ANTI-ORTHOPOXVIRUS RECOMBINANT POLYCLONAL ANTIBODY

Abstract

Disclosed is an anti-orthopoxvirus recombinant polyclonal antibody comprising distinct members which in union are capable of binding at least three orthopoxvirus related antigens, a pharmaceutical composition comprising the antibody, and a method for its production. Also disclosed is a polyclonal cell line capable of producing the recombinant polyclonal antibody as therapeutic methods utilizing the polyclonal antibody. Finally, the invention also pertains to a method for screening for useful V_H and V_L pairs useful when preparing the polyclonal antibody.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No. 11/633,070, filed Dec. 4, 2006, which claims the benefit of Danish Application No. PA 2005 01720, filed Dec. 5, 2005, both of which are incorporated herein by reference in their entireties.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

[0002] The content of the electronically submitted sequence listing in ASCII text file (Name: Sequence_listing_--2488.0020001.ascii.txt; Size: 117,921 bytes; and Date of Creation: Dec. 10, 2010) filed with the application is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates to a recombinant polyclonal anti-orthopoxvirus antibody (anti-orthopoxvirus rpAb), in particular a recombinant polyclonal anti-vaccinia virus antibody (anti-VV rpAb). The invention also relates to polyclonal expression cell lines producing anti-orthopoxvirus rpAb or anti-Vs/rpAb. Further, the application describes diagnostic and pharmacological compositions comprising anti-orthopoxvirus rpAb or anti-VV rpAb and their use in prevention, and treatment of adverse effects of vaccination, or diagnosis and treatment of orthopoxvirus infections.

[0005] 2. Background Art

[0006] Smallpox is caused by airway infection with the orthopoxvirus, variola. The threat of smallpox outbreaks as a result of bioterrorism and the emergence of related viruses such as monkeypox, have revived the need for anti-orthopoxvirus therapeutics and vaccination. Vaccinia virus vaccination mediates moderate to severe adverse reactions in approximately one in every 1000. These are currently treated with anti-vaccinia virus immunoglobulin (VIG) isolated from donors with a high antibody titer. However, the estimated incidence of adverse effects resulting from a general vaccination program using live attenuated vaccinia virus exceeds the current production capacity of VIG, thereby preventing vaccination as an approach for public protection against smallpox. Furthermore, VIG has a very low specific activity resulting in a need for injection of large volumes. There is also the risk of transmission of viral diseases from serum derived VIG products, as well as problems with batch-to-batch variations. Therefore, investigations of possible alternatives for providing protecting against vaccinia virus adverse effects or infections by other orthopoxviruses have been conducted.

[0007] Orthopoxviruses produces two types infectious particles, namely the Intracellular Mature Virions (IMV) and the Extracellular Enveloped Virions (EEV). IMV plays a predominant role in host-to-host transmission and EEV plays a major role in virus propagation within the host. The IMV particle is assembled in the cytoplasm of infected cells and consists of a virally induced membrane surrounding the genome containing a homogenous core particle. EEV particles are generated by wrapping of IMV particles in a host cell-derived membrane followed by egress of the EEV particle. At a later stage the vaccinia virus infection results in cell death and release of the infectious IMV particles. Viral proteins presented at the surface of IMV or EEV particles are potential targets for antibodies, a total of five IMV-specific proteins and two EEV-specific proteins have been reported to elicit virus neutralizing and/or protective effects when used for immunization or vaccination. Additionally, neutralizing and protective effects have been observed for the passive administration of antibodies which specifically bind these proteins (summarized in Table 1).

TABLE-US-00001 TABLE 1 Antigen Virion Antibody effect Immunization/vaccination effect A27L IMV + neutralize¹¹, 12, 13 DNA: + neutralize / protective⁶ (P14) (+) protective¹¹ Protein: + neutralize + protective¹, 8 - protective¹⁵ A17L IMV + neutralize¹³ (P21) L1R IMV + neutralize¹⁰, 14, 15 DNA: + neutralize (+) protective³ (P25-29) + protective¹⁰, 15 Protein: + neutralize + protective² D8L IMV + neutralize¹⁵ Protein: / neutralize + protective¹ (P32) / protective¹⁵ H3L IMV + neutralize⁴, 9 (P35) A33R EEV / neutralize¹⁰, 15 DNA: / neutralize + protective³, 5 (Gp23-28) + protective¹⁰, 15, 16 Protein: + neutralize + protective² B5R EEV + neutralize⁷ DNA: / neutralize (+) protective³, 6 (Gp42) + protective¹⁰, 16 Protein: / neutralize + protective², 3

The column "antibody effect" summarizes results from references describing the effect of an antibody reactive with the named antigen either in in vitro neutralization assays or by in vivo challenging assays to measure protectiveness. The column "immunization/vaccination effect" summarizes results from references where the antigen has been injected into animals either in protein form or as DNA. The neutralizing effect is analyzed by assessing the neutralization titer of the injected animals and the protective effect by challenging the immunized/vaccinated animals with vaccinia virus. [0008] 1. Demkowicz et al. 1992, J. Virol. 66:386-98. [0009] 2. Fogg et al. 2004, J. Virol. 78:10230-7. This reference also describes increased protection when immunization was performed with the following protein combinations B5R+A33R+L1R>A33R+L1R>A33R+B5R>B5R+L1R [0010] 3. Galmiche et al, 1999, Virology 254:71-80. [0011] 4. Gordon et al. 1991, Virology 181:671-86 [0012] 5. Hooper et al. 2000, Virology 266:329-39. This reference also describes increased protection when vaccination was performed with both L1R and A33R encoding DNA. [0013] 6. Hooper et al. 2003, Virology 306:181-95. This reference also describes increased protection when vaccination was performed with the following DNA combinations: B5R+A33R+L1R+A27L and B5R+A27L, where the first combination showed better protection than the second combiniation. [0014] 7. Law et al. 2001, Virology 280:132-42. [0015] 8. Lai et al. 1991, J. Virol. 65:5631-5. [0016] 9. Lin et al. 2000, J. Virol. 74:3353-3365. [0017] 10. Lustig et al 2005, J. Virol. 79:13454-13462. This reference also shows enhanced protection when monoclonal antibodies against L1R, A33R and B5R were combined. [0018] 11. Ramirez et al. 2002, J. Gen. Virol. 83:1059-1067. [0019] 12. Rodriguez et al. 1985, J. Virol. 56:482-488. [0020] 13. Wallengren et al. 2001, Virology 290:143-52. [0021] 14. Wolffe et al. 1995, Virology 211:53-63. [0022] 15. U.S. Pat. No. 6,451,309 illustrates increased protection when monoclonal antibodies against L1R and A33R were combined. Further, L1R and A33R mAbs combined with at least one mAb directed against H3L, D8L, B5R, A27L and A17L is suggested, but there is no evidence of the effect of such a combination. [0023] 16. WO 03/068151 suggests individual or combinations of fully human antibodies which binds an EEV protein, in particular B5R, A33R or B7R, where B7R is a variola ortholog of B5R and shares 92.7% identity with it. The application does not contain any evidence of the neutralizing or protective effect of such compositions.

[0024] Some of the studies cited in table 1 have revealed that protection against virus challenge is generally increased when protein/DNA combinations targeting both IMV and EEV virion proteins are used for immunization/vaccination (ref. 2, 5, 6 and 10). Similarly, U.S. Pat. No. 6,451,309 illustrated that the combination anti-L1R and anti-A33R mAbs administered to mice prior to a vaccinia virus challenge had an increased protective effect compared to the individual mAbs. This correlates with early observations that vaccination with inactivated IMV particles elicited antibody responses, but did not confer protection to virus challenge in animal experiments (Boulter and Appleyard, 1973, Prog. Med Virol. 16, 86-108).

[0025] The fact that a combination of antibodies is better than a single monoclonal antibody is further supported by the observations by Gordon et al. 1991, Virology 181:671-86, where it was shown that when comparing the neutralizing capability of a single mAb with an anti-VV envelope serum which has been purified with respect to the same antigen specificity as the mAb, or a non-purified polyclonal anti-VV envelope serum, both the purified and non-purified polyclonal anti-envelope serum was much more effective than the monoclonal antibody. Thus, both the binding of antibodies to more than one epitope on the same antigen as well as the binding of several antigens on different proteins is likely to be relevant when neutralizing vaccinia virus.

DISCLOSURE OF CONTRIBUTION

[0026] The present invention provides an alternative anti-VV immunoglobulin product which, although it is recombinantly produced, shows reactivity to multiple antigens and epitopes of the orthopoxvirus.

DESCRIPTION OF THE INVENTION

[0027] The present invention provides a smallpox countermeasure and an alternative to the serum derived VIG product, in the form of a polyclonal antibody which is capable of binding to multiple antigens and potentially multiple epitopes on individual antigens related to orthopoxvirus infections. In contrast to serum derived VIG, a polyclonal antibody of the present invention does not contain antibody molecules, which bind to non-orthopoxvirus antigens. Thus, the polyclonal antibody of the present invention is essentially free from immunoglobulin molecules that do not bind to orthopoxvirus antigens. Currently, mixtures of three monoclonal antibodies produced in mice (anti-L1R, anti-A33R and anti-B5R) or mixtures of serum derived polyclonal antibodies from rabbits immunized with a particular antigen (L1R, B5R or A33R) are known (Lustig et al 2005, J. Virol. 79: 13454-13462 and U.S. Pat. No. 6,451,309). The rationale behind selecting antibodies against exactly these three antigens was that they are directed against the IMV and EEV particles. However, since the biology of the orthopoxviruses is complex and not completely understood, it is highly likely that there are other antigens in addition to these three antigens which are important for virus neutralization and/or protection and thereby alternative compositions may provide the same or better effects. Further, it has been shown that affinity purified serum has a greater neutralizing effect than a single monoclonal antibody (Gordon et al. 1991, Virology 181:671-86). This is likely to be due to several antibodies binding to different epitopes on the antigen, thereby increasing the complement activation and removal of the antigen.

[0028] The present invention provides a polyclonal anti-orthopoxvirus antibody.

[0029] Preferably, the polyclonal anti-orthopoxvirus antibody is a recombinant polyclonal antibody (anti-orthopoxvirus rpAb), in particular an anti-VV rpAb which is directed against multiple IMV and/or EEV particle proteins and preferably also against multiple epitopes on individual IMV/EEV proteins. Further, antibodies with reactivity against orthopoxvirus related regulators of complement activation (RCA) are a desired component of an anti-orthopoxvirus rpAb of the present invention.

[0030] Further, the present invention provides pharmaceutical compositions where the active ingredient is an anti-orthopoxvirus polyclonal antibody, as well as uses of such compositions. For example can an anti-VV rpAb of the present invention serve as replacement of the presently used serum derived VIG and facilitate anti-variola activity for the treatment of smallpox as an anti-terror countermeasure.

[0031] The present invention further provides screening procedures suitable for selecting a broad diversity of anti-orthopoxvirus antibodies, and in particular procedures for mirroring the humeral immune response raised upon challenge with an orthopoxvirus, by isolating the original V_H and V_L gene pairs from such challenged individuals, and producing antibodies maintaining this original paring.

Definitions

[0032] The term "antibody" describes a functional component of serum and is often referred to either as a collection of molecules (antibodies or immunoglobulin) or as one molecule (the antibody molecule or immunoglobulin molecule). An antibody molecule is capable of binding to or reacting with a specific antigenic determinant (the antigen or the antigenic epitope), which in turn may lead to induction of immunological effector mechanisms. An individual antibody molecule is usually regarded as monospecific, and a composition of antibody molecules may be monoclonal (i.e., consisting of identical antibody molecules) or polyclonal (i.e., consisting of different antibody molecules reacting with the same or different epitopes on the same antigen or on distinct, different antigens). Each antibody molecule has a unique structure that enables it to bind specifically to its corresponding antigen, and all natural antibody molecules have the same overall basic structure of two identical light chains and two identical heavy chains. Antibodies are also known collectively as immunoglobulin. The terms antibody or antibodies as used herein is used in the broadest sense and covers intact antibodies, chimeric, humanized, fully human and single chain antibodies, as well as binding fragments of antibodies, such as Fab, Fv fragments or scFv fragments, as well as multimeric forms such as dimeric IgA molecules or pentavalent IgM.

[0033] The term "anti-orthopoxvirus recombinant polyclonal antibody" or "anti-orthopoxvirus rpAb" describes a composition of recombinantly produced diverse antibody molecules, where the individual members are capable of binding to at least one epitope on a virus belonging to the genus orthopoxvirus. Preferably, an anti-orthopoxvirus rpAb composition is reactive to more than one virus species or strain belonging to the genus orthopoxvirus. Preferably, the composition is produced from a single manufacturing cell line, but may also be a mixture of monoclonal antibodies or any combination of an anti-orthopoxvirus rpAb composition and one or more monoclonal antibodies. The diversity of the polyclonal antibody is located in the variable regions (V_H and V_L regions), in particular in the CDR1, CDR2 and CDR 3 regions.

[0034] The term "anti-VV recombinant polyclonal antibody" or "anti-VV rpAb" describes a composition of recombinantly produced diverse antibody molecules, where the individual members are capable of binding to at least one epitope on a vaccinia virus species or strain. Preferably, an anti-VV rpAb composition is reactive to at least one IMV and at least one EEV specific antigen, where the reactivity is characterized by distinct members reactive to either an IMV or an EEV specific antigen. Preferably, the composition is produced from a single manufacturing cell line, but it may also be a mixture of monoclonal antibodies. The diversity of the polyclonal antibody is located in the variable regions (V_H and V_L regions), in particular in the CDR1, CDR2 and CDR 3 regions.

[0035] The term "the polyclonal antibody of the present invention is essentially free from immunoglobulin molecules that do not bind to orthopoxvirus antigens" means that more than 80% of the antibodies, preferably more than 90%, more preferably more than 95% and most preferably more than 99%, bind to one of the orthopoxvirus antigens.

[0036] The term "cognate V_H and V_L coding pair" describes an original pair of V_H and V_L coding sequences contained within or derived from the same cell. Thus, a cognate V_H and V_L pair represents the V_H and V_L pairing originally present in the donor from which such a cell is derived. The term "an antibody expressed from a V_H and V_L coding pair" indicates that an antibody or an antibody fragment is produced from a vector, plasmid or similar containing the V_H and V_L coding sequence. When a cognate V_H and V_L coding pair is expressed, either as a complete antibody or as a stable fragment thereof, they preserve the binding affinity and specificity of the antibody originally expressed from the cell they are derived from. A library of cognate pairs is also termed a repertoire or collection of cognate pairs, and may be kept individually or pooled.

[0037] The terms "a distinct member of a recombinant polyclonal antibody" denotes an individual antibody molecule of the recombinant polyclonal antibody composition, comprising one or more stretches within the variable regions, which are characterized by differences in the amino acid sequence compared to the other individual members of the polyclonal protein. These stretches are in particular located in the CDR1, CDR2 and CDR 3 regions.

[0038] The term "epitope" is commonly used to describe a site on a larger molecule (e.g. antigen) to which the antibody will bind. An antigen is a substance that stimulates an immune response, e.g. toxin, virus, bacteria, proteins or DNA. An antigen often has more than one epitope, unless they are very small. Antibodies binding to different epitopes on the same antigen can have varying effects on the activity of the antigen they bind depending on the location of the epitope. An antibody binding to an epitope in an active site of the antigen may block the function of the antigen completely, whereas another antibody binding at a different epitope may have no or little effect on the activity of the antigen. Such antibodies, may however still activate complement and thereby result in the elimination of the antigen.

[0039] The term "fully human" used for example in relation to DNA, RNA or protein sequences describes sequences which are between 98 to 100% human.

[0040] The term "immunoglobulin" commonly is used as a collective designation of the mixture of antibodies found in blood or serum, but may also be used to designate a mixture of antibodies derived from other sources.

[0041] The term "mirrors the humeral immune response" when used in relation to a polyclonal antibody refers to an antibody composition where the nucleic acid sequences encoding the individual antibody members are derived from a donor who either has been subject to vaccination with an orthopoxvirus or who is recovering from an infection with an orthopoxvirus. In order to mirror the affinity and specificity of antibodies raised in a donor upon challenge, the sequences encoding the variable heavy chain (V_H) and the variable light chain (V_L) should be maintained in the gene pairs or combinations originally present in the donor (cognate pairs) when they are isolated. In order to mirror the diversity of a humeral immune response in a donor all the sequences encoding antibodies which bind to an orthopoxvirus are selected based on a screening procedure. The isolated sequences are analyzed with respect to diversity of the variable regions, in particular the CDR regions, but also with respect to the V_H and V_L family. Based on these analyses a population of cognate pairs representing the overall diversity of the orthopoxvirus binding antibodies are selected. Such a polyclonal antibody typically have at least 8, 10, 20, 30, 40, 50, 100, 1000 or 10⁴ distinct members.

[0042] The term "orthopoxvirus" refers to a virus species or strain belonging to the genus orthopoxvirus. Known viruses include Buffalopox, California vole pox, camelpox, cowpox, ectromelia, monkeypox, rabbitpox, raccoon pox, tatera pox, Uasin Gishu pox, vaccinia, variola, and vole pox virus.

[0043] A composition is said to be "pharmacologically acceptable" if its administration can be tolerated by a recipient patient.

[0044] The term "unit dose form" refers to a ready-to-administer form comprising a therapeutically effective amount of the active ingredient.

[0045] The term "polyclonal antibody" describes a composition of different (diverse) antibody molecules which is capable of binding to or reacting with several different specific antigenic determinants on the same or on different antigens. Usually, the variability of a polyclonal antibody is located in the so-called variable regions of the polyclonal antibody, in particular in the CDR regions. In the present invention a polyclonal antibody may either be produced in one pot from a polyclonal cell line, or it may be a mixture of different polyclonal antibodies. A mixture of monoclonal antibodies is not as such considered a polyclonal antibody, since they are produced in individual batches and not necessarily form the same cell line which will result in e.g. post translational modification differences. However, if a mixture of monoclonal antibodies provide the same antigen/epitope coverage as a polyclonal antibody of the present invention it will be considered as an equivalent of the polyclonal antibody. When stating that a member of a polyclonal antibody binds to an antigen, it is herein meant a binding having binding constant that is below 100 nM, preferably below 10 nM, even more preferred below 1 nM.

[0046] The term "recombinant antibody" is used to describe an antibody molecule or several molecules that is/are expressed from a cell or cell line transfected with an expression vector comprising the coding sequence of the antibody which is not naturally associated with the cell. If the antibody molecules in a recombinant antibody composition are diverse or different, the term "recombinant polyclonal antibody" or "rpAb" applies in accordance with the definition of a polyclonal antibody.

[0047] The term "recombinant polyclonal cell line" or "polyclonal cell line" refers to a mixture/population of protein expressing cells that are transfected with a repertoire of variant nucleic acid sequences (e.g. a repertoire of antibody encoding nucleic acid sequences). Preferably, the transfection is performed such that the individual cells, which together constitute the recombinant polyclonal cell line, each carry a transcriptionally active copy of a single distinct nucleic acid sequence of interest, which encodes one member of the recombinant polyclonal antibody of interest. Even more preferred, only a single copy of the distinct nucleic acid sequence is integrated at a specific site in the genome. The cells constituting the recombinant polyclonal cell line are selected for their ability to retain the integrated copy of the distinct nucleic acid sequence of interest, for example by antibiotic selection. Cells which can constitute such a polyclonal cell line can be for example bacteria, fungi, eukaryotic cells, such as yeast, insect cells, plant cells or mammalian cells, especially immortal mammalian cell lines such as CHO cells, COS cells, BHK cells, myeloma cells (e.g., Sp2/0 cells, NS0), NIH 3T3, YB2/0 and immortalized human cells, such as HeLa cells, HEK 293 cells, or PER.C6.

[0048] The terms "sequences encoding V_H and V_L pairs" or "V_H and V_L encoding sequence pairs" indicate nucleic acid molecules, where each molecule comprise a sequence that code for the expression of a variable heavy chain and a variable light chain, such that these can be expressed as a pair from the nucleic acid molecule if suitable promoter and/or IRES regions are present and operably linked to the sequences. The nucleic acid molecule may also code for part of the constant regions or the complete constant region of the heavy chain and/or the light chain, allowing for the expression of a Fab fragment, a full-length antibody or other antibody fragments if suitable promoter and/or IRES regions are present and operably linked to the sequences.

[0049] A recombinant polyclonal antibody is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant, e.g. prevents or attenuates an orthopoxvirus infection in an animal or human.

DESCRIPTION OF THE DRAWINGS

[0050] FIG. 1: Alignment of amino acid sequences of known orthopoxvirus related regulators of complement activation (RCA), including a consensus sequence.

[0051] FIG. 2: Schematic outline of the multiplex overlap-extension RT-PCR (A) and the cloning steps (B). (A) Two sets of primers, CH+VH 1-8 and VK1-6+CK1, specific for V_H and V.sub.κ gene families, respectively, were used for the first PCR step. A homologous region between the V_H or V.sub.κ primers results in the generation of an overlap PCR product. In the second step this product is amplified in the nested PCR. The primers also include recognition sites for restriction enzymes that facilitate cloning. (B) The generated cognate linked V_H and V.sub.κ coding pairs are pooled and inserted into a Fab expression vector by the use of the flanking XhoI and NotI restriction sites. Subsequently a bi-directional promoter is inserted into the AscI-NneI restriction site between the linked V_H and V.sub.κ coding sequences to facilitate Fab expression. PCR primers used are indicated by horizontal arrows. CH1: heavy chain constant domain 1, CL: constant domain, LC: light chain; Ab: antibody; P1-P2: bi-directional promoters.

[0052] FIG. 3: Schematic presentations of suitable Fab expression vectors. A) Shows JSK301, an E. coli Fab expression vector with NotI/XhoI restriction sites for insertion of linked V_H and V_L coding pairs. The vector comprises the following elements: Amp and Amp pro=ampicillin resistance gene and its promoter. pUC19 Ori=origin of replication. Human CH1=sequence encoding human immunoglobulin gamma 1 heavy chain domain 1. Stuffer=an irrelevant sequence insert which is cut out upon insertion of the overlap extension fragments. tac P and lac Z=bacterial promoters which can be excised at the NheI and AscI restriction sites and substituted with other promoter pairs. B) Shows a mammalian Fab expression vector with NotI/XhoI restriction sites for insertion of linked V_H and V_L coding pairs. The vector comprises the following elements: Amp and Amp pro=ampicillin resistance gene and its promoter. pUC origin=origin of replication. Rabbit B-globin A=rabbit beta-globin polyA sequence. IgG1 CH1=sequence encoding human immunoglobulin gamma 1 heavy chain domain 1. VH=sequence encoding variable heavy chain. HC leader=A genomic human heavy chain leader. P1=mammalian promoter driving the expression of the light chain. P2=mammalian promoter driving the expression of the heavy chain. Kappa leader=A murine genomic kappa chain leader. LC=light chain encoding sequence. SV40 term=Simian virus 40 terminator sequence. Neo=Neomycin resistance gene. SV40 PolyA=Simian virus 40 poly A signal sequence.

[0053] FIG. 4: A schematic presentation of a mammalian full-length antibody expression vector 00-VP-530. The vector comprises the following elements: Amp and Amp pro ampicillin resistance gene and its promoter. pUC origen=pUC origin of replication. P1=mammalian promoter driving the expression of the light chain. P2=mammaliaa promoter driving the expression of the heavy chain. Leader IGHV=genomic human heavy chain leader. VH=heavy chain variable region encoding sequence. IgG1 Sequence encoding for genomic immunoglobulin isotype G1 heavy chain constant region. Rabbit B-globin A=rabbit beta-globin polyA sequence. Kappa leader sequence encoding for murine kappa leader. LC=Sequence encoding light chain encoding sequence. SV40 term=Simian virus 40 terminator sequence. FRT=A Flp recognition target site. Neo=neomycin resistance gene. SV40 poly A=Simian virus 40 poly A signal sequence

[0054] FIG. 5: Representative Western blots of the identified antigen groups. Virus particles or recombinant antigens were separated by SDS-PAGE, and blotted. The Western blots were probed with the indicated antibody identified by its clone number. The identified antigens are indicated above each blot. The antigen group assigned A did not give any detectable band in Western blot. M indicates the marker. The size of the marker is 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 80 kDa, 100 kDa, 120 kDa.

[0055] FIG. 6: Flow chart illustrating the antibody screening process. The Fab fragments or full length antibodies (Abs) were screened against inactivated vaccinia virus strains and recombinant vaccinia virus associated protein in a first round of screening generating primary hits. The primary hits were then subjected to a second round of screening. Generally the first round of screening was performed using FLISA and the second round of screening was performed with FLISA and/or ELISA. The figure exemplifies EEV or non-particle associated proteins as recombinant vaccinia virus associated proteins. However recombinant IMV specific proteins such as L1R can also be used.

[0056] FIG. 7: Anti-vaccinia virus reactivity in 10 donors (indicated by D-001 to D-011). Antibody titers against recombinant antigens A27L, A33R, B5R, L1R, and VCP and virus particles from different strains (Lister, IHD-W, IHD-J) were determined as the minimum dilution producing a signal 4-fold above background in ELISA.

[0057] FIG. 8: Anti-vaccinia virus reactivity of the Mini-H and Mini-V compositions compared to the serum derived SymVIG. The binding was detected by ELISA using inactivated Lister strain particles as antigen and the negative control was anti-RhD polyclonal antibody.

[0058] FIG. 9: In vivo inhibition of vaccinia virus replication by Mini-V in the mouse tail lesion vaccinia virus replication model. The indicated amount of Mini-V or SymVIG was injected intraperitoneally 24 hours after viral challenge. Either the Lister strain (dashed) or the NYCBOH strain (solid) was used for challenge. Results are percentages of lesions compared with the control group treated with an irrelevant recombinant polyclonal antibody (anti-RhD rpAb). The small molecule drug Cidofovir was included as positive control and administered intramuscularly (i.m.).

[0059] FIG. 10: IEX profile of anti-VV rpAb compound. The individual clones identified by the clone numbers given in Table 5 are assigned to the individual peaks. Cell lines expressing 02-113 and 02-225 were included in the polyclonal cell line but were not found in the anti-VV rpAb.

[0060] FIG. 11: Vaccinia virus binding of the three anti-VV rpAb batches and the two VIG products, SymVIG and Cangene VIG (VIG). The binding was tested in ELISA against the antigens indicated at the top of each plot.

[0061] FIG. 12: Sym002 anti-VV rpAb (SYM002) inhibits vaccinia virus replication in vivo. Prophylactic (lower panel) or therapeutic (top panel) intramuscular administered Sym002 anti-VV rpAb inhibits both the Lister and NYCBOH vaccinia virus replication in vivo as assayed by the mouse tail pock model. All the four data sets showed an approximately 300-fold increased specific activity of Sym002 anti-VV rpAb as compared to VIG (Cangene).

[0062] FIG. 13: Sym002 mix and Sym002 anti-VV rpAb (SYM002) have identical anti-viral potency in the mouse tail lesion model. Eight mice in each group were challenged with vaccinia virus, NYCOBH strain 24 hours prior to injection I.P. injection of the indicated amount of antibodies.

[0063] FIG. 14: Affinities of antibodies reactive against B5R, A33R, A27L, or VCP determined by surface plasmon resonance using a BIAcore 2000. The obtained affinities are in the nanomolar range indicating affinity maturated antibodies.

DETAILED DESCRIPTION OF THE INVENTION

Target Antigens and Polyclonal Antibody Compositions

[0064] A polyclonal antibody of the present invention is composed of a number of distinct antibody molecules in the same composition. Each molecule is selected based on its ability to bind an orthopoxvirus associated antigen. Preferably, the distinct members of a polyclonal anti-orthopoxvirus antibody of the present invention are capable of binding at least three orthopoxvirus related antigens in union. Further, it is preferred that each distinct antibody of the polyclonal antibody binds an epitope which is not bound by any of the other members of the polyclonal antibody. An antibody of the polyclonal antibody composition may bind an epitope which overlaps epitopes of other distinct antibodies of the composition and still be considered a distinct antibody. An additional feature of an anti-orthopoxvirus polyclonal antibody of the present invention is the capability of binding at least two distinct epitopes on the same orthopoxvirus related antigen, thereby supplementing the binding to at least three different orthopoxvirus associated antigens. Such a polyclonal antibody is, hence, composed of at least 4 distinct antibody members. A polyclonal antibody of the present invention comprises binding reactivity corresponding to the compiled binding reactivity of the distinct antibody molecules constituting the polyclonal antibody composition. Preferably, a polyclonal antibody of the present invention is produced as a single batch or a few batches from a polyclonal cell line which is not naturally expressing antibody molecules (also termed a recombinant polyclonal antibody). One of the advantages of producing a recombinant polyclonal antibody compared to mixing monoclonal antibodies, is the ability to produce an principally unlimited number of distinct antibody molecules at the same time (at a cost similar to that of producing a single monoclonal antibody). Thus, it is possible to include antibodies with reactivity towards a large number of orthopoxvirus associated antigens, known as well as unknown, without increasing the cost of the end product significantly. In particular with a target as complex as the orthopoxviruses where the biology is not completely understood, individual antibodies which have not been shown to neutralize or protect against orthopoxviruses alone, may when combined with other antibodies induce a synergistic effect. Thus, it can be an advantage to include distinct antibodies in a polyclonal antibody composition, where the only criterion is that the individual antibody binds to an orthopoxvirus antigen.

[0065] One way to acquire potentially relevant antibodies that bind orthopoxvirus target antigens which have not been verified as relevant antigens, but none the less may be so, is to generate a polyclonal antibody composition which is composed of individual antibodies raised by the immune response of a donor which has been vaccinated or infected with an orthopoxvirus (full immune response). In addition to broadly obtaining antibodies derived from a full immune response against othopoxviruses, a positive selection for antibodies binding to antigens that are likely to be of particular relevance in the protection, neutralization, and/or elimination of orthopoxvirus infections or in the protection against adverse effects from vaccina virus vaccination, can be performed. Further, if antibodies to a particular antigen, which is known to be of relevance in the protection, neutralization and/or elimination of orthopoxvirus are not identified in the full immune response of the donor, such antibodies may be raised by immunization/vaccination of a donor with that particular antigen (selected immune response). Generally, neutralization is assessed by in vitro neutralization assays such as plaque reduction neutralization assays (PRNT assay) using either IMV or EEV preparations, by comet assay, EEV neutralization assay (EEV specific) (Law et al. 2001, Virology 280:132-42) or by flow cytometric detection of green fluorescent protein (Earl et al. 2003 J. Virol. 77: 10684-88). Protection is generally assessed by in vivo challenging experiments such as the mouse tail lesion model, lethal dose challenge or footpad measurements. The in vivo challenging experiments can either be performed in a prophylactic fashion, where the antibodies are administered prior to the viral challenge or as a treatment, where the antibodies are administered after viral challenge or as a combination of both.

[0066] A polyclonal antibody composition of the present invention can be composed of antibodies capable of binding an orthopoxvirus antigen which is not necessarily known, but where the antibodies are acquired from a full immune response to an orthopoxvirus, e.g. by obtaining nucleic acid sequences encoding the distinct antibodies from one or more donors vaccinated with an orthopoxvirus, or recovering from an orthopoxvirus infection. Secondly, antibodies from the same full immune response, which have been selected based on their ability to bind a particular antigen and/or epitope, can be included in a polyclonal antibody of the present invention. Thirdly, distinct antibodies encoded from V_H and V_L pairs obtained from one or more donors which have been immunized/vaccinated with a particular orthopoxvirus related antigen thereby raising a "selected" immune response in these donors, can be included in a polyclonal antibody composition of the present invention. Thus, antibodies derived by any of the mentioned techniques in the present invention may be combined into a single polyclonal antibody. Preferably the nucleic acids encoding the antibodies of the present invention are obtained from human donors and the antibodies produced are fully human antibodies.

[0067] The motivation behind the polyclonal antibody compositions of the present invention is: if a donor immunized or infected with an orthopoxvirus, raises an humeral immune response against an antigen, these antibodies are likely at least to some extend to contribute to viral clearance, and thereby qualify for inclusion in a polyclonal antibody product.

[0068] One embodiment of the present invention is an anti-orthopoxvirus rpAb wherein the composition of distinct antibody members mirrors the humeral immune response with respect to diversity, affinity and specificity against antigens associated with one or more orthopoxviruses, in particular vaccinia virus, variola virus and/or monkeypox virus. Preferably, the mirror of the humeral response is established by ensuring that one or more of the following are fulfilled i) the nucleic acid sequences coding for the V_H and V_L regions of the individual antibody members in such an anti-orthopoxvirsus rpAb are derived from a donor(s) who has raised a humeral immune response against an orthopoxvirus, for example following vaccination with vaccinia virus or an orthopox virus infection from which the donor is recovering; ii) the V_H and V_L coding sequences are isolated from the donor(s) such that the original pairing of the V_H and V_L coding sequences present in the donor(s) is maintained, iii) the V_H and V_L pairs, coding for the individual members of the rpAb, are selected such that the CDR regions are as diverse as possible; or iv) the specificity of the individual members of the anti-orthopoxvirus rpAb are selected such that the antibody composition collectively binds antigens that elicit significant antibody responses in mammals. Preferably, the antibody composition collectively binds antigens which produce significant antibody titers in a serum sample from said donor(s).

[0069] The antigens of relevance to the present invention are any orthopoxvirus derived protein, polypeptide or nucleic acid, towards which a humeral immune response or a selected immune response can be raised. The relevant antigens can be selected from viral proteins presented at the surface of IMV and/or EEV particles. At least twelve different viral proteins, (here referred to by the vaccinia virus variants) A14.5L, E10R, I5L, A13L, A27L, A17L, L1R, L5R, DBL, H3L, A14L and A17L, are inserted in the IMV outer membrane and may be relevant antigens according to the present invention. Further, at least six other proteins, A33R, A34R, F13L, B5R, A56R, F12L and A36R, are present at the surface membrane of the EEV particle and may likewise be relevant antigens according to the present invention.

[0070] In an additional embodiment of the present invention the anti-orthopoxvirus rpAb comprises binding reactivity against antigens selected from the group of viral proteins associated with IMV and/or EEV particles, in particular proteins presented on the surface of these particles. In a preferred embodiment of the present invention the anti-orthopoxvirus rpAb comprises binding reactivity against antigens selected among the viral proteins A27L, A17L, D8L and H3L, and against antigens selected among the viral proteins A33R and B5R. Additionally, the first group can constitute the viral proteins L1R and the second group can constitute the viral protein A56R.

[0071] Further, additional orthopoxvirus proteins have shown immunoreactivity in humans, macaques and/or mice following vaccination with vaccinia virus. These include the vaccinia virus ortholog A10L, A11R, D13L, H5R, A26L, E3L, L4R, H7R, P4A and A4L (Davies et al. 2005, PNAS 102: 547-552 and Demkowicz et al. 1992, J. Virol. 66:386-98), and are also considered as potentially relevant antigens to which individual members of a polyclonal antibody according to the present invention can bind. In a further embodiment of the present invention, the polyclonal antibody comprises binding reactivity against one or more of the antigens selected from the following group of viral proteins, represented as the vaccinia virus ortholog A14.5L, E10R, ISL, A13L, A27L, A17L, L1R, D8L, H3L, A14L, A17L, A33R, A34R, F13L, B5R, A56R, F12L, A36R, A10L, A11R, D13L, H5R, A26L, E3L, L4R, H7R, P4A and A4L.

[0072] The viral proteins mentioned above are, however, not the only viral proteins with potential relevance in the protection, neutralization and/or elimination of orthopoxvirus infections or prevention of adverse effects due to vaccination with vaccinia virus. The orthopoxviruses encode regulators of complement activation (RCA), that contain four tandem short consensus repeats (SCRs), allowing them to evade the consequences of complement activation in the host (reviewed in Mullick et al. 2003, Trends Immunol. 24:500-7). Several RCA proteins have been identified in the group of orthopoxviruses, namely vaccinia virus complement control protein (VCP), smallpox inhibitor of complement enzyme (SPICE) and inflammatory modulatory protein (IMP), from vaccinia virus, variola virus and cowpox, respectively. Certain monkeypox viral strains also have an ortholog of the VCP, which may be responsible for the violence of these particular strains, compared to other monkeypox strains (Chen et al. 2005, Virology 340:46-63). Further, a sequence relating to a VCP protein from camelpox virus has also been identified under NCBI accession number AAL73730. FIG. 1 shows an alignment of the mentioned RCA proteins, including an orthopoxvirus RCA protein consensus sequence. US 2005/0129700 describes the generation of anti-VCP and anti-SPICE antibodies, preferably with reactivity to both antigens. The monoclonal antibodies are used to pre-inject animals which are subjected to vaccinia virus vaccination, there are however no indication whether the antibodies are protective or not. Further, a series of monoclonal mouse antibodies against VCP has been generated in order to map the SCR domains involved in abolishing complement-enhanced neutralization. Antibodies binding to the SCR2, SCR4 or the junction between the SCR3 and 4 domains blocked the interaction of VCP with complement (Isaacs et al. 2003, J. Virol. 77:8256-62).

[0073] In further embodiments of the present invention the above mentioned polyclonal antibody compositions additionally comprise binding reactivity against an RCA encoded by an orthopoxvirus. In particular a polyclonal anti-orthopoxvirus composition comprising binding reactivity against IMV, EEV and RCA specific antigens is desired. A further anti-orthopoxvirus polyclonal antibody of the present invention comprises binding reactivity against antigens selected among the viral proteins (vaccinia virus orthologs) A27L, A17L, D8L, H3L, A33R, B5R and VCP. Additionally, the group can constitute the viral proteins L1R and/or A56R. In any of the embodiments of the present invention relating to RCA specific antibody members, the RCA binding specificity is preferably directed against a protein selected from the group VCP, SPICE, IMP, MPXV-VCP and CMLV-VCP. In a preferred embodiment the rpAb compositions of the present invention comprises individual members with binding reactivity against the orthopoxvirus RCA protein consensus sequence. In an even more preferred embodiment the RCA related binding reactivity is directed to an epitope located in the SCR2, SCR4 and/or the junction between the SCR3 and 4 domains of one of the mentioned RCA proteins or the RCA protein consensus sequence. Preferably the VCP reactivity is directed against the SCR2, SCR4 and/or the junction between the SCR3 and 4 domains.

[0074] The present invention has identified a series of V_H and V_L pairs that can be expressed as full-length antibodies, Fab fragment or other antibody fragments that have binding specificity to an vaccinia virus associated antigen. The specific V_H and V_L pairs are identified by clone number in Table 5 in Example 2. An antibody containing a V_H and V_L pair as identified in Table 5 is preferably a fully human antibody. However, if desired chimeric antibodies may also be produced.

[0075] A preferred anti-orthopoxvirus recombinant polyclonal antibody of the present invention is composed of distinct members comprising heavy chain and light chain CDR1, CDR2 and CDR3 regions selected from the group of V_H and V_L pairs listed in Table 5. Preferably, the CDR regions are maintained in the pairing indicated in Table 5 and inserted into a desired framework. Alternatively CDR regions from the heavy chain (CDRH) of a first clone are combined with the CDR regions from the light chain (CDRL) of a second clone (scrambling of V_H and V_L pairs). The CDR regions may also be scrambled within the light chain or heavy chain, for example by combining the CDRL1 region from a first clone with the CDRL2 and CDRL3 region from a second clone. Such scrambling is preferably performed among clones that bind the same antigen. The CDR regions of the present invention may also be subjected to affinity maturation, e.g. by point mutations.

[0076] An even more preferred anti-orthopoxvirus recombinant polyclonal antibody of the present invention is comprised of distinct members with heavy chain and light chain CDR1, CDR2 and CDR3 regions corresponding to clone numbers 02-029, 02-037, 02-058, 02-086, 02-147, 02-186, 02-188, 02-195, 02-197, 02-203, 02-211, 02-229, 02-235, 02-286, 02-295, 02-303, 02-339, 02-461, 02-482, 02-488, 02-526, 02-551, 02-586, 02-589, 02-607 and 02-633.

[0077] In a further embodiment, the above composition, comprising 26 individual members, additionally comprise the following two distinct members with heavy chain and light chain CDR1, CDR2 and CDR3 regions corresponding to clone numbers 02-113 and 02-225.

[0078] A further aspect of the present invention is the individual antibodies, identified by the method of the present invention, which bind previously unidentified epitopes of an orthopoxvirus, in particular vaccinia virus and/or variola virus.

[0079] In addition to the antigens mentioned above, individual antibodies with binding specificity towards unidentified antigens can be identified by for example Western blot analysis using inactivated orthopoxvirus particles as antigen source. These unidentified antigens may correspond to known antigens, but they may also correspond to unknown antigens. The identity of an antigen toward which an antibody of the present invention binds, can be assessed by analyzing binding specificity of the identified antibodies to recombinant proteins of known antigens. Alternatively, competition assays against antibodies with a known specificity can be performed. Such competition assays does, however, not exclude that the identified antibody binds to the same antigen as the known antibody, since it may bind to a different epitope.

[0080] The present invention has identified antibodies against the following antigens from the Lister strain by Western blotting: B (˜82 kDa), C (35-40 kDa), D (Three band appearance ˜65, ˜72, ˜95 kDa), E (32-35 kDA), G (Three band appearance 80 kDa, 60 kDa, 31-33 kDa), HI (˜35 Da), J (35-38 kDa) and L (<20 kDa). Antibodies against the known antigen VCP, B5R, A27L and A33R have also been verified by Western blotting. By using in vitro translated proteins the HI antigen was shown to correspond to antigen H3L, the E antigen to D8L, the D antigen to A56R, the remaining antigens does not correspond to VCP, B5R, A27L, A33R or H3L, D8L, A56R and the antibodies binding to these unidentified antigens may potentially bind to previously unknown orthopoxvirus antigens or epitopes.

[0081] An embodiment of the present invention is an antibody binding antigen B of the Lister strain at the same epitope as an antibody comprising three heavy chain CDRs and three light-chain CDRs derivable from clone nr. 02-037, 02-089 and/or 02-058.

[0082] An embodiment of the present invention is an antibody binding antigen C of the Lister strain at the same epitope as an antibody comprising three heavy chain CDRs and three light-chain CDRs derivable from clone nr. 02-243.

[0083] An embodiment of the present invention is an antibody binding antigen D or A56R of the Lister strain at the same epitope as an antibody comprising three heavy chain CDRs and three light-chain CDRs derivable from clone nr. 02-628, 02-431,002-516 and/or 02-551.

[0084] An embodiment of the present invention is an antibody binding antigen E or D8L of the Lister strain at the same epitope as an antibody comprising three heavy chain CDRs and three light-chain CDRs derivable from clone nr. 02-339.

[0085] An embodiment of the present invention is an antibody binding antigen G of the Lister strain at the same epitope as an antibody comprising three heavy chain CDRs and three light-chain CDRs derivable from clone nr. 02-147.

[0086] An embodiment of the present invention is an antibody binding antigen J of the

[0087] Lister strain at the same epitope as an antibody comprising three heavy chain CDRs and three light-chain CDRs derivable from clone nr. 02-640 and/or 02-633.

[0088] An embodiment of the present invention is an antibody binding antigen L of the

[0089] Lister strain at the same epitope as an antibody comprising three heavy chain CDRs and three light-chain CDRs derivable from clone nr. 02-589, 02-156, and/or 02-225.

Isolation and Selection of Variable Heavy Chain and Variable Light Chain Coding Pairs

[0090] The process of generating an anti-orthopoxvirus recombinant polyclonal antibody composition involves the isolation of sequences coding for variable heavy chains (V_H) and variable light chains (V_L) from a suitable source, thereby generating a repertoire of V_H and V_L coding pairs. Generally, a suitable source for obtaining V_H and V_L coding sequences are lymphocyte containing cell fractions such as blood, spleen or bone marrow samples from an animal or human immunized/vaccinated with an orthopoxvirus strain or proteins or DNA derived from such a strain. Preferably, lymphocyte containing fractions are collected from humans or transgenic animals with human immunoglobulin genes, which have been vaccinated with a vaccinia virus strain, such a strain can for example be selected from a group of strains comprising Connaught, IHD-J, IHD-W, Brighton, WT, Lister, NYCBOH, Copenhagen, Ankara, Dairen I, L-IPV, LC16MO, LIVP, Tian Tan, WR 65-16, and Wyeth or proteins or DNA derived from such a strain. Patients recovering from an infection with an orthopoxvirus strain can also be used as source for the V_H and V_L gene isolation. The collected lymphocyte containing cell fraction may be enriched further to obtain a particular lymphocyte population, e.g. cells of the B lymphocyte lineage. Preferably, the enrichment is performed using magnetic bead cell sorting (MACS) and/or fluorescence activated cell sorting (FACS), taking advantage of lineage-specific cell surface marker proteins for example for B cells and/or plasma cells. Preferably, the lymphocyte containing cell fraction is enriched with respect to B cells and/or plasma cells. Even more preferred cells with high CD19 and CD38 expression and intermediate CD45 expression are isolated from blood. These cells are sometimes termed circulating plasma cells, early plasma cells or plasmablasts, for ease, they are just termed plasma cells in the present invention.

[0091] The isolation of V_H and V_L coding sequences can either be performed in the classical way where the V_H and V_L coding sequences are combined randomly in a vector to generate a combinatorial library of V_H and V_L coding sequences pairs. However, in the present invention it is preferred to mirror the diversity, affinity and specificity of the antibodies produced in a humeral immune response upon challenge with an orthopoxvirus. This involves the maintenance of the V_H and V_L pairing originally present in the donor, thereby generating a repertoire of sequence pairs where each pair encodes a variable heavy chain (V_H) and a variable light chain (V_L) corresponding to a V_H and V_L pair originally present in an antibody produced by the donor from which the sequences are isolated. This is also termed a cognate pair of V_H and V_L encoding sequences and the antibody is termed a cognate antibody. Preferably, the V_H and V_L coding pairs of the present invention, combinatorial or cognate, are obtained from human donors, and therefore the sequences are completely human.

[0092] There are several different approaches for the generation of cognate pairs of V_H and V_L encoding sequences, one approach involves the amplification and isolation of V_H and V_L encoding sequences from single cells sorted out from a lymphocyte-containing cell fraction. The V_H and V_L encoding sequences may be amplified separately and paired in a second step or they may be paired during the amplification (Coronella et al. 2000 Nucleic Acids Res. 28: E85; Babcook et al 1996 PNAS 93: 7843-7848 and WO 05/042774). An alternative approach involves in-cell amplification and pairing of the V_H and V_L encoding sequences (Embleton et al. 1992. Nucleic Acids Res. 20: 3831-3837; Chapal et al. 1997 BioTechniques 23: 518-524). In order to obtain a repertoire of V_H and V_L encoding sequence pairs which resemble the diversity of V_H and V_L sequence pairs in the donor, a high-throughput method with as little scrambling (random combination) of the V_H and V_L pairs as possible, is preferred, e.g. as described in WO 05/042774 (hereby incorporated by reference).

[0093] In a preferred embodiment of the present invention a repertoire of V_H and V_L coding pairs, where the member pairs mirror the gene pairs responsible for the humeral immune response upon challenge with an orthopoxvirus, is generated according to a method comprising the steps i) providing a lymphocyte-containing cell fraction from a donor vaccinated with an orthopoxvirus or recovering from an orthopoxvirus infection; ii) optionally enriching B cells or plasma cells from said cell fraction; iii) obtaining a population of isolated single cells, comprising distributing cells from said cell fraction individually into a plurality of vessels; iv) amplifying and effecting linkage of the V_H and V_L coding pairs, in a multiplex overlap extension RT-PCR procedure, using a template derived from said isolated single cells and v) optionally performing a nested PCR of the linked V_H and V_L coding pairs. Preferably the isolated cognate V_H and V_L coding pairs are subjected to a screening procedure as described below.

[0094] Once the V_H and V_L sequence pairs have been generated a screening procedure to identify sequences encoding V_H and V_L pairs with binding reactivity towards an orthopoxvirus associated antigen is performed. If the V_H and V_L sequence pairs are combinatorial a phage display procedure can be applied to enrich for V_H and V_L pairs coding for antibody fragments binding to orthopoxvirus prior to screening.

[0095] in order to mirror the diversity, affinity and specificity of the antibodies produced in a humeral immune response upon challenge with an orthopoxvirus, the present invention has developed a screening procedure for the cognate pairs, in order to obtain the broadest diversity possible. For screening purposes the repertoire of cognate V_H and V_L coding pairs are expressed individually either as antibody fragments (e.g. scFv or Fab) or as full-length antibodies using either a bacterial or mammalian screening vector transfected into a suitable host cell. The repertoire of Fabs/antibodies is first screened for reactivity to one or more orthopoxvirus strains. In parallel the Fabs/antibodies, are screened against selected antigens. These antigens are selected based on the knowledge of the orthopoxvirus biology and the expected neutralizing and/or protective effect antibodies capable of binding to these antigens potentially can provide. This screening procedure can likewise be applied to a combinatorial phage display library.

[0096] In an embodiment of the present invention the screening procedure for selecting V_H and V_L sequence pairs capable of encoding a broad diversity of anti-orthopoxvirus antibodies is performed as follows: an antibody or antibody fragments is expressed from a host cell transfected with a screening vector containing a distinct member of the repertoire of V_H and V_L coding pairs. The antibody or antibody fragment is screened against at least two different vaccinia virus strains in conjunction with a parallel screening against one or more of the following antigens A27L, A17L, D8L, H3L, L1R, A33R, B5R and VCP by contacting the antibody or fragment with these strains/antigens. This process is performed for each member of the repertoire of V_H and V_L coding pairs, and sequences encoding V_H and V_L pairs that bind to either the whole virus and/or one of the specific antigens are selected from the host cells containing them (also termed clones). Preferably a second screening is performed, in order to ensure that none of the selected sequences encode false positives. In the second screening all the vaccinia virus/antigen binding V_H and V_L pairs identified in the first screening are screened again against both the virus strains and the selected antigens. The screening procedure is illustrated in FIG. 6, exemplified with some of the antigens mentioned above. Generally, immunological assays are suitable for the screening performed in the present invention. Such assays are well know in the art and constitute for example ELISPOTS, ELISA, FLISA, membrane assays (e.g. Western blots), arrays on filters, and FACS. The assays can either be performed without any prior enrichment steps, utilizing polypeptides produced from the sequences encoding the V_H and V_L pairs. In the event that the repertoire of V_H and V_L coding pairs are cognate pairs no enrichment by e.g. phage display is needed prior to the screening. However, in the screening of combinatorial libraries, the immunoassays is preferably performed in combination with or following enrichment methods such as phage display, ribosome display, bacterial surface display, yeast display, eukaryotic virus display, RNA display or covalent display (reviewed in FitzGerald, K., 2000. Drug Discov. Today 5, 253-258).

[0097] The V_H and V_L pair encoding sequences selected in the screening are generally subjected to sequencing, and analyzed with respect to diversity of the variable regions. In particular the diversity in the CDR regions is of interest, but also the V_H and V_L family representation is of interest. Based on these analyses, sequences encoding V_H and V_L pairs representing the overall diversity of the orthopoxvirus binding antibodies isolated from one or more donors are selected. Preferably, sequences with differences in all the CDR regions (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2 and CDRL3) are selected. If there are sequences with one or more identical or very similar CDR regions which belong to different V_H or V_L families, these are also selected. The selection of V_H and V_L sequence pairs can also be performed based on the diversity of the CDR3 region of the variable heavy chain. During the priming and amplification of the sequences, mutations may occur in the framework regions of the variable region. Preferably, such errors are corrected in order to ensure that the sequences correspond completely to those of the donor, e.g. such that the sequences are completely human in all conserved regions such as the framework regions of the variable region.

[0098] When it is ensured that the overall diversity of the collection of selected sequences encoding V_H and V_L pairs is highly representative of the diversity seen at the genetic level in a humeral response to an orthopoxvirus challenge, it is expected that the overall specificity of antibodies expressed from a collection of selected V_H and V_L coding pairs, also are representative with respect to the specificity of the antibodies produced in the challenged donors. An indication of whether the specificity of the antibodies expressed from a collection of selected V_H and V_L coding pairs, are representative of the specificity of the antibodies raised by challenged donors can be obtained by comparing the antibody titers towards the virus strains as well as the selected antigens of the donor blood with the specificity of the antibodies expressed from a collection of selected V_H and V_L coding pairs. Additionally, the specificity of the antibodies expressed from a collection of selected V_H and V_L coding pairs can be analyzed further. The degree of specificity correlates with the number of different antigens towards which binding reactivity can be detected. In a further embodiment of the present invention the specificity of the individual antibodies expressed from a collection of selected V_H and V_L coding pairs are analyzed by Western blot. Briefly, the antigens from an orthopoxvirus strain are resolved on polyacrylamide gel, under reducing conditions. The antibodies are analyzed individually in a Western blot procedure, identifying the protein antigens to which they bind. The binding pattern of the individual antibodies is analyzed and compared to the other antibodies expressed from a collection of selected V_H and V_L coding pairs. Preferably, individual members to be comprised in an anti-orthopoxvirus rpAb of the present invention are selected such that the specificity of the antibody composition collectively covers all the antigens which have produced significant antibody titers in a serum sample from the donor(s). Even more preferred, antibodies with different binding pattern in the Western blot analysis are selected to constitute an anti-orthopoxvirus rpAb of the present invention.

Production of a Recombinant Polyclonal Antibody from Selected V_H and V_L Coding Pairs

[0099] A polyclonal antibody of the present invention is produced from a polyclonal expression cell line in one or a few bioreactors or equivalents thereof. Following this approach the anti-orthopoxvirus rpAb can be purified from the reactor as a single preparation without having to separate the individual members constituting the anti-orthopoxvirus rpAb during the process. If the polyclonal antibody is produced in more than one bioreactor, the supernatants from each bioreactor can be pooled prior to the purification, or the purified anti-orthopoxvirus rpAb can be obtained by pooling the antibodies obtained from individually purified supernatants from each bioreactor.

[0100] One way of producing a recombinant polyclonal antibody is described in WO 04/061104 and PCT/DK2005/000501 (these references are hereby incorporated by reference). The method described therein, is based on site-specific integration of the antibody coding sequence into the genome of the individual host cells, ensuring that the V_H and V_L protein chains are maintained in their original pairing during production. Further, the site-specific integration minimizes position effects and therefore the growth and expression properties of the individual cells in the polyclonal cell line are expected to be very similar. Generally, the method involves the following: i) a host cell with one or more recombinase recognition sites; ii) an expression vector with at least one recombinase recognition site compatible with that of the host cell; iii) generation of a collection of expression vectors by transferring the selected V_H and V_L coding pairs from the screening vector to an expression vector such that a full-length antibody or antibody fragment can be expressed from the vector; iv) transfection of the host cell with the collection of expression vectors and a vector coding for a recombinase capable of combining the recombinase recognition sites in the genome of the host cell with that in the vector; v) obtaining/generating a polyclonal cell line from the transfected host cell and vi) expressing and collecting the polyclonal antibody from the polyclonal cell line.

[0101] Preferably mammalian cells such as CHO cells, COS cells, BHK cells, myeloma cells (e.g., Sp2/0 or NSO cells), fibroblasts such as NIH 3T3, and immortalized human cells, such as HeLa cells, HEK 293 cells, or PER.C6, are used. However, non-mammalian eukaryotic or prokaryotic cells, such as plant cells, insect cells, yeast cells, fungi, E. coli etc., can also be employed. A suitable host cell comprises one or more suitable recombinase recognition sites in its genome. The host cell should also contain a mode of selection which is operably linked to the integration site, in order to be able to select for integrants, (i.e., cells having an integrated copy of an anti-orthopoxvirus Ab expression vector or expression vector fragment in the integration site). The preparation of cells having an FRT site at a pre-determined location in the genome was described in e.g. U.S. Pat. No. 5,677,177. Preferably, a host cell only has a single integration site, which is located at a site allowing for high expression of the integrant (a hot-spot).

[0102] A suitable expression vector comprises a recombination recognition site matching the recombinase recognition site(s) of the host cell. Preferably the recombinase recognition site is linked to a suitable selection gene different from the selection gene used for construction of the host cell. Selection genes are well known in the art, and include glutamine synthetase gene (GS) and neomycin. The vector may also contain two different recombinase recognition sites to allow for recombinase-mediated cassette exchange (RMCE) of the antibody coding sequence instead of complete integration of the vector. RMCE is described in Langer et al 2002, Nucleic Acids Res. 30, 3067-3077; Schlake and Bode 1994, Biochemistry 33, 12746-12751 and Belteki et al 2003, Nat. biotech. 21, 321-324. Suitable recombinase recognition sites are well known in the art, and include FRT, lox and attP/attB sites. Preferably the integrating vector is an isotype-encoding vector, where the constant regions (preferably including introns) are present in the vector prior to transfer of the V_H and V_L coding pair from the screening vector. The constant regions present in the vector can either be the entire heavy chain constant region (CH₁ to CH₃ or to CH₄) or the constant region encoding the Fc part of the antibody (CH₂ to CH₃ or to CH₄). The light chain Kappa or Lambda constant region may also be present prior to transfer. The choice of the number of constant regions present, if any, depends on the screening and transfer system used. The heavy chain constant regions can be selected from the isotypes IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD and IgE. Preferred isotypes are IgG1 and/or IgG3. Further, the expression vector for site-specific integration of the anti-orthopoxvirus antibody coding nucleic acid contains suitable promoters or equivalent sequences directing high levels of expression of each of the V_H and V_L chains. FIG. 4 illustrates one possible way to design the expression vector, although numerous other designs are possible.

[0103] The transfer of the selected V_H and V_L coding pairs from the screening vector can be performed by conventional restriction enzyme cleavage and ligation, such that each expression vector molecule contain one V_H and V_L coding pair. Preferably, the V_H and V_L coding pairs are transferred individually, they may, however, also be transferred in-mass if desired. When all the selected V_H and V_L coding pairs are transferred to the expression vector a collection or a library of expression vectors is obtained. Alternative ways of transfer may also be used if desired.

[0104] Methods for transfecting a nucleic acid sequence into a host cell are known in the art. To ensure site-specific integration a suitable recombinase must be provided to the host cell as well. This is preferably assured by co-transfection of a plasmid encoding the recombinase. Suitable recombinases are for example Flp, Cre or phage ΦC31 integrase, when used together with a host cell/vector system with the corresponding recombinase recognition sites. The host cell can either be transfected in bulk, meaning that the library of expression vectors is transfected into the cell line in one single reaction thereby obtaining a polyclonal cell line. Alternatively, the collection of expression vectors can be transfected individually into the host cell, thereby generating a collection of individual cell lines (producing monoclonal antibodies). The cell lines generated upon transfection (monoclonal or polyclonal) are then selected for site specific integrants, and adapted to grow in suspension and serum free media, if they did not already do this prior to transfection. If the transfection was performed individually, the individual cell lines are analyzed further with respect to their grow properties and antibody production. Preferably cell lines with similar proliferation rates and antibody expression levels are selected for the generation of the polyclonal cell line. The polyclonal cell line is then generated by mixing the individual cell lines in a predefined ratio. Generally, a polyclonal master cell bank (pMCB) and/or a polyclonal working cell bank (pWCB) is laid down from the polyclonal cell line.

[0105] One embodiment of the present invention is a polyclonal cell line capable of expressing a recombinant polyclonal anti-orthopoxvirus antibody of the present invention.

[0106] A further embodiment of the present invention is a polyclonal cell line wherein each individual cell is capable of expressing a single V_H and V_L coding pair, and the polyclonal cell line as a whole is capable of expressing a collection of V_H and V_L coding pairs, where each V_H and V_L coding pair encode an anti-orthopoxvirus antibody of the present invention. Preferably the collection of V_H and V_L coding pairs are cognate pairs generated according to the methods of the present invention.

[0107] The recombinant polyclonal antibody is then expressed by culturing one ampoule from the pWCB in an appropriate medium for a period of time allowing for sufficient expression of antibody and where the polyclonal cell line remains stable (The window is approximately between 15 days and 50 days). Culturing methods such as fed batch or perfusion may be used. The recombinant polyclonal antibody is obtained from the culture medium and purified by conventional purification techniques. Affinity chromatography combined with subsequent purification steps such as ion-exchange chromatography, hydrophobic interactions and gel filtration has frequently been used for the purification of IgG. Following purification, the presence of all the individual members in the polyclonal antibody composition is assessed, for example by ion-exchange chromatography. The characterization of a polyclonal antibody composition is described in detail in PCT/DK2005/000504 (hereby incorporated by reference).

[0108] An alternatively method of expressing a mixture of antibodies in a recombinant host is described in WO 04/009618, this method produces antibodies with different heavy chains associated with the same light chain from a single cell line. This approach may be applicable if the anti-orthopoxvirus rpAb is produced from a combinatorial library.

Therapeutic Compositions

[0109] Another aspect of the invention is a pharmaceutical composition comprising as an active ingredient anti-orthopoxvirus rpAb or anti-orthopoxvirus recombinant polyclonal Fab or another anti-orthopoxvirus recombinant polyclonal fragment. Preferably, the active ingredient of such a composition is an anti-orthopoxvirus recombinant polyclonal antibody as claimed by the present invention. Such compositions are intended for prevention, and treatment of adverse effects of vaccination with vaccinia virus or treatment of orthopoxvirus infections, in particular infections with variola virus or monkeypoxvirus. Preferably, the treatment is administered to a human a domestic animal or a pet.

[0110] The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0111] In further embodiments of the present invention, any of the previously described anti-orthopoxvirus Ab compositions (polyclonal or monoclonal) may additionally be combined with other compositions for the treatment of an orthopoxvirus infection, such as Cidofovir, STI-571 (Reeves et al. 2005, Nature Med. 11:731-739) and/or ST-246 (Yang et al. 2005, J. Virol. 79:13139-13149).

[0112] Anti-orthopoxvirus rpAb or polyclonal fragments thereof may be administered within a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dose form. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer to individuals being vaccinated with an orthopoxvirus or patients showing adverse effects following vaccination or patients infected with an orthopoxvirus. In a preferred embodiment the administration is prophylactic. Any appropriate route of administration may be employed, for example, administration may be parenteral, intravenous, intra-arterial, subcutaneous, intramuscular, intraperitoneal, intranasal, aerosol, suppository, or oral administration. For example, therapeutic formulations may be in the form of, liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules chewing gum or pasta, and for intranasal formulations, in the form of powders, nasal drops, or aerosols.

[0113] The pharmaceutical compositions of the present invention are prepared in a manner known per se, for example, by means of conventional dissolving, lyophilizing, mixing, granulating or confectioning processes. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see for example, in Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, 2000, Lippincott Williams & Wilkins, Philadelphia, Pa. and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York, N.Y.).

[0114] Solutions of the active ingredient, and also suspensions, and especially isotonic aqueous solutions or suspensions, are preferably used, it being possible, for example in the case of lyophilized compositions that comprise the active ingredient alone or together with a carrier, for example mannitol, for such solutions or suspensions to be produced prior to use. The pharmaceutical compositions may be sterilized and/or may comprise excipients, for example preservatives, stabilizers, wetting and/or emulsifying agents, solubilizers, salts for regulating the osmotic pressure and/or buffers, and are prepared in a manner known per se, for example by means of conventional dissolving or lyophilizing processes. The said solutions or suspensions may comprise viscosity-increasing substances, such as sodium carboxymethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone or gelatin.

[0115] The injection compositions are prepared in customary manner under sterile conditions; the same applies also to introducing the compositions into ampoules or vials and sealing the containers.

[0116] Pharmaceutical compositions for oral administration can be obtained by combining the active ingredient with solid carriers, if desired granulating a resulting mixture, and processing the mixture, if desired or necessary, after the addition of appropriate excipients, into tablets, pills, or capsules, which may be coated with shellac, sugar or both. It is also possible for them to be incorporated into plastics carriers that allow the active ingredients to diffuse or be released in measured amounts.

[0117] The pharmaceutical compositions comprise from approximately 1% to approximately 95%, preferably from approximately 20% to approximately 90%, active ingredient. Pharmaceutical compositions according to the invention may be, for example, in unit dose form, such as in the form of ampoules, vials, suppositories, tablets, pills, or capsules. The formulations can be administered to human individuals in therapeutically or prophylactic effective amounts (e.g., amounts which prevent, eliminate, or reduce a pathological condition) to provide therapy for a disease or condition. The preferred dosage of therapeutic agent to be administered is likely to depend on such variables as the type and extent of the disorder, the overall health status of the particular patient, the formulation of the compound excipients, and its route of administration.

Therapeutic Uses of the Compositions According to the Invention

[0118] The pharmaceutical compositions according to the present invention may be used for the treatment, amelioration or prophylaxis of a disease in a mammal. Conditions that can be treated or prevented with the present pharmaceutical compositions include prevention, and treatment of adverse effects of vaccination with vaccinia virus or other orthopoxvirus and treatment of individuals infected with an orthopoxvirus, in particular variola virus, monkeypox virus and camelpox virus infections.

[0119] One embodiment of the present invention is a method for treatment or prophylaxis of an orthopoxvirus infection in a human or animal, wherein an effective amount of an anti-orthopoxvirus recombinant polyclonal of the present invention is administered to said human or animal.

[0120] A further embodiment of the present invention is a method for treatment, or prevention of adverse side effects of vaccination with vaccinia virus in a human or an animal, wherein an effective amount of an anti-orthopoxvirus recombinant polyclonal of the present invention administered to said human or animal.

[0121] A further embodiment of the present invention is the use of an anti-orthopoxvirus recombinant polyclonal antibody of the present invention for the preparation of a composition for the treatment, or prevention of adverse side effects of vaccination with vaccinia virus, or for treatment or prophylaxis of orthopoxvirus infections.

Diagnostic Use and Environmental Detection Use

[0122] Another embodiment of the invention is directed to diagnostic kits. Kits according to the present invention comprise an anti-orthopoxvirus rpAb prepared according to the invention which protein may be labeled with a detectable label or non-labeled for non-label detection. The kit may be used to identify individuals infected with orthopoxvirus.

EXAMPLES

Example 1

[0123] This example is a collection of the methods applied to illustrate the present invention.

a. Sorting of Plasma Cells from Donor Blood

[0124] The peripheral blood mononuclear cells (PBMC) were isolated from blood drawn from donors using Lymphoprep (Axis Shield) and gradient centrifugation according to the manufactures instructions. The isolated PBMC were either cryopreserved in FCS; 10% DMSO at -150° C. or used directly. The B cell fraction was labeled with anti-CD19 antibody and isolated from the PBMC fraction using magnetic cell sorting (MACS). The PBMC (1×10⁶ cells) were incubated with anti-CD19-FITC conjugated antibody (BD Pharmingen) for 20 minutes at 4° C. Cells were washed twice in, and resuspended in MACS buffer (Miltenyi Biotec). Anti-FITC MicroBeads (Miltenyi Biotec) were mixed with the labeled cells and incubated for 15 minutes at 4° C. The washing procedure was repeated before the cell-bead suspension was applied to a LS MACS column (Miltenyi Biotec). The CD19 positive cell fraction was eluted from the column according to the manufactures instructions and either stored in FCS-10% DMSO, or proceeded directly to single cell sorting.

[0125] Plasma blasts or circulating plasma cells (hereafter plasma cells) were selected from the CD19.sup.+ B cell fraction by fluorescence activated cell sorting (FACS) based on the expression profile of CD19, CD38, and CD45 cell surface proteins. CD19 is a B-cell market that is also expressed on early plasma cells, while CD38 is highly expressed on plasma cells. The plasma cells apparently, have a somewhat lower expression of CD45 than the rest of the CD19.sup.+ cells, which allow separation of a discrete population. The MACS purified cells were thawed or used directly. The cells were washed in FACS buffer (PBS; 1% BSA) and stained for 20 minute with CD19-FITC, CD38-PE, CD45-PerCP (BD Pharmingen). The cells were washed and re-suspended in FACS buffer.

[0126] The flow rate of the cells during the FACS was set at 200 events/sec and the cell concentration was 5×10⁵/ml to obtain a high plasma cells rescue. The following set of gates, were used. Each gate is a daughter of the former. [0127] Gate 1: FSC/SSC gate. The lymphocyte population having the highest FSC is selected ensuring sorting of living cells. [0128] Gate 2: SSCh/SSCw. This gate ensures sorting of single cells. [0129] Gate 3: CD19.sup.+ cells. In the FL1-FL2 dot plot, only the CD19 positive cells are selected. [0130] Gate 4: In the FL2-FL3 dot plot, a discrete population should be visible, which is CD38high and CD45intermediate.

[0131] The resulting population that fulfills these four criteria was single-cell sorted into 96-well PCR plates containing a sorting buffer (see section c). The cell containing plates were stored at -80° C.

b. ELISpot

[0132] ELISpot was used to estimate the percentages of plasma cells expressing anti-vaccinia virus antibodies in obtained cell samples i.e. PBMC, MACS purified CD19+ cells, or a population of FACS sorted plasma cells. 96-well plates with a nitrocellulose surface (Millipore) were coated with a solution of 20 μg/ml inactivated vaccinia virus particles of either Lister strain of IHD-W strain (AutogenBioclear, UK). The wells were blocked by incubation with RPMI, 10% FCS at 4° C. over night. The plasma cell containing cell sample was added in RPMI culture medium to each well followed by incubation at standard tissue culture conditions for 24 hours. The secreted vaccinia virus specific antibodies will bind to the immobilized virus particles surrounding the antibody producing plasma cell. The cells were removed by washing three times in PBS; 0.01% Tween20 and three times in PBS. HRP-conjugated anti-human IgG (H+L) (CalTag) and HRP-conjugated anti-human IgA (SeroTech) were added and allowed to react with the immobilized antibodies for 1 hour at 37° C. The washing procedure was repeated and the chromogen substrate (3-amino-9-ethylcarbazole solubilized in N,N-DMF (di-methyl formamide) was added. The color development was terminated after 4 minutes by addition of H₂O. Red spots were identified at the sites where antigen specific plasma cells had been located.

c. Linkage of Cognate V_H and V_L Pairs

[0133] The linkage of V_H and V_L coding sequences was performed on the single cells obtained as described in section a), facilitating cognate pairing of the V_H and V_L coding sequences. The procedure was a two step PCR procedure based on a one-step multiplex overlap-extension RT-PCR followed by a nested PCR. The primer mixes used in the present example only amplify Kappa light chains. Primers capable of amplifying Lambda light chains could, however, be added to the multiplex primer mix and nested PCR primer mix if desired. The principle is illustrated in FIG. 2.

[0134] The 96-well PCR plates from produced in step a) were thawed. The single cell served as template for the multiplex overlap-extension RT-PCR. Sorting buffer containing reaction buffer (Phusion HF buffer; Finnzymes), primers for RT-PCR (see Table 2) and RNase inhibitor (RNasin, Promega) was already added to each well before the single-cell sorting. The following was added to each well to obtain the given final concentration: dNTP mix (200 μM each), RNAse inhibitor (20 U/μl), Sensiscript Reverse Transcriptase (320× dilution; Qiagen) and Phusion DNA Polymerase (0.4 U; Finnzymes).

[0135] The plates were incubated for 1 hour at 37° C. to allow reverse transcription of the RNA from each cell. Following the RT, the plates were subjected to the following PCR cycle: 30 sec. at 98° C., 30× (20 sec. at 98° C., 30 sec. at 60° C., 45 sec. at 72° C.), 45 sec. at 72° C.

[0136] The PCR reactions were performed in H20BIT Thermal cycler with Peel Seal Basket for 24×96-well plates (ABgene), to facilitate a high-throughput. The PCR plates were stored at -20° C. after cycling.

TABLE-US-00002 TABLE 2 RT-PCR multiplex overlap-extension primer mix Final Primer Conc. name μM Sequence SEQ ID VH set CH-IgG 0.2 GACSGATGGGCCCTTGGTGG 1 CH-IgA 0.2 GAGTGGCTCCTGGGGGAAGA 2 VH-1 0.04 TATTCCCATGGCGCGCCCAGRTGCAGCTGGTGCART 3 VH-2 0.04 TATTCCCATGGCGCGCCSAGGTCCAGCTGGTRCAGT 4 VH-3 0.04 TATTCCCATGGCGCGCCCAGRTCACCTTGAAGGAGT 5 VH-4 0.04 TATTCCCATGGCGCGCCSAGGTGCAGCTGGTGGAG 6 VH-5 0.04 TATTCCCATGGCGCGCCCAGGTGCAGCTACAGCAGT 7 VH-6 0.04 TATTCCCATGGCGCGCCCAGSTGCAGCTGCAGGAGT 8 VH-7 0.04 TATTCCCATGGCGCGCCGARGTGCAGCTGGTGCAGT 9 VH-8 0.04 TATTCCCATGGCGCGCCCAGGTACAGCTGCAGCAGTC 10 LC set CK1 0.2 ATATATATGCGGCCGCTTATTAACACTCTCCCCTGTTG 11 VL-1 0.04 GGCGCGCCATGGGAATAGCTAGCCGACATCCAGWTGA 12 CCCAGTCT VL-2 0.04 GGCGCGCCATGGGAATAGCTAGCCGATGTTGTGATGA 13 CTCAGTCT VL-3 0.04 GGCGCGCCATGGGAATAGCTAGCCGAAATTGTGWTGA 14 CRCAGTCT VL-4 0.04 GGCGCGCCATGGGAATAGCTAGCCGATATTGTGATGA 15 CCCACACT VL-5 0.04 GGCGCGCCATGGGAATAGCTAGCCGAAACGACACTCA 16 CGCAGT VL-6 0.04 GGCGCGCCATGGGAATAGCTAGCCGAAATTGTGCTGA 17 CTCAGTCT W = A/T, R = A/G, S = G/C

[0137] For the nested PCR step, 96-well PCR plates were prepared with the following mixture in each well to obtain the given final concentration: 1× FastStart buffer (Roche), dNTP mix (200 μM each), nested primer mix (see Table 3), Phusion DNA Polymerase (0.08 U, Finnzymes) and FastStart High Fidelity Enzyme blend (0.8 U; Roche). As template for the nested PCR, 1 μl was transferred from the multiplex overlap-extension PCR reactions. The nested PCR plates were subjected to the following PCR cycle: 35× (30 sec. at 95° C., 30 sec. at 60° C., 90 sec. at 72° C.), 600 sec. at 72° C.

[0138] Selected reactions were analyzed on a 1% agarose gel to verify the presence of an overlap-extension fragment of approximately 1070 bp.

[0139] The plates were stored at -20° C. until further processing of the PCR fragments.

TABLE-US-00003 TABLE 3 Nested primer set Final Primer Conc. SEQ name μM Sequence ID CK2 0.2 ACCGCCTCCACCGGCGGCCGCTTATTAACACTCTCCCCT 18 GTTGAAGCTCTT PJ 1-2 0.2 GGAGGCGCTCGAGACGGTGACCAGGGTGCC 19 PJ 3 0.2 GGAGGCGCTCGAGACGGTGACCATTGTCCC 20 PJ 4-5 0.2 GGAGGCGCTCGAGACGGTGACCAGGGTTCC 21 PJ 6 0.2 GGAGGCGCTCGAGACGGTGACCGTGGTCCC 22

d. Insertion of Cognate V_H and V_L Coding Pairs Into a Screening Vector

[0140] In order to identify antibodies with binding specificity to vaccinia virus particles, the V_H and V_L coding sequences were expressed either as Fabs or full-length antibodies. This involved insertion of the repertoire of V_H and V_L coding pairs into a screening vector and transformation into a host cell.

[0141] A two-step cloning procedure was employed for generation of a repertoire of screening vectors containing the V_H and V_L coding pairs. Statistically, if the repertoire of screening vectors contain ten times as many recombinant plasmids as the number of cognate paired V_H and V_L PCR products used for generation of the screening repertoire, there is 99% likelihood that all unique gene pairs are represented. Thus, if 400 overlap-extension V-gene fragments were obtained in section c) a repertoire of at least 4000 clones was generated for screening.

[0142] Briefly, the repertoire of linked V_H and V_L coding pairs from the nested PCR in section c) were pooled (without mixing pairs from different donors). The PCR fragments were cleaved with XhoI and NotI DNA endonucleases at the recognition sites introduced into the termini of PCR products. The cleaved and purified fragments were ligated into an XhoI/NotI digested Fab-expression vector by standard ligation procedures. Suitable vectors were for example the bacterial or mammalian expression vectors illustrated in FIG. 3. The ligation mix was electroporated into E. coli and added to 2xYT plates containing the appropriated antibiotic and incubated at 37° C. over night. The amplified repertoire of vectors was purified from cells recovered from the plates using standard DNA purification methods (Qiagen). The plasmids were prepared for insertion of promoter-leader fragments by cleavage using AscI and NheI endonucleases. The restriction sites for these enzymes were located between the V_H and V_L coding gene pairs. Following purification of the vector, an AscI-NheI digested bi-directional promoter-leader fragment was inserted into the AscI and NheI restriction sites by standard ligation procedures. The ligated vector was amplified in E. coli and the plasmid was purified using standard methods. Where a bacterial screening vector was used the bi-directional promoters were bacterial promoters, and where the mammalian screening vector was used the bi-directional promoters were mammalian promoters. The generated repertoire of screening vectors was transformed into E. coli for by conventional procedures. Colonies obtained were consolidated into 384-well master plates and stored. The number of arrayed colonies exceeded the number of input PCR products by at least 3 fold, thus giving 95% percent likelihood for presence of all unique V-gene pairs obtained in section c).

e. Screening

[0143] The screening strategy is presented in FIG. 6. Fab expression was performed by inoculation of the colonies from the master plate into a 384-well plate. In cases where Fab expression was performed from E. coli the plates contained 0.9 ml 2xYT, 0.1% glucose, 50 μg/ml Carbencilin and the colonies were incubated with vigorous shaking at 37° C. until the cell density detected as OD600 reached ˜1. The Fab expression was induced by addition of 0.1 ml 2xYT, 0.1 M IPTG, 50 μg/ml Carbenicillin and the temperature decreased to 30° C. The Fab-containing supernatants were cleared by centrifugation and stored for screening reactions. The Fab-containing supernatants were cleared by centrifugation and stored for screening reactions. In cases where Fab expression was performed from mammalian vectors, DNA for transfection was prepared from the E. coli master plates. CHO cells were seeded into 384-well cell culture plates (3000 cells per well) in F12-HAM medium with 10% fetal calf serum (FCS) and after an overnight incubation the cells were transfected with the DNA using Fugene 6 as transfection agent. After 2-3 days in cell culture the Fab-containing supernatants were harvested and stored for screening reactions.

[0144] Screening of individual clones was performed using a fluorescence-linked immunosorbent assay (FLISA) based on the fluorometric microvolume assay technology (FMAT) (Swartzman et al. 1999, Anal. Biochem. 271:143-151). Briefly, inactivated virus particles of the Lister strain, IHD-W strain, and the recombinant protein antigens B5R, VCP and A33R were immobilized individually on polystyrene beads (6.79 μm diameter, Spherotech Inc.) by incubating 16.5 μg protein or 20 μg virus particles with 100 μL 5% w/v beads. The supernatant containing Fab-fragments were screened against all five populations of coated beads. The coated beads, a secondary fluorescently-labeled anti-human antibody (Alexa Dye 647, Molecular Probes) and supernatant containing Fab-fragments were mixed in 384-well plates. Wells containing Fabs with reactivity against the coated antigen resulted in an increased fluorescence at the bead surface, which were detected using FLISA reader (8200 Cellular Detection System; Applied Biosystems). In principle, the assay is equivalent to ELISA but since no washing steps are included, the procedure has a high throughput. Cut-off was set at as low as 50 detected fluorescent beads (counts) during the primary screen in order to minimize losses and to identify as many clones reactive with viral particles or antigen as possible. From the original master plates, the primary hits were retrieved and collected in wells of 96-well plates. The generated primary hit plates were handled as master plates, including storage and re-expression of these primary hits. These re-expressed Fab molecules were tested in a secondary screening using the same antigens in both FLISA and standard ELISA. Clones which produced Fabs with reactivity in both FLISA and ELISA in the secondary screen were submitted for DNA sequencing of the V-gene region.

[0145] The obtained sequences were aligned based on the amino acid sequence of the CDR3 region and grouped into clusters of clones expressing identical Fabs, some of the clusters only contained a single clone (a singleton). Large scale batches of Fab-fragments of representative clones from each cluster were prepared for validation of the anti-vaccinia virus reactivity. These studies consisted of binding analyses by ELISA using inactivated IHD-W, inactivated IHD-J, inactivated Lister strain VV particles, and the recombinant antigens, A27L, L1R, B5R, VCP, and A33R. Clones producing Fab-fragments which were positive to an inactivated vaccinia virus strain and/or one of the recombinant antigens were termed validated

f. Transfer of Selected Clones to Mammalian Expression Vector

[0146] When using a multiplex PCR approach as described in section c), a certain degree of intra-V-gene family cross-priming and inter-V-gene family cross-priming is expected due to the high degree of homology. The cross-priming introduces non-natural occurring amino acids into the sequences with several potential consequences e.g. structural changes and increased immunogenicity, all resulting in a decreased therapeutic activity.

[0147] In order to eliminate these drawbacks and to ensure that selected clones mirror the natural humeral immune response such cross-priming mutations were corrected during the transfer of the V_H and V_L coding pairs (in the form of the complete light chain linked to the variable heavy chain) from the screening vector to the mammalian expression vector. Either all clones, mutations or not, or just the clones with mutations were subjected to the two-step transfer procedure.

[0148] In the first step of the repair transfer procedure, the V_H sequence was PCR amplified with a primer set containing the sequence corresponding to the originating V_H-gene family thereby reverse mutating any cross-priming introduced changes. The PCR fragment was digested with XhoI and AscI and ligated into the XhoI/AscI digested mammalian expression vector (FIG. 3) using conventional ligation procedures. The ligated vector was amplified in E. coli and the plasmid was purified using standard methods. The V_H sequence was sequenced to verify the correction. The vector was digested with NheI/NotI, to prepare it for insertion of the light chain.

[0149] In the second step the complete light chain was PCR amplified with a primer set containing the sequence corresponding to the originating V_L-gene thereby reverse mutating any cross-priming changes. The PCR fragment was digested with NheI/NotI and ligated into the V_H containing vector prepared above. The ligation product was amplified in E. coli and the plasmid was purified. The light chain was sequenced to verify the correction.

[0150] If clones did not need correction of the V_H and V_L coding pair, they could optionally be transferred directly as a pair in a single step using the XhoI/NheI restriction sites in a conventional cloning procedure. In that event a promoter change was necessary if the screening vector was a bacterial vector. This was performed as described in section d). If the transfer was performed from a mammalian screening vector no promoter exchange was necessary.

g. Generation of a Polyclonal Cell Line

[0151] The generation of a polyclonal expression cell line producing a recombinant polyclonal antibody is a multi-step procedure involving the generation of individual expression cell lines (monoclonal cell lines) which each express a unique antibody. The polyclonal cell line is obtained by mixing the individual cell lines thereby generating a polyclonal master cell bank (pMCB) from which a polyclonal working cell bank (pWCB) can be generated simply by continuing amplification.

h. Transfection and Selection of Mammalian Cell Lines

[0152] The Flp-In CHO cell line (Invitrogen) was used as starting cell line. In order to obtain a more homogenous cell line the parental Flp-In CHO cell line was sub-cloned by limited dilution and several clones were selected and expanded. Based on growth behavior one clone, CHO-Flp-In (019), was selected as starting cell line. The CHO-Flp-In (019) cells were cultured as adherent cells in F12-HAM with 10% fetal calf serum (FCS).

[0153] The individual plasmid preparations each containing a selected V_H and V_L coding pair obtained in step 0), were co-transfected with Flp recombinase encoding plasmid into 5×10⁶ CHO-Flp-In (019) cell line using Fugene6 (Roche) (for further details see WO 04/061104) to generate approximately 10,000 independent recombination events for each transformation. The large-scale transformation procedure was applied as uniformly as possibly to ensure that identical expression cell populations were generated for each V_H and V_L coding pair. Cells were trypsinated after 24 hours and transferred to 3×T175 flasks. Recombinant cell lines were selected by culturing in the presence of 450 μg/ml Neomycin, which was added 48 hours after transfection. Approximately two weeks later clones appeared. Clones were counted and cells were trypsinated and hereafter cultured as pools of clones expressing one of the specific anti-VV antibodies.

i. Adaptation to Serum Free Suspension Culture

[0154] The individual adherent anti-VV antibody CHO-Flp-In (019) cell cultures were trypsinated, centrifuged and transferred to separate shaker flasks with 8×10⁵ cells/ml in appropriate serum free medium (Excell 302, JRH Biosciences). Growth and cell morphology were followed over several weeks. After 4-6 weeks the cell lines usually showed good and stable growth behavior with doubling times below 32 hours and the adapted individual cell line was cryopreserved as described above.

j. Characterization of Cell Lines

[0155] All the individual cell lines were characterized with respect to antibody production and proliferation. This was performed with the following assays:

Production:

[0156] The production of recombinant antibodies of the individual expression cell lines were followed during the adaptation by Kappa specific ELISA. ELISA plates were coated overnight with goat-anti-human Kappa antibodies (Caltag) in carbonate buffer, pH 9.6. Plates were washed 6 times with washing buffer (PBS; 0.05% Tween 20) and blocked by incubation for 1 hour in washing buffer containing 2% non fat milk. Cell culture media supernatants were added and the incubated extended for 1 hour. Plates were washed 6 times in washing buffer and secondary antibodies (goat-anti-human IgG (H+L) HRPO, Caltag) were added and the incubation repeated. After vigorous washing the ELISA was developed with TMB substrate and reaction stopped by addition of H₂SO₄. Plates were read at 450 nm.

[0157] Further, intracellular staining was used to determine the general expression level as well as to determine the homogeneity of the cell population in relation to expression of recombinant antibody. 5×10⁵ cells were washed in cold FACS buffer (PBS; 2% FCS) before fixation by incubation in CellFix (BD-Biosciences) for 20 minutes and hereafter washed in saponin buffer (PBS; 0.2% Saponin). The suspension was centrifuged and fluorescently tagged antibody (Goat F(ab')₂ Fragment, Anti-human IgG(H+L)-PE, Beckman Coulter) was added. After 20 minutes on ice the cells were washed twice in saponin buffer and suspended in FACS buffer and analyzed by FACS.

Proliferation:

[0158] Aliquots of the cell suspensions were taken three times a week and cell number, cell size, degree of clumping, and viability was determined by CASY® (Cell Counter+Analyzer System from Scharfe System GmbH) analysis. The doubling time for the cell cultures was calculated by cell number derived form CASY® measurements.

k. Characterization of the Antigen Specificity of the Individual Antibodies

[0159] The antigen specificity of the individual expressed antibodies was assessed in order to allow the generation of an anti-VV rpAb with a well-characterized specificity. As already described in section e) the antibodies identified during screening were validated by assessing their binding specificity to inactivated vaccinia virus strains and recombinantly produced A27L, L1R, B5R, VCP, and A33R antigens. These analyses were repeated using the full-length antibodies and additional analysis of the antigen specificity was performed by Western blotting.

[0160] The vaccinia virus particle associated proteins (antigens) were separated by SDS-PAGE, using acetone precipitated virus particles which subsequently were dissolved in SDS-loading buffer containing 8 M urea and run on a NuPAGE Bis-Tris 4-12% gel or NuPAGE Bis-Tris 10%. This resulted in a clear separation of the vaccinia virus particle associated proteins when visualized by Coomassie blue. For antigen binding analysis, the vaccinia virus particle associated proteins were separated by SDS-PAGE and electroblotted onto a PVDF membrane and purified preparations of the individual antibodies were analyzed by Western blotting. FIG. 5 shows Western blot analyses identifying a series of antigens derived from the Lister strain detected by different antibodies.

[0161] Putative target proteins were expressed by in vitro translation and tested for interaction with the selected antibodies by ELISA. The antigen genes were generated by a gene specific PCR using vaccinia virus, lister strain DNA as template (Autogenbioclear, UK Cat. No.: 08-940-250) followed by a second PCR step for addition of T7 promoter and Poly-A sequences. The Phusion DNA polymerase (Finnzymes, F cat. No: F530L) gene specific PCR reactions consisted of total volume at 50 μl containing 0.2 μM of 5' and 3' gene specific oligo pairs as indicated in Table 4, 1× Phusion HF reaction, 0.2 μM of each dNTP and 0.5 μl vaccinia virus DNA, 1 U Phusion DNA polymerase and were cycled through, 1×98° C. 30 seconds, 25×(98° C. for 10 seconds, 50° C. for 15 seconds 72° C. 20 seconds), 1×72° C. 7 minutes. The T7-Kozak translational initiation and Poly-A sequence were added by a second PCR reaction identical to the above described except for that T7-Koz. Poly-A (Table 4) primers and 0.5 μl of the 1 step PCR reaction were used as template. The PCR products were in vitro translated using the TNT T7 Quick for PCR DNA kit (Promega, USA, Cat. No.: L1170) according to manufacturers procedure. In detail each reaction consists of 40 μl TNT T7 Quick Master Mix, 1 μl mM Methionine 1, 1 μl Transcend Biotin-Lysyl-tRNA (Promega, Cat. No. L5061), 2 μl PCR generated DNA and incubated for 75 minutes at 30° C.

[0162] The in vitro translated antigens were tested for interactions with purified antibodies by ELISA performed in standard ELISA. The purified antibodies were immobilized in an ELISA plate and incubated with dilutions of the in vitro translated antigens. Retained antigen was detected by Streptavidin Peroxidase Polymer (Sigma Cat. No. S-2438) according to standard ELISA procedure similar to the description in Example 1, section g).

TABLE-US-00004 TABLE 4 Primers for generation of in vitro translational antigen genes Primer SEQ Name Sequence ID H3L-5' GGGAACAGCCACCATGGCGGCGGTGAAAACTCCTGTTA 23 H3L-3' GGATCCTCTAGATCATTAAAATGAAATCAGTGGAGTAGTAAAC 24 A56R-5' GGGAACAGCCACCATGACATGACACCTTTTCCTCAGACATCT 25 A56R-3' GGATCCTCTAGATCATTAAGAGGTTGTACTACTACCTAC 26 B5R-5' GGGAACAGCCACCATGACATGTACTGTACCCACTATGA 27 B5R-3' GGATCCTCTAGATCATTATAACGATTCTATTTCTTG 28 Poly-A TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGATCCTCTAGATCATTA 29 T7-Koz. GGATCCTAATACGACTCACTATAGGGAACAGCCACCATG 30 A10L-5' GGGAACAGCCACCATGATGCCTATTAAGTCAATAG 31 A10L-3 GGATCCTCTAGATCATTATTCATCATCAAAAGAG 32 A3L-5' GGGAACAGCCACCATGGAAGCCGTGGTCAATAGCGA 33 A3L-3' GGATCCTCTAGATCATTAAAATAGTTCTGTAATATGTCTA 34 A13L-5' GGGAACAGCCACCATGATTGGTATTCTTTTGTTGAT 35 A13L-3' GGATCCTCTAGATCATTATACAGAAGATTTAACTAGAT 36 D8L-5' GGGAACAGCCACCATGCCGCAACAACTATCTCCT 37 D8L-3' GGATCCTCTAGATCATTATTTATTCCCTTCGATATATTTTTGA 38 A11L-5' GGGAACAGCCACCATGACGACCGTACCAGTGACG 39 A11L-3' GGATCCTCTAGATCATTAAATAATTTTAATTCGTTTAA 40 A17L-5' GGGAACAGCCACCATGAGTTATTTAAGATATTACAAT 41 A17L-3' GGATCCTCTAGATCATTAATAATCGTCAGTATTTAAACT 42 L5R-5' GGGAACAGCCACCATGGAGAATGTTCCTAATGTA 43 L5R-3' GGATCCTCTAGATTATCATCTGCGAAGAACATCGTTA 44 F13L-5' GGGAACAGCCACCATGTGGCCATTTGCATCGGTA 45 F13L-3' GGATCCTCTAGATCATTAAATTTTTAACGATTTACTGT 46 A16L-5' GGGAACAGCCACCATGATGGGGGCAGCTGTTACTCTT 47 A16L-3' GGATCCTCTAGATCATTAAGGCAGTTTTATTTTATCTTTTA 48

l. Characterization of the Biochemical Properties the Individual Antibodies

[0163] Heterogeneity is a common phenomenon in antibodies and recombinant proteins.

[0164] Antibody modifications can occur during expression, through unfavorable storage conditions, and may cause size or charge heterogeneity. Common modifications include N-glycosylation, methionine oxidation, proteolytic fragmentation, and deamidation. Since these parameters need to be well-defined for therapeutic antibodies, they are analyzed prior to the generation of the polyclonal cell line.

[0165] The antibodies expressed during adaptation were purified by affinity chromatography (Protein-A columns) with low pH elution, and used for characterization of the biochemical properties of each individual antibody. The methods used for characterization included reducing and non-reducing SDS-PAGE and weak cation exchange chromatography (IEX). Well-defined heavy and light chain bands in the reducing SDS-PAGE indicated intact antibodies. SDS-PAGE analysis of antibody preparations resulting in well-defined bands, were expected to exert a single peak behavior in IEX analysis, indicating a homogeneously antibody preparation. Antibody preparations resulting in multiple peaks in the IEX analysis and/or aberrant migration of either the light or heavy chain in SDS gels were analyzed in detail for intact N-termini by N-terminal sequencing, as well as for the presence of additional N-glycosylation sites in the variable chains using enzymatic treatment.

m. Establishment of a Polyclonal Cell Line for Anti-VV Recombinant Polyclonal Antibody Production

[0166] Out of the pool of established expression cell lines a subset were selected to constitute the polyclonal expression cell line (pMCB). The selection parameters can be defined according to the use of the polyclonal antibody to be produced from the polyclonal cell line and the performance of the individual cell lines. Generally the following parameters were considered: [0167] Cell line characteristics; to optimize the stability of the polyclonal cell line, individual cell lines with doubling times between 21 and 34 hours and antibody productivity above 1 μg/cell/day. [0168] Antigen reactivity; which antigens should the anti-VV rpAb exert reactivity against, IMV, EEV and/or RCA derived proteins? [0169] Protein chemistry; generally antibodies with well-defined biochemical characteristics were included in the final anti-VV rpAb.

[0170] The selected individual cell lines each expressing a recombinant anti-VV antibody were thawed and expanded at 37° C. in serum free medium in shaker flasks to reach at least 4×10⁸ cells of each clone having a population doubling time of 21-34 hours. The viabilities were in the range of 93% to 96%. The polyclonal cell line was prepared by mixing 2×10⁶ cells from each cell line. The polyclonal cell line was distributed into freeze ampoules containing 5.6×10⁷ cells and cryopreserved. This collection of vials with a polyclonal cell line is termed the polyclonal master cell bank (pMCB) from which the polyclonal working cell bank (pWCB) was generated by expanding one ampoule from the pMCB to reach a sufficient number of cells to lay down a polyclonal working cell bank (pWCB) of approximately 200 ampoules with the same cell density as the ampoules of the pMCB. Samples in the cell banks were tested for mycoplasma and sterility.

n. Expression of a Recombinant Polyclonal Anti-VV Antibody

[0171] Recombinant polyclonal anti-VV antibody batches were produced in 5 liter bioreactors (B. Braun Biotech International, Melsungen, Germany). Briefly, vials from the pWCB were thawed and expanded in shaker flasks (Coming). Cells in seed train were cultured in ExCell 302 medium with G418 and with anti-clumping agent at 37° C., 5% CO₂. The bioreactors were inoculated with 0.6×10⁶ cells/ml suspended in 3 liter ExCell 302 medium without G418 and without anti-clumping agent. The cell numbers/viable cells were daily monitored by CASY counting. At 50 hours 2000 ml ExCell 302 medium was supplemented and after 92 hours a temperature downshift from 37° C. to 32° C. was performed. The cell culture supernatant was harvested after 164 hours and subjected to purification as described in section o).

o. Purification of Anti-VV rpAb

[0172] The antibody expressed as described in section n) was of the IgG1 isotype and affinity purified (Protein-A). The individual antibodies interacted with immobilized Protein A at pH 7.4, whereas contaminating proteins were washed from the column. The bound antibodies were subsequently eluted from the column by lowering of the pH to 2.7. The fractions containing antibodies, determined from absorbance measurements at 280 nm, were pooled and dialyzed against 5 mM sodium acetate, 150 mM NaCl, pH 5 and long time stored at -20° C.

p. In Vitro Neutralization Assays

Preparation of Vaccinia Virus for Use in Vivo and In Vitro

[0173] Lister, NYCBOH, and IHD-J vaccinia virus at 10⁴ pfu/ml was added to 10×175 cm² flasks of sub-confluent BSC-1 cells in 5 ml of serum-free Gibco Earle's Minimal Essential Medium (MEM). The virus was allowed to adsorb for 45 minutes at 37° C. 30 ml of MEM containing 2% (v/v) Gibco foetal calf serum (FCS) was then added to each flask before returning to 37° C. When the infected cells showed full cytopathic effect, the medium was discarded. The cells were detached from the flasks, by tapping, into 25 ml of PBS. This was centrifuged at 3,000 rpm for 10 minutes at 8° C. The virus containing cell pellet was re-suspended in 6 ml of PBS and frozen at -80° C. Once thawed, the cell debris were removed by centrifugation, 3,000 rpm for 10 minutes at 8° C. Aliquots of the supernatant were frozen at -80° C. Virus was titred by plaque assay.

Plaque Reduction and Neutralisation Assays (PRNT)

[0174] The test substances were diluted in serum free MEM and pre-incubated for 1 hour at 37° C. with 10⁴ pfu of Lister strain virus particles. The mixture was applied to a monolayer of Vero cells pre-seeded in 24-well plates. The infected cells were overlayed by addition of 2× MEM including 2% carboxymethylcellulose followed by incubation at 37° C.; 5% CO2 for 3 days. The cells were fixated by incubation with PBS; 10% formalin and stained using 20% Ethanol containing 0.1% crystal violet and the number of plaques in each well was recorded by counting.

[0175] The EEV-neutralization assay was performed using a similar protocol except for that IHD-J strain was used and the carboxymethylcellulose overlay was omitted. The IHD-J/Mini-H mixture was pre-incubated with the cells before addition of the Mini-V dilutions and thereby only detection a Mini-V effect on the EEV mediated virus spreading.

q. In Vivo Protection Assays

[0176] The mouse tail lesion model was used to analyze the in-vivo protection conferred by an anti-VV antibody composition. The experiments were performed at the Health Protection Agency, Porton Down, UK ("HPA"). In brief, the mice were challenged by injection of infectious vaccinia virus particles of either the Lister or the NYCBOH strain into the tail vein of the mouse. The used amount of virus results in a countable number of virus induced lesions on the tail within seven days. Twenty-four hours after or prior to viral challenge, increasing amounts of test antibody compound Mini-V, SymVIG, Sym002 anti-V rp-Ab or an unrelated polyclonal antibody was injected intraperitoneally (I.P.). or intramuscular (LM). In some of the experiments a total of 2.5 mg Cidofovir was injected intramuscularly (1.M) as a positive control for viral inhibition.

r. Composition of Sym002 Mix

[0177] Antibodies for Sym002 mix was purified individually as described in Example 1, section o, and subsequently mixed. The final antibody concentration of Sym002 mix was 1.09 mg/ml and was produced mixing 0.2 mg of each of the antibodies 02-029, 02-058, 02-086, 02-113, 02-147, 02-186, 02-188, 02-195, 02-197, 02-211, 02-225, 02-229, 02-235, 02-286, 02-295, 02-303, 02-339, 02-461, 02-482, 02-488, 02-526, 02-551, 02-586, 02-589, 02-607, 02-633, 0.574 mg of 02-037 and 0.171 mg of 02-203. The antibody identity is given in Table 5.

s. Affinity Measurements of Antibody-Antigen Interactions

[0178] Affinities of antibody-antigen interactions were measured by surface plasmon resonance using a Biacore 2000 (Biacore AB). The antigen (B5R, VCP, A33R, or A27L) was immobilized on a CM5 chip surface using standard amine coupling chemistry to a level resulting in a RU_max of approximately 100 RU or less. Purified antibody Fab fragments (see section 0) were diluted serially in HBS-EP running-buffer (Biacore AB) and passed over the chip at 10-50 μl/min. Rate constants (k_on and k_off) and affinity constants (K_D) were determined using the BIAevaluation software (Biacore AB) by global fitting of four to six different concentrations passed over the same sensor surface.

t. Preparation of Fab Fragment by Papain Digestion of Purified Antibodies

[0179] Fab fragments were produced from purified antibodies by using the ImmunoPure Fab preparation Kit (Pierce, USA cat. No.: 44885) according to manufactures instructions. Briefly, 1.2 mg of the antibodies subject for Papain cleavage were dialyzed against 20 mM sodium phosphate, 10 mM EDTA, pH 7 at 4° C. The dialyzed antibody solution were added to 250 μl gel immobilized Papain, pre-equilibrated and suspended in 20 mM Cysteine/HCl, 20 mM sodium phosphate pH 7. The reaction were incubated at 37° C. with shaking over night, followed by centrifugation for removal of the gel immobilized Papain. Fab fragments were purified by passing the supernatants through protein A columns. The eluates were dialyzed against PBS and up-concentrated by using Spectrum gel/absorbent (Spectrum Laboratories, Cat. No.: 292600). The Fab fragments were analyzed by SDS-PAGE and the concentrations were determined by OD₂₈₀ measurements.

Example 2

[0180] In the present Example the isolation, screening, selection and banking of clones containing cognate V_H and V_L pairs expressed as Fabs or antibodies with anti-vaccinia virus specificity was illustrated.

Donors

[0181] Twelve donors were recruited from a smallpox vaccination program of British first line responders in collaboration with the Health Protection Agency (HPA), United Kingdom. The donors included both primary and secondary vaccinia virus-immunized individuals. Blood was withdrawn in range of 9 to 21 days after vaccination. The B cell fraction was recovered by anti-CD19 MACS column purification and the sub-population of plasma blasts identified by a CD38high and CD45intermediate cell marker expression profile and single-cell sorted by FACS into 96-well plates as described in section a) of Example 1. The percentages of plasma blasts expressing anti-vaccinia virus antibodies were estimated by ELISpot (Example 1, section b). From 0 to 0.6% of the total plasma cells were specific, and plasma cells from the top five donors (0.3-0.6%) were used to isolate cognate V_H and V_L coding pairs. All the selected donors belonged to the group of secondary immunized donors.

Isolation of Cognate V_H and V_L Coding Pairs

[0182] The nucleic acids encoding the antibody repertoires were isolated from the single cell-sorted plasma cells by multiplex overlap-extension RT-PCR (Example 1, section c). The multiplex overlap-extension RT-PCR creates a physical link between the heavy chain variable region gene fragment (V_H) and the full-length light chain (LC). The protocol was designed to amplify antibody genes of all V_H-gene families and the kappa light chain, by using two primer sets, one for V_H amplification and one for the LC amplification. Following the reverse transcription and multiplex overlap-extension PCR, the linked sequences were subjected to a second PCR amplification with a nested primer set.

[0183] Each donor was processed individually, and 400 to 1200 overlap products were generated by the multiplex overlap-extension RT-PCR. The generated collection of cognate linked V_H and V_L coding pairs from each donor were pooled and inserted into a Fab expression vector as described in Example 1 section d). The generated repertoires were transformed into E. coli, and consolidated into ten 384-well master plates and stored.

Screening

[0184] Fab-fragments were obtained starting from the master plates, and screened against inactivated IHD-W and Lister strain as well as against the individual recombinant antigen proteins A33R, VCP and B5R as described in Example 1, section e). Several hundred secondary hits were sequenced and aligned. The majority fell in clusters of two or more members, but there were also clones that only were isolated once, so-called singletons. Representative clones from each cluster and the singletons were subjected to validation studies as described in Example 1, section e).

[0185] A total of 89 clones passed the validation. These are summarized in Table 5. Each clone number specifies a particular V_H and V_L pair. The IGHV and IGKV gene family is indicated for each clone and specifies the frame work regions (FR) of the selected clones. The amino acid sequence of the complementarity determining regions (CDR) of an antibody or Fab-fragment expressed from each clone are shown, where CDRH1, CDRH2, CDRH3 indicate the CDR regions 1, 2 and 3 of the heavy chain and CDRL1, CDRL2 and CDRL3 indicate the CDR regions 1, 2 and 3 of the light chain (definitions according to Kabat et al. 1991, Sequences of Proteins of Immunological Interest, 5th edn. US Department of Health and Human Services, Public Health Service, NIH.). Finally, the antigen specificity of the antibody or Fab-fragment expressed from the V_H and V_L coding pair contained in the clones is indicated. The antigen specificity was either identified according to the binding properties analyzed during validation, by binding to in vitro translated antigens, or by Western blotting. The in vitro translated approach identified H3I, A56R, and D8L as target proteins. Un-identified antigens targeted by antibodies produced from the selected clones using Western blotting, were assigned a letter from B to L according to migration properties in SDS-PAGE (Example 1, section g. subparagraph "Characterization of the antigen specificity of the individual antibodies"). Clones indicated as reactive against antigen A in Table 5, were positive in ELISA and FLISA screens against the IHD-W and/or Lister strain, but did not interact with a particle associated antigen detectable by Western blotting. Where no particular antigen is indicated, no further analysis with respect to binding reactivity has been performed, antibodies expressed from these clones are however reactive against the IHD-W and/or Lister strain.

[0186] The complete variable heavy and light chain sequence can be established from the information in Table 5. The only exception is clone 02-291, since this clone carry an 11-codon insertion in the so-called hyper variable region 4 (framework region 3) of the heavy chain. For this reason the complete heavy chain amino acid sequence is given here for this particular clone:

TABLE-US-00005 (SEQ ID NO: 49) QVQLVQSGAEVKKPGYSVKVSCQASGLTFSNYRVSWVRQAPGQGLEWMGG IIPIFGTTNYAQKFQGRVTISADRSPSRVTSLADKSTITVYMELSSLRSE DTAVYYCARSRGSQDYYGMDVWGQGTTVTVSS.

TABLE-US-00006 TABLE 5 CDRH1 CDRH2 CDRH3 IGHV 3 3 5 6 9 0 0 Clone gene 1abc2345 # 012abc3456789012345 # 234567890abcdefghijklmno123 # 02 029 1-24 E---LAMH 50 GFDP--EDGEAVYAQGFQG 139 CATDVWRRTPEGGTDWF-------DPW 228 02-031 4-34 G---FHWS 51 EIN---HSGSTNYNPSVKS 140 CAGSRSFDLLTAYDLFHRKGNAM-DVW 229 02-037 6-1 NNI-ASWN 52 RTYYR-SKWYDDFALSVKG 141 CARGDVLRYF--------------DYW 230 02-058 3-30 R---YGMH 53 FLSF--DERNKFYPDSLKG 142 CAKGGLGTNEF-------------DHW 231 02-086 5-51 T---YWIG 54 IIYP--GDSDTKYSPSFQG 143 CATLPRYDAYGARIR---------DYW 232 02-089 1-2 G---HYMH 55 WINP--SSGGTNYAQKFQG 144 CARYCSSPTCS-------------IVW 233 02-112 2-5 TSG-VGVD 56 LIY---WDDDKRYSPSLKS 145 CAHSSQRVVTGL------------DFW 234 02-113 2-70 TSG-MCVS 57 FID---WDDDKYYTTSLKT 146 CARIRTCYPDLYGDYNDAF-----DIW 235 02-147 3-7 K---YWMS 58 NINQ--EGSAKHYVDSVKG 147 CARAADYGDY--------------VRP 236 02-156 3-30 S---YGMH 59 LISY--HGNTKYYADSVKG 148 CAKHVAAGGTL-------------DYW 237 02-159 1-46 N---YYIH 60 IINP--SGGSTTYAQRFQG 149 CARVIRKYYTSSNSYLTEQAF---DIW 238 02-160* 3-9 D---YAMH 61 GCSW--NSGFISYADSVKG 150 CVKETVAGRRGAF-----------DYW 239 02-166 4-34 G---YYWT 62 EIN---HSANTDYKPSLKS 151 CARGREWPSNF-------------DSW 240 02-169 5-51 T---YWIG 63 IIYP--GDSDTRYSPSFQG 152 CARRGSTYYY--------------DTW 241 02-172 3-21 S---YTMN 64 AITS--STTYIYYADSVKG 153 CASKPYGGDFG-------------SYW 242 02-183 5-51 K---YWIG 65 IIYP--EDGDTRYSPAFQG 154 CARPPSNWDESF------------DIW 243 02-186 1-3 N---YAVH 66 WINV--GNGQTKFSQRFQG 155 CARDPTQWLLQGDVYDM-------DVW 244 02-188 4-30-4 SGD-YYWN 67 NIY---STGSTYYDPSDQN 156 CAREAWLGEPLLLGDDAF------DIW 245 02-189 3-30 K---YYMH 68 TISY--DVKNKDYADSVKG 157 CARDGAGEWDLLMRRDF-------DYW 246 02-195 1-18 T---YGIS 69 WISA--WDGNTKYGEKFQD 158 CARDPARRPRSGYSVF--------EYW 247 02-197 4-4 SN--NWWS 70 EIY---HSGNTNYNPSLQS 159 CARDNRQSSSWVEGFFYYYGM---DVW 248 02-201 1-46 K---YYIH 71 MINP--SGGTTTYAQKFQG 160 CARLRLGATIGRD-----------DYW 249 02-203 4-30-4 RGD-FYWS 72 YIY---YTGSTYYNPSLKS 161 CARDRASSGYDSRVWF--------DPW 250 02-205 3-30 D---YTTH 73 IVLY--DGKNKNYADSVRG 162 CARTYRVYAKFDPF----------DVW 251 02-211 3-9 D---YAMH 74 GISW--NSEYIGYEDSVKG 163 CGKDGVPGRRGYI-----------EDW 252 02-214* 3-9 D---YAMH 75 TISW--NSGFIDYADSVKG 164 CVKDNIAGRRGSF-----------DSW 253 02-215 1-2 D---YYIH 76 WINP--NFGGTDYAQKFQG 165 CARDYIRATGATPSKYFIYYYGM-GVW 254 02-219 1-18 S---YAIA 77 WISA--YNGNTDYAQKFQG 166 CARARRVTNSPNNWF---------DPW 255 02-225 4-34 G---YYWG 78 EIN---HSGSANYHPSLKS 167 CARAGERSGSGSFVLGRF------DFW 256 02-229 3-49 D---YAVS 79 LIRSRHYGAKTQFAASVQG 168 CTNTSSLAVA--------------GNW 257 02-232 4-39 SRN-YYWG 80 TIY---YTGRTYYNPSLKN 169 CARIPQQRVNYF------------DYW 258 02-235 4-39 SSTNYYWG 81 TVY---LSGRAYYNPSLKS 170 CARLPGQRITFF------------DYW 259 02-237 6-1 TST-AAWN 82 RTYYR-SRWRNDYAGSVRS 171 CARGRRFEDDAF------------DIW 260 02-238* 3-9 D---YAMH 83 GINW--NSGNIVYADSVKG 172 CVKDSVAGRRGGF-----------DHW 261 02-242 1-f E---YYIH 84 LVDP--EDGEPIYAEKFQG 173 CATRDGDF----------------DHW 262 02-243 5-51 S---YWIS 85 IIYG--GDSDTKYSPSFQG 174 CVRHGTRYSFGRSDII--------DIW 263 02-246 5-51 S---YWIA 86 IIFP--GDSDTRYSPSFQG 175 CTKTPARGAYGDYIS---------GSW 264 02-250* 3-9 D---FAMH 87 GVSW--NSDVINYSDSVKG 176 CAKSTKAVRRGSF-----------DYW 265 02-267 1-69 D---YAIS 88 GIIP--VFGTPNYAQQFQG 177 CARGGELYEGNGYYSFHYF-----DYW 266 02-269 2-5 STG-VGVG 89 LIY---WDDEERYSPSLKN 178 CAHTELAF----------------DYW 267 02-271 1-69 S---YAIN 90 SIIP--IFATTNYAQRFQG 179 CARVKGTQNYYGM-----------DVW 268 02-274 4-34 G---YYWS 91 EVN---HSGSTNYNPSLRS 180 CHYYDSTGYYVS------------DFW 269 02-286 1-18 G---YGIS 92 WITY--DKGNTNHAQKFRG 181 CARGVVLIQTILF-----------DYW 270 02-290 1-69 N---SAIN 93 GVVP--IYDTSHYAQKFKG 182 CARTVLDSGAYSYY----------DSR 271 02-291 1-69 N---YRVS 94 GIIP--IFGTTNYAQKFQG 183 CARSRGSQDYYGM-----------DVW 272 02-294 3-23 N---YAMS 95 AISG--SGGKTYHAHSVRG 184 CAKLRDSSVYSAYVFRVIF-----DCW 273 02-295 3-11 D---NYMN 96 YISS--TSGSIYYADSVKG 185 CATLTVASTY--------------DYW 274 02-297 3-9 D---YAMH 97 GLNW--NGANIRYADSVKG 186 CVKDTVALLTSRGGCM--------DVW 275 02-302 2-5 TSG-VGVG 98 LIY---WDDDKRYSPSLKS 187 CAHSPPHGG---------------DYW 276 02-303 3-30 T---YGMH 99 FISS--DGSTKYYADSVKG 188 CAKGLSQALNYYGSGS--------PFL 277 02-335 3-30 N---YGIH 100 FISY--DGSKKYYVDSVKG 189 CAKDRGVSAWYPRDAF--------DIW 278 02-339 4-59 S---DNWS 101 YIY---KTGSTNYNPSLKS 190 CARVPLIEAGITIFAKIGAF----DIW 279 02-349.dagger-dbl. 5-51 S---FWIG 102 VTYP--GDSDTRYSPSFQG 191 CARGSPMIKFYF------------DYW 280 02-351 3-9 N---YAMY 103 GIIW--NSEYIGYADSVKG 192 CARATGAGRRNPL-----------DYW 281 02-431 1-46 S---YYMH 104 LINP--SSGTTSYAQNFQG 193 CARPYRSYSSSPQ-----------DYW 282 02-437 4-31 SPG-YYWN 105 YIY---YSGSTNYNPSLKS 194 CARYYYSSGPKF------------DYW 283 02-446 1-69 S---FAIS 106 SIIP--IFGTAHYAQRFEG 195 CARNNRPLGALFGM----------DVW 284 02-461 1-18 T---YGIS 107 GIRV--HNGNTNYAQKFQG 196 CARGGFNRLV--------------DPW 285 02-482 4-31 SAG-YYWS 108 YIH---YTGTTYYNPSLKS 197 CARNIGIYLGGSPGGIRNNWF---DPW 286 02-488 5-51 S---YWIG 109 IIYP--GDSDTRYSPSFQG 198 CARQQAKTLYYDSSGSKSAF----DIW 287 02-515 4-31 SGG-YYWS 110 YIH---YSGSTYYMPSLKS 199 CARVRGNIVATTAFYYYYGL----DAW 288 02-516 3-11 D---YYMS 111 YTNL--FTGYTNYADSVKG 200 CAKFDYGEGAYHF-----------DFW 289 02-517 4-31 GA--YHWS 112 YIY---YTGNTYFNPSLKS 201 CARDPIALPGRGVF----------DYW 290 02-520 4-34 A---YYWS 113 EIS---HSGSTHYNPSLNS 202 CSSGYYFAGGEF------------DYW 291 02-526 3-49 D---YTMS 114 FIRGKKFGGTKDYAASVKG 203 CTRDRGYSDHTGLYTRFGF-----DSW 292 02-532 1-69 D---HSIG 115 KIIP--IYGRANYAQKFQG 204 CARWRGGYSGYGDYF---------DSW 293 02-536 4-4 SS--HWWN 116 EIY---HSGSTNYNPSLKS 205 CARDPQKPRQHLWPNPYYYSGM--DVW 294 02-551 1-69 Y---YAIN 117 GIVP--MVGPADYAEKFRG 206 CARGRSWRGYL-------------DYW 295 02-559 1-46 N---YYMH 118 LINP--SGDSTTNAQKFQG 207 CARDYGDYCGGDCPYDAF------DIW 296 02-572 1-69 S---FGIS 119 GIIP--IFGTPNYSLKFQD 208 CARDKGESDINGWQTGAFYYGM--DVW 297 02-575 2-26 NDR-MGVS 120 HIF---SNDERSHSSSLKS 209 CARIDSVGWPSSHYYGM-------DVW 298 02-586 4-59 S---YYWS 121 YIF---YSGNTNYNPSLKS 210 CARDRITGYDSSGHAF--------DIW 299 02-589 4-34 G---YYWT 122 EIN---QNGRSNHNPSLKS 211 CARGGKFCGSTSCFTEGRL-----DYW 300 02-607 3-30 S---YGMH 123 VISY--DGRYKFYANSVKG 212 CAKDSGRYSSLGHYYYYGM-----DVW 301 02-611 5-51 N---YWIG 124 IIHP--GDSETRYSPSFQG 213 CARGYYYDTSGYRPGSF-------QHW 302 02-612 3-30 T---YTMH 125 VISY--DGTNKYHTDSVKG 214 CARPLFYGAGDAF-----------DIW 303 02-614 1-69 N---YAII 126 EIIP--KFGTANYAQKFQG 215 CADWVVGNYNGL------------DVW 304 02-617 1-46 N---YYVH 127 LINP--SAGKTTYAQRFQG 216 CAREGKHDFWRGYFSPLGM-----DVW 305 02-621 1-69 S---HGVN 128 GIIP--VFGTTNYAQSLQG 217 CATARNSSNWYEGHYYL-------AHW 306 02-626 4-30-4 TGD-YYWS 129 YVF---NSGSTYYNPSLQS 218 CANMVVVATQPKNWF---------DPW 307 02-628 4-30-4 SGY-YYWN 130 YID---YRGTTYYSPSFKS 219 CASYGSGMGSEYYF----------GHW 308 02-632 1-2 G---YYIH 131 RINP--ITDVTNYAQIFQG 220 CGRVGREAFYYYGM----------DVW 309

02-633 1-2 A---YYIH 132 RINP--DSGGTDFSQKFQG 221 CARASRRLTTHNYF----------DGW 310 02-634 3-48 T---YEMS 133 YIGS--GGVTIYYADSVKG 222 CARVRGGRYF--------------DYW 311 02-640 5-51 T---YWIA 134 IIWP--VDSDTRYSPSFQG 223 CASGSGYDSYYNM-----------DVW 312 02-643 7-4-1 S---YAMN 135 WINT--NTGNPTYAQGFTG 224 CARDSSTVTGLMTEYNWF------DPW 313 02-649 4-31 SGP-YYWS 136 YSS---NRGIAYYNPSLKS 225 CATEKGSGGDVGKF----------DNW 314 02-650 1-69 S---NPVS 137 GIIP--FAQKVLGAQRVRD 226 CATGQQLYSL--------------HYW 315 02-651 1-2 D---YYLH 138 RINP--KSGDTHHVQKFQG 227 CAREGPQFYYDSGDYYSAHSPGDFDHW 316 CDRL1 CDRL2 CDRL3 IGKV 2 3 3 5 8 9 Clone gene 45678901abcdefghi234 # 0123456 # 89012345a678 # 02-029 3-11 RASQSVRR---------SLA 317 DASNRAT 406 CLQRSNWP-ITF 495 02-031 1-39 RASQGISN---------SLN 318 GASGLES 407 CQQSYRTL-YTF 496 02-037 1-5 RASQSISI---------WLA 319 KASTLES 408 CQQYNGYSEVTF 497 02-058 1-5 RASQSIGN---------WLA 320 DASSLKS 409 CQQYDTYP-ITF 498 02-086 1D-33 QASQDISK---------YLN 321 DASNLET 410 CQQYDNLP-PTF 499 02-089 3-20 RASESVRSN--------YLA 322 GASSRAT 411 CQQYGRSP-LTF 500 02-112 1-39 RASQSIST---------YLN 323 AASSLQS 412 CQQSYNTP-ATF 501 02-113 1D-33 QASQDIKY---------YLN 324 DASNLET 413 CQQYENVP-YTF 502 02-147 2-28 RSSKSLLHSNGYN----YLD 325 LASNRAS 414 CMQALQIP-RTF 503 02-156 2-24 RSSESLVNNDGNT----YLS 326 KISNRFS 415 CMQTTHIP-HTF 504 02-159 1-39 RASQNISN---------FLL 327 AASSLQS 416 CQQTYGNP-LTF 505 02-160* 1-39 RASQSINN---------YLN 328 AVSSLQT 417 CQQSFRTP-HTF 506 02-166 3-11 RASQSVDR---------YLN 329 DASNRDT 418 CQQRAIWP-PEF 507 02-169 1-39 RASQSIWT---------FLN 330 TASSLQS 419 CQQSFTSW-WTF 508 02-172 1-5 RASQSISN---------WLA 331 KASNLES 420 CQQYSNYP-ITF 509 02-183 1-17 RASQDISN---------DLG 332 LASSLQS 421 CLQHNSF--LTF 510 02-186 2-30 RSSQSLVYSDGNT----YLH 333 KVSNRDS 422 CMQGTHWP-PAF 511 02-188 1-17 RASQGIGY---------DLG 334 AASSLQS 423 CLQLHTFP-RTF 512 02-189 1-39 RASQSISN---------YLS 335 AASLLQT 424 CQQGYSTP-YTF 513 02-195 3-15 RASQSVSS---------NLA 336 GASTRAT 425 CHQYNYWPPLAF 514 02-197 3-20 RASQSIASA--------YLA 337 GASSRPT 426 CQQYGISP-RTF 515 02-201 2-28 RSSQSLLHSNGYN----YLD 338 LGSTRAS 427 CMQALQTP-HTF 516 02-203 ID-33 QASHDVSN---------FLN 339 DASNLKT 428 CHQYDSLP-FTF 517 02-205 3-20 RASQSVSSN--------YIA 340 GASSRAT 429 CQQFGYSPRFTF 518 02-211 1-39 RASQSIRT---------YLN 341 AASSLQS 430 CQQTYITP-KSF 519 02-214* 1-39 RASQTIST---------YLN 342 AASSLQS 431 CQQSYRTP-LTF 520 02-215 1-27 RASQGISN---------YLA 343 GASTLQS 432 CQKYDSAP-YTF 521 02-219 1-39 RASRSIST---------YLN 344 AASSLQS 433 CQQTYTIP-LTF 522 02-225 1-39 RASQSIHT---------YLN 345 TASNLQS 434 CQQSYSTL-RTF 523 02-229 4-1 KSSQSVLYSSNNNN---YLA 346 WASTRES 435 CQQYYKTP-PTF 524 02-232 1-5 RASQTIST---------WLA 347 DASSLES 436 CQQYNSYP-LTF 525 02-235 1-5 RASQSIST---------WLA 348 DASSLES 437 CQQYNFY--GTF 526 02-237 1-39 RASQSISN---------YLN 349 GASSLES 438 CQQSYSIP-RTF 527 02-238* 1-39 RASLNIRN---------YLN 350 AASTLQI 439 CQQSYSMSPYTF 528 02-242 1-16 RASQVIGK---------YLA 351 ATSILQS 440 CQQYNSFP-LTF 529 02-243 1-17 RASQGIRN---------DLG 352 AASSLQS 441 CLQQNNYP-WTF 530 02-246 1D-33 QASHDINK---------YLN 353 DASNLET 442 CQQYDNFP-YTF 531 02-250* 1-39 RASQSINN---------YLN 354 AASSLHS 443 CQQTYIST-RTF 532 02-267 1D-33 QASQDISN---------YLN 355 DASHLET 444 CQQYDNLP--LF 533 02-269 1D-33 QASQDISF---------YLN 356 DASILET 445 CQQYDNLI--TF 534 02-271 1-5 RASQSISS---------WLA 357 KVSSLES 446 CQQYESDI-FTF 535 02-274 1-16 RASQDISN---------YLA 358 AASSLLS 447 CQQYGRYP-LTF 536 02-286 1-39 RASQSVST---------FLN 359 GVSNLQS 448 CQQSHRTP-YTF 537 02-290 1-5 RASQDVSP---------WLA 360 KASSLES 449 CQQYQTY--STF 538 02-291 1-5 RASQGISD---------WLA 361 KASSLES 450 CQQYESDS-WTF 539 02-294 1D-33 QASQDISN---------YLN 362 DTSNLET 451 CQQYDNLP-FTF 540 02-295 2-30 RSSQSVVYSDGNI----YLN 363 QVSNRDS 452 CMQGTHWP-YSF 541 02-297 1-12 RASQDISS---------WLA 364 AASSLQS 453 CQQAYSFP-WTF 542 02-302 1D-33 QASQDISN---------YLN 365 DASNLET 454 CQQYDNL--PTF 543 02-303 3-20 RASQSVSSL--------YVG 366 GTSSRAT 455 CQQYGTSP-WTF 544 02-335 1-39 RASQSISS---------FLN 367 GATTLQS 456 CHQSYSLP-FTF 545 02-339 3-20 RASQSVSS--------SYLA 368 RASSRAA 457 CQQYVASP-FTF 546 02-349.dagger-dbl. 3-20 RASQSVSSRASQSVSSNYLA 369 GASTRAA 458 CHQYGTSP-RTF 547 02-351 1-39 RASQTIRN---------YLN 370 TASSLHS 459 CQQSYITP-YTF 548 02-431 3-20 RASQSVSNN--------NLA 371 GASSRAA 460 CQQYGSSP-YTF 549 02-437 1-12 RASQGISN---------WLA 372 AASSLQS 461 CQQANSFP-FTF 550 02-446 1-9 RASQGIGG---------ALA 373 AASTLQS 462 CQQLDTYP-LTF 551 02-461 3-20 RASQSVSSN--------YLA 374 GASSRAT 463 CQQYASSP-YTF 552 02-482 1-39 RASQSISR---------HLN 375 AASSLQT 464 CQHSSKTP-FTF 553 02-488 1-5 RASQSIST---------YLA 376 KASSLEP 465 CQQYSSY--LSF 554 02-515 1-12 RASQGVSN---------WVA 377 AASSLQS 466 CQQANGFL-WTF 555 02-516 1D-12 RASQGIST---------FLA 378 AASSLQS 467 CQQAHSFP-VTF 556 02-517 3-20 RASQSVTSN--------YLA 379 GASNRAT 468 CQQYGSSP-LTF 557 02-520 1-12 RASQGIST---------WLA 380 AASTLQH 469 CQQANSFP-RTF 558 02-526 2-28 RSSQSLLHSNGYN----YLD 381 LGSNRAS 470 CMQSLQT--VTF 559 02-532 1-39 RASQSISN---------YLN 382 AASRLQS 471 CQHSYETPPYTF 560 02-536 1-5 RASQSLNN---------WLA 383 DASSLQS 472 CQQYNFYP-WTF 561 02-551 3-20 RASQSVSNN--------YLA 384 GASSRAT 473 CQQYGGSP-QTF 562 02-559 1-27 RASQGIFN---------YLA 385 GASTLRS 474 CQKYNSAP-LTF 563 02-572 1-12 RASQNIGN---------WLA 386 SASSLQN 475 CQQANSFP-VTF 564 02-575 1-12 RASQDIIS---------WLA 387 AASSLQS 476 CQQTHSFPPWTF 565 02-586 1-5 RASQSIYI---------WLA 388 DASSLES 477 CQQYHHYS-PTF 566 02-589 1-39 RASQSISR---------SLN 389 AASTLQS 478 CQQSYSTL-RTF 567 02-607 1-39 RASQPISS---------FLN 390 AASSLQS 479 CQQGYSTP-PTF 568 02-611 1-5 RASQSISS---------WLA 391 HAFSLEG 480 CQQYDSYP-YTF 569 02-612 1-39 RASQSFNG---------YLN 392 AASTLQS 481 CQQSYSTP-RTF 570 02-614 3-20 RASQTVIST--------YLA 393 GASSRAT 482 CQQYSDS--LTF 571 02-617 3-20 RASQSVSSG--------SLD 394 GASNRAS 483 CHQYGGAQ-GTF 572 02-621 1D-33 QASQDISN---------YLN 395 DASNLET 484 CQQYDTLPPITF 573 02-626 1-39 RASQSISS---------YLN 396 AASSLQS 485 CQQSHSSP-WTF 574 02-628 1-39 RASQSIRS---------YLN 397 GASSLQS 486 CQQSYLAP-WTF 575 02-632 1-9 RASQGISS---------YLV 398 AASTLES 487 CQQFNNYP-YTF 576 02-633 1-16 RASQAISN---------YLV 399 GAFILES 488 CQQYHTYP-FTF 577 02-634 3-20 RASQSVSST--------YLA 400 GASNRAT 489 CQKYGRSPTWTF 578 02-640 1-39 RASQSISN---------HLN 401 VASSLQG 490 CQQGFTTP-ITF 579 02-643 1-39 RASQSISS---------YLN 402 AASSLQS 491 CQQSYSTP-YTF 580 02-649 1-39 RANQSIDD---------YLH 403 DASTLHS 492 CQQSYTIPLWTF 581 02-650 2-30 RSSQSLVYADGDT----HLN 404 HVSNRDA 493 CMQGTHWP-PTF 582 02-651 1-39 RASQSITN---------CLN 405 GASTLQS 494 CQQSDSTP-YTF 583 IGHV and IGKV gene names were assigned according to the official HUGO/MGT nomenclature (IMGT; Lefranc & Lefranc, 2001, The Immunoglobulin FactsBook, Academic Press). Numbering and alignments are according to Chothia (Al-Lazikani et al. 1997 J. Mol. Biol. 273: 927-48). *These clones were isolated from different donors but utilize highly similar VDJ and VJ rearrangements. These two clones carry an 11-codon insertion in the so-called hypervariable region 4 (framework region 3). .dagger-dbl.This clone carries an eight-codon insertion in CDRL1.

Mirroring the Humeral Immune Response

[0187] In order to illustrate that the cognate V_H and V_L coding pairs isolated and selected as described above mirrors the natural humeral immune response raised upon challenge with vaccina virus, serum samples from ten of the twelve donors were tested for reactivity against three virus strains (Lister, IHD-W and IHD-J), and five recombinant antigens (B5R, VCP, A27L, A33R and L1R). The antibody titers were determined as the minimum sera dilution required for a four-fold background signal and the determined values are displayed in FIG. 7. The titers varied among the different antigens and donors, this is likely to reflect differences in immunogenicity, efficient up-take of the vaccinia virus, time from vaccination to blood withdrawal, primary and secondary response, etc. Comparison of the specific antibody titers from the donors and identified cognate Fabs/antibodies with the same reactivity, suggested that the antibody diversity recovered in the present experiment included the specificities present in the collected serum samples. The analysis thereby supports the claim that the identified anti-vaccinia virus repertoire reflects the specificity of the natural humeral immune response.

[0188] The vaccinia virus-specific clones also display a high diversity. All immunoglobulin variable region heavy chain gene families (IGHV), including IGHV7, as well the immunoglobulin kappa light chain gene families (IGKV) 1-4, were found among the validated clones. The V-gene families for each isolated and selected clone are indicated in Table 5. The heavy chain V-gene usage correlated with what has been found previously in human antibody repertoires, with a frequent usage of the IGHV3, IGHV1 and IGHV4 families. The light chain V-gene usage was dominated by the IGKV1 family, mainly due to the frequent usage of the IGKV1-39 gene. This gene is also among the most frequently used light chain V-genes in the human immunoglobulin repertoire. As a whole, the IGKV1 genes make up 75% of the repertoire. The only other IGKV gene of prominent usage in the isolated repertoire was the IGKV3-20, which also is the most frequently expressed light chain gene in humans. The IGKV families 5 and 6 were the only families not represented in the repertoire, IGKV5 and 6 are very rarely utilized in a human antibody response, or may not even consist of functional IGKV genes (de Wildt et al. 1999, J. Mol. Biol. 285:895-891; Lefranc & Lefranc 2001, The Immunoglobulin FactsBook, Academic Press). In conclusion, the diversity of the isolated repertoire is evidence of an unbiased recovery of V-genes by multiplex overlap-extension RT-PCR, indicating that the isolated antibodies minor the diversity of the humeral response in humans.

[0189] Further, due to the cognate pairing of the V_H and V_L coding sequences representing the V_H and V_L pairing originally present in the donor from which such a cell is derived, the affinity of the humeral immune response raised upon vaccination with vaccinia virus is also considered to be mirrored by the isolated antibodies.

Generation of a Cell Bank of Individual Antibody Expressing Members

[0190] A subset of 47 unique cognate VH and VL coding pairs corresponding to clone nr 02-029, 02-031, 02-037, 02-058, 02-086, 02-089, 02-112, 02-113, 02-147, 02-156, 02-159, 02-160, 02-169, 02-172, 02-186, 02-188, 02-195, 02-197, 02-201, 02-203, 02-205, 02-211, 02-225, 02-229, 02-232, 02-235, 02-243, 02-271, 02-286, 02-295, 02-297, 02-303, 02-339, 02-431, 02-461, 02-482, 02-488, 02-516, 02-520, 02-526, 02-551, 02-586, 02-589, 02-607, 02-628, 02-633 and 02-640 of Table 5 were selected for expression as complete antibodies. The VH and VL coding pairs were selected from the 89 validated antibodies, according to the Fab reactivity in ELISA, Western blotting, antigen diversity, and sequence diversity. The selected cognate V-gene pairs were transferred to a mammalian expression vector. The transfer process is described in Example 1 section f). The individual expression constructs were co-transfected with a Flp-recombinase expressing plasmid into the CHO-FlpIn recipient cell line (Invitrogen), followed by antibiotic selection of integrants. The transfections, selection, and adaptation to serum free culture was performed as described in Example 1, section g). The adaptation process was continued until the doubling time was below 32 hours. This was on average completed within a 4-6 week period; thereafter the individual cells lines were banked.

Example 3

[0191] In the present Example the biological activity of a mixture of monoclonal anti-VV antibodies was compared to a serum derived VIG product.

[0192] The first sixteen individual cell lines introduced into the bank generated in Example 2, were used to express monoclonal antibodies, which where purified, and characterized individually and mixed to generate the Mini-V preliminary antibody composition, a composition which was composed of a mixture of 16 monoclonal antibodies containing the cognate V_H and V_L pairs corresponding to clone nr 02-029, 02-031, 02-037, 02-058, 02-086, 02-089, 02-112, 02-113, 02-147, 02-156, 02-159, 02-160, 02-169, 02-172, 02-211 and 02-243 of Table 5 in Example 2. The Mini-V composition was produced to resemble a polyclonal antibody product, to verify the biological activity of such a product. In parallel, an oligoclonal composition was compiled consisting of three antibodies specific for IMV antigens as indicated by Western blotting. This oligoclonal antibody composition was termed Mini-H, and was composed of a mixture of 3 monoclonal antibodies corresponding to clone nr 02-31, 02-211 and 02-243 of Table 5.

[0193] The specific binding to inactivated Lister strain of the Mini-V and Mini-H compositions were compared to the serum derived antibodies of the five processed donors, which had been affinity purified using Protein-A columns (termed SyrnVIG) (FIG. 8). The direct binding analyses demonstrated at least a 100-fold increase in the specific binding activity of the recombinant antibody compositions.

[0194] The neutralizing activity of the two compositions was assayed in vitro by plaque reduction and neutralization assay as described in Example 1, section p). The anti-viral potency was indicated as the antibody concentration required for 50% reduction of viral infectivity detected as number of plaques caused by a vaccinia virus infection of a confluent monolayer of adhered cells (IC₅₀). Mini-V showed a 100-fold increased potency compared to SymVIG (Table 6). This correlated with the profound specific activity of Mini-V. Interestingly, Mini-H was 10-fold less potent than Mini-V, indicating superiority of a polyclonal antibody composition, including reactivity against both IMV- and EEV-particles.

TABLE-US-00007 TABLE 6 IMV- EEV- Antibody neutralization neutralization composition (μg/ml) (μg/ml) Mini-V 0.5 25 Mini-H 6.25 n.a. SymVIG 62.5 250

[0195] EEV-specific infection was detected in a plaque reduction assay as described in Example 1, section p) in the presence of 100 μg/ml of the Mini-H IMV-neutralizing antibody composition. The EEV-neutralization potency of Mini-V was only found to be 10-fold more profound than SymVIG (Table 6). The Mini-V composition only contains two EEV-specific antibodies. This is most likely the reason that the mini-V composition has a lower anti-EEV than anti-IMV activity, and this suggests that an increased proportion of anti-EEV antibodies will improve the EEV-neutralization.

[0196] The effect on vaccinia virus replication of Mini-V in vivo has been investigated using the mouse tail lesion model (see Example 1, section q). Both SymVIG and Mini-V significantly reduced the number of tail lesions (FIG. 9). A nearly complete elimination of pocks was observed with the 100 μg to 300 μg Mini-V doses, in contrast to a maximum of ˜65% inhibition observed for the highest concentration of SymVIG. These experiments suggest that the in vivo specific activity of Mini-V is at least 100-fold higher than SymVIG, correlating with the in vitro neutralization and binding studies.

Example 4

[0197] The present Example illustrates the generation of a recombinant polyclonal anti-VV antibody and compares the biological activity of an anti-VV rpAb with that of a mixture of monoclonal antibodies and a serum derived polyclonal VIG product.

Generation of a Polyclonal Cell Line and Production of Anti-VV rpAb

[0198] The aim of the lead identification for the production of the recombinant polyclonal anti-VV antibody was to select a composition with the broadest possible reactivity against vaccinia virus and thus presumably also variola virus. The individual antibody members included in the anti-VV recombinant polyclonal antibody were selected based on several characteristics: [0199] Antigen reactivity; in principle all antigen reactivities identified in Example 2 should be represented in the composition, and the reactivity should be specific and of high affinity. Further, if possible an average of 2-3 antibodies against each identified antigen should be included. [0200] Biochemical properties; only antibodies with well-defined characteristics were included in the final product. The individually expressed antibodies were analyzed by reducing and non-reducing SDS-PAGE, and ion exchange-profiles. Antibodies with an aberrant biochemical behavior were subjected to detailed analysis and most likely excluded from the final composition. [0201] Cell line characteristics; to optimize the stability of the polyclonal cell line, individual cell lines with similar doubling times, as well as productivity after adaptation were preferred.

[0202] Twenty eight individual cell cultures expressing cognate V_H and V_L coding pairs corresponding to clone nr. 02-029, 02-037, 02-058, 02-086, 02-113, 02-147, 02-186, 02-188, 02-195, 02-197, 02-203, 02-211, 02-225, 02-229, 02-235, 02-286, 02-295, 02-303, 02-339, 02-461, 02-482, 02-488, 02-526, 02-551, 02-586, 02-589, 02-607 and 02-633 of Table 5 in Example 2, were selected for the generation of a polyclonal cell line. The individual cell lines were thawed and expanded at 37° C. in serum free medium in shaker flasks to reach at least 4×10⁸ cells of each clone having a population doubling time of 21-34 hours. The viabilities were in the range of 93% to 96%, prior to mixing of the individual cell lines. Further details to the generation of the polyclonal cell line and the establishment of a polyclonal working cell bank (pWCB) are found in Example 1, section g).

[0203] The recombinant polyclonal anti-VV antibody was produced in laboratorial scale bioreactors as described in Example 1, section n) and purified as described in section o). Three batches have been produced independently from three vials from the pWCB.

[0204] The diversity of the anti-VV rpAb produced from the polyclonal cell line was analyzed by IEX chromatography using a PolyCAT column; buffer A: 25 mM sodium acetate, 150 mM sodium chloride, pH 5.0; B: 25 mM sodium acetate, 0.5 mM sodium chloride, pH 5.0 (gradient: 30-100% B). The IEX profiles of the three anti-VV rpAb batches were highly similar. Assignment of the individual peaks in the IEX profile of the anti-VV rpAb product was conducted by running the individual antibodies produced in Example 2, under identical conditions to identify the position of the peaks in the IEX profile (FIG. 10). Antibodies from two of the individual cell lines (clones 02-113 and 02-225) included in the polyclonal cell line were not found in the anti-VV rpAb. These clones produce antibodies which are reactive against VCP and antigen L, respectively. However, since there are other antibodies with similar reactivity in the composition the loss of these clones are considered acceptable for the further analysis.

Biological Activity of the Anti-VV rpAb

[0205] The reactivity of the three anti-VV rpAb batches against different vaccinia virus strains and the recombinant antigens was compared by ELISA (FIG. 11). In correlation with the highly similar IEX profiles, the three batches exert a nearly identical binding reactivity against all the tested antigens supporting a consistent antibody mixture of the produced polyclonal antibody. For this reason it was decided to pool the three batches. Further, the anti-VV rpAb batches were compared to SymVIG and the commercial available serum derived VIG product (Cangene). In comparison to the serum derived VIG, a ˜250 fold increase specific binding activity of the anti-VV rpAb batches was observed more or less independent of the target antigen.

[0206] The SYM002 anti-VV rpAb was tested in two types of plaque reduction and neutralization assays (PRNT; Example 1, section p). SYM002 anti-VV rpAb exerted a 40-fold improved specific antiviral activity as compared to the Mini-V of Example 3 and 800-fold improved specific activity than the commercial available blood derived VIG product (Cangene) (Table 6). The superior antiviral activity of Sym002 anti-VV rpAb was also observed in the modified PRNT assay detecting EEV-mediated infection. The IC50 values obtained in the in vitro assays are connected with a high degree of uncertainty, but the presented data clearly demonstrate a superior specific activity of Sym002 anti-VV rpAb.

TABLE-US-00008 TABLE 6 IMV- EEV- Antibody neutralization neutralization composition (μg/ml) (μg/ml) Sym002 anti-VV 0.125 2 rpAb Mini-V 5.0 10 VIG (cangene) 100 200

[0207] The Sym002 anti-VV rpAb has been tested for in vivo antiviral activity using the tail lesion model challenged with either Lister of NYCBOH vaccinia virus strain. The Sym002 pre-lead product was administered intramuscularly (I.M) 24 hours prior (prophylactic) or post (therapeutic) virus challenges. All data sets demonstrated an approximately 300-fold increase specific activity of Sym002 anti-VV rpAb as compared to blood derived commercial available VIG (FIG. 12). It was anticipated that prophylactic administered Sym002 anti-VV rpAb would elicit a profound anti-virus effect and not as observed a nearly identical antiviral activity. The result is likely to reflect the limitation of the used mouse model, in which a huge amount of infectious virus particles (˜10⁵ PFU) is injected into the tail vein and thereby creating a temporary high local concentration that are giving raise to approximately 50-70 countable pocks in the untreated situation. In this scenario the temporary high concentration of virus is likely to exceed the concentration of administered antibody which thereby is more likely to influence continues spreading of virus than blocking the primary infection. If this is the dynamic of the mouse tail lesion model no profound potency of Sym002 anti-VV rpAb can be obtained by a prophylactic administration of antibody correlating with the observation. Combined the in vitro and in vivo data provide evidence for that all essential antibody reactivities required for in vitro neutralization of vaccinia virus are included in anti-VV rpAv.

[0208] A Sym002 mix was compiled by mixing of individual purified antibodies (Example 1, section i) to obtain a more equal representation of each antibody than present in the anti-VV-rpAb produce from the pWCB (Example 4). The anti-viral activity of the two polyclonal antibody compounds was assayed in the mouse tail lesion model as described above except for that the indicated doses of antibodies were administered intraperitoneal (LP) 24 hours post challenge with the NYCBOH vaccinia virus. Within the tested dose range we observed an identical antiviral activity of the SYM002 anti-VV-rpAb and the Sym002 mix (FIG. 13) proving that the used production method generate fully biological active polyclonal antibody product.

Example 5

Affinity of Selected Antibodies

[0209] The binding kinetic of antibodies can be detected by surface plasmon resonance measurements e.i. by BIAcore. This method requires chemical immobilization of the antigens on a CHIP surface, restricting the analysis to the Sym002 antibodies interacting with A27L, A33R, VCP and B5R, since only these antigens were available as recombinant proteins. Fab fragments were generated from purified IgG1 antibodies by papain digestions (Example 1, section J). The kinetic measurements revealed affinities constants (K_D) between 10^-8 to 10^-10 M^-1 of the majority of the investigated antibodies (FIG. 14). The observed affinities are approaching the theoretical antibody affinity ceiling (KD at 10^-10 M-1) (Foote and Eisen, 1995, Proc Natl Acad Sci USA, 92: 1254-1256) supporting that the selected antibodies repertoire mirrors the natural humeral immune response. In addition to the reported data, some of the tested antibodies did not interact with the immobilized antigen. The explanation to this observation is unknown but it might be due to sterical constrains introduced by the immobilization or that the structure of some epitopes may be denatured during either during the immobilization or the chip cleaning procedures,

Sequence CWU 1

589120DNAArtificial sequenceSynthetic PCR primer 1gacsgatggg cccttggtgg 20220DNAArtificial sequenceSynthetic PCR primer 2gagtggctcc tgggggaaga 20336DNAArtificial sequenceSynthetic PCR primer 3tattcccatg gcgcgcccag rtgcagctgg tgcart 36436DNAArtificial sequenceSynthetic PCR primer 4tattcccatg gcgcgccsag gtccagctgg trcagt 36536DNAArtificial sequenceSynthetic PCR primer 5tattcccatg gcgcgcccag rtcaccttga aggagt 36635DNAArtificial sequenceSynthetic PCR primer 6tattcccatg gcgcgccsag gtgcagctgg tggag 35736DNAArtificial sequenceSynthetic PCR primer 7tattcccatg gcgcgcccag gtgcagctac agcagt 36836DNAArtificial sequenceSynthetic PCR primer 8tattcccatg gcgcgcccag stgcagctgc aggagt 36936DNAArtificial sequenceSynthetic PCR primer 9tattcccatg gcgcgccgar gtgcagctgg tgcagt 361037DNAArtificial sequenceSynthetic PCR primer 10tattcccatg gcgcgcccag gtacagctgc agcagtc 371138DNAArtificial sequenceSynthetic PCR primer 11atatatatgc ggccgcttat taacactctc ccctgttg 381245DNAArtificial sequenceSynthetic PCR primer 12ggcgcgccat gggaatagct agccgacatc cagwtgaccc agtct 451345DNAArtificial sequenceSynthetic PCR primer 13ggcgcgccat gggaatagct agccgatgtt gtgatgactc agtct 451445DNAArtificial sequenceSynthetic PCR primer 14ggcgcgccat gggaatagct agccgaaatt gtgwtgacrc agtct 451545DNAArtificial sequenceSynthetic PCR primer 15ggcgcgccat gggaatagct agccgatatt gtgatgaccc acact 451643DNAArtificial sequenceSynthetic PCR primer 16ggcgcgccat gggaatagct agccgaaacg acactcacgc agt 431745DNAArtificial sequenceSynthetic PCR primer 17ggcgcgccat gggaatagct agccgaaatt gtgctgactc agtct 451851DNAArtificial sequenceSynthetic PCR primer 18accgcctcca ccggcggccg cttattaaca ctctcccctg ttgaagctct t 511930DNAArtificial sequenceSynthetic PCR primer 19ggaggcgctc gagacggtga ccagggtgcc 302030DNAArtificial sequenceSynthetic PCR primer 20ggaggcgctc gagacggtga ccattgtccc 302130DNAArtificial sequenceSynthetic PCR primer 21ggaggcgctc gagacggtga ccagggttcc 302230DNAArtificial sequenceSynthetic PCR primer 22ggaggcgctc gagacggtga ccgtggtccc 302338DNAArtificial sequenceSynthetic PCR primer 23gggaacagcc accatggcgg cggtgaaaac tcctgtta 382443DNAArtificial sequenceSynthetic PCR primer 24ggatcctcta gatcattaaa atgaaatcag tggagtagta aac 432542DNAArtificial sequenceSynthetic PCR primer 25gggaacagcc accatgacat gacacctttt cctcagacat ct 422639DNAArtificial sequenceSynthetic PCR primer 26ggatcctcta gatcattaag aggttgtact actacctac 392738DNAArtificial sequenceSynthetic PCR primer 27gggaacagcc accatgacat gtactgtacc cactatga 382836DNAArtificial sequenceSynthetic PCR primer 28ggatcctcta gatcattata acgattctat ttcttg 362948DNAArtificial sequenceSynthetic PCR primer 29tttttttttt tttttttttt tttttttttt ggatcctcta gatcatta 483039DNAArtificial sequenceSynthetic PCR primer 30ggatcctaat acgactcact atagggaaca gccaccatg 393135DNAArtificial sequenceSynthetic PCR primer 31gggaacagcc accatgatgc ctattaagtc aatag 353234DNAArtificial sequenceSynthetic PCR primer 32ggatcctcta gatcattatt catcatcaaa agag 343336DNAArtificial sequenceSynthetic PCR primer 33gggaacagcc accatggaag ccgtggtcaa tagcga 363440DNAArtificial sequenceSynthetic PCR primer 34ggatcctcta gatcattaaa atagttctgt aatatgtcta 403536DNAArtificial sequenceSynthetic PCR primer 35gggaacagcc accatgattg gtattctttt gttgat 363638DNAArtificial sequenceSynthetic PCR primer 36ggatcctcta gatcattata cagaagattt aactagat 383734DNAArtificial sequenceSynthetic PCR primer 37gggaacagcc accatgccgc aacaactatc tcct 343843DNAArtificial sequenceSynthetic PCR primer 38ggatcctcta gatcattatt tattcccttc gatatatttt tga 433934DNAArtificial sequenceSynthetic PCR primer 39gggaacagcc accatgacga ccgtaccagt gacg 344038DNAArtificial sequenceSynthetic PCR primer 40ggatcctcta gatcattaaa taattttaat tcgtttaa 384137DNAArtificial sequenceSynthetic PCR primer 41gggaacagcc accatgagtt atttaagata ttacaat 374239DNAArtificial sequenceSynthetic PCR primer 42ggatcctcta gatcattaat aatcgtcagt atttaaact 394334DNAArtificial sequenceSynthetic PCR primer 43gggaacagcc accatggaga atgttcctaa tgta 344437DNAArtificial sequenceSynthetic PCR primer 44ggatcctcta gattatcatc tgcgaagaac atcgtta 374534DNAArtificial sequenceSynthetic PCR primer 45gggaacagcc accatgtggc catttgcatc ggta 344638DNAArtificial sequenceSynthetic PCR primer 46ggatcctcta gatcattaaa tttttaacga tttactgt 384737DNAArtificial sequenceSynthetic PCR primer 47gggaacagcc accatgatgg gggcagctgt tactctt 374841DNAArtificial sequenceSynthetic PCR primer 48ggatcctcta gatcattaag gcagttttat tttatctttt a 4149132PRTHomo sapiens 49Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Tyr1 5 10 15Ser Val Lys Val Ser Cys Gln Ala Ser Gly Leu Thr Phe Ser Asn Tyr 20 25 30Arg Val Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro Ile Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Ser Ala Asp Arg Ser Pro Ser Arg Val Thr65 70 75 80Ser Leu Ala Asp Lys Ser Thr Ile Thr Val Tyr Met Glu Leu Ser Ser 85 90 95Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Arg Gly 100 105 110Ser Gln Asp Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val 115 120 125Thr Val Ser Ser 130505PRTHomo sapiens 50Glu Leu Ala Met His1 5515PRTHomo sapiens 51Gly Phe His Trp Ser1 5527PRTHomo sapiens 52Asn Asn Ile Ala Ser Trp Asn1 5535PRTHomo sapiens 53Arg Tyr Gly Met His1 5545PRTHomo sapiens 54Thr Tyr Trp Ile Gly1 5555PRTHomo sapiens 55Gly His Tyr Met His1 5567PRTHomo sapiens 56Thr Ser Gly Val Gly Val Asp1 5577PRTHomo sapiens 57Thr Ser Gly Met Cys Val Ser1 5585PRTHomo sapiens 58Lys Tyr Trp Met Ser1 5595PRTHomo sapiens 59Ser Tyr Gly Met His1 5605PRTHomo sapiens 60Asn Tyr Tyr Ile His1 5615PRTHomo sapiens 61Asp Tyr Ala Met His1 5625PRTHomo sapiens 62Gly Tyr Tyr Trp Thr1 5635PRTHomo sapiens 63Thr Tyr Trp Ile Gly1 5645PRTHomo sapiens 64Ser Tyr Thr Met Asn1 5655PRTHomo sapiens 65Lys Tyr Trp Ile Gly1 5665PRTHomo sapiens 66Asn Tyr Ala Val His1 5677PRTHomo sapiens 67Ser Gly Asp Tyr Tyr Trp Asn1 5685PRTHomo sapiens 68Lys Tyr Tyr Met His1 5695PRTHomo sapiens 69Thr Tyr Gly Ile Ser1 5706PRTHomo sapiens 70Ser Asn Asn Trp Trp Ser1 5715PRTHomo sapiens 71Lys Tyr Tyr Ile His1 5727PRTHomo sapiens 72Arg Gly Asp Phe Tyr Trp Ser1 5735PRTHomo sapiens 73Asp Tyr Thr Thr His1 5745PRTHomo sapiens 74Asp Tyr Ala Met His1 5755PRTHomo sapiens 75Asp Tyr Ala Met His1 5765PRTHomo sapiens 76Asp Tyr Tyr Ile His1 5775PRTHomo sapiens 77Ser Tyr Ala Ile Ala1 5785PRTHomo sapiens 78Gly Tyr Tyr Trp Gly1 5795PRTHomo sapiens 79Asp Tyr Ala Val Ser1 5807PRTHomo sapiens 80Ser Arg Asn Tyr Tyr Trp Gly1 5818PRTHomo sapiens 81Ser Ser Thr Asn Tyr Tyr Trp Gly1 5827PRTHomo sapiens 82Thr Ser Thr Ala Ala Trp Asn1 5835PRTHomo sapiens 83Asp Tyr Ala Met His1 5845PRTHomo sapiens 84Glu Tyr Tyr Ile His1 5855PRTHomo sapiens 85Ser Tyr Trp Ile Ser1 5865PRTHomo sapiens 86Ser Tyr Trp Ile Ala1 5875PRTHomo sapiens 87Asp Phe Ala Met His1 5885PRTHomo sapiens 88Asp Tyr Ala Ile Ser1 5897PRTHomo sapiens 89Ser Thr Gly Val Gly Val Gly1 5905PRTHomo sapiens 90Ser Tyr Ala Ile Asn1 5915PRTHomo sapiens 91Gly Tyr Tyr Trp Ser1 5925PRTHomo sapiens 92Gly Tyr Gly Ile Ser1 5935PRTHomo sapiens 93Asn Ser Ala Ile Asn1 5945PRTHomo sapiens 94Asn Tyr Arg Val Ser1 5955PRTHomo sapiens 95Asn Tyr Ala Met Ser1 5965PRTHomo sapiens 96Asp Asn Tyr Met Asn1 5975PRTHomo sapiens 97Asp Tyr Ala Met His1 5987PRTHomo sapiens 98Thr Ser Gly Val Gly Val Gly1 5995PRTHomo sapiens 99Thr Tyr Gly Met His1 51005PRTHomo sapiens 100Asn Tyr Gly Ile His1 51015PRTHomo sapiens 101Ser Asp Asn Trp Ser1 51025PRTHomo sapiens 102Ser Phe Trp Ile Gly1 51035PRTHomo sapiens 103Asn Tyr Ala Met Tyr1 51045PRTHomo sapiens 104Ser Tyr Tyr Met His1 51057PRTHomo sapiens 105Ser Pro Gly Tyr Tyr Trp Asn1 51065PRTHomo sapiens 106Ser Phe Ala Ile Ser1 51075PRTHomo sapiens 107Thr Tyr Gly Ile Ser1 51087PRTHomo sapiens 108Ser Ala Gly Tyr Tyr Trp Ser1 51095PRTHomo sapiens 109Ser Tyr Trp Ile Gly1 51107PRTHomo sapiens 110Ser Gly Gly Tyr Tyr Trp Ser1 51115PRTHomo sapiens 111Asp Tyr Tyr Met Ser1 51126PRTHomo sapiens 112Gly Ala Tyr His Trp Ser1 51135PRTHomo sapiens 113Ala Tyr Tyr Trp Ser1 51145PRTHomo sapiens 114Asp Tyr Thr Met Ser1 51155PRTHomo sapiens 115Asp His Ser Ile Gly1 51166PRTHomo sapiens 116Ser Ser His Trp Trp Asn1 51175PRTHomo sapiens 117Tyr Tyr Ala Ile Asn1 51185PRTHomo sapiens 118Asn Tyr Tyr Met His1 51195PRTHomo sapiens 119Ser Phe Gly Ile Ser1 51207PRTHomo sapiens 120Asn Asp Arg Met Gly Val Ser1 51215PRTHomo sapiens 121Ser Tyr Tyr Trp Ser1 51225PRTHomo sapiens 122Gly Tyr Tyr Trp Thr1 51235PRTHomo sapiens 123Ser Tyr Gly Met His1 51245PRTHomo sapiens 124Asn Tyr Trp Ile Gly1 51255PRTHomo sapiens 125Thr Tyr Thr Met His1 51265PRTHomo sapiens 126Asn Tyr Ala Ile Ile1 51275PRTHomo sapiens 127Asn Tyr Tyr Val His1 51285PRTHomo sapiens 128Ser His Gly Val Asn1 51297PRTHomo sapiens 129Thr Gly Asp Tyr Tyr Trp Ser1 51307PRTHomo sapiens 130Ser Gly Tyr Tyr Tyr Trp Asn1 51315PRTHomo sapiens 131Gly Tyr Tyr Ile His1 51325PRTHomo sapiens 132Ala Tyr Tyr Ile His1 51335PRTHomo sapiens 133Thr Tyr Glu Met Ser1 51345PRTHomo sapiens 134Thr Tyr Trp Ile Ala1 51355PRTHomo sapiens 135Ser Tyr Ala Met Asn1 51367PRTHomo sapiens 136Ser Gly Pro Tyr Tyr Trp Ser1 51375PRTHomo sapiens 137Ser Asn Pro Val Ser1 51385PRTHomo sapiens 138Asp Tyr Tyr Leu His1 513917PRTHomo sapiens 139Gly Phe Asp Pro Glu Asp Gly Glu Ala Val Tyr Ala Gln Gly Phe Gln1 5 10 15Gly14016PRTHomo sapiens 140Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Val Lys Ser1 5 10 1514118PRTHomo sapiens 141Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asp Asp Phe Ala Leu Ser Val1 5 10 15Lys Gly14217PRTHomo sapiens 142Phe Leu Ser Phe Asp Glu Arg Asn Lys Phe Tyr Pro Asp Ser Leu Lys1 5 10 15Gly14317PRTHomo sapiens 143Ile Ile Tyr Pro Gly Asp Ser Asp Thr Lys Tyr Ser Pro Ser Phe Gln1 5 10 15Gly14417PRTHomo sapiens 144Trp Ile Asn Pro Ser Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln1 5 10 15Gly14516PRTHomo sapiens 145Leu Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Ser Pro Ser Leu Lys Ser1 5 10 1514616PRTHomo sapiens 146Phe Ile Asp Trp Asp Asp Asp Lys Tyr Tyr Thr Thr Ser Leu Lys Thr1 5 10 1514717PRTHomo sapiens 147Asn Ile Asn Gln Glu Gly Ser Ala Lys His Tyr Val Asp Ser Val Lys1 5 10 15Gly14817PRTHomo sapiens 148Leu Ile Ser Tyr His Gly Asn Thr Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly14917PRTHomo sapiens 149Ile Ile Asn Pro Ser Gly Gly Ser Thr Thr Tyr Ala Gln Arg Phe Gln1 5 10 15Gly15017PRTHomo sapiens 150Gly Cys Ser Trp Asn Ser Gly Phe Ile Ser Tyr Ala Asp Ser Val Lys1 5 10 15Gly15116PRTHomo sapiens 151Glu Ile Asn His Ser Ala Asn Thr Asp Tyr Lys Pro Ser Leu Lys Ser1 5 10 1515217PRTHomo sapiens 152Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln1 5 10 15Gly15317PRTHomo sapiens 153Ala Ile Thr Ser Ser Thr Thr Tyr Ile Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly15417PRTHomo sapiens 154Ile Ile Tyr Pro Glu Asp Gly Asp Thr Arg Tyr Ser Pro Ala Phe Gln1 5 10 15Gly15517PRTHomo sapiens 155Trp Ile Asn Val Gly Asn Gly Gln Thr Lys Phe Ser Gln Arg Phe Gln1 5 10 15Gly15616PRTHomo sapiens 156Asn Ile Tyr Ser Thr Gly Ser Thr Tyr Tyr Asp Pro Ser Leu Gln Asn1 5 10 1515717PRTHomo sapiens 157Thr Ile Ser Tyr Asp Val Lys Asn Lys Asp Tyr Ala Asp Ser Val Lys1 5 10 15Gly15817PRTHomo sapiens 158Trp Ile Ser Ala Trp Asp Gly Asn Thr Lys Tyr Gly Glu Lys Phe Gln1 5 10 15Asp15916PRTHomo sapiens 159Glu Ile Tyr His Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Gln Ser1 5 10 1516017PRTHomo sapiens 160Met Ile Asn Pro Ser Gly Gly Thr Thr Thr Tyr Ala Gln Lys Phe Gln1 5 10 15Gly16116PRTHomo sapiens 161Tyr Ile Tyr Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser1 5 10 1516217PRTHomo sapiens 162Ile Val Leu Tyr Asp Gly Lys Asn Lys Asn Tyr Ala Asp Ser Val Arg1 5 10 15Gly16317PRTHomo sapiens 163Gly Ile Ser Trp Asn Ser Glu Tyr Ile Gly Tyr Glu Asp Ser Val Lys1 5 10 15Gly16417PRTHomo sapiens 164Thr Ile Ser Trp Asn Ser Gly Phe Ile Asp Tyr Ala Asp Ser Val Lys1 5 10 15Gly16517PRTHomo sapiens 165Trp Ile Asn Pro Asn Phe Gly Gly Thr Asp Tyr Ala Gln Lys Phe Gln1 5 10 15Gly16617PRTHomo sapiens 166Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asp Tyr Ala Gln Lys Phe Gln1 5 10 15Gly16716PRTHomo sapiens 167Glu Ile Asn His Ser Gly Ser Ala Asn Tyr His Pro Ser Leu Lys Ser1 5 10 1516819PRTHomo sapiens 168Leu Ile Arg Ser Arg His Tyr Gly Ala Lys Thr Gln Phe Ala Ala Ser1 5 10 15Val Gln Gly16916PRTHomo sapiens 169Thr Ile Tyr Tyr Thr Gly Arg Thr Tyr Tyr Asn Pro Ser Leu Lys Asn1 5 10 1517016PRTHomo sapiens 170Thr Val Tyr Leu Ser Gly Arg Ala Tyr Tyr Asn Pro Ser Leu Lys Ser1 5 10 1517118PRTHomo sapiens 171Arg Thr Tyr Tyr Arg Ser Arg Trp Arg Asn Asp Tyr Ala Gly Ser Val1 5 10 15Arg Ser17217PRTHomo sapiens 172Gly Ile Asn Trp Asn Ser Gly Asn Ile Val Tyr Ala Asp Ser Val Lys1 5 10 15Gly17317PRTHomo sapiens 173Leu Val Asp Pro Glu Asp Gly Glu Pro Ile Tyr Ala Glu Lys Phe Gln1 5 10 15Gly17417PRTHomo sapiens 174Ile Ile Tyr Gly Gly Asp Ser Asp Thr Lys Tyr Ser Pro Ser Phe Gln1 5 10 15Gly17517PRTHomo sapiens 175Ile Ile Phe Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln1 5 10 15Gly17617PRTHomo sapiens 176Gly Val Ser Trp Asn Ser Asp Val Ile Asn Tyr Ser Asp Ser Val Lys1 5 10 15Gly17717PRTHomo sapiens 177Gly Ile Ile Pro Val Phe Gly Thr Pro Asn Tyr Ala Gln Gln Phe Gln1 5 10 15Gly17816PRTHomo sapiens 178Leu Ile Tyr Trp Asp Asp Glu

Glu Arg Tyr Ser Pro Ser Leu Lys Asn1 5 10 1517917PRTHomo sapiens 179Ser Ile Ile Pro Ile Phe Ala Thr Thr Asn Tyr Ala Gln Arg Phe Gln1 5 10 15Gly18016PRTHomo sapiens 180Glu Val Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Arg Ser1 5 10 1518117PRTHomo sapiens 181Trp Ile Thr Tyr Asp Lys Gly Asn Thr Asn His Ala Gln Lys Phe Arg1 5 10 15Gly18217PRTHomo sapiens 182Gly Val Val Pro Ile Tyr Asp Thr Ser His Tyr Ala Gln Lys Phe Lys1 5 10 15Gly18317PRTHomo sapiens 183Gly Ile Ile Pro Ile Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe Gln1 5 10 15Gly18417PRTHomo sapiens 184Ala Ile Ser Gly Ser Gly Gly Lys Thr Tyr His Ala His Ser Val Arg1 5 10 15Gly18517PRTHomo sapiens 185Tyr Ile Ser Ser Thr Ser Gly Ser Ile Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly18617PRTHomo sapiens 186Gly Leu Asn Trp Asn Gly Ala Asn Ile Arg Tyr Ala Asp Ser Val Lys1 5 10 15Gly18716PRTHomo sapiens 187Leu Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Ser Pro Ser Leu Lys Ser1 5 10 1518817PRTHomo sapiens 188Phe Ile Ser Ser Asp Gly Ser Thr Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly18917PRTHomo sapiens 189Phe Ile Ser Tyr Asp Gly Ser Lys Lys Tyr Tyr Val Asp Ser Val Lys1 5 10 15Gly19016PRTHomo sapiens 190Tyr Ile Tyr Lys Thr Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser1 5 10 1519117PRTHomo sapiens 191Val Thr Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln1 5 10 15Gly19217PRTHomo sapiens 192Gly Ile Ile Trp Asn Ser Glu Tyr Ile Gly Tyr Ala Asp Ser Val Lys1 5 10 15Gly19317PRTHomo sapiens 193Leu Ile Asn Pro Ser Ser Gly Thr Thr Ser Tyr Ala Gln Asn Phe Gln1 5 10 15Gly19416PRTHomo sapiens 194Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser1 5 10 1519517PRTHomo sapiens 195Ser Ile Ile Pro Ile Phe Gly Thr Ala His Tyr Ala Gln Arg Phe Glu1 5 10 15Gly19617PRTHomo sapiens 196Gly Ile Arg Val His Asn Gly Asn Thr Asn Tyr Ala Gln Lys Phe Gln1 5 10 15Gly19716PRTHomo sapiens 197Tyr Ile His Tyr Thr Gly Thr Thr Tyr Tyr Asn Pro Ser Leu Lys Ser1 5 10 1519817PRTHomo sapiens 198Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln1 5 10 15Gly19916PRTHomo sapiens 199Tyr Ile His Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser1 5 10 1520017PRTHomo sapiens 200Tyr Thr Asn Leu Phe Thr Gly Tyr Thr Asn Tyr Ala Asp Ser Val Lys1 5 10 15Gly20116PRTHomo sapiens 201Tyr Ile Tyr Tyr Thr Gly Asn Thr Tyr Phe Asn Pro Ser Leu Lys Ser1 5 10 1520216PRTHomo sapiens 202Glu Ile Ser His Ser Gly Ser Thr His Tyr Asn Pro Ser Leu Asn Ser1 5 10 1520319PRTHomo sapiens 203Phe Ile Arg Gly Lys Lys Phe Gly Gly Thr Lys Asp Tyr Ala Ala Ser1 5 10 15Val Lys Gly20417PRTHomo sapiens 204Lys Ile Ile Pro Ile Tyr Gly Arg Ala Asn Tyr Ala Gln Lys Phe Gln1 5 10 15Gly20516PRTHomo sapiens 205Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser1 5 10 1520617PRTHomo sapiens 206Gly Ile Val Pro Met Val Gly Pro Ala Asp Tyr Ala Glu Lys Phe Arg1 5 10 15Gly20717PRTHomo sapiens 207Leu Ile Asn Pro Ser Gly Asp Ser Thr Thr Asn Ala Gln Lys Phe Gln1 5 10 15Gly20817PRTHomo sapiens 208Gly Ile Ile Pro Ile Phe Gly Thr Pro Asn Tyr Ser Leu Lys Phe Gln1 5 10 15Asp20916PRTHomo sapiens 209His Ile Phe Ser Asn Asp Glu Arg Ser His Ser Ser Ser Leu Lys Ser1 5 10 1521016PRTHomo sapiens 210Tyr Ile Phe Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys Ser1 5 10 1521116PRTHomo sapiens 211Glu Ile Asn Gln Asn Gly Arg Ser Asn His Asn Pro Ser Leu Lys Ser1 5 10 1521217PRTHomo sapiens 212Val Ile Ser Tyr Asp Gly Arg Tyr Lys Phe Tyr Ala Asn Ser Val Lys1 5 10 15Gly21317PRTHomo sapiens 213Ile Ile His Pro Gly Asp Ser Glu Thr Arg Tyr Ser Pro Ser Phe Gln1 5 10 15Gly21417PRTHomo sapiens 214Val Ile Ser Tyr Asp Gly Thr Asn Lys Tyr His Thr Asp Ser Val Lys1 5 10 15Gly21517PRTHomo sapiens 215Glu Ile Ile Pro Lys Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln1 5 10 15Gly21617PRTHomo sapiens 216Leu Ile Asn Pro Ser Ala Gly Lys Thr Thr Tyr Ala Gln Arg Phe Gln1 5 10 15Gly21717PRTHomo sapiens 217Gly Ile Ile Pro Val Phe Gly Thr Thr Asn Tyr Ala Gln Ser Leu Gln1 5 10 15Gly21816PRTHomo sapiens 218Tyr Val Phe Asn Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Gln Ser1 5 10 1521916PRTHomo sapiens 219Tyr Ile Asp Tyr Arg Gly Thr Thr Tyr Tyr Ser Pro Ser Phe Lys Ser1 5 10 1522017PRTHomo sapiens 220Arg Ile Asn Pro Ile Thr Asp Val Thr Asn Tyr Ala Gln Ile Phe Gln1 5 10 15Gly22117PRTHomo sapiens 221Arg Ile Asn Pro Asp Ser Gly Gly Thr Asp Phe Ser Gln Lys Phe Gln1 5 10 15Gly22217PRTHomo sapiens 222Tyr Ile Gly Ser Gly Gly Val Thr Ile Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly22317PRTHomo sapiens 223Ile Ile Trp Pro Val Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln1 5 10 15Gly22417PRTHomo sapiens 224Trp Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala Gln Gly Phe Thr1 5 10 15Gly22516PRTHomo sapiens 225Tyr Ser Ser Asn Arg Gly Ile Ala Tyr Tyr Asn Pro Ser Leu Lys Ser1 5 10 1522617PRTHomo sapiens 226Gly Ile Ile Pro Phe Ala Gln Lys Val Leu Gly Ala Gln Arg Val Arg1 5 10 15Asp22717PRTHomo sapiens 227Arg Ile Asn Pro Lys Ser Gly Asp Thr His His Val Gln Lys Phe Gln1 5 10 15Gly22820PRTHomo sapiens 228Cys Ala Thr Asp Val Trp Arg Arg Thr Pro Glu Gly Gly Thr Asp Trp1 5 10 15Phe Asp Pro Trp 2022926PRTHomo sapiens 229Cys Ala Gly Ser Arg Ser Phe Asp Leu Leu Thr Ala Tyr Asp Leu Phe1 5 10 15His Arg Lys Gly Asn Ala Met Asp Val Trp 20 2523013PRTHomo sapiens 230Cys Ala Arg Gly Asp Val Leu Arg Tyr Phe Asp Tyr Trp1 5 1023114PRTHomo sapiens 231Cys Ala Lys Gly Gly Leu Gly Thr Asn Glu Phe Asp His Trp1 5 1023218PRTHomo sapiens 232Cys Ala Thr Leu Pro Arg Tyr Asp Ala Tyr Gly Ala Arg Ile Arg Asp1 5 10 15Tyr Trp23314PRTHomo sapiens 233Cys Ala Arg Tyr Cys Ser Ser Pro Thr Cys Ser Ile Val Trp1 5 1023415PRTHomo sapiens 234Cys Ala His Ser Ser Gln Arg Val Val Thr Gly Leu Asp Phe Trp1 5 10 1523522PRTHomo sapiens 235Cys Ala Arg Ile Arg Thr Cys Tyr Pro Asp Leu Tyr Gly Asp Tyr Asn1 5 10 15Asp Ala Phe Asp Ile Trp 2023613PRTHomo sapiens 236Cys Ala Arg Ala Ala Asp Tyr Gly Asp Tyr Val Arg Pro1 5 1023714PRTHomo sapiens 237Cys Ala Lys His Val Ala Ala Gly Gly Thr Leu Asp Tyr Trp1 5 1023824PRTHomo sapiens 238Cys Ala Arg Val Ile Arg Lys Tyr Tyr Thr Ser Ser Asn Ser Tyr Leu1 5 10 15Thr Glu Gln Ala Phe Asp Ile Trp 2023916PRTHomo sapiens 239Cys Val Lys Glu Thr Val Ala Gly Arg Arg Gly Ala Phe Asp Tyr Trp1 5 10 1524014PRTHomo sapiens 240Cys Ala Arg Gly Arg Glu Trp Pro Ser Asn Phe Asp Ser Trp1 5 1024113PRTHomo sapiens 241Cys Ala Arg Arg Gly Ser Thr Tyr Tyr Tyr Asp Thr Trp1 5 1024214PRTHomo sapiens 242Cys Ala Ser Lys Pro Tyr Gly Gly Asp Phe Gly Ser Tyr Trp1 5 1024315PRTHomo sapiens 243Cys Ala Arg Pro Pro Ser Asn Trp Asp Glu Ser Phe Asp Ile Trp1 5 10 1524420PRTHomo sapiens 244Cys Ala Arg Asp Pro Thr Gln Trp Leu Leu Gln Gly Asp Val Tyr Asp1 5 10 15Met Asp Val Trp 2024521PRTHomo sapiens 245Cys Ala Arg Glu Ala Trp Leu Gly Glu Pro Leu Leu Leu Gly Asp Asp1 5 10 15Ala Phe Asp Ile Trp 2024620PRTHomo sapiens 246Cys Ala Arg Asp Gly Ala Gly Glu Trp Asp Leu Leu Met Arg Arg Asp1 5 10 15Phe Asp Tyr Trp 2024719PRTHomo sapiens 247Cys Ala Arg Asp Pro Ala Arg Arg Pro Arg Ser Gly Tyr Ser Val Phe1 5 10 15Glu Tyr Trp24824PRTHomo sapiens 248Cys Ala Arg Asp Asn Arg Gln Ser Ser Ser Trp Val Glu Gly Phe Phe1 5 10 15Tyr Tyr Tyr Gly Met Asp Val Trp 2024916PRTHomo sapiens 249Cys Ala Arg Leu Arg Leu Gly Ala Thr Ile Gly Arg Asp Asp Tyr Trp1 5 10 1525019PRTHomo sapiens 250Cys Ala Arg Asp Arg Ala Ser Ser Gly Tyr Asp Ser Arg Val Trp Phe1 5 10 15Asp Pro Trp25117PRTHomo sapiens 251Cys Ala Arg Thr Tyr Arg Val Tyr Ala Lys Phe Asp Pro Phe Asp Val1 5 10 15Trp25216PRTHomo sapiens 252Cys Gly Lys Asp Gly Val Pro Gly Arg Arg Gly Tyr Ile Glu Asp Trp1 5 10 1525316PRTHomo sapiens 253Cys Val Lys Asp Asn Ile Ala Gly Arg Arg Gly Ser Phe Asp Ser Trp1 5 10 1525426PRTHomo sapiens 254Cys Ala Arg Asp Tyr Ile Arg Ala Thr Gly Ala Thr Pro Ser Lys Tyr1 5 10 15Phe Ile Tyr Tyr Tyr Gly Met Gly Val Trp 20 2525518PRTHomo sapiens 255Cys Ala Arg Ala Arg Arg Val Thr Asn Ser Pro Asn Asn Trp Phe Asp1 5 10 15Pro Trp25621PRTHomo sapiens 256Cys Ala Arg Ala Gly Glu Arg Ser Gly Ser Gly Ser Phe Val Leu Gly1 5 10 15Arg Phe Asp Phe Trp 2025713PRTHomo sapiens 257Cys Thr Asn Thr Ser Ser Leu Ala Val Ala Gly Asn Trp1 5 1025815PRTHomo sapiens 258Cys Ala Arg Ile Pro Gln Gln Arg Val Asn Tyr Phe Asp Tyr Trp1 5 10 1525915PRTHomo sapiens 259Cys Ala Arg Leu Pro Gly Gln Arg Thr Thr Phe Phe Asp Tyr Trp1 5 10 1526015PRTHomo sapiens 260Cys Ala Arg Gly Arg Arg Phe Glu Asp Asp Ala Phe Asp Ile Trp1 5 10 1526116PRTHomo sapiens 261Cys Val Lys Asp Ser Val Ala Gly Arg Arg Gly Gly Phe Asp His Trp1 5 10 1526211PRTHomo sapiens 262Cys Ala Thr Arg Asp Gly Asp Phe Asp His Trp1 5 1026319PRTHomo sapiens 263Cys Val Arg His Gly Thr Arg Tyr Ser Phe Gly Arg Ser Asp Ile Ile1 5 10 15Asp Ile Trp26418PRTHomo sapiens 264Cys Thr Lys Thr Pro Ala Arg Gly Ala Tyr Gly Asp Tyr Ile Ser Gly1 5 10 15Ser Trp26516PRTHomo sapiens 265Cys Ala Lys Ser Thr Lys Ala Val Arg Arg Gly Ser Phe Asp Tyr Trp1 5 10 1526622PRTHomo sapiens 266Cys Ala Arg Gly Gly Lys Leu Tyr Glu Gly Asn Gly Tyr Tyr Ser Phe1 5 10 15His Tyr Phe Asp Tyr Trp 2026711PRTHomo sapiens 267Cys Ala His Thr Glu Leu Ala Phe Asp Tyr Trp1 5 1026816PRTHomo sapiens 268Cys Ala Arg Val Lys Gly Thr Gln Asn Tyr Tyr Gly Met Asp Val Trp1 5 10 1526915PRTHomo sapiens 269Cys His Tyr Tyr Asp Ser Thr Gly Tyr Tyr Val Ser Asp Phe Trp1 5 10 1527016PRTHomo sapiens 270Cys Ala Arg Gly Val Val Leu Ile Gln Thr Ile Leu Phe Asp Tyr Trp1 5 10 1527117PRTHomo sapiens 271Cys Ala Arg Thr Val Leu Asp Ser Gly Ala Tyr Ser Tyr Tyr Asp Ser1 5 10 15Arg27216PRTHomo sapiens 272Cys Ala Arg Ser Arg Gly Ser Gln Asp Tyr Tyr Gly Met Asp Val Trp1 5 10 1527322PRTHomo sapiens 273Cys Ala Lys Leu Arg Asp Ser Ser Val Tyr Ser Ala Tyr Val Phe Arg1 5 10 15Val Ile Phe Asp Cys Trp 2027413PRTHomo sapiens 274Cys Ala Thr Leu Thr Val Ala Ser Thr Tyr Asp Tyr Trp1 5 1027519PRTHomo sapiens 275Cys Val Lys Asp Thr Val Ala Leu Leu Thr Ser Arg Gly Gly Cys Met1 5 10 15Asp Val Trp27612PRTHomo sapiens 276Cys Ala His Ser Pro Pro His Gly Gly Asp Tyr Trp1 5 1027719PRTHomo sapiens 277Cys Ala Lys Gly Leu Ser Gln Ala Leu Asn Tyr Tyr Gly Ser Gly Ser1 5 10 15Pro Phe Leu27819PRTHomo sapiens 278Cys Ala Lys Asp Arg Gly Val Ser Ala Trp Tyr Pro Arg Asp Ala Phe1 5 10 15Asp Ile Trp27923PRTHomo sapiens 279Cys Ala Arg Val Pro Leu Ile Glu Ala Gly Ile Thr Ile Phe Ala Lys1 5 10 15Ile Gly Ala Phe Asp Ile Trp 2028015PRTHomo sapiens 280Cys Ala Arg Gly Ser Pro Met Ile Lys Phe Tyr Phe Asp Tyr Trp1 5 10 1528116PRTHomo sapiens 281Cys Ala Arg Ala Thr Gly Ala Gly Arg Arg Asn Pro Leu Asp Tyr Trp1 5 10 1528216PRTHomo sapiens 282Cys Ala Arg Pro Tyr Arg Ser Tyr Ser Ser Ser Pro Gln Asp Tyr Trp1 5 10 1528315PRTHomo sapiens 283Cys Ala Arg Tyr Tyr Tyr Ser Ser Gly Pro Lys Phe Asp Tyr Trp1 5 10 1528417PRTHomo sapiens 284Cys Ala Arg Asn Asn Arg Pro Leu Gly Ala Leu Phe Gly Met Asp Val1 5 10 15Trp28513PRTHomo sapiens 285Cys Ala Arg Gly Gly Phe Asn Arg Leu Val Asp Pro Trp1 5 1028624PRTHomo sapiens 286Cys Ala Arg Asn Thr Gly Ile Tyr Leu Gly Gly Ser Pro Gly Gly Thr1 5 10 15Arg Asn Asn Trp Phe Asp Pro Trp 2028723PRTHomo sapiens 287Cys Ala Arg Gln Gln Ala Lys Thr Leu Tyr Tyr Asp Ser Ser Gly Ser1 5 10 15Lys Ser Ala Phe Asp Ile Trp 2028823PRTHomo sapiens 288Cys Ala Arg Val Arg Gly Asn Ile Val Ala Thr Thr Ala Phe Tyr Tyr1 5 10 15Tyr Tyr Gly Leu Asp Ala Trp 2028916PRTHomo sapiens 289Cys Ala Lys Phe Asp Tyr Gly Glu Gly Ala Tyr His Phe Asp Phe Trp1 5 10 1529017PRTHomo sapiens 290Cys Ala Arg Asp Pro Ile Ala Leu Pro Gly Arg Gly Val Phe Asp Tyr1 5 10 15Trp29115PRTHomo sapiens 291Cys Ser Ser Gly Tyr Tyr Phe Ala Gly Gly Glu Phe Asp Tyr Trp1 5 10 1529222PRTHomo sapiens 292Cys Thr Arg Asp Arg Gly Tyr Ser Asp His Thr Gly Leu Tyr Thr Arg1 5 10 15Phe Gly Phe Asp Ser Trp 2029318PRTHomo sapiens 293Cys Ala Arg Trp Arg Gly Gly Tyr Ser Gly Tyr Gly Asp Tyr Phe Asp1 5 10 15Ser Trp29425PRTHomo sapiens 294Cys Ala Arg Asp Pro Gln Lys Pro Arg Gln His Leu Trp Pro Asn Pro1 5 10 15Tyr Tyr Tyr Ser Gly Met Asp Val Trp 20 2529514PRTHomo sapiens 295Cys Ala Arg Gly Arg Ser Trp Arg Gly Tyr Leu Asp Tyr Trp1 5 1029621PRTHomo sapiens 296Cys Ala Arg Asp Tyr Gly Asp Tyr Cys Gly Gly Asp Cys Pro Tyr Asp1 5 10 15Ala Phe Asp Ile Trp 2029725PRTHomo sapiens 297Cys Ala Arg Asp Lys Gly Glu Ser Asp Ile Asn Gly Trp Gln Thr Gly1 5 10 15Ala Phe Tyr Tyr Gly Met Asp Val Trp 20 2529820PRTHomo sapiens 298Cys Ala Arg Ile Asp Ser Val Gly Trp Pro Ser Ser His Tyr Tyr Gly1 5 10 15Met Asp Val Trp 2029919PRTHomo sapiens 299Cys Ala Arg Asp Arg Ile Thr Gly Tyr Asp Ser Ser Gly His Ala Phe1 5 10 15Asp Ile Trp30022PRTHomo sapiens 300Cys Ala

Arg Gly Gly Lys Phe Cys Gly Ser Thr Ser Cys Phe Thr Glu1 5 10 15Gly Arg Leu Asp Tyr Trp 2030122PRTHomo sapiens 301Cys Ala Lys Asp Ser Gly Arg Tyr Ser Ser Leu Gly His Tyr Tyr Tyr1 5 10 15Tyr Gly Met Asp Val Trp 2030220PRTHomo sapiens 302Cys Ala Arg Gly Tyr Tyr Tyr Asp Thr Ser Gly Tyr Arg Pro Gly Ser1 5 10 15Phe Gln His Trp 2030316PRTHomo sapiens 303Cys Ala Arg Pro Leu Phe Tyr Gly Ala Gly Asp Ala Phe Asp Ile Trp1 5 10 1530415PRTHomo sapiens 304Cys Ala Asp Trp Val Val Gly Asn Tyr Asn Gly Leu Asp Val Trp1 5 10 1530522PRTHomo sapiens 305Cys Ala Arg Glu Gly Lys His Asp Phe Trp Arg Gly Tyr Phe Ser Pro1 5 10 15Leu Gly Met Asp Val Trp 2030620PRTHomo sapiens 306Cys Ala Thr Ala Arg Asn Ser Ser Asn Trp Tyr Glu Gly His Tyr Tyr1 5 10 15Leu Ala His Trp 2030718PRTHomo sapiens 307Cys Ala Asn Met Val Val Val Ala Thr Gln Pro Lys Asn Trp Phe Asp1 5 10 15Pro Trp30817PRTHomo sapiens 308Cys Ala Ser Tyr Gly Ser Gly Met Gly Ser Glu Tyr Tyr Phe Gly His1 5 10 15Trp30917PRTHomo sapiens 309Cys Gly Arg Val Gly Arg Glu Ala Phe Tyr Tyr Tyr Gly Met Asp Val1 5 10 15Trp31017PRTHomo sapiens 310Cys Ala Arg Ala Ser Arg Arg Leu Thr Thr His Asn Tyr Phe Asp Gly1 5 10 15Trp31113PRTHomo sapiens 311Cys Ala Arg Val Arg Gly Gly Arg Tyr Phe Asp Tyr Trp1 5 1031216PRTHomo sapiens 312Cys Ala Ser Gly Ser Gly Tyr Asp Ser Tyr Tyr Asn Met Asp Val Trp1 5 10 1531321PRTHomo sapiens 313Cys Ala Arg Asp Ser Ser Thr Val Thr Gly Leu Met Thr Glu Tyr Asn1 5 10 15Trp Phe Asp Pro Trp 2031417PRTHomo sapiens 314Cys Ala Thr Glu Lys Gly Ser Gly Gly Asp Val Gly Lys Phe Asp Asn1 5 10 15Trp31513PRTHomo sapiens 315Cys Ala Thr Gly Gln Gln Leu Tyr Ser Leu His Tyr Trp1 5 1031627PRTHomo sapiens 316Cys Ala Arg Glu Gly Pro Gln Phe Tyr Tyr Asp Ser Gly Asp Tyr Tyr1 5 10 15Ser Ala His Ser Pro Gly Asp Phe Asp His Trp 20 2531711PRTHomo sapiens 317Arg Ala Ser Gln Ser Val Arg Arg Ser Leu Ala1 5 1031811PRTHomo sapiens 318Arg Ala Ser Gln Gly Ile Ser Asn Ser Leu Asn1 5 1031911PRTHomo sapiens 319Arg Ala Ser Gln Ser Ile Ser Ile Trp Leu Ala1 5 1032011PRTHomo sapiens 320Arg Ala Ser Gln Ser Ile Gly Asn Trp Leu Ala1 5 1032111PRTHomo sapiens 321Gln Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn1 5 1032212PRTHomo sapiens 322Arg Ala Ser Glu Ser Val Arg Ser Asn Tyr Leu Ala1 5 1032311PRTHomo sapiens 323Arg Ala Ser Gln Ser Ile Ser Thr Tyr Leu Asn1 5 1032411PRTHomo sapiens 324Gln Ala Ser Gln Asp Ile Lys Tyr Tyr Leu Asn1 5 1032516PRTHomo sapiens 325Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp1 5 10 1532616PRTHomo sapiens 326Arg Ser Ser Glu Ser Leu Val Asn Asn Asp Gly Asn Thr Tyr Leu Ser1 5 10 1532711PRTHomo sapiens 327Arg Ala Ser Gln Asn Ile Ser Asn Phe Leu Leu1 5 1032811PRTHomo sapiens 328Arg Ala Ser Gln Ser Ile Asn Asn Tyr Leu Asn1 5 1032911PRTHomo sapiens 329Arg Ala Ser Gln Ser Val Asp Arg Tyr Leu Ala1 5 1033011PRTHomo sapiens 330Arg Ala Ser Gln Ser Ile Trp Thr Phe Leu Asn1 5 1033111PRTHomo sapiens 331Arg Ala Ser Gln Ser Ile Ser Asn Trp Leu Ala1 5 1033211PRTHomo sapiens 332Arg Ala Ser Gln Asp Ile Ser Asn Asp Leu Gly1 5 1033316PRTHomo sapiens 333Arg Ser Ser Gln Ser Leu Val Tyr Ser Asp Gly Asn Thr Tyr Leu His1 5 10 1533411PRTHomo sapiens 334Arg Ala Ser Gln Gly Ile Gly Tyr Asp Leu Gly1 5 1033511PRTHomo sapiens 335Arg Ala Ser Gln Ser Ile Ser Asn Tyr Leu Ser1 5 1033611PRTHomo sapiens 336Arg Ala Ser Gln Ser Val Ser Ser Asn Leu Ala1 5 1033712PRTHomo sapiens 337Arg Ala Ser Gln Ser Ile Ala Ser Ala Tyr Leu Ala1 5 1033816PRTHomo sapiens 338Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp1 5 10 1533911PRTHomo sapiens 339Gln Ala Ser His Asp Val Ser Asn Phe Leu Asn1 5 1034012PRTHomo sapiens 340Arg Ala Ser Gln Ser Val Ser Ser Asn Tyr Ile Ala1 5 1034111PRTHomo sapiens 341Arg Ala Ser Gln Ser Ile Arg Thr Tyr Leu Asn1 5 1034211PRTHomo sapiens 342Arg Ala Ser Gln Thr Ile Ser Thr Tyr Leu Asn1 5 1034311PRTHomo sapiens 343Arg Ala Ser Gln Gly Ile Ser Asn Tyr Leu Ala1 5 1034411PRTHomo sapiens 344Arg Ala Ser Arg Ser Ile Ser Thr Tyr Leu Asn1 5 1034511PRTHomo sapiens 345Arg Ala Ser Gln Ser Ile His Thr Tyr Leu Asn1 5 1034617PRTHomo sapiens 346Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Asn Asn Tyr Leu1 5 10 15Ala34711PRTHomo sapiens 347Arg Ala Ser Gln Thr Ile Ser Thr Trp Leu Ala1 5 1034811PRTHomo sapiens 348Arg Ala Ser Gln Ser Ile Ser Thr Trp Leu Ala1 5 1034911PRTHomo sapiens 349Arg Ala Ser Gln Ser Ile Ser Asn Tyr Leu Asn1 5 1035011PRTHomo sapiens 350Arg Ala Ser Leu Asn Ile Arg Asn Tyr Leu Asn1 5 1035111PRTHomo sapiens 351Arg Ala Ser Gln Val Ile Gly Lys Tyr Leu Ala1 5 1035211PRTHomo sapiens 352Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly1 5 1035311PRTHomo sapiens 353Gln Ala Ser His Asp Ile Asn Lys Tyr Leu Asn1 5 1035411PRTHomo sapiens 354Arg Ala Ser Gln Ser Ile Asn Asn Tyr Leu Asn1 5 1035511PRTHomo sapiens 355Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn1 5 1035611PRTHomo sapiens 356Gln Ala Ser Gln Asp Ile Ser Phe Tyr Leu Asn1 5 1035711PRTHomo sapiens 357Arg Ala Ser Gln Ser Ile Ser Ser Trp Leu Ala1 5 1035811PRTHomo sapiens 358Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Ala1 5 1035911PRTHomo sapiens 359Arg Ala Ser Gln Ser Val Ser Thr Phe Leu Asn1 5 1036011PRTHomo sapiens 360Arg Ala Ser Gln Asp Val Ser Pro Trp Leu Ala1 5 1036111PRTHomo sapiens 361Arg Ala Ser Gln Gly Ile Ser Asp Trp Leu Ala1 5 1036211PRTHomo sapiens 362Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn1 5 1036316PRTHomo sapiens 363Arg Ser Ser Gln Ser Val Val Tyr Ser Asp Gly Asn Ile Tyr Leu Asn1 5 10 1536411PRTHomo sapiens 364Arg Ala Ser Gln Asp Ile Ser Ser Trp Leu Ala1 5 1036511PRTHomo sapiens 365Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn1 5 1036612PRTHomo sapiens 366Arg Ala Ser Gln Ser Val Ser Ser Leu Tyr Val Gly1 5 1036711PRTHomo sapiens 367Arg Ala Ser Gln Ser Ile Ser Ser Phe Leu Asn1 5 1036812PRTHomo sapiens 368Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala1 5 1036920PRTHomo sapiens 369Arg Ala Ser Gln Ser Val Ser Ser Arg Ala Ser Gln Ser Val Ser Ser1 5 10 15Asn Tyr Leu Ala 2037011PRTHomo sapiens 370Arg Ala Ser Gln Thr Ile Arg Asn Tyr Leu Asn1 5 1037112PRTHomo sapiens 371Arg Ala Ser Gln Ser Val Ser Asn Asn Asn Leu Ala1 5 1037211PRTHomo sapiens 372Arg Ala Ser Gln Gly Ile Ser Asn Trp Leu Ala1 5 1037311PRTHomo sapiens 373Arg Ala Ser Gln Gly Ile Gly Gly Ala Leu Ala1 5 1037412PRTHomo sapiens 374Arg Ala Ser Gln Ser Val Ser Ser Asn Tyr Leu Ala1 5 1037511PRTHomo sapiens 375Arg Ala Ser Gln Ser Ile Ser Arg His Leu Asn1 5 1037611PRTHomo sapiens 376Arg Ala Ser Gln Ser Ile Ser Thr Tyr Leu Ala1 5 1037711PRTHomo sapiens 377Arg Ala Ser Gln Gly Val Ser Asn Trp Val Ala1 5 1037811PRTHomo sapiens 378Arg Ala Ser Gln Gly Ile Ser Thr Phe Leu Ala1 5 1037912PRTHomo sapiens 379Arg Ala Ser Gln Ser Val Thr Ser Asn Tyr Leu Ala1 5 1038011PRTHomo sapiens 380Arg Ala Ser Gln Gly Ile Ser Thr Trp Leu Ala1 5 1038116PRTHomo sapiens 381Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp1 5 10 1538211PRTHomo sapiens 382Arg Ala Ser Gln Ser Ile Ser Asn Tyr Leu Asn1 5 1038311PRTHomo sapiens 383Arg Ala Ser Gln Ser Leu Asn Asn Trp Leu Ala1 5 1038412PRTHomo sapiens 384Arg Ala Ser Gln Ser Val Ser Asn Asn Tyr Leu Ala1 5 1038511PRTHomo sapiens 385Arg Ala Ser Gln Gly Ile Phe Asn Tyr Leu Ala1 5 1038611PRTHomo sapiens 386Arg Ala Ser Gln Asn Ile Gly Asn Trp Leu Ala1 5 1038711PRTHomo sapiens 387Arg Ala Ser Gln Asp Ile Ile Ser Trp Leu Ala1 5 1038811PRTHomo sapiens 388Arg Ala Ser Gln Ser Ile Tyr Ile Trp Leu Ala1 5 1038911PRTHomo sapiens 389Arg Ala Ser Gln Ser Ile Ser Arg Ser Leu Asn1 5 1039011PRTHomo sapiens 390Arg Ala Ser Gln Pro Ile Ser Ser Phe Leu Asn1 5 1039111PRTHomo sapiens 391Arg Ala Ser Gln Ser Ile Ser Ser Trp Leu Ala1 5 1039211PRTHomo sapiens 392Arg Ala Ser Gln Ser Phe Asn Gly Tyr Leu Asn1 5 1039312PRTHomo sapiens 393Arg Ala Ser Gln Thr Val Ile Ser Thr Tyr Leu Ala1 5 1039412PRTHomo sapiens 394Arg Ala Ser Gln Ser Val Ser Ser Gly Ser Leu Asp1 5 1039511PRTHomo sapiens 395Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn1 5 1039611PRTHomo sapiens 396Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn1 5 1039711PRTHomo sapiens 397Arg Ala Ser Gln Ser Ile Arg Ser Tyr Leu Asn1 5 1039811PRTHomo sapiens 398Arg Ala Ser Gln Gly Ile Ser Ser Tyr Leu Val1 5 1039911PRTHomo sapiens 399Arg Ala Ser Gln Ala Ile Ser Asn Tyr Leu Val1 5 1040012PRTHomo sapiens 400Arg Ala Ser Gln Ser Val Ser Ser Thr Tyr Leu Ala1 5 1040111PRTHomo sapiens 401Arg Ala Ser Gln Ser Ile Ser Asn His Leu Asn1 5 1040211PRTHomo sapiens 402Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn1 5 1040311PRTHomo sapiens 403Arg Ala Asn Gln Ser Ile Asp Asp Tyr Leu His1 5 1040416PRTHomo sapiens 404Arg Ser Ser Gln Ser Leu Val Tyr Ala Asp Gly Asp Thr His Leu Asn1 5 10 1540511PRTHomo sapiens 405Arg Ala Ser Gln Ser Ile Thr Asn Cys Leu Asn1 5 104067PRTHomo sapiens 406Asp Ala Ser Asn Arg Ala Thr1 54077PRTHomo sapiens 407Gly Ala Ser Gly Leu Glu Ser1 54087PRTHomo sapiens 408Lys Ala Ser Thr Leu Glu Ser1 54097PRTHomo sapiens 409Asp Ala Ser Ser Leu Lys Ser1 54107PRTHomo sapiens 410Asp Ala Ser Asn Leu Glu Thr1 54117PRTHomo sapiens 411Gly Ala Ser Ser Arg Ala Thr1 54127PRTHomo sapiens 412Ala Ala Ser Ser Leu Gln Ser1 54137PRTHomo sapiens 413Asp Ala Ser Asn Leu Glu Thr1 54147PRTHomo sapiens 414Leu Ala Ser Asn Arg Ala Ser1 54157PRTHomo sapiens 415Lys Ile Ser Asn Arg Phe Ser1 54167PRTHomo sapiens 416Ala Ala Ser Ser Leu Gln Ser1 54177PRTHomo sapiens 417Ala Val Ser Ser Leu Gln Thr1 54187PRTHomo sapiens 418Asp Ala Ser Asn Arg Asp Thr1 54197PRTHomo sapiens 419Thr Ala Ser Ser Leu Gln Ser1 54207PRTHomo sapiens 420Lys Ala Ser Asn Leu Glu Ser1 54217PRTHomo sapiens 421Leu Ala Ser Ser Leu Gln Ser1 54227PRTHomo sapiens 422Lys Val Ser Asn Arg Asp Ser1 54237PRTHomo sapiens 423Ala Ala Ser Ser Leu Gln Ser1 54247PRTHomo sapiens 424Ala Ala Ser Leu Leu Gln Thr1 54257PRTHomo sapiens 425Gly Ala Ser Thr Arg Ala Thr1 54267PRTHomo sapiens 426Gly Ala Ser Ser Arg Pro Thr1 54277PRTHomo sapiens 427Leu Gly Ser Thr Arg Ala Ser1 54287PRTHomo sapiens 428Asp Ala Ser Asn Leu Lys Thr1 54297PRTHomo sapiens 429Gly Ala Ser Ser Arg Ala Thr1 54307PRTHomo sapiens 430Ala Ala Ser Ser Leu Gln Ser1 54317PRTHomo sapiens 431Ala Ala Ser Ser Leu Gln Ser1 54327PRTHomo sapiens 432Gly Ala Ser Thr Leu Gln Ser1 54337PRTHomo sapiens 433Ala Ala Ser Ser Leu Gln Ser1 54347PRTHomo sapiens 434Thr Ala Ser Asn Leu Gln Ser1 54357PRTHomo sapiens 435Trp Ala Ser Thr Arg Glu Ser1 54367PRTHomo sapiens 436Asp Ala Ser Ser Leu Glu Ser1 54377PRTHomo sapiens 437Asp Ala Ser Ser Leu Glu Ser1 54387PRTHomo sapiens 438Gly Ala Ser Ser Leu Glu Ser1 54397PRTHomo sapiens 439Ala Ala Ser Thr Leu Gln Ile1 54407PRTHomo sapiens 440Ala Thr Ser Ile Leu Gln Ser1 54417PRTHomo sapiens 441Ala Ala Ser Ser Leu Gln Ser1 54427PRTHomo sapiens 442Asp Ala Ser Asn Leu Glu Thr1 54437PRTHomo sapiens 443Ala Ala Ser Ser Leu His Ser1 54447PRTHomo sapiens 444Asp Ala Ser His Leu Glu Thr1 54457PRTHomo sapiens 445Asp Ala Ser Ile Leu Glu Thr1 54467PRTHomo sapiens 446Lys Val Ser Ser Leu Glu Ser1 54477PRTHomo sapiens 447Ala Ala Ser Ser Leu Leu Ser1 54487PRTHomo sapiens 448Gly Val Ser Asn Leu Gln Ser1 54497PRTHomo sapiens 449Lys Ala Ser Ser Leu Glu Ser1 54507PRTHomo sapiens 450Lys Ala Ser Ser Leu Glu Ser1 54517PRTHomo sapiens 451Asp Thr Ser Asn Leu Glu Thr1 54527PRTHomo sapiens 452Gln Val Ser Asn Arg Asp Ser1 54537PRTHomo sapiens 453Ala Ala Ser Ser Leu Gln Ser1 54547PRTHomo sapiens 454Asp Ala Ser Asn Leu Glu Thr1 54557PRTHomo sapiens 455Gly Thr Ser Ser Arg Ala Thr1 54567PRTHomo sapiens 456Gly Ala Thr Thr Leu Gln Ser1 54577PRTHomo sapiens 457Arg Ala Ser Ser Arg Ala Ala1 54587PRTHomo sapiens 458Gly Ala Ser Thr Arg Ala Ala1 54597PRTHomo sapiens 459Thr Ala Ser Ser Leu His Ser1 54607PRTHomo sapiens 460Gly Ala Ser Ser Arg Ala Ala1 54617PRTHomo sapiens 461Ala Ala Ser Ser Leu Gln Ser1 54627PRTHomo sapiens 462Ala Ala Ser Thr Leu Gln Ser1 54637PRTHomo sapiens 463Gly Ala Ser Ser Arg Ala Thr1 54647PRTHomo sapiens 464Ala Ala Ser Ser Leu Gln Thr1 54657PRTHomo sapiens 465Lys Ala Ser Ser Leu Glu Pro1 54667PRTHomo sapiens 466Ala Ala Ser Ser Leu Gln Ser1 54677PRTHomo sapiens 467Ala Ala Ser Ser Leu Gln Ser1 54687PRTHomo sapiens 468Gly Ala Ser Asn Arg Ala Thr1 54697PRTHomo sapiens 469Ala Ala Ser Thr Leu Gln His1 54707PRTHomo sapiens 470Leu Gly Ser Asn Arg Ala Ser1 54717PRTHomo sapiens 471Ala Ala Ser Arg Leu Gln Ser1 54727PRTHomo sapiens 472Asp Ala Ser Ser Leu Gln Ser1 54737PRTHomo sapiens 473Gly Ala Ser Ser Arg Ala Thr1 54747PRTHomo sapiens 474Gly Ala Ser Thr Leu Arg Ser1 54757PRTHomo sapiens 475Ser Ala Ser Ser Leu Gln Asn1 54767PRTHomo sapiens 476Ala Ala Ser Ser Leu Gln Ser1 54777PRTHomo sapiens 477Asp Ala Ser Ser Leu Glu Ser1 54787PRTHomo sapiens 478Ala Ala Ser Thr Leu Gln Ser1 54797PRTHomo sapiens 479Ala Ala Ser Ser Leu Gln Ser1 54807PRTHomo sapiens 480His Ala Phe Ser Leu Glu Gly1 54817PRTHomo sapiens 481Ala Ala Ser Thr Leu Gln Ser1

54827PRTHomo sapiens 482Gly Ala Ser Ser Arg Ala Thr1 54837PRTHomo sapiens 483Gly Ala Ser Asn Arg Ala Ser1 54847PRTHomo sapiens 484Asp Ala Ser Asn Leu Glu Thr1 54857PRTHomo sapiens 485Ala Ala Ser Ser Leu Gln Ser1 54867PRTHomo sapiens 486Gly Ala Ser Ser Leu Gln Ser1 54877PRTHomo sapiens 487Ala Ala Ser Thr Leu Glu Ser1 54887PRTHomo sapiens 488Gly Ala Phe Ile Leu Glu Ser1 54897PRTHomo sapiens 489Gly Ala Ser Asn Arg Ala Thr1 54907PRTHomo sapiens 490Val Ala Ser Ser Leu Gln Gly1 54917PRTHomo sapiens 491Ala Ala Ser Ser Leu Gln Ser1 54927PRTHomo sapiens 492Asp Ala Ser Thr Leu His Ser1 54937PRTHomo sapiens 493His Val Ser Asn Arg Asp Ala1 54947PRTHomo sapiens 494Gly Ala Ser Thr Leu Gln Ser1 549511PRTHomo sapiens 495Cys Leu Gln Arg Ser Asn Trp Pro Ile Thr Phe1 5 1049611PRTHomo sapiens 496Cys Gln Gln Ser Tyr Arg Thr Leu Tyr Thr Phe1 5 1049712PRTHomo sapiens 497Cys Gln Gln Tyr Asn Gly Tyr Ser Glu Val Thr Phe1 5 1049811PRTHomo sapiens 498Cys Gln Gln Tyr Asp Thr Tyr Pro Ile Thr Phe1 5 1049911PRTHomo sapiens 499Cys Gln Gln Tyr Asp Asn Leu Pro Pro Thr Phe1 5 1050011PRTHomo sapiens 500Cys Gln Gln Tyr Gly Arg Ser Pro Leu Thr Phe1 5 1050111PRTHomo sapiens 501Cys Gln Gln Ser Tyr Asn Thr Pro Ala Thr Phe1 5 1050211PRTHomo sapiens 502Cys Gln Gln Tyr Glu Asn Val Pro Tyr Thr Phe1 5 1050311PRTHomo sapiens 503Cys Met Gln Ala Leu Gln Ile Pro Arg Thr Phe1 5 1050411PRTHomo sapiens 504Cys Met Gln Thr Thr His Ile Pro His Thr Phe1 5 1050511PRTHomo sapiens 505Cys Gln Gln Thr Tyr Gly Asn Pro Leu Thr Phe1 5 1050611PRTHomo sapiens 506Cys Gln Gln Ser Phe Arg Thr Pro His Thr Phe1 5 1050711PRTHomo sapiens 507Cys Gln Gln Arg Ala Ile Trp Pro Pro Glu Phe1 5 1050811PRTHomo sapiens 508Cys Gln Gln Ser Phe Thr Ser Trp Trp Thr Phe1 5 1050911PRTHomo sapiens 509Cys Gln Gln Tyr Ser Asn Tyr Pro Ile Thr Phe1 5 1051010PRTHomo sapiens 510Cys Leu Gln His Asn Ser Phe Leu Thr Phe1 5 1051111PRTHomo sapiens 511Cys Met Gln Gly Thr His Trp Pro Pro Ala Phe1 5 1051211PRTHomo sapiens 512Cys Leu Gln Leu His Thr Phe Pro Arg Thr Phe1 5 1051311PRTHomo sapiens 513Cys Gln Gln Gly Tyr Ser Thr Pro Tyr Thr Phe1 5 1051412PRTHomo sapiens 514Cys His Gln Tyr Asn Tyr Trp Pro Pro Leu Ala Phe1 5 1051511PRTHomo sapiens 515Cys Gln Gln Tyr Gly Ile Ser Pro Arg Thr Phe1 5 1051611PRTHomo sapiens 516Cys Met Gln Ala Leu Gln Thr Pro His Thr Phe1 5 1051711PRTHomo sapiens 517Cys His Gln Tyr Asp Ser Leu Pro Phe Thr Phe1 5 1051812PRTHomo sapiens 518Cys Gln Gln Phe Gly Tyr Ser Pro Arg Phe Thr Phe1 5 1051911PRTHomo sapiens 519Cys Gln Gln Thr Tyr Ile Thr Pro Lys Ser Phe1 5 1052011PRTHomo sapiens 520Cys Gln Gln Ser Tyr Arg Thr Pro Leu Thr Phe1 5 1052111PRTHomo sapiens 521Cys Gln Lys Tyr Asp Ser Ala Pro Tyr Thr Phe1 5 1052211PRTHomo sapiens 522Cys Gln Gln Thr Tyr Thr Ile Pro Leu Thr Phe1 5 1052311PRTHomo sapiens 523Cys Gln Gln Ser Tyr Ser Thr Leu Arg Thr Phe1 5 1052411PRTHomo sapiens 524Cys Gln Gln Tyr Tyr Lys Thr Pro Pro Thr Phe1 5 1052511PRTHomo sapiens 525Cys Gln Gln Tyr Asn Ser Tyr Pro Leu Thr Phe1 5 1052610PRTHomo sapiens 526Cys Gln Gln Tyr Asn Phe Tyr Gly Thr Phe1 5 1052711PRTHomo sapiens 527Cys Gln Gln Ser Tyr Ser Ile Pro Arg Thr Phe1 5 1052813PRTHomo sapiens 528Val Cys Gln Gln Ser Tyr Ser Met Ser Pro Tyr Thr Phe1 5 1052911PRTHomo sapiens 529Cys Gln Gln Tyr Asn Ser Phe Pro Leu Thr Phe1 5 1053011PRTHomo sapiens 530Cys Leu Gln Gln Asn Asn Tyr Pro Trp Thr Phe1 5 1053111PRTHomo sapiens 531Cys Gln Gln Tyr Asp Asn Phe Pro Tyr Thr Phe1 5 1053211PRTHomo sapiens 532Cys Gln Gln Thr Tyr Ile Ser Thr Arg Thr Phe1 5 1053310PRTHomo sapiens 533Cys Gln Gln Tyr Asp Asn Leu Pro Leu Phe1 5 1053410PRTHomo sapiens 534Cys Gln Gln Tyr Asp Asn Leu Ile Thr Phe1 5 1053511PRTHomo sapiens 535Cys Gln Gln Tyr Glu Ser Asp Ile Phe Thr Phe1 5 1053611PRTHomo sapiens 536Cys Gln Gln Tyr Gly Arg Tyr Pro Leu Thr Phe1 5 1053711PRTHomo sapiens 537Cys Gln Gln Ser His Arg Thr Pro Tyr Thr Phe1 5 1053810PRTHomo sapiens 538Cys Gln Gln Tyr Gln Thr Tyr Ser Thr Phe1 5 1053911PRTHomo sapiens 539Cys Gln Gln Tyr Glu Ser Asp Ser Trp Thr Phe1 5 1054011PRTHomo sapiens 540Cys Gln Gln Tyr Asp Asn Leu Pro Phe Thr Phe1 5 1054111PRTHomo sapiens 541Cys Met Gln Gly Thr His Trp Pro Tyr Ser Phe1 5 1054211PRTHomo sapiens 542Cys Gln Gln Ala Tyr Ser Phe Pro Trp Thr Phe1 5 1054310PRTHomo sapiens 543Cys Gln Gln Tyr Asp Asn Leu Pro Thr Phe1 5 1054411PRTHomo sapiens 544Cys Gln Gln Tyr Gly Thr Ser Pro Trp Thr Phe1 5 1054511PRTHomo sapiens 545Cys His Gln Ser Tyr Ser Leu Pro Phe Thr Phe1 5 1054611PRTHomo sapiens 546Cys Gln Gln Tyr Val Ala Ser Pro Phe Thr Phe1 5 1054711PRTHomo sapiens 547Cys His Gln Tyr Gly Thr Ser Pro Arg Thr Phe1 5 1054811PRTHomo sapiens 548Cys Gln Gln Ser Tyr Ile Thr Pro Tyr Thr Phe1 5 1054911PRTHomo sapiens 549Cys Gln Gln Tyr Gly Ser Ser Pro Tyr Thr Phe1 5 1055011PRTHomo sapiens 550Cys Gln Gln Ala Asn Ser Phe Pro Phe Thr Phe1 5 1055111PRTHomo sapiens 551Cys Gln Gln Leu Asp Thr Tyr Pro Leu Thr Phe1 5 1055211PRTHomo sapiens 552Cys Gln Gln Tyr Ala Ser Ser Pro Tyr Thr Phe1 5 1055311PRTHomo sapiens 553Cys Gln His Ser Ser Lys Thr Pro Phe Thr Phe1 5 1055410PRTHomo sapiens 554Cys Gln Gln Tyr Ser Ser Tyr Leu Ser Phe1 5 1055511PRTHomo sapiens 555Cys Gln Gln Ala Asn Gly Phe Leu Trp Thr Phe1 5 1055611PRTHomo sapiens 556Cys Gln Gln Ala His Ser Phe Pro Val Thr Phe1 5 1055711PRTHomo sapiens 557Cys Gln Gln Tyr Gly Ser Ser Pro Leu Thr Phe1 5 1055811PRTHomo sapiens 558Cys Gln Gln Ala Asn Ser Phe Pro Arg Thr Phe1 5 1055910PRTHomo sapiens 559Cys Met Gln Ser Leu Gln Thr Val Thr Phe1 5 1056012PRTHomo sapiens 560Cys Gln His Ser Tyr Glu Thr Pro Pro Tyr Thr Phe1 5 1056111PRTHomo sapiens 561Cys Gln Gln Tyr Asn Phe Tyr Pro Trp Thr Phe1 5 1056211PRTHomo sapiens 562Cys Gln Gln Tyr Gly Gly Ser Pro Gln Thr Phe1 5 1056311PRTHomo sapiens 563Cys Gln Lys Tyr Asn Ser Ala Pro Leu Thr Phe1 5 1056411PRTHomo sapiens 564Cys Gln Gln Ala Asn Ser Phe Pro Val Thr Phe1 5 1056512PRTHomo sapiens 565Cys Gln Gln Thr His Ser Phe Pro Pro Trp Thr Phe1 5 1056611PRTHomo sapiens 566Cys Gln Gln Tyr His His Tyr Ser Pro Thr Phe1 5 1056711PRTHomo sapiens 567Cys Gln Gln Ser Tyr Ser Thr Leu Arg Thr Phe1 5 1056811PRTHomo sapiens 568Cys Gln Gln Gly Tyr Ser Thr Pro Pro Thr Phe1 5 1056911PRTHomo sapiens 569Cys Gln Gln Tyr Asp Ser Tyr Pro Tyr Thr Phe1 5 1057011PRTHomo sapiens 570Cys Gln Gln Ser Tyr Ser Thr Pro Arg Thr Phe1 5 1057110PRTHomo sapiens 571Cys Gln Gln Tyr Ser Asp Ser Leu Thr Phe1 5 1057211PRTHomo sapiens 572Cys His Gln Tyr Gly Gly Ala Gln Gly Thr Phe1 5 1057312PRTHomo sapiens 573Cys Gln Gln Tyr Asp Thr Leu Pro Pro Ile Thr Phe1 5 1057411PRTHomo sapiens 574Cys Gln Gln Ser His Ser Ser Pro Trp Thr Phe1 5 1057511PRTHomo sapiens 575Cys Gln Gln Ser Tyr Leu Ala Pro Trp Thr Phe1 5 1057611PRTHomo sapiens 576Cys Gln Gln Phe Asn Asn Tyr Pro Tyr Thr Phe1 5 1057711PRTHomo sapiens 577Cys Gln Gln Tyr His Thr Tyr Pro Phe Thr Phe1 5 1057812PRTHomo sapiens 578Cys Gln Lys Tyr Gly Arg Ser Pro Thr Trp Thr Phe1 5 1057911PRTHomo sapiens 579Cys Gln Gln Gly Phe Thr Thr Pro Ile Thr Phe1 5 1058011PRTHomo sapiens 580Cys Gln Gln Ser Tyr Ser Thr Pro Tyr Thr Phe1 5 1058112PRTHomo sapiens 581Cys Gln Gln Ser Tyr Thr Ile Pro Leu Trp Thr Phe1 5 1058211PRTHomo sapiens 582Cys Met Gln Gly Thr His Trp Pro Pro Thr Phe1 5 1058311PRTHomo sapiens 583Cys Gln Gln Ser Asp Ser Thr Pro Tyr Thr Phe1 5 10584263PRTVaccinia virus 584Met Lys Val Glu Ser Val Thr Phe Leu Thr Leu Leu Gly Ile Gly Cys1 5 10 15Val Leu Ser Cys Cys Thr Ile Pro Ser Arg Pro Ile Asn Met Lys Phe 20 25 30Lys Asn Ser Val Glu Thr Asp Ala Asn Ala Asn Tyr Asn Ile Gly Asp 35 40 45Thr Ile Glu Tyr Leu Cys Leu Pro Gly Tyr Arg Lys Gln Lys Met Gly 50 55 60Pro Ile Tyr Ala Lys Cys Thr Gly Thr Gly Trp Thr Leu Phe Asn Gln65 70 75 80Cys Ile Lys Arg Arg Cys Pro Ser Pro Arg Asp Ile Asp Asn Gly Gln 85 90 95Leu Asp Ile Gly Gly Val Asp Phe Gly Ser Ser Ile Thr Tyr Ser Cys 100 105 110Asn Ser Gly Tyr His Leu Ile Gly Glu Ser Lys Ser Tyr Cys Glu Leu 115 120 125Gly Ser Thr Gly Ser Met Val Trp Asn Pro Glu Ala Pro Ile Cys Glu 130 135 140Ser Val Lys Cys Gln Ser Pro Pro Ser Ile Ser Asn Gly Arg His Asn145 150 155 160Gly Tyr Glu Asp Phe Tyr Thr Asp Gly Ser Val Val Thr Tyr Ser Cys 165 170 175Asn Ser Gly Tyr Ser Leu Ile Gly Asn Ser Gly Val Leu Cys Ser Gly 180 185 190Gly Glu Trp Ser Asp Pro Pro Thr Cys Gln Ile Val Lys Cys Pro His 195 200 205Pro Thr Ile Ser Asn Gly Tyr Leu Ser Ser Gly Phe Lys Arg Ser Tyr 210 215 220Ser Tyr Asn Asp Asn Val Asp Phe Lys Cys Lys Tyr Gly Tyr Lys Leu225 230 235 240Ser Gly Ser Ser Ser Ser Thr Cys Ser Pro Gly Asn Thr Trp Lys Pro 245 250 255Glu Leu Pro Lys Cys Val Arg 260585265PRTCamelpox virus 585Met Lys Val Glu Ser Val Thr Phe Leu Thr Leu Leu Gly Ile Val Cys1 5 10 15Val Leu Ser Cys Cys Thr Ile Pro Ser Arg Pro Ile Asn Met Lys Phe 20 25 30Lys Asn Ser Val Glu Thr Tyr Ala Asn Thr Asn Thr Asn Tyr Asn Ile 35 40 45Gly Asp Thr Ile Glu Tyr Leu Cys Leu Pro Gly Tyr Arg Lys Gln Lys 50 55 60Met Gly Pro Ile Tyr Ala Lys Cys Thr Gly Thr Gly Trp Thr Leu Phe65 70 75 80Asn Gln Cys Ile Lys Arg Arg Cys Pro Ser Pro Arg Asp Ile Asp Asn 85 90 95Gly Gln Leu Asp Ile Gly Gly Val Asp Phe Gly Ser Ser Ile Thr Tyr 100 105 110Ser Cys Asn Ser Gly Tyr His Leu Ile Gly Glu Ser Lys Ser Tyr Cys 115 120 125Glu Leu Gly Ser Thr Gly Ser Met Val Trp Asn Pro Glu Ala Pro Ile 130 135 140Cys Glu Ser Val Lys Cys Gln Ser Pro Pro Ser Ile Ser Asn Gly Arg145 150 155 160His Asn Gly Tyr Asp Asn Phe Tyr Thr Asp Gly Ser Val Val Thr Tyr 165 170 175Ser Cys Asn Ser Gly Tyr Ser Leu Ile Gly Asn Ser Gly Val Leu Cys 180 185 190Ser Gly Gly Glu Trp Ser Asp Pro Pro Thr Cys Gln Ile Val Lys Cys 195 200 205Pro His Pro Thr Ile Ser Asn Gly Tyr Leu Ser Ser Gly Phe Lys Arg 210 215 220Ser Tyr Ser Tyr Asn Asp Asn Val Asp Phe Thr Cys Lys Tyr Gly Tyr225 230 235 240Lys Leu Ser Gly Ser Ser Ser Ser Thr Cys Ser Pro Gly Asn Thr Trp 245 250 255Gln Pro Glu Leu Pro Lys Cys Val Arg 260 265586263PRTVariola virus 586Met Lys Val Glu Arg Val Thr Phe Leu Thr Leu Leu Gly Ile Gly Cys1 5 10 15Val Leu Ser Cys Cys Thr Ile Pro Ser Arg Pro Ile Asn Met Lys Phe 20 25 30Lys Asn Ser Val Glu Thr Asp Ala Asn Ala Asn Tyr Asn Ile Gly Asp 35 40 45Thr Ile Glu Tyr Leu Cys Leu Pro Gly Tyr Arg Lys Gln Lys Met Gly 50 55 60Pro Ile Tyr Ala Lys Cys Thr Gly Thr Gly Trp Thr Leu Phe Asn Gln65 70 75 80Cys Ile Lys Arg Arg Cys Pro Ser Pro Arg Asp Ile Asp Asn Gly His 85 90 95Leu Asp Ile Gly Gly Val Asp Phe Gly Ser Ser Ile Thr Tyr Ser Cys 100 105 110Asn Ser Gly Tyr Tyr Leu Ile Gly Glu Tyr Lys Ser Tyr Cys Lys Leu 115 120 125Gly Ser Thr Gly Ser Met Val Trp Asn Pro Lys Ala Pro Ile Cys Glu 130 135 140Ser Val Lys Cys Gln Leu Pro Pro Ser Ile Ser Asn Gly Arg His Asn145 150 155 160Gly Tyr Asn Asp Phe Tyr Thr Asp Gly Ser Val Val Thr Tyr Ser Cys 165 170 175Asn Ser Gly Tyr Ser Leu Ile Gly Asn Ser Gly Val Leu Cys Ser Gly 180 185 190Gly Glu Trp Ser Asn Pro Pro Thr Cys Gln Ile Val Lys Cys Pro His 195 200 205Pro Thr Ile Leu Asn Gly Tyr Leu Ser Ser Gly Phe Lys Arg Ser Tyr 210 215 220Ser Tyr Asn Asp Asn Val Asp Phe Thr Cys Lys Tyr Gly Tyr Lys Leu225 230 235 240Ser Gly Ser Ser Ser Ser Thr Cys Ser Pro Gly Asn Thr Trp Gln Pro 245 250 255Glu Leu Pro Lys Cys Val Arg 260587263PRTCowpox virus 587Met Lys Val Glu Ser Val Thr Phe Leu Thr Leu Leu Gly Ile Gly Cys1 5 10 15Val Leu Ser Cys Cys Thr Ile Pro Ser Arg Pro Ile Asn Met Lys Phe 20 25 30Lys Asn Ser Val Gly Thr Asp Ala Asn Ala Asn Tyr Asn Ile Gly Asp 35 40 45Thr Ile Glu Tyr Leu Cys Leu Pro Gly Tyr Arg Lys Gln Lys Met Gly 50 55 60Pro Ile Tyr Ala Lys Cys Thr Gly Thr Gly Trp Thr Leu Phe Asn Gln65 70 75 80Cys Ile Lys Arg Lys Cys Pro Ser Pro Arg Asp Ile Asp Asn Gly Gln 85 90 95Ile Asp Ile Gly Gly Val Glu Phe Gly Ser Ser Ile Thr Tyr Ser Cys 100 105 110Asn Ser Gly Tyr Gln Leu Ile Gly Glu Ser Lys Ser Tyr Cys Glu Leu 115 120 125Gly Tyr Thr Gly Ser Met Val Trp Asn Pro Glu Ala Pro Ile Cys Glu 130 135 140Ser Val Lys Cys Pro Ser Pro Pro Ser Val Thr Asn Gly Arg His Asn145 150 155 160Gly Tyr Glu Asp Phe Tyr Thr Asp Gly Ser Val Val Thr Tyr Ser Cys 165 170 175Asn Ser Gly Tyr Ser Leu Ile Gly Asn Ser Gly Ile Val Cys Ser Gly 180 185 190Gly Glu Trp Ser Asp Pro Pro Thr Cys Gln Ile Val Lys Cys Pro His 195 200 205Pro Ser Ile Thr Asn Gly Tyr Leu Ser Ser Gly Phe Lys Arg Ser Tyr 210 215 220Ser His Asn Asp Asn Val Asp Phe Lys Cys Arg His Gly Tyr Lys Leu225 230 235 240Ser Gly Ser Ser Ser Ser Thr Cys Ser Pro Gly Asn Thr Trp Gln Pro 245

250 255Glu Leu Pro Lys Cys Val Arg 260588216PRTMonkeypox virus 588Met Lys Val Glu Ser Val Thr Phe Leu Thr Leu Leu Gly Ile Gly Cys1 5 10 15Val Leu Ser Tyr Cys Thr Ile Pro Ser Arg Pro Ile Asn Met Lys Phe 20 25 30Lys Asn Ser Val Glu Thr Asp Ala Asn Tyr Asn Ile Gly Asp Thr Ile 35 40 45Glu Tyr Leu Cys Leu Pro Gly Tyr Arg Lys Gln Lys Met Gly Pro Ile 50 55 60Tyr Ala Lys Cys Thr Gly Thr Gly Trp Thr Leu Phe Asn Gln Cys Ile65 70 75 80Lys Arg Arg Cys Pro Ser Pro Arg Asp Ile Asp Asn Gly Gln Leu Asp 85 90 95Ile Gly Gly Val Asp Phe Gly Ser Ser Ile Thr Tyr Ser Cys Asn Ser 100 105 110Gly Tyr His Leu Ile Gly Glu Ser Lys Ser Tyr Cys Glu Leu Gly Ser 115 120 125Thr Gly Ser Met Val Trp Asn Pro Glu Ala Pro Ile Cys Glu Ser Val 130 135 140Lys Cys Gln Ser Pro Pro Ser Ile Ser Asn Gly Arg His Asn Gly Tyr145 150 155 160Glu Asp Phe Tyr Ile Asp Gly Ser Ile Val Thr Tyr Ser Cys Asn Ser 165 170 175Gly Tyr Ser Leu Ile Gly Asn Ser Gly Val Met Cys Ser Gly Gly Glu 180 185 190Trp Ser Asn Pro Pro Thr Cys Gln Ile Val Lys Cys Pro His Pro Ile 195 200 205Ser Asn Gly Lys Leu Leu Ala Ala 210 215589255PRTArtificial sequenceConsensus sequence 589Met Lys Val Glu Ser Val Thr Phe Leu Thr Leu Leu Gly Ile Gly Cys1 5 10 15Val Leu Ser Cys Cys Thr Ile Pro Ser Arg Pro Ile Asn Met Lys Phe 20 25 30Lys Asn Ser Val Glu Thr Asp Ala Asn Asn Tyr Asn Ile Gly Asp Thr 35 40 45Ile Glu Tyr Leu Cys Leu Pro Gly Tyr Arg Lys Gln Lys Met Gly Pro 50 55 60Ile Tyr Ala Lys Cys Thr Gly Thr Gly Trp Thr Leu Phe Asn Gln Cys65 70 75 80Ile Lys Arg Arg Cys Pro Ser Pro Arg Asp Ile Asp Asn Gly Gln Leu 85 90 95Asp Ile Gly Gly Val Xaa Phe Gly Ser Ser Ile Thr Tyr Ser Cys Asn 100 105 110Ser Gly Tyr His Leu Ile Gly Glu Ser Lys Ser Tyr Cys Glu Leu Gly 115 120 125Ser Thr Gly Ser Met Val Trp Asn Pro Glu Ala Pro Ile Cys Glu Ser 130 135 140Val Lys Cys Gln Ser Pro Pro Ser Xaa Ser Asn Gly Arg His Asn Gly145 150 155 160Tyr Xaa Xaa Phe Tyr Thr Asp Gly Ser Xaa Val Thr Tyr Ser Cys Asn 165 170 175Ser Gly Tyr Ser Leu Ile Gly Asn Ser Gly Xaa Cys Ser Gly Gly Glu 180 185 190Trp Ser Xaa Pro Pro Thr Cys Gln Ile Val Lys Cys Pro His Pro Ile 195 200 205Ser Asn Gly Tyr Leu Ser Ser Gly Phe Lys Arg Ser Tyr Ser Asn Asp 210 215 220Asn Val Asp Phe Cys Gly Tyr Lys Leu Ser Gly Ser Ser Ser Ser Thr225 230 235 240Cys Ser Pro Gly Asn Thr Trp Pro Glu Leu Pro Lys Cys Val Arg 245 250 255

Claims

1. An anti-orthopoxvirus recombinant polyclonal antibody comprising distinct members which in union are capable of binding at least three orthopoxvirus related antigens.

2. The anti-orthopoxvirus recombinant polyclonal antibody according to claim 1, wherein at least two distinct epitopes on the same orthopoxvirus related antigen are bound by said polyclonal antibody.

3. The anti-orthopoxvirus recombinant polyclonal antibody according to claim 1, wherein the distinct antibody members mirror the humeral immune response with respect to diversity, affinity and specificity against antigens associated with one or more orthopoxviruses.

4. The anti-orthopoxvirus recombinant polyclonal antibody according to claim 1, wherein the distinct antibodies are encoded by nucleic acid sequences obtained from one or more donors who have raised a humeral immune response against an orthopoxvirus.

5. The anti-orthopoxvirus recombinant polyclonal antibody according to claim 4, wherein the donors have been vaccinated with a vaccinia virus strain or are recovering from an orthopoxvirus infection.

6. The anti-orthopoxvirus recombinant polyclonal antibody according to claim 4, wherein the one or more donors are human, and the polyclonal antibody is a fully human antibody.

7. The anti-orthopoxvirus recombinant polyclonal antibody according to claim 3, wherein the distinct antibody members comprise V_H and V_L pairs originally present in the one or more donors.

8. The anti-orthopoxvirus recombinant polyclonal antibody according to claim 3, wherein the specificity of the individual members of the anti-orthopoxvirus recombinant polyclonal antibody are selected such that the antibody composition collectively binds antigens that elicit significant antibody responses in mammals.

9. The anti-orthopoxvirus recombinant polyclonal antibody according to claim 1, which comprises binding reactivity against Intracellular Mature Virions (IMV) as well as Extracellular Enveloped Virions (EEV) specific antigens, where the binding reactivity is characterized by distinct members capable of binding either an IMV or an EEV specific antigen.

10. The anti-orthopoxvirus recombinant polyclonal antibody according to claim 9, which comprises binding reactivity against antigens selected among the IMV viral proteins A27L, A17L, D8L and H3L and antigens selected among the EEV viral proteins A33R and B5R.

11. The anti-orthopoxvirus recombinant polyclonal antibody according to claim 10, where the group of antigens from IMV additionally comprises the viral protein L1R and the group of antigens from EEV additionally comprises the viral protein A56R.

12. The anti-orthopoxvirus recombinant polyclonal antibody according to claim 1, where said polyclonal antibody comprises at least one individual antibody member with binding reactivity against an orthopoxvirus related regulators of complement activation (RCA) protein encoded by an orthopoxvirus.

13. The anti-orthopoxvirus recombinant polyclonal antibody according to claim 12, wherein the RCA binding reactivity is directed to a protein selected from the group consisting of VCP, SPICE, IMP, MPXV-VCP and CMLV-VCP.

14. The anti-orthopoxvirus recombinant polyclonal antibody according to claim 12, which comprises binding reactivity to at least two of the RCA domains selected from the group consisting of SCR2, SCR4 and the junction between the SCR3 and 4 domains.

15. The anti-orthopoxvirus recombinant polyclonal antibody according to claim 3, which additionally contains one or more distinct antibodies encoded from V_H and V_L pairs selected from one or more donors which have been immunized or vaccinated with a particular orthopoxvirus related antigen.

16-18. (canceled)

19. A method for treatment or prevention of adverse side effects of vaccination with vaccinia virus in a human or an animal, wherein an effective amount of the anti-orthopoxvirus recombinant polyclonal antibody according to claim 1 is administered to said human or animal.

20. A method for treatment or prophylaxis of an orthopoxvirus infection in a human or animal, wherein an effective amount of the anti-orthopoxvirus recombinant polyclonal according to claim 1 is administered to said human or animal.

21. (canceled)

22. A pharmaceutical composition comprising as an active ingredient, an anti-orthopoxvirus recombinant polyclonal antibody according to claim 1 and a pharmaceutically acceptable excipient.

23. A screening procedure for selecting V₁₄ and V_L sequence pairs capable of encoding a broad diversity of anti-orthopoxvirus antibodies comprising: (a) expressing an antibody or antibody fragment from a host cell transfected with a screening vector comprising a distinct member of the repertoire of V_H and V_L coding pairs, (b) contacting said antibody or antibody fragment with at least two different vaccinia virus strains and one or more antigens selected from the group consisting of A27L, A17L, D8L, H3L, L1R, A33R, B5R and VCP in parallel, (c) repeating step a) and b) for each V_H and V_L sequence pair in the repertoire of sequence pairs, and (d) selecting the V_H and V_L sequence pairs encoding an antibody or antibody fragment which bind to at least one of the vaccinia virus strains or one of the antigens.

24. The screening method according to claim 23, wherein the V_H and V_L coding pairs are cognate pairs.

25. The screening method according to claim 23, wherein the screening procedure does not utilize phage display.

26. A method for generating a repertoire of V_H and V_L coding pairs, where the members mirror the gene pairs responsible for the humeral immune response upon challenge with an orthopoxvirus, comprising: (a) providing a lymphocyte-containing cell fraction from a donor vaccinated with an orthopoxvirus or recovering from an orthopoxvirus infection; (b) optionally enriching B cells or plasma cells from said cell fraction; (c) obtaining a population of isolated single cells, comprising distributing cells from said cell fraction individually into a plurality of vessels; (d) amplifying and effecting linkage of the V_H and V_L coding pairs, in a multiplex overlap extension RT-PCR procedure, using a template derived from said isolated single cells; and (e) optionally performing a nested PCR of the linked V_H and V_L coding pairs.

27. The method according to claim 26, wherein the linked V_H and V_L coding pairs are subjected to a screening procedure comprising: (a) expressing an antibody or antibody fragment from a host cell transfected with a screening vector comprising a distinct member of the repertoire of V_H and V_L coding pairs, (b) contacting said antibody or antibody fragment with at least two different vaccinia virus strains and one or more antigens selected from the group consisting of A27L, A17L, D8L, H3L, L1R, A33R, B5R and VCP in parallel, (c) repeating step a) and b) for each V_H and V_L sequence pair in the repertoire of sequence pairs, and (d) selecting the V_H and V_L sequence pairs encoding an antibody or antibody fragment which bind to at least one of the vaccinia virus strains or one of the antigens.

28. A polyclonal cell line capable of expressing a recombinant polyclonal anti-orthopoxvirus antibody according to claim 1.

29. A polyclonal cell line wherein each individual cell is capable of expressing a single V_H and V_L coding pair and the polyclonal cell line as a whole is capable of expressing a collection of V_H and V_L coding pairs, where each V_H and V_L coding pair encode an anti-orthopoxvirus antibody.

30. The polyclonal cell line according to claim 29, wherein said collection of V_H and V_L coding pairs are generated according to the method comprising: (a) providing a lymphocyte-containing cell fraction from a donor vaccinated with an orthopoxvirus or recovering from an orthopoxvirus infection; (b) optionally enriching B cells or plasma cells from said cell fraction; (c) obtaining a population of isolated single cells, comprising distributing cells from said cell fraction individually into a plurality of vessels; (d) amplifying and effecting linkage of the V_H and V_L coding pairs, in a multiplex overlap extension RT-PCR procedure, using a template derived from said isolated single cells; and (e) optionally performing a nested PCR of the linked V_H and V_L coding pairs.

31. The anti-orthopoxvirus recombinant polyclonal antibody according to claim 1, wherein said anti-orthopoxvirus recombinant polyclonal antibody is essentially free from immunoglobulin molecules that do not bind to orthopoxvirus antigens.

32. The anti-orthopoxvirus recombinant polyclonal antibody according to claim 1, wherein the constant region of said anti-orthopoxvirus recombinant polyclonal antibody belongs to the isotype of IgG, IgA, IgE, IgM, or IgD.

33. The anti-orthopoxvirus recombinant polyclonal antibody according to claim 1, wherein the constant region of said anti-orthopoxvirus recombinant polyclonal antibody belongs to one or two particular immunoglobulin subtypes of IgG or IgA.

Images