c, t, g, unknown or othermodified_base(914)..(914)a, c, t, g, unknown or othermodified_base(938)..(938)a, c, t, g, unknown or othermodified_base(952)..(952)a, c, t, g, unknown or othermodified_base(965)..(966)a, c, t, g, unknown or other 25ctatgcgncc ccattcagat cctcttctga gangagtttt tgttctgcgg ccccgtgatg 60gtgatgatga tgtgcggccg cccgtttgat ttccaccttg gtcccttggc cgaacgtagg 120aggcgaagtc tgaacctgtt gacagtagta agttgcaaaa tcttcaggtt gcagactgct 180gatggtgaga gtgaaatctg tcccagatcc actgccactg aaccttgatg ggaccccact 240ttgcaacagg gatgcacgat agatcaggag cttaggggct ttccctggtt tctgctgata 300ccaatttaaa tagctgctaa tgctctggct tgcccggcaa gtgatggtga ctctgtctcc 360tacagatgca gacagggagg atggagactg ggtcatctgg atgtccgtcg acccgccacc 420gccgctgcca cctccgcctg aaccgcctcc accgctcgag acggtgacca gggttccctg 480gccccagtag tcaaactgct taccacgttt cgcacagtaa tatacggccg tgtcctcggc 540tctcaggctg ttcatttgca gatacagcgt gttcttggaa ttgtctctgg agatggtgaa 600ccggcccttc acggagtctg cgtaatgtgt cacagtacca tccggccaaa tacctgagac 660ccactccagc cccttccctg gagcctggcg gacccagctc atggcatagc tgctaaaggt 720gaatccagag gctgcacagg agagtctcag ggacccccca ggctgtacca agcctccccc 780agactccaac agctgcacct cggccatggc cggctgggcc gcgagtaata acaatccagc 840ggctgccgta ngcaataggt atttcattat gactgtctcc ttgaaataga atttgcatgc 900aagcttggcg taancatggt catagctgtt tcctgtgnga aattgttatc cngctcacaa 960ttccnncaca a 97126288PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(6)..(6)Any amino acidMOD_RES(279)..(279)Any amino acidMOD_RES(287)..(287)Any amino acid 26Met Lys Tyr Leu Leu Xaa Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ser Gly Ile Trp Pro Asp Gly Thr Val Thr 65 70 75 80 His Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Lys Arg Gly Lys Gln Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Ile Gln Met Thr Gln 145 150 155 160 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 165 170 175 Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln 180 185 190 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Arg Ala Ser Leu Leu 195 200 205 Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 210 215 220 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 225 230 235 240 Tyr Cys Gln Gln Val Gln Thr Ser Pro Pro Thr Phe Gly Gln Gly Thr 245 250 255 Lys Val Glu Ile Lys Arg Ala Ala Ala His His His His His His Gly 260 265 270 Ala Ala Glu Gln Lys Leu Xaa Ser Glu Glu Asp Leu Asn Gly Xaa Ala 275 280 285 27965DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(7)..(9)a, c, t, g, unknown or othermodified_base(14)..(14)a, c, t, g, unknown or othermodified_base(30)..(30)a, c, t, g, unknown or othermodified_base(67)..(69)a, c, t, g, unknown or othermodified_base(747)..(747)a, c, t, g, unknown or othermodified_base(848)..(848)a, c, t, g, unknown or othermodified_base(855)..(855)a, c, t, g, unknown or othermodified_base(889)..(889)a, c, t, g, unknown or othermodified_base(907)..(908)a, c, t, g, unknown or othermodified_base(918)..(919)a, c, t, g, unknown or othermodified_base(960)..(960)a, c, t, g, unknown or othermodified_base(962)..(962)a, c, t, g, unknown or other 27tatgcgnnna ttcngatcct cttctgagan gagtttttgt tctgcggccc cgtgatggtg 60atgatgnnnt gcggccgccc gtttgatttc caccttggtc ccttggccga acgtattagg 120acaatcagta gtctgttgac agtagtaagt tgcaaaatct tcaggttgca gactgctgat 180ggtgagagtg aaatctgtcc cagatccact gccactgaac cttgatggga ccccactttg 240caaagtggat gcattataga tcaggagctt aggggctttc cctggtttct gctgatacca 300atttaaatag ctgctaatgc tctgacttgc ccggcaagtg atggtgactc tgtctcctac 360agatgcagac agggaggatg gagactgggt catctggatg tccgtcgacc cgccaccgcc 420gctgccacct ccgcctgaac cgcctccacc gctcgagacg gtgaccaggg ttccctggcc 480ccagtagtca aaattagcac cagatttcgc acagtaatat acggccgtgt cctcggctct 540caggctgttc atttgcagat acagcgtgtt cttggaattg tctctggaga tggtgaacct 600gcccttcacg gagtctgcgt aagatgtagc ataaccacta gcagtaatac ctgagaccca 660ctccagcccc ttccctggag cctggcggac ccagctcatg gcatagctgc taaaggtgaa 720tccagaggct gcacaggaga gtctcangga ccccccaggc tgtaccaagc ctcccccaga 780ctccaacagc tgcacctcgg ccatggccgg ctgggccgcg agtaataaca atccagcggc 840tgccgtangc aatangtatt tcattatgac tgtctccttg aaatagaant ttgcatgcaa 900gcttggnnta atcatggnna tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattn 960cncac 96528305PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(3)..(3)Any amino acidMOD_RES(9)..(9)Any amino acidMOD_RES(21)..(21)Any amino acidMOD_RES(23)..(23)Any amino acidMOD_RES(57)..(57)Any amino acidMOD_RES(283)..(283)Any amino acidMOD_RES(296)..(296)Any amino acidMOD_RES(301)..(301)Any amino acidMOD_RES(303)..(303)Any amino acid 28Met Ile Xaa Pro Ser Leu His Ala Xaa Phe Tyr Phe Lys Glu Thr Val 1 5 10 15 Ile Met Lys Tyr Xaa Leu Xaa Thr Ala Ala Ala Gly Leu Leu Leu Leu 20 25 30 Ala Ala Gln Pro Ala Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly 35 40 45 Gly Leu Val Gln Pro Gly Gly Ser Xaa Arg Leu Ser Cys Ala Ala Ser 50 55 60 Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro 65 70 75 80 Gly Lys Gly Leu Glu Trp Val Ser Gly Ile Thr Ala Ser Gly Tyr Ala 85 90 95 Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 100 105 110 Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu 115 120 125 Asp Thr Ala Val Tyr Tyr Cys Ala Lys Ser Gly Ala Asn Phe Asp Tyr 130 135 140 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 145 150 155 160 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Ile Gln Met Thr 165 170 175 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 180 185 190 Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln 195 200 205 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asn Ala Ser Thr 210 215 220 Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 225 230 235 240 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 245 250 255 Tyr Tyr Cys Gln Gln Thr Thr Asp Cys Pro Asn Thr Phe Gly Gln Gly 260 265 270 Thr Lys Val Glu Ile Lys Arg Ala Ala Ala Xaa His His His His His 275 280 285 Gly Ala Ala Glu Gln Lys Leu Xaa Ser Glu Glu Asp Xaa Asn Xaa Arg 290 295 300 Ile 305 29963DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(6)..(8)a, c, t, g, unknown or othermodified_base(12)..(12)a, c, t, g, unknown or othermodified_base(15)..(15)a, c, t, g, unknown or othermodified_base(28)..(28)a, c, t, g, unknown or othermodified_base(53)..(54)a, c, t, g, unknown or othermodified_base(62)..(66)a, c, t, g, unknown or othermodified_base(610)..(610)a, c, t, g, unknown or othermodified_base(839)..(839)a, c, t, g, unknown or othermodified_base(841)..(841)a, c, t, g, unknown or othermodified_base(846)..(847)a, c, t, g, unknown or othermodified_base(884)..(885)a, c, t, g, unknown or othermodified_base(904)..(904)a, c, t, g, unknown or othermodified_base(908)..(908)a, c, t, g, unknown or othermodified_base(915)..(915)a, c, t, g, unknown or othermodified_base(927)..(927)a, c, t, g, unknown or othermodified_base(931)..(931)a, c, t, g, unknown or othermodified_base(935)..(935)a, c, t, g, unknown or othermodified_base(952)..(954)a, c, t, g, unknown or other 29gcggcnnntt cngancctct tctgaganga gtttttgttc tgcggccccg tgnnggtgat 60gnnnnngtgc ggccgcccgt ttgatttcca ccttggtccc ttggccgaac gtattagggg 120tactgtaact ctgttgacag tagtaagttg caaaatcttc aggttgcaga ctgctgatgg 180tgagagtgaa atctgtccca gatccactgc cactgaacct tgatgggacc ccactttgca 240aactggatgc agcatagatc aggagcttag gggctttccc tggtttctgc tgataccaat 300ttaaatagct gctaatgctc tgacttgccc ggcaagtgat ggtgactctg tctcctacag 360atgcagacag ggaggatgga gactgggtca tctggatgtc cgtcgacccg ccaccgccgc 420tgccacctcc gcctgaaccg cctccaccgc tcgagacggt gaccagggtt ccctggcccc 480agtagtcaaa cgccggatga tatttcgcac agtaatatac ggccgtgtcc tcggctctca 540ggctgttcat ttgcagatac agcgtgttct tggaattgtc tctggagatg gtgaaccggc 600ccttcacggn gtctgcgtac tctgtcggca gaccctgcgg cgcaatcgat gagacccact 660ccagcccctt ccctggagcc tggcggaccc agctcatggc atagctgcta aaggtgaatc 720cagaggctgc acaggagagt ctcagggacc ccccaggctg taccaagcct cccccagact 780ccaacagctg cacctcggcc atggccggct gggccgcgag taataacaat ccagcggcng 840ncgtanncaa taggtatttc attatgactg tctccttgaa atannatttg catgcaagct 900tggngtantc atggncatag ctgtttnctg ngtgnaaatt gttatccgct cnnnaatttc 960cac 96330283PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(6)..(6)Any amino acidMOD_RES(8)..(8)Any amino acidMOD_RES(85)..(85)Any amino acidMOD_RES(266)..(267)Any amino acidMOD_RES(270)..(270)Any amino acidMOD_RES(279)..(279)Any amino acid 30Met Lys Tyr Leu Leu Xaa Thr Xaa Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ser Ser Ile Ala Pro Gln Gly Leu Pro Thr 65 70 75 80 Glu Tyr Ala Asp Xaa Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Lys Tyr His Pro Ala Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Ile Gln Met Thr Gln 145 150 155 160 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 165 170 175 Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln 180 185 190 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu 195 200 205 Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 210 215 220 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 225 230 235 240 Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Asn Thr Phe Gly Gln Gly Thr 245 250 255 Lys Val Glu Ile Lys Arg Ala Ala Ala Xaa Xaa His His Xaa His Gly 260 265 270 Ala Ala Glu Gln Lys Leu Xaa Ser Glu Glu Asp 275 280 31953DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(4)..(4)a, c, t, g, unknown or othermodified_base(7)..(8)a, c, t, g, unknown or othermodified_base(63)..(67)a, c, t, g, unknown or othermodified_base(688)..(688)a, c, t, g, unknown or othermodified_base(846)..(846)a, c, t, g, unknown or othermodified_base(853)..(853)a, c, t, g, unknown or othermodified_base(908)..(908)a, c, t, g, unknown or othermodified_base(915)..(915)a, c, t, g, unknown or othermodified_base(924)..(924)a, c, t, g, unknown or othermodified_base(941)..(941)a, c, t, g, unknown or othermodified_base(943)..(943)a, c, t, g, unknown or other 31gcgnccnntt cagatcctct tctgagatga gtttttgttc tgcggccccg tgatggtgat 60gannnnntgc ggccgcccgt ttgatttcca ccttggtccc ttggccgaac gtagaaggag 120aattaccagt ctgttgacag tagtaagttg caaaatcttc aggttgcaga ctgctgatgg 180tgagagtgaa atctgtccca gatccactgc cactgaacct tgatgggacc ccactttgca 240aagcggatgc agtatagatc aggagcttag gggctttccc tggtttctgc tgataccaat 300ttaaatagct gctaatgctc tgacttgccc ggcaagtgat ggtgactctg tctcctacag 360atgcagacag ggaggatgga gactgggtca tctggatgtc cgtcgacccg ccaccgccgc 420tgccacctcc gcctgaaccg cctccaccgc tcgagacggt gaccagggtt ccctggcccc 480agtagtcaaa agtactataa gatttcgcac agtaatatac ggccgtgtcc tcggctctca 540ggctgttcat ttgcagatac agcgtgttct tggaattgtc tctggagatg gtgaaccggc 600ccttcacgga gtctgcgtaa gctgtactag cactactagc agcaatacct gagacccact 660ccagcccctt ccctggagcc tggcggancc agctcatggc atagctgcta aaggtgaatc 720cagaggctgc acaggagagt ctcagggacc ccccaggctg taccaagcct cccccggact 780ccaacagctg cacctcggcc atggccggct gggccgcgag taataacaat ccagcggctg 840ccgtangcaa tangtatttc attatgactg tctccttgaa atagaatttg catgcaagct 900tggcgtantc atggncatag ctgnttcctg tgtgaaattg ntnatccgct cac 95332284PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(4)..(4)Any amino acidMOD_RES(6)..(6)Any amino acidMOD_RES(59)..(59)Any amino acidMOD_RES(266)..(267)Any amino

acid 32Met Lys Tyr Xaa Leu Xaa Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Xaa Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ser Gly Ile Ala Ala Ser Ser Ala Ser Thr 65 70 75 80 Ala Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Lys Ser Tyr Ser Thr Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Ile Gln Met Thr Gln 145 150 155 160 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 165 170 175 Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln 180 185 190 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Thr Ala Ser Ala Leu 195 200 205 Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 210 215 220 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 225 230 235 240 Tyr Cys Gln Gln Thr Gly Asn Ser Pro Ser Thr Phe Gly Gln Gly Thr 245 250 255 Lys Val Glu Ile Lys Arg Ala Ala Ala Xaa Xaa His His His His Gly 260 265 270 Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 275 280 33696DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 33atggccgagg tgcagctgtc ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60agactctcct gtgcagcctc tggattcacc tttagcagct atgccatgag ctgggtccgc 120caggctccag ggaaggggct ggagtgggtc tcaggtatta atagtaatgg tacttctaca 180tcttacgcag actccgtgaa gggccggttc accatctcca gagacaattc caagaacacg 240ctgtatctgc aaatgaacag cctgagagcc gaggacacgg ccgtatatta ctgtgcgaaa 300tctgcttctg attttgacta ctggggccag ggaaccctgg tcaccgtctc gagcggtgga 360ggcggttcag gcggaggtgg cagcggcggt ggcgggtcga cggacatcca gatgacccag 420tctccatcct ccctgtctgc atctgtagga gacagagtca ccatcacttg ccgggcaagt 480cagagcatta gcagctattt aaattggtat cagcagaaac cagggaaagc ccctaagctc 540ctgatctata atgcatccac tttgcaaagt ggggtcccat caaggttcag tggcagtgga 600tctgggacag atttcactct caccatcagc agtctgcaac ctgaagattt tgcaacttac 660tactgtcaac agaatactta tagtcctact acgttc 69634264PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 34Met Lys Tyr Leu Leu Pro Thr Asn Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Asn Pro Ala Met Ala Glu Val Gln Leu Ser Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ser Gly Ile Asn Ser Asn Gly Thr Ser Thr 65 70 75 80 Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Lys Ser Ala Ser Asp Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Ile Gln Met Thr Gln 145 150 155 160 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 165 170 175 Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln 180 185 190 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asn Ala Ser Thr Leu 195 200 205 Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 210 215 220 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 225 230 235 240 Tyr Cys Gln Gln Asn Thr Tyr Ser Pro Thr Thr Phe Gly Asn Asn Asn 245 250 255 Lys Val Glu Ile Lys Arg Ala Ala 260 35732DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(706)..(706)a, c, t, g, unknown or other 35atggccgaga tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60agactctcct gtgcagcctc tggattcacc tttagcagct atgccatgag ctgggtccgc 120caggctccag ggaaggggct ggagtgggtc tcatatatta ctgctaatgg tgatagtaca 180acttacgcag actccgtgaa gggccggttc accatctcca gagacaattc caagaacacg 240ctgtatctgc aaatgaacag cctgagagcc gaggacacgg ccgtatatta ctgtgcgaaa 300agtactactg attttgacta ctggggccag ggaaccctgg tcaccgtctc gagcggtgga 360ggcggttcag gcggaggtgg cagcggcggt ggcgggtcga cggacatcca gatgacccag 420tctccatcct ccctgtctgc atctgtagga gacagagtca ccatcacttg ccgggcaagt 480cagagcatta gcagctattt aaattggtat cagcagaaac cagggaaagc ccctaagctc 540ctgatctata gtgcatccaa tttgcaaagt ggggtcccat caaggttcag tggcagtgga 600tctgggacag atttcactct caccatcagc agtctgcaac ctgaagattt tgcaacttac 660tactgtcaac agacttctta tagtccttct acgttcggcc aagggnccaa ggtggaaatc 720aaacgggcgg cc 73236245PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(236)..(236)Any amino acid 36Met Ala Glu Met Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Tyr Ile Thr Ala Asn Gly Asp Ser Thr Thr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Ser Thr Thr Asp Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Thr Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 145 150 155 160 Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175 Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Asn Leu Gln Ser Gly Val 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 195 200 205 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 210 215 220 Thr Ser Tyr Ser Pro Ser Thr Phe Gly Gln Gly Xaa Lys Val Glu Ile 225 230 235 240 Lys Arg Ala Ala Ala 245 37735DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(54)..(54)a, c, t, g, unknown or other 37atggccgagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg gggntccctg 60agactctcct gtgcagcctc tggattcacc tttagcagct atgccatgag ctgggtccgc 120caggctccag ggaaggggct ggagtgggtc tcaactatta atgctagtgg tggtagtaca 180ggttacgcag actccgtgaa gggccggttc accatctcca gagacaattc caagaacacg 240ctgtatctgc aaatgaacag cctgagagcc gaggacacgg ccgtatatta ctgtgcgaaa 300gctgatgctt attttgacta ctggggccag ggaaccctgg tcaccgtctc gagcggtgga 360ggcggttcag gcggaggtgg cagcggcggt ggcgggtcga cggacatcca gatgacccag 420tctccatcct ccctgtctgc atctgtagga gacagagtca ccatcacttg ccgggcaagt 480cagagcatta gcagctattt aaattggtat cagcagaaac cagggaaagc ccctaagctc 540ctgatctatt ctgcatcctc gttgcaaagt ggggtcccat caaggttcag tggcagtgga 600tctgggacag atttcactct caccatcagc agtctgcaac ctgaagattt tgcaacttac 660tactgtcaac aggatgctag tggtccttct acgttcggcc aagggaccaa ggtggaaatc 720aaacgggcgg ccgca 73538244PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 38Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Thr Ile Asn Ala Ser Gly Gly Ser Thr Gly Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Ala Asp Ala Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Thr Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 145 150 155 160 Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175 Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser Leu Gln Ser Gly Val 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 195 200 205 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 210 215 220 Asp Ala Ser Gly Pro Ser Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 225 230 235 240 Lys Arg Ala Ala 39736DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(706)..(706)a, c, t, g, unknown or othermodified_base(731)..(731)a, c, t, g, unknown or other 39atggccgagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60agactctcct gtgcagcctc tggattcacc tttagcagct atgccatgag ctgggtccgc 120caggctccag ggaaggggct ggagtgggtc tcatatattg ctgatgatgg tgctaataca 180gcttacgcag actccgtgaa gggccggttc accatctcca gagacaattc caagaacacg 240ctgtatctgc aaatgaacag cctgagagcc gaggacacgg ccgtatatta ctgtgcgaaa 300aataatgatg gttttgacta ctggggccag ggaaccctgg tcaccgtctc gagcggtgga 360ggcggttcag gcggaggtgg cagcggcggt ggcgggtcga cgaacatcca gatgacccag 420tctccatcct ccctgtctgc atctgtagga gacagagtca ccatcacttg ccgggcaagt 480cagagcatta gcagctattt aaattggtat cagcagaaac cagggaaagc ccctaagctc 540ctgatctatt ctgcatccac tttgcaaagt ggggtcccat caaggttcag tggcagtgga 600tctgggacag atttcactct caccatcagc agtctgcaac ctgaagattt tgcaacttac 660tactgtcaac aggctgctac tagtccttct acgttcggcc aagggnccaa ggtggaaatc 720aaacgggcgg ncgcac 73640245PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(236)..(236)Any amino acidMOD_RES(244)..(244)Any amino acid 40Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Tyr Ile Ala Asp Asp Gly Ala Asn Thr Ala Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Asn Asn Asp Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Thr Asn Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 145 150 155 160 Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175 Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Thr Leu Gln Ser Gly Val 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 195 200 205 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 210 215 220 Ala Ala Thr Ser Pro Ser Thr Phe Gly Gln Gly Xaa Lys Val Glu Ile 225 230 235 240 Lys Arg Ala Xaa Ala 245 41886DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(27)..(27)a, c, t, g, unknown or othermodified_base(36)..(36)a, c, t, g, unknown or othermodified_base(44)..(45)a, c, t, g, unknown or othermodified_base(76)..(76)a, c, t, g, unknown or othermodified_base(78)..(78)a, c, t, g, unknown or othermodified_base(852)..(852)a, c, t, g, unknown or othermodified_base(875)..(876)a, c, t, g, unknown or othermodified_base(885)..(885)a, c, t, g, unknown or other 41ttcaaggaga cagtcataat gaaatancct attgcntacg gcanncgctg gattgttatt 60actcgcggcc cagccngncc atggccgagg tgcagctgtt ggagtctggg ggaggcttgg 120tacagcctgg ggggtccctg agactctcct gtgcagcctc tggattcacc tttagcagct 180atgccatgag ctgggtccgc caggctccag ggaaggggct ggagtgggtc tcaaatatta 240gttctgatgg tgattctaca gcttacgcag actccgtgaa gggccggttc accatctcca 300gagacaattc caagaacacg ctgtatctgc aaatgaacag cctgagagcc gaggacacgg 360ccgtatatta ctgtgcgaaa gcttctagta attttgacta ctggggccag ggaaccctgg 420tcaccgtctc gagcggtgga ggcggttcag gcggaggtgg cagcggcggt ggcgggtcga 480cggacatcca gatgacccag tctccatcct ccctgtctgc atctgtagga gacagagtca 540ccatcacttg ccgggcaagt cagagcatta gcagctattt aaattggtat cagcagaaac 600cagggaaagc ccctaagctc ctgatctatg ctgcatccaa tttgcaaagt ggggtcccat 660caaggttcag tggcagtgga tctgggacag atttcactct caccatcagc agtctgcaac 720ctgaagattt tgcaacttac tactgtcaac agtctaattc tgatcctact acgttcggcc 780aagggaccaa ggtaatcaaa cgggcggccg cacatcatca tcaccatcac ggggccgcag 840aacaaaaact cntctcagaa gaggatctga atggnnccgc atagnc 88642267PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(258)..(258)Any amino acidMOD_RES(265)..(266)Any amino acid 42Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Asn Ile Ser Ser Asp Gly Asp Ser Thr Ala Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Ala Ser Ser Asn Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Thr Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 145 150 155

160 Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175 Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 195 200 205 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 210 215 220 Ser Asn Ser Asp Pro Thr Thr Phe Gly Gln Gly Thr Lys Val Ile Lys 225 230 235 240 Arg Ala Ala Ala His His His His His His Gly Ala Ala Glu Gln Lys 245 250 255 Leu Xaa Ser Glu Glu Asp Leu Asn Xaa Xaa Ala 260 265 43909DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(13)..(13)a, c, t, g, unknown or othermodified_base(21)..(21)a, c, t, g, unknown or othermodified_base(907)..(907)a, c, t, g, unknown or other 43cagggggggc ggngcctatg naaaaaacgc cagcaacgcg gccttttacg gttcctggcc 60ctttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 120cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 180gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt 240tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 300cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg 360ctcccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc 420tatgaccatg attacgccaa gcttgcatgc aaattctatt tcaaggagac agtcatagct 480agcatgaaaa agatttggct ggcgctggct ggtttagttt tagcgtttag cgcatcggcg 540gactacaaag aggcccagcc ggccatggac ctgggtaaga aactgctgga agctgctcgt 600gctggtcagg acgacgaagt tcgtatcctg atggctaacg gtgctgacgt taacgctcat 660gacgaacagg gtactactcc gctgcacctg gctgctaaag aaggtcacct ggaaatcgtt 720gaagttctgc tgaagtacgg tgctgacgtt aacgctcagg acaaattcgg taagaccgct 780ttcgacatct ccatcgacaa cggtaacgag gacctggctg aaatcctgca agcggccgca 840catcatcatc accatcacgg ggccgcagaa caaaaactca tctcagaaga ggatctgaat 900ggccgcnta 90944183PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(183)..(183)Any amino acid 44Met Leu Pro Ala Arg Met Leu Cys Gly Ile Val Ser Gly Gln Phe His 1 5 10 15 Thr Gly Asn Ser Tyr Asp His Asp Tyr Ala Lys Leu Ala Cys Lys Phe 20 25 30 Tyr Phe Lys Glu Thr Val Ile Ala Ser Met Lys Lys Ile Trp Leu Ala 35 40 45 Leu Ala Gly Leu Val Leu Ala Phe Ser Ala Ser Ala Asp Tyr Lys Glu 50 55 60 Ala Gln Pro Ala Met Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg 65 70 75 80 Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp 85 90 95 Val Asn Ala His Asp Glu Gln Gly Thr Thr Pro Leu His Leu Ala Ala 100 105 110 Lys Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala 115 120 125 Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser 130 135 140 Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Ala Ala Ala 145 150 155 160 His His His His His His Gly Ala Ala Glu Gln Lys Leu Ile Ser Glu 165 170 175 Glu Asp Leu Asn Gly Arg Xaa 180 45930DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(12)..(17)a, c, t, g, unknown or othermodified_base(30)..(30)a, c, t, g, unknown or othermodified_base(64)..(64)a, c, t, g, unknown or othermodified_base(98)..(99)a, c, t, g, unknown or othermodified_base(101)..(101)a, c, t, g, unknown or othermodified_base(139)..(140)a, c, t, g, unknown or othermodified_base(898)..(898)a, c, t, g, unknown or othermodified_base(914)..(914)a, c, t, g, unknown or othermodified_base(918)..(922)a, c, t, g, unknown or othermodified_base(926)..(926)a, c, t, g, unknown or other 45gagctatgag annnnnncca cgcttccccn aagggagaaa ggcggacagg tatcccggta 60agcnggcagg gtcggaacag gagagcgcac gagggagnnt ncagggggaa acgcctggta 120tctttatagt cctgtcggnn tttcgccacc tctgacttga gcgtcgattt tttgtgatgc 180tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg 240gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat 300aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc 360agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg 420cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt 480gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt 540atgctcccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac 600agctatgacc atgattacgc caagcttgca tgcaaattct atttcaagga gacagtcata 660gctagcatga aaaagatttg gctggcgctg gctggtttag ttttagcgtt tagcgcatcg 720gcggactaca aagaggccca gccggccatg gtaggaagac ctgacgttaa cgctcaggac 780aaattcggta agaccgcttt cgacatctcc atcgacaacg gtaacgagga cctggctgaa 840atcctgcaag cggccgcaca tcatcatcac catcacgggg ccgcagaaca aaaactcntc 900tcagaagagg atcngaannn nncgcntaga 93046211PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(207)..(207)Any amino acid 46Met Phe Phe Pro Ala Leu Ser Pro Asp Ser Val Asp Asn Arg Ile Thr 1 5 10 15 Ala Phe Glu Ala Asp Thr Ala Arg Arg Ser Arg Thr Thr Glu Arg Ser 20 25 30 Glu Ser Val Ser Glu Glu Ala Glu Glu Arg Pro Ile Arg Lys Pro Pro 35 40 45 Leu Pro Ala Arg Trp Pro Ile His Cys Ser Trp His Asp Arg Phe Pro 50 55 60 Asp Trp Lys Ala Gly Ser Glu Arg Asn Ala Ile Asn Val Ser Leu Thr 65 70 75 80 His Ala Pro Gln Ala Leu His Phe Met Leu Pro Ala Arg Met Leu Cys 85 90 95 Gly Ile Val Ser Gly Gln Phe His Thr Gly Asn Ser Tyr Asp His Asp 100 105 110 Tyr Ala Lys Leu Ala Cys Lys Phe Tyr Phe Lys Glu Thr Val Ile Ala 115 120 125 Ser Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe 130 135 140 Ser Ala Ser Ala Asp Tyr Lys Glu Ala Gln Pro Ala Met Val Gly Arg 145 150 155 160 Pro Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile 165 170 175 Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Ala Ala 180 185 190 Ala His His His His His His Gly Ala Ala Glu Gln Lys Leu Xaa Ser 195 200 205 Glu Glu Asp 210 47915DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(13)..(13)a, c, t, g, unknown or othermodified_base(17)..(17)a, c, t, g, unknown or othermodified_base(35)..(36)a, c, t, g, unknown or othermodified_base(44)..(44)a, c, t, g, unknown or othermodified_base(46)..(46)a, c, t, g, unknown or othermodified_base(48)..(48)a, c, t, g, unknown or othermodified_base(58)..(61)a, c, t, g, unknown or othermodified_base(77)..(77)a, c, t, g, unknown or othermodified_base(104)..(105)a, c, t, g, unknown or othermodified_base(124)..(124)a, c, t, g, unknown or othermodified_base(141)..(141)a, c, t, g, unknown or othermodified_base(158)..(158)a, c, t, g, unknown or othermodified_base(901)..(902)a, c, t, g, unknown or othermodified_base(906)..(906)a, c, t, g, unknown or othermodified_base(908)..(910)a, c, t, g, unknown or other 47tcgtcagggg ggncggnagc ctatggaaaa aacgnnagca acgngncntt tttacggnnn 60ntggcctttt gctggcnttt gctcacatgt tctttcctgc gttnncccct gattctgtgg 120atanccgtat taccgccttt ngagtgagct gataccgntc gccgcagccg aacgaccgag 180cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc 240gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc 300agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac 360tttatgctcc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga 420aacagctatg accatgatta cgccaagctt gcatgcaaat tctatttcaa ggagacagtc 480atagctagca tgaaaaagat ttggctggcg ctggctggtt tagttttagc gtttagcgca 540tcggcggact acaaagaggc ccagccggcc atggacctgg gtaagaaact gctggaagct 600gctcgtgctg gtcaggacga cgaagttcgt atcctgatgg ctaacggtgc tgacgttaac 660gcttgggaca tgactggtca tactccgctg cacctggctg ctcagttcgg tcacctggaa 720atcgttgaag ttctgctgaa gcacggtgct gacgttaacg ctcaggacaa attcggtaag 780accgctttcg acatctccat cgacaacggt aacgaggacc tggctgaaat cctgcaagcg 840gccgcacatc atcatcacca tcacggggcc gcagaacaaa aactcatctc agaagaggat 900nngaangnnn ccgca 91548178PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 48Met Leu Pro Ala Arg Met Leu Cys Gly Ile Val Ser Gly Gln Phe His 1 5 10 15 Thr Gly Asn Ser Tyr Asp His Asp Tyr Ala Lys Leu Ala Cys Lys Phe 20 25 30 Tyr Phe Lys Glu Thr Val Ile Ala Ser Met Lys Lys Ile Trp Leu Ala 35 40 45 Leu Ala Gly Leu Val Leu Ala Phe Ser Ala Ser Ala Asp Tyr Lys Glu 50 55 60 Ala Gln Pro Ala Met Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg 65 70 75 80 Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp 85 90 95 Val Asn Ala Trp Asp Met Thr Gly His Thr Pro Leu His Leu Ala Ala 100 105 110 Gln Phe Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala 115 120 125 Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser 130 135 140 Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Ala Ala Ala 145 150 155 160 His His His His His His Gly Ala Ala Glu Gln Lys Leu Ile Ser Glu 165 170 175 Glu Asp 49848DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(9)..(9)a, c, t, g, unknown or othermodified_base(11)..(11)a, c, t, g, unknown or othermodified_base(23)..(23)a, c, t, g, unknown or othermodified_base(28)..(28)a, c, t, g, unknown or othermodified_base(30)..(30)a, c, t, g, unknown or othermodified_base(35)..(35)a, c, t, g, unknown or othermodified_base(41)..(41)a, c, t, g, unknown or othermodified_base(47)..(47)a, c, t, g, unknown or othermodified_base(51)..(54)a, c, t, g, unknown or othermodified_base(60)..(61)a, c, t, g, unknown or othermodified_base(89)..(89)a, c, t, g, unknown or othermodified_base(100)..(100)a, c, t, g, unknown or othermodified_base(102)..(102)a, c, t, g, unknown or othermodified_base(106)..(106)a, c, t, g, unknown or othermodified_base(108)..(108)a, c, t, g, unknown or othermodified_base(114)..(115)a, c, t, g, unknown or othermodified_base(118)..(118)a, c, t, g, unknown or othermodified_base(120)..(120)a, c, t, g, unknown or othermodified_base(152)..(152)a, c, t, g, unknown or othermodified_base(164)..(164)a, c, t, g, unknown or othermodified_base(170)..(170)a, c, t, g, unknown or othermodified_base(179)..(179)a, c, t, g, unknown or othermodified_base(221)..(222)a, c, t, g, unknown or othermodified_base(240)..(240)a, c, t, g, unknown or othermodified_base(292)..(292)a, c, t, g, unknown or othermodified_base(337)..(339)a, c, t, g, unknown or othermodified_base(480)..(480)a, c, t, g, unknown or othermodified_base(603)..(603)a, c, t, g, unknown or othermodified_base(645)..(645)a, c, t, g, unknown or othermodified_base(707)..(707)a, c, t, g, unknown or othermodified_base(719)..(719)a, c, t, g, unknown or othermodified_base(753)..(753)a, c, t, g, unknown or other 49tttatagtnc ntgtcgggtt tcnccacntn tgacntgagc ntcgatnttt nnnntgctcn 60ncaggggggc ggagcctatg gaaaaacgnc agcaacgcgn cntttntncg gttnntgncn 120ttttgctggc cttttgctca catgttcttt cntgcgttat cccntgattn tgtggatanc 180cgtattaccg cctttgagtg agctgatacc gctcgccgca nncgaacgac cgagcgcagn 240gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct cnccgcgcgt 300tggccgattc attaatgcag ctggcacgac aggtttnnng actggaaagc gggcagtgag 360cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg 420ctcccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagn 480tatgaccatg attacgccaa gcttgcatgc aaattctatt tcaaggagac agtcatagct 540agcatgaaaa agatttggct ggcgctggct ggtttagttt tagcgtttag cgcatcggcg 600gantacaaag aggcccagcc ggccatgggc ggaaccagca gtttnttacc caggtccatg 660gacctgggtc acctggaaat cgttgaagtt ctgctgaagt acggtgntga cgttaacgnt 720caggacaaat tcggtaagac cgctttcgac atntccatcg acaacggtaa cgaggacctg 780gctgaaatcc tgcaagcggc cgcacatcat catcaccatc atcgggctcg cagaacaaaa 840atcatctc 84850230PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(4)..(4)Any amino acidMOD_RES(8)..(8)Any amino acidMOD_RES(10)..(10)Any amino acidMOD_RES(13)..(13)Any amino acidMOD_RES(26)..(26)Any amino acidMOD_RES(32)..(32)Any amino acidMOD_RES(50)..(50)Any amino acidMOD_RES(64)..(64)Any amino acidMOD_RES(108)..(108)Any amino acidMOD_RES(149)..(149)Any amino acidMOD_RES(163)..(163)Any amino acidMOD_RES(184)..(184)Any amino acidMOD_RES(188)..(188)Any amino acidMOD_RES(199)..(199)Any amino acid 50Met Phe Phe Xaa Ala Leu Ser Xaa Asp Xaa Val Asp Xaa Arg Ile Thr 1 5 10 15 Ala Phe Glu Ala Asp Thr Ala Arg Arg Xaa Arg Thr Thr Glu Arg Xaa 20 25 30 Glu Ser Val Ser Glu Glu Ala Glu Glu Arg Pro Ile Arg Lys Pro Pro 35 40 45 Leu Xaa Ala Arg Trp Pro Ile His Cys Ser Trp His Asp Arg Phe Xaa 50 55 60 Asp Trp Lys Ala Gly Ser Glu Arg Asn Ala Ile Asn Val Ser Leu Thr 65 70 75 80 His Ala Pro Gln Ala Leu His Phe Met Leu Pro Ala Arg Met Leu Cys 85 90 95 Gly Ile Val Ser Gly Gln Phe His Thr Gly Asn Xaa Tyr Asp His Asp 100 105 110 Tyr Ala Lys Leu Ala Cys Lys Phe Tyr Phe Lys Glu Thr Val Ile Ala 115 120 125 Ser Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe 130 135 140 Ser Ala Ser Ala Xaa Tyr Lys Glu Ala Gln Pro Ala Met Gly Gly Thr 145 150 155 160 Ser Ser Xaa Leu Pro Arg Ser Met Asp Leu Gly His Leu Glu Ile Val 165 170 175 Glu Val Leu Leu Lys Tyr Gly Xaa Asp Val Asn Xaa Gln Asp Lys Phe 180 185 190 Gly Lys Thr Ala Phe Asp Xaa Ser Ile Asp Asn Gly Asn Glu Asp Leu 195 200 205 Ala Glu Ile Leu Gln Ala Ala Ala His His His His His His Arg Ala 210 215 220 Arg Arg Thr Lys Ile Ile 225 230 51900DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(33)..(33)a, c, t, g, unknown or othermodified_base(59)..(59)a, c, t, g, unknown or othermodified_base(61)..(61)a, c, t, g, unknown or othermodified_base(63)..(63)a, c, t, g, unknown or othermodified_base(69)..(69)a, c, t, g, unknown or othermodified_base(133)..(136)a, c, t, g, unknown or othermodified_base(833)..(838)a, c, t, g, unknown or othermodified_base(872)..(874)a, c, t, g, unknown or othermodified_base(886)..(887)a, c, t, g, unknown or othermodified_base(891)..(894)a, c, t, g, unknown or other 51gagcctatgg

aaaaaacgcc cagcaacgcg gcntttttac ggttcctggc cttttgctng 60ncnttttgnt cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 120cgcctttgag tgnnnngata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 180gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat 240tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc 300aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgctcccggc 360tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca 420tgattacgcc aagcttgcat gcaaattcta tttcaaggag acagtcatag ctagcatgaa 480aaagatttgg ctggcgctgg ctggtttagt tttagcgttt agcgcatcgg cggactacaa 540agaggcccag ccggccatgg acctgggtaa gaaactgctg gaagctgctc gtgctggtca 600ggacgacgaa gttcgtatcc tgatggctaa cggtgctgac gttaacgctg acgacttctc 660tggtactact ccgctgcacc tggctgctca tcatggtcac ctggaaatcg ttgaagttct 720gctgaagtac ggtgctgacg ttaacgctca ggacaaattc ggtaagaccg ctttcgacat 780ctccatcgac aacggtaacg aggacctggc tgaaatcctg caagcggccg cannnnnnca 840tcaccatcac ggggccgcag aacaaaaact cnnncagaag aggatnngaa nnnncgcata 90052263PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(20)..(21)Any amino acidMOD_RES(250)..(251)Any amino acidMOD_RES(263)..(263)Any amino acid 52Met Phe Phe Pro Ala Leu Ser Pro Asp Ser Val Asp Asn Arg Ile Thr 1 5 10 15 Ala Phe Glu Xaa Xaa Asp Thr Ala Arg Arg Ser Arg Thr Thr Glu Arg 20 25 30 Ser Glu Ser Val Ser Glu Glu Ala Glu Glu Arg Pro Ile Arg Lys Pro 35 40 45 Pro Leu Pro Ala Arg Trp Pro Ile His Cys Ser Trp His Asp Arg Phe 50 55 60 Pro Asp Trp Lys Ala Gly Ser Glu Arg Asn Ala Ile Asn Val Ser Leu 65 70 75 80 Thr His Ala Pro Gln Ala Leu His Phe Met Leu Pro Ala Arg Met Leu 85 90 95 Cys Gly Ile Val Ser Gly Gln Phe His Thr Gly Asn Ser Tyr Asp His 100 105 110 Asp Tyr Ala Lys Leu Ala Cys Lys Phe Tyr Phe Lys Glu Thr Val Ile 115 120 125 Ala Ser Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala 130 135 140 Phe Ser Ala Ser Ala Asp Tyr Lys Glu Ala Gln Pro Ala Met Asp Leu 145 150 155 160 Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val 165 170 175 Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Asp Asp Phe Ser 180 185 190 Gly Thr Thr Pro Leu His Leu Ala Ala His His Gly His Leu Glu Ile 195 200 205 Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Gln Asp Lys 210 215 220 Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp 225 230 235 240 Leu Ala Glu Ile Leu Gln Ala Ala Ala Xaa Xaa His His His His Gly 245 250 255 Ala Ala Glu Gln Lys Leu Xaa 260 53919DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(3)..(3)a, c, t, g, unknown or othermodified_base(5)..(5)a, c, t, g, unknown or othermodified_base(8)..(9)a, c, t, g, unknown or othermodified_base(29)..(29)a, c, t, g, unknown or othermodified_base(52)..(52)a, c, t, g, unknown or othermodified_base(130)..(130)a, c, t, g, unknown or othermodified_base(910)..(912)a, c, t, g, unknown or othermodified_base(916)..(916)a, c, t, g, unknown or other 53gangntcnnc agggggggcg gagcctatng aaaaaacgcc agcaacgcgg cnttttttac 60ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt 120ctgtggatan ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga 180ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc 240tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag 300cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt 360tacactttat gctcccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca 420caggaaacag ctatgaccat gattacgcca agcttgcatg caaattctat ttcaaggaga 480cagtcatagc tagcatgaaa aagatttggc tggcgctggc tggtttagtt ttagcgttta 540gcgcatcggc ggactacaaa gaggcccagc cggccatgga cctgggtaag aaactgctgg 600aagctgctcg tgctggtcag gacgacgaag ttcgtatcct gatggctaac ggtgctgacg 660ttaacgctct ggacgaagtt ggttctactc cgctgcacct ggctgctatg gctggtcacc 720tggaaatcgt tgaagtgctg aagcacggtg ctgacgttaa cgctcaggac aaattcggta 780agaccgcttt cgacatctcc atcgacaacg gtaacgagga cctggctgaa atcctgcaag 840cggccgcaca tcatcatcac catcacgggg ccgcagaaca aaaactcatc tcagaagagg 900atctgaatgn nncgcntag 91954177PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 54Met Leu Pro Ala Arg Met Leu Cys Gly Ile Val Ser Gly Gln Phe His 1 5 10 15 Thr Gly Asn Ser Tyr Asp His Asp Tyr Ala Lys Leu Ala Cys Lys Phe 20 25 30 Tyr Phe Lys Glu Thr Val Ile Ala Ser Met Lys Lys Ile Trp Leu Ala 35 40 45 Leu Ala Gly Leu Val Leu Ala Phe Ser Ala Ser Ala Asp Tyr Lys Glu 50 55 60 Ala Gln Pro Ala Met Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg 65 70 75 80 Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp 85 90 95 Val Asn Ala Leu Asp Glu Val Gly Ser Thr Pro Leu His Leu Ala Ala 100 105 110 Met Ala Gly His Leu Glu Ile Val Glu Val Leu Lys His Gly Ala Asp 115 120 125 Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile 130 135 140 Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Ala Ala Ala His 145 150 155 160 His His His His His Gly Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu 165 170 175 Asp 55934DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(8)..(8)a, c, t, g, unknown or othermodified_base(34)..(34)a, c, t, g, unknown or othermodified_base(37)..(38)a, c, t, g, unknown or othermodified_base(58)..(59)a, c, t, g, unknown or othermodified_base(97)..(97)a, c, t, g, unknown or othermodified_base(923)..(927)a, c, t, g, unknown or othermodified_base(931)..(931)a, c, t, g, unknown or other 55ggacaggnta tccggtaaag cggcagggtc ggancannag agcgcacgag ggagcttnnc 60agggggaaac gcctggtatc tttatagtcc tgtcggnttt cgcccacctc tgacttgagc 120gtcgattttt gtgatgctcg tcaggggggg cggagcctat ggaaaaacgc cagcaacgcg 180gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 240tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 300agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc 360aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc 420gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca 480ccccaggctt tacactttat gctcccggct cgtatgttgt gtggaattgt gagcggataa 540caatttcaca caggaaacag ctatgaccat gattacgcca agcttgcatg caaattctat 600ttcaaggaga cagtcatagc tagcatgaaa aagatttggc tggcgctggc tggtttagtt 660ttagcgttta gcgcatcggc ggactacaaa gaggcccagc cggccatgga cctggctgct 720catgttggtc acctggaaat cgttgaagtt ctgctgaagt acggtgctga cgttaacgct 780caggacaaat tcggtaagac cgctttcgac atctccatcg acaacggtaa cgaggacctg 840gctgaaatcc tgcaagcggc cgcacatcat catcaccatc acggggccgc agaacaaaaa 900ctcatctcag aagaggatct gannnnncgc ntag 93456228PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 56Met Phe Phe Pro Ala Leu Ser Pro Asp Ser Val Asp Asn Arg Ile Thr 1 5 10 15 Ala Phe Glu Ala Asp Thr Ala Arg Arg Ser Arg Thr Thr Glu Arg Ser 20 25 30 Glu Ser Val Ser Glu Glu Ala Glu Glu Arg Pro Ile Arg Lys Pro Pro 35 40 45 Leu Pro Ala Arg Trp Pro Ile His Cys Ser Trp His Asp Arg Phe Pro 50 55 60 Asp Trp Lys Ala Gly Ser Glu Arg Asn Ala Ile Asn Val Ser Leu Thr 65 70 75 80 His Ala Pro Gln Ala Leu His Phe Met Leu Pro Ala Arg Met Leu Cys 85 90 95 Gly Ile Val Ser Gly Gln Phe His Thr Gly Asn Ser Tyr Asp His Asp 100 105 110 Tyr Ala Lys Leu Ala Cys Lys Phe Tyr Phe Lys Glu Thr Val Ile Ala 115 120 125 Ser Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe 130 135 140 Ser Ala Ser Ala Asp Tyr Lys Glu Ala Gln Pro Ala Met Asp Leu Ala 145 150 155 160 Ala His Val Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly 165 170 175 Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile 180 185 190 Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Ala Ala 195 200 205 Ala His His His His His His Gly Ala Ala Glu Gln Lys Leu Ile Ser 210 215 220 Glu Glu Asp Leu 225 57201PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMISC_FEATURE(1)..(100)This region may encompass 3-100 residuesMISC_FEATURE(102)..(201)This region may encompass 3-100 residues 57Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 15 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 20 25 30 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 35 40 45 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 50 55 60 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 65 70 75 80 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 85 90 95 Gly Gly Gly Gly Pro Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 100 105 110 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 115 120 125 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 130 135 140 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 145 150 155 160 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 165 170 175 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 180 185 190 Gly Gly Gly Gly Gly Gly Gly Gly Gly 195 200

Claims

1-9. (canceled)

10. A nucleic acid encoding an antibody that preferentially recognizes TBI-associated tau, wherein the nucleic acid sequence has at least 95% sequence identity of any one of SEQ ID NO: 41, 43, 45, 47, 49, 51, 53 or 55.

11-31. (canceled)