Detection Methods Employing Hcv Core Lipid And Dna Binding Domain Monoclonal Antibodies

Abstract

The present disclosure provides detection methods employing HCV core lipid binding domain and DNA binding domain monoclonal antibodies or antibody fragments. In certain embodiments, the lipid binding domain monoclonal antibody or antibody fragment recognizes an epitope in amino acids 141 to 161 of HCV core protein and the DNA binding domain antibody or antibody fragment recognizes an epitope in amino acids 95-123 (e.g., in amino acids 99-117) of HCV core protein.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a divisional of copending U.S. application Ser. No. 15/189,646, filed Jun. 22, 2016, which is a continuation-in-part of U.S. application Ser. No. 15/079,013, filed Mar. 23, 2016, which is a divisional of U.S. application Ser. No. 14/138,991, filed Dec. 23, 2013, now U.S. Pat. No. 9,371,374, issued Jun. 21, 2016, which claims priority to U.S. Provisional Patent Application No. 61/783,529, filed Mar. 14, 2013, each of which are herein incorporated by reference in their entireties.

STATEMENT REGARDING JOINT RESEARCH AGREEMENT

[0002] The subject matter disclosed and claimed herein was made by or on behalf of the parties to a joint research agreement, Icahn School of Medicine at Mount Sinai and Abbott Laboratories, within the meaning of 35 U.S.C. .sctn. 100(h) and .sctn. 1.9(e). The joint research agreement was in effect on or before the date the claimed invention was made, and the claimed invention was made as a result of activities undertaken within the scope of the joint research agreement.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0003] Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 245,923 Byte ASCII (Text) file named "34340-407_ST25.txt," created on Aug. 13, 2019.

FIELD OF THE INVENTION

[0004] The present disclosure provides detection methods employing HCV core lipid binding domain and DNA binding domain monoclonal antibodies or antibody fragments. In certain embodiments, the lipid binding domain monoclonal antibody or antibody fragment recognizes an epitope in amino acids 141 to 161 of HCV core protein and the DNA binding domain antibody or antibody fragment recognizes an epitope in amino acids 95-123 (e.g., in amino acids 99-117) of HCV core protein.

BACKGROUND OF THE INVENTION

[0005] According to WHO statistics, as many as 170 million people worldwide are infected by hepatitis C virus (HCV), a viral infection of the liver. 75 to 85% of persons infected with HCV progress to chronic infection, approximately 20% of these cases develop complications of chronic hepatitis C, including cirrhosis of the liver or hepatocellular carcinoma after 20 years of infection. The current recommended treatment for HCV infections is a combination of interferon and ribavirin drugs, however the treatment is not effective in all cases and the liver transplantation is indicated in hepatitis C-related end-stage liver disease. At present, there is no vaccine available to prevent HCV infection, therefore all precautions to avoid infection must be taken.

[0006] Thus, patient care, as well as the prevention of transmission of Hepatitis C Virus (HCV) by blood and blood products or by close personal contact requires extreme vigilance using sensitive detection assays. This creates a need for specific methods for screening and identifying carriers of HCV and HCV-contaminated blood or blood products. Serological determination of HCV exposure relies on the detection of HCV present in human blood plasma or sera. This can be accomplished by detection of distinct structural and non-structural proteins encoded by the virus.

[0007] The HCV virus is a (+) sense single-stranded enveloped RNA virus in the Hepacivirus genus of the Flaviviridae family. The viral genome is approximately 10 kb in length and encodes a 3011 amino acid polyprotein precursor. The HCV genome has a large single open reading frame (ORF) coding for a unique polyprotein. This polyprotein is co- and post-translationally processed by cellular and viral proteases into three structural proteins, i.e., core, E1 and E2 and at least six non-structural NS2, NS3, NS4A, NS4B, NS5A and NS5B proteins. (Choo et al., Science 244: 359-362 (1989)).

[0008] Following HCV exposure, the virus enters a susceptible hepatocyte and viral replication occurs. During an eclipse phase period of approximately 10 days, viral presence is not evident (i.e., viral RNA cannot be detected), serum transaminase levels are within normal limits, and no evidence exists of an immune response to HCV (Busch et al., Transfusion 40:143 (2000)). Typically, about 10 days following exposure, HCV RNA can be detected, often with viral loads between 100,000-120,000,000 HCV RNA copies per ml of serum. Typically several weeks later, an increase in ALT levels is observed, indicating inflammation of the liver; antibodies are detected an average of about 70 days after exposure.

[0009] Screening of blood for exposure to HCV, either by the detection of antibodies to HCV or by the detection of viral-specific molecules (e.g., HCV RNA or HCV core proteins) in serum/plasma is an integral and important part of patient care. Blood or blood products derived from individuals identified as having been exposed to HCV, by these tests, are removed from the blood supply and are not utilized for distribution to recipients of blood products (see, e.g., U.S. Pat. No. 6,172,189). These tests may also be utilized in the clinical setting to diagnose liver disease attributable to HCV infection.

[0010] Serologic antibody tests rely on the use of recombinant antigens or synthetic peptides, representing selected fragments of the viral polyprotein. The first generation anti-HCV screening tests were based on detection of antibodies directed against a recombinant protein (HCV genotype 1a) originating from sequences located in the nonstructural NS-4 protein (C100-3) (Choo et al., Science 244:359 (1989); Kuo et al., Science 244:362 (1989)). The first generation assays failed to detect antibodies in approximately 10% of individuals having chronic HCV infection and up to 10-30% of individuals presenting with acute HCV infection. The second generation anti-HCV assays have incorporated recombinant proteins from three different regions of the HCV genome (HCV genotype 1a), including amino acid sequences from the core, NS3, and NS4 protein (Mimms et al., Lancet 336:1590 (1990); Bresters et al., Vox Sang 62:213 (1992)), allowing a marked improvement over the first generation tests in identifying HCV infected blood donors (Aach et al., N Engl J Med 325:1325 (1991); Kleinman et al., Transfusion 32:805 (1992). The second-generation assays detect antibodies in close to 100% of chronic HCV cases (Hino K., Intervirology 37:77 (1994)) and in nearly 100% of the acute cases by 12 weeks post infection (Alter et al., N Engl J Med 327:1899 (1992); Bresters et al., Vox Sang 62:213 (1992)). The third generation test includes a recombinant protein expressing amino acid sequences from the NS5 region, as well as antigens from the core, NS3 and NS4. Some studies have indicated a slight improvement in sensitivity in comparing the third generation tests to second generation tests (Lee et al., Transfusion 35:845 (1995); Courouce et al. Transfusion 34:790-795 (1994)), but this improvement is largely attributed to changes in the NS3 protein rather than the inclusion of NS5 (Courouce et al., Lancet 343:853 (1994)).

[0011] In general, the second and third generation HCV antibody tests detect exposure to HCV about 70 days after exposure. Since HCV establishes persistent, and in many cases lifelong infection, the detection of antibodies to HCV represents a very efficient method for determining exposure to HCV. However, antibody testing alone will frequently fail to detect HCV infected individuals during the first 70 days after exposure.

[0012] It has been suggested that testing for HCV antigen detects exposure to HCV significantly earlier than antibody testing and represents an alternative to nucleic acid testing for detecting exposure to HCV during the pre-seroconversion period. The HCV antigen detection test is rapid, simple, may-not require sample extraction or other pretreatment, and is not as prone to handling errors (e.g., contamination) as may occur in the HCV RNA tests. Thus, HCV core antigen tests present a practical alternative to HCV RNA for screening blood donors or for monitoring antiviral therapy.

[0013] Existing HCV antigen tests rely on detecting the presence of the HCV core antigen in serum or plasma. HCV core protein is a structural protein of HCV comprising the first 191 amino acids of the polyprotein and that forms the internal viral coat encapsidating the genomic RNA. Two different types of serologic assays have been developed which permit detection of HCV core antigens in serum. One assay format detects HCV core antigens in subjects prior to seroconversion and is utilized in screening blood donors, while the other assay format detects core antigens only in hepatitis C patients, regardless of their HCV antibody status, and is utilized in clinical laboratories to diagnose exposure to HCV or to monitor antiviral therapy. The currently available core antigen detection assays all use antibodies against the DNA binding domain of HCV core which is located at amino acids 1-125 of the core protein. The core protein also contains a lipid binding domain that is located between amino acids 134-171. To date there have been no antigens described from that section of core protein and until now it has been assumed that core detection required antibodies against the DNA binding domain.

[0014] Thus, binding proteins that can readily detect HCV core antigen will markedly improve the available methods of detection of HCV exposure in a patient. Thus, there is a recognized need for new antibodies that can readily be employed in screening tests.

SUMMARY OF THE INVENTION

[0015] The present disclosure provides detection methods employing HCV core lipid binding domain and DNA binding domain monoclonal antibodies or antibody fragments. In certain embodiments, the lipid binding domain monoclonal antibody or antibody fragment recognizes an epitope in amino acids 141 to 161 of HCV core protein and the DNA binding domain antibody or antibody fragment recognizes an epitope in amino acids 95-123 (e.g., in amino acids 99-117) of HCV core protein. The DNA binding domain, may also be referred to as the RNA binding domain. Both DNA and RNA binding domain refer to amino acids 1-125 of the HCV core protein.

[0016] In some embodiments, provided herein are methods for the detection of HCV protein in a test sample comprising: (i) contacting a test sample suspected of containing HCV with a first antibody, or antigen-binding portion thereof, directed against an epitope in the DNA (or RNA) binding domain of an HCV core protein to form a complex between the first antibody, or the antigen-binding portion thereof, and the HCV core protein located within the test sample, wherein the epitope in the DNA binding domain is within amino acids 95-123 of full-length HCV core protein; (ii) contacting the complex formed in step (i) with a second antibody, or antigen-binding portion thereof, directed against an epitope in the lipid binding domain of HCV core protein, to form a complex between the second antibody, or the antigen-binding portion thereof, and the complex formed in step (i), wherein the epitope in the lipid binding domain is within amino acids 141-161 of full-length HCV core protein; and (iii) detecting the complex formed in step (ii).

[0017] In some embodiments, provided herein are methods for the detection of HCV protein in a test sample comprising: (i) contacting a test sample suspected of containing HCV with a first antibody, or antigen-binding portion thereof, directed against an epitope in the lipid binding domain of an HCV core protein, to form a complex between the first antibody, or the antigen-binding portion thereof, and the HCV core protein located within the test sample; wherein the epitope in the lipid binding domain is within amino acids 141-161 of full-length HCV core protein, (ii) contacting the complex formed in step (i) with a second antibody, or antigen-binding portion thereof, directed against an epitope in the DNA (or RNA) binding domain of HCV core protein to form a complex between the second antibody, or the antigen-binding portion thereof, and the complex formed in step (i), wherein the epitope in the DNA binding domain is within amino acids 95-123 of full-length HCV core protein; and (iii) detecting the complex formed in step (ii).

[0018] In other embodiments, provide herein are systems or compositions comprising: (i) a first antibody, or antigen-binding portion thereof, directed against an epitope in the DNA binding domain of HCV core antigen, wherein the epitope in the DNA binding domain is within amino acids 95-123 of full-length HCV core protein; and (ii) a second antibody, or antigen-binding portion thereof, directed against an epitope in the lipid binding domain of HCV core antigen, wherein the epitope in the lipid binding domain is within amino acids 141-161 of full-length HCV core antigen. In certain embodiments, the first and second antibodies, or fragments thereof, are in separate or the same container. In further embodiments, the systems further comprise a sample from a subject suspect of being infected with HCV.

[0019] In other embodiments, the first and/or second antibody, or the antigen-binding portion thereof, is detectably labeled with a label, and wherein the detecting the complex in step (iii) comprises detecting the label. In some embodiments, provide herein the HCV core protein in the complex is an HCV mini-core protein. In particular embodiments, the HCV core protein in the complex is a full-length HCV core protein. In additional embodiments, the epitope in the DNA binding domain is within amino acids 95-117 of full-length HCV core protein. In further embodiments, the epitope in the DNA binding domain is within amino acids 103-109 or 103-110 of full-length HCV core protein. In certain embodiments, the epitope in the DNA binding domain is within amino acids 99-123 of full-length HCV core protein. In some embodiments, the epitope in the DNA binding domain is within amino acids 109-113 of full-length HCV core protein.

[0020] In certain embodiments, the present invention provides a monoclonal antibody that is specifically immunoreactive with the lipid binding domain of HCV core antigen. More particularly, the HCV core antigen is amino acid residues 134-171 of HCV. In more particular embodiments, the antibody specifically binds at least one epitope formed by amino acid sequence MGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG (SEQ ID NO:578). In more specific embodiments, the antibody is immunoreactive with an epitope formed by amino acids 141-161, 134-154 and 151-171 of HCV core antigen.

[0021] Another aspect of the invention provides a monoclonal antibody that is specifically immunoreactive with the lipid binding domain of HCV core antigen, wherein said monoclonal antibody has a heavy chain variable domain selected from the group consisting of the antibodies listed in FIG. 1A, and a light chain variable domain selected from the group consisting of the antibodies listed in FIG. 1B.

[0022] It is contemplated that any of the antibodies described herein may be prepared as immunoassay reagents, more particularly, such reagents preferably are labeled with a detectable label.

[0023] In still other embodiments, immunoassay reagents of the invention comprise one or more of the antibodies disclosed herein being bound to a solid phase.

[0024] The immunoassay reagents comprising the antibodies of the invention may further comprise an additional antibody against an HCV antigen. For example, such an additional antibody is an additional anti-core antibody.

[0025] A further aspect of the invention is directed to an immunoassay for the detection of HCV in a test sample, said immunoassay comprising:

(i) contacting a test sample suspected of containing HCV with a first antibody directed against HCV core antigen to form a complex between said first antibody and antigen located within said test sample; (ii) contacting said complex formed in step (i) with an antibody to a core lipid binding domain, to form a complex between said antibody to a core lipid binding domain and antigen in the complex formed in step (i) wherein said antibody to a core lipid binding domain is detectably labeled; and (iii) detecting the label of the complex formed in step (ii).

[0026] In more specific embodiments, the immunoassay may further be characterized in that the first antibody is directed to the DNA binding domain of HCV core antigen. In more particular embodiments the antibody employed in step (ii) is labeled with a fluorescent label. In exemplary embodiments, the label is acridinium.

[0027] In some embodiments, the immunoassay is one in which the antibody of step (i) is coated on solid phase. In specific preferred embodiments, the antibody of step (i) comprises an antibody that is distinct from the antibody of step (ii). Alternatively, the immunoassay is one in which the antibody of step (i) comprises an antibody that is the same as the antibody of step (ii).

[0028] Any of the immunoassays of the invention may be used on a test sample obtained from a patient and the method further comprises diagnosing, prognosticating, or assessing the efficacy of a therapeutic/prophylactic treatment of the patient, wherein, if the method further comprises assessing the efficacy of a therapeutic/prophylactic treatment of the patient, the method optionally further comprises modifying the therapeutic/prophylactic treatment of the patient as needed to improve efficacy.

[0029] As will be described in further detail herein, it will be understood by those skilled in the art that any of the immunoassays of the invention may be readily adapted for use in an automated system or a semi-automated system.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

[0030] FIG. 1A shows the heavy chain variable domains of preferred antibodies of the present invention. FIG. 1A discloses SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, and 100, respectively, in order of appearance.

[0031] FIG. 1B shows the light chain variable domains of preferred antibodies of the present invention. FIG. 1B discloses SEQ ID NOS: 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 324, 326, 328, 330, 332, 334, 336, 338, 340, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 370, 370, 372, 374, 376, 376, 378, 380, 382, 384, 386, 388, 388, 388, respectively, in order of appearance.

[0032] FIGS. 2A and 2B show two representations of a clustering diagram derived from the alignments of FIG. 1A.

[0033] FIG. 3 shows the core proteins encoded by HCV, including the precursor p23, mature core p21, and N-terminally deleted 70 and 91 mini-cores. Both p21 mature core and minicore proteins contain the hydrophobic domain D2 which interacts with lipids. Minicores contain only part of domain D1, the RNA-binding domain (aka, the DNA binding domain). The location of an immunogen used in Example 14 (peptides 98-110; SEQ ID N0582) with respect to full length HCV core and mini-cores 70 and 91 is shown in this figure.

[0034] FIG. 4 shows a flow diagram of an exemplary purification scheme for isolating HCV core and HCV minicores.

[0035] FIGS. 5A and 5B show results from Example 14 where it was shown that HCV RNA is enriched in the heparin/Mn.sup.+2 pellet fraction in the four HCV patients (P1-P4), but not in two normal controls (N1-N2).

[0036] FIG. 6 shows results from Example 14, where minicores are detected by western blot in four HCV patients (lanes P1-P4) and not in two normal controls (lanes N1, N2). Cell culture supernatant of infected cells serves as a positive control (lane C).

[0037] FIG. 7 shows results of Example 14, where duplicate Western blots of sample P4 were probed with antibodies directed either to the C-term or N-term portion of the core protein confirms the identity of mini-cores.

[0038] FIG. 8A shows the amino acid sequence of Neo4 antibody light chain variable region. This figure shows the light chain CDRs underlined, which include CDRL1 (RASKSVNEYGYTYMH; SEQ ID NO:585), CDRL2 (LASNLDS; SEQ ID NO:586), and CDRL3 (QHSRELPYT, SEQ ID NO:587).

[0039] FIG. 8B shows the amino acid sequence of Neo4 antibody heavy chain variable region. This figure shows the heavy chain CDRs underlined, which include CDRH1 (GFSITSSVYCWQ; SEQ ID NO:588), CDRH2 (RICYDGSVDYSPSITS; SEQ ID NO:589), and CDRH3 (ENHIDYYDTTYPSFDV; SEQ ID NO:590).

[0040] FIG. 9A shows the nucleic acid sequence encoding the light chain variable region of Neo4 (SEQ ID NO:591).

[0041] FIG. 9B shows the nucleic acid sequence encoding the heavy chain variable region of Neo4 (SEQ ID NO:592).

[0042] FIG. 10 shows results from Example 18, which demonstrated that when antibodies Neo4 and 208B-750 are used in combination, the signal intensity for core and 91-minicore are enhanced as compared to when the antibodies are used individually.

DETAILED DESCRIPTION OF THE INVENTION

[0043] The present disclosure provides detection methods employing HCV core lipid binding domain and DNA binding domain monoclonal antibodies or antibody fragments. In certain embodiments, the lipid binding domain monoclonal antibody or antibody fragment recognizes an epitope in amino acids 141 to 161 of HCV core protein and the DNA binding domain antibody or antibody fragment recognizes an epitope in amino acids 95-123 (e.g., in amino acids 99-117) of HCV core protein.

[0044] In some embodiments, the present invention describes the development of monoclonal antibodies directed against the Hepatitis C Virus core antigen, specifically, the lipid binding domain of core antigen between amino acids 134-171. The immunogen used was a synthetic peptide and screening of hybridomas utilized both the immunogen peptide and a set of three overlapping smaller peptides within the 134-171 region. In addition, a recombinant core antigen representing amino acids 1-169 was used for screening to determine the efficacy of the identified monoclonal antibodies as reactive with HCV core protein. The antibodies are delineated by their reactivity to the antigen, the immunogen peptide, and smaller overlapping peptides comprising the immunogen. Binding kinetics of the antibodies to the immunogen peptide were determined by SPR (surface plasmon resonance) using a BIAcore 4000 instrument. Immunoreactivity to the recombinant core antigen was determined by standard ELISA.

[0045] In addition, to show that the monoclonal antibodies of the present invention were useful in analyzing the presence of core antigen, core antigen capture microtiter plate assays were performed by using monoclonal antibodies directed to epitopes within the DNA binding domain of HCV core (e.g. amino acids 1-125) as the capture reagent and the 134-171-directed antibodies of the present invention as detection reagents. These assays produced results to demonstrate the utility of the 134-171-directed antibodies of the invention for HCV core antigen detection immunoassays. This is the first demonstration of an antigen capture assay that independently targets two major domains of HCV core for capture and detection. Previously reported core antigen detection assays use antibodies that bind to epitopes within the DNA binding domain (e.g., amino acids 1-125).

[0046] To produce the antibodies of the present invention, mice were immunized with a synthetic peptide comprised of HCV core genotype 1 consensus sequence from amino acids 134-171 linked to BSA. More specifically, the immunogen had the sequence of:

TABLE-US-00001 (SEQ ID NO: 578) MGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG.

[0047] In addition, the binding of the monoclonal antibodies to three specific N-terminally biotinylated epitope regions also was characterized and is further discussed in the Examples. Specifically, the three overlapping epitopes were derived from the above region and had the sequences of:

TABLE-US-00002 HCVc 134-154 (SEQ ID NO: 573) MGYIPLVGAPLGGAARALAHG HCVc 141-161 (SEQ ID NO: 574) GAPLGGAARALAHGVRVLEDG HCVc 151-171 (SEQ ID NO: 575) LAHGVRVLEDGVNYATGNLPG

[0048] The immunogen was conjugated to BSA to produce the antibodies. In other embodiments, the immunogen was conjugated to TT and fibrils. The TT sequence is often used to provide a more robust immune response in mice. The sequence of the TT conjugate was:

TABLE-US-00003 (SEQ ID NO: 577) Ac-Met-Gly-Tyr-Ile-Pro-Leu-Val-Gly-Ala-Pro-Leu- Gly-Gly-Ala-Ala-Arg-Ala-Leu-Ala-His-Gly-Val-Arg- Val-Leu-Glu-Asp-Gly-Val-Asn-Tyr-Ala-Thr-Gly-Asn- Leu-Pro-Gly-Gln-Tyr-Ile-Lys-Ala-Asn-Ser-Lys-Phe- Ile-Gly-Ile-Thr-Glu-Leu-NH2 .

[0049] The sequence of the fibrils conjugate was:

TABLE-US-00004 (SEQ ID NO: 579) Ac-Met-Gly-Tyr-Ile-Pro-Leu-Val-Gly-Ala-Pro-Leu- Gly-Gly-Ala-Ala-Arg-Ala-Leu-Ala-His-Gly-Val-Arg- Val-Leu-Glu-Asp-Gly-Val-Asn-Tyr-Ala-Thr-Gly-Asn- Leu-Pro-Gly-Gln-Gln-Lys-Phe-Gln-Phe-Gln-Phe-Glu- Gln-Gln-NH2.

[0050] B-lymphocytes were fused with a myeloma fusion partner to create hybridomas that were then screened for reactivity against the immunogen peptide, three overlapping peptides within the immunogen peptide sequence, and a recombinant HCV core antigen. Kinetic profiling using a Biacore 4000 allowed for identification of clusters of antibodies wherein the clusters are defined by their ability to bind to the immunogen peptide, or shorter peptides overlapping the 134-171 region. Combining these results with immunoreactivity, or lack thereof, for the recombinant antigen as determined by ELISA, allowed further delineation of the antibodies into groups with similar characteristics (specificities).

[0051] To briefly summarize the screening results discussed in further detail in the Examples, the greatest immune response was seen in mice immunized with the peptide linked to BSA. Additionally, the response from these mice was predominantly focused on the amino acid 141-161 region, although there was also some response to the amino acid 134-154 and 151-171 regions as well. With the HCV peptide linked to TT, an immune response was seen, however, this response was not as strong as with BSA. The response was spread over all 3 epitope regions. On the other hand, mice immunized using the amino acid 134-171 peptide linked to a peptide that would form fibril networks failed to show a significant immune response. The antibodies of the present invention are described in further detail in FIGS. 1A and 1B, and in the Examples.

[0052] The HCV core antigen used for these studies was expressed in E. coli and purified in a two-step process using IMAC followed by reverse-phase HPLC based on previously published methods (Boulant et al., J. Virol. (2005), 79(17):11353-11365).

[0053] In this manner a significant array of monoclonal antibodies that are specific for the lipid binding domain of HCV core have been produced. These monoclonal antibodies have utility in development of diagnostic assays for the detection of HCV core antigen in the serum and plasma of infected individuals. Prior to the present invention, there has been no reported generation of monoclonal antibodies to the multiple epitopes within the core amino acid 134-171 region that have shown binding activity to the HCV full length core peptide. The availability of the monoclonal antibodies of the present invention allows for the development of immunoassays for core antigen detection wherein two major domains of HCV core antigen are targeted. Previous core antigen assays described the use of monoclonals directed to epitopes within amino acid 1-125 (nucleic acid binding domain). Because the previously described monoclonal antibodies were only able to target the nucleic acid binding domain of HCV core, they were at best inefficient and often ineffective at detection of core protein fragments, break-down products, or smaller core proteins derived by internal translation initiation. The present invention for the first time overcomes these inadequacies in the previous assays by providing specific monoclonal antibodies that can be used as reagents to more efficiently and rapidly detect HCV core present in a test sample.

[0054] More particularly, the antibodies described herein are reagents useful for detection of HCV core antigen and are useful reagents to facilitate investigation of the life cycle of HCV. As noted above, HCV encoded proteins are expressed in a concerted process in which ribosomes bind to the internal ribosome entry site (IRES) and initiate translation, leading to synthesis of the viral polyprotein, which is cleaved to produce the classical HCV proteins, p21 core, E1, E2, p'7, and the nonstructural proteins. None of the viral enzymes, including the viral polymerase, can be made without the initiation of translation in the core gene region. Because of this temporal relationship, it is believed that translation events in this region control the expression all HCV proteins. Hence, a complete understanding of the core gene and its gene products is essential to understanding the life cycle of the virus and may shed light on our understanding of mechanisms of virus pathogenicity. Recently, a new family of conserved viral proteins, referred to as minicores have been described (Eng et al., J Virol. 2009 April; 83(7):3104-3114). These proteins are encoded in the same reading frame as the core gene but, are believed to be derived from internal translation initiation events rather than post-translational processing of the full-length core protein. One of the minicore proteins described is termed "91 minicore" named for the presumed initiator codon within core. It is hypothesized that "134 minicore" also exists, being derived from translation initiation at codon 134 which encodes a methionine in many HCV isolates. However, reagents are not available that allow detection of minicores that are, essentially, derived from the lipid binding domain. Such proteins may play an important role in HCV persistence.

[0055] Since the monoclonal antibodies were raised against a linear, synthetic peptide derived from HCV core 134-171, it is unknown whether they will bind to the native, complete core antigen or processed forms of HCV core that exist in infected individuals. However, provided that the monoclonal antibodies of the present invention are able at least to bind one or more epitopes presented by the linear HCV core region from amino acids 134-171, it is contemplated that such binding will be sufficient to render these monoclonal antibodies significantly useful in HCV detection assays. Some of the monoclonal antibodies of the present invention react with recombinant core antigen while others do not, suggesting that within the core amino acid 134-171 region, there are both linear and conformational epitopes. Antibodies recognizing either linear or conformational epitopes are very useful tools for the study of virus assembly within infected cells and the virus life cycle generally.

[0056] Finally, these reagents can also be used in immunoassays where it is desirable to determine the presence of only the lipid binding domain. Since little is known about circulating levels of minicores in infected individuals it is possible that they are present at much higher levels than core proteins containing the region of amino acids 1-125. In providing antibodies that detect HCV core peptides outside of the region of amino acids 1-125, the present invention provides HCV core antigen detection assays with much greater sensitivity than those currently available.

Definitions

[0057] The terms "specific binding" or "specifically binding", as used herein, in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A" and the antibody, will reduce the amount of labeled A bound to the antibody.

[0058] The term "antibody", as used herein, broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art. Nonlimiting embodiments of which are discussed below.

[0059] In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHL CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG-3, IgG4, IgA1 and IgA2) or subclass.

[0060] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, which may be generated by papain digestion of an intact antibody. The Fc region may be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. Replacements of amino acid residues in the Fc portion to alter antibody effector function are known in the art (Winter, et al. U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fc portion of an antibody mediates several important effector functions e.g., cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC) and half-life/clearance rate of antibody and antigen-antibody complexes. In some cases these effector functions are desirable for therapeutic antibody but in other cases might be unnecessary or even deleterious, depending on the therapeutic objectives. Certain human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC via binding to Fc.gamma.R5 and complement Clq, respectively. Neonatal Fc receptors (FcRn) are the critical components determining the circulating half-life of antibodies. At least one amino acid residue is replaced in the constant region of the antibody, for example the Fc region of the antibody, such that effector functions of the antibody are altered. The dimerization of two identical heavy chains of an immunoglobulin is mediated by the dimerization of CH3 domains and is stabilized by the disulfide bonds within the hinge region (Huber et al. Nature; 264: 415-20; Thies et al 1999 J Mol Biol; 293: 67-79.). Mutation of cysteine residues within the hinge regions to prevent heavy chain-heavy chain disulfide bonds will destabilize dimerization of CH3 domains. Residues responsible for CH3 dimerization have been identified (Dall'Acqua 1998 Biochemistry 37: 9266-73.). Therefore, it is possible to generate a monovalent half-Ig. Interestingly, these monovalent half Ig molecules have been found in nature for both IgG and IgA subclasses (Seligman 1978 Ann Immunol 129: 855-70; Biewenga et al 1983 Clin Exp Immunol 51: 395-400). The stoichiometry of FcRn: Ig Fc region has been determined to be 2:1 (West et al. 2000 Biochemistry 39: 9698-708), and half Fc is sufficient for mediating FcRn binding (Kim et al 1994 Eur J Immunol; 24: 542-548.). Mutations to disrupt the dimerization of CH3 domain may not have greater adverse effect on its FcRn binding as the residues important for CH3 dimerization are located on the inner interface of CH3 b sheet structure, whereas the region responsible for FcRn binding is located on the outside interface of CH2-CH3 domains. However the half Ig molecule may have certain advantage in tissue penetration due to its smaller size than that of a regular antibody. At least one amino acid residue may be replaced in the constant region of the binding protein of the present disclosure, for example the Fc region, such that the dimerization of the heavy chains is disrupted, resulting in half DVD Ig molecules. The anti-inflammatory activity of IgG is completely dependent on sialylation of the N-linked glycan of the IgG Fc fragment. The precise glycan requirements for anti-inflammatory activity has been determined, such that an appropriate IgG1 Fc fragment can be created, thereby generating a fully recombinant, sialylated IgG1 Fc with greatly enhanced potency (Anthony, R. M., et al. (2008) Science 320:373-376).

[0061] The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments may also be bispecific, dual specific, or multi-specific formats; specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab').sub.2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546, Winter et al., PCT publication WO 90/05144 A1 herein incorporated by reference), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites. (See, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Such antibody binding portions are known in the art. (See, e.g., Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5)). In addition, single chain antibodies also include "linear antibodies" comprising a pair of tandem Fv segments (VH--CH1-VH--CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al., Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No. 5,641,870).

[0062] The term "multivalent binding protein" is used throughout this specification to denote a binding protein comprising two or more antigen binding sites. In one aspect, the multivalent binding protein is engineered to have three or more antigen binding sites, and is generally not a naturally occurring antibody. Dual variable domain (DVD) binding proteins comprise two or more antigen binding sites and are tetravalent or multivalent binding proteins. DVDs as described herein can be monospecific, i.e., capable of one antigen such as HCV core protein, or multispecific, i.e. capable of binding two or more antigens. DVD binding proteins comprising two heavy chain DVD polypeptides and two light chain DVD polypeptides are referred to as DVD-Ig, and are described for example in U.S. Pat. No. 7,612,181, the disclosure of which is herein incorporated by reference in its entirety. Each half of a DVD-Ig comprises a heavy chain DVD polypeptide, and a light chain DVD polypeptide, and two antigen binding sites. Each binding site comprises a heavy chain variable domain and a light chain variable domain with a total of 6 CDRs involved in antigen binding per antigen binding site.

[0063] A "functional antigen binding site" of a binding protein is one that is capable of binding a target antigen. The antigen binding affinity of the antigen binding site is not necessarily as strong as the parent antibody from which the antigen binding site is derived, but the ability to bind antigen must be measurable using any one of a variety of methods known for evaluating antibody binding to an antigen. Moreover, the antigen binding affinity of each of the antigen binding sites of a multivalent antibody herein need not be quantitatively the same.

[0064] An "immunoglobulin constant domain" refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences are known in the art.

[0065] The term "monoclonal antibody" or "mAb" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method.

[0066] The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0067] The term "recombinant human antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further in Section II C, below), antibodies isolated from a recombinant, combinatorial human antibody library (Hoogenboom H. R. (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W. E. (2002) Clin. Biochem. 35:425-445; Gavilondo J. V., and Larrick J. W. (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A. and Green L. L. (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al. (2000) Immunology Today 21:364-370) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. Such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0068] An "affinity matured" antibody is an antibody with one or more alterations in one or more CDRs thereof which result an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). Exemplary affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. Marks et al. BidlTechnology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and/or framework residues is described by: Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); Hawkins et al, J. Mol. Biol. 226:889-896 (1992) and selective mutation at selective mutagenesis positions, contact or hypermutation positions with an activity enhancing amino acid residue as described in U.S. Pat. No. 6,914,128 B 1.

[0069] The term "chimeric antibody" refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions.

[0070] The term "CDR-grafted antibody" refers to antibodies which comprise heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of another species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) has been replaced with human CDR sequences.

[0071] The term "humanized antibody" refers to antibodies which comprise heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more "human-like", i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding nonhuman CDR sequences. Also "humanized antibody" is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of a CDR refers to a CDR having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab') 2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. In one aspect, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, a humanized antibody contains both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CHL hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, a humanized antibody only contains a humanized light chain. In some embodiments, a humanized antibody only contains a humanized heavy chain. In specific embodiments, a humanized antibody only contains a humanized variable domain of a light chain and/or humanized heavy chain.

[0072] The terms "Kabat numbering", "Kabat definitions" and "Kabat labeling" are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e. hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable region ranges from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3. For the light chain variable region, the hypervariable region ranges from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.

[0073] As used herein, the term "CDR" refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. The term "CDR set" as used herein refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as L1, L2 and L3 or H1, H2 and H3 where the "L" and the "H" designates the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Still other CDR boundary definitions may not strictly follow one of the herein systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use Kabat or Chothia defined CDRs.

[0074] As used herein, the term "framework" or "framework sequence" refers to the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence can be determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, -L2, and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy chain) also divide the framework regions on the light chain and the heavy chain into four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying the particular sub-regions as FR1, FR2, FR3 or FR4, a framework region, as referred by others, represents the combined FR's within the variable region of a single, naturally occurring immunoglobulin chain. As used herein, a FR represents one of the four sub-regions, and FRs represents two or more of the four sub-regions constituting a framework region. As used herein, the term "germline antibody gene" or "gene fragment" refers to an immunoglobulin sequence encoded by non-lymphoid cells that have not undergone the maturation process that leads to genetic rearrangement and mutation for expression of a particular immunoglobulin. (See, e.g., Shapiro et al., Crit. Rev. Immunol. 22(3): 183-200 (2002); Marchalonis et al., Adv Exp Med. Biol. 484:13-30 (2001)). One of the advantages provided by various embodiments of the present disclosure stems from the recognition that germline antibody genes are more likely than mature antibody genes to conserve essential amino acid sequence structures characteristic of individuals in the species, hence less likely to be recognized as from a foreign source when used therapeutically in that species.

[0075] As used herein, the term "humanized antibody" is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of a CDR refers to a CDR having an amino acid sequence at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab') 2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. Preferably, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, a humanized antibody contains both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, a humanized antibody only contains a humanized light chain. In some embodiments, a humanized antibody only contains a humanized heavy chain. In specific embodiments, a humanized antibody only contains a humanized variable domain of a light chain and/or humanized heavy chain.

[0076] As used herein, the term "neutralizing" refers to counteracting the biological activity of an antigen when a binding protein specifically binds the antigen. In one aspect, the neutralizing binding protein binds the cytokine and reduces its biologically activity by at least about 20%, 40%, 60%, 80%, 85% or more.

[0077] The term "epitope" includes any polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, an antibody is said to specifically bind an antigen when it recognizes its target antigen in a complex mixture of proteins and/or macromolecules. Antibodies are said to "bind to the same epitope" if the antibodies cross-compete (one prevents the binding or modulating effect of the other). In addition structural definitions of epitopes (overlapping, similar, identical) are informative, but functional definitions are often more relevant as they encompass structural (binding) and functional (modulation, competition) parameters.

[0078] The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore.RTM. system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, N.J.). For further descriptions, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0079] The term "Kon", as used herein, is intended to refer to the on rate constant for association of a binding protein (e.g., an antibody) to the antigen to form the, e.g., antibody/antigen complex as is known in the art. The "Kon" also is known by the terms "association rate constant", or "ka", as used interchangeably herein. This value indicating the binding rate of an antibody to its target antigen or the rate of complex formation between an antibody and antigen.

[0080] The term "Koff", as used herein, is intended to refer to the off rate constant for dissociation, or "dissociation rate constant", of a binding protein (e.g., an antibody) from the, e.g., antibody/antigen complex as is known in the art. This value indicates the dissociation rate of an antibody from its target antigen or separation of Ab-Ag complex over time into free antibody and antigen.

[0081] The term "KD" as used herein, is intended to refer to the "equilibrium dissociation constant", and refers to the value obtained in a titration measurement at equilibrium, or by dividing the dissociation rate constant (koff) by the association rate constant (kon). The association rate constant, the dissociation rate constant and the equilibrium dissociation constant are used to represent the binding affinity of an antibody to an antigen. Methods for determining association and dissociation rate constants are well known in the art. Using fluorescence-based techniques offers high sensitivity and the ability to examine samples in physiological buffers at equilibrium. Other experimental approaches and instruments such as a BIAcore.RTM. (biomolecular interaction analysis) assay can be used (e.g., instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). Additionally, a KinExA.RTM. (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Id.) can also be used.

[0082] "Label" and "detectable label" mean a moiety attached to a specific binding partner, such as an antibody or an analyte, e.g., to render the reaction between members of a specific binding pair, such as an antibody and an analyte, detectable, and the specific binding partner, e.g., antibody or analyte, so labeled is referred to as "detectably labeled." Thus, the term "labeled binding protein" as used herein, refers to a protein with a label incorporated that provides for the identification of the binding protein. In one aspect, the label is a detectable marker that can produce a signal that is detectable by visual or instrumental means, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, or 153Sm); chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents, such as gadolinium chelates. Representative examples of labels commonly employed for immunoassays include moieties that produce light, e.g., acridinium compounds, and moieties that produce fluorescence, e.g., fluorescein. Other labels are described herein. In this regard, the moiety itself may not be detectably labeled but may become detectable upon reaction with yet another moiety. Use of "detectably labeled" is intended to encompass the latter type of detectable labeling.

[0083] The term "conjugate" refers to a binding protein, such as an antibody, chemically linked to a second chemical moiety, such as a therapeutic or cytotoxic agent. The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials. In one aspect, the therapeutic or cytotoxic agents include, but are not limited to, pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. When employed in the context of an immunoassay, the conjugate antibody may be a detectably labeled antibody used as the detection antibody.

[0084] The terms "isolated polynucleotide" and "isolated nucleotide molecule" as used interchangeably herein mean a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or some combination thereof) that, is not associated with all or a portion of a polynucleotide with which the "isolated polynucleotide" or "isolated nucleotide molecule" is found in nature, or does not occur in nature as part of a larger sequence. An "isolated polynucleotide" or "isolated nucleotide molecule" may be operably linked to a polynucleotide that it is not linked to in nature.

[0085] The terms "regulate" and "modulate" as used interchangeably herein refer to a change or an alteration in the activity of a molecule of interest (e.g., the biological activity of a cytokine). Modulation may be an increase or a decrease in the magnitude of a certain activity or function of the molecule of interest. Exemplary activities and functions of a molecule include, but are not limited to, binding characteristics, enzymatic activity, cell receptor activation, and signal transduction. Correspondingly, the term "modulator," as used herein, is a compound capable of changing or altering an activity or function of a molecule of interest (e.g., the biological activity of a cytokine). For example, a modulator may cause an increase or decrease in the magnitude of a certain activity or function of a molecule compared to the magnitude of the activity or function observed in the absence of the modulator. In certain embodiments, a modulator is an inhibitor, which decreases the magnitude of at least one activity or function of a molecule. Exemplary inhibitors include, but are not limited to, proteins, peptides, antibodies, peptibodies, carbohydrates or small organic molecules. Peptibodies are described, e.g., in WO01/83525.

[0086] "Patient" and "subject" may be used interchangeably herein to refer to an animal, such as a mammal, including a primate (for example, a human, a monkey, and a chimpanzee), a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a whale), a bird (e.g., a duck or a goose), and a shark. Preferably, the patient or subject is a human, such as a human being treated or assessed for a disease, disorder or condition, a human at risk for a disease, disorder or condition, a human having a disease, disorder or condition, and/or human being treated for a disease, disorder or condition.

[0087] The term "sample", as used herein, is used in its broadest sense. A "biological sample", as used herein, includes, but is not limited to, any quantity of a substance from a living thing or formerly living thing. Such living things include, but are not limited to, humans, mice, rats, monkeys, dogs, rabbits and other animals. Such substances include, but are not limited to, blood, (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes and spleen.

[0088] "Component," "components," and "at least one component," refer generally to a capture antibody, a detection or conjugate antibody, a control, a calibrator, a series of calibrators, a sensitivity panel, a container, a buffer, a diluent, a salt, an enzyme, a co-factor for an enzyme, a detection reagent, a pretreatment reagent/solution, a substrate (e.g., as a solution), a stop solution, and the like that can be included in a kit for assay of a test sample, such as a patient urine, serum or plasma sample, in accordance with the methods described herein and other methods known in the art. Thus, in the context of the present disclosure, "at least one component," "component," and "components" can include a polypeptide or other analyte as above, such as a composition comprising an analyte such as polypeptide, which is optionally immobilized on a solid support, such as by binding to an anti-analyte (e.g., anti-polypeptide) antibody. Some components can be in solution or lyophilized for reconstitution for use in an assay.

[0089] "Control" refers to a composition known to not analyte ("negative control") or to contain analyte ("positive control"). A positive control can comprise a known concentration of analyte. "Control," "positive control," and "calibrator" may be used interchangeably herein to refer to a composition comprising a known concentration of analyte. A "positive control" can be used to establish assay performance characteristics and is a useful indicator of the integrity of reagents (e.g., analytes).

[0090] "Predetermined cutoff" and "predetermined level" refer generally to an assay cutoff value that is used to assess diagnostic/prognostic/therapeutic efficacy results by comparing the assay results against the predetermined cutoff/level, where the predetermined cutoff/level already has been linked or associated with various clinical parameters (e.g., severity of disease, progression/nonprogression/improvement, etc.). While the present disclosure may provide exemplary predetermined levels, it is well-known that cutoff values may vary depending on the nature of the immunoassay (e.g., antibodies employed, etc.). It further is well within the ordinary skill of one in the art to adapt the disclosure herein for other immunoassays to obtain immunoassay-specific cutoff values for those other immunoassays based on this disclosure. Whereas the precise value of the predetermined cutoff/level may vary between assays, correlations as described herein (if any) should be generally applicable.

[0091] "Pretreatment reagent," e.g., lysis, precipitation and/or solubilization reagent, as used in a diagnostic assay as described herein is one that lyses any cells and/or solubilizes any analyte that is/are present in a test sample. Pretreatment is not necessary for all samples, as described further herein. Among other things, solubilizing the analyte (e.g., polypeptide of interest) may entail release of the analyte from any endogenous binding proteins present in the sample. A pretreatment reagent may be homogeneous (not requiring a separation step) or heterogeneous (requiring a separation step). With use of a heterogeneous pretreatment reagent there is removal of any precipitated analyte binding proteins from the test sample prior to proceeding to the next step of the assay.

[0092] "Quality control reagents" in the context of immunoassays and kits described herein, include, but are not limited to, calibrators, controls, and sensitivity panels. A "calibrator" or "standard" typically is used (e.g., one or more, such as a plurality) in order to establish calibration (standard) curves for interpolation of the concentration of an analyte, such as an antibody or an analyte. Alternatively, a single calibrator, which is near a predetermined positive/negative cutoff, can be used. Multiple calibrators (i.e., more than one calibrator or a varying amount of calibrator(s)) can be used in conjunction so as to comprise a "sensitivity panel."

[0093] "Risk" refers to the possibility or probability of a particular event occurring either presently or at some point in the future. "Risk stratification" refers to an array of known clinical risk factors that allows physicians to classify patients into a low, moderate, high or highest risk of developing a particular disease, disorder or condition.

[0094] "Specific" and "specificity" in the context of an interaction between members of a specific binding pair (e.g., an antigen (or fragment thereof) and an antibody (or antigenically reactive fragment thereof)) refer to the selective reactivity of the interaction. The phrase "specifically binds to" and analogous phrases refer to the ability of antibodies (or antigenically reactive fragments thereof) to bind specifically to analyte (or a fragment thereof) and not bind specifically to other entities.

[0095] "Specific binding partner" is a member of a specific binding pair. A specific binding pair comprises two different molecules, which specifically bind to each other through chemical or physical means. Therefore, in addition to antigen and antibody specific binding pairs of common immunoassays, other specific binding pairs can include biotin and avidin (or streptavidin), carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzyme inhibitors and enzymes, and the like. Furthermore, specific binding pairs can include members that are analogs of the original specific binding members, for example, an analyte-analog. Immunoreactive specific binding members include antigens, antigen fragments, and antibodies, including monoclonal and polyclonal antibodies as well as complexes, fragments, and variants (including fragments of variants) thereof, whether isolated or recombinantly produced.

[0096] Monoclonal Antibodies

[0097] FIGS. 1A and 1B show the sequences of various antibodies that have been determined to be specific for HCV core antigen and more particularly, have been determined to be specific for the lipid binding domain of HCV core antigen. It has been found that these monoclonal antibodies are specifically immunoreactive with the lipid binding domain of HCV core antigen. More specifically, it is found that the antibodies of the present invention specifically bind at least one epitope formed by amino acid sequence MGYIPLVGAPLGGAARALAHGVRVLED GVNYATGNLPG (SEQ ID NO:578). More particularly, the monoclonal antibodies at least are immunoreactive with an epitope formed by amino acids 141-161, 134-154 and 151-171 of HCV core antigen. Given the disclosure of these monoclonal antibodies, the present invention contemplates the uses thereof in specific immunoassays to facilitate a rapid and efficient detection of the presence of HCV in a test sample by determining the presence of HCV core antigen in such a test sample.

[0098] The anti-HCV core binding proteins, including monoclonal antibodies and any derivative (e.g., a fragment or variant) thereof that comprises the CDRs of the heavy and light chains of the monoclonal antibodies described herein (see FIGS. 1A and 1B) provided that such a derivative retains the property of binding specifically to HCV core protein lipid binding domain, can be used in immunoassays for diagnosing or prognosing hepatitis C virus infection in a mammal. As used throughout the present disclosure, "mammal" includes humans and non-human primates, as well as other animals. It will be understood that a target analyte in the immunoassays and related methods is the lipid domain of HCV core protein, and hence the target analyte is HCV core protein which would be present in the sample, such as for example, after HCV infection. Additionally, it should be understood that the immunoassay may detect two or more target analytes provided that at least one of the analytes is HCV core protein, the second or additional target analyte may be another core protein analyte (e.g., the DNA binding domain of HCV core protein) or may be an analyte that is not HCV core protein.

[0099] The nucleotide (DNA) sequences and deduced protein sequences encoding the heavy and light chain variable domains of anti-HCV core monoclonal antibodies were obtained by immunizing mice with a synthetic peptide comprised of HCV core genotype 1 consensus sequence from amino acids 134-171 and a tetanus toxoid (TT) peptide sequence. In some embodiments, the amino acid 134-171 sequence was conjugated to BSA. However, in other embodiments, the synthetic peptide also was conjugated to the TT sequence as this is often used to provide a more robust immune response in mice, by methods known to those skilled in the art such as those described in detail herein below and in, for example, Goding, J. W. 1983. Monoclonal Antibodies: Principles and Practice, Pladermic Press, Inc., NY, N.Y., pp. 56 97. Briefly, to produce a human-human hybridoma, a human lymphocyte donor is selected. A donor who is known as infected with HCV (where infection has been shown for example by the presence of anti-virus antibodies in the blood or by virus culture) may serve as a suitable lymphocyte donor. Lymphocytes can be isolated from a peripheral blood sample or spleen cells may be used if the donor is subject to splenectomy. Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes or a human fusion partner can be used to produce human-human hybridomas. Primary in vitro immunization with peptides can also be used in the generation of human monoclonal antibodies. Antibodies secreted by the immortalized cells are screened to determine the clones that secrete antibodies of the desired specificity. For monoclonal anti-HCV core antibodies, the antibodies must bind to HCV core protein and more specifically, the lipid binding domain of HCV core protein respectively. Cells producing antibodies of the desired specificity are selected. Other methods for obtaining monoclonal antibodies can be used, as known in the art. The Examples below describes how the anti-HCV core monoclonal antibodies were obtained and characterized following isolation of mRNA from hybridoma cells grown in cell culture. Deduced amino acid sequences of the heavy and light chain variable regions for the anti-HCV core monoclonal antibodies of the present invention are listed in FIG. 1A and FIG. 1B, respectively.

[0100] The deduced amino acid sequences of the heavy and the light chain domains were assigned SEQ ID NOs and the corresponding cDNAs sequences encoding the same are shown in the Sequence Table in Appendix A.

[0101] The cDNA sequences set forth in the Sequence Table represent exemplary embodiments of the disclosed cDNAs. Variations are contemplated in the cDNA sequences shown therein. Such variations include those that will result in a nucleic acid sequence that is capable of directing production of analogs of the corresponding protein shown in the Sequence Table. It will be understood that due to the degeneracy of the genetic code, many substitutions of nucleotides may be made that will lead to a DNA sequence that remains capable of directing production of the corresponding protein or its analogs. All such variant DNA sequences that are functionally equivalent to any of the sequences described herein, are encompassed by the present disclosure.

[0102] A variant of any of the binding proteins (as exemplified by monoclonal antibodies of the invention shown in FIGS. 1A and 1B) described herein means a protein (or polypeptide) that differs from a given protein (e.g., an anti-HCV core monoclonal antibody) in amino acid sequence by the addition (e.g., insertion), deletion, or conservative substitution of amino acids, but that retains the biological activity of the given protein. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity and degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art (see, e.g., Kyte et al., J. Mol. Biol. 157: 105-132 (1982)). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of .+-.2 are substituted. The hydrophilicity of amino acids also can be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity (see, e.g., U.S. Pat. No. 4,554,101, which is incorporated herein by reference). Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. In one aspect, substitutions are performed with amino acids having hydrophilicity values within .+-.2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties. "Variant" also can be used to describe a polypeptide or fragment thereof that has been differentially processed, such as by proteolysis, phosphorylation, or other post-translational modification, yet retains its biological activity or antigen reactivity, e.g., the ability to bind to IL-18. Use of "variant" herein is intended to encompass fragments of a variant unless otherwise contradicted by context.

[0103] The antibodies of the present invention or antigen binding fragments of those antibodies (e.g., fragments that comprise the heavy and light chain CDRs of the antibodies of the present invention) may also be produced by genetic engineering. For example, the technology for expression of both heavy and light chain genes in E. coli is the subject of the PCT patent applications; publication number WO 901443, W0901443, and WO 9014424 and in Huse et al., 1989 Science 246:1275 1281. The present disclosure also encompasses an isolated recombinant vector comprising a nucleic acid molecule as described herein, as well as a host cell comprising such a recombinant vector. A vector is a nucleic acid molecule, which may be a construct, capable of transporting another nucleic acid to which it has been linked. A vector may include any preferred or required operational elements. Preferred vectors are those for which the restriction sites have been described and which contain the operational elements needed for transcription of the nucleic acid sequence. Such operational elements include for example at least one suitable promoter, at least one operator, at least one leader sequence, at least one terminator codon, and any other DNA sequences necessary or preferred for appropriate transcription and subsequent translation of the nucleic acid sequence. Such vectors contain at least one origin of replication recognized by the host organism along with at least one selectable marker and at least one promoter sequence capable of initiating transcription of the nucleic acid sequence. A vector may be a plasmid into which additional DNA segments may be ligated. A vector may be a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as a plasmid is the most commonly used form of vector. However, the present disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0104] Sequences that are operably linked are in a relationship permitting them to function in their intended manner. A control sequence operably linked to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. Operably linked sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences are polynucleotide sequences that are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. Control sequences include components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0105] A host cell may be transformed with a vector that introduces exogenous DNA into a host cell in order to render that cell one that recombinantly produces the antibodies of the present invention. Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, lipofection, and particle bombardment. Transformed cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, and cells which transiently express the inserted DNA or RNA for limited periods of time.

[0106] Suitable host organisms include for example a eukaryotic cell system such as but not limited to cell lines such as HeLa, MRC-5 or CV-1. Host organisms such as host cells are cultured under conditions appropriate for amplification of the vector and expression of the protein, as well known in the art. Expressed recombinant proteins may be detected by any of a number of methods also well known in the art.

[0107] Although the HCV detection aspects of the present invention merely need the antibodies to be monoclonal antibodies such that they specifically recognize HCV core antigen, it may in some embodiments be desirable to produce humanized versions of the antibodies of the present invention. "Humanized" antibodies and production thereof is well known to those of skill in the art. General reviews of "humanized" antibodies are provided by Morrison S., 1985 Science 229:1202 and by Oi et al., 1986 BioTechniques 4:214. Suitable "humanized" antibodies can be alternatively produced by CDR or CEA substitution (Jones et al., 1986 Nature 321:552; Verhoeyan et al., 1988 Science 239:1534; Biedler et al. 1988 J. Immunol. 141:4053, the entire disclosures of which are incorporated herein by reference).

[0108] In other embodiments, the monoclonal antibodies of the present invention may serve as useful starting materials for the production of engineered and derivatized binding proteins including dual variable domain immunoglobulin (DVD-Ig) binding proteins comprising one or more anti-HCV monoclonal antibodies as described herein. For example, DVD-Ig's with unique binding affinities for HCV core protein may be produced, as described for example in U.S. Pat. No. 7,612,181, the entire disclosure of which is hereby incorporated by reference. DVD-Ig binding proteins are capable of binding one or more targets. Preferably the binding protein comprises a polypeptide chain comprising VD1-(X1)n-VD2-C--(X2)n, wherein VD1 is a first variable domain, VD2 is a second variable domain, C is a constant domain, X1 represents an amino acid or polypeptide, X2 represents an Fc region and n is 0 or 1. The binding protein can be generated using various techniques.

[0109] In exemplary techniques, the DVD-Ig can be formed with four polypeptide chains which form four functional antigen binding sites. Thus, for example, the DVD-Ig is capable of binding HCV core protein. The binding protein can be capable of modulating a biological function of HCV core protein, or of neutralizing HCV core protein. Exemplary such binding proteins have at least one heavy chain variable domain comprising an amino acid sequence of at least 90% identity with one of the antibodies of the present invention and at least the corresponding light chain variable domain comprising an amino acid sequence having at least 90% identity with a sequence of that light chain variable domain.

[0110] The variable domains of a DVD binding protein can be obtained from parent antibodies, including polyclonal and monoclonal antibodies capable of binding antigens of interest. The monoclonal antibodies that specifically bind to HCV core protein described herein are suitable parent antibodies. Generally, antibodies used for the DVD binding protein may be naturally occurring or may be generated by recombinant technology.

[0111] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those as described herein for preparing the anti-HCV core protein monoclonal antibodies, and those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Hybridomas are selected, cloned and further screened for desirable characteristics, including robust hybridoma growth, high antibody production and desirable antibody characteristics, as discussed in Example 1 below. Hybridomas may be cultured and expanded in vivo in syngeneic animals, in animals that lack an immune system, e.g., nude mice, or in cell culture in vitro. Methods of selecting, cloning and expanding hybridomas are well known to those of ordinary skill in the art. In a preferred embodiment, the hybridomas are mouse hybridomas. In another preferred embodiment, the hybridomas are produced in a non-human, non-mouse species such as rats, sheep, pigs, goats, cattle or horses. In another embodiment, the hybridomas are human hybridomas, in which a human non-secretory myeloma is fused with a human cell expressing an antibody capable of binding a specific antigen.

[0112] Recombinant monoclonal antibodies are also generated from single, isolated lymphocytes using a procedure referred to in the art as the selected lymphocyte antibody method (SLAM), as described in U.S. Pat. No. 5,627,052, PCT Publication WO 92/02551 and Babcock, J. S. et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848. In this method, single cells secreting antibodies of interest, e.g., lymphocytes derived from an immunized animal, are identified, and, heavy- and light-chain variable region cDNAs are rescued from the cells by reverse transcriptase-PCR and these variable regions can then be expressed, in the context of appropriate immunoglobulin constant regions (e.g., human constant regions), in mammalian host cells, such as COS or CHO cells. The host cells transfected with the amplified immunoglobulin sequences, derived from in vivo selected lymphocytes, can then undergo further analysis and selection in vitro, for example by panning the transfected cells to isolate cells expressing antibodies to the antigen of interest. The amplified immunoglobulin sequences further can be manipulated in vitro, such as by in vitro affinity maturation methods such as those described in PCT Publication WO 97/29131 and PCT Publication WO 00/56772.

[0113] Monoclonal antibodies are also produced by immunizing a non-human animal comprising some, or all, of the human immunoglobulin locus with an antigen of interest. In a preferred embodiment, the non-human animal is a XENOMOUSE.RTM. transgenic mouse, an engineered mouse strain that comprises large fragments of the human immunoglobulin loci and is deficient in mouse antibody production. See, e.g., Green et al. Nature Genetics 7:13-21 (1994) and U.S. Pat. Nos. 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598 and 6,130,364. See also WO 91/10741, published Jul. 25, 1991, WO 94/02602, published Feb. 3, 1994, WO 96/34096 and WO 96/33735, both published Oct. 31, 1996, WO 98/16654, published Apr. 23, 1998, WO 98/24893, published Jun. 11, 1998, WO 98/50433, published Nov. 12, 1998, WO 99/45031, published Sep. 10, 1999, WO 99/53049, published Oct. 21, 1999, WO 00 09560, published Feb. 24, 2000 and WO 00/037504, published Jun. 29, 2000. The XENOMOUSE.RTM. transgenic mouse produces an adult-like human repertoire of fully human antibodies, and generates antigen-specific human Mabs. The XENOMOUSE.RTM. transgenic mouse contains approximately 80% of the human antibody repertoire through introduction of megabase sized, germline configuration YAC fragments of the human heavy chain loci and x light chain loci. See Mendez et al., Nature Genetics 15:146-156 (1997), Green and Jakobovits J. Exp. Med. 188:483-495 (1998), the disclosures of which are hereby incorporated by reference.

[0114] In vitro methods also can be used to make the parent antibodies, wherein an antibody library is screened to identify an antibody having the desired binding specificity. Methods for such screening of recombinant antibody libraries are well known in the art and include methods described in, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No. WO 92/18619; Dower et al. PCT Publication No. WO 91/17271; Winter et al. PCT Publication No. WO 92/20791; Markland et al. PCT Publication No. WO 92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCafferty et al. PCT Publication No. WO 92/01047; Garrard et al. PCT Publication No. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, US patent application publication 20030186374, and PCT Publication No. WO 97/29131, the contents of each of which are incorporated herein by reference.

[0115] Parent antibodies can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. In a particular, such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies as described herein include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

[0116] As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies including human antibodies or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties). Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988).

[0117] Alternative to screening of recombinant antibody libraries by phage display, other methodologies known in the art for screening large combinatorial libraries can be applied to the identification of parent antibodies. One type of alternative expression system is one in which the recombinant antibody library is expressed as RNA-protein fusions, as described in PCT Publication No. WO 98/31700 by Szostak and Roberts, and in Roberts, R. W. and Szostak, J. W. (1997) Proc. Natl. Acad. Sci. USA 94:12297-12302. In this system, a covalent fusion is created between an mRNA and the peptide or protein that it encodes by in vitro translation of synthetic mRNAs that carry puromycin, a peptidyl acceptor antibiotic, at their 3' end. Thus, a specific mRNA can be enriched from a complex mixture of mRNAs (e.g., a combinatorial library) based on the properties of the encoded peptide or protein, e.g., antibody, or portion thereof, such as binding of the antibody, or portion thereof, to the dual specificity antigen. Nucleic acid sequences encoding antibodies, or portions thereof, recovered from screening of such libraries can be expressed by recombinant means as described above (e.g., in mammalian host cells) and, moreover, can be subjected to further affinity maturation by either additional rounds of screening of mRNA-peptide fusions in which mutations have been introduced into the originally selected sequence(s), or by other methods for affinity maturation in vitro of recombinant antibodies, as described above.

[0118] In another approach the parent antibodies can also be generated using yeast display methods known in the art. In yeast display methods, genetic methods are used to tether antibody domains to the yeast cell wall and display them on the surface of yeast. In particular, such yeast can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Examples of yeast display methods that can be used to make the parent antibodies include those disclosed in Wittrup, et al. U.S. Pat. No. 6,699,658 incorporated herein by reference.

[0119] The monoclonal antibodies described herein can be further modified to generate CDR grafted and Humanized parent antibodies. CDR-grafted parent antibodies comprise heavy and light chain variable region sequences from a human antibody wherein one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of murine antibodies capable of binding antigen of interest. A framework sequence from any human antibody may serve as the template for CDR grafting. However, straight chain replacement onto such a framework often leads to some loss of binding affinity to the antigen. The more homologous a human antibody is to the original murine antibody, the less likely the possibility that combining the murine CDRs with the human framework will introduce distortions in the CDRs that could reduce affinity. Therefore, it is preferable that the human variable framework that is chosen to replace the murine variable framework apart from the CDRs has at least a 65% sequence identity with the murine antibody variable region framework. It is more preferable that the human and murine variable regions apart from the CDRs have at least 70% sequence identify. It is even more preferable that the human and murine variable regions apart from the CDRs have at least 75% sequence identity. It is most preferable that the human and murine variable regions apart from the CDRs have at least 80% sequence identity. Methods for producing such antibodies are known in the art (see EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,352).

[0120] Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Known human Ig sequences are disclosed, e.g., www.ncbi.nlm.nih.gov/entrez-/query.fcgi; www.atcc.org/phage/hdb.html; www.sciquest.com/; www.abcam.com/; www.antibodyresource.com/onlinecomp.html; www.public.iastate.edu/.about.pedro/research_tools.html; www.mgen.uni-heidelberg.de/SD/IT/IT.html; www.whfreeman.com/immunology/CH-05/kuby05.html; www.library.thinkquest.org/12429/Immune/Antibody.html; www.hhmi.org/grants/lectures/1996/vlab/; www.path.cam.ac.ukiabout.mrc7/m-ikeimages.html; www.antibodyresource.com/; mcb.harvard.edu/BioLinks/Immunology.html.www.immunologylink.com/; pathbox.wustl.edu/.about.hcenter/index.-html; www.biotech.ufl.edu/.about.hcl/; www.pebio.com/pa/340913/340913.html-; www.nal.usda.gov/awic/pub s/antibody/; www.m.ehime-u.acjp/.about.yasuhito-/Elisa.html; www.biodesign.com/table.asp; www.icnet.uk/axp/facs/davies/lin-ks.html; www.biotech.ufl.edu/.about.fccl/protocol.html; www.isac-net.org/sites_geo.html; aximtl.imt.uni-marburg.de/.about.rek/AEP-Start.html; baserv.uci.kun.nliaboutjraats/linksl.html; www.recab.uni-hd.de/immuno.bme.nwu.edui; www.mrc-cpe.cam.ac.uk/imt-doc/pu-blic/INTRO.html; www.ibt.unam.mx/virV_-mice.html; imgt.cnusc.fr:8104/; www.biochem.ucl.ac.uk/.about.martin/abs/index.html; antibody.bath.ac.uk/; abgen.cvm.tamu.edu/lab/wwwabgen.html; www.unizh.chLabout.honegger/AHOseminar/Slide01.html; www.cryst.bbk.ac.ukfabout.ubcg07s/; www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm; www.path.cam.ac.uk/.about.mrc7/humanisation/TAHHP.html; www.ibt.unam.mx/vir/structure/stataim.html; www.biosci.missouri.edu/smithgp/index.html; www.cryst.bioc.cam.ac.uk/.abo-ut.fmolina/Webpages/Pept/spottech.html; wwwjerini.de/fr roducts.htm; www.patents.ibm.con/ibm.html.Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Dept. Health (1983), each entirely incorporated herein by reference. Such imported sequences can be used to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic, as known in the art.

[0121] Framework residues in the human framework regions may be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding. Antibodies can be humanized using a variety of techniques known in the art, such as but not limited to those described in Jones et al., Nature 321:522 (1986); Verhoeyen et al., Science 239:1534 (1988)), Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994); PCT publication WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596, EP 239,400, U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539; 4,816,567, each entirely incorporated herein by reference, included references cited therein.

[0122] Parent monoclonal antibodies may be selected from various monoclonal antibodies capable of binding specific targets including, or in addition to, HCV proteins, as well known in the art.

[0123] Parent monoclonal antibodies may also be selected from various therapeutic antibodies approved for use, in clinical trials, or in development for clinical use, particularly those that may be applicable in treating symptoms of HCV infection, or in treating conditions or diseases that co-exist with HCV infection, such as cancer, including particularly hepatocellular carcinoma.

[0124] As noted throughout the present invention, it may be desirable to label the antibodies of the present invention. A labeled antibody (or a binding protein derived from one of the antibodies of the present invention) comprises the antibody, which is derivatized or linked to another functional molecule (e.g., another peptide or protein). For example, the monoclonal antibody can be derivatized by functionally linking it (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the binding protein with another molecule (such as a streptavidin core region or a polyhistidine tag).

[0125] Useful detectable agents with which monoclonal antibody may be derivatized include fluorescent compounds. Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and the like. The antibody may also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. When derivatized with a detectable enzyme, the detection is achieved by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, when the detectable agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is detectable. A monoclonal antibody of the invention may also be derivatized with biotin, and detected through indirect measurement of avidin or streptavidin binding, or vice versa.

[0126] While the compositions of the present invention have demonstrated use in diagnostic applications for determining the presence of HCV core antigen in a test sample, it is contemplated that the compositions of the present invention also may serve a diagnostic or therapeutic purpose for in vivo administration to a mammal. Thus, in some embodiments, the present invention provides pharmaceutical and diagnostic compositions comprising one or more anti-HCV core binding proteins disclosed herein as an active ingredient. Pharmaceutical or diagnostic compositions may comprise any monoclonal antibody described herein, or any combination thereof, and a pharmaceutically acceptable carrier, diluent and/or excipient. Generally, the pharmaceutical and diagnostic compositions are prepared by combining the active ingredient with the carrier, diluent and/or excipient.

[0127] The compositions comprising binding proteins as described herein are for use in, but not limited to, diagnosing, detecting, or monitoring a disorder, but may also find use in preventing, treating, managing, or ameliorating of a disorder or one or more symptoms thereof, and/or in research. In a specific embodiment, a composition comprises one or more monoclonal antibodies of the present invention or a binding protein derived from one or more of the monoclonal antibodies of the present invention. In another embodiment, the composition comprises one or more monoclonal antibodies or binding proteins derived therefrom as described herein and one or more diagnostic, prophylactic or therapeutic agents other than monoclonal antibodies or binding proteins derived therefrom as described herein.

[0128] Immunoassays

[0129] Immunoassays according to the present disclosure include such techniques commonly recognized in the art, including for example radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme immunoassay, chemiluminescent assay, immunohistochemical assay, immunoprecipitation and the like. Standard techniques known in the art for ELISA are well-known and described for example in Methods in Immunodiagnosis, 2nd Edition, Rose and Bigazzi, eds., John Wiley and Sons, 1980 and Campbell et al., Methods of Immunology, W. A. Benjamin, Inc., 1964, both of which are incorporated herein by reference Immunoassays may be a direct, indirect, competitive, or noncompetitive immunoassay as described in the art (Oellerich, M. 1984. J. Clin. Chem. Clin. BioChem 22:895 904). Biological samples appropriate for such detection assays include, but are not limited to blood, plasma, serum, liver, saliva, lymphocytes or other mononuclear cells.

[0130] In preferred embodiments, the antibodies described herein are used in immunoassays specific for the detection of HCV. Examples include, but are not limited to, sandwich immunoassay, radioisotope detection (radioimmunoassay (MA)) and enzyme detection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) (e.g., Quantikine ELISA assays, R&D Systems, Minneapolis, Minn.)), competitive inhibition immunoassay (e.g., forward and reverse), fluorescence polarization immunoassay (FPIA), enzyme multiplied immunoassay technique (EMIT), bioluminescence resonance energy transfer (BRET), and homogeneous chemiluminescent assay, etc. In a SELDI-based immunoassay, a capture reagent that specifically binds an analyte of interest such as HCV core (or a fragment thereof) is attached to the surface of a mass spectrometry probe, such as a pre-activated protein chip array. The analyte (or a fragment thereof) is then specifically captured on the biochip, and the captured analyte (or a fragment thereof) is detected by mass spectrometry. Alternatively, the analyte (or a fragment thereof) can be eluted from the capture reagent and detected by traditional MALDI (matrix-assisted laser desorption/ionization) or by SELDI. A chemiluminescent microparticle immunoassay, in particular one employing the ARCHITECT.RTM. automated analyzer (Abbott Laboratories, Abbott Park, Ill.), is an example of a preferred immunoassay.

[0131] An immunoassay for determining the presence or amount of human hepatitis C virus in a sample may comprise, for example, combining an HCV core protein binding protein with the sample for a time sufficient for the binding protein to bind to any human hepatitis C virus that may be present in the sample, and determining the presence or amount of human hepatitis C virus present in the sample based on specific binding of the binding protein to the human hepatitis C virus core protein. The disclosure also encompasses an immunoassay device for detecting the presence or absence of human HCV in a sample, wherein the device comprises any of the antibodies described herein immobilized on a solid support. The anti-HCV core antibodies and any analogs thereof may be prepared in the form of a kit, alone, or in combinations with other reagents such as secondary antibodies, for use in immunoassays.

[0132] Methods well-known in the art for collecting, handling and processing urine, blood, serum and plasma, and other body fluids, are used in the practice of the present disclosure, for instance, when an anti-HCV core antibody of the present invention is employed as an immunodiagnostic reagent and/or in an analyte immunoassay kit. The test sample can comprise further moieties in addition to the HCV core antigen, including for example, antibodies, antigens, haptens, hormones, drugs, enzymes, receptors, proteins, peptides, polypeptides, oligonucleotides and/or polynucleotides. For example, the sample can be a whole blood sample obtained from a subject. It can be necessary or desired that a test sample, particularly whole blood, be treated prior to immunoassay as described herein, e.g., with a pretreatment reagent. Even in cases where pretreatment is not necessary (e.g., most urine samples), pretreatment optionally may be performed (e.g., as part of a regimen on a commercial platform).

[0133] The pretreatment reagent can be any reagent appropriate for use with the immunoassay and kits of the present disclosure. The pretreatment optionally comprises: (a) one or more solvents (e.g., methanol and ethylene glycol) and optionally, salt, (b) one or more solvents and salt, and optionally, detergent, (c) detergent, or (d) detergent and salt. Pretreatment reagents are known in the art, and such pretreatment can be employed as has been previously described, e.g., as used for assays on Abbott TDx, AxSYM.RTM., and ARCHITECT.RTM. analyzers (Abbott Laboratories, Abbott Park, Ill.), as described in the literature (see, e.g., Yatscoff et al., Abbott TDx Monoclonal Antibody Assay Evaluated for Measuring Cyclosporine in Whole Blood, Clin. Chem. 36: 1969-1973 (1990), and Wallemacq et al., Evaluation of the New AxSYM Cyclosporine Assay Comparison with TDx Monoclonal Whole Blood and EMIT Cyclosporine Assays, Clin. Chem. 45: 432-435 (1999)), and/or as commercially available. Additionally, pretreatment can be performed as described in U.S. Pat. No. 5,135,875, European Patent Pub. No. 0 471 293, U.S. Provisional Patent App. 60/878,017, filed Dec. 29, 2006, and U.S. Patent App. Pub. No. 2008/0020401 (incorporated by reference in its entirety for its teachings regarding pretreatment).

[0134] With use of a pretreatment reagent the assay is rendered more sensitive by disruption of preformed/preexisting immune complexes or viral particles in the test sample. In such a pretreated test sample, the anti-HCV core antibody in the sample is separated from the antigen and the remaining antigen in the sample is then tested for the presence of HCV core antigen using the monoclonal antibodies of the present invention. The HCV core antigen in the test sample is thus subjected to an antibody capture step to capture any HCV antigen present in the test sample.

[0135] In some other embodiments, use of the pretreatment does not require such a separation step. The entire mixture of test sample and pretreatment reagent are contacted with an antibody specific for the targeted antigen (in this case HCV core antigen, or more particularly, HCV core antigen lipid binding domain). The pretreatment reagent employed for such an assay typically is diluted in the pretreated test sample mixture, either before or during capture by the first antibody that is used to capture the HCV antigen. Despite such dilution, a certain amount of the pretreatment reagent may still be present in the test sample mixture during capture. The capture reagents may be an antibody of the present invention, alternatively, it may be another anti-HCV core antigen antibody or indeed it may be an antibody directed against a non-core protein antigen of HCV (e.g., an antibody against an envelope protein, E1, or E2 or other portion of HCV).

[0136] In one assay format, after the test sample is obtained from a subject, a first mixture is prepared. The mixture contains the test sample being assessed for the presence of a given antigen (e.g., in the present case, for the presence of HCV core antigen) and a first specific binding partner (typically an antibody that recognizes an HCV epitope), wherein the first specific binding partner and any HCV antigen contained in the test sample form a first antibody-antigen complex. The order in which the test sample and the first specific binding partner are added to form the mixture is not critical. The first specific binding partner may be immobilized on a solid phase, but in alternative embodiments, the first specific binding partner may be in a solution phase. The solid phase used in the immunoassay (for the first specific binding partner and, optionally, the second specific binding partner) can be any solid phase known in the art, such as, but not limited to, a magnetic particle, a bead, a test tube, a microtiter plate, a cuvette, a membrane, a scaffolding molecule, a film, a filter paper, a disc and a chip.

[0137] The methods described are amenable for adaption to systems that utilize microparticle technology including in automated and semi-automated systems wherein the solid phase comprises a microparticle. Such systems include those described in pending U.S. patent application Ser. Nos. 425,651 and 425,643, which correspond to published EPO applications Nos. EP 0 425 633 and EP 0 424 634, respectively, which are incorporated herein by reference.

[0138] After the mixture containing the first specific binding partner-analyte complex is formed, any unbound analyte is removed from the complex using any technique known in the art. For example, the unbound analyte can be removed by washing. Desirably, however, the first specific binding partner is present in excess of any analyte present in the test sample in order to optimize maximal binding of the analyte present in the test sample by the first specific binding partner.

[0139] After removal of unbound analyte, a second specific binding partner is added to the mixture to form a first specific binding partner-analyte-second specific binding partner complex. The second specific binding partner is preferably an anti-analyte antibody that binds to an epitope on the analyte that differs from the epitope on analyte bound by the first specific binding partner. Simply by way of example, assuming that the assay is for detection of HCV core antigen, a first "capture" antibody is used that is specific for the DNA binding domain of HCV core antigen (alternatively, the first antibody is an anti HCV core antibody that is specific for the HCV core antigen lipid binding domain, such as the antibodies described herein), once this first capture antibody captures HCV core protein from the sample, a second anti-core antigen antibody that binds the lipid binding domain of HCV core antigen (where the first antibody bound the DNA binding domain, or alternatively, where the first antibody is first antibody is specific for the HCV core antigen lipid binding domain, the second antibody could be specific for the DNA binding domain of HCV core antigen). Preferably, in such embodiments, the second specific binding partner is labeled with or contains a detectable label as described above in order to facilitate detection of the (capture antibody-antigen-second antibody) complex.

[0140] Any suitable detectable label as is known in the art can be used. For example, the detectable label can be a radioactive label (such as .sup.3H, .sup.125I, .sup.35S, .sup.14C, .sup.32P, and .sup.33P), an enzymatic label (such as horseradish peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase, and the like), a chemiluminescent label (such as acridinium esters, thioesters, or sulfonamides; luminol, isoluminol, phenanthridinium esters, and the like), a fluorescent label (such as fluorescein (e.g., 5-fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, and the like)), rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (e.g., zinc sulfide-capped cadmium selenide), a thermometric label, or an immuno-polymerase chain reaction label. An introduction to labels, labeling procedures and detection of labels is found in Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, N.Y. (1997), and in Haugland, Handbook of Fluorescent Probes and Research Chemicals (1996), which is a combined handbook and catalogue published by Molecular Probes, Inc., Eugene, Oreg. A fluorescent label can be used in FPIA (see, e.g., U.S. Pat. Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093, and 5,352,803, which are hereby incorporated by reference in their entireties). An acridinium compound can be used as a detectable label in a homogeneous chemiluminescent assay (see, e.g., Adamczyk et al., Bioorg. Med. Chem. Lett. 16: 1324-1328 (2006); Adamczyk et al., Bioorg. Med. Chem. Lett. 4: 2313-2317 (2004); Adamczyk et al., Biorg. Med. Chem. Lett. 14: 3917-3921 (2004); and Adamczyk et al., Org. Lett. 5: 3779-3782 (2003)).

[0141] A preferred acridinium compound is an acridinium-9-carboxamide. Methods for preparing acridinium 9-carboxamides are described in Mattingly, J. Biolumin. Chemilumin. 6: 107-114 (1991); Adamczyk et al., J. Org. Chem. 63: 5636-5639 (1998); Adamczyk et al., Tetrahedron 55: 10899-10914 (1999); Adamczyk et al., Org. Lett. 1: 779-781 (1999); Adamczyk et al., Bioconjugate Chem. 11: 714-724 (2000); Mattingly et al., In Luminescence Biotechnology: Instruments and Applications; Dyke, K. V. Ed.; CRC Press: Boca Raton, pp. 77-105 (2002); Adamczyk et al., Org. Lett. 5: 3779-3782 (2003); and U.S. Pat. Nos. 5,468,646, 5,543,524 and 5,783,699 (each of which is incorporated herein by reference in its entirety for its teachings regarding same).

[0142] Another preferred acridinium compound is an acridinium-9-carboxylate aryl ester. An example of an acridinium-9-carboxylate aryl ester of formula II is 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (available from Cayman Chemical, Ann Arbor, Mich.). Methods for preparing acridinium 9-carboxylate aryl esters are described in McCapra et al., Photochem. Photobiol. 4: 1111-21 (1965); Razavi et al., Luminescence 15: 245-249 (2000); Razavi et al., Luminescence 15: 239-244 (2000); and U.S. Pat. No. 5,241,070 (each of which is incorporated herein by reference in its entirety for its teachings regarding same). Such acridinium-9-carboxylate aryl esters are efficient chemiluminescent indicators for hydrogen peroxide produced in the oxidation of an analyte by at least one oxidase in terms of the intensity of the signal and/or the rapidity of the signal. The course of the chemiluminescent emission for the acridinium-9-carboxylate aryl ester is completed rapidly, i.e., in under 1 second, while the acridinium-9-carboxamide chemiluminescent emission extends over 2 seconds. Acridinium-9-carboxylate aryl ester, however, loses its chemiluminescent properties in the presence of protein. Therefore, its use requires the absence of protein during signal generation and detection. Methods for separating or removing proteins in the sample are well-known to those skilled in the art and include, but are not limited to, ultrafiltration, extraction, precipitation, dialysis, chromatography, and/or digestion (see, e.g., Wells, High Throughput Bioanalytical Sample Preparation. Methods and Automation Strategies, Elsevier (2003)). The amount of protein removed or separated from the test sample can be about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. Further details regarding acridinium-9-carboxylate aryl ester and its use are set forth in U.S. patent application Ser. No. 11/697,835, filed Apr. 9, 2007, and published on Oct. 9, 2008, as U.S. Pat. App. Pub. No. 2008/0248493. Acridinium-9-carboxylate aryl esters can be dissolved in any suitable solvent, such as degassed anhydrous N,N-dimethylformamide (DMF) or aqueous sodium cholate.

[0143] Chemiluminescent assays can be performed in accordance with the methods described in Adamczyk et al., Anal. Chim. Acta 579(1): 61-67 (2006). While any suitable assay format can be used, a microplate chemiluminometer (Mithras LB-940, Berthold Technologies U.S.A., LLC, Oak Ridge, Tenn.) enables the assay of multiple samples of small volumes rapidly. The chemiluminometer can be equipped with multiple reagent injectors using 96-well black polystyrene microplates (Costar #3792). Each sample can be added into a separate well, followed by the simultaneous/sequential addition of other reagents as determined by the type of assay employed. Desirably, the formation of pseudobases in neutral or basic solutions employing an acridinium aryl ester is avoided, such as by acidification. The chemiluminescent response is then recorded well-by-well. In this regard, the time for recording the chemiluminescent response will depend, in part, on the delay between the addition of the reagents and the particular acridinium employed.

[0144] The order in which the test sample and the specific binding partner(s) are added to form the mixture for chemiluminescent assay is not critical. If the first specific binding partner is detectably labeled with a chemiluminescent agent such as an acridinium compound, detectably labeled first specific binding partner-analyte complexes form. Alternatively, if a second specific binding partner is used and the second specific binding partner is detectably labeled with a chemiluminescent agent such as an acridinium compound, detectably labeled first specific binding partner-analyte-second specific binding partner complexes form. Any unbound specific binding partner, whether labeled or unlabeled, can be removed from the mixture using any technique known in the art, such as washing.

[0145] Hydrogen peroxide can be generated in situ in the mixture or provided or supplied to the mixture (e.g., the source of the hydrogen peroxide being one or more buffers or other solutions that are known to contain hydrogen peroxide) before, simultaneously with, or after the addition of an above-described acridinium compound. Hydrogen peroxide can be generated in situ in a number of ways such as would be apparent to one skilled in the art.

[0146] Upon the simultaneous or subsequent addition of at least one basic solution to the sample, a detectable signal, namely, a chemiluminescent signal, indicative of the presence of analyte is generated. The basic solution contains at least one base and has a pH greater than or equal to 10, preferably, greater than or equal to 12. Examples of basic solutions include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide, magnesium hydroxide, sodium carbonate, sodium bicarbonate, calcium hydroxide, calcium carbonate, and calcium bicarbonate. The amount of basic solution added to the sample depends on the concentration of the basic solution. Based on the concentration of the basic solution used, one skilled in the art can easily determine the amount of basic solution to add to the sample.

[0147] The chemiluminescent signal that is generated can be detected using routine techniques known to those skilled in the art. Based on the intensity of the signal generated, the amount of analyte in the sample can be quantified. Specifically, the amount of analyte in the sample is proportional to the intensity of the signal generated. The amount of analyte present can be quantified by comparing the amount of light generated to a standard curve for analyte or by comparison to a reference standard. The standard curve can be generated using serial dilutions or solutions of known concentrations of analyte by mass spectroscopy, gravimetric methods, and other techniques known in the art. While the above is described with emphasis on use of an acridinium compound as the chemiluminescent agent, one of ordinary skill in the art can readily adapt this description for use of other chemiluminescent agents.

[0148] Analyte immunoassays generally can be conducted using any format known in the art, such as, but not limited to, a sandwich format. Specifically, in one immunoassay format, at least two antibodies are employed to capture and quantify analyte, such as human analyte, or a fragment thereof in a sample. More specifically, preferably, the at least two antibodies bind to different epitopes on an analyte (or a fragment thereof) forming an immune complex, which is referred to as a "sandwich." Generally, in the immunoassays one or more antibodies can be used to capture the analyte (or a fragment thereof) in the test sample (these antibodies are frequently referred to as a "capture" antibody or "capture" antibodies) and one or more antibodies can be used to bind a detectable (namely, quantifiable) label to the sandwich (these antibodies are frequently referred to as the "detection antibody," the "detection antibodies," the "conjugate," or the "conjugates"). Thus, in the context of a sandwich immunoassay format, an anti-HCV core antibody of the present invention can be used as a capture antibody, a detection antibody, or both. For example, one anti-HCV core antibody having a domain that can bind a first epitope (e.g., the lipid binding domain of HCV core antigen) on an analyte can be used as a capture antibody and/or another anti-HCV core antibody having a domain that can bind a second epitope (e.g., the DNA binding domain of HCV core antigen) can be used as a detection antibody, or vice versa. Alternatively, one antibody having a first domain that can bind an epitope on a HCV core antigen and a second antibody that binds an epitope on a different HCV antigen can be used as a capture antibody and/or a detection antibody to detect, and optionally quantify, two or more analytes.

[0149] Generally speaking, a sample being tested for (for example, suspected of containing) analyte can be contacted with at least one capture antibody (or antibodies) and at least one detection antibody (which can be a second detection antibody or a third detection antibody or even a successively numbered antibody, e.g., as where the capture and/or detection antibody comprise multiple antibodies) either simultaneously or sequentially and in any order. For example, the test sample can be first contacted with at least one capture antibody and then (sequentially) with at least one detection antibody. Alternatively, the test sample can be first contacted with at least one detection antibody and then (sequentially) with at least one capture antibody. In yet another alternative, the test sample can be contacted simultaneously with a capture antibody and a detection antibody.

[0150] In the sandwich assay format, a sample suspected of containing analyte (or a fragment thereof) is first brought into contact with at least one first capture antibody under conditions that allow the formation of a first antibody/analyte complex. If more than one capture antibody is used, a first capture antibody/analyte complex comprising two or more capture antibodies is formed. In a sandwich assay, the antibodies, i.e., preferably, the at least one capture antibody, are used in molar excess amounts of the maximum amount of analyte (or a fragment thereof) expected in the test sample. For example, from about 5 ug to about 1 mg of antibody per mL of buffer (e.g., microparticle coating buffer) can be used.

[0151] Competitive inhibition immunoassays, which are often used to measure small analytes because binding by only one antibody is required, comprise sequential and classic formats. In a sequential competitive inhibition immunoassay a capture antibody to an analyte of interest is coated onto a well of a microtiter plate or other solid support. When the sample containing the analyte of interest is added to the well, the analyte of interest binds to the capture antibody. After washing, a known amount of labeled (e.g., biotin or horseradish peroxidase (HRP)) analyte is added to the well. A substrate for an enzymatic label is necessary to generate a signal. An example of a suitable substrate for HRP is 3,3',5,5'-tetramethylbenzidine (TMB). After washing, the signal generated by the labeled analyte is measured and is inversely proportional to the amount of analyte in the sample. In a classic competitive inhibition immunoassay an antibody to an analyte of interest is coated onto a solid support (e.g., a well of a microtiter plate). However, unlike the sequential competitive inhibition immunoassay, the sample and the labeled analyte are added to the well at the same time. Any analyte in the sample competes with labeled analyte for binding to the capture antibody. After washing, the signal generated by the labeled analyte is measured and is inversely proportional to the amount of analyte in the sample.

[0152] Optionally, prior to contacting the test sample with the at least one capture antibody (for example, the first capture antibody), the at least one capture antibody can be bound to a solid support, which facilitates the separation of the first antibody/analyte (or a fragment thereof) complex from the test sample. The substrate to which the capture antibody is bound can be any suitable solid support or solid phase that facilitates separation of the capture antibody-analyte complex from the sample.

[0153] Examples of solid phases or supports are well known to those of skill in the art and include a well of a plate, such as a microtiter plate, a test tube, a porous gel (e.g., silica gel, agarose, dextran, or gelatin), a polymeric film (e.g., polyacrylamide), beads (e.g., polystyrene beads or magnetic beads), a strip of a filter/membrane (e.g., nitrocellulose or nylon), microparticles (e.g., latex particles, magnetizable microparticles (e.g., microparticles having ferric oxide or chromium oxide cores and homo- or hetero-polymeric coats and radii of about 1-10 microns). The substrate can comprise a suitable porous material with a suitable surface affinity to bind antigens and sufficient porosity to allow access by detection antibodies. A microporous material is generally preferred, although a gelatinous material in a hydrated state can be used. Such porous substrates are preferably in the form of sheets having a thickness of about 0.01 to about 0.5 mm, preferably about 0.1 mm. While the pore size may vary quite a bit, preferably the pore size is from about 0.025 to about 15 microns, more preferably from about 0.15 to about 15 microns. The surface of such substrates can be activated by chemical processes that cause covalent linkage of an antibody to the substrate. Irreversible binding, generally by adsorption through hydrophobic forces, of the antigen or the antibody to the substrate results; alternatively, a chemical coupling agent or other means can be used to bind covalently the antibody to the substrate, provided that such binding does not interfere with the ability of the antibody to bind to analyte. Alternatively, the antibody can be bound with microparticles, which have been previously coated with streptavidin (e.g., DYNAL.RTM. Magnetic Beads, Invitrogen, Carlsbad, Calif.) or biotin (e.g., using Power-Bind.TM.-SA-MP streptavidin-coated microparticles (Seradyn, Indianapolis, Ind.)) or anti-species-specific monoclonal antibodies. If necessary, the substrate can be derivatized to allow reactivity with various functional groups on the antibody. Such derivatization requires the use of certain coupling agents, examples of which include, but are not limited to, maleic anhydride, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. If desired, one or more capture reagents, such as antibodies (or fragments thereof), each of which is specific for analyte(s) can be attached to solid phases in different physical or addressable locations (e.g., such as in a biochip configuration (see, e.g., U.S. Pat. No. 6,225,047; Int'l Patent App. Pub. No. WO 99/51773; U.S. Pat. No. 6,329,209; International Patent App. Pub. No. WO 00/56934, and U.S. Pat. No. 5,242,828). If the capture reagent is attached to a mass spectrometry probe as the solid support, the amount of analyte bound to the probe can be detected by laser desorption ionization mass spectrometry. Alternatively, a single column can be packed with different beads, which are derivatized with the one or more capture reagents, thereby capturing the analyte in a single place (see, antibody-derivatized, bead-based technologies, e.g., the xMAP technology of Luminex (Austin, Tex.)).

[0154] After the test sample being assayed for analyte (or a fragment thereof) is brought into contact with the at least one capture antibody (for example, the first capture antibody), the mixture is incubated in order to allow for the formation of a first antibody (or multiple antibody)-analyte (or a fragment thereof) complex. The incubation can be carried out at a pH of from about 4.5 to about 10.0, at a temperature of from about 2.degree. C. to about 45.degree. C., and for a period from at least about one (1) minute to about eighteen (18) hours, preferably from about 1 to about 24 minutes, most preferably for about 4 to about 18 minutes. The immunoassay described herein can be conducted in one step (meaning the test sample, at least one capture antibody and at least one detection antibody are all added sequentially or simultaneously to a reaction vessel) or in more than one step, such as two steps, three steps, etc.

[0155] After formation of the (first or multiple) capture antibody/analyte complex, the complex is then contacted with at least one detection antibody under conditions which allow for the formation of a (first or multiple) capture antibody/analyte/second detection antibody complex). While captioned for clarity as the "second" antibody (e.g., second detection antibody), in fact, where multiple antibodies are used for capture and/or detection, the at least one detection antibody can be the second, third, fourth, etc. antibodies used in the immunoassay. If the capture antibody/analyte complex is contacted with more than one detection antibody, then a (first or multiple) capture antibody/analyte (or a fragment thereof)/(multiple) detection antibody complex is formed. As with the capture antibody (e.g., the first capture antibody), when the at least one (e.g., second and any subsequent) detection antibody is brought into contact with the capture antibody/analyte (or a fragment thereof) complex, a period of incubation under conditions similar to those described above is required for the formation of the (first or multiple) capture antibody/analyte/(second or multiple) detection antibody complex. Preferably, at least one detection antibody contains a detectable label. The detectable label can be bound to the at least one detection antibody (e.g., the second detection antibody) prior to, simultaneously with, or after the formation of the (first or multiple) capture antibody/analyte/(second or multiple) detection antibody complex. Any detectable label known in the art can be used (see discussion above).

[0156] The detectable label can be bound to the antibodies either directly or through a coupling agent. An example of a coupling agent that can be used is EDAC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, hydrochloride), which is commercially available from Sigma-Aldrich, St. Louis, Mo. Other coupling agents that can be used are known in the art. Methods for binding a detectable label to an antibody are known in the art. Additionally, many detectable labels can be purchased or synthesized that already contain end groups that facilitate the coupling of the detectable label to the antibody, such as CPSP-Acridinium Ester (i.e., 9-[N-tosyl-N-(3-carboxypropyl)]-10-(3-sulfopropyl)acridinium carboxamide) or SP SP-Acridinium Ester (i.e., N10-(3-sulfopropyl)-N-(3-sulfopropyl)-acridinium-9-carboxamide).

[0157] The (first or multiple) capture antibody/analyte/(second or multiple) detection antibody complex can be, but does not have to be, separated from the remainder of the test sample prior to quantification of the label. For example, if the at least one capture antibody (e.g., the first capture antibody) is bound to a solid support, such as a well or a bead, separation can be accomplished by removing the fluid (of the test sample) from contact with the solid support. Alternatively, if the at least first capture antibody is bound to a solid support, it can be simultaneously contacted with the analyte-containing sample and the at least one second detection antibody to form a first (multiple) antibody/analyte/second (multiple) antibody complex, followed by removal of the fluid (test sample) from contact with the solid support. If the at least one first capture antibody is not bound to a solid support, then the (first or multiple) capture antibody/analyte/(second or multiple) detection antibody complex does not have to be removed from the test sample for quantification of the amount of the label.

[0158] After formation of the labeled capture antibody/analyte/detection antibody complex (e.g., the first capture antibody/analyte/second detection antibody complex), the amount of label in the complex is quantified using techniques known in the art. For example, if an enzymatic label is used, the labeled complex is reacted with a substrate for the label that gives a quantifiable reaction such as the development of color. If the label is a radioactive label, the label is quantified using appropriate means, such as a scintillation counter. If the label is a fluorescent label, the label is quantified by stimulating the label with a light of one color (which is known as the "excitation wavelength") and detecting another color (which is known as the "emission wavelength") that is emitted by the label in response to the stimulation. If the label is a chemiluminescent label, the label is quantified by detecting the light emitted either visually or by using luminometers, x-ray film, high speed photographic film, a CCD camera, etc. Once the amount of the label in the complex has been quantified, the concentration of analyte or a fragment thereof in the test sample is determined by appropriate means, such as by use of a standard curve that has been generated using serial dilutions of analyte or a fragment thereof of known concentration. Other than using serial dilutions of analyte or a fragment thereof, the standard curve can be generated gravimetrically, by mass spectroscopy and by other techniques known in the art.

[0159] In a chemiluminescent microparticle assay employing the ARCHITECT.RTM. analyzer, the conjugate diluent pH should be about 6.0+/-0.2, the microparticle coating buffer should be maintained at about room temperature (i.e., at from about 17 to about 27.degree. C.), the microparticle coating buffer pH should be about 6.5+/-0.2, and the microparticle diluent pH should be about 7.8+/-0.2. Solids preferably are less than about 0.2%, such as less than about 0.15%, less than about 0.14%, less than about 0.13%, less than about 0.12%, or less than about 0.11%, such as about 0.10%.

[0160] FPIAs are based on competitive binding immunoassay principles. A fluorescently labeled compound, when excited by a linearly polarized light, will emit fluorescence having a degree of polarization inversely proportional to its rate of rotation. When a fluorescently labeled tracer-antibody complex is excited by a linearly polarized light, the emitted light remains highly polarized because the fluorophore is constrained from rotating between the time light is absorbed and the time light is emitted. When a "free" tracer compound (i.e., a compound that is not bound to an antibody) is excited by linearly polarized light, its rotation is much faster than the corresponding tracer-antibody conjugate produced in a competitive binding immunoassay. FPIAs are advantageous over RIAs inasmuch as there are no radioactive substances requiring special handling and disposal. In addition, FPIAs are homogeneous assays that can be easily and rapidly performed.

[0161] In view of the above, a method of determining the presence, amount, or concentration of HCV core (or a fragment thereof) in a test sample is provided. The method comprises assaying the test sample for an HCV core antigen (or a fragment thereof) by an assay (i) employing (i') at least one of an antibody, a fragment of an antibody that can bind to an analyte, a variant of an antibody that can bind to an analyte, a fragment of a variant of an antibody that can bind to an analyte, or a DVD-Ig (or a fragment, a variant, or a fragment of a variant thereof) that can bind to an HCV core antigen, and (ii') at least one detectable label and (ii) comprising comparing a signal generated by the detectable label as a direct or indirect indication of the presence, amount or concentration of the HCV core antigen (or a fragment thereof) in the test sample to a signal generated as a direct or indirect indication of the presence, amount or concentration of HCV core antigen (or a fragment thereof) in a control or calibrator. The calibrator is optionally part of a series of calibrators, in which each of the calibrators differs from the other calibrators by the concentration of analyte.

[0162] The method can comprise (i) contacting the test sample with at least one first specific binding partner for HCV core (or a fragment thereof) selected from the group consisting of an antibody of the present invention, a fragment of such an antibody that can bind to an HCV core antigen, a variant of an antibody that can bind to an HCV core antigen, a fragment of a variant of an antibody that can bind to an HCV core antigen, or a DVD-Ig (or a fragment, a variant, or a fragment of a variant thereof) that can bind to an HCV core antigen so as to form a first specific binding partner/HCV core antigen (or fragment thereof) complex, (ii) contacting the first specific binding partner/HCV core antigen (or fragment thereof) complex with at least one second specific binding partner for the HCV core antigen (or fragment thereof) selected from the group consisting of a detectably labeled anti-HCV core antibody, a detectably labeled fragment of an anti-HCV core antibody that can bind to HCV core antigen, a detectably labeled variant of an anti-HCV core antibody that can bind to HCV core antigen, a detectably labeled fragment of a variant of an anti-HCV core antibody that can bind to HCV core antigen, and a detectably labeled DVD-Ig (or a fragment, a variant, or a fragment of a variant thereof) so as to form a first specific binding partner/HCV core antigen (or fragment thereof)/second specific binding partner complex, and (iii) determining the presence, amount or concentration of HCV core antigen in the test sample by detecting or measuring the signal generated by the detectable label in the first specific binding partner/HCV core antigen (or fragment thereof)/second specific binding partner complex formed in (ii).

[0163] Alternatively, the method can comprise contacting the test sample with at least one first specific binding partner for HCV core (or a fragment thereof) selected from the group consisting of an antibody, a fragment of an antibody that can bind to an HCV core, a variant of an antibody that can bind to an HCV core, a fragment of a variant of an antibody that can bind to an HCV core, and a DVD-Ig (or a fragment, a variant, or a fragment of a variant thereof) and simultaneously or sequentially, in either order, contacting the test sample with at least one second specific binding partner, which can compete with HCV core (or a fragment thereof) for binding to the at least one first specific binding partner and which is selected from the group consisting of a detectably labeled HCV core, a detectably labeled fragment of HCV core that can bind to the first specific binding partner, a detectably labeled variant of HCV core that can bind to the first specific binding partner, and a detectably labeled fragment of a variant of HCV core that can bind to the first specific binding partner. Any HCV core (or a fragment thereof) present in the test sample and the at least one second specific binding partner compete with each other to form a first specific binding partner/HCV core (or fragment thereof) complex and a first specific binding partner/second specific binding partner complex, respectively. The method further comprises determining the presence, amount or concentration of HCV core in the test sample by detecting or measuring the signal generated by the detectable label in the first specific binding partner/second specific binding partner complex formed in (ii), wherein the signal generated by the detectable label in the first specific binding partner/second specific binding partner complex is inversely proportional to the amount or concentration of HCV core in the test sample.

[0164] The above methods can further comprise diagnosing, prognosticating, or assessing the efficacy of a therapeutic/prophylactic treatment of a patient from whom the test sample was obtained. If the method further comprises assessing the efficacy of a therapeutic/prophylactic treatment of the patient from whom the test sample was obtained, the method optionally further comprises modifying the therapeutic/prophylactic treatment of the patient as needed to improve efficacy. The method can be adapted for use in an automated system or a semi-automated system.

[0165] With regard to the methods of assay (and kit therefor), it may be possible to employ commercially available anti-HCV core antibodies or methods for production of anti-HCV core as described in the literature. Commercial supplies of various antibodies include, but are not limited to, Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.), GenWay Biotech, Inc. (San Diego, Calif.), and R&D Systems (RDS; Minneapolis, Minn.).

[0166] Generally, a predetermined level can be employed as a benchmark against which to assess results obtained upon assaying a test sample for HCV core or a fragment thereof, e.g., for detecting disease or risk of disease. Generally, in making such a comparison, the predetermined level is obtained by running a particular assay a sufficient number of times and under appropriate conditions such that a linkage or association of HCV core presence, amount or concentration with a particular stage or endpoint of a disease, disorder or condition or with particular clinical indicia can be made. Typically, the predetermined level is obtained with assays of reference subjects (or populations of subjects). The HCV core measured can include fragments thereof, degradation products thereof, and/or enzymatic cleavage products thereof.

[0167] In particular, with respect to a predetermined level as employed for monitoring HCV disease progression and/or treatment, the amount or concentration of analyte or a fragment thereof may be "unchanged," "favorable" (or "favorably altered"), or "unfavorable" (or "unfavorably altered"). "Elevated" or "increased" refers to an amount or a concentration in a test sample that is higher than a typical or normal level or range (e.g., predetermined level), or is higher than another reference level or range (e.g., earlier or baseline sample). The term "lowered" or "reduced" refers to an amount or a concentration in a test sample that is lower than a typical or normal level or range (e.g., predetermined level), or is lower than another reference level or range (e.g., earlier or baseline sample). The term "altered" refers to an amount or a concentration in a sample that is altered (increased or decreased) over a typical or normal level or range (e.g., predetermined level), or over another reference level or range (e.g., earlier or baseline sample).

[0168] The typical or normal level or range for HCV core antigen is defined in accordance with standard practice. Because the levels of HCV core in some instances will be very low, a so-called altered level or alteration can be considered to have occurred when there is any net change as compared to the typical or normal level or range, or reference level or range, that cannot be explained by experimental error or sample variation. Thus, the level measured in a particular sample will be compared with the level or range of levels determined in similar samples from a so-called normal subject. In this context, a "normal subject" is an individual with no detectable disease, for example, and a "normal" (sometimes termed "control") patient or population is/are one(s) that exhibit(s) no detectable disease, respectively, for example. Furthermore, given that HCV core is not routinely found at a high level in the majority of the human population, a "normal subject" can be considered an individual with no substantial detectable increased or elevated amount or concentration of HCV core, and a "normal" (sometimes termed "control") patient or population is/are one(s) that exhibit(s) no substantial detectable increased or elevated amount or concentration of HCV core. An "apparently normal subject" is one in which HCV core has not yet been or currently is being assessed. The level of an HCV core is said to be "elevated" when the HCV core is normally undetectable (e.g., the normal level is zero, or within a range of from about 25 to about 75 percentiles of normal populations), but is detected in a test sample, as well as when the HCV core is present in the test sample at a higher than normal level. Thus, inter alia, the disclosure provides a method of screening for a subject having, or at risk of having, a particular disease, disorder, or condition. The method of assay can also involve the assay of other markers and the like.

[0169] Accordingly, the methods described herein also can be used to determine whether or not a subject has or is at risk of developing a HCV disease, disorder or condition. Specifically, such a method can comprise the steps of:

[0170] (a) determining the concentration or amount in a test sample from a subject of HCV core (or a fragment thereof) (e.g., using the methods described herein, or methods known in the art); and

[0171] (b) comparing the concentration or amount of HCV core (or a fragment thereof) determined in step (a) with a predetermined level, wherein, if the concentration or amount of HCV core determined in step (a) is favorable with respect to a predetermined level, then the subject is determined not to have or be at risk for a given disease, disorder or condition. However, if the concentration or amount of HCV core determined in step (a) is unfavorable with respect to the predetermined level, then the subject is determined to have or be at risk for a given disease, disorder or condition.

[0172] Additionally, provided herein is method of monitoring the progression of disease in a subject. Optimally the method comprising the steps of:

[0173] (a) determining the concentration or amount in a test sample from a subject of HCV core;

[0174] (b) determining the concentration or amount in a later test sample from the subject of HCV core; and

[0175] (c) comparing the concentration or amount of HCV core as determined in step (b) with the concentration or amount of HCV core determined in step (a), wherein if the concentration or amount determined in step (b) is unchanged or is unfavorable when compared to the concentration or amount of HCV core determined in step (a), then the disease in the subject is determined to have continued, progressed or worsened. By comparison, if the concentration or amount of HCV core as determined in step (b) is favorable when compared to the concentration or amount of HCV core as determined in step (a), then the disease in the subject is determined to have discontinued, regressed or improved.

[0176] Optionally, the method further comprises comparing the concentration or amount of HCV core as determined in step (b), for example, with a predetermined level. Further, optionally the method comprises treating the subject with one or more pharmaceutical compositions for a period of time if the comparison shows that the concentration or amount of HCV core as determined in step (b), for example, is unfavorably altered with respect to the predetermined level.

[0177] In still other embodiments, any of the assays described herein for monitoring presence or levels of HCV core antigen can advantageously be combined with other assays that also determine HCV infection. For example, any of the HCV core determining methods of the invention may further comprise determining the level of another HCV antigen or HCV antibody directed to an antigen other than core protein, including but not limited to determining the presence of HCV Core, E1, E2, NS2, NS3, NS4a, NS4b and NS5.

[0178] Still further, the methods can be used to monitor treatment in a subject receiving treatment with one or more pharmaceutical compositions. Specifically, such methods involve providing a first test sample from a subject before the subject has been administered one or more pharmaceutical compositions. Next, the concentration or amount in a first test sample from a subject of HCV core is determined (e.g., using the methods described herein or as known in the art). After the concentration or amount of HCV core is determined, optionally the concentration or amount of HCV core is then compared with a predetermined level. If the concentration or amount of HCV core as determined in the first test sample is lower than the predetermined level, then the subject is not treated with one or more pharmaceutical compositions. However, if the concentration or amount of HCV core as determined in the first test sample is higher than the predetermined level, then the subject is treated with one or more pharmaceutical compositions for a period of time. The period of time that the subject is treated with the one or more pharmaceutical compositions can be determined by one skilled in the art (for example, the period of time can be from about seven (7) days to about two years, preferably from about fourteen (14) days to about one (1) year).

[0179] During the course of treatment with the one or more pharmaceutical compositions, second and subsequent test samples are then obtained from the subject. The number of test samples and the time in which said test samples are obtained from the subject are not critical. For example, a second test sample could be obtained seven (7) days after the subject is first administered the one or more pharmaceutical compositions, a third test sample could be obtained two (2) weeks after the subject is first administered the one or more pharmaceutical compositions, a fourth test sample could be obtained three (3) weeks after the subject is first administered the one or more pharmaceutical compositions, a fifth test sample could be obtained four (4) weeks after the subject is first administered the one or more pharmaceutical compositions, etc.

[0180] After each second or subsequent test sample is obtained from the subject, the concentration or amount of HCV core is determined in the second or subsequent test sample is determined (e.g., using the methods described herein or as known in the art). The concentration or amount of HCV core as determined in each of the second and subsequent test samples is then compared with the concentration or amount of HCV core as determined in the first test sample (e.g., the test sample that was originally optionally compared to the predetermined level). If the concentration or amount of HCV core as determined in step (c) is favorable when compared to the concentration or amount of HCV core as determined in step (a), then the disease in the subject is determined to have discontinued, regressed or improved, and the subject should continue to be administered the one or pharmaceutical compositions of step (b). However, if the concentration or amount determined in step (c) is unchanged or is unfavorable when compared to the concentration or amount of HCV core as determined in step (a), then the disease in the subject is determined to have continued, progressed or worsened, and the subject should be treated with a higher concentration of the one or more pharmaceutical compositions administered to the subject in step (b) or the subject should be treated with one or more pharmaceutical compositions that are different from the one or more pharmaceutical compositions administered to the subject in step (b). Specifically, the subject can be treated with one or more pharmaceutical compositions that are different from the one or more pharmaceutical compositions that the subject had previously received to decrease or lower said subject's HCV core level.

[0181] Generally, for assays in which repeat testing may be done (e.g., monitoring disease progression and/or response to treatment), a second or subsequent test sample is obtained at a period in time after the first test sample has been obtained from the subject. Specifically, a second test sample from the subject can be obtained minutes, hours, days, weeks or years after the first test sample has been obtained from the subject. For example, the second test sample can be obtained from the subject at a time period of about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27 weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks, about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36 weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks, about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45 weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2 years, about 2.5 years, about 3.0 years, about 3.5 years, about 4.0 years, about 4.5 years, about 5.0 years, about 5.5. years, about 6.0 years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0 years, about 8.5 years, about 9.0 years, about 9.5 years or about 10.0 years after the first test sample from the subject is obtained.

[0182] When used to monitor disease progression, the above assay can be used to monitor the progression of disease in subjects suffering from acute conditions. Acute conditions, also known as critical care conditions, refer to acute, life-threatening diseases or other critical medical conditions involving, for example, the cardiovascular system or excretory system. Typically, critical care conditions refer to those conditions requiring acute medical intervention in a hospital-based setting (including, but not limited to, the emergency room, intensive care unit, trauma center, or other emergent care setting) or administration by a paramedic or other field-based medical personnel. For critical care conditions, repeat monitoring is generally done within a shorter time frame, namely, minutes, hours or days (e.g., about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days or about 7 days), and the initial assay likewise is generally done within a shorter timeframe, e.g., about minutes, hours or days of the onset of the disease or condition.

[0183] The assays also can be used to monitor the progression of disease in subjects suffering from chronic or non-acute conditions. Non-critical care or, non-acute conditions, refers to conditions other than acute, life-threatening disease or other critical medical conditions involving, for example, the cardiovascular system and/or excretory system. Typically, non-acute conditions include those of longer-term or chronic duration. For non-acute conditions, repeat monitoring generally is done with a longer timeframe, e.g., hours, days, weeks, months or years (e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27 weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks, about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36 weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks, about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45 weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2 years, about 2.5 years, about 3.0 years, about 3.5 years, about 4.0 years, about 4.5 years, about 5.0 years, about 5.5. years, about 6.0 years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0 years, about 8.5 years, about 9.0 years, about 9.5 years or about 10.0 years), and the initial assay likewise generally is done within a longer time frame, e.g., about hours, days, months or years of the onset of the disease or condition.

[0184] Furthermore, the above assays can be performed using a first test sample obtained from a subject where the first test sample is obtained from one source, such as urine, serum or plasma. Optionally, the above assays can then be repeated using a second test sample obtained from the subject where the second test sample is obtained from another source. For example, if the first test sample was obtained from urine, the second test sample can be obtained from serum or plasma. The results obtained from the assays using the first test sample and the second test sample can be compared. The comparison can be used to assess the status of a disease or condition in the subject.

[0185] Moreover, the present disclosure also relates to methods of determining whether a subject predisposed to or suffering from a given disease, disorder or condition will benefit from treatment. In particular, the disclosure relates to HCV core companion diagnostic methods and products. Thus, the method of "monitoring the treatment of disease in a subject" as described herein further optimally also can encompass selecting or identifying candidates for therapy.

[0186] Thus, in particular embodiments, the disclosure also provides a method of determining whether a subject having, or at risk for, a given disease, disorder or condition is a candidate for therapy. Generally, the subject is one who has experienced some symptom of a given disease, disorder or condition or who has actually been diagnosed as having, or being at risk for, a given disease, disorder or condition, and/or who demonstrates an unfavorable concentration or amount of HCV core or a fragment thereof, as described herein.

[0187] The method optionally comprises an assay as described herein, where HCV core is assessed before and following treatment of a subject with one or more pharmaceutical compositions (e.g., particularly with a pharmaceutical related to a mechanism of action involving HCV core), with immunosuppressive therapy, or by immunoabsorption therapy, or where HCV core is assessed following such treatment and the concentration or the amount of HCV core is compared against a predetermined level. An unfavorable concentration of amount of HCV core observed following treatment confirms that the subject will not benefit from receiving further or continued treatment, whereas a favorable concentration or amount of HCV core observed following treatment confirms that the subject will benefit from receiving further or continued treatment. This confirmation assists with management of clinical studies, and provision of improved patient care.

[0188] The method of assay also can be used to identify a compound that ameliorates a given disease, disorder or condition. For example, a cell that expresses HCV core can be contacted with a candidate compound. The level of expression of HCV core in the cell contacted with the compound can be compared to that in a control cell using the method of assay described herein.

[0189] In yet another detection method, each of the binding proteins as described herein can be employed in the detection of HCV antigens in fixed tissue sections, as well as fixed cells by immunohistochemical analysis.

[0190] In addition, these binding proteins can be bound to matrices similar to CNBr-activated Sepharose and used for the affinity purification of specific HCV proteins from cell cultures, or biological tissues such as blood and liver.

[0191] The monoclonal antibodies as described herein can also be used for the generation of chimeric antibodies for therapeutic use, or other similar applications. In addition, as discussed herein throughout the antibodies also could be used in the production of DVD-Ig molecules.

[0192] The monoclonal antibodies or fragments thereof can be provided individually to detect HCV core antigens. It is contemplated that combinations of the monoclonal antibodies (and fragments thereof) provided herein also may be used together as components in a mixture or "cocktail" of at least one anti-HCV core antibody as described herein with antibodies to other HCV regions, each having different binding specificities. Thus, this cocktail can include the monoclonal antibodies as described herein, which are directed to HCV core protein, and other monoclonal antibodies to other antigenic determinants of the HCV genome. Examples of other monoclonal antibodies useful for these contemplated cocktails include those to HCV C-100, HCV 33C, HCV CORE, HCV NS5 and/or HCV putative ENV, which are disclosed in, for example, U.S. Ser. No. 07/610,175 entitled MONOCLONAL ANTIBODIES TO HEPATITIS C VIRUS AND METHOD FOR USING SAME, U.S. Ser. No. 07/610,175 entitled MONOCLONAL ANTIBODIES TO HCV 33C PROTEINS AND METHODS FOR USING SAME, U.S. Ser. No. 07/648,475 entitled MONOCLONAL ANTIBODIES TO PUTATIVE HCV ENVELOPE REGION AND METHODS FOR USING SAME, U.S. Ser. No. 07/648,473 entitled MONOCLONAL ANTIBODIES TO HCV CORE PROTEINS AND METHODS FOR USING SAME and in co-filed patent application entitled MONOCLONAL ANTIBODIES TO HCV NS5 PROTEIN AND METHODS FOR USING SAME, U.S. Ser. No. 07/748,563, all of which enjoy common ownership and are incorporated herein by reference. This cocktail of monoclonal antibodies as described herein would be used in the assay formats detailed herein in place of the monoclonal antibody to HCV core, and thus would be able to detect the HCV core and other HCV antigens.

[0193] The polyclonal antibody or fragment thereof which can be used in the assay formats should specifically bind to HCV core or other HCV proteins used in the assay, such as HCV C-100 protein, HCV 33C protein, HCV ENV, HCV E2/NS1 or HCV NS5 protein. The polyclonal antibody used preferably is of mammalian origin; human, goat, rabbit or sheep anti-HCV polyclonal antibody can be used. Most preferably, the polyclonal antibody is rabbit polyclonal anti-HCV antibody. The polyclonal antibodies used in the assays can be used either alone or as a cocktail of polyclonal antibodies. Since the cocktails used in the assay formats are comprised of either monoclonal antibodies or polyclonal antibodies having different HCV specificity, they would be useful for diagnosis, evaluation and prognosis of HCV infection, as well as for studying HCV protein differentiation and specificity.

[0194] As noted elsewhere herein throughout, the test samples which can be tested by the methods as described herein described herein include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, biological fluids such as cell culture supernatants, fixed tissue specimens and fixed ceil specimens.

[0195] The indicator reagent comprises a signal-generating compound (label) that is capable of generating a measurable signal detectable by external means conjugated (attached) to a specific binding member for HCV core. "Specific binding member" as used herein means a member of a specific binding pair. That is, two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule. In addition to being an antibody member of a specific binding pair for HCV core, the indicator reagent also can be a member of any specific binding pair, including either hapten-anti-hapten systems such as biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme, and the like. An immunoreactive specific binding member can be an antibody, an antigen, or an antibody/antigen complex that is capable of binding either to HCV core as in a sandwich assay, to the capture reagent as in a competitive assay, or to the ancillary specific binding member as in an indirect assay.

[0196] The various signal generating compounds (labels) contemplated include chromogens, catalysts such as enzymes, luminescent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as acridinium, phenanthridinium and dioxetane compounds, radioactive elements, and direct visual labels. Examples of enzymes include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, and the like. The selection of a particular label is not critical, but it will be capable of producing a signal either by itself or in conjunction with one or more additional substances.

[0197] The use of scanning probe microscopy (SPM) for immunoassays also is a technology to which the monoclonal antibodies as described herein are easily adaptable. In scanning probe microscopy, in particular in atomic force microscopy, the capture phase, for example, at least one of the monoclonal antibodies as described herein, is adhered to a solid phase and a scanning probe microscope is utilized to detect antigen/antibody complexes which may be present on the surface of the solid phase. The use of scanning tunnelling microscopy eliminates the need for labels that normally must be utilized in many immunoassay systems to detect antigen/antibody complexes. Such a system is described in pending U.S. patent application Ser. No. 662,147, which enjoys common ownership and is incorporated herein by reference.

[0198] The use of SPM to monitor specific binding reactions can occur in many ways. In one embodiment, one member of a specific binding partner (the HCV core specific substance, which is the monoclonal antibody as described herein) is attached to a surface suitable for scanning. The attachment of the HCV core specific substance may be by adsorption to a test piece, which comprises a solid phase of a plastic or metal surface, following methods known to those of ordinary skill in the art. Or, covalent attachment of a specific binding partner (HCV core specific substance) to a test piece which test piece comprises a solid phase of derivatized plastic, metal, silicon, or glass may be utilized. Covalent attachment methods are known to those skilled in the art and include a variety of means to irreversibly link specific binding partners to the test piece. If the test piece is silicon or glass, the surface must be activated prior to attaching the specific binding partner. Activated silane compounds such as triethoxy amino propyl silane (available from Sigma Chemical Co., St. Louis, Mo.), triethoxy vinyl silane (Aldrich Chemical Co., Milwaukee, Wis.), and (3-mercapto-propyl)trimethoxy silane (Sigma Chemical Co., St. Louis, Mo.) can be used to introduce reactive groups such as amino-, vinyl, and thiol, respectively. Such activated surfaces can be used to link the binding partner directly (in the cases of amino or thiol) or the activated surface can be further reacted with linkers such as glutaraldehyde, bis(succinimidyl) suberate, SPPD 9 succinimidyl 3-[2-pyridyldithio]propionate), SMCC (succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate), STAB (succinimidyl [4-iodoacetyl]aminobenzoate), and SMPB (succinimidyl 4-[1-maleimidophenyl]butyrate) to separate the binding partner from the surface. The vinyl group can be oxidized to provide a means for covalent attachment. It also can be used as an anchor for the polymerization of various polymers such as poly acrylic acid, which can provide multiple attachment points for specific binding partners. The amino surface can be reacted with oxidized dextrans of various molecular weights to provide hydrophilic linkers of different size and capacity. Examples of oxidizable dextrans include Dextran T-40 (molecular weight 40,000 daltons), Dextran T-110 (molecular weight 110,000 daltons), Dextran T-500 (molecular weight 500,000 daltons), Dextran T-2M (molecular weight 2,000,000 daltons) (all of which are available from Pharmacia, Piscataway, N.J.), or Ficoll (molecular weight 70,000 daltons (available from Sigma Chemical Co., St. Louis, Mo.). Also, polyelectrolyte interactions may be used to immobilize a specific binding partner on a surface of a test piece by using techniques and chemistries described by pending U.S. patent application Ser. Nos. 150,278, filed Jan. 29, 1988, and Ser. No. 375,029, filed Jul. 7, 1989, each of which enjoys common ownership and each of which is incorporated herein by reference. The preferred method of attachment is by covalent means. Following attachment of a specific binding member, the surface may be further treated with materials such as serum, proteins, or other blocking agents to minimize non-specific binding. The surface also may be scanned either at the site of manufacture or point of use to verify its suitability for assay purposes. The scanning process is not anticipated to alter the specific binding properties of the test piece.

[0199] While the present disclosure expresses a preference for the use of solid phases, it is contemplated that the monoclonal antibodies as described herein can be utilized in non-solid phase assay systems. These assay systems are known to those skilled in the art, and are considered to be within the scope of the disclosure.

[0200] It is contemplated that the reagent employed for the assay can be provided in the form of a kit with one or more containers such as vials or bottles, with each container containing a separate reagent such as a monoclonal antibody, or a cocktail of monoclonal antibodies, detection reagents and washing reagents employed in the assay.

[0201] The antibodies can also be used as a means of enhancing the immune response. The antibodies can be administered in amount similar to those used for other therapeutic administrations of antibody. For example, normal immune globulin is administered at 0.02 0.1 ml/lb body weight during the early incubation period of other viral diseases such as rabies, measles, and hepatitis B to interfere with viral entry into cells. Thus, antibodies reactive with the HCV core proteins can be passively administered alone or in conjunction with another anti-viral agent to a host infected with an HCV to enhance the immune response and/or the effectiveness of an antiviral drug.

[0202] When used as a means of inducing anti-HCV virus antibodies in an animal, the manner of injecting the antibody is the same as for vaccination purposes, namely intramuscularly, intraperitoneally, subcutaneously or the like in an effective concentration in a physiologically suitable diluent with or without adjuvant. One or more booster injections may be desirable.

[0203] Kits

[0204] Also contemplated herein are kits for assaying a test sample for the presence, amount or concentration of HCV core protein (or a fragment thereof) in a test sample. Such a kit comprises at least one component for assaying the test sample for HCV core protein (or a fragment thereof) and instructions for assaying the test sample for the HCV core (or a fragment thereof). The at least one component for assaying the test sample for the HCV core (or a fragment thereof) can include a composition comprising an anti-HCV core protein monoclonal antibody or an anti-HCV core protein DVD-Ig (or a fragment, a variant, or a fragment of a variant thereof), which is optionally immobilized or capable of being immobilized on a solid phase.

[0205] The kit can comprise at least one component for assaying the test sample for HCV core protein by immunoassay, e.g., chemiluminescent microparticle immunoassay, and instructions for assaying the test sample for an HCV core by immunoassay, e.g., chemiluminescent microparticle immunoassay. For example, the kit can comprise at least one specific binding partner for an HCV core, such as an anti-HCV core, monoclonal/polyclonal antibody (or a fragment thereof that can bind to the HCV core, a variant thereof that can bind to the HCV core, or a fragment of a variant that can bind to the HCV core) or an anti-HCV core DVD-Ig (or a fragment, a variant, or a fragment of a variant thereof), either of which can be detectably labeled. Alternatively or additionally, the kit can comprise detectably labeled HCV core (or a fragment thereof that can bind to an anti-HCV core, monoclonal/polyclonal antibody or an anti-HCV core DVD-Ig (or a fragment, a variant, or a fragment of a variant thereof)), which can compete with any HCV core in a test sample for binding to an anti-HCV core, monoclonal/polyclonal antibody (or a fragment thereof that can bind to the HCV core, a variant thereof that can bind to the HCV core, or a fragment of a variant that can bind to the HCV core) or an anti-HCV core DVD-Ig (or a fragment, a variant, or a fragment of a variant thereof), either of which can be immobilized on a solid support. The kit can comprise a calibrator or control, e.g., isolated or purified HCV core. The kit can comprise at least one container (e.g., tube, microtiter plates or strips, which can be already coated with a first specific binding partner, for example) for conducting the assay, and/or a buffer, such as an assay buffer or a wash buffer, either one of which can be provided as a concentrated solution, a substrate solution for the detectable label (e.g., an enzymatic label), or a stop solution. Preferably, the kit comprises all components, i.e., reagents, standards, buffers, diluents, etc., which are necessary to perform the assay. The instructions can be in paper form or computer-readable form, such as a disk, CD, DVD, or the like.

[0206] Any antibodies, such as an anti-HCV core antibody or an anti-HCV core DVD-Ig, or tracer can incorporate a detectable label as described herein, such as a fluorophore, a radioactive moiety, an enzyme, a biotin/avidin label, a chromophore, a chemiluminescent label, or the like, or the kit can include reagents for carrying out detectable labeling. The antibodies, calibrators and/or controls can be provided in separate containers or pre-dispensed into an appropriate assay format, for example, into microtiter plates.

[0207] Optionally, the kit includes quality control components (for example, sensitivity panels, calibrators, and positive controls). Preparation of quality control reagents is well-known in the art and is described on insert sheets for a variety of immunodiagnostic products. Sensitivity panel members optionally are used to establish assay performance characteristics, and further optionally are useful indicators of the integrity of the immunoassay kit reagents, and the standardization of assays.

[0208] The kit can also optionally include other reagents required to conduct a diagnostic assay or facilitate quality control evaluations, such as buffers, salts, enzymes, enzyme co-factors, enzyme substrates, detection reagents, and the like. Other components, such as buffers and solutions for the isolation and/or treatment of a test sample (e.g., pretreatment reagents), also can be included in the kit. The kit can additionally include one or more other controls. One or more of the components of the kit can be lyophilized, in which case the kit can further comprise reagents suitable for the reconstitution of the lyophilized components.

[0209] The various components of the kit optionally are provided in suitable containers as necessary, e.g., a microtiter plate. The kit can further include containers for holding or storing a sample (e.g., a container or cartridge for a urine sample). Where appropriate, the kit optionally also can contain reaction vessels, mixing vessels, and other components that facilitate the preparation of reagents or the test sample. The kit can also include one or more instruments for assisting with obtaining a test sample, such as a syringe, pipette, forceps, measured spoon, or the like.

[0210] If the detectable label is at least one acridinium compound, the kit can comprise at least one acridinium-9-carboxamide, at least one acridinium-9-carboxylate aryl ester, or any combination thereof. If the detectable label is at least one acridinium compound, the kit also can comprise a source of hydrogen peroxide, such as a buffer, a solution, and/or at least one basic solution. If desired, the kit can contain a solid phase, such as a magnetic particle, bead, test tube, microtiter plate, cuvette, membrane, scaffolding molecule, film, filter paper, disc or chip.

[0211] The kit (or components thereof), as well as the method of determining the presence, amount or concentration of an HCV core in a test sample by an assay, such as an immunoassay as described herein, can be adapted for use in a variety of automated and semi-automated systems (including those wherein the solid phase comprises a microparticle), as described, e.g., in U.S. Pat. Nos. 5,089,424 and 5,006,309, and as commercially marketed, e.g., by Abbott Laboratories (Abbott Park, Ill.) as ARCHITECT.RTM..

[0212] Some of the differences between an automated or semi-automated system as compared to a non-automated system (e.g., ELISA) include the substrate to which the first specific binding partner (e.g., an anti-HCV core, monoclonal/polyclonal antibody (or a fragment thereof, a variant thereof, or a fragment of a variant thereof) or an anti-HCV core DVD-Ig (or a fragment thereof, a variant thereof, or a fragment of a variant thereof) is attached; either way, sandwich formation and HCV core reactivity can be impacted), and the length and timing of the capture, detection and/or any optional wash steps. Whereas a non-automated format, such as an ELISA, may require a relatively longer incubation time with sample and capture reagent (e.g., about 2 hours), an automated or semi-automated format (e.g., ARCHITECT.RTM., Abbott Laboratories) may have a relatively shorter incubation time (e.g., approximately 18 minutes for ARCHITECT.RTM.). Similarly, whereas a non-automated format, such as an ELISA, may incubate a detection antibody, such as the conjugate reagent, for a relatively longer incubation time (e.g., about 2 hours), an automated or semi-automated format (e.g., ARCHITECT.RTM.) may have a relatively shorter incubation time (e.g., approximately 4 minutes for the ARCHITECT.RTM.).

[0213] Other platforms available from Abbott Laboratories include, but are not limited to, AxSYM.RTM., IMx.RTM. (see, e.g., U.S. Pat. No. 5,294,404, which is hereby incorporated by reference in its entirety), PRISM.RTM., EIA (bead), and Quantum.TM. II, as well as other platforms. Additionally, the assays, kits and kit components can be employed in other formats, for example, on electrochemical or other hand-held or point-of-care assay systems. The present disclosure is, for example, applicable to the commercial Abbott Point of Care (i-STAT.RTM., Abbott Laboratories) electrochemical immunoassay system that performs sandwich immunoassays Immunosensors and their methods of manufacture and operation in single-use test devices are described, for example in, U.S. Pat. No. 5,063,081, U.S. Patent App. Pub. No. 2003/0170881, U.S. Patent App. Pub. No. 2004/0018577, U.S. Patent App. Pub. No. 2005/0054078, and U.S. Patent App. Pub. No. 2006/0160164, which are incorporated in their entireties by reference for their teachings regarding same.

[0214] In particular, with regard to the adaptation of an HCV core assay to the I-STAT.RTM. system, the following configuration is preferred. A microfabricated silicon chip is manufactured with a pair of gold amperometric working electrodes and a silver-silver chloride reference electrode. On one of the working electrodes, polystyrene beads (0.2 mm diameter) with immobilized anti-HCV core, monoclonal/polyclonal antibody (or a fragment thereof, a variant thereof, or a fragment of a variant thereof) or anti-HCV core DVD-Ig (or a fragment thereof, a variant thereof, or a fragment of a variant thereof), are adhered to a polymer coating of patterned polyvinyl alcohol over the electrode. This chip is assembled into an I-STAT.RTM. cartridge with a fluidics format suitable for immunoassay. On a portion of the wall of the sample-holding chamber of the cartridge there is a layer comprising a specific binding partner for an HCV core, such as an anti-HCV core, monoclonal/polyclonal antibody (or a fragment thereof, a variant thereof, or a fragment of a variant thereof that can bind the HCV core) or an anti-HCV core DVD-Ig (or a fragment thereof, a variant thereof, or a fragment of a variant thereof that can bind the HCV core), either of which can be detectably labeled. Within the fluid pouch of the cartridge is an aqueous reagent that includes p-aminophenol phosphate.

[0215] In operation, a sample suspected of containing an HCV core is added to the holding chamber of the test cartridge, and the cartridge is inserted into the I-STAT.RTM. reader. After the specific binding partner for an HCV core has dissolved into the sample, a pump element within the cartridge forces the sample into a conduit containing the chip. Here it is oscillated to promote formation of the sandwich. In the penultimate step of the assay, fluid is forced out of the pouch and into the conduit to wash the sample off the chip and into a waste chamber. In the final step of the assay, the alkaline phosphatase label reacts with p-aminophenol phosphate to cleave the phosphate group and permit the liberated p-aminophenol to be electrochemically oxidized at the working electrode. Based on the measured current, the reader is able to calculate the amount of HCV core in the sample by means of an embedded algorithm and factory-determined calibration curve.

[0216] It further goes without saying that the methods and kits as described herein necessarily encompass other reagents and methods for carrying out the immunoassay. For instance, encompassed are various buffers such as are known in the art and/or which can be readily prepared or optimized to be employed, e.g., for washing, as a conjugate diluent, microparticle diluent, and/or as a calibrator diluent. An exemplary conjugate diluent is ARCHITECT.RTM. conjugate diluent employed in certain kits (Abbott Laboratories, Abbott Park, Ill.) and containing 2-(N-morpholino)ethanesulfonic acid (MES), a salt, a protein blocker, an antimicrobial agent, and a detergent. An exemplary calibrator diluent is ARCHITECT.RTM. human calibrator diluent employed in certain kits (Abbott Laboratories, Abbott Park, Ill.), which comprises a buffer containing MES, other salt, a protein blocker, and an antimicrobial agent. Additionally, as described in U.S. Patent Application No. 61/142,048 filed Dec. 31, 2008, improved signal generation may be obtained, e.g., in an I-Stat cartridge format, using a nucleic acid sequence linked to the signal antibody as a signal amplifier.

[0217] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the invention described herein are obvious and may be made using suitable equivalents without departing from the scope of the invention or the embodiments disclosed herein. Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting of the invention.

EXAMPLES

Example 1

[0218] Animal Immunizations.

[0219] Female CAF1/J and RBF/DnJ mice (both from The Jackson Laboratory, Bar Harbor, Me.) were immunized on weeks 0, 4 and 10 with 50 .mu.g of an HCV core peptide corresponding to amino acids (all numbering per HCV-1) 134-171 covalently linked to BSA (ALRZ-8 immunogen).

[0220] HCV core peptide-BSA was prepared by AnaSpec, Inc. (Fremont, Calif.). The immunogen peptide was emulsified in Complete or Incomplete Adjulite Freund's Adjuvant (Pacific Immunology, Ramona, Calif.). Complete Freund's adjuvant was used for the primary immunization and Incomplete Freund's adjuvant for the second and third immunizations. Each inoculum was prepared by first diluting the HCV peptide-BSA to the appropriate concentration in sterile saline (0.9% sodium chloride), adding an equal volume of adjuvant and then mixing by passing back and forth between two syringes via a 3-way stopcock until a thick, stable emulsion was formed. Sera samples were taken 10-14 days following the 3rd immunization. On the 4.sup.th and 3.sup.rd days prior to B cell harvest, RBF/DnJ mice #306 and 315 were administered 50 .mu.g peptide-BSA diluted in sterile saline. This inoculum was delivered into the body cavity near the spleen.

TABLE-US-00005 ALRZ-8 immunogen (SEQ ID NO: 576) Ac-MGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGC-BSA.

Example 2

[0221] Screening of Mouse Sera for Antigen Immunoreactivity.

[0222] Mouse sera samples collected 7-10 days after their final immunization were first tested in a 96-well micro titer enzyme immunoassay (EIA) for reactivity to each of three synthetic (Anaspec, Inc.) carboxy-terminal biotinylated HCV core peptides. The peptides used for screening were derived from the immunogen sequence described in Example 1 and had the following designations and sequences: Peptide 1 (all numbering per HCV-1), amino acids 134-151: MGYIPLVGAPLGGAARALAHG (SEQ ID NO:573); Peptide 2, amino acids 141-161: GAPLGGAARALAHGVRVLEDG (SEQ ID NO:574), Peptide 3, amino acids 151-171, LAHGVRVLEDGVNYATGNLPG (SEQ ID NO:575). Assay plates (NUNC Corporation, Naperville, Ill.) were coated with 100 .mu.L/well of sheep anti-mouse IgG Fc specific antibody (Jackson ImmunoResearch, West Grove, Pa.) diluted to 2 .mu.g/mL in phosphate-buffered saline (PBS). Plates were incubated at 37 deg C. for about 2 hours and then at about 21 deg C. for about 2 hours. The capture antibody was then removed and 200 .mu.L/well of blocking solution (3% w/v [weight/volume] bovine serum albumin (BSA) and 0.5% v/v [volume/volume] polysorbate-20 diluted in PBS added. The plates were incubated for about 30 minutes and then washed with distilled water. Next, serial dilutions (in block solution) of the mouse sera or a positive control were added to the assay plates (100 .mu.L/well), incubated for between 2 and 20 hours and then washed with dH2O. Next, 100 .mu.L/well of normal serum solution (NSS; block solution containing 2% v/v normal mouse serum) was added for additional blocking. This solution helps to prevent non-specific binding in the assay well. The plates were incubated for about 30 minutes and then washed with dH2O. Subsequently, 100 .mu.L/well of a 224 nM solution of each peptide was added to the assay wells for a brief incubation, after which the plates were washed with dH2O (sera samples were tested for reactivity to individual peptides, rather than a mixture of all three). Next, 100 .mu.L/well horse radish peroxidase labeled streptavidin (Jackson ImmunoResearch) diluted to 200 ng/mL in blocking solution was added, allowed to incubate for about 30 minutes and then the plates washed; o-phenylenediamine substrate (OPD) was used as the chromagen to generate signal, and the reaction was quenched using 1 N sulfuric acid. Signal was read at a wavelength of 492 nm.

Example 3

[0223] Screening of Mouse Sera for Relative Affinity.

[0224] Relative affinity testing was completed for each sera sample--peptide combination for which a strong signal was seen in the previous assay. To determine the relative affinity of each serum sample for the individual core peptides, samples were tested for reactivity to limiting concentrations of each biotin labeled peptide. The assay format was identical to that described above, except that instead of preparing serial dilutions of mouse sera test samples, each sample was prepared at a single dilution, in blocking solution. Additionally, the individual peptides were tested at varying concentrations, beginning with a 500 nM solution in blocking solution followed by ten log 2 dilutions, also in blocking solution. Binding curves were generate and used to determine relative affinity for each sera sample. Based on these results, RBF/DnJ mice #306 and 315 were chosen for B cell fusion.

Example 4

[0225] Mouse Splenocyte Fusion.

[0226] On the day of fusion, the mice were euthanized and their splenocytes were harvested and placed into Iscoves Modified Dulbeccos Medium (IMDM) supplemented with Pen Strep (Invitrogen Corporation). A cell fusion was performed as described by Kohler and Milstein (Nature 1975; 256:495-7). Each mouse spleen was placed into a petri dish containing IMDM. The splenocytes were perfused out of each spleen using a syringe containing IMDM and a cell scraper. All splenocytes from mouse #306 and 315 were isolated and pooled in a 50 ml centrifuge tube, then counted using a hemocytometer with trypan blue dye exclusion to determine viability. Approximately 8.0.times.10.sup.8 total cells at 89% viability were recovered from these spleens. Approximately 7.6.times.10.sup.6 cells/ml were estimated to be B-cells based on their physical appearance under the microscope. Approximately 5 mL of this cell suspension was used for a first fusion experiment (fusion 208A), and the remaining cells were processed using magnetic activated cell sorting (MACS) and a Pan B-cell isolation kit (Miltenyi Biotech) to enrich the cell population for B-cells and deplete other cell types. Approximately 6.7.times.10.sup.8 total cells were incubated with the Pan B-cell biotin labeled antibody cocktail followed by the anti-biotin micobeads per manufacturer's instructions. The cell suspension/microbead mixture was washed by centrifugation and passed over a Miltenyi Biotech LS column contained within a magnetic field. B-cells flow freely through the column and other cell types are retained in the column. The columns were washed 3 times with PBS containing 2% FBS to wash out all B-cells. The B-cell suspension was centrifuged and the pellet was resuspended in IMDM, and then counted using a hemocytometer. Approximately 1.4.times.10.sup.8 B-cells were recovered from the enrichment procedure. Approximately 7.0.times.10' B-cells from that suspension were used for a second fusion experiment (fusion 208B) and the remaining B-cells were cryopreserved for later usage.

[0227] The unenriched splenocytes from the spleens (.about.3.8.times.10.sup.7B-cells for fusion 208A) and the enriched B-cell pool (.about.7.0.times.10.sup.7B-cells for fusion 208B) were washed by centrifugation in separate tubes and the cell pellets were re-suspended in IMDM. These splenocytes were mixed with an equal number of NS/0 myeloma cells and centrifuged into a pellet. The fusion was accomplished by exposing the splenocytes and NS/0 cells to 50% Polyethylene glycol (PEG) (American Type Culture Collection--Molecular Weight 1300-1600) in HSFM. One mL of the PEG solution was added to each cell pellet over 30 seconds, followed by one additional minute of incubation. The PEG and cell pellet was diluted by slowly adding 30 mL of IMDM over 30 seconds. The fused cells were then removed from suspension by centrifugation and decanting the supernatant. The cell pellet from each fusion (208A and 208B) was re-suspended into .about.250 mL of IMDM supplemented with .about.10% FBS (Hyclone Laboratories), HAT (Hypoxanthine, Aminopterin, Thymidine) (Sigma Laboratories), HT Supplement (Invitrogen Corporation), BM Condimed H1 (Roche Applied Science), Cholesterol and L-Glutamine (Invitrogen Corporation) in order to select for hybridomas. The fused cells were seeded into T162 culture flasks containing the HAT medium and cultured in bulk for approximately 48 hours at 37.degree. C. with 5% CO2. Following 48 hours of HAT selection, the bulk culture was centrifuged and the pellet was re-suspended into semi-solid tissue culture medium. The semi-solid tissue culture medium consisted of a 50% mixture of 2.times.IMDM (Invitrogen) with Clone Matrix (Molecular Devices) supplemented with 10% FBS, HT Supplement, Penn/Strep, L-Glutamine, and anti-mouse IgG-FITC Clone Detect (Molecular Devices). The semi-solid culture plates were allowed to incubate for 7-10 days before colony selection on the ClonepixFL (Molecular Devices). A colony grown in the semi-solid medium was considered a clone because the single cell initiating it had not been allowed to move and mix with other cells during growth, but all cell lines of interest will be subcloned at a later date to ensure clonality. An immunoprecipitation reaction occurs between the antibody being produced by the colony and the goat anti-mouse IgG Fc-FITC that fluoresces. The brighter the fluorescence signal observed, the more antibody being produced. Colonies were analyzed for fluorescence on the ClonepixFL and the ones with the brightest fluorescent signal were selected for automated transfer to 96 well tissue culture plates containing IMDM supplemented with 10% FBS, HT supplement, cholesterol, L-Glutamine, and Pen Strep. The 96 well tissue culture plates were allowed to grow for 3 to 7 days at 37.degree. C. prior to supernatant screening for antibody production.

Example 5

[0228] Hybridoma Screening and Selection Using Peptides.

[0229] Cell supernatant samples were analyzed for anti-HCV antibodies by EIA. Sheep anti-mouse IgG Fc (Jackson Immunoresearch) was coated on 96 well micro-titer EIA plates at 1 .mu.g/mL. After the capture reagent had been coated on the solid phase, remaining solution was removed and the plates were blocked using 3% BSA in PBS. The wells were washed with distilled water and cell supernatants were added to the blocked plates and allowed to incubate at room temperature for at least one hour. The anti-mouse IgG Fc captures the anti-HCV mouse antibody from the supernatant. Following the incubation, the plates were washed using distilled water. A 3% normal mouse serum in BSA block solution was added to all wells and incubated at room temperature for 30 minutes to block any unbound sheep anti-mouse IgG Fc capture sites coated on the plate. The wells were washed with distilled water and a mixture of the biotinylated HCV peptides described in Example 2 (i.e. corresponding to amino acids 134-154, 141-161, and 151-171 of HCV-1), each at 100 ng/mL, was added and incubated for 30 minutes at room temperature. Following this incubation, the biotinylated antigens were washed from the plates using distilled water and streptavidin-HRPO (Jackson Immunoresearch) diluted to approximately 200 ng/mL was added to the plates and allowed to incubate for 30 minutes. The plates were washed with distilled water and o-phenylenediamine substrate was used as the chromagen to generate signal. Plates were read at 492 nm and the results were analyzed. Wells were considered positive if they had an EIA signal at least 3 times greater than background. Positive wells were expanded to 24 well plates in IMDM supplemented with 10% FBS, HT supplement, cholesterol, and L-Glutamine.

[0230] Following 5-14 days of growth, the 24 well cultures were evaluated by EIA in the same manner as previously described, except the supernatant samples were titrated against each of the biotinylated HCV core peptides individually and BSA to identify clones that might bind nonspecifically to the peptides or the blocking protein. The 24 well cultures generating signal at least 5 times greater than the average BSA background value of 0.08 OD units with at least one of the screening peptides were considered positive and selected for further evaluation. Values are listed in Table 1.

TABLE-US-00006 TABLE 1 Reactivity to Reactivity to Reactivity to Clone # HCV bt-134-154 HCV bt-141-161 HCV bt-151-171 BSA (Pep) HCV 208A-1006 0.15 3.56 0.07 0.08 HCV 208A-1007 0.07 3.53 0.08 0.07 HCV 208A-1015 0.07 0.42 0.11 0.08 HCV 208A-1022 0.08 1.63 0.08 0.09 HCV 208A-1032 0.12 3.73 0.09 0.08 HCV 208A-1064 0.10 3.50 0.12 0.07 HCV 208A-1081 0.06 3.52 0.06 0.05 HCV 208A-110 0.07 2.88 0.11 0.06 HCV 208A-122 0.06 2.65 0.09 0.06 HCV 208A-126 0.20 2.08 0.10 0.10 HCV 208A-133 0.10 3.68 0.13 0.06 HCV 208A-134 2.06 0.08 0.06 0.07 HCV 208A-147 0.67 0.07 0.06 0.20 HCV 208A-152 0.08 3.55 0.09 0.07 HCV 208A-158 0.10 2.70 0.09 0.07 HCV 208A-159 0.15 3.77 0.19 0.08 HCV 208A-160 0.11 3.28 0.09 0.09 HCV 208A-194 0.07 0.20 0.06 0.07 HCV 208A-207 1.24 0.17 0.08 0.07 HCV 208A-208 0.09 3.22 0.29 0.10 HCV 208A-210 0.06 1.15 0.05 0.06 HCV 208A-222 0.06 2.70 0.08 0.07 HCV 208A-227 0.08 0.24 0.43 0.07 HCV 208A-230 0.08 0.42 0.07 0.07 HCV 208A-264 0.05 0.07 0.37 0.09 HCV 208A-286 0.11 2.62 0.11 0.06 HCV 208A-293 0.08 2.42 0.11 0.07 HCV 208A-312 0.22 3.70 0.06 0.06 HCV 208A-334 0.05 3.07 0.05 0.05 HCV 208A-352 1.08 0.07 0.06 0.07 HCV 208A-367 0.08 1.79 0.10 0.08 HCV 208A-381 0.07 3.69 0.12 0.08 HCV 208A-382 0.10 0.07 0.43 0.08 HCV 208A-393 0.06 1.04 0.06 0.05 HCV 208A-422 0.05 0.84 0.10 0.07 HCV 208A-427 0.20 3.82 0.11 0.11 HCV 208A-442 0.08 3.04 0.06 0.06 HCV 208A-460 0.09 2.69 0.10 0.07 HCV 208A-470 0.08 0.10 0.08 0.08 HCV 208A-489 0.06 3.11 0.07 0.06 HCV 208A-493 0.09 3.60 0.07 0.07 HCV 208A-557 0.05 2.41 0.06 0.05 HCV 208A-558 0.12 0.95 0.14 0.10 HCV 208A-562 0.21 3.71 0.12 0.09 HCV 208A-575 0.07 1.55 0.13 0.12 HCV 208A-576 0.08 3.05 0.09 0.06 HCV 208A-584 0.89 0.10 0.06 0.08 HCV 208A-603 0.07 1.35 0.09 0.07 HCV 208A-604 0.05 2.27 0.06 0.05 HCV 208A-605 0.22 3.71 0.16 0.11 HCV 208A-638 0.07 2.96 0.09 0.06 HCV 208A-641 0.10 0.93 0.11 0.09 HCV 208A-658 0.06 1.10 0.08 0.20 HCV 208A-692 3.67 0.38 0.07 0.06 HCV 208A-695 0.08 3.82 0.11 0.07 HCV 208A-719 0.07 3.60 0.09 0.06 HCV 208A-736 3.24 0.12 0.06 0.07 HCV 208A-741 1.20 3.37 0.06 0.07 HCV 208A-744 0.12 3.88 0.10 0.08 HCV 208A-759 0.16 3.95 0.14 0.08 HCV 208A-768 0.11 3.28 0.09 0.09 HCV 208A-774 0.14 3.53 0.12 0.07 HCV 208A-793 0.07 2.96 0.10 0.07 HCV 208A-807 0.79 0.07 0.06 0.06 HCV 208A-826 3.28 0.11 0.11 0.10 HCV 208A-828 0.07 3.35 0.10 0.07 HCV 208A-830 0.07 2.88 0.08 0.06 HCV 208A-850 0.07 3.49 0.10 0.06 HCV 208A-863 0.07 4.00 0.08 0.06 HCV 208A-874 0.05 0.73 0.06 0.05 HCV 208A-877 0.06 1.21 0.05 0.06 HCV 208A-879 0.09 0.17 0.79 0.19 HCV 208A-920 0.17 4.00 0.08 0.08 HCV 208A-926 0.06 3.32 0.07 0.09 HCV 208A-938 0.09 3.49 0.09 0.10 HCV 208A-939 3.33 0.17 0.05 0.05 HCV 208A-967 0.52 0.73 0.18 0.07 HCV 208A-982 0.08 3.40 0.06 0.07 HCV 208A-983 3.03 0.40 0.05 0.05 HCV 208B-1024 0.08 0.75 0.15 0.10 HCV 208B-1029 0.07 0.51 0.10 0.06 HCV 208B-1043 0.16 0.12 3.88 0.07 HCV 208B-1070 0.07 3.50 0.05 0.05 HCV 208B-1072 0.09 2.28 0.09 0.12 HCV 208B-109 0.08 3.46 0.11 0.07 HCV 208B-1094 0.12 3.28 0.13 0.10 HCV 208B-1096 0.34 0.13 3.56 0.08 HCV 208B-131 0.13 0.18 3.89 0.10 HCV 208B-141 1.66 0.09 0.06 0.06 HCV 208B-174 0.08 1.26 0.07 0.08 HCV 208B-178 0.10 0.07 1.69 0.07 HCV 208B-181 0.12 2.44 0.07 0.06 HCV 208B-183 0.31 0.14 0.09 0.11 HCV 208B-189 0.55 0.15 3.95 0.09 HCV 208B-207 0.19 0.09 0.50 0.07 HCV 208B-214 0.16 0.07 0.60 0.06 HCV 208B-230 0.41 0.12 0.08 0.08 HCV 208B-251 0.11 2.35 0.41 0.10 HCV 208B-281 1.48 0.16 0.12 0.07 HCV 208B-309 0.11 2.34 0.09 0.09 HCV 208B-319 0.17 3.43 0.13 0.13 HCV 208B-327 0.07 0.05 0.49 0.05 HCV 208B-348 0.07 0.20 0.07 0.07 HCV 208B-353 0.07 2.47 0.07 0.07 HCV 208B-395 0.54 0.13 3.69 0.11 HCV 208B-408 0.81 0.20 0.16 0.06 HCV 208B-409 0.09 3.21 0.14 0.07 HCV 208B-446 0.06 2.50 0.08 0.06 HCV 208B-457 0.75 0.66 0.24 0.17 HCV 208B-471 1.62 0.11 0.08 0.10 HCV 208B-488 0.08 0.07 0.30 0.08 HCV 208B-515 0.51 0.13 0.06 0.06 HCV 208B-517 0.10 0.41 0.70 0.11 HCV 208B-547 0.12 0.08 2.90 0.08 HCV 208B-556 0.13 3.07 0.18 0.08 HCV 208B-560 0.11 3.90 0.14 0.07 HCV 208B-589 0.27 0.10 2.85 0.12 HCV 208B-591 0.08 3.10 0.07 0.07 HCV 208B-602 0.20 1.87 0.20 0.07 HCV 208B-608 0.19 0.37 0.13 0.07 HCV 208B-612 3.05 3.04 0.15 0.08 HCV 208B-616 0.10 0.71 0.11 0.09 HCV 208B-617 0.45 0.51 0.13 0.10 HCV 208B-646 0.15 3.94 0.18 0.10 HCV 208B-652 0.21 0.55 0.10 0.09 HCV 208B-672 0.08 2.79 0.10 0.08 HCV 208B-739 1.13 0.11 0.07 0.08 HCV 208B-742 0.08 0.44 0.07 0.09 HCV 208B-750 0.10 3.41 0.12 0.09 HCV 208B-762 0.07 0.65 0.05 0.05 HCV 208B-765 0.10 0.64 0.16 0.08 HCV 208B-778 0.11 0.06 0.23 0.06 HCV 208B-780 0.07 1.02 0.17 0.10 HCV 208B-788 0.09 0.49 0.10 0.16 HCV 208B-793 0.09 0.72 0.09 0.13 HCV 208B-796 0.07 3.03 0.07 0.09 HCV 208B-822 0.08 1.35 0.07 0.05 HCV 208B-826 0.53 1.35 0.08 0.07 HCV 208B-853 0.09 0.74 0.12 0.13 HCV 208B-860 0.12 0.73 0.12 0.07 HCV 208B-862 0.21 3.28 0.10 0.09 HCV 208B-894 0.07 0.09 0.48 0.07 HCV 208B-909 0.09 3.11 0.11 0.07 HCV 208B-911 0.09 0.09 0.71 0.10 HCV 208B-922 0.93 0.11 0.11 0.09 HCV 208B-952 0.07 0.64 0.07 0.09 HCV 208B-954 0.06 3.43 0.06 0.06 HCV 208B-956 0.10 0.85 0.05 0.05 HCV 208B-960 0.09 3.38 0.11 0.08 HCV 208B-982 0.20 1.10 0.13 0.09

Example 6

[0231] Cloning and Expression of Recombinant HCV Corel-169.

[0232] The nucleotide sequence encoding amino acids 1-169 of HCV-1 was codon optimized for E. coli expression and cloned into a modified pET32a vector wherein the sequence encoding a thioredoxin fusion protein was eliminated and replaced with Methionine (M). In addition, a carboxy-terminal hexahistidine tag (SEQ ID NO:580) was included immediately after codon 169 of HCV core to facilitate purification via immobilized metal affinity chromatography (IMAC). E. coli BL21(DE3) cells were transformed with purified plasmid DNA and a clone harboring the plasmid pET-HCVCore1-169 identified. The protein expressed therefrom was designated as HCV Corel-169.

[0233] Protein expression was achieved by culturing the pET-HCVCore1-169-transformed E. coli BL21(DE3) cells in terrific broth (TB) medium. Cells were grown in a fermentor to an OD600 nm of 10 and then induced with 1 mM IPTG and grown at 37.degree. C. for approximately three hours until an OD600 nm of approximately 20 was obtained. Cells were harvested by centrifugation and lysed by sonication in 25 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl, 1 mM DTT, 5 mM MgCl.sub.2, lysozyme and benzonase. The lysate was clarified by centrifugation and the insoluble fraction was dissolved in 25 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl, 6 M urea, 1.0% n-dodecyl-B-D-maltoside, 1 mM DTT, and 5 mM MgCl.sub.2. The dissolved lysate was again clarified by centrifugation and the soluble fraction was loaded onto a HisTrap Fast Flow column (GE Healthcare). The column was then washed with 25 column volumes of 25 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl, 1 mM DTT, 5 mM MgCl.sub.2, 6 M urea, 0.1% n-dodecyl-B-D-maltoside, and 10 mM imidazole. Elution was done using the same buffer and a linear gradient of imidazole (0-500 mM). Eluted fractions containing the desired protein of interest (determined by SDS-PAGE) were pooled and dialyzed against 25 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl, 1 mM DTT, 5 mM MgCl.sub.2, with and without 6 M urea and 0.1% n-dodecyl-B-D-maltoside.

TABLE-US-00007 HCV Core1-169 Nucleotide Sequence (SEQ ID NO: 571) Atgtctaccaacccgaaaccgcagaaaaaaaacaaacgtaacaccaaccg tcgtccgcaggacgttaaattcccgggtggtggtcagatcgttggtggtg tttacctgctgccgcgtcgtggtccgcgtctgggtgttcgtgctacgcgt aaaacctctgaacgttctcagccgcgtgggcgtcgtcagccgatcccgaa agctcgtcgtccggaaggtcgtacctgggctcagccgggttacccgtggc cgctgtacggtaacgaaggttgcggttgggctggttggctgctgtctccg cgtggatctcgtccgtcttggggtccgaccgacccgcgtcgtcgttctcg taaccttggtaaagttatcgataccctgacctgcggtttcgctgacctga tgggttacataccgctggttggagctccgctgggtggtgctgctcgtgct ctggcgcatggcgtgcgtgttctggaagatggcgtcaactatgccaccgg taatctg HCV Core1-169 Amino Acid Sequence (SEQ ID NO: 572) Mstnpkpqkknkrntnrrpqdvkfpgggqivggvyllprrgprlgvratr ktsersqprgrrqpipkarrpegrtwaqpgypwplygnegcgwagwllsp rgsrpswgptdprrrsrnlgkvidtltcgfadlmgyiplvgaplggaara lahgvrvledgvnyatgnl.

Example 7

[0234] Hybridoma Screening Using Core Antigen.

[0235] The 24 well cultures were then evaluated by EIA for their ability bind HCV core1-169 (as described in Example 6) coated directly onto microtiter plates (solid phase assay). HCV core1-169 was coated onto 96 well micro-titer EIA plates at 1 .mu.g/mL. After the capture reagent had been coated on the solid phase, the solution was removed and the plates were blocked using 3% BSA in PBS. The wells were washed with distilled water and 5 fold serial dilutions of the cell culture supernatants were added and allowed to incubate at room temperature for at least one hour. The plates were washed with distilled water and a HRP labeled goat anti-mouse IgG FC antibody diluted at approximately 200 ng/ml in BSA block solution was added to the plates and allowed to incubate for 30 minutes at room temperature. The plates were washed with distilled water and o-phenylenediamine substrate was used as the chromagen to generate signal. Plates were read at 492 nm and the results were analyzed.

[0236] Antibodies with BSA background reactivity greater than or equal to the core 1-169 reactivity value were considered negative and not used in calculating the average BSA background value. For the remainder of antibodies to be considered positive for core 1-169, they had to have an EIA signal of, at least, 0.50 OD units, or at least 5 times greater than the average BSA background signal of 0.10 OD units. Values are listed in Table 2.

TABLE-US-00008 TABLE 2 Solid Phase HCV Core BSA Clone # OD (A492) OD (A492) HCV 208A-1006 0.75 0.09 HCV 208A-1007 0.39 0.06 HCV 208A-1015 0.16 0.08 HCV 208A-1022 0.09 0.07 HCV 208A-1032 1.27 0.08 HCV 208A-1064 0.94 0.08 HCV 208A-1081 1.25 0.06 HCV 208A-110 1.60 0.08 HCV 208A-122 1.36 0.06 HCV 208A-126 0.69 0.24 HCV 208A-133 1.73 0.07 HCV 208A-134 0.10 0.07 HCV 208A-147 0.08 0.10 HCV 208A-152 1.31 0.07 HCV 208A-158 1.16 0.06 HCV 208A-159 0.57 0.35 HCV 208A-160 1.65 0.08 HCV 208A-194 0.08 0.07 HCV 208A-207 0.11 0.06 HCV 208A-208 0.77 0.28 HCV 208A-210 0.87 0.06 HCV 208A-222 1.06 0.07 HCV 208A-227 0.06 0.08 HCV 208A-230 0.08 0.06 HCV 208A-264 0.09 0.07 HCV 208A-286 1.20 0.08 HCV 208A-293 1.41 0.07 HCV 208A-312 1.39 0.07 HCV 208A-334 1.28 0.09 HCV 208A-352 0.10 0.08 HCV 208A-367 0.88 0.07 HCV 208A-381 1.43 0.13 HCV 208A-382 0.08 0.07 HCV 208A-393 0.24 0.07 HCV 208A-422 1.68 1.10 HCV 208A-427 0.62 0.11 HCV 208A-442 1.61 0.06 HCV 208A-460 1.68 0.07 HCV 208A-470 0.08 0.07 HCV 208A-489 1.37 0.07 HCV 208A-493 1.58 0.06 HCV 208A-557 1.40 0.07 HCV 208A-558 0.40 0.25 HCV 208A-562 1.29 0.07 HCV 208A-575 0.12 0.06 HCV 208A-576 1.13 0.06 HCV 208A-584 0.07 0.06 HCV 208A-603 0.25 0.07 HCV 208A-604 1.27 0.06 HCV 208A-605 0.55 0.06 HCV 208A-638 1.07 0.06 HCV 208A-641 0.20 0.07 HCV 208A-658 0.83 0.07 HCV 208A-692 0.07 0.06 HCV 208A-695 0.61 0.17 HCV 208A-719 1.12 0.06 HCV 208A-736 0.07 0.07 HCV 208A-741 1.20 0.06 HCV 208A-744 1.57 0.07 HCV 208A-759 0.36 0.22 HCV 208A-768 1.12 0.07 HCV 208A-774 1.41 0.06 HCV 208A-793 1.24 0.06 HCV 208A-807 0.14 0.10 HCV 208A-826 0.09 0.06 HCV 208A-828 1.49 0.07 HCV 208A-830 1.16 0.06 HCV 208A-850 1.18 0.06 HCV 208A-863 1.55 0.07 HCV 208A-874 0.45 0.06 HCV 208A-877 0.90 0.07 HCV 208A-879 0.32 0.30 HCV 208A-920 1.14 0.07 HCV 208A-926 1.14 0.09 HCV 208A-938 0.83 0.13 HCV 208A-939 0.07 0.06 HCV 208A-967 1.08 1.30 HCV 208A-982 1.25 0.07 HCV 208A-983 0.12 0.06 HCV 208B-1024 0.60 0.40 HCV 208B-1029 0.07 0.07 HCV 208B-1043 0.86 0.15 HCV 208B-1070 1.24 0.08 HCV 208B-1072 0.89 0.07 HCV 208B-109 0.62 0.15 HCV 208B-1094 0.90 0.17 HCV 208B-1096 0.49 0.15 HCV 208B-131 0.74 0.06 HCV 208B-141 0.07 0.08 HCV 208B-174 0.15 0.06 HCV 208B-178 0.89 0.07 HCV 208B-181 0.47 0.07 HCV 208B-183 0.84 0.85 HCV 208B-189 0.49 0.11 HCV 208B-207 0.06 0.08 HCV 208B-214 0.09 0.06 HCV 208B-230 0.08 0.09 HCV 208B-251 0.79 0.08 HCV 208B-281 0.09 0.07 HCV 208B-309 0.77 0.07 HCV 208B-319 0.50 0.11 HCV 208B-327 0.14 0.08 HCV 208B-348 0.90 0.67 HCV 208B-353 0.30 0.06 HCV 208B-395 0.50 0.12 HCV 208B-408 0.09 0.08 HCV 208B-409 0.52 0.13 HCV 208B-446 0.78 0.24 HCV 208B-457 0.13 0.11 HCV 208B-471 0.24 0.12 HCV 208B-488 0.34 0.07 HCV 208B-515 0.06 0.07 HCV 208B-517 0.79 0.07 HCV 208B-547 0.46 0.13 HCV 208B-556 0.74 0.36 HCV 208B-560 1.07 0.07 HCV 208B-589 0.68 0.17 HCV 208B-591 0.72 0.08 HCV 208B-602 0.14 0.06 HCV 208B-608 0.07 0.06 HCV 208B-612 0.18 0.06 HCV 208B-616 0.19 0.15 HCV 208B-617 0.93 1.21 HCV 208B-646 1.24 0.07 HCV 208B-652 0.10 0.07 HCV 208B-672 1.77 0.79 HCV 208B-739 0.19 0.17 HCV 208B-742 0.29 0.08 HCV 208B-750 0.98 0.07 HCV 208B-762 1.62 1.49 HCV 208B-765 0.15 0.12 HCV 208B-778 0.10 0.08 HCV 208B-780 1.05 0.97 HCV 208B-788 0.21 0.21 HCV 208B-793 0.85 0.42 HCV 208B-796 0.49 0.14 HCV 208B-822 0.22 0.06 HCV 208B-826 0.65 0.08 HCV 208B-853 0.10 0.06 HCV 208B-860 0.12 0.08 HCV 208B-862 0.49 0.12 HCV 208B-894 0.07 0.07 HCV 208B-909 0.66 0.23 HCV 208B-911 1.18 1.17 HCV 208B-922 0.07 0.08 HCV 208B-952 0.16 0.07 HCV 208B-954 1.10 0.07 HCV 208B-956 0.12 0.07 HCV 208B-960 1.16 0.09 HCV 208B-982 0.46 0.08

Example 8

[0237] Hybridoma Screening Via Core Antigen Capture Assay.

[0238] The cell lines that were identified as positive at the 24 well stage by peptide-based EIA (Example 5) or HCV Corel-169 solid phase immunoassay (Example 7) were expanded for cryopreservation, followed by generation of high-density spent-cell supernatant. The high density exhausted supernatant from fusion 208A and 208B cell lines were tested for their ability to detect HCV Corel-169 captured from solution by a monoclonal antibody (14-153-229, U.S. Pat. No. 7,858,752) directed against an epitope within the nucleic acid binding domain of HCV Core (e.g. amino acids 1-125), also known as Domain 1. An anti-domain 1 monoclonal antibody was coated on the solid phase of 96 well micro-titer EIA plates at 1 .mu.g/mL. After the capture reagent had been coated on the solid phase, it was removed and the plates were blocked for 30 minutes at room temperature using a 5.times.PBS buffer containing 2% fish gelatin, 0.5% Tween20, and 0.1% n-Dodecyl-N,N-Dimethylamine-N-Oxide (Affymetrix). The plates were washed with distilled water and a 50 ng/ml solution of Corel-169 antigen diluted in the fish gelatin/detergent solution was added to all wells and allowed to incubate for at least 30 minutes at room temperature. The wells were washed with distilled water and cell supernatants were titrated down the blocked plates and allowed to incubate at room temperature for at least 30 minutes at room temperature. The plates were washed with distilled water and a HRP labeled goat anti-mouse IgG FC antibody diluted to approximately 200 ng/ml in BSA block solution was added to the plates and allowed to incubate for 30 minutes at room temperature. The plates were washed with distilled water and o-phenylenediamine substrate was used as the chromagen to generate signal. Plates were read at 492 nm and the results were analyzed. Antibodies were considered positive for Corel-169 if they had EIA signal of, at least, 0.50 OD units, or at least 5 times greater than the average BSA background signal of 0.10 OD units. Values are listed in Table 3.

TABLE-US-00009 TABLE 3 Captured HCV Core BSA Clone # OD (A492) OD (A492) HCV 208A-1006 0.97 0.10 HCV 208A-1007 1.25 0.13 HCV 208A-1015 0.15 0.14 HCV 208A-1022 0.33 0.22 HCV 208A-1032 1.53 0.08 HCV 208A-1064 1.57 0.09 HCV 208A-1081 1.95 0.09 HCV 208A-110 1.87 0.07 HCV 208A-122 2.10 0.07 HCV 208A-126 1.20 0.07 HCV 208A-133 1.46 0.08 HCV 208A-134 0.13 0.09 HCV 208A-147 0.14 0.08 HCV 208A-152 1.60 0.06 HCV 208A-158 1.84 0.08 HCV 208A-159 1.29 0.08 HCV 208A-160 1.54 0.08 HCV 208A-194 0.14 0.14 HCV 208A-207 0.41 0.09 HCV 208A-208 1.77 0.07 HCV 208A-210 0.87 0.08 HCV 208A-222 2.07 0.08 HCV 208A-227 0.09 0.08 HCV 208A-230 0.07 0.08 HCV 208A-264 0.09 0.08 HCV 208A-286 2.06 0.14 HCV 208A-293 2.31 0.07 HCV 208A-312 2.07 0.08 HCV 208A-334 1.90 0.10 HCV 208A-352 0.53 0.08 HCV 208A-367 1.41 0.08 HCV 208A-381 2.18 0.07 HCV 208A-382 0.11 0.08 HCV 208A-393 0.07 0.09 HCV 208A-422 0.18 0.11 HCV 208A-427 1.84 0.08 HCV 208A-442 0.36 0.14 HCV 208A-460 1.50 0.08 HCV 208A-470 0.09 0.09 HCV 208A-489 1.46 0.07 HCV 208A-493 1.44 0.08 HCV 208A-557 2.39 0.08 HCV 208A-558 0.23 0.18 HCV 208A-562 2.15 0.07 HCV 208A-575 0.30 0.10 HCV 208A-576 1.44 0.07 HCV 208A-584 1.10 1.20 HCV 208A-603 1.55 0.09 HCV 208A-604 1.75 0.07 HCV 208A-605 2.21 0.08 HCV 208A-638 2.24 0.08 HCV 208A-641 0.11 0.10 HCV 208A-658 0.91 0.08 HCV 208A-692 0.57 0.09 HCV 208A-695 1.77 0.09 HCV 208A-719 1.68 0.08 HCV 208A-736 0.32 0.10 HCV 208A-741 1.90 0.08 HCV 208A-744 1.62 0.10 HCV 208A-759 0.63 0.08 HCV 208A-768 2.29 0.08 HCV 208A-774 1.91 0.09 HCV 208A-793 1.19 0.08 HCV 208A-807 0.09 0.08 HCV 208A-826 0.17 0.08 HCV 208A-828 1.55 0.08 HCV 208A-830 2.09 0.08 HCV 208A-850 1.50 0.09 HCV 208A-863 1.88 0.08 HCV 208A-874 0.18 0.10 HCV 208A-877 1.25 0.08 HCV 208A-879 0.15 0.11 HCV 208A-920 1.82 0.08 HCV 208A-926 1.83 0.08 HCV 208A-938 1.66 0.08 HCV 208A-939 0.31 0.09 HCV 208A-967 0.18 0.15 HCV 208A-982 1.44 0.09 HCV 208A-983 0.64 0.09 HCV 208B-1024 0.31 0.12 HCV 208B-1029 0.14 0.15 HCV 208B-1043 1.21 0.07 HCV 208B-1070 1.25 0.08 HCV 208B-1072 1.45 0.08 HCV 208B-109 1.28 0.10 HCV 208B-1094 1.51 0.08 HCV 208B-1096 1.75 0.07 HCV 208B-131 1.52 0.08 HCV 208B-141 0.17 0.11 HCV 208B-174 0.75 0.09 HCV 208B-178 1.09 0.08 HCV 208B-181 1.65 0.07 HCV 208B-183 0.11 0.10 HCV 208B-189 1.81 0.12 HCV 208B-207 0.07 0.08 HCV 208B-214 0.08 0.08 HCV 208B-230 0.13 0.09 HCV 208B-251 1.39 0.10 HCV 208B-281 0.11 0.12 HCV 208B-309 1.23 0.08 HCV 208B-319 1.45 0.08 HCV 208B-327 0.11 0.09 HCV 208B-348 0.21 0.23 HCV 208B-353 0.91 0.10 HCV 208B-395 1.95 0.08 HCV 208B-408 0.09 0.10 HCV 208B-409 1.34 0.10 HCV 208B-446 1.08 0.08 HCV 208B-457 0.09 0.07 HCV 208B-471 0.35 0.08 HCV 208B-488 0.10 0.08 HCV 208B-515 0.08 0.08 HCV 208B-517 0.97 0.12 HCV 208B-547 1.62 0.07 HCV 208B-556 1.35 0.18 HCV 208B-560 1.30 0.07 HCV 208B-589 1.29 0.08 HCV 208B-591 1.66 0.08 HCV 208B-602 0.24 0.07 HCV 208B-608 0.09 0.08 HCV 208B-612 1.20 0.08 HCV 208B-616 0.09 0.08 HCV 208B-617 0.07 0.08 HCV 208B-646 1.29 0.08 HCV 208B-652 0.10 0.09 HCV 208B-672 0.99 0.08 HCV 208B-739 0.88 0.09 HCV 208B-742 0.12 0.08 HCV 208B-750 1.53 0.08 HCV 208B-762 0.28 0.21 HCV 208B-765 0.36 0.44 HCV 208B-778 0.08 0.07 HCV 208B-780 0.10 0.08 HCV 208B-788 0.09 0.07 HCV 208B-793 0.17 0.20 HCV 208B-796 1.19 0.09 HCV 208B-822 0.12 0.11 HCV 208B-826 0.72 0.41 HCV 208B-853 0.08 0.09 HCV 208B-860 0.09 0.09 HCV 208B-862 1.07 0.07 HCV 208B-894 0.09 0.09 HCV 208B-909 0.39 0.10 HCV 208B-911 0.14 0.17 HCV 208B-922 0.08 0.08 HCV 208B-952 0.09 0.08 HCV 208B-954 1.53 0.07 HCV 208B-956 0.14 0.12 HCV 208B-960 1.19 0.08 HCV 208B-982 0.99 0.08

Example 9

[0239] Determination of Anti-HCV Core Antibody Binding Kinetics.

[0240] The affinities/kinetics of the anti-HCV core peptide 134-171 monoclonal antibodies were determined using a Biacore 4000 instrument (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).

[0241] First, after pre-treating a CM5 Series S biosensor chip (GE Healthcare) with duplicate injections of 100 mM HCl, 50 mM NaOH, and 0.1% SDS, a rabbit anti-mouse IgG Capture Biosensor was created by amine-coupling rabbit anti-mouse IgG antibody (GE Healthcare, Piscataway, N.J.) on Spots 1, 2, 4, and 5 in all four flow cells of the biosensor chip via EDC/NHS/ethanolamine chemistry provided in an Amine Coupling Kit (GE Healthcare). Clarified anti-HCV Core antibody exhausted hybridoma supernatants and HCV Core peptide were diluted into a filtered buffer composed of 10.times.HBS-EP+ buffer (GE Healthcare; hereinafter "running buffer") diluted 10-fold into distilled H.sub.2O, supplemented with 0.1% BSA and 0.1% CM-Dextran, and 0.2 .mu.m filtered. Each HCV Core antibody supernatant was diluted 1:1 with running buffer and 0.2 .mu.m filtered again. A 53 amino acid custom peptide (ALRZ-9 peptide, Anaspec, Fremont, Calif.) was chemically synthesized to contain HCV Core residues 134-171 and a carboxy-terminal tetanus toxoid (TT) immunogenic T-cell epitope peptide (Eur. J. Immunol. (1989), 19:2237-2242) with the terminal amino and carboxy groups acetylated and amidated, respectively. The lyophilized HCV core 134-171-tetanus toxoid synthetic peptide immunogen was diluted in distilled water to a stock concentration of 0.7 or 1 mg/mL and further diluted into running buffer to concentrations of either 0.457 to 3,000 nM or 0.412 to 2,700 nM, both using a 3-fold dilution series. All antigen solutions were 0.2 .mu.m filtered prior to use.

[0242] The HCV Core 134-171-TT peptide procedure was as follows: 10 .mu.L of HCV Core antibody was separately injected over Spots 1 and 5 in all four flow cells at 10 .mu.L/minute. After all the spots contained captured antibody, the flow rate was increased to 30 .mu.L/minute and the biosensor was equilibrated at this new flow rate for 2 minutes; then, a 3 minute injection (90 .mu.L) of HCV Core peptide followed by 4 minutes of running buffer. All biosensor surfaces were regenerated with one 30 .mu.L injection of 10 mM glycine, pH 1.7 (GE Healthcare), at a flow rate of 10 .mu.L/minute. All concentrations were tested in duplicate. The binding kinetics (association and dissociation) were monitored via sensorgrams during antigen injection followed by running buffer. The sensorgrams were double-referenced and fit to a 1:1 binding model using Biacore 4000 Evaluation software (GE Healthcare Bio-Sciences AB) to determine association and dissociation rates, as well as overall K.sub.D. Kinetic and affinity values are listed in Table 4. If values are not present, then either binding kinetics could not be determined or the antibody did not interact with the HCV Core 134-171-TT peptide in this assay.

TABLE-US-00010 ALRZ-9 peptide (SEQ ID NO: 577) Ac-MGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGQYIKANSKF IGITEL-NH2

TABLE-US-00011 TABLE 4 Binding Interactions with HCV Core 134-171-TT Peptide Clone # ka (1/Ms) kd (Vs) KD (M) HCV 208A-1006 1.8E+06 6.2E-04 3.5E-10 HCV 208A-1007 1.8E+06 7.4E-04 4.1E-10 HCV 208A-1022 1.6E+05 2.5E-03 1.6E-08 HCV 208A-1032 1.2E+06 5.5E-04 4.5E-10 HCV 208A-1064 1.6E+06 3.4E-04 2.1E-10 HCV 208A-1081 1.3E+06 5.7E-04 4.5E-10 HCV 208A-110 1.7E+06 5.5E-04 3.3E-10 HCV 208A-122 1.8E+06 2.1E-04 1.2E-10 HCV 208A-133 1.2E+06 5.0E-04 4.1E-10 HCV 208A-134 1.8E+04 3.8E-05 2.1E-09 HCV 208A-152 7.7E+05 4.0E-04 5.2E-10 HCV 208A-158 1.3E+06 4.4E-04 3.3E-10 HCV 208A-159 1.4E+06 6.5E-04 4.5E-10 HCV 208A-160 1.6E+06 6.1E-04 3.8E-10 HCV 208A-207 3.1E+05 <1E-05 <3.2E-11 HCV 208A-208 1.6E+06 5.4E-04 3.4E-10 HCV 208A-222 1.1E+06 3.5E-04 3.1E-10 HCV 208A-286 2.0E+06 2.5E-04 1.2E-10 HCV 208A-293 2.1E+06 2.3E-04 1.1E-10 HCV 208A-312 2.1E+06 2.6E-04 1.3E-10 HCV 208A-334 1.7E+06 5.7E-04 3.4E-10 HCV 208A-352 1.0E+05 5.4E-05 5.2E-10 HCV 208A-367 1.5E+06 5.9E-04 3.9E-10 HCV 208A-381 2.0E+06 2.7E-04 1.3E-10 HCV 208A-427 1.2E+06 5.6E-04 4.5E-10 HCV 208A-460 1.3E+06 5.6E-04 4.4E-10 HCV 208A-489 1.3E+06 6.6E-04 4.9E-10 HCV 208A-493 1.4E+06 5.7E-04 4.1E-10 HCV 208A-557 2.4E+06 2.1E-04 9.1E-11 HCV 208A-562 1.7E+06 6.6E-04 4.0E-10 HCV 208A-576 1.2E+06 5.8E-04 4.9E-10 HCV 208A-603 1.5E+06 5.6E-04 3.9E-10 HCV 208A-604 1.3E+06 5.8E-04 4.5E-10 HCV 208A-605 8.7E+05 3.7E-04 4.3E-10 HCV 208A-638 2.7E+06 5.3E-04 2.0E-10 HCV 208A-695 2.0E+06 7.1E-04 3.5E-10 HCV 208A-719 1.9E+06 5.7E-04 3.0E-10 HCV 208A-736 2.2E+04 1.7E-05 7.6E-10 HCV 208A-741 1.7E+06 6.9E-04 4.1E-10 HCV 208A-744 1.3E+06 6.0E-04 4.5E-10 HCV 208A-768 1.1E+06 3.6E-04 3.3E-10 HCV 208A-774 1.3E+06 5.9E-04 4.5E-10 HCV 208A-793 1.8E+06 6.6E-04 3.7E-10 HCV 208A-826 1.8E+04 7.4E-05 4.2E-09 HCV 208A-828 1.4E+06 5.7E-04 4.2E-10 HCV 208A-830 3.4E+06 5.4E-04 1.6E-10 HCV 208A-850 1.6E+06 6.0E-04 3.9E-10 HCV 208A-863 1.3E+06 6.1E-04 4.6E-10 HCV 208A-877 1.1E+06 2.4E-03 2.2E-09 HCV 208A-920 1.5E+06 5.6E-04 3.9E-10 HCV 208A-926 1.7E+06 5.6E-04 3.3E-10 HCV 208A-938 1.6E+06 5.2E-04 3.2E-10 HCV 208A-939 2.5E+04 2.2E-04 9.0E-09 HCV 208A-982 1.3E+06 6.2E-04 4.9E-10 HCV 208A-983 9.7E+04 4.6E-05 4.6E-10 HCV 208B-1024 1.0E+06 1.4E-02 1.5E-08 HCV 208B-1043 1.1E+06 6.0E-04 5.4E-10 HCV 208B-1070 1.6E+06 5.2E-04 3.2E-10 HCV 208B-1072 1.3E+06 5.5E-04 4.2E-10 HCV 208B-109 1.4E+06 5.1E-04 3.7E-10 HCV 208B-1094 1.5E+06 2.5E-04 1.6E-10 HCV 208B-1096 1.3E+06 6.0E-04 4.6E-10 HCV 208B-131 1.3E+06 1.7E-03 1.3E-09 HCV 208B-141 3.1E+04 1.6E-04 5.3E-09 HCV 208B-174 7.6E+05 1.8E-04 2.5E-10 HCV 208B-181 2.0E+06 3.6E-04 1.8E-10 HCV 208B-189 8.8E+05 1.5E-04 1.7E-10 HCV 208B-251 1.1E+06 6.5E-04 5.8E-10 HCV 208B-281 2.2E+03 2.5E-04 9.6E-08 HCV 208B-309 1.2E+06 6.8E-04 5.7E-10 HCV 208B-319 2.2E+06 2.8E-04 1.3E-10 HCV 208B-395 1.3E+06 4.7E-04 3.6E-10 HCV 208B-409 1.9E+06 2.8E-04 1.5E-10 HCV 208B-446 2.1E+06 2.8E-04 1.4E-10 HCV 208B-471 2.2E+05 5.5E-05 2.5E-10 HCV 208B-547 1.6E+06 4.9E-03 3.2E-09 HCV 208B-556 1.5E+06 6.0E-04 4.0E-10 HCV 208B-560 1.1E+06 2.6E-04 2.3E-10 HCV 208B-589 9.6E+05 6.1E-04 6.3E-10 HCV 208B-591 2.2E+06 2.0E-04 9.3E-11 HCV 208B-612 1.8E+05 6.5E-05 3.5E-10 HCV 208B-646 1.8E+06 6.0E-04 3.4E-10 HCV 208B-672 2.2E+06 3.4E-04 1.6E-10 HCV 208B-739 2.8E+05 5.5E-05 2.0E-10 HCV 208B-750 2.0E+06 2.6E-04 1.3E-10 HCV 208B-796 1.5E+06 6.8E-04 4.6E-10 HCV 208B-862 1.4E+06 1.0E-04 7.0E-11 HCV 208B-954 1.6E+06 5.5E-04 3.4E-10 HCV 208B-960 1.6E+06 5.6E-04 3.5E-10 HCV 208B-982 2.2E+06 8.0E-04 3.5E-10

Example 10

[0243] BIACore Antibody Binding Pair Analysis with Nucleic Acid Binding Domain mAbs

[0244] The ability of the anti-HCV core peptide 134-171 monoclonal antibodies to form antibody binding pairs with anti-HCV Core C11-3, C11-7, C11-9, and C11-14 (U.S. Pat. No. 6,727,092; Morota, et al, J. Virol. Meth., 2009, 157:8-14) antibodies and recombinant HCV Corel-169 antigen was determined using a Biacore 4000 instrument (GE Healthcare Bio-Sciences AB). First, after pre-treating a CM5 Series S biosensor chip (GE Healthcare) with duplicate injections of 100 mM HCl, 50 mM NaOH, and 0.1% SDS, a rabbit anti-mouse IgG Capture Biosensor was created by amine-coupling rabbit anti-mouse IgG antibody (GE Healthcare, Piscataway, N.J.) on Spots 1, 2, 4, and 5 in all four flow cells of the biosensor chip via EDC/NHS/ethanolamine chemistry provided in an Amine Coupling Kit (GE Healthcare).

[0245] Clarified anti-HCV Core (peptide aa 134-171) antibody exhausted hybridoma supernatants, recombinant HCV Corel-169 antigen, 3 different purified mouse monoclonal IgG representing isotypes IgG1, IgG2a, and IgG2b non-reactive to HCV Core used as blocking reagents, and anti-HCV Core C11-3, C11-7, C11-9, and C11-14 mAbs were diluted into a filtered running buffer (hereinafter "running buffer") composed of 10.times.PBS buffer (GE Healthcare) diluted 5-fold into distilled H.sub.2O, supplemented with 3 mM EDTA, 0.1% BSA, 0.1% CM-Dextran, 0.1% n-Dodecyl-N,N-Dimethylamine-N-Oxide, an extra 500 mM NaCl, and 0.2 .mu.m filtered. Each HCV Core antibody supernatant was diluted 1:1, the recombinant HCV Corel-169 antigen was diluted to 500 nM per the calculated dimer molecular weight (39,453 Da), the anti-HCV Core C11-3, C11-7, C11-9, and C11-14 purified monoclonal antibodies were individually diluted to 20 .mu.g/mL, and the 3 mouse IgG blocking reagents were all diluted as a pool with each isotype having a concentration of at least 100 .mu.g/mL in running buffer. All dilutions were 0.2 .mu.m filtered prior to use.

[0246] The HCV Core antibody-antigen-antibody sandwich procedure was as follows. 20 .mu.L of the HCV Core C11 antibodies were injected over Spots 1 and 5 in all four flow cells at 10 .mu.L/minute: C11-3 in flow cell 1, C11-7 in flow cell 2, C11-9 in flow cell 3, and c11-14 in flow cell 4. The flow rate was increased to 30 .mu.L/minute and the remaining available anti-mouse IgG binding sites on the biosensor were blocked with the mouse IgG1, IgG2a, and IgG2b isotype pool by injecting 60 .mu.L over Spots 1 and 2 and then 60 .mu.L over Spots 4 and 5 in all flow cells. 60 .mu.L of HCV Corel-169 antigen was injected over Spot 1 and then another 60 .mu.L over Spot 5 in all flow cells. 60 .mu.L of one HCV Core antibody diluted supernatant was inject over Spots 1 and 2 over all flow cells and another diluted supernatant over Spots 4 and 5 in all flow cells. The flow rate was decreased 10 .mu.L/minute and all biosensor surfaces were regenerated with one 30 .mu.L injection of 10 mM glycine, pH 1.7 (GE Healthcare).

[0247] Using the Biacore 4000 Evaluation Epitope Mapping software module (GE Healthcare Bio-Sciences AB), the binding response for each C11 antibody, antigen, and HCV Core antibody supernatant was determined after each injection and used to calculate an expected response reference value using the dimeric antigen and antibody (150,000 Da) molecular weights. An expected percent binding value was determined using individual samples versus the reference value. Any expected percent binding values that were greater than 5.0 were considered positive for the ability to form an antibody sandwich with the recombinant HCV Corel-169 antigen. Expected percent values are listed in Table 5.

TABLE-US-00012 TABLE 5 Secondary Conjugate Antibody Primary Capture Antibody Anti-HCV Core 134-171 Anti-HCV Core antibody antibodies C11-3 C11-7 C11-9 C11-14 HCV 208A-1006 2.4 3.1 <1.0 2.5 HCV 208A-1007 4.4 6.1 2.7 4.2 HCV 208A-1015 <1.0 <1.0 <1.0 <1.0 HCV 208A-1022 <1.0 1.3 <1.0 <1.0 HCV 208A-1032 19.5 26.7 15.7 21.8 HCV 208A-1064 16.3 19.6 10.5 16.3 HCV 208A-1081 19.0 25.9 14.9 20.8 HCV 208A-110 15.7 21.1 9.4 14.8 HCV 208A-122 17.3 19.7 9.6 14.8 HCV 208A-126 1.2 2.0 <1.0 1.3 HCV 208A-133 9.8 13.9 5.8 9.3 HCV 208A-134 <1.0 <1.0 <1.0 <1.0 HCV 208A-147 <1.0 <1.0 <1.0 <1.0 HCV 208A-152 10.0 12.5 6.9 9.8 HCV 208A-158 14.0 16.5 7.8 12.4 HCV 208A-159 4.6 7.0 3.3 5.5 HCV 208A-160 15.5 20.9 8.7 13.8 HCV 208A-194 <1.0 <1.0 <1.0 <1.0 HCV 208A-207 <1.0 <1.0 <1.0 <1.0 HCV 208A-208 2.6 3.2 <1.0 2.5 HCV 208A-210 1.2 1.6 <1.0 <1.0 HCV 208A-222 15.6 18.8 10.7 15.0 HCV 208A-227 <1.0 1.1 <1.0 <1.0 HCV 208A-230 <1.0 <1.0 <1.0 <1.0 HCV 208A-264 <1.0 1.1 <1.0 <1.0 HCV 208A-286 18.9 22.1 13.0 18.5 HCV 208A-293 17.9 20.9 11.0 16.4 HCV 208A-312 16.3 19.7 11.8 16.4 HCV 208A-334 17.2 20.6 10.3 15.9 HCV 208A-352 <1.0 <1.0 <1.0 <1.0 HCV 208A-367 9.7 14.5 6.1 9.3 HCV 208A-381 17.2 20.3 12.3 17.4 HCV 208A-382 <1.0 1.5 <1.0 <1.0 HCV 208A-393 <1.0 <1.0 <1.0 <1.0 HCV 208A-422 <1.0 <1.0 <1.0 <1.0 HCV 208A-427 15.5 22.1 11.7 16.5 HCV 208A-442 <1.0 1.6 <1.0 <1.0 HCV 208A-460 17.5 24.3 12.9 18.3 HCV 208A-470 <1.0 <1.0 <1.0 <1.0 HCV 208A-489 15.9 21.9 11.5 16.6 HCV 208A-493 12.6 17.7 7.8 12.2 HCV 208A-557 17.7 21.4 13.3 17.6 HCV 208A-558 <1.0 1.1 <1.0 <1.0 HCV 208A-562 13.8 17.1 9.5 14.8 HCV 208A-575 <1.0 <1.0 <1.0 <1.0 HCV 208A-576 12.6 16.9 9.1 13.3 HCV 208A-584 <1.0 1.6 <1.0 <1.0 HCV 208A-603 6.7 9.1 4.0 6.8 HCV 208A-604 15.4 20.8 9.8 14.8 HCV 208A-605 15.9 19.3 11.9 15.8 HCV 208A-638 14.1 16.9 9.2 13.3 HCV 208A-641 <1.0 <1.0 <1.0 <1.0 HCV 208A-658 1.8 2.5 <1.0 <1.0 HCV 208A-692 <1.0 <1.0 <1.0 <1.0 HCV 208A-695 14.2 20.4 9.5 15.0 HCV 208A-719 7.5 10.0 4.9 8.0 HCV 208A-736 <1.0 <1.0 <1.0 <1.0 HCV 208A-741 19.4 22.7 14.2 21.2 HCV 208A-744 16.7 22.5 10.5 16.1 HCV 208A-759 <1.0 <1.0 <1.0 <1.0 HCV 208A-768 12.7 17.1 8.8 12.6 HCV 208A-774 18.5 24.5 13.4 19.3 HCV 208A-793 3.8 6.2 2.5 3.9 HCV 208A-807 <1.0 <1.0 <1.0 <1.0 HCV 208A-826 <1.0 1.3 <1.0 <1.0 HCV 208A-828 14.8 20.7 10.9 16.5 HCV 208A-830 11.9 14.7 7.9 11.7 HCV 208A-850 7.3 9.7 4.4 7.5 HCV 208A-863 18.0 24.9 12.1 18.0 HCV 208A-874 <1.0 <1.0 <1.0 <1.0 HCV 208A-877 9.4 12.1 5.9 10.7 HCV 208A-879 <1.0 <1.0 <1.0 <1.0 HCV 208A-920 19.3 22.9 13.2 19.1 HCV 208A-926 19.3 26.3 14.7 21.1 HCV 208A-938 14.1 21.0 10.4 15.5 HCV 208A-939 <1.0 <1.0 <1.0 <1.0 HCV 208A-967 <1.0 <1.0 <1.0 <1.0 HCV 208A-982 19.2 26.3 15.0 20.6 HCV 208A-983 <1.0 <1.0 <1.0 <1.0 HCV 208B-1024 10.9 14.3 7.1 12.2 HCV 208B-1029 <1.0 <1.0 <1.0 <1.0 HCV 208B-1043 17.4 20.7 12.0 17.9 HCV 208B-1070 22.0 28.6 17.7 25.3 HCV 208B-1072 25.0 30.2 19.3 27.4 HCV 208B-109 12.5 16.0 8.6 13.3 HCV 208B-1094 24.4 28.4 19.8 27.8 HCV 208B-1096 15.1 16.9 11.4 16.3 HCV 208B-131 11.4 12.4 6.8 11.6 HCV 208B-141 <1.0 <1.0 <1.0 <1.0 HCV 208B-174 1.6 1.5 <1.0 1.6 HCV 208B-178 <1.0 1.7 <1.0 <1.0 HCV 208B-181 7.4 10.0 5.4 8.1 HCV 208B-183 <1.0 <1.0 <1.0 <1.0 HCV 208B-189 2.3 2.9 <1.0 2.7 HCV 208B-207 <1.0 <1.0 <1.0 <1.0 HCV 208B-214 <1.0 <1.0 <1.0 <1.0 HCV 208B-230 <1.0 <1.0 <1.0 <1.0 HCV 208B-251 16.6 20.1 12.3 18.2 HCV 208B-281 <1.0 <1.0 <1.0 <1.0 HCV 208B-309 13.6 17.5 10.2 15.5 HCV 208B-319 6.0 8.5 4.2 6.3 HCV 208B-327 <1.0 <1.0 <1.0 <1.0 HCV 208B-348 <1.0 1.5 <1.0 <1.0 HCV 208B-353 1.9 2.6 <1.0 2.4 HCV 208B-395 7.6 9.4 5.2 7.8 HCV 208B-408 <1.0 <1.0 <1.0 <1.0 HCV 208B-409 1.8 2.7 <1.0 2.0 HCV 208B-446 10.0 12.2 7.8 10.3 HCV 208B-457 <1.0 <1.0 <1.0 <1.0 HCV 208B-471 <1.0 <1.0 <1.0 <1.0 HCV 208B-488 <1.0 <1.0 <1.0 <1.0 HCV 208B-515 <1.0 <1.0 <1.0 <1.0 HCV 208B-517 <1.0 <1.0 <1.0 <1.0 HCV 208B-547 7.2 8.3 4.9 8.2 HCV 208B-556 11.5 15.3 8.3 12.7 HCV 208B-560 10.6 15.5 8.3 12.0 HCV 208B-589 9.4 11.7 6.7 9.9 HCV 208B-591 19.1 23.0 15.1 21.7 HCV 208B-602 1.3 1.8 <1.0 <1.0 HCV 208B-608 <1.0 <1.0 <1.0 <1.0 HCV 208B-612 3.8 4.4 <1.0 3.1 HCV 208B-616 <1.0 <1.0 <1.0 <1.0 HCV 208B-617 <1.0 <1.0 <1.0 <1.0 HCV 208B-646 22.0 30.2 17.8 25.6 HCV 208B-652 <1.0 1.3 <1.0 <1.0 HCV 208B-672 2.6 2.8 <1.0 2.7 HCV 208B-739 <1.0 <1.0 <1.0 <1.0 HCV 208B-742 <1.0 <1.0 <1.0 <1.0 HCV 208B-750 22.8 27.3 17.2 24.1 HCV 208B-762 <1.0 <1.0 <1.0 <1.0 HCV 208B-765 <1.0 <1.0 <1.0 <1.0 HCV 208B-778 <1.0 <1.0 <1.0 <1.0 HCV 208B-780 <1.0 <1.0 <1.0 <1.0 HCV 208B-788 <1.0 <1.0 <1.0 <1.0 HCV 208B-793 <1.0 <1.0 <1.0 <1.0 HCV 208B-796 20.2 27.7 17.3 25.2 HCV 208B-822 <1.0 <1.0 <1.0 <1.0 HCV 208B-826 <1.0 <1.0 <1.0 <1.0 HCV 208B-853 <1.0 <1.0 <1.0 <1.0 HCV 208B-860 <1.0 <1.0 <1.0 <1.0 HCV 208B-862 11.2 15.2 8.9 12.6 HCV 208B-894 <1.0 <1.0 <1.0 <1.0 HCV 208B-909 1.6 2.5 <1.0 <1.0 HCV 208B-911 <1.0 <1.0 <1.0 <1.0 HCV 208B-922 <1.0 <1.0 <1.0 <1.0 HCV 208B-952 <1.0 <1.0 <1.0 <1.0 HCV 208B-954 23.9 32.7 18.4 26.0 HCV 208B-956 <1.0 <1.0 <1.0 <1.0 HCV 208B-960 21.0 28.1 15.8 22.9 HCV 208B-982 4.5 5.4 <1.0 4.9

Example 11

[0248] Immunoglobulin Purification and Labeling.

[0249] Anti-HCV core hybridomas were expanded in Hybridoma Serum Free Medium (Invitrogen Corporation) supplemented with L-glutamine and 10% Ultra Low IgG FBS (Invitrogen Corporation) and seeded into roller bottles at approximately 0.5.times.10E.sup.5 cells/mL. The cultures were incubated at 37.degree. C. while rotating at approximately 1 revolution per minute for 10-14 days, or until a terminal end culture was obtained. The terminal roller bottle supernatant was harvested and clarified with a 0.45 micron filter. The clarified supernatant was diluted with an equal volume of 1.5 M glycine, 3M NaCl buffer, pH 8.9, then loaded onto a pre-equilibrated 5 ml Protein A column using the AKTA automated purification system (Amersham/Pharmacia/GE). The column was then washed with approximately 5 column volumes of binding buffer and when a stable baseline is achieved, the mAb was eluted with 0.1 M sodium citrate buffer, pH 2.8. The IgG was then transferred to a desalting column and exchanges into PBS, pH 7.2-7.4, and then further dialyzed in PBS pH 7.2-7.4, using 10,000 molecular weight cut-off dialysis membrane (Pierce Chemical). Selected antibodies were biotinylated by using Sulfo-NHS-LC-Biotin (Pierce) at a 20-fold molar excess and incubated for 30 minutes at room temperature. Unbound biotin was removed through dialysis in PBS pH 7.2-7.4. All biotinylated monoclonals were tested by EIA to confirm successful labeling.

Example 12

[0250] HCV Core Antigen Capture Assays.

[0251] Purified anti-HCV core134-171 monoclonal antibodies were evaluated for their ability to form binding pairs with themselves and two other domain 1 monoclonal antibodies using HCV core1-169 recombinant antigen in an EIA format. Anti-HCV Domain 1 monoclonal antibodies, C11-7 and C11-9, and anti-HCV core134-171 monoclonals were coated onto microtiter plates at approximately 1000 ng/ml and allowed to incubate overnight at 2-8 degrees C. After the capture reagent had been coated on the solid phase, the plates were blocked using a 5.times.PBS buffer containing 2% fish gelatin, 0.5 Tween 20, and 0.1% n-dodecyl-N,N-dimethylamine-N-oxide. The wells were washed with distilled water and purified core 1-169 antigen was added to the blocked plates in serial dilutions from 50 to 0.78 ng/ml diluted in fish gelatin block, and then allowed to incubate at room temperature for approximately 30 minutes. The wells were washed with distilled water and biotin labeled anti-HCV core monoclonals were added to the plates at concentrations ranging from 100 to 5000 ng/ml, and then incubated for 30 minutes at room temperature. The plates were washed with distilled water and streptavidin-HRPO diluted to approximately 200 ng/mL was added to the plates and allowed to incubate for 30 minutes at room temperature. The plates were washed with distilled water and o-phenylenediamine substrate was used as the chromagen to generate signal and the optical density at 492 nm was measured.

[0252] Table 6 summarizes the assay signal (OD492 nm) for each antibody pair combination using 25 ng/ml of core1-169 antigen, which indicates whether or not each binding pair is capable of forming a sandwich. An OD492 value of at least 3.times. greater than the value generated by a negative control (NC) monoclonal antibody as a capture or conjugate reagent are considered positive for core antigen detection.

TABLE-US-00013 TABLE 6 208A- 208A- 208A- 208A- 208A- 208B- 110 692 293 557 207-271 741 (aa (aa (aa (aa (aa (aa 134-154 141-161) 134-154) 141-161) 141-161) 134-154) & 141-161) 208A-110-Bt 0.18 0.14 0.20 0.17 0.17 0.20 208A-692-Bt 0.54 0.35 0.51 0.53 0.39 0.44 208A-293-Bt 0.52 0.34 0.49 0.52 0.39 0.50 208A-557-Bt 0.44 0.13 0.41 0.32 0.18 0.38 208A-207-271-Bt 0.38 0.42 0.42 0.82 0.36 0.30 208B-741-Bt 0.42 0.32 0.42 0.40 0.33 0.36 208B-1096-Bt 0.19 0.15 0.24 0.17 0.18 0.23 208B-395-334-Bt 0.21 0.15 0.26 0.21 0.20 0.21 208B-612-226-Bt 0.26 0.18 0.28 0.23 0.24 0.23 NC mAb-Bt 0.16 0.15 0.23 0.18 0.17 0.18 C11-7-Bt 1.78 0.18 1.55 0.64 0.22 1.76 C11-9-Bt 2.59 0.58 1.84 1.18 0.44 2.32 208B- 208B- 208B- 1096 395-334 612-226 NC C11-7 C11-9 (aa (aa (aa 134-154 capture (aa 115- (aa 29- 151-171) 151-171) & 141-161) mAb 121) 37) 208A-110-Bt 0.21 0.22 0.20 0.15 0.18 0.13 208A-692-Bt 0.38 0.44 0.37 0.40 2.92 2.66 208A-293-Bt 0.41 0.44 0.37 0.37 2.87 2.60 208A-557-Bt 0.35 0.28 0.25 0.18 0.22 0.14 208A-207-271-Bt 0.33 0.37 0.33 0.37 2.47 2.20 208B-741-Bt 0.52 0.34 0.29 0.29 2.36 2.13 208B-1096-Bt 0.26 0.20 0.24 0.17 1.06 0.33 208B-395-334-Bt 0.23 0.23 0.26 0.20 0.89 0.32 208B-612-226-Bt 0.70 0.83 0.23 0.21 1.52 0.64 NC mAb-Bt 0.21 0.22 0.22 0.18 0.17 0.14 C11-7-Bt 0.45 0.46 0.53 0.22 0.23 1.04 C11-9-Bt 0.82 0.95 0.63 0.62 1.91 2.75

Example 13

[0253] Sequences of Anti-Core 134-171 Variable Domains.

[0254] A subset of the anti-HCV core 134-171 hybridomas were selected for determination of variable heavy (VH) and variable light (VL) chain nucleotide and deduced amino acid sequences. Total RNA was extracted from the hybridoma cells using Trizol (Invitrogen) or Tri-Reagent (Sigma) according to the manufacturer's recommendations. The heavy chain and light chain cDNA was generated from the extracted total RNA using Superscript III (Life Technologies) and oligo dT primers following standard protocols. The 5' RACE (rapid amplification of cDNA ends) protocol was used to amplify the variable heavy and light chain cDNA sequences using a dC anchor primer (5'-AAGCAGTGGTATCAACGCAGAGTACCCCCCCCCCCCCCCCC-3'; SEQ ID NO:581) and a generic primer specific to the constant region of the mouse heavy or light chain (Novogen). Amplicons were cloned into a commercially available vector (pCR2.1-TOPO cloning kit, Invitrogen) per the manufacturer's directions and transformed into TOP 10 E. coli. At least eight colonies were selected for PCR amplification of cloned variable domain sequences using M13 forward and reverse primers. Amplicons were treated with ExoSap (Affymetrix) prior to sequencing using M13 forward primer and BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, Calif.). Sequences were obtained using ABI3130.times.1 automated sequencer and assembled and analyzed using Vector NTI software (Invitrogen).

[0255] Deduced amino acid sequences were aligned using ClustalW (Higgins et al., Nucleic Acids Res. 22:4673-4680, 1994) as implemented in the MEGAS software package (Tamura et al., Molecular Biology and Evolution 28: 2731-2739, 2011). MEGAS software was used to determine groupings or clusters of related heavy chain amino acid sequences from the alignments and phylogenetic tree construction using the Neighbor Joining method with complete deletion of sequence gaps in the alignment. Tree topology, and hence clusters or groups therein, was examined for reliability by using a bootstrap test from 1000 replicates. As a general rule, if the bootstrap value for a given interior branch is 95% or higher, then the topology at that branch is considered "correct" (Nei and Kumar, Molecular Evolution and Phylogenetics, 2000; Oxford University Press, New York). Analysis of heavy chain variable domain sequences from 52 anti-HCV core 134-171 monoclonals revealed the existence of 4 main groups with bootstrap values >95%. Antibodies comprising three of these groups exhibited specificity for binding to one of each of the peptides used for screening, i.e. Group B with peptide 1 (134-154), Group A with peptide 2 (141-161), and Group C with peptide 3 (151-171). FIG. 2 provides two representations of the tree topology.

Example 14

Generating the Neo4 Antibody

[0256] Six peptides from the HCV core region (genotype 1b) were synthesized. The HCV core amino acid (aa) positions covered by each peptide are as follows: peptide 1, 98-110; peptide 2, 104-113; peptide 3, 104-121; peptide 4, 110-124; peptide 5, 101-112, and peptide 6, 153-165. Balb/C mice were each immunized with a combination of 2 different peptides; peptides 1 with 6; peptides 2 with 3; peptides 4 with 5; and peptides 1 with 5. All of the peptides were conjugated to keyhole limpet hemocyanin (KLH) at their C-terminus through an added cysteine residue. Mice were boosted four times. Spleens were collected for 2 independent fusions using the PEG 1500 chemical method. The Myeloma fusion partner used was SP2/0. Subcloning began after fusion screening. 150 cells were plated onto a 96 well plate which usually nets about 1 cell per well. Three rounds of subcloning were performed. Media used for the growth of cells from stable clones was Iscove's Modified Dulbecco's Medium (IMDM) with 10% FBS. Supernatants were analyzed by ELISA using the above core peptides. Hybridoma supernatants and ascites generated from ELISA-positive clones were then screened by western blot for their ability to detect minicores. One hybridoma clone (Neo4) was identified that gave an exceptionally strong signal at detecting minicore and p21 core proteins. The isotype of this antibody was determined to be IgG1. Antibodies were subsequently purified with Protein G sepharose and used for the detection of minicores.

[0257] Study Population.

[0258] Hepatologists and other providers at the Icahn School of Medicine at Mount Sinai recruited patients with a HCV viral load of 10 million IU/mL or above. Written informed consent was obtained and up to 16 mL of blood were collected, and serum was prepared. Data on age, sex, HCV genotype, viral load, and other medical conditions were extracted from medical records. The study was conducted in compliance with the Icahn School

[0259] Blood was obtained from four patients with high-titer HCV and two non-infected volunteers (Table 7). All four HCV-infected patients were male and had a HCV viral load over 10 million IU/mL. Patients with a high HCV viral load were selected for this study because they were expected to have relatively high blood levels of HCV proteins, enabling the development of methods for detection.

TABLE-US-00014 TABLE 7 Clinical characteristics of patients HCV Viral Patient Load HCV Age ID (IU/mL) Genotype (years) Sex Other P1 7.46 .times. 10.sup.7 1b 49 M HIV co-infected P2 2.91 .times. 10.sup.7 1b 63 M 5 years post kidney transplant P3 1.23 .times. 10.sup.7 1a 67 M -- P4 >1 .times. 10.sup.8 * 1a 60 M 1 year post liver transplant and B cell lymphoma N1 -- -- 55 M Healthy control N2 -- -- 65 F Healthy control * Value was above the upper limit of detection of the assay

[0260] Isolation and Characterization of Minicores and p21 Core in Blood.

[0261] The general procedure for isolating minicore and mature p21 core protein from patient blood is shown in FIG. 4. An equal volume of 50 mM Tris (pH 7.3) and 150 mM NaCl was added to 3.5 mL of serum and passed through a 0.45 .mu.m syringe filter to remove aggregates. ApoB-associated lipoproteins were then precipitated by mixing an equal volume of heparin/Mn.sup.+2 solution containing 60 mM Tris (pH 7.3), 110 mM MnCl.sub.2.4H.sub.2O, 154 mM NaCl, and 400 USP/mL heparin. Solution was incubated for 1 h on ice in the dark. The precipitate was recovered by centrifugation at 3,000.times.g (30 min; 4.degree. C.) and washed with gentle resuspension three times with 3.5 mL of an ice-cold solution containing 50 mM Tris (pH 7.3), 55 mM MnCl.sub.2.4H.sub.2O, 154 mM NaCl, and 200 USP units/mL heparin. Heparin/Mn.sup.+2 was then removed: Manganese was removed from the heparin/Mn.sup.2 pellet by resuspension in 2.0 or 2.5 mL of 10% NaHCO.sub.3, which precipitates an insoluble Mn(HCO.sub.3).sub.2 that is removed by centrifugation at 1,500.times.g (15 min; 4.degree. C.). Heparin was removed by dialyzing 3 times (in 24 h) with one liter of 5% BaCl.sub.2 in 20 mM Tris (pH 7) in a Slide-A-Lyzer Cassette G2 (Thermo Scientific) with a 20,000 MWCO. The supernatant was collected from the dialysis unit and the heparin-BaCl.sub.2 insoluble complex was removed by centrifugation at 1,500.times.g (15 min; 4.degree. C.). Excess BaCl2 was removed from the supernatant by dialyzing 3 times (in 24 h; 4.degree. C.) with one liter of THE buffer consisting of 20 mM Tris (pH 7.0), 0.15 M NaCl and 1 mM EDTA. The supernatant was collected from the dialysis unit and stored at -80.degree. C. After thawing, the supernatant was concentrated using a 15 mL Amicon Ultra centrifugal filtration unit with a 100 kDa MWCO (Millipore) (3,200.times.g; 90 min; 4.degree. C.). Concentrated volumes ranged between 130 .mu.L and 210 .mu.L. Finally, samples were delipidated to allow for loading onto a Western blot gel. Samples were delipidated using an extraction method previously described for whole serum and plasma which partitions proteins into an aqueous phase and lipids into an organic phase using a mixture of butanol and diisopropyl ether (butanol:DIPE at 40:60; v/v) (see, Cham et al., J Lipid Res 1976; 17:176-81, herein incorporated by reference). The original protocol was modified by using only a 1.times. volume of the 40:60 butanol:DIPE mixture to our sample rather than a 2.times. volume. It was found that the 2.times. volume can greatly dehydrate the aqueous phase. Also, instead of extracting by end-over-end tube rotation of the samples for 30 minutes, the samples were just briefly vortexed. The lipid-containing organic and protein-containing aqueous phases were separated by centrifugation at 400.times.g (2 min; 25.degree. C.). The lower aqueous phase was immediately collected, placed on ice for 1 h, and then stored at -80.degree. C. Incubation of the protein-containing aqueous phase on ice for 1 h followed by freeze-thawing led to the formation of a precipitate that was pelleted by gentle centrifugation at 1,000.times.g (30 sec; 4.degree. C.). The pellet was solubilized in a solution containing 8 M Urea and 1% SDS, sonicated, adjusted with NuPage sample buffer, heated and loaded onto NuPAGE gels. This pellet contained core and minicore proteins. These proteins were not detected in the supernatant. After electrophoresis, gels were processed for western analysis. Protease inhibitors, Complete-EDTA free (Roche) and 1 mM phenylmethylsulfonyl fluoride (PMSF) were used throughout the purification procedure.

[0262] HCV-Infected Cell Culture Lysate.

[0263] Huh-7.5 cells infected with a Con1/JFH chimeric HCV were were lysed directly in culture dishes after three washes with Dulbecco's phosphate buffered saline with 2.times.LDS NuPage sample buffer (Life Technologies) containing 4% lithium dodecyl sulfate and 10% 2-mercaptoethanol.

[0264] Western Blot Analysis.

[0265] Samples were electrophoresed in 10% NuPAGE Bis-Tris gels (Life Technologies) and the proteins were transferred to 0.2 uM pore size, polyvinylidene difluoride (PVDF) membranes. Antibodies targeting the C-terminal portion of p21 core and used to detect minicores were a combination of Neo4 monoclonal antibody at a concentration of 2 ug/ml mixed with mAb1 (Cll-3) at 1 ug/ml, that we previously described (see, Eng et al., J Virol 2009; 83:3104-14, herein incorporated by reference). Antibody targeting the N-terminal portion of core is C11-10 (Abbott Labs, epitope 32-36).

[0266] Quantification of HCV RNA.

[0267] QIAamp Viral RNA mini kit (Qiagen) was used to purify RNA from serum, Hep/Mn.sup.+2 pellet and supernatant. For the heparin/Mn.sup.+2 pellet and supernatatant, heparin was removed from the purified RNAs by treating with heparinase I (Sigma) (Johnson et al., Biotechniques 2003; 35:1140-2, 1144, herein incorporated by reference). Reverse transcription (RT) of RNA was performed using SuperScript III First-Strand Synthesis (Invitrogen) and random hexamers. RT-reaction products were then used for quantitative-PCR (q-PCR) using the LightCycler 480 SYBR Green I Master kit and the LightCycler 480 instrument (Roche).

[0268] Minicores were present in the blood of all four patients (FIG. 6). The relative amounts of p21 core, 70 minicore and 91 minicore varied from patient to patient. Patient samples P1, P2, and P4 had prominent p21 core bands. Interestingly, P1, P2, and P4 were immune compromised as a result of HIV infection or immunosuppressive drugs, while P3 had no known immunological deficiency (Table 7). P1, P2, and P3 had prominent 70 minicore bands, and P4 had a prominent 91 minicore band. A cell culture lysate of HCV-infected Huh-7.5 cells provided molecular weight markers of the core protein isoforms (FIGS. 6 and 7, lanes C). To confirm the identity of the core isoform bands, duplicate Western blots of sample P4 were probed with antibodies specific for either the N- or the C-terminal portion of the core protein (FIG. 7), allowing detection of p21 core only (N-term) or p21 plus the 70 and 91 minicores (C-term).

[0269] This study provides direct evidence that HCV-expressed non-classical proteins (minicores) are present in blood during natural infections in addition to the conventional proteins. Minicores were detected in all four high HCV viral load patients. The presence of relatively large quantities of minicores in blood suggests that they enhance viral transmission and/or pathogenesis/persistence.

Example 15

Neo4 Antibody Sequencing

[0270] This example describes how the nucleotide and deduced amino acid sequences of the light and heavy chain variable domains of the Neo4 monoclonal antibody were determined. Total cellular RNA was extracted from the anti-HCV core Neo4 hybridoma cells using Trizol (Tri-ZOL, Invitrogen) per manufacturer's recommendations. To sequence the variable domains, cDNA was generated from the extracted RNA using Superscript III (Life Technologies) and an oligo-dT primer (Novagen). Amplification of the respective immunoglobulin variable sequences was performed by 5'RACE PCR using a dC-anchor primer and a murine consensus heavy or light chain primer. Purified products were cloned into pCR.TM.2.1-TOPO.RTM. (Life Technologies) which was then used to transform E. coli. For sequence analysis, variable heavy and variable light chain fragments from multiple E. coli colonies were PCR amplified using M13 forward and reverse primers. Sequencing was performed using the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) on the ABI 3130.times.1 Genetic Analyzer. At least 5 heavy or light chain clones were viewed, analyzed, and annotated using Vector NTI Advance (Life Technologies) to create the consensus nucleotide sequences. FIG. 8 shows the amino acid sequence of the light chain variable region (FIG. 8A, SEQ ID NO:583) and the heavy chain variable region (FIG. 8B, SEQ ID NO:584) of Neo4. The CDRs were identified by visual inspection of the deduced amino acid sequences relative to the locations of conserved features within the respective domains (see, Kabat et al., (1987) Sequences of Proteins of Immunological Interest, 4th ed., U. S. Govt. Printing Office No. 165-492, Bethesda, Md.). FIG. 8A shows the light chain CDRs underlined, which include CDRL1 (RASKSVNEYGYTYMH; SEQ ID NO:585), CDRL2 (LASNLDS; SEQ ID NO:586), and CDRL3 (QHSRELPYT, SEQ ID NO:587). FIG. 8B shows the heavy chain CDRs underlined, which include CDRH1 (GFSITSSVYCWQ; SEQ ID NO:588), CDRH2 (RICYDGSVDYSPSITS; SEQ ID NO:589), and CDRH3 (ENHIDYYDTTYPSFDV; SEQ ID NO:590). FIG. 9A shows the nucleic acid sequence encoding the light chain variable region of Neo4 (SEQ ID NO:591), and FIG. 9B shows the nucleic acid sequence encoding the heavy chain variable region of Neo4 (SEQ ID NO:592).

Example 16

Neo4 and CLL-3 (ABT-4) Antibody Epitope Mapping

[0271] Purified Neo4 and C11-3 (ABT-4) monoclonal IgG samples were analyzed for reactivity to overlapping HCV core antigen peptides by EIA. Biotin-(SGSG)-15 mer HCV core-amide peptides were synthesized that overlapped by two amino acids and encompassed the entire length of HCV core protein amino acids 1-191 (Mimotopes). Each of the 89 synthesized peptides was coated in an individual micro-titer well as indicated on the peptide plate key (Table 8) as shown. The coated plates were kept dry in a sealed foil pouch and stored at <10 degrees C. until ready for use. A peptide coated plate for each test antibody was blocked using 3% BSA in PBS with 0.5% Tween 20. The blocked plates were washed with distilled water and a 2 ug/mL solution of purified IgG from each test sample diluted in BSA block solution was added to each peptide coated well of the blocked plate and allowed to incubate at room temperature for at least one hour. Following incubation the plates were washed with distilled water. A 200 ng/mL solution of peroxidase labeled F(ab')2 fragment goat anti-mouse IgG Fc fragment specific (Jackson Immunoresearch) in BSA block solution was added to all wells and incubated at room temperature for 30 minutes. The plates were washed with distilled water and o-phenylenediamine substrate was used as the chromagen to generate signal. Plates were read at 492 nm and the results were analyzed. Wells were considered positive if they had an EIA signal at least 3 times greater than background. The reactivity of ABT-4 to the peptides is shown with underlining in Table 9, while the reactivity of the Neo4 antibody to the peptides is shown with underlining in Table 10. As shown in these tables, the ABT-4 mAb reacts with peptide numbers 48-53 while the Neo4 reacts with peptide numbers 50-55.

TABLE-US-00015 TABLE 8 Peptide Plate Key 1 2 3 4 5 6 7 8 9 10 11 12 A 1 9 17 25 33 41 49 57 65 73 81 89 B 2 10 18 26 34 42 50 58 66 74 82 Blank C 3 11 19 27 35 43 51 59 67 75 83 Blank D 4 12 20 28 36 44 52 60 68 76 84 Blank E 5 13 21 29 37 45 53 61 69 77 85 Blank F 6 14 22 30 38 46 54 62 70 78 86 Blank G 7 15 23 31 39 47 55 63 71 79 87 Blank H 8 16 24 32 40 48 56 64 72 80 88 Blank

TABLE-US-00016 TABLE 9 HCV ABT-4 1 2 3 4 5 6 7 8 9 10 11 12 A 0.05 0.06 0.07 0.06 0.07 0.07 0.97 0.07 0.07 0.07 0.07 0.07 B 0.06 0.06 0.06 0.06 0.06 0.07 0.97 0.07 0.07 0.07 0.07 0.07 C 0.06 0.07 0.06 0.06 0.06 0.06 1.00 0.07 0.07 0.07 0.07 0.07 D 0.06 0.07 0.06 0.06 0.06 0.07 0.96 0.07 0.06 0.06 0.06 0.06 E 0.05 0.06 0.06 0.06 0.06 0.07 0.36 0.06 0.06 0.06 0.06 0.06 F 0.07 0.06 0.06 0.06 0.06 0.07 0.08 0.06 0.06 0.06 0.06 0.06 G 0.06 0.06 0.06 0.06 0.06 0.07 0.06 0.06 0.06 0.06 0.07 0.06 H 0.06 0.06 0.07 0.07 0.08 0.81 0.07 0.07 0.08 0.07 0.09 0.07

TABLE-US-00017 TABLE 10 HCV Neo4 1 2 3 4 6 6 7 8 9 10 11 12 A 0.06 0.05 0.04 0.04 0.05 0.04 0.05 0.05 0.04 0.04 0.04 0.05 B 0.06 0.05 0.05 0.04 0.04 0.04 1.03 0.05 0.05 0.05 0.04 0.05 C 0.05 0.05 0.05 0.04 0.04 0.05 0.96 0.05 0.05 0.10 0.05 0.05 D 0.06 0.07 0.05 0.04 0.05 0.05 0.93 0.06 0.04 0.05 0.05 0.05 E 0.05 0.05 0.05 0.04 0.04 0.04 0.96 0.05 0.05 0.05 0.05 0.04 F 0.06 0.06 0.07 0.05 0.05 0.06 1.01 0.05 0.04 0.06 0.05 0.05 G 0.07 0.06 0.05 0.05 0.05 0.05 0.97 0.04 0.05 0.05 0.05 0.04 H 0.06 0.06 0.05 0.06 0.05 0.04 0.04 0.04 0.04 0.05 0.05 0.05

[0272] A summary of the peptide reactivity of ABT-4 (C11-3) and Neo4 is provided in Table 11 below.

TABLE-US-00018 TABLE 11 Peptide Reactivity Summary HCV Core monoclonal HCV antibody Core SEQ ABT-4 Neo4 Peptide HCV ID HCV OD OD # Core Peptide Sequence NO Core Region (A492nm) (A492nm) 48 SGSGGWLLSPRGSRPSWGP 593 aa 95-109 0.95 0.04 49 SGSGLLSPRGSRPSWGPTD 594 aa 97-111 1.03 0.05 50 SGSGSPRGSRPSWGPTDPR 595 aa 99-113 1.03 1.03 51 SGSGRGSRPSWGPTDPRRR 596 aa 101-115 1.01 0.96 52 SGSGSRPSWGPTDPRRRSR 597 aa 103-117 1.01 0.93 53 SGSGPSWGPTDPRRRSRNL 598 aa 105-119 0.36 0.96 54 SGSGWGPTDPRRRSRNLGK 599 aa 107-121 0.08 1.01 55 SGSGPTDPRRRSRNLGKVI 600 aa 109-123 0.08 0.97

[0273] Shown in Table 12 below, are the reactive HCV core peptides aligned, with the minimal overlap underlined. For Abt-4, the reactive sequence is shown to span from amino acid 95 to amino acid 117 of the core peptide sequence, with the minimum reactive sequence spanning from amino acid 103 to amino acid 109. For Neo4, the reactive sequence is shown to span from amino acid 99 to amino acid 123 of the core peptide sequence, with the minimum reactive sequence spanning from amino acid 109 to amino acid 113.

TABLE-US-00019 TABLE 12 Abt4 (C11-3) Neo4 GWLLSPRGSRPSWGP (SEQ ID NO: 601) SPRGSRPSWGPTDPR (SEQ ID NO: 606) LLSPRGSRPSWGPTD (SEQ ID NO: 602) RGSRPSWGPTDPRRR (SEQ ID NO: 607) SPRGSRPSWGPTDPR (SEQ ID NO: 603) SRPSWGPTDPRRRSR (SEQ ID NO: 608) RGSRPSWGPTDPRRR (SEQ ID NO: 604) PSWGPTDPRRRSRNL (SEQ ID NO: 609) SRPSWGPTDPRRRSR (SEQ ID: 605) WGPTDPRRRSRNLGK (SEQ ID NO: 610) PTDPRRRSRNLGKVI (SEQ ID: 611)

Example 17

HCV Core Peptide Reactivity of Anti-Core Monoclonal Antibodies

[0274] Assay wells were coated with sheep anti-mouse IgG Fc specific antibody and incubated overnight. The coating solution was then removed; the wells blocked using BSA/tween in PBS and then washed with dH2O. Serially diluted antibody (in block) test samples (Neo4 antibody and Abt-4 antibody) were then added, the plates incubated for at least 1 hour and then washed. Next, biotin labeled peptides (diluted to 500 ng/mL in block) were added, the plates incubated for 10 minutes while shaking at 700 rpm and then washed. Peroxidase conjugated streptavidin (diluted to 200 ng/mL in block) was then added to all assay wells, incubated for 20 minutes while shaking at 700 rpm and then washed. Finally, color was developed using OPD and signal quenched using 1N H2SO4. Signal was read at 492 nm. Data are tabulated and summarized in Table 13 below.

TABLE-US-00020 TABLE 13 normal mouse serum sera dil (1:X) bt-pep 500 1,000 2,000 4,000 8,000 16,000 32,000 64,000 128,000 256,000 512,000 1.1 MM WT1STHV 0.0625 0.0552 0.0511 0.053 0.0537 0.06 0.0635 0.0628 0.067 0.0675 0.0743 0.0585 Mut1T110N 0.2575 0.0516 0.0617 0.0501 0.057 0.0626 0.0667 0.0702 0.0651 0.0677 0.0759 0.0631 Mut6T110S 0.2025 0.0545 0.0531 0.0505 0.0523 0.0611 0.0591 0.0631 0.069 0.0674 0.065 0.0621 C113WT 0.0603 0.056 0.0534 0.0532 0.0589 0.0537 0.0644 0.0623 0.066 0.066 0.0708 0.0586 C113T109N 0.0619 0.051 0.0534 0.0536 0.0607 0.0714 0.0751 0.0674 0.068 0.0629 0.0931 0.0726 92-133 0.0636 0.0521 0.0603 0.0533 0.0756 0.1009 0.0822 0.0656 0.0668 0.0615 0.0715 0.0691 blank 0.0563 0.0488 0.0538 0.0486 0.0508 0.0549 0.0679 0.0596 0.0631 0.065 0.0772 0.0783 blank 0.0691 0.0503 0.048 0.0494 0.0543 0.0534 0.0694 0.0584 0.0622 0.0654 0.0637 0.0772 HCV C11-3 ng/mL Ab bt-pep 2000 1000 500 250 125 62.5 31.25 15.625 7.813 3.906 1.953 0.977 WT1STHV 2.1334 2.0583 2.1436 1.8662 2.038 2.098 1.8905 1.5079 1.1164 0.6026 0.3531 0.1881 Mut1T110N 0.0915 0.0721 0.0765 0.0857 0.1268 0.1549 0.1066 0.1103 0.0966 0.079 0.0854 0.0755 Mut6T110S 1.7329 2.1499 2.237 1.8305 2.0224 2.1105 1.5367 0.8075 0.3435 0.2271 0.0964 0.1005 C113WT 1.821 2.3479 2.3525 1.8679 2.3715 2.1987 1.758 1.0506 0.6131 0.2717 0.1721 0.1029 C113T109N 0.0707 0.0806 0.0882 0.0963 0.1475 0.2015 0.1599 0.1161 0.1192 0.0833 0.1143 0.1041 92-133 1.4473 1.8692 1.9517 1.6777 1.8343 1.9204 1.5476 1.0138 0.5623 0.1832 0.1342 0.1163 blank 0.0657 0.071 0.0995 0.0639 0.0736 0.0987 0.1178 0.1047 0.101 0.0831 0.0937 0.0959 blank 0.0743 0.0711 0.0655 0.0644 0.0791 0.1032 0.1452 0.0979 0.1179 0.0856 0.0763 0.142 HCV Neo4 ng/mL Ab bt-pep 2000 1000 500 250 125 62.5 31.25 15.625 7.813 3.906 1.953 0.977 WT1STHV 1.8978 2.0033 1.9126 1.9886 1.9279 1.8543 1.6701 1.4209 1.0414 0.6691 0.4092 0.2204 Mut1T110N 0.0882 0.0678 0.065 0.089 0.2042 0.1391 0.0938 0.0771 0.0729 0.0719 0.0728 0.0619 Mut6T110S 1.1667 1.165 1.1716 1.1549 1.2728 1.1072 0.4805 0.2502 0.1316 0.0847 0.0717 0.0644 C113WT 0.0766 0.2219 0.0561 0.0607 0.0599 0.0626 0.0622 0.0697 0.0731 0.0694 0.0765 0.0584 C113T109N 0.059 0.0591 0.0803 0.1105 0.0858 0.1106 0.0815 0.0736 0.0751 0.0717 0.0853 0.0784 92-133 1.3067 1.4138 1.4754 1.4019 1.3064 1.4726 1.1055 0.8058 0.45 0.2166 0.1293 0.0978 blank 0.0643 0.0581 0.0663 0.0538 0.0534 0.0641 0.0689 0.0674 0.0696 0.0728 0.093 0.0715 blank 0.0676 0.0515 0.051 0.0526 0.0512 0.0642 0.0723 0.0687 0.0708 0.0687 0.0715 0.0767

[0275] Sequences of HCV core peptides are shown below where # denotes position 110 of the HCV core protein sequence.

TABLE-US-00021 # WT1STHV GSRPSWGPTDPRHRSRNVGKVID (SEQ ID NO: 612) MUT1T110N ##STR00001## (SEQ ID NO: 613) MUT6T110S ##STR00002## (SEQ ID NO: 614) C113WT RGSRPSWGPTD (SEQ ID NO: 615) C113T110N ##STR00003## (SEQ ID NO: 616)

[0276] Differences in sequence relative to genotype 1 at position 110 are highlighted. Reactivity of C11-3 (Abt4) and Neo4 at the 15.6 ng/mL dilution are summarized below in Table 14.

TABLE-US-00022 TABLE 14 HCV HCV C11-3 Neo4 bt-pep 15.6 ng/mL 15.6 ng/mL WT1STHV 1.5079 1.4209 Mut1T110N 0.1103 0.0771 Mut6T110S 0.8075 0.2502 C113WT 1.0506 0.0697 C113T109N 0.1161 0.0736 92-133 1.0138 0.8058 blank 0.1047 0.0674 blank 0.0979 0.0687

[0277] Both Neo4 and C11-3 (Abt-4) are sensitive to the T110N mutation common among non-genotype 1 and 2 isolates. However, Neo4 appears to more susceptible to the T110S mutation compared to Abt4. Neo4 does not bind to peptides terminating at position 111 indicating that the Neo4 epitope involves HCV core sequences downstream of position 111. This is consistent with the epitope mapping using the core peptide library wherein the Neo4 and Abt4 epitopes overlap but the Neo4 epitope is shifted downstream (C-terminally) relative to Abt 4 by about 3-4 amino acids.

Example 18

Monoclonal Antibodies Neo4 and 750 Used in Combination Enhance the Detection of HCV Core and 91-Minicore Proteins

[0278] Core and 91-minicore proteins were expressed in 293T cells by transfecting plasmids which encode the respective proteins. The core and minicore plasmid constructs also contained the 5' two-thirds of the HCV E1 gene which normally follows the core gene in the HCV genome. Core protein sequences are cleaved away from E1 protein by cellular signal peptidase and signal peptide peptidase to yield the mature core or minicore proteins. The 91-minicore construct begins with a start codon at codon 91 of the core gene. HCV sequences in both constructs were codon-optimized for enhanced expression. Cell extracts were prepared 48 hours post-transfection using a 2.times. lithium dodecyl sulfate (LDS) gel loading buffer followed by sonication. Extracts were diluted with 1.times.LDS loading buffer and were run on 10% Bis-Tris NuPage gels. Proteins were transferred to 0.2 uM pore size polyvinylidene difluoride (PVDF) membranes for Western blot analysis. The HCV core epitopes targeted by Neo4 and 208A-750 monoclonal antibodies (aa98-110 and aa141-161, respectively as immunogens) are downstream of amino acid 91 and are thus capable of detecting both core and 91-minicore. The concentration of each antibody used in the Western blot analysis was at 2 .mu.g/ml.

[0279] As shown in FIG. 10, when antibodies Neo4 and 208B-750 are used in combination, the signal intensity for core and 91-minicore are enhanced as compared to when the antibodies are used individually.

TABLE-US-00023 APPENDIX A DESCRIPTION OF SEQUENCES mAb SEQ SEQ mAb Clone ID Nucleic Acid Seq ID Clone Amino Acid Seq 208A- 1 GAGGTCCAGCTGCAACAGTCTGGACCTGAGTT 2 208A-1064H EVQLQQSGPELVKPGASVKISCK 1064H GGTGAAGCCTGGGGCCTCAGTGAAGATATCTT TSGYTFTEYAMHWMKQSHGKSLE GCAAGACTTCTGGATACACTTTCACTGAATAC WIGGINPTNGDTIYNQHFKDKAK GCCATGCACTGGATGAAGCAGAGCCATGGAAA LTVDRSSSTAYMELRSLTSDDSA GAGCCTTGAGTGGATTGGAGGTATCAATCCTA LFYCARRELDYFASWGQGTTLTV CTAATGGTGATACAATCTACAACCAGAAGTTC SS AAGGACAAGGCCAAATTGACTGTAGACAGGTC CTCCAGCACAGCCTACATGGAGCTCCGCAGCC TGACATCTGACGATTCTGCATTATTTTATTGT GCAAGACGGGAACTGGACTACTTTGCCTCCTG GGGCCAAGGCACCACTCTCACAGTCTCCTCA 208B- 3 GAGGTCCAGCTGCAACAGTCTGGACCTGAGCT 4 208B-1094H EVQLQQSGPELVKPGASVKISCK 1094H GGTGAAGCCTGGGGCCTCAGTGAAGATATCCT TSGYTFTEYAMHWMKQSHGKSLE GCAAGACTTCTGGATACACTTTCACTGAATAC WIGGINPTNGDTIYNQRFKDKAK GCCATGCACTGGATGAAGCAGAGCCATGGAAA LTVDRSSSTAYMELRSLTSDDSA GAGCCTTGAGTGGATTGGCGGTATCAATCCTA LFYCARRELDYFASWGQGTTLTV CTAATGGTGATACAATCTACAACCAGAGGTTC SS AAGGACAAGGCCAAATTGACTGTAGACAGGTC CTCCAGCACAGCCTACATGGAGCTCCGCAGCC TGACATCTGACGATTCTGCATTATTTTATTGT GCAAGACGGGAACTGGACTACTTTGCCTCCTG GGGCCAAGGCACCACTCTCACAGTCTCCTCA 208A-293H 5 GAGGTCCAGCTGCAACAGTCTGGACCTGAACT 6 208A-293H EVQLQQSGPELVKPGASVKISCK GGTGAAGCCTGGGGCCTCAGTGAAGATATCCT ASGFTFTEYAMHWMKQSHGKSLE GTAAGGCTTCGGGATTCACTTTCACTGAATAC WIGGINPTNGDAIYNQHFKDKAK GCCATGCACTGGATGAAACAGAGCCATGGAAA LTVDRSSSTAYMELRSLTSDDSA GAGCCTTGAGTGGATTGGAGGTATCAATCCTA LFYCARRELDYFPSWGQGTTLTV CTAACGGTGATGCAATCTACAACCAGAAGTTC SS AAGGACAAGGCCAAGTTGACTGTAGACAGGTC CTCCAGCACAGCCTACATGGAGCTCCGCAGCC TGACATCTGACGATTCTGCATTATTTTATTGT GCAAGACGGGAACTGGACTACTTTCCCTCCTG GGGCCAAGGCACCACTCTCACAGTCTCCTCA 208A-222H 7 GAGGTCCAGCTGCAACAGTCTGGACCTGAACT 8 208A-222H EVQLQQSGPELEKPGASVRISCK GGAAAAGCCTGGGGCTTCAGTGAGGATATCCT TSGYTFTEYAMHWVKQSHGKSLE GCAAGACTTCTGGATACACATTCACTGAATAC WIGGINPNNGNAIYNQIFKDKAT GCCATGCACTGGGTGAAGCAGAGCCATGGAAA LTVDRSSSTAYMGLRSLTFGDSG GAGCCTTGAGTGGATTGGAGGTATTAATCCTA VYFCVRRQLDYFDYWGQGASLTV ACAATGGCAATGCTATCTACAACCAGATATTC SS AAGGACAAGGCCACACTGACTGTGGACAGGTC CTCCAGCACAGCCTACATGGGCCTCCGCAGCC TGACATTCGGGGATTCTGGAGTCTACTTCTGT GTAAGACGACAACTGGACTACTTTGACTATTG GGGCCAGGGCGCCTCTCTCACAGTCTCCTCA 208A-605H 9 GAGGTCCAGCTGCAACAGTCTGGACCTGAGCT 10 208A-605H EVQLQQSGPELEKPGASVKISCK GGAAAAGCCTGGGGCTTCAGTGAAGATATCCT TSGYTFTEYAIHWVKQSHGMSLE GCAAGACTTCTGGATACACATTCACTGAATAC WIGGINPSNGNAIYNQIFKDKAT GCCATACACTGGGTGAAGCAGAGCCATGGAAT LTVDRSSSTAYMGLRSLTFGDSG GAGCCTTGAGTGGATTGGAGGTATTAATCCTA VYFCVRRQLDFFDYWGQGASLTV GCAATGGCAATGCTATCTACAACCAAATATTC SS AAGGACAAGGCCACACTGACTGTGGACAGGTC CTCCAGCACAGCCTACATGGGCCTCCGCAGCC TGACATTTGGGGATTCTGGAGTCTACTTCTGT GTAAGACGACAACTGGACTTCTTTGACTATTG GGGCCAGGGCGCCTCTCTCACAGTCTCCTCA 208B-560H 11 GAGGTCCAGCTGCAACAGTCTGGACCTGAGCT 12 208B-560H EVQLQQSGPELEKPGASVKISCK GGAAAAGCCTGGGGCTTCAGTGAAGATATCCT TSGYTFTEYAMHWVKQSHGMSLE GCAAGACTTCTGGATACACATTCACTGAATAC WIGGINPSNGNAIYNQIFKDKAT GCCATGCACTGGGTGAAGCAGAGCCATGGAAT LTVDRSSSTAYMGLRSLTFGDSG GAGCCTTGAGTGGATTGGAGGTATTAATCCTA VYFCVRRQLDFFDYWGQGASLTV GCAATGGCAATGCTATCTACAACCAGATATTC SS AAGGACAAGGCCACACTGACTGTGGACAGGTC CTCCAGCACAGCCTACATGGGCCTCCGCAGCC TGACATTTGGGGATTCTGGAGTCTACTTCTGT GTAAGACGACAACTGGACTTCTTTGACTATTG GGGCCAGGGCGCCTCTCTCACAGTCTCCTCA 208A-830H 13 GAGGTCCGGCTGCAGCAGCCTGGACCTGAGGT 14 208A-830H EVRLQQPGPEVEKPGASVKISCK GGAAAAGCCTGGGGCTTCAGTGAAGATATCCT TSGYTFTEYAIHWVEQSHGESLE GCAAGACTTCTGGATACACATTCACTGAATAC WIGGINPSNGDPIYNQIFKDKAT GCCATCCACTGGGTGAAACAGAGCCATGGAGA LTVDRSSNTAYMGLRSLTVGDSG GAGCCTTGAGTGGATTGGAGGTATTAATCCTA VYFCVRRQLDYFDFWGQGASLTV GCAATGGCGATCCTATCTATAACCAGATATTC SS AAGGACAAGGCCACACTGACTGTGGACAGGTC CTCCAACACAGCCTACATGGGCCTCCGCAGCC TGACAGTTGGGGATTCTGGAGTCTACTTCTGT GTTAGACGACAACTGGACTACTTTGACTTTTG GGGCCAGGGCGCCTCTCTCACAGTCTCCTCA 208A-134H 15 CAGGGTCAGATGCAGCAGTCTGGAGCTGAACT 16 208A-134H QGQMQQSGAELAKPGASVKLSCK GGCGAAGCCTGGGGCTTCAGTGAAGCTGTCCT TSGFTFSSSYISWLEQFPGQSLE GCAAGACTTCTGGCTTCACCTTCAGCAGTAGT WIAWIYAGTGNTNYNQKFTDKAQ TATATAAGTTGGTTGAAGCAAAAGCCTGGACA LTVDTSSSTAYMQLSSLTTEDSA GAGTCTTGAGTGGATTGCATGGATTTATGCTG IYYCAISGTGFTYWGQGTLVTVS GAACTGGTAATACTAACTATAATCAGAAGTTC A ACAGACAAGGCCCAACTGACTGTAGACACATC CTCCAGTACAGCCTACATGCAACTCAGCAGCC TGACAACTGAGGACTCTGCCATCTATTACTGT GCGATAAGTGGGACGGGATTTACTTACTGGGG CCAAGGGACTCTGGTCACTGTCTCTGCAACA 208A-692H 17 CAGGGTCAGATGCAGCAGTCTGGAGCTGAACT 18 208A-692H QGQMQQSGAELAKPGASVKLSCK GGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCT TSGFTFSSSYISWLEQFPGQSLE GCAAGACTTCTGGCTTCACCTTCAGCAGTAGT WIAWIFAGTGNTNYNQKFTDKAQ TATATAAGTTGGTTGAAGCAAAAGCCTGGACA LTVDTSSSTAYMQLSSLTTEDSA GAGTCTTGAGTGGATTGCATGGATTTATGCTG IYYCAISGTGFTYWGQGTLVTVS GAACTGGTAATACTAACTATAATCAGAAGTTC A ACAGACAAGGCCCAACTGACTGTAGACACATC CTCCAGTACAGCCTACATGCAACTCAGCAGCC TGACAACTGAGGACTCTGCCATCTATTACTGT GCGATAAGTGGGACGGGATTTACTTACTGGGG CCAAGGGACTCTGGTCACTGTCTCTGCAACA 208A-557H 19 CAGGGTCAGATGCAGCAGTCTGGAGCTGAGCT 20 208A-557H QGQMQQSGAELVEPGASVKLSCK GGCGAAGCCTGGGGCTTCAGTGAAACTGTCCT TSGFTFSSSYISWLEQFPGQSLE GCAAGACTTCTGGCTTCACCTTCAGCAGTAGT WIAWIYAGTGNTNYNQKFTDKAQ TATATAAGTTGGTTGAAGCAAAAGCCTGGACA LTVDTSSSTAYMQLSSLTTEDSA GAGTCTTGAGTGGATTGCATGGATTTTTGCTG IYYCAISGTGFTYWGQGTLVTVS GAACTGGTAATACTAATTATAATCAGAAGTTC A ACAGACAAGGCCCAACTGACTGTAGACACATC CTCCAGTACAGCCTACATGCAACTCAGCAGCC TGACAACTGAGGACTCTGCCATCTATTACTGT GCGATAAGTGGGACGGGATTTACTTACTGGGG CCAAGGGACTCTGGTCACTGTCTCTGCA 208A-352H 21 CAGGGTCAGATGCAGCAGTCTGGAGCTGAGCT 22 208A-352H QGQMQQSGAELVEPGASVKLSCK GGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCT TSGFTFSSSFISWLEQFPGQSLE GCAAGACTTCTGGCTTCACCTTCAGCAGTAGT WIAWIYAGTGNTNYNQKFTDKAQ TTTATAAGTTGGTTGAAGCAAAAGCCTGGACA LTVDTSSSTAYMQFSSLTTEDSA GAGTCTTGAGTGGATTGCATGGATTTATGCTG IYYCAISGTGFTYWGQGTLVTVS GAACTGGAAATACTAACTATAATCAGAAGTTC A ACAGACAAGGCCCAACTGACTGTAGACACATC CTCCAGCACAGCCTACATGCAATTCAGCAGCC TGACGACTGAGGACTCTGCCATCTATTACTGT GCGATAAGTGGGACGGGGTTTACTTACTGGGG CCAAGGGACTCTGGTCACTGTCTCTGCA 208A-983H 23 CAGGGTCAGATGCAGCAGTCTGGAGCTGAGCT 24 208A-983H QGQMQQSGAELVEPGASVKLSCK GGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCT TSGFTFSSSYFSWLEQFPGQSLE GCAAGACTTCTGGCTTCACCTTCAGCAGTAGT WIAWIYAGTGNTIYNQKFTDKAQ TATTTTAGTTGGTTGAAGCAAAAGCCTGGACA LTVDTASSTAFMQLSSLTIEDSA GAGTCTTGAGTGGATTGCATGGATTTATGCTG IYYCAISGTGFTYWGQGTLVTVS GAACTGGTAATACTATCTATAATCAGAAGTTC A ACAGACAAGGCCCAACTGACTGTAGACACAGC CTCCAGCACAGCCTTCATGCAACTCAGCAGCC TGACAATTGAGGACTCTGCCATCTACTACTGT GCGATAAGTGGGACGGGGTTTACTTACTGGGG CCAAGGGACTCTGGTCACTGTCTCTGCAACA 208B-281H 25 CAGGGTCAGCTGCAGCAGTCTGGAGCTGAGCT 26 208B-281H QGQLQQSGAELVEPGASVKLSCK GGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCT TSGFTFSSSYISWLEQRPGQSLE GCAAGACTTCTGGCTTCACCTTCAGCAGTAGT WIAWIYAGTGGTNYNQKFTDKAQ TATATAAGTTGGTTGAAGCAAAGGCCTGGACA LTVDTSSSTAYMQFSSLTTEDSA GAGTCTTGAGTGGATTGCATGGATTTATGCTG IYYCAISGTGFIYWGQGTLVTVS GAACTGGTGGTACTAACTATAATCAGAAGTTC A ACAGACAAGGCCCAACTGACTGTAGACACATC CTCCAGCACAGCCTACATGCAATTCAGCAGCC TGACAACTGAGGACTCTGCCATCTATTACTGT GCGATAAGTGGGACGGGGTTTATTTACTGGGG CCAAGGGACTCTGGTCACTGTCTCTGCA 208B-471H 27 CAGGGTCAGTTGCAGCAGTCTGGACCAGTACT 28 208B-471H QGQLQQSGPVLVKPGASEILYCK GGTGAAGCCTGGGGCTTCAGAAATACTATACT TSGFTESSTYISWLKQKPGQSLE GCAAGACTTCTGGCTTCACCTTCAGCAGTACC WIAWIYAGTGATNYNQKFTGKAQ TATATAAGTTGGTTGAAGCAAAAGCCTGGACA LTVDASSNTAYMHFSGLTPEDSA GAGTCTTGAGTGGATTGCGTGGATTTATGCTG IYYCAISGAGVYWGQGTLVTVSA GAACTGGTGCTACTAATTATAATCAGAAGTTC ACAGGCAAGGCCCAACTGACTGTAGACGCTTC CTCCAACACAGCCTACATGCACTTCAGCGGCC TGACACCTGAGGACTCTGCCATCTATTACTGT GCAATTTCTGGGGCGGGGGTTTACTGGGGCCA AGGGACTCTGGTCACTGTCTCTGCA 208A-207H 29 CAGGGCCAACTGCAGCAGCCTGGGGCTGAGTT 30 208A-207H QGQLQQPGAEFVKPGASLKLSCR TGTGAAGCCTGGGGCTTCACTGAAGCTGTCCT ASGYTFTSYWIHWVKQRPGQGLE GCAGGGCTTCTGGCTACACCTTCACCAGCTAC WIGEIDPSDSYINQNQHFRGKAT TGGATACACTGGGTGAAGCAGAGGCCTGGACA LTVDKSSSTAYMELSGLTSEDSA AGGCCTTGAGTGGATTGGAGAAATTGATCCTT VYYCARHYYGVLDSWGQGTTLTV CTGACAGTTATATTAACCAGAATCAAAAGTTC SS AGGGGCAAGGCCACATTGACTGTGGACAAATC CTCCAGCACAGCCTACATGGAACTCAGCGGCC TGACATCTGAAGACTCTGCGGTCTATTACTGT GCAAGACATTACTACGGTGTTCTTGACTCCTG GGGCCAAGGTACCACTCTCACAGTCTCCTCAA CA 208A-638H 31 CAGGGCCAACTGCAGCAGCCTGGGGCTGAGTT 32 208A-638H QGQLQQPGAEFVKPGASLKLSCR TGTGAAGCCTGGGGCTTCACTGAAGCTGTCCT ASGYTFTSYWIHWVKQRPGQGLE GCAGGGCTTCTGGCTACACCTTCACCAGCTAC WIGEVDPSDSYINQNEKERGKAT TGGATTCACTGGGTGAAGCAGAGGCCTGGACA LTVDKSSSTAYMQLGSLTSEDSA AGGCCTTGAGTGGATCGGAGAAGTTGATCCTT VYYCARHYYGVLDSWGQGTALTV CTGACAGTTATATTAACCAGAATGAAAAGTTC SS AGGGGCAAGGCCACATTGACTGTGGACAAATC CTCCAGCACAGCCTACATGCAGCTCGGCAGCC TGACATCTGAAGACTCTGCGGTCTATTACTGT GCAAGACATTACTACGGTGTTCTTGACTCCTG GGGCCAAGGCACCGCTCTCACAGTCTCCTCA 208B-515H 33 CAGGTCCAACTGCAGCAGCCTGGGGCTGAACT 34 208B-515H QVQLQQPGAELVKPGASLKLSCR TGTGAAGCCTGGGGCTTCACTGAAGCTGTCCT ASGYTFTSYWIHWVKQRPGQGLE GCAGGGCCTCTGGCTACACCTTCACCAGCTAC WIGEIDPSDSYTNYNQHFKGKAT TGGATTCACTGGGTGAAGCAGAGGCCTGGACA LTVDKSSRAAYMQLSSLTSEDSA AGGCCTTGAGTGGATCGGAGAGATTGATCCTT VYYCARHYYGVEDSWGQGTHLTV CTGATAGTTATACTAACTACAATCAAAAGTTC SS AAGGGCAAGGCCACATTGACTGTAGACAAATC CTCCAGGGCAGCCTACATGCAGCTCAGCAGCC TGACATCTGAGGACTCTGCGGTCTATTACTGT GCAAGACATTACTACGGTGTCTTTGACTCCTG GGGCCAAGGCACCAAACTCACAGTCTCCTCA 208A-874H 35 CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCT 36 208A-874H QVQLQQPGAELVKPGASVKLSCK TGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCT ASGYTLSSYWMHWVKQRPGQGLE GCAAGGCTTCTGGCTACACCCTCAGTAGCTAT WIGEIHPSDSYTSYNQHFKDKAT TGGATGCACTGGGTGAAGCAGAGGCCTGGACA LTVDKSSSTAYMQLSSLTSEDSA AGGCCTTGAGTGGATCGGAGAGATTCATCCTT VYYCARGGYYRYDEFAYWGQGTL CTGATAGTTATACTAGCTACAATCAAAAGTTC VTVSA AAGGACAAGGCCACATTGACTGTAGACAAATC CTCCAGCACAGCCTACATGCAGCTCAGCAGCC TGACATCTGAGGACTCTGCAGTCTATTACTGT GCAAGGGGGGGCTACTATAGGTACGACGAGTT TGCTTACTGGGGCCAAGGGACTCTGGTCACTG TCTCTGCA 208B-911H 37 CAGGTCCATCTGCAGCAGCCTGGGGCTGAGCT 38 208B-911H QVHLQQPGAELVRPGVSVKLSCK GGTGAGGCCTGGGGTTTCAGTGAAGCTGTCCT ASGYTETTYSINWMKQRPGQGLE GCAAGGCTTCTGGCTACACCTTCACCACCTAC WIGNIYPSTSHTNYNQHFRDKAT TCGATAAACTGGATGAAGCAGAGGCCTGGACA MTVDKSSSTAYMQLSSPTSEDSA AGGCCTTGAGTGGATCGGAAATATTTATCCTT VYYCTINAYSMDYWGQGTSVTVS CTACCAGTCATACTAACTACAATCAAAAGTTC S AGGGACAAGGCCACAATGACTGTAGACAAATC

CTCCAGCACAGCCTACATGCAGCTCAGCAGCC CGACATCTGAGGACTCTGCGGTCTATTATTGT ACAATAAATGCCTATTCTATGGACTACTGGGG TCAAGGAACCTCAGTCACCGTCTCCTCA 208B- 39 CAGGTCCAGCTTCAGCAGTCTGGGGCTGGACT 40 208B-1096H QVQLQQSGAGLAKPGASVKMSCK 1096H GGCAAAACCTGGGGCCTCAGTGAAGATGTCCT ASGYTFTANKMHWAKQRPGQGLE GCAAGGCTTCTGGCTACACCTTTACTGCCAAC WIGYIDPSSGYTEYNHEIQYKAT AAGATGCACTGGGCAAAACAGCGGCCTGGACA LTADTSSSTAYMQLSTLTFEDSA GGGTCTGGAATGGATTGGATACATTGATCCTA VYYCTNFAYWGQGTLVTVSA GCTCTGGTTATACTGAATACAATCATAAGATC CAGTACAAGGCCACTTTGACTGCAGACACATC CTCCAGCACAGCCTACATGCAACTGAGCACCC TAACATTTGAAGACTCTGCAGTCTATTACTGT ACAAATTTTGCTTACTGGGGCCAAGGGACTCT GGTCACTGTCTCAGCAACA 208B-589H 41 CAGGTCCAGCTTCAGCAGTCTGGGGCTGGACT 42 208B-589H QVQLQQSGAGLAKPGASVKMSCK GGCAAAACCTGGGGCCTCAGTGAAGATGTCCT ASGYTFTANKMHWAKQRPGQGLE GCAAGGCTTCTGGCTACACCTTTACTGCCAAC WIGYIDPSSGYTEYNHEIQDKAT AAGATGCACTGGGCAAAACAGCGGCCTGGACA LTADTSSSTAYMQLSSLTFEDSA GGGTCTGGAATGGATTGGATACATTGATCCTA VYYCTNFAYWGQGTLVTVSA GCTCTGGTTATACTGAATACAATCATAAGATC CAGGACAAGGCCACATTGACTGCAGACACATC CTCCAGCACAGCCTACATGCAACTGAGCAGCC TAACATTTGAAGACTCTGCAGTCTATTACTGT ACAAATTTTGCTTACTGGGGCCAAGGGACTCT GGTCACTGTCTCAGCA 208B-395H 43 CAGGTCCAGCTTCAGCAGTCTGGGGCTGGACT 44 208B-395H QVQLQQSGAGLAKPGASVKMSCK GGCAAAACCTGGGGCCTCAGTGAAGATGTCCT ASGYTFTANKMHWTHQRPGQGLE GCAAGGCTTCTGGCTATACCTTTACTGCCAAC WIGYIDPSSGYTQYNHEIQDKAT AAGATGCACTGGACAAAACAGCGGCCTGGACA LTADTSSSTAYMQLSSLTFEDSA GGGTCTGGAATGGATTGGATACATTGATCCTA VYYCTNFAYWGQGTLVTVSA GCTCTGGTTATACTCAATACAATCATAAGATC CAGGACAAGGCCACATTGACTGCAGACACATC CTCCAGCACAGCCTACATGCAACTGAGCAGCC TAACATTTGAAGACTCTGCAGTCTATTACTGT ACAAATTTTGCTTACTGGGGCCAAGGGACTCT GGTCACTGTCTCAGCA 208B-189H 45 CAGGTCCACCTTCAGCAGTCTGGGGCTGAACT 46 208B-189H QVHLQQSGAELAKPGASVQMSCK GGCCAAACCTGGGGCCTCAGTGCAGATGTCCT ASGYTFTANKMHWARQRPRQGLE GCAAGGCTTCTGGCTACACCTTTACTGCCAAC WIGYIDPASGYTEYNQKIKDRAT AAGATGCACTGGGCAAGACAGCGGCCTAGACA LTADESSSTAYMQLSSLTSEDSA GGGTCTGGAATGGATTGGATACATTGATCCTG VYYCTNFAYWGQGTLVTVST CCTCTGGCTATACTGAATACAATCAGAAGATC AAGGACAGGGCCACATTGACTGCAGACAAATC CTCCAGCACAGCCTACATGCAACTGAGCAGCC TGACATCTGAGGACTCTGCAGTCTATTACTGT ACAAATTTTGCTTACTGGGGCCAAGGGACTCT GGTCACTGTCTCTACAACA 208B-547H 47 CAGGTCCAGCTTCAGCAGTCTGGGGCTGAACT 48 208B-547H QVQLQQSGAELAKPGASVKMSCK GGCAAAACCTGGGGCCTCAGTGAAGATGTCCT ASGYTFTSNEMHWAKQRPGQGLE GCAAGGCTTCTGGCTACACCTTTACTAGCAAC WIGYIDPSSGYTEYNQKIEDKAT AAGATGCACTGGGCAAAACAGCGGCCTGGACA LTADESSSTAYMQLSSLTSEDSA GGGTCTGGAATGGATTGGATACATTGATCCTA VYYCTNFAYWGQGTLVTVSA GCTCTGGTTATACTGAATACAATCAGAAGATC AAGGACAAGGCCACATTGACTGCAGACAAATC CTCCAGCACAGCCTACATGCAACTGAGCAGCC TGACATCTGAGGACTCTGCAGTCTATTACTGT ACAAATTTTGCTTACTGGGGCCAAGGGACTCT GGTCACTGTCTCTGCAACA 208A-210H 49 CAGGTCCAGTTGCAGCAGTCTGGACCTGAGTT 50 208A-210H QVQLQQSGPELVKPGASMRISCK GGTGAAGCCTGGGGCTTCAATGAGGATATCCT ASGYTFTSYYVHWIEQRPGQGLE GCAAGGCTTCTGGCTACACCTTCACAAGCTAC WIGCTYPGDVNTDYNEFFEGKAT TATGTACACTGGATAAAGCAGAGGCCTGGACA LTADESSSTAYMQVSTLTSEDSA GGGACTTGAGTGGATTGGATGTATTTATCCTG IYFCVLYYYGSFAYWGQGTLVTV GAGATGTTAATACTGACTATAATGAGAAGTTC SA AAGGGCAAGGCCACGCTGACTGCAGACAAATC CTCCAGCACAGCCTACATGCAGGTCAGCACCC TGACCTCTGAGGACTCTGCGATCTATTTCTGT GTCCTTTATTACTACGGTAGTTTTGCTTACTG GGGCCAAGGGACTCTGGTCACTGTCTCTGCA 208A-422H 51 GATGTACAGCTTCAGGAGTCAGGACCTGGCCT 52 208A-422H DVQLQESGPGLVNPSQSLSLTCS CGTGAATCCTTCTCAGTCTCTGTCTCTCACCT VTGYSITSGYYWIWIQQSPGNKL GCTCTGTCACTGGCTACTCCATCACCAGTGGT EWMGYIKYDGGNNYSPSLKNRIS TATTACTGGATCTGGATCCAGCAGTCTCCAGG IARDTSKNQCFLKLNSVTIEDTA AAACAAACTGGAATGGATGGGCTACATAAAGT TYYCTRGSDSFDYWGQGTTLTVS ACGACGGTGGCAATAACTACAGCCCATCTCTC S AAAAATCGAATCTCCATCGCTCGTGACACATC TAAGAACCAGTGTTTCCTGAAGTTGAATTCTG TGACTATTGAGGACACAGCTACATATTACTGT ACAAGAGGGTCGGACTCCTTTGACTACTGGGG CCAAGGCACCACTCTCACAGTCTCCTCA 208A-442H 53 GATGTACAGCTTCAGGAGTCAGGACCTGGCCT 54 208A-442H DVQLQESGPGLVNPSQSLSLTCS CGTGAATCCTTCTCAGTCTCTGTCTCTCACCT VTGYSITSGYYWIWIRQFPGNKL GCTCTGTCACTGGCTACTCCATCACCAGTGGT EWMGYIKYDGGNNYSPSLKNRIS TATTACTGGATCTGGATCCGGCAGTTTCCAGG IARDTSKNQFFLKLNSVTIEDTA AAACAAACTGGAATGGATGGGCTACATAAAGT TYYCTRGSDSFDYWGQGTTLTVS ACGACGGTGGCAATAACTACAGCCCATCTCTC S AAAAATCGAATCTCCATCGCTCGTGACACATC TAAGAACCAGTTTTTCCTGAAGTTGAATTCTG TGACTATTGAGGACACAGCTACATATTACTGT ACAAGAGGGTCGGACTCCTTTGACTACTGGGG CCAAGGCACCACTCTCACAGTCTCCTCA 208B-862H 55 GATGTGCAGCTTCAGGAGTCGGGACCTGGCCT 56 208B-862H DVQLQESGPGLVKPSQSLSLTCT GGTGAAACCTTCTCAGTCTCTGTCCCTCACCT VTGYSITSDYAWNWIRQFPGNKL GCACTGTCACTGGCTACTCAATCACCAGTGAT EWMGYISYSGTTVYSPSLKSRIS TATGCCTGGAACTGGATCCGGCAGTTTCCTGG ITRDTSKNQFFLQLNSVTIEDSA AAACAAACTGGAGTGGATGGGCTACATAAGCT TYYCGGNYWGQGTLVTVSA ACAGTGGTACCACTGTCTACAGCCCATCTCTC AAAAGTCGAATCTCCATCACTCGGGACACATC CAAAAACCAGTTCTTCCTGCAATTGAATTCTG TGACTATTGAGGACTCAGCCACGTATTATTGT GGGGGTAATTACTGGGGCCAAGGGACTCTGGT CACTGTCTCTGCA 208A-967H 57 CAGGTGCAGCTGGAGGAGTCAGGACCTGGCCT 58 208A-967H QVQLEESGPGLVAPSQSLSITCT GGTGGCGCCCTCACAGAGCCTGTCCATCACTT VSGFSLTSYGVHWVRQPPGKGLE GCACTGTCTCTGGATTTTCATTAACCAGCTAT WLGVIWAVGSINYNSALMSRLSI GGTGTACACTGGGTTCGCCAGCCTCCAGGAAA SKDNSKSQVFLKMNSLRTDDTAM GGGTCTGGAGTGGCTGGGAGTAATATGGGCTG YYCARDRTTATPFFDYWGQGTTL TTGGAAGTATAAATTATAATTCGGCTCTCATG TVSS TCCAGACTGAGCATCAGCAAAGACAACTCCAA GAGCCAAGTTTTCTTAAAAATGAACAGTCTAC GAACTGATGACACAGCCATGTACTACTGTGCC AGAGATCGGACTACGGCTACCCCCTTCTTTGA CTACTGGGGCCAAGGCACCACTCTCACAGTCT CCTCATCCAAAACA 208B-517H 59 CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCT 60 208B-517H QVQLKQSGPGLVQPSQSLSITCT AGTGCAGCCCTCACAGAGCCTGTCCATCACCT VSGFSLITHGVHWVRQSPGKGLE GCACAGTCTCTGGTTTCTCATTAATTACCCAT WLGVIWSGGSTDYNAAFISRLSI GGTGTACACTGGGTTCGCCAGTCTCCAGGAAA SKDTSKSQVFLKMSSLQADDTAI GGGTCTGGAGTGGCTGGGAGTGATATGGAGTG YYCARNGGATAFDYWGQGTTLTV GTGGAAGCACAGACTATAATGCAGCTTTCATA SS TCCAGACTGAGCATCAGCAAGGACACCTCCAA GAGCCAAGTTTTCCTTAAAATGAGCAGTCTGC AAGCTGATGACACAGCCATATACTACTGTGCC AGAAATGGGGGGGCTACGGCCTTTGACTACTG GGGCCAAGGCACCACTCTCACAGTCTCCTCA 208B-822H 61 CAGGTACAGCTGAAGCAGTCAGGACCTGGCCT 62 208B-822H QVQLKQSGPGLVQPSQSLSITCT AGTGCAGCCCTCACAGAGCCTGTCCATCACCT VSGFSLITYGVHWVRQSPGKGLE GCACAGTCTCTGGTTTCTCATTAATTACCTAT WLGVIWGGGSTGYNAAFVSRLNI GGTGTACACTGGGTTCGCCAGTCTCCAGGAAA TKDNSKSQVFFKMNSLQPDDTAI GGGTCTGGAGTGGCTGGGAGTGATATGGGGTG YYCARNGGATVFDYWGQGTTLTV GTGGAAGCACAGGCTATAATGCAGCTTTCGTA SS TCCAGACTGAACATCACCAAGGACAACTCCAA GAGCCAAGTTTTCTTTAAAATGAACAGTCTGC AACCTGATGACACAGCCATATACTACTGTGCC AGAAATGGAGGGGCTACGGTCTTTGACTACTG GGGCCAAGGCACCACTCTCACAGTCTCCTCA 208B- 63 GAGGTGCAGCTGGTGGAGTCTGGGGGAGACTT 64 208B-1024H EVQLVESGGDLVKPGGSLKLSCA 1024H AGTGAAGCCTGGAGGGTCCCTGAAACTCTCCT ASGFTFSNYGMSWVRQTPDHRLE GTGCAGCCTCTGGATTCACTTTCAGTAACTAT WVATISSGGSYSYYPDSVKGRFT GGCATGTCTTGGGTTCGCCAGACTCCAGACAA ISRDNAKNILYLQMSSLKSEDTA GAGGCTGGAGTGGGTCGCAACCATTAGTAGTG MYYCASLYYGYGDYWGQGTAFTV GTGGTAGTTATAGCTACTATCCAGACAGTGTA SS AAGGGGCGGTTCACCATCTCCAGAGACAATGC CAAGAACATCCTGTACCTGCAAATGAGCAGTC TGAAGTCTGAGGACACAGCCATGTATTACTGT GCAAGTCTCTACTACGGCTACGGGGACTACTG GGGCCAAGGCACCGCTTTCACAGTCTCCTCA 208B-327H 65 GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTT 66 208B-327H EVQLVESGGGLVKPGGSLKLSCA GGTGAAGCCTGGAGGGTCCCTGAAACTCTCCT ASGFTFSDSYMYWVRQTPDQRLE GTGCAGCCTCTGGATTCACTTTCAGTGACTCT WVATISDGGSYTFYPDSVKGRFT TATATGTATTGGGTTCGCCAGACTCCGGACCA ISRDNAQNNLYLQMSSLKSEDTA GAGGCTGGAGTGGGTCGCAACCATTAGTGATG MYYCASPHAGYFGWFAYWGRGTL GTGGTAGTTACACCTTCTATCCAGACAGTGTG VTVSA AAGGGACGATTCACCATCTCCAGAGACAATGC CCAGAACAACCTGTACCTGCAAATGAGCAGTC TGAAGTCTGAGGACACAGCCATGTATTACTGT GCATCCCCCCATGCTGGCTACTTCGGCTGGTT TGCTTACTGGGGCCGAGGGACTCTGGTCACTG TCTCTGCA 208B-353H 67 GAGGTGCAGCTGGTGGAGTCTGGGGGAGACTT 68 208B-353H EVQLVESGGDLVKPGGSLKLSCA AGTGAAGCCTGGAGGGTCCCTGAAACTCTCCT ASGFTFNHYGMSWVRQPPDKRLE GTGCAGCCTCTGGATTCACTTTCAATCACTAT WVATISSGGGYTYYPDSVKGRFT GGCATGTCTTGGGTTCGCCAGCCTCCAGACAA ISRDNAKDTLSLQMSSLRSGDTA GAGACTGGAGTGGGTCGCAACCATTAGTAGTG VYYCASLYGSLFAYWGQGTLVTV GTGGTGGTTACACCTACTATCCAGACAGTGTG SA AAGGGGCGCTTCACCATCTCCAGAGACAATGC CAAGGACACCCTGTCCCTGCAAATGAGCAGTC TGAGGTCTGGGGACACAGCCGTGTATTACTGT GCAAGCCTATACGGTAGCCTGTTTGCTTACTG GGGCCAAGGGACTCTGGTCACTGTCTCTGCA 208B-178H 69 GACGTGAAGCTCGTGGAGTCTGGGGGAGGCTT 70 208B-178H DVKLVESGGGLVKLGGSLKLSCA AGTGAAGCTTGGAGGGTCCCTGAAACTCTCCT ASGFTFSSYYMSWVRQTPEKRLE GTGCAGCCTCTGGATTCACTTTCAGTAGCTAT LVAAINSNGGSTYYPDTVKGRFT TACATGTCTTGGGTTCGCCAGACTCCAGAGAA ISRDNAHNTLYLQMSSLKSEDTA GAGGCTGGAGTTGGTCGCAGCCATTAATAGTA LYYCARHGGLGRRDWYFDVWGAG ATGGTGGTAGCACCTACTATCCAGACACTGTG TTVTVSS AAGGGCCGATTCACCATCTCCAGAGACAATGC CAAGAACACCCTGTACCTGCAAATGAGCAGTC TGAAGTCTGAGGACACAGCCTTGTATTACTGT GCAAGACATGGGGGACTGGGACGTAGGGACTG GTACTTCGATGTCTGGGGCGCAGGGACCACGG TCACCGTCTCCTCA 208B-672H 71 GAAGTGAAACTGGTGGAGTCTGGGGGAAGTTT 72 208B-672H EVKLVESGGSLVQPGGSLKLSCA AGTGCAGCCTGGAGGGTCCCTGAAACTCTCCT ASGFNFNTYAMSWVRQTPEKRLE GCGCAGCCTCTGGATTCAATTTCAATACCTAT WVAYISNGGGNTYYVDTVKGRFT GCCATGTCTTGGGTTCGCCAGACTCCAGAGAA ISRDNAHNTLYLRMSSLKSEDTA GAGGCTGGAGTGGGTCGCATACATTAGTAATG MYYCARHGLYWGYSMDYWGQGTS GTGGTGGTAACACCTACTATGTAGACACTGTA VTVSS AAGGGCCGATTCACCATCTCCAGAGACAATGC CAAGAACACCCTGTACCTGCGAATGAGCAGTC TGAAGTCTGAGGACACGGCCATGTATTACTGT GCAAGACATGGGCTCTACTGGGGCTATTCTAT GGACTACTGGGGTCAAGGAACCTCAGTCACCG TCTCCTCA 208B-793H 73 GAAGTGAAGCTGGTGGAGTCTGGGGGAGGTTT 74 208B-793H EVKLVESGGGLVQPGGSLKLSCA AGTGCAGCCAGGAGGGTCCCTGAAACTCTCCT ASGFTFSSYAMSWVRQTPERRLE GTGCAGCCTCTGGATTCACTTTCAGTAGCTAT WVTYISNGGGSTYYSDTVKGRFT GCCATGTCTTGGGTTCGCCAGACTCCAGAGAG FSRDNAHNTLYLQMSSLKSEDTA GAGGCTGGAGTGGGTCACATACATTAGTAATG MYYCARHGLGRTGFASWGQGTLV GTGGTGGTAGCACCTACTATTCAGACACTGTA TVSA AAGGGCCGATTCACCTTCTCCAGAGACAATGC CAAGAACACCCTGTACCTGCAAATGAGCAGTC TGAAGTCTGAGGACACGGCCATGTATTACTGT GCAAGACATGGACTGGGAAGGACAGGGTTTGC TTCCTGGGGCCAAGGGACTCTGGTCACTGTCT CTGCA 208B-826H 75 GATGTGCAGCTGGTGGAGTCTGGGGGAGGCCT 76 208B-826H DVQLVESGGGLVQAGGSRKLSCA AGTGCAGGCTGGAGGGTCCCGGAAACTCTCCT ASGFPFSSFGMHWVRQAPEKGLE GTGCAGCCTCTGGATTCCCTTTCAGTTCCTTT WVASISSRTSKIYYADNLKGRFT GGAATGCACTGGGTTCGTCAGGCTCCAGAGAA ISRDNPRNTLFLQMTSLGSEDTA GGGGCTGGAGTGGGTCGCCTCCATTAGTAGTC MYYCVRSVFGNSYWFFDVWGAGT GCACTAGTAAGATCTACTATGCAGACAACCTG TVTVSS

AAGGGCCGATTCACCATCTCCAGAGACAATCC CAAGAACACCCTGTTCCTGCAAATGACCAGTC TTGGATCTGAGGACACGGCCATGTATTACTGT GTAAGATCCGTCTTTGGTAATTCTTACTGGTT TTTCGATGTCTGGGGCGCAGGGACCACGGTCA CCGTCTCCTCA 208B-174H 77 GAAGTGAAGCTTGAGGAGTCTGGAGGAGGCTT 78 208B-174H EVKLEESGGGLVQPGGSMKLSCV GGTACAACCTGGGGGATCCATGAAACTCTCCT ASGFSFSSYWMSWVRQSPEKGLD GTGTAGCCTCTGGATTTTCTTTCAGTAGCTAC WVAEIRLRSDNYATHYAESVKGR TGGATGTCTTGGGTCCGCCAGTCTCCAGAGAA FTISRDDSISRLYLQMNTLRAED GGGGCTTGACTGGGTTGCTGAAATTAGATTGA TGIYYCTWMTYWGQGTLVTVSA GATCTGATAATTATGCAACCCATTATGCGGAG TCTGTGAAAGGGAGGTTCACCATCTCAAGAGA TGATTCCATAAGTCGTCTCTACCTGCAAATGA ACACCTTAAGAGCTGAAGACACTGGAATTTAT TACTGTACATGGATGACGTACTGGGGCCAAGG GACTCTGGTCACTGTCTCTGCAACA 208B-408H 79 GAGGTGAAGCTGGTGGAGTCTGGAGGAGGCTT 80 208B-408H EVKLVESGGGLVQPGGSLRLSCA GGTACAGCCTGGGGGTTCTCTGAGACTCTCCT SSGFTFTDYYMSWVRQPPGKALE GTGCAAGTTCTGGGTTCACCTTCACTGATTAC WLGFIRNKAYGYTTEFSASVNGR TACATGAGCTGGGTCCGCCAGCCTCCAGGAAA FTISRDDSQSVPYLQMNTLRAED GGCACTTGAGTGGTTGGGTTTTATTAGAAACA SATYYCARVLYYDYGGFAYWGQG AAGCTTATGGTTACACGACCGAGTTCAGTGCA TLVTVST TCTGTGAACGGTCGGTTCACCATCTCCAGAGA TGATTCCCAAAGCGTCCCCTATCTTCAAATGA ACACCCTGAGAGCTGAGGACAGTGCCACTTAT TACTGTGCGAGAGTCCTCTACTATGATTACGG GGGATTTGCTTACTGGGGCCAAGGGACTCTGG TCACTGTCTCTACA 208B-612H 81 GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCT 82 208B-612H EVQLQQSGPELVKPGASVMSCK GGTAAAGCCTGGGGCTTCAGTGAAGATGTCCT ASGYRFTSYVMHWVRQKPGQGLE GTAAGGCTTCCGGATACAGATTCACTAGCTAT WIGYIDPHND-- GTTATGCACTGGGTGAGGCAGAAGCCTGGACA DTKYSEKFRGKATLTSDKSSTTA GGGCCTTGAGTGGATTGGATATATTGATCCTC YMELSSLTSEDSAVYYCVRYSYD ACAATGATGATACAAAATACAGTGAGAAGTTC RDYSPMDYWGQGTSVTVSS AGAGGTAAGGCCACACTGACTTCAGACAAGTC CTCCACCACAGCCTACATGGAGCTCAGCAGCC TGACCTCTGAGGACTCTGCGGTCTATTACTGT GTGAGATATTCTTACGACAGGGATTACAGTCC TATGGACTACTGGGGTCAAGGAACCTCAGTCA CCGTCTCCTCA 208A-877H 83 CAGCTGCAACAGTCTGGACCTGAGCTGGTGAA 84 208A-877H QLQQSGPELVKPGASVKISCKTS GCCTGGGGCTTCAGTGAAAATTTCCTGCAAGA GYTFTENAMHWVKQSRGKSLEWI CTTCTGGATACACATTCACTGAAAACGCCATG GGVNPNNGDTVYTQKFKGKATLT CACTGGGTGAAGCAGAGCCGTGGAAAGAGCCT VAKSSSTAYMELRTMTCEESTVY TGAGTGGATTGGAGGTGTTAATCCTAACAATG YCASREPDFFDYWGQGSSVTVSS GTGATACTGTCTACACCCAGAAGTTCAAGGGC AAGGCCACATTGACTGTAGCCAAGTCTTCCAG CACAGCCTACATGGAGCTCCGCACCATGACAT GTGAGGAATCTACAGTGTATTACTGTGCAAGC CGGGAACCGGACTTCTTTGACTACTGGGGCCA AGGCTCCTCTGTCACAGTCTCCTCA 208B-251H 85 CAGCTGCAACAGTCTGGACCTGTACTGGTGAA 86 208B-251H QLQQSGPVLVKPGASVKISCKTS GCCTGGGGCTTCAGTGAAAATTTCCTGCAAGA GYTFTENAMHWVKQSHGKSLEWI CTTCTGGATACACATTCACTGAAAACGCCATG GGVNPNNGDTIYNQKFKGKATLT CACTGGGTGAAGCAGAGCCATGGAAAGAGCCT VDKSSSTAFMELRSLTSEESPVY TGAGTGGATTGGAGGTGTTAATCCTAACAATG FCVRRELDFFDYWGQGTSVTVSS GTGATACTATCTACAACCAGAAGTTCAAGGGC AAGGCCACATTGACTGTAGACAAGTCCTCCAG CACAGCCTTCATGGAGCTCCGCAGCCTGACAT CTGAAGAATCCCCAGTCTATTTCTGTGTAAGA CGGGAACTGGACTTCTTTGACTACTGGGGCCA AGGCACCTCTGTCACAGTCTCCTCA 208A-110H 87 CAGCTGCAACAGTCTGGCCCTGTCCTGGTGAA 88 208A-110H QLQQSGPVLVKSGTSVKISCKTS 208B- GTCTGGGACTTCAGTTAAAATTTCCTGCAAGA 208B-1070H GYTFTENAMHWVKQSHGQSLEWI 1070H CTTCTGGATACACATTCACTGAAAACGCCATG GGVNPNNGDTIFNQKFKGKATLT CACTGGGTGAAACAGAGCCATGGACAGAGCCT VDKSSSTAFMELRSLTSEESTVY TGAGTGGATTGGAGGTGTTAATCCTAACAATG YCVRRELDFFDYWGQGTSVTVSS GTGATACTATCTTCAACCAGAAGTTCAAGGGC AAGGCCACATTGACTGTAGACAAGTCCTCCAG CACAGCCTTCATGGAGCTCCGCAGCCTGACAT CTGAAGAATCCACAGTCTATTACTGTGTAAGA CGGGAACTGGACTTCTTTGACTACTGGGGTCA AGGCACCTCTGTCACAGTCTCCTCA 208A-334H 89 CAGCTGCAACAGTCTGGCCCTGTCCTGGTGAA 90 208A-334H QLQQSGPVLVKPGTSVKISCKTS 208A-920H GCCTGGGACTTCAGTGAAAATTTCCTGCAAGA 208A-920H GYTFTENAMHWVKQSHGKSLEWI CTTCTGGATACACATTCACTGAAAACGCCATG GGVNPNNGDTIFNQHFKGKATLT CACTGGGTGAAACAGAGCCATGGAAAGAGCCT VDKSSSTAFMELRSLTSEESTVY TGAGTGGATTGGAGGTGTTAATCCTAACAATG YCVRRELDFFDYWGQGTSVTVSS GTGATACTATCTTCAACCAGAAGTTCAAGGGC AAGGCCACATTGACTGTAGACAAGTCCTCCAG CACAGCCTTCATGGAGCTCCGCAGCCTGACAT CTGAAGAATCCACAGTCTATTACTGTGTAAGA CGGGAACTGGACTTCTTTGACTACTGGGGCCA AGGCACCTCTGTCACAGTCTCCTCA 208A-159H 91 CAGCTGCAACAGTCTGGACCTGCCCAGGTGAA 92 208A-159H QLQQSGPAQVKPGASVMISCKTS GCCTGGGGCTTCAGTGATGATTTCCTGCAAGA GYTFTENAMHWVKQSHGKSLEWI CTTCTGGATACACATTCACTGAAAACGCCATG GGVNPNNGDTIFNQHFKDKAALT CACTGGGTGAAACAGAGCCATGGAAAGAGCCT VDKSSSTAFMELRSLTSEESTVY TGAGTGGATTGGAGGTGTTAATCCTAACAATG YCVRRELDFFDYWGQGTSVTVSS GTGATACTATTTTCAACCAGAAGTTCAAGGAC AAGGCCGCATTGACTGTAGACAAGTCCTCCAG CACAGCCTTCATGGAGCTCCGCAGCCTGACAT CTGAGGAGTCCACAGTCTATTACTGTGTAAGA CGGGAATTGGACTTCTTTGACTACTGGGGCCA AGGCACCTCTGTCACAGTCTCCTCA 208A-126H 93 CAGCTGCAACAGTCTGGACCTGTCCTGGTGAA 94 208A-126H QLQQSGPVLVKPGASVMISCKTS GCCTGGGGCTTCAGTGATGATTTCCTGCAAGA GYTFTENAMHWVKQSHGKSLEWI CTTCTGGATACACATTCACTGAAAACGCCATG GGVNPNNGDTIFNQHFKGKATLT CACTGGGTGAAACAGAGCCATGGAAAGAGCCT VDKSSNTAFTELRSLPSEESTVY TGAGTGGATTGGAGGTGTTAATCCTAACAATG YCVRRGLDFFDYWGQGTSVTVSS GTGATACTATTTTCAACCAGAAGTTCAAGGGC AAGGCCACATTGACTGTAGACAAGTCCTCCAA CACAGCCTTCACGGAGCTCCGCAGCCTGCCAT CTGAAGAATCCACAGTCTATTACTGTGTTAGA CGGGGATTGGACTTCTTTGACTACTGGGGCCA AGGCACCTCTGTCACAGTCTCCTCA 208A-133H 95 CAGCTGCAACAGTCTGGCCCTGTCCTGGTGGG 96 208A-133H QLQQSGPVLVGPGASVMISCKTS GCCTGGGGCTTCAGTGATGATTTCCTGCAAGA GYTFTENAMHWVEQSHGKSLEWI CTTCTGGATACACATTCACTGAAAACGCCATG GGVNPNNGDTIFNQHFKGKATLT CACTGGGTGGAACAGAGCCATGGAAAGAGCCT VDKSSSTAFMEFRSLTSEESTVY TGAGTGGATTGGAGGTGTTAATCCTAACAATG YCVRRELDFFDYWGQGTSVTVSS GTGATACTATTTTCAACCAGAAGTTCAAGGGC AAGGCCACATTGACTGTAGACAAGTCCTCCAG CACAGCCTTCATGGAGTTCCGCAGCCTGACAT CTGAAGAATCCACAGTCTATTACTGTGTAAGA CGGGAATTGGACTTCTTTGACTACTGGGGCCA AGGCACCTCTGTCACAGTCTCCTCA 208B-556H 97 CAGCTGCAACAGTCTGGACCTGTCCTGGTGAA 98 208B-556H QLQQSGPVLVKPGASVMISCKTS GCCTGGGGCTTCAGTGATGATTTCCTGCAAGA GYTFTENAMHWVKQSHGKSLEWI CTTCTGGATACACATTCACTGAAAACGCCATG GGVNPNNGDTIFNQHFKGKATLT CACTGGGTGAAACAGAGCCATGGAAAGAGCCT VDKSSSTAFMEFRSLTSEESTVY TGAGTGGATTGGAGGTGTTAATCCTAACAATG YCVRRELDFFDYWGQGTSVTVSS GTGATACTATTTTCAACCAGAAGTTCAAGGGC AAGGCCACATTGACTGTAGACAAGTCCTCCAG CACAGCCTTCATGGAGTTCCGCAGCCTGACAT CTGAAGAATCCACAGTCTATTACTGTGTAAGA CGGGAATTGGACTTCTTTGACTACTGGGGCCA AGGCACCTCTGTCACAGTCTCCTCAACA 208A-741H 99 CAGCTGCAACAGTCTGGACCTGTCCTGGTGAA 100 208A-741H QLQQSGPVLVKPGASVMISCKTS GCCTGGGGCTTCAGTGATGATTTCCTGCAAGA GYTFTENAMHWVKQSHGKSLEWI CTTCTGGATACACATTCACTGAAAACGCCATG GGVNPNNGDTIFNQHFKGKATLT CACTGGGTGAAACAGAGCCATGGAAAGAGCCT VDKSSSTAFMELRSLTSEESTVY TGAGTGGATTGGAGGTGTTAATCCTAACAATG YCVRRELDFFDYWGQGTSVTVSS GTGATACTATTTTCAACCAGAAGTTCAAGGGC AAGGCCACATTGACTGTAGACAAGTCCTCCAG CACAGCCTTCATGGAGCTCCGCAGCCTGACAT CTGAAGAATCCACAGTCTATTACTGTGTAAGA CGGGAATTGGACTTCTTTGACTACTGGGGCCA AGGCACCTCTGTCACAGTCTCCTCA 208A-1064 101 GAATACGCCATGCAC 102 208A-1064 EYAMH CDR H1 CDR H1 208B-1094 208B-1094 CDR H1 CDR H1 208A-293 208A-293 CDR H1 CDR H1 208A-222 208A-222 CDR H1 CDR H1 208B-560 208B-560 CDR H1 CDR H1 208A-605 103 GAATACGCCATACAC 104 208A-605 EYAIH CDR H1 CDR H1 208A-830 208A-830 CDR H1 CDR H1 208A-134 105 AGTAGTTATATAAGT 106 208A-134 SSYIS CDR H1 CDR H1 208A-692 208A-692 CDR H1 CDR H1 208A-557 208A-557 CDR H1 CDR H1 208B-281 208B-281 CDR H1 CDR H1 208A-352 107 AGTAGTTTTATAAGT 108 208A-352 SSFIS CDR H1 CDR H1 208A-983 109 AGTAGTTATTTTAGT 110 208A-983 SSYFS CDR H1 CDR H1 208B-471 111 AGCAGTACCTATATAAGT 112 208B-471 STYIS CDR H1 CDR H1 208A-207 113 AGCTACTGGATACAC 114 208A-207 SYWIH CDR H1 CDR H1 208A-638 208A-638 CDR H1 CDR H1 208B-515 208B-515 CDR H1 CDR H1 208A-874 115 AGCTATTGGATGCAC 116 208A-874 SYWMH CDR H1 CDR H1 208B-911 117 CCTACTCGATAAAC 118 208B-911 TYSIN CDR H1 CDR H1 208B-1096 119 GCCAACAAGATGCAC 120 208B-1096 ANKMH CDR H1 CDR H1 208B-589 208B-589 CDR H1 CDR H1 208B-395 208B-395 CDR H1 CDR H1 208B-189 208B-189 CDR H1 CDR H1 208B-547 121 AGCAACAAGATGCAC 122 208B-547 SNKMH CDR H1 CDR H1 208A-210 123 AGCTACTATGTACAC 124 208A-210 SYYVH CDR H1 CDR H1 208A-422 125 AGTGGTTATTACTGGATC 126 208A-422 SGYYWI CDR H1 CDR H1 208A-442 208A-442 CDR H1 CDR H1 208B-862 127 AGTGATTATGCCTGGAAC 128 208B-862 SDYAWN CDR H1 CDR H1 208A-967 129 AGCTATGGTGTACAC 130 208A-967 SYGVH CDR H1 CDR H1 208B-517 131 ACCCATGGTGTACAC 132 208B-517 THGVH CDR H1 CDR H1 208B-822 133 ACCTATGGTGTACAC 134 208B-822 TYGVH CDR H1 CDR H1 208B-1024 135 AACTATGGCATGTCT 136 208B-1024 NYGMS CDR H1 CDR H1 208B-327 137 GACTCTTATATGTAT 138 208B-327 DSYMY CDR H1 CDR H1

208B-353 139 CACTATGGCATGTCT 140 208B-353 HYGMS CDR H1 CDR H1 208B-178 141 AGCTATTACATGTCT 142 208B-178 SYYMS CDR H1 CDR H1 208B-672 143 ACCTATGCCATGTCT 144 208B-672 TYAMS CDR H1 CDR H1 208B-793 145 AGCTATGCCATGTCT 146 208B-793 SYAMS CDR H1 CDR H1 208B-826 147 TCCTTTGGAATGCAC 148 208B-826 SFGMH CDR H1 CDR H1 208B-174 149 AGCTACTGGATGTCT 150 208B-174 SYWMS CDR H1 CDR H1 208B-408 151 GATTACTACATGAGC 152 208B-408 DYYMS CDR H1 CDR H1 208B-612 153 AGCTATGTTATGCAC 154 208B-612 SYVMH CDR H1 CDR H1 208A-877 155 GAAAACGCCATGCAC 156 208A-877 ENAMH CDR H1 CDR H1 208B-251 208B-251 CDR H1 CDR H1 208A-110 208A-110 CDR H1 CDR H1 208B-1070 208B-1070 CDR H1 CDR H1 208A-334 208A-334 CDR H1 CDR H1 208A-920 208A-920 CDR H1 CDR H1 208A-159 208A-159 CDR H1 CDR H1 208A-126 208A-126 CDR H1 CDR H1 208A-133 208A-133 CDR H1 CDR H1 208B-556 208B-556 CDR H1 CDR H1 208A-741 208A-741 CDR H1 CDR H1 208A-1064 157 GGTATCAATCCTACTAATGGTGATACAATCTA 158 208A-1064 GINPTNGDTIYNQHFKD CDR H2 CAACCAGAAGTTCAAGGAC CDR H2 208A-293 208A-293 CDR H2 CDR H2 208B-1094 159 GGTATCAATCCTACTAATGGTGATACAATCTA 160 208B-1094 GINPTNGDTIYNQRFKD CDR H2 CAACCAGAGGTTCAAGGAC CDR H2 208A-222 161 GGTATTAATCCTAACAATGGCAATGCTATCTA 162 208A-222 GINPNNGNAIYNQIFKD CDR H2 CAACCAGATATTCAAGGAC CDR H2 208A-605 163 GGTATTAATCCTAGCAATGGCAATGCTATCTA 164 208A-605 GINPSNGNAIYNQIFKD CDR H2 CAACCAAATATTCAAGGAC CDR H2 208B-560 208B-560 CDR H2 CDR H2 208A-830 165 GGTATTAATCCTAGCAATGGCGATCCTATCTA 166 208A-830 GINPSNGDPITNQIFKD CDR H2 TAACCAGATATTCAAGGAC CDR H2 208A-134 167 TGGATTTATGCTGGAACTGGTAATACTAACTA 168 208A-134 WIYAGTGNTNYNQKFTD CDR H2 TAATCAGAAGTTCACAGAC CDR H2 208A-557 208A-557 CDR H2 CDR H2 208A-352 208A-352 CDR H2 CDR H2 208A-692 169 TGGATTTTTGCTGGAACTGGTAATACTAATTA 170 208A-692 WIFAGTGNTNYNQKFTD CDR H2 TAATCAGAAGTTCACAGAC CDR H2 208A-983 171 TGGATTTATGCTGGAACTGGTAATACTATCTA 172 208A-983 WIYAGTGNTIYNQKFTD CDR H2 TAATCAGAAGTTCACAGAC CDR H2 208B-281 173 TGGATTTATGCTGGAACTGGTGGTACTAACTA 174 208B-281 WIYAGTGGTNYNQKFTD CDR H2 TAATCAGAAGTTCACAGAC CDR H2 208B-471 175 TGGATTTATGCTGGAACTGGTGCTACTAATTA 176 208B-471 WIYAGTGATNYNQKFTG CDR H2 TAATCAGAAGTTCACAGGC CDR H2 208A-207 177 GAAATTGATCCTTCTGACAGTTATATTAACCA 178 208A-207 EIDPSDSYINQNQHFRG CDR H2 GAATCAAAAGTTCAGGGGC CDR H2 208A-638 179 GAAGTTGATCCTTCTGACAGTTATATTAACCA 180 208A-638 EVDPSDSYINQNEKERG CDR H2 GAATGAAAAGTTCAGGGGC CDR H2 208B-515 181 GAGATTGATCCTTCTGATAGTTATACTAACTA 182 208B-515 EIDPSDSYTNYNQHFKG CDR H2 CAATCAAAAGTTCAAGGGC CDR H2 208A-874 183 GAGATTCATCCTTCTGATAGTTATACTAGCTA 184 208A-874 EIHPSDSYTSYNQHFKD CDR H2 CAATCAAAAGTTCAAGGAC CDR H2 208B-911 185 AATATTTATCCTTCTACCAGTCATACTAACTA 186 208B-911 NIYPSTSHTNYNQKFRD CDR H2 CAATCAAAAGTTCAGGGAC CDR H2 208B-1096 187 TACATTGATCCTAGCTCTGGTTATACTGAATA 188 208B-1096 YIDPSSGYTEYNHKIQY CDR H2 CAATCATAAGATCCAGTAC CDR H2 208B-589 189 TACATTGATCCTAGCTCTGGTTATACTGAATA 190 208B-589 YIDPSSGYTEYNHKIQD CDR H2 CAATCATAAGATCCAGGAC CDR H2 208B-395 191 TACATTGATCCTAGCTCTGGTTATACTCAATA 192 208B-395 YIDPSSGYTQYNHKIQD CDR H2 CAATCATAAGATCCAGGAC CDR H2 208B-189 193 TACATTGATCCTGCCTCTGGCTATACTGAATA 194 208B-189 YIDPASGYTEYNQKIKD CDR H2 CAATCAGAAGATCAAGGAC CDR H2 208B-547 195 TACATTGATCCTAGCTCTGGTTATACTGAATA 196 208B-547 YIDPSSGYTEYNQKIKD CDR H2 CAATCAGAAGATCAAGGAC CDR H2 208A-210 197 TGTATTTATCCTGGAGATGTTAATACTGACTA 198 208A-210 CIYPGDVNTDYNEKFKG CDR H2 TAATGAGAAGTTCAAGGGC CDR H2 208A-422 199 TACATAAAGTACGACGGTGGCAATAACTACAG 200 208A-422 YIKYDGGNNYSPSLKN CDR H2 CCCATCTCTCAAAAAT CDR H2 208A-442 208A-442 CDR H2 CDR H2 208B-862 201 TACATAAGCTACAGTGGTACCACTGTCTACAG 202 208B-862 YISYSGTTVYSPSLKS CDR H2 CCCATCTCTCAAAAGT CDR H2 208A-967 203 GTAATATGGGCTGTTGGAAGTATAAATTATAA 204 208A-967 VIWAVGSINYNSALMS CDR H2 TTCGGCTCTCATGTCC CDR H2 208B-517 205 GTGATATGGAGTGGTGGAAGCACAGACTATAA 206 208B-517 VIWSGGSTDYNAAFIS CDR H2 TGCAGCTTTCATATCC CDR H2 208B-822 207 GTGATATGGGGTGGTGGAAGCACAGGCTATAA 208 208B-822 VIWGGGSTGYNAAFVS CDR H2 TGCAGCTTTCGTATCC CDR H2 208B-1024 209 ACCATTAGTAGTGGTGGTAGTTATAGCTACTA 210 208B-1024 TISSGGSYSYYPDSVKG CDR H2 TCCAGACAGTGTAAAGGGG CDR H2 208B-327 211 ACCATTAGTGATGGTGGTAGTTACACCTTCTA 212 208B-327 TISDGGSYTFYPDSVKG CDR H2 TCCAGACAGTGTGAAGGGA CDR H2 208B-353 213 ACCATTAGTAGTGGTGGTGGTTACACCTACTA 214 208B-353 TISSGGGYTYYPDSVKG CDR H2 TCCAGACAGTGTGAAGGGG CDR H2 208B-178 215 GCCATTAATAGTAATGGTGGTAGCACCTACTA 216 208B-178 AINSNGGSTYYPDTVKG CDR H2 TCCAGACACTGTGAAGGGC CDR H2 208B-672 217 TACATTAGTAATGGTGGTGGTAACACCTACTA 218 208B-672 YISNGGGNTYYVDTVKG CDR H2 TGTAGACACTGTAAAGGGC CDR H2 208B-793 219 TACATTAGTAATGGTGGTGGTAGCACCTACTA 220 208B-793 YISNGGGSTYYSDTVKG CDR H2 TTCAGACACTGTAAAGGGC CDR H2 208B-826 221 TCCATTAGTAGTCGCACTAGTAAGATCTACTA 222 208B-826 SISSRTSKITYADNLKG CDR H2 TGCAGACAACCTGAAGGGC CDR H2 208B-174 223 GAAATTAGATTGAGATCTGATAATTATGCAAC 224 208B-174 EIRLRSDNYATHYAESVKG CDR H2 CCATTATGCGGAGTCTGTGAAAGGG CDR H2 208B-408 225 TTTATTAGAAACAAAGCTTATGGTTACACGAC 226 208B-408 FIRNKAYGYTTEFSASVNG CDR H2 CGAGTTCAGTGCATCTGTGAACGGT CDR H2 208B-612 227 TATATTGATCCTCACAATGATGATACAAAATA 228 208B-612 YIDPHNDDTKYSEKFRG CDR H2 CAGTGAGAAGTTCAGAGGT CDR H2 208A-877 229 GGTGTTAATCCTAACAATGGTGATACTGTCTA 230 208A-877 GVNPNNGDTVYTQKFKG CDR H2 CACCCAGAAGTTCAAGGGC CDR H2 208B-251 231 GGTGTTAATCCTAACAATGGTGATACTATCTA 232 208B-251 GVNPNNGDTIYNQKFKG CDR H2 CAACCAGAAGTTCAAGGGC CDR H2 208A-110 233 GGTGTTAATCCTAACAATGGTGATACTATCTT 234 208A-110 GVNPNNGDTIFNQKFKG CDR H2 CAACCAGAAGTTCAAGGGC CDR H2 208B-1070 208B-1070 CDR H2 CDR H2 208A-334 208A-334 CDR H2 CDR H2 208A-920 208A-920 CDR H2 CDR H2 208A-126 208A-126 CDR H2 CDR H2 208A-133 208A-133 CDR H2 CDR H2 208B-556 208B-556 CDR H2 CDR H2 208A-741 208A-741 CDR H2 CDR H2 208A-159 235 GGTGTTAATCCTAACAATGGTGATACTATTTT 236 208A-159 GVNPNNGDTIFNQKFKD CDR H2 CAACCAGAAGTTCAAGGAC CDR H2 208A-1064 237 CGGGAACTGGACTACTTTGCCTCC 238 208A-1064 RELDYFAS CDR H3 CDR H3 208B-1094 208B-1094 CDR H3 CDR H3 208A-293 239 CGGGAACTGGACTACTTTCCCTCC 240 208A-293 RELDYFPS CDR H3 CDR H3 208A-222 241 CGACAACTGGACTACTTTGACTAT 242 208A-222 RQLDYFDY CDR H3 CDR H3 208A-605 243 CGACAACTGGACTTCTTTGACTAT 244 208A-605 RQLDFFDY CDR H3 CDR H3 208B-560 208B-560 CDR H3 CDR H3 208A-830 245 CGACAACTGGACTACTTTGACTTT 246 208A-830 RQLDYFDF CDR H3 CDR H3

208A-134 247 AGTGGGACGGGGTTTACTTAC 248 208A-134 SGTGFTY CDR H3 CDR H3 208A-692 208A-692 CDR H3 CDR H3 208A-557 208A-557 CDR H3 CDR H3 208A-352 208A-352 CDR H3 CDR H3 208A-983 208A-983 CDR H3 CDR H3 208B-281 249 AGTGGGACGGGGTTTATTTAC 250 208B-281 SGTGFIY CDR H3 CDR H3 208B-471 251 TCTGGGGCGGGGGTTTAC 252 208B-471 SGAGVY CDR H3 CDR H3 208A-207 253 CATTACTACGGTGTTCTTGACTCC 254 208A-207 HYYGVLDS CDR H3 CDR H3 208A-638 208A-638 CDR H3 CDR H3 208B-515 255 CATTACTACGGTGTCTTTGACTCC 256 208B-515 HYYGVFDS CDR H3 CDR H3 208A-874 257 GGGGGCTACTATAGGTACGACGAGTTTGCTTA 258 208A-874 GGYYRYDEFAY CDR H3 C CDR H3 208B-911 259 AATGCCTATTCTATGGACTAC 260 208B-911 NAYSMDY CDR H3 CDR H3 208B-1096 261 TTTGCTTAC 262 208B-1096 FAY CDR H3 CDR H3 208B-589 208B-589 CDR H3 CDR H3 208B-395 208B-395 CDR H3 CDR H3 208B-189 208B-189 CDR H3 CDR H3 208B-547 208B-547 CDR H3 CDR H3 208A-210 263 TATTACTACGGTAGTTTTGCTTAC 264 208A-210 YYYGSFAY CDR H3 CDR H3 208A-422 265 GGGTCGGACTCCTTTGACTAC 266 208A-422 GSDSFDY CDR H3 CDR H3 208A-442 208A-442 CDR H3 CDR H3 208B-862 267 AATTAC 268 208B-862 NY CDR H3 CDR H3 208A-967 269 GATCGGACTACGGCTACCCCCTTCTTTGACTA 270 208A-967 DRTTATPFFDY CDR H3 C CDR H3 208B-517 271 AATGGGGGGGCTACGGCCTTTGACTAC 272 208B-517 NGGATAFDY CDR H3 CDR H3 208B-822 273 AATGGAGGGGCTACGGTCTTTGACTAC 274 208B-822 NGGATVFDY CDR H CDR H 208B-1024 275 CTCTACTACGGCTACGGGGACTAC 276 208B-1024 LYYGYGDY CDR H3 CDR H3 208B-327 277 CCCCATGCTGGCTACTTCGGCTGGTTTGCTTA 278 208B-327 PHAGYFGWFAY CDR H3 C CDR H3 208B-353 279 CTATACGGTAGCCTGTTTGCTTAC 280 208B-353 LYGSLFAY CDR H3 CDR H3 208B-178 281 CATGGGGGACTGGGACGTAGGGACTGGTACTT 282 208B-178 HGGLGRRDWYFDV CDR H3 CGATGTC CDR H3 208B-672 283 CATGGGCTCTACTGGGGCTATTCTATGGACTA 284 208B-672 HGLYWGYSMDY CDR H3 C CDR H3 208B-793 285 CATGGACTGGGAAGGACAGGGTTTGCTTCC 286 208B-793 HGLGRTGFAS CDR H3 CDR H3 208B-826 287 TCCGTCTTTGGTAATTCTTAC 288 208B-826 SVFGNSYWFFDV CDR H3 CDR H3 208B-174 289 ATGACGTAC 290 208B-174 MTY CDR H3 CDR H3 208B-408 291 GTCCTCTACTATGATTACGGGGGATTTGCTTA 292 208B-408 VLYYDYGGFAY CDR H3 C CDR H3 208B-612 293 TATTCTTACGACAGGGATTACAGTCCTATGGA 294 208B-612 YSYDRDYSPMDY CDR H3 CTAC CDR H3 208A-877 295 CGGGAACCGGACTTCTTTGACTAC 296 208A-877 REPDFFDY CDR H3 CDR H3 208B-251 297 CGGGAATTGGACTTCTTTGACTAC 298 208B-251 RELDFFDY CDR H3 CDR H3 208A-110 208A-110 CDR H3 CDR H3 208B-1070 208B-1070 CDR H3 CDR H3 208A-334 208A-334 CDR H3 CDR H3 208A-920 208A-920 CDR H3 CDR H3 208A-159 208A-159 CDR H3 CDR H3 208A-133 208A-133 CDR H3 CDR H3 208B-556 208B-556 CDR H3 CDR H3 208A-741 208A-741 CDR H3 CDR H3 208A-126 299 CGGGGATTGGACTTCTTTGACTAC 300 208A-126 RGLDFFDY CDR H3 CDR H3 208A- 301 GACATTGTGCTGACACAGTCTCCTGCTTCCTT 302 208A-1064L DIVLTQSPASLIVSLGQTATISC 1064L AATTGTTTCTCTGGGGCAGACGGCCACCATCT RASQSVSTSRFSYLHWIQQFPGQ CATGCAGGGCCAGCCAAAGTGTCAGTACATCT PPKLLIKYASNLESGVPVRFSGS AGGTTTAGTTATCTGCACTGGATCCAACAGAA GSGTDFTLNIHPVEEEDTATYFC ACCAGGGCAGCCACCCAAACTCCTCATCAAGT QHSWEFPFTFGSGTKLKIKR ATGCATCCAACCTTGAATCTGGGGTCCCTGTC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACCCTCAACATCCATCCTGTGGAGGAGGAGG ATACTGCAACATATTTCTGTCAGCACAGTTGG GAGTTTCCATTCACGTTCGGCTCGGGGACAAA GTTGAAAATAAAACGGGCTGATGCTGC 208B- 303 GACATTGTGCTGACACAGTCTCCTGCTTCCTT 304 208B-1094L DIVLTQSPASLIVSLGQTATISC 1094L AATTGTTTCTCTGGGGCAGACGGCCACCATCT RASQSLSTSRFSYVHWIQQFPGQ CATGCAGGGCCAGCCAAAGTCTCAGTACATCT PPKLLIKYASNLESGVPVRFSGS AGGTTTAGCTATGTGCACTGGATCCAACAGAA GSGTDFTLNIHPVEEEDTATYFC ACCAGGGCAGCCACCCAAACTCCTCATCAAGT QHSWEFPFTFGSGTKLKIKR ATGCATCCAACCTTGAATCTGGGGTCCCTGTC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACCCTCAACATCCATCCTGTGGAGGAGGAGG ATACTGCAACATATTTCTGTCAGCACAGTTGG GAGTTTCCATTCACGTTCGGCTCGGGGACAAA GTTGAAAATAAAACGGGCTGATGCTGC 208A-293L 305 GACATTGTGCTGACACAGTCTCCTGCTTCCTT 306 208A-293L DIVLTQSPASLIVSLGQRATISC AATTGTATCTCTGGGGCAGAGGGCCACCATCT RASQSVSTSRFSYVHWIQQFPGQ CATGTAGGGCCAGCCAAAGTGTCAGTACATCC PPKLLIKYASNLESGVPVRFSGS AGGTTTAGTTATGTGCACTGGATCCAACAGAA GSGTDFILNIHPVEEEDTATYFC ACCAGGGCAGCCACCCAAACTCCTCATCAAGT QHSWEFPFTFGSGTKLEIKR ATGCATCCAACCTTGAATCTGGGGTCCCTGTC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CATCCTCAACATCCATCCTGTGGAGGAGGAGG ATACTGCAACATATTTCTGTCAGCACAGTTGG GAGTTTCCATTCACGTTCGGCTCGGGGACAAA GTTGGAAATAAAACGGGCTGATGCTGC 208A-134L 307 GAAGTTTTGATGACCCAAAGTCCACTCTCCCT 308 208A-134L EVLMTQSPLSLPVSLGDQASISC GCCTGTCAGTCTTGGAGATCAGGCCTCCATCT RSSQSLEHTNGNTYLEWFLQRPG CTTGCAGATCTAGTCAGAGCCTTGAACATACT QPPKLLITKVSSRFSGVPDRFSG AATGGAAACACCTATTTAGAGTGGTTCCTGCA SGSGTDFTLKISRVEAEDLGVYY GAGACCAGGCCAGCCTCCAAAGCTCCTGATCT CFQGSHVPFTFGSGTKLAIKR ACAAAGTTTCCAGCCGATTTTCTGGGGTCCCA GACAGGTTCAGTGGCAGTGGATCAGGGACAGA TTTCACACTCAAGATCAGCAGAGTGGAGGCTG AGGATCTGGGAGTTTATTACTGTTTTCAAGGT TCACATGTTCCATTCACGTTCGGCTCGGGGAC AAAGTTGGCAATAAAACGGGCTGATGCTGC 208A-692L 309 GAAGTTTTGATGACCCAAAGTCCACTCTCCCT 310 208A-692L EVLMTQSPLSLPVSLGDQASISC GCCTGTCAGTCTTGGAGATCAGGCCTCCATCT RSSQSLEHSNGNTYLEWFLQRPG CTTGCAGATCTAGTCAGAGCCTTGAACATAGT QPPKLLIYKVSSRFSGVPDRFSG AATGGAAACACCTATTTAGAGTGGTTCCTGCA SGSGTDFTLKISRVEAEDLGVYY GAGACCAGGCCAGCCTCCAAAGCTCCTGATCT CFQGSHVPFTFGSGTKLAIKR ACAAAGTTTCCAGCCGATTTTCTGGGGTCCCA GACAGGTTCAGTGGCAGTGGATCAGGGACAGA TTTCACACTCAAGATCAGCAGAGTGGAGGCTG AGGATCTGGGAGTTTATTACTGTTTTCAAGGT TCACATGTTCCATTCACGTTCGGCTCGGGGAC AAAGTTGGCAATAAAACGGGCTGATGCTGC 208A-352L 311 GATGTTTTGATGGCCCAAACTCCACTCTCCCT 312 208A-352L DVLMAQTPLSLPVTLGDQASISC GCCTGTCACCCTTGGAGATCAAGCCTCCATCT RSSQSLVHSNGNTYLEWFLQKPG CTTGCAGATCTAGTCAGAGCCTTGTACATAGT QSPKLLIYNVSNRFSGVPDRFSG AATGGAAACACCTATTTAGAGTGGTTCCTGCA SGSGTDFTLKISRVEAEDLGVYY GAAACCAGGCCAGTCTCCAAAGCTCCTGATCT CFQGSHVPLTFGSGTKLEIKR ACAACGTTTCCAACCGATTTTCTGGGGTCCCA GACAGGTTCAGTGGCAGTGGATCAGGGACAGA TTTCACACTCAAGATCAGCAGAGTGGAGGCTG AGGATCTGGGAGTTTATTACTGCTTTCAAGGT TCACATGTTCCACTCACGTTCGGCTCGGGGAC AAAGTTGGAAATAAAACGGGCTGATGCTGC 208A-983L 313 GATGTTTTGATGACCCAAACTCCACTCTCCCT 314 208A-983L DVLMTQTPLSLPVNLGDQASISC GCCTGTCAATCTTGGAGATCAGGCCTCCATCT RSSQSLLHSNGNTYLEWFLQKPG CTTGCAGATCTAGTCAGAGCCTTCTACATAGT QSPKLLIYNVSNRFSGVPDRFSG AATGGAAACACCTATTTAGAGTGGTTCCTGCA SGSGTDFTLKISRVEAEDLGVYY GAAACCAGGCCAGTCTCCAAAGCTCCTGATCT CFQGSHVPFTFGSGTKLAIKR ACAATGTTTCCAACCGATTTTCTGGGGTCCCA GACAGGTTCAGTGGCAGTGGATCAGGGACAGA TTTCACACTCAAGATCAGCAGAGTGGAGGCTG AGGATCTGGGAGTTTATTACTGCTTTCAAGGT TCACATGTTCCATTCACGTTCGGCTCGGGGAC AAAGTTGGCAATAAAACGGGCTGATGCTGC 208B-281L 315 GATGTTTTGATGACCCAAACTCCACTCTCCCT 316 208B-281L DVLMTQTPLSLPVSLGDQASISC GCCTGTCAGTCTTGGAGATCAAGCCTCCATCT RSSQSLVHNNGNTYLEWFLQKPG CTTGCAGATCTAGTCAGAGCCTTGTACATAAT QSPKLLIYKVSNRFSGVPDRFSG AATGGAAACACCTATTTAGAATGGTTCCTGCA SGSGTDFTLKISRVEAEDLGVYY GAAACCAGGCCAGTCTCCAAAGCTCCTGATCT CFQGSHVPFTFGSGTKLEIKR ACAAAGTTTCCAACCGATTTTCTGGGGTCCCA GACAGGTTCAGTGGCAGTGGATCAGGGACAGA TTTCACACTCAAGATCAGCAGAGTGGAGGCTG AGGATCTGGGAGTTTATTACTGCTTTCAAGGT TCACATGTTCCATTCACGTTCGGCTCGGGGAC AAAGTTGGAAATAAAACGGGCTGATGCTGC 208B- 317 GATGTTTTGATGACCCAAACTCCACTCTCCCT 318 208B-1024L DVLMTQTPLSLPVSLGDQASISC 1024L GCCTGTCAGTCTTGGAGATCAAGCCTCCATCT RSSQSIVHSNGNTYLEWYLQKLG CTTGCAGATCTAGTCAGAGCATTGTACACAGT QSPKLLITRVSNRFSGVPDRFSG AATGGAAACACCTATTTAGAGTGGTACCTGCA SGSGTDFTLKISRVEAEDLGVYY GAAACTAGGCCAGTCTCCAAAGCTCCTGATCT CFQGSHVPWTFGGGTKLEIKR ACAGAGTTTCCAACCGATTTTCTGGGGTCCCA GACAGGTTCAGTGGCAGTGGATCAGGGACAGA TTTCACACTCAAGATCAGCAGAGTGGAGGCTG AGGATCTGGGAGTTTATTACTGCTTTCAAGGT TCACATGTTCCGTGGACGTTCGGTGGAGGCAC CAAGCTGGAAATCAAACGGGCTGATGCTGC 208B-471L 319 GATGTTGTGATGACCCAAACTCCACTCTCCCT 320 208B-471L DVVMTQTPLSLPVSLGDQASISC GCCTGTCAGTCTTGGAGATCAAGCCTCCATCT RSSQSLLHSNGNTYLEWYLQKPG CTTGCAGATCTAGTCAGAGCCTTCTACATAGT QSPNLLIYNVSNRFSGVPDRFSG AATGGAAACACCTATTTAGAATGGTACCTGCA SGSGTDFALKISRVGAEDLGVYY GAAACCTGGCCAGTCTCCAAACCTCCTGATCT CFQGSHVPLTFGAGTKLELKR ACAATGTTTCCAACCGATTTTCTGGGGTCCCA GACAGGTTCAGTGGCAGTGGATCAGGGACAGA TTTCGCACTCAAGATCAGCAGAGTGGGGGCTG AGGATCTGGGAGTTTATTACTGCTTTCAAGGT TCACATGTTCCGCTCACGTTCGGTGCTGGGAC CAAGCTGGAGCTGAAACGGGCTGATGCTGC 208B-862L 321 GATGTTGTGCTGACCCAAACTCCACTCTCCCT 322 208B-862L DVVLTQTPLSLPFSFGNKASIFC GCCTCTCAGTCTTGGAGATCAGGCCTCCATCT KFSQTLLHRDGNPFLLWYLQKPG CTTGCAGATCTAGTCAGACCCTTCTACACAGT QSPKLLIYKLSNRFFGVPKRFRG

GATGGAGACACCTATTTACATTGGTACCTGCA RGSGTNFPLKISKGEAEDLGVFF GAAGCCAGGCCAGTCTCCAAAGCTCCTGATCT CSQSTHVPYTFGGGTKLEIKR ACAAACTTTCCAACCGATTTTCTGGGGTCCCA GACAGGTTCAGTGGCAGTGGATCAGGGACAGA TTTCACACTCAAGATCAGCAGAGTGGAGGCTG AGGATCTGGGAGTTTATTTCTGCTCTCAAAGT ACACATGTTCCGTACACGTTCGGAGGGGGGAC CAAGCTGGAAATAAAACGGGCTGATGCTGC 208B-589L 323 GATATTGTGATGACCCAGACTCCACTCACTTT 324 208B-589L DIVMTQTPLTLSVTIGQPASISC 208B- GTCGGTTACCATTGGACAACCAGCTTCCATCT 208B-1096L KSSQSLLFTNGKTYLNWFLQRPG 1096L CTTGCAAGTCAAGTCAGAGCCTCTTATTTACT QSPKRLIYLLSKLDSGVPDRFSG AATGGAAAAACCTATTTAAATTGGTTTTTACA SGSGTDFTLKISRVEAEDLGVYY GAGGCCAGGCCAGTCTCCAAAACGCCTAATCT CLQSTYFPLTFGAGTKLELKR ATCTGCTGTCTAAATTGGACTCTGGAGTCCCT GACAGGTTCAGTGGCAGTGGATCAGGGACAGA TTTCACACTGAAAATCAGCAGAGTGGAGGCTG AGGATTTGGGAGTTTATTACTGCTTGCAGAGT ACATATTTTCCTCTCACGTTCGGTGCTGGGAC CAAGCTGGAGCTGAAACGGGCTGATGCTGC 208B-189L 325 GATGTTGTGATGACCCAGACTCCACTCACTTT 326 208B-189L DVVMTQTPLTLSVTIGQPASISC GTCGGTTACCATTGGACAACCAGCTTCCATCT KSSQSLLYTNGKTYLNWLLQRPG CTTGCAAGTCAAGTCAGAGCCTCTTATATACT QSPKRLIYLVSKLDSGVPDRFSG AATGGAAAGACCTATTTGAATTGGTTATTACA SGSGTDFTLKISRVEAEDLGVYY GAGGCCAGGCCAGTCTCCAAAACGCCTAATCT CLQSIHFPYTFGGGTKLDIKR ATCTGGTGTCAAAATTGGACTCTGGAGTCCCT GACAGGTTCAGTGGCAGTGGATCAGGGACAGA TTTCACACTGAAAATCAGCAGAGTGGAGGCTG AGGATTTGGGAGTTTATTACTGCTTGCAGAGT ATACATTTTCCGTACACGTTCGGAGGGGGGAC CAAGCTGGACATAAAACGGGCTGATGCTGC 208B-327L 327 GATATTGTGATGACGCAGGCTGCATTCTCCAA 328 208B-327L DIVMTQAAFSNPVTPGTSVSISC TCCAGTCACTCCTGGAACATCAGTTTCCATCT RSSKSLLHSNGITYLYWYLQKPG CCTGCAGGTCTAGTAAGAGTCTCCTACATAGT QSPQLLIYQMSKIASGVPDRFRS AATGGCATCACTTATTTGTATTGGTATCTGCA SGSGTDFTLRISRVEAADVGVYY GAAGCCAGGCCAGTCTCCTCAGCTCCTGATTT CAQNLELPWTFGGGTKLEIKR ATCAGATGTCCAAGATTGCCTCAGGAGTCCCA GACAGGTTCAGGAGCAGTGGGTCAGGAACTGA TTTCACACTGAGAATCAGCAGAGTGGAGGCTG CGGATGTGGGTGTTTATTACTGTGCTCAAAAT CTAGAACTTCCGTGGACGTTCGGTGGAGGCAC CAAGCTGGAAATCAAACGGGCTGATGCTGC 208A-874L 329 GACATTGTGATGACCCAGTCTCACAAATTCAT 330 208A-874L DIVMTQSHKFMSTSIGDRVSITC GTCCACATCAATAGGAGACAGGGTCAGCATCA KASQNVSPAVAWYQQKPGQSPKL CCTGCAAGGCCAGTCAGAATGTGAGTCCTGCT LIYSASSRYTGVPDRFTGSGSGT GTAGCCTGGTATCAACAGAAACCAGGACAATC AFTFTISSVQAEDLAVYFCQQHF TCCTAAACTACTGATTTACTCGGCATCCTCCC STPWTFGGGTMLEIKR GATACACTGGAGTCCCTGATCGCTTCACTGGC AGTGGATCTGGGACGGCTTTCACTTTCACCAT CAGCAGTGTGCAGGCTGAAGACCTGGCAGTTT ATTTCTGTCAGCAACATTTTAGTACTCCGTGG ACGTTCGGTGGAGGCACCATGCTGGAAATCAA ACGGGCTGATGCTGC 208B-353L 331 GACATTGTGATGACCCAGTCTCAAAAATTCAT 332 208B-353L DIVMTQSQKFMSTTVGDRVRVTC GTCCACAACAGTTGGGGACAGGGTCAGAGTCA KASQNVGTAVAWYQQKPGQSPKL CCTGCAAGGCCAGTCAGAATGTGGGTACTGCT LIYSASNRYTGVPDRFTGSGSGT GTAGCCTGGTATCAACAGAAACCAGGACAATC DFTLTITNMQSEDLADYFCQQYS TCCTAAACTACTGATTTACTCAGCATCCAATC TYPLTFGSGAKLEIKR GGTACACTGGAGTCCCTGATCGCTTCACAGGC AGTGGATCTGGGACAGATTTCACTCTCACCAT TACCAATATGCAGTCTGAAGACCTGGCAGATT ATTTCTGTCAGCAATATAGCACCTATCCTCTC ACGTTCGGCTCGGGGGCAAAGTTGGAAATAAA ACGGGCTGATGCTGC 208B-793L 333 GACATTGTGATGACCCAGTCTCACAAATTCAT 334 208B-793L DIVMTQSHKFMSTSVGDRVSITC GTCCACATCAGTAGGAGACAGGGTCAGCATCA KASQDVGTAVAWYQQKPGQSPKL CCTGCAAGGCCAGTCAGGATGTGGGTACTGCT LIYWASTRHTGVPDRFTGSGSGT GTAGCCTGGTATCAACAGAAACCAGGACAATC DFTLTISNVQSEDLADYFCQQYS TCCTAAACTACTGATTTACTGGGCATCCACCC NYLTFGAGTKLEVKR GGCACACTGGAGTCCCTGATCGCTTCACAGGC AGTGGATCTGGGACAGATTTCACTCTCACCAT TAGCAATGTGCAGTCTGAAGACTTGGCAGATT ATTTCTGTCAGCAATATAGCAACTATCTCACG TTCGGTGCTGGGACCAAGCTGGAGGTGAAACG GGCTGATGCTGC 208B-672L 335 GACATCCAGATGACCCAGTCTCCATCCTCCTT 336 208B-672L DIQMTQSPSSLSASLGERVSLTC ATCTGCCTCTCTGGGCGAAAGAGTCAGTCTCA RASQDIGGSINWLQQEPDGTIER CTTGTCGGGCAAGTCAGGACATTGGTGGTAGC LIYATSSLDSGVPKRFSGSRSGS ATAAACTGGCTTCAGCAGGAACCAGATGGAAC DYSLTISSLESEDFVDYYCLQYA TATTAAACGCCTGATCTACGCCACATCCAGTT SSPPTFGGGTKLEIKR TAGATTCTGGTGTCCCCAAAAGGTTCAGTGGC AGTAGGTCTGGGTCAGATTATTCTCTCACCAT CAGCAGCCTTGAGTCTGAAGATTTTGTAGACT ATTACTGTCTACAATATGCTAGTTCTCCTCCG ACGTTCGGTGGAGGCACCAAACTGGAAATCAA ACGGGCTGATGCTGC 208B-408L 337 GACATCCAGATGACTCAGTCTCCAGCCTCCCT 338 208B-408L DIQMTQSPASLSASVGETVTITC ATCTGCATCTGTGGGAGAAACTGTCACCATCA RASGNIHTYLAWYQQKQGESPQL CATGTCGAGCAAGTGGGAATATTCACACTTAT LVYNANTLADGVPSRFSGSGSGT TTAGCATGGTATCAGCAGAAACAGGGAAAATC QFSLKINSLQPDDEGSYYCQHFW TCCTCAGCTCCTGGTCTACAATGCAAACACCT SAPWTFGGGTQLEIKR TGGCAGATGGTGTGCCATCAAGGTTCAGTGGC AGTGGATCAGGAACACAATTTTCTCTCAAGAT CAACAGTCTGCAGCCTGACGATTTTGGGAGTT ATTACTGTCAACATTTTTGGAGTGCTCCGTGG ACGTTCGGTGGAGGCACCCAGCTGGAAATCAA ACGGGCTGATGCTGC 208A-207L 339 GATATTGTGTTAACTCAGTCTCCAGCCACCCT 340 208A-207L DIVLTQSPATLSVTPGDRVSLSC 208B-826L GTCTGTGACTCCAGGAGATAGAGTCAGTCTTT 208B-826L RASQRIYNYLHWYQQESHESPRL CCTGCAGGGCCAGTCAAAGAATTTACAACTAC LTHYASQSISGIPSRFSGSGSGT CTACACTGGTATCAACAAAAATCACATGAGTC DFILTINSVETEDFGMYFCQQSN TCCAAGGCTTCTCACCAAGTATGCTTCCCAGT SWPLTFGAGTKLELRR CCATCTCTGGGATCCCCTCCAGGTTCAGTGGC AGTGGCTCAGGGACAGATTTCATTCTCACTAT CAACAGTGTGGAGACTGAAGATTTTGGAATGT ATTTCTGTCAACAGAGTAACAGCTGGCCTCTC ACGTTCGGTGCTGGGACCAAGCTGGAGCTGAG ACGGGCTGATGCTGC 208B-395L 341 GATATTGTGTTAACTCAGTCTCCAGCCACCCT 342 208B-395L DIVLTQSPATLSVTPGDRVSLSC GTCTGTGACTCCAGGAGATAGAGTCAGTCTTT RASQRIYNYLHWYQQESHESPRL CCTGCAGGGCCAGTCAAAGAATTTACAACTAC LIKYASQSISGIPSRFSGSGSGT CTACACTGGTATCAACAAAAATCACATGAGTC DFILTINSVETEDFGMYFCQQSN TCCAAGGCTTCTCATCAAGTATGCTTCCCAGT SWPLTFGAGTKLELRR CCATCTCTGGGATCCCCTCCAGGTTCAGTGGC AGTGGCTCAGGGACAGATTTCATTCTCACTAT CAACAGTGTGGAGACTGAAGATTTTGGAATGT ATTTCTGTCAACAGAGTAACAGCTGGCCTCTC ACGTTCGGTGCTGGGACCAAGCTGGAGCTGAG ACGGGCTGATGCTGC 208B-517L 343 GACATTGTGATGACTCAGTCTCCAGCCACCCT 344 208B-517L DIVMTQSPATLSVTPGDRVSLSC GTCTGTGACTCCAGGAGATAGAGTCTCTCTTT RASQSISDYLHWYQQESHESPRL CCTGCAGGGCCAGCCAGAGTATTAGCGACTAC LIKYASQSISGIPSRFRGSGSGS TTACACTGGTATCAACAAAAATCACATGAGTC HFTLSINSVEPEDVGVYYCQNGH TCCAAGGCTTCTCATCAAATATGCTTCCCAAT SFPWTFGGGTKLEIKR CCATCTCTGGGATCCCCTCCAGGTTCCGTGGC AGTGGATCAGGGTCACATTTCACTCTCAGTAT CAACAGTGTGGAACCTGAAGATGTTGGAGTGT ATTACTGTCAAAATGGTCACAGTTTTCCGTGG ACGTTCGGTGGAGGCACCAAGCTGGAAATCAA ACGGGCTGATGCTGC 208B-822L 345 GACATTGTGATGACTCAGTCTCCAGCCACCCT 346 208B-822L DIVMTQSPATLSVTPGDRVSLSC GTCTGTGACTCCAGGAGATAGAGTCTCTCTTT RASQTISDYLHWYQQESHESPRL CCTGCAGGGCCAGCCAGACTATTAGCGACTAC LIKYASQSISGIPSRFSGSGSGS TTACACTGGTATCAACAAAAATCGCATGAGTC HFTLSINSVEPEDVGVYYCQNGH TCCAAGGCTTCTCATCAAATATGCTTCCCAAT SFPWTFGGGTKLEIKR CCATCTCTGGGATCCCCTCCAGGTTCAGTGGC AGTGGATCAGGGTCACATTTCACTCTCAGTAT CAACAGTGTGGAACCTGAAGATGTTGGAGTGT ATTACTGTCAAAATGGTCACAGTTTTCCGTGG ACGTTCGGTGGAGGCACCAAGCTGGAAATCAA ACGGGCTGATGCTGC 208A-210L 347 GAGATTGTGCTCACTCAGTCTCCAGCCATCAC 348 208A-210L EIVLTQSPAITAASLGQNVTITC AGCTGCATCTCTGGGGCAAAACGTCACCATCA RASSSVSYMHWYRQESGTSPQLW CCTGCAGAGCCAGCTCAAGTGTAAGTTACATG IYEISRRASGVPARFRASGSGTS CATTGGTACCGGCAGAAGTCCGGCACCTCCCC YSLTISSMEAEDAAIYYCQQWNY CCAACTATGGATTTATGAGATATCCAGACGGG PLTFGAGTKLEVKR CTTCTGGAGTCCCAGCTCGCTTCCGTGCCAGT GGGTCTGGGACCTCTTATTCTCTCACAATCAG CAGCATGGAGGCTGAAGATGCTGCCATTTATT ACTGCCAGCAGTGGAATTATCCTCTCACGTTC GGTGCTGGGACCAAGCTGGAGGTGAAACGGGC TGATGCTGC 208B-547L 349 GAAGTTGTGCTCACTCAGTCTCCAGCCATCAC 350 208B-547L EVVLTQSPAITAASLGQKVTITC AGCTGCATCTCTGGGGCAAAAGGTCACCATCA RASSSVSYMHWYRQESGTSPQPW CCTGCAGAGCCAGCTCAAGTGTAAGTTACATG IYEISTLASGVPTRFRASGSGTS CACTGGTACCGGCAGAAGTCAGGCACCTCCCC YSLTISSMEAEDAAIYYCQQWNY CCAGCCATGGATTTATGAAATATCCACACTGG PLTFGAGTKLELKR CTTCTGGAGTCCCAACTCGCTTCCGTGCCAGT GGGTCTGGGACCTCTTATTCTCTCACAATCAG CAGCATGGAGGCTGAAGATGCTGCCATTTATT ACTGCCAGCAGTGGAATTATCCTCTCACGTTC GGTGCTGGGACCAAGCTGGAACTGAAACGGGC TGATGCTGC 208A-638L 351 CAAATTGTTCTCACCCAGTCTCCAACAATCAT 352 208A-638L QIVLTQSPTIMSASLGERVTMTC GTCTGCATCTCTAGGGGAACGGGTCACCATGA TASSSVSSSYLHWFQQFPGSSPE CCTGCACTGCCAGCTCAAGTGTGAGTTCCAGT LWIYSTSNLASGVPPRFSGSGSG TACTTGCACTGGTTCCAGCAGAAGCCAGGATC TSYSLSISSVEAEDVATYYCLQF CTCCCCCAAACTCTGGATTTATAGCACATCCA HRSPWTFEGGGAKLEIKR ACCTGGCTTCTGGAGTCCCACCTCGCTTCAGT GGCAGTGGGTCTGGGACCTCTTACTCTCTCTC AATCAGCAGCGTGGAGGCTGAAGATGTTGCCA CTTATTACTGCCTCCAGTTTCATCGTTCCCCG TGGACGTTCGGTGGAGGCGCCAAGTTGGAAAT CAAACGGGCTGATGCTGC 208B-515L 353 CAAATTGTTTTCACCCAGTATCCAGCAATAAT 354 208B-515L QIVFTQYPAIMSASLGERVTMTC GTCTGCATCTCTAGGGGAACGGGTCACCATGA TASSSVTSSYLHWFQQFPGSSPK CCTGCACAGCCAGCTCAAGTGTAACTTCCAGT LWIYSTSNPGSGVPARFSGRGSG TACTTGCACTGGTTCCAGCAGAAGCCAGGATC TSYSLSISSMEAEDAATYYCLQF CTCCCCCAAACTCTGGATTTATAGCACGTCCA HRSPWTFGGGTKLEIRR ACCCGGGTTCTGGAGTCCCAGCTCGCTTCAGT GGCAGAGGATCTGGGACCTCTTACTCTCTCTC AATCAGCAGCATGGAGGCTGAAGATGCTGCCA CTTATTACTGCCTCCAGTTTCATCGTTCCCCG TGGACGTTCGGTGGAGGCACCAAGCTGGAAAT CAGACGGGCTGATGCTGC 208A-877L 355 CAAATTGTTCTCACCCAGTCTCCAGCAATCAT 356 208A-877L QIVLTQSPAIMSVSPGEKVSMTC GTCTGTATCTCCAGGGGAGAAGGTCTCCATGA SASSSVTYMHWYQQESGTSPERW CCTGCAGTGCCAGCTCAAGTGTCACTTACATG IYDTSELASGVPARFSGSGSGTT CACTGGTATCAGCAGAAGTCAGGCACCTCCCC YSLTISSMEAEDAATYYCQQWSN CAAAAGATGGATTTATGACACATCCGAGCTGG KPLTFGAGTKLELKR CTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGT GGGTCTGGGACCACTTACTCTCTCACAATCAG CAGCATGGAGGCTGAAGATGCTGCCACTTATT ACTGCCAGCAGTGGAGTAATAAACCGCTCACG TTCGGTGCTGGGACCAAGCTGGAGCTGAAACG GGCTGATGCTGC 208B-174L 357 CAAAATGTTCTCACCCAGTCTCCAGCAATCAT 358 208B-174L QNVLTQSPAIMSASPGEKVTMSC GTCTGCATCTCCAGGGGAGAAGGTCACCATGT SASSSVTYMEWYQLEPESSPRLL CCTGCAGTGCCAGCTCAAGTGTCACTTACATG IYDTSNLASGVPVRFSGSGSGTS TTCTGGTACCAGCTGAAGCCAGAATCCTCCCC YSLTISRMEAEDAATYYCQEWSS CAGACTCCTGATTTATGACACATCCAATTTGG YPLTFGAGTKLDLKR CTTCTGGCGTCCCTGTTCGCTTCAGTGGCAGT GGGTCTGGGACCTCTTACTCTCTCACAATCAG CCGTATGGAGGCTGAAGATGCTGCCACTTATT ACTGCCAGGAGTGGAGTAGTTACCCACTCACG TTCGGTGCTGGGACCAAGCTGGACCTGAAACG GGCTGATGCTGC 208B-612L 359 CAAATTGTTCTCACCCAGTCTCCAACACTCAT 360 208B-612L QIVLTQSPTLMSASPGEKVTMTC GTCTGCATCGCCAGGAGAAAAGGTCACCATGA SASSTVTYIYWYQQFPGSSPRLW CCTGCAGTGCCAGCTCAACTGTGACTTACATT MYDTFNLVSGVPARFSGSRSGTS TACTGGTACCAACAGAAGCCCGGCTCCTCCCC YFLTISSMEGEDAATYYCQQYSD CAGACTCTGGATGTATGACACATTCAACCTGG SPYTFGGGTKLEIKR

TTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGT AGGTCTGGGACCTCTTATTTTCTCACAATCAG TAGCATGGAGGGTGAAGATGCTGCCACTTATT ACTGCCAACAGTACAGTGATTCCCCGTACACG TTCGGAGGGGGGACCAAGCTGGAGATAAAACG GGCTGATGCTGC 208B-911L 361 CAAATTGTTCTCACCCAGTCTCCAGAGATCAT 362 208B-911L QIVLTQSPEIMSASPGEKVTITC GTCTGCATCTCCAGGGGAGAAGGTCACCATAA SASSSVSFMYWFQQFPGTSPELW CCTGCAGTGCCAGCTCAAGTGTAAGTTTCATG IYITSNLASGVPTRFSGSGSGTS TATTGGTTCCAGCAGAAGCCAGGCACTTCTCC YSLTISRMEAEDAATYYCQQRSS CAAACTCTGGATTTATATCACATCCAACCTGG FPYTFGGGTKLEMKR CTTCTGGAGTCCCTACTCGCTTCAGTGGCAGT GGATCTGGGACCTCTTACTCTCTCACAATCAG CCGAATGGAGGCTGAAGATGCTGCCACTTATT ACTGCCAGCAAAGGAGTAGTTTCCCGTACACG TTCGGAGGGGGGACCAAACTGGAAATGAAACG GGCTGATGCTGC 208A-422L 363 GACATTGTGCTGACCCAATCTCCAGCTTCTTT 364 208A-422L DIVLTQSPASLAVSLGQRAIISC 208A-442L GGCTGTGTCTCTAGGGCAGAGGGCCATCATCT 208A-442L KASQSVSFAGTNLMHWYQQFPGQ CCTGCAAGGCCAGCCAAAGTGTCAGTTTTGCT QPELLITRASNLETGVPTRFSGS GGTACTAATTTAATGCACTGGTACCAACAGAA GSRTDFTLNIHPVEEDDAATYYC ACCAGGACAGCAACCCAAACTCCTCATCTATC QQSREYYTFGGGTKLEIKR GTGCATCCAACCTAGAAACTGGGGTTCCTACC AGGTTTAGTGGCAGTGGGTCTAGGACAGACTT CACCCTCAATATCCATCCTGTGGAGGAAGATG ATGCTGCAACCTATTACTGTCAGCAAAGTAGG GAATATTACACGTTCGGAGGGGGGACCAAGCT GGAAATAAAACGGGCTGATGCTGC 208A-967L 365 GACATTGTGCTGACCCAATCTCCAGCTTCTTT 366 208A-967L DIVLTQSPASLAGSLGERAPISC GGCTGGGTCTCTAGGGAAAAGGGCCCCCATCT KASESVNEFGTNLIHWYQQFPGQ CCTGCAAAGCCAGCGAAAGTGTCAATTTTTTT PPELLIYHASNLKTGVPARFRGR GGTACTAATTTAATACACTGGTACCAACAAAA GSKTNFPLPIDPVEENDVAITYC ACCAGGACAGCCCCCCAAACTCCTCATCTATC LQNRKIPLTFGVGTKLELKR ATGCATCCAACCTAAAAACTGGAGTCCCTGCC AGGTTCAGGGGCAGGGGGTCTAAAACAAACTT CCCCCTCCCCATTGATCCTGTGGAGGAAAATG ATGTTGCAATCTATTACTGTCTGCAAAATAGG AAAATTCCTCTCACGTTCGGGGTTGGGACCAA GCTGGAGCTGAAACGGGCTGATGCTGC 208B-178L 367 GACATTGTGCTGACCCAATCTCCAGCTTCTTT 368 208B-178L DIVLTQSPASLAVSLGQRATISC GGCTGTGTCTCTAGGGCAGAGGGCCACCATCT RASESVDNYGISFMHWYQQFPGQ CCTGCAGAGCCAGCGAAAGTGTTGATAATTAT PPELLITRASNLESGIPARFSGS GGCATTAGTTTTATGCACTGGTACCAGCAGAA GSRTDFTLTINPVETDDVATYYC ACCAGGACAGCCACCCAAACTCCTCATCTATC QQSNEDPFTFGSGAKLEIER GTGCATCCAACCTAGAATCTGGGATCCCTGCC AGGTTCAGTGGCAGTGGGTCTAGGACAGACTT CACCCTCACCATTAATCCTGTGGAGACTGATG ATGTTGCAACCTATTACTGTCAGCAAAGTAAT AAGGATCCATTCACGTTCGGCTCGGGGGCAAA GTTGGAAATAAAACGGGCTGATGCTGC 208A-222L 369 GACATTGTGGTGACACAGTCTCCTGCTTCCTT 370 208A-222L DIVVTQSPASLAVSLGQRATISC 208A-605L AGCTGTATCTCTGGGGCAGAGGGCCACCATCT 208A-605L RASQSVSTSRYSYLHWYQQFPGQ 208B-560L CATGCAGGGCCAGCCAAAGTGTCAGTACATCT 208B-560L PPELLIKYASNLESGVPARFSGS AGATATAGTTATCTGCACTGGTACCAACAGAA GSGTDFTLNIHPVGEEDTATYYC ACCAGGACAACCTCCCAAACTCCTCATCAAGT QHSWEFPFTFGSGTKLEIKR ATGCATCCAACCTAGAATCTGGGGTCCCTGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACCCTCAATATCCATCCTGTGGGGGAGGAGG ATACTGCAACATATTACTGTCAGCACAGTTGG GAGTTTCCATTCACGTTCGGCTCGGGGACAAA GTTGGAAATAAAACGGGCTGATGCTGC 208A-830L 371 GACATTGTACTGACACAGTCTCCTGCTTCCTT 372 208A-830L DIVLTQSPASLAVSLGQRATISC AGCTGTATCTCTGGGGCAGAGGGCCACCATCT RSSQSVSTSRYSYLHWYQQFPGQ CATGCAGGTCCAGCCAAAGTGTCAGTACATCT PPELLIKYASNLESGVPARFSGS AGATATAGTTATTTGCACTGGTACCAACAGAA GSGTDFTLNIHPVGEEDPATYYC ACCAGGACAACCTCCCAAACTCCTCATCAAGT QHSWEFPFTFGSGTKLEIKR ATGCATCCAACCTAGAATCTGGGGTCCCTGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACCCTCAACATCCATCCTGTGGGGGAGGAGG ATCCTGCAACATATTACTGTCAGCACAGTTGG GAGTTTCCATTCACGTTCGGCTCGGGGACAAA GTTGGAAATAAAACGGGCTGATGCTGC 208A-557L 373 GACATTGCCCTGACACAGTCTCCTGCTTCGTT 374 208A-557L DIALTQSPASLAVSLGQRATISC AGCTGTATCTCTGGGGCAGAGGGCCACCATCT RASQSVSTSRYSYMHWYQQFPGQ CATGCAGGGCCAGCCAAAGTGTCAGTACATCT PPELLIKYASNLESGVPARFSGS AGGTATAGTTATATGCACTGGTACCAACAGAA GSGTDFTLNIHPVGEEDTATYYC ACCAGGACAACCACCCGAACTCCTCATCAAGT QHSWDFPFTFGSGTKLEIKR ATGCATCCAACCTAGAATCTGGGGTCCCTGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACCCTCAACATCCATCCTGTGGGGGAGGAGG ATACTGCAACATATTACTGTCAGCACAGTTGG GATTTTCCATTCACGTTCGGCTCGGGGACAAA GTTGGAAATAAAACGGGCTGATGCTGC 208A-133L 375 GACATTGTGCTGACACAGTCTCCTGCTTCCTT 376 208A-133L DIVLTQSPASLAVSLGQRATISC 208B-556L AGCTGTTTCTCTGGGGCAGAGGGCCACCATCT 208B-556L RASQSVSTSRYSYMHWYQQFPGQ CATGCAGGGCCAGCCAAAGTGTCAGTACATCT PPELLIKYASNLESGVPARFSGS AGGTATAGCTACATGCACTGGTACCAACAGAA GSGTDFTLNIHPVAEEDTATYYC ACCAGGACAGCCACCCAAACTCCTCATCAAGT QHSWEFPFTFGSGTKLEIKR ATGCATCCAACCTAGAATCTGGGGTCCCTGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACCCTCAACATCCATCCTGTGGCGGAGGAGG ATACTGCAACATATTACTGTCAGCACAGTTGG GAGTTTCCATTCACGTTCGGCTCGGGGACAAA GTTGGAAATAAAACGGGCTGATGCTGC 208B- 377 GACATTGTGCTGACACAGTCTCTTGCTTCCTT 378 208B-1070L DIVLTQSLASLAVSLGQRATISC 1070L AGCTGTTTCTCTGGGGCAGAGGGCCACCATCT RASQSVSTSRYSYMHWYQQFPGQ CATGCAGGGCCAGCCAAAGTGTCAGTACATCT PPELLIKYASNLECGVRARFSGS AGGTATAGTTATATGCACTGGTACCAACAGAA GCGTDFTLNIHPVEEEDTAAYYC ACCAGGACAGCCACCCAAACTCCTCATCAAGT QHSWEFPFTFGSGTKLEIKR ATGCATCCAACCTTGAATGTGGGGTCCGTGCC AGGTTCAGTGGCAGTGGGTGTGGGACAGACTT CACCCTCAACATCCATCCTGTGGAGGAGGAGG ATACTGCAGCATATTACTGTCAGCACAGTTGG GAGTTTCCATTCACGTTCGGCTCGGGGACAAA ATTGGAAATAAAACGGGCTGATGCTGC 208A-159L 379 GACATTGTACTGACACAGTCTCCTGCTTCCTT 380 208A-159L DIVLTQSPASLAVSLGQRATISC AGCTGTTTCTCTGGGGCAGAGGGCCACCATCT RASQSVSTSRYSYVHWYQQFPGQ CCTGCAGGGCCAGCCAAAGTGTCAGTACATCT PPELLIKYAANLESGVPARFSGS AGGTATAGTTATGTGCACTGGTATCAACAGAA GSGTDFTLNIHPVAEEDAAAYYC ACCAGGACAGCCACCCAAACTCCTCATCAAGT QHSWEFPFTFGSGTKLEIKR ATGCAGCCAACCTAGAATCTGGGGTCCCTGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACCCTCAACATCCATCCTGTGGCGGAGGAGG ATGCTGCAGCATATTACTGTCAGCACAGTTGG GAGTTTCCATTCACGTTCGGCTCGGGGACAAA GTTGGAAATAAAACGGGCTGATGCTGC 208A-741L 381 GACATTGTGCTGACACAGTCTCCTGCTTCCTT 382 208A-741L DIVLTQSPASLAVSLGQRTTISC AGCTGTTTCTCTGGGGCAGAGGACCACCATCT GASQSVSTSRFSYMHWYQQFPGQ CATGCGGGGCCAGCCAAAGTGTCAGTACATCT PPELLIKYASNLESGVPARFSGS AGGTTTAGTTATATGCACTGGTACCAACAGAA GSGTDFTLNIHPVAEEDTAAYYC ACCAGGACAGCCACCCAAACTCCTCATCAAGT QHSWEFPFTFGSGTKLEIKR ATGCATCCAACCTAGAATCTGGGGTCCCTGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACCCTCAACATCCATCCTGTGGCGGAGGAGG ATACTGCAGCATATTACTGTCAGCACAGTTGG GAGTTTCCATTCACGTTCGGCTCGGGGACAAA GTTGGAAATAAAACGGGCTGATGCTGC 208A-334L 383 GACATTGTGCTGACACAGTCTCCTGCTTCCTT 384 208A-334L DIVLTQSPASLAVSLGQRATISC AGCTGTTTCTCTGGGGCAGAGGGCCACCATCT RASQSVSTSRYSYMHWYQQFPGQ CATGCAGGGCCAGCCAAAGTGTCAGTACATCT PPELLIKYASNLESGVPARFSGS AGGTATAGTTATATGCACTGGTACCAACAGAA GSGTDFTLNIHPVAEEDTAAYYC ACCAGGACAGCCACCCAAACTCCTCATCAAGT QHSWGFPFTFGSGTKLEIKR ATGCATCCAACCTAGAATCTGGGGTCCCTGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACCCTCAACATCCATCCTGTGGCGGAGGAGG ATACTGCAGCATATTACTGTCAGCACAGTTGG GGGTTTCCATTCACGTTCGGCTCGGGGACAAA GTTGGAAATAAAGCGGGCTGATGCTGC 208B-251L 385 GACATTGTGCTGACACAGTCTCCTGCTTCCTT 386 208B-251L DIVLTQSPASLAVSLGEKATISC AGCTGTATCTTTGGGAAAAAAGGCCACCATCT RASQSVSTSRYSYMHWYQQFPGH CATGCAGGGCCAGCCAAAGTGTCAGTACATCT PPELLIKYASNLESGVPARFSGS AGGTATAGTTATATGCACTGGTACCAACAGAA GSGTDFTLNIHPVAEEDTAAYYC ACCAGGACACCCACCCAAACTCCTCATCAAAT QHSWEFPFTFGSGTKLEIKR ATGCATCCAACCTAGAATCTGGGGTCCCTGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACCCTCAACATCCATCCTGTGGCGGAGGAGG ATACTGCAGCATATTACTGTCAGCACAGTTGG GAGTTTCCATTCACGTTCGGCTCGGGGACAAA GTTGGAAATAAAACGGGCTGATGCTGC 208A-1101 387 GACATTGTGCTGACACAGTCTCCTGCTTCCTT 388 208A-1101 DIVLTQSPASLAVSLGQRATISC 208A-126 AGCTGTTTCTCTGGGGCAGAGGGCCACCATCT 208A-126 RASQSVSTSRYSYMHWYQQKPGQ 208A-9201 CATGCAGGGCCAGCCAAAGTGTCAGTACATCT 208A-9201 PPKLLIKYASNLESGVPARFSGS AGGTATAGTTATATGCACTGGTACCAACAGAA GSGTDFTLNIHPVAEEDTAAYYC ACCAGGACAGCCACCCAAACTCCTCATCAAGT QHSWEFPFTFGSGTKLEIKR ATGCATCCAACCTTGAATCTGGGGTCCCTGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACCCTCAACATCCATCCTGTGGCGGAGGAGG ATACTGCAGCATATTACTGTCAGCACAGTTGG GAGTTTCCATTCACGTTCGGCTCGGGGACAAA ATTGGAAATAAAACGGGCTGATGCTGC 208A- 389 AGATCTAGTCAGAGCCTTGAACATAGTAATGG 390 208A- RSSQSLEHSNGNTYLE 692CDRL1 AAACACCTATTTAGAG 692CDRL1 208A- 391 AGATCTAGTCAGAGCCTTGTACATAGTAATGG 392 208A- RSSQSLVHSNGNTYLE 352CDRL1 AAACACCTATTTAGAG 352CDRL1 208A- 393 AGATCTAGTCAGAGCCTTGTACATAGTAATGG 394 208A- RSSQSLLHSNGNTYLE 983CDRL1 AAACACCTATTTAGAG 983CDRL1 208B- 208B- 471CDRL1 471CDRL1 208B- 395 AGATCTAGTCAGAGCATTGTACACAGTAATGG 396 208B- RSSQSIVHSNGNTYLE 1024CDRL1 AAACACCTATTTAGAG 1024CDRL1 208A- 397 AGATCTAGTCAGAGCCTTGAACATACTAATGG 398 208A- RSSQSLEHTNGNTYLE 134CDRL1 AAACACCTATTTAGAG 134CDRL1 208B- 399 AGATCTAGTCAGAGCCTTGTACATAATAATGG 400 208B- RSSQSLVHNNGNTYLE 281CDRL1 AAACACCTATTTAGAA 281CDRL1 208B- 401 AGGTCTAGTAAGAGTCTCCTACATAGTAATGG 402 208B- RSSKSLLHSNGITYLY 327CDRL1 CATCACTTATTTGTAT 327CDRL1 208B- 403 AGATCTAGTCAGACCCTTCTACACAGTGATGG 404 208B- KESQTLLHRDGNPFLL 862CDRL1 AGACACCTATTTACAT 862CDRL1 208B- 405 AAGTCAAGTCAGAGCCTCTTATTTACTAATGG 406 208B- KSSQSLLFTNGKTYLN 589CDRL1 AAAAACCTATTTAAAT 589CDRL1 208B- 208B- 1096CDRL1 1096CDRL1 208B- 407 AAGTCAAGTCAGAGCCTCTTATATACTAATGG 408 208B- KSSQSLLYTNGKTYLN 189CDRL1 AAAGACCTATTTGAAT 189CDRL1 208A- 409 AAGGCCAGTCAGAATGTGAGTCCTGCTGTAGC 410 208A- KASQNVSPAVA 874CDRL1 C 874CDRL1 208B- 411 AAGGCCAGTCAGAATGTGGGTACTGCTGTAGC 412 208B- KASQNVGTAVA 353CDRL1 C 353CDRL1 208B- 413 AAGGCCAGTCAGGATGTGGGTACTGCTGTAGC 414 208B- KASQDVGTAVA 793CDRL1 C 793CDRL1 208B- 415 CGGGCAAGTCAGGACATTGGTGGTAGCATAAA 416 208B- RASQDIGGSIN 672CDRL1 C 672CDRL1 208B- 417 CGAGCAAGTGGGAATATTCACACTTATTTAGC 418 208B- RASGNIHTYLA 408CDRL1 A 408CDRL1 208A- 419 AGGGCCAGTCAAAGAATTTACAACTACCTACA 420 208A- RASQRIYNYLH 207CDRL1 C 207CDRL1 208B- 208B- 826CDRL1 826CDRL1 208B- 208B- 395CDRL1 395CDRL1

208B- 421 AGGGCCAGCCAGAGTATTAGCGACTACTTACA 422 208B- RASQSISDYLH 517CDRL1 C 517CDRL1 208B- 423 AGGGCCAGCCAGACTATTAGCGACTACTTACA 424 208B- RASQTISDYLH 822CDRL1 C 822CDRL1 208A- 425 AGAGCCAGCTCAAGTGTAAGTTACATGCAT 426 208A- RASSSVSYMH 210CDRL1 210CDRL1 208B- 427 AGAGCCAGCTCAAGTGTAAGTTACATGCAT 428 208B- RASSSVSYMH 547CDRL1 547CDRL1 208A- 429 ACTGCCAGCTCAAGTGTGAGTTCCAGTTACTT 430 208A- TASSSVSSSYLH 638CDRL1 GCAC 638CDRL1 208B- 431 ACAGCCAGCTCAAGTGTAACTTCCAGTTACTT 432 208B- TASSSVTSSYLH 515CDRL1 GCAC 515CDRL1 208A- 433 AGTGCCAGCTCAAGTGTCACTTACATGCAC 434 208A- SASSSVTYMH 877CDRL1 877CDRL1 208B- 435 AGTGCCAGCTCAAGTGTCACTTACATGTTC 436 208B- SASSSVTYMF 174CDRL1 174CDRL1 208B- 437 AGTGCCAGCTCAACTGTGACTTACATTTAC 438 208B- SASSTVTYIY 612CDRL1 612CDRL1 208B- 439 AGTGCCAGCTCAAGTGTAAGTTTCATGTAT 440 208B- SASSSVSFMY 911CDRL1 911CDRL1 208A- 441 AAGGCCAGCCAAAGTGTCAGTTTTGCTGGTAC 442 208A- KASQSVSFAGTNLMH 422CDRL1 TAATTTAATGCAC 422CDRL1 208A- 208A- 442CDRL1 442CDRL1 208A- 443 AAAGCCAGCGAAAGTGTCAATTTTTTTGGTAC 444 208A- KASESVNFFGTNLIH 967CDRL1 TAATTTAATACAC 967CDRL1 208B- 445 AGAGCCAGCGAAAGTGTTGATAATTATGGCAT 446 208B- RASESVDNYGISFMH 178CDRL1 TAGTTTTATGCAC 178CDRL1 208A- 447 AGGGCCAGCCAAAGTGTCAGTACATCTAGATA 448 208A- RASQSVSTSRYSYLH 222CDRL1 TAGTTATCTGCAC 222CDRL1 208A- 208A- 605CDRL1 605CDRL1 208B- 208B- 560CDRL1 560CDRL1 208A- 449 AGGGCCAGCCAAAGTGTCAGTACATCTAGGTA 450 208A- RASQSVSTSRYSYMH 557CDRL1 TAGTTATATGCAC 557CDRL1 208A- 208A- 133CDRL1 133CDRL1 208B- 208B- 556CDRL1 556CDRL1 208B- 208B- 1070CDRL1 1070CDRL1 208B- 208B- 251CDRL1 251CDRL1 208A- 208A- 110CDRL1 110CDRL1 208A- 208A- 126CDRL1 126CDRL1 208A- 208A- 920CDRL1 920CDRL1 208A- 208A- 334CDRL1 334CDRL1 208A- 451 GGGGCCAGCCAAAGTGTCAGTACATCTAGGTT 452 208A- GASQSVSTSRFSYMH 741CDRL1 TAGTTATATGCAC 741CDRL1 208A- 453 AGGTCCAGCCAAAGTGTCAGTACATCTAGATA 454 208A- RSSQSVSTSRYSYLH 830CDRL1 TAGTTATTTGCAC 830CDRL1 208A- 455 AGGGCCAGCCAAAGTGTCAGTACATCTAGGTT 456 208A- RASQSVSTSRFSYLH 1064CDRL1 TAGTTATCTGCAC 1064CDRL1 208A- 457 AGGGCCAGCCAAAGTGTCAGTACATCTAGGTA 458 208A- RASQSVSTSRYSYVH 159CDRL1 TAGTTATGTGCACTGG 159CDRL1 208A- 459 AGGGCCAGCCAAAGTGTCAGTACATCCAGGTT 460 208A- RASQSVSTSRFSYVH 293CDRL1 TAGTTATGTGCAC 293CDRL1 208B- 461 AGGGCCAGCCAAAGTCTCAGTACATCTAGGTT 462 208B- RASQSLSTSRFSYVH 1094CDRL1 TAGCTATGTGCAC 1094CDRL1 208A- 463 AAAGTTTCCAGCCGATTTTCT 464 208A- KVSSRFS 692CDRL2 692CDRL2 208A- 208A- 134CDRL2 134CDRL2 208B- 465 AAAGTTTCCAACCGATTTTCT 466 208B- KVSNRFS 281CDRL2 281CDRL2 208B- 467 AAACTTTCCAACCGATTTTCT 468 208B- KLSNRFF 862CDRL2 862CDRL2 208A- 469 AATGTTTCCAACCGATTTTCT 470 208A- NVSNRFS 352CDRL2 352CDRL2 208A- 208A- 983CDRL2 983CDRL2 208B- 208B- 471CDRL2 471CDRL2 208B- 471 AGAGTTTCCAACCGATTTTCT 472 208B- RVSNRFS 1024CDRL2 1024CDRL2 208B- 473 CAGATGTCCAAGATTGCCTCA 474 208B- QMSKIAS 327CDRL2 327CDRL2 208B- 475 CTGCTGTCTAAATTGGACTCT 476 208B- LLSKLDS 589CDRL2 589CDRL2 208B- 208B- 1096CDRL2 1096CDRL2 208B- 477 CTGGTGTCAAAATTGGACTCT 478 208B- LVSELDS 189CDRL2 189CDRL2 208A- 479 TCGGCATCCTCCCGATACACT 480 208A- SASSRYT 874CDRL2 874CDRL2 208B- 481 TCAGCATCCAATCGGTACACT 482 208B- SASNRYT 353CDRL2 353CDRL2 208B- 483 TGGGCATCCACCCGGCACACT 484 208B- WASTRHT 793CDRL2 793CDRL2 208B- 485 GCCACATCCAGTTTAGATTCT 486 208B- ATSSLDS 672CDRL2 672CDRL2 208B- 487 AATGCAAACACCTTGGCAGAT 488 208B- NANTLAD 408CDRL2 408CDRL2 208A- 489 GCTTCCCAGTCCATCTCTGGG 490 208A- ASQSISG 207CDRL2 207CDRL2 208B- 208B- 826CDRL2 826CDRL2 208B- 208B- 395CDRL2 395CDRL2 208B- 208B- 517CDRL2 517CDRL2 208B- 491 TATGCTTCCCAATCCATCTCT 492 208B- YASQSIS 822CDRL2 822CDRL2 208A- 493 GAGATATCCAGACGGGCTTCT 494 208A- EISRRAS 210CDRL2 210CDRL2 208B- 495 GAAATATCCACACTGGCTTCT 496 208B- EISTLAS 547CDRL2 547CDRL2 208A- 497 AGCACATCCAACCTGGCTTCT 498 208A- STSNLAS 638CDRL2 638CDRL2 208B- 499 AGCACGTCCAACCCGGGTTCT 500 208B- STSNPGS 515CDRL2 515CDRL2 208A- 501 GACACATCCGAGCTGGCTTCT 502 208A- DTSELAS 877CDRL2 877CDRL2 208B- 503 GACACATCCAATTTGGCTTCT 504 208B- DTSNLAS 174CDRL2 174CDRL2 208B- 505 GACACATTCAACCTGGTTTCT 506 208B- DTFNLVS 612CDRL2 612CDRL2 208B- 507 ATCACATCCAACCTGGCTTCT 508 208B- ITSNLAS 911CDRL2 911CDRL2 208A- 509 CGTGCATCCAACCTAGAAACT 510 208A- RASNLET 422CDRL2 422CDRL2 208A- 208A- 442CDRL2 442CDRL2 208A- 511 CATGCATCCAACCTAAAAACT 512 208A- HASNLET 967CDRL2 967CDRL2 208B- 513 CGTGCATCCAACCTAGAATCT 514 208B- RASNLES 178CDRL2 178CDRL2 208A- 515 TATGCATCCAACCTAGAATCT 516 208A- YASNLES 222CDRL2 222CDRL2 208A- 208A- 605CDRL2 605CDRL2 208B- 208B- 560CDRL2 560CDRL2 208A- 208A- 557CDRL2 557CDRL2 208A- 208A- 133CDRL2 133CDRL2 208B- 208B- 556CDRL2 556CDRL2 208B- 208B- 251CDRL2 251CDRL2 208A- 208A- 110CDRL2 110CDRL2 208A- 208A- 126CDRL2 126CDRL2 208A- 208A- 920CDRL2 920CDRL2 208A- 208A- 334CDRL2 334CDRL2 208A- 208A- 741CDRL2 741CDRL2 208A- 208A- 830CDRL2 830CDRL2 208A- 208A- 1064CDRL2 1064CDRL2 208A- 208A- 159CDRL2 159CDRL2 208A- 208A- 293CDRL2 293CDRL2 208B- 208B- 1094CDRL2 1094CDRL2 208B- 517 TATGCATCCAACCTTGAATGT 518 208B- YASNLEC 1070CDRL2 1070CDRL2 208A- 519 TTTCAAGGTTCACATGTTCCATTCACG 520 208A- FQGSHVPFT 692CDRL3 692CDRL3 208A- 208A- 983CDRL3 983CDRL3 208B- 208B- 281CDRL3 281CDRL3 208A- 208A- 134CDRL3 134CDRL3 208A- 521 TTTCAAGGTTCACATGTTCCACTCACG 522 208A- FQGSHVPLT 352CDRL3 352CDRL3 208B- 208B- 471CDRL3 471CDRL3 208B- 523 TTTCAAGGTTCACATGTTCCGTGGACG 524 208B- FQGSHVPWTF 1024CDRL3 1024CDRL3 208B- 525 GCTCAAAATCTAGAACTTCCGTGGACG 526 208B- AQNLELPWT 327CDRL3 327CDRL3 208B- 527 TCTCAAAGTACACATGTTCCGTACACG 528 208B- SQSTHVPYT 862CDRL3 862CDRL3 208B- 529 TTGCAGAGTACATATTTTCCTCTCACG 530 208B- LQSTYFPLT 589CDRL3 589CDRL3 208B- 208B- 1096CDRL3 1096CDRL3 208B- 531 TTGCAGAGTATACATTTTCCGTACACG 532 208B- LQSIHFPYT 189CDRL3 189CDRL3 208B- 533 CTACAATATGCTAGTTCTCCTCCGACG 534 208B- LQYASSPPT 672CDRL3 672CDRL3

208A- 535 CTCCAGTTTCATCGTTCCCCGTGGACG 536 208A- LQFHRSPWT 638CDRL3 638CDRL3 208B- 208B- 515CDRL3 515CDRL3 208A- 537 CTGCAAAATAGGAAAATTCCTCTCACG 538 208A- LQNRKIPLT 967CDRL3 967CDRL3 208A- 539 CAGCAACATTTTAGTACTCCGTGGACG 540 208A- QQHFSTPWT 874CDRL3 874CDRL3 208B- 541 CAGCAATATAGCACCTATCCTCTCACG 542 208B- QQYSTYPLT 353CDRL3 353CDRL3 208B- 543 CAGCAATATAGCAACTATCTCACG 544 208B- QQYSNYLT 793CDRL3 793CDRL3 208A- 545 CAACAGAGTAACAGCTGGCCTCTCACG 546 208A- QQSNSWPLT 207CDRL3 207CDRL3 208B- 208B- 826CDRL3 826CDRL3 208B- 208B- 395CDRL3 395CDRL3 208A- 547 CAGCAGTGGAATTATCCTCTCACG 548 208A- QQWNYPLT 210CDRL3 210CDRL3 208B- 208B- 547CDRL3 547CDRL3 208A- 549 CAGCAGTGGAGTAATAAACCGCTCACG 550 208A- QQWSNIKPLT 877CDRL3 877CDRL3 208B- 551 CAACAGTACAGTGATTCCCCGTACACG 552 208B- QQYSDSPYT 612CDRL3 612CDRL3 208B- 553 CAGCAAAGGAGTAGTTTCCCGTACACG 554 208B- QQRSSFPYT 911CDRL3 911CDRL3 208A- 555 CAGCAAAGTAGGGAATATTACACG 556 208A- QQSREYYT 422CDRL3 422CDRL3 208A- 208A- 442CDRL3 442CDRL3 208B- 557 CAGCAAAGTAATAAGGATCCATTCACG 558 208B- QQSNEDPFT 178CDRL3 178CDRL3 208B- 559 CAAAATGGTCACAGTTTTCCGTGGACG 560 208B- QNGHSFPWT 517CDRL3 517CDRL3 208B- 208B- 822CDRL3 822CDRL3 208B- 561 CAACATTTTTGGAGTGCTCCGTGGACG 562 208B- QHFWSAPWT 408CDRL3 408CDRL3 208B- 563 CAGGAGTGGAGTAGTTACCCACTCACG 564 208B- QEWSSYPLT 174CDRL3 174CDRL3 208A- 565 CAGCACAGTTGGGAGTTTCCATTCACG 566 208A- QHSWEFPFT 222CDRL3 222CDRL3 208A- 208A- 605CDRL3 605CDRL3 208B- 208B- 560CDRL3 560CDRL3 208A- 208A- 133CDRL3 133CDRL3 208B- 208B- 556CDRL3 556CDRL3 208B- 208B- 1070CDRL3 1070CDRL3 208B- 208B- 251CDRL3 251CDRL3 208A- 208A- 110CDRL3 110CDRL3 208A- 208A- 126CDRL3 126CDRL3 208A- 208A- 920CDRL3 920CDRL3 208A- 208A- 741CDRL3 741CDRL3 208A- 208A- 830CDRL3 830CDRL3 208A- 208A- 1064CDRL3 1064CDRL3 208A- 208A- 159CDRL3 159CDRL3 208A- 208A- 293CDRL3 293CDRL3 208B- 208B- 1094CDRL3 1094CDRL3 208A- 567 CAGCACAGTTGGGATTTTCCATTCACG 568 208A- QHSWDFPFT 557CDRL3 557CDRL3 208A- 569 CAGCACAGTTGGGATTTTCCATTCACG 570 208A- QHSWGFPFT 334CDRL3 334CDRL3 HCV core 571 ATGTCTACCAACCCGAAACCGCAGAAAAAAAA 572 HCV core 1- MSTNPKPQKKNKRNTNRRPQDVK 1-169 CAAACGTAACACCAACCGTCGTCCGCAGGACG 169 amino FPGGGQIVGGVYLLPRRGPRLGV amino TTAAATTCCCGGGTGGTGGTCAGATCGTTGGT acid RATRKTSERSQPRGRRQPIPKAR acid GGTGTTTACCTGCTGCCGCGTCGTGGTCCGCG sequence RPEGRTWAQPGYPWPLYGNEGCG sequence TCTGGGTGTTCGTGCTACGCGTAAAACCTCTG WAGWLLSPRGSRPSWGPTDPRRR AACGTTCTCAGCCGCGTGGGCGTCGTCAGCCG SRNLGKVIDTLTCGFADLMGYIP ATCCCGAAAGCTCGTCGTCCGGAAGGTCGTAC LVGAPLGGAARALAHGVRVLEDG CTGGGCTCAGCCGGGTTACCCGTGGCCGCTGT VNYATGNL ACGGTAACGAAGGTTGCGGTTGGGCTGGTTGG CTGCTGTCTCCGCGTGGATCTCGTCCGTCTTG GGGTCCGACCGACCCGCGTCGTCGTTCTCGTA ACCTTGGTAAAGTTATCGATACCCTGACCTGC GGTTTCGCTGACCTGATGGGTTACATACCGCT GGTTGGAGCTCCGCTGGGTGGTGCTGCTCGTG CTCTGGCGCATGGCGTGCGTGTTCTGGAAGAT GGCGTCAACTATGCCACCGGTAATCTG 573 HCV 134-154 MGYIPLVGAPLGGAARALAHG Peptide 1 574 HCV 141-161 GAPLGGAARALAHGVRVLEDG Peptide 2 575 HCV 151-171 LAHGVRVLEDGVNYATGNLPG Peptide 3 576 ALRZ-8 MGYIPLVGAPLGGAARALAHGVR VLEDGVNYATGNLPGC 577 ALRZ-9 MGYIPLVGAPLGGAARALAHGVR VLEDGVNYATGNLPGQYIKANSK FIGITEL

Sequence CWU 1

1

6161351DNAArtificial Sequencesynthetic 1gaggtccagc tgcaacagtc tggacctgag ttggtgaagc ctggggcctc agtgaagata 60tcttgcaaga cttctggata cactttcact gaatacgcca tgcactggat gaagcagagc 120catggaaaga gccttgagtg gattggaggt atcaatccta ctaatggtga tacaatctac 180aaccagaagt tcaaggacaa ggccaaattg actgtagaca ggtcctccag cacagcctac 240atggagctcc gcagcctgac atctgacgat tctgcattat tttattgtgc aagacgggaa 300ctggactact ttgcctcctg gggccaaggc accactctca cagtctcctc a 3512117PRTArtificial Sequencesynthetic 2Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30Ala Met His Trp Met Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45Gly Gly Ile Asn Pro Thr Asn Gly Asp Thr Ile Tyr Asn Gln Lys Phe 50 55 60Lys Asp Lys Ala Lys Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Thr Ser Asp Asp Ser Ala Leu Phe Tyr Cys 85 90 95Ala Arg Arg Glu Leu Asp Tyr Phe Ala Ser Trp Gly Gln Gly Thr Thr 100 105 110Leu Thr Val Ser Ser 1153351DNAArtificial Sequencesynthetic 3gaggtccagc tgcaacagtc tggacctgag ctggtgaagc ctggggcctc agtgaagata 60tcctgcaaga cttctggata cactttcact gaatacgcca tgcactggat gaagcagagc 120catggaaaga gccttgagtg gattggcggt atcaatccta ctaatggtga tacaatctac 180aaccagaggt tcaaggacaa ggccaaattg actgtagaca ggtcctccag cacagcctac 240atggagctcc gcagcctgac atctgacgat tctgcattat tttattgtgc aagacgggaa 300ctggactact ttgcctcctg gggccaaggc accactctca cagtctcctc a 3514117PRTArtificial Sequencesynthetic 4Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30Ala Met His Trp Met Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45Gly Gly Ile Asn Pro Thr Asn Gly Asp Thr Ile Tyr Asn Gln Arg Phe 50 55 60Lys Asp Lys Ala Lys Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Thr Ser Asp Asp Ser Ala Leu Phe Tyr Cys 85 90 95Ala Arg Arg Glu Leu Asp Tyr Phe Ala Ser Trp Gly Gln Gly Thr Thr 100 105 110Leu Thr Val Ser Ser 1155351DNAArtificial Sequencesynthetic 5gaggtccagc tgcaacagtc tggacctgaa ctggtgaagc ctggggcctc agtgaagata 60tcctgtaagg cttcgggatt cactttcact gaatacgcca tgcactggat gaaacagagc 120catggaaaga gccttgagtg gattggaggt atcaatccta ctaacggtga tgcaatctac 180aaccagaagt tcaaggacaa ggccaagttg actgtagaca ggtcctccag cacagcctac 240atggagctcc gcagcctgac atctgacgat tctgcattat tttattgtgc aagacgggaa 300ctggactact ttccctcctg gggccaaggc accactctca cagtctcctc a 3516117PRTArtificial Sequencesynthetic 6Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Glu Tyr 20 25 30Ala Met His Trp Met Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45Gly Gly Ile Asn Pro Thr Asn Gly Asp Ala Ile Tyr Asn Gln Lys Phe 50 55 60Lys Asp Lys Ala Lys Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Thr Ser Asp Asp Ser Ala Leu Phe Tyr Cys 85 90 95Ala Arg Arg Glu Leu Asp Tyr Phe Pro Ser Trp Gly Gln Gly Thr Thr 100 105 110Leu Thr Val Ser Ser 1157351DNAArtificial Sequencesynthetic 7gaggtccagc tgcaacagtc tggacctgaa ctggaaaagc ctggggcttc agtgaggata 60tcctgcaaga cttctggata cacattcact gaatacgcca tgcactgggt gaagcagagc 120catggaaaga gccttgagtg gattggaggt attaatccta acaatggcaa tgctatctac 180aaccagatat tcaaggacaa ggccacactg actgtggaca ggtcctccag cacagcctac 240atgggcctcc gcagcctgac attcggggat tctggagtct acttctgtgt aagacgacaa 300ctggactact ttgactattg gggccagggc gcctctctca cagtctcctc a 3518117PRTArtificial Sequencesynthetic 8Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala1 5 10 15Ser Val Arg Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30Ala Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45Gly Gly Ile Asn Pro Asn Asn Gly Asn Ala Ile Tyr Asn Gln Ile Phe 50 55 60Lys Asp Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gly Leu Arg Ser Leu Thr Phe Gly Asp Ser Gly Val Tyr Phe Cys 85 90 95Val Arg Arg Gln Leu Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Ala Ser 100 105 110Leu Thr Val Ser Ser 1159351DNAArtificial Sequencesynthetic 9gaggtccagc tgcaacagtc tggacctgag ctggaaaagc ctggggcttc agtgaagata 60tcctgcaaga cttctggata cacattcact gaatacgcca tacactgggt gaagcagagc 120catggaatga gccttgagtg gattggaggt attaatccta gcaatggcaa tgctatctac 180aaccaaatat tcaaggacaa ggccacactg actgtggaca ggtcctccag cacagcctac 240atgggcctcc gcagcctgac atttggggat tctggagtct acttctgtgt aagacgacaa 300ctggacttct ttgactattg gggccagggc gcctctctca cagtctcctc a 35110117PRTArtificial Sequencesynthetic 10Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30Ala Ile His Trp Val Lys Gln Ser His Gly Met Ser Leu Glu Trp Ile 35 40 45Gly Gly Ile Asn Pro Ser Asn Gly Asn Ala Ile Tyr Asn Gln Ile Phe 50 55 60Lys Asp Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gly Leu Arg Ser Leu Thr Phe Gly Asp Ser Gly Val Tyr Phe Cys 85 90 95Val Arg Arg Gln Leu Asp Phe Phe Asp Tyr Trp Gly Gln Gly Ala Ser 100 105 110Leu Thr Val Ser Ser 11511351DNAArtificial Sequencesynthetic 11gaggtccagc tgcaacagtc tggacctgag ctggaaaagc ctggggcttc agtgaagata 60tcctgcaaga cttctggata cacattcact gaatacgcca tgcactgggt gaagcagagc 120catggaatga gccttgagtg gattggaggt attaatccta gcaatggcaa tgctatctac 180aaccagatat tcaaggacaa ggccacactg actgtggaca ggtcctccag cacagcctac 240atgggcctcc gcagcctgac atttggggat tctggagtct acttctgtgt aagacgacaa 300ctggacttct ttgactattg gggccagggc gcctctctca cagtctcctc a 35112117PRTArtificial Sequencesynthetic 12Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30Ala Met His Trp Val Lys Gln Ser His Gly Met Ser Leu Glu Trp Ile 35 40 45Gly Gly Ile Asn Pro Ser Asn Gly Asn Ala Ile Tyr Asn Gln Ile Phe 50 55 60Lys Asp Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gly Leu Arg Ser Leu Thr Phe Gly Asp Ser Gly Val Tyr Phe Cys 85 90 95Val Arg Arg Gln Leu Asp Phe Phe Asp Tyr Trp Gly Gln Gly Ala Ser 100 105 110Leu Thr Val Ser Ser 11513351DNAArtificial Sequencesynthetic 13gaggtccggc tgcagcagcc tggacctgag gtggaaaagc ctggggcttc agtgaagata 60tcctgcaaga cttctggata cacattcact gaatacgcca tccactgggt gaaacagagc 120catggagaga gccttgagtg gattggaggt attaatccta gcaatggcga tcctatctat 180aaccagatat tcaaggacaa ggccacactg actgtggaca ggtcctccaa cacagcctac 240atgggcctcc gcagcctgac agttggggat tctggagtct acttctgtgt tagacgacaa 300ctggactact ttgacttttg gggccagggc gcctctctca cagtctcctc a 35114117PRTArtificial Sequencesynthetic 14Glu Val Arg Leu Gln Gln Pro Gly Pro Glu Val Glu Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30Ala Ile His Trp Val Lys Gln Ser His Gly Glu Ser Leu Glu Trp Ile 35 40 45Gly Gly Ile Asn Pro Ser Asn Gly Asp Pro Ile Tyr Asn Gln Ile Phe 50 55 60Lys Asp Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Asn Thr Ala Tyr65 70 75 80Met Gly Leu Arg Ser Leu Thr Val Gly Asp Ser Gly Val Tyr Phe Cys 85 90 95Val Arg Arg Gln Leu Asp Tyr Phe Asp Phe Trp Gly Gln Gly Ala Ser 100 105 110Leu Thr Val Ser Ser 11515351DNAArtificial Sequencesynthetic 15cagggtcaga tgcagcagtc tggagctgaa ctggcgaagc ctggggcttc agtgaagctg 60tcctgcaaga cttctggctt caccttcagc agtagttata taagttggtt gaagcaaaag 120cctggacaga gtcttgagtg gattgcatgg atttatgctg gaactggtaa tactaactat 180aatcagaagt tcacagacaa ggcccaactg actgtagaca catcctccag tacagcctac 240atgcaactca gcagcctgac aactgaggac tctgccatct attactgtgc gataagtggg 300acgggattta cttactgggg ccaagggact ctggtcactg tctctgcaac a 35116116PRTArtificial Sequencesynthetic 16Gln Gly Gln Met Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Phe Thr Phe Ser Ser Ser 20 25 30Tyr Ile Ser Trp Leu Lys Gln Lys Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45Ala Trp Ile Tyr Ala Gly Thr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60Thr Asp Lys Ala Gln Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Thr Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95Ala Ile Ser Gly Thr Gly Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ala 11517351DNAArtificial Sequencesynthetic 17cagggtcaga tgcagcagtc tggagctgaa ctggtgaagc ctggggcttc agtgaagctg 60tcctgcaaga cttctggctt caccttcagc agtagttata taagttggtt gaagcaaaag 120cctggacaga gtcttgagtg gattgcatgg atttatgctg gaactggtaa tactaactat 180aatcagaagt tcacagacaa ggcccaactg actgtagaca catcctccag tacagcctac 240atgcaactca gcagcctgac aactgaggac tctgccatct attactgtgc gataagtggg 300acgggattta cttactgggg ccaagggact ctggtcactg tctctgcaac a 35118116PRTArtificial Sequencesynthetic 18Gln Gly Gln Met Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Phe Thr Phe Ser Ser Ser 20 25 30Tyr Ile Ser Trp Leu Lys Gln Lys Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45Ala Trp Ile Phe Ala Gly Thr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60Thr Asp Lys Ala Gln Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Thr Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95Ala Ile Ser Gly Thr Gly Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ala 11519348DNAArtificial Sequencesynthetic 19cagggtcaga tgcagcagtc tggagctgag ctggcgaagc ctggggcttc agtgaaactg 60tcctgcaaga cttctggctt caccttcagc agtagttata taagttggtt gaagcaaaag 120cctggacaga gtcttgagtg gattgcatgg atttttgctg gaactggtaa tactaattat 180aatcagaagt tcacagacaa ggcccaactg actgtagaca catcctccag tacagcctac 240atgcaactca gcagcctgac aactgaggac tctgccatct attactgtgc gataagtggg 300acgggattta cttactgggg ccaagggact ctggtcactg tctctgca 34820116PRTArtificial Sequencesynthetic 20Gln Gly Gln Met Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Phe Thr Phe Ser Ser Ser 20 25 30Tyr Ile Ser Trp Leu Lys Gln Lys Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45Ala Trp Ile Tyr Ala Gly Thr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60Thr Asp Lys Ala Gln Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Thr Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95Ala Ile Ser Gly Thr Gly Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ala 11521348DNAArtificial Sequencesynthetic 21cagggtcaga tgcagcagtc tggagctgag ctggtgaagc ctggggcttc agtgaagctg 60tcctgcaaga cttctggctt caccttcagc agtagtttta taagttggtt gaagcaaaag 120cctggacaga gtcttgagtg gattgcatgg atttatgctg gaactggaaa tactaactat 180aatcagaagt tcacagacaa ggcccaactg actgtagaca catcctccag cacagcctac 240atgcaattca gcagcctgac gactgaggac tctgccatct attactgtgc gataagtggg 300acggggttta cttactgggg ccaagggact ctggtcactg tctctgca 34822116PRTArtificial Sequencesynthetic 22Gln Gly Gln Met Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Phe Thr Phe Ser Ser Ser 20 25 30Phe Ile Ser Trp Leu Lys Gln Lys Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45Ala Trp Ile Tyr Ala Gly Thr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60Thr Asp Lys Ala Gln Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Phe Ser Ser Leu Thr Thr Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95Ala Ile Ser Gly Thr Gly Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ala 11523351DNAArtificial Sequencesynthetic 23cagggtcaga tgcagcagtc tggagctgag ctggtgaagc ctggggcttc agtgaagctg 60tcctgcaaga cttctggctt caccttcagc agtagttatt ttagttggtt gaagcaaaag 120cctggacaga gtcttgagtg gattgcatgg atttatgctg gaactggtaa tactatctat 180aatcagaagt tcacagacaa ggcccaactg actgtagaca cagcctccag cacagccttc 240atgcaactca gcagcctgac aattgaggac tctgccatct actactgtgc gataagtggg 300acggggttta cttactgggg ccaagggact ctggtcactg tctctgcaac a 35124116PRTArtificial Sequencesynthetic 24Gln Gly Gln Met Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Phe Thr Phe Ser Ser Ser 20 25 30Tyr Phe Ser Trp Leu Lys Gln Lys Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45Ala Trp Ile Tyr Ala Gly Thr Gly Asn Thr Ile Tyr Asn Gln Lys Phe 50 55 60Thr Asp Lys Ala Gln Leu Thr Val Asp Thr Ala Ser Ser Thr Ala Phe65 70 75 80Met Gln Leu Ser Ser Leu Thr Ile Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95Ala Ile Ser Gly Thr Gly Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ala 11525348DNAArtificial Sequencesynthetic 25cagggtcagc tgcagcagtc tggagctgag ctggtgaagc ctggggcttc agtgaagctg 60tcctgcaaga cttctggctt caccttcagc agtagttata taagttggtt gaagcaaagg 120cctggacaga gtcttgagtg gattgcatgg atttatgctg gaactggtgg tactaactat 180aatcagaagt tcacagacaa ggcccaactg actgtagaca catcctccag cacagcctac 240atgcaattca gcagcctgac aactgaggac tctgccatct attactgtgc gataagtggg 300acggggttta tttactgggg ccaagggact ctggtcactg tctctgca 34826116PRTArtificial Sequencesynthetic 26Gln Gly Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Phe Thr Phe Ser Ser Ser 20 25 30Tyr Ile Ser Trp Leu Lys Gln Arg Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45Ala Trp Ile Tyr Ala Gly Thr Gly Gly Thr Asn Tyr Asn Gln Lys Phe 50 55 60Thr Asp Lys Ala Gln Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Phe Ser Ser Leu Thr Thr Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95Ala Ile Ser Gly Thr Gly Phe Ile Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ala 11527345DNAArtificial Sequencesynthetic 27cagggtcagt tgcagcagtc tggaccagta ctggtgaagc ctggggcttc agaaatacta 60tactgcaaga cttctggctt caccttcagc agtacctata taagttggtt gaagcaaaag 120cctggacaga gtcttgagtg gattgcgtgg atttatgctg gaactggtgc tactaattat 180aatcagaagt tcacaggcaa

ggcccaactg actgtagacg cttcctccaa cacagcctac 240atgcacttca gcggcctgac acctgaggac tctgccatct attactgtgc aatttctggg 300gcgggggttt actggggcca agggactctg gtcactgtct ctgca 34528115PRTArtificial Sequencesynthetic 28Gln Gly Gln Leu Gln Gln Ser Gly Pro Val Leu Val Lys Pro Gly Ala1 5 10 15Ser Glu Ile Leu Tyr Cys Lys Thr Ser Gly Phe Thr Phe Ser Ser Thr 20 25 30Tyr Ile Ser Trp Leu Lys Gln Lys Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45Ala Trp Ile Tyr Ala Gly Thr Gly Ala Thr Asn Tyr Asn Gln Lys Phe 50 55 60Thr Gly Lys Ala Gln Leu Thr Val Asp Ala Ser Ser Asn Thr Ala Tyr65 70 75 80Met His Phe Ser Gly Leu Thr Pro Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95Ala Ile Ser Gly Ala Gly Val Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ala 11529354DNAArtificial Sequencesynthetic 29cagggccaac tgcagcagcc tggggctgag tttgtgaagc ctggggcttc actgaagctg 60tcctgcaggg cttctggcta caccttcacc agctactgga tacactgggt gaagcagagg 120cctggacaag gccttgagtg gattggagaa attgatcctt ctgacagtta tattaaccag 180aatcaaaagt tcaggggcaa ggccacattg actgtggaca aatcctccag cacagcctac 240atggaactca gcggcctgac atctgaagac tctgcggtct attactgtgc aagacattac 300tacggtgttc ttgactcctg gggccaaggt accactctca cagtctcctc aaca 35430117PRTArtificial Sequencesynthetic 30Gln Gly Gln Leu Gln Gln Pro Gly Ala Glu Phe Val Lys Pro Gly Ala1 5 10 15Ser Leu Lys Leu Ser Cys Arg Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Asp Pro Ser Asp Ser Tyr Ile Asn Gln Asn Gln Lys Phe 50 55 60Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg His Tyr Tyr Gly Val Leu Asp Ser Trp Gly Gln Gly Thr Thr 100 105 110Leu Thr Val Ser Ser 11531351DNAArtificial Sequencesynthetic 31cagggccaac tgcagcagcc tggggctgag tttgtgaagc ctggggcttc actgaagctg 60tcctgcaggg cttctggcta caccttcacc agctactgga ttcactgggt gaagcagagg 120cctggacaag gccttgagtg gatcggagaa gttgatcctt ctgacagtta tattaaccag 180aatgaaaagt tcaggggcaa ggccacattg actgtggaca aatcctccag cacagcctac 240atgcagctcg gcagcctgac atctgaagac tctgcggtct attactgtgc aagacattac 300tacggtgttc ttgactcctg gggccaaggc accgctctca cagtctcctc a 35132117PRTArtificial Sequencesynthetic 32Gln Gly Gln Leu Gln Gln Pro Gly Ala Glu Phe Val Lys Pro Gly Ala1 5 10 15Ser Leu Lys Leu Ser Cys Arg Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Glu Val Asp Pro Ser Asp Ser Tyr Ile Asn Gln Asn Glu Lys Phe 50 55 60Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Gly Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg His Tyr Tyr Gly Val Leu Asp Ser Trp Gly Gln Gly Thr Ala 100 105 110Leu Thr Val Ser Ser 11533351DNAArtificial Sequencesynthetic 33caggtccaac tgcagcagcc tggggctgaa cttgtgaagc ctggggcttc actgaagctg 60tcctgcaggg cctctggcta caccttcacc agctactgga ttcactgggt gaagcagagg 120cctggacaag gccttgagtg gatcggagag attgatcctt ctgatagtta tactaactac 180aatcaaaagt tcaagggcaa ggccacattg actgtagaca aatcctccag ggcagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attactgtgc aagacattac 300tacggtgtct ttgactcctg gggccaaggc accaaactca cagtctcctc a 35134117PRTArtificial Sequencesynthetic 34Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Leu Lys Leu Ser Cys Arg Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Arg Ala Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg His Tyr Tyr Gly Val Phe Asp Ser Trp Gly Gln Gly Thr Lys 100 105 110Leu Thr Val Ser Ser 11535360DNAArtificial Sequencesynthetic 35caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccctcagt agctattgga tgcactgggt gaagcagagg 120cctggacaag gccttgagtg gatcggagag attcatcctt ctgatagtta tactagctac 180aatcaaaagt tcaaggacaa ggccacattg actgtagaca aatcctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcagtct attactgtgc aagggggggc 300tactataggt acgacgagtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca 36036120PRTArtificial Sequencesynthetic 36Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Leu Ser Ser Tyr 20 25 30Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile His Pro Ser Asp Ser Tyr Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Tyr Tyr Arg Tyr Asp Glu Phe Ala Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ala 115 12037348DNAArtificial Sequencesynthetic 37caggtccatc tgcagcagcc tggggctgag ctggtgaggc ctggggtttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc acctactcga taaactggat gaagcagagg 120cctggacaag gccttgagtg gatcggaaat atttatcctt ctaccagtca tactaactac 180aatcaaaagt tcagggacaa ggccacaatg actgtagaca aatcctccag cacagcctac 240atgcagctca gcagcccgac atctgaggac tctgcggtct attattgtac aataaatgcc 300tattctatgg actactgggg tcaaggaacc tcagtcaccg tctcctca 34838116PRTArtificial Sequencesynthetic 38Gln Val His Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Val1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30Ser Ile Asn Trp Met Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Asn Ile Tyr Pro Ser Thr Ser His Thr Asn Tyr Asn Gln Lys Phe 50 55 60Arg Asp Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Thr Ile Asn Ala Tyr Ser Met Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110Thr Val Ser Ser 11539339DNAArtificial Sequencesynthetic 39caggtccagc ttcagcagtc tggggctgga ctggcaaaac ctggggcctc agtgaagatg 60tcctgcaagg cttctggcta cacctttact gccaacaaga tgcactgggc aaaacagcgg 120cctggacagg gtctggaatg gattggatac attgatccta gctctggtta tactgaatac 180aatcataaga tccagtacaa ggccactttg actgcagaca catcctccag cacagcctac 240atgcaactga gcaccctaac atttgaagac tctgcagtct attactgtac aaattttgct 300tactggggcc aagggactct ggtcactgtc tcagcaaca 33940112PRTArtificial Sequencesynthetic 40Gln Val Gln Leu Gln Gln Ser Gly Ala Gly Leu Ala Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ala Asn 20 25 30Lys Met His Trp Ala Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asp Pro Ser Ser Gly Tyr Thr Glu Tyr Asn His Lys Ile 50 55 60Gln Tyr Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Thr Leu Thr Phe Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Thr Asn Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 11041336DNAArtificial Sequencesynthetic 41caggtccagc ttcagcagtc tggggctgga ctggcaaaac ctggggcctc agtgaagatg 60tcctgcaagg cttctggcta cacctttact gccaacaaga tgcactgggc aaaacagcgg 120cctggacagg gtctggaatg gattggatac attgatccta gctctggtta tactgaatac 180aatcataaga tccaggacaa ggccacattg actgcagaca catcctccag cacagcctac 240atgcaactga gcagcctaac atttgaagac tctgcagtct attactgtac aaattttgct 300tactggggcc aagggactct ggtcactgtc tcagca 33642112PRTArtificial Sequencesynthetic 42Gln Val Gln Leu Gln Gln Ser Gly Ala Gly Leu Ala Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ala Asn 20 25 30Lys Met His Trp Ala Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asp Pro Ser Ser Gly Tyr Thr Glu Tyr Asn His Lys Ile 50 55 60Gln Asp Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Thr Asn Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 11043336DNAArtificial Sequencesynthetic 43caggtccagc ttcagcagtc tggggctgga ctggcaaaac ctggggcctc agtgaagatg 60tcctgcaagg cttctggcta tacctttact gccaacaaga tgcactggac aaaacagcgg 120cctggacagg gtctggaatg gattggatac attgatccta gctctggtta tactcaatac 180aatcataaga tccaggacaa ggccacattg actgcagaca catcctccag cacagcctac 240atgcaactga gcagcctaac atttgaagac tctgcagtct attactgtac aaattttgct 300tactggggcc aagggactct ggtcactgtc tcagca 33644112PRTArtificial Sequencesynthetic 44Gln Val Gln Leu Gln Gln Ser Gly Ala Gly Leu Ala Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ala Asn 20 25 30Lys Met His Trp Thr Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asp Pro Ser Ser Gly Tyr Thr Gln Tyr Asn His Lys Ile 50 55 60Gln Asp Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Thr Asn Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 11045339DNAArtificial Sequencesynthetic 45caggtccacc ttcagcagtc tggggctgaa ctggccaaac ctggggcctc agtgcagatg 60tcctgcaagg cttctggcta cacctttact gccaacaaga tgcactgggc aagacagcgg 120cctagacagg gtctggaatg gattggatac attgatcctg cctctggcta tactgaatac 180aatcagaaga tcaaggacag ggccacattg actgcagaca aatcctccag cacagcctac 240atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtac aaattttgct 300tactggggcc aagggactct ggtcactgtc tctacaaca 33946112PRTArtificial Sequencesynthetic 46Gln Val His Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala1 5 10 15Ser Val Gln Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ala Asn 20 25 30Lys Met His Trp Ala Arg Gln Arg Pro Arg Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asp Pro Ala Ser Gly Tyr Thr Glu Tyr Asn Gln Lys Ile 50 55 60Lys Asp Arg Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Thr Asn Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Thr 100 105 11047339DNAArtificial Sequencesynthetic 47caggtccagc ttcagcagtc tggggctgaa ctggcaaaac ctggggcctc agtgaagatg 60tcctgcaagg cttctggcta cacctttact agcaacaaga tgcactgggc aaaacagcgg 120cctggacagg gtctggaatg gattggatac attgatccta gctctggtta tactgaatac 180aatcagaaga tcaaggacaa ggccacattg actgcagaca aatcctccag cacagcctac 240atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtac aaattttgct 300tactggggcc aagggactct ggtcactgtc tctgcaaca 33948112PRTArtificial Sequencesynthetic 48Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn 20 25 30Lys Met His Trp Ala Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asp Pro Ser Ser Gly Tyr Thr Glu Tyr Asn Gln Lys Ile 50 55 60Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Thr Asn Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 11049351DNAArtificial Sequencesynthetic 49caggtccagt tgcagcagtc tggacctgag ttggtgaagc ctggggcttc aatgaggata 60tcctgcaagg cttctggcta caccttcaca agctactatg tacactggat aaagcagagg 120cctggacagg gacttgagtg gattggatgt atttatcctg gagatgttaa tactgactat 180aatgagaagt tcaagggcaa ggccacgctg actgcagaca aatcctccag cacagcctac 240atgcaggtca gcaccctgac ctctgaggac tctgcgatct atttctgtgt cctttattac 300tacggtagtt ttgcttactg gggccaaggg actctggtca ctgtctctgc a 35150117PRTArtificial Sequencesynthetic 50Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Met Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Val His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Cys Ile Tyr Pro Gly Asp Val Asn Thr Asp Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Val Ser Thr Leu Thr Ser Glu Asp Ser Ala Ile Tyr Phe Cys 85 90 95Val Leu Tyr Tyr Tyr Gly Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ala 11551348DNAArtificial Sequencesynthetic 51gatgtacagc ttcaggagtc aggacctggc ctcgtgaatc cttctcagtc tctgtctctc 60acctgctctg tcactggcta ctccatcacc agtggttatt actggatctg gatccagcag 120tctccaggaa acaaactgga atggatgggc tacataaagt acgacggtgg caataactac 180agcccatctc tcaaaaatcg aatctccatc gctcgtgaca catctaagaa ccagtgtttc 240ctgaagttga attctgtgac tattgaggac acagctacat attactgtac aagagggtcg 300gactcctttg actactgggg ccaaggcacc actctcacag tctcctca 34852116PRTArtificial Sequencesynthetic 52Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Asn Pro Ser Gln1 5 10 15Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30Tyr Tyr Trp Ile Trp Ile Gln Gln Ser Pro Gly Asn Lys Leu Glu Trp 35 40 45Met Gly Tyr Ile Lys Tyr Asp Gly Gly Asn Asn Tyr Ser Pro Ser Leu 50 55 60Lys Asn Arg Ile Ser Ile Ala Arg Asp Thr Ser Lys Asn Gln Cys Phe65 70 75 80Leu Lys Leu Asn Ser Val Thr Ile Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95Thr Arg Gly Ser Asp Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110Thr Val Ser Ser 11553348DNAArtificial Sequencesynthetic 53gatgtacagc ttcaggagtc aggacctggc ctcgtgaatc cttctcagtc tctgtctctc 60acctgctctg tcactggcta ctccatcacc agtggttatt actggatctg gatccggcag 120tttccaggaa acaaactgga atggatgggc tacataaagt acgacggtgg caataactac 180agcccatctc tcaaaaatcg aatctccatc gctcgtgaca catctaagaa ccagtttttc 240ctgaagttga attctgtgac tattgaggac acagctacat attactgtac aagagggtcg 300gactcctttg actactgggg ccaaggcacc actctcacag tctcctca 34854116PRTArtificial Sequencesynthetic 54Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Asn Pro Ser Gln1 5 10 15Ser Leu Ser Leu Thr Cys Ser

Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30Tyr Tyr Trp Ile Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45Met Gly Tyr Ile Lys Tyr Asp Gly Gly Asn Asn Tyr Ser Pro Ser Leu 50 55 60Lys Asn Arg Ile Ser Ile Ala Arg Asp Thr Ser Lys Asn Gln Phe Phe65 70 75 80Leu Lys Leu Asn Ser Val Thr Ile Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95Thr Arg Gly Ser Asp Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110Thr Val Ser Ser 11555333DNAArtificial Sequencesynthetic 55gatgtgcagc ttcaggagtc gggacctggc ctggtgaaac cttctcagtc tctgtccctc 60acctgcactg tcactggcta ctcaatcacc agtgattatg cctggaactg gatccggcag 120tttcctggaa acaaactgga gtggatgggc tacataagct acagtggtac cactgtctac 180agcccatctc tcaaaagtcg aatctccatc actcgggaca catccaaaaa ccagttcttc 240ctgcaattga attctgtgac tattgaggac tcagccacgt attattgtgg gggtaattac 300tggggccaag ggactctggt cactgtctct gca 33356111PRTArtificial Sequencesynthetic 56Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp 20 25 30Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45Met Gly Tyr Ile Ser Tyr Ser Gly Thr Thr Val Tyr Ser Pro Ser Leu 50 55 60Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe65 70 75 80Leu Gln Leu Asn Ser Val Thr Ile Glu Asp Ser Ala Thr Tyr Tyr Cys 85 90 95Gly Gly Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 11057366DNAArtificial Sequencesynthetic 57caggtgcagc tggaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccatc 60acttgcactg tctctggatt ttcattaacc agctatggtg tacactgggt tcgccagcct 120ccaggaaagg gtctggagtg gctgggagta atatgggctg ttggaagtat aaattataat 180tcggctctca tgtccagact gagcatcagc aaagacaact ccaagagcca agttttctta 240aaaatgaaca gtctacgaac tgatgacaca gccatgtact actgtgccag agatcggact 300acggctaccc ccttctttga ctactggggc caaggcacca ctctcacagt ctcctcatcc 360aaaaca 36658119PRTArtificial Sequencesynthetic 58Gln Val Gln Leu Glu Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1 5 10 15Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Ala Val Gly Ser Ile Asn Tyr Asn Ser Ala Leu Met 50 55 60Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu65 70 75 80Lys Met Asn Ser Leu Arg Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95Arg Asp Arg Thr Thr Ala Thr Pro Phe Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr Leu Thr Val Ser Ser 11559351DNAArtificial Sequencesynthetic 59caggtgcagc tgaagcagtc aggacctggc ctagtgcagc cctcacagag cctgtccatc 60acctgcacag tctctggttt ctcattaatt acccatggtg tacactgggt tcgccagtct 120ccaggaaagg gtctggagtg gctgggagtg atatggagtg gtggaagcac agactataat 180gcagctttca tatccagact gagcatcagc aaggacacct ccaagagcca agttttcctt 240aaaatgagca gtctgcaagc tgatgacaca gccatatact actgtgccag aaatgggggg 300gctacggcct ttgactactg gggccaaggc accactctca cagtctcctc a 35160117PRTArtificial Sequencesynthetic 60Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln1 5 10 15Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Thr His 20 25 30Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile 50 55 60Ser Arg Leu Ser Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Phe Leu65 70 75 80Lys Met Ser Ser Leu Gln Ala Asp Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95Arg Asn Gly Gly Ala Thr Ala Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110Leu Thr Val Ser Ser 11561351DNAArtificial Sequencesynthetic 61caggtacagc tgaagcagtc aggacctggc ctagtgcagc cctcacagag cctgtccatc 60acctgcacag tctctggttt ctcattaatt acctatggtg tacactgggt tcgccagtct 120ccaggaaagg gtctggagtg gctgggagtg atatggggtg gtggaagcac aggctataat 180gcagctttcg tatccagact gaacatcacc aaggacaact ccaagagcca agttttcttt 240aaaatgaaca gtctgcaacc tgatgacaca gccatatact actgtgccag aaatggaggg 300gctacggtct ttgactactg gggccaaggc accactctca cagtctcctc a 35162117PRTArtificial Sequencesynthetic 62Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln1 5 10 15Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Thr Tyr 20 25 30Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Gly Gly Gly Ser Thr Gly Tyr Asn Ala Ala Phe Val 50 55 60Ser Arg Leu Asn Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Phe65 70 75 80Lys Met Asn Ser Leu Gln Pro Asp Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95Arg Asn Gly Gly Ala Thr Val Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110Leu Thr Val Ser Ser 11563351DNAArtificial Sequencesynthetic 63gaggtgcagc tggtggagtc tgggggagac ttagtgaagc ctggagggtc cctgaaactc 60tcctgtgcag cctctggatt cactttcagt aactatggca tgtcttgggt tcgccagact 120ccagacaaga ggctggagtg ggtcgcaacc attagtagtg gtggtagtta tagctactat 180ccagacagtg taaaggggcg gttcaccatc tccagagaca atgccaagaa catcctgtac 240ctgcaaatga gcagtctgaa gtctgaggac acagccatgt attactgtgc aagtctctac 300tacggctacg gggactactg gggccaaggc accgctttca cagtctcctc a 35164117PRTArtificial Sequencesynthetic 64Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Ser Gly Gly Ser Tyr Ser Tyr Tyr Pro Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ile Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Ser Leu Tyr Tyr Gly Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Ala 100 105 110Phe Thr Val Ser Ser 11565360DNAArtificial Sequencesynthetic 65gaagtgcagc tggtggagtc tgggggaggc ttggtgaagc ctggagggtc cctgaaactc 60tcctgtgcag cctctggatt cactttcagt gactcttata tgtattgggt tcgccagact 120ccggaccaga ggctggagtg ggtcgcaacc attagtgatg gtggtagtta caccttctat 180ccagacagtg tgaagggacg attcaccatc tccagagaca atgcccagaa caacctgtac 240ctgcaaatga gcagtctgaa gtctgaggac acagccatgt attactgtgc atccccccat 300gctggctact tcggctggtt tgcttactgg ggccgaggga ctctggtcac tgtctctgca 36066120PRTArtificial Sequencesynthetic 66Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Tyr Met Tyr Trp Val Arg Gln Thr Pro Asp Gln Arg Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Asp Gly Gly Ser Tyr Thr Phe Tyr Pro Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Asn Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Ser Pro His Ala Gly Tyr Phe Gly Trp Phe Ala Tyr Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val Ser Ala 115 12067351DNAArtificial Sequencesynthetic 67gaggtgcagc tggtggagtc tgggggagac ttagtgaagc ctggagggtc cctgaaactc 60tcctgtgcag cctctggatt cactttcaat cactatggca tgtcttgggt tcgccagcct 120ccagacaaga gactggagtg ggtcgcaacc attagtagtg gtggtggtta cacctactat 180ccagacagtg tgaaggggcg cttcaccatc tccagagaca atgccaagga caccctgtcc 240ctgcaaatga gcagtctgag gtctggggac acagccgtgt attactgtgc aagcctatac 300ggtagcctgt ttgcttactg gggccaaggg actctggtca ctgtctctgc a 35168117PRTArtificial Sequencesynthetic 68Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn His Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Pro Pro Asp Lys Arg Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Ser Gly Gly Gly Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asp Thr Leu Ser65 70 75 80Leu Gln Met Ser Ser Leu Arg Ser Gly Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Leu Tyr Gly Ser Leu Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ala 11569366DNAArtificial Sequencesynthetic 69gacgtgaagc tcgtggagtc tgggggaggc ttagtgaagc ttggagggtc cctgaaactc 60tcctgtgcag cctctggatt cactttcagt agctattaca tgtcttgggt tcgccagact 120ccagagaaga ggctggagtt ggtcgcagcc attaatagta atggtggtag cacctactat 180ccagacactg tgaagggccg attcaccatc tccagagaca atgccaagaa caccctgtac 240ctgcaaatga gcagtctgaa gtctgaggac acagccttgt attactgtgc aagacatggg 300ggactgggac gtagggactg gtacttcgat gtctggggcg cagggaccac ggtcaccgtc 360tcctca 36670122PRTArtificial Sequencesynthetic 70Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Leu Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Leu Val 35 40 45Ala Ala Ile Asn Ser Asn Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Ala Arg His Gly Gly Leu Gly Arg Arg Asp Trp Tyr Phe Asp Val Trp 100 105 110Gly Ala Gly Thr Thr Val Thr Val Ser Ser 115 12071360DNAArtificial Sequencesynthetic 71gaagtgaaac tggtggagtc tgggggaagt ttagtgcagc ctggagggtc cctgaaactc 60tcctgcgcag cctctggatt caatttcaat acctatgcca tgtcttgggt tcgccagact 120ccagagaaga ggctggagtg ggtcgcatac attagtaatg gtggtggtaa cacctactat 180gtagacactg taaagggccg attcaccatc tccagagaca atgccaagaa caccctgtac 240ctgcgaatga gcagtctgaa gtctgaggac acggccatgt attactgtgc aagacatggg 300ctctactggg gctattctat ggactactgg ggtcaaggaa cctcagtcac cgtctcctca 36072120PRTArtificial Sequencesynthetic 72Glu Val Lys Leu Val Glu Ser Gly Gly Ser Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asn Thr Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45Ala Tyr Ile Ser Asn Gly Gly Gly Asn Thr Tyr Tyr Val Asp Thr Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Arg Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg His Gly Leu Tyr Trp Gly Tyr Ser Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Ser Val Thr Val Ser Ser 115 12073357DNAArtificial Sequencesynthetic 73gaagtgaagc tggtggagtc tgggggaggt ttagtgcagc caggagggtc cctgaaactc 60tcctgtgcag cctctggatt cactttcagt agctatgcca tgtcttgggt tcgccagact 120ccagagagga ggctggagtg ggtcacatac attagtaatg gtggtggtag cacctactat 180tcagacactg taaagggccg attcaccttc tccagagaca atgccaagaa caccctgtac 240ctgcaaatga gcagtctgaa gtctgaggac acggccatgt attactgtgc aagacatgga 300ctgggaagga cagggtttgc ttcctggggc caagggactc tggtcactgt ctctgca 35774119PRTArtificial Sequencesynthetic 74Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Thr Pro Glu Arg Arg Leu Glu Trp Val 35 40 45Thr Tyr Ile Ser Asn Gly Gly Gly Ser Thr Tyr Tyr Ser Asp Thr Val 50 55 60Lys Gly Arg Phe Thr Phe Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg His Gly Leu Gly Arg Thr Gly Phe Ala Ser Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ala 11575363DNAArtificial Sequencesynthetic 75gatgtgcagc tggtggagtc tgggggaggc ctagtgcagg ctggagggtc ccggaaactc 60tcctgtgcag cctctggatt ccctttcagt tcctttggaa tgcactgggt tcgtcaggct 120ccagagaagg ggctggagtg ggtcgcctcc attagtagtc gcactagtaa gatctactat 180gcagacaacc tgaagggccg attcaccatc tccagagaca atcccaagaa caccctgttc 240ctgcaaatga ccagtcttgg atctgaggac acggccatgt attactgtgt aagatccgtc 300tttggtaatt cttactggtt tttcgatgtc tggggcgcag ggaccacggt caccgtctcc 360tca 36376121PRTArtificial Sequencesynthetic 76Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Ser Phe 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Ile Ser Ser Arg Thr Ser Lys Ile Tyr Tyr Ala Asp Asn Leu 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe65 70 75 80Leu Gln Met Thr Ser Leu Gly Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Val Arg Ser Val Phe Gly Asn Ser Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110Ala Gly Thr Thr Val Thr Val Ser Ser 115 12077345DNAArtificial Sequencesynthetic 77gaagtgaagc ttgaggagtc tggaggaggc ttggtacaac ctgggggatc catgaaactc 60tcctgtgtag cctctggatt ttctttcagt agctactgga tgtcttgggt ccgccagtct 120ccagagaagg ggcttgactg ggttgctgaa attagattga gatctgataa ttatgcaacc 180cattatgcgg agtctgtgaa agggaggttc accatctcaa gagatgattc cataagtcgt 240ctctacctgc aaatgaacac cttaagagct gaagacactg gaatttatta ctgtacatgg 300atgacgtact ggggccaagg gactctggtc actgtctctg caaca 34578114PRTArtificial Sequencesynthetic 78Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Ser Phe Ser Ser Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Asp Trp Val 35 40 45Ala Glu Ile Arg Leu Arg Ser Asp Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Ile Ser Arg65 70 75 80Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Gly Ile Tyr 85 90 95Tyr Cys Thr Trp Met Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110Ser Ala79366DNAArtificial Sequencesynthetic 79gaggtgaagc tggtggagtc tggaggaggc ttggtacagc ctgggggttc tctgagactc 60tcctgtgcaa gttctgggtt caccttcact gattactaca tgagctgggt ccgccagcct 120ccaggaaagg cacttgagtg gttgggtttt attagaaaca aagcttatgg ttacacgacc 180gagttcagtg catctgtgaa cggtcggttc accatctcca gagatgattc ccaaagcgtc 240ccctatcttc aaatgaacac cctgagagct gaggacagtg ccacttatta ctgtgcgaga 300gtcctctact atgattacgg gggatttgct tactggggcc aagggactct ggtcactgtc 360tctaca 36680122PRTArtificial Sequencesynthetic 80Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5

10 15Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45Gly Phe Ile Arg Asn Lys Ala Tyr Gly Tyr Thr Thr Glu Phe Ser Ala 50 55 60Ser Val Asn Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Val65 70 75 80Pro Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Ser Ala Thr Tyr 85 90 95Tyr Cys Ala Arg Val Leu Tyr Tyr Asp Tyr Gly Gly Phe Ala Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Thr 115 12081363DNAArtificial Sequencesynthetic 81gaggtccagc tgcagcagtc tggacctgag ctggtaaagc ctggggcttc agtgaagatg 60tcctgtaagg cttccggata cagattcact agctatgtta tgcactgggt gaggcagaag 120cctggacagg gccttgagtg gattggatat attgatcctc acaatgatga tacaaaatac 180agtgagaagt tcagaggtaa ggccacactg acttcagaca agtcctccac cacagcctac 240atggagctca gcagcctgac ctctgaggac tctgcggtct attactgtgt gagatattct 300tacgacaggg attacagtcc tatggactac tggggtcaag gaacctcagt caccgtctcc 360tca 36382121PRTArtificial Sequencesynthetic 82Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Ser Tyr 20 25 30Val Met His Trp Val Arg Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asp Pro His Asn Asp Asp Thr Lys Tyr Ser Glu Lys Phe 50 55 60Arg Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Thr Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Val Arg Tyr Ser Tyr Asp Arg Asp Tyr Ser Pro Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Ser Val Thr Val Ser Ser 115 12083345DNAArtificial Sequencesynthetic 83cagctgcaac agtctggacc tgagctggtg aagcctgggg cttcagtgaa aatttcctgc 60aagacttctg gatacacatt cactgaaaac gccatgcact gggtgaagca gagccgtgga 120aagagccttg agtggattgg aggtgttaat cctaacaatg gtgatactgt ctacacccag 180aagttcaagg gcaaggccac attgactgta gccaagtctt ccagcacagc ctacatggag 240ctccgcacca tgacatgtga ggaatctaca gtgtattact gtgcaagccg ggaaccggac 300ttctttgact actggggcca aggctcctct gtcacagtct cctca 34584115PRTArtificial Sequencesynthetic 84Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val1 5 10 15Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Asn Ala Met 20 25 30His Trp Val Lys Gln Ser Arg Gly Lys Ser Leu Glu Trp Ile Gly Gly 35 40 45Val Asn Pro Asn Asn Gly Asp Thr Val Tyr Thr Gln Lys Phe Lys Gly 50 55 60Lys Ala Thr Leu Thr Val Ala Lys Ser Ser Ser Thr Ala Tyr Met Glu65 70 75 80Leu Arg Thr Met Thr Cys Glu Glu Ser Thr Val Tyr Tyr Cys Ala Ser 85 90 95Arg Glu Pro Asp Phe Phe Asp Tyr Trp Gly Gln Gly Ser Ser Val Thr 100 105 110Val Ser Ser 11585345DNAArtificial Sequencesynthetic 85cagctgcaac agtctggacc tgtactggtg aagcctgggg cttcagtgaa aatttcctgc 60aagacttctg gatacacatt cactgaaaac gccatgcact gggtgaagca gagccatgga 120aagagccttg agtggattgg aggtgttaat cctaacaatg gtgatactat ctacaaccag 180aagttcaagg gcaaggccac attgactgta gacaagtcct ccagcacagc cttcatggag 240ctccgcagcc tgacatctga agaatcccca gtctatttct gtgtaagacg ggaactggac 300ttctttgact actggggcca aggcacctct gtcacagtct cctca 34586115PRTArtificial Sequencesynthetic 86Gln Leu Gln Gln Ser Gly Pro Val Leu Val Lys Pro Gly Ala Ser Val1 5 10 15Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Asn Ala Met 20 25 30His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly Gly 35 40 45Val Asn Pro Asn Asn Gly Asp Thr Ile Tyr Asn Gln Lys Phe Lys Gly 50 55 60Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe Met Glu65 70 75 80Leu Arg Ser Leu Thr Ser Glu Glu Ser Pro Val Tyr Phe Cys Val Arg 85 90 95Arg Glu Leu Asp Phe Phe Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr 100 105 110Val Ser Ser 11587345DNAArtificial Sequencesynthetic 87cagctgcaac agtctggccc tgtcctggtg aagtctggga cttcagttaa aatttcctgc 60aagacttctg gatacacatt cactgaaaac gccatgcact gggtgaaaca gagccatgga 120cagagccttg agtggattgg aggtgttaat cctaacaatg gtgatactat cttcaaccag 180aagttcaagg gcaaggccac attgactgta gacaagtcct ccagcacagc cttcatggag 240ctccgcagcc tgacatctga agaatccaca gtctattact gtgtaagacg ggaactggac 300ttctttgact actggggtca aggcacctct gtcacagtct cctca 34588115PRTArtificial Sequencesynthetic 88Gln Leu Gln Gln Ser Gly Pro Val Leu Val Lys Ser Gly Thr Ser Val1 5 10 15Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Asn Ala Met 20 25 30His Trp Val Lys Gln Ser His Gly Gln Ser Leu Glu Trp Ile Gly Gly 35 40 45Val Asn Pro Asn Asn Gly Asp Thr Ile Phe Asn Gln Lys Phe Lys Gly 50 55 60Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe Met Glu65 70 75 80Leu Arg Ser Leu Thr Ser Glu Glu Ser Thr Val Tyr Tyr Cys Val Arg 85 90 95Arg Glu Leu Asp Phe Phe Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr 100 105 110Val Ser Ser 11589345DNAArtificial Sequencesynthetic 89cagctgcaac agtctggccc tgtcctggtg aagcctggga cttcagtgaa aatttcctgc 60aagacttctg gatacacatt cactgaaaac gccatgcact gggtgaaaca gagccatgga 120aagagccttg agtggattgg aggtgttaat cctaacaatg gtgatactat cttcaaccag 180aagttcaagg gcaaggccac attgactgta gacaagtcct ccagcacagc cttcatggag 240ctccgcagcc tgacatctga agaatccaca gtctattact gtgtaagacg ggaactggac 300ttctttgact actggggcca aggcacctct gtcacagtct cctca 34590115PRTArtificial Sequencesynthetic 90Gln Leu Gln Gln Ser Gly Pro Val Leu Val Lys Pro Gly Thr Ser Val1 5 10 15Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Asn Ala Met 20 25 30His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly Gly 35 40 45Val Asn Pro Asn Asn Gly Asp Thr Ile Phe Asn Gln Lys Phe Lys Gly 50 55 60Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe Met Glu65 70 75 80Leu Arg Ser Leu Thr Ser Glu Glu Ser Thr Val Tyr Tyr Cys Val Arg 85 90 95Arg Glu Leu Asp Phe Phe Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr 100 105 110Val Ser Ser 11591345DNAArtificial Sequencesynthetic 91cagctgcaac agtctggacc tgcccaggtg aagcctgggg cttcagtgat gatttcctgc 60aagacttctg gatacacatt cactgaaaac gccatgcact gggtgaaaca gagccatgga 120aagagccttg agtggattgg aggtgttaat cctaacaatg gtgatactat tttcaaccag 180aagttcaagg acaaggccgc attgactgta gacaagtcct ccagcacagc cttcatggag 240ctccgcagcc tgacatctga ggagtccaca gtctattact gtgtaagacg ggaattggac 300ttctttgact actggggcca aggcacctct gtcacagtct cctca 34592115PRTArtificial Sequencesynthetic 92Gln Leu Gln Gln Ser Gly Pro Ala Gln Val Lys Pro Gly Ala Ser Val1 5 10 15Met Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Asn Ala Met 20 25 30His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly Gly 35 40 45Val Asn Pro Asn Asn Gly Asp Thr Ile Phe Asn Gln Lys Phe Lys Asp 50 55 60Lys Ala Ala Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe Met Glu65 70 75 80Leu Arg Ser Leu Thr Ser Glu Glu Ser Thr Val Tyr Tyr Cys Val Arg 85 90 95Arg Glu Leu Asp Phe Phe Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr 100 105 110Val Ser Ser 11593345DNAArtificial Sequencesynthetic 93cagctgcaac agtctggacc tgtcctggtg aagcctgggg cttcagtgat gatttcctgc 60aagacttctg gatacacatt cactgaaaac gccatgcact gggtgaaaca gagccatgga 120aagagccttg agtggattgg aggtgttaat cctaacaatg gtgatactat tttcaaccag 180aagttcaagg gcaaggccac attgactgta gacaagtcct ccaacacagc cttcacggag 240ctccgcagcc tgccatctga agaatccaca gtctattact gtgttagacg gggattggac 300ttctttgact actggggcca aggcacctct gtcacagtct cctca 34594115PRTArtificial Sequencesynthetic 94Gln Leu Gln Gln Ser Gly Pro Val Leu Val Lys Pro Gly Ala Ser Val1 5 10 15Met Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Asn Ala Met 20 25 30His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly Gly 35 40 45Val Asn Pro Asn Asn Gly Asp Thr Ile Phe Asn Gln Lys Phe Lys Gly 50 55 60Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Phe Thr Glu65 70 75 80Leu Arg Ser Leu Pro Ser Glu Glu Ser Thr Val Tyr Tyr Cys Val Arg 85 90 95Arg Gly Leu Asp Phe Phe Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr 100 105 110Val Ser Ser 11595345DNAArtificial Sequencesynthetic 95cagctgcaac agtctggccc tgtcctggtg gggcctgggg cttcagtgat gatttcctgc 60aagacttctg gatacacatt cactgaaaac gccatgcact gggtggaaca gagccatgga 120aagagccttg agtggattgg aggtgttaat cctaacaatg gtgatactat tttcaaccag 180aagttcaagg gcaaggccac attgactgta gacaagtcct ccagcacagc cttcatggag 240ttccgcagcc tgacatctga agaatccaca gtctattact gtgtaagacg ggaattggac 300ttctttgact actggggcca aggcacctct gtcacagtct cctca 34596115PRTArtificial Sequencesynthetic 96Gln Leu Gln Gln Ser Gly Pro Val Leu Val Gly Pro Gly Ala Ser Val1 5 10 15Met Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Asn Ala Met 20 25 30His Trp Val Glu Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly Gly 35 40 45Val Asn Pro Asn Asn Gly Asp Thr Ile Phe Asn Gln Lys Phe Lys Gly 50 55 60Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe Met Glu65 70 75 80Phe Arg Ser Leu Thr Ser Glu Glu Ser Thr Val Tyr Tyr Cys Val Arg 85 90 95Arg Glu Leu Asp Phe Phe Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr 100 105 110Val Ser Ser 11597348DNAArtificial Sequencesynthetic 97cagctgcaac agtctggacc tgtcctggtg aagcctgggg cttcagtgat gatttcctgc 60aagacttctg gatacacatt cactgaaaac gccatgcact gggtgaaaca gagccatgga 120aagagccttg agtggattgg aggtgttaat cctaacaatg gtgatactat tttcaaccag 180aagttcaagg gcaaggccac attgactgta gacaagtcct ccagcacagc cttcatggag 240ttccgcagcc tgacatctga agaatccaca gtctattact gtgtaagacg ggaattggac 300ttctttgact actggggcca aggcacctct gtcacagtct cctcaaca 34898115PRTArtificial Sequencesynthetic 98Gln Leu Gln Gln Ser Gly Pro Val Leu Val Lys Pro Gly Ala Ser Val1 5 10 15Met Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Asn Ala Met 20 25 30His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly Gly 35 40 45Val Asn Pro Asn Asn Gly Asp Thr Ile Phe Asn Gln Lys Phe Lys Gly 50 55 60Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe Met Glu65 70 75 80Phe Arg Ser Leu Thr Ser Glu Glu Ser Thr Val Tyr Tyr Cys Val Arg 85 90 95Arg Glu Leu Asp Phe Phe Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr 100 105 110Val Ser Ser 11599345DNAArtificial Sequencesynthetic 99cagctgcaac agtctggacc tgtcctggtg aagcctgggg cttcagtgat gatttcctgc 60aagacttctg gatacacatt cactgaaaac gccatgcact gggtgaaaca gagccatgga 120aagagccttg agtggattgg aggtgttaat cctaacaatg gtgatactat tttcaaccag 180aagttcaagg gcaaggccac attgactgta gacaagtcct ccagcacagc cttcatggag 240ctccgcagcc tgacatctga agaatccaca gtctattact gtgtaagacg ggaattggac 300ttctttgact actggggcca aggcacctct gtcacagtct cctca 345100115PRTArtificial Sequencesynthetic 100Gln Leu Gln Gln Ser Gly Pro Val Leu Val Lys Pro Gly Ala Ser Val1 5 10 15Met Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Asn Ala Met 20 25 30His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly Gly 35 40 45Val Asn Pro Asn Asn Gly Asp Thr Ile Phe Asn Gln Lys Phe Lys Gly 50 55 60Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe Met Glu65 70 75 80Leu Arg Ser Leu Thr Ser Glu Glu Ser Thr Val Tyr Tyr Cys Val Arg 85 90 95Arg Glu Leu Asp Phe Phe Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr 100 105 110Val Ser Ser 11510115DNAArtificial Sequencesynthetic 101gaatacgcca tgcac 151025PRTArtificial Sequencesynthetic 102Glu Tyr Ala Met His1 510315DNAArtificial Sequencesynthetic 103gaatacgcca tacac 151045PRTArtificial Sequencesynthetic 104Glu Tyr Ala Ile His1 510515DNAArtificial Sequencesynthetic 105agtagttata taagt 151065PRTArtificial Sequencesynthetic 106Ser Ser Tyr Ile Ser1 510715DNAArtificial Sequencesynthetic 107agtagtttta taagt 151085PRTArtificial Sequencesynthetic 108Ser Ser Phe Ile Ser1 510915DNAArtificial Sequencesynthetic 109agtagttatt ttagt 151105PRTArtificial Sequencesynthetic 110Ser Ser Tyr Phe Ser1 511118DNAArtificial Sequencesynthetic 111agcagtacct atataagt 181125PRTArtificial Sequencesynthetic 112Ser Thr Tyr Ile Ser1 511315DNAArtificial Sequencesynthetic 113agctactgga tacac 151145PRTArtificial Sequencesynthetic 114Ser Tyr Trp Ile His1 511515DNAArtificial Sequencesynthetic 115agctattgga tgcac 151165PRTArtificial Sequencesynthetic 116Ser Tyr Trp Met His1 511714DNAArtificial Sequencesynthetic 117cctactcgat aaac 141185PRTArtificial Sequencesynthetic 118Thr Tyr Ser Ile Asn1 511915DNAArtificial Sequencesynthetic 119gccaacaaga tgcac 151205PRTArtificial Sequencesynthetic 120Ala Asn Lys Met His1 512115DNAArtificial Sequencesynthetic 121agcaacaaga tgcac 151225PRTArtificial Sequencesynthetic 122Ser Asn Lys Met His1 512315DNAArtificial Sequencesynthetic 123agctactatg tacac 151245PRTArtificial Sequencesynthetic 124Ser Tyr Tyr Val His1 512518DNAArtificial Sequencesynthetic 125agtggttatt actggatc 181266PRTArtificial Sequencesynthetic 126Ser Gly Tyr Tyr Trp Ile1 512718DNAArtificial Sequencesynthetic 127agtgattatg cctggaac 181286PRTArtificial Sequencesynthetic 128Ser Asp Tyr Ala Trp Asn1 512915DNAArtificial Sequencesynthetic 129agctatggtg tacac 151305PRTArtificial Sequencesynthetic 130Ser Tyr Gly Val His1 513115DNAArtificial Sequencesynthetic 131acccatggtg tacac 151325PRTArtificial Sequencesynthetic 132Thr His Gly Val His1 513315DNAArtificial Sequencesynthetic 133acctatggtg tacac

151345PRTArtificial Sequencesynthetic 134Thr Tyr Gly Val His1 513515DNAArtificial Sequencesynthetic 135aactatggca tgtct 151365PRTArtificial Sequencesynthetic 136Asn Tyr Gly Met Ser1 513715DNAArtificial Sequencesynthetic 137gactcttata tgtat 151385PRTArtificial Sequencesynthetic 138Asp Ser Tyr Met Tyr1 513915DNAArtificial Sequencesynthetic 139cactatggca tgtct 151405PRTArtificial Sequencesynthetic 140His Tyr Gly Met Ser1 514115DNAArtificial Sequencesynthetic 141agctattaca tgtct 151425PRTArtificial Sequencesynthetic 142Ser Tyr Tyr Met Ser1 514315DNAArtificial Sequencesynthetic 143acctatgcca tgtct 151445PRTArtificial Sequencesynthetic 144Thr Tyr Ala Met Ser1 514515DNAArtificial Sequencesynthetic 145agctatgcca tgtct 151465PRTArtificial Sequencesynthetic 146Ser Tyr Ala Met Ser1 514715DNAArtificial Sequencesynthetic 147tcctttggaa tgcac 151485PRTArtificial Sequencesynthetic 148Ser Phe Gly Met His1 514915DNAArtificial Sequencesynthetic 149agctactgga tgtct 151505PRTArtificial Sequencesynthetic 150Ser Tyr Trp Met Ser1 515115DNAArtificial Sequencesynthetic 151gattactaca tgagc 151525PRTArtificial Sequencesynthetic 152Asp Tyr Tyr Met Ser1 515315DNAArtificial Sequencesynthetic 153agctatgtta tgcac 151545PRTArtificial Sequencesynthetic 154Ser Tyr Val Met His1 515515DNAArtificial Sequencesynthetic 155gaaaacgcca tgcac 151565PRTArtificial Sequencesynthetic 156Glu Asn Ala Met His1 515751DNAArtificial Sequencesynthetic 157ggtatcaatc ctactaatgg tgatacaatc tacaaccaga agttcaagga c 5115817PRTArtificial Sequencesynthetic 158Gly Ile Asn Pro Thr Asn Gly Asp Thr Ile Tyr Asn Gln Lys Phe Lys1 5 10 15Asp15951DNAArtificial Sequencesynthetic 159ggtatcaatc ctactaatgg tgatacaatc tacaaccaga ggttcaagga c 5116017PRTArtificial Sequencesynthetic 160Gly Ile Asn Pro Thr Asn Gly Asp Thr Ile Tyr Asn Gln Arg Phe Lys1 5 10 15Asp16151DNAArtificial Sequencesynthetic 161ggtattaatc ctaacaatgg caatgctatc tacaaccaga tattcaagga c 5116217PRTArtificial Sequencesynthetic 162Gly Ile Asn Pro Asn Asn Gly Asn Ala Ile Tyr Asn Gln Ile Phe Lys1 5 10 15Asp16351DNAArtificial Sequencesynthetic 163ggtattaatc ctagcaatgg caatgctatc tacaaccaaa tattcaagga c 5116417PRTArtificial Sequencesynthetic 164Gly Ile Asn Pro Ser Asn Gly Asn Ala Ile Tyr Asn Gln Ile Phe Lys1 5 10 15Asp16551DNAArtificial Sequencesynthetic 165ggtattaatc ctagcaatgg cgatcctatc tataaccaga tattcaagga c 5116617PRTArtificial Sequencesynthetic 166Gly Ile Asn Pro Ser Asn Gly Asp Pro Ile Tyr Asn Gln Ile Phe Lys1 5 10 15Asp16751DNAArtificial Sequencesynthetic 167tggatttatg ctggaactgg taatactaac tataatcaga agttcacaga c 5116817PRTArtificial Sequencesynthetic 168Trp Ile Tyr Ala Gly Thr Gly Asn Thr Asn Tyr Asn Gln Lys Phe Thr1 5 10 15Asp16951DNAArtificial Sequencesynthetic 169tggatttttg ctggaactgg taatactaat tataatcaga agttcacaga c 5117017PRTArtificial Sequencesynthetic 170Trp Ile Phe Ala Gly Thr Gly Asn Thr Asn Tyr Asn Gln Lys Phe Thr1 5 10 15Asp17151DNAArtificial Sequencesynthetic 171tggatttatg ctggaactgg taatactatc tataatcaga agttcacaga c 5117217PRTArtificial Sequencesynthetic 172Trp Ile Tyr Ala Gly Thr Gly Asn Thr Ile Tyr Asn Gln Lys Phe Thr1 5 10 15Asp17351DNAArtificial Sequencesynthetic 173tggatttatg ctggaactgg tggtactaac tataatcaga agttcacaga c 5117417PRTArtificial Sequencesynthetic 174Trp Ile Tyr Ala Gly Thr Gly Gly Thr Asn Tyr Asn Gln Lys Phe Thr1 5 10 15Asp17551DNAArtificial Sequencesynthetic 175tggatttatg ctggaactgg tgctactaat tataatcaga agttcacagg c 5117617PRTArtificial Sequencesynthetic 176Trp Ile Tyr Ala Gly Thr Gly Ala Thr Asn Tyr Asn Gln Lys Phe Thr1 5 10 15Gly17751DNAArtificial Sequencesynthetic 177gaaattgatc cttctgacag ttatattaac cagaatcaaa agttcagggg c 5117817PRTArtificial Sequencesynthetic 178Glu Ile Asp Pro Ser Asp Ser Tyr Ile Asn Gln Asn Gln Lys Phe Arg1 5 10 15Gly17951DNAArtificial Sequencesynthetic 179gaagttgatc cttctgacag ttatattaac cagaatgaaa agttcagggg c 5118017PRTArtificial Sequencesynthetic 180Glu Val Asp Pro Ser Asp Ser Tyr Ile Asn Gln Asn Glu Lys Phe Arg1 5 10 15Gly18151DNAArtificial Sequencesynthetic 181gagattgatc cttctgatag ttatactaac tacaatcaaa agttcaaggg c 5118217PRTArtificial Sequencesynthetic 182Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe Lys1 5 10 15Gly18351DNAArtificial Sequencesynthetic 183gagattcatc cttctgatag ttatactagc tacaatcaaa agttcaagga c 5118417PRTArtificial Sequencesynthetic 184Glu Ile His Pro Ser Asp Ser Tyr Thr Ser Tyr Asn Gln Lys Phe Lys1 5 10 15Asp18551DNAArtificial Sequencesynthetic 185aatatttatc cttctaccag tcatactaac tacaatcaaa agttcaggga c 5118617PRTArtificial Sequencesynthetic 186Asn Ile Tyr Pro Ser Thr Ser His Thr Asn Tyr Asn Gln Lys Phe Arg1 5 10 15Asp18751DNAArtificial Sequencesynthetic 187tacattgatc ctagctctgg ttatactgaa tacaatcata agatccagta c 5118817PRTArtificial Sequencesynthetic 188Tyr Ile Asp Pro Ser Ser Gly Tyr Thr Glu Tyr Asn His Lys Ile Gln1 5 10 15Tyr18951DNAArtificial Sequencesynthetic 189tacattgatc ctagctctgg ttatactgaa tacaatcata agatccagga c 5119017PRTArtificial Sequencesynthetic 190Tyr Ile Asp Pro Ser Ser Gly Tyr Thr Glu Tyr Asn His Lys Ile Gln1 5 10 15Asp19151DNAArtificial Sequencesynthetic 191tacattgatc ctagctctgg ttatactcaa tacaatcata agatccagga c 5119217PRTArtificial Sequencesynthetic 192Tyr Ile Asp Pro Ser Ser Gly Tyr Thr Gln Tyr Asn His Lys Ile Gln1 5 10 15Asp19351DNAArtificial Sequencesynthetic 193tacattgatc ctgcctctgg ctatactgaa tacaatcaga agatcaagga c 5119417PRTArtificial Sequencesynthetic 194Tyr Ile Asp Pro Ala Ser Gly Tyr Thr Glu Tyr Asn Gln Lys Ile Lys1 5 10 15Asp19551DNAArtificial Sequencesynthetic 195tacattgatc ctagctctgg ttatactgaa tacaatcaga agatcaagga c 5119617PRTArtificial Sequencesynthetic 196Tyr Ile Asp Pro Ser Ser Gly Tyr Thr Glu Tyr Asn Gln Lys Ile Lys1 5 10 15Asp19751DNAArtificial Sequencesynthetic 197tgtatttatc ctggagatgt taatactgac tataatgaga agttcaaggg c 5119817PRTArtificial Sequencesynthetic 198Cys Ile Tyr Pro Gly Asp Val Asn Thr Asp Tyr Asn Glu Lys Phe Lys1 5 10 15Gly19948DNAArtificial Sequencesynthetic 199tacataaagt acgacggtgg caataactac agcccatctc tcaaaaat 4820016PRTArtificial Sequencesynthetic 200Tyr Ile Lys Tyr Asp Gly Gly Asn Asn Tyr Ser Pro Ser Leu Lys Asn1 5 10 1520148DNAArtificial Sequencesynthetic 201tacataagct acagtggtac cactgtctac agcccatctc tcaaaagt 4820216PRTArtificial Sequencesynthetic 202Tyr Ile Ser Tyr Ser Gly Thr Thr Val Tyr Ser Pro Ser Leu Lys Ser1 5 10 1520348DNAArtificial Sequencesynthetic 203gtaatatggg ctgttggaag tataaattat aattcggctc tcatgtcc 4820416PRTArtificial Sequencesynthetic 204Val Ile Trp Ala Val Gly Ser Ile Asn Tyr Asn Ser Ala Leu Met Ser1 5 10 1520548DNAArtificial Sequencesynthetic 205gtgatatgga gtggtggaag cacagactat aatgcagctt tcatatcc 4820616PRTArtificial Sequencesynthetic 206Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile Ser1 5 10 1520748DNAArtificial Sequencesynthetic 207gtgatatggg gtggtggaag cacaggctat aatgcagctt tcgtatcc 4820816PRTArtificial Sequencesynthetic 208Val Ile Trp Gly Gly Gly Ser Thr Gly Tyr Asn Ala Ala Phe Val Ser1 5 10 1520951DNAArtificial Sequencesynthetic 209accattagta gtggtggtag ttatagctac tatccagaca gtgtaaaggg g 5121017PRTArtificial Sequencesynthetic 210Thr Ile Ser Ser Gly Gly Ser Tyr Ser Tyr Tyr Pro Asp Ser Val Lys1 5 10 15Gly21151DNAArtificial Sequencesynthetic 211accattagtg atggtggtag ttacaccttc tatccagaca gtgtgaaggg a 5121217PRTArtificial Sequencesynthetic 212Thr Ile Ser Asp Gly Gly Ser Tyr Thr Phe Tyr Pro Asp Ser Val Lys1 5 10 15Gly21351DNAArtificial Sequencesynthetic 213accattagta gtggtggtgg ttacacctac tatccagaca gtgtgaaggg g 5121417PRTArtificial Sequencesynthetic 214Thr Ile Ser Ser Gly Gly Gly Tyr Thr Tyr Tyr Pro Asp Ser Val Lys1 5 10 15Gly21551DNAArtificial Sequencesynthetic 215gccattaata gtaatggtgg tagcacctac tatccagaca ctgtgaaggg c 5121617PRTArtificial Sequencesynthetic 216Ala Ile Asn Ser Asn Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val Lys1 5 10 15Gly21751DNAArtificial Sequencesynthetic 217tacattagta atggtggtgg taacacctac tatgtagaca ctgtaaaggg c 5121817PRTArtificial Sequencesynthetic 218Tyr Ile Ser Asn Gly Gly Gly Asn Thr Tyr Tyr Val Asp Thr Val Lys1 5 10 15Gly21951DNAArtificial Sequencesynthetic 219tacattagta atggtggtgg tagcacctac tattcagaca ctgtaaaggg c 5122017PRTArtificial Sequencesynthetic 220Tyr Ile Ser Asn Gly Gly Gly Ser Thr Tyr Tyr Ser Asp Thr Val Lys1 5 10 15Gly22151DNAArtificial Sequencesynthetic 221tccattagta gtcgcactag taagatctac tatgcagaca acctgaaggg c 5122217PRTArtificial Sequencesynthetic 222Ser Ile Ser Ser Arg Thr Ser Lys Ile Tyr Tyr Ala Asp Asn Leu Lys1 5 10 15Gly22357DNAArtificial Sequencesynthetic 223gaaattagat tgagatctga taattatgca acccattatg cggagtctgt gaaaggg 5722419PRTArtificial Sequencesynthetic 224Glu Ile Arg Leu Arg Ser Asp Asn Tyr Ala Thr His Tyr Ala Glu Ser1 5 10 15Val Lys Gly22557DNAArtificial Sequencesynthetic 225tttattagaa acaaagctta tggttacacg accgagttca gtgcatctgt gaacggt 5722619PRTArtificial Sequencesynthetic 226Phe Ile Arg Asn Lys Ala Tyr Gly Tyr Thr Thr Glu Phe Ser Ala Ser1 5 10 15Val Asn Gly22751DNAArtificial Sequencesynthetic 227tatattgatc ctcacaatga tgatacaaaa tacagtgaga agttcagagg t 5122817PRTArtificial Sequencesynthetic 228Tyr Ile Asp Pro His Asn Asp Asp Thr Lys Tyr Ser Glu Lys Phe Arg1 5 10 15Gly22951DNAArtificial Sequencesynthetic 229ggtgttaatc ctaacaatgg tgatactgtc tacacccaga agttcaaggg c 5123017PRTArtificial Sequencesynthetic 230Gly Val Asn Pro Asn Asn Gly Asp Thr Val Tyr Thr Gln Lys Phe Lys1 5 10 15Gly23151DNAArtificial Sequencesynthetic 231ggtgttaatc ctaacaatgg tgatactatc tacaaccaga agttcaaggg c 5123217PRTArtificial Sequencesynthetic 232Gly Val Asn Pro Asn Asn Gly Asp Thr Ile Tyr Asn Gln Lys Phe Lys1 5 10 15Gly23351DNAArtificial Sequencesynthetic 233ggtgttaatc ctaacaatgg tgatactatc ttcaaccaga agttcaaggg c 5123417PRTArtificial Sequencesynthetic 234Gly Val Asn Pro Asn Asn Gly Asp Thr Ile Phe Asn Gln Lys Phe Lys1 5 10 15Gly23551DNAArtificial Sequencesynthetic 235ggtgttaatc ctaacaatgg tgatactatt ttcaaccaga agttcaagga c 5123617PRTArtificial Sequencesynthetic 236Gly Val Asn Pro Asn Asn Gly Asp Thr Ile Phe Asn Gln Lys Phe Lys1 5 10 15Asp23724DNAArtificial Sequencesynthetic 237cgggaactgg actactttgc ctcc 242388PRTArtificial Sequencesynthetic 238Arg Glu Leu Asp Tyr Phe Ala Ser1 523924DNAArtificial Sequencesynthetic 239cgggaactgg actactttcc ctcc 242408PRTArtificial Sequencesynthetic 240Arg Glu Leu Asp Tyr Phe Pro Ser1 524124DNAArtificial Sequencesynthetic 241cgacaactgg actactttga ctat 242428PRTArtificial Sequencesynthetic 242Arg Gln Leu Asp Tyr Phe Asp Tyr1 524324DNAArtificial Sequencesynthetic 243cgacaactgg acttctttga ctat 242448PRTArtificial Sequencesynthetic 244Arg Gln Leu Asp Phe Phe Asp Tyr1 524524DNAArtificial Sequencesynthetic 245cgacaactgg actactttga cttt 242468PRTArtificial Sequencesynthetic 246Arg Gln Leu Asp Tyr Phe Asp Phe1 524721DNAArtificial Sequencesynthetic 247agtgggacgg ggtttactta c 212487PRTArtificial Sequencesynthetic 248Ser Gly Thr Gly Phe Thr Tyr1 524921DNAArtificial Sequencesynthetic 249agtgggacgg ggtttattta c 212507PRTArtificial Sequencesynthetic 250Ser Gly Thr Gly Phe Ile Tyr1 525118DNAArtificial Sequencesynthetic 251tctggggcgg gggtttac 182526PRTArtificial Sequencesynthetic 252Ser Gly Ala Gly Val Tyr1 525324DNAArtificial Sequencesynthetic 253cattactacg gtgttcttga ctcc 242548PRTArtificial Sequencesynthetic 254His Tyr Tyr Gly Val Leu Asp Ser1 525524DNAArtificial Sequencesynthetic 255cattactacg gtgtctttga ctcc 242568PRTArtificial Sequencesynthetic 256His Tyr Tyr Gly Val Phe Asp Ser1 525733DNAArtificial Sequencesynthetic 257gggggctact ataggtacga cgagtttgct tac 3325811PRTArtificial Sequencesynthetic 258Gly Gly Tyr Tyr Arg Tyr Asp Glu Phe Ala Tyr1 5 1025921DNAArtificial Sequencesynthetic 259aatgcctatt ctatggacta c 212607PRTArtificial Sequencesynthetic 260Asn Ala Tyr Ser Met Asp Tyr1 52619DNAArtificial Sequencesynthetic 261tttgcttac 92623PRTArtificial Sequencesynthetic 262Phe Ala Tyr126324DNAArtificial Sequencesynthetic 263tattactacg gtagttttgc ttac 242648PRTArtificial Sequencesynthetic 264Tyr Tyr Tyr Gly Ser Phe Ala Tyr1 526521DNAArtificial Sequencesynthetic 265gggtcggact cctttgacta c 212667PRTArtificial Sequencesynthetic 266Gly Ser Asp Ser Phe Asp Tyr1 52676DNAArtificial Sequencesynthetic 267aattac 62682PRTArtificial Sequencesynthetic 268Asn Tyr126933DNAArtificial Sequencesynthetic 269gatcggacta cggctacccc cttctttgac tac 3327011PRTArtificial Sequencesynthetic 270Asp Arg Thr Thr Ala Thr Pro Phe Phe Asp Tyr1 5 1027127DNAArtificial Sequencesynthetic 271aatggggggg ctacggcctt tgactac 272729PRTArtificial Sequencesynthetic 272Asn Gly Gly Ala Thr Ala Phe Asp Tyr1 527327DNAArtificial Sequencesynthetic 273aatggagggg ctacggtctt tgactac 272749PRTArtificial Sequencesynthetic 274Asn Gly Gly Ala Thr Val Phe Asp Tyr1 527524DNAArtificial Sequencesynthetic 275ctctactacg

gctacgggga ctac 242768PRTArtificial Sequencesynthetic 276Leu Tyr Tyr Gly Tyr Gly Asp Tyr1 527733DNAArtificial Sequencesynthetic 277ccccatgctg gctacttcgg ctggtttgct tac 3327811PRTArtificial Sequencesynthetic 278Pro His Ala Gly Tyr Phe Gly Trp Phe Ala Tyr1 5 1027924DNAArtificial Sequencesynthetic 279ctatacggta gcctgtttgc ttac 242808PRTArtificial Sequencesynthetic 280Leu Tyr Gly Ser Leu Phe Ala Tyr1 528139DNAArtificial Sequencesynthetic 281catgggggac tgggacgtag ggactggtac ttcgatgtc 3928213PRTArtificial Sequencesynthetic 282His Gly Gly Leu Gly Arg Arg Asp Trp Tyr Phe Asp Val1 5 1028333DNAArtificial Sequencesynthetic 283catgggctct actggggcta ttctatggac tac 3328411PRTArtificial Sequencesynthetic 284His Gly Leu Tyr Trp Gly Tyr Ser Met Asp Tyr1 5 1028530DNAArtificial Sequencesynthetic 285catggactgg gaaggacagg gtttgcttcc 3028610PRTArtificial Sequencesynthetic 286His Gly Leu Gly Arg Thr Gly Phe Ala Ser1 5 1028721DNAArtificial Sequencesynthetic 287tccgtctttg gtaattctta c 2128812PRTArtificial Sequencesynthetic 288Ser Val Phe Gly Asn Ser Tyr Trp Phe Phe Asp Val1 5 102899DNAArtificial Sequencesynthetic 289atgacgtac 92903PRTArtificial Sequencesynthetic 290Met Thr Tyr129133DNAArtificial Sequencesynthetic 291gtcctctact atgattacgg gggatttgct tac 3329211PRTArtificial Sequencesynthetic 292Val Leu Tyr Tyr Asp Tyr Gly Gly Phe Ala Tyr1 5 1029336DNAArtificial Sequencesynthetic 293tattcttacg acagggatta cagtcctatg gactac 3629412PRTArtificial Sequencesynthetic 294Tyr Ser Tyr Asp Arg Asp Tyr Ser Pro Met Asp Tyr1 5 1029524DNAArtificial Sequencesynthetic 295cgggaaccgg acttctttga ctac 242968PRTArtificial Sequencesynthetic 296Arg Glu Pro Asp Phe Phe Asp Tyr1 529724DNAArtificial Sequencesynthetic 297cgggaattgg acttctttga ctac 242988PRTArtificial Sequencesynthetic 298Arg Glu Leu Asp Phe Phe Asp Tyr1 529924DNAArtificial Sequencesynthetic 299cggggattgg acttctttga ctac 243008PRTArtificial Sequencesynthetic 300Arg Gly Leu Asp Phe Phe Asp Tyr1 5301347DNAArtificial Sequencesynthetic 301gacattgtgc tgacacagtc tcctgcttcc ttaattgttt ctctggggca gacggccacc 60atctcatgca gggccagcca aagtgtcagt acatctaggt ttagttatct gcactggatc 120caacagaaac cagggcagcc acccaaactc ctcatcaagt atgcatccaa ccttgaatct 180ggggtccctg tcaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240cctgtggagg aggaggatac tgcaacatat ttctgtcagc acagttggga gtttccattc 300acgttcggct cggggacaaa gttgaaaata aaacgggctg atgctgc 347302112PRTArtificial Sequencesynthetic 302Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ile Val Ser Leu Gly1 5 10 15Gln Thr Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30Arg Phe Ser Tyr Leu His Trp Ile Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Val 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His65 70 75 80Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Phe Cys Gln His Ser Trp 85 90 95Glu Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Lys Ile Lys Arg 100 105 110303347DNAArtificial Sequencesynthetic 303gacattgtgc tgacacagtc tcctgcttcc ttaattgttt ctctggggca gacggccacc 60atctcatgca gggccagcca aagtctcagt acatctaggt ttagctatgt gcactggatc 120caacagaaac cagggcagcc acccaaactc ctcatcaagt atgcatccaa ccttgaatct 180ggggtccctg tcaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240cctgtggagg aggaggatac tgcaacatat ttctgtcagc acagttggga gtttccattc 300acgttcggct cggggacaaa gttgaaaata aaacgggctg atgctgc 347304112PRTArtificial Sequencesynthetic 304Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ile Val Ser Leu Gly1 5 10 15Gln Thr Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Leu Ser Thr Ser 20 25 30Arg Phe Ser Tyr Val His Trp Ile Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Val 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His65 70 75 80Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Phe Cys Gln His Ser Trp 85 90 95Glu Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Lys Ile Lys Arg 100 105 110305347DNAArtificial Sequencesynthetic 305gacattgtgc tgacacagtc tcctgcttcc ttaattgtat ctctggggca gagggccacc 60atctcatgta gggccagcca aagtgtcagt acatccaggt ttagttatgt gcactggatc 120caacagaaac cagggcagcc acccaaactc ctcatcaagt atgcatccaa ccttgaatct 180ggggtccctg tcaggttcag tggcagtggg tctgggacag acttcatcct caacatccat 240cctgtggagg aggaggatac tgcaacatat ttctgtcagc acagttggga gtttccattc 300acgttcggct cggggacaaa gttggaaata aaacgggctg atgctgc 347306112PRTArtificial Sequencesynthetic 306Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ile Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30Arg Phe Ser Tyr Val His Trp Ile Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Val 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ile Leu Asn Ile His65 70 75 80Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Phe Cys Gln His Ser Trp 85 90 95Glu Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110307350DNAArtificial Sequencesynthetic 307gaagttttga tgacccaaag tccactctcc ctgcctgtca gtcttggaga tcaggcctcc 60atctcttgca gatctagtca gagccttgaa catactaatg gaaacaccta tttagagtgg 120ttcctgcaga gaccaggcca gcctccaaag ctcctgatct acaaagtttc cagccgattt 180tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240agcagagtgg aggctgagga tctgggagtt tattactgtt ttcaaggttc acatgttcca 300ttcacgttcg gctcggggac aaagttggca ataaaacggg ctgatgctgc 350308113PRTArtificial Sequencesynthetic 308Glu Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu His Thr 20 25 30Asn Gly Asn Thr Tyr Leu Glu Trp Phe Leu Gln Arg Pro Gly Gln Pro 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Ser Arg Phe 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 Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95Ser His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Ala Ile Lys 100 105 110Arg309350DNAArtificial Sequencesynthetic 309gaagttttga tgacccaaag tccactctcc ctgcctgtca gtcttggaga tcaggcctcc 60atctcttgca gatctagtca gagccttgaa catagtaatg gaaacaccta tttagagtgg 120ttcctgcaga gaccaggcca gcctccaaag ctcctgatct acaaagtttc cagccgattt 180tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240agcagagtgg aggctgagga tctgggagtt tattactgtt ttcaaggttc acatgttcca 300ttcacgttcg gctcggggac aaagttggca ataaaacggg ctgatgctgc 350310113PRTArtificial Sequencesynthetic 310Glu Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu His Ser 20 25 30Asn Gly Asn Thr Tyr Leu Glu Trp Phe Leu Gln Arg Pro Gly Gln Pro 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Ser Arg Phe 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 Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95Ser His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Ala Ile Lys 100 105 110Arg311350DNAArtificial Sequencesynthetic 311gatgttttga tggcccaaac tccactctcc ctgcctgtca cccttggaga tcaagcctcc 60atctcttgca gatctagtca gagccttgta catagtaatg gaaacaccta tttagagtgg 120ttcctgcaga aaccaggcca gtctccaaag ctcctgatct acaacgtttc caaccgattt 180tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttcca 300ctcacgttcg gctcggggac aaagttggaa ataaaacggg ctgatgctgc 350312113PRTArtificial Sequencesynthetic 312Asp Val Leu Met Ala Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu Glu Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Asn Val Ser Asn Arg Phe 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 Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95Ser His Val Pro Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110Arg313350DNAArtificial Sequencesynthetic 313gatgttttga tgacccaaac tccactctcc ctgcctgtca atcttggaga tcaggcctcc 60atctcttgca gatctagtca gagccttcta catagtaatg gaaacaccta tttagagtgg 120ttcctgcaga aaccaggcca gtctccaaag ctcctgatct acaatgtttc caaccgattt 180tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttcca 300ttcacgttcg gctcggggac aaagttggca ataaaacggg ctgatgctgc 350314113PRTArtificial Sequencesynthetic 314Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Asn Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30Asn Gly Asn Thr Tyr Leu Glu Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Asn Val Ser Asn Arg Phe 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 Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95Ser His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Ala Ile Lys 100 105 110Arg315350DNAArtificial Sequencesynthetic 315gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60atctcttgca gatctagtca gagccttgta cataataatg gaaacaccta tttagaatgg 120ttcctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttcca 300ttcacgttcg gctcggggac aaagttggaa ataaaacggg ctgatgctgc 350316113PRTArtificial Sequencesynthetic 316Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Asn 20 25 30Asn Gly Asn Thr Tyr Leu Glu Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe 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 Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95Ser His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110Arg317350DNAArtificial Sequencesynthetic 317gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60atctcttgca gatctagtca gagcattgta cacagtaatg gaaacaccta tttagagtgg 120tacctgcaga aactaggcca gtctccaaag ctcctgatct acagagtttc caaccgattt 180tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttccg 300tggacgttcg gtggaggcac caagctggaa atcaaacggg ctgatgctgc 350318113PRTArtificial Sequencesynthetic 318Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Leu Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe 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 Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110Arg319350DNAArtificial Sequencesynthetic 319gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60atctcttgca gatctagtca gagccttcta catagtaatg gaaacaccta tttagaatgg 120tacctgcaga aacctggcca gtctccaaac ctcctgatct acaatgtttc caaccgattt 180tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcgc actcaagatc 240agcagagtgg gggctgagga tctgggagtt tattactgct ttcaaggttc acatgttccg 300ctcacgttcg gtgctgggac caagctggag ctgaaacggg ctgatgctgc 350320113PRTArtificial Sequencesynthetic 320Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Asn Leu Leu Ile Tyr Asn Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ala Leu Lys Ile65 70 75 80Ser Arg Val Gly Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110Arg321350DNAArtificial Sequencesynthetic 321gatgttgtgc tgacccaaac tccactctcc ctgcctctca gtcttggaga tcaggcctcc 60atctcttgca gatctagtca gacccttcta cacagtgatg gagacaccta tttacattgg 120tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaactttc caaccgattt 180tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttccg 300tacacgttcg gaggggggac caagctggaa ataaaacggg ctgatgctgc 350322113PRTArtificial Sequencesynthetic 322Asp Val Val Leu Thr Gln Thr Pro Leu Ser Leu Pro Phe Ser Phe Gly1 5 10 15Asn Lys Ala Ser Ile Phe Cys Lys Phe Ser Gln Thr Leu Leu His Arg 20 25 30Asp Gly Asn Pro Phe Leu Leu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Lys Leu Ser Asn Arg Phe Phe Gly Val Pro 50 55 60Lys Arg Phe Arg Gly Arg Gly Ser Gly Thr Asn Phe Pro Leu Lys Ile65 70 75 80Ser Lys Gly Glu Ala Glu Asp Leu Gly Val Phe Phe Cys Ser Gln Ser 85 90 95Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110Arg323350DNAArtificial Sequencesynthetic 323gatattgtga tgacccagac tccactcact ttgtcggtta ccattggaca accagcttcc 60atctcttgca agtcaagtca gagcctctta tttactaatg gaaaaaccta tttaaattgg 120tttttacaga ggccaggcca gtctccaaaa cgcctaatct atctgctgtc taaattggac 180tctggagtcc ctgacaggtt cagtggcagt ggatcaggga cagatttcac actgaaaatc 240agcagagtgg aggctgagga tttgggagtt tattactgct tgcagagtac atattttcct

300ctcacgttcg gtgctgggac caagctggag ctgaaacggg ctgatgctgc 350324113PRTArtificial Sequencesynthetic 324Asp Ile Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Phe Thr 20 25 30Asn Gly Lys Thr Tyr Leu Asn Trp Phe Leu Gln Arg Pro Gly Gln Ser 35 40 45Pro Lys Arg Leu Ile Tyr Leu Leu Ser Lys Leu Asp 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 Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Leu Gln Ser 85 90 95Thr Tyr Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110Arg325350DNAArtificial Sequencesynthetic 325gatgttgtga tgacccagac tccactcact ttgtcggtta ccattggaca accagcttcc 60atctcttgca agtcaagtca gagcctctta tatactaatg gaaagaccta tttgaattgg 120ttattacaga ggccaggcca gtctccaaaa cgcctaatct atctggtgtc aaaattggac 180tctggagtcc ctgacaggtt cagtggcagt ggatcaggga cagatttcac actgaaaatc 240agcagagtgg aggctgagga tttgggagtt tattactgct tgcagagtat acattttccg 300tacacgttcg gaggggggac caagctggac ataaaacggg ctgatgctgc 350326113PRTArtificial Sequencesynthetic 326Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Thr 20 25 30Asn Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp 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 Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Leu Gln Ser 85 90 95Ile His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Asp Ile Lys 100 105 110Arg327350DNAArtificial Sequencesynthetic 327gatattgtga tgacgcaggc tgcattctcc aatccagtca ctcctggaac atcagtttcc 60atctcctgca ggtctagtaa gagtctccta catagtaatg gcatcactta tttgtattgg 120tatctgcaga agccaggcca gtctcctcag ctcctgattt atcagatgtc caagattgcc 180tcaggagtcc cagacaggtt caggagcagt gggtcaggaa ctgatttcac actgagaatc 240agcagagtgg aggctgcgga tgtgggtgtt tattactgtg ctcaaaatct agaacttccg 300tggacgttcg gtggaggcac caagctggaa atcaaacggg ctgatgctgc 350328113PRTArtificial Sequencesynthetic 328Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Pro Gly1 5 10 15Thr Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Gln Met Ser Lys Ile Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Arg Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile65 70 75 80Ser Arg Val Glu Ala Ala Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95Leu Glu Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110Arg329335DNAArtificial Sequencesynthetic 329gacattgtga tgacccagtc tcacaaattc atgtccacat caataggaga cagggtcagc 60atcacctgca aggccagtca gaatgtgagt cctgctgtag cctggtatca acagaaacca 120ggacaatctc ctaaactact gatttactcg gcatcctccc gatacactgg agtccctgat 180cgcttcactg gcagtggatc tgggacggct ttcactttca ccatcagcag tgtgcaggct 240gaagacctgg cagtttattt ctgtcagcaa cattttagta ctccgtggac gttcggtgga 300ggcaccatgc tggaaatcaa acgggctgat gctgc 335330108PRTArtificial Sequencesynthetic 330Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Ile Gly1 5 10 15Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Ser Pro Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Ser Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Ala Phe Thr Phe Thr Ile Ser Ser Val Gln Ala65 70 75 80Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln His Phe Ser Thr Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Met Leu Glu Ile Lys Arg 100 105331335DNAArtificial Sequencesynthetic 331gacattgtga tgacccagtc tcaaaaattc atgtccacaa cagttgggga cagggtcaga 60gtcacctgca aggccagtca gaatgtgggt actgctgtag cctggtatca acagaaacca 120ggacaatctc ctaaactact gatttactca gcatccaatc ggtacactgg agtccctgat 180cgcttcacag gcagtggatc tgggacagat ttcactctca ccattaccaa tatgcagtct 240gaagacctgg cagattattt ctgtcagcaa tatagcacct atcctctcac gttcggctcg 300ggggcaaagt tggaaataaa acgggctgat gctgc 335332108PRTArtificial Sequencesynthetic 332Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Thr Val Gly1 5 10 15Asp Arg Val Arg Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Met Gln Ser65 70 75 80Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Thr Tyr Pro Leu 85 90 95Thr Phe Gly Ser Gly Ala Lys Leu Glu Ile Lys Arg 100 105333332DNAArtificial Sequencesynthetic 333gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagc 60atcacctgca aggccagtca ggatgtgggt actgctgtag cctggtatca acagaaacca 120ggacaatctc ctaaactact gatttactgg gcatccaccc ggcacactgg agtccctgat 180cgcttcacag gcagtggatc tgggacagat ttcactctca ccattagcaa tgtgcagtct 240gaagacttgg cagattattt ctgtcagcaa tatagcaact atctcacgtt cggtgctggg 300accaagctgg aggtgaaacg ggctgatgct gc 332334107PRTArtificial Sequencesynthetic 334Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly1 5 10 15Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser65 70 75 80Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Asn Tyr Leu Thr 85 90 95Phe Gly Ala Gly Thr Lys Leu Glu Val Lys Arg 100 105335335DNAArtificial Sequencesynthetic 335gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggcga aagagtcagt 60ctcacttgtc gggcaagtca ggacattggt ggtagcataa actggcttca gcaggaacca 120gatggaacta ttaaacgcct gatctacgcc acatccagtt tagattctgg tgtccccaaa 180aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240gaagattttg tagactatta ctgtctacaa tatgctagtt ctcctccgac gttcggtgga 300ggcaccaaac tggaaatcaa acgggctgat gctgc 335336108PRTArtificial Sequencesynthetic 336Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly1 5 10 15Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp Ile Gly Gly Ser 20 25 30Ile Asn Trp Leu Gln Gln Glu Pro Asp Gly Thr Ile Lys Arg Leu Ile 35 40 45Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser65 70 75 80Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Ser Pro Pro 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105337335DNAArtificial Sequencesynthetic 337gacatccaga tgactcagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60atcacatgtc gagcaagtgg gaatattcac acttatttag catggtatca gcagaaacag 120ggaaaatctc ctcagctcct ggtctacaat gcaaacacct tggcagatgg tgtgccatca 180aggttcagtg gcagtggatc aggaacacaa ttttctctca agatcaacag tctgcagcct 240gacgattttg ggagttatta ctgtcaacat ttttggagtg ctccgtggac gttcggtgga 300ggcacccagc tggaaatcaa acgggctgat gctgc 335338108PRTArtificial Sequencesynthetic 338Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly1 5 10 15Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Thr Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45Tyr Asn Ala Asn Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro65 70 75 80Asp Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser Ala Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Gln Leu Glu Ile Lys Arg 100 105339335DNAArtificial Sequencesynthetic 339gatattgtgt taactcagtc tccagccacc ctgtctgtga ctccaggaga tagagtcagt 60ctttcctgca gggccagtca aagaatttac aactacctac actggtatca acaaaaatca 120catgagtctc caaggcttct caccaagtat gcttcccagt ccatctctgg gatcccctcc 180aggttcagtg gcagtggctc agggacagat ttcattctca ctatcaacag tgtggagact 240gaagattttg gaatgtattt ctgtcaacag agtaacagct ggcctctcac gttcggtgct 300gggaccaagc tggagctgag acgggctgat gctgc 335340108PRTArtificial Sequencesynthetic 340Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly1 5 10 15Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Arg Ile Tyr Asn Tyr 20 25 30Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Thr 35 40 45Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Ile Leu Thr Ile Asn Ser Val Glu Thr65 70 75 80Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro Leu 85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Arg Arg 100 105341335DNAArtificial Sequencesynthetic 341gatattgtgt taactcagtc tccagccacc ctgtctgtga ctccaggaga tagagtcagt 60ctttcctgca gggccagtca aagaatttac aactacctac actggtatca acaaaaatca 120catgagtctc caaggcttct catcaagtat gcttcccagt ccatctctgg gatcccctcc 180aggttcagtg gcagtggctc agggacagat ttcattctca ctatcaacag tgtggagact 240gaagattttg gaatgtattt ctgtcaacag agtaacagct ggcctctcac gttcggtgct 300gggaccaagc tggagctgag acgggctgat gctgc 335342108PRTArtificial Sequencesynthetic 342Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly1 5 10 15Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Arg Ile Tyr Asn Tyr 20 25 30Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Ile Leu Thr Ile Asn Ser Val Glu Thr65 70 75 80Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro Leu 85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Arg Arg 100 105343335DNAArtificial Sequencesynthetic 343gacattgtga tgactcagtc tccagccacc ctgtctgtga ctccaggaga tagagtctct 60ctttcctgca gggccagcca gagtattagc gactacttac actggtatca acaaaaatca 120catgagtctc caaggcttct catcaaatat gcttcccaat ccatctctgg gatcccctcc 180aggttccgtg gcagtggatc agggtcacat ttcactctca gtatcaacag tgtggaacct 240gaagatgttg gagtgtatta ctgtcaaaat ggtcacagtt ttccgtggac gttcggtgga 300ggcaccaagc tggaaatcaa acgggctgat gctgc 335344108PRTArtificial Sequencesynthetic 344Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly1 5 10 15Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr 20 25 30Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Arg Gly 50 55 60Ser Gly Ser Gly Ser His Phe Thr Leu Ser Ile Asn Ser Val Glu Pro65 70 75 80Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105345335DNAArtificial Sequencesynthetic 345gacattgtga tgactcagtc tccagccacc ctgtctgtga ctccaggaga tagagtctct 60ctttcctgca gggccagcca gactattagc gactacttac actggtatca acaaaaatcg 120catgagtctc caaggcttct catcaaatat gcttcccaat ccatctctgg gatcccctcc 180aggttcagtg gcagtggatc agggtcacat ttcactctca gtatcaacag tgtggaacct 240gaagatgttg gagtgtatta ctgtcaaaat ggtcacagtt ttccgtggac gttcggtgga 300ggcaccaagc tggaaatcaa acgggctgat gctgc 335346108PRTArtificial Sequencesynthetic 346Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly1 5 10 15Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Thr Ile Ser Asp Tyr 20 25 30Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Ser His Phe Thr Leu Ser Ile Asn Ser Val Glu Pro65 70 75 80Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105347329DNAArtificial Sequencesynthetic 347gagattgtgc tcactcagtc tccagccatc acagctgcat ctctggggca aaacgtcacc 60atcacctgca gagccagctc aagtgtaagt tacatgcatt ggtaccggca gaagtccggc 120acctcccccc aactatggat ttatgagata tccagacggg cttctggagt cccagctcgc 180ttccgtgcca gtgggtctgg gacctcttat tctctcacaa tcagcagcat ggaggctgaa 240gatgctgcca tttattactg ccagcagtgg aattatcctc tcacgttcgg tgctgggacc 300aagctggagg tgaaacgggc tgatgctgc 329348106PRTArtificial Sequencesynthetic 348Glu Ile Val Leu Thr Gln Ser Pro Ala Ile Thr Ala Ala Ser Leu Gly1 5 10 15Gln Asn Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30His Trp Tyr Arg Gln Lys Ser Gly Thr Ser Pro Gln Leu Trp Ile Tyr 35 40 45Glu Ile Ser Arg Arg Ala Ser Gly Val Pro Ala Arg Phe Arg Ala Ser 50 55 60Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu65 70 75 80Asp Ala Ala Ile Tyr Tyr Cys Gln Gln Trp Asn Tyr Pro Leu Thr Phe 85 90 95Gly Ala Gly Thr Lys Leu Glu Val Lys Arg 100 105349329DNAArtificial Sequencesynthetic 349gaagttgtgc tcactcagtc tccagccatc acagctgcat ctctggggca aaaggtcacc 60atcacctgca gagccagctc aagtgtaagt tacatgcact ggtaccggca gaagtcaggc 120acctcccccc agccatggat ttatgaaata tccacactgg cttctggagt cccaactcgc 180ttccgtgcca gtgggtctgg gacctcttat tctctcacaa tcagcagcat ggaggctgaa 240gatgctgcca tttattactg ccagcagtgg aattatcctc tcacgttcgg tgctgggacc 300aagctggaac tgaaacgggc tgatgctgc 329350106PRTArtificial Sequencesynthetic 350Glu Val Val Leu Thr Gln Ser Pro Ala Ile Thr Ala Ala Ser Leu Gly1 5 10 15Gln Lys Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30His Trp Tyr Arg Gln Lys Ser Gly Thr Ser Pro Gln Pro Trp Ile Tyr 35 40 45Glu Ile Ser Thr Leu Ala Ser Gly Val Pro Thr Arg Phe Arg Ala Ser 50 55 60Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu65 70 75 80Asp Ala Ala Ile Tyr Tyr Cys Gln Gln Trp Asn Tyr Pro Leu Thr Phe 85 90 95Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105351338DNAArtificial Sequencesynthetic 351caaattgttc tcacccagtc tccaacaatc atgtctgcat ctctagggga acgggtcacc 60atgacctgca ctgccagctc aagtgtgagt tccagttact tgcactggtt ccagcagaag

120ccaggatcct cccccaaact ctggatttat agcacatcca acctggcttc tggagtccca 180cctcgcttca gtggcagtgg gtctgggacc tcttactctc tctcaatcag cagcgtggag 240gctgaagatg ttgccactta ttactgcctc cagtttcatc gttccccgtg gacgttcggt 300ggaggcgcca agttggaaat caaacgggct gatgctgc 338352109PRTArtificial Sequencesynthetic 352Gln Ile Val Leu Thr Gln Ser Pro Thr Ile Met Ser Ala Ser Leu Gly1 5 10 15Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser 20 25 30Tyr Leu His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp 35 40 45Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Pro Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Ser Ile Ser Ser Val Glu65 70 75 80Ala Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Phe His Arg Ser Pro 85 90 95Trp Thr Phe Gly Gly Gly Ala Lys Leu Glu Ile Lys Arg 100 105353338DNAArtificial Sequencesynthetic 353caaattgttt tcacccagta tccagcaata atgtctgcat ctctagggga acgggtcacc 60atgacctgca cagccagctc aagtgtaact tccagttact tgcactggtt ccagcagaag 120ccaggatcct cccccaaact ctggatttat agcacgtcca acccgggttc tggagtccca 180gctcgcttca gtggcagagg atctgggacc tcttactctc tctcaatcag cagcatggag 240gctgaagatg ctgccactta ttactgcctc cagtttcatc gttccccgtg gacgttcggt 300ggaggcacca agctggaaat cagacgggct gatgctgc 338354109PRTArtificial Sequencesynthetic 354Gln Ile Val Phe Thr Gln Tyr Pro Ala Ile Met Ser Ala Ser Leu Gly1 5 10 15Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Thr Ser Ser 20 25 30Tyr Leu His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp 35 40 45Ile Tyr Ser Thr Ser Asn Pro Gly Ser Gly Val Pro Ala Arg Phe Ser 50 55 60Gly Arg Gly Ser Gly Thr Ser Tyr Ser Leu Ser Ile Ser Ser Met Glu65 70 75 80Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Gln Phe His Arg Ser Pro 85 90 95Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Arg Arg 100 105355332DNAArtificial Sequencesynthetic 355caaattgttc tcacccagtc tccagcaatc atgtctgtat ctccagggga gaaggtctcc 60atgacctgca gtgccagctc aagtgtcact tacatgcact ggtatcagca gaagtcaggc 120acctccccca aaagatggat ttatgacaca tccgagctgg cttctggagt ccctgctcgc 180ttcagtggca gtgggtctgg gaccacttac tctctcacaa tcagcagcat ggaggctgaa 240gatgctgcca cttattactg ccagcagtgg agtaataaac cgctcacgtt cggtgctggg 300accaagctgg agctgaaacg ggctgatgct gc 332356107PRTArtificial Sequencesynthetic 356Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Val Ser Pro Gly1 5 10 15Glu Lys Val Ser Met Thr Cys Ser Ala Ser Ser Ser Val Thr Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45Asp Thr Ser Glu Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Thr Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Asn Lys Pro Leu Thr 85 90 95Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105357332DNAArtificial Sequencesynthetic 357caaaatgttc tcacccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60atgtcctgca gtgccagctc aagtgtcact tacatgttct ggtaccagct gaagccagaa 120tcctccccca gactcctgat ttatgacaca tccaatttgg cttctggcgt ccctgttcgc 180ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagccgtat ggaggctgaa 240gatgctgcca cttattactg ccaggagtgg agtagttacc cactcacgtt cggtgctggg 300accaagctgg acctgaaacg ggctgatgct gc 332358107PRTArtificial Sequencesynthetic 358Gln Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly1 5 10 15Glu Lys Val Thr Met Ser Cys Ser Ala Ser Ser Ser Val Thr Tyr Met 20 25 30Phe Trp Tyr Gln Leu Lys Pro Glu Ser Ser Pro Arg Leu Leu Ile Tyr 35 40 45Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Glu Trp Ser Ser Tyr Pro Leu Thr 85 90 95Phe Gly Ala Gly Thr Lys Leu Asp Leu Lys Arg 100 105359332DNAArtificial Sequencesynthetic 359caaattgttc tcacccagtc tccaacactc atgtctgcat cgccaggaga aaaggtcacc 60atgacctgca gtgccagctc aactgtgact tacatttact ggtaccaaca gaagcccggc 120tcctccccca gactctggat gtatgacaca ttcaacctgg tttctggagt ccctgctcgc 180ttcagtggca gtaggtctgg gacctcttat tttctcacaa tcagtagcat ggagggtgaa 240gatgctgcca cttattactg ccaacagtac agtgattccc cgtacacgtt cggagggggg 300accaagctgg agataaaacg ggctgatgct gc 332360107PRTArtificial Sequencesynthetic 360Gln Ile Val Leu Thr Gln Ser Pro Thr Leu Met Ser Ala Ser Pro Gly1 5 10 15Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Thr Val Thr Tyr Ile 20 25 30Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Trp Met Tyr 35 40 45Asp Thr Phe Asn Leu Val Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60Arg Ser Gly Thr Ser Tyr Phe Leu Thr Ile Ser Ser Met Glu Gly Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asp Ser Pro Tyr Thr 85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105361332DNAArtificial Sequencesynthetic 361caaattgttc tcacccagtc tccagagatc atgtctgcat ctccagggga gaaggtcacc 60ataacctgca gtgccagctc aagtgtaagt ttcatgtatt ggttccagca gaagccaggc 120acttctccca aactctggat ttatatcaca tccaacctgg cttctggagt ccctactcgc 180ttcagtggca gtggatctgg gacctcttac tctctcacaa tcagccgaat ggaggctgaa 240gatgctgcca cttattactg ccagcaaagg agtagtttcc cgtacacgtt cggagggggg 300accaaactgg aaatgaaacg ggctgatgct gc 332362107PRTArtificial Sequencesynthetic 362Gln Ile Val Leu Thr Gln Ser Pro Glu Ile Met Ser Ala Ser Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Phe Met 20 25 30Tyr Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr 35 40 45Ile Thr Ser Asn Leu Ala Ser Gly Val Pro Thr Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Phe Pro Tyr Thr 85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Met Lys Arg 100 105363344DNAArtificial Sequencesynthetic 363gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccatc 60atctcctgca aggccagcca aagtgtcagt tttgctggta ctaatttaat gcactggtac 120caacagaaac caggacagca acccaaactc ctcatctatc gtgcatccaa cctagaaact 180ggggttccta ccaggtttag tggcagtggg tctaggacag acttcaccct caatatccat 240cctgtggagg aagatgatgc tgcaacctat tactgtcagc aaagtaggga atattacacg 300ttcggagggg ggaccaagct ggaaataaaa cgggctgatg ctgc 344364111PRTArtificial Sequencesynthetic 364Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Ile Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Phe Ala 20 25 30Gly Thr Asn Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Thr Gly Val Pro Thr 50 55 60Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Asn Ile His65 70 75 80Pro Val Glu Glu Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Arg 85 90 95Glu Tyr Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110365347DNAArtificial Sequencesynthetic 365gacattgtgc tgacccaatc tccagcttct ttggctgggt ctctagggaa aagggccccc 60atctcctgca aagccagcga aagtgtcaat ttttttggta ctaatttaat acactggtac 120caacaaaaac caggacagcc ccccaaactc ctcatctatc atgcatccaa cctaaaaact 180ggagtccctg ccaggttcag gggcaggggg tctaaaacaa acttccccct ccccattgat 240cctgtggagg aaaatgatgt tgcaatctat tactgtctgc aaaataggaa aattcctctc 300acgttcgggg ttgggaccaa gctggagctg aaacgggctg atgctgc 347366112PRTArtificial Sequencesynthetic 366Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Gly Ser Leu Gly1 5 10 15Lys Arg Ala Pro Ile Ser Cys Lys Ala Ser Glu Ser Val Asn Phe Phe 20 25 30Gly Thr Asn Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr His Ala Ser Asn Leu Lys Thr Gly Val Pro Ala 50 55 60Arg Phe Arg Gly Arg Gly Ser Lys Thr Asn Phe Pro Leu Pro Ile Asp65 70 75 80Pro Val Glu Glu Asn Asp Val Ala Ile Tyr Tyr Cys Leu Gln Asn Arg 85 90 95Lys Ile Pro Leu Thr Phe Gly Val Gly Thr Lys Leu Glu Leu Lys Arg 100 105 110367347DNAArtificial Sequencesynthetic 367gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60atctcctgca gagccagcga aagtgttgat aattatggca ttagttttat gcactggtac 120cagcagaaac caggacagcc acccaaactc ctcatctatc gtgcatccaa cctagaatct 180gggatccctg ccaggttcag tggcagtggg tctaggacag acttcaccct caccattaat 240cctgtggaga ctgatgatgt tgcaacctat tactgtcagc aaagtaataa ggatccattc 300acgttcggct cgggggcaaa gttggaaata aaacgggctg atgctgc 347368112PRTArtificial Sequencesynthetic 368Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr 20 25 30Gly Ile Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn65 70 75 80Pro Val Glu Thr Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Lys Asp Pro Phe Thr Phe Gly Ser Gly Ala Lys Leu Glu Ile Lys Arg 100 105 110369347DNAArtificial Sequencesynthetic 369gacattgtgg tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60atctcatgca gggccagcca aagtgtcagt acatctagat atagttatct gcactggtac 120caacagaaac caggacaacc tcccaaactc ctcatcaagt atgcatccaa cctagaatct 180ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caatatccat 240cctgtggggg aggaggatac tgcaacatat tactgtcagc acagttggga gtttccattc 300acgttcggct cggggacaaa gttggaaata aaacgggctg atgctgc 347370112PRTArtificial Sequencesynthetic 370Asp Ile Val Val Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30Arg Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His65 70 75 80Pro Val Gly Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln His Ser Trp 85 90 95Glu Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110371347DNAArtificial Sequencesynthetic 371gacattgtac tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60atctcatgca ggtccagcca aagtgtcagt acatctagat atagttattt gcactggtac 120caacagaaac caggacaacc tcccaaactc ctcatcaagt atgcatccaa cctagaatct 180ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240cctgtggggg aggaggatcc tgcaacatat tactgtcagc acagttggga gtttccattc 300acgttcggct cggggacaaa gttggaaata aaacgggctg atgctgc 347372112PRTArtificial Sequencesynthetic 372Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ser Ser Gln Ser Val Ser Thr Ser 20 25 30Arg Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His65 70 75 80Pro Val Gly Glu Glu Asp Pro Ala Thr Tyr Tyr Cys Gln His Ser Trp 85 90 95Glu Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110373347DNAArtificial Sequencesynthetic 373gacattgccc tgacacagtc tcctgcttcg ttagctgtat ctctggggca gagggccacc 60atctcatgca gggccagcca aagtgtcagt acatctaggt atagttatat gcactggtac 120caacagaaac caggacaacc acccgaactc ctcatcaagt atgcatccaa cctagaatct 180ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240cctgtggggg aggaggatac tgcaacatat tactgtcagc acagttggga ttttccattc 300acgttcggct cggggacaaa gttggaaata aaacgggctg atgctgc 347374112PRTArtificial Sequencesynthetic 374Asp Ile Ala Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30Arg Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Glu Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His65 70 75 80Pro Val Gly Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln His Ser Trp 85 90 95Asp Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110375347DNAArtificial Sequencesynthetic 375gacattgtgc tgacacagtc tcctgcttcc ttagctgttt ctctggggca gagggccacc 60atctcatgca gggccagcca aagtgtcagt acatctaggt atagctacat gcactggtac 120caacagaaac caggacagcc acccaaactc ctcatcaagt atgcatccaa cctagaatct 180ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240cctgtggcgg aggaggatac tgcaacatat tactgtcagc acagttggga gtttccattc 300acgttcggct cggggacaaa gttggaaata aaacgggctg atgctgc 347376112PRTArtificial Sequencesynthetic 376Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30Arg Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His65 70 75 80Pro Val Ala Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln His Ser Trp 85 90 95Glu Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110377347DNAArtificial Sequencesynthetic 377gacattgtgc tgacacagtc tcttgcttcc ttagctgttt ctctggggca gagggccacc 60atctcatgca gggccagcca aagtgtcagt acatctaggt atagttatat gcactggtac 120caacagaaac caggacagcc acccaaactc ctcatcaagt atgcatccaa ccttgaatgt 180ggggtccgtg ccaggttcag tggcagtggg tgtgggacag acttcaccct caacatccat 240cctgtggagg aggaggatac tgcagcatat tactgtcagc acagttggga gtttccattc 300acgttcggct cggggacaaa attggaaata aaacgggctg atgctgc 347378112PRTArtificial Sequencesynthetic 378Asp Ile Val Leu Thr Gln Ser Leu Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30Arg Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Cys Gly Val Arg Ala 50 55 60Arg Phe Ser Gly Ser Gly Cys Gly Thr Asp Phe Thr Leu Asn Ile His65

70 75 80Pro Val Glu Glu Glu Asp Thr Ala Ala Tyr Tyr Cys Gln His Ser Trp 85 90 95Glu Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110379347DNAArtificial Sequencesynthetic 379gacattgtac tgacacagtc tcctgcttcc ttagctgttt ctctggggca gagggccacc 60atctcctgca gggccagcca aagtgtcagt acatctaggt atagttatgt gcactggtat 120caacagaaac caggacagcc acccaaactc ctcatcaagt atgcagccaa cctagaatct 180ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240cctgtggcgg aggaggatgc tgcagcatat tactgtcagc acagttggga gtttccattc 300acgttcggct cggggacaaa gttggaaata aaacgggctg atgctgc 347380112PRTArtificial Sequencesynthetic 380Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30Arg Tyr Ser Tyr Val His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Lys Tyr Ala Ala Asn Leu Glu Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His65 70 75 80Pro Val Ala Glu Glu Asp Ala Ala Ala Tyr Tyr Cys Gln His Ser Trp 85 90 95Glu Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110381347DNAArtificial Sequencesynthetic 381gacattgtgc tgacacagtc tcctgcttcc ttagctgttt ctctggggca gaggaccacc 60atctcatgcg gggccagcca aagtgtcagt acatctaggt ttagttatat gcactggtac 120caacagaaac caggacagcc acccaaactc ctcatcaagt atgcatccaa cctagaatct 180ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240cctgtggcgg aggaggatac tgcagcatat tactgtcagc acagttggga gtttccattc 300acgttcggct cggggacaaa gttggaaata aaacgggctg atgctgc 347382112PRTArtificial Sequencesynthetic 382Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Thr Thr Ile Ser Cys Gly Ala Ser Gln Ser Val Ser Thr Ser 20 25 30Arg Phe Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His65 70 75 80Pro Val Ala Glu Glu Asp Thr Ala Ala Tyr Tyr Cys Gln His Ser Trp 85 90 95Glu Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110383347DNAArtificial Sequencesynthetic 383gacattgtgc tgacacagtc tcctgcttcc ttagctgttt ctctggggca gagggccacc 60atctcatgca gggccagcca aagtgtcagt acatctaggt atagttatat gcactggtac 120caacagaaac caggacagcc acccaaactc ctcatcaagt atgcatccaa cctagaatct 180ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240cctgtggcgg aggaggatac tgcagcatat tactgtcagc acagttgggg gtttccattc 300acgttcggct cggggacaaa gttggaaata aagcgggctg atgctgc 347384112PRTArtificial Sequencesynthetic 384Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30Arg Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His65 70 75 80Pro Val Ala Glu Glu Asp Thr Ala Ala Tyr Tyr Cys Gln His Ser Trp 85 90 95Gly Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110385347DNAArtificial Sequencesynthetic 385gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctttgggaaa aaaggccacc 60atctcatgca gggccagcca aagtgtcagt acatctaggt atagttatat gcactggtac 120caacagaaac caggacaccc acccaaactc ctcatcaaat atgcatccaa cctagaatct 180ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240cctgtggcgg aggaggatac tgcagcatat tactgtcagc acagttggga gtttccattc 300acgttcggct cggggacaaa gttggaaata aaacgggctg atgctgc 347386112PRTArtificial Sequencesynthetic 386Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Lys Lys Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30Arg Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly His Pro Pro 35 40 45Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His65 70 75 80Pro Val Ala Glu Glu Asp Thr Ala Ala Tyr Tyr Cys Gln His Ser Trp 85 90 95Glu Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110387347DNAArtificial Sequencesynthetic 387gacattgtgc tgacacagtc tcctgcttcc ttagctgttt ctctggggca gagggccacc 60atctcatgca gggccagcca aagtgtcagt acatctaggt atagttatat gcactggtac 120caacagaaac caggacagcc acccaaactc ctcatcaagt atgcatccaa ccttgaatct 180ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240cctgtggcgg aggaggatac tgcagcatat tactgtcagc acagttggga gtttccattc 300acgttcggct cggggacaaa attggaaata aaacgggctg atgctgc 347388112PRTArtificial Sequencesynthetic 388Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30Arg Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His65 70 75 80Pro Val Ala Glu Glu Asp Thr Ala Ala Tyr Tyr Cys Gln His Ser Trp 85 90 95Glu Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105 11038948DNAArtificial Sequencesynthetic 389agatctagtc agagccttga acatagtaat ggaaacacct atttagag 4839016PRTArtificial Sequencesynthetic 390Arg Ser Ser Gln Ser Leu Glu His Ser Asn Gly Asn Thr Tyr Leu Glu1 5 10 1539148DNAArtificial Sequencesynthetic 391agatctagtc agagccttgt acatagtaat ggaaacacct atttagag 4839216PRTArtificial Sequencesynthetic 392Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu Glu1 5 10 1539348DNAArtificial Sequencesynthetic 393agatctagtc agagccttgt acatagtaat ggaaacacct atttagag 4839416PRTArtificial Sequencesynthetic 394Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Glu1 5 10 1539548DNAArtificial Sequencesynthetic 395agatctagtc agagcattgt acacagtaat ggaaacacct atttagag 4839616PRTArtificial Sequencesynthetic 396Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu1 5 10 1539748DNAArtificial Sequencesynthetic 397agatctagtc agagccttga acatactaat ggaaacacct atttagag 4839816PRTArtificial Sequencesynthetic 398Arg Ser Ser Gln Ser Leu Glu His Thr Asn Gly Asn Thr Tyr Leu Glu1 5 10 1539948DNAArtificial Sequencesynthetic 399agatctagtc agagccttgt acataataat ggaaacacct atttagaa 4840016PRTArtificial Sequencesynthetic 400Arg Ser Ser Gln Ser Leu Val His Asn Asn Gly Asn Thr Tyr Leu Glu1 5 10 1540148DNAArtificial Sequencesynthetic 401aggtctagta agagtctcct acatagtaat ggcatcactt atttgtat 4840216PRTArtificial Sequencesynthetic 402Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr1 5 10 1540348DNAArtificial Sequencesynthetic 403agatctagtc agacccttct acacagtgat ggagacacct atttacat 4840416PRTArtificial Sequencesynthetic 404Lys Phe Ser Gln Thr Leu Leu His Arg Asp Gly Asn Pro Phe Leu Leu1 5 10 1540548DNAArtificial Sequencesynthetic 405aagtcaagtc agagcctctt atttactaat ggaaaaacct atttaaat 4840616PRTArtificial Sequencesynthetic 406Lys Ser Ser Gln Ser Leu Leu Phe Thr Asn Gly Lys Thr Tyr Leu Asn1 5 10 1540748DNAArtificial Sequencesynthetic 407aagtcaagtc agagcctctt atatactaat ggaaagacct atttgaat 4840816PRTArtificial Sequencesynthetic 408Lys Ser Ser Gln Ser Leu Leu Tyr Thr Asn Gly Lys Thr Tyr Leu Asn1 5 10 1540933DNAArtificial Sequencesynthetic 409aaggccagtc agaatgtgag tcctgctgta gcc 3341011PRTArtificial Sequencesynthetic 410Lys Ala Ser Gln Asn Val Ser Pro Ala Val Ala1 5 1041133DNAArtificial Sequencesynthetic 411aaggccagtc agaatgtggg tactgctgta gcc 3341211PRTArtificial Sequencesynthetic 412Lys Ala Ser Gln Asn Val Gly Thr Ala Val Ala1 5 1041333DNAArtificial Sequencesynthetic 413aaggccagtc aggatgtggg tactgctgta gcc 3341411PRTArtificial Sequencesynthetic 414Lys Ala Ser Gln Asp Val Gly Thr Ala Val Ala1 5 1041533DNAArtificial Sequencesynthetic 415cgggcaagtc aggacattgg tggtagcata aac 3341611PRTArtificial Sequencesynthetic 416Arg Ala Ser Gln Asp Ile Gly Gly Ser Ile Asn1 5 1041733DNAArtificial Sequencesynthetic 417cgagcaagtg ggaatattca cacttattta gca 3341811PRTArtificial Sequencesynthetic 418Arg Ala Ser Gly Asn Ile His Thr Tyr Leu Ala1 5 1041933DNAArtificial Sequencesynthetic 419agggccagtc aaagaattta caactaccta cac 3342011PRTArtificial Sequencesynthetic 420Arg Ala Ser Gln Arg Ile Tyr Asn Tyr Leu His1 5 1042133DNAArtificial Sequencesynthetic 421agggccagcc agagtattag cgactactta cac 3342211PRTArtificial Sequencesynthetic 422Arg Ala Ser Gln Ser Ile Ser Asp Tyr Leu His1 5 1042333DNAArtificial Sequencesynthetic 423agggccagcc agactattag cgactactta cac 3342411PRTArtificial Sequencesynthetic 424Arg Ala Ser Gln Thr Ile Ser Asp Tyr Leu His1 5 1042530DNAArtificial Sequencesynthetic 425agagccagct caagtgtaag ttacatgcat 3042610PRTArtificial Sequencesynthetic 426Arg Ala Ser Ser Ser Val Ser Tyr Met His1 5 1042730DNAArtificial Sequencesynthetic 427agagccagct caagtgtaag ttacatgcat 3042810PRTArtificial Sequencesynthetic 428Arg Ala Ser Ser Ser Val Ser Tyr Met His1 5 1042936DNAArtificial Sequencesynthetic 429actgccagct caagtgtgag ttccagttac ttgcac 3643012PRTArtificial Sequencesynthetic 430Thr Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu His1 5 1043136DNAArtificial Sequencesynthetic 431acagccagct caagtgtaac ttccagttac ttgcac 3643212PRTArtificial Sequencesynthetic 432Thr Ala Ser Ser Ser Val Thr Ser Ser Tyr Leu His1 5 1043330DNAArtificial Sequencesynthetic 433agtgccagct caagtgtcac ttacatgcac 3043410PRTArtificial Sequencesynthetic 434Ser Ala Ser Ser Ser Val Thr Tyr Met His1 5 1043530DNAArtificial Sequencesynthetic 435agtgccagct caagtgtcac ttacatgttc 3043610PRTArtificial Sequencesynthetic 436Ser Ala Ser Ser Ser Val Thr Tyr Met Phe1 5 1043730DNAArtificial Sequencesynthetic 437agtgccagct caactgtgac ttacatttac 3043810PRTArtificial Sequencesynthetic 438Ser Ala Ser Ser Thr Val Thr Tyr Ile Tyr1 5 1043930DNAArtificial Sequencesynthetic 439agtgccagct caagtgtaag tttcatgtat 3044010PRTArtificial Sequencesynthetic 440Ser Ala Ser Ser Ser Val Ser Phe Met Tyr1 5 1044145DNAArtificial Sequencesynthetic 441aaggccagcc aaagtgtcag ttttgctggt actaatttaa tgcac 4544215PRTArtificial Sequencesynthetic 442Lys Ala Ser Gln Ser Val Ser Phe Ala Gly Thr Asn Leu Met His1 5 10 1544345DNAArtificial Sequencesynthetic 443aaagccagcg aaagtgtcaa tttttttggt actaatttaa tacac 4544415PRTArtificial Sequencesynthetic 444Lys Ala Ser Glu Ser Val Asn Phe Phe Gly Thr Asn Leu Ile His1 5 10 1544545DNAArtificial Sequencesynthetic 445agagccagcg aaagtgttga taattatggc attagtttta tgcac 4544615PRTArtificial Sequencesynthetic 446Arg Ala Ser Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met His1 5 10 1544745DNAArtificial Sequencesynthetic 447agggccagcc aaagtgtcag tacatctaga tatagttatc tgcac 4544815PRTArtificial Sequencesynthetic 448Arg Ala Ser Gln Ser Val Ser Thr Ser Arg Tyr Ser Tyr Leu His1 5 10 1544945DNAArtificial Sequencesynthetic 449agggccagcc aaagtgtcag tacatctagg tatagttata tgcac 4545015PRTArtificial Sequencesynthetic 450Arg Ala Ser Gln Ser Val Ser Thr Ser Arg Tyr Ser Tyr Met His1 5 10 1545145DNAArtificial Sequencesynthetic 451ggggccagcc aaagtgtcag tacatctagg tttagttata tgcac 4545215PRTArtificial Sequencesynthetic 452Gly Ala Ser Gln Ser Val Ser Thr Ser Arg Phe Ser Tyr Met His1 5 10 1545345DNAArtificial Sequencesynthetic 453aggtccagcc aaagtgtcag tacatctaga tatagttatt tgcac 4545415PRTArtificial Sequencesynthetic 454Arg Ser Ser Gln Ser Val Ser Thr Ser Arg Tyr Ser Tyr Leu His1 5 10 1545545DNAArtificial Sequencesynthetic 455agggccagcc aaagtgtcag tacatctagg tttagttatc tgcac 4545615PRTArtificial Sequencesynthetic 456Arg Ala Ser Gln Ser Val Ser Thr Ser Arg Phe Ser Tyr Leu His1 5 10 1545748DNAArtificial Sequencesynthetic 457agggccagcc aaagtgtcag tacatctagg tatagttatg tgcactgg 4845815PRTArtificial Sequencesynthetic 458Arg Ala Ser Gln Ser Val Ser Thr Ser Arg Tyr Ser Tyr Val His1 5 10 1545945DNAArtificial Sequencesynthetic 459agggccagcc aaagtgtcag tacatccagg tttagttatg tgcac 4546015PRTArtificial Sequencesynthetic 460Arg Ala Ser Gln Ser Val Ser Thr Ser Arg Phe Ser Tyr Val His1 5 10 1546145DNAArtificial Sequencesynthetic 461agggccagcc aaagtctcag tacatctagg tttagctatg tgcac 4546215PRTArtificial Sequencesynthetic 462Arg Ala Ser Gln Ser Leu Ser Thr Ser Arg Phe Ser Tyr Val His1 5 10 1546321DNAArtificial Sequencesynthetic 463aaagtttcca gccgattttc t 214647PRTArtificial Sequencesynthetic 464Lys Val Ser Ser Arg Phe Ser1 546521DNAArtificial Sequencesynthetic 465aaagtttcca accgattttc t 214667PRTArtificial Sequencesynthetic 466Lys Val Ser Asn Arg Phe Ser1 546721DNAArtificial Sequencesynthetic 467aaactttcca accgattttc t 214687PRTArtificial Sequencesynthetic 468Lys Leu Ser Asn Arg Phe Phe1 546921DNAArtificial Sequencesynthetic 469aatgtttcca accgattttc t 214707PRTArtificial Sequencesynthetic 470Asn Val Ser Asn Arg Phe Ser1 547121DNAArtificial Sequencesynthetic 471agagtttcca accgattttc t 214727PRTArtificial Sequencesynthetic 472Arg Val Ser Asn Arg Phe Ser1 547321DNAArtificial Sequencesynthetic 473cagatgtcca agattgcctc a 214747PRTArtificial

Sequencesynthetic 474Gln Met Ser Lys Ile Ala Ser1 547521DNAArtificial Sequencesynthetic 475ctgctgtcta aattggactc t 214767PRTArtificial Sequencesynthetic 476Leu Leu Ser Lys Leu Asp Ser1 547721DNAArtificial Sequencesynthetic 477ctggtgtcaa aattggactc t 214787PRTArtificial Sequencesynthetic 478Leu Val Ser Lys Leu Asp Ser1 547921DNAArtificial Sequencesynthetic 479tcggcatcct cccgatacac t 214807PRTArtificial Sequencesynthetic 480Ser Ala Ser Ser Arg Tyr Thr1 548121DNAArtificial Sequencesynthetic 481tcagcatcca atcggtacac t 214827PRTArtificial Sequencesynthetic 482Ser Ala Ser Asn Arg Tyr Thr1 548321DNAArtificial Sequencesynthetic 483tgggcatcca cccggcacac t 214847PRTArtificial Sequencesynthetic 484Trp Ala Ser Thr Arg His Thr1 548521DNAArtificial Sequencesynthetic 485gccacatcca gtttagattc t 214867PRTArtificial Sequencesynthetic 486Ala Thr Ser Ser Leu Asp Ser1 548721DNAArtificial Sequencesynthetic 487aatgcaaaca ccttggcaga t 214887PRTArtificial Sequencesynthetic 488Asn Ala Asn Thr Leu Ala Asp1 548921DNAArtificial Sequencesynthetic 489gcttcccagt ccatctctgg g 214907PRTArtificial Sequencesynthetic 490Ala Ser Gln Ser Ile Ser Gly1 549121DNAArtificial Sequencesynthetic 491tatgcttccc aatccatctc t 214927PRTArtificial Sequencesynthetic 492Tyr Ala Ser Gln Ser Ile Ser1 549321DNAArtificial Sequencesynthetic 493gagatatcca gacgggcttc t 214947PRTArtificial Sequencesynthetic 494Glu Ile Ser Arg Arg Ala Ser1 549521DNAArtificial Sequencesynthetic 495gaaatatcca cactggcttc t 214967PRTArtificial Sequencesynthetic 496Glu Ile Ser Thr Leu Ala Ser1 549721DNAArtificial Sequencesynthetic 497agcacatcca acctggcttc t 214987PRTArtificial Sequencesynthetic 498Ser Thr Ser Asn Leu Ala Ser1 549921DNAArtificial Sequencesynthetic 499agcacgtcca acccgggttc t 215007PRTArtificial Sequencesynthetic 500Ser Thr Ser Asn Pro Gly Ser1 550121DNAArtificial Sequencesynthetic 501gacacatccg agctggcttc t 215027PRTArtificial Sequencesynthetic 502Asp Thr Ser Glu Leu Ala Ser1 550321DNAArtificial Sequencesynthetic 503gacacatcca atttggcttc t 215047PRTArtificial Sequencesynthetic 504Asp Thr Ser Asn Leu Ala Ser1 550521DNAArtificial Sequencesynthetic 505gacacattca acctggtttc t 215067PRTArtificial Sequencesynthetic 506Asp Thr Phe Asn Leu Val Ser1 550721DNAArtificial Sequencesynthetic 507atcacatcca acctggcttc t 215087PRTArtificial Sequencesynthetic 508Ile Thr Ser Asn Leu Ala Ser1 550921DNAArtificial Sequencesynthetic 509cgtgcatcca acctagaaac t 215107PRTArtificial Sequencesynthetic 510Arg Ala Ser Asn Leu Glu Thr1 551121DNAArtificial Sequencesynthetic 511catgcatcca acctaaaaac t 215127PRTArtificial Sequencesynthetic 512His Ala Ser Asn Leu Lys Thr1 551321DNAArtificial Sequencesynthetic 513cgtgcatcca acctagaatc t 215147PRTArtificial Sequencesynthetic 514Arg Ala Ser Asn Leu Glu Ser1 551521DNAArtificial Sequencesynthetic 515tatgcatcca acctagaatc t 215167PRTArtificial Sequencesynthetic 516Tyr Ala Ser Asn Leu Glu Ser1 551721DNAArtificial Sequencesynthetic 517tatgcatcca accttgaatg t 215187PRTArtificial Sequencesynthetic 518Tyr Ala Ser Asn Leu Glu Cys1 551927DNAArtificial Sequencesynthetic 519tttcaaggtt cacatgttcc attcacg 275209PRTArtificial Sequencesynthetic 520Phe Gln Gly Ser His Val Pro Phe Thr1 552127DNAArtificial Sequencesynthetic 521tttcaaggtt cacatgttcc actcacg 275229PRTArtificial Sequencesynthetic 522Phe Gln Gly Ser His Val Pro Leu Thr1 552327DNAArtificial Sequencesynthetic 523tttcaaggtt cacatgttcc gtggacg 2752410PRTArtificial Sequencesynthetic 524Phe Gln Gly Ser His Val Pro Trp Thr Phe1 5 1052527DNAArtificial Sequencesynthetic 525gctcaaaatc tagaacttcc gtggacg 275269PRTArtificial Sequencesynthetic 526Ala Gln Asn Leu Glu Leu Pro Trp Thr1 552727DNAArtificial Sequencesynthetic 527tctcaaagta cacatgttcc gtacacg 275289PRTArtificial Sequencesynthetic 528Ser Gln Ser Thr His Val Pro Tyr Thr1 552927DNAArtificial Sequencesynthetic 529ttgcagagta catattttcc tctcacg 275309PRTArtificial Sequencesynthetic 530Leu Gln Ser Thr Tyr Phe Pro Leu Thr1 553127DNAArtificial Sequencesynthetic 531ttgcagagta tacattttcc gtacacg 275329PRTArtificial Sequencesynthetic 532Leu Gln Ser Ile His Phe Pro Tyr Thr1 553327DNAArtificial Sequencesynthetic 533ctacaatatg ctagttctcc tccgacg 275349PRTArtificial Sequencesynthetic 534Leu Gln Tyr Ala Ser Ser Pro Pro Thr1 553527DNAArtificial Sequencesynthetic 535ctccagtttc atcgttcccc gtggacg 275369PRTArtificial Sequencesynthetic 536Leu Gln Phe His Arg Ser Pro Trp Thr1 553727DNAArtificial Sequencesynthetic 537ctgcaaaata ggaaaattcc tctcacg 275389PRTArtificial Sequencesynthetic 538Leu Gln Asn Arg Lys Ile Pro Leu Thr1 553927DNAArtificial Sequencesynthetic 539cagcaacatt ttagtactcc gtggacg 275409PRTArtificial Sequencesynthetic 540Gln Gln His Phe Ser Thr Pro Trp Thr1 554127DNAArtificial Sequencesynthetic 541cagcaatata gcacctatcc tctcacg 275429PRTArtificial Sequencesynthetic 542Gln Gln Tyr Ser Thr Tyr Pro Leu Thr1 554324DNAArtificial Sequencesynthetic 543cagcaatata gcaactatct cacg 245448PRTArtificial Sequencesynthetic 544Gln Gln Tyr Ser Asn Tyr Leu Thr1 554527DNAArtificial Sequencesynthetic 545caacagagta acagctggcc tctcacg 275469PRTArtificial Sequencesynthetic 546Gln Gln Ser Asn Ser Trp Pro Leu Thr1 554724DNAArtificial Sequencesynthetic 547cagcagtgga attatcctct cacg 245488PRTArtificial Sequencesynthetic 548Gln Gln Trp Asn Tyr Pro Leu Thr1 554927DNAArtificial Sequencesynthetic 549cagcagtgga gtaataaacc gctcacg 275509PRTArtificial Sequencesynthetic 550Gln Gln Trp Ser Asn Lys Pro Leu Thr1 555127DNAArtificial Sequencesynthetic 551caacagtaca gtgattcccc gtacacg 275529PRTArtificial Sequencesynthetic 552Gln Gln Tyr Ser Asp Ser Pro Tyr Thr1 555327DNAArtificial Sequencesynthetic 553cagcaaagga gtagtttccc gtacacg 275549PRTArtificial Sequencesynthetic 554Gln Gln Arg Ser Ser Phe Pro Tyr Thr1 555524DNAArtificial Sequencesynthetic 555cagcaaagta gggaatatta cacg 245568PRTArtificial Sequencesynthetic 556Gln Gln Ser Arg Glu Tyr Tyr Thr1 555727DNAArtificial Sequencesynthetic 557cagcaaagta ataaggatcc attcacg 275589PRTArtificial Sequencesynthetic 558Gln Gln Ser Asn Lys Asp Pro Phe Thr1 555927DNAArtificial Sequencesynthetic 559caaaatggtc acagttttcc gtggacg 275609PRTArtificial Sequencesynthetic 560Gln Asn Gly His Ser Phe Pro Trp Thr1 556127DNAArtificial Sequencesynthetic 561caacattttt ggagtgctcc gtggacg 275629PRTArtificial Sequencesynthetic 562Gln His Phe Trp Ser Ala Pro Trp Thr1 556327DNAArtificial Sequencesynthetic 563caggagtgga gtagttaccc actcacg 275649PRTArtificial Sequencesynthetic 564Gln Glu Trp Ser Ser Tyr Pro Leu Thr1 556527DNAArtificial Sequencesynthetic 565cagcacagtt gggagtttcc attcacg 275669PRTArtificial Sequencesynthetic 566Gln His Ser Trp Glu Phe Pro Phe Thr1 556727DNAArtificial Sequencesynthetic 567cagcacagtt gggattttcc attcacg 275689PRTArtificial Sequencesynthetic 568Gln His Ser Trp Asp Phe Pro Phe Thr1 556927DNAArtificial Sequencesynthetic 569cagcacagtt gggattttcc attcacg 275709PRTArtificial Sequencesynthetic 570Gln His Ser Trp Gly Phe Pro Phe Thr1 5571507DNAArtificial Sequencesynthetic 571atgtctacca acccgaaacc gcagaaaaaa aacaaacgta acaccaaccg tcgtccgcag 60gacgttaaat tcccgggtgg tggtcagatc gttggtggtg tttacctgct gccgcgtcgt 120ggtccgcgtc tgggtgttcg tgctacgcgt aaaacctctg aacgttctca gccgcgtggg 180cgtcgtcagc cgatcccgaa agctcgtcgt ccggaaggtc gtacctgggc tcagccgggt 240tacccgtggc cgctgtacgg taacgaaggt tgcggttggg ctggttggct gctgtctccg 300cgtggatctc gtccgtcttg gggtccgacc gacccgcgtc gtcgttctcg taaccttggt 360aaagttatcg ataccctgac ctgcggtttc gctgacctga tgggttacat accgctggtt 420ggagctccgc tgggtggtgc tgctcgtgct ctggcgcatg gcgtgcgtgt tctggaagat 480ggcgtcaact atgccaccgg taatctg 507572169PRTArtificial Sequencesynthetic 572Met Ser Thr Asn Pro Lys Pro Gln Lys Lys Asn Lys Arg Asn Thr Asn1 5 10 15Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 25 30Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 35 40 45Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro 50 55 60Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly65 70 75 80Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Cys Gly Trp Ala Gly Trp 85 90 95Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro 100 105 110Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys 115 120 125Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu 130 135 140Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp145 150 155 160Gly Val Asn Tyr Ala Thr Gly Asn Leu 16557321PRTArtificial Sequencesynthetic 573Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg1 5 10 15Ala Leu Ala His Gly 2057421PRTArtificial Sequencesynthetic 574Gly Ala Pro Leu Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg1 5 10 15Val Leu Glu Asp Gly 2057521PRTArtificial Sequencesynthetic 575Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala Thr1 5 10 15Gly Asn Leu Pro Gly 2057639PRTArtificial Sequencesynthetic 576Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg1 5 10 15Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala 20 25 30Thr Gly Asn Leu Pro Gly Cys 3557753PRTArtificial Sequencesynthetic 577Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg1 5 10 15Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala 20 25 30Thr Gly Asn Leu Pro Gly Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile 35 40 45Gly Ile Thr Glu Leu 5057838PRTArtificial SequenceSynthetic 578Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg1 5 10 15Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala 20 25 30Thr Gly Asn Leu Pro Gly 3557948PRTArtificial SequenceSynthetic 579Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg Ala1 5 10 15Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala Thr 20 25 30Gly Asn Leu Pro Gly Gln Gln Lys Phe Gln Phe Gln Phe Glu Gln Gln 35 40 455806PRTArtificial SequenceSynthetic 580His His His His His His1 558141DNAArtificial SequenceSynthetic 581aagcagtggt atcaacgcag agtacccccc cccccccccc c 4158213PRTArtificial Sequence;Synthetic 582Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr1 5 10583112PRTArtificial Sequence;Synthetic 583Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ile Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Asn Glu Tyr 20 25 30Gly Tyr Thr Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Leu Pro Pro 35 40 45Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Asp Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His65 70 75 80Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90 95Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110584126PRTArtificial Sequence;Synthetic 584Gln Ile Gln Leu Lys Glu Ser Gly Pro Ala Val Ile Lys Pro Ser Gln1 5 10 15Ser Leu Ser Leu Thr Cys Ile Val Ser Gly Phe Ser Ile Thr Ser Ser 20 25 30Val Tyr Cys Trp Gln Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45Trp Met Gly Arg Ile Cys Tyr Asp Gly Ser Val Asp Tyr Ser Pro Ser 50 55 60Ile Thr Ser Arg Gly Thr Ile Ser Arg Asp Thr Ser Leu Asn Lys Val65 70 75 80Phe Phe Gln Leu Ser Ser Val Thr Asn Glu Asp Thr Ala Met Tyr Tyr 85 90 95Cys Ser Arg Glu Asn His Ile Asp Tyr Tyr Asp Thr Thr Tyr Pro Ser 100 105 110Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 12558515PRTArtificial Sequence;Synthetic 585Arg Ala Ser Lys Ser Val Asn Glu Tyr Gly Tyr Thr Tyr Met His1 5 10 155867PRTArtificial Sequence;Synthetic 586Leu Ala Ser Asn Leu Asp Ser1 55879PRTArtificial Sequence;Synthetic 587Gln His Ser Arg Glu Leu Pro Tyr Thr1 558812PRTArtificial Sequence;Synthetic 588Gly Phe Ser Ile Thr Ser Ser Val Tyr Cys Trp Gln1 5 1058916PRTArtificial Sequence;Synthetic 589Arg Ile Cys Tyr Asp Gly Ser Val Asp Tyr Ser Pro Ser Ile Thr Ser1 5 10 1559016PRTArtificial Sequence;Synthetic 590Glu Asn His Ile Asp Tyr Tyr Asp Thr Thr Tyr Pro Ser Phe Asp Val1 5 10 15591336DNAArtificial Sequence;Synthetic 591gacattgtgc tgacacagtc tcctgcttcc ttaattgtat ctctggggca gagggccacc 60atctcgtgca gggccagcaa aagtgtcaat gaatatggct atacttatat gcactggtac 120caacagaaac caggactgcc acccaaactc ctcatctatc ttgcatccaa tctagattct 180ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240cctgtggagg aggaggatgc tgcaacctat tactgtcaac acagtaggga gcttccgtac 300acgttcggag gggggaccaa gctggaaata aaacgg 336592378DNAArtificial Sequence;Synthetic 592cagattcagc tgaaggagtc tggacctgct gtcatcaagc catcacagtc actgtctctc 60acgtgcatag tctctggatt ctccatcaca agtagtgttt attgctggca gtggatccgc 120cagcccccag gaaagggatt

agagtggatg ggacgcatct gttatgacgg ttcagttgac 180tatagtccat ccatcacaag ccgcggcacc atctccagag acacatctct gaacaaagtc 240tttttccagc tgagctctgt gacaaatgag gacacagcca tgtactactg ttccagggaa 300aaccatattg attactacga tactacttat ccgtccttcg atgtctgggg cgcagggacc 360acggtcaccg tctcctca 37859319PRTArtificial Sequence;Synthetic 593Ser Gly Ser Gly Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser1 5 10 15Trp Gly Pro59419PRTArtificial Sequence;Synthetic 594Ser Gly Ser Gly Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly1 5 10 15Pro Thr Asp59519PRTArtificial Sequence;Synthetic 595Ser Gly Ser Gly Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr1 5 10 15Asp Pro Arg59619PRTArtificial Sequence;Synthetic 596Ser Gly Ser Gly Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro1 5 10 15Arg Arg Arg59719PRTArtificial Sequence;Synthetic 597Ser Gly Ser Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg1 5 10 15Arg Ser Arg59819PRTArtificial Sequence;Synthetic 598Ser Gly Ser Gly Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg Arg Ser1 5 10 15Arg Asn Leu59919PRTArtificial Sequence;Synthetic 599Ser Gly Ser Gly Trp Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg Asn1 5 10 15Leu Gly Lys60019PRTArtificial Sequence;Synthetic 600Ser Gly Ser Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly1 5 10 15Lys Val Ile60115PRTArtificial Sequence;Synthetic 601Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro1 5 10 1560215PRTArtificial Sequence;Synthetic 602Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp1 5 10 1560315PRTArtificial Sequence;Synthetic 603Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg1 5 10 1560415PRTArtificial Sequence;Synthetic 604Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg Arg1 5 10 1560515PRTArtificial Sequence;Synthetic 605Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg1 5 10 1560615PRTArtificial Sequence;Synthetic 606Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg1 5 10 1560715PRTArtificial Sequence;Synthetic 607Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg Arg1 5 10 1560815PRTArtificial Sequence;Synthetic 608Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg1 5 10 1560915PRTArtificial Sequence;Synthetic 609Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg Asn Leu1 5 10 1561015PRTArtificial Sequence;Synthetic 610Trp Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly Lys1 5 10 1561115PRTArtificial Sequence;Synthetic 611Pro Thr Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile1 5 10 1561223PRTArtificial Sequence;Synthetic 612Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg His Arg Ser Arg1 5 10 15Asn Val Gly Lys Val Ile Asp 2061323PRTArtificial Sequence;Synthetic 613Gly Ser Arg Pro Ser Trp Gly Pro Asn Asp Pro Arg His Arg Ser Arg1 5 10 15Asn Val Gly Lys Val Ile Asp 2061423PRTArtificial Sequence;Synthetic 614Gly Ser Arg Pro Ser Trp Gly Pro Ser Asp Pro Arg His Arg Ser Arg1 5 10 15Asn Val Gly Lys Val Ile Asp 2061511PRTArtificial Sequence;Synthetic 615Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp1 5 1061611PRTArtificial Sequence;Synthetic 616Arg Gly Ser Arg Pro Ser Trp Gly Pro Asn Asp1 5 10

Claims

1.-16. (canceled)

17. A system comprising: (a) a first antibody, or antigen-binding portion thereof, which comprises heavy chain CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NO: 588, SEQ ID NO: 589, and SEQ ID NO: 590, respectively, and light chain CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NO: 585, SEQ ID NO: 586, and SEQ ID NO: 587; and (b) a second antibody, or antigen-binding portion thereof, which specifically binds to the amino acid sequence of SEQ ID NO: 574 in the lipid binding domain of HCV core protein.

18.-21. (canceled)

22. The system of claim 17, wherein the first and/or second antibody, or the antigen-binding portion thereof, comprises a detectable label attached thereto.

23. A kit comprising: (a) a first antibody, or antigen-binding portion thereof, which comprises heavy chain CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NO: 588, SEQ ID NO: 589, and SEQ ID NO: 590, respectively, and light chain CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NO: 585, SEQ ID NO: 586, and SEQ ID NO: 587; (b) a second antibody, or antigen-binding portion thereof, which specifically binds to the amino acid sequence of SEQ ID NO: 574 in the lipid binding domain of HCV core protein; and (c) instructions for detecting an HCV core protein in a sample.

24. The kit of claim 23, wherein the first and/or second antibody, or the antigen-binding portion thereof, comprises a detectable label attached thereto.

Images