# Patent

## Title
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 VH and VL pairs useful when preparing the polyclonal antibody.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of Danish Application No. PA 2005 01720, filed Dec. 5, 2005, herein incorporated by reference.
REFERENCE TO A SEQUENCE LISTING SUBMITTED ON A COMPACT DISC
This application includes a “Sequence Listing,” which is provided as an electronic document on a compact disc (CD-R). This compact disc contains the file “Sequence Listing ASCII—2488.0020000.txt” (116 kilobytes, created on Mar. 16, 2007), which is hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
1. Field of the Invention
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-VV 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.
2. Background Art
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.
Orthopoxviruses produces two types infectious particles, namely the Intracellular Mature Virions (EMV) 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 1





Antigen
Virion
Antibody effect
Immunization/vaccination effect







A27L
IMV
+ neutralize11,12,15
DNA: + neutralize ÷ protective6


(P14)

(+) protective11
Protein: + neutralize + protective1,8




− protective15


A17L
IMV
+ neutralize13


(P21)


L1R
IMV
+ neutralize10,14,15
DNA: + neutralize (+) protective5


(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.

    1. Demkowicz et al. 1992, J. Virol. 66:386-98.
    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
    3. Galmiche et al, 1999, Virology 254:71-80.
    4. Gordon et al. 1991, Virology 181:671-86
    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.
    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 combination.
    7. Law et al. 2001, Virology 280:132-42.
    8. Lai et al. 1991, J. Virol. 65:5631-5.
    9. Lin et al. 2000, J. Virol. 74:3353-3365.
    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.
    11. Ramirez et al. 2002, J. Gen. Virol. 83:1059-1067.
    12. Rodriguez et al. 1985, J. Virol. 56:482-488.
    13. Wallengren et al. 2001, Virology 290:143-52.
    14. Wolffe et al. 1995, Virology 211:53-63.
    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.
    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.


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).
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
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
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.
The present invention provides a polyclonal anti-orthopoxvirus antibody. 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.
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.
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 VH and VL gene pairs from such challenged individuals, and producing antibodies maintaining this original paring.
DEFINITIONS
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.
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 (VH and VL regions), in particular in the CDR1, CDR2 and CDR3 regions.
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 (VH and VL regions), in particular in the CDR1, CDR2 and CDR3 regions.
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.
The term “cognate VH and VL coding pair” describes an original pair of VH and VL coding sequences contained within or derived from the same cell. Thus, a cognate VH and VL pair represents the VH and VL pairing originally present in the donor from which such a cell is derived. The term “an antibody expressed from a VH and VL coding pair” indicates that an antibody or an antibody fragment is produced from a vector, plasmid or similar containing the VH and VL coding sequence. When a cognate VH and VL 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.
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.
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.
The term “fully human” used for example in relation to DNA, RNA or protein sequences describes sequences which are between 98 to 100% human.
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.
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 (VH) and the variable light chain (VL) 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 VH and VL 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.
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.
A composition is said to be “pharmacologically acceptable” if its administration can be tolerated by a recipient patient.
The term “unit dose form” refers to a ready-to-administer form comprising a therapeutically effective amount of the active ingredient.
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.
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.
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.
The terms “sequences encoding VH and VL pairs” or “VH and VL 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.
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
FIG. 1: Alignment of amino acid sequences of known orthopoxvirus related regulators of complement activation (RCA), including a consensus sequence.
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 VH and Vκ gene families, respectively, were used for the first PCR step. A homologous region between the VH or Vκ 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 VH and Vκ 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-NheI restriction site between the linked VH and Vκ 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.
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 VH and VL 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 VH and VL 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.
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=mammalian 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
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.
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.
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.
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.
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.).
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.
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.
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).
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.
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
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.
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.
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 VH and VL 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.
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.
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 VH and VL regions of the individual antibody members in such an anti-orthopoxyirsus 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 VH and VL coding sequences are isolated from the donor(s) such that the original pairing of the VH and VL coding sequences present in the donor(s) is maintained, iii) the VH and VL 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).
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, 15L, A13L, A27L, A17L, L1R, L5R, D8L, 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.
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.
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, 15L, 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.
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. U.S. 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).
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.
The present invention has identified a series of VH and VL 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 VH and VL pairs are identified by clone number in Table 5 in Example 2. An antibody containing a VH and VL pair as identified in Table 5 is preferably a fully human antibody. However, if desired chimeric antibodies may also be produced.
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 VH and VL 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 VH and VL 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
An embodiment of the present invention is an antibody binding antigen J 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. 02-640 and/or 02-633.
An embodiment of the present invention is an antibody binding antigen L 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. 02-589, 02-156, and/or 02-225.
Isolation and Selection of Variable Heavy Chain and Variable Light Chain Coding Pairs
The process of generating an anti-orthopoxvirus recombinant polyclonal antibody composition involves the isolation of sequences coding for variable heavy chains (VH) and variable light chains (VL) from a suitable source, thereby generating a repertoire of VH and VL coding pairs. Generally, a suitable source for obtaining VH and VL 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 VH and VL 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.
The isolation of VH and VL coding sequences can either be performed in the classical way where the VH and VL coding sequences are combined randomly in a vector to generate a combinatorial library of VH and VL 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 VH and VL pairing originally present in the donor, thereby generating a repertoire of sequence pairs where each pair encodes a variable heavy chain (VH) and a variable light chain (VL) corresponding to a VH and VL pair originally present in an antibody produced by the donor from which the sequences are isolated. This is also termed a cognate pair of VH and VL encoding sequences and the antibody is termed a cognate antibody. Preferably, the VH and VL coding pairs of the present invention, combinatorial or cognate, are obtained from human donors, and therefore the sequences are completely human.
There are several different approaches for the generation of cognate pairs of VH and VL encoding sequences, one approach involves the amplification and isolation of VH and VL encoding sequences from single cells sorted out from a lymphocyte-containing cell fraction. The VH and VL 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 VH and VL 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 VH and VL encoding sequence pairs which resemble the diversity of VH and VL sequence pairs in the donor, a high-throughput method with as little scrambling (random combination) of the VH and VL pairs as possible, is preferred, e.g. as described in WO 05/042774 (hereby incorporated by reference).
In a preferred embodiment of the present invention a repertoire of VH and VL 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 VH and VL 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 VH and VL coding pairs. Preferably the isolated cognate VH and VL coding pairs are subjected to a screening procedure as described below.
Once the VH and VL sequence pairs have been generated a screening procedure to identify sequences encoding VH and VL pairs with binding reactivity towards an orthopoxvirus associated antigen is performed. If the VH and VL sequence pairs are combinatorial a phage display procedure can be applied to enrich for VH and VL pairs coding for antibody fragments binding to orthopoxvirus prior to screening.
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 VH and VL 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.
In an embodiment of the present invention the screening procedure for selecting VH and VL 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 VH and VL 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 VH and VL coding pairs, and sequences encoding VH and VL 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 VH and VL 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 VH and VL pairs. In the event that the repertoire of VH and VL 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).
The VH and VL 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 VH and VL family representation is of interest. Based on these analyses, sequences encoding VH and VL 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 VH or VL families, these are also selected. The selection of VH and VL 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.
When it is ensured that the overall diversity of the collection of selected sequences encoding VH and VL 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 VH and VL 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 VH and VL 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 VH and VL coding pairs. Additionally, the specificity of the antibodies expressed from a collection of selected VH and VL 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 VH and VL 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 VH and VL 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 VH and VL Coding Pairs
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.
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 VH and VL 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 VH and VL 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.
Preferably mammalian cells such as CHO cells, COS cells, BHK cells, myeloma cells (e.g., Sp2/0 or NS0 cells), fibroblasts such as NIH 3T3, and immortalized human cells, such as HeLa cells, HEK 293 cells, or PER.C6, are used. However, non-mammalian eukaryotic or prokaryotic cells, such as plant cells, insect cells, yeast cells, fingi, 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).
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, 10× 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 VH and VL 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 VH and VL chains. FIG. 4 illustrates one possible way to design the expression vector, although numerous other designs are possible.
The transfer of the selected VH and VL coding pairs from the screening vector can be performed by conventional restriction enzyme cleavage and ligation, such that each expression vector molecule contain one VH and VL coding pair. Preferably, the VH and VL coding pairs are transferred individually, they may, however, also be transferred in-mass if desired. When all the selected VH and VL 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.
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.
One embodiment of the present invention is a polyclonal cell line capable of expressing a recombinant polyclonal anti-orthopoxvirus antibody of the present invention.
A further embodiment of the present invention is a polyclonal cell line wherein each individual cell is capable of expressing a single VH and VL coding pair, and the polyclonal cell line as a whole is capable of expressing a collection of VH and VL coding pairs, where each VH and VL coding pair encode an anti-orthopoxvirus antibody of the present invention. Preferably the collection of VH and VL coding pairs are cognate pairs generated according to the methods of the present invention.
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).
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
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.
The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
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).
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.
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.).
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.
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.
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.
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
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.
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.
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.
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
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
This example is a collection of the methods applied to illustrate the present invention.
a. Sorting of Plasma Cells from Donor Blood
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.
Plasma blasts or circulating plasma cells (hereafter plasma cells) were selected from the CD19+ 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 marker 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+ 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.
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.

    Gate 1: FSC/SSC gate. The lymphocyte population having the highest FSC is selected ensuring sorting of living cells.
    Gate 2: SSCh/SSCw. This gate ensures sorting of single cells.
    Gate 3: CD19+ cells. In the FL1-FL2 dot plot, only the CD19 positive cells are selected.
    Gate 4: In the FL2-FL3 dot plot, a discrete population should be visible, which is CD38high and CD45intermediate.

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



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 VH and VL Pairs
The linkage of VH and VL coding sequences was performed on the single cells obtained as described in section a), facilitating cognate pairing of the VH and VL 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.
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).
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.
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 2







RT-PCR multiplex overlap-extension


primer mix













Final





Primer
Conc.





name
μM
Sequence
SEQ ID















VH set






CH-IgG
0.2
GACSGATGGGCCCTTGGTGG



 



CH-IgA
0.2
GAGTGGCTCCTGGGGGAAGA



 



VH-1
0.04
TATTCCCATGGCGCGCCCAG






RTGCAGCTGGTGCART



 



VH-2
0.04
TATTCCCATGGCGCGCCSAG






GTCCAGCTGGTRCAGT



 



VH-3
0.04
TATTCCCATGGCGCGCCCAG






RTCACCTTGAAGGAGT



 



VH-4
0.04
TATTCCCATGGCGCGCCSAG






GTGCAGCTGGTGGAG



 



VH-5
0.04
TATTCCCATGGCGCGCCCAG






GTGCAGCTACAGCAGT



 



VH-6
0.04
TATTCCCATGGCGCGCCCAG






STGCAGCTGCAGGAGT



 



VH-7
0.04
TATTCCCATGGCGCGCCGAR






GTGCAGCTGGTGCAGT



 



VH-8
0.04
TATTCCCATGGCGCGCCCAG






GTACAGCTGCAGCAGTC



 



LC set






CK1
0.2
ATATATATGCGGCCGCTTAT






TAACACTCTCCCCTGTTG



 



VL-1
0.04
GGCGCGCCATGGGAATAGCT






AGCCGACATCCAGWTGACCC






AGTCT



 



VL-2
0.04
GGCGCGCCATGGGAATAGCT






AGCCGATGTTGTGATGACTC






AGTCT



 



VL-3
0.04
GGCGCGCCATGGGAATAGCT






AGCCGAAATTGTGWTGACRC






AGTCT



 



VL-4
0.04
GGCGCGCCATGGGAATAGCT






AGCCGATATTGTGATGACCC






ACACT



 



VL-5
0.04
GGCGCGCCATGGGAATAGCT






AGCCGAAACGACACTCACGC






AGT



 



VL-6
0.04
GGCGCGCCATGGGAATAGCT






AGCCGAAATTGTGCTGACTC






AGTCT





W = A/T, R = A/G, S = G/C






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.
Selected reactions were analyzed on a 1% agarose gel to verify the presence of an overlap-extension fragment of approximately 1070 bp.
The plates were stored at −20° C. until further processing of the PCR fragments.







TABLE 3







Nested primer set











Final




Primer
Conc.

SEQ


name
μM
Sequence
ID












CK2
0.2
ACCGCCTCCACCGGCGGCCGCTTATTAACA




CTCTCCCCTGTTGAAGCTCTT


 


PJ 1-2
0.2
GGAGGCGCTCGAGACGGTGACCAGGGTGCC


 


PJ 3
0.2
GGAGGCGCTCGAGACGGTGACCATTGTCCC


 


PJ 4-5
0.2
GGAGGCGCTCGAGACGGTGACCAGGGTTCC


 


PJ 6
0.2
GGAGGCGCTCGAGACGGTGACCGTGGTCCC









d. Insertion of cognate VH and VL coding pairs into a screening vector
In order to identify antibodies with binding specificity to vaccinia virus particles, the VH and VL coding sequences were expressed either as Fabs or full-length antibodies. This involved insertion of the repertoire of VH and VL coding pairs into a screening vector and transformation into a host cell.
A two-step cloning procedure was employed for generation of a repertoire of screening vectors containing the VH and VL coding pairs. Statistically, if the repertoire of screening vectors contain ten times as many recombinant plasmids as the number of cognate paired VH and VL 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.
Briefly, the repertoire of linked VH and VL 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 2×YT 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 VH and VL 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
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 2×YT, 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 2×YT, 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.
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.
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
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.
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 VH and VL 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.
In the first step of the repair transfer procedure, the VH sequence was PCR amplified with a primer set containing the sequence corresponding to the originating VH-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 VH sequence was sequenced to verify the correction. The vector was digested with NheI/NotI, to prepare it for insertion of the light chain.
In the second step the complete light chain was PCR amplified with a primer set containing the sequence corresponding to the originating VL-gene thereby reverse mutating any cross-priming changes. The PCR fragment was digested with NheI/NotI and ligated into the VH 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.
If clones did not need correction of the VH and VL 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
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
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).
The individual plasmid preparations each containing a selected VH and VL 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 VH and VL 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
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 (Excell302, 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
All the individual cell lines were characterized with respect to antibody production and proliferation. This was performed with the following assays:
Production:
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.
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:
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 Schärfe 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
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.
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.
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.
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 desription in Example 1, section g).







TABLE 4







Primers for generation of in vitro


translational antigen genes











Primer

SEQ



Name
Sequence
ID













H3L-5′
GGGAACAGCCACCATGGCG




GCGGTGAAAACTCCTGTTA


 



H3L-3′
GGATCCTCTAGATCATTAA




AATGAAATCAGTGGAGTAG




TAAAC


 



A56R-5′
GGGAACAGCCACCATGACA




TGACACCTTTTCCTCAGAC




ATCT


 



A56R-3′
GGATCCTCTAGATCATTAA




GAGGTTGTACTACTACC




TAC


 



BSR-5′
GGGAACAGCCACCATGACA




TGTACTGTACCCACTATGA


 



B5R-3′
GGATCCTCTAGATCATTAT




AACGATTCTATTTCTTG


 



Poly-A
TTTTTTTTTTTTTTTTTTT




TTTTTTTTTTTGGATCCTC




TAGATCATTA


 



T7-Koz.
GGATCCTAATACGACTCAC




TATAGGGAACAGCCACC




ATG


 



A10L-5′
GGGAACAGCCACCATGATG




CCTATTAAGTCAATAG


 



A10L-3
GGATCCTCTAGATCATTAT




TCATCATCAAAAGAG


 



A3L-5′
GGGAACAGCCACCATGGAA




GCCGTGGTCAATAGCGA


 



A3L-3′
GGATCCTCTAGATCATTAA




AATAGTTCTGTAATATGT




CTA


 



A13L-5′
GGGAACAGCCACCATGATT




GGTATTCTTTTGTTGAT


 



A13L-3′
GGATCCTCTAGATCATTAT




ACAGAAGATTTAACTAGAT


 



D8L-5′
GGGAACAGCCACCATGCCG




CAACAACTATCTCCT


 



D8L-3′
GGATCCTCTAGATCATTAT




TTATTCCCTTCGATATATT




TTTGA


 



A11L-5′
GGGAACAGCCACCATGACG




ACCGTACCAGTGACG


 



A11L-3′
GGATCCTCTAGATCATTAA




ATAATTTTAATTCGTTTAA


 



A17L-5′
GGGAACAGCCACCATGAGT




TATTTAAGATATTACAAT


 



A17L-3′
GGATCCTCTAGATCATTAA




TAATCGTCAGTATTTAA




ACT


 



L5R-5′
GGGAACAGCCACCATGGAG




AATGTTCCTAATGTA


 



L5R-3′
GGATCCTCTAGATTATCAT




CTGCGAAGAACATCGTTA


 



F13L-5′
GGGAACAGCCACCATGTGG




CCATTTGCATCGGTA


 



F13L-3′
GGATCCTCTAGATCATTAA




ATTTTTAACGATTTACTGT


 



A16L-5′
GGGAACAGCCACCATGATG




GGGGCAGCTGTTACTCTT


 



A16L-3′
GGATCCTCTAGATCATTAA




GGCAGTTTTATTTTATCTT




TTA









1. Characterization of the Biochemical Properties the Individual Antibodies

Heterogeneity is a common phenomenon in antibodies and recombinant proteins. 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.
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
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:

    
    
        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 pg/cell/day.
        Antigen reactivity; which antigens should the anti-VV rpAb exert reactivity against, IMV, EEV and/or RCA derived proteins?
        Protein chemistry; generally antibodies with well-defined biochemical characteristics were included in the final anti-VV rpAb.
    
    


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
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 (Corning). 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
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
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)
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.
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
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 (I.M). In some of the experiments a total of 2.5 mg Cidofovir was injected intramuscularly (I.M) as a positive control for viral inhibition.
r. Composition of Sym002 Mix
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
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 RUmax 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 (kon and koff) and affinity constants (KD) 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
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
In the present Example the isolation, screening, selection and banking of clones containing cognate VH and VL pairs expressed as Fabs or antibodies with anti-vaccinia virus specificity was illustrated.
Donors
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 VH and VL coding pairs. All the selected donors belonged to the group of secondary immunized donors.
Isolation of Cognate VH and VL Coding Pairs
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 (VH) and the full-length light chain (LC). The protocol was designed to amplify antibody genes of all VH- gene families and the kappa light chain, by using two primer sets, one for VH 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.
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 VH and VL 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
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).
A total of 89 clones passed the validation. These are summarized in Table 5. Each clone number specifies a particular VH and VL 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 VH and VL 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 H31, 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.
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:







QVQLVQSGAEVKKPGYSVKVSCQASGLTFSNYRVSWVRQAPGQGLEWMGG


 


IIPIFGTTNYAQKFQGRVTISADRSPSRVTSLADKSTITVYMELSSLRSE


 


DTAVYYCARSRGSQDYYGMDVWGQGTTVTVSS.





















TABLE 5









CDRH1

CDRH2

CDRH3





IGHV
3   3

5            6

9       0               0

IGKV


Clone
gene
1abc2345
#
012abc3456789012345
#
234567890abcdefghijklmno123
#
gene





02-029
1-24
E---LAMH
50
GFDP--EDGEAVYAQGFQG
139
CATDVWRRTPEGGTDWF-------DPW
228
3-11


 


02-031
4-34
G---FHWS
51
EIN---HSGSTNYNPSVKS
140
CAGSRSFDLLTAYDLFHRKGNAM-DVW
229
1-39


 


02-037
6-1
NNI-ASWN
52
RTYYR-SKWYDDFALSVKG
141
CARGDVLRYF--------------DYW
230
1-5


 


02-058
3-30
R---YGMH
53
FLSF--DERNKFYPDSLKG
142
CAKGGLGTNEF-------------DHW
231
1-5


 


02-086
5-51
T---YWIG
54
IIYP--GDSDTKYSPSFQG
143
CATLPRYDAYGARIR---------DYW
232
1D-33


 


02-089
1-2
G---HYMH
55
WINP--SSGGTNYAQKFQG
144
CARYCSSPTCS-------------IVW
233
3-20


 


02-112
2-5
TSG-VGVD
56
LIY---WDDDKRYSPSLKS
145
CAHSSQRVVTGL------------DFW
234
1-39


 


02-113
2-70
TSG-MCVS
57
FID---WDDDKYYTTSLKT
146
CARIRTCYPDLYGDYNDAF-----DIW
235
1D-33


 


02-147
3-7
K---YWMS
58
NINQ--EGSAKHYVDSVKG
147
CARAADYGDY--------------VRP
236
2-28


 


02-156
3-30
S---YGMH
59
LISY--HGNTKYYADSVKG
148
CAKHVAAGGTL-------------DYW
237
2-24


 


02-159
1-46
N---YYIH
60
IINP--SGGSTTYAQRFQG
149
CARVIRKYYTSSNSYLTEQAF---DIW
238
1-39


 


02-160*
3-9
D---YAMH
61
GCSW--NSGFISYADSVKG
150
CVKETVAGRRGAF-----------DYW
239
1-39


 


02-166
4-34
G---YYWT
62
EIN---HSANTDYKPSLKS
151
CARGREWPSNF-------------DSW
240
3-11


 


02-169
5-51
T---YWIG
63
IIYP--GDSDTRYSPSFQG
152
CARRGSTYYY--------------DTW
241
1-39


 


02-172
3-21
S---YTMN
64
AITS--STTYIYYADSVKG
153
CASKPYGGDFG-------------SYW
242
1-5


 


02-183
5-51
K---YWIG
65
IIYP--EDGDTRYSPAFQG
154
CARPPSNWDESF------------DIW
243
1-17


 


02-186
1-3
N---YAVH
66
WINV--GNGQTKFSQRFQG
155
CARDPTQWLLQGDVYDM-------DVW
244
2-30


 


02-188
4-30-4
SGD-YYWN
67
NIY---STGSTYYDPSLQN
156
CAREAWLGEPLLLGDDAF------DIW
245
1-17


 


02-189
3-30
K---YYMH
68
TISY--DVKNKDYADSVKG
157
CARDGAGEWDLLMRRDF-------DYW
246
1-39


 


02-195
1-18
T---YGIS
69
WISA--WDGNTKYGEKFQD
158
CARDPARRPRSGYSVF--------EYW
247
3-15


 


02-197
4-4
SN--NWWS
70
EIY---HSGNTNYNPSLQS
159
CARDNRQSSSWVEGFFYYYGM---DVW
248
3-20


 


02-201
1-46
K---YYIH
71
MINP--SGGTTTYYAQKFQG
160
CARLRLGATIGRD-----------DYW
249
2-28


 


02-203
4-30-4
RGD-FYWS
72
YIY---YTGSTYYNPSLKS
161
CARDRASSGYDSRVWF--------DPW
250
1D-33


 


02-205
3-30
D---YTTH
73
IVLY--DGKNKNYADSVRG
162
CARTYRVYAKFDPF----------DVW
251
3-20


 


02-211
3-9
D---YAMH
74
GISW--NSEYIGYEDSVKG
163
CGKGVPGRRGYI------------EDW
252
1-39


 


02-214*
3-9
D---YAMH
75
TISW--NSGFIDYADSVKG
164
CVKDNIAGRRGSF-----------DSW
253
1-39


 


02-215
1-2
D---YYIH
76
WINP--NFGGTDYAQKFQG
165
CARDYIRATGATPSKYFIYYYGM-GVW
254
1-27


 


02-219
1-18
S---YAIA
77
WISA--YNGNTDYAQKFQG
166
CARARRVTNSPNNWF---------DPW
255
1-39


 


02-225
4-34
G---YYWG
78
EIN---HSGSANYHPSLKS
167
CARAGERSGSGSFVLGRF------DFW
256
1-39


 


02-229
3-49
D---YAVS
79
LIRSRHYGAKTQFAASVQG
168
CTNTSSLAVA--------------GNW
257
4-1


 


02-232
4-39
SRN-YYWG
80
TIY---YTGRTYYNPSLKN
169
CARIPQQRVNYF------------DYW
258
1-5


 


02-235
4-39
SSTNYYWG
81
TVY---LSGRAYYNPSLKS
170
CARLPGQRTTFF------------DYW
259
1-5


 


02-237
6-1
TST-AAWN
82
RTYYR-SRWRNDYAGSVRS
171
CARGRRFEDDAF------------DIW
260
1-39


 


02-238*
3-9
D---YAMH
83
GINW--NSGNIVYADSVKG
172
CVKDSVAGRRGGF-----------DHW
261
1-39


 


02-242
1-f
E---YYIH
84
LVDP--EDGEPIYAEKFQG
173
CATRDGDF----------------DHW
262
1-16


 


02-243
5-51
S---YWIS
85
IIYG--GDSDTKYSPSFQG
174
CVRHGTRYSFGRSDII--------DIW
263
1-17


 


02-246
5-51
S---YWIA
86
IIFP--GDSDTRYSPSFQG
175
CTKTPARGAYGDYIS---------GSW
264
1D-33


 


02-250*
3-9
D---FAMH
87
GVSW--NSDVINYSDSVKG
176
CAKSTKAVRRGSF-----------DYW
265
1-39


 


02-267
1-69
D---YAIS
88
GIIP--VFGTPNYAQQFQG
177
CARGGKLYEGNGYYSFHYF-----DYW
266
1D-33


 


02-269
2-5
STG-VGVG
89
LIY---WDDEERYSPSLKN
178
CAHTELAF----------------DYW
267
1D-33


 


02-271
1-69
S---YAIN
90
SIIP--IFATTNYAQRFQG
179
CARVKGTQNYYGM-----------DVW
268
1-5


 


02-274
4-34
G---YYWS
91
EVN---HSGSTNYNPSLRS
180
CHYYDSTGYYVS------------DFW
269
1-16


 


02-286
1-18
G---YGIS
92
WITY--DKGNTNHAQKFRG
181
CARGVVLIQTILF-----------DYW
270
1-39


 


02-290
1-69
N---SAIN
93
GVVP--IYDTSHYAQKFKG
182
CARTVLDSGAYSYY----------DSR
271
1-5


 


02-291 
1-69
N---YRVS
94
GIIP--IFGTTNYAQKFQG
183
CARSRGSQDYYGM-----------DVW
272
1-5


 


02-294
3-23
N---YAMS
95
AISG--SGGKTYHAHSVRG
184
CAKLRDSSVYSAYVFRVIF-----DGW
273
1D-33


 


02-295
3-11
D---NYMN
96
YISS--TSGSIYYADSVKG
185
CATLTVASTY--------------DYW
274
2-30


 


02-297
3-9
D---YAMH
97
GLNW--NGANIRYADSVKG
186
CVKDTVALLTSRGGCM--------DVW
275
1-12


 


02-302
2-5
TSG-VGVG
98
LIY---WDDDKRYSPSLKS
187
CAHSPPHGG---------------DYW
276
1D-33


 


02-303
3-30
T---YGMH
99
FISS--DGSTKYYADSVKG
188
CAKGLSQALNYYGSGS--------PFL
277
3-20


 


02-335
3-30
N---YGIH
100
FISY--DGSKKYYVDSVKG
189
CAKDRGVSAWYPRDAF--------DIW
278
1-39


 


02-339
4-59
S---DNWS
101
YIY---KTGSTNYNPSLKS
190
CARVPLIEAGITIFAKIGAF----DIW
279
3-20


 


02-349‡
5-51
S---FWIG
102
VTYP--GDSDTRYSPSFQG
191
CARGSPMIKFYF------------DYW
280
3-20


 


02-351
3-9
N---YAMY
103
GIIW--NSEYIGYADSVKG
192
CARATGAGRRNPL-----------DYW
281
1-39


 


02-431
1-46
S---YYMH
104
LINP--SSGTTSYAQNFQG
193
CARPYRSYSSSPQ-----------DYW
282
3-20


 


02-437
4-31
SPG-YYWN
105
YIY---YSGSTNYNPSLKS
194
CARYYYSSGPKF------------DYW
283
1-12


 


02-446
1-69
S---FAIS
106
SIIP--IFGTAHYAQRFEG
195
CARNNRPLGALFGM----------DVW
284
1-9


 


02-461
1-18
T---YGIS
107
GIRV--HNGNTNYAQKFQG
196
CARGGFNRLV--------------DPW
285
3-20


 


02-482
4-31
SAG-YYWS
108
YIH---YTGTTYYNPSLKS
197
CARNTGIYLGGSPGGTRNNWF---DPW
286
1-39


 


02-488
5-51
S---YWIG
109
IIYP--GDSDTRYSPSFQG
198
CARQQAKTLYYDSSGSKSAF----DIW
287
1-5


 


02-515
4-31
SGG-YYWS
110
YIH---YSGSTYYNPSLKS
199
CARVRGNIVATTAFYYYYGL----DAW
288
1-12


 


02-516
3-11
D---YYMS
111
YTNL--FTGYTNYADSVKG
200
CAKFDYGEGAYHF-----------DFW
289
1D-12


 


02-517
4-31
GA--YHWS
112
YIY---YTGNTYFNPSLKS
201
CARDPIALPGRGVF----------DYW
290
3-20


 


02-520
4-34
A---YYWS
113
EIS---HSGSTHYNPSLNS
202
CSSGYYFAGGEF------------DYW
291
1-12


 


02-526
3-49
D---YTNS
114
FIRGKKFGGTKDYAASVKG
203
CTRDRGYSDHTGLYTRFGF-----DSW
292
2-28


 


02-532
1-69
D---HSIG
115
KIIP--IYGRANYAQKFQG
204
CARWRGGYSGYGDYF---------DSW
293
1-39


 


02-536
4-4
SS--HWWN
116
EIY---HSGSTNYNPSLKS
205
CARDPQKPRQHLWPNPYYYSGM--DVW
294
1-5


 


02-551
1-69
Y---YAIN
117
GIVP--MVGPADYAEKFRG
206
CARGRSWRGYL-------------DYW
295
3-20


 


02-559
1-46
N---YYMH
118
LINP--SGDSTTNAQKFQG
207
CARDYGDYCGGDCPYDAF------DIW
296
1-27


 


02-572
1-69
S---FGIS
119
GIIP--IFGTPNYSLKFQD
208
CARDKGESDINGWQTGAFYYGM--DVW
297
1-12


 


02-575
2-26
NDR-MGVS
120
HIF---SNDERSHSSSLKS
209
CARIDSVGWPSSHYYGM-------DVW
298
1-12


 


02-586
4-59
S---YYWS
121
YIF---YSGNTNYNPSLKS
210
CARDRITGYDSSGHAF--------DIW
299
1-5


 


02-589
4-34
G---YYWT
122
EIN---QNGRSNHNPSLKS
211
CARGGKFCGSTSCFTEGRL-----DYW
300
1-39


 


02-607
3-30
S---YGMH
123
VISY--DGRYKFYANSVKG
212
CAKDSGRYSSLGHYYYYGM-----DVW
301
1-39


 


02-611
5-51
N---YWIG
124
IIHP--GDSETRYSPSFQG
213
CARGYYYDTSGYRPGSF-------QHW
302
1-5


 


02-612
3-30
T---YTMH
125
VISY--DGTNKYHTDSVKG
214
CARPLFYGAGDAF-----------DIW
303
1-39


 


02-614
1-69
N---YAII
126
EIIP--KFGTANYAQKFQG
215
CADWVVGNYNGL------------DVW
304
3-20


 


02-617
1-46
N---YYVH
127
LINP--SAGKTTYAQRFQG
216
CAREGKHDFWRGYFSPLGM-----DVW
305
3-20


 


02-621
1-69
S---HGVN
128
GIIP--VFGTTNYAQSLQG
217
CATARNSSNWYEGHYYL-------AHW
306
1D-33


 


02-626
4-30-4
TGD-YYWS
129
YVF---NSGSTYYNPSLQS
218
CANMVVVATQPKNWF---------DPW
307
1-39


 


02-628
4-30-4
SGY-YYWN
130
YID---YRGTTYYSPSFKS
219
CASYGSGMGSEYYF----------GHW
308
1-39


 


02-632
1-2
G---YYIH
131
RINP--ITDVTNYAQIFQG
220
CGRVGREAFYYYGM----------DVW
309
1-9


 


02-633
1-2
A---YYIH
132
RINP--DSGGTDFSQKFQG
221
CARASRRLTTHNYF----------DGW
310
1-16


 


02-634
3-48
T---YEMS
133
YIGS--GGVTIYYADSVKG
222
CARVRGGRYF--------------DYW
311
3-20


 


02-640
5-51
T---YWIA
134
IIWP--VDSDTRYSPSFQG
223
CASGSGYDSYYNM-----------DVW
312
1-39


 


02-643
7-4-1
S---YAMN
135
WINT--NGNPTYAQGFTG
224
CARDSSTVTGLMTEYNWF------DPW
313
1-39


 


02-649
4-31
SGP-YYWS
136
YSS---NRGIAYYNPSLKS
225
CATEKGSGGDVGKF----------DNW
314
1-39


 


02-650
1-69
S---NPVS
137
GIIP--FAQKVLGAORVRD
226
CATGQQLYSL--------------HYW
315
2-30


 


02-651
1-2
D---YYLH
138
RINP--KSGDTHHVQKFQG
227
CAREGPQFYYDSGDYYSAHSPGDFDHW
316
1-39























CDRL1


CDRL2

CDRL3







2     3          3


5

8 9






Clone
45678901abcdefghi234
#

0123456
#
89012345a678
#
Spec.







02-029
RASQSVRR---------SLA
317

DASNRAT
406
CLQRSNWP-ITF
495
H3L


 



















02-031
RASQGISN---------SLN
318

GASGLES
407
CQQSYRTL-YTF
496
H3L


 




02-037
RASQSISI---------WLA
319

KASTLES
408
CQQYNGYSEVTF
497
B


 




02-058
RASQSIGN---------WLA
320

DASSLKS
409
CQQYDTYP-ITF
498
B


 




02-086
QASQDISK---------YLN
321

DASNLET
410
CQQYDNLP-PTF
499
C


 




02-089
RASESVRSN--------YLA
322

GASSRAT
411
CQQYGRSP-LTF
500
B


 




02-112
RASQSIST---------YLN
323

AASSLQS
412
CQQSYNTP-ATF
501
A27L


 




02-113
QASQDIKY---------YLN
324

DASNLET
413
CQQYENVP-YTF
502
VCP


 




02-147
RSSKSLLHSNGYN----YLD
325

LASNRAS
414
CMQALQIP-RTF
503
G


 




02-156
RSSESLVNNDGNT----YLS
326

KISNRFS
415
CMQTTHIP-HTF
504
L


 




02-159
RASQNISN---------FLL
327

AASSLQS
416
CQQTYGNP-LTF
505
A27L


 




02-160*
RASQSINN---------YLN
328

AVSSLQT
417
CQQSFRTP-NTF
506
H3L


 




02-166
RASQSVDR---------YLA
329

DASNRDT
418
CQQRAIWP-PEF
507



 




02-169
RASQSIWT---------FLN
330

TASSLQS
419
CQQSFTSW-WTF
508
B5R


 




02-172
RASQSISN---------WLA
331

KASNLES
420
CQQYSNYP-ITF
509
B5R


 




02-183
RASQDISN---------DLG
332

LASSLQS
421
CLQHNSF--LTF
510



 




02-186
RSSQSLVYSDGNT----YLH
333

KVSNRDS
422
CMQGTHWP-PAF
511
A27L


 




02-188
RASQGIGY---------DLG
334

AASSLQS
423
CLQLHTFP-RTF
512
A27L


 




02-189
RASQSISN---------YLS
335

AASLLQT
424
CQQGYSTP-YTF
513



 




02-195
RASQSVSS---------NLA
336

GASTRAT
425
CHQYNYWPPLAF
514
H3L


 




02-197
RASQSIASA--------YLA
337

GASSRPT
426
CQQYGISP-RTF
515
H3L


 




02-201
RSSQSLLHSNGYN----YLD
338

LGSTRAS
427
GMQALQTP-HTF
516
H3L


 




02-203
QASHDVSN---------FLN
339

DASNLKT
428
CHQYDSLP-FTF
517
A27L


 




02-205
RASQSVSSN--------YIA
340

GASSRAT
429
CQQFGYSPRFTF
518
H3L


 




02-211
RASQSIRT---------YLN
341

AASSLQS
430
CQQTYITP-KSF
519
H3L


 




02-214*
RASQTIST---------YLN
342

AASSLQS
431
CQQSYRTP-LTF
520



 




02-215
RASQGISN---------YLA
343

GASTLQS
432
CQKYDSAP-YTF
521



 




02-219
RASRSIST---------YLN
344

AASSLQS
433
CQQTYTIP-LTF
522



 




02-225
RASQSIHT---------YLN
345

TASNLQS
434
CQQSYSTL-RTF
523
L


 




02-229
KSSQSVLYSSNNNN---YLA
346

WASTRES
435
CQQYYKTP-PTF
524
A27L


 




02-232
RASQTIST---------WLA
347

DASSLES
436
CQQYNSYP-LTF
525
H3L


 




02-235
RASQSIST---------WLA
348

DASSLES
437
CQQYNFY--GTF
526
H3L


 




02-237
RASQSISN---------YLN
349

GASSLES
438
CQQSYSIP-RTF
527



 




02-238*
RASLNIRN---------YLN
350

AASTLQI
439
CQQSYSMSPYTF
528



 




02-242
RASQVIGK---------YLA
351

ATSILQS
440
CQQYNSFP-LTF
529



 




02-243
RASQGIRN---------DLG
352

AASSLQS
441
CLQQNNYP-WTF
530
C


 




02-246
QASHDINK---------YLN
353

DASNLET
442
CQQYDNFP-YTF
531



 




02-250*
RASQSINN---------YLN
354

AASSLHS
443
CQQTYIST-RTF
532



 




02-267
QASQDISN---------YLN
355

DASHLET
444
CQQYDNLP--LF
533
VCP


 




02-269
QASQDISF---------YLN
356

DASILET
445
CQQYDNLI--TF
534
VCP


 




02-271
RASQSISS---------WLA
357

KVSSLES
446
CQQYESDI-FTF
535
VCP


 




02-274
RASQDISN---------YLA
358

AASSLLS
447
CQQYGRYP-LTF
536
VCP


 




02-286
RASQSVST---------FLN
359

GVSNLQS
448
CQQSHRTP-YTF
537
VCP


 




02-290
RASQDVSP---------WLA
360

KASSLES
449
CQQYQTY--STF
538
VCP


 




02-291 
RASQGISD---------WLA
361

KASSLES
450
CQQYESDS-WTF
539
VCP


 




02-294
QASQDISN---------YLN
362

DTSNLET
451
CQQYDNLP-FTF
540
VCP


 




02-295
RSSQSVVYSDGNI----YLN
363

QVSNRDS
452
CMQGTHWP-YSF
541
B5R


 




02-297
RASQDISS---------WLA
364

AASSLQS
453
CQQAYSFP-WTF
542
VCP


 




02-302
QASQDISN---------YLN
365

DASNLET
454
CQQYDNL--PTF
543
VCP


 




02-303
RASQSVSSL--------YVG
366

GTSSRAT
455
CQQYGTSP-WTF
544
B5R


 




02-335
RASQSISS---------FLN
367

GATTLQS
456
CHQSYSLP-FTF
545



 




02-339
RASQSVSS--------SYLA
368

RASSRAA
457
CQQYVASP-FTF
546
D8L


 




02-349‡
RASQSVSSRASQSVSSNYLA
369

GASTRAA
458
CHQYGTSP-RTF
547



 




02-351
RASQTIRN---------YLN
370

TASSLHS
459
CQQSYITP-YTF
548



 




02-431
RASQSVSNN--------NLA
371

GASSRAA
460
CQQYGSSP-YTF
549
A56R


 




02-437
RASQGISN---------WLA
372

AASSLQS
461
CQQANSFP-FTF
550



 




02-446
RASQGIGG---------ALA
373

AASTLQS
462
CQQLDTYP-LTF
551



 




02-461
RASQSVSSN--------YLA
374

GASSRAT
463
CQQYASSP-YTF
552
A33R


 




02-482
RASQSISR---------HLN
375

AASSLQT
464
CQHSSKTP-FTF
553
A33R


 




02-488
RASQSIST---------YLA
376

KASSLEP
465
CQQYSSY--LSF
554
A27L


 




02-515
RASQGVSN---------WVA
377

AASSLQS
466
CQQANGFL-WTF
555



 




02-516
RASQGIST---------FLA
378

AASSLQS
467
CQQAHSFP-VTF
556
A56R


 




02-517
RASQSVTSN--------YLA
379

GASNRAT
468
CQQYGSSP-LTF
557



 




02-520
RASQGIST---------WLA
380

AASTLQH
469
CQQANSFP-RTF
558
A


 




02-526
RSSQSLLHSNGYN----YLD
381

LGSNRAS
470
CMQSLQT--VTF
559
B5R


 




02-532
RASQSISN---------YLN
382

AASRLQS
471
CQHSYETPPYTF
560



 




02-536
RASQSLNN---------WLA
383

DASSLQS
472
CQQYNFYP-WTF
561



 




02-551
RASQSVSNN--------YLA
384

GASSRAT
473
CQQYGGSP-QTF
562
A56R


 




02-559
RASQGIFN---------YLA
385

GASTLRS
474
CQKYNSAP-LTF
563



 




02-572
RASQNIGN---------WLA
386

SASSLQN
475
CQQANSFP-VTF
564



 




02-575
RASQDIIS---------WLA
387

AASSLQS
476
CQQTHSFPPWTF
565



 




02-586
RASQSIYI---------WLA
388

DASSLES
477
CQQYHHYS-PTF
566
A


 




02-589
RASQSISR---------SLN
389

AASTLQS
478
CQQSYSTL-RTF
567
L


 




02-607
RASQPISS---------FLN
390

AASSLQS
479
CQQGYSTP-PTF
568
A


 




02-611
RASQSISS---------WLA
391

HAFSLEG
480
CQQYDSYP-YTF
569



 




02-612
RASQSFNG---------YLN
392

AASTLQS
481
CQQSYSTP-RTF
570



 




02-614
RASQTVIST--------YLA
393

GASSRAT
482
CQQYSDS--LTF
571



 




02-617
RASQSVSSG--------SLD
394

GASNRAS
483
CHQYGGAQ-GTF
572



 




02-621
QASQDISN---------YLN
395

DASNLET
484
CQQYDTLPPITF
573



 




02-626
RASQSISS---------YLN
396

AASSLQS
485
CQQSHSSP-WTF
574



 




02-628
RASQSIRS---------YLN
397

GASSLQS
486
CQQSYLAP-WTF
575



 




02-632
RASQGISS---------YLV
398

AASTLES
487
CQQFNNYP-YTF
576



 




02-633
RASQAISN---------YLN
399

GAFILES
488
CQQYHTYP-FTF
577
J


 




02-634
RASQSVSST--------YLA
400

GASNRAT
489
CQKYGRSPTWTF
578



 




02-640
RASQSISN---------HLN
401

VASSLQG
490
CQQGFTTP-ITF
579
J


 




02-643
RASQSISS---------YLN
402

AASSLQS
491
CQQSYSTP-YTF
580



 




02-649
RANQSIDD---------YLH
403

DASTLHS
492
CQQSYTIPLWTF
581



 




02-650
RSSQSLVYADGDT----HLN
404

HVSNRDA
493
CMQGTHWP-PTF
582



 




02-651
RASQSITN---------CLN
405

GASTLQS
494
CQQSDSTP-YTF
583






IGHV and IGKV gene names were assigned according to the official HUGO/IMGT 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).? 


‡) This clone carries an eight-codon insertion in CDRL1.






Mirroring the Humeral Immune Response

In order to illustrate that the cognate VH and VL 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.
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 mirror the diversity of the humeral response in humans.
Further, due to the cognate pairing of the VH and VL coding sequences representing the VH and VL 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
A subset of 47 unique cognate VH and VL coding pairs corresponding to clone nr O2-029, 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
In the present Example the biological activity of a mixture of monoclonal anti-VV antibodies was compared to a serum derived VIG product.
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 VH and VL 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.
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 SymVIG) (FIG. 8). The direct binding analyses demonstrated at least a 100-fold increase in the specific binding activity of the recombinant antibody compositions.
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 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










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.
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
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
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:

    
    
        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.
        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.
        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.
    
    


Twenty eight individual cell cultures expressing cognate VH and VL 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).
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.
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
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 SyinVIG 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.
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 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










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.
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 (I.P) 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
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 (KD) 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.

## Claims

1. An anti-orthopoxvirus polyclonal recombinant antibody composition, comprising two or more distinct antibody molecules binding to different orthopoxvirus epitopes, wherein each distinct antibody comprises the following:
(a) a VH CDR1 selected from SEQ ID NOs: 50-138;
(b) a VH CDR2 selected from SEQ ID NOs: 139-227;
(c) a VH CDR3 selected from SEQ ID NOs: 228-316;
(d) a VL CDR1 selected from SEQ ID NOs: 317-405;
(e) a VL CDR2 selected from SEQ ID NOs: 406-494; and
(f) a VL CDR3 selected from SEQ ID NOs: 495-583;

wherein the CDRs are combined according to Table 5.

2. The composition of claim 1, comprising two or more distinct antibody molecules binding to different epitopes of the same orthopoxvirus antigen.

3. The composition of claim 1, comprising three or more distinct antibody molecules binding to three different orthopoxvirus antigens.

4. The composition of claim 3, further comprising two or more distinct antibody molecules binding to different epitopes of the same orthopoxvirus antigen.

5. An anti-orthopoxvirus polyclonal recombinant antibody composition, comprising two or more antibodies selected from the group consisting of antibodies comprising heavy and light chain CDR1, CDR2, and CDR3 regions:
(a) SEQ ID NOs: 50, 139, 228, 317, 406, and 495;
(b) SEQ ID NOs: 52, 141, 230, 319, 408, and 497;
(c) SEQ ID NOs: 53, 142, 231, 320, 409 and 498;
(d) SEQ ID NOs: 54, 143, 232, 321, 410, and 499;
(e) SEQ ID NOs: 58, 147, 236, 325, 414, and 503;
(f) SEQ ID NOs: 66, 155, 244, 333, 422, and 511;
(g) SEQ ID NOs: 67, 156, 245, 334, 423 and 512;
(h) SEQ ID NOs: 69, 158, 247, 336, 425, and 514;
(i) SEQ ID NOs: 70, 159, 248, 337, 426, and 515;
(j) SEQ ID NOs: 72, 161, 250, 339, 428, and 517;
(k) SEQ ID NOs: 74, 163, 252, 341, 430, and 519;
(l) SEQ ID NOs: 79, 168, 257, 346, 435, and 524;
(m) SEQ ID NOs: 81, 170, 259, 348, 437 and 526;
(n) SEQ ID NOs: 92, 181, 270, 359, 448, and 537;
(o) SEQ ID NOs: 96, 185, 274, 363, 452, and 541;
(p) SEQ ID NOs: 99, 188, 277, 366, 455, and 544;
(q) SEQ ID NOs: 101, 190, 279, 368, 457, and 546;
(r) SEQ ID NOs: 107, 196, 285, 374, 463, and 552;
(s) SEQ ID NOs: 108, 197, 286, 375, 464 and 553;
(t) SEQ ID NOs: 109, 198, 287, 376, 465, and 554;
(u) SEQ ID NOs: 114, 203, 292, 381, 470, and 559;
(v) SEQ ID NOs: 117, 206, 295, 384, 473, and 562;
(w) SEQ ID NOs: 121, 210, 299, 388, 477, and 566;
(x) SEQ ID NOs: 122, 211, 300, 389, 478, and 567;
(y) SEQ ID NOs: 123, 212, 301, 390, 479, and 568; and
(z) SEQ ID NOs: 132, 221, 310, 399, 488, and 577.

6. An anti-orthopoxvirus polyclonal recombinant antibody composition, comprising antibodies comprising heavy and light chain CDR1, CDR2, and CDR3 regions:
(a) SEQ ID NOs: 57, 146, 235, 324, 413, and 502; and
(b) SEQ ID NOs: 78, 167, 256, 345, 434, and 523.