Patent application title: Anti-Rhesus D Recombinant Polyclonal Antibody and Methods of Manufacture
Inventors:
Søren Kofoed Rasmussen (Roskilde, DK)
Anne Bondgaard Tolstrup (Hillerod, DK)
Søren Bregenholt Frederiksen (Soborg, DK)
John Haurum (Copenhagen O, DK)
Assignees:
SYMPHOGEN A/S
IPC8 Class: AC40B5006FI
USPC Class:
506 14
Class name: Combinatorial chemistry technology: method, library, apparatus library, per se (e.g., array, mixture, in silico, etc.) library contained in or displayed by a micro-organism (e.g., bacteria, animal cell, etc.) or library contained in or displayed by a vector (e.g., plasmid, etc.) or library containing only micro-organisms or vectors
Publication date: 2012-12-20
Patent application number: 20120322690
Abstract:
The invention relates to a method for manufacturing an anti-RhD
recombinant polyclonal antibody composition (anti-RhD rpAb). The method
comprises obtaining a collection of cells transfected with a library of
anti-RhD antibody expression vectors, wherein each cell in the collection
is capable of expressing from a VH and VL comprising nucleic acid
segment, one member of the library, which encodes a distinct member of
anti-RhD recombinant polyclonal antibody composition and is located at
the same site in the genome of individual cells in said collection. The
cells are cultured under suitable conditions for expression of the
recombinant polyclonal antibody, which is obtained from the cells or
culture supernatant. Nucleic acid segments encoding the anti-RhD rpAb are
introduced into the cells by transfection with a library of vectors for
site-specific integration. The method is suitable for manufacturing
anti-RhD rpAb, thereby making available a superior replacement of
plasma-derived prophylactic and therapeutic immunoglobulin products.Claims:
1-53. (canceled)
54. A method for generating a polyclonal working cell bank, said method comprising: a) providing a collection of cell lines, obtained from host cells which have been individually transfected with an individual member of a library comprised of variable region-encoding nucleic acid segments, and where each individual cell line upon transfection produces a different member of a polyclonal protein, b) mixing a predefined number of cells expanded from each of said cell lines, and c) freezing aliquots of the mixture.
55. The method according to claim 54, wherein an aliquot obtained in step c) is thawed and expanded for a number of generations sufficient to produce a total number of cells, which are frozen down in a new series of aliquots (sub-pWCB), with approximately the same number of cells in each aliquot of said sub-pWCB as in said thawed aliquot.
56. The method according to claim 54, wherein said library comprised of variable region-encoding nucleic acid segments is a library comprised of antibody VH- and VL-encoding nucleic acid segments.
57. The method according to claim 56, wherein said library comprised of antibody VH- and VL-encoding nucleic acid segments encodes an anti-RhD recombinant polyclonal antibody.
58. The method according to claim 54, wherein said individual cell lines are selected, prior to the mixing of the cells, such that they have similar proliferation rates or productivity.
59. The method according to claim 58, wherein said individual cell lines are selected for similar productivity by FACS analysis.
60. The method according to claim 58, wherein said individual cell lines are selected for similar productivity or proliferation rates by means of a robot.
61. The method according to claim 58, wherein said selected cell lines have a proliferation rate between 22 and 40 hours or a productivity exceeding 1.5 pg/(cell×day).
62. The method according to claim 54, wherein said individual cell lines are cloned cell lines.
63. The method according to claim 54, wherein each individual cell line produces a full-length antibody with properties that differ from the properties of the antibodies produced by the other members of the polyclonal working cell bank.
64. The method according to claim 54, wherein said individual cell lines are mixed at equal ratios.
65. The method according to claim 54, wherein said individual cell lines are mixed at different ratios.
66. The method according to claim 65, wherein the ratio of one or more individual cell lines producing an antibody which binds a particular antigen is increased compared to the other members of the polyclonal working cell bank.
67. The method according to claim 65, wherein the ratio of one or more individual cell lines characterized by having a slower proliferation rate is increased compared to other members of the polyclonal working cell bank characterized by a faster proliferation rate.
68. A polyclonal working cell bank comprising a mixture of a predefined number of cells from a collection of individual cell lines, where each individual cell line is obtained from host cells which have been individually transfected with an individual member of a library comprised of variable region-encoding nucleic acid segments, and where each individual cell line produces a different member of a polyclonal protein, and where said pWCB has been frozen down in aliquots.
69. The polyclonal working cell bank according to claim 68, wherein said individual cell lines have similar proliferation rates or productivity.
70. The polyclonal working cell bank according to claim 68, wherein said individual cell lines are cloned cells.
71. The polyclonal working cell bank according to claim 68, wherein said individual cell lines are mixed at different ratios.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. Appl. No. 11/632,937, §371(c) date: Aug. 30, 2007, which is the National Stage of International Appl. No. PCT/DK2005/000501, filed Jul. 18, 2005, which claims the benefit of Danish Appl. No. PA 2004 01133, filed Jul. 20, 2004, and Danish Appl. No. PA 2004 01992, filed Dec. 22, 2004.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] The content of the electronically submitted sequence listing (Name: SequenceListing.txt; Size: 263,218 bytes; and Date of Creation: Jun. 6, 2012) is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention describes the production of an anti-Rhesus D recombinant polyclonal antibody (anti-RhD rpAb), as well as the general approach of generating a polyclonal working cell bank for later production of a desired polyclonal antibody. The invention also relates to libraries encoding anti-RhD rpAb and to cell lines producing anti-RhD rpAb. Further, the application describes pharmacological and diagnostic compositions comprising anti-RhD rpAb and their use in prophylaxis of hemolytic disease of the newborn (HDN), treatment of idiopathic thrombocytopenic purpura (ITP) and prevention of sensitization to the Rhesus D antigen after mistransfusions of RhD(+) blood to RhD(-) individuals.
BACKGROUND OF THE INVENTION
[0004] The Rhesus Mood group antigens are located on transmembrane erythrocyte proteins encompassing the so-called C, c, F, e and D antigens. Approximately 16% of the Caucasian population is Rhesus D negative (RhD(˜)) due to an inherited polymorphism. In addition, multiple genetic and serological variants of RhD exist (divided into category II-VII) of which RhDVI is the most clinically relevant. Since category VI positive red blood cells (RBC) carry fewer of the various epitopes of the D protein than RBC of other categories, RhDVI(+) individuals may form alloantibodies against RBC from other RhD positive (RhD(+)) individuals (Issitt, P. D. and Anstee, D. J., 1998. The Rh Blood Group System, Montgomery Scientific Publications, Durham, N.C., pp. 315-423). {Issitt & Anstee 1998 11809/id}
[0005] RhD negativity in itself is not associated with any medical conditions, but has important medical implications when a RhD(-) female carries a RhD(+) or RhDVI(+) fetus or a RhDVI(+) female carries a RhD(+) fetus. Fetomaternal RhD alloimmunization may then occur if fetal erythrocytes enter the maternal circulation, usually perinatally (during delivery), and thereby causes the induction of a maternal anti-RED antibody response. In subsequent pregnancies RhD-specific IgG-molecules from the mother will cross the placenta into the fetal circulation and mediate lysis of fetal erythrocytes, thereby causing Hemolytic Disease of Newborns (HDN). It has been estimated that on average 20% of RhD(-) women delivering a RhD(+) infant for the second time, and Who are not protected appropriately with anti-D prophylaxis, will generate an anti-RhD antibody response. When untreated, approximately 30% of the newborn will have moderate anemia, jaundice, and hepatomegaly, and 20% develop severe anemia and hydrops fetalis, and severely affected newborns are at risk of neonatal death or permanent handicaps.
[0006] Polyclonal immunoglobulin preparations against RhD are used worldwide to prevent alloimmunization of pregnant RhD(-) and RhDVI(+) women, thereby preventing hemolytic disease of the newborn. Polyclonal immunoglobulin preparations against RhD (anti-D) are currently obtained by pooling of blood plasma obtained from donors who have become hyperimmune, either through natural RhD alloimmunization or through vaccination of RhD negative volunteer males with RID positive erythrocytes. The efficacy of anti-RhD immunoglobulin preparations for prophylaxis of HDN is well established and has been in routine use for many years. As a result this severe disease has become a rarity.
[0007] Nevertheless the underlying cause of the disease, i.e. alloimmunization of pregnant RhD(-) and RhDVI(+) women, still remains and thus requires a continual supply of anti-D immunoglobulin preparations.
[0008] In addition to the prophylaxis of HDN, anti-D immunoglobulin has also proven useful in the treatment of idiopathic thrombocytopenic purpura (ITP) (George, J. N., 2002. Blood Rev. 16, 37-38). ITP is a hematological disorder, where autoantibodies results in an accelerated platelet clearance in the spleen and liver. Symptoms are decreased platelet levels resulting in bruising and bleeding. In severe cases the spleen is removed. This is however, not possible in infants due to severe side effect, thus alternative treatments like anti-D immunoglobulin are needed. Further, anti-D immunoglobulin is used after mistransfusions of RhD(+) blood to RhD(-) recipients in order to prevent sensitization to the Rhesus D antigen.
[0009] The current methods for production of anti-D require, as already mentioned, repeated immunization of an increasingly reluctant pool of donors for the production of high titer antiserum. There are also associated risk factors and technical problems, such as the use of Rhesus positive RBC for repeated immunization carrying the risk of transmission of viral diseases like hepatitis B, AIDS and other as yet unknown viruses. Further, there are problems with batch-to-batch variations. Therefore, an alternative method for production of anti-RhD antibodies is required.
[0010] Cellular approaches for generating anti-RhD monoclonal antibodies were first developed as an alternative to hyperimmune serum. These techniques encompassed Epstein Barr Virus transformation of lymphocytes creating B lymphoblastoid cell lines (Crawford et al. 1983. Lancet 1, 386-8). However, these cell lines are unstable and require extensive cloning. Production of human antibodies by the hybridoma technique was also restricted by the lack of a suitable human myeloma cell fusion partner (Kozbor, D. and Roder, J. C., 1983. Immunol. Today. 4, 72).
[0011] As substitute for these techniques a molecular approach involving repertoire cloning of VH and VL and the construction of phage display libraries was developed (Barbas, C. F. et al. 1991. Proc Natl. Acad. Sci. USA 88, 7978-7982). The phage display technique was also applicable for the isolation of Rhesus D antigen binders. A large number of monoclonal antibodies (mAbs) with Rhesus D antigen binding specificity have been isolated with this technique (WO 97/49809 and Siegel, D. L et al. 2002. Transfus. Clin. Biol. 9, 83-97).
[0012] Recent clinical trials with a recombinant anti-RhDVI mAb have shown that it is possible to prevent RhD immunization after a large challenge with RhD(+) RBC (Miescher, S., et al. 2004, Blood 103, 4028-4035). However, the trial also showed that the mAb was less efficient with respect to clearance of the RBC than an anti-D immunoglobulin. The cause of this decreased clearance rate is not known. It is possible that a single antibody is not as efficient as the diversity of antibodies present in the anti-D immunoglobulin product, or that the presence of more than one immunoglobulin isotype i.e. IgG1 and IgG3 {Siegel, Czerwinski, et al. 2002 10320 id}increases RBC clearance.
[0013] In addition to the efficiency issue, another issue with respect to HDN prophylaxis is the situation where a RhDVI(+) female carries a RhD(+) fetus. In this situation an anti-RhDVI mAb will not be able to prevent alloimmunization of the female. Thus, in order to protect both RhD(-) and RhDVI(+) females, a product with antibodies against Rhesus D category VI antigen as well as antibodies that do not bind category VI antigen but other common Rhesus D antigens is needed.
[0014] Another possible issue with mAbs is that they might be immunogenic. Although the mAbs are human, a first time treatment might result in an antibody response from the female treated with the mAb. Theoretically this may happen because the CDR regions of the mAb, which have never been seen by the immune system of the treated individual before, may be recognized as foreign if presented in a sufficiently large dose. Such a reaction will render the anti-RhD mAb useless in repeated prophylactic treatment.
[0015] It is possible that some of these potential problems with mAbs could be overcome by mixing monoclonal antibodies. However, this would mean separate production and purification of an undefined number of antibodies, which will be quite costly. Further, different batch properties of the individual monoclonal antibodies of such a mixture may affect the final product.
Disclosure of Contribution
[0016] The present invention provides a method for generating a manufacturing cell line which can express an anti-RhD recombinant polyclonal antibody (anti-RhD rpAb) as a single batch.
DESCRIPTION OF THE INVENTION
[0017] The present invention provides methods for the consistent manufacturing of anti-RhD recombinant polyclonal antibody (anti-RID rpAb). It is contemplated that the present invention will open up the possibility for large-scale manufacturing and production of a new class of prophylactic and therapeutic anti-RhD antibody products.
[0018] An anti-RhD rpAb of the present invention potentially has some advantages over monoclonal anti-Rhesus D antibodies. First of all every potential Rhesus D epitope will be covered by more than one antibody, thus an anti-RhD rpAb composition can be used in the prophylactic treatment of both RhD(-) and RhDVI females bearing a RhD(+) child. Hence, it will not be necessary to mix mAb from different production and purification batches in order to obtain full prophylactic effect.
[0019] Further, in the instance where mAbs should prove to be immunogenic due to the high concentration of one single or a few molecules, an anti-RhD rpAb may be a good alternative. Since an anti-RhD rpAb according to the present invention is composed of between 5 and 56 variant antibody molecules, their individual concentration will be lower, and if one of the antibodies should be depleted due to immunogenicity, there will be plenty of others to cover the Rhesus D antigen, thus prophylaxis will still be efficient.
[0020] The production of an anti-RhD rpAb antibody of the present invention can be performed from a single cell line, as a single batch. The generation of a polyclonal manufacturing cell line for the anti-RhD rpAb production will be demonstrated in the detailed description and by a working example.
DEFINITIONS
[0021] An "antibiotic resistance gene" is a gene encoding a protein that can overcome the inhibitory or toxic effect that an antibiotic has on a cell ensuring the survival and continued proliferation of cells in the presence of the antibiotic.
[0022] The term "antibody" describes a functional component of serum and is often referred to either as a collection of molecules (antibodies or immunoglobulin) or as one molecule (the antibody molecule or immunoglobulin molecule). An antibody molecule is capable of binding to or reacting with a specific antigenic determinant (the antigen or the antigenic epitope), which in turn may lead to induction of immunological effector mechanisms. An individual antibody molecule is usually regarded as monospecific, and a composition of antibody molecules may be monoclonal (i.e., consisting of identical antibody molecules) or polyclonal (i.e., consisting of different antibody molecules reacting with the same or different epitopes on the same antigen or even on distinct, different antigens). Each antibody molecule has a unique structure that enables it to bind specifically to its corresponding antigen, and all natural antibody molecules have the same overall basic structure of two identical light chains and two identical heavy chains. Antibodies are also known collectively as immunoglobulins. The terms antibody or antibodies as used herein are also intended to include chimeric and single chain antibodies, as well as binding fragments of antibodies, such as Fab, Fab' or F(ab)2 molecules, Fv fragments or scFv fragments or any other stable fragment, as well as full-length antibody molecules and multimeric forms such as dimeric IgA molecules or pentavalent IgM.
[0023] The term "anti-RhD antibody-encoding nucleic acid segment" describes a nucleic acid segment comprising a pair of VH and VL genetic elements. The segment may further comprise light chain and/or heavy chain constant region genetic elements, e.g. Kappa or Lambda light chain constant region and/or one or more of the constant region domains CH1, CH2, CH3 or CH4 selected from one of the isotypes IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD and IgE. The preferred isotypes are IgG1 and/or IgG3. The nucleic acid segment may also comprise one or more promoter cassettes, either facilitating bi-directional or uni-directional transcription of the VH and VL-encoding sequences. Additional transcriptional or translational elements, such as functional leader sequences directing the gene product to the secretory pathway, poly A signal sequences, UCOE's and/or an IRES may also be present in the segment.
[0024] The term "anti-RhD recombinant polyclonal antibody" or "anti-RhD rpAb" describes a composition of recombinantly produced diverse antibody molecules, where the individual members are capable of binding to at least one epitope on the Rhesus D antigen. Preferably, the composition is produced from a single manufacturing cell line. The diversity of the polyclonal antibody is located in the variable regions (VH and VL regions), in particular in the CDR1, CDR2 and CDR 3 regions.
[0025] The term "bias" is used to denote the phenomenon during recombinant polyclonal antibody production, wherein the composition of an expression library, polyclonal cell line, or polyclonal protein alters over time due to random genetic mutations, differences in proliferation kinetics between individual cells, differences in expression levels between different expression construct sequences, or differences in the cloning efficiency of DNA.
[0026] The terms "a distinct member of the anti-RhD rpAb" denotes an individual antibody molecule of the recombinant polyclonal antibody composition, comprising one or more stretches within the variable regions, which are characterized by differences in the amino acid sequence compared to the other individual members of the polyclonal protein. These stretches are in particular located in the CDR1, CDR2 and CDR 3 regions.
[0027] As used herein, the term "genome" is not to be taken literally as the normal complement of chromosomes present in a cell, but also extra-chromosomal elements that can be introduced into and maintained in a cell. Such extra-chromosomal elements can include, but are not limited to, mini-chromosomes, YACs (yeast artificial chromosomes), MACs (mouse artificial chromosomes), or HACs (human artificial chromosomes).
[0028] The term "head-to-head promoters" refers to a promoter pair being placed in close proximity so that transcription of two genetic elements driven by the promoters occurs in opposite directions (bi-directional transcription). Construction of such a system is described in details in example 3 of U.S. Pat. No. 5,789,208, which is hereby incorporated by reference. A head-to-head promoter can also be constructed with a stuffer composed of irrelevant nucleic acids between the two promoters. Such a stuffer fragment can easily contain more than 500 nucleotides.
[0029] The term "hot-spot" as in "hot-spot cell line" refers to a pre-established locus of the genome of the cell that has been selected or generated and characterized for highly efficient transcription of an integrated nucleic acid segment of interest upon integration of the expression vector into that site.
[0030] The term "immunoglobulin" commonly is used as a collective designation of the mixture of antibodies found in blood or serum, but may also be used to designate a mixture of antibodies derived from other sources or is used in the term "immunoglobulin molecule".
[0031] The term "internal ribosome entry site" or "IRES" describes a structure different from the normal 5' cap-structure on an mRNA. Both structures can be recognized by a ribosome to initiate scanning for an AUG codon to initiate translation. By using one promoter sequence and two initiating AUG's, a first and a second polypeptide sequence can be translated from a single mRNA. Thus, to enable co-translation of a first and a second polynucleotide sequence from a single dicistronic mRNA, the first and second polynucleotide sequence can be transcriptionally fused via a linker sequence including an IRES sequence that enables translation of the polynucleotide sequence downstream of the IRES sequence. In this case, a transcribed dicistronic RNA molecule will be translated from both the capped 5' end and from the internal IRES sequence of the dicistronic RNA molecule to thereby produce both the first and the second polypeptide.
[0032] As used herein the term "library" refers to a collection of variant nucleic acid sequences. For example a collection of nucleic acid sequences encoding a diverse population of antibody variable heavy chains and/or variable light chains. Where a member of the variant nucleic acid sequence is comprised of two variant genetic elements, e.g. VH and VL, it will often be termed a nucleic acid segment. The collection of variant nucleic acid sequences/segments can either be in the form of a pool of such nucleic acid sequences, or it can be a collection of separate nucleic acid sequences (e.g. one unique sequence in each well of a 96 well plate). A library of the present invention typically have at least 3, 5, 10, 20, 50, 1000, 104, 105 or 106 distinct members. In "library of vectors" the variant nucleic acid sequences/segments have been inserted into a vector. However, the terms library and library of vectors can also be used interchangeably.
[0033] The term "a library of anti-RhD antibody expression vectors" refers to a collection of variant anti-RhD antibody-encoding nucleic acid sequences inserted into a vector carrying regulatory elements for transcription of the anti-RhD antibodies. The regulatory elements can either be Located in the inserted nucleic acid segments or in the vector framework. Preferably the anti-RhD antibody expression vectors also carry at least one recombinase recognition sequences, e.g. a FRT site, it may also carry two different recombinase recognition sequences such as a FRT and a FRT site.
[0034] The term "a majority of the individual cells" refers to a percentage of the cells such as more than 80%, preferably more than 85%, more preferably 90%, 95%, or even 99% or higher.
[0035] The term "mass transfer" or "transfer in-mass" is used to describe the transfer of nucleic acid segments of interest from one population of vectors to another population of vectors and doing so for each nucleic acid segments simultaneously without resorting to isolation of the individual segments of interest. Such populations of vectors can be libraries containing for example variable regions, promoters, leaders or enhancing elements of interest. These sequences can then be moved without prior isolation from for example a phage vector to a mammalian expression vector. Especially for antibody sequences this technique ensures that the linkage between VH and VL diversity is not lost while moving libraries from, for example, a selection vector (e.g., a phage display vector) to a mammalian expression vector. Hereby the original pairing of VH and VL is retained.
[0036] As used herein, the term "operably linked" refers to a segment being linked to another segment when placed into a functional relationship with the other segment. For example, DNA encoding a signal sequence is operably linked to DNA encoding a polypeptide if it is expressed as a leader that participates in the transfer of the polypeptide to the endoplasmic reticulum. Also, a promoter or enhancer is operably linked to a coding sequence if it stimulates the transcription of the sequence.
[0037] The term "polyclonal antibody" describes a composition of different (diverse) antibody molecules which is capable of binding to or reacting with several different specific antigenic determinants on the same or on different antigens. Usually, the variability of a polyclonal antibody is located in the so-called variable regions of the polyclonal antibody, in particular in the CDR regions. When stating that a member of a polyclonal antibody binds to an antigen, it is herein meant a binding having binding constant that is below 1 mM, preferably below 100 nM, even more preferred below 10 nM.
[0038] The term "recombinant polyclonal manufacturing cell line" refers to a mixture/population of protein expressing cells that are transfected with a library of variant nucleic acid segments of interest such that the individual cells, which together constitute the recombinant polyclonal manufacturing cell line, each carry only one transcriptionally active copy of a distinct nucleic acid segment of interest, which encodes one member of the recombinant polyclonal antibody of interest, and that each copy is integrated into the same site of the genome of each cell. The cells constituting the recombinant polyclonal manufacturing cell line are selected for their ability to retain the integrated copy of the distinct nucleic acid segment of interest, for example by antibiotic selection. Cells which can constitute such a manufacturing cell line can be for example bacteria, fungi, eukaryotic cells, such as yeast, insect cells or mammalian cells, especially immortal mammalian cell lines such as CHO cells, COS cells, BHK cells, myeloma cells (e.g., Sp2/0 cells, NS0), NIH 3T3, YB2/0 and immortalized human cells, such as HeLa cells, HEK 293 cells, or PER.C6.
[0039] The term "recombinant antibody" is used to describe an antibody molecule or several molecules that is/are expressed from a cell or cell line transfected with an expression vector comprising the coding sequence of the protein which is not naturally associated with the cell, if the antibody molecules are diverse or different, the term "recombinant polyclonal antibody" applies in accordance with the definition of a polyclonal antibody.
[0040] The term "recombinase" refers to an enzyme that catalyses recombination between two or more recombination sites or recombination recognition sequences. Recombinases useful in the present invention catalyze recombination at specific recombination sites that are specific nucleic acid sequences recognized by a particular recombinase.
[0041] The terms "recombinase recognition site" or "recombination site" describe a nucleic acid sequence which serves as site for both recognition and recombination by a site-specific recombinase enzyme. A recombinase recognition site is generally comprised of short inverted repeat elements (11-13 bp in length) that flank a core sequence (6-8 bp in length). Recombinase recognition sites are also termed recombinase target sites, recombination sites or integration sites and include as examples the FLP-site, loxP-site, attP/attB-sites, six-site, gix-site, R-site and Res-site. Recombinase recognition sites between which a recombinase can catalyze an integration, excision or inversion event are termed matching recombinase recognition sites, for example are two wild type FRT sites considered to match, as well as an attB site and an attP site constitute a matching pair of recombinase recognition sites, whereas, a wildtype FRT site and a mutant FRT site will not necessarily constitute a matching pair of recombinase recognition sites; this will depend on the mutation. These terms are also used interchangeably with the term integration site.
[0042] The term "RFLP analysis" refers to "restriction fragment length polymorphism analysis", a method whereby the migratory gel pattern of nucleic acid molecule fragments is analyzed after cleavage with restriction enzymes.
[0043] The term "scrambling" describes situations where two or more distinct members of a polyclonal protein, where each member is comprised of two different polypeptide chains, e.g. VH and VL chains, is expressed from an individual cell. This situation may arise when the individual cell has integrated into the genome, more than one pair of genetic elements, where each pair of genetic elements encodes a distinct member of the polyclonal protein. In such situations unintended combinations of the polypeptide chains expressed from the genetic elements can be made, "VH-VL chain scrambling" is an example of the scrambling defined above. The scrambling occurs when unintended combinations of VH and VL polypeptides are produced from a cell where two different VH and VL-encoding nucleic acid segments are integrated into transcriptional active sites in the same cell. Such a scrambled antibody molecule is not likely to retain the original specificity, and thus might not have any therapeutic effect.
[0044] The term "selection" is used to describe a method where cells have acquired a certain characteristic that enable the isolation from cells that have not acquired that characteristic. Such characteristics can be resistance to a cytotoxic agent or production of an essential nutrient, enzyme, or color.
[0045] The terms "selectable marker gene", "selection marker gene", "selection gene" and "marker gene" are used to describe a gene encoding a selectable marker (e.g., a gene conferring resistance against some cytotoxic drug such as certain antibiotics, a gene capable of producing an essential nutrient which can be depleted from the growth medium, a gene encoding an enzyme producing analyzable metabolites or a gene encoding a colored protein which for example can be sorted by FACS) which is co-introduced into the cells together with the gene(s) of interest.
[0046] The term "transfection" is herein used as a broad term for introducing foreign DNA into a cell. The term is also meant to cover other functional equivalent methods for introducing foreign DNA into a cell, such as e.g., transformation, infection, transduction or fusion of a donor cell and an acceptor cell.
[0047] As used herein, the term "vector" refers to a nucleic acid molecule into which a nucleic acid sequence can be inserted for transport between different genetic environments and/or for expression in a host cell. A vector capable of integrating into the genome of a host cell at a pre-determined, specific locus in the genome is herein named "a vector for site-specific integration". If the vector carries regulatory elements for transcription of the nucleic acid sequence inserted in the vector (at least a suitable promoter), the vector is herein called "an expression vector". The term "an isotype-encoding vector" refers to a vector carrying nucleic acid sequences encoding an antibody isotype. In the present specification, "phagemid vector" and "phage vector" are used interchangeably. The terms "plasmid" and "vector" are used interchangeably. The invention is intended to include such other forms of vectors, which serve equivalent functions for example plasmids, phagemids and virus genomes or any nucleic acid molecules capable of directing the production of a desired protein in a proper host.
[0048] The following style of writing "VH:LC" and "VH:VL" indicate a particular pair of a variable heavy chain sequence with a light chain or a variable light chain sequence. Such particular pairs of VH and VL sequences can either be nucleic acid sequences or polypeptides. In the present invention particular VH and VL pairs confer binding specificity towards the rhesus D antigen.
[0049] Abbreviations: Ab=antibody. Anti-RhD rpAb=anti-Rhesus D recombinant polyclonal antibody. CASY=Cell Counter+Analyzer System. ELISA=Enzyme-Linked Immunosorbent Assay. FRT=Flp Recombinase Target. GFP=Green Flourescent Proteins. HDN=hemolytic disease of the newborn. ITP=idiopathic thrombocytopenic purpura. LTR=Long Terminal Repeat mAb=monoclonal antibody pMCB=polyclonal master cell bank, PVDF=polyvinylidene difluorid. pWCB=polyclonal working cell hank. RBC=red blood cells. RhD=Rhesus D, RhD(-)=Rhesus D negative. RhD(+)=Rhesus D positive. RhDVI=Rhesus D category VI antigen. AntiD=polyclonal immunoglobulin preparation against RhD from hyperimmune donors. SV40 poly A=Simian Virus 40 poly A signal sequence. UCOE=ubiquitous chromatin opening elements. 5'UTR=5' untranslated region of the mRNA.
DESCRIPTION OF THE DRAWINGS
[0050] FIG. 1A: Flow chart outlining the generation of a recombinant polyclonal manufacturing cell line and the production of a recombinant polyclonal antibody, 1) Illustrates a bulk transfection strategy; 2) illustrates a semi-hulk transfection strategy and 3) illustrates an individual transfection strategy. A) Illustrates the library of anti-RhD antibody expression vectors (horizontal lines), the arrowheads illustrate the grouping of the vectors. In strategy 1 the vectors are grouped in bulk, in strategy 2 they are grouped in smaller fractions (semi-bulk), whereas in strategy 3 they are kept separate from each other (individual). B) Illustrates the transfection, where the number of tubes depends on the grouping of the vectors constituting the library. C) Illustrates selection of cells that site-specifically have integrated an anti-RhD antibody-encoding nucleic acid segment into the host cell genome, D) illustrates the generation of a polyclonal anti-RhD antibody library stock, where the selected cells constituting the integrated anti-RhD antibody-encoding nucleic acid segments are stored in a freezer. It is optional to bank individual clones or pool the clones. E) Illustrates the beginning of the manufacturing phase, where clones from the stock are thawed (either individually, from smaller fractions or from a pool). F) Illustrates the stage in the production where the polyclonal cell line is propagated for seeding of a larger bioreactor (intermediate seeding steps are an option although not illustrated). In strategy 2 and 3, this is the stage where the polyclonal cell clone stock no longer is kept as individual clones or semi-bulk fractions, but pooled into a collection of cells, forming a recombinant polyclonal manufacturing cell line (this polyclonal manufacturing cell line may also be stored as a frozen stock). G) Illustrates the final production obtained from the bioreactor manufacturing. Following the production phase, the polyclonal protein composition is harvested for purification and characterization of the product.
[0051] FIG. 1B: Flow chart outlining the generation a pWCB/pMCB and a sub-pWCB from individually transfected host cells and the seeding of a polyclonal manufacturing cell line. A) Illustrates a library comprised of variable region-encoding nucleic acid segments, the arrowheads illustrate the individual members of the library. B) Illustrates the transfection, where each individual member of the library is used to transfect a host cell. The transfection requires as many separate tubes as there are individual members of the library. C) Illustrates selection of cells that have integrated a variable region-encoding nucleic acid segment into their genome in a stable manner, D) Illustrates the selection of individual cell lines that have similar proliferation rates and/or productivity, e.g. by cloning and analysis of single cells sorted by FACS. This step is optional in the generation of a pWCB/pMCB and may also be performed after step E. E) Illustrates the generation of a frozen library stock, constituted of n times individual cell lines each expressing one member of the library comprised of variable region-encoding nucleic acid segments used for transfection. It is optional to bank individual clones into a frozen library stock prior to the generation of a pWCB/pMCB. F) Illustrates the mixing of the individual cell lines, where ampoules from the individual library stock are thawed and expanded in separate cell cultures, followed by the mixing of a predefined number of cells from each culture into a single cell culture. G) Illustrates generation of a pWCB/pMCB by freezing down aliquots from the mixed cell culture in F, thereby generating a collection of vials. H) Illustrates the generation of a sub-pWCB by expanding a single vial from the pMCB and freezing down aliquots with approximately the same number of cells as in the vial from the pMCB. I) Illustrates the generation of a polyclonal manufacturing cell line from a seed train (intermediate seeding steps which are not illustrated) initiated either from the pWCB or the sub-pWCB.
[0052] FIG. 2: Phage display vector: Em351, an E. coli vector used to generate an anti-RhD Fab phage display library by inserting heavy chain variable region and the light chain fragments amplified from a suitable donor into the vector at the indicated AscI/XhoI and NheI/NotI restriction sites, respectively. The vector comprises the following elements: pro Amp and Amp=promoter and ampicillin resistance gene. pUC Ori=origin of replication. Human CH1=sequence encoding human immunoglobulin gamma 1 heavy chain domain 1. Stuffer=irrelevant sequence inserts which are cut out during insertion of the heavy and light chain fragments. p tac and p lac Z=bacterial promoters. PelB=modified bacterial PelB leaders for targeting expression of the Fab to the periplasmic space of the E. coli. Mycut=proteinase recognition site. Amber stop=amber stop codon. gIII=phage M13 truncated geneIII (from by 198 to the C-terminal).
[0053] FIG. 3A-C: Alignment of the nucleic acid sequences encoding the variable heavy chain (VH) of the 56 selected RhD clones. The individual clone names are indicated to the right of the alignment, and the position of CDR regions are indicated above the alignments.
[0054] FIG. 4A-E: Alignment of the nucleic acid sequences encoding the entire light chain of the 56 selected RhD clones. The individual clone names together with an indication of whether it is a Kappa or Lambda chain are indicated to the right of the alignment, and the position of CDR regions are indicated above the alignments.
[0055] FIG. 5: Alignment of the amino acid sequences corresponding to VH of the 56 selected RhD clones. The individual clone names are indicated to the right of the alignment, and the position of CDR regions are indicated above the alignments.
[0056] FIG. 6A-B: Alignment of the amino acid sequences corresponding to VL of the 56 selected RhD clones, wherein (A) corresponds to the Kappa chains and (B) to the Lambda chains. The individual clone names are indicated to the right of the alignment, and the position of CDR regions are indicated above the alignments.
[0057] FIG. 7: Neo exp. vector: Schematic representation of the mammalian expression vector used to facilitate site-specific integration into the genome of a host cell of the anti-RhD antibody-encoding nucleic acid segments. The vector comprises the following elements: pro amp and AMP=promoter and ampicillin resistance gene. pUC origin=pUC origin of replication. Restriction enzyme sites: XhoI, AscI, NheI and NotI. P1/P2=promoter set driving the expression of the light chain and IgG heavy chain, respectively. LH=heavy chain leader sequence. VH=Sequence coding for the variable heavy chain of an anti-RhD Ab. Human IgG1 constant heavy=Sequences coding for the human constant IgG1 heavy chain. RBG polyA=Rabbit β-globin polyA signal sequence. BGH polyA=Bovine Growth Hormone polyA signal sequence. LK=kappa chain leader sequence. Light chain=Sequence coding for the light chain of an anti-RhD Ab. FRT site=Flp recombinase recognition sequence. Neomycin=Neomycin resistance gene. SV40 poly A=Simian virus 40 polyA signal sequence.
[0058] FIG. 8: Cation exchange chromatograms of anti-RhD rpAb composition from aliquots 3948 and 3949 after 9 weeks cultivation. The lower diagram corresponds to aliquot 3949 and the upper one to aliquot 3948. The Y-axis of the top diagram has been displaced in order to separate it from the lower diagram. Peaks A-J comprise antibodies differing in net charge and individual antibodies appearing charge heterogeneous.
[0059] FIG. 9: Gel picture showing HinfI RFLP analysis on RT-PCR product derived from polyclonal cell line aliquots 3948+ and 3949+ (FCW065) producing anti-RhD rpAb after 11 weeks cultivation. Bands which can be assigned to specific clones are identified.
[0060] FIG. 10: T-RFLP patterns of anti-Rhesus D antibody light chains from a polyclonal cell culture expressing anti-RhD rpAb with eight different anti-Rhesus D antibodies. The eight different anti-Rhesus D clones have been assigned to the peaks indicated by arrows.
[0061] FIG. 11: T-RFLP patterns of anti-Rhesus D antibody heavy chain variable regions from a polyclonal cell culture expressing anti-RhD rpAb with twenty-five different anti-Rhesus D antibodies at a given time point. The twenty-five different anti-Rhesus D clones have been assigned to the peaks indicated by arrows.
[0062] FIG. 12: cDNA distribution estimated by T-RFLP of eight different anti-Rhesus D heavy chain-encoding sequences from a polyclonal cell culture which was cultivated for five weeks.
[0063] FIG. 13: Shows the relative content (%) of an anti-RhD rpAb with eight different antibodies analyzed using cation-exchange chromatography. Integrated chromatographic peaks were assigned to individual antibodies from the retention times and peak patterns obtained from single antibodies analyzed individually using cation-exchange chromatography under identical conditions.
[0064] FIG. 14: Cation-exchange chromatogram of an anti-RhD rpAb with twenty-five individual members from a sample obtained after 4 weeks cultivation. Peaks AC1 to 25 comprise antibodies differing in net charge and individual antibodies appearing charge heterogeneous.
[0065] FIG. 15: (A) Shows a comparison of the potency of three batches, Sym04:21, Sym04:23, and Sym04:24, of anti-RhD pAb with 25 individual members, produced by fed batch cultivation in 5 L scale. Binding of pAb to RhD-positive erythrocytes was measured by FACS and the mean fluorescence intensity (MFI) is shown as a function of pAb concentration in ng/ml. Further, the functional activity of an anti-RhD pAb with 25 individual members was measured on Sym04:21 and Sym04:24 in a combined ADCC/phagocytosis assay. (B) Shows the ADCC results as percentage of specific lysis of RhD-positive and RhD-negative erythrocytes as a function of pAb concentration in ng/ml. (c) Shows the percentage of phagocytosis of RhD-positive and RhD-negative erythrocytes as a function of pAb concentration in ng/ml.
[0066] FIG. 16: Cation-exchange chromatography profiles showing samples taken at different stages during down-stream processing of an anti-RhD rpAb sample containing 25 individual members represented by material collected following capture elution (A), Sephadex G-25 (B), DEAE-Sepharose (C), and MEP Hypercel (D)
DETAILED DESCRIPTION OF THE INVENTION
The Recombinant Polyclonal Protein Expression System
[0067] The present invention provides a recombinant polyclonal antibody expression system for the consistent manufacturing of anti-RhD recombinant polyclonal antibody (anti-RhD rpAb) from one or a few cell lines.
[0068] One of the major advantages of the manufacturing method of the present invention is that all the members constituting the anti-RhD rpAb can be produced in one or a few bioreactors or equivalents thereof. Further, the anti-RhD rpAb composition can be purified from the reactor as a single preparation without having to separate the individual members constituting the anti-RhD rpAb during the process. In contrast, if one wanted to mimic an anti-RhD rpAb composition by mixing purified anti-RhD monoclonal antibodies (anti-RED mAbs) (as for example proposed in WO 97/49809) it would require the separate manufacturing in a bioreactor, of each anti-RhD mAb to be included in the composition and most likely the antibodies would be purified individually as well. Such a production of an anti-RhD mAb mixture would be very costly, and time and space consuming compared to the method of the present invention for producing an anti-RhD recombinant polyclonal. Thus, the method as described in WO 97/49809 would naturally result in a practical limit to the number of anti-RhD mAbs that could be included in such a mixture, whereas the technology as described herein generally can produce a polyclonal antibody with as many individual members as desired. Further, the individual members of an anti-RhD rpAb of the present invention are produced under exact same conditions (in the same manufacturing reactor), thus uniform posttranslational modifications are ensured compared to a mixture of anti-RhD mAbs where slight production differences from batch to hatch may change the product properties.
[0069] In order to obtain a recombinant polyclonal manufacturing cell line which is capable of expressing anti-RhD rpAb without significant loss of the diversity characterizing the polyclonality during the production period, the individual cells within the mixture of cells composing the polyclonal manufacturing cell line will need to be as uniform as possible.
[0070] Conventional monoclonal antibody expression techniques using random integration are undesirable for the production of a recombinant polyclonal antibody, since the random nature of the process will cause the number and positions of the integrated nucleic acid sequences to vary from cell to cell. Thus, if recombinant polyclonal antibody is produced by such traditional protocols, it is likely to result in a heterogeneous cell culture with variable expression rates of individual members of the polyclonal protein, and genetic instability due to positional effects of the integrated nucleic acid segment. This will most likely result in a biased expression of the members constituting the polyclonal protein.
[0071] Introduction of the anti-RhD antibody-encoding nucleic acid segment into a predefined genomic site is therefore desirable, this can in principle be achieved by homologous recombination. However, owing to the dominance of illegitimate recombination events, homologous recombination is very inefficient and may also result in introduction of several copies of variant anti-RhD antibody-encoding nucleic acid segments into the genome of a single cell.
[0072] To circumvent these problems the expression system of the present invention uses site-specific integration into the genome of the individual host cells. The system of the present invention encompasses a library of anti-RFD antibody expression vectors for site-specific integration comprising the variant nucleic acid segments encoding the anti-RhD rpAb. Individual nucleic acid segments from the library are inserted into individual cells at the same pre-established chromosomal location by site-specific integration at a predefined recombination recognition site or by a recombinase-mediated cassette exchange procedure, thereby generating a cell line, wherein the individual cells expresses a distinct member of the anti-RhD rpAb. As described below, multiple integrations might occur in some of the cells constituting the recombinant polyclonal manufacturing cell line. This, however, is not considered to pose a problem as long as a majority of the individual cells express a single distinct member of the anti-RhD rpAb. Preferably this is achieved by ensuring a single integrant in the genome of the majority of the individual cells or if there are more integrants, ensuring that only one is transcribed.
[0073] Recombinases such as Cre, Flp, beta-recombinase, Gin, Pin, PinB, PinD, R/RS, Tn3 resolvase, XerC/D integrase/recombinase, lambda integrase, or phage ΦC31 integrase can be used. Suitable recombinases for integration into the chromosomal location can be provided either (i) by expression from the cell's own genome into which said nucleic acid segment is introduced, (ii) by being operatively encoded by the vector inserted into the cell, (iii) through expression from a second nucleic acid molecule, or (iv) as a protein. In a preferred embodiment, the anti-RhD antibody-encoding nucleic acid segment contained in an individual vector of the library is integrated into a locus that mediates high-level transcription and expression of the anti-RhD antibody nucleic acid segment, a so-called "hot-spot".
[0074] The host cell line used is preferably a mammalian cell line comprising those typically used for biopharmaceutical protein expression, e.g., CHO cells, COS cells, BHK cells, myeloma cells (e.g., Sp2/0 cells, NS0), YB2/0, NIH 3T3, and immortalized human cells, such as HeLa cells, HEK 293 cells, or PER.C6. In the present invention CHO cells were used. However, a person of ordinary skill in the art would easily be able to substitute CHO cells with other mammalian cells as described, or even utilize other types of cells, including plant cells, yeast cells, insect cells, fungi and bacteria. Thus, the choice of cell type is not intended to be limiting to the invention. In a preferred embodiment, a mammalian cell line containing a pre-characterized hot-spot, mediating high expression levels of the anti-RhD rpAb is used for the manufacture. In an even more preferred embodiment, the mammalian cell line contains a single recombinase recognition site located in a pre-identified hot-spot.
[0075] In a further embodiment of the present invention, variant anti-RhD antibody-encoding nucleic acid segments are integrated in a site-specific manner utilizing the same chromosomal integration site in the host cells. Such incorporation into a single specific site minimizes positional effects otherwise seen with random integration or integration into multiple sites in a genome. Further, scrambling among VH and VL chains is not likely to occur when using a single specific site for integration.
[0076] In a host cell line comprising a site-specific integration system, the individual transfected host cells are expressing the same overall antibody apart from the differences observed in the variable region of the antibody. Therefore, a majority of cells within such a pool of cells should display similar characteristics with respect to productivity and genetic stability and hence this technology offers the possibility of a controlled production of an anti-RhD rpAb.
[0077] In addition to the variability of the VH and VL regions, in particular the CDR regions, the constant regions may also be varied with respect to isotype. This implies that one particular VH and VL pair may be produced with varying constant heavy chain isotypes, e.g. the human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD and IgE. Thus, an anti-RhD rpAb may comprise antibody molecules that are characterized by sequence differences between the individual antibody molecules in the variable region (V region) as well as in the constant region. The anti-RhD rpAb composition can be composed of antibodies with any heavy chain isotype mentioned above or combinations thereof. Preferred anti-RhD rpAb compositions contain IgG1 constant regions, IgG3 constant regions or IgG1 and IgG3 constant regions. In a preferred embodiment of the present invention each or some of the VH and VL pairs are expressed with a human IgG1, IgG3, IgA1 and/or IgA2 constant heavy chain.
[0078] In order to provide a library of anti-RhD antibody-encoding nucleic acid segments a number of methods known in the art may be utilized. A first library comprising VH and VL-encoding segments may either be generated by combinatorial techniques (e.g. EP 0 368 684) or techniques maintaining the cognate pairing (pairs of variable region-encoding sequences derived from the same cell, described in WO 05/042774 claiming the priority of the unpublished patent application DK 200400782). Further, VH and VL-encoding segment libraries may be generated by incorporating isolated CDR gene fragments, into an appropriate framework (e.g. Soderlind, E. et al., 2000. Nat. Biotechnol. 18, 852-856), or by mutation of one or more anti-RhD VH. and VL-encoding sequences. This first library is screened for VH and VL-encoding nucleic acid segments producing antibodies or fragments with binding specificity towards RhD, thereby generating a library of anti-RhD Ab-encoding nucleic acid segments. In particular with combinatorial libraries the screening is preceded by an enrichment step for example a so-called biopanning step. Known biopanning technologies are phage display (Kang, A. S. et al. 1991. Proc Natl Acad Sci USA 88, 4363-4366), ribosome display (Schaffitzel, C. et al. 1999. J. Immunol. Methods 231, 119-135), DNA display (Cull, M. G. et al. 1992, Proc Natl Acad Sci USA 89, 1865-1869), RNA-peptide display (Roberts, R. W., Szostak, J. W., 1997. Proc Natl Acad Sci USA 94, 12297-12302), covalent display (WO 98/37186), bacterial surface display (Fuchs, P. et al. 1991. Biotechnology 9, 1369-1372), yeast surface display (Boder, E. T., Wittrup, K. D., 1997. Nat Biotechnol 15, 553-557) and eukaryotic virus display (Grabherr, R., Ernst, W., 2001. Comb. Chem., High Throughput. Screen. 4, 185-192). FACS and magnetic bead sorting are also applicable for enrichment (panning) purposes using labeled antigen. The screening for Rhesus D binders are generally performed with immunodetection assays such as agglutination, FACS, ELBA, ELISA and/or immunodot assays.
[0079] Following screening, the generated sub-library of VH and VL-encoding nucleic acid segments, generally needs to be transferred from the screening vector to an expression vectors suitable for site-specific integration and expression in the desired host cell. It is important that the sequences encoding the individual VH:VL pairs are maintained during the transfer. This can either be achieved by having the individual members of the sub-library separate and moving VH and VL-encoding sequences one by one. Alternatively, the vectors constituting the sub-library are pooled, and the sequences encoding the VH:VL pairs are moved as segments, keeping the VH and VL-encoding sequences together during the transfer. This process is also termed mass transfer, and enables an easy transfer of all the selected VH:VL pairs from one vector to another.
[0080] In a further embodiment of the present invention, an anti-RhD recombinant polyclonal antibody composition comprises a defined subset of individual antibodies, based on the common feature that they exhibit binding to at least one epitope on the Rhesus D antigen e.g. epD1, epD2, epD3, epD4, epD5, epD6/7, epD8 and/or epD9, but not or very weakly to Rhesus C, c, E, e antigens. Preferably the anti-RhD rpAb composition is composed of at least one antibody which bind to epi)3, epD4 and epD9 (RhD category VI antigen binding antibody) and further antibodies which at least in combination binds to the remaining epitopes epD1, epD2, epD5, epD6 7 and epD8, e.g. an antibody against RhD category II or III antigen, or a RhD category IV or V antigen binding antibody combined with an antibody against category VII antigen. Typically an anti-RhD rpAb composition has at least 5, 10, 20, 50, 100 or 500 distinct variant members. The preferred number of variant members range between 5 and 100, even more preferred between 5 and 50 and most preferred between 10 and 25.
[0081] A further embodiment of the present invention is a recombinant polyclonal manufacturing cell line, comprising a collection of cells transfected with a library of anti-RhD polyclonal antibody-encoding nucleic acid segments, wherein each cell in the collection is capable of expressing one member of the library, which encodes a distinct member of an anti-RhD rpAb or fragment and which is located at the same site in the genome of individual cells in said collection, wherein said nucleic acid segment is not naturally associated with said cell in the collection.
[0082] In an additional embodiment the variant nucleic acid segments encoding the anti-RhD rpAb are all derived from naturally occurring sequences, for example isolated from a donor, either as combinatorial VH:VL pairs or as cognate pairs, and not derived by mutation.
[0083] Compositions of cells that contain variant nucleic acids located at a single specific site in the genome within each cell have been described in WO 02/44361. This document discloses the use of the cells to identify molecules having desirable properties, but the reference does not deal with the provision of a production system or with the provision of polyclonal antibody characterized by a specific binding to an antigen.
The Host Cell
[0084] A suitable host cell comprises, in a region of its genome, one or more suitable recombination sites, i.e., nucleic acid sequences recognizable by one or more recombinase enzymes, hence also termed recombinase recognition sequences. To be able to select for integrants, (i.e., cells having an integrated copy of an anti-RhD antibody-encoding nucleic acid segment in an integration site) the recombination site is operably linked to a first selection gene (e.g., an antibiotic resistance gene) situated 3' (downstream) to the recombination site. Furthermore, a weak promoter (e.g., a truncated SV40 early promoter) and a transcription start codon may be situated 5 (upstream) to the recombination site that constitutes an integral part of the resistance marker-coding region. Thus, the transcription start codon initiates the start of transcription of the selection gene in the host cell before transfection with the library of anti-RhD antibody expression vectors encoding the anti-RhD rpAb. Preferably, the host cell line only has one recombination site, and if it has more than one recombinase recognition sequence, these should be non-homologous as described in the section "The vector for site-specific integration", only allow for a single integration into the genome.
[0085] Host cells for site-specific integration as described above can be generated from any cell which can integrate DNA into their chromosomes or retain extra-chromosomal elements such as mini-chromosomes, YACs (Yeast artificial chromosomes), MACs (Mouse artificial chromosomes), or HACs (Human artificial chromosomes). MACs and HACs are described in detail in WO 97/40183, hereby incorporated by reference. Preferably mammalian cells such as CHO cells, COS cells, BHK cells, myeloma cells (e.g., Sp2/0 or NS0 cells), fibroblasts such as NIH 3T3, and immortalized human cells, such as HeLa cells, HEK 293 cells, or PER.C6, are used. However, non-mammalian eukaryotic or prokaryotic cells, such as plant cells, insect cells, yeast cells, fungi. E. coli etc., can also be employed.
[0086] In one embodiment of the present invention, the cell line which is to be used as starting material is sub-cloned by performing a so-called limiting dilution of the cell line down to a single cell level, followed by growing each single cell to a new population of cells prior to transfection with the library of vectors of interest. Such sub-cloning can also be performed later in the process of selecting the right cell line, if desired.
[0087] The host cells for site-specific integration may be obtained by transfection with a randomly integrating plasmid comprising a weak promoter (e.g., a truncated SV40 early promoter), a transcription start codon, a recombination site situated 3' to the start codon. Preferably, the integrating plasmid also comprises a marker gene coupled to a first selection gene. One example of such an integrating plasmid is the pFRT/LacZeo2 from Invitrogen (Carlsbad, Calif.). The marker gene can be used to evaluate the relative strength of expression at the genomic location used for inserting a nucleic acid sequence of interest. A marker gene, (e.g., beta-galactosidase (LacZ), green fluorescent protein (GFP) or a cell surface marker) can be linked to the first selection gene in a gene fusion or transcriptionally linked by an IRES (internal ribosomal entry site) such that co-expression of the first selection gene and marker gene occurs. The use of a selection gene that establishes a survival pressure on the cells (e.g. drug resistance or nutritional depletion) combined with a marker allowing for evaluation of the relative expression levels from cell line to cell line is an efficient method to ensure high producing cells which maintain the integrated plasmid within the genome. Cells with the recombination sequence inserted at a spot with particularly active transcription will lead to high expression of the marker gene e.g. GFP or LacZ. High expressers can be selected by fluorescence activated cell sorting (FACS) and cloned. At this point it should also be analyzed whether the integrant is a single integrant. This can be performed by real-time PCR and Southern blotting. The preparation of cells having an FRT site at a pre-determined location in the genome was described in e.g. U.S. Pat. No. 5,677,177.
[0088] Another method for evaluating relative expression levels from cells transfected with an integrating plasmid is to perform an additional integration-excision step on the cells generated as described above. This pool of selected cells are transfected again, with a plasmid encoding a recombinase corresponding to the recombination site of the integrating plasmid and a second plasmid containing a second selection marker without a start codon, the coding region of which is preceded by a recombination sequence likewise corresponding to the first integrating plasmid. This second plasmid also contains the coding sequence for a fluorescent marker protein (e.g., GFP (or equivalent fluorescent proteins) driven by a suitable promoter. The recombinase mediates integration of this plasmid into the host cell genome where a similar recombination sequence previously has been inserted by the integrating plasmid. Cells with the recombination sequence inserted at a spot with particularly active transcription will lead to high expression of the fluorescent protein. High expressers are selected by fluorescence activated cell sorting (FACS) and cloned. Clones with consistently high expression and containing one copy of the inserted plasmid are transfected with the recombinase and selected by the first selection marker, identifying cells where the second plasmid sequence has been removed by the recombinase, making the first selection marker work again. These cells still contain the first recombination sequence inserted at a transcriptional hot-spot and can now be used for the expression of genes of interest.
[0089] Cell lines, which achieve high expression of the marker gene upon integration of a single copy of the plasmid, are used for transfection with the anti-RhD antibody expression library. The recombination site in the host cell is preferably located in a gene or region of particularly active expression, i.e., in a so-called hot-spot.
The Vector for Site-Specific Integration
[0090] A suitable vector comprises a suitable recombination site linked to a suitable selection gene different from the selection gene used for construction of the host cell. Suitable selection genes for use in mammalian cell expression include, but are not limited to, genes enabling for nutritional selection, such as the thymidine kinase gene (TK), glutamine synthetase gene (GS), tryptophan synthase gene (trpB) or histidinol dehydrogenase gene (hisD). Further, selection markers are antimetabolite resistance genes conferring drug resistance, such as the dihydrofolate reductase gene (dhfr) which can be selected for with hypoxanthine and thymidine deficient medium and further selected for with methotrexate, the xanthine-guanine phosphoribosyltransferase gene (gpt), which can be selected for with mycophenolic acid, the neomycin phosphotransferase gene (neo) which can be selected for with G418 in eukaryotic cells and neomycin or kanamycin in prokaryotic cells, the hygromycin B phosphotransferase (hyg, hph, hpt) gene which can be selected for with hygromycin, the puromycin N-acetyl-transferase gene (pac) which can be selected for with puromycin or the Blasticidin S deaminase gene (Bsd) which can be selected for with blasticidin. Finally, genes encoding proteins that enables sorting e.g. by flow cytometry can also be used as selection markers, such as green fluorescent protein (GFP), the nerve growth factor receptor (NGFR) or other membrane proteins, or beta-galactosidase (LacZ).
[0091] In one aspect of the present invention, the selectable gene is neither preceded by a promoter nor equipped with a translation initiating codon. The promoter and ATG codon is provided at the selected site-specific recombination site. If the vector is integrated at a location other than the selected recombination site in the genome of the host cell, no expression of this second selection gene can occur due to lack of promoter and initiation codon. If integration occurs at the selected recombination site in the genome of the host cell, the second selection gene is expressed and expression of the first selection gene is lost.
[0092] Integration may e.g., be carried out using a so-called FRT site/Flp recombinase recognition sequence (5'-gaagttcctattccgaagttcctattactagaaagtataggaacttc-3' (SEQ ID NO 1) or variants thereof) in the genome and on the vector for site-specific integration together with the Flp recombinase or mutants thereof from Saccharomyces cerevisiae. However, other recombinase systems may equally well be used, including those of Cre recombinase and a variety of lox sites such as loxP from bacteriophage P1 or variants or mutants thereof, e.g., lox66, lox71, lox76, lox75, lox43, lox44 and lox511 (C. Gorman and C. Bullock, Curr. Opinion in Biotechnology 2000, 11: 455-460) or by using phage integrase ΦC31 or lambda integrase, which carries out recombination between the attP site and the attB site (A. C. Groth et al. PNAS 2000, 97: 5995-6000). Further recombinase systems that could be utilized in the present invention are, but are not limited to, the β-recombinase-six system from bacterial plasmid. pSM19035 (Rojo and Alonso 1995), the Gin-gix system from bacteriophage Mu (Crisona et al 1994), the R-RS system from Zygosaccharomyces rouxii (Onouchi et al 1995), or Tn3 resolvase which recognize res recombination sites (Stark et al 1994) or the XerC/D system from F coli (Blakely and Sherratt 1994).
[0093] A further variant of the site-specific recombination system, termed recombinase cassette mediated exchange (RMCE),uses non-homologous recombination sites. In such a system, two non-identical recombination sites are introduced into the host genome for the generation of specific target sites. Recombination sites corresponding to those flanking the target site also flank the construct containing the gene of interest. Such a system has been described in WO 99/25854, which is hereby incorporated by reference in its entirety. The use of non-homologous recombination sites was shown to suppress excision of the gene of interest from the chromosome. The non-identical recombination sites can be composed of any of the recombination sites described above as long as the corresponding recombinases are provided and the sites cannot recombine with each other. For example, non-identical recombination sites could consist of a FRT site and a mutant FRT site utilizing a Flp recombinase for integration (Schlake and Bode 1994, Biochemistry 33, 12746-12754 a loxP site and a mutant non-compatible loxP site utilizing the Cre recombinase (Langer et al 2002, Nucleic Acids Res. 30, 3067-3077) or a FRT site and a loxP site utilizing Hp and Cre recombinases for the integration (Lauth et al 2002, Nucleic Acids Res. 30, 21, e115).
[0094] Further, a system using two different FRT sites has been described in Verhoeyen et al., Hum. Gene Ther. 2001 12, 933-44. In this approach the integrating plasmid is transferred to the host cells by retroviral infection. The plasmid consists of a combination of a reporter gene and a first selection marker gene as well as the retroviral elements required for infection. The retroviral 3'LTR contains two different FRT sites. A non functional second selection marker gene, which lacks a promoter and the translation initiating codon is located 3' to these sites. During the process of retroviral infection the 3'LTR sequence is copied to the 5'LTR. This results in the flanking of the reporter gene and the first selection marker gene by two different FRT sites on each side. The sequence between the outer FRT sites can be exchanged against an anti-RhD antibody-encoding nucleic acid segment under the control of a strong promoter. The cassette containing the anti-RhD antibody-encoding nucleic acid segment is flanked by the same set of FRT sites. The reaction is catalyzed by the Flp recombinase. In the transfected exchange plasmid an IRES element and a translation initiating codon are located further downstream of the nucleic acid segment. After replacement of the integrated cassette the non functional selection marker gene located in the 3' LTR sequence outside the FRT sites is activated by the translation initiating codon provided by the cassette constituting the anti-RhD antibody-encoding nucleic acid segment. The exchange status can further be enriched if a negative selection marker (e.g. thymidine kinase) is present in the integrating vector.
[0095] The integrating vector can also be transferred to the host cells by standard transfection. In this case the integrating cassette is flanked by an FRT site at the 5' end and a different FRT' site at the 3' end. The ATG-deficient second resistance marker gene is positioned further downstream of the 3' FRT' site. The exchange for an anti-RhD antibody-encoding nucleic acid segment proceeds as described for the retroviral system.
[0096] Another system that prevents excision of the anti-RhD antibody-encoding nucleic acid segment after its site-specific integration into the chromosome is the ΦC31 integrase, also mentioned above. This system has been described thoroughly in patent applications WO 01/07572 and WO 02/08409, hereby incorporated by reference in their entirety.
[0097] Preferably the integrating vector is an isotype-encoding vector, where the constant regions (preferably including introns) are present in the vector prior to insertion of the VH and VL comprising segment from the screening vector. The constant regions present in the vector can either be the entire heavy chain constant region (CH1 to CH3 or to CH4) or the constant region encoding the Fc part of the antibody (CH2 to CH3 or to CH4). The light chain Kappa or Lambda constant region may also be present prior to transfer. The choice of the number of constant regions present, if any, depends on the screening and transfer system used. The heavy chain constant regions can be selected from the isotypes IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD and IgE. Preferred isotypes are IgG1 and/or IgG3.
[0098] Further, the vector for site-specific integration of the anti-RhD antibody-encoding nucleic acid segment contains suitable promoters or equivalent sequences directing high levels of expression of each of the VH and VL chains. Preferably the promoters are of mammalian origin. The VH and VL-encoding sequences are placed as pairs in the vector used for integration (one pair per vector molecule), thereby ensuring that they will be kept together throughout the integration process. Preferably, the promoters are located within the anti-RhD antibody-encoding nucleic acid segment. For bi-directional expression a head-to-head promoter configuration in the expression vector is used (FIG. 7). For unidirectional expression two promoters, one in front of the VH genetic element and one in front of the VL genetic element, or one promoter in front of VH or VL combined with an IRES sequence between the heavy and light genetic elements, can be used to achieve expression.
[0099] A nucleic acid sequence encoding a functional leader sequence can be included in the expression vector to direct the gene product to the endoplasmic reticulum or a specific location within the cell such as an organelle. A strong polyadenylation signal sequence can be situated 3' of the heavy chain and light chain-encoding sequences. The polyadenylation signal ensures termination and polyadenylation of the nascent RNA transcript and is correlated with message stability.
[0100] The expression vector for site-specific integration can carry additional transcriptional regulatory elements, such as enhancers or UCOE (ubiquitous chromatin opening elements) for increased expression at the site of integration. Enhancers are nucleic acid sequences that interact specifically with cellular proteins involved in transcription. The UCOE opens chromatin or maintains chromatin in an open state and facilitates reproducible expression of an operably-linked gene (described in more detail in WO 00/05393, hereby incorporated by reference in its entirety). When one or more of the regulatory elements described in the above are integrated into the chromosome of a host cell they are termed heterologous regulatory elements.
Establishing an Expression System for High-Level Expression of a Polyclonal Protein
[0101] Methods for introducing a nucleic acid sequence into a cell are known in the art. These methods typically include the use of a DNA vector to introduce the sequence of interest into the cell, the genome or an extra-chromosomal element. Transfection of cells may be accomplished by a number of methods known to those skilled in the art, including calcium phosphate precipitation, electroporation, microinjection, liposome fusion, RBC ghost fusion, protoplast fusion, and the like.
[0102] For the transfection of a host cell line, a library of anti-RhD antibody expression vectors, wherein each individual vector comprises one single copy of a nucleic acid segment, encoding a distinct member of the anti-RhD rpAb, is used. This library of anti-RhD antibody expression vectors collectively encodes the anti-RhD rpAb. Suitable vectors for site-specific integration were described in the previous section. The individual vectors constituting the library of anti-RhD antibody-encoding nucleic acid segments can either be mixed together into a single composition, or the individual vectors encoding an individual member of the anti-RhD rpAb can be kept in separate compositions or in mixtures of approximately 5 to 50 individual vectors of the library in a composition.
[0103] The generation of a recombinant polyclonal manufacturing cell line and the production of a recombinant polyclonal antibody from such a cell line can be obtained by several different transfection and manufacturing strategies. These strategies are outlined in FIG. 1A and described in more detail below.
[0104] One way of generating the recombinant polyclonal manufacturing cell line, is to use a library of vectors mixed together into a single composition for the transfection of the host cell line. This method is termed bulk transfection or transfection in bulk (all the individual members of the library are transfected into the host cell line in one tube). Generally, the vector and host cell design previously described will ensure that a polyclonal cell line will be obtained upon appropriate selection. In such a cell line a majority of the individual cells have integrated one copy of a nucleic acid segment, encoding a distinct member of the anti-RhD rpAb from the library of anti-RID antibody expression vectors into the genome. The single copy of the nucleic acid segment is integrated into a single specific site of the genome of each cell in the collection of cells, thereby generating a polyclonal cell line comprised of individual cells expressing individual members of the anti-RhD rpAb. Preferably a frozen stock of the polyclonal cell line is generated before initiation of the anti-RhD rpAb manufacturing.
[0105] Another way of generating the recombinant polyclonal manufacturing cell line is to split the library of anti-RhD antibody expression vectors into fractions, containing approximately 5 to 50 individual vectors of the library before transfection. Preferably, a fraction of the library constitutes 10 to 15 individual vectors. Each composition is then transfected into an aliquot of host cells. This method is termed semi-bulk transfection. The number of aliquots transfected will depend on the size of the library and the number of individual vectors in each fraction. If the library for example constitutes 50 distinct variant members, which are split into fractions containing 10 distinct variant members in a composition, 5 aliquots of host cells would need to be transfected with a library composition constituting a distinct fraction of the original library. The aliquots of host cells are selected for site-specific integration. Preferably, the distinct aliquots are selected separately. However, they can also be pooled before selection. To obtain the desired polyclonal cell line for manufacturing, the aliquots can be mixed before generating the frozen stock, immediately after they have been retrieved from the stock or after a short proliferation time. Optionally, the aliquots of cells are kept separate throughout production, and the polyclonal antibody composition is assembled by combining the products of each aliquot rather than the aliquots of cells before production.
[0106] A third way of generating the recombinant polyclonal manufacturing cell line, is a high throughput method in which host cells are transfected separately using the individual vectors constituting the library of anti-RID antibody expression vectors. This method is termed individual transfection. The individually transfected host cells are preferably selected for site-specific integration separately. However, they can also be pooled before selection. The individual cell clones generated upon selection may be analyzed with respect to proliferation time and integration pattern and preferably, those with similar growth rates and a single site-specific integrant are used to generate a frozen library stock. The individual cell clones can be mixed to obtain the desired polyclonal cell line before generating the stock, immediately after they have been retrieved from the stock, or after a short proliferation time. Alternatively, the individually transfected host cells are mixed even earlier, namely before selection is performed.
[0107] A shared feature in the manufacturing strategies outlined in the above is that all the individual members constituting the anti-RhD rpAb can be produced in one, or a limited number of bioreactors, with approximately 5 to 10 as the maximum. The only difference is the stage at which one chooses to generate the collection of cells that constitutes the recombinant polyclonal manufacturing cell line.
[0108] The host cell line to be used for expression and production of an anti-RhD rpAb has at least one nucleic acid sequence recognizable by a recombinase enzyme. The preparation of such a host cell line was described in the section "The host cell".
[0109] The vector for site-specific integration is preferably integrated in a predefined genomic locus that mediates high-level expression, a so-called hot-spot.
[0110] If expression levels need to be increased, gene amplification can be performed using selection for a DHFR gene or a glutamine synthetase (GS) gene. This requires the use of vectors comprising such a selection marker,
[0111] The following description is one example of how to obtain a recombinant polyclonal antibody manufacturing cell line, where scrambling of the chains is minimal if existing at all.
[0112] Nucleic acid segments containing a universal promoter cassette for constitutive expression having two promoters placed in opposite transcriptional direction, such as a head-to-head construction surrounded by the variable heavy chain and the whole of the kappa light chain is constructed, allowing transfer of the whole construct into a vector for site-specific integration said vector comprising a FRT site and a neomycin resistance gene and the heavy chain constant region. It is contemplated that a promoter cassette for inducible expression can also be used. Furthermore, the promoters can be placed head-to-tail for unidirectional transcription. CHO-Flp-In cells (Invitrogen, Carlsbad, Calif.) which stably express the lacZ-Zeocin fusion gene, are used for the experiment, rendering the cells resistant to the antibiotic Zeocin. The cells are maintained in a suitable media medium containing Zeocin. The cells are co-transfected in bulk with a plasmid expressing the Flp recombinase and the library of anti-RhD antibody expression vectors for site-specific integration encoding the anti-RhD rpAb and a different selection marker (neomycin). After transfection, the cells are cultivated in the presence of neomycin. Cells that exhibit resistance to neomycin are then preferably adapted to growth in suspension as well as serum free conditions, this can be performed in one or two steps and with or without selection pressure. Alternatively, the cells are adapted to grow in suspension under serum free conditions prior to transfection of the cells. When the polyclonal cell line has been adapted to the appropriate conditions scaling up can be initiated using different culture systems, such as conventional small culture flasks, Nunc multilayer cell factories, small high yield bioreactors (MiniPerm, INTEGRA-CELLine, wavebags, BelloCell) and spinner flasks to hollow fiber- and bioreactors. The suitable production time and choice of final bioreactor size are dependent on the desired yield of protein from the batch and expression levels from the cell line. Times might vary from a couple of days up to three month. The cells are tested for antibody production using ELISA. The expressed anti-RhD rpAb is isolated from the supernatant. The anti-RIM rpAb is purified and characterized. Examples of purification and characterization procedures are described later.
Clonal Diversity/Polyclonality
[0113] One of the characteristics of a polyclonal antibody is that it is constituted of a number of individual antibody molecules where each antibody molecule is homologous to the other molecules of the polyclonal antibody, but also has a variability that is characterized by differences in the amino acid sequence between the individual members of the polyclonal antibody. These differences are normally confined to the variable region in particular the CDR regions, CDR1, CDR2 and CDR3. This variability of a polyclonal antibody can also be described as diversity on the functional level, e.g., different specificity and affinity with respect to different antigenic determinants on the same or different antigens located on one or more targets. In a recombinant polyclonal antibody the diversity constitutes a sub-set of the diversity observed in a donor derived immunoglobulin product. Such a sub-set is carefully selected and characterized with respect to its ability to bind desired target antigens, in this particular case the Rhesus D antigen.
[0114] One of the concerns with respect to production of a recombinant polyclonal antibody may be whether the clonal diversity is maintained in the final product. The clonal diversity may be analyzed by RFLP or sequencing of (RT)-PCR products from the cells expressing the anti-RhD rpAb. The diversity can also be analyzed on protein level by functional tests (e.g., ELISA) on the anti-RhD rpAb produced by the cell line, by anti-idiotypic antibodies to individual members or by chromatographic methods.
[0115] Clonal bias, if it exists, can be estimated by comparing the clonal diversity of the initial library, used for transfection, with the diversity found in the pool of cells (polyclonal cell line) expressing the anti-RhD rpAb.
[0116] Clonal diversity of an anti-RhD rpAb can be assessed as the distribution of individual members of the polyclonal composition. This distribution can be assessed as the total number of different individual members in the final polyclonal antibody composition compared to the number of different encoding sequences originally introduced into the cell line during transfection. In this case sufficient diversity is considered to be acquired when at least 50% of the encoding sequences originally used in the transfection can be identified as different individual members of the final anti-RhD rpAb. Preferably at least 75% of the anti-RhD antibody-encoding sequences used for transfection can be identified as antibodies in the final composition. Even more preferred at least 85% to 95%, and most preferred a 100% of the anti-RhD antibody-encoding sequences used for transfection can be identified as antibodies in the final composition.
[0117] The distribution of individual members of the anti-RhD rpAb composition can also be assessed with respect to the mutual distribution among the individual members. In this case sufficient clonal diversity is considered to be acquired if no single member of the composition constitutes more than 75% of the total number of individual members in the final anti-RhD rpAb composition. Preferably, no individual member exceeds more that 50%, even more preferred 25% and most preferred 10% of the total number of individual members in the final polyclonal composition. The assessment of clonal diversity based on the distribution of the individual members in the polyclonal composition can be performed by RFLP analysis, sequence analysis or protein analysis such as the approaches described later on for characterization of a polyclonal composition.
[0118] Clonal diversity may be reduced as a result of clonal bias which can arise a) during the cloning process, b) as a result of variations in cellular proliferation, or c) through scrambling of multiple integrants. If such biases arise, each of these sources of a loss of clonal diversity is easily remedied by minor modifications to the methods as described herein.
[0119] In order to limit bias introduced by cloning of the variable domains into the appropriate vectors, the transfer of the genes of interest from one vector to another may be designed in such a way that cloning bias is limited. Mass transfer techniques and a careful selection of the E. coli strain used for amplification can reduce the cloning bias. Another possibility is to perform an individual transfer of each polynucleotide encoding an individual member of the polyclonal antibody, between screening vectors and vectors for site-specific integration.
[0120] It is possible that variations in cellular proliferation rates of the individual cells in the cell line could, over a prolonged period of time, introduce a bias into the anti-RhD rpAb expression, increasing or reducing the presence of some members of the anti-RhD rpAb expressed by the cell line. One reason for such variations in proliferation rates could be that the population of cells constituting the starting cell line used for the initial transfection is heterogeneous. It is known that individual cells in a cell line develop differently over a prolonged period of time. To ensure a more homogeneous starting material, sub-cloning of the cell line prior to transfection with the library of interest may be performed using a limiting dilution of the cell line down to the single cell level and growing each single cell to a new population of cells (so-called cellular sub-cloning by limiting dilution). One or more of these populations of cells are then selected as starting material based on their proliferation and expression properties.
[0121] Further, the selection pressure used to ensure that only cells that have received site-specific integrants will survive, might affect proliferation rates of individual cells within a polyclonal cell line. This might be due to the favoring of cells that undergo certain genetic changes in order to adapt to the selection pressure. Thus, the choice of selection marker might also influence proliferation rate-induced bias. If this occurs, different selection markers should be tested. In cases where selection is based on a substance that is toxic to the cells, the optimal concentration should be tested carefully, as well as whether selection is needed throughout the entire production period or only in the initial phase.
[0122] An additional approach to ensure a well defined cell population is to use fluorescence activated cell sorting (FACS) after the transfection and selection procedures. Fluorescence labeled antibodies can be used to enrich for highly productive cells derived from a pool of cells transfected with IgG constructs (Brezinsky et al. J. 2003. Immunol Methods 277, 141-155). This method can also be used to sort cells expressing similar levels of immunoglobulin, thereby creating a homogenous cell population with respect to productivity. Likewise, by using labeling with the fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) cells showing similar proliferation rates can be selected by FACS methods. Further, differences in expression levels of the individual members of the anti-RhD rpAb may also introduce a bias into the final product over a prolonged period of time.
[0123] If the polyclonal cell line is generated by mixing separately transfected clones after selection (the 3rd approach in FIG. 1A), the following selection criteria may be set up for the individual clones at the cell culture level prior to mixing: proliferation rates have to be between 24 and 32 hours, the productivity should exceed 1.5 μg antibody per cell per day, and the culture should show a homogenous cell population assessed by an intra cellular staining method. If desired a more homogenous cell population for each individual clone can be obtained with the surface staining method described by Brezinsky prior to mixing the individual clones by gating on a particular area of the population in connection with the FACS analysis.
[0124] Even if a proliferation rate-induced, or productivity-induced bias occurs, the loss or over-representation of individual members might not necessarily be critical, depending on the diversity requirements of the final anti-RhD rpAb product.
[0125] In cells with site-specific single integrants, the cells will only differ in the sequence of the variable regions of the antibodies to be expressed. Therefore, the different cellular effects imposed by variation in integration site and gene regulatory elements are eliminated and the integrated segments have minimal effects on the cellular proliferation rate. Neither scrambling nor multiple integrations is likely to cause problems in the proliferation rate of the manufacturing cell line, since these are rare events. Random integrations generally occur with an efficiency of approximately 10-5, whereas site-specific integration occurs with an efficiency of approximately 10-3. If multiple integrations should unexpectedly pose a problem, an alternative is to repeat the transfection with the library of anti-RhD antibody expression vectors, because the likelihood that the event will reoccur is very small, as described above.
[0126] Considering statistics, bulk transfection of a large number of cells also constitutes a way to circumvent an undesired clonal bias. In this approach, a host cell line is transfected in bulk with the library of anti-RhD antibody expression vectors. Such a library constitutes many copies of each distinct member of the library. These copies should preferably be integrated into a large number of host cells. Preferably at least 100, 1000, 10000 or 100000 individual cells are transfected with copies of distinct members of the library of variant nucleic acid segments. Thus, if a library of distinct variant nucleic acid segments is composed of 1000 distinct members which are each integrated into 1000 individual cells, 106 clones containing a site-specifically integrated anti-RED antibody-encoding segment should arise from the transfection. In this manner the gausian curve of individual cell doubling rates should influence the general population only in very small degrees. This will increase the probability of keeping the clonal composition constant, even if a low percentage of the manufacturing cells should exhibit aberrant growth and/or expression properties.
[0127] Alternatively the semi-bulk transfection or individual transfection methods previously described may be used.
Establishment of a Polyclonal Working Cell Bank (pWCB)
[0128] The section "Establishing an expression system for high-level expression of a polyclonal protein" describes three alternative ways of establishing a polyclonal manufacturing cell line. The section describes the generation of a frozen library stock which is constituted of a collection of cells, obtained by bulk or semi-bulk transfection, where each individual cell in the library stock is capable of expressing an individual member from a library of anti-RhD antibody expression vectors. Preferably, the clonal diversity requirements already described is fulfilled by the collection of cells, such that essentially all members of the library can be expressed from a frozen library stock ampoule, when thawn and expanded to establish a polyclonal manufacturing cell line. In the bulk transfection and semi-bulk transfection approaches the frozen library stock, can also be considered as a polyclonal working cell bank (pWCB), in that a single vial from the frozen library stock can be thawn and expanded into a polyclonal manufacturing cell line.
[0129] Alternatively, in the previously described third approach for the generation of a recombinant polyclonal manufacturing cell line, the frozen library stock is composed of separate cell lines, which have been individually transfected with an individual member of a library of anti-RhD antibody expression vectors. The transfectants are selected for stable expression of the integrated vector-derived nucleic acid segment from their genome. Preferably, the nucleic acid segments are integrated site-specifically into one or more sites in the genome of the transfectants, and even more preferred in a single site of the genome. The transfected cells obtained e.g. from clonal colonies upon selection may either be isolated and maintained as single clones or pooled to generate a pool of clones expressing the same anti-RhD antibody. In the present invention a single clone of cells as well as pool of clones expressing the same antibody is termed an individual cell line. Thus, if the library of anti-RhD antibody expression vectors constituted 25 individual members, the frozen library stock, in this third approach, would be composed of 25 individual cell lines (not a mixture of cell lines) each expressing an individual member from the library of anti-RhD antibody expression vectors. Hence, one vial from this library stock will result in the generation of a monoclonal anti-RhD antibody if used for manufacturing.
[0130] The present invention exemplifies a library of anti-RhD antibody expression vectors. However, the generation of a frozen library stock is independent of the antigen specificity of the polyclonal protein produced from a library comprised of variable region-encoding nucleic acid segments and may be used with any other library comprised of antibody VH and VL-encoding nucleic acid segments, or T cell receptor (TcR) α and β-, or γ and δ-encoding nucleic acid segments. A library comprised of variable region-encoding nucleic acid segments can in addition to the variable regions also encode one or more constant regions. Thus, a library comprised of antibody VH and VL-encoding nucleic acid segments may result in Fv, scFv, Fab molecules or full-length antibody molecules, and a library comprised of TcR variable region-encoding segments may result in molecules composed of TcR variable domain fragments, soluble TcRs or full-length TcRs.
[0131] In situations where the frozen library stock is composed of individual cell lines it will be appropriate to generate a pWCB which can be used for the establishment of the polyclonal manufacturing cell line by thawing and expanding the contents of a single ampoule. The individual cell lines used to generate such a pWCB are either obtained from i) a single clone or ii) a pool of clones (a pool of single colonies obtained after selection). The clones have been obtained from host cells individually transfected with, and selected for stable expression of an individual member of a library comprising variable region-encoding nucleic acid segments, such as antibody VH and VL-encoding segments or TcR α and β-, or γ and δ-encoding segments. Selection for stable expression is performed by procedures known in the art, e.g. using selection marker genes. In a preferred embodiment of the present invention the individual cell lines are obtained from cloned or subcloned cells, e.g. by subjecting a cell line originating from i) or ii) (see previous description) to limiting dilution or single cell FACS analysis and selection, or by selecting high expression clones e.g. using a robot like the ClonePix FL (see below). The individual cell lines used to generate the pWCB as described above may be pre-stored in a frozen library stock of individual cell lines, from which an ampoule of each individual cell line is thawn and expanded prior to the generation of a pWCB. Preferably, the individual cell lines express full-length antibodies with properties that differ from the properties of the antibodies produced by the other members of the pWBC, e.g. different antigen specificity, different affinity, different variable or CDR regions and/or different constant regions.
[0132] Each cell line used to generate the pWCB, produces a different member of a polyclonal protein. Preferably, each distinct member of the polyclonal protein binds a particular antigen. Additionally, it is preferred that each distinct member is produced from a single specific site in the genome of each host cell. A pWCB is generated by mixing a predefined number of cells from each individual cell line. Preferably, the cells are mixed in equal numbers (a 1:1 ratio), although other ratios also may be desired (see later). The mixture of cells is frozen down in aliquots, in that they are distributed into a number of vials with a defined number of cells in each vial. These vials are frozen and stored as the pWCB for later manufacturing purposes. Preferably, the number of vials constituting the pWCB exceeds 1.0, 25, 50, 5, 100, 200, 500 or 1000 vials. The individual vials in a pWCB may be thawn at different points in time generating different batches of the polyclonal manufacturing cell line which are capable of producing a polyclonal protein with essentially the same composition from batch to batch (See Example 5).
[0133] In an alternative approach of the present invention, the polyclonal manufacturing cell line may be expanded from a sub-pWCB, which is derived from a pWCB. The sub-pWCB is generated by thawing a single vial from a pWCB and expanding the cells for a number of generations sufficient to produce a total number of cells which can be frozen down in a new series of aliquots (the sub-pWCB), with approximately the same number of cells in each sub-pWCB aliquot as in the pWCB vial originally used to generate the sub-pWCB. The advantage of this approach is that the pWCB now serves as a master cell bank as known from other recombinant protein production protocols. Thus, in this approach the pWCB may also be termed a polyclonal master cell bank (pMCB). When the sub-pWCB has been exhausted, it is possible to generate a new sub-pWCB from an aliquot of the pWCB/pMCB. This approach will therefore require a significantly lower amount of work than would be required to expand the individual cell lines from the frozen library stock and mixing a new pWCB. Further, in the event that the sub-pWCB is exhausted, the chance of producing further batches of the polyclonal manufacturing cell line, which are capable of producing a polyclonal protein with essentially the same composition from batch to batch is increased. The principle of generating a pWCB/pMCB and a sub-pWCB from individually transfected host cells is illustrated in FIG. 1B.
[0134] The advantage of producing a pWCB or pMCB by mixing individual cell lines which have been obtained by individual transfection, compared to the direct generation of a pWCB of pMCB by bulk transfection or semi-bulk transfection, is that it is possible to perform additional analysis and selections of the individually transfected cell lines prior to generation of the pWBC or pMCB. This may ensure a more stable polyclonal manufacturing cell line which fulfills the diversity requirements already described. In the following pWCB is to be understood as pWCB or pMCB.
[0135] In an additional embodiment of the present invention, individual cell lines which have been selected for stable expression of an individual member of a library of variable region-encoding nucleic acid segments as described above, are further characterized with respect to their proliferation rates and/or productivity prior to generation of a pWCB. In a preferred embodiment cell lines with similar proliferation rates or productivity are selected for the generation of a pWCB. Even more preferred, cell lines with similar productivity as well as similar proliferation rates are selected for the generation of the pWCB. Preferably, the cell lines are adapted to serum free suspension culture prior to the characterization of proliferation rates and/or productivity. Alternatively, the parental cells used for transfection are adapted to serum free suspension culture prior to transfection.
[0136] Proliferation rates can be assessed by methods known in the art, for example as described in example 2 of the current invention. Proliferation rates for mammalian cell lines should be between 18 and 100 hours, preferably between 22 and 40 hours and most preferred between 24 and 32 hours. The productivity should exceed 0.5 μg protein per cell per day (pg/(cell*day)), preferably it should exceed 1, 1.5, 3, 5 or 8 μg/(cell*day). Further, the cell line should show a homogenous cell population with respect to expression when assessed by an intra-cellular staining method. If desired a more homogeneous cell population for each individual cell line can be obtained by cloning e.g. by the FACS sorting methods described below.
[0137] In further embodiments of the present invention, the individual cell lines are
[0138] FACS sorted to identify cells with a homogeneous expression level, after the transfection and selection procedures. The possibility of sorting for individual high-expressing clones or a sub-pool of cells with high expression levels by gating on a particular area of the population in connection with the FACS analysis is therefore an additional embodiment of the present invention. The generation of cloned cells by FACS analysis and selection is particularly useful when the individual cell lines are generated from a pool of clones.
[0139] Fluorescence labeled antibodies can be used to sort for cells expressing high levels of the desired protein e.g. antibody or TcR, thereby creating a homogeneous cell population with respect to productivity. This technique is based on the observation that secreted proteins can be detected on the surface of the cell secreting them, and the amount of surface protein apparently corresponds to the expression levels of the individual cell. The high producing cells can therefore be single cell sorted upon staining with a labeled antibody, followed by analysis by FACS. The technique has been described by Brezinsky (Brezinsky et al. J. 2003. Immunol Methods 277, 141-155).
[0140] An alternative sorting technique is based on the coupling of a ligand, with specificity to the protein expressed from the cells, to the surface of the cells. For example an anti-Fc antibody or an anti-idiotype antibody can be coupled to the surface of the protein secreting cell population via biotin. The antibodies secreted by an individual cell are then captured by the anti-Fc antibodies on the surface of that cell. Following this, the high producing cells can be sorted by FACS upon staining with a labeled antibody. This technique has been described in EP 667896.
[0141] To obtain cell lines with a homogeneous high expression levels, single cells having a high expression level are analyzed based on the FACS profile obtained by one of the described techniques. The individual cell clones are then expanded and potentially analyzed with respect to proliferation rates and productivity as described above. Alternatively, a sub-pool of cells having the highest expression level as identified by the FACS profile is collected by sorting. The sub-pool of cells from the individual cell line can likewise be analyzed with respect to proliferation rates and productivity if desired.
[0142] In an alternative embodiment of this invention, a robot such as the ClonePixFL robot (Genetix, UK) is used to select clones exhibiting high expression levels and/or similar growth properties. This is done as follows: The colonies obtained after transfection and selection are grown in a semi-solid medium which allows for detection of high-producing colonies by capturing the secreted protein product in the immediate proximity of the colony. The production level from each colony is determined by means of immunofluorescence labeling of the protein expressed by the cells followed by image software selection of the best clones based on predetermined selection criteria such as expression level and growth properties. Furthermore, the size (reflecting the cell proliferation rate) of each colony can be assessed by the robot using light detection imaging. Colonies with the desired production and/or growth properties are then isolated by the robot and transferred to 96-well plates for further propagation.
[0143] Preferably, individual cell lines with similar productivity are selected for the generation of the pWCB. In a preferred embodiment individual cell lines constituting the pWCB are generated from cloned cells, e.g. obtained by single cell sorting, limiting dilution or robot picking, with a high expression level or from a pool of cells with high expression level.
[0144] In the present invention, both individual cell lines obtained from a single colony of cells isolated after transfection and selection as well as individual cell lines obtained from a clone obtained e.g. by single cell FACS sorting, are termed cloned cell lines. In a preferred embodiment such cloned cell lines are used to generate the pWCB.
[0145] In further embodiments of the present invention, the individual cell lines are mixed at different ratios upon generation of a pWCB. The individual cell lines can be mixed according to predetermined criteria based on the properties of the individual cell lines and/or individual protein member expressed by said cell line, e.g. specific productivity or binding affinity. For example, individual cell lines expressing certain antibodies binding particularly critical antigens or epitopes can be supplied in excess of the remaining member cell lines of the pWCB, e.g. in 2-fold, 3-fold, 5-fold or 10-fold higher amounts. One member cell line may for example be added in a 2:1 ratio over all the other members, e.g. 4×106 cells of member 1 and 2×106 cells of each of the remaining member cell lines.
[0146] In a preferred embodiment of the present invention, a pWCB for production of an Anti-RhD rpAb is generated. Even more preferred such a pWCB is generated such that cell lines which produce antibodies with reactivity against a RhD category VI antigen constitute at least 5%, 8%, 10%, 12%, 15%, 20% or 25% of the total amount of cells included in the pWCB.
[0147] This approach of differentiated ratios of the individual cell lines in the pWCB may also be adopted to circumvent differences in proliferation rates and productivity among the individual cell lines, in particular if these have not been selected for similarity in these traits. Hence, if one or more of the individual cell lines have a slower proliferation rate, i.e. longer doubling times, compared to other members of the polyclonal working cell bank which are characterized by a faster proliferation rate, but this slower proliferation rate is not associated with a particular high productivity, this particular member(s) may be added to the pWCB in an increased amount to compensate for its slow growth. For example may a cell line with a proliferation rate of 50 hours be added in a 2:1 ration if the remaining cell lines constituting the pWCB have proliferation rates between 22 and 30 hours. Likewise, the ratio of cell lines with short doubling times may be reduced to ensure that these will not take over during manufacturing. Further, the ratios of the individual cell lines in a pWCB may be adjusted upon analysis of the polyclonal protein products produced from the polyclonal manufacturing cell lines generated from the pWCB. Such adjustments may for example be made based on IEX profiles or equivalent characterization tools. If such an analysis shows that one or more particular protein members are produced in an increased amount compared to the remaining members, a new pWCB may be generated, wherein the ratio of the cell lines producing these particular protein members are reduced. And visa versa, if a particular member is produced in a low amount, a pWCB with an increased ratio of the cell line producing this member may be generated.
Purification of an Anti-RhD rpAb from Culture Supernatant
[0148] Isolation of anti-RhD rpAb from culture supernatants is possible using various chromatographic techniques that utilize differences in the physico-chemical properties of proteins, e.g. differences in molecular weight, net charge, hydrophobicity, or affinity towards a specific ligand or protein. Proteins may thus be separated according to molecular weight using gel filtration chromatography or according to net charge using ion-exchange (cation/anion) chromatography or alternatively using chromatofocusing.
[0149] Affinity chromatography combined with subsequent purification steps such as ion-exchange chromatography, hydrophobic interactions and gel filtration has frequently been used for the purification of IgG (polyclonal as well as monoclonal) from different sources e.g., ascites fluid, cell culture supernatants and serum. Affinity purification, where the separation is based on a reversible interaction between the anti-RhD antibodies and a specific ligand coupled to a chromatographic matrix, is an easy and rapid method, which offers high selectivity, usually high capacity and concentration into a smaller volume. Specific ligands in the form of peptides capable of binding to anti-RhD antibodies may be obtained according to the method described in EP 1 106 625 using peptide phage display. Protein A and protein G, two bacterial cell surface proteins, have high affinity for the Fc region, and have, in an immobilized form, been used for many routine applications, including purification of polyclonal IgG and its subclasses from various species and absorption and purification of immune complexes.
[0150] Following affinity chromatography, downstream chromatography steps, e.g. ion-exchange and/or hydrophobic interaction chromatography, can be performed to remove host cell proteins, leaked Protein A, and DNA. With the protein A affinity and cation exchange chromatography it has been observed that pH-values above 5 may cause precipitation of the anti-RhD rpAb. Thus buffers should be adjusted carefully with appropriate buffering agents, e.g. Tris or acetate.
[0151] Gel filtration, as a final purification step, can be used to remove contaminant molecules such as dimers and other aggregates, and transfer the sample into storage buffer. Depending on the source and expression conditions it may be necessary to include an additional purification step to achieve the required level of antibody purity. Hydrophobic interaction chromatography or ion-exchange chromatography are thus frequently used, in combination with Protein A and gelfiltration chromatography, to purify antibodies for therapeutic use.
[0152] In order to purify other classes of antibodies, alternative affinity chromatography media have to be used since proteins A and G do not bind IgA and IgM. An immuno-affinity purification can be used (anti-IgA or anti-IgM monoclonal antibodies coupled to solid phase) or, alternatively, multistep purification strategies including ion-exchange and hydrophobic interaction can be employed.
Structural Characterization of Anti-RhD rpAb
[0153] Structural characterization of polyclonal antibodies requires high resolution due to the complexity of the mixture (clonal diversity, heterogeneity and glycosylation). Traditional approaches such as gel filtration, ion-exchange chromatography or electrophoresis may not have sufficient resolution to differentiate among the individual antibodies in the anti-RhD rpAb. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has been used for profiling of complex protein mixtures followed by mass spectrometry (MS) or liquid chromatography (LC)-MS (e.g., proteomics). 2D-PAGE, which combines separation on the basis of a protein's charge and mass, has proven useful for differentiating among polyclonal, oligoclonal and monoclonal immunoglobulin in serum samples. However, this method has some limitations. Chromatographic techniques, in particular capillary and LC coupled to electrospray ionization MS are increasingly being applied for the analysis of complex peptide mixtures. LC-MS has been used for the characterization of monoclonal antibodies and recently also for profiling of polyclonal antibody light chains. The analysis of very complex samples requires more resolving power of the chromatographic system, which can be obtained by separation in two dimensions (or more). Such an approach is based on ion-exchange in the first dimension and reversed-phase chromatography (or hydrophobic interaction) in the second dimension optionally coupled to MS.
Functional Characterization of Anti-RhD rpAb
[0154] An anti-RhD rpAb antibody can for example be characterized functionally through comparability studies with anti-D immunoglobulin products or anti-RhD mAbs. Such studies can be performed in vitro as well as in vivo.
[0155] in vitro functional characterization methods of anti-RhD rpAb could for example be phagocytosis assays (51Cr-based or FACS based), antibody dependent cellular cytotoxicity (ADCC) and rosetting assay. Briefly described the assays are performed as follows:
ADCC Assay (51Cr Based):
[0156] Human PBMC are used as effector cells and RhD negative and positive RBC (0 in the AB0 system) are used as targets. First, the RBC (RhD(+) and RhD(-)) are 51Cr labelled, washed and then sensitized with anti-RhD antibodies (e.g. anti-RhD rpAb, anti D or anti-RhD mAb) in various dilutions. The effector cells (PMBC) are added to the sensitized RBC (ratio of 20:1) and incubation is performed overnight. Cells are spun down and the supernatants from the wells are transferred to a Lumaplate (PerkinElmer). Controls for spontaneous release are included (RBC with 51Cr only) and for total release (addition of Triton-X-100 to 51Cr-labeled RBC). The Lumaplate is dried and counted in a Topcounter (PerkinElmer).
Phagocytosis Assay (51Cr Based):
[0157] Phagocytosis can be measured in combination with the ADCC assay. After harvesting the supernatant in the ADCC assay, the remaining supernatant is removed and the red blood cells are lysed by addition of a hypotonic buffer. The cells are washed and the supernatant is removed. PBS+1% Triton-X-100 is added to all wells and fixed amounts are transferred to a Lumaplate, dried and counted as before.
Phagocytosis Assay (FACS Based):
[0158] This assay is based on adherence of the phagocytic cells. The human leukemic monoblast cell line U937 can be used for this assay. U937 cells are differentiated using 10 nM PMA. Two days later 60% of the medium is removed and replaced by medium without PMA. The cell membrane of red blood cells (RhD(+) and RhD(-)) are stained with PKH26 (PE) according to the manufactures protocol (Sigma). The RBC's are sensitized with anti-RhD antibodies in various dilutions and excess antibodies are removed by washing. On day three, the non-adherent cells U937 cell are removed by washing and sensitized RBC (RhD(+) and RhD(-)) are added to the wells. The plates are incubated overnight in the incubator. Non-phagocytozed RBC are washed away by several steps. Attached but not phagocytozed RBC are lysed by addition of hypotonic buffer followed by additional washing. The U937 cells detached from the wells by incubation with trypsin. Cells are analyzed on the FACS.
Rosetting Assay
[0159] A rosetting assay is merely an Fc receptor binding assay. Sensitized red blood cells are incubated with differentiated U937 cells prepared as described above. RBC (RhD (-) and RhD(+)) are sensitized with anti-RhD antibodies in various dilutions and excess antibodies are removed by washing before they are mixed with U937 cells. Incubation is performed for one hour and non-bound RBC are washed away. The percentage of cells with two or more RBC attached to the surface is counted.
[0160] An in vivo functional characterization of anti-RhD antibodies is described by Miescher (Miescher, S., et al. 2004, Blood 103, 4028-4035), an involves injection of RhD(+) cells into RhD(-) individuals followed by administration of anti-RhD antibody. RBC clearance and anti-RhD antibody sensation of the donors was analyzed.
Therapeutic Compositions
[0161] In an embodiment of the invention, a pharmaceutical composition comprising anti-RhD rpAb or anti-RhD recombinant polyclonal Fab or another anti-RhD recombinant polyclonal fragment as active ingredient is intended for the prophylaxis of hemolytic disease of the newborn, treatment of idiopathic thrombocytopenic purpura (ITP) or prevention of sensitization to the Rhesus D antigen after mistransfusions of RhD(+) blood to RhD(-) individuals.
[0162] The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
[0163] Anti-RhD rpAb or polyclonal fragments thereof may be administered within a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer to female mothers or patients. In a preferred embodiment the administration is prophylactic. Any appropriate route of administration may be employed, for example, administration may be parenteral, intravenous, intra-arterial, subcutaneous, intramuscular, intraperitoneal, intranasal, aerosol, suppository, or oral administration. For example, therapeutic formulations may be in the form of, liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules chewing gum or pasta, and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
[0164] The pharmaceutical compositions of the present invention are prepared in a manner known per se, for example, by means of conventional dissolving, lyophilizing, mixing, granulating or confectioning processes. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see for example, in Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, 2000, Lippincott Williams & Wilkins, Philadelphia, Pa. and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York, N.Y.).
[0165] Solutions of the active ingredient, and also suspensions, and especially isotonic aqueous solutions or suspensions, are preferably used, it being possible, for example in the case of lyophilized compositions that comprise the active ingredient alone or together with a carrier, for example mannitol, for such solutions or suspensions to be produced prior to use. The pharmaceutical compositions may be sterilized and/or may comprise excipients, for example preservatives, stabilizers, wetting and/or emulsifying agents, solubilizers, salts for regulating the osmotic pressure and/or buffers, and are prepared in a manner known per se, for example by means of conventional dissolving or lyophilizing processes. The said solutions or suspensions may comprise viscosity-increasing substances, such as sodium carboxymethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone or gelatin.
[0166] The injection compositions are prepared in customary manner under sterile conditions; the same applies also to introducing the compositions into ampoules or vials and sealing the containers.
[0167] Pharmaceutical compositions for oral administration can be obtained by combining the active ingredient with solid carriers, if desired granulating a resulting mixture, and processing the mixture, if desired or necessary, after the addition of appropriate excipients, into tablets, pills, or capsules, which may be coated with shellac, sugar or both. It is also possible for them to be incorporated into plastics carriers that allow the active ingredients to diffuse or be released in measured amounts.
[0168] The pharmaceutical compositions comprise from approximately 1% to approximately 95%, preferably from approximately 20% to approximately 90%, active ingredient. Pharmaceutical compositions according to the invention may be, for example, in unit dose form, such as in the form of ampoules, vials, suppositories, tablets, pills, or capsules The formulations can be administered to human individuals in therapeutically or prophylactic effective amounts (e.g., amounts which prevent, eliminate, or reduce a pathological condition) to provide therapy for a disease or condition. The preferred dosage of therapeutic agent to be administered is likely to depend on such variables as the type and extent of the disorder, the overall health status of the particular patient, the formulation of the compound excipients, and its route of administration.
Therapeutic Uses of the Compositions According to the Invention
[0169] The pharmaceutical compositions according to the present invention may be used for the treatment, amelioration or prophylaxis of a disease in a mammal. Conditions that can be treated or prevented with the present pharmaceutical compositions include prevention of hemolytic disease of the newborn, treatment of idiopathic thrombocytopenic purpura (ITP) or prevention of sensitization to the Rhesus D antigen after mistransfusions of RhD(+) blood to RhD(-) individuals.
[0170] One aspect of the present invention is a method for disease treatment, amelioration or prophylaxis in an animal, wherein an effective amount of anti-RhD rpAb or fragment is administered.
[0171] A further embodiment of the present invention is the use of an anti-RhD recombinant polyclonal antibody or polyclonal antibody fragment for the preparation of a composition for the prophylaxis of hemolytic disease of the newborn or treatment of idiopathic thrombocytopenic purpura (ITP).
Diagnostic Use and Environmental Detection Use
[0172] Another embodiment of the invention is directed to diagnostic kits. Kits according to the present invention comprise an anti-RhD rpAb prepared according to the invention which protein may be labeled with a detectable label or non-labeled for non-label detection. The kit may be used to identify RhD(+) individuals, or individuals with a particular Rhesus D category. Identification of the later can be achieved by having a anti-RhD rpAb composition which only react with that particular Rhesus D category.
EXAMPLES
[0173] The following examples describe how anti-RhD rpAb is expressed and produced in a high-producer cell line, where VH and VL comprising nucleic acid segments or vector(s) have been inserted by site-specific integration into a pre-characterized chromosomal "hot-spot" site.
[0174] In the examples, CHO cells were utilized as host cell. The advantages thereof include the availability of suitable growth medium, their ability to grow efficiently to a high density in culture, and their ability to express mammalian proteins such as antibodies in a biologically active form.
[0175] In general, transformation of E. coli and transfection of mammalian cells according to the subject invention will be performed according to conventional methods.
[0176] The following examples illustrate the invention, but should not be viewed as limiting the scope of the invention.
Example 1
Production of an Anti-Rhesus D Recombinant Polyclonal Antibody
Donors
[0177] Donors were enrolled at Aalborg Sygehus Nord. A total of eight RhD(-) women were immunized with RhD(+) erythrocytes derived from RhD(+) individuals. The donors had a varying history of the immunizations with respect to the number of boosts and the origin of RhD(+) erythrocytes for the immunization. The immunization history of the different donors is given in the table 1.
TABLE-US-00001 TABLE 1 Donor # # of boost # of boosts from different origin 1 3 2 2 6 2 3 2 1 4 4 4 5 2 2 6 3 2 7 2 2 8 2 2
[0178] Mononuclear cells were harvested by leukopheresis 5-7 days after the last boost. The cells were pelleted and immediately transferred to the cell lysis solution from a commercially available RNA preparation kit (NucleoSpin RNA L, Machery-Nagel, cat. no. 740 962.20). After lysis of the cells, the suspension was frozen before further processing.
Generation of Anti-Rhesus D Fab Display Library
[0179] The material obtained from each donor was kept separate throughout the procedure of library generation and panning. The cell lysates were thawed and RNA was prepared according to kit instructions (NucleoSpin RNA L). The integrity of the RNA was analyzed by agarose gel electrophoresis, thus verifying that the 18S/28S ribosomal RNAs were not degraded.
[0180] RNA was subjected to cDNA synthesis in an oligo(dT) primed reaction using approximately 10 μg total RNA in a reaction using ThermoScript (Invitrogen), according to the manufacturer's instructions. The cDNA was used as template in PCR reactions using the following primers:
VH Forward Primers (XhoI Site in Bold):
TABLE-US-00002 [0181] J SEQ region ID Primer sequence JH1/ 2 2 GGAGGCGCTC GAGACGGTGA CCAGGGTGCC JH3 3 GGAGGCGCTC GAGACGGTGA CCATTGTCCC JH4/5 4 GGAGGCGCTC GAGACGGTGA GCAGGGTTCC JH6 5 GGAGGCGCTC GAGACGGTGA CCGTGGTCCC
VH Reverse Primers (AscI Site in Bold):
TABLE-US-00003 [0182] V gene family SEQ ID Primer sequence 1B/7A 6 CCAGCCGGGG CGCGCCCAGR TGCAGCTGGT GCARTCTGG 1C 7 CCAGCCGGGG CGCGCCSAGG TCCAGCTGGT RCAGTCTGG 2B 8 CCAGCCGGGG CGCGCCCAGR TCACCTTGAA GGAGTCTGG 3B 9 CCAGCCGGGG CGCGCCSAGG TGCAGCTGGT GGAGTCTGG 3C 10 CCAGCCGGGG CGCGCCGAGG TGCAGCTGGT GGAGWCYGG 4B 11 CCAGCCGGGG CGCGCCCAGG TGCAGCTACA GCAGTGGGG 4C 12 CCAGCCGGGG CGCGCCCAGS TGCAGCTGCA GGAGTCSGG 5B 13 CCAGCCGGGG CGCGCCGARG TGCAGCTGGT GCAGTCTGG 6A 14 CCAGCCGGGG CGCGCCCAGG TACAGCTGCA GCAGTCAGG
C.sub.κ Forward Primer (NotI Site in Bold):
TABLE-US-00004 [0183] SEQ ID Primer sequence 15 ACCGCCTCCA CCGGCGGCCG CTTATTAACA CTCTCCCCTG TTGAAGCTCT T
V.sub.κ Reverse Primers (NheI Site in Bold):
TABLE-US-00005 [0184] V gene family SEQ ID Prime sequence 1B 16 CAACCAGCGC TAGCCGACAT CCAGWTGACC CAGTCTCC 2 17 CAACCAGCGC TAGCCGATGT TGTGATGACT CAGTCTCC 3B 18 CAACCAGCGC TAGCCGAAAT TGTGWTGACR CAGTCTCC 4B 19 CAACCAGCGC TAGCCGATAT TGTGATGACC CACACTCC 5 20 CAACCAGCGC TAGCCGAAAC GACACTCACG CAGTCTCC 6 21 CAACCAGCGC TAGCCGAAAT TGTGCTGACT CAGTCTCC
C.sub.λ Forward Primer (NotI Site in Bold):
TABLE-US-00006 [0185]λ SEQ family ID Primer sequence 2 22 ACCGCCTCCACCGGCGGCCGCTTATTATGAACATTCTGTAGGGCCACTG 7 23 ACCGCCTCCACCGGCGGCCGCTTATTAAGAGCATTCTGCAGGGGCCACTG
V.sub.λ Reverse Primers (NheI in Bold):
TABLE-US-00007 [0186] V gene SEQ family ID Primer sequence 1A 24 CAACCAGCGC TAGCCCAGTC TGTGCTGACT CAGCCACC 1B 25 CAACCAGCGC TAGCCCAGTC TGTGYTGACG CAGCCGCC 1C 26 CAACCAGCGC TAGCCCAGTC TGTCGTGACG CAGCCGCC 2 27 CAACCAGCGC TAGCCCARTC TGCCCTGACT CAGCCT 3A 28 CAACCAGCGC TAGCCCTTTC CTATGWGCTG ACTCAGCCACC 3B 29 CAACCAGCGC TAGCCCTTTC TTCTGAGCTG ACTCAGGACCC 4 30 CAACCAGCGC TAGCCCACGT TATACTGACT CAACCGCC 5 31 CAACCAGCGC TAGCCCAGGC TGTGCTGACT CAGCCGTC 6 32 CAACCAGCGC TAGCCCTTAA TTTTATGCTG ACTCAGCCCCA 7/8 33 CAACCAGCGC TAGCCCAGRC TGTGGTGACY CAGGAGCC 9 34 CAACCAGCGC TAGCCCWGCC TGTGCTGACT CAGCCMCC
[0187] PCR was performed with individual primer pairs amounting to 36 VH reactions, 6 Kappa reactions and 22 Lambda reactions. All VH, Kappa, and Lambda PCR products were pooled separately and following purification using NucleoSpin columns (Machery-Nagel, cat. no. 740 590.250), the products were digested prior to cloning (VH: AscI/XhoI, Kappa and Lambda: NheI/NotI) followed by a gel purification step of the bands of interest (PerfectPrep Gel Cleanup kit, Eppendorf, cat. no. 0032 007.759). The light chains (Kappa and Lambda separately) were inserted into a NheI/NotI treated Em351 phage display vector (FIG. 2), by ligation and amplified in E. coli XL1 Blue (Stratagene). Plasmid DNA constituting the light chain library was isolated from the E. coli cells selected over night on Carbenicillin agar plates (two libraries for each donor, Kappa and Lambda, respectively). This library DNA was subjected to digest with AscI/XhoI, and after gel purification, the VH PCR products (subjected to digest with the same enzymes and gel purified) were ligated into the two light chain libraries from each donor and amplified in E. coli TG1 cells (Stratagene) using Carbenicillin selection on agar plates. After overnight growth, bacteria were scraped off the plates, and glycerol stocks were prepared for proper library storage. A plasmid DNA preparation containing the combinatorial variable heavy chain-light chain (VH:LC) library was also performed to secure the library for the future. The combinatorial libraries contained in the TG1 cells (two from each donor) were now ready for phage display and panning. The sizes of the combinatorial libraries (16 in total) were 106 or larger.
Enrichment for Phages Displaying Rhesus D Antigen Binding Fab Fragments
[0188] Phages displaying Fabs on their surface were generated as follows: 50 mL 2×YT/1% glucose/100 μg/mL Carbenicillin was inoculated with TG1 cells containing the combinatorial VH:VL library to obtain an OD600 of approximately 0.08. The culture was shaking for 11/2 h, and helper phage was added (VSCM13). The culture was incubated at 37° C. for 1/2 h without shaking and for 1/2 h with shaking. The bacteria were pelleted (3200×g, 10 minutes, 4° C.), and re-suspended in 50 mL 2×YT/100 μg/mL Carbenicillin/70 μg/mL Kanamycin, and the culture was shaken overnight at 30° C. The phages were precipitated from the culture supernatant by adding 1/5 volume of 20% PEG/1.5 M NaCl, incubating on ice for 30 minutes, and centrifugation at 8000×g for 30 minutes at 4° C. Precipitated phages were resuspended in PBS and used directly for panning.
[0189] Panning for Rhesus D antigen binding Fab fragments was performed in a two-step procedure. 108 RhD(-) red blood cells (RBC) were washed three times in PBS (centrifugation at 2000×g, 45 sec), and re-suspended in 150 μl panning buffer (2% skim milk in 0.85×PBS). Fifty μl freshly prepared phages were added to the RhD(-) cells (re-suspended in panning buffer) in order to perform a negative selection step, and incubated for 1 h on an end-over-end rotator at 4° C. Following the one hour incubation, the cells were pelleted by centrifugation (2000×g, 45 sec), and the phage-containing supernatant was incubated with 2×107RhD(+) RBC (washed three times in PBS). The phage:RhD(+) RBC mix was incubated for one hour on an end-over-end rotator at 4° C. Unbound phages were removed by washing five times with 1 mL panning buffer, and five times with PBS. Bound phages were eluted by addition of 200 μl H2O (which lyses the cells). One hundred μl of the eluate was added to exponentially growing TG1 cells, the remainder was stored at -80° C. TG1 cells infected with eluated phages were plated on Carb/glu agar dishes and incubated overnight at 37° C. The following day, the colonies were scraped off the plates, and 10 mL culture medium was inoculated for preparation of phages for the second round of panning. The second round of panning was performed as described for the first round.
Enrichment for Phages Displaying Rhesus D Category VI Antigen Binding Fab Fragments
[0190] In a separate set of pannings, selections were performed in order to retrieve clones with reactivity towards the RhD category VI antigen. The negative selection was performed on RhD(-) blood as described, and the positive selection was performed on RhDVI positive erythrocytes. The procedure was otherwise as described above.
Screening for Anti-RhD Binding Fabs
[0191] After each round of panning single colonies were picked for analysis of their binding properties to red blood cells in agglutination assays. Briefly, single colonies were inoculated into 2×YT/100 μg/mL carbenicillin/1% glucose and shaken overnight at 37° C. The next day, DeepWell plates were inoculated using 900 μl 2×YT/100 μg/mL carbenicillin/0.1% glucose and 10 μl overnight culture. The plates were shaken for two hours at 37° C., before Fab induction was performed with addition of 300 μl 2×YT/100 μg/mL carbenicillin/0.25 mM IPTG per well. The plate was shaken overnight at 30° C.
[0192] The following day, the bacteria were pelleted by centrifugation (3200×g, 4° C., 10 minutes), and re-suspended in 100 μl of 0.8 M NaCl, 0.2×PBS, 8 mM EDTA, and incubated for 15 minutes on ice in order to perform a periplasmic extraction of the Fab fragments. The plate was transferred to -20° C. and finally the suspension was thawed and centrifugation was performed for 10 minutes at 4° C. and 3200×g. The periplasmic extract was used in ELISA assays for analysis of Fab content and in agglutination assays to evaluate the binding potential of the individual Fab fragments.
[0193] The agglutination assay was performed as follows: RhD(-) and RhD(+) RBC were mixed in a 1:1 ratio, and washed 3 times in PBS. After the final wash, the cell mix was re-suspended in 1% BSA in PBS at a density of 1% cells, 50 μl was added to each well of a 96-plate. Periplasmic extracts were added to the wells. As a positive control Rhesogamma P immunoglobulin (Aventis) was used according to the manufacturer's instructions. The plates were incubated for one hour at room temperature with gentle shaking. The cells were washed three times with PBS, before the secondary antibody was added (goat anti-human Fab/FITC conjugate, Sigma F5512) in a 1:100 dilution. The plates were left for agglutination for one hour at room temperature without shaking. Fab fragments positive in the agglutination assay was determined by visual inspection, and recorded by taking a picture. Quantization of the binding activity of the Fab fragments was performed by FACS analysis of the agglutination samples.
[0194] When performing screening for clones with reactivity towards RhDVI+ erythrocytes, such cells were used in conjunction with RhD(-) cells in a procedure otherwise identical to that described above.
Selection of Diverse Anti-RhD Fab-Encoding Sequences.
[0195] A total of 1700 RhD antigen binding clones were identified. All the positive clones were submitted for DNA sequencing. From these 56 clones were selected based on their unique set of heavy chain CDR sequences. For multiple clones which used the same heavy chain with different light chains, the clone which showed the highest binding activity in the FACS assay was selected. Thereby a sub-library comprised of pairs of variable heavy chain (VH) and light chain (LC)-encoding sequences, representing a broad diversity with high RhD antigen specificity, was selected from all the positive clones.
[0196] The binding activity of these 56 clones was re-confirmed in agglutination assays, to ensure no false positive clones were selected.
[0197] The selected clones were further analyzed with respect to mutations due to for instance inter-family cross-priming, since such mutations may lead to overall structural changes of the expressed antibody possibly creating new epitopes and thereby result in an increased immunogenicity of the final product. Clones with such mutations were repaired as described in the following section relating to VH:LC transfer from the phagemid vector to the mammalian expression vector.
[0198] Alignments of the corrected nucleic acid sequences for the VH and light chains (LC) are shown in FIGS. 3 to 6, respectively. Further alignments of the VH and VL polypeptide chains are shown in FIGS. 5 and 6, respectively. The polypeptide alignments were performed and numbered according to structural criteria defined by Chothia (Chothia et al. 1992 J. Mol. Biol. 227, 776-798; Tomlinson et al. 1995 EMBO J. 14, 4628-4638 and Williams et al. 1996 J. Mol. Biol., 264, 220-232). The figures further indicate the position of the three CDR regions within the variable regions. The CDR region positions within the amino acid sequences are summarized in table 2. The numbering of the CDR3 regions in the polypeptide alignments (FIGS. 5 and 6) does not follow Chothia (transition marked with an asterisk in the figures). In order to enable identification of the CDR3 region with respect to amino acid position, a continued numbering has been assigned after the asterisk. The CDR3 region sequence for each individual clone can be derived from the figures based on this numbering.
TABLE-US-00008 TABLE 2 VL Kappa VL Lambda VH a.a. position a.a. position a.a. position FIG. 5 6A 6B CDR1 31-35 24-34 25-35 CDR2 50-65 50-56 53-57 CDR3 95-125 89-110 90-113
[0199] The pairs of variable heavy chain and complete light chain which have been screened as Fabs and selected for their ability to bind RhD antigen can be identified by their identical clone numbers. All the 56 VH:LC pairs are listed by clone number, the nucleic acid (nuc.) SEQ IDs and the amino acid (a.a.) SEQ IDs in table 3.
TABLE-US-00009 TABLE 3 VH nuc. LC nuc. VH a.a. LC a.a Clone Name SEQ ID SEQ ID SEQ ID SEQ ID RhD157.119D11 35 91 147 203 RhD158.119B06 36 92 148 204 RhD159.119B09 37 93 149 205 RhD160.119C07 38 94 150 206 RhD161.119E09 39 95 151 207 RhD162.119G12 40 96 152 208 RhD163.119A02 41 97 153 209 RhD189.181E07 42 98 154 210 RhD190.119F05 43 99 155 211 RhD191.119E08 44 100 156 212 RhD192.119G06 45 101 157 213 RhD193.126G05 46 102 158 214 RhD194.126G10 47 103 159 215 RhD195.127A07 48 104 160 216 RhD196.126H11 49 105 161 217 RhD197.127A08 50 106 162 218 RhD198.127F10 51 107 163 219 RhD199.164E03 52 108 164 220 RhD200.164G10 53 109 165 221 RhD201.164H12 54 110 166 222 RhD202.158E07 55 111 167 223 RhD203.179F07 56 112 168 224 RhD204.128A03 57 113 169 225 RhD205.160B12 58 114 170 226 RhD206.160C06 59 115 171 227 RhD207.127A11 60 116 172 228 RhD208.179B11 61 117 173 229 RhD239.126F09 62 118 174 230 RhD240.125A09 63 119 175 231 RhD241.119B05 64 120 176 232 RhD242.181A03 65 121 177 233 RhD243.109A05 66 122 178 234 RhD244.158B10 67 123 179 235 RhD245.164E06 68 124 180 236 RhD246.179B10 69 125 181 237 RhD292.109A02 70 126 182 238 RhD293.109A09 71 127 183 239 RhD294.119E10 72 128 184 240 RhD295.119B11 73 129 185 241 RhD296.126A03 74 130 186 242 RhD297.126E06 75 131 187 243 RhD298.126E10 76 132 188 244 RhD299.127A12 77 133 189 245 RhD300.134H09 78 134 190 246 RhD301.160A04 79 135 191 247 RhD302.160B10 80 136 192 248 RhD303.160B11 81 137 193 249 RhD304.164B06 82 138 194 250 RhD305.181E06 83 139 195 251 RhD306.223E11 84 140 196 252 RhD307.230E11 85 141 197 253 RhD317.144A02 86 142 198 254 RhD319.187A11 87 143 199 255 RhD321.187G08 88 144 200 256 RhD323.229B07 89 145 201 257 RhD324.231F07 90 146 202 258
Transfer of the Selected VH and Light Chain-Encoding Sequences to a Mammalian Expression Vector.
[0200] Due to the mutations resulting from, for instance, inter-family cross-priming it was necessary to repair of a large number of the selected sequences. This was done in connection with exchange of expression system from phage display to mammalian expression. For this reason the transfer was performed separately for each individual clone.
[0201] The transfer and repair was performed as follows: First the VH-encoding sequence situated in the Em351 vector was re-amplified by PCR using the high fidelity polymerase, Phusion (Finnzymes) and a proper set of correcting primers. The VH PCR fragment was digested with AscI and XhoI and subjected to gel purification. The Neo exp. vector (FIG. 7) was digested with the corresponding enzymes and gel purified thereby removing the nucleic acid sequence situated between the leader sequence and the heavy chain constant regions. The corrected V fragment and the Neo exp. vector were ligated and amplified in E. coli Top10 cells. Plasmid DNA of the VH containing Neo exp. vector was isolated from the E. coli cells selected over night on Carbenicillin.
[0202] Following transfer of the VH-encoding sequence the corresponding LC sequence was re-amplified by PCR using the high fidelity polymerase. Phusion (Finnzymes) and a proper set of correcting primers. The LC PCR fragment was digested with NheI and NotI and subjected to gel purification. The VH containing Neo exp. vector was digested with the corresponding enzymes and gel purified thereby removing the nucleic acid sequence situated between the kappa leader sequence and the BGHpolyA signal sequence. The corrected LC fragment and the VH containing Neo exp. vector were ligated and amplified in E. coli Top10 cells. Glycerol stocks were prepared for each individual clone, and a high quality plasmid preparation suitable for transfection of mammalian cells was prepared from the bacterial cultures as well.
[0203] By performing the transfer separately for each clone the VH:LC pairs originally selected by phage display were regenerated in the mammalian expression vector. In the instances where repair was not necessary the nucleic acid segment was transferred without performing PCR prior to the digestion with the appropriate restriction enzymes.
[0204] The mammalian expression vectors generated by the transfer described are suitable for expressing a full-length anti-RhD recombinant polyclonal antibody. Although the vectors are kept separate at this point it is still considered as a library of anti-RhD antibody expression vectors.
Transfection and Selection of Mammalian Cell Lines
[0205] The Flp-In CHO cell line (Invitrogen) was used as starting cell line for establishment of a recombinant polyclonal manufacturing cell line. However, to obtain a more homogenous cell line the parental Flp-In CHO cell line was sub-cloned. Briefly, the parental cell line was sub-cloned by limited dilution and several clones were selected and expanded. Based on growth behavior one clone, CHO-Flp-In (019), was selected as production cell line.
[0206] All the 56 plasmid preparations were transfected individually into the CHO-Flp-In (019) cell line as follows: the CHO-Flp-In (019) cells were cultured as adherent cells in F12-HAM with 10% fetal calf serum (FCS). 2.5×106 cells were transfected with plasmid representing one clone using Fugene6 (Roche). Cells were trypsinated 24 hours after transfection and transferred to 3×T175 flasks. Selection pressure, in this case 450 μg/ml Neomycin, was added 48 hours after transfection. Approximately two weeks later clones appeared. Clones were counted and cells were trypsinated and hereafter cultured as pools of clones expressing one of the 56 specific anti-Rhesus-D antibodies.
Adaptation to Serum Free Suspension Culture
[0207] The individual adherent anti-Rhesus-D antibody CHO-Flp-In (019) cell cultures were trypsinated, centrifuged and transferred to separate shaker flasks with 8×105 cells/ml in appropriate serum free medium (Excell302, JRH Biosciences).
[0208] Growth and cell morphology were followed over several weeks. When cells showed good and stable growth behavior and had doubling time below 32 hours 50 aliquots of each culture with 10×106 cells/tube were frozen down (56×50 aliquots).
Characterization of Cell Lines
[0209] All the individual cell lines were characterized with respect to antibody production and proliferation. This was performed with the following assays:
Production:
[0210] The production of recombinant antibodies in the individual cultures were followed over time by Kappa or Lambda specific ELISA. ELISA plates were coated overnight with goat-anti-human Kappa (Caltag) or goat-anti-human Lambda (Caltag) antibodies in carbonate buffer, pH 9.6. Plates were washed 6 times with washing buffer (1×PBS and 0.05% Tween 20) and blocked for 1 hour with washing buffer with 2% milk. Samples were added to wells and plates were incubated for 1 hour. Plates were washed 6× and secondary antibodies (goat-anti-human IgG (H+L) HRPO, Caltag) were added for 1 hour followed by 6× wash. ELISA was developed with TMB substrate and reaction stopped by addition of H2SO4. Plates were read at 450 nm.
[0211] Further, intracellular FACS staining, using fluorescently tagged antibodies was used to measure the production of recombinant antibodies in the cell culture system. 5×105 cells were washed in cold FACS PBS (1×PBS ad 2% FCS) and centrifuged. Cells were fixed in Cell:Fix (BD-Biosciences) for 20 min and hereafter washed in saponin buffer (1×PBS and 0.2% Saponin). The suspension was centrifuged and fluorescently tagged antibody (Goat F(ab)2 Fragment, Anti-human IgG(H+L)-PE, Beckman Coulter) was added for 20 min on ice. Cells were washed twice in saponin buffer and resuspended in FACS buffer and analyzed by FACS. This intracellular staining was used to determine the general expression level as well as to determine the homogeneity of the cell population in relation to expression of recombinant antibodies.
Proliferation:
[0212] Aliquots of cell suspension were taken three times a week and cell number, cell size, degree of clumping and percentage of dead cells were determined by CASY® (Cell Counter+Analyzer System from Scharfe System GmbH) analysis. The doubling time for the cell cultures was calculated by cell number derived form CASY® measurements.
Establishment of a Manufacturing Cell Line for Anti-Rhesus D Recombinant Polyclonal Antibody Production
[0213] Ten cell lines each expressing. a distinct recombinant anti-Rhesus-D antibody (RhD157.119D11, RhD158.119B06, RhD159.119B09, RhD161.119E09, RhD163.119A02, RhD190.119F05, RhD191.119E08, RhD192.119G06, RhD197.127A08 and RhD204.12.8A03) were selected to constitute the recombinant polyclonal manufacturing cell line. The Rhd197 and RhD204 were lambda clones whereas all the others were kappa clones.
[0214] After the cell cultures expressing the individual anti-Rhesus antibodies were fully adapted to serum free suspension culture in shaker flasks they were mixed in equal cell number, thereby generating a polyclonal CHO-Flp-In (019) cell line. The mixed cell culture was centrifuged and frozen down in aliquots of 10×106 cells/tube.
[0215] Two tubes (3948 FCW065 and 3949 FCW065) were thawed and cultured individually for 11 weeks in 1000 ml shaker flasks containing 100 ml Excell302 medium with neomycin.
[0216] The supernatant was harvested and filtered prior to purification of the anti-RhD rpAb.
Clonal Diversity
[0217] The clonal diversity was assayed both on the protein level as well as on the mRNA level. The supernatant sample used to analyze the antibody composition was taken after 9 weeks of cultivation, whereas the cell sample used to analyze the mRNA composition was taken at the harvest after 11 weeks of cultivation.
Antibody Composition:
[0218] The anti-RED rpAb expressed from the polyclonal CHO-Flp-In (019) cell line is an IgG1 isotype antibody. Anti-RhD rpAb was purified from both aliquots (3948 and 3949) using a column with immobilized Protein A. The individual antibodies interacted with immobilized Protein A at pH 7.4, whereas contaminating proteins were washed from the column. The bound antibodies were subsequently eluted from the column at low pH value (pH 2.7). The fractions containing antibodies, determined from absorbance measurements at 280 um, were pooled and dialyzed against 5 mM sodium acetate pH 5.
[0219] The anti-RhD rpAb compositions obtained from aliquot 3948 and 3949 (FCW065) after 9 weeks of cultivation were analyzed using cation exchange chromatography. The Protein A purified anti-RED rpAb was applied onto a PolyCatA column (4.6×100 mm) in 25 mM sodium acetate, 150 mM sodium chloride, pH 5.0 at a flow rate of 60 ml operated at room temperature. The antibody components were subsequently eluted using a linear gradient from 150-350 mM sodium chloride in 25 mM sodium acetate, pH 5.0 at a flow rate of 60 ml h-1. The antibody components were detected spectrophotometrically at 280 nm. The chromatogram (FIG. 8) was subsequently integrated and the area of the individual peaks A-J was subsequently used to quantitate antibody components (table 4). The total area of the peaks was set to 100%. The chromatograms from the two aliquots showed an identical peak distribution, as well as similar concentrations of the components in each peak. From these results it can be concluded that aliquots of the same polyclonal cell line grown under identical conditions will produce anti-RhD rpAb with a similar clonal diversity.
[0220] The individual members of the anti-RhD rpAb were allocated to one or more particular peaks (summarized in table 4). The allocation is based on chromatograms obtained for antibody products from each individual clone. No individual chromatogram was obtained for antibodies produced from RhD158.119B06, thus this clone was not assigned to any of the peaks. However it is considered likely that peak D constitute RhD158.119B06, the clone may also be represented in some of the other peaks due to heterogeneity. In particular the antibody product from clone RhD197.127A08 has a high degree of heterogeneity. Clone RhD190.119F05 should have been visible at 15.3 min. However, it was not detectable, indicating that this clone has been lost from the recombinant polyclonal manufacturing cell line. The loss of clone RhD190.119F05 corresponds to a 10% reduction of diversity which is considered acceptable with respect to diversity of the final anti-RhD rpAb composition.
TABLE-US-00010 TABLE 4 Quantity Quantity 3948 3949 Peak (% area) (% area) Clone name Comment A 5.1 5.1 RhD157.119D11 Clone is also present in peak B B 12.0 10.2 RhD157.119D11 This peak represent at least three RhD159.119B09 different clones RhD192.119G06 C 5.2 5.3 RhD191.119E08 D 1.2 0.8 (RhD158.119B06) Not actually allocated to this peak, but it is likely to be. May also be represented in other peaks. E 10.9 14.4 RhD204.128A03 F 24.3 23.0 RhD197.127A08 This clone split into several peaks, due G 13.6 12.5 RhD197.127A08 to heterogeneity. H 3.3 4.0 RhD197.127A08 I 14.0 13.7 RhD161.119E09 J 10.5 10.5 RhD163.119A02 RhD190.119F05 The clone has been lost
mRNA Composition:
[0221] The clonal diversity within the polyclonal CHO-Flp-In (019) cell line after 11 weeks of cultivation was estimated by RT-PCR-RFLP analysis. Briefly, a cell suspension corresponding to 200 cells were subjected to a freeze-thaw procedure and these lysates were used as template in a RT-PCR using One-STEP RT-PCR kit (Qiagen) with primers amplifying the light chain. The primer sequences were:
TABLE-US-00011 (SEQ ID 259) forward primer 5'-CGTTCTTTTTCGCAACGGGTTTG (SEQ ID 260) reverse primer 5'-AAGACCGATGGGCCCTTGGTGGA
[0222] The RT-PCR products were digested with HinfI and analyzed by agarose gel electrophoresis, visualizing the restriction product with ethidium bromide staining (FIG. 9).
[0223] The expected size of the restriction fragments obtained by HinfI digestion of the RT-PCR amplified light chains are shown for each individual clone in table 5. Six unique fragment sizes on the gel, which could be assigned to specific Rhesus D antibody producing clones, are indicated in bold. Not all unique fragments could be identified on the gel, these are indicated in italic. This does, however not necessarily mean that these clones are not represented in the culture, the fragments may either not have been separated sufficiently from other fragments to be identifiable, or their concentration is to weak compared to the stronger bands. This may be more pronounced for shorter fragments, since they bind a smaller number of ethidium bromide molecules and therefore are less visible.
TABLE-US-00012 TABLE 5 RhD # 157 158 159 161 163 190 191 192 197 204 HinfI 825 671 505 696 505 502 475 671 743 521 fragment 138 138 320 138 166 191 268 149 138 167 size 76 126 138 126 154 138 138 138 85 138 76 77 76 138 126 85 76 76 88 22 76 76 76
[0224] The two aliquots (3948 and 3949) of the same polyclonal cell line, show a similar expression pattern in the gel, although the intensity of the bands are not completely identical, this also indicates that aliquots of the same polyclonal cell line grown under identical conditions will produce anti-RhD rpAb with a similar clonal diversity.
Summary
[0225] The present. experiment succeeded in generating a library of anti-Rhesus D antibody expression vectors comprising 56 variant anti-Rhesus D-encoding nucleic acid segments (Table 3).
[0226] Plasmids containing individual members of the library were used to transfect the CHO-Flp-In (019) cell line, generating 56 individual cell lines capable of expressing a specific anti-RhD antibody.
[0227] 10 of these cell lines were mixed in order to generate a anti-RhD rpAb manufacturing cell line, which after 9 weeks cultivation still maintained 90% of the initial diversity. After 11 weeks of cultivation mRNA from six different clones could be unambiguously identified and several other clones are likely to be represented in the band an approximately 500 bp.
[0228] The fact that two aliquots of the polyclonal CHO-Flp-In (019) cell lines showed similar results with respect to clonal diversity, illustrated that reproducible results can be obtained.
Example 2
Generation of a Working Cell Bank for Larger Scale Production
[0229] Twenty seven cell cultures were selected to constitute the polyclonal cell line
TABLE-US-00013 (RhD157.119D11, RhD159.119B09, RhD160.119C07, RhD161.119E09, RhD162.119G12, RhD163.119A02, RhD189.181E07, RhD191.119E08, RhD192.119G06, RhD196.126H11, RhD197.127A08, RhD199.164E03, RhD201.164H12, RhD202.158E07, RhD203.179F07, RhD207.127A11, RhD240.125A09, RhD241.119B05, RhD244.158B10, RhD245.164E06, RhD293.109A09, RhD301.160A04, RhD305.181E06, RhD306.223E11, RhD307.230E11, RhD319.187A11 and RhD324.231F07).
[0230] In addition to the high degree of diversity among the individual clones, the clone selections were also based on growth and production characteristics of the individual cell cultures.
[0231] Included in the selection criteria at the cell culture level were:
I. Doubling time; had to be between 24 and 32 hours II: Intracellular staining; had to show a homogenous cell population III: Productivity; had to exceed 1.5 pg per cell per day
[0232] The 27 different cell cultures will be equally mixed in regard to cell number and this mix will constitute the working cell bank for a pilot plant production of anti-RhD rpAb.
Example 3
[0233] The present example illustrates the characterization of a polyclonal cell culture with eight members over time. The clonal diversity of the culture was assessed at the genetic level using RFLP analysis and at the protein level using a chromatographic technique in one dimension.
[0234] The polyclonal cell line of the present example was constituted of the following eight members: RhD191.119E08, RhD196.126H11, RhD201.164H12, RhD203.179F07, RhD244.158B10, RhD306.223E11, RhD319.187A11 and RhD324.231F07
[0235] In the example they will simply be written as follows RhD191, RhD201, RhD203, RhD244, RhD306, RhD319 and RhD324.
RFLP Analysis to Estimate Clone Diversity in Polyclonal Cell Cultures
[0236] The distribution of the individual clones in a polyclonal cell culture expressing eight different anti-Rhesus D antibodies was estimated by terminal RFLP (T-RFLP) analysis of RT-PCR products derived from the polyclonal cell line. In the T-RFLP procedure the forward and/or reverse primer(s) are fluorescently labeled and therefore a proportion of the restriction fragments generated from the amplicons will contain the label. The labeled fragments can subsequently be separated by capillary electrophoresis and detected by fluorescence. The analysis can be performed both on the light chain and the variable region of the heavy chain-encoding sequences, depending on the primers applied.
[0237] Briefly, a cell suspension corresponding to 200 cells was washed one time in PBS and subjected to a freeze-thaw procedure generating lysates used as template in a RT-PCR amplification using a One-Step RT-PCR kit (Qiagen) and suitable primers.
[0238] The RT-PCR was carried out on a standard thermal cycler with the following conditions:
TABLE-US-00014 Reverse transcription 55° C. for 30 min Denature 95° C. for 15 min Start cycle loop (35 cycles) Denature 95° C. for 30 sec Anneal 60° C. for 30 sec Elongate 72° C. for 5 min End cycle loop Elongate 72° C. for 15 min Finish 8° C. forever
[0239] For analysis of the light chain the following primers were used for the RT-PCR amplification. The reverse primer was 6-carboxyflorescein (FAM) labeled and the primer sequences were as follows:
TABLE-US-00015 VL Forward primer: 5'-TCTCTTCCGCATCGCTGTCT CL Reverse primer: 5'-FAM-AGGAAAGGACAGTGGGAGTGGCAC
[0240] Twenty μl of the RT-PCR product was digested with 1 U of NheI, 1 U of PstI and 1 U of HinfI (all from New England Biolabs) in NEB1 for 2 hours.
[0241] The labeled fragments were detected by fluorescence capillary electrophoresis on an ABI3700 (Applied Biosystems) at Statens Serum Institute, Copenhagen, DK.
[0242] The expected fragments for each of the anti-RhD antibody producing cell clones are shown in Table 6 and the FAM labeled fragments are indicated in bold.
TABLE-US-00016 TABLE 6 RhD # 191 196 201 203 244 306 319 324 NheI/PstI/HinfI 475 696 516 422 690 682 761 513 fragment size 210 138 166 318 138 138 138 166 138 76 138 138 76 76 76 138 76 67 76 76 67 67 67 76 67 59 76 67 41 59 76 58 67 18 18 17 67 18
[0243] The T-RFLP pattern is shown in FIG. 10 and all eight anti-Rhesus D antibody producing clones have been assigned to specific peaks. Under the assumption that there was no template/primer competition during the RT-PCR, the relative peak area will correspond to the relative amount of mRNA transcribed from each antibody light chain gene represented in the polyclonal cell line.
[0244] For analysis of the heavy chain variable region within the same polyclonal cell line the RT-PCR amplification was carried out with VH-specific primers. The primer sequences were as follows:
TABLE-US-00017 VH Forward primer: 5'-FAM CGTAGCTCTTTTAAGAGGTG VH Reverse primer: 5'-HEX-ACCGATGGGCCCTTGGTGGA
[0245] Twenty μl of the RT-PCR product was digested with 1 U of RsaI and 1 U of NdeI (all from New England Biolabs) in NEB2 for 2 hours.
[0246] The labeled fragments were detected by fluorescence capillary electrophoresis on an ABI3700. The analysis was performed by Statens Serum Institute, Copenhagen, DK.
[0247] The expected T-RFLP patterns are shown in Table 7, where the FAM labeled fragments are shown in bold and the HEX (6-Carboxy-2',4,4',5,7,7'-hexachlorofluorescein succinimidyl ester) labeled fragments are underscored.
TABLE-US-00018 TABLE 7 RhD # 191 196 201 203 244 306 319 324 RsaI/NdeI 203 429 186 350 435 328 232 266 Fragment 166 142 88 79 118 157 size 63 79 22 79 22 9 9 9
[0248] The polyclonal cell line was cultivated over 5 weeks and once a week samples were taken for T-RFLP analyses. The analysis was performed on the variable heavy chain, but could have been performed on the light chain as well if desired.
[0249] After capillary electrophoresis of the restriction fragments, the relative peak areas were integrated and used to estimate the clonal diversity of the polyclonal cell culture. The relative quantities over time are shown in FIG. 12.
[0250] Based on these results, it seems that RhD196 increase whereas RhD203 seems to decrease over time. The quantities of the other clones are quite stable during the cultivation period and all eight cDNA could be detected after five weeks of cultivation.
[0251] By performing T-RFLP on both light chain and heavy chain as well as on both mRNA and DNA it should be possible to obtain a precise fingerprint of the clonal diversity within the polyclonal cell culture, for example in cells at the limit of in vitro cell age or at any given time point during cultivation.
[0252] The technique can therefore be used to monitor the stability of the clonal diversity in a cell culture over time during antibody production. The technique can also be applied to monitor the batch-to-batch consistency for example of different ampoules frozen down from the same pWCB or in cells harvested after two or more manufacturing runs.
Cation-Exchange Chromatographic Analysis to Estimate Clonal Diversity in a Polyclonal Cell Culture
[0253] The polyclonal antibody produced from the same polyclonal cell culture as used in the T-RFLP analysis described above was analyzed using cation-exchange chromatography. The protein A purified recombinantly produced polyclonal antibody was applied onto a PolyCatA column (4.6×100 mm) in 25 mM sodium acetate, 150 mM sodium chloride, pH 5.0 at a flow rate of 60 ml h-1 operated at room temperature. The antibody components were subsequently eluted using a linear gradient from 150-350 mM sodium chloride in 25 mM sodium acetate, pH 5.0 at a flow rate of 60 ml h-1. The antibody components were detected spectrophotometrically at 280 nm and the Chromatogram was subsequently integrated and the area of individual peaks was then used to quantitate antibody components. The relative quantities over time are shown in FIG. 13.
Summary
[0254] The results obtained at the genetic level by the RFLP analysis and at the protein level by cation-exchange chromatography are comparable. FIGS. 12 and 13 clearly illustrate that most of the individual clones in the polyclonal cell line as well as the individual antibodies of the polyclonal antibody expressed from the cell line follow the same trends during the S weeks of cultivation. Thus, analyses at the genetic as well as at protein level are good equivalents for assessing the compositional diversity of a cell line at the genetic level and of the recombinant polyclonal protein produced from the cell line.
Example 4
[0255] The present example illustrates the characterization of a polyclonal cell culture with twenty-five members over time. The clonal diversity of the culture was assessed at the genetic level using T-RFLP analysis and at the protein level using a chromatographic technique in one dimension.
[0256] The polyclonal cell line of the present example was constituted of the twenty five members indicated in Table 8. Further, the growth characteristics of the individual clones are shown in Table 8.
TABLE-US-00019 TABLE 8 Doubling time Productivity Doubling Productivity Clone name (h) pg/(cell * day)a Clone name time pg/(cell * day) RhD157.119D11 25.6 4 RhD207.127A11 34.4 3.8 RhD159.119B09 26.1 1.4 RhD240.125A09 29.6 3.6 RhD160.119C07 25.4 3.8 RhD241.119B05 32.8 4.1 RhD162.119G12 27.7 5.9 RhD245.164E06 28 1.5 RhD189.181E07 27.8 3.2 RhD293.109A09 30.7 7.1 RhD191.119E08 30.8 1.2 RhD301.160A04 29.7 5.1 RhD192.119G06 25.4 1.2 RhD305.181E06 31 6.7 RhD196.126H11 32 8.7 RhD306.223E11 30.9 1.7 RhD197.127A08 30.1 1.6 RhD317.144A02 27 10.4 RhD199.164E03 27.3 2.9 RhD319.187A11 28 5.6 RhD201.164H12 27.6 10.6 RhD321.187G08 31 2.7 RhD202.158E07 28.8 3.1 RhD324.231F07 31.2 6.4 RhD203.179F07 31.5 N.Ac aData represent the average of two ELISA measurements bRhDVI reactive cData not available
[0257] In the following the clone names are generally only identified by their first three digits, e.g. RhD157.119D11 is written as RhD157.
T-RFLP Analysis of the Variable Part of the Heavy Chain Genes Derived from a Polyclonal Cell Culture Expressing Twenty-Five Different Anti-Rhesus D Antibodies Over a 5 Weeks Cultivation Period.
[0258] The polyclonal cell culture examined in the present example was composed of a mixture of cell cultures expressing twenty-five different anti-Rhesus D antibodies (generated as described in Example 1). The polyclonal cell culture was cultivated over 5 weeks and once a week samples were taken for T-RFLP analyses.
[0259] The RT-PCR was carried out with the VH-specific primers described in Example 3 and restriction fragmentation was carried out likewise.
[0260] T-RFLP of the twenty-five different anti-Rhesus D-encoding sequences will, if all genotypes are present, result in seventeen different FAM labeled fragments. Some fragments will represent up to three different genotypes whereas others will represent a single genotype. The expected sizes of FAM labeled fragments are shown in Table 9 together with the relative quantities of the different FAM labeled fragments over time. Further, one example of a T-RFLP profile is shown in FIG. 11.
TABLE-US-00020 TABLE 9 RsaI/NdeI FAM fragment Week 1 Week 2 Week 3 Week 4 Week 5 RhD # size (bp) Group Area % Area % Area % Area % Area % Rhd157 63 1 9.5 5.0 5.3 4.8 4.6 Rhd159 63 1 Rhd191 63 1 Rhd319 118 2 0.8 0.2 0.2 0.2 0.0 Rhd201 186 3 1.5 0.8 0.9 1.1 0.7 Rhd192 187 3 Rhd199 203 4 0.9 0.3 0.3 0.4 0.4 Rhd162 260 5 7.4 3.6 1.7 1.0 0.0 Rhd324 266 6 1.0 0.8 0.6 0.5 0.0 Rhd306 328 7 10.3 8.0 7.2 7.9 7.8 Rhd203 350 8 6.0 3.4 3.8 5.9 8.9 Rhd305 350 8 Rhd197 356 9 5.1 1.8 1.7 1.8 1.3 Rhd202 359 10 3.8 4.3 5.6 5.2 3.7 Rhd240 369 11 3.3 1.8 1.3 0.8 0.0 Rhd207 414 12 11.7 10.5 10.1 9.9 11.1 Rhd160 426 13 11.3 17.1 17.5 18.1 17.2 Rhd293 426 13 Rhd196 426 13 Rhd245 429 14 6.5 7.1 8.3 11.0 16.8 Rhd321 432 15 6.8 9.4 8.3 7.5 4.9 Rhd241 435 16 4.8 13.7 12.5 7.2 4.0 Rhd189 438 17 9.4 12.3 14.8 16.8 18.7 Rhd301 438 17 Rhd317 438 17
[0261] It was possible to separate the restriction fragments to an extent that allowed information to be obtained for twelve individual clones of the twenty-five clones constituting the cell line. The remaining fractions could potentially be subjected to sequencing in order to obtain more information on the remaining clones.
Cation-Exchange Chromatographic Analysis to Estimate Clonal Diversity in a Polyclonal Cell Culture Expressing Twenty-Five Different Anti-Rhesus D Antibodies
[0262] The polyclonal antibody produced from the same polyclonal cell culture as used in the T-RFLP analysis described above, was analyzed using cation-exchange chromatography. The protein A purified recombinantly produced polyclonal antibody was applied onto a PolyCatA column (4.6×100 mm) in 25 mM sodium acetate, 150 mM sodium chloride, pH 5.0 at a flow rate of 60 ml h-1 operated at room temperature. The antibody components were subsequently eluted using a linear gradient from 150-350 mM sodium chloride in 25 mM sodium acetate, pH 5.0 at a flow rate of 60 ml h-1. The antibody components were detected spectrophotometrically at 280 nm and the chromatogram was subsequently integrated and the area of individual peaks was used to quantitate the different antibody components. FIG. 14 shows the chromatogram produced from the sample obtained a week 4, the antibody containing peaks being numbered from 1 to 25. It is pure concurrence that the chromatogram contains an identical number of peaks as the number of individual antibodies in the polyclonal antibody analyzed. Table 10 show the relative content in percent of the total antibody components (AC1 to 25), as well as the representation of the individual antibodies in each antibody component (peak). The assignment of individual antibodies to the integrated chromatographic peaks was based on the retention times and peak patterns obtained from monoclonal antibodies analyzed using cation-exchange chromatography under identical conditions.
TABLE-US-00021 TABLE 10 RhD# Ab Week 1 Week 2 Week 3 Week 4 Week 5 Peak represented Rel. Area % Rel. Area % Rel. Area % Rel. Area % Rel. Area % AC 1 293, 319 2.06 2.3 1.7 1.06 0.81 AC 2 157, 293 3.63 3.83 3.97 3.89 3.06 AC 3 157, 192 2.66 2.8 2.89 2.83 2.34 AC 4 159, 189, 6.11 5.52 5.1 4.1 2.99 199 AC 5 319 2.18 1.94 1.33 1.08 1.26 AC 6 241, 191 6.01 6.4 6.32 5.42 4.1 AC 7 189, 192, 3.89 4.21 3.38 2.95 2.63 199, 201 AC 8 160 12.1 15.77 18.71 17.59 15.56 AC 9 203, 191 2.65 3.89 3.69 3.99 4.14 AC 10 162, 202 6.78 10.22 13.52 12.29 9.75 AC 11 203, 306, 2.86 3.63 4.35 3.66 3.92 301 AC 12 245 1.43 1.63 1.5 2.27 2.02 AC 13 301, 321 2.5 3.35 3.92 4.16 3.64 AC 14 305 2.44 2.61 3.12 4.23 6.07 AC 15 196, 197, 8.33 7.22 7.36 8.49 4.01 240, 305, 321 AC 16 197 3.82 2.71 2.15 1.86 7.86 AC 17 196, 240, 7.57 5.12 4.86 6.89 7.79 324 AC 18 197, 321 2.27 1.44 1.51 1.39 2.83 AC 19 196, 240 3.8 2.63 2.87 3.98 6.35 AC 20 317 4.58 1.39 0.77 0.71 0.86 AC 21 317 2.86 0.59 0.36 0.83 0.42 AC 22 207 2.07 2.61 1.58 1.65 1.93 AC 23 207 3.33 3.87 2.56 2.41 2.87 AC 24 207 2.46 3.48 1.73 1.52 1.92 AC 25 Unknown 1.58 0.83 2 0.75 0.87
[0263] Cation-exchange chromatography separates individual antibody members from a polyclonal antibody used on differences in net charge between the individual members and in addition separates forms of individual antibodies that appear charge heterogeneous. Several antibodies were therefore represented in a single peak, e.g. AC 1 containing RhD293 and RhD319 (see Table 10) and some individual antibodies were further represented in several chromatographic peaks, e.g. RhD319 which is present both in AC1 and 5 (see Table 10).
[0264] Peaks which contain more than one individual antibody could be subjected to additional protein chemical characterization techniques, such as quantitative analysis with anti-idiotype peptides, proteolytic peptide mapping, N-terminal sequencing or a second dimension chromatography.
Summary
[0265] The present example illustrates the combined use of T-RFLP analyses and cation exchange chromatography for assessing the distribution of the primary transcripts and of antibody components, respectively, over a period of cultivation. The T-RFLP analysis allows for unique identification of 12 individual clones of the 25 clones expressed in the polyclonal cell line and in the present example it is illustrated that these 12 clones could be detected during 4 weeks cultivation with the T-RFLP analysis. Potentially, more clones could be identified by sequence analysis of fragments representing more than one clone. The distribution of antibody components was analyzed using cation-exchange chromatography and in the present example it is seen that the distribution of the 25 analyzed components is relatively stable during cultivation. Although unique identification of all individual antibodies is difficult due to the inherent charge heterogeneous nature of the expressed antibodies it was demonstrated in the present example that antibody component 8 representing the RhD160 antibody showed the highest antibody level during the cultivation period in accordance with the high T-RFLP values obtained for group 13 representing the RhD160, 293, and 196 clones. Furthermore, the RhD 207 component, which could be uniquely identified by T-RFLP as well as by cation-exchange chromatography, showed T-RFLP levels of 10-11% and slightly lower levels of 5.5-10% obtained at antibody level. Overall, the two techniques together demonstrate a relatively stable production at the mRNA and antibody level during cultivation; however, potential discrepancies between the two techniques could also be seen, illustrated by the apparent loss of transcription of some clones at weeks 5 of cultivation contrasting the results obtained at the antibody level. Thus, the present example justifies the complementary use of both techniques to define cultivation intervals within which stable production of complex polyclonal protein can be obtained.
Example 5
[0266] The present example demonstrates the generation of pWCB containing anti-RhD rpAb with 25 individual members and provides confirmation of a minimal batch-to-batch variation of rpAb products purified from different vials from the pWCB.
Generation of the pWCB
[0267] To generate a pWCB containing anti-RhD rpAb with 25 individual members, one vial of each of 25 banked monoclonal anti-RhD antibody production cell lines (RhD157, 159, 160, 162, 189, 191, 192, 196, 197, 199, 201, 202, 203, 207, 240, 241, 245, 293, 301, 305, 306, 317, 319, 321, 324) were thawed in ExCell 302 medium containing 4 mM glutamine and expanded for 3 weeks in the same medium supplemented with 500 μg/ml G418 and anti-clumping agent diluted 1:250. Equal numbers of cells (2×106) from each culture were then carefully mixed together, and frozen in liquid nitrogen (5×107 cells/vial) using standard freezing procedures.
Cultivation in Bioreactors
[0268] Vials from the pWCB were thawed in T75 flasks (Nunc, Roskilde, Denmark) and expanded in spinner flasks (Techne, Cambridge, UK). 5 L bioreactors (Applikon, Schiedam, Netherlands) were inoculated with 0.6×106 cells/ml in 1.5 L. During the reactor runs, cells were fed on a daily basis with ExCell 302 medium supplemented with concentrated feed solution, glutamine and glucose to a final volume of 4.5 L. The bioreactor runs were terminated after 16-17 days. The three batches are termed Sym04:21, Sym04:23 and Sym04:24. The hatches were cultured at different points in time.
Analysis of Batch-to-Batch Variation
[0269] The recombinant polyclonal antibody samples were purified by affinity chromatography using HiTrap® rProtein A columns (GE Healthcare, UK).
[0270] The purified recombinant polyclonal antibody samples were analyzed using cation-exchange chromatography employing a PolyCAT A column (4.6×100 mm, from PolyLC Inc., MA, US) in 25 mM sodium acetate, 150 mM sodium chloride, pH 5.0 at a flow rate of 60 ml/h (room temperature). The antibody peaks were subsequently eluted using a linear gradient from 150 mM to 350 or 500 mM NaCl in 25 mM sodium acetate, pH 5.0 at a flow rate of 60 ml/h. The antibody peaks were detected spectrophotometrically at 280 nm. The chromatograms were integrated and the area of individual peaks used for quantification. As already mentioned some of the individual antibodies displayed charge heterogeneity and two antibodies may contribute to the same peak in the IEX chromatogram.
[0271] Table 11 show the relative content in percent of the total antibody components (AC). In the present example the relative area has been calculated for 35 AC, whereas Example 4 only calculated the relative area for 25 AC. This difference is strictly due to a different assignment of the peaks in the chromatogram and not to actual differences in the profile as such.
TABLE-US-00022 TABLE 11 Average Standard Peak Rel. Area % deviation AC 1 1.71 0.35 AC 2 2.36 0.13 AC 3 4.40 0.78 AC 4 3.58 0.78 AC 5 5.83 0.60 AC 6 2.11 0.25 AC 7 4.16 0.33 AC 8 4.21 0.59 AC 9 3.41 0.97 AC 10 14.22 2.91 AC 11 4.24 0.79 AC 12 2.98 0.47 AC 13 2.31 0.16 AC 14 2.44 0.26 AC 15 9.17 0.52 AC 16 5.08 0.43 AC 17 1.98 0.26 AC 18 3.04 0.26 AC 19 1.79 0.16 AC 20 1.39 0.07 AC 21 1.32 0.15 AC 22 2.60 0.23 AC 23 1.59 0.25 AC 24 0.62 0.12 AC 25 1.12 0.06 AC 26 1.31 0.04 AC 27 0.58 0.12 AC 28 1.30 0.25 AC 29 1.05 0.39 AC 30 0.66 0.24 AC 31 0.70 0.44 AC 32 1.64 0.10 AC 33 2.30 0.16 AC 34 1.77 0.24 AC 35 1.03 0.44
[0272] Table 11 shows that the reproducibility between the harvested antibody products from the three batches was high. The variation in the size of individual antibody peaks was within 20% for most antibody components, whereas the variation for some of the smallest peaks was slightly larger.
Example 6
[0273] The present example demonstrates that different batches of an anti-RhD rpAb with 25 individual members (same composition as in Example 4) bind to RhD-positive erythrocytes with similar potency and show comparable biological activity with respect to the relevant effector mechanisms. Antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis.
Preparation of Red Blood Cells
[0274] Red blood cells (RBC) were prepared from whole blood obtained from healthy donors after informed consent at the Blood Bank, Aalborg Hospital, DK, by washing the blood three times in PBS (Gibco, Invitrogen, United Kingdom) containing 1% bovine serum albumin (BSA, Sigma-Aldrich, Germany). The erythrocytes were resuspended and stored at 4° C. as a 10% solution in ID-Cellstab (DiaMed, Switzerland).
Preparation of PBMC
[0275] Buffy coats containing blood from healthy donors were obtained from the Blood Bank at the National Hospital, Copenhagen, Denmark and peripheral blood mononuclear cells (PBMC) were purified on Lymphoprep (Axis-Shield, Norway).
Potency Assay
[0276] The potency assay was adopted from the European Pharmacopoeia 4 (section 2.7A3 method C). The binding capacity of an anti-RhD rpAb with 25 individual members was measured using RhD-positive erythrocytes at 5×104 cells/μl in PBS, 1% BSA. Anti RhD rpAb batches, Sym04:21, Sym04:23, and Sym04:24, were obtained from individual 5 L fed batch bioreactor runs. Dilutions (11/2-fold) of the Anti-RhD rpAb batches were made in PBS, 1% BSA in triplicate in 96 well plates (Becton Dickinson Labware, N.J., USA). Fifty μl of the anti-RhD rpAb dilutions were mixed with 50 μl of erythrocytes and incubated at 37° C. for 40 min. The cells were washed twice (300×g, 2 min) in PBS, 1% BSA. Eighty μl of phycoerythrin-conjugated goat anti-human IgG, (Beckman Coulter, Calif., USA) diluted 1:20 in PBS, 1% BSA was added to each sample and left at 4° C. for 30 min. The samples were washed in PBS, 1% BSA and in FacsFlow (Becton Dickinson, Belgium) (300×g, 2 min), and resuspended in 200 μl FACSFlow. The samples were run on a FACSCalibur (Becton Dickinson, Calif., USA) and data analysis performed using CellQuest Pro and Excel. The three individual Anti-RhD rpAb batches displayed essentially identical binding potency to RhD-positive erythrocytes (FIG. 15A)
Combined ADCC and Phagocytosis Assay
[0277] This assay was adapted from Berkman et al. 2002. Autoimmunity 35, 415-419. Briefly, RhD positive (RhD+) and RhD negative (RhD-) red blood cells (RBC) were labeled with radioactive Chromium. For Cr51 labeling, 1×108 RhD+ and RhD- RBC, respectively, were centrifuged (600×g for 10 min) and 100 μl Dulbeccos'modified eagles medium (DMEM) and 200 μl sodium chromate (0.2 μCi) (GE Healthcare, UK) were added to each tube before incubation for 1.5 hours at 37° C. The suspension was washed twice in 50 ml PBS and resuspended in 1 ml complete DMEM (containing 2 mM glutamine, 1% Penicillin-Streptomycin and 10% fetal calf serum) (Invitrogen, CA, US). Cells were adjusted to 4×106 cells/ml and 50 μl/well were added to 96-well cell culture plates (Nunc). Fifty μl of two-fold. dilutions of Anti-RhD rpAb from batch Sym04:21 or Sym04:24, was then added to each well, except control. wells. Control wells were supplied with complete DMEM and used for either spontaneous lysis/retention or maximum lysis.
[0278] The PBMC were adjusted to 2×107 cells/ml, and 100 μl were added to each well and incubated at 37° C. overnight. One hundred ti 1% Triton-X-100 (Merck, Germany) was added to the maximum lysis control wells. The plates were centrifuged (600×g for 2 min) and 50 μl of the supernatant was transferred to ADCC Lumaplates (Perkin Elmer, Belgium).
[0279] Following transfer of the supernatants, the cell culture plates were centrifuged (300×g for 2 min) and 50 μl supernatant from the maximum lysis wells were transferred to another LumaPlate (phagocytosis LumaPlate). In the cell culture plate, the supernatant was removed from the remaining wells and lysis buffer (140 mM NH4Cl, 17 mM Tris-HCl) was added, followed by 5 min incubation at 37° C. NH4Cl lyses the RBC, but leaves the PBMC fraction and thereby the phagocytozed RBC intact. After RBC lysis, the plates were centrifuged (4° C., 2 min, 300 g), pellets were washed twice in PBS, and resuspended in 100 μl PBS. One hundred μl 1% Triton-X-100 was added to the wells to lyse the phagocytic PBMC, and 50 μl of the lysate was transferred to the phagocytosis LumaPlates. The Lumaplates were dried overnight at 40° C. and counted in a TopCount NXT (Packard, Conn., USA). All data were imported into Excell and analyzed as described by Berkman et al. 2002. Autoimmunity 35, 415-419. Briefly, the computations were performed as follows:
ADCC: Immune lysis (%)=(mean test Cr51 released-mean spontaneous Cr51 released)/(total Cr51 in target erythrocytes-machine background)×100
Phagocytosis: Immune phagocytosis (%)=(mean test Cr51 retention-mean spontaneous Cr51 retention)/(total Cr51 in target erythrocytes-machine background)×100
[0280] All data were normalized to the combined maximum plateau values
[0281] The functional activity of anti-RhD rpAb from the two consecutive reactor runs showed nearly identical functional activity in both in vitro assays (FIGS. 15B and 15C) reflecting the high consistency between the batches.
Example 7
[0282] The present example demonstrates that the clonal diversity of an anti-RhD rpAb with 25 individual members (same composition as in Example 4) is maintained during down-stream processing (DSP). Cation-exchange chromatographic analysis is used to estimate clonal diversity during DSP of the recombinant polyclonal antibody.
Down-Stream Processing
[0283] An anti-RhD rpAb sample, containing 25 individual members, from a developmental bioreactor run was purified using the following DSP steps:
1. capture of the antibodies using a MAbSelect column 2. virus inactivation at pH 3 3. buffer exchange using a Sephadex G-25 column 4. anion-exchange chromatography using a DEAE-Sepharose column 5. virus filtration using a Planova 15N filter, and 6. hydrophobic charge induction chromatography using a MEP Hypercel column 7. ultra filtration/diafiltration using a Millipore biomax filter Analysis of Clonal Diversity after Individual DSP Steps
[0284] Cation-exchange chromatography was used to analyze the clonal diversity during DSP of a recombinant polyclonal antibody composition. Samples taken after step 1, 3, 4 and 6 during DSP of a anti-RhD rpAb was applied onto a PolyCatA column (4.6×100 mm) in 25 mM sodium acetate, 150 mM sodium chloride, pH 5.0 at a flow rate of 60 ml h-1 operated at room temperature. The antibody components were subsequently eluted using a linear gradient from 150-500 mM sodium chloride in 25 mM sodium acetate, pH 5.0 at a flow rate of 60 ml h-1. The antibody components were detected spectrophotometrically at 280 nm and the chromatograms were compared (FIG. 16) to detect the potential loss of clonal diversity during DSP. In the present example it was demonstrated, using cation-exchange chromatography that the clonal diversity is essentially unchanged during DSP of a recombinant polyclonal antibody.
Sequence CWU
1
260148DNASaccharomyces cerevisiae 1gaagttccta ttccgaagtt cctattctct
agaaagtata ggaacttc 48230DNAArtificialSynthetic primer
sequence 2ggaggcgctc gagacggtga ccagggtgcc
30330DNAArtificialSynthetic primer sequence 3ggaggcgctc gagacggtga
ccattgtccc
30430DNAArtificialSynthetic primer sequence 4ggaggcgctc gagacggtga
ccagggttcc
30530DNAArtificialSynthetic primer sequence 5ggaggcgctc gagacggtga
ccgtggtccc
30639DNAArtificialSynthetic primer sequence 6ccagccgggg cgcgcccagr
tgcagctggt gcartctgg
39739DNAArtificialSynthetic primer sequence 7ccagccgggg cgcgccsagg
tccagctggt rcagtctgg
39839DNAArtificialSynthetic primer sequence 8ccagccgggg cgcgcccagr
tcaccttgaa ggagtctgg
39939DNAArtificialSynthetic primer sequence 9ccagccgggg cgcgccsagg
tgcagctggt ggagtctgg
391039DNAArtificialSynthetic primer sequence 10ccagccgggg cgcgccgagg
tgcagctggt ggagwcygg
391139DNAArtificialSynthetic primer sequence 11ccagccgggg cgcgcccagg
tgcagctaca gcagtgggg
391239DNAArtificialSynthetic primer sequence 12ccagccgggg cgcgcccags
tgcagctgca ggagtcsgg
391339DNAArtificialSynthetic primer sequence 13ccagccgggg cgcgccgarg
tgcagctggt gcagtctgg
391439DNAArtificialSynthetic primer sequence 14ccagccgggg cgcgcccagg
tacagctgca gcagtcagg
391551DNAArtificialSynthetic primer sequence 15accgcctcca ccggcggccg
cttattaaca ctctcccctg ttgaagctct t
511638DNAArtificialSynthetic primer sequence 16caaccagcgc tagccgacat
ccagwtgacc cagtctcc
381738DNAArtificialSynthetic primer sequence 17caaccagcgc tagccgatgt
tgtgatgact cagtctcc
381838DNAArtificialSynthetic primer sequence 18caaccagcgc tagccgaaat
tgtgwtgacr cagtctcc
381938DNAArtificialSynthetic primer sequence 19caaccagcgc tagccgatat
tgtgatgacc cacactcc
382038DNAArtificialSynthetic primer sequence 20caaccagcgc tagccgaaac
gacactcacg cagtctcc
382138DNAArtificialSynthetic primer sequence 21caaccagcgc tagccgaaat
tgtgctgact cagtctcc
382250DNAArtificialSynthetic primer sequence 22accgcctcca ccggcggccg
cttattatga acattctgta ggggccactg
502350DNAArtificialSynthetic primer sequence 23accgcctcca ccggcggccg
cttattaaga gcattctgca ggggccactg
502438DNAArtificialSynthetic primer sequence 24caaccagcgc tagcccagtc
tgtgctgact cagccacc
382538DNAArtificialSynthetic primer sequence 25caaccagcgc tagcccagtc
tgtgytgacg cagccgcc
382638DNAArtificialSynthetic primer sequence 26caaccagcgc tagcccagtc
tgtcgtgacg cagccgcc
382736DNAArtificialSynthetic primer sequence 27caaccagcgc tagcccartc
tgccctgact cagcct
362841DNAArtificialSynthetic primer sequence 28caaccagcgc tagccctttc
ctatgwgctg actcagccac c
412941DNAArtificialSynthetic primer sequence 29caaccagcgc tagccctttc
ttctgagctg actcaggacc c
413038DNAArtificialSynthetic primer sequence 30caaccagcgc tagcccacgt
tatactgact caaccgcc
383138DNAArtificialSynthetic primer sequence 31caaccagcgc tagcccaggc
tgtgctgact cagccgtc
383241DNAArtificialSynthetic primer sequence 32caaccagcgc tagcccttaa
ttttatgctg actcagcccc a
413338DNAArtificialSynthetic primer sequence 33caaccagcgc tagcccagrc
tgtggtgacy caggagcc
383438DNAArtificialSynthetic primer sequence 34caaccagcgc tagcccwgcc
tgtgctgact cagccmcc 3835381DNAHomo Sapiens
35gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggagggtc cctgagactc
60tcctgtgtag cctctggatt caccttcagg agttttgaca tgaactgggt ccgccaggct
120ccagggaagg ggctggagtg ggtttcatac attaatagta ggggtagtac catatactac
180gcagactctg tgaagggccg attcaccatc tccagagaga acgccaagaa ctcactgtat
240ctgcaaatga acagcctgag agccgaggac acggctgttt attactgtgc gagagatttg
300tacggtgact atgaccctaa gtcctactat tactacggta tgggcgtctg gggccaaggg
360accacggtca ccgtctcgag t
38136375DNAHomo Sapiens 36gaggtgcagc tggtggagac cgggggaggc gtggtccagc
ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt ctccttcagt aactttggct
tccactggat ccgccagtct 120ccaggcaagg ggctcgaatg ggtggcagtt atttggtatg
atggaagcaa cagattctat 180gcagattccg tgaagggccg attcaccatc tccagagata
gttcgaagaa catgctgttt 240ctgcaaatgg acagcctgag agccgaggac acggctgtgt
attactgtgc gagagaaatt 300tccatgaaag tagtgatccg cagacactac gttatggacg
tctggggcca cgggaccacg 360gtcaccgtct cgagt
37537381DNAHomo sapiens 37gaggtgcagc tggtggagac
cgggggaggc ttagtacagc ctggagggtc cctgagactc 60tcctgtgcag cctctggatt
cagcttcagg agttatgaca tgaactgggt ccgccaggct 120ccagggaagg ggctggagtg
ggtgtcatac atcagtggca gaggtagtac aacatattac 180gcagactctg tgaagggccg
attcaccatc tccagagaca acgccaagaa ctcactgtat 240ctgcaaatga acagtctgag
agccgaggac acggctgttt attactgtgc gagagatttg 300tacggtgact acgatcctaa
gtcctactat tactacgcta tggacgtctg gggccacggg 360accacggtca ccgtctcgag t
38138375DNAHomo Sapiens
38gaggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc
60tcctgtgcag cgtctggatt caccttcagt aattatggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcggtt atatggtatg atggaagtaa tagattctat
180gcggactccg tgaagggccg attcaccatc tccagagaca attccaagaa tacgctgtat
240ctgcaaatga acaacctgag agccgaggac acggctctct attcctgtgc gagagagatt
300actacgacag tagtggtccg aagacactac cttatggaca tctggggcca agggaccacg
360gtcaccgtct cgagt
37539378DNAHomo Sapiens 39gaggtgcagc tggtggagtc tgggggaggc gtggtccagt
ctgggaggtc cctgagactc 60tcatgtgcag cctctggatt caccttcagt aacaatggca
tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcattt atttggtatg
atggaagtaa taaatactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacactgtat 240ctgcaaatgg acggcctgag agccgaggac tcggctgtgt
attactgtgc gagagaggaa 300atagcagctc gtctttattc tcgctaccac tacgctatgg
acgtctgggg ccaagggaca 360atggtcaccg tctcgagt
37840375DNAHomo Sapiens 40caggtgcagc tggtggagtc
tgggggaggc ctggtcaagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
cggcttcagt gcttatagca tgaactgggt ccgccaggct 120ccggggaagg ggctggagtg
ggtctcatcc attactagca ctactacata ctacgcagac 180tcagtgaagg gccgattcag
catctccaga gacaacgcca agagcacact gtacctgcga 240atgaacagcc tgagagccga
ggacacggct gtatattatt gtgtgagaga aatcgccttt 300agggggagca cttattctcg
gtggtcgtac tactttgact tctggggcca gggaaccctg 360gtcaccgtct cgagt
37541384DNAHomo Sapiens
41caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc
60acctgcactg tctctggtgg ctccatcagt agttactact ggagctgggt ccggcagccc
120ccagggaagg gactggagtg gattgggtat atctattaca gtgggagcac caactacaac
180ccctccctca agagtcgagt caccatagca ttagacacgt ccaagaacca gttctccctg
240aagctgaggt ctgtgaccgc tgcggacacg gccgtgtatt tctgtacgag agactggagg
300caatatgggt cggcgatccg aggttctcga tactactacg ggatggacgt ctggggccaa
360gggaccacgg tcaccgtctc gagt
38442387DNAHomo Sapiens 42caggtgcagc tggtggagtc tgggggaggc gtggtccagc
ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt caccttcagt agctatggca
tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atatggtatg
atggaagtaa taaatactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggctgtgt
attactgtgc gagagacatg 300gttactatgg ttcggggagc ctacagaaac tactactact
acggtatgga cgtctggggc 360aaagggacca cggtcaccgt ctcgagt
38743381DNAHomo Sapiens 43gaggtgcagc tggtggagac
cgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt
cagcttcagt aactatggca tgcactgggt ccgccaggct 120ccaggcaagg ggttggagtg
ggtggcagtt atttggtttg atggaagtat taaatattat 180gtagactccg tgaaaggccg
attcaccatc tccagagaca attccaagaa cacactctat 240ctgcaaatga acagcctgag
agccgaggag acggctatat atttctgtgc gagagaaaat 300agtgttctag tcccaggtac
tatacggagg cgatattatt tggactactg gggccaggga 360accctggtca ccgtctcgag t
38144381DNAHomo Sapiens
44caggtgcagc tggtggagtc tgggggagac ctggtacagc ctggagggtc cctgagactc
60tcctgtgcag cctctggatt caccttcagg agttatgaaa tgaactgggt ccgccaggct
120ccagggaagg ggctggagtg ggtttcatac attaatagta gaggtaatac caaatactac
180gcagactctg tgaagggccg attcaccatc tccagagaca acgccaagaa gtcactgtac
240ctgcaaatga gcagcctgag agccgaggac acggctgttt attactgtgc gagaaatttg
300ttcggtgact acgatcttaa gtcctactac tataacgcta tggacgtctg gggccaaggc
360accctggtca ccgtctcgag t
38145381DNAHomo Sapiens 45caggtgcagc tggtggagtc tgggggaggc ctggtcaagc
ctggggggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt agttatgcca
tgaactgggt ccgccaggct 120ccagggaagg gactggagtg ggtctcatcc attagcggta
ctagtagtta catatactat 180gcagactcag tgaagggccg atttaccatt ttcagagaca
acgccaagag ctcagtttat 240ctgcaaatga acagcctgag agtcgaggac acggctgtct
attactgcgc gagagataga 300tggtggggca tggttcggag agtttttccc acctatccct
ttgactactg gggccaggga 360accctggtca ccgtctcgag t
38146375DNAHomo Sapiens 46caggtgcagc tggtggagac
cgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt
caccttcagt agctatggca tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg
ggtggcagtt atatggtatg atggaagtaa taaagactat 180gcagaccccg tgaagggccg
attcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctcag
agccgaggac acggctgtgt attactgtgc gagagagatc 300gcctcccgtg gatatagtcg
ctacttatac tactttgact cctggggcca gggaaccctg 360gtcaccgtct cgagt
37547387DNAHomo Sapiens
47caggtcacct tgaaggagtc tggtcctgtg ctggtgaaac ccacagagac cctcacgctg
60acctgcaccg tctctgggtt ctcactcagc aatgctagaa tgggtgtgag ctggatccgt
120cagcccccag ggaaggccct ggagtggctt gcacacattt tttcgaatga cgaaaaatcc
180tacagcacat ctctgaagag caggctcacc atctccaagg acacctccaa aagccaggtg
240gtccttacca tgaccaacat ggaccctgtg gacacagcca catattactg tgcacggatg
300aggcttacta tggttcgggg agttattacg tactactact acagtatgga cgtctggggc
360caagggacca cggtcaccgt ctcgagt
38748381DNAHomo Sapiens 48caggtgcagc tgcaggagtc gggcccagga ctggtgaagc
cttcacagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagc agtggtagtt
actactggag ctggatccgg 120cagcccgccg ggaagggacc ggagtggatt gggcgtatct
ataccagtgg gagtaccaac 180tacaacccct ccctcaagag tcgactcacc atatcagtag
acacgtccaa gaaccagttc 240tccctgaagc tgacctctgt gaccgccgca gacacggccg
tgtattactg tgcgagggcc 300ccttcttact atgatagtag tggttatcgt tactggtaca
tcgatctctg gggccgtggc 360accctggtca ccgtctcgag t
38149378DNAHomo Sapiens 49caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt
caccttcagt agctatggca tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg
ggtggcagtt atatggtatg atggaagtaa taaagactat 180gcagactccg tgaagggccg
attcaccatc tccagagaca actccaagaa cacgctgtat 240ctgcaaatga acagcgtgag
agccgaggac acggctgttt attactgtgc gagagaattg 300agcacgcaac gtggatacag
ccgctaccac tatgttatgg acgtctgggg ccaagggacc 360acggtcaccg tctcgagt
37850363DNAHomo Sapiens
50caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc
60tcctgtgcag cgtctggatt caccttcagt aactatggca tgcactgggt ccgccaggct
120cctggcaagg ggctggaatg ggtggcagtt atctggtttg atggaagtaa tagagactat
180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa gacgctctat
240ctgcaaatga acagcctgag agccgaggac acggctgttt attactgtgc gacagagttg
300gccagagggc ggctacgagc cctagagtac tggggccagg gaaccctggt caccgtctcg
360agt
36351378DNAHomo Sapiens 51caggtgcagc tggtgcagtc tgggggaggc gtggtccagc
ctgggaagtc cctgagactc 60tcctgtgcag cgtctggatt caccttcagt acctatggca
tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atatggtatg
atggaagtaa taaatactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
cttccaagaa cacgctgtat 240ctgcaaatga acagcgtgag agtcgaggac acggctgttt
attactgtgc gagagatttg 300accacgcaac gtggatacag ccgctatcac tatgttatgg
acgtctgggg ccaagggacc 360acggtcaccg tctcgagt
37852378DNAHomo Sapiens 52caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt
caccttcagt agctatggca tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg
ggtggcagtt atatggtatg acggaagtaa taagtactat 180gcagactccg tgaagggccg
attcaccatc tccagagaca attccaagaa cacgctgcat 240ctgcaaatga acagcctgag
agccgaggac acggctgtgt attactgtgc gagagaagtg 300ggttttggca gtggctggtc
acgatactac tacggtatgg acgtctgggg ccaagggacc 360acggtcaccg tctcgagt
37853390DNAHomo Sapiens
53gaggtgcagc tggtggagtc tggaggagga ggcgtggtcc agcctgggag gtccctgagg
60ctctcctgtg cagcgtctgg attcaccttc agtagctatg gcatgcactg ggtccgccag
120gctccaggca aggggctgga gtgggtggca gttatatggt atgatggaag taataaatac
180tatgcagact ccgtgaaggg ccgattcacc atctccagag acaattccaa gaacacgctg
240tatctgcaaa tgaacagcct gagagctgag gacacggctg tgtattactg tgcgagagag
300agtactctgt atagcagcag ctggtacagg aggtactact actacggtat ggacgtctgg
360ggccaaggga ccacggtcac cgtctcgagt
39054387DNAHomo Sapiens 54caggtgcagc tggtggagtc tgggggaggc gtggtccagc
ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt agctatgcta
tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atatcatatg
atggaagcaa taaatactac 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agctgaggac acggctgtgt
attactgtgc gagagagagt 300actctatata gcagcagctg gtacaggagg tactactact
acagtatgga cgtctggggc 360caagggacca cggtcaccgt ctcgagt
38755384DNAHomo Sapiens 55caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcgg cgtctggatt
caccttcagt agctatggca tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg
ggtggcagtt atatggttcg atggaagtaa tagatactat 180ggagactccg tgaagggccg
agtcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcgaatga acagcctgag
agccgaggac acggctgtgt attactgtgc gagagagttc 300tatacacgta gcgggctatg
gtcacaaggg tactcctatt acatggacgt ctggggcaaa 360gggaccacgg tcaccgtctc
gagt 38456387DNAHomo Sapiens
56gaggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc
60tcctgtgcag cgtctggatt caccttcagt aactatggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaatactat
180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat
240ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagagatg
300gtctcctata gcagcagctg gtaccgccgc tactactact acgttatgga cgtctggggc
360aaagggacca cggtcaccgt ctcgagt
38757369DNAHomo Sapiens 57gaggtgcagc tggtggagtc tgggggaggc gtggtccagc
ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt cagcttcagt aactatggca
tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atatcatatg
atggaagtga gaagtactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa ggcgctgtat 240ctgcaaatga acagcctgag agctgaggac acggctatgt
attattgtaa gaataaagtg 300ggagctaccc ggcgggcagt cgttgctttt gatatctggg
gccaagggac cacggtcacc 360gtctcgagt
36958381DNAHomo Sapiens 58gaggtgcagc tggtggagac
cgggggaggc ctggtcaagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
caccttcagg agttatagca tgaactgggt ccgccaggct 120ccagggaagg ggctggagtg
ggtctcatcc attggtagta gtagtactta cacatactcc 180gcagactcag tgaagggccg
attcaccatc tccagagaca acgccaagaa ctcactgtat 240ctgcaaatga acagcctgag
agccgaggac acggctgtgt attactgtgc gagaggagag 300cctctaaact atgattacat
ttggggaggg tatcgtttca ctatccactg gggccaggga 360accctggtca ccgtctcgag t
38159387DNAHomo Sapiens
59caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc
60tcctgtgcag cgtctggatt caccttcagt agttatggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggaatg ggtggcaatt atatggtttg atggaagtaa taaatactat
180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat
240ctgcaaataa acagcctgag agctgaggac acggctgtgt attactgtgc gaaagagcac
300ggctattata gcagcagctg gtaccgaaac tactattact acgctatgga cgtctggggc
360caagggacca cggtcaccgt ctcgagt
38760363DNAHomo Sapiens 60gaggtgcagc tggtggagtc cgggggaggc gtggtccagc
ctgggaggtc cctgagactc 60tcctgtgcgg cgtctggatt caccttcagt agctatggca
tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atatggtatg
atggaagtaa taaagactat 180gtagactctg tgaagggccg attcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagtctgag agccgaggac acagctgtgt
attactgtgc gagagagttg 300gccaaagggc ggctacgaga cctagaccac tggggccagg
gaaccctggt caccgtctcg 360agt
36361387DNAHomo Sapiens 61gaggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt
caccttcagg acttctgcca tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg
ggtggcagtt atatggtatg atggaagtaa taaatactat 180gcagactccg tgaagggccg
attcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag
agccgaggac acggctgtgt attactgtgc gagagagatg 300gtctcctata gcagcagctg
gtaccgccgc tactactact acaatatgga cgtctggggc 360aaagggacca cggtcaccgt
ctcgagt 38762375DNAHomo Sapiens
62caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggacgtc cctgagattg
60tcctgtgcag cgtctgggtt cacctttaga acctatggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcatat atatggtatg atggaagtaa taaatactat
180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagag cacgctgaat
240ctgcaaatga acagcctcag agccgaggac acggctgtgt attactgtgc gagagagatc
300gcctcccgtg gatatagtcg ctacttatac tactttgact cctggggcca gggcaccctg
360gtcaccgtct cgagt
37563387DNAHomo Sapiens 63cagctgcagc tgcaggagtc gggcccagga ctggtgaagc
cttcggggac cctgtccctc 60acctgcgctg tctctggtgg ctccatgagg agtagtaact
ggtggacttg ggtccgccag 120cccccaggga aggggctgga atggattggg gaaatccatc
atggtgggag caccaactac 180aacccgtccc tccagagtcg agtcacgata tcagtagaca
agtccaagaa ccggttctcc 240ctgaagctga gctctgtgac cgccgcggac acggccgtat
atcactgtgc gagggggagg 300agttattatg atagtagtgg gcattccttt cgcggtctgg
taccttttga tatctggggc 360caagggacaa tggtcaccgt ctcgagt
38764384DNAHomo Sapiens 64caggtgcagc tgcaggagtc
gggcccagga ctggtgaagc cttcggagac cctgtccctc 60atctgcactg tctctggtgg
ctccatcagt agtaactact ggagctggat ccggcagccc 120ccagggaagg gactggagtg
gattgggtat atctattaca gtgggaacac caactacaac 180ccctccctca agagtcgagt
caccatatca ttagacacgt ccaagaacca gttctccctg 240aagctgagat ctgtgaccgc
tgcggacacg gccgtgtatt actgtgcgag agagtggagg 300cagtatggct cggggatccg
aggttctcga tactactacg gtatggacgt ctggggccag 360ggcaccctgg tcaccgtctc
gagt 38465387DNAHomo Sapiens
65gaggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc
60tcctgtgcag cgtctggatt caccttcagt aaccatggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaatactat
180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat
240ctgcaaatga acagcctgag ggccgaggac acggctgtgt attactgtgc gagagagatg
300gcctcctata gcagcagctg gtaccgccgc tactactact acgttatgga cgtctggggc
360aaagggacca cggtcaccgt ctcgagt
38766375DNAHomo Sapiens 66caggtgcagc tggtggagtc tgggggaggc gtggtccagc
ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt caccttcagt acctatggca
tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atatggtatg
atggaagtca gaaatactat 180gtagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggctgtct
attactgtgc gagagaggtt 300gcggttcggg gagttattcg ctactactac ggtatggacg
tctggggcca agggaccacg 360gtcaccgtct cgagt
37567384DNAHomo Sapiens 67caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc tctgagactc 60tcctgtgcag cctcgggatt
cagcttcagt agttatggca tgcactgggt ccgccaggct 120ccaggcaagg gactggagtg
ggtggcaatt atttggtatg atgggagtaa caaactctat 180gcagactccg tgaagggccg
attcaccatc tccagagaca attccaggaa tacgttgtat 240ctgcaaatga gcagtgtgag
agccgaggac acggctgtgt attactgtgc gagagactct 300gttcggggag tcagtagatg
ggggactcag aaatattacg ctatggacgt ctggggccaa 360gggaccacgg tcaccgtctc
gagt 38468378DNAHomo Sapiens
68gaggtgcagc tggtgcaatc tgggggaggc gtggtccagc ctgggaggtc cccgagactc
60tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcattt atatggtatg atggaagtaa taaatactat
180gcagactctg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat
240ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagatctc
300cggaaccacg ttttttggag tggttattct acctcttttg actactgggg ccagggaacc
360ctggtcaccg tctcgagt
37869387DNAHomo Sapiens 69gaggtgcagc tggtggagac cgggggaggc gtggtccagc
ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt caccttcagt agctatggca
tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atatggtatg
atggaagtaa taaatactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggctgtgt
attactgtgc gagagagatg 300gtctcctata gcagcagctg gtaccgccgc tactactact
acaatatgga cgtctggggc 360aaagggacca cggtcaccgt ctcgagt
38770387DNAHomo Sapiens 70caggtgcagc tggtggagac
cgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt
caccttcagt agctatggca tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg
ggtggcagtt atatggtctg atggaagtaa taaatactat 180gcagactccg tgaagggccg
attcaccatc tccagagaca attccaggaa cacgctgtat 240ctgcaaatga acagcctgag
agccgaggac acggctgtgt tttactgtgc gagagagcaa 300ggggggtata gcagcagttg
gtaccgccgc tactactact actatatgga cgtctggggc 360caagggacca cggtcaccgt
ctcgagt 38771375DNAHomo Sapiens
71gaggtgcagc tggtggagtc tgggggaggc gtggtccagc ctggaaggtc cctgagactc
60tcctgtgcag cgtctggatt caccttcaat acctatggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaatactat
180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat
240ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagaggtt
300gcggttcggg gagttattcg ctactactac gctatggacg tctggggcca agggaccacg
360gtcaccgtct cgagt
37572369DNAHomo Sapiens 72caggtgcagc tggtggagtc tgggggaggc gtggtccagc
ctgggaggtc tctgagactc 60tcctgtgcag cctctggatt cagcttcagt aactatggca
tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atatcatatg
atggaagtga gaagtactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa aacgctgtat 240ctgcaaatga acagcctgag agctgaggac acggctatgt
attattgtaa gaataaagtg 300ggagctaccc ggcgggcagt tgttgctgtt gatatttggg
gccaagggac aatggtcacc 360gtctcgagt
36973381DNAHomo Sapiens 73caggtgcagc tggtggagtc
tgggggaggc ttggtacagc ctggagggtc cctgagactc 60tcctgtgtag gctctggatt
caccttcagg agttttgata tgaactgggt ccgccaggct 120ccagggaagg ggctggagtg
gatttcatat attaatagta gggggaacac cagatactat 180gtagactctg tgaagggccg
attcaccatc tccagagaca acgccaagaa ctcattgtat 240ctgcaaatga acagcctgcg
agccgaggac acggctgttt actactgtgc gagagatttg 300tacggtgact acgatcctaa
gtcctactat tactacggta tggacgtctg gggccaaggg 360acaatggtca ccgtctcgag t
38174375DNAHomo Sapiens
74gaggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc
60tcctgtgaag cgtctggatt caccttcagt aattatggca tgcactggtt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcaatt atatggtatg atggaagtaa taaacactat
180gcagactccg tgaagggccg attcaccatc tccagagaca attcgaagaa cacgctgtat
240ctgcaaatga acagcctcag agccgaggac acggctgtgt atttctgtgc gagagagatc
300gcctcccgtg gatatagtcg ctacttatac tactttgact cctggggcca gggaaccctg
360gtcaccgtct cgagt
37575375DNAHomo Sapiens 75caggtgcagc tggtggagtc tgggggaggc gtggtccagc
ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt caccttcagg acctatggca
tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atatggtatg
atggaagtaa taaagactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacgctgcat 240ctgcaaatga acagcctgag agccgaggac acggctgtgt
attactgtgc gagagagatc 300gcctcccgtg gatatagtcg gtacttatac taccttgact
tctggggcca gggcaccctg 360gtcaccgtct cgagt
37576378DNAHomo Sapiens 76caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt
caccttcagt agctatggca tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg
ggtggcagtt atatggtatg atggaagtaa taaatattat 180gcagactccg tgaagggccg
attcaccatc tccagagaca attccaagaa cacactatat 240ctgcaaatga acagcgtgag
agccgaggac acggctgttt attactgtgc gagagatttg 300agcacgcaac gtggatacag
ccgctactac tatgttatgg acgtctgggg ccaagggacc 360acggtcaccg tctcgagt
37877363DNAHomo Sapiens
77caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc
60tcctgtgcag cgtctggatt caccttcagt aactttggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaagactat
180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagag gacgctgtat
240ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagagttg
300gccagagggc ggctacgaga cctagactac tggggccagg gcaccctggt caccgtctcg
360agt
36378384DNAHomo Sapiens 78caggtgcagc tggtggagtc tgggggaggc gtggtccagc
ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt caccttcagt agctatggca
tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atatggtatg
atggaagtaa taaatactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggctgtgt
attactgtgc gagagagttc 300tatacacgta gcgggctatg gtcacaaggg tactcctatt
acatggacgt ctggggcaaa 360gggaccacgg tcaccgtctc gagt
38479387DNAHomo Sapiens 79caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt
caccttcagt agctatggca tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg
ggtggcagtt atatggtttg atggaggtaa taaatactat 180gcagactccg cgaagggccg
attcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag
agccgaggac acggctgtgt attactgtgc gagagatgcg 300tcggtgcttt ctggattggt
tactcgaagg ttagtctact acggtatgga cgtctggggc 360caagggacca cggtcaccgt
ctcgagt 38780387DNAHomo Sapiens
80caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc
60tcctgtgcag cctctggatt caccttcagt agctatggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcgttt atatcatatg atggaagtaa taaatactat
180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtct
240ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gaaagagcac
300ggctattatc gcagcagctg gtaccgaaac tactactact atgggatgga cgtctggggc
360caagggacca cggtcaccgt ctcgagt
38781387DNAHomo Sapiens 81caggtgcagc tggtggagtc tgggggaggc gtggtgcagc
ctgagaggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt tcctatggca
tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atatcatatg
atggaagtaa taaatactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agctgaggac acggctgtgt
attactgtgc gaaagatgaa 300gtggggtata gcagcagctg gtacaggcgc tactactact
acgctatgga cgtctggggc 360caagggacca cggtcaccgt ctcgagt
38782384DNAHomo Sapiens 82caggtgcagc tggtgcagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt
caccttcagt agctatggca tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg
ggtggcagtt atatggtatg atggaagtaa taaatactat 180gcagactccg tgaagggccg
attcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag
agccgaggac acggctgtgt attactgtgc gagagagaca 300gtggtggtag ctgccaaaat
acgaaaccac tactactacg ctatggacgt ctggggccaa 360gggaccacgg tcaccgtctc
gagt 38483387DNAHomo Sapiens
83caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc
60tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcaggt atatggtatg atggaagtaa taaatactat
180gcagactccg tgaagggccg attcaccatc tccagagaca attccaggaa cacgatgtat
240ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagagcaa
300ggggggtata gcagcagttg gtaccgccgc tactactact acaatatgga cctctggggc
360caagggacca cggtcaccgt ctcgagt
38784387DNAHomo Sapiens 84caggtgcagc tggtggagtc tgggggaggc gtggtccagc
ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt caccttcagt agctatggca
tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atatggtatg
atggaagtaa taaatactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acaccctgag agacgacgac acggctgtgt
attactgtgc gagagagggt 300actctgtata gcagcagctg gtacaggagg tactactact
acggtatgga cgcctggggc 360caagggacca cggtcaccgt ctcgagt
38785387DNAHomo Sapiens 85caggtgcagc tggtggagac
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt
caccttcagt agctatggca tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg
ggtggcaggt atatggtatg atggaagtaa taaatactat 180ggagactccg tgaagggccg
attcaccatc tccagagaca attccaggaa tacgctgtat 240ctgcatatga acagcctcag
agccgacgac acggctgtgt attactgtgc gagggaacac 300ggcgggtcta ggagtggctg
gtacacttta cgtctcgcgt actactttga ctactggggc 360cagggcaccc tggtcaccgt
ctcgagt 38786387DNAHomo Sapiens
86caggtgcagc tgcaggagtc cggcccagga ctggtgaagc cttcggggac cctgtccctc
60acctgcgctg tctctggtgg ctccatcagg ggtagtaatt ggtggagttg ggtccgccag
120cccccaggga aggggctgga gtggattggg gaaatccatc atggtgggag caccaactac
180aacccgtccc tcaagagtcg agtcacgata tcagtagaca agtccaagaa ccggttctcc
240ctgaagctga gctctgtgac cgccgcggac acggccgtgt attactgtgc gagggggacg
300tcttattatg atagtagtgg ttattccttt cgcggtctgg tagcttttga tatctggggc
360caagggacaa tggtcaccgt ctcgagt
38787387DNAHomo Sapiens 87caggtgcagc tggtggagtc tgggggaggc gtggtccagc
ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt caccttcagt acctatggca
tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atatggtatg
atggaagtaa taaaaactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggctgtgt
attactgtgc gagagaccta 300caagggtata gaagcagctg gtaccggatg tactactact
acggtatgga cgtctggggc 360caagggacca cggtcaccgt ctcgagt
38788381DNAHomo Sapiens 88gaggtgcagc tggtggagtc
tgggggaggc ctggtcaagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
caccttcagg agttatagca tgaactgggt ccgccaggct 120ccagggaagg ggctggagtg
ggtctcatcc attggtagta gtagtattta cacatactcc 180gcagactcag tgaagggccg
attcaccatc tccagagaca acgccaagaa ctcactgtat 240ctgcaaatga acagcctgag
agccgaggac acggctgtgt attactgtgc gagaggagag 300cctctaaact atgattacat
ttggggaagg tctcgtctca ctatccactg gggccaggga 360accctggtca ccgtctcgag t
38189387DNAHomo Sapiens
89caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc
60tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaatattat
180gcagactccg tgaagggccg attcaccatc tcccgagaca attccaagaa cacgctgtat
240ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gagagattgg
300gtcactcgca gcagcaactg gtacaggaac tactactact acggtatgga cgtctggggc
360caagggacca cggtcaccgt ctcgagt
38790372DNAHomo Sapiens 90gaggtgcagc tggtggagtc tgggggaggc ttagttcagc
cggggaggtc cctgagactc 60tcctgtgtag cctctggatt caccttcagt agctactgga
tgcactgggt ccgccaagtt 120ccagggaagg ggctggtgtg ggtctcacgt attaatgttg
atgggaagag cacaagctac 180gcggactccg tgaagggccg attcaccatc tccagagaca
acgccaagaa cacgctgtac 240ctgcaaatga acagtctgag agccgaggac acggctgtgt
attactgtgc aagagatccc 300cgacgatttt tggagtgggc ccgctacggt atggacgtct
ggggccgagg gaccacggtc 360accgtctcga gt
37291654DNAHomo Sapiens 91gaaattgtgt tgacgcagtc
tccagccacc ctgtctttgt ctccagggga gagagccacc 60ctctcctgca gggccagtca
gagtgttagc agctacttag cctggtacca acagaaacgt 120ggccaggctc ccaggctcct
catctttaat gcatccaaca gggccactgg catcccagcc 180aggttcagtg gcagtgggtc
tgggacagac ttcactctca ccatcagcag cctagagcct 240gaagattttg cagtttatta
ctgtcagcag cgtagcagct ggcctccgat gtacactttt 300ggccagggga ccaagctgga
gatcaaacga actgtggctg caccatctgt cttcatcttc 360ccgccatctg atgagcagtt
gaaatctgga actgcctctg ttgtgtgcct gctgaataac 420ttctatccca gagaggccaa
agtacagtgg aaggtggata acgccctcca atcgggtaac 480tcccaggaga gtgtcacaga
gcaggacagc aaggacagca cctacagcct cagcagcacc 540ctgacgctga gcaaagcaga
ctacgagaaa cacaaagtct acgcctgcga agtcacccat 600cagggcctga gctcgcccgt
cacaaagagc ttcaacaggg gagagtgtta ataa 65492648DNAHomo Sapiens
92gacatccaga tgacccagtc tccatccccc ctgtctgcat ctgtaggaga cagagtctcc
60atcacttgcc gggcgagtcg gggcattagc aattctttag cctggtatca gcagaaacca
120gggaaagttc ctaagctcct catctatgct gcatccactt tgcaatcagg ggtcccatct
180cggttcagtg gcggtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct
240gaagatgttg caacttatta ctgtcacacg tataacagtg cccccttcgc tttcggccct
300gggaccaaag tggatatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca
360tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat
420cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag
480gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg
540ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc
600ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttaataa
64893654DNAHomo Sapiens 93gaaattgtga tgacacagtc tccaggcacc ctgtctttgt
ctccagggga aagagccacc 60ctctcctgca gggccagtca cactgtcagc agcggctact
tagcctggta tcagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca
acagggccac tggcgtccca 180gacaggttcg gtggcagtgg gtctgggaca gacttcactc
tcaccatcag cagactggag 240cctgaagatt ctgcagtgta tttttgtcag caatatggaa
cctcaccggg ggtcactttc 300ggccaaggga cacgactgga aattgaacga actgtggctg
caccatctgt cttcatcttc 360ccgccatctg atgagcagtt gaaatctggg actgcctctg
ttgtgtgcct gctgaataac 420ttctatccca gagaggccaa agtacagtgg aaggtggata
acgccctcca atcgggtaac 480tcccaggaga gtgtcacaga gcaggacagc aaggacagca
cctacagcct cagcagcacc 540ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct
acgcctgcga agtcacccat 600cagggcctga gctcgcccgt cacaaagagc ttcaacaggg
gagagtgtta ataa 65494648DNAHomo Sapiens 94gacatccagt tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga cagagtctcc 60atcacttgcc gggcgagtca
gggcattagc aattctttag cctggtatca gcagaaacca 120gggaaagttc ctaagctcct
catctatgct gcatccactt tgcaatcagg ggtcccatct 180cggttcagtg gcggtggatc
tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagatgttg caacttatta
ctgtcaaaag tataacagtg cccccttcgc tttcggccct 300gggaccaaag tggatatcaa
acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360tctgatgagc agttgaaatc
tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420cccagagagg ccaaagtaca
gtggaaggtg gataacgccc tccaatcggg taactcccag 480gagagtgtca cagagcagga
cagcaaggac agcacctaca gcctcagcag caccctgacg 540ctgagcaaag cagactacga
gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa
gagcttcaac aggggagagt gttaataa 64895651DNAHomo Sapiens
95gacatccaga tgacccagtc tccatccttc ctgtctgcat ctgtaggaga cagagtcacc
60atcacttgcc gggccagtca gggcattaga cgttatttag cctggtttca gcaaaaacca
120gggaaagccc ctaaactcct gatcttttct gcatccactt tgcaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct
240gaggattttg caacttatta ctgtcaacag cttagtagtt accctccgta cacttttggc
300caggggacca agctggagat caaacgaact gtggctgcac catctgtctt catcttcccg
360ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc
420tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc
480caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg
540acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag
600ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgttaata a
65196648DNAHomo Sapiens 96gacatccagt tgacccagtc tccatcttct gtgtctgctt
ctgtagggga cagcgttacc 60atcacttgtc gggcgagtca ggctgtgagc gggtgggtag
cctggtatca gcagaaacca 120gggaaagccc ctaaactcct gatctttggt ttgtccaatt
tggaggatgg ggtcccatca 180aggttcagcg gcagtggatc tgcgacagac ttcactctca
ccatcaccgg cctgcagcct 240gaagatttgg caacgtacta ctgtctacag gctaacaggt
tccccctctc tttcggcgga 300gggaccaggg tagagatcaa acgaactgtg gctgcaccat
ctgtcttcat cttcccgcca 360tctgatgagc agttgaaatc tggaactgcc tctgttgtgt
gcctgctgaa taacttctat 420cccagagagg ccaaagtaca gtggaaggtg gataacgccc
tccaatcggg taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca
gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa gtctacgcct
gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt
gttaataa 64897654DNAHomo Sapiens 97gaaattgtgt tgacgcagtc
tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca
gaatcctaga cgcaactttt tagcctggta ccaacagaaa 120cctggccagg ctcccaggct
tctcatctat gctgcatcca ccagggccac cggcatccca 180gacaggttca gtggcagtgg
gtctgggaca gacttcactc tcaccatcga cagactggag 240cctgaagatt ctgcagtgta
ttactgtcag gtctatggta gctcacctct gtacactttt 300ggccagggga ccaaggtgga
gatgaaacga actgtggctg caccatctgt cttcatcttc 360ccgccatctg atgagcagtt
gaaatctgga actgcctctg ttgtgtgcct gctgaataac 420ttctatccca gagaggccaa
agtacagtgg aaggtggata acgccctcca atcgggtaac 480tcccaggaga gtgtcacaga
gcaggacagc aaggacagca cctacagcct cagcagcacc 540ctgacgctga gcaaagcaga
ctacgagaaa cacaaagtct acgcctgcga agtcacccat 600cagggcctga gctcgcccgt
cacaaagagc ttcaacaggg gagagtgtta ataa 65498654DNAHomo Sapiens
98gaaattgtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc
60ctctcctgca gggccagtca gagtgttagc agcaacttag cctggtacca gcagaaacct
120ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg tatcccagcc
180aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct
240gaagattttg cagtttatta ctgtcagcag tataataact ggccgaccct gtacactttt
300ggccagggga ccaagctgga gatcaaacga actgtggctg caccatctgt cttcatcttc
360ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac
420ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac
480tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc
540ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat
600cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta ataa
65499648DNAHomo Sapiens 99gacatccagt tgacccagtc tccatcctcc ctgtctgcat
ctgtgggaga cagagtcacc 60atcacttgcc gggcaagtca gagcattagc agctatttaa
attggtatca gcagaaacca 120ggggaagccc ccaaactcct gatctatgtt gcatccactt
tgcaaagtgg ggccccatca 180aggttcagtg gcagtggatc tgggacagat tacactctaa
ccattagcag tctgcaacct 240gaagattctg caactttcta ctgtcaacag acttacagtc
ccccttacac ttttggccag 300ggaaccaagc tggagatcaa acgaactgtg gctgcaccat
ctgtcttcat cttcccgcca 360tctgatgagc agttgaaatc tggaactgcc tctgttgtgt
gcctgctgaa taacttctat 420cccagagagg ccaaagtaca gtggaaggtg gataacgccc
tccaatcggg taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca
gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa gtctacgcct
gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt
gttaataa 648100657DNAHomo Sapiens 100gaaattgtgc
tgactcagtc tccaggcacc ctgtcttcgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca aagtgttact agcagatact tagcctggta ccagcagaaa 120catggccagg
ctcccaggct cctcatctat ggtacatcca cgagggccac tggcatccca 180gacaggttca
gtggcggagg gtctcagaca gacttcactc tcaccatcag cagactggag 240cctgaagatt
ttgcagtgta ttattgtcag cactatgatg actcaatttc gacgtacatt 300tttggcccgg
ggaccgagct ggagatcaag cgaactgtgg ctgcaccatc tgtcttcatc 360ttcccgccat
ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat 420aacttctatc
ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggt 480aactcccagg
agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc 540accctgacgc
tgagcaaagc agactacgag aaacacaaag tctacgcctg cgaagtcacc 600catcagggcc
tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg ttaataa
657101648DNAHomo Sapiens 101gacatccagt tgacccagtc tccatcttct gtgtctgctt
ctgtaggaga cagagtgacc 60atcacttgtc gggcgagtca gggtattaac aacttattag
cctggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctacgct gcatccaatt
tgcaaagtgg ggtcccatcg 180aggttcagcg gcagtggatc tgggacagat ttcactctca
ctatcaacag cctgcagcct 240gaagattttg caacctacta ttgtcaacag gctaacagtt
tccctctcac tttcggcgga 300gggaccaagg tggagatcaa acgaactgtg gctgcaccat
ctgtcttcat cttcccgcca 360tctgatgagc agttgaaatc tggaactgcc tctgttgtgt
gcctgctgaa taacttctat 420cccagagagg ccaaagtaca gtggaaggtg gataacgccc
tccaatcggg taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca
gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa gtctacgcct
gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt
gttaataa 648102651DNAHomo Sapiens 102gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
gggcaagtca gagcattagc agctatttaa attggtatca gcagaatcca 120gggaaagccc
ctaagctcct gatctatggt gcatccaatt tgcaaagtgg ggtcccatca 180aggttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg
caacttacta ctgtcaacag agttacagta ccctcgcgct cactttcggc 300ggagggacca
aggtggagat caaacgaact gtggctgcac catctgtctt catcttcccg 360ccatctgatg
agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420tatcccagag
aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480caggagagtg
tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540acgctgagca
aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600ggcctgagct
cgcccgtcac aaagagcttc aacaggggag agtgttaata a
651103651DNAHomo Sapiens 103gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gagcattagc acctatttaa
attggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gcatccactt
tgcaaagtgg ggtcccatca 180aggttcagtg gcagtggatc tgggacacat ttcactctca
ccatcagctc tctgcaacgt 240gaagattttg caacttacta ctgtcaacag acttacagaa
cccccacgtg gacgttcggc 300caagggacca aggtggaaat caaacgaact gtggctgcac
catctgtctt catcttcccg 360ccatctgatg agcagttgaa atctggaact gcctctgttg
tgtgcctgct gaataacttc 420tatcccagag aggccaaagt acagtggaag gtggataacg
ccctccaatc gggtaactcc 480caggagagtg tcacagagca ggacagcaag gacagcacct
acagcctcag cagcaccctg 540acgctgagca aagcagacta cgagaaacac aaagtctacg
cctgcgaagt cacccatcag 600ggcctgagct cgcccgtcac aaagagcttc aacaggggag
agtgttaata a 651104645DNAHomo Sapiens 104gacatccagt
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
gggcaagtca gaacattaac aggtatttaa attggtatca gcacaaacca 120gggagagccc
ctgagctcct gatctatgct gcgtccactt tacgaagggg ggtcccatca 180aggttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg
caacttacta ctgtcaacag agttacagta gagggacgtt cggccaaggg 300actaaggtgg
aaatcaaacg aactgtggct gcaccatctg tcttcatctt cccgccatct 360gatgagcagt
tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 420agagaggcca
aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 480agtgtcacag
agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 540agcaaagcag
actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 600agctcgcccg
tcacaaagag cttcaacagg ggagagtgtt aataa
645105651DNAHomo Sapiens 105gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gagcattagc acctatttaa
attggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gcatccactt
tgcaaagtgg ggtcccatca 180aggttcagtg gcagtggatc tgggacacat ttcactctca
ccatcagcag tctgcaacgt 240gaagattttg caacttacta ctgtcaacag agttacggaa
cccccacgtg gacgttcggc 300caagggacca aggtggaaat caagcgaact gtggctgcac
catctgtctt catcttcccg 360ccatctgatg agcagttgaa atctggaact gcctctgttg
tgtgcctgct gaataacttc 420tatcccagag aggccaaagt acagtggaag gtggataacg
ccctccaatc gggtaactcc 480caggagagtg tcacagagca ggacagcaag gacagcacct
acagcctcag cagcaccctg 540acgctgagca aagcagacta cgagaaacac aaagtctacg
cctgcgaagt cacccatcag 600ggcctgagct cgcccgtcac aaagagcttc aacaggggag
agtgttaata a 651106657DNAHomo Sapiens 106cagactgtgg
tgactcagga gccctcactg actgtgtccc caggagggac agtcaccctc 60acctgtgctt
ccagcactgg agcagtcacc actggttact atccaaactg gttccagcag 120aaacctggac
aagcacccag ggcactgatt tatagtacaa gcaagaaaca ctcctggacc 180cctgcccggt
tctcaggctc cctccttggg ggcaaagctg ccctgacact gtcaggtgtg 240cagcctgagg
acgaggctga gtattactgc ctgctcttct atggtggtgc tcagctgggg 300gtgttcggcg
gagggaccaa gctgaccgtc ctaggtcagc ccaaggctgc cccctcggtc 360actctgttcc
cgccctcctc tgaggagctt caagccaaca aggccacact ggtgtgtctc 420ataagtgact
tctacccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480aaggcgggag
tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540agctacctga
gcctgacgcc tgagcagtgg aagtcccaca aaagctacag ctgccaggtc 600acgcatgaag
ggagcaccgt ggagaagaca gtggccccta cagaatgttc ataataa
657107651DNAHomo Sapiens 107gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gagcattggc aactatttaa
attggtatca gcagaaacca 120ggaaaagccc ctaagctcct gatctctgct gcatccagtt
tgcaaagtgg ggtcccgtca 180aggttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcatcgt 240gaagactatg caacttacta ctgtcaacag agttacagta
cccccccgta cacttttggc 300caggggacca agctggagat caaacgaact gtggctgcac
catctgtctt catcttcccg 360ccatctgatg agcagttgaa atctggaact gcctctgttg
tgtgcctgct gaataacttc 420tatcccagag aggccaaggt acagtggaag gtggataacg
ccctccaatc gggtaactcc 480caggagagtg tcacagagca ggacagcaag gacagcacct
acagcctcag cagcaccctg 540acgctgagca aagcagacta cgagaaacac aaagtctacg
cctgcgaagt cacccatcag 600ggcctgagct cgcccgtcac aaagagcttc aacaggggag
agtgttaata a 651108651DNAHomo Sapiens 108gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120gggaaagccc
ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg
caacttacta ctgtcaacag agttacagta cctccacgtg gacgttcggc 300caagggacca
aggtggaaat caaacgaact gtggctgcac catctgtctt catcttcccg 360ccatctgatg
agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420tatcccagag
aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480caggagagtg
tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540acgctgagca
aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600ggcctgagct
cgcccgtcac aaagagcttc aacaggggag agtgttaata a
651109648DNAHomo Sapiens 109gacatccagt tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gagcattagc agctatttaa
attggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gcatccagtt
tgcaaagtgg ggtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg caacttacta ctgtcaacag agttacagta
cctcgtggac gttcggccaa 300gggaccaagg tggaaatcaa acgaactgtg gctgcaccat
ctgtcttcat cttcccgcca 360tctgatgagc agttgaaatc tggaactgcc tctgttgtgt
gcctgctgaa taacttctat 420cccagagagg ccaaagtaca gtggaaggtg gataacgccc
tccaatcggg taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca
gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa gtctacgcct
gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt
gttaataa 648110654DNAHomo Sapiens 110gaaattgtgt
tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagtgttacc agcaacttag cctggtacca gcagaaacct 120ggccaggctc
ccaggctcct catctatggt gcatccacca gggccactgg tgtcccagcc 180aggttcagtg
gcagtgggtc tgggacagag ttcagtctca ccatcagcag cctgcagtct 240gaagattttg
cagtttatta ctgtcagcag tataataact ggcctcccat attcactttc 300ggccctggga
ccaaactgga tatcaaacga actgtggctg caccatctgt cttcatcttc 360ccgccatctg
atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac 420ttctatccca
gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac 480tcccaggaga
gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc 540ctgacgctga
gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat 600cagggcctga
gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta ataa
654111651DNAHomo Sapiens 111gaaattgtgc tgactcagtc tccaggcacc ctgtctttgt
ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcagctact
tagcctggta ccagcataaa 120cctggccagg ctcccaggct cctcatctac ggttcatcca
acagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca gacttcactg
tcaccatcag cagactggag 240cctgaagatt ttgcagtgta ttactgtcag cagtatggta
ctgcacccta cacttttggc 300caggggacca agctggagat caaacgaact gtggctgcac
catctgtctt catcttcccg 360ccatctgatg agcagttgaa atctggaact gcctctgttg
tgtgcctgct gaataacttc 420tatcccagag aggccaaagt acagtggaag gtggataacg
ccctccaatc gggtaactcc 480caggagagtg tcacagagca ggacagcaag gacagcacct
acagcctcag cagcaccctg 540acgctgagca aagcagacta cgagaaacac aaagtctacg
cctgcgaagt cacccatcag 600ggcctgagct cgcccgtcac aaagagcttc aacaggggag
agtgttaata a 651112654DNAHomo Sapiens 112cagtctgccc
tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60tcctgcactg
caaccagcag tgatattggg gcttataact atgtctcctg gtaccaacac 120cacccaggta
aagcccccaa agtcatcatt actgatgtta ataagcggcc ctcaggggtc 180cctgatcgct
tctctggctc caagtctggc aacacggcct ccctgaccat ctcagggctc 240cagcctgagg
atgaggctga gtattcctgc tgctcatatg caggcaccta cagttatgtc 300ttcggaactg
ggaccaaggt caccgtcctg agtcagccca aggccaaccc cactgtcact 360ctgttcccgc
cctcctctga ggagctccaa gccaacaagg ccacactagt gtgtctgatc 420agtgacttct
acccgggagc tgtgacagtg gcctggaagg cagatggcag ccccgtcaag 480gcgggagtgg
agaccaccaa accctccaaa cagagcaaca acaagtacgc ggccagcagc 540tacctgagcc
tgacgcccga gcagtggaag tcccacagaa gctacagctg ccaggtcacg 600catgaaggga
gcaccgtgga gaagacagtg gcccctacag aatgttcata ataa
654113654DNAHomo Sapiens 113ctttcttctg agctgactca ggaccctgct gtgtctgtgg
ccttgggaca gacagtcagg 60atcacatgcc aaggagacag cctcagaagt tattatgcaa
actggtacca gcagaagcca 120ggacaggccc ctctatcagt catctatggt aaaaacaacc
ggccctcagg gatcccggac 180cgattctctg gctccaactc aggaaacaca gctttcttga
ccatcactgg gactcaggcg 240gaagatgagg ctgactatta ctgtaactcc cgggacagca
gtggtaatta tcgggagcta 300ttcggcggag ggaccaagct gaccgtcctt ggtcagccca
aggctgcccc ctcggtcact 360ctgttcccgc cctcctctga ggagcttcaa gccaacaagg
ccacactggt gtgtctcata 420agtgacttct acccgggagc cgtgacagtg gcctggaagg
cagatagcag ccccgtcaag 480gcgggagtgg agaccaccac accctccaaa caaagcaaca
acaagtacgc ggccagcagc 540tatctgagcc tgacgcctga gcagtggaag tcccacagaa
gctacagctg ccaggtcacg 600catgaaggga gcaccgtgga gaagacagtg gcccctgcag
aatgctctta ataa 654114651DNAHomo Sapiens 114gacatccagt
tgacccagtc tccatccttc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
gggccagtca tggcattagc agttatttag cctggtatca acagaaacca 120gggaaagccc
ctaacctcct gatctttcct gcatccactt tgcaaagtgg ggtcccgtca 180agattcagcg
gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcggcct 240gaagattttg
caacttatta ctgtcaacaa cttaatagtt attccaggtg ggcgttcggc 300caagggacca
aggtggaagt caaacgaact gtggctgcac catctgtctt catcttcccg 360ccatctgatg
agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420tatcccagag
aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480caggagagtg
tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540acgctgagca
aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600ggcctgagct
cgcccgtcac aaagagcttc aacaggggag agtgttaata a
651115651DNAHomo Sapiens 115gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgtc gggcaagtca gagcattagg aggtatttaa
attggtatca gaagaaacca 120gggaaagccc ctaagctcct gatctatgct gcatccagtt
tgcaaagtgg ggtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg caacttacta ctgtcaacag agttaccgta
cccaaggtct cactttcggc 300ggagggacca aggtggagat caaacgaact gtggctgcac
catctgtctt catcttcccg 360ccatctgatg agcagttgaa atctggaact gcctctgttg
tgtgcctgct gaataacttc 420tatcccagag aggccaaagt acagtggaag gtggataacg
ccctccaatc gggtaactcc 480caggagagtg tcacagagca ggacagcaag gacagcacct
acagcctcag cagcaccctg 540acgctgagca aagcagacta cgagaaacac aaagtctacg
cctgcgaagt cacccatcag 600ggcctgagct cgcccgtcac aaagagcttc aacaggggag
agtgttaata a 651116657DNAHomo Sapiens 116caggctgtgg
tgactcagga gccctcactg actgtgtccc caggagggac agtcactctc 60acctgtgctt
ccagcactgg agcagtcacc actggttact atccaaactg gttccagcag 120aaacctggac
aagcacccag ggcactggtt catagtacaa gcaagaaaca ctcctggacc 180cctgcccggt
tctcaggctc cctccttggg ggcaaagctg ccctgacact gtcaggtgtg 240cagcctgagg
acgaggctga gtattactgc ctgctcttct atggtggtgc tcaactgggg 300gtgttcggcg
gagggaccaa actgaccgtc ctaggtcagc ccaaggctgc cccctcggtc 360actctgttcc
cgccctcctc tgaggagctt caagccaaca aggccacact ggtgtgtctc 420ataagtgact
tctacccggg agccgtgaca gtggcctgga aggcggatag cagccccgtc 480aaggcgggag
tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540agctacctga
gcctgacgcc tgagcagtgg aagtcccaca aaagctacag ctgccaggtc 600acgcatgaag
ggagcaccgt ggagaagaca gtggcccctg cagaatgctc ttaataa
657117657DNAHomo Sapiens 117caggctgtgg tgactcagga gccctcactg actgtgtccc
caggagggac agtcactctc 60acctgtgctt ccagcactgg atcagtcacc agtggttact
atccaaactg gttccagcag 120aaacctggac aagcacccag gccactgatt tctggtacaa
gcaacaaact ctcctggacc 180cctgcccggt tctcaggctc cctccttggg ggcaaagctg
ccctcacagt gtcaggtgtg 240cagcctgagg acgaggctgt gtattactgc ctgctctact
atggtgttcc tcagccagtg 300gtattcggcg gagggaccaa gctgaccgtc ctaggtcagc
ccaaggctgc cccctcggtc 360actctgttcc cgccctcctc tgaggagctt caagccaaca
aggccacact ggtgtgtctc 420ataagtgact tctacccggg agccgtgaca gtggcctgga
aggcagatag cagccccgtc 480aaggcgggag tggagaccac cacaccctcc aaacaaagca
acaacaagta cgcggccagc 540agctacctga gcctgacgcc tgagcagtgg aagtcccaca
gaagctacag ctgccaggtc 600acgcatgaag ggagcaccgt ggagaagaca gtggccccta
cagaatgttc ataataa 657118651DNAHomo Sapiens 118gacatccagt
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120ggaaaagccc
ctaagctcct gatctctgct gcatccagtt tgcaaagtgg ggtcccgtca 180aggttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacgt 240gaagactatg
caacttacta ctgtcaacag agttacagta cccccccgta cacttttggc 300caggggacca
agctggagat caaacgaact gtggctgcac catctgtctt catcttcccg 360ccatctgatg
agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420tatcccagag
aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480caggagagtg
tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540acgctgagca
aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600ggcctgagct
cgcccgtcac aaagagcttc aacaggggag agtgttaata a
651119648DNAHomo Sapiens 119gacatccaga tgacccagtc tccttccacc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca gagtattagc agttggttgg
cctggtatca acagaaacca 120gggaaagccc ctaagctcct ggtctataag acgtctagtt
tagaaggtgg ggtcccatcc 180aggttcagcg gcagtggatc tgggacagaa ttcagtctca
caatcttcag actgcagtct 240gatgattttg caacttatta ctgccaacag tataatagtt
ttccgtacac ctttggccag 300gggaccaagc tggagttcac acgaactgtg gctgcaccat
ctgtcttcat cttcccgcca 360tctgatgagc agttgaaatc tggaactgcc tctgttgtgt
gcctgctgaa taacttctat 420cccagagagg ccaaagtaca gtggaaggtg gataacgccc
tccaatcggg taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca
gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa gtctacgcct
gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt
gttaataa 648120654DNAHomo Sapiens 120gaaattgtgt
tgacgcagtc tccagtcacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgta
gggccagtca gagtgttagc agcggctact tagcctggta ccagcagaaa 120cctggccagg
ctcccaggct cctcatctat ggtacatcca tcagggccac tggcatccca 180gacaggttca
gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt
ttgcagtgta ttactgtcag cagtatggta gctcacctct atactctttt 300ggccagggga
ccaaggtgga catcaaacga actgtggctg caccatctgt cttcatcttc 360ccgccatctg
atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac 420ttctatccca
gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac 480tcccaggaga
gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc 540ctgacgctga
gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat 600cagggcctga
gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta ataa
654121651DNAHomo Sapiens 121gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt
ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcagctact
tagcctggta ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca
gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca gacttcactc
tcaccatcag cagactggag 240cctgaagatt ttgcagtgta ttactgtcag cagtatggta
gctcacggta cacttttggc 300caggggacca agctggagat caaacgaact gtggctgcac
catctgtctt catcttcccg 360ccatctgatg agcagttgaa atctggaact gcctctgttg
tgtgcctgct gaataacttc 420tatcccagag aggccaaagt acagtggaag gtggataacg
ccctccaatc gggtaactcc 480caggagagtg tcacagagca ggacagcaag gacagcacct
acagcctcag cagcaccctg 540acgctgagca aagcagacta cgagaaacac aaagtctacg
cctgcgaagt cacccatcag 600ggcctgagct cgcccgtcac aaagagcttc aacaggggag
agtgttaata a 651122648DNAHomo Sapiens 122gacatccagt
tgacccagtc tccgtcctcc ctggctgcat ctgtgggaga cagagtcatt 60attacttgcc
ggtcaggtca gggcattagg aactatttaa attggtatca gcagaaacct 120gggaaagccc
ctaaactcct gatctatgct gcgtcctttt tgcaaagtgg ggtcccatca 180aggttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaggattttg
caacttacaa ctgtcaacag agttacagtg acccgtggac gttcggccaa 300gggaccaagg
tggaaatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360tctgatgagc
agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420cccagagagg
ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480gagagtgtca
cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540ctgagcaaag
cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcgc
ccgtcacaaa gagcttcaac aggggagagt gttaataa
648123645DNAHomo Sapiens 123gacatccaga tgacccagtc tccatcttcc ctgtctgcat
ctgtaggaga cagagtcatt 60atcacttgcc gggcaagtca gagcgttaac aggtatttaa
attggtatca gcagaaacca 120gggaaagccc ctaaactcct catctatgct gcatccagtt
tgcaaggtgg ggtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacgt 240gaagattttg caacttacta ctgccaacag agttacagaa
ctcggacgtt cggccaaggg 300accaaggtgg aaatcaaacg aactgtggct gcaccatctg
tcttcatctt cccgccatct 360gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc
tgctgaataa cttctatccc 420agagaggcca aagtacagtg gaaggtggat aacgccctcc
aatcgggtaa ctcccaggag 480agtgtcacag agcaggacag caaggacagc acctacagcc
tcagcagcac cctgacgctg 540agcaaagcag actacgagaa acacaaagtc tacgcctgcg
aagtcaccca tcagggcctg 600agctcgcccg tcacaaagag cttcaacagg ggagagtgtt
aataa 645124648DNAHomo Sapiens 124gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cggaatcacc 60atcacttgcc
gggcaagtca aagcgttagg agctatttaa attggtatca gcagaaacca 120gggaaagccc
ctgagctcct gatctatgct gcatcccgtt tgcaaagtgg ggtcccatca 180aggttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcagcct 240gaagattttg
caacttacta ctgtcaacat agttacagta cccctgtcac gttcggccaa 300gggaccaagg
tggaagtcaa gcgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360tctgatgagc
agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420cccagagagg
ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480gagagtgtca
cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540ctgagcaaag
cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcgc
ccgtcacaaa gagcttcaac aggggagagt gttaataa
648125651DNAHomo Sapiens 125ctttcttctg agctgactca ggaccctgct gtgtctgtga
ccttgggaca gacggtcaga 60atcacatgcc aaggagacag cctcagacac tcttatgcaa
gctggtacca gcagaagcca 120gggcaggctc ctatacttgt catctatggt aaaaacatcc
ggccctcagg gatcccagac 180cgattctctg gctccacctc ggggaacaca gcttccttga
ccatcactgg ggctcaggcg 240gaagatggcg gtgactatta ctgtaactcc cgggacacca
gtactgacca ttatgtcttc 300ggagatggga ccagggtcac cgtcgtaggt cagcccaagg
ccaaccccac tgtcactctg 360ttcccgccct cctctgagga gctccaagcc aacaaggcca
cactagtgtg tctgatcagt 420gacttctacc cgggagctgt gacagtggcc tggaaggcag
atggcagccc cgtcaaggcg 480ggagtggaga ccaccaaacc ctccaaacag agcaacaaca
agtacgcggc cagcagctac 540ctgagcctga cgcccgagca gtggaagtcc cacagaagct
acagctgcca ggtcacgcat 600gaagggagca ccgtggagaa gacagtggcc cctacagaat
gttcataata a 651126651DNAHomo Sapiens 126gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
gggcaagtca gagcattagc agctggttaa attggtatca gcagaaacca 120gggaaagccc
ctaacctcct gatctttgct gcatccactt tgcaaagtgg ggtcccgtca 180aggttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg
caacttacta ctgtcaacag agttacagta gttctgtgta cacttttggc 300caggggacca
agctggagat caaacgaact gtggctgcac catctgtctt catcttcccg 360ccatctgatg
agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420tatcccagag
aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480caggagagcg
tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540acgctgagca
aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600ggcctgagct
cgcccgtcac aaagagcttc aacaggggag agtgttaata a
651127648DNAHomo Sapiens 127gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gagcattagc agctatttaa
attggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gcatccagtt
tgcaaagtgg ggtcccatca 180aggttcagtg gcagtggatc tgggatagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg caacttacta ctgtcaacag agttacacta
ccctctggac gttcggccaa 300gggaccaagg tggaaatcaa acgaactgtg gctgcaccat
ctgtcttcat cttcccgcca 360tctgatgagc agttgaaatc tggaactgcc tctgttgtgt
gcctgctgaa taacttctat 420cccagagagg ccaaagtaca gtggaaggtg gataacgccc
tccaatcggg taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca
gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa gtctacgcct
gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt
gttaataa 648128657DNAHomo Sapiens 128gaaattgtgc
tgactcagtc tccaggcacc ctgtctttgt ctccaaggga aagagccacc 60ctctcctgca
gggccaatca gtatgttaac agcaaccact tagcctggta ccagcagaaa 120cctggccagg
ctcccaggct cctcctttat ggtgcatcaa ggagggccac tggcatccca 180gacagattca
gtggcagtgg gactgggaca gacttcactc tcatcatcag cagactggag 240cctgaagatt
ttgccgtata tttctgtcag ctgtatgatc actcacgtcc gatgtacact 300tttggccagg
ggactaagct ggagatcaaa cgaactgtgg ctgcaccatc tgtcttcatc 360ttcccgccat
ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat 420aacttctatc
ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggt 480aactcccagg
agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc 540accctgacgc
tgagcaaagc agactacgag aaacacaaag tctacgcctg cgaagtcacc 600catcagggcc
tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg ttaataa
657129654DNAHomo Sapiens 129gaaattgtgc tgactcagtc tccaggcacc ctgtctttgt
ctccagggga aagagccacc 60ctctcctgca gggccagtca gagttttagc agcggctact
tagcctggta ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca
acagggccac tggcatccca 180gacaggttca gtggcagtgg gtctggaaca gacttcactc
tcaccatcag cagactggag 240cctgaagatt ttgcagtgta ttactgtcag cactatggta
gctcacctcc catcaccttc 300ggccaaggga cacgactgga gattaaacga actgtggctg
caccatctgt cttcatcttc 360ccgccatctg atgagcagtt gaaatctgga actgcctctg
ttgtgtgcct gctgaataac 420ttctatccca gggaggccaa agtacagtgg aaggtggata
acgccctcca atcgggtaac 480tcccaggaga gtgtcacaga gcaggacagc aaggacagca
cctacagcct cagcagcacc 540ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct
acgcctgcga agtcacccat 600cagggcctga gctcgcccgt cacaaagagc ttcaacaggg
gagagtgtta ataa 654130651DNAHomo Sapiens 130gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
gggcaagtca gagcattagc agctatttaa attggtatca acagaaacca 120gggaaagccc
ctaagctcct gatctttgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagtg
gcagtggatc tgggacagat ttcagtctca ccatcagcag tctgcaacct 240gaagactttg
caacttacta ctgtcaacag agttacagtt ccctcgcgct cactttcggc 300ggagggacca
aggtggagat caaacgaact gtggctgcac catctgtctt catcttcccg 360ccatctgatg
agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420tatcccagag
aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480caggagagtg
tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540acgctgagca
aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600ggcctgagct
cgcccgtcac aaagagcttc aacaggggag agtgttaata a
651131651DNAHomo Sapiens 131gacatccagt tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gagcattagc agctatttaa
attggtatca gcagaatcca 120gggaaagccc ctaagctcct gatctatggt gcatccaatt
tgcaaagtgg ggtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg caacttacta ctgtcaacag agttacagta
ccctcgcgct cactttcggc 300ggagggacca aggtggagat caaacgaact gtggctgcac
catctgtctt catcttcccg 360ccatctgatg agcagttgaa atctggaact gcctctgttg
tgtgcctgct gaataacttc 420tatcccagag aggccaaagt acagtggaag gtggataacg
ccctccaatc gggtaactcc 480caggagagtg tcacagagca ggacagcaag gacagcacct
acagcctcag cagcaccctg 540acgctgagca aagcagacta cgagaaacac aaagtctacg
cctgcgaagt cacccatcag 600ggcctgagct cgcccgtcac aaagagcttc aacaggggag
agtgttaata a 651132651DNAHomo Sapiens 132gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
gggcaagtca gagcattagc acctatttaa attggtatca gcagaaacca 120ggaaaagccc
ctaagctcct gatctctgct gcatccagtt tgcaaagtgg ggtcccgtca 180aggttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacgt 240gaagactatg
cagcttacta ctgtcaacag agttacagta cccccccgta cacttttggc 300caggggacca
agctggagat caagcgaact gtggctgcac catctgtctt catcttcccg 360ccatctgatg
agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420tatcccagag
aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480caggagagtg
tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540acgctgagca
aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600ggcctgagct
cgcccgtcac aaagagcttc aacaggggag agtgttaata a
651133657DNAHomo Sapiens 133caggctgtgg tgactcagga gccctcactg actgtgtccc
caggagggac agtcactctc 60acctgtgctt ccagcactgg agcagtcacc actggttact
atccaaactg gttccagcag 120aaacctggac aagcacccag ggcactgatt tatagtacaa
gcaagaaaca ctcctggacc 180cctgcccggt tctcaggctc cctccttggg ggcaaagctg
ccctgacact gtcaggtgtg 240cagcctgagg acgaggctga gtattactgc ctgctcttct
atggtggtgc tcagctgggg 300gtgttcggcg gagggaccaa gctgaccgtc ctaggtcagc
ccaaggctgc cccctcggtc 360actctgttcc cgccctcctc tgaggagctt caagccaaca
aggccacact ggtgtgtctc 420ataagtgact tctacccggg agccgtgaca gtggcctgga
aggcagatag cagccccgtc 480aaggcgggag tggagaccac cacaccctcc aaacaaagca
acaacaagta cgcggccagc 540agctacctga gcctgacgcc tgagcagtgg aagtcccaca
aaagctacag ctgccaggtc 600acgcatgaag ggagcaccgt ggagaagaca gtggccccta
cagaatgttc ataataa 657134654DNAHomo Sapiens 134cagactgtgg
tgacccagga gccatcgttc tcagtgtccc ctggagggac ggtcacactc 60acttgtggct
tgagctctgg ctcagtctct gctcgttact accccagctg gtaccagcag 120accccaggcc
agcctccacg cacgctcatc cacagcacaa atactcggtc ttctggggtc 180cctgatcgct
tctctggctc catccttggg aacaaagctg ccctcaccat cacgggggcc 240caggcagatg
atgaatctga ttattactgt gtgctgtata tgggtagtgg cccttgggtg 300ttcggcggag
ggaccaagct gaccgtccta ggtcagccca aggctgcccc ctcggtcact 360ctgttcccac
cctcctctga ggagcttcaa gccaacaagg ccacactggt gtgtctcata 420agtgacttct
acccgggagc cgtgacagtg gcctggaagg cagatagcag ccccgtcaag 480gcgggagtgg
agaccaccac accctccaaa caaagcaaca acaagtacgc ggccagcagc 540tacctgagcc
tgacgcctga gcagtggaag tcccacaaaa gctacagctg ccaggtcacg 600catgaaggga
gcaccgtgga gaagacagtg gcccctacag aatgttcata ataa
654135663DNAHomo Sapiens 135gatgttgtga tgactcagtc tccactctcc ctgcccgtca
cccctggaga gccggcctcc 60atctcctgca ggtctagtca gagcctcctg cataggaatg
gatacaacta tttgaattgg 120tacctgcaga agccagggca gtctccacag ctcctgatct
atttgggttc taatcgggcc 180tccggggtcc ctgacaggtt cagtggcagt ggatcaggca
cagattttac actgaaaatc 240agcggagtgg aggctgagga tgttgcgttt tattactgca
tgcaaggtct acgaactccg 300tacactttcg gccaggggac caagctggag atcaagcgaa
ctgtggctgc accatctgtc 360ttcatcttcc cgccatctga tgagcagttg aaatctggaa
ctgcctctgt tgtgtgcctg 420ctgaataact tctatcccag agaggccaaa gtacagtgga
aggtggataa cgccctccaa 480tcgggtaact cccaggagag tgtcacagag caggacagca
aggacagcac ctacagcctc 540agcagcaccc tgacgctgag caaagcagac tacgagaaac
acaaagtcta cgcctgcgaa 600gtcacccatc agggcctgag ctcgcccgtc acaaagagct
tcaacagggg agagtgttaa 660taa
663136648DNAHomo Sapiens 136gacatccagt tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca
gagtatcagc agctatttaa attggtataa gcagagacca 120gggaaagccc ctaagctcct
gatctatgct gcatccactt tgcagagtgg ggtcccatca 180aggttcagtg gcagtggatc
tgggacagat ttcgctctca ccatcagcag tctgcaagct 240gaagattttg caacttacta
ctgtcaacag acttacagta ccctttggac gttcggccaa 300gggaccaagg tggaaatcac
acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360tctgatgagc agttgaaatc
tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420cccagagagg ccaaagtaca
gtggaaggtg gataacgccc tccaatcggg taactcccag 480gagagtgtca cagagcagga
cagcaaggac agcacctaca gcctcagcag caccctgacg 540ctgagcaaag cagactacga
gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa
gagcttcaac aggggagagt gttaataa 648137651DNAHomo Sapiens
137gaaattgtgt tgacacagtc tccaggtacc ttgtctctgt ctccagggga aacagccacc
60ctgtcctgca gggccagtca gagtgtcagt gatcgcgact tggcctggta tcaacaaaag
120tctggccagt ctcccagact tctcatgtat ggtggatcca ccagggcccc tggtataccg
180gtcaggttca gtggcagtgg gtctgggaca gagttcactc tcaccatcag cagcctgcag
240tctgaagatt ttgcaattta ttactgtcaa cactatcatg actggcctcc gaccttcggc
300caagggacac gactggagat taaacgaact gtggctgcac catctgtctt catcttcccg
360ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc
420tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc
480caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg
540acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag
600ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgttaata a
651138648DNAHomo Sapiens 138gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctttaggggg cacggtcacc 60ctcacttgcc ggtcaagtca gttcattagt cgctatttaa
attggtatca acaacaccca 120gggaaagtcc ctagactcct catttctggc gcatcaagat
tgcaaagggg ggtcccgtca 180aggttcactg gcggcgggtc tgggacagac ttcacactca
ccataaagaa tgtacagcct 240gacgatattg caacatactt ctgtcagcac tcttacagaa
gtgggcgggc gttcggccaa 300gggaccacgg tggaggtgaa acgaactgtg gctgcaccat
ctgtcttcat cttcccgcca 360tctgatgagc agttgaaatc tggaactgcc tccgttgtgt
gcctgctgaa taacttctat 420cccagagagg ccaaagtaca gtggaaggtg gataacgccc
tccaatcggg taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca
gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa gtctacgcct
gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt
gttaataa 648139651DNAHomo Sapiens 139gaaattgtgt
tgacgcagtc tccaggcagc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120cctggccagg
ctcccaggct cctcatctat ggtccatcca gcagggccac tggcatccca 180gacaggttca
gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt
ttgcagtgta ttactgtcag cattttggta actcacgggg aacgttcggc 300caagggacca
aggtggaaat cagacgaact gtggctgcac catctgtctt catcttcccg 360ccatctgatg
agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420tatcccagag
aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480caggagagtg
tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540acgctgagca
aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600ggcctgagct
cgcccgtcac aaagagcttc aacaggggag agtgttaata a
651140654DNAHomo Sapiens 140gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt
ctccagggga aagagtcaca 60ctctcctgca ggcccagtcg gtatattgcc agcgactact
tagcctggta ccaactaaga 120cctggccagg ctcccaaact cctcatctat ggtgcctcca
gcagggccac tggcatccca 180gacaggttca gtggcgttgg gtctccgaca gacttcactc
tcaccatcag cagactggag 240cctgaagatt ttgcgatgta ttactgtcac tattctggtg
gctcacctcc gtaccctttt 300ggccagggga ccaggctgga catcaaacga actgtggctg
caccatctgt cttcatcttc 360ccgccatctg atgagcagtt gaaatctgga actgcctctg
ttgtgtgcct gctgaataac 420ttctatccca gagaggccaa agtacagtgg aaggtggata
acgccctcca atcgggtaac 480tcccaggaga gtgtcacaga gcaggacagc aaggacagca
cctacagcct cagcagcacc 540ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct
acgcctgcga agtcacccat 600cagggcctga gctcgcccgt cacaaagagc ttcaacaggg
gagagtgtta ataa 654141648DNAHomo Sapiens 141gacatccagt
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
gggcaagtca gtacattaac gtctacttaa attggtatca gcacaaagca 120gggagagccc
ctaagctcct gatctatgct gcatccaatt tgcaaagtgg ggtcccacca 180aggttcattg
gcagtggatc tgggacagat ttcactctta ccatcagcag tctgcaatct 240gaagatttcg
caacttacta ctgtctccag agtttcactg tccctcggac tttcggccct 300gggaccaaag
tggatgtcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360tctgatgagc
agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420cccagagagg
ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480gagagtgtca
cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540ctgagcaaag
cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcgc
ccgtcacaaa gagcttcaac aggggagagt gttaataa
648142651DNAHomo Sapiens 142gaaattgtgt tgacacagtc tccaggcacc ctgtctttgt
ctccagggga gagagccacc 60ctctcctgca gggccagtca gagtgttagc agcgccttct
tagcctggta ccagcagaaa 120cctggccagg ctcccagact cctcatctat ggtgcctcca
gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca gacttcactc
tcaccatcag cagactggag 240cctgaagatt ttgcagtgta ttactgtcag cagtatggca
gcttttcgat caccttcggc 300caagggacac gactggagat taaacgaact gtggctgcac
catctgtctt catcttcccg 360ccatctgatg agcagttgaa atctggaact gcctctgttg
tgtgcctgct gaataacttc 420tatcccagag aggccaaagt acagtggaag gtggataacg
ccctccaatc gggtaactcc 480caggagagtg tcacagagca ggacagcaag gacagcacct
acagcctcag cagcaccctg 540acgctgagca aagcagacta cgagaaacac aaagtctacg
cctgcgaagt cacccatcag 600ggcctgagct cgcccgtcac aaagagcttc aacaggggag
agtgttaata a 651143657DNAHomo Sapiens 143cagactgtgg
tgacccagga gccctcactg actgtgtccc caggagggac agtcactctc 60acctgtgctt
ccagcactgg agcagtcacc agtggttact atccaaactg gttccagcag 120aaacctggac
aagcacccag ggcactgatt tatagtacaa gcaacaaaca ctcctggacc 180cctgcccggt
tctcaggctc cctccttggg ggcaaagctg ccctgacact gtcaggtgtg 240cagcctgagg
acgaggctga gtattactgc ctgctctact atggtggtgc tcagcgttgg 300gtgttcggcg
gagggaccat cctgaccgtc ctaggtcagc ccaaggctgc cccctcggtc 360actctgttcc
cgccctcctc tgaggagctt caagccaaca aggccacact ggtgtgtctc 420ataagtgact
tctacccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480aaggcgggag
tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540agctacctga
gcctgacgcc tgagcagtgg aagtcccaca aaagctacag ctgccaggtc 600acgcatgaag
ggagcaccgt ggagaagaca gtggccccta cagaatgttc ataataa
657144657DNAHomo Sapiens 144cttaatttta tgctgactca gccccactct gtgtcggagt
ctccggggaa gacggtaacc 60atctcctgca cccgcagcag tggcagcatt gccagcaact
atatgcagtg gtaccagcag 120cgcccgggca gttcccccac cactgtgatc tatgaggata
atcggagacc ctctggggtc 180cctgatcgct tctctggctc catcgacagc tcctccaact
ctgcctccct caccatctct 240ggactgaaga ctgaggacga ggctgactac tactgtcagt
cttatgatag taacaattgg 300gtgttcggcg gagggaccaa gctgaccgtc ctaggtcagc
ccaaggctgc cccctcggtc 360actctgttcc cgccctcctc tgaggagctt caagccaaca
aggccacact ggtgtgtctc 420ataagtgact tctacccggg agccgtgaca gtggcctgga
aggcagatag cagccccgtc 480aaggcgggag tggagaccac cacaccctcc aaacaaagca
acaacaagta cgcggccagc 540agctacctga gcctgacgcc tgagcagtgg aagtcccaca
aaagctacag ctgccaggtc 600acgcatgaag ggagcaccgt ggagaagaca gtggccccta
cagaatgttc ataataa 657145651DNAHomo Sapiens 145gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120gggaaagccc
ctaagctcct gatctatgct gcatccactt tgcaaagtgg ggtcccatca 180aggttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg
caacttacta ctgtcaacag tattacagaa cgcccacgtg gacgttcggc 300caagggacca
aggtggaaat caaacgaact gtggctgcac catctgtctt catcttcccg 360ccatctgatg
agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420tatcccagag
aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480caggagagtg
tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540acgctgagca
aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600ggcctgagct
cgcccgtcac aaagagcttc aacaggggag agtgttaata a
651146651DNAHomo Sapiens 146gaaattgtgt tgacgcagtc tccagccacc ctgtctgtgt
ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcaacttag
cctggtacca gcagaaacct 120ggccaggctc ccaggctcct catctatggt gcatccacca
gggccactgg tatcccagcc 180aggttcagtg gcagtgggtc tgggacagag ttcactctca
ccatcagcag cctgcagtct 240gaagattttg cagtttatta ctgtcagcag tataataact
ggccccggta cacttttggc 300caggggacca agctggagat caaacgaact gtggctgcac
catctgtctt catcttcccg 360ccatctgatg agcagttgaa atctggaact gcctctgttg
tgtgcctgct gaataacttc 420tatcccagag aggccaaagt acagtggaag gtggataacg
ccctccaatc gggtaactcc 480caggagagtg tcacagagca ggacagcaag gacagcacct
acagcctcag cagcaccctg 540acgctgagca aagcagacta cgagaaacac aaagtctacg
cctgcgaagt cacccatcag 600ggcctgagct cgcccgtcac aaagagcttc aacaggggag
agtgttaata a 651147127PRTHomo Sapiens 147Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe
Thr Phe Arg Ser Phe 20 25
30Asp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Tyr Ile Asn Ser Arg Gly Ser
Thr Ile Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Arg Asp Leu Tyr Gly Asp Tyr Asp Pro Lys
Ser Tyr Tyr Tyr Tyr 100 105
110Gly Met Gly Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
120 125148125PRTHomo Sapiens 148Glu Val
Gln Leu Val Glu Thr Gly Gly Gly Val Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Ser Phe Ser Asn Phe 20 25
30Gly Phe His Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Val Ile Trp Tyr
Asp Gly Ser Asn Arg Phe Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Ser Lys Asn Met
Leu Phe65 70 75 80Leu
Gln Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Ile Ser Met Lys
Val Val Ile Arg Arg His Tyr Val Met 100 105
110Asp Val Trp Gly His Gly Thr Thr Val Thr Val Ser Ser
115 120 125149127PRTHomo Sapiens 149Glu
Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Ser Phe Arg Ser Tyr 20 25
30Asp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Tyr Ile Ser
Gly Arg Gly Ser Thr Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Leu Tyr Gly
Asp Tyr Asp Pro Lys Ser Tyr Tyr Tyr Tyr 100
105 110Ala Met Asp Val Trp Gly His Gly Thr Thr Val Thr
Val Ser Ser 115 120
125150125PRTHomo Sapiens 150Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Val Ile Trp Tyr Asp Gly Ser Asn Arg Phe Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70
75 80Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr
Ala Leu Tyr Ser Cys 85 90
95Ala Arg Glu Ile Thr Thr Thr Val Val Val Arg Arg His Tyr Leu Met
100 105 110Asp Ile Trp Gly Gln Gly
Thr Thr Val Thr Val Ser Ser 115 120
125151126PRTHomo Sapiens 151Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Ser Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Asn
20 25 30Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Phe Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70
75 80Leu Gln Met Asp Gly Leu Arg Ala Glu Asp Ser
Ala Val Tyr Tyr Cys 85 90
95Ala Arg Glu Glu Ile Ala Ala Arg Leu Tyr Ser Arg Tyr His Tyr Ala
100 105 110Met Asp Val Trp Gly Gln
Gly Thr Met Val Thr Val Ser Ser 115 120
125152125PRTHomo Sapiens 152Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Lys Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Phe Ser Ala Tyr
20 25 30Ser Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Ser Ile Thr Ser Thr Thr Thr Tyr Tyr Ala Asp Ser Val
Lys Gly 50 55 60Arg Phe Ser Ile Ser
Arg Asp Asn Ala Lys Ser Thr Leu Tyr Leu Arg65 70
75 80Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys Val Arg 85 90
95Glu Ile Ala Phe Arg Gly Ser Thr Tyr Ser Arg Trp Ser Tyr Tyr Phe
100 105 110Asp Phe Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 115 120
125153128PRTHomo Sapiens 153Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Glu1 5 10
15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr
20 25 30Tyr Trp Ser Trp Val Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser
Leu Lys 50 55 60Ser Arg Val Thr Ile
Ala Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70
75 80Lys Leu Arg Ser Val Thr Ala Ala Asp Thr
Ala Val Tyr Phe Cys Thr 85 90
95Arg Asp Trp Arg Gln Tyr Gly Ser Ala Ile Arg Gly Ser Arg Tyr Tyr
100 105 110Tyr Gly Met Asp Val
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
120 125154129PRTHomo Sapiens 154Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30Gly
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ala Val Ile Trp Tyr Asp Gly Ser Asn
Lys Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Arg Asp Met Val Thr Met Val Arg Gly Ala
Tyr Arg Asn Tyr Tyr 100 105
110Tyr Tyr Gly Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser
115 120 125Ser155127PRTHomo Sapiens
155Glu Val Gln Leu Val Glu Thr Gly Gly Gly Val Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Ser Phe Ser Asn Tyr 20 25
30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ala Val Ile
Trp Phe Asp Gly Ser Ile Lys Tyr Tyr Val Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Glu Thr Ala Ile Tyr Phe Cys
85 90 95Ala Arg Glu Asn Ser Val
Leu Val Pro Gly Thr Ile Arg Arg Arg Tyr 100
105 110Tyr Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120
125156127PRTHomo Sapiens 156Gln Val Gln Leu Val Glu Ser Gly Gly Asp Leu
Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr
20 25 30Glu Met Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Tyr Ile Asn Ser Arg Gly Asn Thr Lys Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr65 70
75 80Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95Ala Arg Asn Leu Phe Gly Asp Tyr Asp Leu Lys Ser Tyr Tyr Tyr Asn
100 105 110Ala Met Asp Val Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
125157127PRTHomo Sapiens 157Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Lys Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30Ala Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ser Ser Ile Ser Gly Thr Ser Ser Tyr Ile Tyr Tyr
Ala Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Ile Phe Arg Asp Asn Ala Lys Ser Ser Val Tyr65 70
75 80Leu Gln Met Asn Ser Leu Arg Val
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Asp Arg Trp Trp Gly Met Val Arg Arg Val Phe Pro
Thr Tyr 100 105 110Pro Phe Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 125158125PRTHomo Sapiens 158Gln Val Gln Leu Val Glu
Thr Gly Gly Gly Val Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30Gly
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ala Val Ile Trp Tyr Asp Gly Ser Asn
Lys Asp Tyr Ala Asp Pro Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Arg Glu Ile Ala Ser Arg Gly Tyr Ser Arg
Tyr Leu Tyr Tyr Phe 100 105
110Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 125159129PRTHomo Sapiens 159Gln Val Thr Leu
Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu1 5
10 15Thr Leu Thr Leu Thr Cys Thr Val Ser Gly
Phe Ser Leu Ser Asn Ala 20 25
30Arg Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu
35 40 45Trp Leu Ala His Ile Phe Ser Asn
Asp Glu Lys Ser Tyr Ser Thr Ser 50 55
60Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val65
70 75 80Val Leu Thr Met Thr
Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85
90 95Cys Ala Arg Met Arg Leu Thr Met Val Arg Gly
Val Ile Thr Tyr Tyr 100 105
110Tyr Tyr Ser Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser
115 120 125Ser160127PRTHomo Sapiens
160Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25
30Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Ala Gly Lys
Gly Pro Glu 35 40 45Trp Ile Gly
Arg Ile Tyr Thr Ser Gly Ser Thr Asn Tyr Asn Pro Ser 50
55 60Leu Lys Ser Arg Leu Thr Ile Ser Val Asp Thr Ser
Lys Asn Gln Phe65 70 75
80Ser Leu Lys Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95Cys Ala Arg Ala Pro Ser
Tyr Tyr Asp Ser Ser Gly Tyr Arg Tyr Trp 100
105 110Tyr Ile Asp Leu Trp Gly Arg Gly Thr Leu Val Thr
Val Ser Ser 115 120
125161126PRTHomo Sapiens 161Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Asp Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70
75 80Leu Gln Met Asn Ser Val Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95Ala Arg Glu Leu Ser Thr Gln Arg Gly Tyr Ser Arg Tyr His Tyr Val
100 105 110Met Asp Val Trp Gly Gln
Gly Thr Thr Val Thr Val Ser Ser 115 120
125162121PRTHomo Sapiens 162Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Val Ile Trp Phe Asp Gly Ser Asn Arg Asp Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Lys Thr Leu Tyr65 70
75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90
95Ala Thr Glu Leu Ala Arg Gly Arg Leu Arg Ala Leu Glu Tyr Trp Gly
100 105 110Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120163126PRTHomo Sapiens
163Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Lys1
5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25
30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ala Val Ile
Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys
Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Val Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Leu Thr Thr
Gln Arg Gly Tyr Ser Arg Tyr His Tyr Val 100
105 110Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val
Ser Ser 115 120 125164126PRTHomo
Sapiens 164Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly
Arg1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20
25 30Gly Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40
45Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu His65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95Ala Arg
Glu Val Gly Phe Gly Ser Gly Trp Ser Arg Tyr Tyr Tyr Gly 100
105 110Met Asp Val Trp Gly Gln Gly Thr Thr
Val Thr Val Ser Ser 115 120
125165130PRTHomo Sapiens 165Glu Val Gln Leu Val Glu Ser Gly Gly Gly Gly
Val Val Gln Pro Gly1 5 10
15Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
20 25 30Tyr Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40
45Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp
Ser 50 55 60Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu65 70
75 80Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr 85 90
95Cys Ala Arg Glu Ser Thr Leu Tyr Ser Ser Ser Trp Tyr Arg Arg Tyr
100 105 110Tyr Tyr Tyr Gly Met Asp
Val Trp Gly Gln Gly Thr Thr Val Thr Val 115 120
125Ser Ser 130166129PRTHomo Sapiens 166Gln Val Gln Leu
Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25
30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Val Ile Ser Tyr Asp Gly Ser
Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Arg Glu Ser Thr Leu Tyr Ser Ser Ser Trp
Tyr Arg Arg Tyr Tyr 100 105
110Tyr Tyr Ser Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser
115 120 125Ser167128PRTHomo Sapiens
167Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ala Val Ile
Trp Phe Asp Gly Ser Asn Arg Tyr Tyr Gly Asp Ser Val 50
55 60Lys Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75
80Leu Arg Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Phe Tyr Thr
Arg Ser Gly Leu Trp Ser Gln Gly Tyr Ser 100
105 110Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val
Thr Val Ser Ser 115 120
125168129PRTHomo Sapiens 168Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70
75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95Ala Arg Glu Met Val Ser Tyr Ser Ser Ser Trp Tyr Arg Arg Tyr Tyr
100 105 110Tyr Tyr Val Met Asp Val
Trp Gly Lys Gly Thr Thr Val Thr Val Ser 115 120
125Ser169123PRTHomo Sapiens 169Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe
Ser Asn Tyr 20 25 30Gly Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ala Val Ile Ser Tyr Asp Gly Ser Glu Lys
Tyr Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Ala Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys 85
90 95Lys Asn Lys Val Gly Ala Thr Arg Arg Ala Val Val
Ala Phe Asp Ile 100 105 110Trp
Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
120170127PRTHomo Sapiens 170Glu Val Gln Leu Val Glu Thr Gly Gly Gly Leu
Val Lys Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr
20 25 30Ser Met Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Ser Ile Gly Ser Ser Ser Thr Tyr Thr Tyr Ser Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70
75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95Ala Arg Gly Glu Pro Leu Asn Tyr Asp Tyr Ile Trp Gly Gly Tyr Arg
100 105 110Phe Thr Ile His Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
125171129PRTHomo Sapiens 171Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30Gly Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ala Ile Ile Trp Phe Asp Gly Ser Asn Lys Tyr Tyr
Ala Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70
75 80Leu Gln Ile Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Lys Glu His Gly Tyr Tyr Ser Ser Ser Trp Tyr Arg Asn
Tyr Tyr 100 105 110Tyr Tyr Ala
Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115
120 125Ser172121PRTHomo Sapiens 172Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25
30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Val Ile Trp Tyr Asp Gly Ser
Asn Lys Asp Tyr Val Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Arg Glu Leu Ala Lys Gly Arg Leu Arg Asp
Leu Asp His Trp Gly 100 105
110Gln Gly Thr Leu Val Thr Val Ser Ser 115
120173129PRTHomo Sapiens 173Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Thr Ser
20 25 30Ala Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70
75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95Ala Arg Glu Met Val Ser Tyr Ser Ser Ser Trp Tyr Arg Arg Tyr Tyr
100 105 110Tyr Tyr Asn Met Asp Val
Trp Gly Lys Gly Thr Thr Val Thr Val Ser 115 120
125Ser174125PRTHomo Sapiens 174Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Thr1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Arg Thr Tyr 20 25 30Gly Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ala Tyr Ile Trp Tyr Asp Gly Ser Asn Lys
Tyr Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Thr Leu Asn65
70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Arg Glu Ile Ala Ser Arg Gly Tyr Ser Arg Tyr
Leu Tyr Tyr Phe 100 105 110Asp
Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 125175129PRTHomo Sapiens 175Gln Leu Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Gly1 5
10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser
Met Arg Ser Ser 20 25 30Asn
Trp Trp Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35
40 45Ile Gly Glu Ile His His Gly Gly Ser
Thr Asn Tyr Asn Pro Ser Leu 50 55
60Gln Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Arg Phe Ser65
70 75 80Leu Lys Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr His Cys 85
90 95Ala Arg Gly Arg Ser Tyr Tyr Asp Ser Ser Gly
His Ser Phe Arg Gly 100 105
110Leu Val Pro Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser
115 120 125Ser176128PRTHomo Sapiens
176Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1
5 10 15Thr Leu Ser Leu Ile Cys
Thr Val Ser Gly Gly Ser Ile Ser Ser Asn 20 25
30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu
Glu Trp Ile 35 40 45Gly Tyr Ile
Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys 50
55 60Ser Arg Val Thr Ile Ser Leu Asp Thr Ser Lys Asn
Gln Phe Ser Leu65 70 75
80Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Glu Trp Arg Gln Tyr
Gly Ser Gly Ile Arg Gly Ser Arg Tyr Tyr 100
105 110Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120
125177129PRTHomo Sapiens 177Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn His
20 25 30Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70
75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95Ala Arg Glu Met Ala Ser Tyr Ser Ser Ser Trp Tyr Arg Arg Tyr Tyr
100 105 110Tyr Tyr Val Met Asp Val
Trp Gly Lys Gly Thr Thr Val Thr Val Ser 115 120
125Ser178125PRTHomo Sapiens 178Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Thr Tyr 20 25 30Gly Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ala Val Ile Trp Tyr Asp Gly Ser Gln Lys
Tyr Tyr Val Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Arg Glu Val Ala Val Arg Gly Val Ile Arg Tyr
Tyr Tyr Gly Met 100 105 110Asp
Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
120 125179128PRTHomo Sapiens 179Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser
Phe Ser Ser Tyr 20 25 30Gly
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ala Ile Ile Trp Tyr Asp Gly Ser Asn
Lys Leu Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Thr Leu Tyr65
70 75 80Leu Gln Met Ser Ser
Val Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Arg Asp Ser Val Arg Gly Val Ser Arg Trp
Gly Thr Gln Lys Tyr 100 105
110Tyr Ala Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125180126PRTHomo Sapiens 180Glu
Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1
5 10 15Ser Pro Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Phe Ile Trp
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Leu Arg Asn
His Val Phe Trp Ser Gly Tyr Ser Thr Ser 100
105 110Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 115 120 125181129PRTHomo
Sapiens 181Glu Val Gln Leu Val Glu Thr Gly Gly Gly Val Val Gln Pro Gly
Arg1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20
25 30Gly Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40
45Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95Ala Arg
Glu Met Val Ser Tyr Ser Ser Ser Trp Tyr Arg Arg Tyr Tyr 100
105 110Tyr Tyr Asn Met Asp Val Trp Gly Lys
Gly Thr Thr Val Thr Val Ser 115 120
125Ser182129PRTHomo Sapiens 182Gln Val Gln Leu Val Glu Thr Gly Gly Gly
Val Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Val Ile Trp Ser Asp Gly Ser Asn Lys Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Arg Asn Thr Leu Tyr65 70
75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Phe Tyr Cys 85 90
95Ala Arg Glu Gln Gly Gly Tyr Ser Ser Ser Trp Tyr Arg Arg Tyr Tyr
100 105 110Tyr Tyr Tyr Met Asp
Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115
120 125Ser183125PRTHomo Sapiens 183Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Asn Thr Tyr 20 25
30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Val Ile Trp Tyr Asp Gly Ser
Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Arg Glu Val Ala Val Arg Gly Val Ile Arg
Tyr Tyr Tyr Ala Met 100 105
110Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
120 125184123PRTHomo Sapiens 184Gln Val Gln Leu
Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Ser Phe Ser Asn Tyr 20 25
30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Val Ile Ser Tyr Asp Gly Ser
Glu Lys Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys 85
90 95Lys Asn Lys Val Gly Ala Thr Arg Arg Ala Val
Val Ala Val Asp Ile 100 105
110Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115
120185127PRTHomo Sapiens 185Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Val Gly Ser Gly Phe Thr Phe Arg Ser Phe
20 25 30Asp Met Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45Ser Tyr Ile Asn Ser Arg Gly Asn Thr Arg Tyr Tyr Val Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70
75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95Ala Arg Asp Leu Tyr Gly Asp Tyr Asp Pro Lys Ser Tyr Tyr Tyr Tyr
100 105 110Gly Met Asp Val Trp Gly
Gln Gly Thr Met Val Thr Val Ser Ser 115 120
125186125PRTHomo Sapiens 186Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Asn
Tyr 20 25 30Gly Met His Trp
Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ala Ile Ile Trp Tyr Asp Gly Ser Asn Lys His Tyr
Ala Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70
75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Phe Cys 85 90
95Ala Arg Glu Ile Ala Ser Arg Gly Tyr Ser Arg Tyr Leu Tyr
Tyr Phe 100 105 110Asp Ser Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
125187125PRTHomo Sapiens 187Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg
Thr Tyr 20 25 30Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Asp
Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu His65
70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Glu Ile Ala Ser Arg Gly Tyr Ser Arg Tyr Leu
Tyr Tyr Leu 100 105 110Asp Phe
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 125188126PRTHomo Sapiens 188Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30Gly
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ala Val Ile Trp Tyr Asp Gly Ser Asn
Lys Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser
Val Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Arg Asp Leu Ser Thr Gln Arg Gly Tyr Ser
Arg Tyr Tyr Tyr Val 100 105
110Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
120 125189121PRTHomo Sapiens 189Gln Val Gln
Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asn Phe 20 25
30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Val Ile Trp Tyr Asp Gly
Ser Asn Lys Asp Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Arg Thr Leu Tyr65
70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Arg Glu Leu Ala Arg Gly Arg Leu Arg
Asp Leu Asp Tyr Trp Gly 100 105
110Gln Gly Thr Leu Val Thr Val Ser Ser 115
120190128PRTHomo Sapiens 190Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70
75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95Ala Arg Glu Phe Tyr Thr Arg Ser Gly Leu Trp Ser Gln Gly Tyr Ser
100 105 110Tyr Tyr Met Asp Val Trp
Gly Lys Gly Thr Thr Val Thr Val Ser Ser 115 120
125191129PRTHomo Sapiens 191Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Ser Tyr 20 25 30Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ala Val Ile Trp Phe Asp Gly Gly Asn Lys Tyr
Tyr Ala Asp Ser Ala 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Asp Ala Ser Val Leu Ser Gly Leu Val Thr Arg
Arg Leu Val 100 105 110Tyr Tyr
Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115
120 125Ser192129PRTHomo Sapiens 192Gln Val Gln
Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25
30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Phe Ile Ser Tyr Asp Gly
Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Ser65
70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Lys Glu His Gly Tyr Tyr Arg Ser Ser
Trp Tyr Arg Asn Tyr Tyr 100 105
110Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser
115 120 125Ser193129PRTHomo Sapiens
193Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Glu Arg1
5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ala Val Ile
Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Asp Glu Val Gly
Tyr Ser Ser Ser Trp Tyr Arg Arg Tyr Tyr 100
105 110Tyr Tyr Ala Met Asp Val Trp Gly Gln Gly Thr Thr
Val Thr Val Ser 115 120
125Ser194128PRTHomo Sapiens 194Gln Val Gln Leu Val Gln Ser Gly Gly Gly
Val Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70
75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Glu Thr Val Val Val Ala Ala Lys Ile Arg Asn His Tyr Tyr
100 105 110Tyr Ala Met Asp Val
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
120 125195129PRTHomo Sapiens 195Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30Gly
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ala Gly Ile Trp Tyr Asp Gly Ser Asn
Lys Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Thr Met Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Arg Glu Gln Gly Gly Tyr Ser Ser Ser Trp
Tyr Arg Arg Tyr Tyr 100 105
110Tyr Tyr Asn Met Asp Leu Trp Gly Gln Gly Thr Thr Val Thr Val Ser
115 120 125Ser196129PRTHomo Sapiens
196Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ala Val Ile
Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Thr Leu Arg Asp Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Gly Thr Leu
Tyr Ser Ser Ser Trp Tyr Arg Arg Tyr Tyr 100
105 110Tyr Tyr Gly Met Asp Ala Trp Gly Gln Gly Thr Thr
Val Thr Val Ser 115 120
125Ser197129PRTHomo Sapiens 197Gln Val Gln Leu Val Glu Thr Gly Gly Gly
Val Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Gly Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Gly Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Arg Asn Thr Leu Tyr65 70
75 80Leu His Met Asn Ser Leu Arg Ala Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Glu His Gly Gly Ser Arg Ser Gly Trp Tyr Thr Leu Arg Leu
100 105 110Ala Tyr Tyr Phe Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115
120 125Ser198129PRTHomo Sapiens 198Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly1 5
10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly
Ser Ile Arg Gly Ser 20 25
30Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
35 40 45Ile Gly Glu Ile His His Gly Gly
Ser Thr Asn Tyr Asn Pro Ser Leu 50 55
60Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Arg Phe Ser65
70 75 80Leu Lys Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Arg Gly Thr Ser Tyr Tyr Asp Ser Ser Gly
Tyr Ser Phe Arg Gly 100 105
110Leu Val Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser
115 120 125Ser199129PRTHomo Sapiens
199Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25
30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ala Val Ile
Trp Tyr Asp Gly Ser Asn Lys Asn Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Leu Gln Gly
Tyr Arg Ser Ser Trp Tyr Arg Met Tyr Tyr 100
105 110Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr
Val Thr Val Ser 115 120
125Ser200127PRTHomo Sapiens 200Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Lys Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr
20 25 30Ser Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Ser Ile Gly Ser Ser Ser Ile Tyr Thr Tyr Ser Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70
75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Gly Glu Pro Leu Asn Tyr Asp Tyr Ile Trp Gly Arg Ser Arg
100 105 110Leu Thr Ile His Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
125201129PRTHomo Sapiens 201Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Ser Tyr 20 25 30Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Asp Trp Val Thr Arg Ser Ser Asn Trp Tyr Arg
Asn Tyr Tyr 100 105 110Tyr Tyr
Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115
120 125Ser202124PRTHomo Sapiens 202Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Val Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25
30Trp Met His Trp Val Arg Gln Val Pro Gly Lys Gly Leu Val Trp Val
35 40 45Ser Arg Ile Asn Val Asp Gly
Lys Ser Thr Ser Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Arg Asp Pro Arg Arg Phe Leu Glu Trp
Ala Arg Tyr Gly Met Asp 100 105
110Val Trp Gly Arg Gly Thr Thr Val Thr Val Ser Ser 115
120203216PRTHomo Sapiens 203Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30Leu Ala Trp Tyr Gln Gln
Lys Arg Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45Phe Asn Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70
75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Arg Ser Ser Trp Pro Pro 85 90
95Met Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val
100 105 110Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115
120 125Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr Pro Arg 130 135 140Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn145
150 155 160Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165
170 175Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys 180 185 190Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195
200 205Lys Ser Phe Asn Arg Gly Glu Cys
210 215204214PRTHomo Sapiens 204Asp Ile Gln Met Thr Gln
Ser Pro Ser Pro Leu Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Arg Gly
Ile Ser Asn Ser 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35
40 45Tyr Ala Ala Ser Thr Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55
60Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Val Ala Thr
Tyr Tyr Cys His Thr Tyr Asn Ser Ala Pro Phe 85
90 95Ala Phe Gly Pro Gly Thr Lys Val Asp Ile Lys
Arg Thr Val Ala Ala 100 105
110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln145 150 155 160Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185
190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205Phe Asn Arg Gly
Glu Cys 210205216PRTHomo Sapiens 205Glu Ile Val Met Thr Gln Ser Pro
Gly Thr Leu Ser Leu Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser His Thr Val Ser
Ser Gly 20 25 30Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35
40 45Ile Tyr Gly Ala Ser Asn Arg Ala Thr Gly Val
Pro Asp Arg Phe Gly 50 55 60Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65
70 75 80Pro Glu Asp Ser Ala Val Tyr
Phe Cys Gln Gln Tyr Gly Thr Ser Pro 85 90
95Gly Val Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Glu
Arg Thr Val 100 105 110Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115
120 125Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg 130 135 140Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn145
150 155 160Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165
170 175Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys 180 185 190Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195
200 205Lys Ser Phe Asn Arg Gly Glu Cys
210 215206214PRTHomo Sapiens 206Asp Ile Gln Leu Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Gln Gly
Ile Ser Asn Ser 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35
40 45Tyr Ala Ala Ser Thr Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55
60Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Val Ala Thr
Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala Pro Phe 85
90 95Ala Phe Gly Pro Gly Thr Lys Val Asp Ile Lys
Arg Thr Val Ala Ala 100 105
110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln145 150 155 160Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185
190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205Phe Asn Arg Gly
Glu Cys 210207215PRTHomo Sapiens 207Asp Ile Gln Met Thr Gln Ser Pro
Ser Phe Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg
Arg Tyr 20 25 30Leu Ala Trp
Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45Phe Ser Ala Ser Thr Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Leu Ser Ser Tyr Pro Pro 85 90
95Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
Thr Val Ala 100 105 110Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115
120 125Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro Arg Glu 130 135 140Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser145
150 155 160Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165
170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val 180 185 190Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195
200 205Ser Phe Asn Arg Gly Glu Cys 210
215208214PRTHomo Sapiens 208Asp Ile Gln Leu Thr Gln Ser Pro
Ser Ser Val Ser Ala Ser Val Gly1 5 10
15Asp Ser Val Thr Ile Thr Cys Arg Ala Ser Gln Ala Val Ser
Gly Trp 20 25 30Val Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45Phe Gly Leu Ser Asn Leu Glu Asp Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Ala Thr Asp Phe Thr Leu Thr Ile Thr Gly Leu Gln Pro65
70 75 80Glu Asp Leu Ala Thr Tyr Tyr
Cys Leu Gln Ala Asn Arg Phe Pro Leu 85 90
95Ser Phe Gly Gly Gly Thr Arg Val Glu Ile Lys Arg Thr
Val Ala Ala 100 105 110Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115
120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr Pro Arg Glu Ala 130 135 140Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145
150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165
170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys Val Tyr 180 185 190Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195
200 205Phe Asn Arg Gly Glu Cys
210209216PRTHomo Sapiens 209Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu
Ser Leu Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Pro Arg Arg Asn
20 25 30Phe Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40
45Ile Tyr Ala Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Asp Arg Leu Glu65 70
75 80Pro Glu Asp Ser Ala Val Tyr Tyr Cys Gln Val
Tyr Gly Ser Ser Pro 85 90
95Leu Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Met Lys Arg Thr Val
100 105 110Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120
125Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg 130 135 140Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn145 150
155 160Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr Tyr Ser 165 170
175Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
180 185 190Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195
200 205Lys Ser Phe Asn Arg Gly Glu Cys 210
215210216PRTHomo Sapiens 210Glu Ile Val Met Thr Gln Ser Pro Ala Thr
Leu Ser Val Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn
20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser65 70
75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Tyr Asn Asn Trp Pro Thr 85 90
95Leu Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val
100 105 110Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115
120 125Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr Pro Arg 130 135 140Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn145
150 155 160Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165
170 175Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys 180 185 190Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195
200 205Lys Ser Phe Asn Arg Gly Glu Cys
210 215211214PRTHomo Sapiens 211Asp Ile Gln Leu Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
Ile Ser Ser Tyr 20 25 30Leu
Asn Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Lys Leu Leu Ile 35
40 45Tyr Val Ala Ser Thr Leu Gln Ser Gly
Ala Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Ser Ala Thr
Phe Tyr Cys Gln Gln Thr Tyr Ser Pro Pro Tyr 85
90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
Arg Thr Val Ala Ala 100 105
110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln145 150 155 160Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185
190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205Phe Asn Arg Gly
Glu Cys 210212217PRTHomo Sapiens 212Glu Ile Val Leu Thr Gln Ser Pro
Gly Thr Leu Ser Ser Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr
Ser Arg 20 25 30Tyr Leu Ala
Trp Tyr Gln Gln Lys His Gly Gln Ala Pro Arg Leu Leu 35
40 45Ile Tyr Gly Thr Ser Thr Arg Ala Thr Gly Ile
Pro Asp Arg Phe Ser 50 55 60Gly Gly
Gly Ser Gln Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65
70 75 80Pro Glu Asp Phe Ala Val Tyr
Tyr Cys Gln His Tyr Asp Asp Ser Ile 85 90
95Ser Thr Tyr Ile Phe Gly Pro Gly Thr Glu Leu Glu Ile
Lys Arg Thr 100 105 110Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115
120 125Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro 130 135 140Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly145
150 155 160Asn Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165
170 175Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His 180 185 190Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195
200 205Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215213214PRTHomo Sapiens 213Asp Ile Gln Leu Thr Gln
Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly
Ile Asn Asn Leu 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45Tyr Ala Ala Ser Asn Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Leu 85
90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
Arg Thr Val Ala Ala 100 105
110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln145 150 155 160Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185
190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205Phe Asn Arg Gly
Glu Cys 210214215PRTHomo Sapiens 214Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
Ser Tyr 20 25 30Leu Asn Trp
Tyr Gln Gln Asn Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45Tyr Gly Ala Ser Asn Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ser Tyr Ser Thr Leu Ala 85 90
95Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
Thr Val Ala 100 105 110Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115
120 125Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro Arg Glu 130 135 140Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser145
150 155 160Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165
170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val 180 185 190Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195
200 205Ser Phe Asn Arg Gly Glu Cys 210
215215215PRTHomo Sapiens 215Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
Thr Tyr 20 25 30Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr His Phe Thr Leu Thr Ile Ser Ser Leu Gln Arg65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Thr Tyr Arg Thr Pro Thr 85 90
95Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
Thr Val Ala 100 105 110Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115
120 125Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro Arg Glu 130 135 140Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser145
150 155 160Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165
170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val 180 185 190Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195
200 205Ser Phe Asn Arg Gly Glu Cys 210
215216213PRTHomo Sapiens 216Asp Ile Gln Leu Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Asn
Arg Tyr 20 25 30Leu Asn Trp
Tyr Gln His Lys Pro Gly Arg Ala Pro Glu Leu Leu Ile 35
40 45Tyr Ala Ala Ser Thr Leu Arg Arg Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ser Tyr Ser Arg Gly Thr 85 90
95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val
Ala Ala Pro 100 105 110Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115
120 125Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala Lys 130 135 140Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu145
150 155 160Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165
170 175Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr Ala 180 185 190Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195
200 205Asn Arg Gly Glu Cys
210217215PRTHomo Sapiens 217Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr Tyr
20 25 30Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr His
Phe Thr Leu Thr Ile Ser Ser Leu Gln Arg65 70
75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser
Tyr Gly Thr Pro Thr 85 90
95Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
100 105 110Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120
125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu 130 135 140Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser145 150
155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu 165 170
175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195
200 205Ser Phe Asn Arg Gly Glu Cys 210
215218217PRTHomo Sapiens 218Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr
Val Ser Pro Gly Gly1 5 10
15Thr Val Thr Leu Thr Cys Ala Ser Ser Thr Gly Ala Val Thr Thr Gly
20 25 30Tyr Tyr Pro Asn Trp Phe Gln
Gln Lys Pro Gly Gln Ala Pro Arg Ala 35 40
45Leu Ile Tyr Ser Thr Ser Lys Lys His Ser Trp Thr Pro Ala Arg
Phe 50 55 60Ser Gly Ser Leu Leu Gly
Gly Lys Ala Ala Leu Thr Leu Ser Gly Val65 70
75 80Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Leu
Leu Phe Tyr Gly Gly 85 90
95Ala Gln Leu Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110Gln Pro Lys Ala Ala Pro
Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 115 120
125Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser
Asp Phe 130 135 140Tyr Pro Gly Ala Val
Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val145 150
155 160Lys Ala Gly Val Glu Thr Thr Thr Pro Ser
Lys Gln Ser Asn Asn Lys 165 170
175Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
180 185 190His Lys Ser Tyr Ser
Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 195
200 205Lys Thr Val Ala Pro Thr Glu Cys Ser 210
215219215PRTHomo Sapiens 219Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Asn
Tyr 20 25 30Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45Ser Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu His Arg65 70
75 80Glu Asp Tyr Ala Thr Tyr Tyr Cys
Gln Gln Ser Tyr Ser Thr Pro Pro 85 90
95Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr
Val Ala 100 105 110Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115
120 125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr Pro Arg Glu 130 135 140Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser145
150 155 160Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165
170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val 180 185 190Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195
200 205Ser Phe Asn Arg Gly Glu Cys 210
215220215PRTHomo Sapiens 220Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
Ser Tyr 20 25 30Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ser Tyr Ser Thr Ser Thr 85 90
95Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
Thr Val Ala 100 105 110Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115
120 125Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro Arg Glu 130 135 140Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser145
150 155 160Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165
170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val 180 185 190Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195
200 205Ser Phe Asn Arg Gly Glu Cys 210
215221214PRTHomo Sapiens 221Asp Ile Gln Leu Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
Ser Tyr 20 25 30Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ser Tyr Ser Thr Ser Trp 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr
Val Ala Ala 100 105 110Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115
120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr Pro Arg Glu Ala 130 135 140Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145
150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165
170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys Val Tyr 180 185 190Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195
200 205Phe Asn Arg Gly Glu Cys
210222216PRTHomo Sapiens 222Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
Ser Val Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ser Asn
20 25 30Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45Tyr Gly Ala Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Glu
Phe Ser Leu Thr Ile Ser Ser Leu Gln Ser65 70
75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr
Asn Asn Trp Pro Pro 85 90
95Ile Phe Thr Phe Gly Pro Gly Thr Lys Leu Asp Ile Lys Arg Thr Val
100 105 110Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120
125Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg 130 135 140Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn145 150
155 160Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr Tyr Ser 165 170
175Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
180 185 190Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195
200 205Lys Ser Phe Asn Arg Gly Glu Cys 210
215223215PRTHomo Sapiens 223Glu Ile Val Leu Thr Gln Ser Pro Gly Thr
Leu Ser Leu Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
20 25 30Tyr Leu Ala Trp Tyr Gln
His Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40
45Ile Tyr Gly Ser Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg
Phe Ser 50 55 60Gly Ser Gly Ser Gly
Thr Asp Phe Thr Val Thr Ile Ser Arg Leu Glu65 70
75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Tyr Gly Thr Ala Pro 85 90
95Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
100 105 110Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115
120 125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu 130 135 140Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser145
150 155 160Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165
170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val 180 185 190Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195
200 205Ser Phe Asn Arg Gly Glu Cys 210
215224216PRTHomo Sapiens 224Gln Ser Ala Leu Thr Gln Pro Ala
Ser Val Ser Gly Ser Pro Gly Gln1 5 10
15Ser Ile Thr Ile Ser Cys Thr Ala Thr Ser Ser Asp Ile Gly
Ala Tyr 20 25 30Asn Tyr Val
Ser Trp Tyr Gln His His Pro Gly Lys Ala Pro Lys Val 35
40 45Ile Ile Thr Asp Val Asn Lys Arg Pro Ser Gly
Val Pro Asp Arg Phe 50 55 60Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65
70 75 80Gln Pro Glu Asp Glu Ala Glu
Tyr Ser Cys Cys Ser Tyr Ala Gly Thr 85 90
95Tyr Ser Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val
Leu Ser Gln 100 105 110Pro Lys
Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115
120 125Leu Gln Ala Asn Lys Ala Thr Leu Val Cys
Leu Ile Ser Asp Phe Tyr 130 135 140Pro
Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys145
150 155 160Ala Gly Val Glu Thr Thr
Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165
170 175Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln
Trp Lys Ser His 180 185 190Arg
Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195
200 205Thr Val Ala Pro Thr Glu Cys Ser
210 215225216PRTHomo Sapiens 225Leu Ser Ser Glu Leu Thr
Gln Asp Pro Ala Val Ser Val Ala Leu Gly1 5
10 15Gln Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu
Arg Ser Tyr Tyr 20 25 30Ala
Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Leu Ser Val Ile 35
40 45Tyr Gly Lys Asn Asn Arg Pro Ser Gly
Ile Pro Asp Arg Phe Ser Gly 50 55
60Ser Asn Ser Gly Asn Thr Ala Phe Leu Thr Ile Thr Gly Thr Gln Ala65
70 75 80Glu Asp Glu Ala Asp
Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn 85
90 95Tyr Arg Glu Leu Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly Gln 100 105
110Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu
115 120 125Leu Gln Ala Asn Lys Ala Thr
Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135
140Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val
Lys145 150 155 160Ala Gly
Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr
165 170 175Ala Ala Ser Ser Tyr Leu Ser
Leu Thr Pro Glu Gln Trp Lys Ser His 180 185
190Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
Glu Lys 195 200 205Thr Val Ala Pro
Ala Glu Cys Ser 210 215226215PRTHomo Sapiens 226Asp
Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser His Gly Ile Ser Ser Tyr 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu
Leu Ile 35 40 45Phe Pro Ala Ser
Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser
Leu Arg Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Ser Arg
85 90 95Trp Ala Phe Gly Gln Gly
Thr Lys Val Glu Val Lys Arg Thr Val Ala 100
105 110Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu Lys Ser 115 120 125Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130
135 140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser145 150 155
160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
165 170 175Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180
185 190Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys 195 200 205Ser
Phe Asn Arg Gly Glu Cys 210 215227215PRTHomo Sapiens
227Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Ser Ile Arg Arg Tyr 20 25
30Leu Asn Trp Tyr Gln Lys Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Arg Thr Gln Gly
85 90 95Leu Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100
105 110Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu Lys Ser 115 120 125Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130
135 140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser145 150 155
160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
165 170 175Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180
185 190Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys 195 200 205Ser
Phe Asn Arg Gly Glu Cys 210 215228217PRTHomo Sapiens
228Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1
5 10 15Thr Val Thr Leu Thr Cys
Ala Ser Ser Thr Gly Ala Val Thr Thr Gly 20 25
30Tyr Tyr Pro Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala
Pro Arg Ala 35 40 45Leu Val His
Ser Thr Ser Lys Lys His Ser Trp Thr Pro Ala Arg Phe 50
55 60Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr
Leu Ser Gly Val65 70 75
80Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Leu Leu Phe Tyr Gly Gly
85 90 95Ala Gln Leu Gly Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100
105 110Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro
Pro Ser Ser Glu 115 120 125Glu Leu
Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130
135 140Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala
Asp Ser Ser Pro Val145 150 155
160Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
165 170 175Tyr Ala Ala Ser
Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180
185 190His Lys Ser Tyr Ser Cys Gln Val Thr His Glu
Gly Ser Thr Val Glu 195 200 205Lys
Thr Val Ala Pro Ala Glu Cys Ser 210 215229217PRTHomo
Sapiens 229Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly
Gly1 5 10 15Thr Val Thr
Leu Thr Cys Ala Ser Ser Thr Gly Ser Val Thr Ser Gly 20
25 30Tyr Tyr Pro Asn Trp Phe Gln Gln Lys Pro
Gly Gln Ala Pro Arg Pro 35 40
45Leu Ile Ser Gly Thr Ser Asn Lys Leu Ser Trp Thr Pro Ala Arg Phe 50
55 60Ser Gly Ser Leu Leu Gly Gly Lys Ala
Ala Leu Thr Val Ser Gly Val65 70 75
80Gln Pro Glu Asp Glu Ala Val Tyr Tyr Cys Leu Leu Tyr Tyr
Gly Val 85 90 95Pro Gln
Pro Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100
105 110Gln Pro Lys Ala Ala Pro Ser Val Thr
Leu Phe Pro Pro Ser Ser Glu 115 120
125Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
130 135 140Tyr Pro Gly Ala Val Thr Val
Ala Trp Lys Ala Asp Ser Ser Pro Val145 150
155 160Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln
Ser Asn Asn Lys 165 170
175Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
180 185 190His Arg Ser Tyr Ser Cys
Gln Val Thr His Glu Gly Ser Thr Val Glu 195 200
205Lys Thr Val Ala Pro Thr Glu Cys Ser 210
215230215PRTHomo Sapiens 230Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr
20 25 30Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Ser Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Arg65 70
75 80Glu Asp Tyr Ala Thr Tyr Tyr Cys Gln Gln Ser
Tyr Ser Thr Pro Pro 85 90
95Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
100 105 110Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120
125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu 130 135 140Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser145 150
155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu 165 170
175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195
200 205Ser Phe Asn Arg Gly Glu Cys 210
215231214PRTHomo Sapiens 231Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp
20 25 30Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Val 35 40
45Tyr Lys Thr Ser Ser Leu Glu Gly Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Glu
Phe Ser Leu Thr Ile Phe Arg Leu Gln Ser65 70
75 80Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Asn Ser Phe Pro Tyr 85 90
95Thr Phe Gly Gln Gly Thr Lys Leu Glu Phe Thr Arg Thr Val Ala Ala
100 105 110Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150
155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser 165 170
175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195
200 205Phe Asn Arg Gly Glu Cys 210232216PRTHomo
Sapiens 232Glu Ile Val Leu Thr Gln Ser Pro Val Thr Leu Ser Leu Ser Pro
Gly1 5 10 15Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Gly 20
25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40
45Ile Tyr Gly Thr Ser Ile Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50
55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser
Ser Pro 85 90 95Leu Tyr
Ser Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg Thr Val 100
105 110Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys 115 120
125Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
130 135 140Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn145 150
155 160Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser 165 170
175Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
180 185 190Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200
205Lys Ser Phe Asn Arg Gly Glu Cys 210
215233215PRTHomo Sapiens 233Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu
Ser Leu Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
20 25 30Tyr Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40
45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70
75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Tyr Gly Ser Ser Arg 85 90
95Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
100 105 110Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120
125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu 130 135 140Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser145 150
155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu 165 170
175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195
200 205Ser Phe Asn Arg Gly Glu Cys 210
215234214PRTHomo Sapiens 234Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu
Ala Ala Ser Val Gly1 5 10
15Asp Arg Val Ile Ile Thr Cys Arg Ser Gly Gln Gly Ile Arg Asn Tyr
20 25 30Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr Ala Ala Ser Phe Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70
75 80Glu Asp Phe Ala Thr Tyr Asn Cys Gln Gln Ser
Tyr Ser Asp Pro Trp 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150
155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser 165 170
175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195
200 205Phe Asn Arg Gly Glu Cys 210235213PRTHomo
Sapiens 235Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly1 5 10 15Asp Arg Val
Ile Ile Thr Cys Arg Ala Ser Gln Ser Val Asn Arg Tyr 20
25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40
45Tyr Ala Ala Ser Ser Leu Gln Gly Gly Val Pro Ser Arg Phe Ser Gly 50
55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Arg65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Arg Thr
Arg Thr 85 90 95Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100
105 110Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly Thr 115 120
125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser Gly Asn Ser Gln Glu145 150
155 160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser Ser 165 170
175Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200
205Asn Arg Gly Glu Cys 210236214PRTHomo Sapiens 236Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Gly Ile Thr Ile Thr Cys
Arg Ala Ser Gln Ser Val Arg Ser Tyr 20 25
30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu
Leu Ile 35 40 45Tyr Ala Ala Ser
Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ser Tyr Ser Thr Pro Val
85 90 95Thr Phe Gly Gln Gly Thr
Lys Val Glu Val Lys Arg Thr Val Ala Ala 100
105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
Leu Lys Ser Gly 115 120 125Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130
135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser Gly Asn Ser Gln145 150 155
160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180
185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser 195 200 205Phe
Asn Arg Gly Glu Cys 210237215PRTHomo Sapiens 237Leu Ser Ser Glu Leu
Thr Gln Asp Pro Ala Val Ser Val Thr Leu Gly1 5
10 15Gln Thr Val Arg Ile Thr Cys Gln Gly Asp Ser
Leu Arg His Ser Tyr 20 25
30Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Ile Leu Val Ile
35 40 45Tyr Gly Lys Asn Ile Arg Pro Ser
Gly Ile Pro Asp Arg Phe Ser Gly 50 55
60Ser Thr Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala65
70 75 80Glu Asp Gly Gly Asp
Tyr Tyr Cys Asn Ser Arg Asp Thr Ser Thr Asp 85
90 95His Tyr Val Phe Gly Asp Gly Thr Arg Val Thr
Val Val Gly Gln Pro 100 105
110Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu
115 120 125Gln Ala Asn Lys Ala Thr Leu
Val Cys Leu Ile Ser Asp Phe Tyr Pro 130 135
140Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys
Ala145 150 155 160Gly Val
Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala
165 170 175Ala Ser Ser Tyr Leu Ser Leu
Thr Pro Glu Gln Trp Lys Ser His Arg 180 185
190Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
Lys Thr 195 200 205Val Ala Pro Thr
Glu Cys Ser 210 215238215PRTHomo Sapiens 238Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser Ser Trp 20 25
30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu
Ile 35 40 45Phe Ala Ala Ser Thr
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ser Ser Val
85 90 95Tyr Thr Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105
110Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser 115 120 125Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130
135 140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser Gly Asn Ser145 150 155
160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
165 170 175Ser Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180
185 190Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys 195 200 205Ser Phe
Asn Arg Gly Glu Cys 210 215239214PRTHomo Sapiens
239Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25
30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60Ser Gly Ser Gly Ile Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Leu Trp
85 90 95Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100
105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
Leu Lys Ser Gly 115 120 125Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130
135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser Gly Asn Ser Gln145 150 155
160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180
185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser 195 200 205Phe
Asn Arg Gly Glu Cys 210240217PRTHomo Sapiens 240Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Arg1 5
10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Asn Gln
Tyr Val Asn Ser Asn 20 25
30His Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Leu Tyr Gly Ala Ser Arg Arg Ala
Thr Gly Ile Pro Asp Arg Phe Ser 50 55
60Gly Ser Gly Thr Gly Thr Asp Phe Thr Leu Ile Ile Ser Arg Leu Glu65
70 75 80Pro Glu Asp Phe Ala
Val Tyr Phe Cys Gln Leu Tyr Asp His Ser Arg 85
90 95Pro Met Tyr Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys Arg Thr 100 105
110Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
115 120 125Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135
140Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly145 150 155 160Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
165 170 175Ser Leu Ser Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185
190Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val 195 200 205Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 215241216PRTHomo Sapiens
241Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser
Cys Arg Ala Ser Gln Ser Phe Ser Ser Gly 20 25
30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu 35 40 45Ile Tyr Gly
Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50
55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Ser Ser Pro
85 90 95Pro Ile Thr Phe Gly Gln
Gly Thr Arg Leu Glu Ile Lys Arg Thr Val 100
105 110Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys 115 120 125Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130
135 140Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn145 150 155
160Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
165 170 175Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180
185 190Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr 195 200 205Lys
Ser Phe Asn Arg Gly Glu Cys 210 215242215PRTHomo
Sapiens 242Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20
25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40
45Phe Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60Ser Gly Ser Gly Thr Asp Phe Ser Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ser
Leu Ala 85 90 95Leu Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100
105 110Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser 115 120
125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
130 135 140Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser145 150
155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu 165 170
175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200
205Ser Phe Asn Arg Gly Glu Cys 210
215243215PRTHomo Sapiens 243Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr
20 25 30Leu Asn Trp Tyr Gln Gln Asn
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr Gly Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70
75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser
Tyr Ser Thr Leu Ala 85 90
95Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
100 105 110Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120
125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu 130 135 140Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser145 150
155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu 165 170
175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195
200 205Ser Phe Asn Arg Gly Glu Cys 210
215244215PRTHomo Sapiens 244Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr Tyr
20 25 30Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Ser Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Arg65 70
75 80Glu Asp Tyr Ala Ala Tyr Tyr Cys Gln Gln Ser
Tyr Ser Thr Pro Pro 85 90
95Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
100 105 110Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120
125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu 130 135 140Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser145 150
155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu 165 170
175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195
200 205Ser Phe Asn Arg Gly Glu Cys 210
215245217PRTHomo Sapiens 245Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr
Val Ser Pro Gly Gly1 5 10
15Thr Val Thr Leu Thr Cys Ala Ser Ser Thr Gly Ala Val Thr Thr Gly
20 25 30Tyr Tyr Pro Asn Trp Phe Gln
Gln Lys Pro Gly Gln Ala Pro Arg Ala 35 40
45Leu Ile Tyr Ser Thr Ser Lys Lys His Ser Trp Thr Pro Ala Arg
Phe 50 55 60Ser Gly Ser Leu Leu Gly
Gly Lys Ala Ala Leu Thr Leu Ser Gly Val65 70
75 80Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Leu
Leu Phe Tyr Gly Gly 85 90
95Ala Gln Leu Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110Gln Pro Lys Ala Ala Pro
Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 115 120
125Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser
Asp Phe 130 135 140Tyr Pro Gly Ala Val
Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val145 150
155 160Lys Ala Gly Val Glu Thr Thr Thr Pro Ser
Lys Gln Ser Asn Asn Lys 165 170
175Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
180 185 190His Lys Ser Tyr Ser
Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 195
200 205Lys Thr Val Ala Pro Thr Glu Cys Ser 210
215246216PRTHomo Sapiens 246Gln Thr Val Val Thr Gln Glu Pro Ser
Phe Ser Val Ser Pro Gly Gly1 5 10
15Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Ala
Arg 20 25 30Tyr Tyr Pro Ser
Trp Tyr Gln Gln Thr Pro Gly Gln Pro Pro Arg Thr 35
40 45Leu Ile His Ser Thr Asn Thr Arg Ser Ser Gly Val
Pro Asp Arg Phe 50 55 60Ser Gly Ser
Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala65 70
75 80Gln Ala Asp Asp Glu Ser Asp Tyr
Tyr Cys Val Leu Tyr Met Gly Ser 85 90
95Gly Pro Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
Gly Gln 100 105 110Pro Lys Ala
Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115
120 125Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu
Ile Ser Asp Phe Tyr 130 135 140Pro Gly
Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys145
150 155 160Ala Gly Val Glu Thr Thr Thr
Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165
170 175Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln
Trp Lys Ser His 180 185 190Lys
Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195
200 205Thr Val Ala Pro Thr Glu Cys Ser
210 215247219PRTHomo Sapiens 247Asp Val Val Met Thr Gln
Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5
10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser
Leu Leu His Arg 20 25 30Asn
Gly Tyr Asn Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45Pro Gln Leu Leu Ile Tyr Leu Gly Ser
Asn Arg Ala Ser Gly Val Pro 50 55
60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65
70 75 80Ser Gly Val Glu Ala
Glu Asp Val Ala Phe Tyr Tyr Cys Met Gln Gly 85
90 95Leu Arg Thr Pro Tyr Thr Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys 100 105
110Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125Gln Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe 130 135
140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln145 150 155 160Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175Thr Tyr Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185
190Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser 195 200 205Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 210 215248214PRTHomo
Sapiens 248Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20
25 30Leu Asn Trp Tyr Lys Gln Arg Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40
45Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60Ser Gly Ser Gly Thr Asp Phe Ala Leu
Thr Ile Ser Ser Leu Gln Ala65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Ser Thr
Leu Trp 85 90 95Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Thr Arg Thr Val Ala Ala 100
105 110Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150
155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 165 170
175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 210249215PRTHomo Sapiens
249Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Thr Ala Thr Leu Ser
Cys Arg Ala Ser Gln Ser Val Ser Asp Arg 20 25
30Asp Leu Ala Trp Tyr Gln Gln Lys Ser Gly Gln Ser Pro
Arg Leu Leu 35 40 45Met Tyr Gly
Gly Ser Thr Arg Ala Pro Gly Ile Pro Val Arg Phe Ser 50
55 60Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile
Ser Ser Leu Gln65 70 75
80Ser Glu Asp Phe Ala Ile Tyr Tyr Cys Gln His Tyr His Asp Trp Pro
85 90 95Pro Thr Phe Gly Gln Gly
Thr Arg Leu Glu Ile Lys Arg Thr Val Ala 100
105 110Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu Lys Ser 115 120 125Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130
135 140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser145 150 155
160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
165 170 175Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180
185 190Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys 195 200 205Ser
Phe Asn Arg Gly Glu Cys 210 215250214PRTHomo Sapiens
250Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly1
5 10 15Gly Thr Val Thr Leu Thr
Cys Arg Ser Ser Gln Phe Ile Ser Arg Tyr 20 25
30Leu Asn Trp Tyr Gln Gln His Pro Gly Lys Val Pro Arg
Leu Leu Ile 35 40 45Ser Gly Ala
Ser Arg Leu Gln Arg Gly Val Pro Ser Arg Phe Thr Gly 50
55 60Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Lys
Asn Val Gln Pro65 70 75
80Asp Asp Ile Ala Thr Tyr Phe Cys Gln His Ser Tyr Arg Ser Gly Arg
85 90 95Ala Phe Gly Gln Gly Thr
Thr Val Glu Val Lys Arg Thr Val Ala Ala 100
105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
Leu Lys Ser Gly 115 120 125Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130
135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser Gly Asn Ser Gln145 150 155
160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180
185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser 195 200 205Phe
Asn Arg Gly Glu Cys 210251215PRTHomo Sapiens 251Glu Ile Val Leu Thr
Gln Ser Pro Gly Ser Leu Ser Leu Ser Pro Gly1 5
10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Ser 20 25
30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Pro Ser Ser Arg Ala
Thr Gly Ile Pro Asp Arg Phe Ser 50 55
60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65
70 75 80Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Gln His Phe Gly Asn Ser Arg 85
90 95Gly Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Arg Arg Thr Val Ala 100 105
110Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
115 120 125Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135
140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser145 150 155 160Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
165 170 175Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys His Lys Val 180 185
190Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys 195 200 205Ser Phe Asn Arg
Gly Glu Cys 210 215252216PRTHomo Sapiens 252Glu Ile
Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5
10 15Glu Arg Val Thr Leu Ser Cys Arg
Pro Ser Arg Tyr Ile Ala Ser Asp 20 25
30Tyr Leu Ala Trp Tyr Gln Leu Arg Pro Gly Gln Ala Pro Lys Leu
Leu 35 40 45Ile Tyr Gly Ala Ser
Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55
60Gly Val Gly Ser Pro Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu65 70 75 80Pro
Glu Asp Phe Ala Met Tyr Tyr Cys His Tyr Ser Gly Gly Ser Pro
85 90 95Pro Tyr Pro Phe Gly Gln Gly
Thr Arg Leu Asp Ile Lys Arg Thr Val 100 105
110Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
Leu Lys 115 120 125Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130
135 140Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn145 150 155
160Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
165 170 175Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180
185 190Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr 195 200 205Lys Ser
Phe Asn Arg Gly Glu Cys 210 215253214PRTHomo Sapiens
253Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Tyr Ile Asn Val Tyr 20 25
30Leu Asn Trp Tyr Gln His Lys Ala Gly Arg Ala Pro Lys
Leu Leu Ile 35 40 45Tyr Ala Ala
Ser Asn Leu Gln Ser Gly Val Pro Pro Arg Phe Ile Gly 50
55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Ser65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Ser Phe Thr Val Pro Arg
85 90 95Thr Phe Gly Pro Gly Thr
Lys Val Asp Val Lys Arg Thr Val Ala Ala 100
105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
Leu Lys Ser Gly 115 120 125Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130
135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser Gly Asn Ser Gln145 150 155
160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180
185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser 195 200 205Phe
Asn Arg Gly Glu Cys 210254215PRTHomo Sapiens 254Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5
10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Ala 20 25
30Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala
Thr Gly Ile Pro Asp Arg Phe Ser 50 55
60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65
70 75 80Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Phe Ser 85
90 95Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile
Lys Arg Thr Val Ala 100 105
110Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
115 120 125Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135
140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser145 150 155 160Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
165 170 175Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys His Lys Val 180 185
190Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys 195 200 205Ser Phe Asn Arg
Gly Glu Cys 210 215255217PRTHomo Sapiens 255Gln Thr
Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1 5
10 15Thr Val Thr Leu Thr Cys Ala Ser
Ser Thr Gly Ala Val Thr Ser Gly 20 25
30Tyr Tyr Pro Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg
Ala 35 40 45Leu Ile Tyr Ser Thr
Ser Asn Lys His Ser Trp Thr Pro Ala Arg Phe 50 55
60Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser
Gly Val65 70 75 80Gln
Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Leu Leu Tyr Tyr Gly Gly
85 90 95Ala Gln Arg Trp Val Phe Gly
Gly Gly Thr Ile Leu Thr Val Leu Gly 100 105
110Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser
Ser Glu 115 120 125Glu Leu Gln Ala
Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130
135 140Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp
Ser Ser Pro Val145 150 155
160Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
165 170 175Tyr Ala Ala Ser Ser
Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180
185 190His Lys Ser Tyr Ser Cys Gln Val Thr His Glu Gly
Ser Thr Val Glu 195 200 205Lys Thr
Val Ala Pro Thr Glu Cys Ser 210 215256217PRTHomo
Sapiens 256Leu Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro
Gly1 5 10 15Lys Thr Val
Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser 20
25 30Asn Tyr Met Gln Trp Tyr Gln Gln Arg Pro
Gly Ser Ser Pro Thr Thr 35 40
45Val Ile Tyr Glu Asp Asn Arg Arg Pro Ser Gly Val Pro Asp Arg Phe 50
55 60Ser Gly Ser Ile Asp Ser Ser Ser Asn
Ser Ala Ser Leu Thr Ile Ser65 70 75
80Gly Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser
Tyr Asp 85 90 95Ser Asn
Asn Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100
105 110Gln Pro Lys Ala Ala Pro Ser Val Thr
Leu Phe Pro Pro Ser Ser Glu 115 120
125Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
130 135 140Tyr Pro Gly Ala Val Thr Val
Ala Trp Lys Ala Asp Ser Ser Pro Val145 150
155 160Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln
Ser Asn Asn Lys 165 170
175Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
180 185 190His Lys Ser Tyr Ser Cys
Gln Val Thr His Glu Gly Ser Thr Val Glu 195 200
205Lys Thr Val Ala Pro Thr Glu Cys Ser 210
215257215PRTHomo Sapiens 257Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr
20 25 30Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70
75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Tyr Arg Thr Pro Thr 85 90
95Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
100 105 110Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120
125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu 130 135 140Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser145 150
155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu 165 170
175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195
200 205Ser Phe Asn Arg Gly Glu Cys 210
215258215PRTHomo Sapiens 258Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
Ser Val Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn
20 25 30Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Glu
Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser65 70
75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr
Asn Asn Trp Pro Arg 85 90
95Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
100 105 110Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120
125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu 130 135 140Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser145 150
155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu 165 170
175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195
200 205Ser Phe Asn Arg Gly Glu Cys 210
21525923DNAArtificialSynthetic primer sequence 259cgttcttttt cgcaacgggt
ttg
2326023DNAArtificialSynthetic primer sequence 260aagaccgatg ggcccttggt
gga 23
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