US20120316071A1 - Methods for affinity maturation-based antibody optimization - Google Patents

Abstract

Provided herein is a rational method of affinity maturation to evolve the activity of an antibody or portion thereof based on the structure/affinity or activity relationship of an antibody. The resulting affinity matured antibodies exhibit improved or optimized binding affinity for a target antigen.

Description

RELATED APPLICATIONS
• Benefit of priority is claimed to U.S. Provisional Application Ser. No. 61/280,618, entitled “Methods for Affinity Maturation-Based Antibody Optimization,” filed Nov. 4, 2009, and to U.S. Provisional Application Ser. No. 61/395,670, entitled “Methods for Affinity Maturation-Based Antibody Optimization, Antibody Conversion and Antibodies,” filed May 13, 2010. Where permitted, the subject matter of the above-noted applications are incorporated by reference in its entirety.
• This application also is related to International PCT Application No. PCT/US2009/063299, entitled “Combinatorial Antibody Libraries and Uses Thereof,” filed Nov. 4, 2009, which claims priority to U.S. Provisional Application No. 61/198,764 filed Nov. 7, 2008 and to U.S. Provisional Application No. 61/211,204 filed Mar. 25, 2009, each entitled “Combinatorial Antibody Libraries and Uses Thereof.” This application also is related to International PCT Application No. PCT/US09/63303, entitled Anti-DLL4 Antibodies and Uses Thereof, which also claims priority to each of U.S. Provisional Application Nos. 61/198,764 and 61/211,204.
• The subject matter of each of the above-noted applications is incorporated by reference in its entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ON COMPACT DISCS
• An electronic version of the Sequence Listing is filed herewith, the contents of which are incorporated by reference in their entirety. The electronic file is 2.66 megabytes in size, and titled 702seqPC1.txt.
FIELD OF THE INVENTION
• Provided herein is a rational method of affinity maturation to evolve the activity of an antibody or portion thereof based on the structure/affinity or activity relationship of an antibody. The resulting affinity matured antibodies exhibit improved or optimized binding affinity for a target antigen.
BACKGROUND
• Numerous therapeutic and diagnostic monoclonal antibodies (MAbs) are used in the clinical setting to treat and diagnose human diseases, for example, cancer and autoimmune diseases. For example, exemplary therapeutic antibodies include Rituxan (Rituximab), Herceptin (Trastuzumab), Avastin (Bevacizumab) and Remicade (Infliximab). In designing antibody therapeutics, it is desirable to create antibodies, for example, antibodies that modulate a functional activity of a target, and/or improved antibodies such as antibodies with higher specificity and/or affinity and/or and antibodies that are more bioavailable, or stable or soluble in particular cellular or tissue environments. It is among the objects herein to provide methods for optimizing and improving the binding affiniites of antibodies and for selecting antibodies with desired affinities.
SUMMARY
• Provided herein are methods of affinity maturation of antibodies or fragments thereof based on structure/activity relationship (SAR). The methods result in the optimization of antibodies to have increased and improved activity (e.g. binding specificity or affinity) for a target antigen compared to the starting antibody that is affinity matured.
• Provided herein is a method of affinity maturation of a first antibody or portion thereof for a target antigen. In the method, a related antibody or portion thereof is identified that exhibits a reduced activity for the target antigen than the corresponding form of a first antibody, whereby the related antibody or portion thereof contains a related variable heavy chain or a related variable light chain that is either 1) one in which the corresponding variable heavy chain or variable light chain of the related antibody exhibits at least 75% amino acid sequence identity to the variable heavy chain or variable light chain of the first antibody but does not exhibit 100% sequence identity therewith; or 2) one in which at least one of the V_H, D_H and J_H germline segments of the nucleic acid molecule encoding the variable heavy chain of the related antibody is identical to one of the V_H, D_H and J_H germline segments of the nucleic acid molecule encoding the variable heavy chain of the first antibody and/or at least one of the V_κ and J_κ or at least one of the V_λ and J_λ germline segments of the nucleic acid molecule encoding the variable light chain is identical to one of the V_κ and J_κ or V_λ and J_λ germline segments of the nucleic acid molecule encoding the variable light chain of the first antibody. Further, in the method, the amino acid sequence of the variable heavy chain or variable light chain of the first antibody is compared to the amino acid sequence of the corresponding related variable heavy chain or variable light chain of the related antibody. Following comparison, a target region within the variable heavy chain or variable light chain of a first antibody is identified, whereby a target region is a region in the first antibody that exhibits at least one amino acid difference compared to the same region in the related antibody. After identifying a target region, a plurality of modified antibodies are produced each containing a variable heavy chain and a variable light chain, or a portion thereof, where at least one of the variable heavy chain or variable light chain is modified in its target region by replacement of a single amino acid residue, such that the target region in each of the plurality of antibodies contains replacement of an amino acid to a different amino acid compared to the first antibody. The resulting plurality of mutated antibodies are screened for an activity to the target antigen. Modified antibodies that exhibit increased activity for the target antigen compared to the first antibody. In one example of the method, the plurality of modified antibodies are produced by producing a plurality of nucleic acid molecules that encode modified forms of a variable heavy chain or a variable light chain of the first antibody, wherein the nucleic acid molecules contain one codon encoding an amino acid in the target region that encodes a different amino acid from the unmodified variable heavy or variable light chain, such that each nucleic acid molecule of the plurality encodes a variable heavy chain or variable light chain that is modified in its target region by replacement of a single amino acid residue.
• In the method provided herein, the target region in the first antibody exhibits 1, 2, 3, 4, 5, 6 7, 8, 9 or 10 amino acid differences compared to the corresponding region in the related antibody. Further, in the method, the first antibody can be compared to 1, 2, 3, 4, or 5 related antibodies. In the method herein, the target region is selected from among a CDR1, CDR2, CDR3, FR1, FR2, FR3 and FR4. For example, the target region is a CDR1, CDR2 or CDR3.
• In the method provided herein, an activity that is assessed can be binding, signal transduction, differentiation, alteration of gene expression, cellular proliferation, apoptosis, chemotaxis, cytotoxicity, cancer cell invasion, endothelial cell proliferation or tube formation. In one example, the activity is binding and binding is assessed by an immunoassay, whole cell panning or surface plasmon resonance (SPR). For example, binding can be assessed by immunoassay such as by a radioimmunoassay, enzyme linked immunosorbent assay (ELISA) or electrochemiluminescence assay. In particular, binding is assessed using an electrochemiluminescence assay such as meso scale discovery (MSD).
• In the method herein, the first antibody that is affinity matured binds to the target antigen with a binding affinity that is at or about 10⁻⁴ M, 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, or lower, when the antibody is in a Fab form.
• In one example, the affinity maturation method provided herein involves comparison to a related antibody or portion thereof that exhibits 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less activity than the corresponding form of the first antibody. For example, the related antibody can exhibit the same or similar level of activity to the target antigen compared to a negative control. In another example, the related antibody exhibits a binding affinity that is less than the binding affinity of the first antibody, whereby the binding affinity is at or about 10⁻⁴ M, 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M or lower in its Fab form.
• In one example of the method provided herein, a target region is identified within the variable heavy chain of the first antibody, and the method is performed therefrom. In another example of a method provided herein, a target region is identified within the variable light chain of the first antibody, the method is performed therefrom. In a further example of the method herein, a target region is identified within the variable heavy chain of the first antibody and steps the method is performed therefrom; and separately and independently a target region is identified within the variable light chain of the first antibody, and the method is performed therefrom.
• In one aspect of the method herein, a related antibody that contains the related corresponding variable heavy chain is different than a related antibody that contains the related corresponding variable light chain. In another aspect of the method herein, a related antibody that contains the related corresponding variable heavy chain is the same as a related antibody that contains the related corresponding variable light chain.
• In one example of the method herein, the variable heavy chain or variable light chain of the first antibody exhibits 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the corresponding related variable heavy chain or variable light chain of the related antibody. In particular, the variable heavy chain or variable light chain of the first antibody exhibits at least 95% sequence identity with the corresponding related variable heavy chain or variable light chain of the related antibody.
• In another example, the related antibody contains a related variable heavy chain or variable light chain that is one in which at least one of the V_H, D_H and J_H germline segments of the nucleic acid molecule encoding the variable heavy chain of the first antibody is identical to one of the V_H, D_H and J_H germline segments of the nucleic acid molecule encoding the variable heavy chain of the related antibody; and/or at least one of the V_κ and J_κ or at least one of the V_λ and J_λ germline segments of the nucleic acid molecule encoding the variable light chain of the first antibody is identical to one of the V_κ and J_κ or V_λ and J_λ, germline segments of the nucleic acid molecule encoding the variable light chain of the related antibody. For example, the related antibody contains a related variable heavy chain or variable light that is one in which at least one of the V_H, D_H and V_H germline segments of the nucleic acid molecule encoding the variable heavy chain of the first antibody is from the same gene family as one of the V_H, D_H and J_H germline segments of the nucleic acid molecule encoding the variable heavy chain of the related antibody; and/or at least one of the V_κ and J_κ or at least one of the V_λ and J_λ germline segments of the nucleic acid molecule encoding the variable light chain of the first antibody is from the same gene family as one of the V_κ and J_κ or V_λ and J_λ germline segments of the nucleic acid molecule encoding the variable light chain of the related antibody. In such examples, the variable heavy chain or variable light chain of the first antibody exhibits 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the corresponding related variable heavy chain or variable light chain of the related antibody.
• In the method herein, the first antibody is identified by screening a combinatorial antibody library, where the combinatorial antibody library is produced by combining a V_H, a D_H and a J_H human germline segment or portion thereof in frame to generate a sequence of a nucleic acid molecule encoding a VH chain or a portion thereof; and combining a V_κ and a J_κ human germline segment or portion thereof, or a V_λ and a J_λ germline segment or portion thereof in frame to generate a sequence of a nucleic acid molecule encoding a VL chain or a portion thereof. In the steps of combining, each of the portions of the V_H, D_H, J_H, V_κ, J_κ, V_λ or J_λ are sufficient to produce an antibody or portion thereof containing a VH or VL or portion thereof that forms a sufficient antigen binding site. The steps of combining are repeated a plurality of times to generate sequences of a plurality of different nucleic acid molecules. The nucleic acid molecules are synthesized to produce two libraries. The first library contains nucleic acid molecules encoding a VH chain or a portion thereof; and the second library contains nucleic acid molecules encoding a VL chain or a portion thereof. The nucleic acid molecules from the first and second library are introduced into a cell, which is repeated a plurality of times to produce a library of cells, wherein each cell contains nucleic acid molecules encoding a different combination of VH and VL from every other cell in the library of cells. Finally, in the method of generating a combinatorial library, the cells are grown to express the antibodies or portions thereof in each cell, thereby producing a plurality of antibodies or portion thereof, wherein each antibody or portion thereof in the library comprises a different combination of a VH and a VL chain or a sufficient portion thereof to form an antigen binding site from all other antibodies or portions thereof in the library. To identify a first antibody, the library is screened by contacting an antibody or portion thereof in the library with a target protein, assessing binding of the antibody or portion thereof with the target protein and/or whether the antibody or portion thereof modulates a functional activity of the target protein; and identifying an antibody or portion thereof that exhibits an activity for the target protein, wherein the identified antibody or portion thereof is a first antibody. Similarly, a related antibody also can be identified by screening such a combinatorial antibody library for the target antigen to identify a related antibody that exhibits reduced activity for the target antigen compared to the first antibody.
• The combinatorial library that is screened can be an addressable library. In an addressable library, the synthesized nucleic acid sequences are individually addressed, thereby generating a first addressed nucleic acid library and a second addressed nucleic acid library. The cells also are addressed such that each locus contains a cell that contains nucleic acid molecules encoding a different combination of a VH and a VL from every other cell in the addressed library of cells. Finally, the plurality of antibodies or portions thereof are addressed, such that the antibodies or portions thereof at each locus in the library are the same antibody and are different from those at each and every other locus; and the identity of the antibody or portion thereof is known by its address. The addressable library can be arranged in a spatial array, wherein each individual locus of the array corresponds to a different antibody member. The spatial array can be a multiwell plate. In another example, the antibodies in the addressable library can be attached to a solid support that is a filter, chip, slide, bead or cellulose, and the different antibody members are immobilized to the surface thereof.
• In the affinity maturation method herein, the target antigen is a polypeptide, carbohydrate, lipid, nucleic acid or a small molecule. The target antigen can expressed on the surface of a virus, bacteria, tumor or other cell, or is a recombinant protein or peptide. In one example, the target antigen is a protein that is a target for therapeutic intervention. For example, the target antigen is involved in cell proliferation and differentiation, cell migration, apoptosis or angiogenesis. Exemplary of target antigens include, but are not limited to, a VEGFR-1, VEGFR-2, VEGFR-3 (vascular endothelial growth factor receptors 1, 2, and 3), a epidermal growth factor receptor (EGFR), ErbB-2, ErbB-3, IGF-R1, C-Met (also known as hepatocyte growth factor receptor; HGFR), DLL4, DDR1 (discoidin domain receptor), KIT (receptor for c-kit), FGFR1, FGFR2, FGFR4 (fibroblast growth factor receptors 1, 2, and 4), RON (recepteur d'origine nantais; also known as macrophage stimulating 1 receptor), TEK (endothelial-specific receptor tyrosine kinase), TIE (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains receptor), CSF1R (colony stimulating factor 1 receptor), PDGFRB (platelet-derived growth factor receptor B), EPHA1, EPHA2, EPHB1 (erythropoietin-producing hepatocellular receptor A1, A2 and B1), TNF-R1, TNF-R2, HVEM, LT-βR, CD20, CD3, CD25, NOTCH, G-CSF-R, GM-CSF-R, EPO-R., a cadherin, an integrin, CD52, CD44, VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF, EGF, HGF, TNF-α, LIGHT, BTLA, lymphotoxin (LT), IgE, G-CSF, GM-CSF and EPO.
• In the affinity maturation method provided herein, a subset of the amino acid residues in the target region are modified by amino acid replacement. In one example, only the amino acid residues that differ between the first antibody and related antibody in the target region are modified by amino acid replacement. In another example, only the amino acid residues that are the same between the first antibody and the related antibody in the target region are modified by amino acid replacement. In some instances in the method provided herein, all of the amino acids residues in the target region are modified by amino acid replacement. For amino acid that is modified, the amino acid replacement can be to all 19 other amino acid residues, or a restricted subset thereof.
• In the method provided herein, that antibody is mutated by PCR mutagenesis, cassette mutagenesis, site-directed mutagenesis, random point mutagenesis, mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA, point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, and double-strand break repair. The antibody can be mutated by NNK, NNS, NNN, NNY or NNR mutagenesis.
• In one aspect of the method, scanning mutagenesis of the target region is performed to further elucidate amino acid residues to mutagenenize. In such a method, scanning mutagenesis is performed on the first antibody by producing a plurality of modified antibodies comprising a variable heavy chain and a variable light chain, or a portion thereof, where at least one of the variable heavy chain or variable light chain is one that is modified by replacement of a single amino acid residue with another amino acid residue in the target region, whereby each of the plurality of antibodies contains replacement of an amino acid in the target region compared to the first antibody. Each of the plurality of modified antibodies are screened for an activity to the target antigen. A second antibody is selected from among the modified antibodies that exhibits retained or increased activity for the target antigen compared to the first antibody not containing the amino acid replacement, whereby the second antibody is used in place of the first antibody in the affinity maturation method herein above. In such an example, the plurality of modified antibodies can be produced by producing a plurality of nucleic acid molecules that encode modified forms of a variable heavy chain or a variable light chain of the first antibody containing the target region, wherein the nucleic acid molecules contain one codon that encodes an amino acid in the target region compared to the corresponding codon of the unmodified variable heavy or variable light chain that does not encode the neutral amino acid, whereby each nucleic acid molecule of the plurality encodes a variable heavy chain or variable light chain that is modified by replacement of a single amino acid residue to a neutral amino acid residue in the target region.
• Further, in a method where scanning mutagenesis is performed on a target region, a second antibody can be selected that exhibits an activity that is at least or about 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 130%, 140%, 150%, 200% or more of the activity of the corresponding form of the first antibody. After selecting the antibody that exhibits retained or increased activity, the amino acid residue position that is modified in the second antibody to contain a scanned acid compared to the first antibody not containing the amino acid replacement can be determined.
• In examples of the affinity maturation method herein where scanning mutagenesis is employed, the scanned amino acid can be alanine, threonine, proline or glycine. For example, the scanned amino acid is alanine. The scanned amino acid also can be a non-natural amino acid.
• Further, when performing scanning mutagenesis in the methods herein, a subset of the amino acid residues in the target region are modified by amino acid replacement to a scanned amino acid. In one example, only the amino acid residues that differ between the first antibody and related antibody in the target region are modified by amino acid replacement to a scanned amino acid. In another example, only the amino acid residues that are the same between the first antibody and the related antibody in the target region are modified by amino acid replacement to a scanned amino acid. In an additional example, all of the amino acids in the target region are modified by amino acid replacement to a neutral amino acid.
• In the affinity maturation methods herein, the selected modified antibody exhibits 2-fold, 5-fold, 10-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 10000-fold or more improved activity for the target antigen compared to the first antibody. For example, the modified antibody exhibits a binding affinity that is greater than the binding affinity of the first antibody and is or is about 1×10⁻⁹ M, 2×10⁻⁹ M, 3×10⁻⁹ M, 4×10⁻⁹ M, 5×10⁻⁹ M, 6×10⁻⁹ M, 7×10⁻⁹ M, 8×10⁻⁹ M, 9×10⁻⁹ M, 1×10¹⁰ M, 2×10⁻¹⁰ M, 3×10⁻¹⁰ M, 4×10⁻¹⁰ M, 5×10⁻¹⁰ M, 6×10⁻¹⁰ M, 7×10⁻¹⁰ M, 8×10⁻¹⁰ M, 9×10⁻¹⁰M or less.
• In the methods herein, the amino acid modifications that are altered in the modified antibody compared to the first antibody not containing the amino acid replacements can be determined. Further, the method of affinity maturation provided herein can be repeated iteratively where a modified antibody is selected and is used as the first for subsequent affinity maturation thereof. In addition, in the methods herein, one or more amino acid replacements in the target region of one or more variable heavy chains or one or more variable light chains of selected modified antibodies are combined to generate a further modified antibody, whereby the further modified antibodies are screened for an activity to the target antigen to identify a further modified antibody that exhibits an increased activity for the target antigen compared to the first antibody and to the selected modified antibodies.
• In the affinity maturation methods herein, the method can be performed on the variable heavy chain of the first antibody and first modified antibodies selected each containing an amino acid replacement in the target region. Then, independent and separately, the method can be performed on the variable light chain of the first antibody and a second modified antibodies each containing an amino acid replacement in the target region can be selected. The variable heavy chain of a first modified antibody can be combined with the variable light chain of a second modified antibody to generate a plurality of different third modified antibodies each comprising an amino acid replacement in the target region of the variable heavy chain and variable light chain. Such third antibodies can be screened for an activity to the target antigen, and further modified antibodies that exhibit an increased activity for the target antigen compared to the first and second modified antibodies can be selected.
• Further, in any of the methods herein, other regions of the antibody can be optimized. For example, after selecting a modified antibody, another different region within the variable heavy chain or variable light chain of the first modified antibody can be selected for further mutagenesis. In such an example, a plurality of nucleic acid molecules that encode modified forms of the variable heavy chain or variable light chain of the first modified antibody can be produced, wherein the nucleic acid molecules contain one codon encoding an amino acid in the selected region that encodes a different amino acid from the first modified variable heavy or variable light chain, whereby each nucleic acid molecule of the plurality encodes a variable heavy chain or variable light chain that is modified in the selected region by replacement of a single amino acid residue. A plurality of further modified antibodies then are produced each containg a variable heavy chain and a variable light chain, or a portion thereof, wherein at least one of the variable heavy chain or variable light chain is modified, whereby the selected region in each of the plurality of antibodies contains replacement of an amino acid to a different amino acid compared to the first modified antibody. The further modified antibodies are screen for activity for the target antigen those further modified antibodies that exhibit increased activity for the target antigen compared to the first modified antibody are selected. In such examples, the different region that is modified can be a CDR1, CDR2, CDR3, FR1, FR2, FR3 or FR4.
• In any of the affinity maturation methods herein, any of the antibodoes can include an antibody or portion thereof. Such antibodies can be a Fab, Fab′, F(ab′)₂, single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd′ fragments, Fab fragments, Fd fragments, scFv fragments, and scFab fragments.
• Provided herein is a method of affinity maturation based on scanning mutagenesis. In the method, scanning mutagenesis of a first antibody is performed by producing a plurality of nucleic acid molecules that encode modified forms of a variable heavy chain or a variable light chain of a first antibody, wherein the nucleic acid molecules contain one codon that encodes another amino acid compared to the corresponding codon of the unmodified variable heavy or variable light chain that does not encode the other amino acid, whereby each nucleic acid molecule of the plurality encodes a variable heavy chain or variable light chain that is modified by replacement of a single amino acid residue to another amino acid such that every position across the full-length of the encoded variable heavy or light chain is replaced or every position in a selected region of the encoded variable heavy or variable light chain is replaced, whereby each replacement is to the same amino acid residue. A plurality of modified antibodies are then produced each containing a variable heavy chain and a variable light chain, or a portion thereof, whereby each of the plurality of antibodies contains replacement of an amino acid position with another amino acid compared to the first antibody. The plurality of modified antibodies are screened for an activity to the target antigen. A second antibody is selected from among the modified antibodies that exhibits retained or increased activity for the target antigen compared to the first antibody not containing the amino acid replacement. Further mutagenesis of the second antibody is performed by producing a plurality of nucleic acid molecules that encode modified forms of a variable heavy chain or a variable light chain of the second antibody, wherein the nucleic acid molecules contain one codon encoding an amino acid at the scanned amino acid position that encodes a different amino acid than the scanned amino acid in the second antibody, whereby each nucleic acid molecule of the plurality encodes a variable heavy chain or variable light chain that is modified at the scanned amino acid position by a single amino acid residue. A plurality of further modified antibodies are produced each containing a variable heavy chain and a variable light chain, or a portion thereof whereby the scanned amino acid position contains replacement to a different amino acid compared to the second antibody. The further modified antibodies are screened for an activity to the target antigen. From among the further modified antibodies, a third antibody is selected that exhibits increased activity for the target antigen compared to the first antibody or compared to the second antibody.
• In one example of the scanning affinity maturation method provided herein, every position in a region of the encoded variable heavy or variable light chain is replaced. The selected region can be a complementary determining region in the variable heavy chain or variable light chain selected that is a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3.
• In the method herein, a second antibody containing a scanning mutation is selected that exhibits retained or increased binding compared to the first antibody. Generally, the second antibody that is selected exhibits an activity that is at least or about 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 130%, 140%, 150%, 200% or more of the activity of the corresponding form of the first antibody.
• In the affinity maturation method provided herein, the amino acid residue position that is modified in the second antibody to contain a scanned amino acid compared to the first antibody not containing the amino acid replacement can be determined.
• In the scanning methods of affinity maturation provided herein, the scanning amino acid residue can be an alaninie, threonine, proline and glycine. For example, the amino acid is an alanine. In other examples, the scanning amino acid is a non-natural amino acid. In the methods herein, each of the plurality of nucleic acid molecules encodes a variable heavy chain or variable light chain that is modified by replacement of a single amino acid residue to the same scanned amino acid. In the method, the scanned amino acid position is modified by amino acid replacement to all other amino acid residues, or to a restricted subset thereof.
• In the scanning methods of affinity maturation provided herein, once a second antibody is selected, further modification of the antibody is effected. In the method, modification does not include amino acid replacement to the scanned amino acid or to the original amino acid at that position in the first antibody. The further modification of the second antibody can be effected by a method that is PCR mutagenesis, cassette mutagenesis, site-directed mutagenesis, random point mutagenesis, mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA, point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, and double-strand break repair. In one example, further mutations are made by NNK, NNS, NNN, NNY or NNR mutagenesis.
• In the scanning methods of affinity maturation provided herein, the activity that is assessed is binding, signal transduction, differentiation, alteration of gene expression, cellular proliferation, apoptosis, chemotaxis, cytotoxicity, cancer cell invasion, endothelial cell proliferation and tube formation. For example, where the activity is binding, binding is assessed by immunoassay, whole cell panning and surface plasmon resonance (SPR). The immunoassay can be a radioimmunoassay, enzyme linked immunosorbent assay (ELISA) or electrochemiluminescence assay. For example, the electrochemiluminescence assay can be meso scale discovery (MSD).
• In the scanning methods of affinity maturation provided herein, the target antigen is a polypeptide, carbohydrate, lipid, nucleic acid or a small molecule. The target antigen can be expressed on the surface of a virus, bacteria, tumor or other cell, or is a recombinant protein or peptide. The target antigen can a protein that is a target for therapeutic intervention. For example, the target antigen is involved in cell proliferation and differentiation, cell migration, apoptosis or angiogenesis. Exemplary target antigen include a VEGFR-1, VEGFR-2, VEGFR-3 (vascular endothelial growth factor receptors 1, 2, and 3), a epidermal growth factor receptor (EGFR), ErbB-2, ErbB-3, IGF-R1, C-Met (also known as hepatocyte growth factor receptor; HGFR), DLL4, DDR1 (discoidin domain receptor), KIT (receptor for c-kit), FGFR1, FGFR2, FGFR4 (fibroblast growth factor receptors 1, 2, and 4), RON (recepteur d'origine nantais; also known as macrophage stimulating 1 receptor), TEK (endothelial-specific receptor tyrosine kinase), TIE (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains receptor), CSF1R (colony stimulating factor 1 receptor), PDGFRB (platelet-derived growth factor receptor B), EPHA1, EPHA2, EPHB1 (erythropoietin-producing hepatocellular receptor A1, A2 and B1), TNF-R1, TNF-R2, HVEM, LT-βR, CD20, CD3, CD25, NOTCH, G-CSF-R, GM-CSF-R, EPO-R., a cadherin, an integrin, CD52, CD44, VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF, EGF, HGF, TNF-α, LIGHT, BTLA, lymphotoxin (LT), IgE, G-CSF, GM-CSF and EPO.
• In the scanning methods herein, the third antibody exhibits 2-fold, 5-fold, 10-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 10000-fold or more improved activity for the target antigen compared to the first antibody or the second antibody. For example, where the first antibody binds to the target antigen with a binding affinity that is at or about 10⁻⁴ M, 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, or lower, when the antibody is in a Fab form, the further optimized antibodies, such as the selected third antibody, are those that are optimized to have an improved binding affinity compared to the first antibody. For example, the third antibody exhibits a binding affinity that is greater than the binding affinity of the first antibody and is or is about 1×10⁻⁹ M, 2×10⁻⁹ M, 3×10⁻⁹ M, 4×10⁻⁹ M, 5×10⁻⁹ M, 6×10⁻⁹ M, 7×10⁻⁹ M, 8×10⁻⁹ M, 9×10⁻⁹ M, 1×10⁻¹⁰ M, 2×10⁻¹⁰ M, 3×10⁻¹⁰ M, 4×10⁻¹⁰ M, 5×10⁻¹⁰ M, 6×10⁻¹⁰ M, 7×10⁻¹⁰ M, 8×10⁻¹⁰ M, 9×10⁻¹⁰ M or less.
• In one aspect of the method, scanning mutagenesis is performed within the variable heavy chain of the first antibody, and the method performed therefrom. In another aspect, scanning mutagenesis is performed within the variable light chain of the first antibody, and steps of the method are performed therefrom. In an additional aspect of the method, scanning mutagenesis is performed within the variable heavy chain of the first antibody and steps of the method performed therefrom; and separately and independently scanning mutagenesis is performed within the variable light chain of the first antibody, and steps of the method are performed therefrom.
• In the method herein, further optimization can be achieved. The method can include determining the amino acid modifications that are altered in the third antibody compared to the first antibody not containing the amino acid replacements. Combination mutants can be generated. Also provided in the method herein, is a method that is repeated iteratively, wherein the third antibody identified in that is selected and used as the first antibody for subsequent maturation thereof, whereby the amino acid residue that is modified is not further modified in subsequent iterations of the method. In another example of optimization, one or more amino acid replacement in one or more variable heavy chains or one or more variable light chains of selected third antibodies are combined to generate a further modified antibody, whereby the further modified antibodies are screened for an activity to the target antigen to identify a further modified antibody that exhibits an increased activity for the target antigen compared to the first antibody, second antibody and to the selected third antibodies. For example, the steps of the method can be performed on the variable heavy chain of the first antibody and third antibodies selected each containing an amino acid replacement in the variable heavy chain compared to the corresponding variable heavy chain of the first antibody. Independently and separately, the steps of the method are performed on the variable light chain of the first antibody and different third modified antibodies are selected each containing an amino replacement in the variable light chain compared to the corresponding variable light chain of the first antibody. The variable heavy chain of a third antibody can be combined with the variable light chain of a different third antibody to generate a plurality of different further modified antibodies each containing an amino acid replacement of the variable heavy chain and variable light chain compared to the corresponding variable heavy chain and variable light chain of the first antibody. The further modified antibodies can be screened for activity (e.g. binding) to the target antigen; and those fourth antibodies that exhibit an increased activity for the target antigen compared to the first antibody, second antibody, and third antibodies are selected.
• In another example, after selecting a third antibody another different region within the variable heavy chain or variable light chain of the third antibody is selected for further mutagenesis. In such a method, a plurality of nucleic acid molecules are produced that encode modified forms of the variable heavy chain or variable light chain of the third antibody, wherein the nucleic acids molecules contain one codon encoding an amino acid in the selected region that encodes a different amino acid from the first modified variable heavy or variable light chain, whereby each nucleic acid molecule of the plurality encodes a variable heavy chain or variable light chain that is modified in the selected region by replacement of a single amino acid residue. Then, a plurality of further modified antibodies are produced each containing a variable heavy chain and a variable light chain, or a portion thereof, whereby the selected region in each of the plurality of antibodies contains replacement of an amino acid to a different amino acid compared to the third antibody. The further modified antibodies are screened for an activity (e.g. binding) to the target antigen and those further modified antibodies that exhibit increased activity for the target antigen compared to the third antibody are selected. In such an example, the different region that is subject to further mutagenesis can be a CDR1, CDR2, CDR3, FR1, FR2, FR3 and FR4.
• In any of the methods herein, the antibody can be an antibody or fragment thereof containing a variable heavy chain and a variable light chain, or a portion thereof. For example, the antibody can be a full-length antibody or a fragment thereof that is a Fab, Fab′, F(ab′)₂, single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd′ fragments, Fab fragments, Fd fragments, scFv fragments, and scFab fragments.
• Also provided herein is a method of antibody conversion, whereby, following mutageneis of a first or reference antibody having a known activity, an antibody is selected that exhibits an activity that is changed or inverted compared to the activity of the first or reference antibody for the same target antigen. In one example of the method, an activity of an antibody is converted from an antagonist to an activator. In the method, a first antibody or fragment thereof that is an antagonist antibody is selected, whereby the antibody inhibits a functional activity associated with its target antigen. A plurality of modified antibodies is produced each containing a variable heavy chain and a variable light chain, or a portion thereof sufficient to bind antigen, where at least one of the variable heavy chain or variable light chain is modified such that it contains at least one amino acid modification compared to the first antibody. For example, amino acid modification is replacement of at least a single amino acid residue, such that each of the plurality of antibodies contains replacement of an amino acid(s) to a different amino acid(s) compared to the first antibody. In one example of the method, the plurality of modified antibodies are produced by producing a plurality of nucleic acid molecules that encode modified forms of a variable heavy chain or a variable light chain of the first antibody, wherein the nucleic acid molecules contain at least one codon that encodes a different amino acid from the unmodified variable heavy or variable light chain, such that each nucleic acid molecule of the plurality encodes a variable heavy chain or variable light chain that is modified by replacement of a single amino acid residue. Following mutagenesis, the plurality of modified antibodies are each screened for an activity to the target antigen. Antibodies are selected or identified that result in an increase in a functional activity associated with the target antigen compared to activity in the presence of the first antibody, thereby converting the first antibody to an activator.
• In some examples of the method of converting an antagonist antibody to an activator, before the antibodies are screened for a functional activity the plurality of antibodies are each assessed for binding affinity for the target antigen. Antibodies that exhibit a binding affinity that is greater then the corresponding form of the first antibody for the target antigen are identified or selected. Then, that subset of antibodies are further screened for a functional activity to identify or select those that have a converted activator activity.
• In another example of the method of antibody conversion, an activity of an antibody is converted from an activator to an antagonist. In the method, a first antibody or fragment thereof that is an activator antibody is selected, whereby the antibody increases a functional activity associated with its target antigen. A plurality of modified antibodies is produced each containing a variable heavy chain and a variable light chain, or a portion thereof sufficient to bind antigen, where at least one of the variable heavy chain or variable light chain is modified such that it contains at least one amino acid modification compared to the first antibody. For example, amino acid modification is replacement of at least a single amino acid residue, such that each of the plurality of antibodies contains replacement of an amino acid(s) to a different amino acid(s) compared to the first antibody. In one example of the method, the plurality of modified antibodies are produced by producing a plurality of nucleic acid molecules that encode modified forms of a variable heavy chain or a variable light chain of the first antibody, wherein the nucleic acid molecules contain at least one codon that encodes a different amino acid from the unmodified variable heavy or variable light chain, such that each nucleic acid molecule of the plurality encodes a variable heavy chain or variable light chain that is modified by replacement of a single amino acid residue. Following mutagenesis, the plurality of modified antibodies are each screened for an activity to the target antigen. Antibodies are selected or identified that result in a decrease in a functional activity associated with the target antigen compared to activity in the presence of the first antibody, thereby converting the first antibody to an antagonist.
• In some examples of the method of converting an activator antibody to an antagonist, before the antibodies are screened for a functional activity the plurality of antibodies are each assessed for binding affinity for the target antigen. Antibodies that exhibit a binding affinity that is lower then the corresponding form of the first antibody for the target antigen are identified or selected. Then, that subset of antibodies are further screened for a functional activity to identify or select those that have a converted antagonist activity.
• In each of the conversion methods above, the target antigen is a VEGFR-1, VEGFR-2, VEGFR-3 (vascular endothelial growth factor receptors 1, 2, and 3), a epidermal growth factor receptor (EGFR), ErbB-2, ErbB-b3, IGF-R1, C-Met (also known as hepatocyte growth factor receptor; HGFR), DLL4, DDR1 (discoidin domain receptor), KIT (receptor for c-kit), FGFR1, FGFR2, FGFR4 (fibroblast growth factor receptors 1, 2, and 4), RON (recepteur d'origine nantais; also known as macrophage stimulating 1 receptor), TEK (endothelial-specific receptor tyrosine kinase), TIE (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains receptor), CSF1R (colony stimulating factor 1 receptor), PDGFRB (platelet-derived growth factor receptor B), EPHA1, EPHA2, EPHB1 (erythropoietin-producing hepatocellular receptor A1, A2 and B1), TNF-R1, TNF-R2, HVEM, LT-βR, CD20, CD3, CD25, NOTCH, G-CSF-R, GM-CSF-R or EPO-R.
• Provided herein is an anti-DLL4 antibody multimer that has a binding affinity for DLL4 that is 10⁻⁸ M or lower binding affinity as measured by surface plasmon resonance (SPR) as a monomeric Ig fragment and that is an activator of DLL4 activity. For example, the binding affinity is between 10⁻⁶ M to 10⁻⁸ M. The antibody multimer can be, for example, a full-length antibody, a F(ab′)₂ or a scFv dimer. In some examples, that antibody multimer is a full-length antibody that contains a constant region from a constant region of IgG1, IgG2, IgG3, IgA or IgM. For example, the constant region is an IgG1 constant region, or modified form thereof.
• In one example, the antibody multimer contains a heavy chain CDR1 (CDRH1) set forth in SEQ ID NO:2908, a heavy chain CDR2 (CDRH2) set forth in SEQ ID NO:2909, a heavy chain CDR3 (CDRH3) set forth in SEQ ID NO: 2910, a light chain CDR1 (CDRL1) set forth in SEQ ID NO:2911, a light chain CDR2 (CDRL2) set forth in SEQ ID NO:2912, and a light chain CDR3 (CDRL3) set forth in SEQ ID NO:2913; or contains a sequences of amino acids that exhibits at least 70% sequence identity to any of SEQ ID NOS: 2908-2913, whereby the antibody binds to DLL4 and is an activator of DLL4 activity. For example, the antibody multimer contains a heavy chain having a variable region set forth in SEQ ID NO: 88 and a light chain comprising a variable region set forth in SEQ ID NO:107.
• In another example, the antibody multimer contains a heavy chain CDR1 (CDRH1) set forth in SEQ ID NO:2914, a heavy chain CDR2 (CDRH2) set forth in SEQ ID NO:2915, a heavy chain CDR3 (CDRH3) set forth in SEQ ID NO: 2916, a light chain CDR1 (CDRL1) set forth in SEQ ID NO:2917, a light chain CDR2 (CDRL2) set forth in SEQ ID NO:2918, and a light chain CDR3 (CDRL3) set forth in SEQ ID NO:2919; or contains a sequences of amino acids that exhibits at least 70% sequence identity to any of SEQ ID NOS: 2914-2919, whereby the antibody binds to DLL4 and is an activator of DLL4 activity. For example, the antibody multimer contains a heavy chain having a variable region set forth in SEQ ID NO: 89 and a light chain comprising a variable region set forth in SEQ ID NO:108.
• In examples of antibody multimers provided herein, the heavy chain can contain an IgG1 constant region (e.g. set forth in SEQ ID NO: 2922) a light chain constant region, lambda or kappa (e.g. set forth in SEQ ID NO: 2923 or 2924).
• Provided herein is a method of treating aberrant angiogenesis associated with an angiogenic disease or condition by administering any of the antibody multimers provided herein to a subject, whereby the activity of a DLL4 receptor is increased. For example, the DLL4 receptor is Notch-1 or Notch-4. The angiogenic disease or condition can be a cancer, diabetic retinopathies and other diabetic complications, inflammatory diseases, endometriosis and age-related macular degeneration.
BRIEF DESCRIPTION OF THE FIGURES
• FIG. 1: FIG. 1 is a flow chart that illustrates the method of structure-affinity/activity relationship (SAR) based affinity maturation.
• FIG. 2: Amino acid alignments of “Hit” Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6-IGKJ1*01. FIG. 2A shows the alignment of the variable heavy chain of “Hit” Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKE*01 (SEQ ID NOS:88 and 107) with the variable heavy chain of “non-Hit” Fab VH1-46_IGHD6-13*01_IGHJ4*01 & L6_IGKJ1*01 (SEQ ID NOS:93 and 107). FIG. 2B shows the alignment of the variable light chain of “Hit” Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 (SEQ ID NOS:88 and 107) with the variable light chains of “non-Hit” Fabs VH1-46_IGHD6-6*01_IGHJ1*01 & A27_IGKJ1*01 (SEQ ID NOS:8 and 110), VH1-46_IGHD6-6*01_IGHJ1*01 & L25_IGKJ1*01 (SEQ ID NOS:88 and 120) and VH1-46_IGHD6-6*01_IGHJ1*01 & L2_IGKJ1*01 (SEQ ID NOS:88 and 112). The regions of variation are highlighted in grey. The amino acid sequence of the “Hit” Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 is shown in bold.
• FIG. 3: Amino acid alignment of the variable heavy chain of “Hit” Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01. FIG. 3 shows the alignment of the variable heavy chain of “Hit” Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01 (SEQ ID NOS:89 and 108) with the variable heavy chain of “non-Hit” Fab VH5-51_IGHD6-25*01_IGHJ4*01 & V3-4_IGLJ1*01 (SEQ ID NOS:106 and 108). The regions of variation are highlighted in grey. The amino acid sequence of the “Hit” Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01 is shown in bold.
• FIG. 4: Amino acid alignments of germline swapped variable heavy chains. FIG. 4A shows the alignment of the variable heavy chain of “Hit” Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 (SEQ ID NOS:88 and 107) with the variable heavy chains of J segment germline swapped Fabs VH1-46_IGHD6-6*01_IGHJ2*01 & L6_IGKJ1*01 (SEQ ID NOS:585 and 107), VH1-46_IGHD6-6*01_IGHJ4*01 & L6_IGKJ1*01 (SEQ ID NOS:586 and 107) and VH1-46_IGHD6-6*01_IGHJ5*01 & L6_IGKJ1*01 (SEQ ID NOS:587 and 107). FIG. 4B shows the alignment of the variable heavy chain of “Hit” Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01 (SEQ ID NOS:89 and 108) with the variable heavy chains of J segment germline swapped Fabs VH5-51_IGHD5-18*01>3_IGHJ1*01 & V3-4_IGLJ1*01 (SEQ ID NOS:588 and 108), VH5-51_IGHD5-18*01>3_IGHJ3*01 & V3-4_IGLJ4*01 (SEQ ID NOS:589 and 108) and VH5-51_IGHD5-18*01>3_IGHJ5*01 & V3-4_IGLJ4*01 (SEQ ID NOS:590 and 108). FIG. 4C shows the alignment of the variable heavy chain of “Hit” Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01 (SEQ ED NOS:89 and 108) with the variable heavy chains of D segment germline swapped Fabs VH5-51_IGHD5-12*01_IGHJ4*01 & V3-4_IGLJ1*01 (SEQ ID NOS:591 and 108) and VH5-51_IGHD5-24*01_IGHJ4*01 & V3-4_IGLJ1*01 (SEQ ID NOS:592 and 108). The regions of variation are highlighted in grey. The amino acid sequence of the “Hit” Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 is shown in bold.
• FIG. 5: Amino acid alignment of the variable heavy chain of “Hit” Fab VH3-23_IGHD2-21*01>3_IGHJ6*01 & V2-13_IGLJ2*01. FIG. 5 shows the alignment of the variable heavy chain of “Hit” Fab VH3-23_IGHD2-21*01>3_IGHJ6*01 & V2-13_IGLJ2*01 (SEQ ID NOS:1729 and 594) with the variable heavy chains of related “Hit” Fabs VH3-23_IGHD2-2*01>3_IGHJ6*01 & V2-13_IGLJ2*01 (SEQ ID NOS:1723 and 594), VH3-23_IGHD2-8*01>3_IGHJ6*01 & V2-13_IGLJ2*01 (SEQ ID NOS:1725 and 594) and VH3-23_IGHD2-15*01>3_IGHJ6*01 & V2-13_IGLJ2*01 (SEQ ID NOS:1727 and 594). The regions of variation are highlighted in grey. The amino acid sequence of the “Hit” Fab VH3-23_IGHD2-21*01>3_IGHJ6*01 & V2-13_IGLJ2*01 is shown in bold.
DETAILED DESCRIPTION Outline
• A. Definitions
• B. Overview of Methods
• • 1. Antibody Polypeptides
    • a. Antibody Structure and Function
    • b. Antibody Sequence and Specificity
  • 2. Methods of Identifying Antibodies
  • 3. Existing Methods of Optimizing Antibodies
• C. Method for Affinity Maturation of Antibodies
• • 1. Comparison of Structure and Activity
    • a. Selection of a First Antibody for Affinity Maturation
      • i. Immunization and Hybridoma Screening
      • ii. Screening Assays for Identification of a “Hit”
        • 1) Display Libraries
        • 2) Phage Display Libraries
        • 3) Addressable Libraries
    • b. Identification of a Related Antibody
    • c. Comparison of the amino acid sequences of the First Antibody and Related Antibodies
    • d. Mutagenesis of an Identified Region
  • 2. SAR by Scanning Mutagenesis
  • 3. Further Optimization
    • a. Complementarity Determining Regions
    • b. Framework Regions
    • c. Germline Swapping
• D. Method of Antibody Conversion
• • 1. Choosing the Starting or Reference Antibody
  • 2. Mutagenesis
  • 3. Selecting for a Converted Antibody
    • a. Binding
    • b. Functional Activity
• E. Assays
• • 1. Binding Assays
  • 2. Functional Activity
    • a. Differentiation
    • b. Alteration of Gene Expression
    • c. Cytotoxicity Assay
  • 3. In Vivo Assays
• F. Methods of Production of Antibodies
• • 1. Vectors
  • 2. Cells and Expression System
    • a. Prokaryotic Expression
    • b. Yeast
    • c. Insects
    • d. Mammalian cells
    • e. Plants
  • 3. Purification
• G. Anti-DLL4 Activator/Modulator Antibodies and Uses Thereof
• • 1. DLL4
    • a. Structure
    • b. Expression
    • c. Function
  • 2. Activator/Modulator Anti-DLL4 Multimer Antibodies
    • Exemplary Antibodies
  • 3. Modifications
    • a. Modifications to Reduce Immunogenicity
    • b. Glycosylation
    • c. Fc Modifications
    • d. PEGylation
  • 4. Compositions, Formulations, Administration and Articles of Manufacture/Kits
    • a. Compositions and Formulations
    • b. Articles of Manufacture and Kits
  • 5. Methods of Treatment and Uses
    • Combination Therapy
• H. Examples
A. DEFINITIONS
• Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong. All patents, patent applications, published applications and publications, Genbank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. In the event that there are a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.
• As used herein, an antibody refers to immunoglobulins and immunoglobulin portions, whether natural or partially or wholly synthetic, such as recombinantly, produced, including any portion thereof containing at least a portion of the variable region of the immunoglobulin molecule that is sufficient to form an antigen binding site. Hence, an antibody or portion thereof includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen binding site. For example, an antibody refers to an antibody that contains two heavy chains (which can be denoted H and H′) and two light chains (which can be denoted L and L′), where each heavy chain can be a full-length immunoglobulin heavy chain or a portion thereof sufficient to form an antigen binding site (e.g. heavy chains include, but are not limited to, VH, chains VH-CH1 chains and VH-CH1-CH2-CH3 chains), and each light chain can be a full-length light chain or a portion thereof sufficient to form an antigen binding site (e.g. light chains include, but are not limited to, VL chains and VL-CL chains). Each heavy chain (H and H′) pairs with one light chain (L and L′, respectively). Typically, antibodies minimally include all or at least a portion of the variable heavy (VH) chain and/or the variable light (VL) chain. The antibody also can include all or a portion of the constant region.
• For purposes herein, the term antibody includes full-length antibodies and portions thereof including antibody fragments, such as, but not limited to, Fab, Fab′, F(ab′)₂, single-chain Fvs (scFv), Fv, dsFv, diabody, Fd and Fd′ fragments Fab fragments, Fd fragments and scFv fragments. Other known fragments include, but are not limited to, scFab fragments (Hust et al., BMC Biotechnology (2007), 7:14). Antibodies include members of any immunoglobulin class, including IgG, IgM, IgA, IgD and IgE.
• As used herein, a full-length antibody is an antibody having two full-length heavy chains (e.g. VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and hinge regions, such as human antibodies produced by antibody secreting B cells and antibodies with the same domains that are produced synthetically.
• As used herein, antibody fragment or antibody portion with reference to a “portion thereof” or “fragment thereof” of an antibody refers to any portion of a full-length antibody that is less than full length but contains at least a portion of the variable region of the antibody sufficient to form an antigen binding site (e.g. one or more CDRs) and thus retains the a binding specificity and/or an activity of the full-length antibody; antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically, e.g. recombinantly produced derivatives. Examples of antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)₂, single-chain Fvs (scFv), Fv, dsFv, diabody, Fd and Fd′ fragments (see, for example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragment can include multiple chains linked together, such as by disulfide bridges and/or by peptide linkers. An antibody fragment generally contains at least about 50 amino acids and typically at least 200 amino acids.
• Hence, reference to an “antibody or portion thereof that is sufficient to form an antigen binding site” means that the antibody or portion thereof contains at least 1 or 2, typically 3, 4, 5 or all 6 CDRs of the VH and VL sufficient to retain at least a portion of the binding specificity of the corresponding full-length antibody containing all 6 CDRs. Generally, a sufficient antigen binding site at least requires CDR3 of the heavy chain (CDRH3). It typically further requires the CDR3 of the light chain (CDRL3). As described herein, one of skill in the art knows and can identify the CDRs based on kabat or Chothia numbering (see e.g., Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). For example, based on Kabat numbering, CDR-L1 corresponds to residues L24-L34; CDR-L2 corresponds to residues L50-L56; CDR-L3 corresponds to residues L89-L97; CDR-H1 corresponds to residues H31-H35, 35a or 35b depending on the length; CDR-H2 corresponds to residues H50-H65; and CDR-H3 corresponds to residues H95-H102.
• As used herein, “antigen-binding site” refers to the interface formed by one or more complementary determining regions (CDRs; also called hypervariable region). Each antigen binding site contains three CDRs from the heavy chain variable region and three CDRs from the light chain variable region. An antibody molecule typically has two antigen combining sites, each containing portions of a heavy chain variable region and portions of a light chain variable region. The antigen combining sites can contain other portions of the variable region domains in addition to the CDRs.
• As used herein, an Fv antibody fragment is composed of one variable heavy domain (VH) and one variable light (VL) domain linked by noncovalent interactions.
• As used herein, a dsFv refers to an Fv with an engineered intermolecular disulfide bond, which stabilizes the VH-VL pair.
• As used herein, an Fd fragment is a fragment of an antibody containing a variable domain (VH) and one constant region domain (CH1) of an antibody heavy chain.
• As used herein, “Fab fragment” is an antibody fragment that contains the portion of the full-length antibody that results from digestion of a full-length immunoglobulin with papain, or a fragment having the same structure that is produced synthetically, e.g. recombinantly. A Fab fragment contains a light chain (containing a VL and CL portion) and another chain containing a variable domain of a heavy chain (VH) and one constant region domain portion of the heavy chain (CH1); it can be recombinantly produced.
• As used herein, a F(ab′)₂ fragment is an antibody fragment that results from digestion of an immunoglobulin with pepsin at pH 4.0-4.5, or a synthetically, e.g. recombinantly, produced antibody having the same structure. The F(ab′)2 fragment contains two Fab fragments but where each heavy chain portion contains an additional few amino acids, including cysteine residues that form disulfide linkages joining the two fragments; it can be recombinantly produced.
• A Fab′ fragment is a fragment containing one half (one heavy chain and one light chain) of the F(ab′)2 fragment.
• As used herein, an Fd′ fragment is a fragment of an antibody containing one heavy chain portion of a F(ab′)2 fragment.
• As used herein, an Fv′ fragment is a fragment containing only the V_H and V_L domains of an antibody molecule.
• As used herein, a scFv fragment refers to an antibody fragment that contains a variable light chain (VL) and variable heavy chain (VH), covalently connected by a polypeptide linker in any order. The linker is of a length such that the two variable domains are bridged without substantial interference. Exemplary linkers are (Gly-Ser)_n residues with some Glu or Lys residues dispersed throughout to increase solubility.
• As used herein, diabodies are dimeric scFv; diabodies typically have shorter peptide linkers than scFvs, and they preferentially dimerize.
• As used herein, hsFv refers to antibody fragments in which the constant domains normally present in a Fab fragment have been substituted with a heterodimeric coiled-coil domain (see, e.g., Arndt et al. (2001) J Mol Biol. 7:312:221-228).
• As used herein, an “antibody multimer” refers to an antibody containing at least two or more antigen-binding sites. Antibody multimers include dimers, trimer, tetramers pentamers, and higher ordered oligomers. Formation of an antibody as a multimer can be achieved based on the knowledge of one of skill in the art. For example, multimeric forms include antibody oligomers that form via a multimerization domain that coordinates or facilitates the intereaction of at least two polypeptides or a covalent bond.
• As used herein, a multimerization domain refers to a sequence of amino acids that promotes stable interaction of a polypeptide molecule with one or more additional polypeptide molecules, each containing a complementary multimerization domain, which can be the same or a different multimerization domain to form a stable multimer with the first domain. Generally, a polypeptide is joined directly or indirectly to the multimerization domain. Exemplary multimerization domains include the immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions, and compatible protein-protein interaction domains. The multimerization domain, for example, can be an immunoglobulin constant region or domain, such as, for example, the constant domain or portions thereof from IgG, including IgG1, IgG2, IgG3 or IgG4 subtypes, IgA, IgE, IgD and IgM and modified forms thereof.
• As used herein, a “monospecific” is an antibody that contains two or more antigen-binding sites, where each antigen-binding site immunospecifically binds to the same epitope.
• As used herein, a “multispecific” antibody is an antibody that contains two or more antigen-binding sites, where at least two of the antigen-binding sites immunospecifically bind to different epitopes.
• As used herein, a “bispecific” antibody is a multispecific antibody that contains two or more antigen-binding sites and can immunospecifically bind to two different epitopes. A “trispecific” antibody is a multispecific antibody that contains three or more antigen-binding sites and can immunospecifically bind to three different epitopes, a “tetraspecific” antibody is a multispecific antibody that contains four or more antigen-binding sites and can immunospecifically bind to four different epitopes, and so on.
• As used herein, reference to a “monomeric Ig fragment” refers to an antibody portion that contains only one antigen-binding site. For example, a monomeric Ig fragment includes, for example, a Fab, Fv or a scFv.
• As used herein, a polypeptide domain is a part of a polypeptide (a sequence of three or more, generally 5 or 7 or more amino acids) that is a structurally and/or functionally distinguishable or definable. Exemplary of a polypeptide domain is a part of the polypeptide that can form an independently folded structure within a polypeptide made up of one or more structural motifs (e.g. combinations of alpha helices and/or beta strands connected by loop regions) and/or that is recognized by a particular functional activity, such as enzymatic activity or antigen binding. A polypeptide can have one, typically more than one, distinct domains. For example, the polypeptide can have one or more structural domains and one or more functional domains. A single polypeptide domain can be distinguished based on structure and function. A domain can encompass a contiguous linear sequence of amino acids. Alternatively, a domain can encompass a plurality of non-contiguous amino acid portions, which are non-contiguous along the linear sequence of amino acids of the polypeptide. Typically, a polypeptide contains a plurality of domains. For example, each heavy chain and each light chain of an antibody molecule contains a plurality of immunoglobulin (Ig) domains, each about 110 amino acids in length.
• As used herein, an Ig domain is a domain, recognized as such by those in the art, that is distinguished by a structure, called the Immunoglobulin (Ig) fold, which contains two beta-pleated sheets, each containing anti-parallel beta strands of amino acids connected by loops. The two beta sheets in the Ig fold are sandwiched together by hydrophobic interactions and a conserved intra-chain disulfide bond. Individual immunoglobulin domains within an antibody chain further can be distinguished based on function. For example, a light chain contains one variable region domain (VL) and one constant region domain (CL), while a heavy chain contains one variable region domain (VH) and three or four constant region domains (CH). Each VL, CL, VH, and CH domain is an example of an immunoglobulin domain.
• As used herein, a “variable domain” with reference to an antibody is a specific Ig domain of an antibody heavy or light chain that contains a sequence of amino acids that varies among different antibodies. Each light chain and each heavy chain has one variable region domain (VL, and, VH). The variable domains provide antigen specificity, and thus are responsible for antigen recognition. Each variable region contains CDRs that are part of the antigen binding site domain and framework regions (FRs).
• As used herein, reference to a variable heavy (VH) chain or a variable light (VL) chain (also termed VH domain or VL domain) refers to the polypeptide chains that make up the variable domain of an antibody.
• As used herein, a “region” of an antibody refers to a domain of an antibody or a portion of a domain is associated with a particular function or structure. In an antibody, regions of an antibody include the complementarity-determining region, the framework region, and/or the constant region. Generally, for purposes herein, a region of an antibody is a complementarity determining region CDR1, CDR2 and/or CDR3 of the variable light chain or variable heavy chain (CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, or CDRH3), or is a framework region FR1, FR2 or FR3 of the variable light chain or variable heavy chain.
• As used herein, “hypervariable region,” “HV,” “complementarity-determining region” and “CDR” and “antibody CDR” are used interchangeably to refer to one of a plurality of portions within each variable region that together form an antigen binding site of an antibody. Each variable region domain contains three CDRs, named CDR1, CDR2, and CDR3. The three CDRs are non-contiguous along the linear amino acid sequence, but are proximate in the folded polypeptide. The CDRs are located within the loops that join the parallel strands of the beta sheets of the variable domain.
• As used herein, framework regions (FRs) are the regions within the antibody variable region domains that are located within the beta sheets; the FR regions are comparatively more conserved, in terms of their amino acid sequences, than the hypervariable regions.
• As used herein, a constant region domain is a domain in an antibody heavy or light chain that contains a sequence of amino acids that is comparatively more conserved among antibodies than the variable region domain. Each light chain has a single light chain constant region (CL) domain and each heavy chain contains one or more heavy chain constant region (CH) domains, which include, CH1, CH2, CH3 and CH4. Full-length IgA, IgD and IgG isotypes contain CH1, CH2 CH3 and a hinge region, while IgE and IgM contain CH1, CH2 CH3 and CH4. CH1 and CL domains extend the Fab arm of the antibody molecule, thus contributing to the interaction with antigen and rotation of the antibody arms. Antibody constant regions can serve effector functions, such as, but not limited to, clearance of antigens, pathogens and toxins to which the antibody specifically binds, e.g. through interactions with various cells, biomolecules and tissues.
• As used herein, humanized antibodies refer to antibodies that are modified to include “human” sequences of amino acids so that administration to a human does not provoke an immune response. Methods for preparation of such antibodies are known. For example, the antibody in which the amino acid composition of the non-variable regions can be based on human antibodies. Computer programs have been designed to identify such regions.
• As used herein, “antibody conversion” refers to a process in which the functionl activity of an antibody or fragment thereof for a target antigen or substrate is changed, typically by mutation of one or more amino acid residues, to have an inverse functional activity of the starting or reference antibody. For example, if the starting or reference antibody exhibits antagonist activity for a target antigen, antibody coversion changes the antibody to an agonist or activator/modulator activity. In another example, if the starting or reference antibody exhibits activator/modulator activity for a target antigen, antibody conversion changes the antibody to an antagonist activity.
• As used herein, “affinity maturation” refers to a process in which an antibody is evolved from a reference antibody (also referred to herein as a template or parent antibody), typically by mutation of one or more amino acid residues, to have increased activity for a target antigen than a corresponding form of the reference antibody has for the same target antigen. Hence, the evolved antibody is optimized compared to the reference or template antibody.
• As used herein, reference to an affinity matured antibody refers to an antibody that has an increased activity for a target antigen relative to a reference antibody. For example, the affinity matured antibody exhibits increased binding to the target antigen compared to the reference or parent antibody. Typically, the affinity matured antibody binds to the same epitope as the reference antibody.
• As used herein, an optimized antibody refers to an antibody, or portion thereof, that has an increased activity for a target protein or antigen compared to a reference antibody, for example, improved binding affinity for a target protein and/or an improved functional activity. Typically, the antibody is optimized by virtue of one or more amino acid modifications (amino acid deletion, replacement or insertion) compared to a parent antibody not containing the one or more amino acid modifications. Generally, an activity, for example binding affinity, is increased by at or about 1.5-fold to 1000-fold, generally at least or about 2-fold to 100-fold, for example at or about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold or more compared to an activity of the parent antibody (e.g. germline antibody Hit not containing the modification(s)).
• As used herein, “structure affinity/activity relationship” (SAR) refers to the relationship between structure (e.g. sequence) and function of a molecule, whereby the activity of an antibody can be correlated to it sequence. Thus, knowledge of the SAR elucidates a region of a sequence, including particular amino acid residues, that contribute to the activity of an antibody. Methods of determining SAR are described herein.
• As used herein, activity towards a target protein or target antigen refers to binding specificity or binding affinity and/or modulation of a functional activity of a target protein, or other measurements that reflects the activity of an antibody or portion thereof towards a target protein. Activity of an antibody can be measured using a binding or affinity based assay, such as an ELISA, electrochemiluminescence assay (e.g. Meso Scale Discovery), or surface plasmon resonance, or can measured using a cell based assay as described herein.
• As used herein, “functional activity” refer to activities of a polypeptide (e.g. target protein) or portion thereof associated with a full-length (complete) protein. Functional activities include, but are not limited to, biological activity, catalytic or enzymatic activity, antigenicity (ability to bind to or compete with a polypeptide for binding to an anti-polypeptide antibody), immunogenicity, ability to form multimers, the ability to specifically bind to a receptor or ligand for the polypeptide and signaling and downstream effector functions. For purposes herein, modulation (i.e. activation or inhibition) of a functional activity of a polypeptide by an antibody or portion thereof herein means that a functional activity of the polypeptide is changed or altered in the presence of the antibody compared to the absence of the antibody or portion thereof.
• As used herein, binding activity refer to characteristics of a molecule, e.g. a polypeptide, relating to whether or not, and how, it binds one or more binding partners. Binding activities include ability to bind the binding partner(s), the affinity with which it binds to the binding partner (e.g. high affinity), the avidity with which it binds to the binding partner, the strength of the bond with the binding partner and specificity for binding with the binding partner.
• As used herein, “affinity” or “binding affinity” refers to the strength with which an antibody molecule or portion thereof binds to an epitope on a target protein or antigen. Affinity is often measured by equilibrium association constant (K_A) or equilibrium dissociation constant (K_D). Low-affinity antibody-antigen interaction is weak, and the molecules tend to dissociate rapidly, while high affinity antibody-antigen binding is strong and the molecules remain bound for a longer amount of time. Generally, affinity of an antibody to a target protein is with an equilibrium association constant (K_A) of greater than or equal to about 10⁶M⁻¹, greater than or equal to about 10⁷M⁻¹, greater than or equal to about 10⁸M⁻¹, or greater than or equal to about 10⁹M⁻¹, 10¹⁰M⁻¹, 10¹¹M⁻¹ or 10¹²M⁻¹. Antibodies also can be characterized by an equilibrium dissociation constant (K_D) 10⁻⁴ M, 10⁻⁶ M to 10⁻⁷ M, or 10⁻⁸ M, 10⁻¹⁰M, 10⁻¹¹M or 10⁻¹²M or lower dissociation constant. It is understood that a lower dissociation constant means that the antibody is characterized by a higher binding affinity. Generally, antibodies having a nanomolar or sub-nanomolar dissociaton constant are deemed to be high affinity antibodies. Such affinities can be readily determined using conventional techniques, such as by equilibrium dialysis; by using the BIAcore 2000 instrument, using general procedures outlined by the manufacturer; by radioimmunoassay using radiolabeled target antigen; or by another method known to the skilled artisan. The affinity data can be analyzed, for example, by the method of Scatchard et al., Ann N.Y. Acad. ScL, 51:660 (1949).
• As used herein, “specifically bind” or “immunospecifically bind” with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to the ability of the antibody or antigen-binding fragment to form one or more noncovalent bonds with a cognate antigen, by noncovalent interactions between the antibody combining site(s) of the antibody and the antigen (e.g. human DLL4). Typically, an antibody that immunospecifically binds (or that specifically binds) to an antigen is one that binds to the antigen with an affinity constant Ka of about or 1×10⁷M⁻¹ or 1×10⁸M⁻¹ or greater (or a dissociation constant (K_d) of 1×10⁻⁷M or 1×10⁻⁸M or less). Affinity constants can be determined by standard kinetic methodology for antibody reactions, for example, immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC) or other kinetic interaction assays known in the art (see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); see also U.S. Pat. No. 7,229,619 for a description of exemplary SPR and ITC methods for calculating the binding affinity of anti-RSV antibodies). Instrumentation and methods for real time detection and monitoring of binding rates are known and are commercially available (e.g., BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335).
• As used herein, the term “bind selectively” or “selectively binds,” in reference to a polypeptide or an antibody provided herein, means that the polypeptide or antibody binds with a selected epitope without substantially binding to another epitope. Typically, an antibody or fragment thereof that selectively binds to a selected epitope specifically binds to the epitope, such as with an affinity constant Ka of about or 1×10⁷M⁻¹ or 1×10⁸M⁻¹ or greater.
• As used herein, “epitope” refers to the localized region on the surface of an antigen or protein that is recognized by an antibody. Peptide epitopes include those that are continuous epitopes or discontinuous epitopes. An epitope is generally determined by the three dimensional structure of a protein as opposed to the linear amino acid sequence.
• As used herein, “binds to the same epitope” with reference to two or more antibodies means that the antibodies compete for binding to an antigen and bind to the same, overlapping or encompassing continuous or discontinuous segments of amino acids. Those of skill in the art understand that the phrase “binds to the same epitope” does not necessarily mean that the antibodies bind to exactly the same amino acids. The precise amino acids to which the antibodies bind can differ. For example, a first antibody can bind to a segment of amino acids that is completely encompassed by the segment of amino acids bound by a second antibody. In another example, a first antibody binds one or more segments of amino acids that significantly overlap the one or more segments bound by the second antibody. For the purposes herein, such antibodies are considered to “bind to the same epitope.”
• Antibody competition assays can be used to determine whether an antibody “binds to the same epitope” as another antibody. Such assays are well known on the art. Typically, competition of 70% or more, such as 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or more, of an antibody known to interact with the epitope by a second antibody under conditions in which the second antibody is in excess and the first saturates all sites, is indicative that the antibodies “bind to the same epitope.” To assess the level of competition between two antibodies, for example, radioimmunoassays or assays using other labels for the antibodies, can be used. For example, a DLL4 antigen can be incubated with a saturating amount of a first anti-DLL4 antibody or antigen-binding fragment thereof conjugated to a labeled compound (e.g., ³H, ¹²⁵I, biotin, or rubidium) in the presence the same amount of a second unlabeled anti-DLL4 antibody. The amount of labeled antibody that is bound to the antigen in the presence of the unlabeled blocking antibody is then assessed and compared to binding in the absence of the unlabeled blocking antibody. Competition is determined by the percentage change in binding signals in the presence of the unlabeled blocking antibody compared to the absence of the blocking antibody. Thus, if there is a 70% inhibition of binding of the labeled antibody in the presence of the blocking antibody compared to binding in the absence of the blocking antibody, then there is competition between the two antibodies of 70%. Thus, reference to competition between a first and second antibody of 70% or more, such as 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or more, means that the first antibody inhibits binding of the second antibody (or vice versa) to the antigen by 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or more (compared to binding of the antigen by the second antibody in the absence of the first antibody). Thus, inhibition of binding of a first antibody to an antigen by a second antibody of 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or more indicates that the two antibodies bind to the same epitope.
• As used herein, the term “surface plasmon resonance” refers to an optical phenomenon that allows for the analysis of real-time interactions by detection of alterations in protein concentrations within a biosensor matrix, for example, using the BiaCore system (GE Healthcare Life Sciences).
• As used herein, a “bispecific” antibody is a multispecific antibody that contains two or more antigen-binding sites and can immunospecifically bind to two different epitopes. A “trispecific” antibody is a multispecific antibody that contains three or more antigen-binding sites and can immunospecifically bind to three different epitopes, a “tetraspecific” antibody is a multispecific antibody that contains four or more antigen-binding sites and can immunospecifically bind to four different epitopes, and so on.
• As used herein, “epitope mapping” is the process of identification of the molecular determinants for antibody-antigen recognition.
• As used herein, a “target protein” or “target antigen” refers to candidate proteins or peptides that are specifically recognized by an antibody or portion thereof and/or whose activity is modulated by an antibody or protion thereof. A target protein includes any peptide or protein that contains an epitope for antibody recognition. Target proteins include proteins involved in the etiology of a disease or disorder by virtue of expression or activity. Exemplary target proteins are described herein.
• As used herein, a “Hit” refers to an antibody or portion thereof generated, identified, recognized or selected as having an activity for a target antigen. For example, a “Hit” can be identified in a screening assay. Generally, a “Hit” is identified based on its binding activity or affinity for the target antigen. For purposes herein, a “Hit” is generally recognized to be an antibody or portion thereof that has a binding affinity for a target antigen that is at least about or is 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, or lower. For purposes herein, a Hit typically is a first antibody or a reference or parent antibody that is further optimized using affinity maturation methods herein. Thus, the terms “Hit”, first antibody, reference antibody or parent antibody are used interchangeably herein.
• As used herein, a “modified antibody” refers to an antibody, or portion thereof, that contains one or more amino acid modifications compared to a parent or reference antibody. An amino acid modification includes an amino acid deletion, replacement (or substitution), or addition. A modified antibody can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid modifications. Typically, an amino acid modification is an amino acid replacement. Generally, the amino acid modifications are present in a region or target region of an antibody, but also can be present in other regions of the antibody or portion thereof.
• As used herein, a “related antibody” is an antibody that exhibits structural and functional similarity to a corresponding form of a reference antibody (e.g. a Hit antibody or first antibody), but that does not exhibit the same activity or structure (e.g. sequence) as the reference antibody. For example, a related antibody is one that exhibits sequence simiarlity but is not identical to the reference antibody, and exhibits reduced activity or less activity than the activity of a reference antibody towards a target protein or antigen, such as reduced binding affinity. For purposes herein, an antibody is a related antibody if 1) it exhibits sequence similarity to a reference antibody such that it contains a variable heavy chain and/or a variable light chain that exhibits at least 75% amino acid sequence identity to the corresponding variable heavy chain or variable light chain of the first antibody, where the related antibody (variable heavy chain and variable light chain) does not exhibit 100% sequence identity to the reference antibody; and 2) it exhibits reduced activity compared to a corresponding form of the reference antibody. The sequence similiarity or sequence identity can be In another example, an antibody is a related antibody if 1) it exhibits sequence similarity to a reference antibody such that at least one of the V_H, D_H and J_H germline segments of the nucleic acid molecule encoding the variable heavy chain of the related antibody is identical to one of the V_H, D_H and J_H germline segments of the nucleic acid molecule encoding the variable heavy chain of the first antibody and/or at least one of the V_κ and J_κ or at least one of the V_λ and J_λ germline segments of the nucleic acid molecule encoding the variable light chain is identical to one of the V_κ and J_κ or V_λ and J_λ germline segments of the nucleic acid molecule encoding the variable light chain of the first antibody; and 2) it exhibits reduced activity compared to a corresponding form of the reference antibody.
• As used herein “reduced activity” or “less activity” for a target antigen means that an antibody, or portion thereof, exhibits an activity towards a target antigen (e.g. binding or other functional activity) that is not as high or of the same degree as the activity of a reference antibody for the same target antigen. It is understood that in comparing an activity to a reference antibody, the activity is compared to the corresponding form of the antibody using the same assay to assess activity under the same or similar conditions. Hence, the requisite level of activity between and among two or more antibodies is compared under similar parameters or conditions. For purposes herein, an antibody that has a “reduced activity” or “less activity” for a target antigen generally exhibits 80% or lower the activity towards a target antigen as a reference antibody, such as 5% to 80% of the activity, for example, at or about 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or lower the activity towards a target antigen as a reference antibody.
• As used herein, a “related variable heavy chain” or a “related variable light chain” is one that exhibits sequence identity to the corresponding variable heavy chain and/or variable light chain of a reference antibody, but that is not identical (e.g. does not exhibit 100% sequence identity) to the corresponding variable heavy chain and/or variable light chain of a reference antibody. Generally, a related variable heavy chain or a variable light chain is one that exhibits at least 60% sequence identity to the corresponding chain of the reference antibody, generally at least 75% sequence identity. For example, a related variable heavy chain or a variable light chain is one that exhibits 60% to 99% sequence identity, for example, at or about 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the corresponding chain of the reference antibody. For example, a related antibody includes an antibody in which at least one of the V_H, D_H and J_H germline segments of the nucleic acid molecule encoding the variable heavy chain of the related antibody is identical to one of the V_H, D_H and J_H germline segments of the nucleic acid molecule encoding the variable heavy chain of the first antibody and/or at least one of the V_κ and J_κ or at least one of the V_λ and J_λ germline segments of the nucleic acid molecule encoding the variable light chain is identical to one of the V_κ and J_κ or V_λ and J_λ germline segments of the nucleic acid molecule encoding the variable light chain of the first antibody. Generally, a related variable heavy chain and/or variable light chain of an antibody exhibits at least 75% amino acid sequence identity to the corresponding variable heavy chain or variable light of a reference antibody.
• As used herein, a form of an antibody refers to a particular structure of an antibody. Antibodies herein include full length antibodies and portions thereof, such as, for example, a Fab fragment or other antibody fragment. Thus, a Fab is a particular form of an antibody.
• As used herein, reference to a “corresponding form” of an antibody means that when comparing a property or activity of two antibodies, the property is compared using the same form of the antibody. For example, if its stated that an antibody has less activity compared to the activity of the corresponding form of a first antibody, that means that a particular form, such as a Fab of that antibody, has less activity compared to the Fab form of the first antibody.
• As used herein, “sequence diversity” or “sequence similarity” refers to a representation of nucleic acid sequence similarity and is determined using sequence alignments, diversity scores, and/or sequence clustering. Any two sequences can be aligned by laying the sequences side-by-side and analyzing differences within nucleotides at every position along the length of the sequences. Sequence alignment can be assessed in silico using Basic Local Alignment Search Tool (BLAST), an NCBI tool for comparing nucleic acid and/or protein sequences. The use of BLAST for sequence alignment is well known to one of skill in the art. The Blast search algorithm compares two sequences and calculates the statistical significance of each match (a Blast score). Sequences that are most similar to each other will have a high Blast score, whereas sequences that are most varied will have a low Blast score.
• As used herein, Basic Local Alignment Search Tool (BLAST) is a search algorithm developed by Altschul et al. (1990) to separately search protein or DNA databases, for example, based on sequence identity. For example, blastn is a program that compares a nucleotide query sequence against a nucleotide sequence database (e.g. GenBank). BlastP is a program that compares an amino acid query sequence against a protein sequence database.
• As used herein, a “target region” refers to a region of a variable heavy chain or variable light chain of an antibody (e.g. a Hit antibody) or portion thereof that exhibits at least one amino acid differences compared to the corresponding region of related antibody or antibodies. Thus, a target region includes one or more of a CDR1, CDR2, CDR3, FR1, FR2, FR3 or FR4 of the variable heavy chain or variable light chain of a an antibody that contains at least one amino acid difference compared to the corresponding region of a related antibody. Generally, a target region is a region of an antibody that is associated with the structure/activity relationship (SAR) of the antibody. Thus, for purposes of practice of the method herein, a target region is one that is targeted for further mutagenesis. As described herein, it is within the level of one of skill in the art to identify such regions and to determine if amino acid differences exist. One of skill in the art knows and can identify a region in an antibody, for example a CDR or FR, based on Kabat or Chothia numbering (see e.g., Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917).
• As used herein, “saturation mutagenesis” refers to the process of systematically generating a plurality of mutants by replacing at least one amino acid residue of a protein sequence to all or a subset of the remaining amino acid residues or to effect replacement of a number of amino acid residues (within or across the full length of the protein or within or across a region of a protein) each to all or a subset of the remaining amino acid residues. Saturation mutagenesis can be full or partial.
• As used herein, “full saturation mutagenesis” refers to the process of systematically generating a plurality of mutants by replacing an amino acid residue in a protein sequence with the other 19 other naturally-occurring amino acids. A single amino acid residue in a protein sequence can be subject to mutagenesis. Alternatively, all or a subset of amino acid residues across the full length sequence of a protein or a region of the protein sequence (e.g. target region) can be subjected to full saturation mutagenesis.
• As used herein, “partial saturation mutagenesis” refers to the process of systematically generating a plurality of mutant sequences by replacing an amino acid residue in a protein sequence to a subset of the other 19 other naturally-occurring amino acids. A single amino acid residue in a protein sequence can be subject to mutagenesis. Alternatively, all or a subset of amino acid residues across the full length sequence of a protein or a region of the protein sequence (e.g. target region) can be subjected to partial saturation mutagenesis.
• As used herein, “scanning mutagenesis” refers to the process of systematically replacing all or a subset of amino acids in a protein or in a region of a protein (e.g. target region) with a selected amino acid, typically alanine, glycine or serine, as long as each residue is replaced with the same residue. Typically, the replacing amino acid is an alanine.
• As used herein, reference to an antibody that is an “Up mutant” or an antibody that “exhibits retained or increased activity”, refers to an antibody subjected to scanning mutagenesis whose activity when containing a single amino acid mutation to a scanned amino acid is retained or increased compared to the parent antibody not contained the scanned amino acid mutation. The antibody that retains an activity to a target antigen can exhibit some increase or decrease in binding, but generally exhibits the same binding as the first antibody not containing the scanned mutation, for example, exhibits at least 75% of the binding activity, such as 75% to 120% of the binding, for example, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110% or 115% of the binding. An antibody that exhibits increased activity to a target antigen generally exhibits greater than 115% of the activity, such as greater than 115%, 120%, 130%, 140%, 150%, 200% or more activity than the first antibody not containing the mutation.
• As used herein “iterative” with respect to performing the steps of the method means that the method is repeated a plurality of times, such as 2, 3, 4, 5 or more times, until a modified “Hit” is identified whose activity is optimized or improved compared to prior iterations.
• As used herein, an “intermediate” with reference to an antibody or portion thereof refers to an antibody that is derived from or evolved from a reference antibody, template or parent antibody, for example, by the process of affinity maturation, but that is itself further evolved. For example, once a modified Hit is selected in the affinity maturation method herein, it can itself be used as a template in order to further evolve or optimize the antibody. Hence, the modified Hit is an intermediate antibody in order to identify or select a further modified Hit.
• As used herein, an “antibody library” refers to a collection of antibody members or portions thereof, for example, 2 or more, typically 5 or more, and typically 10 or more, such as, for example, at or about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴ or more of such molecules. In some examples, the members of the collection are analogous to each other in that members within a collection are varied compared to a target or template antibody. An antibody library, however, encompasses a collection of any antibody members, or portions thereof. Thus, it is not necessary that each member within the collection is varied compared to a template member. Generally, collections contain different members (i.e. based on sequence), although in some cases collections of antibodies can contain some members that are the same. Typically, collections contain at least 10⁴ or about 10⁴,10⁵ or about 10⁵, 10⁶ or about 10⁶, at least 10⁸ or about 10⁸, at least 10⁹ or about 10⁹, at least 10¹⁰ or about 10¹⁰, or more different antibody members. Thus, the collections typically have a diversity of at least 10⁴ or about 10⁴,10⁵ or about 10⁵, 10⁶ or about 10⁶, at least 10⁸ or about 10⁸, at least 10⁹ or about 10⁹, at least 10¹⁰ or about 10¹⁰, at least 10¹¹ or about 10¹¹, at least 10¹² or about 10¹², at least 10¹³ or about 10¹³, at least 10¹⁴ or about 10¹⁴, or more. Thus, an antibody library having a diversity of 10⁷ means that it contains 10⁷ different members.
• As used herein, “diversity” with respect to members in a collection or library refers to the number of unique members in a collection. Hence, diversity refers to the number of different amino acid sequences or nucleic acid sequences, respectively, among the analogous polypeptide members of that collection. For example, a collection of polynucleotides having a diversity of 10⁴ contains 10⁴ different nucleic acid sequences among the analogous polynucleotide members. In one example, the provided collections of polynucleotides and/or polypeptides have diversities of at least at or about 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰ or more.
• As used herein, “a diversity ratio” refers to a ratio of the number of different members in the library over the number of total members of the library. Thus, a library with a larger diversity ratio than another library contains more different members per total members, and thus more diversity per total members. The provided libraries include libraries having high diversity ratios, such as diversity ratios approaching 1, such as, for example, at or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 0.91, 0.92, 0.93, 0.94, 0.95. 0.96, 0.97, 0.98, or 0.99.
• As used herein, “combinatorial library” refers to collections of compounds formed by reacting different combinations of interchangeable chemical “building blocks” to produce a collection of compounds based on permutations of the building blocks. For an antibody combinatorial library, the building blocks are the component V, D and J regions (or modified forms thereof) from which antibodies are formed. For purposes herein, the terms “library” or “collection” are used interchangeably.
• As used herein, a combinatorial antibody library is a collection of antibodies (or portions thereof, such as Fabs), where the antibodies are encoded by nucleic acid molecules produced by the combination of V, D and J gene segments, particularly human V, D and J germline segments. The combinatorial libraries herein typically contain at least 50 different antibody (or antibody portions or fragment) members, typically at least or about 50 to 10¹⁰ or more different members, generally at least or about 10² to 10⁶ or more different members, for example, at least or about 50, 100, 500, 10³, 1×10³, 2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, or more different members. The resulting libraries or collections of antibodies or portions thereof, can be screened for binding to a target protein or modulation of a functional activity.
• As used herein, a human combinatorial antibody library is a collection of antibodies or portions thereof, whereby each member contains a VL and VH chains or a sufficient portion thereof to form an antigen binding site encoded by nucleic acid containing human germline segments produced as described in U.S. Provisional Application Nos. 61/198,764 and 61/211,204, incorporated by reference herein.
• As used herein, a locus in a library refers to a location or position, that can contain a member or members of library. The position does not have to be a physical position. For example, if the collection is provided as an array on a solid support, the support contains loci that can or do present members of the array.
• As used herein, an address refers to a unique identifier for each locus in a collection whereby an addressed member (e.g. an antibody) can be identified. An addressed moiety is one that can be identified by virtue of its locus or location. Addressing can be effected by position on a surface, such as a well of a microplate. For example, an address for a protein in a microwell plate that is F9 means that the protein is located in row F, column 9 of the microwell plate. Addressing also can be effected by other identifiers, such as a tag encoded with a bar code or other symbology, a chemical tag, an electronic, such RF tag, a color-coded tag or other such identifier.
• As used herein, an array refers to a collection of elements, such as antibodies, containing three or more members.
• As used herein, a “spatial array” is an array where members are separated or occupy a distinct space in an array. Hence, spatial arrays are a type of addressable array. Examples of spatial arrays include microtiter plates where each well of a plate is an address in the array. Spacial arrays include any arrangement wherein a plurality of different molecules, e.g, polypeptides, are held, presented, positioned, situated, or supported. Arrays can include microtiter plates, such as 48-well, 96-well, 144-well, 192-well, 240-well, 288-well, 336-well, 384-well, 432-well, 480-well, 576-well, 672-well, 768-well, 864-well, 960-well, 1056-well, 1152-well, 1248-well, 1344-well, 1440-well, or 1536-well plates, tubes, slides, chips, flasks, or any other suitable laboratory apparatus. Furthermore, arrays can also include a plurality of sub-arrays. A plurality of sub-arrays encompasses an array where more than one arrangement is used to position the polypeptides. For example, multiple 96-well plates can constitute a plurality of sub-arrays and a single array.
• As used herein, an addressable library is a collection of molecules such as nucleic acid molecules or protein agents, such as antibodies, in which each member of the collection is identifiable by virtue of its address.
• As used herein, an addressable array is one in which the members of the array are identifiable by their address, the position in a spatial array, such as a well of a microtiter plate, or on a solid phase support, or by virtue of an identifiable or detectable label, such as by color, fluorescence, electronic signal (i.e. RF, microwave or other frequency that does not substantially alter the interaction of the molecules of interest), bar code or other symbology, chemical or other such label. Hence, in general the members of the array are located at identifiable loci on the surface of a solid phase or directly or indirectly linked to or otherwise associated with the identifiable label, such as affixed to a microsphere or other particulate support (herein referred to as beads) and suspended in solution or spread out on a surface.
• As used herein, “an addressable antibody library” or “an addressable combinatorial antibody library” refers to a collection of antibodies in which member antibodies are identifiable and all antibodies with the same identifier, such as position in a spatial array or on a solid support, or a chemical or RF tag, bind to the same antigen, and generally are substantially the same in amino acid sequence. For purposes herein, reference to an “addressable arrayed combinatorial antibody library” means that the antibody members are addressed in an array.
• As used herein, a support (also referred to as a matrix support, a matrix, an insoluble support or solid support) refers to any solid or semisolid or insoluble support to which a molecule of interest, typically a biological molecule, organic molecule or biospecific ligand is linked or contacted. Such materials include any materials that are used as affinity matrices or supports for chemical and biological molecule syntheses and analyses, such as, but are not limited to: polystyrene, polycarbonate, polypropylene, nylon, glass, dextran, chitin, sand, pumice, agarose, polysaccharides, dendrimers, buckyballs, polyacrylamide, silicon, rubber, and other materials used as supports for solid phase syntheses, affinity separations and purifications, hybridization reactions, immunoassays and other such applications. The matrix herein can be particulate or can be in the form of a continuous surface, such as a microtiter dish or well, a glass slide, a silicon chip, a nitrocellulose sheet, nylon mesh, or other such materials. When particulate, typically the particles have at least one dimension in the 5-10 mm range or smaller. Such particles, referred collectively herein as “beads”, are often, but not necessarily, spherical. Such reference, however, does not constrain the geometry of the matrix, which can be any shape, including random shapes, needles, fibers, and elongated. Roughly spherical “beads”, particularly microspheres that can be used in the liquid phase, also are contemplated. The “beads” can include additional components, such as magnetic or paramagnetic particles (see, e.g., Dynabeads® (Dynal, Oslo, Norway)) for separation using magnets, as long as the additional components do not interfere with the methods and analyses herein.
• As used herein, matrix or support particles refers to matrix materials that are in the form of discrete particles. The particles have any shape and dimensions, but typically have at least one dimension that is 100 mm or less, 50 mm or less, 10 mm or less, 1 mm or less, 100 μm or less, 50 μm or less and typically have a size that is 100 mm³ or less, 50 mm³ or less, 10 mm³ or less, and 1 mm³ or less, 100 μm³ or less and can be on the order of cubic microns. Such particles are collectively called “beads.”
• As used herein, germline gene segments refer to immunoglobulin (Ig) variable (V), diversity (D) and junction (J) or constant (C) genes from the germline that encode immunoglobulin heavy or light (kappa and lambda) chains. There are multiple V, D, J and C gene segments in the germline, but gene rearrangement results in only one segment of each occurring in each functional rearranged gene. For example, a functionally rearranged heavy chain contains one V, one D and one J and a functionally rearranged light chain gene contains one V and one J. Hence, these gene segments are carried in the germ cells but cannot be transcribed and translated into heavy and light chains until they are arranged into functional genes. During B-cell differentiation in the bone marrow, these gene segments are randomly shuffled by a dynamic genetic system capable of generating more than 10¹⁰ specificities.
• For purposes herein, heavy chain germline segments are designated as V_H, D_H and J_H, and compilation thereof results in a nucleic acid encoding a VH chain. Light chain germline segments are designated as V_L or J_L, and include kappa and lambda light chains (V_κ and J_κ; V_λ and J_λ) and compilation thereof results in a nucleic acid encoding a VL chain. It is understood that a light chain is either a kappa or lambda light chain, but does not include a kappa/lambda combination by virtue of compilation of a V_κ and J_κ.
• Reference to a variable germline segment herein refers to V, D and J groups, subgroups, genes or alleles thereof. Gene segment sequences are accessible from known database (e.g., National Center for Biotechnology Information (NCBI), the international ImMunoGeneTics information System® (IMGT), the Kabat database and the Tomlinson's VBase database (Lefranc (2003) Nucleic Acids Res., 31:307-310; Martin et al., Bioinformatics Tools for Antibody Engineering in Handbook of Therapeutic Antibodies, Wiley-VCH (2007), pp. 104-107).
• As used herein, a “group” with reference to a germline segment refers to a core coding region from an immunoglobulin, i.e. a variable (V) gene, diversity (D) gene, joining (J) gene or constant (C) gene encoding a heavy or light chain. Exemplary of germline segment groups include V_H, D_H, J_H, V_κ, J_κ, V_λ and J_λ.
• As used herein, a “subgroup” with reference to a germline segment refers to a set of sequences that are defined by nucleotide sequence similarity or identity. Generally, a subgroup is a set of genes that belong to the same group [V, D, J or C], in a given species, and that share at least 75% identity at the nucleotide level. Subgroups are classified based on IMGT nomenclature (imgt.cines.fr; see e.g., Lefranc et al. (2008) Briefings in Bioinformatics, 9:263-275). Generally, a subgroup represent a multigene family.
• As used herein, an allele of a gene refer to germline sequences that have sequence polymorphism due to one or more nucleotide differences in the coding region compared to a reference gene sequence (e.g. substitutions, insertions or deletions). Thus, IG sequences that belong to the same subgroup can be highly similar in their coding sequence, but nonetheless exhibit high polymorphism. Subgroup alleles are classified based on IMGT nomenclature with an asterisk (*) followed by a two figure number.
• As used herein, a “family” with reference to a germline segment refers to sets of germline segment sequences that are defined by amino acid sequence similarity or identity. Generally, a germline family includes all alleles of a gene.
• As used herein, inverted sequence with reference to nucleotides of a germline segment means that the gene segment has a sequence of nucleotides that is the reverse complement of a reference sequence of nucleotides.
• As used herein, “compilation,” “compile,” “combine,” “combination,” “rearrange,” “rearrangement,” or other similar terms or grammatical variations thereof refers to the process by which germline segments are ordered or assembled into nucleic acid sequences representing genes. For example, in the combinatorial method, variable heavy chain germline segments are assembled such that the V_H segment is 5′ to the D_H segment which is 5′ to the J_H segment, thereby resulting in a nucleic acid sequence encoding a VH chain. Variable light chain germline segments are assembled such that the V_L segment is 5′ to the J_L segment, thereby resulting in a nucleic acid sequence encoding a VL chain. A constant gene segment or segments also can be assembled onto the 3′ end of a nucleic acid encoding a VH or VL chain.
• As used herein, “linked,” or “linkage” or other grammatical variations thereof with reference to germline segments refers to the joining of germline segments. Linkage can be direct or indirect. Germline segments can be linked directly without additional nucleotides between segments, or additional nucleotides can be added to render the entire segment in-frame, or nucleotides can be deleted to render the resulting segment in-frame. In the method of generating a combinatorial antibody library, it is understood that the choice of linker nucleotides is made such that the resulting nucleic acid molecule is in-frame and encodes a functional and productive antibody.
• As used herein, “in-frame” or “linked in-frame” with reference to linkage of human germline segments means that there are insertions and/or deletions in the nucleotide germline segments at the joined junctions to render the resulting nucleic acid molecule in-frame with the 5′ start codon (ATG), thereby producing a “productive” or functional full-length polypeptide. The choice of nucleotides inserted or deleted from germline segments, particularly at joints joining various VD, DJ and VJ segments, is in accord with the rules provided in the method herein for V(D)J joint generation described in detail in U.S. Provisional Application Nos. 61/198,764 and 61/211,204. For example, germline segments are assembled such that the V_H segment is 5′ to the D_H segment which is 5′ to the J_H segment. At the junction joining the V_H and the D_H and at the junction joining the D_H and J_H segments, nucleotides can be inserted or deleted from the individual V_H, D_H or J_H segments, such that the resulting nucleic acid molecule containing the joined VDJ segments are in-frame with the 5′ start codon (ATG).
• As used herein, a “functional antibody” or “productive antibody” with reference to a nucleic acid encoding an antibody or portion thereof refers to an antibody or portion thereof, such as Fab, that is encoded by the nucleic acid molecule produced by the combinatorial method. In a functional or productive antibody, the V(D)J germline segments are compiled (i.e. rearranged) such that the encoded antibody or portion thereof is not truncated and/or the amino acid sequence is not out of frame. This means that the nucleic acid molecule does not contain internal stop codons that result in the protein translation machinery terminating protein assembly prematurely.
• As used herein, corresponding with reference to corresponding residues, for example “amino acid residues corresponding to”, refers to residues compared among or between two polypeptides that are related sequences (e.g. allelic variants, genes of the same family, species variants). One of skill in the art can readily identify residues that correspond between or among polypeptides. For example, by aligning two sequences, one of skill in the art can identify corresponding residues, using conserved and identical amino acids as guides. One of skill in the art can manually align a sequence or can use any of the numerous alignment programs available (for example, BLAST). Hence, an amino acid residues or positions that correspond to each other are those residues that are determined to correspond to one another based on sequence and/or structural alignments with a specified reference polypeptide.
• As used herein, “screening” refers to identification or selection of an antibody or portion thereof from a plurality of antibodies, such as a collection or library of antibodies and/or portions thereof, based on determination of the activity or property of an antibody or portion thereof. Screening can be performed in any of a variety of ways, including, for example, by assays assessing direct binding (e.g. binding affinity) of the antibody to a target protein or by functional assays assessing modulation of an activity of a target protein.
• As used herein the term assessing is intended to include quantitative and qualitative determination in the sense of obtaining an absolute value for the binding of an antibody or portion thereof with a target protein and/or modulation of an activity of a target protein by an antibody or portion thereof, and also of obtaining an index, ratio, percentage, visual or other value indicative of the level of the binding or activity. Assessment can be direct or indirect. For example, binding can be determined by directly labeling of an antibody or portion thereof with a detectable label and/or by using a secondary antibody that itself is labeled. In addition, functional activities can be determined using any of a variety of assays known to one of skill in the art, for example, proliferation, cytotoxicity and others as described herein, and comparing the activity of the target protein in the presence versus the absence of an antibody or portion thereof.
• As used herein, “modulate” or “modulation” and other various grammatical forms thereof with reference to the effect of an antibody or portion thereof on the functional activity of a target protein refers to increased activity such as induction or potentiation of activity, as well as inhibition of one or more activities of the target protein. Hence, modulation can include an increase in the activity (i.e., up-regulation or agonist activity) a decrease in activity (i.e., down-regulation or inhibition) or any other alteration in an activity (such as a change in periodicity, frequency, duration, kinetics or other parameter). Modulation can be context dependent and typically modulation is compared to a designated state, for example, the wildtype protein, the protein in a constitutive state, or the protein as expressed in a designated cell type or condition. The functional activity of a target protein by an antibody or portion thereof can be modulated by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the activity of the target protein in the abasence of the antibody or portion thereof.
• As used herein, Delta-like 4 (DLL4) refers to a protein that is a ligand for Notch receptors 1 and 4. DLL4 includes any DLL4 polypeptide, including but not limited to, a recombinantly produced polypeptide, a sythentically produced polypeptide, a native DLL4 polypeptide, and a DLL4 polypeptide extracted from cells or tissues, including endothelial cells. DLL4 also includes related polypeptides from different species including, but not limited to animals of human and non-human origin. Human DLL4 includes DLL4, allelic variant isoforms, synthetic molecules from nucleic acids, protein isolated from human tissue and cells, and modified forms thereof. An exemplary DLL4 includes human DLL4 having a sequence of amino acids set forth in SEQ ID NO:2904 and encoded by a sequence of nucleotides set forth in SEQ ID NO:2905. For purposes herein, reference to DLL4 is typically with reference to human DLL4, unless stated otherwise.
• As used herein, an “activator”, such as an “agonist” or “activator/modulator,” refers to an antibody or portion thereof that modulates signal transduction or other functional activity of a receptor by potentiating, inducing or otherwise enhancing the signal transduction activity or other functional activity of a receptor. An activator, such as an agonists or activator/modulator, can modulate or increase signal transduction or other functional activity when used alone or can alter signal transduction or other functional activity in the presence of the natural ligand of the receptor or other receptor stimulator to enhance signaling by the receptor compared to the ligand alone. An activator includes an agonist or activator/modulator.
• As used herein, an “agonist” refers to an antibody or portion thereof that mimics the activity of an endogenous ligand, and can replace the endogenous ligand.
• As used herein, a “modulator/activator” refers to an antibody or portion thereof that binds an allosteric site of a target substrate and alters, such as increases, the activation of a receptor by its ligand.
• As used herein, an “allosteric site” is a site on the target substrate that is not the site conferring ligand/receptor interaction, but that when bound by an antibody or a portion thereof alters the activity of the target substrate.
• As used herein, “antagonist” refers to an antibody or portion thereof that modulates signal transduction or other functional activity of a receptor by blocking or decreasing the signal transduction activity or other functional activity of a receptor.
• As used herein, off-rate (k_off) is the rate at which an antibody dissociates from its antigen.
• As used herein, on-rate (k_on) is the rate at which an antibody binds antigen.
• As used herein, “half-life” (t_1/2) or “dissociation half-life” refers to the time in which half of the initially present protein-ligand or substrate-antibody complexes have disassociated. It is designated as Ln(2)/k_off.
• As used herein, reference to an “antibody or portion thereof that is sufficient to form an antigen binding site” means that the antibody or portion thereof contains at least 1 or 2, typically 3, 4, 5 or all 6 CDRs of the VH and VL sufficient to retain at least a portion of the binding specificity of the corresponding full-length antibody containing all 6 CDRs. Generally, a sufficient antigen binding site at least requires CDR3 of the heavy chain (CDRH3). It typically further requires the CDR3 of the light chain (CDRL3). As described herein, one of skill in the art knows and can identify the CDRs based on Kabat or Chothia numbering (see e.g., Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). For example, based on Kabat numbering, CDR-L1 corresponds to residues L24-L34; CDR-L2 corresponds to residues L50-L56; CDR-L3 corresponds to residues L89-L97; CDR-H1 corresponds to residues H31-H35, 35a or 35b depending on the length; CDR-H2 corresponds to residues H50-H65; and CDR-H3 corresponds to residues H95-H102.
• As used herein, a label is a detectable marker that can be attached or linked directly or indirectly to a molecule or associated therewith. The detection method can be any method known in the art.
• As used herein, a human protein is one encoded by a nucleic acid molecule, such as DNA, present in the genome of a human, including all allelic variants and conservative variations thereof. A variant or modification of a protein is a human protein if the modification is based on the wildtype or prominent sequence of a human protein.
• As used herein, “naturally occurring amino acids” refer to the 20 L-amino acids that occur in polypeptides. The residues are those 20 α-amino acids found in nature which are incorporated into protein by the specific recognition of the charged tRNA molecule with its cognate mRNA codon in humans.
• As used herein, non-naturally occurring amino acids refer to amino acids that are not genetically encoded. For example, a non-natural amino acid is an organic compound that has a structure similar to a natural amino acid but has been modified structurally to mimic the structure and reactivity of a natural amino acid. Non-naturally occurring amino acids thus include, for example, amino acids or analogs of amino acids other than the 20 naturally-occurring amino acids and include, but are not limited to, the D-isostereomers of amino acids. Exemplary non-natural amino acids are known to those of skill in the art.
• As used herein, nucleic acids include DNA, RNA and analogs thereof, including peptide nucleic acids (PNA) and mixtures thereof. Nucleic acids can be single or double-stranded. When referring to probes or primers, which are optionally labeled, such as with a detectable label, such as a fluorescent or radiolabel, single-stranded molecules are contemplated. Such molecules are typically of a length such that their target is statistically unique or of low copy number (typically less than 5, generally less than 3) for probing or priming a library. Generally a probe or primer contains at least 14, 16 or 30 contiguous nucleotides of sequence complementary to or identical to a gene of interest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleic acids long.
• As used herein, a peptide refers to a polypeptide that is from 2 to 40 amino acids in length.
• As used herein, the amino acids which occur in the various sequences of amino acids provided herein are identified according to their known, three-letter or one-letter abbreviations (Table 1). The nucleotides which occur in the various nucleic acid fragments are designated with the standard single-letter designations used routinely in the art.
• As used herein, an “amino acid” is an organic compound containing an amino group and a carboxylic acid group. A polypeptide contains two or more amino acids. For purposes herein, amino acids include the twenty naturally-occurring amino acids, non-natural amino acids and amino acid analogs (i.e., amino acids wherein the α-carbon has a side chain).
• As used herein, “amino acid residue” refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are presumed to be in the “L” isomeric form. Residues in the “D” isomeric form, which are so designated, can be substituted for any L-amino acid residue as long as the desired functional property is retained by the polypeptide. NH₂ refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide. In keeping with standard polypeptide nomenclature described in J. Biol. Chem., 243: 3557-3559 (1968), and adopted 37 C.F.R. §§1.821-1.822, abbreviations for amino acid residues are shown in Table 1:

• TABLE 1
  Table of Correspondence

  SYMBOL

  	1-Letter	3-Letter	AMINO ACID
  	Y	Tyr	Tyrosine
  	G	Gly	Glycine
  	F	Phe	Phenylalanine
  	M	Met	Methionine
  	A	Ala	Alanine
  	S	Ser	Serine
  	I	Ile	Isoleucine
  	L	Leu	Leucine
  	T	Thr	Threonine
  	V	Val	Valine
  	P	Pro	Proline
  	K	Lys	Lysine
  	H	His	Histidine
  	Q	Gln	Glutamine
  	E	Glu	Glutamic acid
  	Z	Glx	Glu and/or Gln
  	W	Trp	Tryptophan
  	R	Arg	Arginine
  	D	Asp	Aspartic acid
  	N	Asn	Asparagine
  	B	Asx	Asn and/or Asp
  	C	Cys	Cysteine
  	X	Xaa	Unknown or other

• It should be noted that all amino acid residue sequences represented herein by formulae have a left to right orientation in the conventional direction of amino-terminus to carboxyl-terminus. In addition, the phrase “amino acid residue” is broadly defined to include the amino acids listed in the Table of Correspondence (Table 1) and modified and unusual amino acids, such as those referred to in 37 C.F.R. §§1.821-1.822, and incorporated herein by reference. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues, to an amino-terminal group such as NH₂ or to a carboxyl-terminal group such as COOH. The abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972) Biochem. 11:1726). Each naturally occurring L-amino acid is identified by the standard three letter code (or single letter code) or the standard three letter code (or single letter code) with the prefix “L-”; the prefix “D-” indicates that the stereoisomeric form of the amino acid is D.
• As used herein, an isokinetic mixture is one in which the molar ratios of amino acids has been adjusted based on their reported reaction rates (see, e.g., Ostresh et al., (1994) Biopolymers 34:1681).
• As used herein, modification is in reference to modification of a sequence of amino acids of a polypeptide or a sequence of nucleotides in a nucleic acid molecule and includes deletions, insertions, and replacements of amino acids and nucleotides, respectively. Methods of modifying a polypeptide are routine to those of skill in the art, such as by using recombinant DNA methodologies.
• As used herein, suitable conservative substitutions of amino acids are known to those of skill in this art and can be made generally without altering the biological activity of the resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. co., p. 224). Such substitutions can be made in accordance with those set forth in TABLE 2 as follows:

• 	TABLE 2
  	Original	Exemplary conservative
  	residue	substitution
  	Ala (A)	Gly; Ser
  	Arg (R)	Lys
  	Asn (N)	Gln; His
  	Cys (C)	Ser
  	Gln (Q)	Asn
  	Glu (E)	Asp
  	Gly (G)	Ala; Pro
  	His (H)	Asn; Gln
  	Ile (I)	Leu; Val
  	Leu (L)	Ile; Val
  	Lys (K)	Arg; Gln; Glu
  	Met (M)	Leu; Tyr; Ile
  	Phe (F)	Met; Leu; Tyr
  	Ser (S)	Thr
  	Thr (T)	Ser
  	Trp (W)	Tyr
  	Tyr (Y)	Trp; Phe
  	Val (V)	Ile; Leu

  
  Other substitutions also are permissible and can be determined empirically or in accord with known conservative substitutions.
• As used herein, a DNA construct is a single or double stranded, linear or circular DNA molecule that contains segments of DNA combined and juxtaposed in a manner not found in nature. DNA constructs exist as a result of human manipulation, and include clones and other copies of manipulated molecules.
• As used herein, a DNA segment is a portion of a larger DNA molecule having specified attributes. For example, a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, which, when read from the 5′ to 3′ direction, encodes the sequence of amino acids of the specified polypeptide.
• As used herein, the term “nucleic acid” refers to single-stranded and/or double-stranded polynucleotides such as deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) as well as analogs or derivatives of either RNA or DNA. Also included in the term “nucleic acid” are analogs of nucleic acids such as peptide nucleic acid (PNA), phosphorothioate DNA, and other such analogs and derivatives or combinations thereof. Nucleic acid can refer to polynucleotides such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The term also includes, as equivalents, derivatives, variants and analogs of either RNA or DNA made from nucleotide analogs, single (sense or antisense) and double-stranded polynucleotides. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine. For RNA, the uracil base is uridine.
• As used herein, “nucleic acid molecule encoding” refers to a nucleic acid molecule which directs the expression of a specific protein or peptide. The nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein or peptide. The nucleic acid molecule includes both the full length nucleic acid sequences as well as non-full length sequences derived from the full length mature polypeptide, such as for example a full length polypeptide lacking a precursor sequence. For purposes herein, a nucleic acid sequence also includes the degenerate codons of the native sequence or sequences which can be introduced to provide codon preference in a specific host.
• As used herein, the term “polynucleotide” refers to an oligomer or polymer containing at least two linked nucleotides or nucleotide derivatives, including a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), and a DNA or RNA derivative containing, for example, a nucleotide analog or a “backbone” bond other than a phosphodiester bond, for example, a phosphotriester bond, a phosphoramidate bond, a phosphorothioate bond, a thioester bond, or a peptide bond (peptide nucleic acid). The term “oligonucleotide” also is used herein essentially synonymously with “polynucleotide,” although those in the art recognize that oligonucleotides, for example, PCR primers, generally are less than about fifty to one hundred nucleotides in length.
• Polynucleotides can include nucleotide analogs, including, for example, mass modified nucleotides, which allow for mass differentiation of polynucleotides; nucleotides containing a detectable label such as a fluorescent, radioactive, luminescent or chemiluminescent label, which allow for detection of a polynucleotide; or nucleotides containing a reactive group such as biotin or a thiol group, which facilitates immobilization of a polynucleotide to a solid support. A polynucleotide also can contain one or more backbone bonds that are selectively cleavable, for example, chemically, enzymatically or photolytically. For example, a polynucleotide can include one or more deoxyribonucleotides, followed by one or more ribonucleotides, which can be followed by one or more deoxyribonucleotides, such a sequence being cleavable at the ribonucleotide sequence by base hydrolysis. A polynucleotide also can contain one or more bonds that are relatively resistant to cleavage, for example, a chimeric oligonucleotide primer, which can include nucleotides linked by peptide nucleic acid bonds and at least one nucleotide at the 3′ end, which is linked by a phosphodiester bond or other suitable bond, and is capable of being extended by a polymerase. Peptide nucleic acid sequences can be prepared using well-known methods (see, for example, Weiler et al. Nucleic acids Res. 25: 2792-2799 (1997)).
• As used herein, “similarity” between two proteins or nucleic acids refers to the relatedness between the sequence of amino acids of the proteins or the nucleotide sequences of the nucleic acids. Similarity can be based on the degree of identity and/or homology of sequences of residues and the residues contained therein. Methods for assessing the degree of similarity between proteins or nucleic acids are known to those of skill in the art. For example, in one method of assessing sequence similarity, two amino acid or nucleotide sequences are aligned in a manner that yields a maximal level of identity between the sequences. “Identity” refers to the extent to which the amino acid or nucleotide sequences are invariant. Alignment of amino acid sequences, and to some extent nucleotide sequences, also can take into account conservative differences and/or frequent substitutions in amino acids (or nucleotides). Conservative differences are those that preserve the physico-chemical properties of the residues involved. Alignments can be global (alignment of the compared sequences over the entire length of the sequences and including all residues) or local (the alignment of a portion of the sequences that includes only the most similar region or regions).
• “Identity” per se has an art-recognized meaning and can be calculated using published techniques. (See, e.g.: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exists a number of methods to measure identity between two polynucleotide or polypeptides, the term “identity” is well known to skilled artisans (Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988)).
• As used herein, homologous (with respect to nucleic acid and/or amino acid sequences) means about greater than or equal to 25% sequence homology, typically greater than or equal to 25%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence homology; the precise percentage can be specified if necessary. For purposes herein the terms “homology” and “identity” are often used interchangeably, unless otherwise indicated. In general, for determination of the percentage homology or identity, sequences are aligned so that the highest order match is obtained (see, e.g.: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carillo et al. (1988) SIAM J Applied Math 48:1073). By sequence homology, the number of conserved amino acids is determined by standard alignment algorithms programs, and can be used with default gap penalties established by each supplier. Substantially homologous nucleic acid molecules hybridize typically at moderate stringency or at high stringency all along the length of the nucleic acid of interest. Also contemplated are nucleic acid molecules that contain degenerate codons in place of codons in the hybridizing nucleic acid molecule.
• Whether any two molecules have nucleotide sequences or amino acid sequences that are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” or “homologous” can be determined using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403 (1990)); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo et al. (1988) SIAM J Applied Math 48:1073). For example, the BLAST function of the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include, DNAStar “MegAlign” program (Madison, Wis.) and the University of Wisconsin Genetics Computer Group (UWG) “Gap” program (Madison Wis.). Percent homology or identity of proteins and/or nucleic acid molecules can be determined, for example, by comparing sequence information using a GAP computer program (e.g., Needleman et al. (1970) J. Mol. Biol. 48:443, as revised by Smith and Waterman ((1981) Adv. Appl. Math. 2:482). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids), which are similar, divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, as described by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
• Therefore, as used herein, the term “identity” or “homology” represents a comparison between a test and a reference polypeptide or polynucleotide. As used herein, the term at least “90% identical to” refers to percent identities from 90 to 99.99 relative to the reference nucleic acid or amino acid sequence of the polypeptide. Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polypeptide length of 100 amino acids are compared. No more than 10% (i.e., 10 out of 100) of the amino acids in the test polypeptide differs from that of the reference polypeptide. Similar comparisons can be made between test and reference polynucleotides. Such differences can be represented as point mutations randomly distributed over the entire length of a polypeptide or they can be clustered in one or more locations of varying length up to the maximum allowable, e.g. 10/100 amino acid difference (approximately 90% identity). Differences are defined as nucleic acid or amino acid substitutions, insertions or deletions. At the level of homologies or identities above about 85-90%, the result should be independent of the program and gap parameters set; such high levels of identity can be assessed readily, often by manual alignment without relying on software.
• As used herein, an aligned sequence refers to the use of homology (similarity and/or identity) to align corresponding positions in a sequence of nucleotides or amino acids. Typically, two or more sequences that are related by 50% or more identity are aligned. An aligned set of sequences refers to 2 or more sequences that are aligned at corresponding positions and can include aligning sequences derived from RNAs, such as ESTs and other cDNAs, aligned with genomic DNA sequence.
• As used herein, “primer” refers to a nucleic acid molecule that can act as a point of initiation of template-directed DNA synthesis under appropriate conditions (e.g., in the presence of four different nucleoside triphosphates and a polymerization agent, such as DNA polymerase, RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. It will be appreciated that a certain nucleic acid molecules can serve as a “probe” and as a “primer.” A primer, however, has a 3′ hydroxyl group for extension. A primer can be used in a variety of methods, including, for example, polymerase chain reaction (PCR), reverse-transcriptase (RT)-PCR, RNA PCR, LCR, multiplex PCR, panhandle PCR, capture PCR, expression PCR, 3′ and 5′ RACE, in situ PCR, ligation-mediated PCR and other amplification protocols.
• As used herein, “primer pair” refers to a set of primers that includes a 5′ (upstream) primer that hybridizes with the 5′ end of a sequence to be amplified (e.g. by PCR) and a 3′ (downstream) primer that hybridizes with the complement of the 3′ end of the sequence to be amplified.
• As used herein, “specifically hybridizes” refers to annealing, by complementary base-pairing, of a nucleic acid molecule (e.g. an oligonucleotide) to a target nucleic acid molecule. Those of skill in the art are familiar with in vitro and in vivo parameters that affect specific hybridization, such as length and composition of the particular molecule. Parameters particularly relevant to in vitro hybridization further include annealing and washing temperature, buffer composition and salt concentration. Exemplary washing conditions for removing non-specifically bound nucleic acid molecules at high stringency are 0.1×SSPE, 0.1% SDS, 65° C., and at medium stringency are 0.2×SSPE, 0.1% SDS, 50° C. Equivalent stringency conditions are known in the art. The skilled person can readily adjust these parameters to achieve specific hybridization of a nucleic acid molecule to a target nucleic acid molecule appropriate for a particular application.
• As used herein, substantially identical to a product means sufficiently similar so that the property of interest is sufficiently unchanged so that the substantially identical product can be used in place of the product.
• As used herein, it also is understood that the terms “substantially identical” or “similar” varies with the context as understood by those skilled in the relevant art.
• As used herein, an allelic variant or allelic variation references any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and can result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or can encode polypeptides having altered amino acid sequence. The term “allelic variant” also is used herein to denote a protein encoded by an allelic variant of a gene. Typically the reference form of the gene encodes a wildtype form and/or predominant form of a polypeptide from a population or single reference member of a species. Typically, allelic variants, which include variants between and among species typically have at least 80%, 90% or greater amino acid identity with a wildtype and/or predominant form from the same species; the degree of identity depends upon the gene and whether comparison is interspecies or intraspecies. Generally, intraspecies allelic variants have at least about 80%, 85%, 90% or 95% identity or greater with a wildtype and/or predominant form, including 96%, 97%, 98%, 99% or greater identity with a wildtype and/or predominant form of a polypeptide. Reference to an allelic variant herein generally refers to variations n proteins among members of the same species.
• As used herein, “allele,” which is used interchangeably herein with “allelic variant” refers to alternative forms of a gene or portions thereof. Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for that gene or allele. When a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene. Alleles of a specific gene can differ from each other in a single nucleotide or several nucleotides, and can include substitutions, deletions and insertions of nucleotides. An allele of a gene also can be a form of a gene containing a mutation.
• As used herein, species variants refer to variants in polypeptides among different species, including different mammalian species, such as mouse and human.
• As used herein, a splice variant refers to a variant produced by differential processing of a primary transcript of genomic DNA that results in more than one type of mRNA.
• As used herein, the term promoter means a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5′ non-coding region of genes.
• As used herein, isolated or purified polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. Preparations can be determined to be substantially free if they appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification does not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound, however, can be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound.
• The term substantially free of cellular material includes preparations of proteins in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly-produced. In one embodiment, the term substantially free of cellular material includes preparations of protease proteins having less that about 30% (by dry weight) of non-protease proteins (also referred to herein as a contaminating protein), generally less than about 20% of non-protease proteins or 10% of non-protease proteins or less that about 5% of non-protease proteins. When the protease protein or active portion thereof is recombinantly produced, it also is substantially free of culture medium, i.e., culture medium represents less than about or at 20%, 10% or 5% of the volume of the protease protein preparation.
• As used herein, the term substantially free of chemical precursors or other chemicals includes preparations of protease proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. The term includes preparations of protease proteins having less than about 30% (by dry weight) 20%, 10%, 5% or less of chemical precursors or non-protease chemicals or components.
• As used herein, synthetic, with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant methods and/or by chemical synthesis methods.
• As used herein, production by recombinant means by using recombinant DNA methods means the use of the well known methods of molecular biology for expressing proteins encoded by cloned DNA.
• As used herein, vector (or plasmid) refers to discrete elements that are used to introduce a heterologous nucleic acid into cells for either expression or replication thereof. The vectors typically remain episomal, but can be designed to effect integration of a gene or portion thereof into a chromosome of the genome. Also contemplated are vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes. Selection and use of such vehicles are well known to those of skill in the art.
• As used herein, an expression vector includes vectors capable of expressing DNA that is operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
• As used herein, vector also includes “virus vectors” or “viral vectors.” Viral vectors are engineered viruses that are operatively linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells.
• As used herein, operably or operatively linked when referring to DNA segments means that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
• As used herein, biological sample refers to any sample obtained from a living or viral source and includes any cell type or tissue of a subject from which nucleic acid or protein or other macromolecule can be obtained. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples from animals and plants. Also included are soil and water samples and other environmental samples, viruses, bacteria, fungi, algae, protozoa and components thereof. Hence bacterial and viral and other contamination of food products and environments can be assessed. The methods herein are practiced using biological samples and in some embodiments, such as for profiling, also can be used for testing any sample.
• As used herein, macromolecule refers to any molecule having a molecular weight from the hundreds up to the millions. Macromolecules include peptides, proteins, nucleotides, nucleic acids, and other such molecules that are generally synthesized by biological organisms, but can be prepared synthetically or using recombinant molecular biology methods.
• As used herein, a composition refers to any mixture. It can be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
• As used herein, a combination refers to any association between or among two or more items. The combination can be two or more separate items, such as two compositions or two collections, can be a mixture thereof, such as a single mixture of the two or more items, or any variation thereof.
• As used herein, kit refers to a packaged combination, optionally including instructions and/or reagents for their use.
• As used herein, a pharmaceutical effect or therapeutic effect refers to an effect observed upon administration of an agent intended for treatment of a disease or disorder or for amelioration of the symptoms thereof.
• As used herein, “disease or disorder” refers to a pathological condition in an organism resulting from cause or condition including, but not limited to, infections, acquired conditions, genetic conditions, and characterized by identifiable symptoms. Diseases and disorders of interest herein are those involving a specific target protein including those mediated by a target protein and those in which a target protein plays a role in the etiology or pathology. Exemplary target proteins and associated diseases and disorders are described elsewhere herein.
• As used herein, angiogenic diseases (or angiogenesis-related diseases) are diseases in which the balance of angiogenesis is altered or the timing thereof is altered. Angiogenic diseases include those in which an alteration of angiogenesis, such as undesirable vascularization, occurs. Such diseases include, but are not limited to cell proliferative disorders, including cancers, diabetic retinopathies and other diabetic complications, inflammatory diseases, endometriosis, age-related macular degeneration and other diseases in which excessive vascularization is part of the disease process, including those known in the art or noted elsewhere herein.
• As used herein, “treating” a subject with a disease or condition means that the subject's symptoms are partially or totally alleviated, or remain static following treatment.
• Hence treatment encompasses prophylaxis, therapy and/or cure. Prophylaxis refers to prevention of a potential disease and/or a prevention of worsening of symptoms or progression of a disease. Treatment also encompasses any pharmaceutical use of a modified interferon and compositions provided herein.
• As used herein, a therapeutic agent, therapeutic regimen, radioprotectant, or chemotherapeutic mean conventional drugs and drug therapies, including vaccines, which are known to those skilled in the art. Radiotherapeutic agents are well known in the art.
• As used herein, treatment means any manner in which the symptoms of a condition, disorder or disease or other indication, are ameliorated or otherwise beneficially altered.
• As used herein therapeutic effect means an effect resulting from treatment of a subject that alters, typically improves or ameliorates the symptoms of a disease or condition or that cures a disease or condition. A therapeutically effective amount refers to the amount of a composition, molecule or compound which results in a therapeutic effect following administration to a subject.
• As used herein, the term “subject” refers to an animal, including a mammal, such as a human being.
• As used herein, a patient refers to a human subject.
• As used herein, amelioration of the symptoms of a particular disease or disorder by a treatment, such as by administration of a pharmaceutical composition or other therapeutic, refers to any lessening, whether permanent or temporary, lasting or transient, of the symptoms that can be attributed to or associated with administration of the composition or therapeutic.
• As used herein, prevention or prophylaxis refers to methods in which the risk of developing disease or condition is reduced.
• As used herein, an effective amount is the quantity of a therapeutic agent necessary for preventing, curing, ameliorating, arresting or partially arresting a symptom of a disease or disorder.
• As used herein, administration refers to any method in which an antibody or protion thereof is contacted with its target protein. Administration can be effected in vivo or ex vivo or in vitro. For example, for ex vivo administration a body fluid, such as blood, is removed from a subject and contacted outside the body with the antibody or portion thereof. For in vivo administration, the antibody or portion thereof can be introduced into the body, such as by local, topical, systemic and/or other route of introduction. In vitro administration encompasses methods, such as cell culture methods.
• As used herein, unit dose form refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art.
• As used herein, a single dosage formulation refers to a formulation for direct administration.
• As used herein, an “article of manufacture” is a product that is made and sold. As used throughout this application, the term is intended to encompass compiled germline antibodies or antibodies obtained therefrom contained in articles of packaging.
• As used herein, fluid refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions.
• As used herein, animal includes any animal, such as, but are not limited to primates including humans, gorillas and monkeys; rodents, such as mice and rats; fowl, such as chickens; ruminants, such as goats, cows, deer, sheep; mammals, such as pigs and other animals. Non-human animals exclude humans as the contemplated animal. The germline segments, and resulting antibodies, provided herein are from any source, animal, plant, prokaryotic and fungal. Most germline segments, and resulting antibodies, are of animal origin, including mammalian origin.
• As used herein, a control refers to a sample that is substantially identical to the test sample, except that it is not treated with a test parameter, or, if it is a sample plasma sample, it can be from a normal volunteer not affected with the condition of interest. A control also can be an internal control.
• As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to compound, comprising “an extracellular domain” includes compounds with one or a plurality of extracellular domains.
• As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 5 bases” means “about 5 bases” and also “5 bases.”
• As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally substituted group means that the group is unsubstituted or is substituted.
• As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972) Biochem. 11:1726).
B. OVERVIEW OF METHODS
• Provided herein are methods of selecting antibodies with desired affinities and activities. The methods include affinity maturation and antibody conversion methods. The methods can be used to engineer antibodies to thereby identify or select antibodies that are antagonist antibodies, partial antagonist antibodies, agonist antibodies and/or activator/modulator antibodies. The ability to “tune” a particular pathway as opposed to completely inhibiting it would be an advantage for protein therapeutics. For example, pharmacologically, the ability to turn a pathway “on” or “off” by a high affinity interaction, might be less desirable than modulation of a pathway through “rheostat” based therapeutics. In other examples, an antibody with a high affinity is desired.
• The resulting affinity-based or activity-based antibodies generated by practice of the methods can be used for any application or purpose as desired, including for example, in a variety of in vitro and in vivo applications by virtue of their specificity for one or more target proteins. Because of their diversity, specificity and effector functions, antibodies are attractive candidates for protein-based therapeutics. Accordingly, the methods provided herein for generating antibodies with desired affinities, specificities and/or activities permits their use as therapeutic antibodies. For example, the antibodies can be used in methods of treatment and other uses for treating a disease or disorder which is associated with expression or activation of a particular target protein, for which the antibody can modulate.
• 1. Antibody Polypeptides
• In the methods provided herein, mutagenesis is typically performed on the variable region of the antibody. Accordingly, the parent antibody selected for affinity conversion or affinity maturation using the methods provided herein typically minimally include all or a portion of a variable heavy chain (VH) and/or a variable light (VL) chain so long as the antibody contains a sufficient antibody binding site. It is understood, however, that any antibody used or obtained by practice of the methods can be generated to include all or a portion of the constant heavy chain (e.g. one or more CH domains such as CH1, CH2, CH3 and CH4 and/or a constant light chain (CL)). Hence, the antibodies subjected to affinity conversion or affinity maturation herein include those that are full-length antibodies, and also include fragments or portions thereof including, for example, Fab, Fab′, F(ab′)₂, single-chain Fvs (scFv), Fv, dsFv, diabody, Fd and Fd′ fragments, Fab fragments, scFv fragments, and scFab fragments. For example, antibodies affinity converted or affinity matured herein include Fabs.
• A skilled artisan understands the structure, sequence and function of antibodies. A general description of the structure, sequence and function of antibodies is provided below.
• a. Antibody Structure and Function
• Antibodies are produced naturally by B cells in membrane-bound and secreted forms. In addition to naturally produced antibodies, antibodies also include synthetically, i.e. recombinantly, produced antibodies, such as antibody fragments. Antibodies specifically recognize and bind antigen epitopes through cognate interactions. Antibody binding to cognate antigens can initiate multiple effector functions, which cause neutralization and clearance of toxins, pathogens and other infectious agents. Diversity in antibody specificity arises naturally due to recombination events during B cell development. Through these events, various combinations of multiple antibody V, D and J gene segments, which encode variable regions of antibody molecules, are joined with constant region genes to generate a natural antibody repertoire with large numbers of diverse antibodies. A human antibody repertoire contains more than 10¹⁰ different antigen specificities and thus theoretically can specifically recognize any foreign antigen.
• A full-length antibody contains four polypeptide chains, two identical heavy (H) chains (each usually containing about 440 amino acids) and two identical light (L) chains (each containing about 220 amino acids). The light chains exist in two distinct forms called kappa (κ) and lambda (λ). Each chain is organized into a series of domains organized as immunoglobulin (Ig) domains, including variable (V) and constant (C) region domains. Light chains have two domains, corresponding to the C region (CL) and the V region (VL). Heavy chains have four domains, the V region (VH) and three or four domains in the C region (CH1, CH2, CH3 and CH4), and, in some cases, hinge region. The four chains (two heavy and two light) are held together by a combination of covalent (disulfide) and non-covalent bonds.
• Antibodies include those that are full-lengths and those that are fragments thereof, namely Fab, Fab′, F(ab′)₂, single-chain Fvs (scFv), Fv, dsFv, diabody, Fd and Fd′ fragments. The fragments include those that are in single-chain or dimeric form. The Fv fragment, which contains only the VH and VL domain, is the smallest immunoglobulin fragment that retains the whole antigen-binding site (see, for example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). Stabilization of Fv are achieved by direct linkage of the VH and VL chains, such as for example, by linkage with peptides (to generate single-chain Fvs (scFv)), disulfide bridges or knob-into-hole mutations. Fab fragments, in contrast, are stable because of the presence of the CH1 and CL domains that hold together the variable chains. Fd antibodies, which contain only the VH domain, lack a complete antigen-binding site and can be insoluble.
• In folded antibody polypeptides, binding specificity is conferred by antigen binding site domains, which contain portions of heavy and/or light chain variable region domains. Other domains on the antibody molecule serve effector functions by participating in events such as signal transduction and interaction with other cells, polypeptides and biomolecules. These effector functions cause neutralization and/or clearance of the infecting agent recognized by the antibody.
• b. Antibody Sequence and Specificity
• The variable region of the heavy and light chains are encoded by multiple germline gene segments separated by non-coding regions, or introns, and often are present on different chromosomes. During B cell differentiation germline DNA is rearranged whereby one and one J_H gene segment of the heavy chain locus are recombined, which is followed by the joining of one V_H gene segment forming a rearranged VDJ gene that encodes a VH chain. The rearrangement occurs only on a single heavy chain allele by the process of allelic exclusion. Allelic exclusion is regulated by in-frame or “productive” recombination of the VDJ segments, which occurs in only about one-third of VDJ recombinations of the variable heavy chain. When such productive recombination events first occur in a cell, this results in production of a μ90 heavy chain that gets expressed on the surface of a pre-B cell and transmits a signal to shut off further heavy chain recombination, thereby preventing expression of the allelic heavy chain locus. The surface-expressed μ heavy chain also acts to activate the kappa (κ) locus for rearrangement. The lambda (λ) locus is only activated for rearrangement if the κ recombination is unproductive on both loci. The light chain rearrangement events are similar to heavy chain, except that only the V_L and J_L segments are recombined. Before primary transcription of each, the corresponding constant chain gene is added. Subsequent transcription and RNA splicing leads to mRNA that is translated into an intact light chain or heavy chain.
• The variable regions of antibodies confer antigen binding and specificity due to recombination events of individual germline V, D and J segments, whereby the resulting recombined nucleic acid sequences encoding the variable region domains differ among antibodies and confer antigen-specificity to a particular antibody. The variation, however, is limited to three complementarity determining regions (CDR1, CDR2, and CDR3) found within the N-terminal domain of the heavy (H) and (L) chain variable regions. The CDRs are interspersed with regions that are more conserved, termed “framework regions” (FR). The extent of the framework region and CDRs has been precisely defined (see e.g., Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). Each VH and VL is typically composed of three CDRs and four FRs arranged from the amino terminus to carboxy terminus in the following order: FR1 CDR1, FR2, CDR2, FR3, CDR3 and FR4. Sequence variability among VL and VH domains is generally limited to the CDRs, which are the regions that form the antigen binding site. For example, for the heavy chain, generally, V_H genes encode the N-terminal three framework regions, the first two complete CDRs and the first part of the third CDR; the D_H gene encodes the central portion of the third CDR, and the J_H gene encodes the last part of the third CDR and the fourth framework region. For the light chain, the V_L genes encode the first CDR and second CDR. The third CDR (CDRL3) is formed by the joining of the V_L and J_L gene segments. Hence, CDRs 1 and 2 are exclusively encoded by germline V gene segment sequences. The VH and VL chain CDR3s form the center of the Ag-binding site, while CDRs 1 and 2 form the outside boundaries; the FRs support the scaffold by orienting the H and L CDRs. On average, an antigen binding site typically requires that at least four of the CDRs make contact with the antigen's epitope, with CDR3 of both the heavy and light chain being the most variable and contributing the most specificity to antigen binding (see e.g., Janis Kuby, Immunology, Third Edition, New York, W.H. Freeman and Company, 1998, pp. 115-118). CDRH3, which includes all of the D gene segment, is the most diverse component of the Ab-binding site, and typically plays a critical role in defining the specificity of the Ab. In addition to sequence variation, there is variation in the length of the CDRs between the heavy and light chains
• The constant regions, on the other hand, are encoded by sequences that are more conserved among antibodies. These domains confer functional properties to antibodies, for example, the ability to interact with cells of the immune system and serum proteins in order to cause clearance of infectious agents. Different classes of antibodies, for example IgM, IgD, IgG, IgE and IgA, have different constant regions, allowing them to serve distinct effector functions.
• These natural recombination events of V, D, and J, can provide nearly 2×10⁷ different antibodies with both high affinity and specificity. Additional diversity is introduced by nucleotide insertions and deletions in the joining segments and also by somatic hypermutation of V regions. The result is that there are approximately 10¹⁰ antibodies present in an individual with differing antigen specificities.
• 2. Methods of Identifying Antibodies
• Antibodies can be identified that have a binding specificity and/or activity against a target protein or antigen by any method known to one of skill in the art. For example, antibodies can be generated against a target antigen by conventional immunization methods resulting in the generation of hybridoma cells secreting the antibody (see e.g. Kohler et al. (1975) Nature, 256:495; Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Macademic Press, 1986), Kozbor, J. Immunol., (1984) 133:3001; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987). In another method, antibodies specific for a target antigen are identified by screening antibody libraries for the desired binding or activity. Antibody libraries can be provided as “one-pot” libraries containing a diverse population of antibody members, for example, as display libraries such as phage display libraries. In such libraries, the identity of each member of the library is typically unknown preceding sequencing of a positive clone with a desired binding activity.
• In other examples, antibody libraries include addressable combinatorial antibody libraries as described in U.S. Provisional Application Nos. 61/198,764 and 61/211,204, and published International PCT Appl. No. WO2010054007, incorporated by reference herein. In the addressable libaries, the nucleic acid molecules encoding each VH chain and/or VL chain are individually synthesized, using standard DNA synthesis techniques, in an addressable format, whereby the identity of the nucleic acid sequence of each VH chain and/or VL chain in each locus is known. VH chains and VL chains are then paired, also in an addressable format, such that the identity of each member of the library is known based on its locus or “address”. The addressable combinatorial antibody libraries can be screened for binding or activity against a target protein to identify antibodies or portions thereof that bind to a target protein and/or modulate an activity of a target protein. By virtue of the fact that these libraries are arrayed, the identity of each individual member in the collection is known during screening, thereby allowing facile comparison of “Hit” antibody.
• 3. Existing Methods of Optimizing Antibodies
• Typically, the antibodies generated and/or identified by any of the above methods are of moderate affinity (e.g. Kd⁻¹ of about 10⁶ to 10⁷ M⁻¹). As discussed herein, existing methods of antibody discovery and engineering seek high-affinity antagonist antibodies. Thus, methods of affinity maturation to optimize and improve the binding affinity are employed to further optimize the antibody. An affinity matured antibody generally is one that contains one or more amino acid alterations that result in improvement of an activity, such as antigen binding affinity. Known method for affinity maturing and antibody include, for example, generating and screening antibody libraries using the previously identified antibody as a template by introducing mutations at random in vitro by using error-prone PCR (Zhou et al., Nucleic Acids Research (1991) 19(21):6052; and US2004/0110294); randomly mutating one or more CDRs or FRs (see e.g., WO 96/07754; Barbas et al. (1994) Proc. Natl. Acad. Sci., 91:3809-3813; Cumbers et al. (2002) Nat. Biotechnol., 20:1129-1134; Hawkins et al. (1992) J. Mol. Biol., 226:889-896; Jackson et al., (1995) J. Immunol., 154:3310-3319; Wu et al. (1998) Proc. Natl. Acad. Sci., 95: 6037-6042; McCall et al. (1999) Molecular Immunology, 36:433-445); oligonucleotide directed mutagenesis (Rosok et al., The Journal of Immunology, (1998) 160:2353-2359); codon cassette mutagenesis (Kegler-Ebo et al., Nucleic Acids Research, (1994) 22(9):1593-1599); degenerate primer PCR, including two-step PCR and overlap PCR (U.S. Pat. Nos. 5,545,142, 6,248,516, and 7,189,841; Higuchi et al., Nucleic Acids Research (1988); 16(15):7351-7367; and Dubreuil et al., The Journal of Biological Chemistry (2005) 280(26):24880-24887); domain shuffling by recombining the VH or VL domains selected by phage display with repertoires of naturally occurring V domain variants obtained from unimmunized donors and screening for higher affinity in several rounds of chain reshuffling as described in Marks et al., Biotechnology, 10: 779-783 (1992).
• Each of the available approaches for optimizing antibodies has limitations. First, the approaches fail to recognize that antibodies with low affinity are candidate therapeutics acting as agonists, partial agonist/antagonists or activator/modulators. Where generating a high affinity antibody is desired, for example to generate an antagonist antibody, the existing affinity maturation approaches also are limited. For example, many available approaches carry the risk of introducing unwanted mutations (e.g. mutations at undesired positions) and/or biases against selection of particular mutants. Limitations in library size and completeness exist, since it is unfeasible to generate all possible combinations of mutants. Additionally, competition must be avoided to prevent abundant low-affinity variants from excluding rarer high-affinity variants. In addition, many of the affinity matured antibodies are produced either by VH and VL domain shuffling or by random mutagenesis of CDR and/or framework residues. These methods, however, require some type of displayed selection because of the vast number of clones to be evaluated. Finally, very high affinity antibodies are difficult to isolate by panning, since the elution conditions required to break a very strong antibody-antigen interaction are generally harsh enough (e.g., low pH, high salt) to denature the phage particle sufficiently to render it non-infective.
• The methods provided herein overcome some or all of these limitations.
C. METHOD FOR AFFINITY MATURATION OF ANTIBODIES
• Provided herein is a rational method for affinity maturation of an antibody to improve its activity towards a target antigen based on the structure/activity relationship (SAR) of the antibody that is being affinity matured. The SAR can be used to identify a region or regions or particular amino acid residues in the antibody that are important for its activity (e.g. binding to a target antigen). For example, in the method, knowledge of the structure (e.g. sequence) of a “Hit” or parent antibody to be affinity matured is correlated to an activity (e.g. binding) for a target antigen. Such knowledge can be used to elucidate the region and/or amino acid residues that are involved in the activity toward the target antigen. The region(s) or amino acid residues are targeted for further mutagenesis. Thus, the SAR information provides guidance for further optimization by providing rational identification of region(s) of the antibody polypeptides to be mutagenized. The resulting mutant antibodies can be screened to identify those antibodies that are optimized compared to the starting or reference antibody.
• In the methods provided herein, affinity maturation of a “Hit” or parent antibody is based on its structure-affinity/activity-relationship. Thus, the method is a rational and targeted mutagenesis approach with much smaller libraries guided by SARs to identify regions and residues that modulate activity.
• The SAR of an antibody can be determined by various approaches. For example, SAR can be determined by comparing the sequence of an antibody that has a desired activity for a target antigen to a related antibody that has reduced activity for the same target antigen to identify those amino acid residues that differ between the antibodies. The region of the antibody that exhibits amino acid differences is identified as a structure that is important in the activity of the antibody, and is targeted for further mutagenesis.
• In particular, the SAR can be quickly elucidated using a spatially addressed combinatorial antibody library as described in U.S. Provisional Application No. 61/198,764 and U.S. Provisional Application No. 61/211,204; and in published International PCT Appl. No. WO2010054007. In the spatially addressed format, activities and binding affinities can be correlated to structure (e.g. sequence) coincident with a screening assay, since the sequences of addressed members are known a priori. In the spatially addressed format, the binding affinities of the hit versus nearby non-hit antibody can be compared in sequence space because their sequence identities are known a priori. Comparisons of sequence can be made between “Hits” and related antibodies that have less activity or no activity in the same assay. Such comparisons can reveal SARs and identify important regions or amino acid residues involved in the activity of the antibody. For example, such comparisons can reveal SARs of important CDRs and potentially important residues within the CDRs for binding the target. SAR also can be determined using other methods that identify regions of an antibody or amino acid residues therein that contribute to the activity of an antibody. For example, mutagenesis methods, for example, scanning mutagenesis, can be used to determine SAR.
• The rational approach described herein facilitates identifying SARs that aid in the optimization of preliminary hits, mimicking the approach used in small molecule medicinal chemistry. This has advantages over existing methods of affinity maturation. Currently many of the in vitro affinity matured antibodies are produced either by VH and VL domain shuffling or by random mutagenesis of CDR and/or framework residues. Many of these methods, however, require some type of displayed selection because of the vast number of clones to be evaluated. In the method herein, a more rational and targeted mutagenesis approach is employed, using much smaller libraries guided by SARs and scanning mutagenesis to identify regions and residues that modulate affinity. True SARs can be identified because active hits can be compared with related, but less active or inactive antibodies present in the library. In addition, the methods herein can be practiced to avoid generating simultaneous mutations to circumvent exponential expansion of the library size. For example, for a given CDR or target region, one the best substitution is identified in each of the mutated positions, the mutations can be combined in a new antibody in order to generate further improvement in activity. In one example, binding affinity is increased. The increase in affinity, measured as a decrease in K_d, can be achieved through either an increase in association rate (k_on), a reduction in dissociation rate (k_off), or both.
• In one aspect of the method, residues to mutagenize in the “Hit” antibody are identified by comparison of the amino acid sequence of the variable heavy or light chain of the “Hit” antibody with a respective variable heavy or light chain of a related antibody that exhibits reduced activity for the target antigen compared to the Hit antibody that is being affinity matured. In some examples, the related antibody is a non-Hit antibody that exhibits significantly less activity towards the target antigen than the Hit antibody, such as less than 80% of the activity, generally less than 50% of the activity, for example 5% to 50% of the activity, such as 50%, 40%, 30%, 20%, 10%, 5% or less the activity. For example, a no-Hit antibody can be one that exhibits no detectable activity or shows only negligible activity towards the target antigen. In practicing the method, a requisite level of relatedness between the “Hit” and a related antibody is required in order to permit rational analysis of the contributing regions to activity. This structure-affinity/activity relationship analysis between the “Hit” antibody and related antibodies reveals target regions of the antibody polypeptide that are important for activity.
• In another aspect of the method provided herein, scanning mutagenesis can be used to reveal more explicit information about the structure/activity relationship of an antibody. In such a method, scanning mutagenesis is generally employed to identify residues to further mutate. Hence, scanning mutagenesis can be employed as the means to determine SAR. Alternatively or optionally, scanning mutagenesis can be used to in combination with the comparison method above. In such an example, once a target region is identified that is involved with an activity, scanning mutagenesis is used to further elucidate the role of individual amino acid residues in an activity in order to rationally select amino acid residues for mutagenesis. As discussed in detail below, in the scanning mutagenesis method herein only those scanned mutant residues that do not negatively impact the activity of the antibody (e.g. either preserve or increase an activity to the target antigen) are subjected to further mutagenesis by further mutating the scanned residue individually to other amino acids.
• Once the SAR is determined, a target region containing residues important for activity are revealed in the variable heavy chain and/or variable light chain of an antibody. Once a target region is identified for either the variable heavy chain or light chain, mutagenesis of amino acid residues within the region is employed and mutants are screened for an activity towards the target antigen. In the methods herein, the mutagenized antibodies can be individually generated, such as by DNA synthesis or by recombinant DNA techniques, expressed, and assayed for their activity for a target antigen. By individually mutating each antibody, for example using cassette mutagenesis, simultaneous mutations can be avoided to avoid exponential expansion of the library. In addition, unwanted mutations can be avoided. In other examples, if desired, mutations can be effected by other mutagenesis approaches, for example by using various doping strategies, and the identity of the mutant identified upon screening and sequencing. Affinity maturation can be performed separately and independently on the variable heavy chain and variable light chain of a reference Hit antibody. The resulting affinity matured variable heavy and light chains can then be paired for further optimization of the antibody.
• The affinity maturation method provided herein can be performed iteratively to further optimize binding affinity. For example, further optimization can be performed by mutagenesis and iterative screening of additional regions of the antibody polypeptide. At each step of the method, the affinity matured antibody can be tested for an activity (e.g. binding) to the target antigen. Antibodies are identified that have improved activity for the target antigen compared to the parent antibody or any intermediate antibody therefrom. Also, once the best substitutions in a region of an antibody are identified for improving an activity towards a target antigen, they can be combined to create a new antibody to further improve and optimize the antibodies activity. Such combination mutants can provide an additive improvement. Accordingly, the method of affinity maturation herein permits a rational optimization of antibody binding affinity
• 1. Comparision of Structure and Activity
• Provided herein is a method of affinity maturation based on the SAR of a Hit antibody by comparison of its structure and activity to a related antibody. In practicing the method, the amino acid sequence of the heavy chain and/or light chain of a “Hit” antibody is compared to the corresponding sequence of a related antibody that exhibits reduced or less activity for the target antigen compared to the “Hit” antibody. As discussed below, for purposes of practice of the method herein, the related antibody is sufficiently related in sequence to the “Hit” antibody in order to limit regions of the primary sequences that exhibit amino acid differences between the “Hit” and related antibody when compared (e.g. by sequence alignment). Thus, the method permits identification of a region of the “Hit” antibody that is involved in an activity to the target antigen. For example, alignment of the primary sequence (e.g. variable heavy chain and/or variable light chain) of the “Hit” and related antibody can identify one or more regions where amino acid differences exist between the “Hit” and the related antibody. The region(s) can be one or more of CDR1, CDR2 or CDR3 and/or can be amino acid residues within the framework regions of the antibody (e.g. FR1, FR2, FR3 or FR4). A region of the antibody that exhibits at least one amino acid difference compared to the corresponding region in the related antibody is a target region targeted for further mutagenesis.
• In the method, mutagenesis to any other amino acid or to a subset of amino acids is performed on amino acid residues within the identified target region. For example, some or up to all amino acid residues of the selected region in the heavy chain and/or light chain of the “Hit” antibody are mutated, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues. Each amino acid residue selected for mutagenesis can be mutated to all 19 other amino acid residues, or to a restricted subset thereof. The resulting mutant antibodies are screened for activity to the target antigen as compared to the starting “Hit” antibody. As discussed below, in some examples, prior to mutagenesis of individual amino acid residues, scanning-mutagenesis of all or select amino acid residues within the target region can be used to identify particular residues for mutagenesis. The subset of identifed residues are then subjected to mutagenesis to improve or optimize an activity towards the target antigen.
• Typically, the method is performed on the variable heavy chain and/or variable light chain of the antibody. Typically, affinity maturation is separately performed for one or both of the heavy and/or light chain(s) of the “Hit” antibody independently of the other. The heavy and light chains can be affinity matured independently such as sequentially in any order. Alternatively, the heavy and light chain are subjected to affinity maturation in parallel. Mutant DNA molecules encoding the variable heavy chain and/or variable light chain are designed, generated by mutagenesis and cloned. In some examples, the modified variable heavy and light chains can be synthetically generated or generated by other recombinant means. Various combinations of heavy and light chains can be paired to generate libraries of variant antibodies. The resulting antibodies or fragments thereof are tested for an activity to the target antigen. Antibodies exhibiting an optimized or improved binding affinity as compared to the starting “Hit” antibody are selected.
• Iterative screening can be performed to further optimize an activity to the target antigen. For example, mutations that increase an activity to the target antigen within a variable heavy or light chain can be combined, thereby creating an antibody that has an improved activity as compared to the starting “Hit” antibody and/or intermediate single mutant antibodies. Also, pairing of an affinity matured heavy chain with an affinity matured light chain can further optimize and improve the activity of resulting antibodies produced by practice of the method. Further, mutagenesis, e.g. scanning mutagenesis or full or partial saturation mutagenesis, of amino acid residues in one or more additional regions of the variable heavy or light chain can be performed to identify further mutations that further optimize an activity to the target antigen.
• At any step in the method, the affinity matured antibodies can be further evaluated for activity. Any activity can be assessed, such as any exemplified in Section E herein. In one example, binding is assessed. Any method known to one of skill in the art can be used to measure the binding or binding affinity of an antibody. In one example, binding affinity is determined using surface Plasmon resonance (SPR). In another example, binding affinity is determined by dose response using ELISA. The resulting antibodies also can be tested for a functional activity as discussed elsewhere herein.
• The resulting affinity matured antibodies are selected to have improved and/or optimized activity towards a target antigen compared to the parent “Hit” antibody. By practice of the method, the activity of an antibody for a target antigen can be improved at least 1.5-fold, generally at least 2-fold, for example at least 2-fold to 10000-fold, such as at least 2-fold, 5-fold, 10-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 10000-fold or more. For example, the affinity matured antibodies generated by practice of the method can have a binding affinity for a target antigen that is improved, for example, that is or is about 1 1×10⁻⁹ M to 1×10⁻¹¹M, generally 5×10⁻⁹ M to 5×10¹⁰ M, such as at or about 1×10⁻⁹ M, 2×10⁻⁹ M, 3×10⁻⁹ M, 4×10⁻⁹ M, 5×10⁻⁹ M, 6×10⁻⁹ M, 7×10⁻⁹ M, 8×10⁻⁹ M, 9×10⁻⁹ M, 1×10⁻¹⁰ M, 2×10¹⁰ M, 3×10⁻¹⁰ M, 4×10⁻¹⁰ M, 5×10⁻¹⁰ M, 6×10⁻¹⁰ M, 7×10⁻¹⁰ M, 8×10⁻¹⁰ M, 9×10⁻¹⁰ M or less.
• A summary of the steps of the method is set forth in FIG. 1. A detailed description of each step of the method is provided below. It is understood that the steps of the affinity maturation method provided herein are the same whether the method is performed on the variable heavy chain or variable light chain sequence of an antibody. Hence, for purposes herein, the description below applies to practice of the method on either one or both of the heavy and light chain sequences, unless explicitly stated otherwise. As discussed elsewhere herein, typically, affinity maturation is performed for one or both of the heavy and/or light chain(s) of the antibody independently of the other. If desired, an affinity matured heavy chain can be paired with an affinity matured light chain to further optimize or improve activity of the antibody.
• a. Selection of a First Antibody for Affinity Maturation
• The antibody chosen to be affinity matured is any antibody that is known in the art or identified as having an activity for a target antigen or antigens. For example, the antibody can be a “Hit” antibody, such as one identified in a screening assay. Generally, the antibody is an antibody that exhibits an activity for a target antigen such that it not ideal for use as a therapeutic because its affinity is not sufficiently high or such that improvement of its activity is achievable or desirable. For example, an antibody chosen for affinity maturation typically has a binding affinity for the target antigen that is at or about 10⁻⁵ M to 10⁻⁸ M, for example that is at or about 10⁻⁵ M, 10⁻⁶ M, M, 10⁻⁸ M, or lower. Generally, an antibody selected for affinity maturation specifically binds to the target antigen. Assays to assess activity of an antibody for a target antigen are known in the art. Exemplary assays are provided in Section E.
• Thus, the first antibody is an antibody that is known to have an activity to a target antigen. The target antigen can be a polypeptide, carbohydrate, lipid, nucleic acid or a small molecule (e.g. neurotransmitter). The antibody can exhibit activity for the antigen expressed on the surface of a virus, bacterial, tumor or other cell, or exhibits an activity (e.g. binding) for the purified antigen. Typically, the target antigen is a purified protein or peptide, including, for example, a recombinant protein.
• Generally, the target antigen is a protein that is a target for a therapeutic intervention. Exemplary target antigens include, but are not limited to, targets involved in cell proliferation and differentiation, cell migration, apoptosis and angiogenesis. Such targets include, but are not limited to, growth factors, cytokines, lymphocytic antigens, other cellular activators and receptors thereof. Exemplary of such targets include, membrane bound receptors, such as cell surface receptors, including, but are not limited to, a VEGFR-1, VEGFR-2, VEGFR-3 (vascular endothelial growth factor receptors 1, 2, and 3), a epidermal growth factor receptor (EGFR), ErbB-2, ErbB-b3, IGF-R1, C-Met (also known as hepatocyte growth factor receptor; HGFR), DLL4, DDR1 (discoidin domain receptor), KIT (receptor for c-kit), FGFR1, FGFR2, FGFR4 (fibroblast growth factor receptors 1, 2, and 4), RON (recepteur d'origine nantais; also known as macrophage stimulating 1 receptor), TEK (endothelial-specific receptor tyrosine kinase), TIE (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains receptor), CSF1R (colony stimulating factor 1 receptor), PDGFRB (platelet-derived growth factor receptor B), EPHA1, EPHA2, EPHB1 (erythropoietin-producing hepatocellular receptor A1, A2 and B1), TNF-R1, TNF-R2, HVEM, LT-βR, CD20, CD3, CD25, NOTCH, G-CSF-R, GM-CSF-R and EPO-R. Other targets include membrane-bound proteins such as selected from among a cadherin, integrin, CD52 or CD44. Exemplary ligands that can be targets of the screening methods herein, include, but are not limited to, VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF, EGF, HGF, TNF-α, LIGHT, BTLA, lymphotoxin (LT), IgE, G-CSF, GM-CSF and EPO. In some examples, the “Hit” antibody can bind to one or more antigens. For example, as exemplified in Example 1, “Hit” antibodies have been identified that binds to only one target antigen, e.g., DLL4, or that bind to two or more different target antigens, e.g., P-cadherin and erythropoietin (EPO).
• In practicing the method provided herein, typically only the variable heavy chain and/or variable light chain of the antibody is affinity matured. Thus, the antibody that is chosen typically contains a variable heavy chain and a variable light chain, or portion thereof sufficient to form an antigen binding site. It is understood, however, that the antibody also can include all or a portion of the constant heavy chain (e.g. one or more CH domains, such as CH1, CH2, CH3 and CH4, and/or a constant light chain (CL)). Hence, the antibody can include those that are full-length antibodies, and also include fragments or portions thereof including, for example, Fab, Fab′, F(ab′)₂, single-chain Fvs (scFv), Fv, dsFv, diabody, Fd and Fd′ fragments, Fab fragments, scFv fragments, and scFab fragments. For example, affinity maturation of antibodies exemplified in the examples herein are Fabs. It is understood that once the antibody is affinity matured as provided herein, the resulting antibody can be produced as a full-length antibody or a fragment thereof, such as a Fab, Fab′, F(ab′)₂, single-chain Fvs (scFv), Fv, dsFv, diabody, Fd and Fd′ fragments, Fab fragments, scFv fragments, and scFab fragments. Further, the constant region of any isotype can be used in the generation of full or partial antibody fragments, including IgG, IgM, IgA, IgD and IgE constant regions. Such constant regions can be obtained from any human or animal species. It is understood that activities and binding affinities can differ depending on the structure of an antibody. For example, generally a bivalent antibody, for example a bivalent F(ab′)₂ fragment or full-length IgG, has a better binding affinity then a monovalent Fab antibody. As a result, where a Fab has a specified binding affinity for a particular target, it is excepted that the binding affinity is even greater for a full-length IgG that is bivalent. Thus, comparison of binding affinities between a first antibody and an affinity matured antibody are typically made between antibodies that have the same structure, e.g. Fab compared to Fab.
• An antibody for affinity maturation can include an existing antibody known to one of skill in the art. In other examples, an antibody is generated or identified empirically depending on a desired target. For example, an antibody can be generated using conventional immunization and hybridoma screening methods. In other examples, an antibody is identified by any of a variety of screening methods known to one of skill in the art.
• i. Immunization and Hybridoma Screening
• Antibodies specific for a target antigen can be made using the hybridoma method first described by Kohler et al. (1975) Nature, 256:495, or made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
• In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Antibodies to a target antigen can be raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of protein antigen and an adjuvant. Two weeks later, animals are boosted. 7 to 14 days later animals are bled and the serum is assayed for antibody titer specific for the target antigen. Animals are boosted until titers plateau.
• Alternatively, lymphocytes can be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).
• The hybridoma cells that are prepared are seeded and grown in a suitable culture medium that contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
• Myeloma cells include those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif., USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection (ATCC), Rockville, Md., USA. Human myeloma and mouse-human heterocyeloma cells lines also have been described for the production of human monoclonal antibodies (Kozbor, (1984) J. Immunol., 133:3001; and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
• Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the target antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by any method known to one of skill in the art (e.g. as described in Section E.1), for example, by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoadsorbent assay (ELISA). The binding affinity also can be determined, for example, using Scatchard analysis.
• After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can be grown in vivo as ascites tumors in an animal.
• The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
• DNA-encoding the hybridoma-derived monoclonal antibody can be readily isolated and sequenced using conventional procedures. For example, sequencing can be effected using oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions of interest from the hybridoma. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein to obtain the synthesis of the desired monoclonal antibodies in the recombinant host cells.
• ii. Screening Assays for Identification of a “Hit”
• Antibodies that are affinity matured by the method herein can be identified by using combinatorial libraries to screen for synthetic antibody clones with the desired activity or activities. Antibodies with a desired activity can be selected as “Hits.” Such “Hit” antibodies can be further affinity matured to optimize the activity.
1) Display Libraries
• Typical of screening methods are high throughput screening of antibody libraries. For example, antibody libraries are screened using a display technique, such that there is a physical link between the individual molecules of the library (phenotype) and the genetic information encoding them (genotype). These methods include, but are not limited to, cell display, including bacterial display, yeast display and mammalian display, phage display (Smith, G. P. (1985) Science 228:1315-1317), mRNA display, ribosome display and DNA display. Using display techniques, the identity of each of the individual antibodies is unknown prior to screening, but the phenotype-genotype link allows for facile identification of selected antibodies. Prior to practice of the method herein, the sequence of a “Hit” antibody is determined.
• Typically, in the libraries, nucleic acids encoding antibody gene fragments are obtained from immune cells harvested from humans or animals. If a library biased in favor of an antigen-specific antibody is desired, the subject is immunized with the target antigen to generate an antibody response, and spleen cells and/or circulating B cells or other peripheral blood lymphocytes (PBLs) are recovered for library construction. Additional enrichment for antigen-specific antibody reactive cell populations can be obtained using a suitable screening procedure to isolate B cells expressing antigen-specific membrane bound antibody, e.g. by cell separation with antigen affinity chromatography or adsorption of cells to fluorochrome-labeled antigen followed by fluorescence-activated cell sorting (FACs).
• Alternatively, the use of spleen cells and/or B cells or other PBLs from an unimmunized donor provides a better representation of the possible antibody repertoire, and also permits the construction of an antibody library using any animal (human or non-human) species in which the target antigen is not antigenic. For libraries incorporating in vitro antibody gene construction, stem cells are harvested from the subject to provide nucleic acids encoding unrearranged antibody gene segments. The immune cells of interest can be obtained from a variety of animal species, such as human, mouse, rat, lagomorpha, lupine, canine, feline, porcine, bovine, equine, and avian species.
• Nucleic acid encoding antibody variable gene segments (including VH and VL segments) can be recovered from the cells of interest and amplified. In the case of rearranged VH and VL gene libraries, the desired DNA can be obtained by isolating genomic DNA or mRNA from lymphocytes followed by polymerase chain reaction (PCR) with primers matching the 5′ and 3′ ends of rearranged VH and VL genes as described in Orlandi et al., (1989) Proc. Natl. Acad. Sci. (USA), 86:3833-3837, thereby making diverse V gene repertoires for expression. The V genes can be amplified from cDNA and genomic DNA, with back primers at the 5′ end of the exon encoding the mature V-domain and forward primers based within the J-segment as described in Orlandi et al., (1989) and in Ward et al., (1989) Nature, 341:544-546. For amplifying from cDNA, however, back primers can also be based in the leader exon as described in Jones et al., (1991) Biotechnology, 9:88-89, and forward primers within the constant region as described in Sastry et al., (1989) Proc. Natl. Acad. Sci. (USA), 86:5728-5732. To maximize complementarity, degeneracy can be incorporated in the primers as described in Orlandi et al. (1989) or Sastry et al. (1989). The library diversity can be maximized by using PCR primers targeted to each V-gene family in order to amplify all available VH and VL arrangements present in the immune cell nucleic acid sample, e.g. as described in the method of Marks et al., (1991) J. Mol. Biol., 222:581-597, or as described in the method of Orum et al., (1993) Nucleic Acids Res., 21:4491-4498. For cloning of the amplified DNA into expression vectors, rare restriction sites can be introduced within the PCR primer as a tag at one end as described in Orlandi et al. (1989), or by further PCR amplification with a tagged primer as described in Clackson et al., (1991) Nature, 352:624-628.
• In another example of generating an antibody library, repertoires of synthetically rearranged V genes can be derived in vitro from V gene segments. Most of the human VH-gene segments have been cloned and sequenced (see e.g. Tomlinson et al., (1992) J. Mol. Biol., 227:776-798), and mapped (see e.g. Matsuda et al., (1993) Nature Genet., 3:988-94). These segments can be used to generate diverse VH gene repertoires with PCR primers encoding H3 loops of diverse sequence and length as described in Hoogenboom and Winter (1992) J. Mol. Biol., 227:381-388. VH repertoires also can be made with all of the sequence diversity focused in a long H3 loop of a single length as described in Barbas et al., (1992) Proc. Natl. Acad. Sci. USA, 89:4457-4461. Human Vκ and Vλ segments have been cloned and sequenced (see e.g. Williams and Winter (1993) Eur. J. Immunol., 23:1456-1461) and can be used to make synthetic light chain repertoires. Synthetic V gene repertoires, based on a range of VH and VL folds, and L3 and H3 lengths, encode antibodies of considerable structural diversity. Following amplification of V-gene encoding DNAs, germline V-gene segments can be rearranged in vitro according to the methods of Hoogenboom and Winter (1992) J. Mol. Biol., 227:381-388.
• Repertoires of antibody fragments can be constructed by combining VH and VL gene repertoires together in several ways. Each repertoire can be created in different vectors, and the vectors recombined in vitro (see e.g. Hogrefe et al., (1993) Gene, 128:119-126), or in vivo by combinatorial infection, for example, using the lox P system (Waterhouse et al., (1993) Nucl. Acids Res., 21:2265-2266). The in vivo recombination approach exploits the two-chain nature of Fab fragments to overcome the limit on library size imposed by E. coli transformation efficiency. Alternatively, the repertoires can be cloned sequentially into the same vector (see e.g. Barbas et al., (1991) Proc. Natl. Acad. Sci. USA, 88:7978-7982), or assembled together by PCR and then cloned (see e.g. Clackson et al., (1991) Nature, 352:624-628). PCR assembly can also be used to join VH and VL DNAs with DNA encoding a flexible peptide spacer to form single chain Fv (scFv) repertoires. In another technique, “in cell PCR assembly” can be used to combine VH and VL genes within lymphocytes by PCR and then clone repertoires of linked genes (see e.g. Embleton (1992) Nucl. Acids Res., 20:3831-3837).
• In typical display libraries, the repertoire of VH and VL chains are constructed as one-pot libraries, such that the sequence of each member of the library is not known. Accordingly, sequencing is required following identification of a “Hit” antibody in order to obtain any knowledge of the SAR relationship as required for practice of the method herein. Thus, as above for hybridoma-generated antibodies, DNA-encoding antibody clones identified from a display library can be readily isolated and sequenced using conventional procedures. For example, sequencing can be effected using oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions of interest from a DNA template, e.g. phage DNA template.
• Exemplary of such antibody libraries that can be used for screening are those described in any of the following: European Patent Application Nos. EP0368684 and EP89311731; International Published Patent Application Nos. WO92/001047, WO 02/38756, WO 97/08320, WO 2005/023993, WO 07/137,616 and WO 2007/054816; U.S. Pat. No. 6,593,081 and U.S. Pat. No. 6,989,250; United States Published Patent Application Nos. US 2002/0102613, US 2003/153038, US 2003/0022240, US 2005/0119455, US 2005/0079574 and US 2006/0234302; and Orlandi et al. (1989) Proc Natl. Acad. Sci. U.S.A., 86:3833-3837; Ward et al. (1989) Nature, 341:544-546; Huse et al. (1989) Science, 246:1275-1281; Burton et al. (1991) Proc. Natl. Acad. Sci., U.S.A., 88:10134-10137; Marks et al. (1991) J Mol Biol, 222:581-591; Hoogenboom et al. (1991) J Mol Biol, 227:381-388; Nissim et al. (1994) EMBO J, 13:692-698; Barbas et al. (1992) Proc. Natl. Acad. Sci., U.S.A., 89:4457-4461; Akamatsu et al. (1993) J. Immunol., 151:4651-1659; Griffiths et al. (1994) EMBO J, 13:3245-3260; Fellouse (2004) PNAS, 101:12467-12472; Persson et al. (2006) J. Mol. Biol. 357:607-620; Knappik et al. (2000) J. Mol. Biol. 296:57-86; Rothe et al. (2008) J. Mol. Biol. 376:1182-1200; Mondon et al. (2008) Frontiers in Bioscience, 13:1117-1129; and Behar, I. (2007) Expert Opin. Biol. Ther., 7:763-779.
2) Phage Display Libraries
• For example, natural or synthetic antibodies are selected by screening phage libraries containing phage that display various fragments of antibody variable region (Fv) fused to phage coat protein. Variable domains can be displayed functionally on phage, either as single-chain Fv (scFv) fragments, in which VH and VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non-covalently, as described in Winter et al., (1994) Ann. Rev. Immunol., 12:433-455. Such phage libraries are panned by affinity chromatography against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are bound to the antigen and thus separated from the non-binding clones in the library. The binding clones are then eluted from the antigen, and can be further enriched by additional cycles of antigen binding/elution. Any antibody can be obtained by designing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of a full length antibody clone using the Fv sequences from the phage clone of interest and suitable constant region (Fe) sequences described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.
• Repertoires of VH and VL genes can be separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be searched for antigen-binding clones as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned to provide a single source of human antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J. 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning the unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
• VH and VL repertoires are cloned separately, one into a phagemid and the other into a phage vector. The two libraries are then combined by phage infection of phagemid-containing bacteria so that each cell contains a different combination and the library size is limited only by the number of cells present (about 10¹² clones). Both vectors contain in vivo recombination signals so that the VH and VL genes are recombined onto a single replicon and are co-packaged into phage virions. The libraries can provide a large number of diverse antibodies of good affinity (Kd-¹ of about 10⁻⁸ M).
• Filamentous phage is used to display antibody fragments by fusion to a coat protein, for example, the minor coat protein pIII. The antibody fragments can be displayed as single chain Fv fragments, in which VH and VL domains are connected on the same polypeptide chain by a flexible polypeptide spacer, e.g. as described by Marks et al., J. Mol. Biol., 222: 581-597 (1991), or as Fab fragments, in which one chain is fused to pIII and the other is secreted into the bacterial host cell periplasm where assembly of a Fab-coat protein structure which becomes displayed on the phage surface by displacing some of the wild type coat proteins, e.g. as described in Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991).
3) Addressable Libraries
• Another method of identifying antibodies, or fragments thereof, that have a desired specificity and/or activity for a target protein includes addressable combinatorial antibody libraries as described in U.S. Provisional Application Nos. 61/198,764 and 61/211,204, and in International published PCT Appl. No. WO2010054007, incorporated by reference herein. These include, for example, spatially addressed combinatorial antibody libraries. An advantage of addressable combinatorial libraries compared to display libraries is that each loci represents a different library member whose identity is known by virtue of its address. In such libraries, each individual member of the library is individually generated, and thus the sequence of each member is known. Display of the members of the library can be achieved on any desired format, which permits screening the members not only for binding but also for function. The “Hits” can be quickly identified, including by sequence, coincident with the screening results. Sequencing is not required to obtain structural information about an identified antibody since the sequence of an identified “Hit” is known a priori. Accordingly, affinity maturation of a “Hit” antibody can be performed immediately after screening and identification of a “Hit” antibody.
• Addressable combinatorial antibody libraries contain antibodies with variable heavy chain and variable light chains composed of recombined human germline segments. Antibody combinatorial diversity in the library exists from recombination of individual V, D and J segments that make up the variable heavy chains and of individual V (V_κ or V_λ) and J (J_κ or J_λ) segments that make up the variable light chains. Additional combinatorial diversity derives from the pairing of different variable heavy chains and variable light chains
• The nucleic acid molecules encoding each VH chain and/or VL chain are individually synthesized, using standard DNA synthesis techniques, in an addressable format, whereby the identity of the nucleic acid sequence of each VH chain and/or VL chain in each locus is known. VH chains and VL chains are then paired, also in an addressable format, such that the identity of each member of the library is known based on its locus or “address”. The addressable combinatorial antibody libraries can be screened for binding or activity against a target protein to identify antibodies or portions thereof that bind to a target protein and/or modulate an activity of a target protein. By virtue of the fact that these libaries are arrayed, the identity of each individual member in the collection is known during screening, thereby allowing facile comparison of “Hit” and related “non-Hit” antibodies.
• U.S. Provisional Appl. Nos. 61/198,764 and 61/211,204, and published International PCT Appl. No. WO2010054007, incorporated by reference herein, provide a method of generating a combinatorial antibody library where the identity of every antibody is known at the time of screening by virtue of the combinatorial generation of antibody members. In the combinatorial addressable libraries, variable heavy (VH) and variable light (VL) chain members of the libraries are generated, recombinantly or synthetically by DNA synthesis, from known germline antibody sequences or modified sequences thereof. Antibody combinatorial diversity in the library exists from recombination of individual V, D and J segments that make up the variable heavy chains and of individual V (V_κ or V_λ) and J (J_κ or J_λ) segments that make up the variable light chains. Additional combinatorial diversity derives from the pairing of different variable heavy chains and variable light chains.
• Each VL chain of the antibodies in the library is encoded by a nucleic acid molecule that contains a V_κ and a J_κ human germline segment or degenerate codons thereof, or a V_λ and a J_λ human germline segment or degenerate codons thereof, whereby the segments are linked in-frame. The germline segments are joined such that the V_L segment is 5′ of the J_L segment. Each VH chain of the antibodies in the library is encoded by a nucleic acid molecule that contains a V_H, D_H and a J_H germline segment, whereby the segments are linked in-frame. The germline segments are joined such that the V_H segment is 5′ of the D_H segment, which is 5′ of the J_H segment.
• The recombination is effected so that each gene segment is in-frame, such that resulting recombined nucleic acid molecules encodes a functional VH or VL polypeptide. For example, recombined segments are joined such that the recombined full length nucleic acid is in frame with the 5′ start codon (ATG), thereby allowing expression of a full length polypeptide. Any combination of a V(D)J can be made, and junctions modified accordingly in order to generate a compiled V(D)J sequence that is in-frame, while preserving reading frames of each segment. The choice of junction modification is a function of the combination of V(D)J that will be joined, and the proper reading frame of each gene segment. In some examples, the nucleic acid molecule encoding a VH chain and/or a VL chain are further modified to remove stop codons and/or restriction enzyme sites so that the resulting encoded polypeptide is in-frame and functional.
• A nucleic acid that encodes a variable heavy chain or a variable light chain is generated as follows. In the first step, individual germline segments (V_H, D_H and J_H for a heavy chain or V_κ and a J_κ, or V_λ and J_λ for a light chain) are selected for recombination. The germline segments can be human germline segments, or dgenerate sequences thereof, or alternatively the germline segments can be modified. For example, the D_H segment of a variable heavy chain can be translated in any open reading frame, or alternatively, the D_H segment can be the reverse complement of a D_H germline segment. Once selected, the germline segments are joined such that the recombined full length nucleic acid is in frame with the 5′ start codon (ATG), thereby allowing expression of a full length polypeptide. Any combination of a V(D)J can be made, and junctions modified accordingly in order to generate a compiled V(D)J sequence that is in-frame, while preserving reading frames of each segment. The V segment is always reading frame 1. The reading frame of the J segment is selected so the correct amino acids are encoded. The D segment can be in any reading frame, but typically, the reading frame is chosen such that the resulting amino acids are predominately hydrophobic. As necessary, nucleic acid modifications are made at the junctions between the gene segments such that each segment is in the desired reading frame. For example, at the V-D junction, one or more nucleotides can be deleted from the 5′ end of the D, one or more nucleotides can be deleted from the 3′ end of the V or one or more nucleotides can be inserted between the V and D (e.g. a nucleotide can be added to the 3′ end of the V). Once the junctions are formed, the sequence is modified to remove any stop codons by substitution of nucleotides, such that stop codon TAA is replaced by codon TAT; stop codon TAG is replaced by codon TAT, and stop codon TGA is replaced by codon TCA. Finally, the nucleic acid can be further modified to, for example, remove unwanted restriction sites, splicing donor or acceptor sites, or other nucleotide sequences potentially detrimental to efficient translation. Modifications of the nucleic acid sequences include replacements or substitutions, insertions, or deletions of nucleotides, or any combination thereof.
• The nucleic acid molecules encoding each VH chain and/or VL chain are individually synthesized, using standard DNA synthesis techniques, in an addressable format, whereby the identity of the nucleic acid sequence of each VH chain and/or VL chain in each locus is known.
• VH chains and VL chains are then paired, also in an addressable format, such that the identity of each member of the library is known based on its locus or “address”. For example, resulting members of the library are produced by co-expression of nucleic acid molecules encoding the recombined variable region genes together, such that when expressed, a combinatorial antibody member is generated minimally containing a VH and VL chain, or portions thereof. In some examples of the methods, the nucleic acid molecule encoding the VH and VL chain can be expressed as a single nucleic acid molecule, whereby the genes encoding the heavy and light chain are joined by a linker. In another example of the methods, the nucleic acid molecules encoding the VH and VL chain can be separately provided for expression together. Thus, upon expression from the recombined nucleic acid molecules, each different member of the library represents a germline encoded antibody, whereby diversity is achieved by combinatorial diversity of V(D)J segments and pairing diversity of heavy and light chains.
• The antibodies within the combinatorial addressable germline antibody libraries contain all or a portion of the variable heavy chain (VH) and variable light chain (VL), as long as the resulting antibody is sufficient to form an antigen binding site. Typically, the combinatorial addressable germline antibodies are Fabs. Exemplary nucleic acids encoding variable heavy chains and light chains are set forth in Table 3 below. A library of antibodies can be generated upon co-expression of a nucleic acid molecule encoding the VH chain and a nucleic acid encoding the VL chain to generate a combinatorial library containing a plurality of different members. An exemplary paired nucleic acid library is set forth in Table 4 below, where each row sets forth a different loci of the library. The combinatorial addressable antibody library can be screened to identify a “Hit” antibody against any target antigen. Related non-Hit antibodies that do not bind the target antigen also can be readily identified, since the identity by sequence structure of each “Hit” or “non-Hit” are immediately known coincident with the binding results.

• TABLE 3
  Exemplary Variable Heavy Chains and Light Chains

  		SEQ
  Number	Name	ID NO.

  Heavy Chain

  1	gnl|Fabrus|VH1-18_IGHD1-26*01_IGHJ2*01	1828
  2	gnl|Fabrus|VH1-18_IGHD2-21*01_IGHJ2*01	1829
  3	gnl|Fabrus|VH1-18_IGHD3-16*01_IGHJ6*01	1830
  4	gnl|Fabrus|VH1-18_IGHD3-22*01_IGHJ4*01	1831
  5	gnl|Fabrus|VH1-18_IGHD4-23*01_IGHJ1*01	1832
  6	gnl|Fabrus|VH1-18_IGHD5-12*01_IGHJ4*01	1833
  7	gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01	1834
  8	gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01	1835
  9	gnl|Fabrus|VH1-24_IGHD1-7*01_IGHJ4*01	1836
  10	gnl|Fabrus|VH1-24_IGHD2-15*01_IGHJ2*01	1837
  11	gnl|Fabrus|VH1-24_IGHD3-10*01_IGHJ4*01	1838
  12	gnl|Fabrus|VH1-24_IGHD3-16*01_IGHJ4*01	1839
  13	gnl|Fabrus|VH1-24_IGHD4-23*01_IGHJ2*01	1840
  14	gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01	1841
  15	gnl|Fabrus|VH1-24_IGHD5-18*01_IGHJ6*01	1842
  16	gnl|Fabrus|VH1-24_IGHD6-19*01_IGHJ4*01	1843
  17	gnl|Fabrus|VH1-3_IGHD2-15*01_IGHJ2*01	1844
  18	gnl|Fabrus|VH1-3_IGHD2-2*01_IGHJ5*01	1845
  19	gnl|Fabrus|VH1-3_IGHD3-9*01_IGHJ6*01	1846
  20	gnl|Fabrus|VH1-3_IGHD4-23*01_IGHJ4*01	101
  21	gnl|Fabrus|VH1-3_IGHD5-18*01_IGHJ4*01	1847
  22	gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01	1848
  23	gnl|Fabrus|VH1-3_IGHD7-27*01_IGHJ4*01	1849
  24	gnl|Fabrus|VH1-45_IGHD1-26*01_IGHJ4*01	1850
  25	gnl|Fabrus|VH1-45_IGHD2-15*01_IGHJ6*01	1851
  26	gnl|Fabrus|VH1-45_IGHD2-8*01_IGHJ3*01	1852
  27	gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01	1853
  28	gnl|Fabrus|VH1-45_IGHD3-16*01_IGHJ2*01	1854
  29	gnl|Fabrus|VH1-45_IGHD4-23*01_IGHJ4*01	1855
  30	gnl|Fabrus|VH1-45_IGHD5-24*01_IGHJ4*01	1856
  31	gnl|Fabrus|VH1-45_IGHD6-19*01_IGHJ4*01	1857
  32	gnl|Fabrus|VH1-45_IGHD7-27*01_IGHJ6*01	1858
  33	gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01	1859
  34	gnl|Fabrus|VH1-46_IGHD2-15*01_IGHJ2*01	99
  35	gnl|Fabrus|VH1-46_IGHD3-10*01_IGHJ4*01	92
  36	gnl|Fabrus|VH1-46_IGHD4-17*01_IGHJ4*01	1860
  37	gnl|Fabrus|VH1-46_IGHD5-18*01_IGHJ4*01	1861
  38	gnl|Fabrus|VH1-46_IGHD6-13*01_IGHJ4*01	93
  39	gnl|Fabrus|VH1-46_IGHD6-6*01_IGHJ1*01	88
  40	gnl|Fabrus|VH1-46_IGHD7-27*01_IGHJ2*01	97
  41	gnl|Fabrus|VH1-58_IGHD1-26*01_IGHJ4*01	1862
  42	gnl|Fabrus|VH1-58_IGHD2-15*01_IGHJ2*01	1863
  43	gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01	1864
  44	gnl|Fabrus|VH1-58_IGHD4-17*01_IGHJ4*01	1865
  45	gnl|Fabrus|VH1-58_IGHD5-18*01_IGHJ4*01	1866
  46	gnl|Fabrus|VH1-58_IGHD6-6*01_IGHJ1*01	1867
  47	gnl|Fabrus|VH1-58_IGHD7-27*01_IGHJ5*01	1868
  48	gnl|Fabrus|VH1-69_IGHD1-1*01_IGHJ6*01	98
  49	gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01	1869
  50	gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01	1870
  51	gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01	1871
  52	gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01	1872
  53	gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01	1873
  54	gnl|Fabrus|VH1-69_IGHD3-9*01_IGHJ6*01	1874
  55	gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01	1875
  56	gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01	1876
  57	gnl|Fabrus|VH1-69_IGHD5-24*01_IGHJ6*01	1877
  58	gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01	1878
  59	gnl|Fabrus|VH1-69_IGHD6-6*01_IGHJ1*01	1879
  60	gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01	1880
  61	gnl|Fabrus|VH1-8_IGHD1-26*01_IGHJ4*01	1881
  62	gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01	1882
  63	gnl|Fabrus|VH1-8_IGHD2-2*01_IGHJ6*01	102
  64	gnl|Fabrus|VH1-8_IGHD3-10*01_IGHJ4*01	1883
  65	gnl|Fabrus|VH1-8_IGHD4-17*01_IGHJ4*01	1884
  66	gnl|Fabrus|VH1-8_IGHD5-5*01_IGHJ4*01	1885
  67	gnl|Fabrus|VH1-8_IGHD7-27*01_IGHJ4*01	1886
  68	gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01	1887
  69	gnl|Fabrus|VH2-26_IGHD2-15*01_IGHJ2*01	1888
  70	gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01	1889
  71	gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01	1890
  72	gnl|Fabrus|VH2-26_IGHD3-9*01_IGHJ6*01	1891
  73	gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01	1892
  74	gnl|Fabrus|VH2-26_IGHD5-12*01_IGHJ4*01	1893
  75	gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01	1894
  76	gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01	1895
  77	gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01	1896
  78	gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01	1897
  79	gnl|Fabrus|VH2-5_IGHD1-1*01_IGHJ5*01	1898
  80	gnl|Fabrus|VH2-5_IGHD2-15*01_IGHJ6*01	1899
  81	gnl|Fabrus|VH2-5_IGHD3-16*01_IGHJ4*01	1900
  82	gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01	1901
  83	gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01	1902
  84	gnl|Fabrus|VH2-5_IGHD6-13*01_IGHJ4*01	1903
  85	gnl|Fabrus|VH2-5_IGHD7-27*01_IGHJ2*01	96
  86	gnl|Fabrus|VH2-70_IGHD1-1*01_IGHJ2*01	1904
  87	gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01	1905
  88	gnl|Fabrus|VH2-70_IGHD3-22*01_IGHJ4*01	1906
  89	gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01	1907
  90	gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01	1908
  91	gnl|Fabrus|VH2-70_IGHD7-27*01_IGHJ2*01	1909
  92	gnl|Fabrus|VH3-11_IGHD1-26*01_IGHJ4*01	1910
  93	gnl|Fabrus|VH3-11_IGHD2-2*01_IGHJ6*01	1911
  94	gnl|Fabrus|VH3-11_IGHD3-16*01_IGHJ4*01	1912
  95	gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01	1913
  96	gnl|Fabrus|VH3-11_IGHD4-23*01_IGHJ5*01	1914
  97	gnl|Fabrus|VH3-11_IGHD5-18*01_IGHJ4*01	1915
  98	gnl|Fabrus|VH3-11_IGHD6-19*01_IGHJ6*01	1916
  99	gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01	1917
  100	gnl|Fabrus|VH3-11_IGHD7-27*01_IGHJ4*01	1918
  101	gnl|Fabrus|VH3-13_IGHD1-26*01_IGHJ4*01	1919
  102	gnl|Fabrus|VH3-13_IGHD2-8*01_IGHJ5*01	1920
  103	gnl|Fabrus|VH3-13_IGHD3-3*01_IGHJ1*01	1921
  104	gnl|Fabrus|VH3-13_IGHD3-9*01_IGHJ6*01	1922
  105	gnl|Fabrus|VH3-13_IGHD4-23*01_IGHJ5*01	1923
  106	gnl|Fabrus|VH3-13_IGHD5-5*01_IGHJ4*01	1924
  107	gnl|Fabrus|VH3-13_IGHD6-6*01_IGHJ1*01	1925
  108	gnl|Fabrus|VH3-13_IGHD7-27*01_IGHJ5*01	1926
  109	gnl|Fabrus|VH3-15_IGHD1-26*01_IGHJ4*01	1927
  110	gnl|Fabrus|VH3-15_IGHD2-15*01_IGHJ2*01	1928
  111	gnl|Fabrus|VH3-15_IGHD2-15*01_IGHJ6*01	1929
  112	gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01	1930
  113	gnl|Fabrus|VH3-15_IGHD3-9*01_IGHJ2*01	1931
  114	gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01	1932
  115	gnl|Fabrus|VH3-15_IGHD6-6*01_IGHJ1*01	1933
  116	gnl|Fabrus|VH3-16_IGHD1-1*01_IGHJ1*01	1934
  117	gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01	1935
  118	gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01	1936
  119	gnl|Fabrus|VH3-16_IGHD2-2*01_IGHJ2*01	1937
  120	gnl|Fabrus|VH3-16_IGHD3-10*01_IGHJ4*01	1938
  121	gnl|Fabrus|VH3-16_IGHD4-4*01_IGHJ2*01	1939
  122	gnl|Fabrus|VH3-16_IGHD5-24*01_IGHJ4*01	1940
  123	gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01	1941
  124	gnl|Fabrus|VH3-16_IGHD7-27*01_IGHJ2*01	1942
  125	gnl|Fabrus|VH3-20_IGHD1-14*01_IGHJ4*01	1943
  126	gnl|Fabrus|VH3-20_IGHD2-15*01_IGHJ2*01	1944
  127	gnl|Fabrus|VH3-20_IGHD2-8*01_IGHJ4*01	1945
  128	gnl|Fabrus|VH3-20_IGHD3-10*01_IGHJ4*01	1946
  129	gnl|Fabrus|VH3-20_IGHD3-9*01_IGHJ6*01	1947
  130	gnl|Fabrus|VH3-20_IGHD4-23*01_IGHJ4*01	1948
  131	gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01	1949
  132	gnl|Fabrus|VH3-20_IGHD6-13*01_IGHJ4*01	1950
  133	gnl|Fabrus|VH3-20_IGHD7-27*01_IGHJ2*01	1951
  134	gnl|Fabrus|VH3-21_IGHD1-26*01_IGHJ4*01	1952
  135	gnl|Fabrus|VH3-21_IGHD2-2*01_IGHJ5*01	1953
  136	gnl|Fabrus|VH3-21_IGHD3-22*01_IGHJ4*01	1954
  137	gnl|Fabrus|VH3-21_IGHD4-23*01_IGHJ5*01	1955
  138	gnl|Fabrus|VH3-21_IGHD5-24*01_IGHJ5*01	1956
  139	gnl|Fabrus|VH3-21_IGHD6-19*01_IGHJ1*01	1957
  140	gnl|Fabrus|VH3-21_IGHD7-27*01_IGHJ4*01	1958
  141	gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ1*01	1959
  142	gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01	1960
  143	gnl|Fabrus|VH3-23_IGHD1-20*01_IGHJ3*01	1961
  144	gnl|Fabrus|VH3-23_IGHD1-26*01_IGHJ4*01	1962
  145	gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01	1963
  146	gnl|Fabrus|VH3-23_IGHD2-21*01_IGHJ1*01	1964
  147	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	1965
  148	gnl|Fabrus|VH3-23_IGHD3-16*01_IGHJ4*01	1966
  149	gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01	1967
  150	gnl|Fabrus|VH3-23_IGHD3-3*01_IGHJ5*01	1968
  151	gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01	1969
  152	gnl|Fabrus|VH3-23_IGHD4-23*01_IGHJ2*01	1970
  153	gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01	1971
  154	gnl|Fabrus|VH3-23_IGHD5-24*01_IGHJ1*01	1972
  155	gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01	1973
  156	gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01	1974
  157	gnl|Fabrus|VH3-23_IGHD6-25*01_IGHJ2*01	1975
  158	gnl|Fabrus|VH3-23_IGHD6-6*01_IGHJ1*01	1976
  159	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01	1977
  160	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01	1978
  161	gnl|Fabrus|VH3-30_IGHD1-1*01_IGHJ6*01	1979
  162	gnl|Fabrus|VH3-30_IGHD1-26*01_IGHJ1*01	1980
  163	gnl|Fabrus|VH3-30_IGHD1-26*01_IGHJ4*01	1981
  164	gnl|Fabrus|VH3-30_IGHD2-15*01_IGHJ2*01	1982
  165	gnl|Fabrus|VH3-30_IGHD2-2*01_IGHJ6*01	1983
  166	gnl|Fabrus|VH3-30_IGHD3-10*01_IGHJ1*01	1984
  167	gnl|Fabrus|VH3-30_IGHD3-16*01_IGHJ6*01	1985
  168	gnl|Fabrus|VH3-30_IGHD4-17*01_IGHJ4*01	1986
  169	gnl|Fabrus|VH3-30_IGHD5-12*01_IGHJ4*01	1987
  170	gnl|Fabrus|VH3-30_IGHD5-18*01_IGHJ1*01	1988
  171	gnl|Fabrus|VH3-30_IGHD6-13*01_IGHJ4*01	1989
  172	gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01	1990
  173	gnl|Fabrus|VH3-35_IGHD1-1*01_IGHJ2*01	1991
  174	gnl|Fabrus|VH3-35_IGHD1-20*01_IGHJ6*01	1992
  175	gnl|Fabrus|VH3-35_IGHD2-15*01_IGHJ2*01	1993
  176	gnl|Fabrus|VH3-35_IGHD2-21*01_IGHJ6*01	1994
  177	gnl|Fabrus|VH3-35_IGHD3-10*01_IGHJ4*01	1995
  178	gnl|Fabrus|VH3-35_IGHD3-9*01_IGHJ6*01	1996
  179	gnl|Fabrus|VH3-35_IGHD5-12*01_IGHJ4*01	1997
  180	gnl|Fabrus|VH3-35_IGHD6-13*01_IGHJ4*01	1998
  181	gnl|Fabrus|VH3-35_IGHD7-27*01_IGHJ1*01	1999
  182	gnl|Fabrus|VH3-38_IGHD1-14*01_IGHJ5*01	2000
  183	gnl|Fabrus|VH3-38_IGHD1-20*01_IGHJ6*01	2001
  184	gnl|Fabrus|VH3-38_IGHD2-15*01_IGHJ6*01	2002
  185	gnl|Fabrus|VH3-38_IGHD2-2*01_IGHJ1*01	2003
  186	gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01	2004
  187	gnl|Fabrus|VH3-38_IGHD3-16*01_IGHJ1*01	2005
  188	gnl|Fabrus|VH3-38_IGHD4-17*01_IGHJ2*01	2006
  189	gnl|Fabrus|VH3-38_IGHD5-24*01_IGHJ3*01	2007
  190	gnl|Fabrus|VH3-38_IGHD6-6*01_IGHJ1*01	2008
  191	gnl|Fabrus|VH3-38_IGHD7-27*01_IGHJ6*01	2009
  192	gnl|Fabrus|VH3-43_IGHD1-26*01_IGHJ5*01	2010
  193	gnl|Fabrus|VH3-43_IGHD1-7*01_IGHJ6*01	2011
  194	gnl|Fabrus|VH3-43_IGHD2-2*01_IGHJ3*01	2012
  195	gnl|Fabrus|VH3-43_IGHD2-21*01_IGHJ6*01	2013
  196	gnl|Fabrus|VH3-43_IGHD3-16*01_IGHJ6*01	2014
  197	gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01	2015
  198	gnl|Fabrus|VH3-43_IGHD4-23*01_IGHJ3*01	2016
  199	gnl|Fabrus|VH3-43_IGHD5-18*01_IGHJ5*01	2017
  200	gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01	2018
  201	gnl|Fabrus|VH3-43_IGHD7-27*01_IGHJ1*01	2019
  202	gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01	2020
  203	gnl|Fabrus|VH3-49_IGHD1-26*01_IGHJ4*01	2021
  204	gnl|Fabrus|VH3-49_IGHD1-7*01_IGHJ6*01	2022
  205	gnl|Fabrus|VH3-49_IGHD2-2*01_IGHJ6*01	2023
  206	gnl|Fabrus|VH3-49_IGHD2-8*01_IGHJ4*01	2024
  207	gnl|Fabrus|VH3-49_IGHD3-22*01_IGHJ4*01	2025
  208	gnl|Fabrus|VH3-49_IGHD3-9*01_IGHJ6*01	2026
  209	gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01	2027
  210	gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01	2028
  211	gnl|Fabrus|VH3-49_IGHD7-27*01_IGHJ1*01	2029
  212	gnl|Fabrus|VH3-53_IGHD1-14*01_IGHJ6*01	2030
  213	gnl|Fabrus|VH3-53_IGHD1-7*01_IGHJ1*01	2031
  214	gnl|Fabrus|VH3-53_IGHD2-2*01_IGHJ2*01	2032
  215	gnl|Fabrus|VH3-53_IGHD3-22*01_IGHJ3*01	2033
  216	gnl|Fabrus|VH3-53_IGHD4-23*01_IGHJ1*01	2034
  217	gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01	2035
  218	gnl|Fabrus|VH3-53_IGHD6-13*01_IGHJ3*01	2036
  219	gnl|Fabrus|VH3-53_IGHD7-27*01_IGHJ4*01	2037
  220	gnl|Fabrus|VH3-64_IGHD1-26*01_IGHJ4*01	2038
  221	gnl|Fabrus|VH3-64_IGHD1-7*01_IGHJ6*01	2039
  222	gnl|Fabrus|VH3-64_IGHD2-2*01_IGHJ5*01	2040
  223	gnl|Fabrus|VH3-64_IGHD3-3*01_IGHJ4*01	2041
  224	gnl|Fabrus|VH3-64_IGHD4-17*01_IGHJ4*01	2042
  225	gnl|Fabrus|VH3-64_IGHD5-12*01_IGHJ4*01	2043
  226	gnl|Fabrus|VH3-64_IGHD6-19*01_IGHJ1*01	2044
  227	gnl|Fabrus|VH3-64_IGHD7-27*01_IGHJ4*01	2045
  228	gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01	2046
  229	gnl|Fabrus|VH3-7_IGHD1-20*01_IGHJ3*01	2047
  230	gnl|Fabrus|VH3-7_IGHD1-7*01_IGHJ6*01	2048
  231	gnl|Fabrus|VH3-7_IGHD2-21*01_IGHJ5*01	2049
  232	gnl|Fabrus|VH3-7_IGHD2-8*01_IGHJ6*01	2050
  233	gnl|Fabrus|VH3-7_IGHD3-22*01_IGHJ3*01	2051
  234	gnl|Fabrus|VH3-7_IGHD3-9*01_IGHJ6*01	2052
  235	gnl|Fabrus|VH3-7_IGHD4-17*01_IGHJ4*01	2053
  236	gnl|Fabrus|VH3-7_IGHD5-12*01_IGHJ4*01	2054
  237	gnl|Fabrus|VH3-7_IGHD5-24*01_IGHJ4*01	2055
  238	gnl|Fabrus|VH3-7_IGHD6-19*01_IGHJ6*01	2056
  239	gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01	2057
  240	gnl|Fabrus|VH3-7_IGHD7-27*01_IGHJ2*01	2058
  241	gnl|Fabrus|VH3-72_IGHD1-1*01_IGHJ4*01	2059
  242	gnl|Fabrus|VH3-72_IGHD2-15*01_IGHJ1*01	2060
  243	gnl|Fabrus|VH3-72_IGHD3-22*01_IGHJ4*01	2061
  244	gnl|Fabrus|VH3-72_IGHD3-9*01_IGHJ6*01	2062
  245	gnl|Fabrus|VH3-72_IGHD4-23*01_IGHJ2*01	2063
  246	gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01	2064
  247	gnl|Fabrus|VH3-72_IGHD5-24*01_IGHJ6*01	2065
  248	gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01	2066
  249	gnl|Fabrus|VH3-72_IGHD7-27*01_IGHJ2*01	2067
  250	gnl|Fabrus|VH3-73_IGHD1-1*01_IGHJ5*01	2068
  251	gnl|Fabrus|VH3-73_IGHD2-8*01_IGHJ2*01	2069
  252	gnl|Fabrus|VH3-73_IGHD3-22*01_IGHJ4*01	2070
  253	gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01	2071
  254	gnl|Fabrus|VH3-73_IGHD4-11*01_IGHJ6*01	2072
  255	gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01	2073
  256	gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01	2074
  257	gnl|Fabrus|VH3-73_IGHD6-19*01_IGHJ1*01	2075
  258	gnl|Fabrus|VH3-73_IGHD7-27*01_IGHJ5*01	2076
  259	gnl|Fabrus|VH3-74_IGHD1-1*01_IGHJ6*01	2077
  260	gnl|Fabrus|VH3-74_IGHD1-26*01_IGHJ4*01	2078
  261	gnl|Fabrus|VH3-74_IGHD2-2*01_IGHJ5*01	2079
  262	gnl|Fabrus|VH3-74_IGHD3-22*01_IGHJ5*01	2080
  263	gnl|Fabrus|VH3-74_IGHD4-17*01_IGHJ1*01	2081
  264	gnl|Fabrus|VH3-74_IGHD5-12*01_IGHJ4*01	2082
  265	gnl|Fabrus|VH3-74_IGHD6-6*01_IGHJ1*01	2083
  266	gnl|Fabrus|VH3-74_IGHD7-27*01_IGHJ4*01	2084
  267	gnl|Fabrus|VH3-9_IGHD1-1*01_IGHJ6*01	2085
  268	gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01	2086
  269	gnl|Fabrus|VH3-9_IGHD2-2*01_IGHJ4*01	2087
  270	gnl|Fabrus|VH3-9_IGHD3-16*01_IGHJ6*01	2088
  271	gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01	2089
  272	gnl|Fabrus|VH3-9_IGHD4-11*01_IGHJ4*01	2090
  273	gnl|Fabrus|VH3-9_IGHD5-24*01_IGHJ1*01	2091
  274	gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01	2092
  275	gnl|Fabrus|VH3-9_IGHD6-25*01_IGHJ6*01	2093
  276	gnl|Fabrus|VH3-9_IGHD7-27*01_IGHJ2*01	2094
  277	gnl|Fabrus|VH4-28_IGHD1-20*01_IGHJ1*01	2095
  278	gnl|Fabrus|VH4-28_IGHD1-7*01_IGHJ6*01	2096
  279	gnl|Fabrus|VH4-28_IGHD2-15*01_IGHJ6*01	2097
  280	gnl|Fabrus|VH4-28_IGHD3-16*01_IGHJ2*01	2098
  281	gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01	2099
  282	gnl|Fabrus|VH4-28_IGHD4-4*01_IGHJ4*01	2100
  283	gnl|Fabrus|VH4-28_IGHD5-5*01_IGHJ1*01	2101
  284	gnl|Fabrus|VH4-28_IGHD6-13*01_IGHJ4*01	2102
  285	gnl|Fabrus|VH4-28_IGHD7-27*01_IGHJ1*01	94
  286	gnl|Fabrus|VH4-31_IGHD1-26*01_IGHJ2*01	91
  287	gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01	103
  288	gnl|Fabrus|VH4-31_IGHD2-2*01_IGHJ6*01	2103
  289	gnl|Fabrus|VH4-31_IGHD3-10*01_IGHJ4*01	2104
  290	gnl|Fabrus|VH4-31_IGHD3-9*01_IGHJ6*01	2105
  291	gnl|Fabrus|VH4-31_IGHD4-17*01_IGHJ5*01	2106
  292	gnl|Fabrus|VH4-31_IGHD5-12*01_IGHJ4*01	2107
  293	gnl|Fabrus|VH4-31_IGHD6-13*01_IGHJ4*01	2108
  294	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	2109
  295	gnl|Fabrus|VH4-31_IGHD7-27*01_IGHJ5*01	95
  296	gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01	2110
  297	gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01	2111
  298	gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01	2112
  299	gnl|Fabrus|VH4-34_IGHD3-22*01_IGHJ6*01	2113
  300	gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01	2114
  301	gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01	2115
  302	gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01	2116
  303	gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01	2117
  304	gnl|Fabrus|VH4-34_IGHD6-6*01_IGHJ6*01	2118
  305	gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01	100
  306	gnl|Fabrus|VH4-39_IGHD1-14*01_IGHJ1*01	2119
  307	gnl|Fabrus|VH4-39_IGHD1-20*01_IGHJ6*01	2120
  308	gnl|Fabrus|VH4-39_IGHD2-21*01_IGHJ3*01	2121
  309	gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01	2122
  310	gnl|Fabrus|VH4-39_IGHD3-16*01_IGHJ2*01	2123
  311	gnl|Fabrus|VH4-39_IGHD3-9*01_IGHJ6*01	2124
  312	gnl|Fabrus|VH4-39_IGHD4-23*01_IGHJ2*01	2125
  313	gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01	2126
  314	gnl|Fabrus|VH4-39_IGHD6-6*01_IGHJ1*01	2127
  315	gnl|Fabrus|VH4-4_IGHD1-20*01_IGHJ3*01	2128
  316	gnl|Fabrus|VH4-4_IGHD2-8*01_IGHJ4*01	2129
  317	gnl|Fabrus|VH4-4_IGHD3-22*01_IGHJ2*01	2130
  318	gnl|Fabrus|VH4-4_IGHD4-23*01_IGHJ4*01	2131
  319	gnl|Fabrus|VH4-4_IGHD5-12*01_IGHJ5*01	2132
  320	gnl|Fabrus|VH4-4_IGHD6-6*01_IGHJ4*01	2133
  321	gnl|Fabrus|VH4-4_IGHD7-27*01_IGHJ6*01	2134
  322	gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01	2135
  323	gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01	2136
  324	gnl|Fabrus|VH5-51_IGHD1-26*01_IGHJ6*01	2137
  325	gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01	2138
  326	gnl|Fabrus|VH5-51_IGHD3-10*01_IGHJ6*01	2139
  327	gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01	2140
  328	gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01	2141
  329	gnl|Fabrus|VH5-51_IGHD5-18*01>3_IGHJ4*01	89
  330	gnl|Fabrus|VH5-51_IGHD5-18*01>1_IGHJ4*01	2142
  331	gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01	106
  332	gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01	2143
  333	gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01	2144
  334	gnl|Fabrus|VH6-1_IGHD1-20*01_IGHJ6*01	2145
  335	gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01	2146
  336	gnl|Fabrus|VH6-1_IGHD2-21*01_IGHJ6*01	2147
  337	gnl|Fabrus|VH6-1_IGHD3-16*01_IGHJ5*01	2148
  338	gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01	90
  339	gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01	2149
  340	gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01	2150
  341	gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01	2151
  342	gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01	2152
  343	gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01	2153
  344	gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01	2154
  345	gnl|Fabrus|VH7-81_IGHD1-14*01_IGHJ4*01	2155
  346	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01	2156
  347	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01	2157
  348	gnl|Fabrus|VH7-81_IGHD3-16*01_IGHJ6*01	2158
  349	gnl|Fabrus|VH7-81_IGHD4-23*01_IGHJ1*01	2159
  350	gnl|Fabrus|VH7-81_IGHD5-12*01_IGHJ6*01	2160
  351	gnl|Fabrus|VH7-81_IGHD6-25*01_IGHJ4*01	2161
  352	gnl|Fabrus|VH7-81_IGHD7-27*01_IGHJ4*01	2162
  353	gi|Fabrus|VH3-23_IGHD1-1*01>1_IGHJ1*01	2211
  355	gi|Fabrus|VH3-23_IGHD1-1*01>2_IGHJ1*01	2212
  356	gi|Fabrus|VH3-23_IGHD1-1*01>3_IGHJ1*01	2213
  357	gi|Fabrus|VH3-23_IGHD1-7*01>1_IGHJ1*01	2214
  358	gi|Fabrus|VH3-23_IGHD1-7*01>3_IGHJ1*01	2215
  359	gi|Fabrus|VH3-23_IGHD1-14*01>1_IGHJ1*01	2216
  360	gi|Fabrus|VH3-23_IGHD1-14*01>3_IGHJ1*01	2217
  361	gi|Fabrus|VH3-23_IGHD1-20*01>1_IGHJ1*01	2218
  362	gi|Fabrus|VH3-23_IGHD1-20*01>3_IGHJ1*01	2219
  363	gi|Fabrus|VH3-23_IGHD1-26*01>1_IGHJ1*01	2220
  364	gi|Fabrus|VH3-23_IGHD1-26*01>3_IGHJ1*01	2221
  365	gi|Fabrus|VH3-23_IGHD2-2*01>2_IGHJ1*01	2222
  366	gi|Fabrus|VH3-23_IGHD2-2*01>3_IGHJ1*01	2223
  367	gi|Fabrus|VH3-23_IGHD2-8*01>2_IGHJ1*01	2224
  368	gi|Fabrus|VH3-23_IGHD2-8*01>3_IGHJ1*01	2225
  369	gi|Fabrus|VH3-23_IGHD2-15*01>2_IGHJ1*01	2226
  370	gi|Fabrus|VH3-23_IGHD2-15*01>3_IGHJ1*01	2227
  371	gi|Fabrus|VH3-23_IGHD2-21*01>2_IGHJ1*01	2228
  372	gi|Fabrus|VH3-23_IGHD2-21*01>3_IGHJ1*01	2229
  373	gi|Fabrus|VH3-23_IGHD3-3*01>1_IGHJ1*01	2230
  374	gi|Fabrus|VH3-23_IGHD3-3*01>2_IGHJ1*01	2231
  375	gi|Fabrus|VH3-23_IGHD3-3*01>3_IGHJ1*01	2232
  376	gi|Fabrus|VH3-23_IGHD3-9*01>2_IGHJ1*01	2233
  377	gi|Fabrus|VH3-23_IGHD3-10*01>2_IGHJ1*01	2234
  378	gi|Fabrus|VH3-23_IGHD3-10*01>3_IGHJ1*01	2235
  379	gi|Fabrus|VH3-23_IGHD3-16*01>2_IGHJ1*01	2236
  380	gi|Fabrus|VH3-23_IGHD3-16*01>3_IGHJ1*01	2237
  381	gi|Fabrus|VH3-23_IGHD3-22*01>2_IGHJ1*01	2238
  382	gi|Fabrus|VH3-23_IGHD3-22*01>3_IGHJ1*01	2239
  383	gi|Fabrus|VH3-23_IGHD4-4*01(1)>2_IGHJ1*01	2240
  384	gi|Fabrus|VH3-23_IGHD4-4*01(1)>3_IGHJ1*01	2241
  385	gi|Fabrus|VH3-23_IGHD4-11*01(1)>2_IGHJ1*01	2242
  386	gi|Fabrus|VH3-23_IGHD4-11*01(1)>3_IGHJ1*01	2243
  387	gi|Fabrus|VH3-23_IGHD4-17*01>2_IGHJ1*01	2244
  388	gi|Fabrus|VH3-23_IGHD4-17*01>3_IGHJ1*01	2245
  389	gi|Fabrus|VH3-23_IGHD4-23*01>2_IGHJ1*01	2246
  390	gi|Fabrus|VH3-23_IGHD4-23*01>3_IGHJ1*01	2247
  391	gi|Fabrus|VH3-23_IGHD5-5*01(2)>1_IGHJ1*01	2248
  392	gi|Fabrus|VH3-23_IGHD5-5*01(2)>2_IGHJ1*01	2249
  393	gi|Fabrus|VH3-23_IGHD5-5*01(2)>3_IGHJ1*01	2250
  394	gi|Fabrus|VH3-23_IGHD5-12*01>1_IGHJ1*01	2251
  395	gi|Fabrus|VH3-23_IGHD5-12*01>3_IGHJ1*01	2252
  396	gi|Fabrus|VH3-23_IGHD5-18*01(2)>1_IGHJ1*01	2253
  397	gi|Fabrus|VH3-23_IGHD5-18*01(2)>2_IGHJ1*01	2254
  398	gi|Fabrus|VH3-23_IGHD5-18*01(2)>3_IGHJ1*01	2255
  399	gi|Fabrus|VH3-23_IGHD5-24*01>1_IGHJ1*01	2256
  400	gi|Fabrus|VH3-23_IGHD5-24*01>3_IGHJ1*01	2257
  401	gi|Fabrus|VH3-23_IGHD6-6*01>1_IGHJ1*01	2258
  402	gi|Fabrus|VH3-23_IGHD6-6*01>2_IGHJ1*01	2259
  403	gi|Fabrus|VH3-23_IGHD6-13*01>1_IGHJ1*01	2260
  404	gi|Fabrus|VH3-23_IGHD6-13*01>2_IGHJ1*01	2261
  405	gi|Fabrus|VH3-23_IGHD6-19*01>1_IGHJ1*01	2262
  406	gi|Fabrus|VH3-23_IGHD6-19*01>2_IGHJ1*01	2263
  407	gi|Fabrus|VH3-23_IGHD6-25*01>1_IGHJ1*01	2264
  408	gi|Fabrus|VH3-23_IGHD6-25*01>2_IGHJ1*01	2265
  409	gi|Fabrus|VH3-23_IGHD7-27*01>1_IGHJ1*01	2266
  410	gi|Fabrus|VH3-23_IGHD7-27*01>3_IGHJ1*01	2267
  411	gi|Fabrus|VH3-23_IGHD1-1*01>1′_IGHJ1*01	2268
  412	gi|Fabrus|VH3-23_IGHD1-1*01>2′_IGHJ1*01	2269
  413	gi|Fabrus|VH3-23_IGHD1-1*01>3′_IGHJ1*01	2270
  414	gi|Fabrus|VH3-23_IGHD1-7*01>1′_IGHJ1*01	2271
  415	gi|Fabrus|VH3-23_IGHD1-7*01>3′_IGHJ1*01	2272
  416	gi|Fabrus|VH3-23_IGHD1-14*01>1′_IGHJ1*01	2273
  417	gi|Fabrus|VH3-23_IGHD1-14*01>2′_IGHJ1*01	2274
  418	gi|Fabrus|VH3-23_IGHD1-14*01>3′_IGHJ1*01	2275
  419	gi|Fabrus|VH3-23_IGHD1-20*01>1′_IGHJ1*01	2276
  420	gi|Fabrus|VH3-23_IGHD1-20*01>2′_IGHJ1*01	2277
  421	gi|Fabrus|VH3-23_IGHD1-20*01>3′_IGHJ1*01	2278
  422	gi|Fabrus|VH3-23_IGHD1-26*01>1′_IGHJ1*01	2279
  423	gi|Fabrus|VH3-23_IGHD1-26*01>3′_IGHJ1*01	2280
  424	gi|Fabrus|VH3-23_IGHD2-2*01>1′_IGHJ1*01	2281
  425	gi|Fabrus|VH3-23_IGHD2-2*01>3′_IGHJ1*01	2282
  426	gi|Fabrus|VH3-23_IGHD2-8*01>1′_IGHJ1*01	2283
  427	gi|Fabrus|VH3-23_IGHD2-15*01>1′_IGHJ1*01	2284
  428	gi|Fabrus|VH3-23_IGHD2-15*01>3′_IGHJ1*01	2285
  429	gi|Fabrus|VH3-23_IGHD2-21*01>1′_IGHJ1*01	2286
  430	gi|Fabrus|VH3-23_IGHD2-21*01>3′_IGHJ1*01	2287
  431	gi|Fabrus|VH3-23_IGHD3-3*01>1′_IGHJ1*01	2288
  432	gi|Fabrus|VH3-23_IGHD3-3*01>3′_IGHJ1*01	2289
  433	gi|Fabrus|VH3-23_IGHD3-9*01>1′_IGHJ1*01	2290
  434	gi|Fabrus|VH3-23_IGHD3-9*01>3′_IGHJ1*01	2291
  435	gi|Fabrus|VH3-23_IGHD3-10*01>1′_IGHJ1*01	2292
  436	gi|Fabrus|VH3-23_IGHD3-10*01>3′_IGHJ1*01	2293
  437	gi|Fabrus|VH3-23_IGHD3-16*01>1′_IGHJ1*01	2294
  438	gi|Fabrus|VH3-23_IGHD3-16*01>3′_IGHJ1*01	2295
  439	gi|Fabrus|VH3-23_IGHD3-22*01>1′_IGHJ1*01	2296
  440	gi|Fabrus|VH3-23_IGHD4-4*01(1)>1′_IGHJ1*01	2297
  441	gi|Fabrus|VH3-23_IGHD4-4*01(1)>3′_IGHJ1*01	2298
  442	gi|Fabrus|VH3-23_IGHD4-11*01(1)>1′_IGHJ1*01	2299
  443	gi|Fabrus|VH3-23_IGHD4-11*01(1)>3′_IGHJ1*01	2300
  444	gi|Fabrus|VH3-23_IGHD4-17*01>1′_IGHJ1*01	2301
  445	gi|Fabrus|VH3-23_IGHD4-17*01>3′_IGHJ1*01	2302
  446	gi|Fabrus|VH3-23_IGHD4-23*01>1′_IGHJ1*01	2303
  447	gi|Fabrus|VH3-23_IGHD4-23*01>3′_IGHJ1*01	2304
  448	gi|Fabrus|VH3-23_IGHD5-5*01(2)>1′_IGHJ1*01	2305
  449	gi|Fabrus|VH3-23_IGHD5-5*01(2)>3′_IGHJ1*01	2306
  450	gi|Fabrus|VH3-23_IGHD5-12*01>1′_IGHJ1*01	2307
  451	gi|Fabrus|VH3-23_IGHD5-12*01>3′_IGHJ1*01	2308
  452	gi|Fabrus|VH3-23_IGHD5-18*01(2)>1′_IGHJ1*01	2309
  453	gi|Fabrus|VH3-23_IGHD5-18*01(2)>3′_IGHJ1*01	2310
  454	gi|Fabrus|VH3-23_IGHD5-24*01>1′_IGHJ1*01	2311
  455	gi|Fabrus|VH3-23_IGHD5-24*01>3′_IGHJ1*01	2312
  456	gi|Fabrus|VH3-23_IGHD6-6*01>1′_IGHJ1*01	2313
  457	gi|Fabrus|VH3-23_IGHD6-6*01>2′_IGHJ1*01	2314
  458	gi|Fabrus|VH3-23_IGHD6-6*01>3′_IGHJ1*01	2315
  459	gi|Fabrus|VH3-23_IGHD6-13*01>1′_IGHJ1*01	2316
  460	gi|Fabrus|VH3-23_IGHD6-13*01>2′_IGHJ1*01	2317
  461	gi|Fabrus|VH3-23_IGHD6-13*01>3′_IGHJ1*01	2318
  462	gi|Fabrus|VH3-23_IGHD6-19*01>1′_IGHJ1*01	2319
  463	gi|Fabrus|VH3-23_IGHD6-19*01>2′_IGHJ1*01	2320
  464	gi|Fabrus|VH3-23_IGHD6-19*01>3′_IGHJ1*01	2321
  465	gi|Fabrus|VH3-23_IGHD6-25*01>1′_IGHJ1*01	2322
  466	gi|Fabrus|VH3-23_IGHD6-25*01>3′_IGHJ1*01	2323
  467	gi|Fabrus|VH3-23_IGHD7-27*01>1′_IGHJ1*01	2324
  468	gi|Fabrus|VH3-23_IGHD7-27*01>2′_IGHJ1*01	2325
  469	gi|Fabrus|VH3-23_IGHD1-1*01>1_IGHJ2*01	2326
  470	gi|Fabrus|VH3-23_IGHD1-1*01>2_IGHJ2*01	2327
  471	gi|Fabrus|VH3-23_IGHD1-1*01>3_IGHJ2*01	2328
  472	gi|Fabrus|VH3-23_IGHD1-7*01>1_IGHJ2*01	2329
  473	gi|Fabrus|VH3-23_IGHD1-7*01>3_IGHJ2*01	2330
  474	gi|Fabrus|VH3-23_IGHD1-14*01>1_IGHJ2*01	2331
  475	gi|Fabrus|VH3-23_IGHD1-14*01>3_IGHJ2*01	2332
  476	gi|Fabrus|VH3-23_IGHD1-20*01>1_IGHJ2*01	2333
  477	gi|Fabrus|VH3-23_IGHD1-20*01>3_IGHJ2*01	2334
  478	gi|Fabrus|VH3-23_IGHD1-26*01>1_IGHJ2*01	2335
  479	gi|Fabrus|VH3-23_IGHD1-26*01>3_IGHJ2*01	2336
  480	gi|Fabrus|VH3-23_IGHD2-2*01>2_IGHJ2*01	2337
  481	gi|Fabrus|VH3-23_IGHD2-2*01>3_IGHJ2*01	2338
  482	gi|Fabrus|VH3-23_IGHD2-8*01>2_IGHJ2*01	2339
  483	gi|Fabrus|VH3-23_IGHD2-8*01>3_IGHJ2*01	2340
  484	gi|Fabrus|VH3-23_IGHD2-15*01>2_IGHJ2*01	2341
  485	gi|Fabrus|VH3-23_IGHD2-15*01>3_IGHJ2*01	2342
  486	gi|Fabrus|VH3-23_IGHD2-21*01>2_IGHJ2*01	2343
  487	gi|Fabrus|VH3-23_IGHD2-21*01>3_IGHJ2*01	2344
  488	gi|Fabrus|VH3-23_IGHD3-3*01>1_IGHJ2*01	2345
  489	gi|Fabrus|VH3-23_IGHD3-3*01>2_IGHJ2*01	2346
  490	gi|Fabrus|VH3-23_IGHD3-3*01>3_IGHJ2*01	2347
  491	gi|Fabrus|VH3-23_IGHD3-9*01>2_IGHJ2*01	2348
  492	gi|Fabrus|VH3-23_IGHD3-10*01>2_IGHJ2*01	2349
  493	gi|Fabrus|VH3-23_IGHD3-10*01>3_IGHJ2*01	2350
  494	gi|Fabrus|VH3-23_IGHD3-16*01>2_IGHJ2*01	2351
  495	gi|Fabrus|VH3-23_IGHD3-16*01>3_IGHJ2*01	2352
  496	gi|Fabrus|VH3-23_IGHD3-22*01>2_IGHJ2*01	2353
  497	gi|Fabrus|VH3-23_IGHD3-22*01>3_IGHJ2*01	2354
  498	gi|Fabrus|VH3-23_IGHD4-4*01(1)>2_IGHJ2*01	2355
  499	gi|Fabrus|VH3-23_IGHD4-4*01(1)>3_IGHJ2*01	2356
  500	gi|Fabrus|VH3-23_IGHD4-11*01(1)>2_IGHJ2*01	2357
  501	gi|Fabrus|VH3-23_IGHD4-11*01(1)>3_IGHJ2*01	2358
  502	gi|Fabrus|VH3-23_IGHD4-17*01>2_IGHJ2*01	2359
  503	gi|Fabrus|VH3-23_IGHD4-17*01>3_IGHJ2*01	2360
  504	gi|Fabrus|VH3-23_IGHD4-23*01>2_IGHJ2*01	2361
  505	gi|Fabrus|VH3-23_IGHD4-23*01>3_IGHJ2*01	2362
  506	gi|Fabrus|VH3-23_IGHD5-5*01(2)>1_IGHJ2*01	2363
  507	gi|Fabrus|VH3-23_IGHD5-5*01(2)>2_IGHJ2*01	2364
  508	gi|Fabrus|VH3-23_IGHD5-5*01(2)>3_IGHJ2*01	2365
  509	gi|Fabrus|VH3-23_IGHD5-12*01>1_IGHJ2*01	2366
  510	gi|Fabrus|VH3-23_IGHD5-12*01>3_IGHJ2*01	2367
  511	gi|Fabrus|VH3-23_IGHD5-18*01(2)>1_IGHJ2*01	2368
  512	gi|Fabrus|VH3-23_IGHD5-18*01(2)>2_IGHJ2*01	2369
  513	gi|Fabrus|VH3-23_IGHD5-18*01(2)>3_IGHJ2*01	2370
  514	gi|Fabrus|VH3-23_IGHD5-24*01>1_IGHJ2*01	2371
  515	gi|Fabrus|VH3-23_IGHD5-24*01>3_IGHJ2*01	2372
  516	gi|Fabrus|VH3-23_IGHD6-6*01>1_IGHJ2*01	2373
  517	gi|Fabrus|VH3-23_IGHD6-6*01>2_IGHJ2*01	2374
  518	gi|Fabrus|VH3-23_IGHD6-13*01>1_IGHJ2*01	2375
  519	gi|Fabrus|VH3-23_IGHD6-13*01>2_IGHJ2*01	2376
  520	gi|Fabrus|VH3-23_IGHD6-19*01>1_IGHJ2*01	2377
  521	gi|Fabrus|VH3-23_IGHD6-19*01>2_IGHJ2*01	2378
  522	gi|Fabrus|VH3-23_IGHD6-25*01>1_IGHJ2*01	2379
  523	gi|Fabrus|VH3-23_IGHD6-25*01>2_IGHJ2*01	2380
  524	gi|Fabrus|VH3-23_IGHD7-27*01>1_IGHJ2*01	2381
  525	gi|Fabrus|VH3-23_IGHD7-27*01>3_IGHJ2*01	2382
  526	gi|Fabrus|VH3-23_IGHD1-1*01>1′_IGHJ2*01	2383
  527	gi|Fabrus|VH3-23_IGHD1-1*01>2′_IGHJ2*01	2384
  528	gi|Fabrus|VH3-23_IGHD1-1*01>3′_IGHJ2*01	2385
  529	gi|Fabrus|VH3-23_IGHD1-7*01>1′_IGHJ2*01	2386
  530	gi|Fabrus|VH3-23_IGHD1-7*01>3′_IGHJ2*01	2387
  531	gi|Fabrus|VH3-23_IGHD1-14*01>1′_IGHJ2*01	2388
  532	gi|Fabrus|VH3-23_IGHD1-14*01>2′_IGHJ2*01	2389
  533	gi|Fabrus|VH3-23_IGHD1-14*01>3′_IGHJ2*01	2390
  534	gi|Fabrus|VH3-23_IGHD1-20*01>1′_IGHJ2*01	2391
  535	gi|Fabrus|VH3-23_IGHD1-20*01>2′_IGHJ2*01	2392
  536	gi|Fabrus|VH3-23_IGHD1-20*01>3′_IGHJ2*01	2393
  537	gi|Fabrus|VH3-23_IGHD1-26*01>1′_IGHJ2*01	2394
  538	gi|Fabrus|VH3-23_IGHD1-26*01>3′_IGHJ2*01	2395
  539	gi|Fabrus|VH3-23_IGHD2-2*01>1′_IGHJ2*01	2396
  540	gi|Fabrus|VH3-23_IGHD2-2*01>3′_IGHJ2*01	2397
  541	gi|Fabrus|VH3-23_IGHD2-8*01>1′_IGHJ2*01	2398
  542	gi|Fabrus|VH3-23_IGHD2-15*01>1′_IGHJ2*01	2399
  543	gi|Fabrus|VH3-23_IGHD2-15*01>3′_IGHJ2*01	2400
  544	gi|Fabrus|VH3-23_IGHD2-21*01>1′_IGHJ2*01	2401
  545	gi|Fabrus|VH3-23_IGHD2-21*01>3′_IGHJ2*01	2402
  546	gi|Fabrus|VH3-23_IGHD3-3*01>1′_IGHJ2*01	2403
  547	gi|Fabrus|VH3-23_IGHD3-3*01>3′_IGHJ2*01	2404
  548	gi|Fabrus|VH3-23_IGHD3-9*01>1′_IGHJ2*01	2405
  549	gi|Fabrus|VH3-23_IGHD3-9*01>3′_IGHJ2*01	2406
  550	gi|Fabrus|VH3-23_IGHD3-10*01>1′_IGHJ2*01	2407
  551	gi|Fabrus|VH3-23_IGHD3-10*01>3′_IGHJ2*01	2408
  552	gi|Fabrus|VH3-23_IGHD3-16*01>1′_IGHJ2*01	2409
  553	gi|Fabrus|VH3-23_IGHD3-16*01>3′_IGHJ2*01	2410
  554	gi|Fabrus|VH3-23_IGHD3-22*01>1′_IGHJ2*01	2411
  555	gi|Fabrus|VH3-23_IGHD4-4*01(1)>1′_IGHJ2*01	2412
  556	gi|Fabrus|VH3-23_IGHD4-4*01(1)>3′_IGHJ2*01	2413
  557	gi|Fabrus|VH3-23_IGHD4-11*01(1)>1′_IGHJ2*01	2414
  558	gi|Fabrus|VH3-23_IGHD4-11*01(1)>3′_IGHJ2*01	2415
  559	gi|Fabrus|VH3-23_IGHD4-17*01>1′_IGHJ2*01	2416
  560	gi|Fabrus|VH3-23_IGHD4-17*01>3′_IGHJ2*01	2417
  561	gi|Fabrus|VH3-23_IGHD4-23*01>1′_IGHJ2*01	2418
  562	gi|Fabrus|VH3-23_IGHD4-23*01>3′_IGHJ2*01	2419
  563	gi|Fabrus|VH3-23_IGHD5-5*01(2)>1′_IGHJ2*01	2420
  564	gi|Fabrus|VH3-23_IGHD5-5*01(2)>3′_IGHJ2*01	2421
  565	gi|Fabrus|VH3-23_IGHD5-12*01>1′_IGHJ2*01	2422
  566	gi|Fabrus|VH3-23_IGHD5-12*01>3′_IGHJ2*01	2423
  567	gi|Fabrus|VH3-23_IGHD5-18*01(2)>1′_IGHJ2*01	2424
  568	gi|Fabrus|VH3-23_IGHD5-18*01(2)>3′_IGHJ2*01	2425
  569	gi|Fabrus|VH3-23_IGHD5-24*01>1′_IGHJ2*01	2426
  570	gi|Fabrus|VH3-23_IGHD5-24*01>3′_IGHJ2*01	2427
  571	gi|Fabrus|VH3-23_IGHD6-6*01>1′_IGHJ2*01	2428
  572	gi|Fabrus|VH3-23_IGHD6-6*01>2′_IGHJ2*01	2429
  573	gi|Fabrus|VH3-23_IGHD6-6*01>3′_IGHJ2*01	2430
  574	gi|Fabrus|VH3-23_IGHD6-13*01>1′_IGHJ2*01	2431
  575	gi|Fabrus|VH3-23_IGHD6-13*01>2′_IGHJ2*01	2432
  576	gi|Fabrus|VH3-23_IGHD6-13*01>3′_IGHJ2*01	2433
  577	gi|Fabrus|VH3-23_IGHD6-19*01>1′_IGHJ2*01	2434
  578	gi|Fabrus|VH3-23_IGHD6-19*01>2′_IGHJ2*01	2435
  579	gi|Fabrus|VH3-23_IGHD6-19*01>3′_IGHJ2*01	2436
  580	gi|Fabrus|VH3-23_IGHD6-25*01>1′_IGHJ2*01	2437
  581	gi|Fabrus|VH3-23_IGHD6-25*01>3′_IGHJ2*01	2438
  582	gi|Fabrus|VH3-23_IGHD7-27*01>1′_IGHJ2*01	2439
  583	gi|Fabrus|VH3-23_IGHD7-27*01>2′_IGHJ2*01	2440
  584	gi|Fabrus|VH3-23_IGHD1-1*01>1_IGHJ3*01	2441
  585	gi|Fabrus|VH3-23_IGHD1-1*01>2_IGHJ3*01	2442
  586	gi|Fabrus|VH3-23_IGHD1-1*01>3_IGHJ3*01	2443
  587	gi|Fabrus|VH3-23_IGHD1-7*01>1_IGHJ3*01	2444
  588	gi|Fabrus|VH3-23_IGHD1-7*01>3_IGHJ3*01	2445
  589	gi|Fabrus|VH3-23_IGHD1-14*01>1_IGHJ3*01	2446
  590	gi|Fabrus|VH3-23_IGHD1-14*01>3_IGHJ3*01	2447
  591	gi|Fabrus|VH3-23_IGHD1-20*01>1_IGHJ3*01	2448
  592	gi|Fabrus|VH3-23_IGHD1-20*01>3_IGHJ3*01	2449
  593	gi|Fabrus|VH3-23_IGHD1-26*01>1_IGHJ3*01	2450
  594	gi|Fabrus|VH3-23_IGHD1-26*01>3_IGHJ3*01	2451
  595	gi|Fabrus|VH3-23_IGHD2-2*01>2_IGHJ3*01	2452
  596	gi|Fabrus|VH3-23_IGHD2-2*01>3_IGHJ3*01	2453
  597	gi|Fabrus|VH3-23_IGHD2-8*01>2_IGHJ3*01	2454
  598	gi|Fabrus|VH3-23_IGHD2-8*01>3_IGHJ3*01	2455
  599	gi|Fabrus|VH3-23_IGHD2-15*01>2_IGHJ3*01	2456
  600	gi|Fabrus|VH3-23_IGHD2-15*01>3_IGHJ3*01	2457
  601	gi|Fabrus|VH3-23_IGHD2-21*01>2_IGHJ3*01	2458
  602	gi|Fabrus|VH3-23_IGHD2-21*01>3_IGHJ3*01	2459
  603	gi|Fabrus|VH3-23_IGHD3-3*01>1_IGHJ3*01	2460
  604	gi|Fabrus|VH3-23_IGHD3-3*01>2_IGHJ3*01	2461
  605	gi|Fabrus|VH3-23_IGHD3-3*01>3_IGHJ3*01	2462
  606	gi|Fabrus|VH3-23_IGHD3-9*01>2_IGHJ3*01	2463
  607	gi|Fabrus|VH3-23_IGHD3-10*01>2_IGHJ3*01	2464
  608	gi|Fabrus|VH3-23_IGHD3-10*01>3_IGHJ3*01	2465
  609	gi|Fabrus|VH3-23_IGHD3-16*01>2_IGHJ3*01	2466
  610	gi|Fabrus|VH3-23_IGHD3-16*01>3_IGHJ3*01	2467
  611	gi|Fabrus|VH3-23_IGHD3-22*01>2_IGHJ3*01	2468
  612	gi|Fabrus|VH3-23_IGHD3-22*01>3_IGHJ3*01	2469
  613	gi|Fabrus|VH3-23_IGHD4-4*01(1)>2_IGHJ3*01	2470
  614	gi|Fabrus|VH3-23_IGHD4-4*01(1)>3_IGHJ3*01	2471
  615	gi|Fabrus|VH3-23_IGHD4-11*01(1)>2_IGHJ3*01	2472
  616	gi|Fabrus|VH3-23_IGHD4-11*01(1)>3_IGHJ3*01	2473
  617	gi|Fabrus|VH3-23_IGHD4-17*01>2_IGHJ3*01	2474
  618	gi|Fabrus|VH3-23_IGHD4-17*01>3_IGHJ3*01	2475
  619	gi|Fabrus|VH3-23_IGHD4-23*01>2_IGHJ3*01	2476
  620	gi|Fabrus|VH3-23_IGHD4-23*01>3_IGHJ3*01	2477
  621	gi|Fabrus|VH3-23_IGHD5-5*01(2)>1_IGHJ3*01	2478
  622	gi|Fabrus|VH3-23_IGHD5-5*01(2)>2_IGHJ3*01	2479
  623	gi|Fabrus|VH3-23_IGHD5-5*01(2)>3_IGHJ3*01	2480
  624	gi|Fabrus|VH3-23_IGHD5-12*01>1_IGHJ3*01	2481
  625	gi|Fabrus|VH3-23_IGHD5-12*01>3_IGHJ3*01	2482
  626	gi|Fabrus|VH3-23_IGHD5-18*01(2)>1_IGHJ3*01	2483
  627	gi|Fabrus|VH3-23_IGHD5-18*01(2)>2_IGHJ3*01	2484
  628	gi|Fabrus|VH3-23_IGHD5-18*01(2)>3_IGHJ3*01	2485
  629	gi|Fabrus|VH3-23_IGHD5-24*01>1_IGHJ3*01	2486
  630	gi|Fabrus|VH3-23_IGHD5-24*01>3_IGHJ3*01	2487
  631	gi|Fabrus|VH3-23_IGHD6-6*01>1_IGHJ3*01	2488
  632	gi|Fabrus|VH3-23_IGHD6-6*01>2_IGHJ3*01	2489
  633	gi|Fabrus|VH3-23_IGHD6-13*01>1_IGHJ3*01	2490
  634	gi|Fabrus|VH3-23_IGHD6-13*01>2_IGHJ3*01	2491
  635	gi|Fabrus|VH3-23_IGHD6-19*01>1_IGHJ3*01	2492
  636	gi|Fabrus|VH3-23_IGHD6-19*01>2_IGHJ3*01	2493
  637	gi|Fabrus|VH3-23_IGHD6-25*01>1_IGHJ3*01	2494
  638	gi|Fabrus|VH3-23_IGHD6-25*01>2_IGHJ3*01	2495
  639	gi|Fabrus|VH3-23_IGHD7-27*01>1_IGHJ3*01	2496
  640	gi|Fabrus|VH3-23_IGHD7-27*01>3_IGHJ3*01	2497
  641	gi|Fabrus|VH3-23_IGHD1-1*01>1′_IGHJ3*01	2498
  642	gi|Fabrus|VH3-23_IGHD1-1*01>2′_IGHJ3*01	2499
  643	gi|Fabrus|VH3-23_IGHD1-1*01>3′_IGHJ3*01	2500
  644	gi|Fabrus|VH3-23_IGHD1-7*01>1′_IGHJ3*01	2501
  645	gi|Fabrus|VH3-23_IGHD1-7*01>3′_IGHJ3*01	2502
  646	gi|Fabrus|VH3-23_IGHD1-14*01>1′_IGHJ3*01	2503
  647	gi|Fabrus|VH3-23_IGHD1-14*01>2′_IGHJ3*01	2504
  648	gi|Fabrus|VH3-23_IGHD1-14*01>3′_IGHJ3*01	2505
  649	gi|Fabrus|VH3-23_IGHD1-20*01>1′_IGHJ3*01	2506
  650	gi|Fabrus|VH3-23_IGHD1-20*01>2′_IGHJ3*01	2507
  651	gi|Fabrus|VH3-23_IGHD1-20*01>3′_IGHJ3*01	2508
  652	gi|Fabrus|VH3-23_IGHD1-26*01>1′_IGHJ3*01	2509
  653	gi|Fabrus|VH3-23_IGHD1-26*01>3′_IGHJ3*01	2510
  654	gi|Fabrus|VH3-23_IGHD2-2*01>1′_IGHJ3*01	2511
  655	gi|Fabrus|VH3-23_IGHD2-2*01>3′_IGHJ3*01	2512
  656	gi|Fabrus|VH3-23_IGHD2-8*01>1′_IGHJ3*01	2513
  657	gi|Fabrus|VH3-23_IGHD2-15*01>1′_IGHJ3*01	2514
  658	gi|Fabrus|VH3-23_IGHD2-15*01>3′_IGHJ3*01	2515
  659	gi|Fabrus|VH3-23_IGHD2-21*01>1′_IGHJ3*01	2516
  660	gi|Fabrus|VH3-23_IGHD2-21*01>3′_IGHJ3*01	2517
  661	gi|Fabrus|VH3-23_IGHD3-3*01>1′_IGHJ3*01	2518
  662	gi|Fabrus|VH3-23_IGHD3-3*01>3′_IGHJ3*01	2519
  663	gi|Fabrus|VH3-23_IGHD3-9*01>1′_IGHJ3*01	2520
  664	gi|Fabrus|VH3-23_IGHD3-9*01>3′_IGHJ3*01	2521
  665	gi|Fabrus|VH3-23_IGHD3-10*01>1′_IGHJ3*01	105
  666	gi|Fabrus|VH3-23_IGHD3-10*01>3′_IGHJ3*01	2522
  667	gi|Fabrus|VH3-23_IGHD3-16*01>1′_IGHJ3*01	2523
  668	gi|Fabrus|VH3-23_IGHD3-16*01>3′_IGHJ3*01	2524
  669	gi|Fabrus|VH3-23_IGHD3-22*01>1′_IGHJ3*01	2525
  670	gi|Fabrus|VH3-23_IGHD4-4*01(1)>1′_IGHJ3*01	2526
  671	gi|Fabrus|VH3-23_IGHD4-4*01(1)>3′_IGHJ3*01	2527
  672	gi|Fabrus|VH3-23_IGHD4-11*01(1)>1′_IGHJ3*01	2528
  673	gi|Fabrus|VH3-23_IGHD4-11*01(1)>3′_IGHJ3*01	2529
  674	gi|Fabrus|VH3-23_IGHD4-17*01>1′_IGHJ3*01	2530
  675	gi|Fabrus|VH3-23_IGHD4-17*01>3′_IGHJ3*01	2531
  676	gi|Fabrus|VH3-23_IGHD4-23*01>1′_IGHJ3*01	2532
  677	gi|Fabrus|VH3-23_IGHD4-23*01>3′_IGHJ3*01	2533
  678	gi|Fabrus|VH3-23_IGHD5-5*01(2)>1′_IGHJ3*01	2534
  679	gi|Fabrus|VH3-23_IGHD5-5*01(2)>3′_IGHJ3*01	2535
  680	gi|Fabrus|VH3-23_IGHD5-12*01>1′_IGHJ3*01	2536
  681	gi|Fabrus|VH3-23_IGHD5-12*01>3′_IGHJ3*01	2537
  682	gi|Fabrus|VH3-23_IGHD5-18*01(2)>1′_IGHJ3*01	2538
  683	gi|Fabrus|VH3-23_IGHD5-18*01(2)>3′_IGHJ3*01	2539
  684	gi|Fabrus|VH3-23_IGHD5-24*01>1′_IGHJ3*01	2540
  685	gi|Fabrus|VH3-23_IGHD5-24*01>3′_IGHJ3*01	2541
  686	gi|Fabrus|VH3-23_IGHD6-6*01>1′_IGHJ3*01	2542
  687	gi|Fabrus|VH3-23_IGHD6-6*01>2′_IGHJ3*01	2543
  688	gi|Fabrus|VH3-23_IGHD6-6*01>3′_IGHJ3*01	2544
  689	gi|Fabrus|VH3-23_IGHD6-13*01>1′_IGHJ3*01	2545
  690	gi|Fabrus|VH3-23_IGHD6-13*01>2′_IGHJ3*01	2546
  691	gi|Fabrus|VH3-23_IGHD6-13*01>3′_IGHJ3*01	2547
  692	gi|Fabrus|VH3-23_IGHD6-19*01>1′_IGHJ3*01	2548
  693	gi|Fabrus|VH3-23_IGHD6-19*01>2′_IGHJ3*01	2549
  694	gi|Fabrus|VH3-23_IGHD6-19*01>3′_IGHJ3*01	2550
  695	gi|Fabrus|VH3-23_IGHD6-25*01>1′_IGHJ3*01	2551
  696	gi|Fabrus|VH3-23_IGHD6-25*01>3′_IGHJ3*01	2552
  697	gi|Fabrus|VH3-23_IGHD7-27*01>1′_IGHJ3*01	2553
  698	gi|Fabrus|VH3-23_IGHD7-27*01>2′_IGHJ3*01	2554
  699	gi|Fabrus|VH3-23_IGHD1-1*01>1_IGHJ4*01	2555
  700	gi|Fabrus|VH3-23_IGHD1-1*01>2_IGHJ4*01	2556
  701	gi|Fabrus|VH3-23_IGHD1-1*01>3_IGHJ4*01	2557
  702	gi|Fabrus|VH3-23_IGHD1-7*01>1_IGHJ4*01	2558
  703	gi|Fabrus|VH3-23_IGHD1-7*01>3_IGHJ4*01	2559
  704	gi|Fabrus|VH3-23_IGHD1-14*01>1_IGHJ4*01	2560
  705	gi|Fabrus|VH3-23_IGHD1-14*01>3_IGHJ4*01	2561
  706	gi|Fabrus|VH3-23_IGHD1-20*01>1_IGHJ4*01	2562
  707	gi|Fabrus|VH3-23_IGHD1-20*01>3_IGHJ4*01	2563
  708	gi|Fabrus|VH3-23_IGHD1-26*01>1_IGHJ4*01	2564
  709	gi|Fabrus|VH3-23_IGHD1-26*01>3_IGHJ4*01	2565
  710	gi|Fabrus|VH3-23_IGHD2-2*01>2_IGHJ4*01	2566
  711	gi|Fabrus|VH3-23_IGHD2-2*01>3_IGHJ4*01	2567
  712	gi|Fabrus|VH3-23_IGHD2-8*01>2_IGHJ4*01	2568
  713	gi|Fabrus|VH3-23_IGHD2-8*01>3_IGHJ4*01	2569
  714	gi|Fabrus|VH3-23_IGHD2-15*01>2_IGHJ4*01	2570
  715	gi|Fabrus|VH3-23_IGHD2-15*01>3_IGHJ4*01	2571
  716	gi|Fabrus|VH3-23_IGHD2-21*01>2_IGHJ4*01	2572
  717	gi|Fabrus|VH3-23_IGHD2-21*01>3_IGHJ4*01	2573
  718	gi|Fabrus|VH3-23_IGHD3-3*01>1_IGHJ4*01	2574
  719	gi|Fabrus|VH3-23_IGHD3-3*01>2_IGHJ4*01	2575
  720	gi|Fabrus|VH3-23_IGHD3-3*01>3_IGHJ4*01	2576
  721	gi|Fabrus|VH3-23_IGHD3-9*01>2_IGHJ4*01	2577
  722	gi|Fabrus|VH3-23_IGHD3-10*01>2_IGHJ4*01	2578
  723	gi|Fabrus|VH3-23_IGHD3-10*01>3_IGHJ4*01	2579
  724	gi|Fabrus|VH3-23_IGHD3-16*01>2_IGHJ4*01	2580
  725	gi|Fabrus|VH3-23_IGHD3-16*01>3_IGHJ4*01	2581
  726	gi|Fabrus|VH3-23_IGHD3-22*01>2_IGHJ4*01	2582
  727	gi|Fabrus|VH3-23_IGHD3-22*01>3_IGHJ4*01	2583
  728	gi|Fabrus|VH3-23_IGHD4-4*01(1)>2_IGHJ4*01	2584
  729	gi|Fabrus|VH3-23_IGHD4-4*01(1)>3_IGHJ4*01	2585
  730	gi|Fabrus|VH3-23_IGHD4-11*01(1)>2_IGHJ4*01	2586
  731	gi|Fabrus|VH3-23_IGHD4-11*01(1)>3_IGHJ4*01	2587
  732	gi|Fabrus|VH3-23_IGHD4-17*01>2_IGHJ4*01	2588
  733	gi|Fabrus|VH3-23_IGHD4-17*01>3_IGHJ4*01	2589
  734	gi|Fabrus|VH3-23_IGHD4-23*01>2_IGHJ4*01	2590
  735	gi|Fabrus|VH3-23_IGHD4-23*01>3_IGHJ4*01	2591
  736	gi|Fabrus|VH3-23_IGHD5-5*01(2)>1_IGHJ4*01	2592
  737	gi|Fabrus|VH3-23_IGHD5-5*01(2)>2_IGHJ4*01	2593
  738	gi|Fabrus|VH3-23_IGHD5-5*01(2)>3_IGHJ4*01	2594
  739	gi|Fabrus|VH3-23_IGHD5-12*01>1_IGHJ4*01	2595
  740	gi|Fabrus|VH3-23_IGHD5-12*01>3_IGHJ4*01	2596
  741	gi|Fabrus|VH3-23_IGHD5-18*01(2)>1_IGHJ4*01	2597
  742	gi|Fabrus|VH3-23_IGHD5-18*01(2)>2_IGHJ4*01	2598
  743	gi|Fabrus|VH3-23_IGHD5-18*01(2)>3_IGHJ4*01	2599
  744	gi|Fabrus|VH3-23_IGHD5-24*01>1_IGHJ4*01	2600
  745	gi|Fabrus|VH3-23_IGHD5-24*01>3_IGHJ4*01	2601
  746	gi|Fabrus|VH3-23_IGHD6-6*01>1_IGHJ4*01	2602
  747	gi|Fabrus|VH3-23_IGHD6-6*01>2_IGHJ4*01	2603
  748	gi|Fabrus|VH3-23_IGHD6-13*01>1_IGHJ4*01	2604
  749	gi|Fabrus|VH3-23_IGHD6-13*01>2_IGHJ4*01	2605
  750	gi|Fabrus|VH3-23_IGHD6-19*01>1_IGHJ4*01	2606
  751	gi|Fabrus|VH3-23_IGHD6-19*01>2_IGHJ4*01	2607
  752	gi|Fabrus|VH3-23_IGHD6-25*01>1_IGHJ4*01	2608
  753	gi|Fabrus|VH3-23_IGHD6-25*01>2_IGHJ4*01	2609
  754	gi|Fabrus|VH3-23_IGHD7-27*01>1_IGHJ4*01	2610
  755	gi|Fabrus|VH3-23_IGHD7-27*01>3_IGHJ4*01	2611
  756	gi|Fabrus|VH3-23_IGHD1-1*01>1′_IGHJ4*01	2612
  757	gi|Fabrus|VH3-23_IGHD1-1*01>2′_IGHJ4*01	2613
  758	gi|Fabrus|VH3-23_IGHD1-1*01>3′_IGHJ4*01	2614
  759	gi|Fabrus|VH3-23_IGHD1-7*01>1′_IGHJ4*01	2615
  760	gi|Fabrus|VH3-23_IGHD1-7*01>3′_IGHJ4*01	2616
  761	gi|Fabrus|VH3-23_IGHD1-14*01>1′_IGHJ4*01	2617
  762	gi|Fabrus|VH3-23_IGHD1-14*01>2′_IGHJ4*01	2618
  763	gi|Fabrus|VH3-23_IGHD1-14*01>3′_IGHJ4*01	2619
  764	gi|Fabrus|VH3-23_IGHD1-20*01>1′_IGHJ4*01	2620
  765	gi|Fabrus|VH3-23_IGHD1-20*01>2′_IGHJ4*01	2621
  766	gi|Fabrus|VH3-23_IGHD1-20*01>3′_IGHJ4*01	2622
  767	gi|Fabrus|VH3-23_IGHD1-26*01>1′_IGHJ4*01	2623
  768	gi|Fabrus|VH3-23_IGHD1-26*01>3′_IGHJ4*01	2624
  769	gi|Fabrus|VH3-23_IGHD2-2*01>1′_IGHJ4*01	2625
  770	gi|Fabrus|VH3-23_IGHD2-2*01>3′_IGHJ4*01	2626
  771	gi|Fabrus|VH3-23_IGHD2-8*01>1′_IGHJ4*01	2627
  772	gi|Fabrus|VH3-23_IGHD2-15*01>1′_IGHJ4*01	2628
  773	gi|Fabrus|VH3-23_IGHD2-15*01>3′_IGHJ4*01	2629
  774	gi|Fabrus|VH3-23_IGHD2-21*01>1′_IGHJ4*01	2630
  775	gi|Fabrus|VH3-23_IGHD2-21*01>3′_IGHJ4*01	2631
  776	gi|Fabrus|VH3-23_IGHD3-3*01>1′_IGHJ4*01	2632
  777	gi|Fabrus|VH3-23_IGHD3-3*01>3′_IGHJ4*01	2633
  778	gi|Fabrus|VH3-23_IGHD3-9*01>1′_IGHJ4*01	2634
  779	gi|Fabrus|VH3-23_IGHD3-9*01>3′_IGHJ4*01	2635
  780	gi|Fabrus|VH3-23_IGHD3-10*01>1′_IGHJ4*01	2636
  781	gi|Fabrus|VH3-23_IGHD3-10*01>3′_IGHJ4*01	2637
  782	gi|Fabrus|VH3-23_IGHD3-16*01>1′_IGHJ4*01	2638
  783	gi|Fabrus|VH3-23_IGHD3-16*01>3′_IGHJ4*01	2639
  784	gi|Fabrus|VH3-23_IGHD3-22*01>1′_IGHJ4*01	2640
  785	gi|Fabrus|VH3-23_IGHD4-4*01(1)>1′_IGHJ4*01	2641
  786	gi|Fabrus|VH3-23_IGHD4-4*01(1)>3′_IGHJ4*01	2642
  787	gi|Fabrus|VH3-23_IGHD4-11*01(1)>1′_IGHJ4*01	2643
  788	gi|Fabrus|VH3-23_IGHD4-11*01(1)>3′_IGHJ4*01	2644
  789	gi|Fabrus|VH3-23_IGHD4-17*01>1′_IGHJ4*01	2645
  790	gi|Fabrus|VH3-23_IGHD4-17*01>3′_IGHJ4*01	2646
  791	gi|Fabrus|VH3-23_IGHD4-23*01>1′_IGHJ4*01	2647
  792	gi|Fabrus|VH3-23_IGHD4-23*01>3′_IGHJ4*01	2648
  793	gi|Fabrus|VH3-23_IGHD5-5*01(2)>1′_IGHJ4*01	2649
  794	gi|Fabrus|VH3-23_IGHD5-5*01(2)>3′_IGHJ4*01	2650
  795	gi|Fabrus|VH3-23_IGHD5-12*01>1′_IGHJ4*01	2651
  796	gi|Fabrus|VH3-23_IGHD5-12*01>3′_IGHJ4*01	2652
  797	gi|Fabrus|VH3-23_IGHD5-18*01(2)>1′_IGHJ4*01	2653
  798	gi|Fabrus|VH3-23_IGHD5-18*01(2)>3′_IGHJ4*01	2654
  799	gi|Fabrus|VH3-23_IGHD5-24*01>1′_IGHJ4*01	2655
  800	gi|Fabrus|VH3-23_IGHD5-24*01>3′_IGHJ4*01	2656
  801	gi|Fabrus|VH3-23_IGHD6-6*01>1′_IGHJ4*01	2657
  802	gi|Fabrus|VH3-23_IGHD6-6*01>2′_IGHJ4*01	2658
  803	gi|Fabrus|VH3-23_IGHD6-6*01>3′_IGHJ4*01	2659
  804	gi|Fabrus|VH3-23_IGHD6-13*01>1′_IGHJ4*01	2660
  805	gi|Fabrus|VH3-23_IGHD6-13*01>2′_IGHJ4*01	2661
  806	gi|Fabrus|VH3-23_IGHD6-13*01>3′_IGHJ4*01	2662
  807	gi|Fabrus|VH3-23_IGHD6-19*01>1′_IGHJ4*01	2663
  808	gi|Fabrus|VH3-23_IGHD6-19*01>2′_IGHJ4*01	2664
  809	gi|Fabrus|VH3-23_IGHD6-19*01>3′_IGHJ4*01	2665
  810	gi|Fabrus|VH3-23_IGHD6-25*01>1′_IGHJ4*01	2666
  811	gi|Fabrus|VH3-23_IGHD6-25*01>3′_IGHJ4*01	2667
  812	gi|Fabrus|VH3-23_IGHD7-27*01>1′_IGHJ4*01	2668
  813	gi|Fabrus|VH3-23_IGHD7-27*01>2′_IGHJ4*01	2669
  814	gi|Fabrus|VH3-23_IGHD1-1*01>1_IGHJ5*01	2670
  815	gi|Fabrus|VH3-23_IGHD1-1*01>2_IGHJ5*01	2671
  816	gi|Fabrus|VH3-23_IGHD1-1*01>3_IGHJ5*01	2672
  817	gi|Fabrus|VH3-23_IGHD1-7*01>1_IGHJ5*01	2673
  818	gi|Fabrus|VH3-23_IGHD1-7*01>3_IGHJ5*01	2674
  819	gi|Fabrus|VH3-23_IGHD1-14*01>1_IGHJ5*01	2675
  820	gi|Fabrus|VH3-23_IGHD1-14*01>3_IGHJ5*01	2676
  821	gi|Fabrus|VH3-23_IGHD1-20*01>1_IGHJ5*01	2677
  822	gi|Fabrus|VH3-23_IGHD1-20*01>3_IGHJ5*01	2678
  823	gi|Fabrus|VH3-23_IGHD1-26*01>1_IGHJ5*01	2679
  824	gi|Fabrus|VH3-23_IGHD1-26*01>3_IGHJ5*01	2680
  825	gi|Fabrus|VH3-23_IGHD2-2*01>2_IGHJ5*01	2681
  826	gi|Fabrus|VH3-23_IGHD2-2*01>3_IGHJ5*01	2682
  827	gi|Fabrus|VH3-23_IGHD2-8*01>2_IGHJ5*01	2683
  828	gi|Fabrus|VH3-23_IGHD2-8*01>3_IGHJ5*01	2684
  829	gi|Fabrus|VH3-23_IGHD2-15*01>2_IGHJ5*01	2685
  830	gi|Fabrus|VH3-23_IGHD2-15*01>3_IGHJ5*01	2686
  831	gi|Fabrus|VH3-23_IGHD2-21*01>2_IGHJ5*01	2687
  832	gi|Fabrus|VH3-23_IGHD2-21*01>3_IGHJ5*01	2688
  833	gi|Fabrus|VH3-23_IGHD3-3*01>1_IGHJ5*01	2689
  834	gi|Fabrus|VH3-23_IGHD3-3*01>2_IGHJ5*01	2690
  835	gi|Fabrus|VH3-23_IGHD3-3*01>3_IGHJ5*01	2691
  836	gi|Fabrus|VH3-23_IGHD3-9*01>2_IGHJ5*01	2692
  837	gi|Fabrus|VH3-23_IGHD3-10*01>2_IGHJ5*01	2693
  838	gi|Fabrus|VH3-23_IGHD3-10*01>3_IGHJ5*01	2694
  839	gi|Fabrus|VH3-23_IGHD3-16*01>2_IGHJ5*01	2695
  840	gi|Fabrus|VH3-23_IGHD3-16*01>3_IGHJ5*01	2696
  841	gi|Fabrus|VH3-23_IGHD3-22*01>2_IGHJ5*01	2697
  842	gi|Fabrus|VH3-23_IGHD3-22*01>3_IGHJ5*01	2698
  843	gi|Fabrus|VH3-23_IGHD4-4*01(1)>2_IGHJ5*01	2699
  844	gi|Fabrus|VH3-23_IGHD4-4*01(1)>3_IGHJ5*01	2700
  845	gi|Fabrus|VH3-23_IGHD4-11*01(1)>2_IGHJ5*01	2701
  846	gi|Fabrus|VH3-23_IGHD4-11*01(1)>3_IGHJ5*01	2702
  847	gi|Fabrus|VH3-23_IGHD4-17*01>2_IGHJ5*01	2703
  848	gi|Fabrus|VH3-23_IGHD4-17*01>3_IGHJ5*01	2704
  849	gi|Fabrus|VH3-23_IGHD4-23*01>2_IGHJ5*01	2705
  850	gi|Fabrus|VH3-23_IGHD4-23*01>3_IGHJ5*01	2706
  851	gi|Fabrus|VH3-23_IGHD5-5*01(2)>1_IGHJ5*01	2707
  852	gi|Fabrus|VH3-23_IGHD5-5*01(2)>2_IGHJ5*01	2708
  853	gi|Fabrus|VH3-23_IGHD5-5*01(2)>3_IGHJ5*01	2709
  854	gi|Fabrus|VH3-23_IGHD5-12*01>1_IGHJ5*01	2710
  855	gi|Fabrus|VH3-23_IGHD5-12*01>3_IGHJ5*01	2711
  856	gi|Fabrus|VH3-23_IGHD5-18*01(2)>1_IGHJ5*01	2712
  857	gi|Fabrus|VH3-23_IGHD5-18*01(2)>2_IGHJ5*01	2713
  858	gi|Fabrus|VH3-23_IGHD5-18*01(2)>3_IGHJ5*01	2714
  859	gi|Fabrus|VH3-23_IGHD5-24*01>1_IGHJ5*01	2715
  860	gi|Fabrus|VH3-23_IGHD5-24*01>3_IGHJ5*01	2716
  861	gi|Fabrus|VH3-23_IGHD6-6*01>1_IGHJ5*01	2717
  862	gi|Fabrus|VH3-23_IGHD6-6*01>2_IGHJ5*01	2718
  863	gi|Fabrus|VH3-23_IGHD6-13*01>1_IGHJ5*01	2719
  864	gi|Fabrus|VH3-23_IGHD6-13*01>2_IGHJ5*01	2720
  865	gi|Fabrus|VH3-23_IGHD6-19*01>1_IGHJ5*01	2721
  866	gi|Fabrus|VH3-23_IGHD6-19*01>2_IGHJ5*01	2722
  867	gi|Fabrus|VH3-23_IGHD6-25*01>1_IGHJ5*01	2723
  868	gi|Fabrus|VH3-23_IGHD6-25*01>2_IGHJ5*01	2724
  869	gi|Fabrus|VH3-23_IGHD7-27*01>1_IGHJ5*01	2725
  870	gi|Fabrus|VH3-23_IGHD7-27*01>3_IGHJ5*01	2726
  871	gi|Fabrus|VH3-23_IGHD1-1*01>1′_IGHJ5*01	2727
  872	gi|Fabrus|VH3-23_IGHD1-1*01>2′_IGHJ5*01	2728
  873	gi|Fabrus|VH3-23_IGHD1-1*01>3′_IGHJ5*01	2729
  874	gi|Fabrus|VH3-23_IGHD1-7*01>1′_IGHJ5*01	2730
  875	gi|Fabrus|VH3-23_IGHD1-7*01>3′_IGHJ5*01	2731
  876	gi|Fabrus|VH3-23_IGHD1-14*01>1′_IGHJ5*01	2732
  877	gi|Fabrus|VH3-23_IGHD1-14*01>2′_IGHJ5*01	2733
  878	gi|Fabrus|VH3-23_IGHD1-14*01>3′_IGHJ5*01	2734
  879	gi|Fabrus|VH3-23_IGHD1-20*01>1′_IGHJ5*01	2735
  880	gi|Fabrus|VH3-23_IGHD1-20*01>2′_IGHJ5*01	2736
  881	gi|Fabrus|VH3-23_IGHD1-20*01>3′_IGHJ5*01	2737
  882	gi|Fabrus|VH3-23_IGHD1-26*01>1′_IGHJ5*01	2738
  883	gi|Fabrus|VH3-23_IGHD1-26*01>3′_IGHJ5*01	2739
  884	gi|Fabrus|VH3-23_IGHD2-2*01>1′_IGHJ5*01	2740
  885	gi|Fabrus|VH3-23_IGHD2-2*01>3′_IGHJ5*01	2741
  886	gi|Fabrus|VH3-23_IGHD2-8*01>1′_IGHJ5*01	2742
  887	gi|Fabrus|VH3-23_IGHD2-15*01>1′_IGHJ5*01	2743
  888	gi|Fabrus|VH3-23_IGHD2-15*01>3′_IGHJ5*01	2744
  889	gi|Fabrus|VH3-23_IGHD2-21*01>1′_IGHJ5*01	2745
  890	gi|Fabrus|VH3-23_IGHD2-21*01>3′_IGHJ5*01	2746
  891	gi|Fabrus|VH3-23_IGHD3-3*01>1′_IGHJ5*01	2747
  892	gi|Fabrus|VH3-23_IGHD3-3*01>3′_IGHJ5*01	2748
  893	gi|Fabrus|VH3-23_IGHD3-9*01>1′_IGHJ5*01	2749
  894	gi|Fabrus|VH3-23_IGHD3-9*01>3′_IGHJ5*01	2750
  895	gi|Fabrus|VH3-23_IGHD3-10*01>1′_IGHJ5*01	2751
  896	gi|Fabrus|VH3-23_IGHD3-10*01>3′_IGHJ5*01	2752
  897	gi|Fabrus|VH3-23_IGHD3-16*01>1′_IGHJ5*01	2753
  898	gi|Fabrus|VH3-23_IGHD3-16*01>3′_IGHJ5*01	2754
  899	gi|Fabrus|VH3-23_IGHD3-22*01>1′_IGHJ5*01	2755
  900	gi|Fabrus|VH3-23_IGHD4-4*01(1)>1′_IGHJ5*01	2756
  901	gi|Fabrus|VH3-23_IGHD4-4*01(1)>3′_IGHJ5*01	2757
  902	gi|Fabrus|VH3-23_IGHD4-11*01(1)>1′_IGHJ5*01	2758
  903	gi|Fabrus|VH3-23_IGHD4-11*01(1)>3′_IGHJ5*01	2759
  904	gi|Fabrus|VH3-23_IGHD4-17*01>1′_IGHJ5*01	2760
  905	gi|Fabrus|VH3-23_IGHD4-17*01>3′_IGHJ5*01	2761
  906	gi|Fabrus|VH3-23_IGHD4-23*01>1′_IGHJ5*01	2762
  907	gi|Fabrus|VH3-23_IGHD4-23*01>3′_IGHJ5*01	2763
  908	gi|Fabrus|VH3-23_IGHD5-5*01(2)>1′_IGHJ5*01	2764
  909	gi|Fabrus|VH3-23_IGHD5-5*01(2)>3′_IGHJ5*01	2765
  910	gi|Fabrus|VH3-23_IGHD5-12*01>1′_IGHJ5*01	2766
  911	gi|Fabrus|VH3-23_IGHD5-12*01>3′_IGHJ5*01	2767
  912	gi|Fabrus|VH3-23_IGHD5-18*01(2)>1′_IGHJ5*01	2768
  913	gi|Fabrus|VH3-23_IGHD5-18*01(2)>3′_IGHJ5*01	2769
  914	gi|Fabrus|VH3-23_IGHD5-24*01>1′_IGHJ5*01	2770
  915	gi|Fabrus|VH3-23_IGHD5-24*01>3′_IGHJ5*01	2771
  916	gi|Fabrus|VH3-23_IGHD6-6*01>1′_IGHJ5*01	2772
  917	gi|Fabrus|VH3-23_IGHD6-6*01>2′_IGHJ5*01	2773
  918	gi|Fabrus|VH3-23_IGHD6-6*01>3′_IGHJ5*01	2774
  919	gi|Fabrus|VH3-23_IGHD6-13*01>1′_IGHJ5*01	2775
  920	gi|Fabrus|VH3-23_IGHD6-13*01>2′_IGHJ5*01	2776
  921	gi|Fabrus|VH3-23_IGHD6-13*01>3′_IGHJ5*01	2777
  922	gi|Fabrus|VH3-23_IGHD6-19*01>1′_IGHJ5*01	2778
  923	gi|Fabrus|VH3-23_IGHD6-19*01>2′_IGHJ5*01	2779
  924	gi|Fabrus|VH3-23_IGHD6-19*01>3′_IGHJ5*01	2780
  925	gi|Fabrus|VH3-23_IGHD6-25*01>1′_IGHJ5*01	2781
  926	gi|Fabrus|VH3-23_IGHD6-25*01>3′_IGHJ5*01	2782
  927	gi|Fabrus|VH3-23_IGHD7-27*01>1′_IGHJ5*01	2783
  928	gi|Fabrus|VH3-23_IGHD7-27*01>2′_IGHJ5*01	2784
  929	gi|Fabrus|VH3-23_IGHD1-1*01>1_IGHJ6*01	2785
  930	gi|Fabrus|VH3-23_IGHD1-1*01>2_IGHJ6*01	2786
  931	gi|Fabrus|VH3-23_IGHD1-1*01>3_IGHJ6*01	2787
  932	gi|Fabrus|VH3-23_IGHD1-7*01>1_IGHJ6*01	2788
  933	gi|Fabrus|VH3-23_IGHD1-7*01>3_IGHJ6*01	2789
  934	gi|Fabrus|VH3-23_IGHD1-14*01>1_IGHJ6*01	2790
  935	gi|Fabrus|VH3-23_IGHD1-14*01>3_IGHJ6*01	2791
  936	gi|Fabrus|VH3-23_IGHD1-20*01>1_IGHJ6*01	2792
  937	gi|Fabrus|VH3-23_IGHD1-20*01>3_IGHJ6*01	2793
  938	gi|Fabrus|VH3-23_IGHD1-26*01>1_IGHJ6*01	2794
  939	gi|Fabrus|VH3-23_IGHD1-26*01>3_IGHJ6*01	2795
  940	gi|Fabrus|VH3-23_IGHD2-2*01>2_IGHJ6*01	2796
  941	gi|Fabrus|VH3-23_IGHD2-2*01>3_IGHJ6*01	2797
  942	gi|Fabrus|VH3-23_IGHD2-8*01>2_IGHJ6*01	2798
  943	gi|Fabrus|VH3-23_IGHD2-8*01>3_IGHJ6*01	2799
  944	gi|Fabrus|VH3-23_IGHD2-15*01>2_IGHJ6*01	2800
  945	gi|Fabrus|VH3-23_IGHD2-15*01>3_IGHJ6*01	2801
  946	gi|Fabrus|VH3-23_IGHD2-21*01>2_IGHJ6*01	2802
  947	gi|Fabrus|VH3-23_IGHD2-21*01>3_IGHJ6*01	2803
  948	gi|Fabrus|VH3-23_IGHD3-3*01>1_IGHJ6*01	2804
  949	gi|Fabrus|VH3-23_IGHD3-3*01>2_IGHJ6*01	2805
  950	gi|Fabrus|VH3-23_IGHD3-3*01>3_IGHJ6*01	2806
  951	gi|Fabrus|VH3-23_IGHD3-9*01>2_IGHJ6*01	2807
  952	gi|Fabrus|VH3-23_IGHD3-10*01>2_IGHJ6*01	2808
  953	gi|Fabrus|VH3-23_IGHD3-10*01>3_IGHJ6*01	104
  954	gi|Fabrus|VH3-23_IGHD3-16*01>2_IGHJ6*01	2809
  955	gi|Fabrus|VH3-23_IGHD3-16*01>3_IGHJ6*01	2810
  956	gi|Fabrus|VH3-23_IGHD3-22*01>2_IGHJ6*01	2811
  957	gi|Fabrus|VH3-23_IGHD3-22*01>3_IGHJ6*01	2812
  958	gi|Fabrus|VH3-23_IGHD4-4*01(1)>2_IGHJ6*01	2813
  959	gi|Fabrus|VH3-23_IGHD4-4*01(1)>3_IGHJ6*01	2814
  960	gi|Fabrus|VH3-23_IGHD4-11*01(1)>2_IGHJ6*01	2815
  961	gi|Fabrus|VH3-23_IGHD4-11*01(1)>3_IGHJ6*01	2816
  962	gi|Fabrus|VH3-23_IGHD4-17*01>2_IGHJ6*01	2817
  963	gi|Fabrus|VH3-23_IGHD4-17*01>3_IGHJ6*01	2818
  964	gi|Fabrus|VH3-23_IGHD4-23*01>2_IGHJ6*01	2819
  965	gi|Fabrus|VH3-23_IGHD4-23*01>3_IGHJ6*01	2820
  966	gi|Fabrus|VH3-23_IGHD5-5*01(2)>1_IGHJ6*01	2821
  967	gi|Fabrus|VH3-23_IGHD5-5*01(2)>2_IGHJ6*01	2822
  968	gi|Fabrus|VH3-23_IGHD5-5*01(2)>3_IGHJ6*01	2823
  969	gi|Fabrus|VH3-23_IGHD5-12*01>1_IGHJ6*01	2824
  970	gi|Fabrus|VH3-23_IGHD5-12*01>3_IGHJ6*01	2825
  971	gi|Fabrus|VH3-23_IGHD5-18*01(2)>1_IGHJ6*01	2826
  972	gi|Fabrus|VH3-23_IGHD5-18*01(2)>2_IGHJ6*01	2827
  973	gi|Fabrus|VH3-23_IGHD5-18*01(2)>3_IGHJ6*01	2828
  974	gi|Fabrus|VH3-23_IGHD5-24*01>1_IGHJ6*01	2829
  975	gi|Fabrus|VH3-23_IGHD5-24*01>3_IGHJ6*01	2830
  976	gi|Fabrus|VH3-23_IGHD6-6*01>1_IGHJ6*01	2831
  977	gi|Fabrus|VH3-23_IGHD6-6*01>2_IGHJ6*01	2832
  978	gi|Fabrus|VH3-23_IGHD6-13*01>1_IGHJ6*01	2833
  979	gi|Fabrus|VH3-23_IGHD6-13*01>2_IGHJ6*01	2834
  980	gi|Fabrus|VH3-23_IGHD6-19*01>1_IGHJ6*01	2835
  981	gi|Fabrus|VH3-23_IGHD6-19*01>2_IGHJ6*01	2836
  982	gi|Fabrus|VH3-23_IGHD6-25*01>1_IGHJ6*01	2837
  983	gi|Fabrus|VH3-23_IGHD6-25*01>2_IGHJ6*01	2838
  984	gi|Fabrus|VH3-23_IGHD7-27*01>1_IGHJ6*01	2839
  985	gi|Fabrus|VH3-23_IGHD7-27*01>3_IGHJ6*01	2840
  986	gi|Fabrus|VH3-23_IGHD1-1*01>1′_IGHJ6*01	2841
  987	gi|Fabrus|VH3-23_IGHD1-1*01>2′_IGHJ6*01	2842
  988	gi|Fabrus|VH3-23_IGHD1-1*01>3′_IGHJ6*01	2843
  989	gi|Fabrus|VH3-23_IGHD1-7*01>1′_IGHJ6*01	2844
  990	gi|Fabrus|VH3-23_IGHD1-7*01>3′_IGHJ6*01	2845
  991	gi|Fabrus|VH3-23_IGHD1-14*01>1′_IGHJ6*01	2846
  992	gi|Fabrus|VH3-23_IGHD1-14*01>2′_IGHJ6*01	2847
  993	gi|Fabrus|VH3-23_IGHD1-14*01>3′_IGHJ6*01	2848
  994	gi|Fabrus|VH3-23_IGHD1-20*01>1′_IGHJ6*01	2849
  995	gi|Fabrus|VH3-23_IGHD1-20*01>2′_IGHJ6*01	2850
  996	gi|Fabrus|VH3-23_IGHD1-20*01>3′_IGHJ6*01	2851
  997	gi|Fabrus|VH3-23_IGHD1-26*01>1′_IGHJ6*01	2852
  998	gi|Fabrus|VH3-23_IGHD1-26*01>3′_IGHJ6*01	2853
  999	gi|Fabrus|VH3-23_IGHD2-2*01>1′_IGHJ6*01	2854
  1000	gi|Fabrus|VH3-23_IGHD2-2*01>3′_IGHJ6*01	2855
  1001	gi|Fabrus|VH3-23_IGHD2-8*01>1′_IGHJ6*01	2856
  1002	gi|Fabrus|VH3-23_IGHD2-15*01>1′_IGHJ6*01	2857
  1003	gi|Fabrus|VH3-23_IGHD2-15*01>3′_IGHJ6*01	2858
  1004	gi|Fabrus|VH3-23_IGHD2-21*01>1′_IGHJ6*01	2859
  1005	gi|Fabrus|VH3-23_IGHD2-21*01>3′_IGHJ6*01	2860
  1006	gi|Fabrus|VH3-23_IGHD3-3*01>1′_IGHJ6*01	2861
  1007	gi|Fabrus|VH3-23_IGHD3-3*01>3′_IGHJ6*01	2862
  1008	gi|Fabrus|VH3-23_IGHD3-9*01>1′_IGHJ6*01	2863
  1009	gi|Fabrus|VH3-23_IGHD3-9*01>3′_IGHJ6*01	2864
  1010	gi|Fabrus|VH3-23_IGHD3-10*01>1′_IGHJ6*01	2865
  1011	gi|Fabrus|VH3-23_IGHD3-10*01>3′_IGHJ6*01	2866
  1012	gi|Fabrus|VH3-23_IGHD3-16*01>1′_IGHJ6*01	2867
  1013	gi|Fabrus|VH3-23_IGHD3-16*01>3′_IGHJ6*01	2868
  1014	gi|Fabrus|VH3-23_IGHD3-22*01>1′_IGHJ6*01	2869
  1015	gi|Fabrus|VH3-23_IGHD4-4*01(1)>1′_IGHJ6*01	2870
  1016	gi|Fabrus|VH3-23_IGHD4-4*01(1)>3′_IGHJ6*01	2871
  1017	gi|Fabrus|VH3-23_IGHD4-11*01(1)>1′_IGHJ6*01	2872
  1018	gi|Fabrus|VH3-23_IGHD4-11*01(1)>3′_IGHJ6*01	2873
  1019	gi|Fabrus|VH3-23_IGHD4-17*01>1′_IGHJ6*01	2874
  1020	gi|Fabrus|VH3-23_IGHD4-17*01>3′_IGHJ6*01	2875
  1021	gi|Fabrus|VH3-23_IGHD4-23*01>1′_IGHJ6*01	2876
  1022	gi|Fabrus|VH3-23_IGHD4-23*01>3′_IGHJ6*01	2877
  1023	gi|Fabrus|VH3-23_IGHD5-5*01(2)>1′_IGHJ6*01	2878
  1024	gi|Fabrus|VH3-23_IGHD5-5*01(2)>3′_IGHJ6*01	2879
  1025	gi|Fabrus|VH3-23_IGHD5-12*01>1′_IGHJ6*01	2880
  1026	gi|Fabrus|VH3-23_IGHD5-12*01>3′_IGHJ6*01	2881
  1027	gi|Fabrus|VH3-23_IGHD5-18*01(2)>1′_IGHJ6*01	2882
  1028	gi|Fabrus|VH3-23_IGHD5-18*01(2)>3′_IGHJ6*01	2883
  1029	gi|Fabrus|VH3-23_IGHD5-24*01>1′_IGHJ6*01	2884
  1030	gi|Fabrus|VH3-23_IGHD5-24*01>3′_IGHJ6*01	2885
  1031	gi|Fabrus|VH3-23_IGHD6-6*01>1′_IGHJ6*01	2886
  1032	gi|Fabrus|VH3-23_IGHD6-6*01>2′_IGHJ6*01	2887
  1033	gi|Fabrus|VH3-23_IGHD6-6*01>3′_IGHJ6*01	2888
  1034	gi|Fabrus|VH3-23_IGHD6-13*01>1′_IGHJ6*01	2889
  1035	gi|Fabrus|VH3-23_IGHD6-13*01>2′_IGHJ6*01	2890
  1036	gi|Fabrus|VH3-23_IGHD6-13*01>3′_IGHJ6*01	2891
  1037	gi|Fabrus|VH3-23_IGHD6-19*01>1′_IGHJ6*01	2892
  1038	gi|Fabrus|VH3-23_IGHD6-19*01>2′_IGHJ6*01	2893
  1039	gi|Fabrus|VH3-23_IGHD6-19*01>3′_IGHJ6*01	2894
  1040	gi|Fabrus|VH3-23_IGHD6-25*01>1′_IGHJ6*01	2895
  1041	gi|Fabrus|VH3-23_IGHD6-25*01>3′_IGHJ6*01	2896
  1042	gi|Fabrus|VH3-23_IGHD7-27*01>1′_IGHJ6*01	2897
  1043	gi|Fabrus|VH3-23_IGHD7-27*01>2′_IGHJ6*01	2898

  Light Chains

  1	gnl|Fabrus|A14_IGKJ1*01	2163
  2	gnl|Fabrus|A17_IGKJ1*01	113
  3	gnl|Fabrus|A2_IGKJ1*01	2164
  4	gnl|Fabrus|A20_IGKJ1*01	2165
  5	gnl|Fabrus|A23_IGKJ1*01	2166
  6	gnl|Fabrus|A26_IGKJ1*01	2167
  7	gnl|Fabrus|A27_IGKJ1*01	110
  8	gnl|Fabrus|A27_IGKJ3*01	2168
  9	gnl|Fabrus|A30_IGKJ1*01	2169
  10	gnl|Fabrus|B2_IGKJ1*01	2170
  11	gnl|Fabrus|B2_IGKJ3*01	2171
  12	gnl|Fabrus|B3_IGKJ1*01	111
  14	gnl|Fabrus|L11_IGKJ1*01	2173
  15	gnl|Fabrus|L12_IGKJ1*01	115
  16	gnl|Fabrus|L14_IGKJ1*01	2174
  17	gnl|Fabrus|L2_IGKJ1*01	112
  18	gnl|Fabrus|L22_IGKJ3*01	2175
  19	gnl|Fabrus|L23_IGKJ1*01	2176
  20	gnl|Fabrus|L25_IGKJ1*01	120
  21	gnl|Fabrus|L25_IGKJ3*01	2177
  22	gnl|Fabrus|L4/18a_IGKJ1*01	2178
  23	gnl|Fabrus|L5_IGKJ1*01	114
  24	gnl|Fabrus|L6_IGKJ1*01	107
  25	gnl|Fabrus|L8_IGKJ1*01	2179
  26	gnl|Fabrus|L9_IGKJ2*01	2180
  27	gnl|Fabrus|O1_IGKJ1*01	116
  28	gnl|Fabrus|O12_IGKJ1*01	119
  29	gnl|Fabrus|O18_IGKJ1*01	2181
  31	gnl|Fabrus|V1-11_IGLJ2*01	2183
  32	gnl|Fabrus|V1-13_IGLJ5*01	2184
  33	gnl|Fabrus|V1-16_IGLJ6*01	2185
  34	gnl|Fabrus|V1-18_IGLJ2*01	2186
  35	gnl|Fabrus|V1-2_IGLJ7*01	2187
  36	gnl|Fabrus|V1-20_IGLJ6*01	2188
  37	gnl|Fabrus|V1-3_IGLJ1*01	2189
  38	gnl|Fabrus|V1-4_IGLJ4*01	117
  39	gnl|Fabrus|V1-5_IGLJ2*01	2190
  40	gnl|Fabrus|V1-7_IGLJ1*01	2191
  41	gnl|Fabrus|V1-9_IGLJ6*01	2192
  42	gnl|Fabrus|V2-1_IGLJ6*01	2193
  43	gnl|Fabrus|V2-11_IGLJ7*01	2194
  44	gnl|Fabrus|V2-13_IGLJ2*01	2195
  45	gnl|Fabrus|V2-14_IGLJ4*01	2196
  46	gnl|Fabrus|V2-15_IGLJ7*01	2197
  47	gnl|Fabrus|V2-17_IGLJ2*01	2198
  48	gnl|Fabrus|V2-19_IGLJ4*01	2199
  49	gnl|Fabrus|V2-6_IGLJ4*01	2200
  50	gnl|Fabrus|V2-7_IGLJ2*01	2201
  51	gnl|Fabrus|V2-7_IGLJ7*01	2202
  52	gnl|Fabrus|V2-8_IGLJ6*01	2203
  53	gnl|Fabrus|V3-2_IGLJ4*01	2204
  54	gnl|Fabrus|V3-3_IGLJ7*01	2205
  55	gnl|Fabrus|V3-4_IGLJ1*01	108
  56	gnl|Fabrus|V4-1_IGLJ4*01	2206
  57	gnl|Fabrus|V4-2_IGLJ4*01	2207
  58	gnl|Fabrus|V4-3_IGLJ4*01	109
  59	gnl|Fabrus|V4-4_IGLJ5*01	2208
  60	gnl|Fabrus|V4-6_IGLJ4*01	118
  61	gnl|Fabrus|V5-4_IGLJ2*01	2209
  62	gnl|Fabrus|V5-6_IGLJ1*01	2210

• TABLE 4
  Exemplary Paired Nucleic Acid Library

  		SEQ		SEQ
  		ID		ID
  	HEAVY CHAIN	NO	LIGHT CHAIN	NO

  1	gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01	863	gnl|Fabrus|O12_IGKJ1*01	1101
  2	gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01	866	gnl|Fabrus|O12_IGKJ1*01	1101
  3	gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01	870	gnl|Fabrus|O12_IGKJ1*01	1101
  4	gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01	872	gnl|Fabrus|O12_IGKJ1*01	1101
  5	gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01	874	gnl|Fabrus|O12_IGKJ1*01	1101
  6	gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01	876	gnl|Fabrus|O12_IGKJ1*01	1101
  7	gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01	877	gnl|Fabrus|O12_IGKJ1*01	1101
  8	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01	880	gnl|Fabrus|O12_IGKJ1*01	1101
  9	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01	881	gnl|Fabrus|O12_IGKJ1*01	1101
  10	gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01	770	gnl|Fabrus|O12_IGKJ1*01	1101
  11	gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01	771	gnl|Fabrus|O12_IGKJ1*01	1101
  12	gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01	772	gnl|Fabrus|O12_IGKJ1*01	1101
  13	gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01	773	gnl|Fabrus|O12_IGKJ1*01	1101
  14	gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01	774	gnl|Fabrus|O12_IGKJ1*01	1101
  15	gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01	776	gnl|Fabrus|O12_IGKJ1*01	1101
  16	gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01	777	gnl|Fabrus|O12_IGKJ1*01	1101
  17	gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01	779	gnl|Fabrus|O12_IGKJ1*01	1101
  18	gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01	781	gnl|Fabrus|O12_IGKJ1*01	1101
  19	gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01	1017	gnl|Fabrus|O12_IGKJ1*01	1101
  20	gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01	1018	gnl|Fabrus|O12_IGKJ1*01	1101
  21	gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01	1019	gnl|Fabrus|O12_IGKJ1*01	1101
  22	gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01	1021	gnl|Fabrus|O12_IGKJ1*01	1101
  23	gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01	1022	gnl|Fabrus|O12_IGKJ1*01	1101
  24	gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01	1023	gnl|Fabrus|O12_IGKJ1*01	1101
  25	gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01	1024	gnl|Fabrus|O12_IGKJ1*01	1101
  26	gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01	1026	gnl|Fabrus|O12_IGKJ1*01	1101
  27	gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01	789	gnl|Fabrus|O12_IGKJ1*01	1101
  28	gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01	791	gnl|Fabrus|O12_IGKJ1*01	1101
  29	gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01	792	gnl|Fabrus|O12_IGKJ1*01	1101
  30	gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01	794	gnl|Fabrus|O12_IGKJ1*01	1101
  31	gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01	796	gnl|Fabrus|O12_IGKJ1*01	1101
  32	gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01	797	gnl|Fabrus|O12_IGKJ1*01	1101
  33	gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01	798	gnl|Fabrus|O12_IGKJ1*01	1101
  34	gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01	1044	gnl|Fabrus|O12_IGKJ1*01	1101
  35	gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01	1046	gnl|Fabrus|O12_IGKJ1*01	1101
  36	gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01	1048	gnl|Fabrus|O12_IGKJ1*01	1101
  37	gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01	1049	gnl|Fabrus|O12_IGKJ1*01	1101
  38	gnl|Fabrus|VH5-51_IGHD5-	1050	gnl|Fabrus|O12_IGKJ1*01	1101
  	18*01>3_IGHJ4*01
  39	gnl|Fabrus|VH5-51_IGHD5-	1051	gnl|Fabrus|O12_IGKJ1*01	1101
  	18*01>1_IGHJ4*01
  40	gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01	1052	gnl|Fabrus|O12_IGKJ1*01	1101
  41	gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01	1053	gnl|Fabrus|O12_IGKJ1*01	1101
  42	gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01	1054	gnl|Fabrus|O12_IGKJ1*01	1101
  43	gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01	1056	gnl|Fabrus|O12_IGKJ1*01	1101
  44	gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01	1059	gnl|Fabrus|O12_IGKJ1*01	1101
  45	gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01	1061	gnl|Fabrus|O12_IGKJ1*01	1101
  46	gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01	1060	gnl|Fabrus|O12_IGKJ1*01	1101
  47	gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01	1062	gnl|Fabrus|O12_IGKJ1*01	1101
  48	gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01	1063	gnl|Fabrus|O12_IGKJ1*01	1101
  49	gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01	1064	gnl|Fabrus|O12_IGKJ1*01	1101
  50	gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01	1065	gnl|Fabrus|O12_IGKJ1*01	1101
  51	gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01	1043	gnl|Fabrus|O12_IGKJ1*01	1101
  52	gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01	923	gnl|Fabrus|O12_IGKJ1*01	1101
  53	gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01	893	gnl|Fabrus|O12_IGKJ1*01	1101
  54	gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01	949	gnl|Fabrus|O12_IGKJ1*01	1101
  55	gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01	938	gnl|Fabrus|O12_IGKJ1*01	1101
  56	gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01	804	gnl|Fabrus|O12_IGKJ1*01	1101
  57	gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01	811	gnl|Fabrus|O12_IGKJ1*01	1101
  58	gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01	835	gnl|Fabrus|O12_IGKJ1*01	1101
  59	gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01	833	gnl|Fabrus|O12_IGKJ1*01	1101
  60	gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01	930	gnl|Fabrus|O12_IGKJ1*01	1101
  61	gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01	931	gnl|Fabrus|O12_IGKJ1*01	1101
  62	gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01	967	gnl|Fabrus|O12_IGKJ1*01	1101
  63	gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01	969	gnl|Fabrus|O12_IGKJ1*01	1101
  64	gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01	977	gnl|Fabrus|O12_IGKJ1*01	1101
  65	gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01	976	gnl|Fabrus|O12_IGKJ1*01	1101
  66	gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01	918	gnl|Fabrus|O12_IGKJ1*01	1101
  67	gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01	921	gnl|Fabrus|O12_IGKJ1*01	1101
  68	gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01	992	gnl|Fabrus|O12_IGKJ1*01	1101
  69	gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01	989	gnl|Fabrus|O12_IGKJ1*01	1101
  70	gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01	995	gnl|Fabrus|O12_IGKJ1*01	1101
  71	gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01	1030	gnl|Fabrus|O12_IGKJ1*01	1101
  72	gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01	1034	gnl|Fabrus|O12_IGKJ1*01	1101
  73	gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01	728	gnl|Fabrus|O12_IGKJ1*01	1101
  74	gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01	735	gnl|Fabrus|O12_IGKJ1*01	1101
  75	gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01	729	gnl|Fabrus|O12_IGKJ1*01	1101
  76	gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01	743	gnl|Fabrus|O12_IGKJ1*01	1101
  77	gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01	748	gnl|Fabrus|O12_IGKJ1*01	1101
  78	gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01	754	gnl|Fabrus|O12_IGKJ1*01	1101
  79	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01	1068	gnl|Fabrus|O12_IGKJ1*01	1101
  80	gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01	810	gnl|Fabrus|O12_IGKJ1*01	1101
  81	gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01	764	gnl|Fabrus|O12_IGKJ1*01	1101
  82	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01	1067	gnl|Fabrus|O12_IGKJ1*01	1101
  83	gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01	1002	gnl|Fabrus|O12_IGKJ1*01	1101
  84	gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01	1008	gnl|Fabrus|O12_IGKJ1*01	1101
  85	gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01	803	gnl|Fabrus|O12_IGKJ1*01	1101
  86	gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01	783	gnl|Fabrus|O12_IGKJ1*01	1101
  87	gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01	808	gnl|Fabrus|O12_IGKJ1*01	1101
  88	gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01	907	gnl|Fabrus|O12_IGKJ1*01	1101
  89	gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01	838	gnl|Fabrus|O12_IGKJ1*01	1101
  90	gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01	974	gnl|Fabrus|O12_IGKJ1*01	1101
  91	gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01	816	gnl|Fabrus|O12_IGKJ1*01	1101
  92	gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01	820	gnl|Fabrus|O12_IGKJ1*01	1101
  93	gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01	852	gnl|Fabrus|O12_IGKJ1*01	1101
  94	gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01	839	gnl|Fabrus|O12_IGKJ1*01	1101
  95	gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01	960	gnl|Fabrus|O12_IGKJ1*01	1101
  96	gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01	844	gnl|Fabrus|O12_IGKJ1*01	1101
  97	gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01	863	gnl|Fabrus|O12_IGKJ1*01	1102
  98	gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01	866	gnl|Fabrus|O12_IGKJ1*01	1102
  99	gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01	870	gnl|Fabrus|O12_IGKJ1*01	1102
  100	gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01	872	gnl|Fabrus|O12_IGKJ1*01	1102
  101	gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01	874	gnl|Fabrus|O12_IGKJ1*01	1102
  102	gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01	876	gnl|Fabrus|O12_IGKJ1*01	1102
  103	gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01	877	gnl|Fabrus|O12_IGKJ1*01	1102
  104	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01	880	gnl|Fabrus|O12_IGKJ1*01	1102
  105	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01	881	gnl|Fabrus|O18_IGKJ1*01	1102
  106	gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01	770	gnl|Fabrus|O18_IGKJ1*01	1102
  107	gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01	771	gnl|Fabrus|O18_IGKJ1*01	1102
  108	gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01	772	gnl|Fabrus|O18_IGKJ1*01	1102
  109	gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01	773	gnl|Fabrus|O18_IGKJ1*01	1102
  110	gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01	774	gnl|Fabrus|O18_IGKJ1*01	1102
  111	gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01	776	gnl|Fabrus|O18_IGKJ1*01	1102
  112	gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01	777	gnl|Fabrus|O18_IGKJ1*01	1102
  113	gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01	779	gnl|Fabrus|O18_IGKJ1*01	1102
  114	gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01	781	gnl|Fabrus|O18_IGKJ1*01	1102
  115	gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01	1017	gnl|Fabrus|O18_IGKJ1*01	1102
  116	gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01	1018	gnl|Fabrus|O18_IGKJ1*01	1102
  117	gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01	1019	gnl|Fabrus|O18_IGKJ1*01	1102
  118	gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01	1021	gnl|Fabrus|O18_IGKJ1*01	1102
  119	gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01	1022	gnl|Fabrus|O18_IGKJ1*01	1102
  120	gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01	1023	gnl|Fabrus|O18_IGKJ1*01	1102
  121	gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01	1024	gnl|Fabrus|O18_IGKJ1*01	1102
  122	gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01	1026	gnl|Fabrus|O18_IGKJ1*01	1102
  123	gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01	789	gnl|Fabrus|O18_IGKJ1*01	1102
  124	gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01	791	gnl|Fabrus|O18_IGKJ1*01	1102
  125	gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01	792	gnl|Fabrus|O18_IGKJ1*01	1102
  126	gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01	794	gnl|Fabrus|O18_IGKJ1*01	1102
  127	gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01	796	gnl|Fabrus|O18_IGKJ1*01	1102
  128	gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01	797	gnl|Fabrus|O18_IGKJ1*01	1102
  129	gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01	798	gnl|Fabrus|O18_IGKJ1*01	1102
  130	gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01	1044	gnl|Fabrus|O18_IGKJ1*01	1102
  131	gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01	1046	gnl|Fabrus|O18_IGKJ1*01	1102
  132	gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01	1048	gnl|Fabrus|O18_IGKJ1*01	1102
  133	gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01	1049	gnl|Fabrus|O18_IGKJ1*01	1102
  134	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1050	gnl|Fabrus|O18_IGKJ1*01	1102
  135	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1051	gnl|Fabrus|O18_IGKJ1*01	1102
  136	gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01	1052	gnl|Fabrus|O18_IGKJ1*01	1102
  137	gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01	1053	gnl|Fabrus|O18_IGKJ1*01	1102
  138	gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01	1054	gnl|Fabrus|O18_IGKJ1*01	1102
  139	gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01	1056	gnl|Fabrus|O18_IGKJ1*01	1102
  140	gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01	1059	gnl|Fabrus|O18_IGKJ1*01	1102
  141	gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01	1061	gnl|Fabrus|O18_IGKJ1*01	1102
  142	gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01	1060	gnl|Fabrus|O18_IGKJ1*01	1102
  143	gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01	1062	gnl|Fabrus|O18_IGKJ1*01	1102
  144	gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01	1063	gnl|Fabrus|O18_IGKJ1*01	1102
  145	gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01	1064	gnl|Fabrus|O18_IGKJ1*01	1102
  146	gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01	1065	gnl|Fabrus|O18_IGKJ1*01	1102
  147	gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01	1043	gnl|Fabrus|O18_IGKJ1*01	1102
  148	gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01	923	gnl|Fabrus|O18_IGKJ1*01	1102
  149	gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01	893	gnl|Fabrus|O18_IGKJ1*01	1102
  150	gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01	949	gnl|Fabrus|O18_IGKJ1*01	1102
  151	gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01	938	gnl|Fabrus|O18_IGKJ1*01	1102
  152	gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01	804	gnl|Fabrus|O18_IGKJ1*01	1102
  153	gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01	811	gnl|Fabrus|O18_IGKJ1*01	1102
  154	gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01	835	gnl|Fabrus|O18_IGKJ1*01	1102
  155	gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01	833	gnl|Fabrus|O18_IGKJ1*01	1102
  156	gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01	930	gnl|Fabrus|O18_IGKJ1*01	1102
  157	gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01	931	gnl|Fabrus|O18_IGKJ1*01	1102
  158	gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01	967	gnl|Fabrus|O18_IGKJ1*01	1102
  159	gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01	969	gnl|Fabrus|O18_IGKJ1*01	1102
  160	gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01	977	gnl|Fabrus|O18_IGKJ1*01	1102
  161	gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01	976	gnl|Fabrus|O18_IGKJ1*01	1102
  162	gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01	918	gnl|Fabrus|O18_IGKJ1*01	1102
  163	gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01	921	gnl|Fabrus|O18_IGKJ1*01	1102
  164	gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01	992	gnl|Fabrus|O18_IGKJ1*01	1102
  165	gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01	989	gnl|Fabrus|O18_IGKJ1*01	1102
  166	gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01	995	gnl|Fabrus|O18_IGKJ1*01	1102
  167	gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01	1030	gnl|Fabrus|O18_IGKJ1*01	1102
  168	gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01	1034	gnl|Fabrus|O18_IGKJ1*01	1102
  169	gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01	728	gnl|Fabrus|O18_IGKJ1*01	1102
  170	gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01	735	gnl|Fabrus|O18_IGKJ1*01	1102
  171	gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01	729	gnl|Fabrus|O18_IGKJ1*01	1102
  172	gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01	743	gnl|Fabrus|O18_IGKJ1*01	1102
  173	gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01	748	gnl|Fabrus|O18_IGKJ1*01	1102
  174	gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01	754	gnl|Fabrus|O18_IGKJ1*01	1102
  175	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01	1068	gnl|Fabrus|O18_IGKJ1*01	1102
  176	gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01	810	gnl|Fabrus|O18_IGKJ1*01	1102
  177	gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01	764	gnl|Fabrus|O18_IGKJ1*01	1102
  178	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01	1067	gnl|Fabrus|O18_IGKJ1*01	1102
  179	gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01	1002	gnl|Fabrus|O18_IGKJ1*01	1102
  180	gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01	1008	gnl|Fabrus|O18_IGKJ1*01	1102
  181	gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01	803	gnl|Fabrus|O18_IGKJ1*01	1102
  182	gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01	783	gnl|Fabrus|O18_IGKJ1*01	1102
  183	gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01	808	gnl|Fabrus|O18_IGKJ1*01	1102
  184	gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01	907	gnl|Fabrus|O18_IGKJ1*01	1102
  185	gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01	838	gnl|Fabrus|O18_IGKJ1*01	1102
  186	gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01	974	gnl|Fabrus|O18_IGKJ1*01	1102
  187	gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01	816	gnl|Fabrus|O18_IGKJ1*01	1102
  188	gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01	820	gnl|Fabrus|O18_IGKJ1*01	1102
  189	gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01	852	gnl|Fabrus|O18_IGKJ1*01	1102
  190	gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01	839	gnl|Fabrus|O18_IGKJ1*01	1102
  191	gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01	960	gnl|Fabrus|O18_IGKJ1*01	1102
  192	gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01	844	gnl|Fabrus|O18_IGKJ1*01	1102
  193	gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01	863	gnl|Fabrus|A20_IGKJ1*01	1077
  194	gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01	866	gnl|Fabrus|A20_IGKJ1*01	1077
  195	gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01	870	gnl|Fabrus|A20_IGKJ1*01	1077
  196	gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01	872	gnl|Fabrus|A20_IGKJ1*01	1077
  197	gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01	874	gnl|Fabrus|A20_IGKJ1*01	1077
  198	gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01	876	gnl|Fabrus|A20_IGKJ1*01	1077
  199	gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01	877	gnl|Fabrus|A20_IGKJ1*01	1077
  200	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01	880	gnl|Fabrus|A20_IGKJ1*01	1077
  201	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01	881	gnl|Fabrus|A20_IGKJ1*01	1077
  202	gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01	770	gnl|Fabrus|A20_IGKJ1*01	1077
  203	gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01	771	gnl|Fabrus|A20_IGKJ1*01	1077
  204	gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01	772	gnl|Fabrus|A20_IGKJ1*01	1077
  205	gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01	773	gnl|Fabrus|A20_IGKJ1*01	1077
  206	gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01	774	gnl|Fabrus|A20_IGKJ1*01	1077
  207	gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01	776	gnl|Fabrus|A20_IGKJ1*01	1077
  208	gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01	777	gnl|Fabrus|A20_IGKJ1*01	1077
  209	gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01	779	gnl|Fabrus|A20_IGKJ1*01	1077
  210	gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01	781	gnl|Fabrus|A20_IGKJ1*01	1077
  211	gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01	1017	gnl|Fabrus|A20_IGKJ1*01	1077
  212	gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01	1018	gnl|Fabrus|A20_IGKJ1*01	1077
  213	gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01	1019	gnl|Fabrus|A20_IGKJ1*01	1077
  214	gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01	1021	gnl|Fabrus|A20_IGKJ1*01	1077
  215	gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01	1022	gnl|Fabrus|A20_IGKJ1*01	1077
  216	gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01	1023	gnl|Fabrus|A20_IGKJ1*01	1077
  217	gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01	1024	gnl|Fabrus|A20_IGKJ1*01	1077
  218	gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01	1026	gnl|Fabrus|A20_IGKJ1*01	1077
  219	gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01	789	gnl|Fabrus|A20_IGKJ1*01	1077
  220	gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01	791	gnl|Fabrus|A20_IGKJ1*01	1077
  221	gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01	792	gnl|Fabrus|A20_IGKJ1*01	1077
  222	gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01	794	gnl|Fabrus|A20_IGKJ1*01	1077
  223	gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01	796	gnl|Fabrus|A20_IGKJ1*01	1077
  224	gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01	797	gnl|Fabrus|A20_IGKJ1*01	1077
  225	gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01	798	gnl|Fabrus|A20_IGKJ1*01	1077
  226	gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01	1044	gnl|Fabrus|A20_IGKJ1*01	1077
  227	gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01	1046	gnl|Fabrus|A20_IGKJ1*01	1077
  228	gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01	1048	gnl|Fabrus|A20_IGKJ1*01	1077
  229	gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01	1049	gnl|Fabrus|A20_IGKJ1*01	1077
  230	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1050	gnl|Fabrus|A20_IGKJ1*01	1077
  231	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1051	gnl|Fabrus|A20_IGKJ1*01	1077
  232	gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01	1052	gnl|Fabrus|A20_IGKJ1*01	1077
  233	gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01	1053	gnl|Fabrus|A20_IGKJ1*01	1077
  234	gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01	1054	gnl|Fabrus|A20_IGKJ1*01	1077
  235	gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01	1056	gnl|Fabrus|A20_IGKJ1*01	1077
  236	gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01	1059	gnl|Fabrus|A20_IGKJ1*01	1077
  237	gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01	1061	gnl|Fabrus|A20_IGKJ1*01	1077
  238	gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01	1060	gnl|Fabrus|A20_IGKJ1*01	1077
  239	gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01	1062	gnl|Fabrus|A20_IGKJ1*01	1077
  240	gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01	1063	gnl|Fabrus|A20_IGKJ1*01	1077
  241	gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01	1064	gnl|Fabrus|A20_IGKJ1*01	1077
  242	gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01	1065	gnl|Fabrus|A20_IGKJ1*01	1077
  243	gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01	1043	gnl|Fabrus|A20_IGKJ1*01	1077
  244	gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01	923	gnl|Fabrus|A20_IGKJ1*01	1077
  245	gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01	893	gnl|Fabrus|A20_IGKJ1*01	1077
  246	gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01	949	gnl|Fabrus|A20_IGKJ1*01	1077
  247	gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01	938	gnl|Fabrus|A20_IGKJ1*01	1077
  248	gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01	804	gnl|Fabrus|A20_IGKJ1*01	1077
  249	gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01	811	gnl|Fabrus|A20_IGKJ1*01	1077
  250	gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01	835	gnl|Fabrus|A20_IGKJ1*01	1077
  251	gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01	833	gnl|Fabrus|A20_IGKJ1*01	1077
  252	gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01	930	gnl|Fabrus|A20_IGKJ1*01	1077
  253	gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01	931	gnl|Fabrus|A20_IGKJ1*01	1077
  254	gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01	967	gnl|Fabrus|A20_IGKJ1*01	1077
  255	gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01	969	gnl|Fabrus|A20_IGKJ1*01	1077
  256	gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01	977	gnl|Fabrus|A20_IGKJ1*01	1077
  257	gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01	976	gnl|Fabrus|A20_IGKJ1*01	1077
  258	gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01	918	gnl|Fabrus|A20_IGKJ1*01	1077
  259	gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01	921	gnl|Fabrus|A20_IGKJ1*01	1077
  260	gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01	992	gnl|Fabrus|A20_IGKJ1*01	1077
  261	gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01	989	gnl|Fabrus|A20_IGKJ1*01	1077
  262	gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01	995	gnl|Fabrus|A20_IGKJ1*01	1077
  263	gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01	1030	gnl|Fabrus|A20_IGKJ1*01	1077
  264	gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01	1034	gnl|Fabrus|A20_IGKJ1*01	1077
  265	gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01	728	gnl|Fabrus|A20_IGKJ1*01	1077
  266	gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01	735	gnl|Fabrus|A20_IGKJ1*01	1077
  267	gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01	729	gnl|Fabrus|A20_IGKJ1*01	1077
  268	gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01	743	gnl|Fabrus|A20_IGKJ1*01	1077
  269	gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01	748	gnl|Fabrus|A20_IGKJ1*01	1077
  270	gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01	754	gnl|Fabrus|A20_IGKJ1*01	1077
  271	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01	1068	gnl|Fabrus|A20_IGKJ1*01	1077
  272	gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01	810	gnl|Fabrus|A20_IGKJ1*01	1077
  273	gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01	764	gnl|Fabrus|A20_IGKJ1*01	1077
  274	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01	1067	gnl|Fabrus|A20_IGKJ1*01	1077
  275	gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01	1002	gnl|Fabrus|A20_IGKJ1*01	1077
  276	gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01	1008	gnl|Fabrus|A20_IGKJ1*01	1077
  277	gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01	803	gnl|Fabrus|A20_IGKJ1*01	1077
  278	gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01	783	gnl|Fabrus|A20_IGKJ1*01	1077
  279	gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01	808	gnl|Fabrus|A20_IGKJ1*01	1077
  280	gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01	907	gnl|Fabrus|A20_IGKJ1*01	1077
  281	gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01	838	gnl|Fabrus|A20_IGKJ1*01	1077
  282	gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01	974	gnl|Fabrus|A20_IGKJ1*01	1077
  283	gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01	816	gnl|Fabrus|A20_IGKJ1*01	1077
  284	gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01	820	gnl|Fabrus|A20_IGKJ1*01	1077
  285	gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01	852	gnl|Fabrus|A20_IGKJ1*01	1077
  286	gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01	839	gnl|Fabrus|A20_IGKJ1*01	1077
  287	gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01	960	gnl|Fabrus|A20_IGKJ1*01	1077
  288	gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01	844	gnl|Fabrus|A20_IGKJ1*01	1077
  289	gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01	863	gnl|Fabrus|A30_IGKJ1*01	1082
  290	gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01	866	gnl|Fabrus|A30_IGKJ1*01	1082
  291	gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01	870	gnl|Fabrus|A30_IGKJ1*01	1082
  292	gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01	872	gnl|Fabrus|A30_IGKJ1*01	1082
  293	gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01	874	gnl|Fabrus|A30_IGKJ1*01	1082
  294	gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01	876	gnl|Fabrus|A30_IGKJ1*01	1082
  295	gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01	877	gnl|Fabrus|A30_IGKJ1*01	1082
  296	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01	880	gnl|Fabrus|A30_IGKJ1*01	1082
  297	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01	881	gnl|Fabrus|A30_IGKJ1*01	1082
  298	gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01	770	gnl|Fabrus|A30_IGKJ1*01	1082
  299	gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01	771	gnl|Fabrus|A30_IGKJ1*01	1082
  300	gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01	772	gnl|Fabrus|A30_IGKJ1*01	1082
  301	gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01	773	gnl|Fabrus|A30_IGKJ1*01	1082
  302	gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01	774	gnl|Fabrus|A30_IGKJ1*01	1082
  303	gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01	776	gnl|Fabrus|A30_IGKJ1*01	1082
  304	gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01	777	gnl|Fabrus|A30_IGKJ1*01	1082
  305	gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01	779	gnl|Fabrus|A30_IGKJ1*01	1082
  306	gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01	781	gnl|Fabrus|A30_IGKJ1*01	1082
  307	gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01	1017	gnl|Fabrus|A30_IGKJ1*01	1082
  308	gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01	1018	gnl|Fabrus|A30_IGKJ1*01	1082
  309	gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01	1019	gnl|Fabrus|A30_IGKJ1*01	1082
  310	gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01	1021	gnl|Fabrus|A30_IGKJ1*01	1082
  311	gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01	1022	gnl|Fabrus|A30_IGKJ1*01	1082
  312	gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01	1023	gnl|Fabrus|A30_IGKJ1*01	1082
  313	gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01	1024	gnl|Fabrus|A30_IGKJ1*01	1082
  314	gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01	1026	gnl|Fabrus|A30_IGKJ1*01	1082
  315	gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01	789	gnl|Fabrus|A30_IGKJ1*01	1082
  316	gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01	791	gnl|Fabrus|A30_IGKJ1*01	1082
  317	gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01	792	gnl|Fabrus|A30_IGKJ1*01	1082
  318	gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01	794	gnl|Fabrus|A30_IGKJ1*01	1082
  319	gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01	796	gnl|Fabrus|A30_IGKJ1*01	1082
  320	gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01	797	gnl|Fabrus|A30_IGKJ1*01	1082
  321	gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01	798	gnl|Fabrus|A30_IGKJ1*01	1082
  322	gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01	1044	gnl|Fabrus|A30_IGKJ1*01	1082
  323	gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01	1046	gnl|Fabrus|A30_IGKJ1*01	1082
  324	gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01	1048	gnl|Fabrus|A30_IGKJ1*01	1082
  325	gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01	1049	gnl|Fabrus|A30_IGKJ1*01	1082
  326	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1050	gnl|Fabrus|A30_IGKJ1*01	1082
  327	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1051	gnl|Fabrus|A30_IGKJ1*01	1082
  328	gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01	1052	gnl|Fabrus|A30_IGKJ1*01	1082
  329	gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01	1053	gnl|Fabrus|A30_IGKJ1*01	1082
  330	gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01	1054	gnl|Fabrus|A30_IGKJ1*01	1082
  331	gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01	1056	gnl|Fabrus|A30_IGKJ1*01	1082
  332	gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01	1059	gnl|Fabrus|A30_IGKJ1*01	1082
  333	gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01	1061	gnl|Fabrus|A30_IGKJ1*01	1082
  334	gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01	1060	gnl|Fabrus|A30_IGKJ1*01	1082
  335	gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01	1062	gnl|Fabrus|A30_IGKJ1*01	1082
  336	gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01	1063	gnl|Fabrus|A30_IGKJ1*01	1082
  337	gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01	1064	gnl|Fabrus|A30_IGKJ1*01	1082
  338	gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01	1065	gnl|Fabrus|A30_IGKJ1*01	1082
  339	gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01	1043	gnl|Fabrus|A30_IGKJ1*01	1082
  340	gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01	923	gnl|Fabrus|A30_IGKJ1*01	1082
  341	gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01	893	gnl|Fabrus|A30_IGKJ1*01	1082
  342	gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01	949	gnl|Fabrus|A30_IGKJ1*01	1082
  343	gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01	938	gnl|Fabrus|A30_IGKJ1*01	1082
  344	gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01	804	gnl|Fabrus|A30_IGKJ1*01	1082
  345	gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01	811	gnl|Fabrus|A30_IGKJ1*01	1082
  346	gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01	835	gnl|Fabrus|A30_IGKJ1*01	1082
  347	gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01	833	gnl|Fabrus|A30_IGKJ1*01	1082
  348	gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01	930	gnl|Fabrus|A30_IGKJ1*01	1082
  349	gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01	931	gnl|Fabrus|A30_IGKJ1*01	1082
  350	gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01	967	gnl|Fabrus|A30_IGKJ1*01	1082
  351	gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01	969	gnl|Fabrus|A30_IGKJ1*01	1082
  352	gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01	977	gnl|Fabrus|A30_IGKJ1*01	1082
  353	gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01	976	gnl|Fabrus|A30_IGKJ1*01	1082
  354	gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01	918	gnl|Fabrus|A30_IGKJ1*01	1082
  355	gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01	921	gnl|Fabrus|A30_IGKJ1*01	1082
  356	gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01	992	gnl|Fabrus|A30_IGKJ1*01	1082
  357	gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01	989	gnl|Fabrus|A30_IGKJ1*01	1082
  358	gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01	995	gnl|Fabrus|A30_IGKJ1*01	1082
  359	gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01	1030	gnl|Fabrus|A30_IGKJ1*01	1082
  360	gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01	1034	gnl|Fabrus|A30_IGKJ1*01	1082
  361	gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01	728	gnl|Fabrus|A30_IGKJ1*01	1082
  362	gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01	735	gnl|Fabrus|A30_IGKJ1*01	1082
  363	gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01	729	gnl|Fabrus|A30_IGKJ1*01	1082
  364	gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01	743	gnl|Fabrus|A30_IGKJ1*01	1082
  365	gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01	748	gnl|Fabrus|A30_IGKJ1*01	1082
  366	gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01	754	gnl|Fabrus|A30_IGKJ1*01	1082
  367	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01	1068	gnl|Fabrus|A30_IGKJ1*01	1082
  368	gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01	810	gnl|Fabrus|A30_IGKJ1*01	1082
  369	gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01	764	gnl|Fabrus|A30_IGKJ1*01	1082
  370	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01	1067	gnl|Fabrus|A30_IGKJ1*01	1082
  371	gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01	1002	gnl|Fabrus|A30_IGKJ1*01	1082
  372	gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01	1008	gnl|Fabrus|A30_IGKJ1*01	1082
  373	gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01	803	gnl|Fabrus|A30_IGKJ1*01	1082
  374	gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01	783	gnl|Fabrus|A30_IGKJ1*01	1082
  375	gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01	808	gnl|Fabrus|A30_IGKJ1*01	1082
  376	gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01	907	gnl|Fabrus|A30_IGKJ1*01	1082
  377	gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01	838	gnl|Fabrus|A30_IGKJ1*01	1082
  378	gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01	974	gnl|Fabrus|A30_IGKJ1*01	1082
  379	gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01	816	gnl|Fabrus|A30_IGKJ1*01	1082
  380	gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01	820	gnl|Fabrus|A30_IGKJ1*01	1082
  381	gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01	852	gnl|Fabrus|A30_IGKJ1*01	1082
  382	gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01	839	gnl|Fabrus|A30_IGKJ1*01	1082
  383	gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01	960	gnl|Fabrus|A30_IGKJ1*01	1082
  384	gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01	844	gnl|Fabrus|A30_IGKJ1*01	1082
  385	gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01	863	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  386	gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01	866	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  387	gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01	870	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  388	gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01	872	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  389	gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01	874	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  390	gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01	876	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  391	gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01	877	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  392	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01	880	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  393	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01	881	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  394	gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01	770	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  395	gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01	771	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  396	gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01	772	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  397	gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01	773	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  398	gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01	774	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  399	gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01	776	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  400	gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01	777	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  401	gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01	779	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  402	gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01	781	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  403	gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01	1017	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  404	gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01	1018	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  405	gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01	1019	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  406	gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01	1021	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  407	gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01	1022	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  408	gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01	1023	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  409	gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01	1024	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  410	gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01	1026	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  411	gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01	789	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  412	gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01	791	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  413	gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01	792	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  414	gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01	794	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  415	gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01	796	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  416	gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01	797	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  417	gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01	798	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  418	gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01	1044	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  419	gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01	1046	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  420	gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01	1048	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  421	gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01	1049	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  422	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1050	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  423	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1051	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  424	gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01	1052	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  425	gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01	1053	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  426	gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01	1054	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  427	gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01	1056	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  428	gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01	1059	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  429	gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01	1061	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  430	gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01	1060	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  431	gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01	1062	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  432	gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01	1063	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  433	gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01	1064	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  434	gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01	1065	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  435	gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01	1043	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  436	gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01	923	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  437	gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01	893	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  438	gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01	949	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  439	gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01	938	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  440	gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01	804	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  441	gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01	811	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  442	gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01	835	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  443	gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01	833	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  444	gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01	930	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  445	gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01	931	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  446	gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01	967	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  447	gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01	969	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  448	gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01	977	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  449	gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01	976	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  450	gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01	918	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  451	gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01	921	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  452	gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01	992	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  453	gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01	989	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  454	gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01	995	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  455	gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01	1030	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  456	gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01	1034	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  457	gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01	728	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  458	gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01	735	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  459	gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01	729	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  460	gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01	743	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  461	gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01	748	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  462	gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01	754	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  463	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01	1068	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  464	gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01	810	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  465	gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01	764	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  466	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01	1067	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  467	gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01	1002	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  468	gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01	1008	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  469	gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01	803	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  470	gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01	783	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  471	gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01	808	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  472	gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01	907	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  473	gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01	838	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  474	gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01	974	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  475	gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01	816	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  476	gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01	820	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  477	gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01	852	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  478	gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01	839	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  479	gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01	960	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  480	gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01	844	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  481	gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01	863	gnl|Fabrus|L5_IGKJ1*01	1096
  482	gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01	866	gnl|Fabrus|L5_IGKJ1*01	1096
  483	gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01	870	gnl|Fabrus|L5_IGKJ1*01	1096
  484	gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01	872	gnl|Fabrus|L5_IGKJ1*01	1096
  485	gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01	874	gnl|Fabrus|L5_IGKJ1*01	1096
  486	gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01	876	gnl|Fabrus|L5_IGKJ1*01	1096
  487	gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01	877	gnl|Fabrus|L5_IGKJ1*01	1096
  488	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01	880	gnl|Fabrus|L5_IGKJ1*01	1096
  489	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01	881	gnl|Fabrus|L5_IGKJ1*01	1096
  490	gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01	770	gnl|Fabrus|L5_IGKJ1*01	1096
  491	gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01	771	gnl|Fabrus|L5_IGKJ1*01	1096
  492	gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01	772	gnl|Fabrus|L5_IGKJ1*01	1096
  493	gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01	773	gnl|Fabrus|L5_IGKJ1*01	1096
  494	gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01	774	gnl|Fabrus|L5_IGKJ1*01	1096
  495	gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01	776	gnl|Fabrus|L5_IGKJ1*01	1096
  496	gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01	777	gnl|Fabrus|L5_IGKJ1*01	1096
  497	gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01	779	gnl|Fabrus|L5_IGKJ1*01	1096
  498	gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01	781	gnl|Fabrus|L5_IGKJ1*01	1096
  499	gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01	1017	gnl|Fabrus|L5_IGKJ1*01	1096
  500	gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01	1018	gnl|Fabrus|L5_IGKJ1*01	1096
  501	gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01	1019	gnl|Fabrus|L5_IGKJ1*01	1096
  502	gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01	1021	gnl|Fabrus|L5_IGKJ1*01	1096
  503	gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01	1022	gnl|Fabrus|L5_IGKJ1*01	1096
  504	gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01	1023	gnl|Fabrus|L5_IGKJ1*01	1096
  505	gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01	1024	gnl|Fabrus|L5_IGKJ1*01	1096
  506	gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01	1026	gnl|Fabrus|L5_IGKJ1*01	1096
  507	gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01	789	gnl|Fabrus|L5_IGKJ1*01	1096
  508	gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01	791	gnl|Fabrus|L5_IGKJ1*01	1096
  509	gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01	792	gnl|Fabrus|L5_IGKJ1*01	1096
  510	gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01	794	gnl|Fabrus|L5_IGKJ1*01	1096
  511	gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01	796	gnl|Fabrus|L5_IGKJ1*01	1096
  512	gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01	797	gnl|Fabrus|L5_IGKJ1*01	1096
  513	gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01	798	gnl|Fabrus|L5_IGKJ1*01	1096
  514	gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01	1044	gnl|Fabrus|L5_IGKJ1*01	1096
  515	gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01	1046	gnl|Fabrus|L5_IGKJ1*01	1096
  516	gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01	1048	gnl|Fabrus|L5_IGKJ1*01	1096
  517	gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01	1049	gnl|Fabrus|L5_IGKJ1*01	1096
  518	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1050	gnl|Fabrus|L5_IGKJ1*01	1096
  519	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1051	gnl|Fabrus|L5_IGKJ1*01	1096
  520	gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01	1052	gnl|Fabrus|L5_IGKJ1*01	1096
  521	gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01	1053	gnl|Fabrus|L5_IGKJ1*01	1096
  522	gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01	1054	gnl|Fabrus|L5_IGKJ1*01	1096
  523	gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01	1056	gnl|Fabrus|L5_IGKJ1*01	1096
  524	gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01	1059	gnl|Fabrus|L5_IGKJ1*01	1096
  525	gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01	1061	gnl|Fabrus|L5_IGKJ1*01	1096
  526	gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01	1060	gnl|Fabrus|L5_IGKJ1*01	1096
  527	gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01	1062	gnl|Fabrus|L5_IGKJ1*01	1096
  528	gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01	1063	gnl|Fabrus|L5_IGKJ1*01	1096
  529	gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01	1064	gnl|Fabrus|L5_IGKJ1*01	1096
  530	gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01	1065	gnl|Fabrus|L5_IGKJ1*01	1096
  531	gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01	1043	gnl|Fabrus|L5_IGKJ1*01	1096
  532	gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01	923	gnl|Fabrus|L5_IGKJ1*01	1096
  533	gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01	893	gnl|Fabrus|L5_IGKJ1*01	1096
  534	gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01	949	gnl|Fabrus|L5_IGKJ1*01	1096
  535	gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01	938	gnl|Fabrus|L5_IGKJ1*01	1096
  536	gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01	804	gnl|Fabrus|L5_IGKJ1*01	1096
  537	gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01	811	gnl|Fabrus|L5_IGKJ1*01	1096
  538	gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01	835	gnl|Fabrus|L5_IGKJ1*01	1096
  539	gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01	833	gnl|Fabrus|L5_IGKJ1*01	1096
  540	gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01	930	gnl|Fabrus|L5_IGKJ1*01	1096
  541	gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01	931	gnl|Fabrus|L5_IGKJ1*01	1096
  542	gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01	967	gnl|Fabrus|L5_IGKJ1*01	1096
  543	gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01	969	gnl|Fabrus|L5_IGKJ1*01	1096
  544	gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01	977	gnl|Fabrus|L5_IGKJ1*01	1096
  545	gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01	976	gnl|Fabrus|L5_IGKJ1*01	1096
  546	gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01	918	gnl|Fabrus|L5_IGKJ1*01	1096
  547	gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01	921	gnl|Fabrus|L5_IGKJ1*01	1096
  548	gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01	992	gnl|Fabrus|L5_IGKJ1*01	1096
  549	gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01	989	gnl|Fabrus|L5_IGKJ1*01	1096
  550	gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01	995	gnl|Fabrus|L5_IGKJ1*01	1096
  551	gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01	1030	gnl|Fabrus|L5_IGKJ1*01	1096
  552	gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01	1034	gnl|Fabrus|L5_IGKJ1*01	1096
  553	gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01	728	gnl|Fabrus|L5_IGKJ1*01	1096
  554	gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01	735	gnl|Fabrus|L5_IGKJ1*01	1096
  555	gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01	729	gnl|Fabrus|L5_IGKJ1*01	1096
  556	gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01	743	gnl|Fabrus|L5_IGKJ1*01	1096
  557	gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01	748	gnl|Fabrus|L5_IGKJ1*01	1096
  558	gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01	754	gnl|Fabrus|L5_IGKJ1*01	1096
  559	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01	1068	gnl|Fabrus|L5_IGKJ1*01	1096
  560	gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01	810	gnl|Fabrus|L5_IGKJ1*01	1096
  561	gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01	764	gnl|Fabrus|L5_IGKJ1*01	1096
  562	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01	1067	gnl|Fabrus|L5_IGKJ1*01	1096
  563	gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01	1002	gnl|Fabrus|L5_IGKJ1*01	1096
  564	gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01	1008	gnl|Fabrus|L5_IGKJ1*01	1096
  565	gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01	803	gnl|Fabrus|L5_IGKJ1*01	1096
  566	gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01	783	gnl|Fabrus|L5_IGKJ1*01	1096
  567	gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01	808	gnl|Fabrus|L5_IGKJ1*01	1096
  568	gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01	907	gnl|Fabrus|L5_IGKJ1*01	1096
  569	gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01	838	gnl|Fabrus|L5_IGKJ1*01	1096
  570	gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01	974	gnl|Fabrus|L5_IGKJ1*01	1096
  571	gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01	816	gnl|Fabrus|L5_IGKJ1*01	1096
  572	gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01	820	gnl|Fabrus|L5_IGKJ1*01	1096
  573	gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01	852	gnl|Fabrus|L5_IGKJ1*01	1096
  574	gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01	839	gnl|Fabrus|L5_IGKJ1*01	1096
  575	gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01	960	gnl|Fabrus|L5_IGKJ1*01	1096
  576	gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01	844	gnl|Fabrus|L5_IGKJ1*01	1096
  577	gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01	863	gnl|Fabrus|L8_IGKJ1*01	1098
  578	gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01	866	gnl|Fabrus|L8_IGKJ1*01	1098
  579	gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01	870	gnl|Fabrus|L8_IGKJ1*01	1098
  580	gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01	872	gnl|Fabrus|L8_IGKJ1*01	1098
  581	gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01	874	gnl|Fabrus|L8_IGKJ1*01	1098
  582	gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01	876	gnl|Fabrus|L8_IGKJ1*01	1098
  583	gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01	877	gnl|Fabrus|L8_IGKJ1*01	1098
  584	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01	880	gnl|Fabrus|L8_IGKJ1*01	1098
  585	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01	881	gnl|Fabrus|L8_IGKJ1*01	1098
  586	gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01	770	gnl|Fabrus|L8_IGKJ1*01	1098
  587	gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01	771	gnl|Fabrus|L8_IGKJ1*01	1098
  588	gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01	772	gnl|Fabrus|L8_IGKJ1*01	1098
  589	gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01	773	gnl|Fabrus|L8_IGKJ1*01	1098
  590	gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01	774	gnl|Fabrus|L8_IGKJ1*01	1098
  591	gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01	776	gnl|Fabrus|L8_IGKJ1*01	1098
  592	gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01	777	gnl|Fabrus|L8_IGKJ1*01	1098
  593	gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01	779	gnl|Fabrus|L8_IGKJ1*01	1098
  594	gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01	781	gnl|Fabrus|L8_IGKJ1*01	1098
  595	gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01	1017	gnl|Fabrus|L8_IGKJ1*01	1098
  596	gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01	1018	gnl|Fabrus|L8_IGKJ1*01	1098
  597	gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01	1019	gnl|Fabrus|L8_IGKJ1*01	1098
  598	gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01	1021	gnl|Fabrus|L8_IGKJ1*01	1098
  599	gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01	1022	gnl|Fabrus|L8_IGKJ1*01	1098
  600	gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01	1023	gnl|Fabrus|L8_IGKJ1*01	1098
  601	gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01	1024	gnl|Fabrus|L8_IGKJ1*01	1098
  602	gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01	1026	gnl|Fabrus|L8_IGKJ1*01	1098
  603	gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01	789	gnl|Fabrus|L8_IGKJ1*01	1098
  604	gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01	791	gnl|Fabrus|L8_IGKJ1*01	1098
  605	gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01	792	gnl|Fabrus|L8_IGKJ1*01	1098
  606	gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01	794	gnl|Fabrus|L8_IGKJ1*01	1098
  607	gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01	796	gnl|Fabrus|L8_IGKJ1*01	1098
  608	gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01	797	gnl|Fabrus|L8_IGKJ1*01	1098
  609	gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01	798	gnl|Fabrus|L8_IGKJ1*01	1098
  610	gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01	1044	gnl|Fabrus|L8_IGKJ1*01	1098
  611	gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01	1046	gnl|Fabrus|L8_IGKJ1*01	1098
  612	gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01	1048	gnl|Fabrus|L8_IGKJ1*01	1098
  613	gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01	1049	gnl|Fabrus|L8_IGKJ1*01	1098
  614	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1050	gnl|Fabrus|L8_IGKJ1*01	1098
  615	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1051	gnl|Fabrus|L8_IGKJ1*01	1098
  616	gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01	1052	gnl|Fabrus|L8_IGKJ1*01	1098
  617	gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01	1053	gnl|Fabrus|L8_IGKJ1*01	1098
  618	gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01	1054	gnl|Fabrus|L8_IGKJ1*01	1098
  619	gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01	1056	gnl|Fabrus|L8_IGKJ1*01	1098
  620	gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01	1059	gnl|Fabrus|L8_IGKJ1*01	1098
  621	gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01	1061	gnl|Fabrus|L8_IGKJ1*01	1098
  622	gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01	1060	gnl|Fabrus|L8_IGKJ1*01	1098
  623	gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01	1062	gnl|Fabrus|L8_IGKJ1*01	1098
  624	gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01	1063	gnl|Fabrus|L8_IGKJ1*01	1098
  625	gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01	1064	gnl|Fabrus|L8_IGKJ1*01	1098
  626	gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01	1065	gnl|Fabrus|L8_IGKJ1*01	1098
  627	gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01	1043	gnl|Fabrus|L8_IGKJ1*01	1098
  628	gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01	923	gnl|Fabrus|L8_IGKJ1*01	1098
  629	gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01	893	gnl|Fabrus|L8_IGKJ1*01	1098
  630	gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01	949	gnl|Fabrus|L8_IGKJ1*01	1098
  631	gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01	938	gnl|Fabrus|L8_IGKJ1*01	1098
  632	gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01	804	gnl|Fabrus|L8_IGKJ1*01	1098
  633	gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01	811	gnl|Fabrus|L8_IGKJ1*01	1098
  634	gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01	835	gnl|Fabrus|L8_IGKJ1*01	1098
  635	gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01	833	gnl|Fabrus|L8_IGKJ1*01	1098
  636	gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01	930	gnl|Fabrus|L8_IGKJ1*01	1098
  637	gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01	931	gnl|Fabrus|L8_IGKJ1*01	1098
  638	gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01	967	gnl|Fabrus|L8_IGKJ1*01	1098
  639	gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01	969	gnl|Fabrus|L8_IGKJ1*01	1098
  640	gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01	977	gnl|Fabrus|L8_IGKJ1*01	1098
  641	gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01	976	gnl|Fabrus|L8_IGKJ1*01	1098
  642	gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01	918	gnl|Fabrus|L8_IGKJ1*01	1098
  643	gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01	921	gnl|Fabrus|L8_IGKJ1*01	1098
  644	gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01	992	gnl|Fabrus|L8_IGKJ1*01	1098
  645	gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01	989	gnl|Fabrus|L8_IGKJ1*01	1098
  646	gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01	995	gnl|Fabrus|L8_IGKJ1*01	1098
  647	gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01	1030	gnl|Fabrus|L8_IGKJ1*01	1098
  648	gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01	1034	gnl|Fabrus|L8_IGKJ1*01	1098
  649	gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01	728	gnl|Fabrus|L8_IGKJ1*01	1098
  650	gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01	735	gnl|Fabrus|L8_IGKJ1*01	1098
  651	gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01	729	gnl|Fabrus|L8_IGKJ1*01	1098
  652	gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01	743	gnl|Fabrus|L8_IGKJ1*01	1098
  653	gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01	748	gnl|Fabrus|L8_IGKJ1*01	1098
  654	gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01	754	gnl|Fabrus|L8_IGKJ1*01	1098
  655	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01	1068	gnl|Fabrus|L8_IGKJ1*01	1098
  656	gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01	810	gnl|Fabrus|L8_IGKJ1*01	1098
  657	gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01	764	gnl|Fabrus|L8_IGKJ1*01	1098
  658	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01	1067	gnl|Fabrus|L8_IGKJ1*01	1098
  659	gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01	1002	gnl|Fabrus|L8_IGKJ1*01	1098
  660	gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01	1008	gnl|Fabrus|L8_IGKJ1*01	1098
  661	gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01	803	gnl|Fabrus|L8_IGKJ1*01	1098
  662	gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01	783	gnl|Fabrus|L8_IGKJ1*01	1098
  663	gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01	808	gnl|Fabrus|L8_IGKJ1*01	1098
  664	gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01	907	gnl|Fabrus|L8_IGKJ1*01	1098
  665	gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01	838	gnl|Fabrus|L8_IGKJ1*01	1098
  666	gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01	974	gnl|Fabrus|L8_IGKJ1*01	1098
  667	gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01	816	gnl|Fabrus|L8_IGKJ1*01	1098
  668	gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01	820	gnl|Fabrus|L8_IGKJ1*01	1098
  669	gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01	852	gnl|Fabrus|L8_IGKJ1*01	1098
  670	gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01	839	gnl|Fabrus|L8_IGKJ1*01	1098
  671	gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01	960	gnl|Fabrus|L8_IGKJ1*01	1098
  672	gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01	844	gnl|Fabrus|L8_IGKJ1*01	1098
  673	gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01	863	gnl|Fabrus|L11_IGKJ1*01	1087
  674	gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01	866	gnl|Fabrus|L11_IGKJ1*01	1087
  675	gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01	870	gnl|Fabrus|L11_IGKJ1*01	1087
  676	gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01	872	gnl|Fabrus|L11_IGKJ1*01	1087
  677	gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01	874	gnl|Fabrus|L11_IGKJ1*01	1087
  678	gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01	876	gnl|Fabrus|L11_IGKJ1*01	1087
  679	gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01	877	gnl|Fabrus|L11_IGKJ1*01	1087
  680	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01	880	gnl|Fabrus|L11_IGKJ1*01	1087
  681	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01	881	gnl|Fabrus|L11_IGKJ1*01	1087
  682	gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01	770	gnl|Fabrus|L11_IGKJ1*01	1087
  683	gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01	771	gnl|Fabrus|L11_IGKJ1*01	1087
  684	gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01	772	gnl|Fabrus|L11_IGKJ1*01	1087
  685	gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01	773	gnl|Fabrus|L11_IGKJ1*01	1087
  686	gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01	774	gnl|Fabrus|L11_IGKJ1*01	1087
  687	gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01	776	gnl|Fabrus|L11_IGKJ1*01	1087
  688	gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01	777	gnl|Fabrus|L11_IGKJ1*01	1087
  689	gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01	779	gnl|Fabrus|L11_IGKJ1*01	1087
  690	gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01	781	gnl|Fabrus|L11_IGKJ1*01	1087
  691	gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01	1017	gnl|Fabrus|L11_IGKJ1*01	1087
  692	gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01	1018	gnl|Fabrus|L11_IGKJ1*01	1087
  693	gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01	1019	gnl|Fabrus|L11_IGKJ1*01	1087
  694	gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01	1021	gnl|Fabrus|L11_IGKJ1*01	1087
  695	gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01	1022	gnl|Fabrus|L11_IGKJ1*01	1087
  696	gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01	1023	gnl|Fabrus|L11_IGKJ1*01	1087
  697	gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01	1024	gnl|Fabrus|L11_IGKJ1*01	1087
  698	gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01	1026	gnl|Fabrus|L11_IGKJ1*01	1087
  699	gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01	789	gnl|Fabrus|L11_IGKJ1*01	1087
  700	gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01	791	gnl|Fabrus|L11_IGKJ1*01	1087
  701	gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01	792	gnl|Fabrus|L11_IGKJ1*01	1087
  702	gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01	794	gnl|Fabrus|L11_IGKJ1*01	1087
  703	gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01	796	gnl|Fabrus|L11_IGKJ1*01	1087
  704	gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01	797	gnl|Fabrus|L11_IGKJ1*01	1087
  705	gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01	798	gnl|Fabrus|L11_IGKJ1*01	1087
  706	gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01	1044	gnl|Fabrus|L11_IGKJ1*01	1087
  707	gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01	1046	gnl|Fabrus|L11_IGKJ1*01	1087
  708	gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01	1048	gnl|Fabrus|L11_IGKJ1*01	1087
  709	gnl|Fabrus|HV5-51_IGHD4-17*01_IGHJ4*01	1049	gnl|Fabrus|L11_IGKJ1*01	1087
  710	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1050	gnl|Fabrus|L11_IGKJ1*01	1087
  711	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1051	gnl|Fabrus|L11_IGKJ1*01	1087
  712	gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01	1052	gnl|Fabrus|L11_IGKJ1*01	1087
  713	gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01	1053	gnl|Fabrus|L11_IGKJ1*01	1087
  714	gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01	1054	gnl|Fabrus|L11_IGKJ1*01	1087
  715	gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01	1056	gnl|Fabrus|L11_IGKJ1*01	1087
  716	gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01	1059	gnl|Fabrus|L11_IGKJ1*01	1087
  717	gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01	1061	gnl|Fabrus|L11_IGKJ1*01	1087
  718	gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01	1060	gnl|Fabrus|L11_IGKJ1*01	1087
  719	gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01	1062	gnl|Fabrus|L11_IGKJ1*01	1087
  720	gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01	1063	gnl|Fabrus|L11_IGKJ1*01	1087
  721	gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01	1064	gnl|Fabrus|L11_IGKJ1*01	1087
  722	gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01	1065	gnl|Fabrus|L11_IGKJ1*01	1087
  723	gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01	1043	gnl|Fabrus|L11_IGKJ1*01	1087
  724	gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01	923	gnl|Fabrus|L11_IGKJ1*01	1087
  725	gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01	893	gnl|Fabrus|L11_IGKJ1*01	1087
  726	gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01	949	gnl|Fabrus|L11_IGKJ1*01	1087
  727	gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01	938	gnl|Fabrus|L11_IGKJ1*01	1087
  728	gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01	804	gnl|Fabrus|L11_IGKJ1*01	1087
  729	gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01	811	gnl|Fabrus|L11_IGKJ1*01	1087
  730	gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01	835	gnl|Fabrus|L11_IGKJ1*01	1087
  731	gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01	833	gnl|Fabrus|L11_IGKJ1*01	1087
  732	gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01	930	gnl|Fabrus|L11_IGKJ1*01	1087
  733	gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01	931	gnl|Fabrus|L11_IGKJ1*01	1087
  734	gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01	967	gnl|Fabrus|L11_IGKJ1*01	1087
  735	gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01	969	gnl|Fabrus|L11_IGKJ1*01	1087
  736	gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01	977	gnl|Fabrus|L11_IGKJ1*01	1087
  737	gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01	976	gnl|Fabrus|L11_IGKJ1*01	1087
  738	gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01	918	gnl|Fabrus|L11_IGKJ1*01	1087
  739	gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01	921	gnl|Fabrus|L11_IGKJ1*01	1087
  740	gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01	992	gnl|Fabrus|L11_IGKJ1*01	1087
  741	gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01	989	gnl|Fabrus|L11_IGKJ1*01	1087
  742	gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01	995	gnl|Fabrus|L11_IGKJ1*01	1087
  743	gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01	1030	gnl|Fabrus|L11_IGKJ1*01	1087
  744	gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01	1034	gnl|Fabrus|L11_IGKJ1*01	1087
  745	gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01	728	gnl|Fabrus|L11_IGKJ1*01	1087
  746	gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01	735	gnl|Fabrus|L11_IGKJ1*01	1087
  747	gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01	729	gnl|Fabrus|L11_IGKJ1*01	1087
  748	gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01	743	gnl|Fabrus|L11_IGKJ1*01	1087
  749	gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01	748	gnl|Fabrus|L11_IGKJ1*01	1087
  750	gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01	754	gnl|Fabrus|L11_IGKJ1*01	1087
  751	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01	1068	gnl|Fabrus|L11_IGKJ1*01	1087
  752	gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01	810	gnl|Fabrus|L11_IGKJ1*01	1087
  753	gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01	764	gnl|Fabrus|L11_IGKJ1*01	1087
  754	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01	1067	gnl|Fabrus|L11_IGKJ1*01	1087
  755	gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01	1002	gnl|Fabrus|L11_IGKJ1*01	1087
  756	gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01	1008	gnl|Fabrus|L11_IGKJ1*01	1087
  757	gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01	803	gnl|Fabrus|L11_IGKJ1*01	1087
  758	gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01	783	gnl|Fabrus|L11_IGKJ1*01	1087
  759	gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01	808	gnl|Fabrus|L11_IGKJ1*01	1087
  760	gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01	907	gnl|Fabrus|L11_IGKJ1*01	1087
  761	gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01	838	gnl|Fabrus|L11_IGKJ1*01	1087
  762	gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01	974	gnl|Fabrus|L11_IGKJ1*01	1087
  763	gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01	816	gnl|Fabrus|L11_IGKJ1*01	1087
  764	gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01	820	gnl|Fabrus|L11_IGKJ1*01	1087
  765	gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01	852	gnl|Fabrus|L11_IGKJ1*01	1087
  766	gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01	839	gnl|Fabrus|L11_IGKJ1*01	1087
  767	gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01	960	gnl|Fabrus|L11_IGKJ1*01	1087
  768	gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01	844	gnl|Fabrus|L11_IGKJ1*01	1087
  769	gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01	863	gnl|Fabrus|L12_IGKJ1*01	1088
  770	gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01	866	gnl|Fabrus|L12_IGKJ1*01	1088
  771	gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01	870	gnl|Fabrus|L12_IGKJ1*01	1088
  772	gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01	872	gnl|Fabrus|L12_IGKJ1*01	1088
  773	gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01	874	gnl|Fabrus|L12_IGKJ1*01	1088
  774	gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01	876	gnl|Fabrus|L12_IGKJ1*01	1088
  775	gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01	877	gnl|Fabrus|L12_IGKJ1*01	1088
  776	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01	880	gnl|Fabrus|L12_IGKJ1*01	1088
  777	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01	881	gnl|Fabrus|L12_IGKJ1*01	1088
  778	gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01	770	gnl|Fabrus|L12_IGKJ1*01	1088
  779	gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01	771	gnl|Fabrus|L12_IGKJ1*01	1088
  780	gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01	772	gnl|Fabrus|L12_IGKJ1*01	1088
  781	gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01	773	gnl|Fabrus|L12_IGKJ1*01	1088
  782	gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01	774	gnl|Fabrus|L12_IGKJ1*01	1088
  783	gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01	776	gnl|Fabrus|L12_IGKJ1*01	1088
  784	gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01	777	gnl|Fabrus|L12_IGKJ1*01	1088
  785	gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01	779	gnl|Fabrus|L12_IGKJ1*01	1088
  786	gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01	781	gnl|Fabrus|L12_IGKJ1*01	1088
  787	gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01	1017	gnl|Fabrus|L12_IGKJ1*01	1088
  788	gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01	1018	gnl|Fabrus|L12_IGKJ1*01	1088
  789	gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01	1019	gnl|Fabrus|L12_IGKJ1*01	1088
  790	gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01	1021	gnl|Fabrus|L12_IGKJ1*01	1088
  791	gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01	1022	gnl|Fabrus|L12_IGKJ1*01	1088
  792	gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01	1023	gnl|Fabrus|L12_IGKJ1*01	1088
  793	gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01	1024	gnl|Fabrus|L12_IGKJ1*01	1088
  794	gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01	1026	gnl|Fabrus|L12_IGKJ1*01	1088
  795	gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01	789	gnl|Fabrus|L12_IGKJ1*01	1088
  796	gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01	791	gnl|Fabrus|L12_IGKJ1*01	1088
  797	gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01	792	gnl|Fabrus|L12_IGKJ1*01	1088
  798	gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01	794	gnl|Fabrus|L12_IGKJ1*01	1088
  799	gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01	796	gnl|Fabrus|L12_IGKJ1*01	1088
  800	gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01	797	gnl|Fabrus|L12_IGKJ1*01	1088
  801	gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01	798	gnl|Fabrus|L12_IGKJ1*01	1088
  802	gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01	1044	gnl|Fabrus|L12_IGKJ1*01	1088
  803	gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01	1046	gnl|Fabrus|L12_IGKJ1*01	1088
  804	gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01	1048	gnl|Fabrus|L12_IGKJ1*01	1088
  805	gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01	1049	gnl|Fabrus|L12_IGKJ1*01	1088
  806	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1050	gnl|Fabrus|L12_IGKJ1*01	1088
  807	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1051	gnl|Fabrus|L12_IGKJ1*01	1088
  808	gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01	1052	gnl|Fabrus|L12_IGKJ1*01	1088
  809	gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01	1053	gnl|Fabrus|L12_IGKJ1*01	1088
  810	gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01	1054	gnl|Fabrus|L12_IGKJ1*01	1088
  811	gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01	1056	gnl|Fabrus|L12_IGKJ1*01	1088
  812	gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01	1059	gnl|Fabrus|L12_IGKJ1*01	1088
  813	gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01	1061	gnl|Fabrus|L12_IGKJ1*01	1088
  814	gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01	1060	gnl|Fabrus|L12_IGKJ1*01	1088
  815	gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01	1062	gnl|Fabrus|L12_IGKJ1*01	1088
  816	gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01	1063	gnl|Fabrus|L12_IGKJ1*01	1088
  817	gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01	1064	gnl|Fabrus|L12_IGKJ1*01	1088
  818	gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01	1065	gnl|Fabrus|L12_IGKJ1*01	1088
  819	gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01	1043	gnl|Fabrus|L12_IGKJ1*01	1088
  820	gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01	923	gnl|Fabrus|L12_IGKJ1*01	1088
  821	gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01	893	gnl|Fabrus|L12_IGKJ1*01	1088
  822	gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01	949	gnl|Fabrus|L12_IGKJ1*01	1088
  823	gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01	938	gnl|Fabrus|L12_IGKJ1*01	1088
  824	gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01	804	gnl|Fabrus|L12_IGKJ1*01	1088
  825	gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01	811	gnl|Fabrus|L12_IGKJ1*01	1088
  826	gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01	835	gnl|Fabrus|L12_IGKJ1*01	1088
  827	gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01	833	gnl|Fabrus|L12_IGKJ1*01	1088
  828	gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01	930	gnl|Fabrus|L12_IGKJ1*01	1088
  829	gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01	931	gnl|Fabrus|L12_IGKJ1*01	1088
  830	gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01	967	gnl|Fabrus|L12_IGKJ1*01	1088
  831	gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01	969	gnl|Fabrus|L12_IGKJ1*01	1088
  832	gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01	977	gnl|Fabrus|L12_IGKJ1*01	1088
  833	gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01	976	gnl|Fabrus|L12_IGKJ1*01	1088
  834	gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01	918	gnl|Fabrus|L12_IGKJ1*01	1088
  835	gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01	921	gnl|Fabrus|L12_IGKJ1*01	1088
  836	gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01	992	gnl|Fabrus|L12_IGKJ1*01	1088
  837	gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01	989	gnl|Fabrus|L12_IGKJ1*01	1088
  838	gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01	995	gnl|Fabrus|L12_IGKJ1*01	1088
  839	gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01	1030	gnl|Fabrus|L12_IGKJ1*01	1088
  840	gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01	1034	gnl|Fabrus|L12_IGKJ1*01	1088
  841	gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01	728	gnl|Fabrus|L12_IGKJ1*01	1088
  842	gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01	735	gnl|Fabrus|L12_IGKJ1*01	1088
  843	gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01	729	gnl|Fabrus|L12_IGKJ1*01	1088
  844	gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01	743	gnl|Fabrus|L12_IGKJ1*01	1088
  845	gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01	748	gnl|Fabrus|L12_IGKJ1*01	1088
  846	gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01	754	gnl|Fabrus|L12_IGKJ1*01	1088
  847	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01	1068	gnl|Fabrus|L12_IGKJ1*01	1088
  848	gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01	810	gnl|Fabrus|L12_IGKJ1*01	1088
  849	gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01	764	gnl|Fabrus|L12_IGKJ1*01	1088
  850	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01	1067	gnl|Fabrus|L12_IGKJ1*01	1088
  851	gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01	1002	gnl|Fabrus|L12_IGKJ1*01	1088
  852	gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01	1008	gnl|Fabrus|L12_IGKJ1*01	1088
  853	gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01	803	gnl|Fabrus|L12_IGKJ1*01	1088
  854	gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01	783	gnl|Fabrus|L12_IGKJ1*01	1088
  855	gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01	808	gnl|Fabrus|L12_IGKJ1*01	1088
  856	gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01	907	gnl|Fabrus|L12_IGKJ1*01	1088
  857	gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01	838	gnl|Fabrus|L12_IGKJ1*01	1088
  858	gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01	974	gnl|Fabrus|L12_IGKJ1*01	1088
  859	gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01	816	gnl|Fabrus|L12_IGKJ1*01	1088
  860	gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01	820	gnl|Fabrus|L12_IGKJ1*01	1088
  861	gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01	852	gnl|Fabrus|L12_IGKJ1*01	1088
  862	gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01	839	gnl|Fabrus|L12_IGKJ1*01	1088
  863	gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01	960	gnl|Fabrus|L12_IGKJ1*01	1088
  864	gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01	844	gnl|Fabrus|L12_IGKJ1*01	1088
  865	gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01	863	gnl|Fabrus|O1_IGKJ1*01	1100
  866	gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01	866	gnl|Fabrus|O1_IGKJ1*01	1100
  867	gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01	870	gnl|Fabrus|O1_IGKJ1*01	1100
  868	gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01	872	gnl|Fabrus|O1_IGKJ1*01	1100
  869	gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01	874	gnl|Fabrus|O1_IGKJ1*01	1100
  870	gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01	876	gnl|Fabrus|O1_IGKJ1*01	1100
  871	gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01	877	gnl|Fabrus|O1_IGKJ1*01	1100
  872	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01	880	gnl|Fabrus|O1_IGKJ1*01	1100
  873	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01	881	gnl|Fabrus|O1_IGKJ1*01	1100
  874	gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01	770	gnl|Fabrus|O1_IGKJ1*01	1100
  875	gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01	771	gnl|Fabrus|O1_IGKJ1*01	1100
  876	gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01	772	gnl|Fabrus|O1_IGKJ1*01	1100
  877	gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01	773	gnl|Fabrus|O1_IGKJ1*01	1100
  878	gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01	774	gnl|Fabrus|O1_IGKJ1*01	1100
  879	gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01	776	gnl|Fabrus|O1_IGKJ1*01	1100
  880	gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01	777	gnl|Fabrus|O1_IGKJ1*01	1100
  881	gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01	779	gnl|Fabrus|O1_IGKJ1*01	1100
  882	gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01	781	gnl|Fabrus|O1_IGKJ1*01	1100
  883	gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01	1017	gnl|Fabrus|O1_IGKJ1*01	1100
  884	gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01	1018	gnl|Fabrus|O1_IGKJ1*01	1100
  885	gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01	1019	gnl|Fabrus|O1_IGKJ1*01	1100
  886	gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01	1021	gnl|Fabrus|O1_IGKJ1*01	1100
  887	gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01	1022	gnl|Fabrus|O1_IGKJ1*01	1100
  888	gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01	1023	gnl|Fabrus|O1_IGKJ1*01	1100
  889	gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01	1024	gnl|Fabrus|O1_IGKJ1*01	1100
  890	gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01	1026	gnl|Fabrus|O1_IGKJ1*01	1100
  891	gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01	789	gnl|Fabrus|O1_IGKJ1*01	1100
  892	gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01	791	gnl|Fabrus|O1_IGKJ1*01	1100
  893	gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01	792	gnl|Fabrus|O1_IGKJ1*01	1100
  894	gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01	794	gnl|Fabrus|O1_IGKJ1*01	1100
  895	gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01	796	gnl|Fabrus|O1_IGKJ1*01	1100
  896	gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01	797	gnl|Fabrus|O1_IGKJ1*01	1100
  897	gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01	798	gnl|Fabrus|O1_IGKJ1*01	1100
  898	gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01	1044	gnl|Fabrus|O1_IGKJ1*01	1100
  899	gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01	1046	gnl|Fabrus|O1_IGKJ1*01	1100
  900	gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01	1048	gnl|Fabrus|O1_IGKJ1*01	1100
  901	gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01	1049	gnl|Fabrus|O1_IGKJ1*01	1100
  902	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1050	gnl|Fabrus|O1_IGKJ1*01	1100
  903	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1051	gnl|Fabrus|O1_IGKJ1*01	1100
  904	gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01	1052	gnl|Fabrus|O1_IGKJ1*01	1100
  905	gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01	1053	gnl|Fabrus|O1_IGKJ1*01	1100
  906	gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01	1054	gnl|Fabrus|O1_IGKJ1*01	1100
  907	gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01	1056	gnl|Fabrus|O1_IGKJ1*01	1100
  908	gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01	1059	gnl|Fabrus|O1_IGKJ1*01	1100
  909	gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01	1061	gnl|Fabrus|O1_IGKJ1*01	1100
  910	gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01	1060	gnl|Fabrus|O1_IGKJ1*01	1100
  911	gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01	1062	gnl|Fabrus|O1_IGKJ1*01	1100
  912	gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01	1063	gnl|Fabrus|O1_IGKJ1*01	1100
  913	gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01	1064	gnl|Fabrus|O1_IGKJ1*01	1100
  914	gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01	1065	gnl|Fabrus|O1_IGKJ1*01	1100
  915	gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01	1043	gnl|Fabrus|O1_IGKJ1*01	1100
  916	gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01	923	gnl|Fabrus|O1_IGKJ1*01	1100
  917	gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01	893	gnl|Fabrus|O1_IGKJ1*01	1100
  918	gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01	949	gnl|Fabrus|O1_IGKJ1*01	1100
  919	gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01	938	gnl|Fabrus|O1_IGKJ1*01	1100
  920	gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01	804	gnl|Fabrus|O1_IGKJ1*01	1100
  921	gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01	811	gnl|Fabrus|O1_IGKJ1*01	1100
  922	gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01	835	gnl|Fabrus|O1_IGKJ1*01	1100
  923	gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01	833	gnl|Fabrus|O1_IGKJ1*01	1100
  924	gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01	930	gnl|Fabrus|O1_IGKJ1*01	1100
  925	gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01	931	gnl|Fabrus|O1_IGKJ1*01	1100
  926	gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01	967	gnl|Fabrus|O1_IGKJ1*01	1100
  927	gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01	969	gnl|Fabrus|O1_IGKJ1*01	1100
  928	gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01	977	gnl|Fabrus|O1_IGKJ1*01	1100
  929	gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01	976	gnl|Fabrus|O1_IGKJ1*01	1100
  930	gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01	918	gnl|Fabrus|O1_IGKJ1*01	1100
  931	gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01	921	gnl|Fabrus|O1_IGKJ1*01	1100
  932	gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01	992	gnl|Fabrus|O1_IGKJ1*01	1100
  933	gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01	989	gnl|Fabrus|O1_IGKJ1*01	1100
  934	gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01	995	gnl|Fabrus|O1_IGKJ1*01	1100
  935	gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01	1030	gnl|Fabrus|O1_IGKJ1*01	1100
  936	gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01	1034	gnl|Fabrus|O1_IGKJ1*01	1100
  937	gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01	728	gnl|Fabrus|O1_IGKJ1*01	1100
  938	gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01	735	gnl|Fabrus|O1_IGKJ1*01	1100
  939	gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01	729	gnl|Fabrus|O1_IGKJ1*01	1100
  940	gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01	743	gnl|Fabrus|O1_IGKJ1*01	1100
  941	gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01	748	gnl|Fabrus|O1_IGKJ1*01	1100
  942	gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01	754	gnl|Fabrus|O1_IGKJ1*01	1100
  943	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01	1068	gnl|Fabrus|O1_IGKJ1*01	1100
  944	gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01	810	gnl|Fabrus|O1_IGKJ1*01	1100
  945	gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01	764	gnl|Fabrus|O1_IGKJ1*01	1100
  946	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01	1067	gnl|Fabrus|O1_IGKJ1*01	1100
  947	gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01	1002	gnl|Fabrus|O1_IGKJ1*01	1100
  948	gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01	1008	gnl|Fabrus|O1_IGKJ1*01	1100
  949	gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01	803	gnl|Fabrus|O1_IGKJ1*01	1100
  950	gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01	783	gnl|Fabrus|O1_IGKJ1*01	1100
  951	gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01	808	gnl|Fabrus|O1_IGKJ1*01	1100
  952	gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01	907	gnl|Fabrus|O1_IGKJ1*01	1100
  953	gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01	838	gnl|Fabrus|O1_IGKJ1*01	1100
  954	gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01	974	gnl|Fabrus|O1_IGKJ1*01	1100
  955	gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01	816	gnl|Fabrus|O1_IGKJ1*01	1100
  956	gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01	820	gnl|Fabrus|O1_IGKJ1*01	1100
  957	gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01	852	gnl|Fabrus|O1_IGKJ1*01	1100
  958	gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01	839	gnl|Fabrus|O1_IGKJ1*01	1100
  959	gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01	960	gnl|Fabrus|O1_IGKJ1*01	1100
  960	gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01	844	gnl|Fabrus|O1_IGKJ1*01	1100
  961	gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01	863	gnl|Fabrus|L25_IGKJ3*01	1094
  962	gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01	866	gnl|Fabrus|L25_IGKJ3*01	1094
  963	gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01	870	gnl|Fabrus|L25_IGKJ3*01	1094
  964	gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01	872	gnl|Fabrus|L25_IGKJ3*01	1094
  965	gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01	874	gnl|Fabrus|L25_IGKJ3*01	1094
  966	gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01	876	gnl|Fabrus|L25_IGKJ3*01	1094
  967	gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01	877	gnl|Fabrus|L25_IGKJ3*01	1094
  968	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01	880	gnl|Fabrus|L25_IGKJ3*01	1094
  969	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01	881	gnl|Fabrus|L25_IGKJ3*01	1094
  970	gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01	770	gnl|Fabrus|L25_IGKJ3*01	1094
  971	gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01	771	gnl|Fabrus|L25_IGKJ3*01	1094
  972	gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01	772	gnl|Fabrus|L25_IGKJ3*01	1094
  973	gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01	773	gnl|Fabrus|L25_IGKJ3*01	1094
  974	gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01	774	gnl|Fabrus|L25_IGKJ3*01	1094
  975	gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01	776	gnl|Fabrus|L25_IGKJ3*01	1094
  976	gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01	777	gnl|Fabrus|L25_IGKJ3*01	1094
  977	gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01	779	gnl|Fabrus|L25_IGKJ3*01	1094
  978	gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01	781	gnl|Fabrus|L25_IGKJ3*01	1094
  979	gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01	1017	gnl|Fabrus|L25_IGKJ3*01	1094
  980	gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01	1018	gnl|Fabrus|L25_IGKJ3*01	1094
  981	gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01	1019	gnl|Fabrus|L25_IGKJ3*01	1094
  982	gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01	1021	gnl|Fabrus|L25_IGKJ3*01	1094
  983	gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01	1022	gnl|Fabrus|L25_IGKJ3*01	1094
  984	gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01	1023	gnl|Fabrus|L25_IGKJ3*01	1094
  985	gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01	1024	gnl|Fabrus|L25_IGKJ3*01	1094
  986	gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01	1026	gnl|Fabrus|L25_IGKJ3*01	1094
  987	gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01	789	gnl|Fabrus|L25_IGKJ3*01	1094
  988	gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01	791	gnl|Fabrus|L25_IGKJ3*01	1094
  989	gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01	792	gnl|Fabrus|L25_IGKJ3*01	1094
  990	gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01	794	gnl|Fabrus|L25_IGKJ3*01	1094
  991	gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01	796	gnl|Fabrus|L25_IGKJ3*01	1094
  992	gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01	797	gnl|Fabrus|L25_IGKJ3*01	1094
  993	gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01	798	gnl|Fabrus|L25_IGKJ3*01	1094
  994	gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01	1044	gnl|Fabrus|L25_IGKJ3*01	1094
  995	gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01	1046	gnl|Fabrus|L25_IGKJ3*01	1094
  996	gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01	1048	gnl|Fabrus|L25_IGKJ3*01	1094
  997	gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01	1049	gnl|Fabrus|L25_IGKJ3*01	1094
  998	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1050	gnl|Fabrus|L25_IGKJ3*01	1094
  999	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1051	gnl|Fabrus|L25_IGKJ3*01	1094
  1000	gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01	1052	gnl|Fabrus|L25_IGKJ3*01	1094
  1001	gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01	1053	gnl|Fabrus|L25_IGKJ3*01	1094
  1002	gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01	1054	gnl|Fabrus|L25_IGKJ3*01	1094
  1003	gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01	1056	gnl|Fabrus|L25_IGKJ3*01	1094
  1004	gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01	1059	gnl|Fabrus|L25_IGKJ3*01	1094
  1005	gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01	1061	gnl|Fabrus|L25_IGKJ3*01	1094
  1006	gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01	1060	gnl|Fabrus|L25_IGKJ3*01	1094
  1007	gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01	1062	gnl|Fabrus|L25_IGKJ3*01	1094
  1008	gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01	1063	gnl|Fabrus|L25_IGKJ3*01	1094
  1009	gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01	1064	gnl|Fabrus|L25_IGKJ3*01	1094
  1010	gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01	1065	gnl|Fabrus|L25_IGKJ3*01	1094
  1011	gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01	1043	gnl|Fabrus|L25_IGKJ3*01	1094
  1012	gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01	923	gnl|Fabrus|L25_IGKJ3*01	1094
  1013	gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01	893	gnl|Fabrus|L25_IGKJ3*01	1094
  1014	gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01	949	gnl|Fabrus|L25_IGKJ3*01	1094
  1015	gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01	938	gnl|Fabrus|L25_IGKJ3*01	1094
  1016	gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01	804	gnl|Fabrus|L25_IGKJ3*01	1094
  1017	gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01	811	gnl|Fabrus|L25_IGKJ3*01	1094
  1018	gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01	835	gnl|Fabrus|L25_IGKJ3*01	1094
  1019	gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01	833	gnl|Fabrus|L25_IGKJ3*01	1094
  1020	gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01	930	gnl|Fabrus|L25_IGKJ3*01	1094
  1021	gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01	931	gnl|Fabrus|L25_IGKJ3*01	1094
  1022	gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01	967	gnl|Fabrus|L25_IGKJ3*01	1094
  1023	gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01	969	gnl|Fabrus|L25_IGKJ3*01	1094
  1024	gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01	977	gnl|Fabrus|L25_IGKJ3*01	1094
  1025	gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01	976	gnl|Fabrus|L25_IGKJ3*01	1094
  1026	gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01	918	gnl|Fabrus|L25_IGKJ3*01	1094
  1027	gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01	921	gnl|Fabrus|L25_IGKJ3*01	1094
  1028	gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01	992	gnl|Fabrus|L25_IGKJ3*01	1094
  1029	gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01	989	gnl|Fabrus|L25_IGKJ3*01	1094
  1030	gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01	995	gnl|Fabrus|L25_IGKJ3*01	1094
  1031	gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01	1030	gnl|Fabrus|L25_IGKJ3*01	1094
  1032	gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01	1034	gnl|Fabrus|L25_IGKJ3*01	1094
  1033	gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01	728	gnl|Fabrus|L25_IGKJ3*01	1094
  1034	gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01	735	gnl|Fabrus|L25_IGKJ3*01	1094
  1035	gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01	729	gnl|Fabrus|L25_IGKJ3*01	1094
  1036	gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01	743	gnl|Fabrus|L25_IGKJ3*01	1094
  1037	gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01	748	gnl|Fabrus|L25_IGKJ3*01	1094
  1038	gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01	754	gnl|Fabrus|L25_IGKJ3*01	1094
  1039	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01	1068	gnl|Fabrus|L25_IGKJ3*01	1094
  1040	gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01	810	gnl|Fabrus|L25_IGKJ3*01	1094
  1041	gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01	764	gnl|Fabrus|L25_IGKJ3*01	1094
  1042	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01	1067	gnl|Fabrus|L25_IGKJ3*01	1094
  1043	gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01	1002	gnl|Fabrus|L25_IGKJ3*01	1094
  1044	gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01	1008	gnl|Fabrus|L25_IGKJ3*01	1094
  1045	gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01	803	gnl|Fabrus|L25_IGKJ3*01	1094
  1046	gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01	783	gnl|Fabrus|L25_IGKJ3*01	1094
  1047	gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01	808	gnl|Fabrus|L25_IGKJ3*01	1094
  1048	gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01	907	gnl|Fabrus|L25_IGKJ3*01	1094
  1049	gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01	838	gnl|Fabrus|L25_IGKJ3*01	1094
  1050	gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01	974	gnl|Fabrus|L25_IGKJ3*01	1094
  1051	gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01	816	gnl|Fabrus|L25_IGKJ3*01	1094
  1052	gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01	820	gnl|Fabrus|L25_IGKJ3*01	1094
  1053	gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01	852	gnl|Fabrus|L25_IGKJ3*01	1094
  1054	gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01	839	gnl|Fabrus|L25_IGKJ3*01	1094
  1055	gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01	960	gnl|Fabrus|L25_IGKJ3*01	1094
  1056	gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01	844	gnl|Fabrus|L25_IGKJ3*01	1094
  1057	gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01	863	gnl|Fabrus|A27_IGKJ1*01	1080
  1058	gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01	866	gnl|Fabrus|A27_IGKJ1*01	1080
  1059	gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01	870	gnl|Fabrus|A27_IGKJ1*01	1080
  1060	gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01	872	gnl|Fabrus|A27_IGKJ1*01	1080
  1061	gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01	874	gnl|Fabrus|A27_IGKJ1*01	1080
  1062	gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01	876	gnl|Fabrus|A27_IGKJ1*01	1080
  1063	gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01	877	gnl|Fabrus|A27_IGKJ1*01	1080
  1064	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01	880	gnl|Fabrus|A27_IGKJ1*01	1080
  1065	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01	881	gnl|Fabrus|A27_IGKJ1*01	1080
  1066	gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01	770	gnl|Fabrus|A27_IGKJ1*01	1080
  1067	gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01	771	gnl|Fabrus|A27_IGKJ1*01	1080
  1068	gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01	772	gnl|Fabrus|A27_IGKJ1*01	1080
  1069	gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01	773	gnl|Fabrus|A27_IGKJ1*01	1080
  1070	gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01	774	gnl|Fabrus|A27_IGKJ1*01	1080
  1071	gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01	776	gnl|Fabrus|A27_IGKJ1*01	1080
  1072	gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01	777	gnl|Fabrus|A27_IGKJ1*01	1080
  1073	gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01	779	gnl|Fabrus|A27_IGKJ1*01	1080
  1074	gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01	781	gnl|Fabrus|A27_IGKJ1*01	1080
  1075	gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01	1017	gnl|Fabrus|A27_IGKJ1*01	1080
  1076	gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01	1018	gnl|Fabrus|A27_IGKJ1*01	1080
  1077	gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01	1019	gnl|Fabrus|A27_IGKJ1*01	1080
  1078	gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01	1021	gnl|Fabrus|A27_IGKJ1*01	1080
  1079	gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01	1022	gnl|Fabrus|A27_IGKJ1*01	1080
  1080	gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01	1023	gnl|Fabrus|A27_IGKJ1*01	1080
  1081	gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01	1024	gnl|Fabrus|A27_IGKJ1*01	1080
  1082	gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01	1026	gnl|Fabrus|A27_IGKJ1*01	1080
  1083	gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01	789	gnl|Fabrus|A27_IGKJ1*01	1080
  1084	gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01	791	gnl|Fabrus|A27_IGKJ1*01	1080
  1085	gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01	792	gnl|Fabrus|A27_IGKJ1*01	1080
  1086	gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01	794	gnl|Fabrus|A27_IGKJ1*01	1080
  1087	gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01	796	gnl|Fabrus|A27_IGKJ1*01	1080
  1088	gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01	797	gnl|Fabrus|A27_IGKJ1*01	1080
  1089	gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01	798	gnl|Fabrus|A27_IGKJ1*01	1080
  1090	gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01	1044	gnl|Fabrus|A27_IGKJ1*01	1080
  1091	gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01	1046	gnl|Fabrus|A27_IGKJ1*01	1080
  1092	gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01	1048	gnl|Fabrus|A27_IGKJ1*01	1080
  1093	gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01	1049	gnl|Fabrus|A27_IGKJ1*01	1080
  1094	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1050	gnl|Fabrus|A27_IGKJ1*01	1080
  1095	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1051	gnl|Fabrus|A27_IGKJ1*01	1080
  1096	gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01	1052	gnl|Fabrus|A27_IGKJ1*01	1080
  1097	gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01	1053	gnl|Fabrus|A27_IGKJ1*01	1080
  1098	gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01	1054	gnl|Fabrus|A27_IGKJ1*01	1080
  1099	gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01	1056	gnl|Fabrus|A27_IGKJ1*01	1080
  1100	gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01	1059	gnl|Fabrus|A27_IGKJ1*01	1080
  1101	gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01	1061	gnl|Fabrus|A27_IGKJ1*01	1080
  1102	gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01	1060	gnl|Fabrus|A27_IGKJ1*01	1080
  1103	gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01	1062	gnl|Fabrus|A27_IGKJ1*01	1080
  1104	gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01	1063	gnl|Fabrus|A27_IGKJ1*01	1080
  1105	gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01	1064	gnl|Fabrus|A27_IGKJ1*01	1080
  1106	gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01	1065	gnl|Fabrus|A27_IGKJ1*01	1080
  1107	gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01	1043	gnl|Fabrus|A27_IGKJ1*01	1080
  1108	gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01	923	gnl|Fabrus|A27_IGKJ1*01	1080
  1109	gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01	893	gnl|Fabrus|A27_IGKJ1*01	1080
  1110	gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01	949	gnl|Fabrus|A27_IGKJ1*01	1080
  1111	gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01	938	gnl|Fabrus|A27_IGKJ1*01	1080
  1112	gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01	804	gnl|Fabrus|A27_IGKJ1*01	1080
  1113	gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01	811	gnl|Fabrus|A27_IGKJ1*01	1080
  1114	gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01	835	gnl|Fabrus|A27_IGKJ1*01	1080
  1115	gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01	833	gnl|Fabrus|A27_IGKJ1*01	1080
  1116	gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01	930	gnl|Fabrus|A27_IGKJ1*01	1080
  1117	gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01	931	gnl|Fabrus|A27_IGKJ1*01	1080
  1118	gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01	967	gnl|Fabrus|A27_IGKJ1*01	1080
  1119	gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01	969	gnl|Fabrus|A27_IGKJ1*01	1080
  1120	gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01	977	gnl|Fabrus|A27_IGKJ1*01	1080
  1121	gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01	976	gnl|Fabrus|A27_IGKJ1*01	1080
  1122	gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01	918	gnl|Fabrus|A27_IGKJ1*01	1080
  1123	gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01	921	gnl|Fabrus|A27_IGKJ1*01	1080
  1124	gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01	992	gnl|Fabrus|A27_IGKJ1*01	1080
  1125	gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01	989	gnl|Fabrus|A27_IGKJ1*01	1080
  1126	gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01	995	gnl|Fabrus|A27_IGKJ1*01	1080
  1127	gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01	1030	gnl|Fabrus|A27_IGKJ1*01	1080
  1128	gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01	1034	gnl|Fabrus|A27_IGKJ1*01	1080
  1129	gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01	728	gnl|Fabrus|A27_IGKJ1*01	1080
  1130	gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01	735	gnl|Fabrus|A27_IGKJ1*01	1080
  1131	gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01	729	gnl|Fabrus|A27_IGKJ1*01	1080
  1132	gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01	743	gnl|Fabrus|A27_IGKJ1*01	1080
  1133	gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01	748	gnl|Fabrus|A27_IGKJ1*01	1080
  1134	gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01	754	gnl|Fabrus|A27_IGKJ1*01	1080
  1135	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01	1068	gnl|Fabrus|A27_IGKJ1*01	1080
  1136	gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01	810	gnl|Fabrus|A27_IGKJ1*01	1080
  1137	gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01	764	gnl|Fabrus|A27_IGKJ1*01	1080
  1138	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01	1067	gnl|Fabrus|A27_IGKJ1*01	1080
  1139	gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01	1002	gnl|Fabrus|A27_IGKJ1*01	1080
  1140	gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01	1008	gnl|Fabrus|A27_IGKJ1*01	1080
  1141	gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01	803	gnl|Fabrus|A27_IGKJ1*01	1080
  1142	gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01	783	gnl|Fabrus|A27_IGKJ1*01	1080
  1143	gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01	808	gnl|Fabrus|A27_IGKJ1*01	1080
  1144	gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01	907	gnl|Fabrus|A27_IGKJ1*01	1080
  1145	gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01	838	gnl|Fabrus|A27_IGKJ1*01	1080
  1146	gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01	974	gnl|Fabrus|A27_IGKJ1*01	1080
  1147	gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01	816	gnl|Fabrus|A27_IGKJ1*01	1080
  1148	gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01	820	gnl|Fabrus|A27_IGKJ1*01	1080
  1149	gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01	852	gnl|Fabrus|A27_IGKJ1*01	1080
  1150	gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01	839	gnl|Fabrus|A27_IGKJ1*01	1080
  1151	gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01	960	gnl|Fabrus|A27_IGKJ1*01	1080
  1152	gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01	844	gnl|Fabrus|A27_IGKJ1*01	1080
  1153	gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01	863	gnl|Fabrus|A2_IGKJ1*01	1076
  1154	gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01	866	gnl|Fabrus|A2_IGKJ1*01	1076
  1155	gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01	870	gnl|Fabrus|A2_IGKJ1*01	1076
  1156	gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01	872	gnl|Fabrus|A2_IGKJ1*01	1076
  1157	gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01	874	gnl|Fabrus|A2_IGKJ1*01	1076
  1158	gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01	876	gnl|Fabrus|A2_IGKJ1*01	1076
  1159	gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01	877	gnl|Fabrus|A2_IGKJ1*01	1076
  1160	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01	880	gnl|Fabrus|A2_IGKJ1*01	1076
  1161	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01	881	gnl|Fabrus|A2_IGKJ1*01	1076
  1162	gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01	770	gnl|Fabrus|A2_IGKJ1*01	1076
  1163	gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01	771	gnl|Fabrus|A2_IGKJ1*01	1076
  1164	gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01	772	gnl|Fabrus|A2_IGKJ1*01	1076
  1165	gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01	773	gnl|Fabrus|A2_IGKJ1*01	1076
  1166	gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01	774	gnl|Fabrus|A2_IGKJ1*01	1076
  1167	gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01	776	gnl|Fabrus|A2_IGKJ1*01	1076
  1168	gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01	777	gnl|Fabrus|A2_IGKJ1*01	1076
  1169	gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01	779	gnl|Fabrus|A2_IGKJ1*01	1076
  1170	gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01	781	gnl|Fabrus|A2_IGKJ1*01	1076
  1171	gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01	1017	gnl|Fabrus|A2_IGKJ1*01	1076
  1172	gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01	1018	gnl|Fabrus|A2_IGKJ1*01	1076
  1173	gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01	1019	gnl|Fabrus|A2_IGKJ1*01	1076
  1174	gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01	1021	gnl|Fabrus|A2_IGKJ1*01	1076
  1175	gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01	1022	gnl|Fabrus|A2_IGKJ1*01	1076
  1176	gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01	1023	gnl|Fabrus|A2_IGKJ1*01	1076
  1177	gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01	1024	gnl|Fabrus|A2_IGKJ1*01	1076
  1178	gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01	1026	gnl|Fabrus|A2_IGKJ1*01	1076
  1179	gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01	789	gnl|Fabrus|A2_IGKJ1*01	1076
  1180	gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01	791	gnl|Fabrus|A2_IGKJ1*01	1076
  1181	gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01	792	gnl|Fabrus|A2_IGKJ1*01	1076
  1182	gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01	794	gnl|Fabrus|A2_IGKJ1*01	1076
  1183	gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01	796	gnl|Fabrus|A2_IGKJ1*01	1076
  1184	gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01	797	gnl|Fabrus|A2_IGKJ1*01	1076
  1185	gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01	798	gnl|Fabrus|A2_IGKJ1*01	1076
  1186	gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01	1044	gnl|Fabrus|A2_IGKJ1*01	1076
  1187	gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01	1046	gnl|Fabrus|A2_IGKJ1*01	1076
  1188	gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01	1048	gnl|Fabrus|A2_IGKJ1*01	1076
  1189	gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01	1049	gnl|Fabrus|A2_IGKJ1*01	1076
  1190	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1050	gnl|Fabrus|A2_IGKJ1*01	1076
  1191	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1051	gnl|Fabrus|A2_IGKJ1*01	1076
  1192	gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01	1052	gnl|Fabrus|A2_IGKJ1*01	1076
  1193	gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01	1053	gnl|Fabrus|A2_IGKJ1*01	1076
  1194	gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01	1054	gnl|Fabrus|A2_IGKJ1*01	1076
  1195	gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01	1056	gnl|Fabrus|A2_IGKJ1*01	1076
  1196	gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01	1059	gnl|Fabrus|A2_IGKJ1*01	1076
  1197	gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01	1061	gnl|Fabrus|A2_IGKJ1*01	1076
  1198	gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01	1060	gnl|Fabrus|A2_IGKJ1*01	1076
  1199	gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01	1062	gnl|Fabrus|A2_IGKJ1*01	1076
  1200	gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01	1063	gnl|Fabrus|A2_IGKJ1*01	1076
  1201	gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01	1064	gnl|Fabrus|A2_IGKJ1*01	1076
  1202	gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01	1065	gnl|Fabrus|A2_IGKJ1*01	1076
  1203	gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01	1043	gnl|Fabrus|A2_IGKJ1*01	1076
  1204	gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01	923	gnl|Fabrus|A2_IGKJ1*01	1076
  1205	gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01	893	gnl|Fabrus|A2_IGKJ1*01	1076
  1206	gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01	949	gnl|Fabrus|A2_IGKJ1*01	1076
  1207	gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01	938	gnl|Fabrus|A2_IGKJ1*01	1076
  1208	gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01	804	gnl|Fabrus|A2_IGKJ1*01	1076
  1209	gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01	811	gnl|Fabrus|A2_IGKJ1*01	1076
  1210	gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01	835	gnl|Fabrus|A2_IGKJ1*01	1076
  1211	gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01	833	gnl|Fabrus|A2_IGKJ1*01	1076
  1212	gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01	930	gnl|Fabrus|A2_IGKJ1*01	1076
  1213	gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01	931	gnl|Fabrus|A2_IGKJ1*01	1076
  1214	gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01	967	gnl|Fabrus|A2_IGKJ1*01	1076
  1215	gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01	969	gnl|Fabrus|A2_IGKJ1*01	1076
  1216	gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01	977	gnl|Fabrus|A2_IGKJ1*01	1076
  1217	gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01	976	gnl|Fabrus|A2_IGKJ1*01	1076
  1218	gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01	918	gnl|Fabrus|A2_IGKJ1*01	1076
  1219	gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01	921	gnl|Fabrus|A2_IGKJ1*01	1076
  1220	gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01	992	gnl|Fabrus|A2_IGKJ1*01	1076
  1221	gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01	989	gnl|Fabrus|A2_IGKJ1*01	1076
  1222	gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01	995	gnl|Fabrus|A2_IGKJ1*01	1076
  1223	gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01	1030	gnl|Fabrus|A2_IGKJ1*01	1076
  1224	gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01	1034	gnl|Fabrus|A2_IGKJ1*01	1076
  1225	gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01	728	gnl|Fabrus|A2_IGKJ1*01	1076
  1226	gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01	735	gnl|Fabrus|A2_IGKJ1*01	1076
  1227	gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01	729	gnl|Fabrus|A2_IGKJ1*01	1076
  1228	gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01	743	gnl|Fabrus|A2_IGKJ1*01	1076
  1229	gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01	748	gnl|Fabrus|A2_IGKJ1*01	1076
  1230	gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01	754	gnl|Fabrus|A2_IGKJ1*01	1076
  1231	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01	1068	gnl|Fabrus|A2_IGKJ1*01	1076
  1232	gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01	810	gnl|Fabrus|A2_IGKJ1*01	1076
  1233	gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01	764	gnl|Fabrus|A2_IGKJ1*01	1076
  1234	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01	1067	gnl|Fabrus|A2_IGKJ1*01	1076
  1235	gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01	1002	gnl|Fabrus|A2_IGKJ1*01	1076
  1236	gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01	1008	gnl|Fabrus|A2_IGKJ1*01	1076
  1237	gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01	803	gnl|Fabrus|A2_IGKJ1*01	1076
  1238	gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01	783	gnl|Fabrus|A2_IGKJ1*01	1076
  1239	gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01	808	gnl|Fabrus|A2_IGKJ1*01	1076
  1240	gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01	907	gnl|Fabrus|A2_IGKJ1*01	1076
  1241	gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01	838	gnl|Fabrus|A2_IGKJ1*01	1076
  1242	gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01	974	gnl|Fabrus|A2_IGKJ1*01	1076
  1243	gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01	816	gnl|Fabrus|A2_IGKJ1*01	1076
  1244	gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01	820	gnl|Fabrus|A2_IGKJ1*01	1076
  1245	gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01	852	gnl|Fabrus|A2_IGKJ1*01	1076
  1246	gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01	839	gnl|Fabrus|A2_IGKJ1*01	1076
  1247	gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01	960	gnl|Fabrus|A2_IGKJ1*01	1076
  1248	gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01	844	gnl|Fabrus|A2_IGKJ1*01	1076
  1249	gnl|Fabrus|VH3-23_IGHD1-1*01_IGHJ4*01	863	gnl|Fabrus|HerceptinLC	1086
  1250	gnl|Fabrus|VH3-23_IGHD2-15*01_IGHJ4*01	866	gnl|Fabrus|HerceptinLC	1086
  1251	gnl|Fabrus|VH3-23_IGHD3-22*01_IGHJ4*01	870	gnl|Fabrus|HerceptinLC	1086
  1252	gnl|Fabrus|VH3-23_IGHD4-11*01_IGHJ4*01	872	gnl|Fabrus|HerceptinLC	1086
  1253	gnl|Fabrus|VH3-23_IGHD5-12*01_IGHJ4*01	874	gnl|Fabrus|HerceptinLC	1086
  1254	gnl|Fabrus|VH3-23_IGHD5-5*01_IGHJ4*01	876	gnl|Fabrus|HerceptinLC	1086
  1255	gnl|Fabrus|VH3-23_IGHD6-13*01_IGHJ4*01	877	gnl|Fabrus|HerceptinLC	1086
  1256	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ4*01	880	gnl|Fabrus|HerceptinLC	1086
  1257	gnl|Fabrus|VH3-23_IGHD7-27*01_IGHJ6*01	881	gnl|Fabrus|HerceptinLC	1086
  1258	gnl|Fabrus|VH1-69_IGHD1-14*01_IGHJ4*01	770	gnl|Fabrus|HerceptinLC	1086
  1259	gnl|Fabrus|VH1-69_IGHD2-2*01_IGHJ4*01	771	gnl|Fabrus|HerceptinLC	1086
  1260	gnl|Fabrus|VH1-69_IGHD2-8*01_IGHJ6*01	772	gnl|Fabrus|HerceptinLC	1086
  1261	gnl|Fabrus|VH1-69_IGHD3-16*01_IGHJ4*01	773	gnl|Fabrus|HerceptinLC	1086
  1262	gnl|Fabrus|VH1-69_IGHD3-3*01_IGHJ4*01	774	gnl|Fabrus|HerceptinLC	1086
  1263	gnl|Fabrus|VH1-69_IGHD4-17*01_IGHJ4*01	776	gnl|Fabrus|HerceptinLC	1086
  1264	gnl|Fabrus|VH1-69_IGHD5-12*01_IGHJ4*01	777	gnl|Fabrus|HerceptinLC	1086
  1265	gnl|Fabrus|VH1-69_IGHD6-19*01_IGHJ4*01	779	gnl|Fabrus|HerceptinLC	1086
  1266	gnl|Fabrus|VH1-69_IGHD7-27*01_IGHJ4*01	781	gnl|Fabrus|HerceptinLC	1086
  1267	gnl|Fabrus|VH4-34_IGHD1-7*01_IGHJ4*01	1017	gnl|Fabrus|HerceptinLC	1086
  1268	gnl|Fabrus|VH4-34_IGHD2-2*01_IGHJ4*01	1018	gnl|Fabrus|HerceptinLC	1086
  1269	gnl|Fabrus|VH4-34_IGHD3-16*01_IGHJ4*01	1019	gnl|Fabrus|HerceptinLC	1086
  1270	gnl|Fabrus|VH4-34_IGHD4-17*01_IGHJ4*01	1021	gnl|Fabrus|HerceptinLC	1086
  1271	gnl|Fabrus|VH4-34_IGHD5-12*01_IGHJ4*01	1022	gnl|Fabrus|HerceptinLC	1086
  1272	gnl|Fabrus|VH4-34_IGHD6-13*01_IGHJ4*01	1023	gnl|Fabrus|HerceptinLC	1086
  1273	gnl|Fabrus|VH4-34_IGHD6-25*01_IGHJ6*01	1024	gnl|Fabrus|HerceptinLC	1086
  1274	gnl|Fabrus|VH4-34_IGHD7-27*01_IGHJ4*01	1026	gnl|Fabrus|HerceptinLC	1086
  1275	gnl|Fabrus|VH2-26_IGHD1-20*01_IGHJ4*01	789	gnl|Fabrus|HerceptinLC	1086
  1276	gnl|Fabrus|VH2-26_IGHD2-2*01_IGHJ4*01	791	gnl|Fabrus|HerceptinLC	1086
  1277	gnl|Fabrus|VH2-26_IGHD3-10*01_IGHJ4*01	792	gnl|Fabrus|HerceptinLC	1086
  1278	gnl|Fabrus|VH2-26_IGHD4-11*01_IGHJ4*01	794	gnl|Fabrus|HerceptinLC	1086
  1279	gnl|Fabrus|VH2-26_IGHD5-18*01_IGHJ4*01	796	gnl|Fabrus|HerceptinLC	1086
  1280	gnl|Fabrus|VH2-26_IGHD6-13*01_IGHJ4*01	797	gnl|Fabrus|HerceptinLC	1086
  1281	gnl|Fabrus|VH2-26_IGHD7-27*01_IGHJ4*01	798	gnl|Fabrus|HerceptinLC	1086
  1282	gnl|Fabrus|VH5-51_IGHD1-14*01_IGHJ4*01	1044	gnl|Fabrus|HerceptinLC	1086
  1283	gnl|Fabrus|VH5-51_IGHD2-8*01_IGHJ4*01	1046	gnl|Fabrus|HerceptinLC	1086
  1284	gnl|Fabrus|VH5-51_IGHD3-3*01_IGHJ4*01	1048	gnl|Fabrus|HerceptinLC	1086
  1285	gnl|Fabrus|VH5-51_IGHD4-17*01_IGHJ4*01	1049	gnl|Fabrus|HerceptinLC	1086
  1286	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1050	gnl|Fabrus|HerceptinLC	1086
  1287	gnl|Fabrus|VH5-51_IGHD5-18*01_IGHJ4*01	1051	gnl|Fabrus|HerceptinLC	1086
  1288	gnl|Fabrus|VH5-51_IGHD6-25*01_IGHJ4*01	1052	gnl|Fabrus|HerceptinLC	1086
  1289	gnl|Fabrus|VH5-51_IGHD7-27*01_IGHJ4*01	1053	gnl|Fabrus|HerceptinLC	1086
  1290	gnl|Fabrus|VH6-1_IGHD1-1*01_IGHJ4*01	1054	gnl|Fabrus|HerceptinLC	1086
  1291	gnl|Fabrus|VH6-1_IGHD2-15*01_IGHJ4*01	1056	gnl|Fabrus|HerceptinLC	1086
  1292	gnl|Fabrus|VH6-1_IGHD3-3*01_IGHJ4*01	1059	gnl|Fabrus|HerceptinLC	1086
  1293	gnl|Fabrus|VH6-1_IGHD4-23*01_IGHJ4*01	1061	gnl|Fabrus|HerceptinLC	1086
  1294	gnl|Fabrus|VH6-1_IGHD4-11*01_IGHJ6*01	1060	gnl|Fabrus|HerceptinLC	1086
  1295	gnl|Fabrus|VH6-1_IGHD5-5*01_IGHJ4*01	1062	gnl|Fabrus|HerceptinLC	1086
  1296	gnl|Fabrus|VH6-1_IGHD6-13*01_IGHJ4*01	1063	gnl|Fabrus|HerceptinLC	1086
  1297	gnl|Fabrus|VH6-1_IGHD6-25*01_IGHJ6*01	1064	gnl|Fabrus|HerceptinLC	1086
  1298	gnl|Fabrus|VH6-1_IGHD7-27*01_IGHJ4*01	1065	gnl|Fabrus|HerceptinLC	1086
  1299	gnl|Fabrus|VH4-59_IGHD6-25*01_IGHJ3*01	1043	gnl|Fabrus|HerceptinLC	1086
  1300	gnl|Fabrus|VH3-48_IGHD6-6*01_IGHJ1*01	923	gnl|Fabrus|HerceptinLC	1086
  1301	gnl|Fabrus|VH3-30_IGHD6-6*01_IGHJ1*01	893	gnl|Fabrus|HerceptinLC	1086
  1302	gnl|Fabrus|VH3-66_IGHD6-6*01_IGHJ1*01	949	gnl|Fabrus|HerceptinLC	1086
  1303	gnl|Fabrus|VH3-53_IGHD5-5*01_IGHJ4*01	938	gnl|Fabrus|HerceptinLC	1086
  1304	gnl|Fabrus|VH2-5_IGHD5-12*01_IGHJ4*01	804	gnl|Fabrus|HerceptinLC	1086
  1305	gnl|Fabrus|VH2-70_IGHD5-12*01_IGHJ4*01	811	gnl|Fabrus|HerceptinLC	1086
  1306	gnl|Fabrus|VH3-15_IGHD5-12*01_IGHJ4*01	835	gnl|Fabrus|HerceptinLC	1086
  1307	gnl|Fabrus|VH3-15_IGHD3-10*01_IGHJ4*01	833	gnl|Fabrus|HerceptinLC	1086
  1308	gnl|Fabrus|VH3-49_IGHD5-18*01_IGHJ4*01	930	gnl|Fabrus|HerceptinLC	1086
  1309	gnl|Fabrus|VH3-49_IGHD6-13*01_IGHJ4*01	931	gnl|Fabrus|HerceptinLC	1086
  1310	gnl|Fabrus|VH3-72_IGHD5-18*01_IGHJ4*01	967	gnl|Fabrus|HerceptinLC	1086
  1311	gnl|Fabrus|VH3-72_IGHD6-6*01_IGHJ1*01	969	gnl|Fabrus|HerceptinLC	1086
  1312	gnl|Fabrus|VH3-73_IGHD5-12*01_IGHJ4*01	977	gnl|Fabrus|HerceptinLC	1086
  1313	gnl|Fabrus|VH3-73_IGHD4-23*01_IGHJ5*01	976	gnl|Fabrus|HerceptinLC	1086
  1314	gnl|Fabrus|VH3-43_IGHD3-22*01_IGHJ4*01	918	gnl|Fabrus|HerceptinLC	1086
  1315	gnl|Fabrus|VH3-43_IGHD6-13*01_IGHJ4*01	921	gnl|Fabrus|HerceptinLC	1086
  1316	gnl|Fabrus|VH3-9_IGHD3-22*01_IGHJ4*01	992	gnl|Fabrus|HerceptinLC	1086
  1317	gnl|Fabrus|VH3-9_IGHD1-7*01_IGHJ5*01	989	gnl|Fabrus|HerceptinLC	1086
  1318	gnl|Fabrus|VH3-9_IGHD6-13*01_IGHJ4*01	995	gnl|Fabrus|HerceptinLC	1086
  1319	gnl|Fabrus|VH4-39_IGHD3-10*01_IGHJ4*01	1030	gnl|Fabrus|HerceptinLC	1086
  1320	gnl|Fabrus|VH4-39_IGHD5-12*01_IGHJ4*01	1034	gnl|Fabrus|HerceptinLC	1086
  1321	gnl|Fabrus|VH1-18_IGHD6-6*01_IGHJ1*01	728	gnl|Fabrus|HerceptinLC	1086
  1322	gnl|Fabrus|VH1-24_IGHD5-12*01_IGHJ4*01	735	gnl|Fabrus|HerceptinLC	1086
  1323	gnl|Fabrus|VH1-2_IGHD1-1*01_IGHJ3*01	729	gnl|Fabrus|HerceptinLC	1086
  1324	gnl|Fabrus|VH1-3_IGHD6-6*01_IGHJ1*01	743	gnl|Fabrus|HerceptinLC	1086
  1325	gnl|Fabrus|VH1-45_IGHD3-10*01_IGHJ4*01	748	gnl|Fabrus|HerceptinLC	1086
  1326	gnl|Fabrus|VH1-46_IGHD1-26*01_IGHJ4*01	754	gnl|Fabrus|HerceptinLC	1086
  1327	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ6*01	1068	gnl|Fabrus|HerceptinLC	1086
  1328	gnl|Fabrus|VH2-70_IGHD3-9*01_IGHJ6*01	810	gnl|Fabrus|HerceptinLC	1086
  1329	gnl|Fabrus|VH1-58_IGHD3-10*01_IGHJ6*01	764	gnl|Fabrus|HerceptinLC	1086
  1330	gnl|Fabrus|VH7-81_IGHD2-21*01_IGHJ2*01	1067	gnl|Fabrus|HerceptinLC	1086
  1331	gnl|Fabrus|VH4-28_IGHD3-9*01_IGHJ6*01	1002	gnl|Fabrus|HerceptinLC	1086
  1332	gnl|Fabrus|VH4-31_IGHD2-15*01_IGHJ2*01	1008	gnl|Fabrus|HerceptinLC	1086
  1333	gnl|Fabrus|VH2-5_IGHD3-9*01_IGHJ6*01	803	gnl|Fabrus|HerceptinLC	1086
  1334	gnl|Fabrus|VH1-8_IGHD2-15*01_IGHJ6*01	783	gnl|Fabrus|HerceptinLC	1086
  1335	gnl|Fabrus|VH2-70_IGHD2-15*01_IGHJ2*01	808	gnl|Fabrus|HerceptinLC	1086
  1336	gnl|Fabrus|VH3-38_IGHD3-10*01_IGHJ4*01	907	gnl|Fabrus|HerceptinLC	1086
  1337	gnl|Fabrus|VH3-16_IGHD1-7*01_IGHJ6*01	838	gnl|Fabrus|HerceptinLC	1086
  1338	gnl|Fabrus|VH3-73_IGHD3-9*01_IGHJ6*01	974	gnl|Fabrus|HerceptinLC	1086
  1339	gnl|Fabrus|VH3-11_IGHD3-9*01_IGHJ6*01	816	gnl|Fabrus|HerceptinLC	1086
  1340	gnl|Fabrus|VH3-11_IGHD6-6*01_IGHJ1*01	820	gnl|Fabrus|HerceptinLC	1086
  1341	gnl|Fabrus|VH3-20_IGHD5-12*01_IGHJ4*01	852	gnl|Fabrus|HerceptinLC	1086
  1342	gnl|Fabrus|VH3-16_IGHD2-15*01_IGHJ2*01	839	gnl|Fabrus|HerceptinLC	1086
  1343	gnl|Fabrus|VH3-7_IGHD6-6*01_IGHJ1*01	960	gnl|Fabrus|HerceptinLC	1086
  1344	gnl|Fabrus|VH3-16_IGHD6-13*01_IGHJ4*01	844	gnl|Fabrus|HerceptinLC	1086
  1345	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|O12_IGKJ1*01	1101
  1346	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|O18_IGKJ1*01	1102
  1347	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|A20_IGKJ1*01	1077
  1348	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|A30_IGKJ1*01	1082
  1349	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|L14_IGKJ1*01	1089
  1350	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  1351	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|L5_IGKJ1*01	1096
  1352	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|L8_IGKJ1*01	1097
  1353	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|L23_IGKJ1*01	1092
  1354	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|L11_IGKJ1*01	1087
  1355	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|L12_IGKJ1*01	1088
  1356	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|O1_IGKJ1*01	1100
  1357	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|A17_IGKJ1*01	1075
  1358	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|A2_IGKJ1*01	1076
  1359	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|A23_IGKJ1*01	1078
  1360	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|A27_IGKJ3*01	1081
  1361	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|L2_IGKJ1*01	1090
  1362	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|L6_IGKJ1*01	1097
  1363	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|L25_IGKJ1*01	1094
  1364	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|B3_IGKJ1*01	1085
  1365	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|B2_IGKJ1*01	1083
  1366	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|A26_IGKJ1*01	1079
  1367	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|A14_IGKJ1*01	1074
  1368	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|L9_IGKJ2*01	1099
  1369	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|A27_IGKJ1*01	1080
  1370	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|B2_IGKJ3*01	1084
  1371	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|L25_IGKJ3*01	1094
  1372	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|RituxanLC	1103
  1373	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|L22_IGKJ3*01	1091
  1374	gnl|Fabrus|VH3-23_IGHD3-10*01_IGHJ4*01	868	gnl|Fabrus|HerceptinLC	1086
  1375	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|O12_IGKJ1*01	1101
  1376	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|O18_IGKJ1*01	1102
  1377	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|A20_IGKJ1*01	1077
  1378	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|A30_IGKJ1*01	1082
  1379	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|L14_IGKJ1*01	1089
  1380	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  1381	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|L5_IGKJ1*01	1096
  1382	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|L8_IGKJ1*01	1097
  1383	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|L23_IGKJ1*01	1092
  1384	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|L11_IGKJ1*01	1087
  1385	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|L12_IGKJ1*01	1088
  1386	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|O1_IGKJ1*01	1100
  1387	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|A17_IGKJ1*01	1075
  1388	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|A2_IGKJ1*01	1076
  1389	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|A23_IGKJ1*01	1078
  1390	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|A27_IGKJ3*01	1081
  1391	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|L2_IGKJ1*01	1090
  1392	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|L6_IGKJ1*01	1097
  1393	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|L25_IGKJ1*01	1094
  1394	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|B3_IGKJ1*01	1085
  1395	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|B2_IGKJ1*01	1083
  1396	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|A26_IGKJ1*01	1079
  1397	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|A14_IGKJ1*01	1074
  1398	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|L9_IGKJ2*01	1099
  1399	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|A27_IGKJ1*01	1080
  1400	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|B2_IGKJ3*01	1084
  1401	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|L25_IGKJ3*01	1094
  1402	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|RituxanLC	1103
  1403	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|L22_IGKJ3*01	1091
  1404	gnl|Fabrus|VH4-31_IGHD6-6*01_IGHJ1*01	1015	gnl|Fabrus|HerceptinLC	1086
  1405	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|O12_IGKJ1*01	1101
  1406	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|O18_IGKJ1*01	1102
  1407	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|A20_IGKJ1*01	1077
  1408	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|A30_IGKJ1*01	1082
  1409	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|L14_IGKJ1*01	1089
  1410	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  1411	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|L5_IGKJ1*01	1096
  1412	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|L8_IGKJ1*01	1097
  1413	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|L23_IGKJ1*01	1092
  1414	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|L11_IGKJ1*01	1087
  1415	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|L12_IGKJ1*01	1088
  1416	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|O1_IGKJ1*01	1100
  1417	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|A17_IGKJ1*01	1075
  1418	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|A2_IGKJ1*01	1076
  1419	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|A23_IGKJ1*01	1078
  1420	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|A27_IGKJ3*01	1081
  1421	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|L2_IGKJ1*01	1090
  1422	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|L6_IGKJ1*01	1097
  1423	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|L25_IGKJ1*01	1094
  1424	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|B3_IGKJ1*01	1085
  1425	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|B2_IGKJ1*01	1083
  1426	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|A26_IGKJ1*01	1079
  1427	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|A14_IGKJ1*01	1074
  1428	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|L9_IGKJ2*01	1099
  1429	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|A27_IGKJ1*01	1080
  1430	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|B2_IGKJ3*01	1084
  1431	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|L25_IGKJ3*01	1094
  1432	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|RituxanLC	1103
  1433	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|L22_IGKJ3*01	1091
  1434	gnl|Fabrus|RituxanHC	721	gnl|Fabrus|HerceptinLC	1086
  1435	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|O12_IGKJ1*01	1101
  1436	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|O18_IGKJ1*01	1102
  1437	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|A20_IGKJ1*01	1077
  1438	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|A30_IGKJ1*01	1082
  1439	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|L14_IGKJ1*01	1089
  1440	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|L4/18a_IGKJ1*01	1095
  1441	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|L5_IGKJ1*01	1096
  1442	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|L8_IGKJ1*01	1097
  1443	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|L23_IGKJ1*01	1092
  1444	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|L11_IGKJ1*01	1087
  1445	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|L12_IGKJ1*01	1088
  1446	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|O1_IGKJ1*01	1100
  1447	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|A17_IGKJ1*01	1075
  1448	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|A2_IGKJ1*01	1076
  1449	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|A23_IGKJ1*01	1078
  1450	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|A27_IGKJ3*01	1081
  1451	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|L2_IGKJ1*01	1090
  1452	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|L6_IGKJ1*01	1097
  1453	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|L25_IGKJ1*01	1094
  1454	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|B3_IGKJ1*01	1085
  1455	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|B2_IGKJ1*01	1083
  1456	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|A26_IGKJ1*01	1079
  1457	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|A14_IGKJ1*01	1074
  1458	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|L9_IGKJ2*01	1099
  1459	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|A27_IGKJ1*01	1080
  1460	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|B2_IGKJ3*01	1084
  1461	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|L25_IGKJ3*01	1094
  1462	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|RituxanLC	1103
  1463	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|L22_IGKJ3*01	1091
  1464	gnl|Fabrus|HerceptinHC	720	gnl|Fabrus|HerceptinLC	1086
  1465	VH3-23_IGHD1-1*01>1_IGHJ1*01	1136	gnl|Fabrus|O12_IGKJ1*01	1101
  1466	VH3-23_IGHD1-1*01>2_IGHJ1*01	1137	gnl|Fabrus|O12_IGKJ1*01	1101
  1467	VH3-23_IGHD1-1*01>3_IGHJ1*01	1138	gnl|Fabrus|O12_IGKJ1*01	1101
  1468	VH3-23_IGHD1-7*01>1_IGHJ1*01	1139	gnl|Fabrus|O12_IGKJ1*01	1101
  1469	VH3-23_IGHD1-7*01>3_IGHJ1*01	1140	gnl|Fabrus|O12_IGKJ1*01	1101
  1470	VH3-23_IGHD1-14*01>1_IGHJ1*01	1141	gnl|Fabrus|O12_IGKJ1*01	1101
  1471	VH3-23_IGHD1-14*01>3_IGHJ1*01	1142	gnl|Fabrus|O12_IGKJ1*01	1101
  1472	VH3-23_IGHD1-20*01>1_IGHJ1*01	1143	gnl|Fabrus|O12_IGKJ1*01	1101
  1473	VH3-23_IGHD1-20*01>3_IGHJ1*01	1144	gnl|Fabrus|O12_IGKJ1*01	1101
  1474	VH3-23_IGHD1-26*01>1_IGHJ1*01	1145	gnl|Fabrus|O12_IGKJ1*01	1101
  1475	VH3-23_IGHD1-26*01>3_IGHJ1*01	1146	gnl|Fabrus|O12_IGKJ1*01	1101
  1476	VH3-23_IGHD2-2*01>2_IGHJ1*01	1147	gnl|Fabrus|O12_IGKJ1*01	1101
  1477	VH3-23_IGHD2-2*01>3_IGHJ1*01	1148	gnl|Fabrus|O12_IGKJ1*01	1101
  1478	VH3-23_IGHD2-8*01>2_IGHJ1*01	1149	gnl|Fabrus|O12_IGKJ1*01	1101
  1479	VH3-23_IGHD2-8*01>3_IGHJ1*01	1150	gnl|Fabrus|O12_IGKJ1*01	1101
  1480	VH3-23_IGHD2-15*01>2_IGHJ1*01	1151	gnl|Fabrus|O12_IGKJ1*01	1101
  1481	VH3-23_IGHD2-15*01>3_IGHJ1*01	1152	gnl|Fabrus|O12_IGKJ1*01	1101
  1482	VH3-23_IGHD2-21*01>2_IGHJ1*01	1153	gnl|Fabrus|O12_IGKJ1*01	1101
  1483	VH3-23_IGHD2-21*01>3_IGHJ1*01	1154	gnl|Fabrus|O12_IGKJ1*01	1101
  1484	VH3-23_IGHD3-3*01>1_IGHJ1*01	1155	gnl|Fabrus|O12_IGKJ1*01	1101
  1485	VH3-23_IGHD3-3*01>2_IGHJ1*01	1156	gnl|Fabrus|O12_IGKJ1*01	1101
  1486	VH3-23_IGHD3-3*01>3_IGHJ1*01	1157	gnl|Fabrus|O12_IGKJ1*01	1101
  1487	VH3-23_IGHD3-9*01>2_IGHJ1*01	1158	gnl|Fabrus|O12_IGKJ1*01	1101
  1488	VH3-23_IGHD3-10*01>2_IGHJ1*01	1159	gnl|Fabrus|O12_IGKJ1*01	1101
  1489	VH3-23_IGHD3-10*01>3_IGHJ1*01	1160	gnl|Fabrus|O12_IGKJ1*01	1101
  1490	VH3-23_IGHD3-16*01>2_IGHJ1*01	1161	gnl|Fabrus|O12_IGKJ1*01	1101
  1491	VH3-23_IGHD3-16*01>3_IGHJ1*01	1162	gnl|Fabrus|O12_IGKJ1*01	1101
  1492	VH3-23_IGHD3-22*01>2_IGHJ1*01	1163	gnl|Fabrus|O12_IGKJ1*01	1101
  1493	VH3-23_IGHD3-22*01>3_IGHJ1*01	1164	gnl|Fabrus|O12_IGKJ1*01	1101
  1494	VH3-23_IGHD4-4*01 (1) >2_IGHJ1*01	1165	gnl|Fabrus|O12_IGKJ1*01	1101
  1495	VH3-23_IGHD4-4*01 (1) >3_IGHJ1*01	1166	gnl|Fabrus|O12_IGKJ1*01	1101
  1496	VH3-23_IGHD4-11*01 (1) >2_IGHJ1*01	1167	gnl|Fabrus|O12_IGKJ1*01	1101
  1497	VH3-23_IGHD4-11*01 (1) >3_IGHJ1*01	1168	gnl|Fabrus|O12_IGKJ1*01	1101
  1498	VH3-23_IGHD4-17*01>2_IGHJ1*01	1169	gnl|Fabrus|O12_IGKJ1*01	1101
  1499	VH3-23_IGHD4-17*01>3_IGHJ1*01	1170	gnl|Fabrus|O12_IGKJ1*01	1101
  1500	VH3-23_IGHD4-23*01>2_IGHJ1*01	1171	gnl|Fabrus|O12_IGKJ1*01	1101
  1501	VH3-23_IGHD4-23*01>3_IGHJ1*01	1172	gnl|Fabrus|O12_IGKJ1*01	1101
  1502	VH3-23_IGHD5-5*01 (2) >1_IGHJ1*01	1173	gnl|Fabrus|O12_IGKJ1*01	1101
  1503	VH3-23_IGHD5-5*01 (2) >2_IGHJ1*01	1174	gnl|Fabrus|O12_IGKJ1*01	1101
  1504	VH3-23_IGHD5-5*01 (2) >3_IGHJ1*01	1175	gnl|Fabrus|O12_IGKJ1*01	1101
  1505	VH3-23_IGHD5-12*01>1_IGHJ1*01	1176	gnl|Fabrus|O12_IGKJ1*01	1101
  1506	VH3-23_IGHD5-12*01>3_IGHJ1*01	1177	gnl|Fabrus|O12_IGKJ1*01	1101
  1507	VH3-23_IGHD5-18*01 (2) >1_IGHJ1*01	1178	gnl|Fabrus|O12_IGKJ1*01	1101
  1508	VH3-23_IGHD5-18*01 (2) >2_IGHJ1*01	1179	gnl|Fabrus|O12_IGKJ1*01	1101
  1509	VH3-23_IGHD5-18*01 (2) >3_IGHJ1*01	1180	gnl|Fabrus|O12_IGKJ1*01	1101
  1510	VH3-23_IGHD5-24*01>1_IGHJ1*01	1181	gnl|Fabrus|O12_IGKJ1*01	1101
  1511	VH3-23_IGHD5-24*01>3_IGHJ1*01	1182	gnl|Fabrus|O12_IGKJ1*01	1101
  1512	VH3-23_IGHD6-6*01>1_IGHJ1*01	1183	gnl|Fabrus|O12_IGKJ1*01	1101
  1513	VH3-23_IGHD1-1*01>1′_IGHJ1*01	1193	gnl|Fabrus|O12_IGKJ1*01	1101
  1514	VH3-23_IGHD1-1*01>2′_IGHJ1*01	1194	gnl|Fabrus|O12_IGKJ1*01	1101
  1515	VH3-23_IGHD1-1*01>3′_IGHJ1*01	1195	gnl|Fabrus|O12_IGKJ1*01	1101
  1516	VH3-23_IGHD1-7*01>1′_IGHJ1*01	1196	gnl|Fabrus|O12_IGKJ1*01	1101
  1517	VH3-23_IGHD1-7*01>3′_IGHJ1*01	1197	gnl|Fabrus|O12_IGKJ1*01	1101
  1518	VH3-23_IGHD1-14*01>1′_IGHJ1*01	1198	gnl|Fabrus|O12_IGKJ1*01	1101
  1519	VH3-23_IGHD1-14*01>2′_IGHJ1*01	1199	gnl|Fabrus|O12_IGKJ1*01	1101
  1520	VH3-23_IGHD1-14*01>3′_IGHJ1*01	1200	gnl|Fabrus|O12_IGKJ1*01	1101
  1521	VH3-23_IGHD1-20*01>1′_IGHJ1*01	1201	gnl|Fabrus|O12_IGKJ1*01	1101
  1522	VH3-23_IGHD1-20*01>2′_IGHJ1*01	1202	gnl|Fabrus|O12_IGKJ1*01	1101
  1523	VH3-23_IGHD1-20*01>3′_IGHJ1*01	1203	gnl|Fabrus|O12_IGKJ1*01	1101
  1524	VH3-23_IGHD1-26*01>1′_IGHJ1*01	1204	gnl|Fabrus|O12_IGKJ1*01	1101
  1525	VH3-23_IGHD1-26*01>3′_IGHJ1*01	1205	gnl|Fabrus|O12_IGKJ1*01	1101
  1526	VH3-23_IGHD2-2*01>1′_IGHJ1*01	1206	gnl|Fabrus|O12_IGKJ1*01	1101
  1527	VH3-23_IGHD2-2*01>3′_IGHJ1*01	1207	gnl|Fabrus|O12_IGKJ1*01	1101
  1528	VH3-23_IGHD2-8*01>1′_IGHJ1*01	1208	gnl|Fabrus|O12_IGKJ1*01	1101
  1529	VH3-23_IGHD2-15*01>1′_IGHJ1*01	1209	gnl|Fabrus|O12_IGKJ1*01	1101
  1530	VH3-23_IGHD2-15*01>3′_IGHJ1*01	1210	gnl|Fabrus|O12_IGKJ1*01	1101
  1531	VH3-23_IGHD2-21*01>1′_IGHJ1*01	1211	gnl|Fabrus|O12_IGKJ1*01	1101
  1532	VH3-23_IGHD2-21*01>3′_IGHJ1*01	1212	gnl|Fabrus|O12_IGKJ1*01	1101
  1533	VH3-23_IGHD3-3*01>1′_IGHJ1*01	1213	gnl|Fabrus|O12_IGKJ1*01	1101
  1534	VH3-23_IGHD3-3*01>3′_IGHJ1*01	1214	gnl|Fabrus|O12_IGKJ1*01	1101
  1535	VH3-23_IGHD3-9*01>1′_IGHJ1*01	1215	gnl|Fabrus|O12_IGKJ1*01	1101
  1536	VH3-23_IGHD3-9*01>3′_IGHJ1*01	1216	gnl|Fabrus|O12_IGKJ1*01	1101
  1537	VH3-23_IGHD3-10*01>1′_IGHJ1*01	1217	gnl|Fabrus|O12_IGKJ1*01	1101
  1538	VH3-23_IGHD3-10*01>3′_IGHJ1*01	1218	gnl|Fabrus|O12_IGKJ1*01	1101
  1539	VH3-23_IGHD3-16*01>1′_IGHJ1*01	1219	gnl|Fabrus|O12_IGKJ1*01	1101
  1540	VH3-23_IGHD3-16*01>3′_IGHJ1*01	1220	gnl|Fabrus|O12_IGKJ1*01	1101
  1541	VH3-23_IGHD3-22*01>1′_IGHJ1*01	1221	gnl|Fabrus|O12_IGKJ1*01	1101
  1542	VH3-23_IGHD4-4*01 (1) >1′_IGHJ1*01	1222	gnl|Fabrus|O12_IGKJ1*01	1101
  1543	VH3-23_IGHD4-4*01 (1) >3′_IGHJ1*01	1223	gnl|Fabrus|O12_IGKJ1*01	1101
  1544	VH3-23_IGHD4-11*01 (1) >1′_IGHJ1*01	1224	gnl|Fabrus|O12_IGKJ1*01	1101
  1545	VH3-23_IGHD4-11*01 (1) >3′_IGHJ1*01	1225	gnl|Fabrus|O12_IGKJ1*01	1101
  1546	VH3-23_IGHD4-17*01>1′_IGHJ1*01	1226	gnl|Fabrus|O12_IGKJ1*01	1101
  1547	VH3-23_IGHD4-17*01>3′_IGHJ1*01	1227	gnl|Fabrus|O12_IGKJ1*01	1101
  1548	VH3-23_IGHD4-23*01>1′_IGHJ1*01	1228	gnl|Fabrus|O12_IGKJ1*01	1101
  1549	VH3-23_IGHD4-23*01>3′_IGHJ1*01	1229	gnl|Fabrus|O12_IGKJ1*01	1101
  1550	VH3-23_IGHD5-5*01 (2) >1′_IGHJ1*01	1230	gnl|Fabrus|O12_IGKJ1*01	1101
  1551	VH3-23_IGHD5-5*01 (2) >3′_IGHJ1*01	1231	gnl|Fabrus|O12_IGKJ1*01	1101
  1552	VH3-23_IGHD5-12*01>1′_IGHJ1*01	1232	gnl|Fabrus|O12_IGKJ1*01	1101
  1553	VH3-23_IGHD5-12*01>3′_IGHJ1*01	1233	gnl|Fabrus|O12_IGKJ1*01	1101
  1554	VH3-23_IGHD5-18*01 (2) >1′_IGHJ1*01	1234	gnl|Fabrus|O12_IGKJ1*01	1101
  1555	VH3-23_IGHD5-18*01 (2) >3′_IGHJ1*01	1235	gnl|Fabrus|O12_IGKJ1*01	1101
  1556	VH3-23_IGHD5-24*01>1′_IGHJ1*01	1236	gnl|Fabrus|O12_IGKJ1*01	1101
  1557	VH3-23_IGHD5-24*01>3′_IGHJ1*01	1237	gnl|Fabrus|O12_IGKJ1*01	1101
  1558	VH3-23_IGHD6-6*01>1′_IGHJ1*01	1238	gnl|Fabrus|O12_IGKJ1*01	1101
  1559	VH3-23_IGHD6-6*01>2′_IGHJ1*01	1239	gnl|Fabrus|O12_IGKJ1*01	1101
  1560	VH3-23_IGHD6-6*01>3′_IGHJ1*01	1240	gnl|Fabrus|O12_IGKJ1*01	1101
  1561	VH3-23_IGHD6-6*01>2_IGHJ1*01	1184	gnl|Fabrus|O12_IGKJ1*01	1101
  1562	VH3-23_IGHD6-13*01>1_IGHJ1*01	1185	gnl|Fabrus|O12_IGKJ1*01	1101
  1563	VH3-23_IGHD6-13*01>2_IGHJ1*01	1186	gnl|Fabrus|O12_IGKJ1*01	1101
  1564	VH3-23_IGHD6-19*01>1_IGHJ1*01	1187	gnl|Fabrus|O12_IGKJ1*01	1101
  1565	VH3-23_IGHD6-19*01>2_IGHJ1*01	1188	gnl|Fabrus|O12_IGKJ1*01	1101
  1566	VH3-23_IGHD6-25*01>1_IGHJ1*01	1189	gnl|Fabrus|O12_IGKJ1*01	1101
  1567	VH3-23_IGHD6-25*01>2_IGHJ1*01	1190	gnl|Fabrus|O12_IGKJ1*01	1101
  1568	VH3-23_IGHD7-27*01>1_IGHJ1*01	1191	gnl|Fabrus|O12_IGKJ1*01	1101
  1569	VH3-23_IGHD7-27*01>3_IGHJ1*01	1192	gnl|Fabrus|O12_IGKJ1*01	1101
  1570	VH3-23_IGHD6-13*01>1′_IGHJ1*01	1241	gnl|Fabrus|O12_IGKJ1*01	1101
  1571	VH3-23_IGHD6-13*01>2′_IGHJ1*01	1242	gnl|Fabrus|O12_IGKJ1*01	1101
  1572	VH3-23_IGHD6-13*01>2_IGHJ1*01_B	1243	gnl|Fabrus|O12_IGKJ1*01	1101
  1573	VH3-23_IGHD6-19*01>1′_IGHJ1*01	1244	gnl|Fabrus|O12_IGKJ1*01	1101
  1574	VH3-23_IGHD6-19*01>2′_IGHJ1*01	1245	gnl|Fabrus|O12_IGKJ1*01	1101
  1575	VH3-23_IGHD6-19*01>2_IGHJ1*01_B	1246	gnl|Fabrus|O12_IGKJ1*01	1101
  1576	VH3-23_IGHD6-25*01>1′_IGHJ1*01	1247	gnl|Fabrus|O12_IGKJ1*01	1101
  1577	VH3-23_IGHD6-25*01>3′_IGHJ1*01	1248	gnl|Fabrus|O12_IGKJ1*01	1101
  1578	VH3-23_IGHD7-27*01>1′_IGHJ1*01_B	1249	gnl|Fabrus|O12_IGKJ1*01	1101
  1579	VH3-23_IGHD7-27*01>2′_IGHJ2*01	1250	gnl|Fabrus|O12_IGKJ1*01	1101
  1580	VH3-23_IGHD1-1*01>1_IGHJ2*01	1251	gnl|Fabrus|O12_IGKJ1*01	1101
  1581	VH3-23_IGHD1-1*01>2_IGHJ2*01	1252	gnl|Fabrus|O12_IGKJ1*01	1101
  1582	VH3-23_IGHD1-1*01>3_IGHJ2*01	1253	gnl|Fabrus|O12_IGKJ1*01	1101
  1583	VH3-23_IGHD1-7*01>1_IGHJ2*01	1254	gnl|Fabrus|O12_IGKJ1*01	1101
  1584	VH3-23_IGHD1-7*01>3_IGHJ2*01	1255	gnl|Fabrus|O12_IGKJ1*01	1101
  1585	VH3-23_IGHD1-14*01>1_IGHJ2*01	1256	gnl|Fabrus|O12_IGKJ1*01	1101
  1586	VH3-23_IGHD1-14*01>3_IGHJ2*01	1257	gnl|Fabrus|O12_IGKJ1*01	1101
  1587	VH3-23_IGHD1-20*01>1_IGHJ2*01	1258	gnl|Fabrus|O12_IGKJ1*01	1101
  1588	VH3-23_IGHD1-20*01>3_IGHJ2*01	1259	gnl|Fabrus|O12_IGKJ1*01	1101
  1589	VH3-23_IGHD1-26*01>1_IGHJ2*01	1260	gnl|Fabrus|O12_IGKJ1*01	1101
  1590	VH3-23_IGHD1-26*01>3_IGHJ2*01	1261	gnl|Fabrus|O12_IGKJ1*01	1101
  1591	VH3-23_IGHD2-2*01>2_IGHJ2*01	1262	gnl|Fabrus|O12_IGKJ1*01	1101
  1592	VH3-23_IGHD2-2*01>3_IGHJ2*01	1263	gnl|Fabrus|O12_IGKJ1*01	1101
  1593	VH3-23_IGHD2-8*01>2_IGHJ2*01	1264	gnl|Fabrus|O12_IGKJ1*01	1101
  1594	VH3-23_IGHD2-8*01>3_IGHJ2*01	1265	gnl|Fabrus|O12_IGKJ1*01	1101
  1595	VH3-23_IGHD2-15*01>2_IGHJ2*01	1266	gnl|Fabrus|O12_IGKJ1*01	1101
  1596	VH3-23_IGHD2-15*01>3_IGHJ2*01	1267	gnl|Fabrus|O12_IGKJ1*01	1101
  1597	VH3-23_IGHD2-21*01>2_IGHJ2*01	1268	gnl|Fabrus|O12_IGKJ1*01	1101
  1598	VH3-23_IGHD2-21*01>3_IGHJ2*01	1269	gnl|Fabrus|O12_IGKJ1*01	1101
  1599	VH3-23_IGHD3-3*01>1_IGHJ2*01	1270	gnl|Fabrus|O12_IGKJ1*01	1101
  1600	VH3-23_IGHD3-3*01>2_IGHJ2*01	1271	gnl|Fabrus|O12_IGKJ1*01	1101
  1601	VH3-23_IGHD3-3*01>3_IGHJ2*01	1272	gnl|Fabrus|O12_IGKJ1*01	1101
  1602	VH3-23_IGHD3-9*01>2_IGHJ2*01	1273	gnl|Fabrus|O12_IGKJ1*01	1101
  1603	VH3-23_IGHD3-10*01>2_IGHJ2*01	1274	gnl|Fabrus|O12_IGKJ1*01	1101
  1604	VH3-23_IGHD3-10*01>3_IGHJ2*01	1275	gnl|Fabrus|O12_IGKJ1*01	1101
  1605	VH3-23_IGHD3-16*01>2_IGHJ2*01	1276	gnl|Fabrus|O12_IGKJ1*01	1101
  1606	VH3-23_IGHD3-16*01>3_IGHJ2*01	1277	gnl|Fabrus|O12_IGKJ1*01	1101
  1607	VH3-23_IGHD3-22*01>2_IGHJ2*01	1278	gnl|Fabrus|O12_IGKJ1*01	1101
  1608	VH3-23_IGHD3-22*01>3_IGHJ2*01	1279	gnl|Fabrus|O12_IGKJ1*01	1101
  1609	VH3-23_IGHD4-4*01 (1) >2_IGHJ2*01	1280	gnl|Fabrus|O12_IGKJ1*01	1101
  1610	VH3-23_IGHD4-4*01 (1) >3_IGHJ2*01	1281	gnl|Fabrus|O12_IGKJ1*01	1101
  1611	VH3-23_IGHD4-11*01 (1) >2_IGHJ2*01	1282	gnl|Fabrus|O12_IGKJ1*01	1101
  1612	VH3-23_IGHD4-11*01 (1) >3_IGHJ2*01	1283	gnl|Fabrus|O12_IGKJ1*01	1101
  1613	VH3-23_IGHD4-17*01>2_IGHJ2*01	1284	gnl|Fabrus|O12_IGKJ1*01	1101
  1614	VH3-23_IGHD4-17*01>3_IGHJ2*01	1285	gnl|Fabrus|O12_IGKJ1*01	1101
  1615	VH3-23_IGHD4-23*01>2_IGHJ2*01	1286	gnl|Fabrus|O12_IGKJ1*01	1101
  1616	VH3-23_IGHD4-23*01>3_IGHJ2*01	1287	gnl|Fabrus|O12_IGKJ1*01	1101
  1617	VH3-23_IGHD5-5*01 (2) >1_IGHJ2*01	1288	gnl|Fabrus|O12_IGKJ1*01	1101
  1618	VH3-23_IGHD5-5*01 (2) >2_IGHJ2*01	1289	gnl|Fabrus|O12_IGKJ1*01	1101
  1619	VH3-23_IGHD5-5*01 (2) >3_IGHJ2*01	1290	gnl|Fabrus|O12_IGKJ1*01	1101
  1620	VH3-23_IGHD5-12*01>1_IGHJ2*01	1291	gnl|Fabrus|O12_IGKJ1*01	1101
  1621	VH3-23_IGHD5-12*01>3_IGHJ2*01	1292	gnl|Fabrus|O12_IGKJ1*01	1101
  1622	VH3-23_IGHD5-18*01 (2) >1_IGHJ2*01	1293	gnl|Fabrus|O12_IGKJ1*01	1101
  1623	VH3-23_IGHD5-18*01 (2) >2_IGHJ2*01	1294	gnl|Fabrus|O12_IGKJ1*01	1101
  1624	VH3-23_IGHD5-18*01 (2) >3_IGHJ2*01	1295	gnl|Fabrus|O12_IGKJ1*01	1101
  1625	VH3-23_IGHD5-24*01>1_IGHJ2*01	1296	gnl|Fabrus|O12_IGKJ1*01	1101
  1626	VH3-23_IGHD5-24*01>3_IGHJ2*01	1297	gnl|Fabrus|O12_IGKJ1*01	1101
  1627	VH3-23_IGHD6-6*01>1_IGHJ2*01	1298	gnl|Fabrus|O12_IGKJ1*01	1101
  1628	VH3-23_IGHD1-1*01>1′_IGHJ2*01	1308	gnl|Fabrus|O12_IGKJ1*01	1101
  1629	VH3-23_IGHD1-1*01>2′_IGHJ2*01	1309	gnl|Fabrus|O12_IGKJ1*01	1101
  1630	VH3-23_IGHD1-1*01>3′_IGHJ2*01	1310	gnl|Fabrus|O12_IGKJ1*01	1101
  1631	VH3-23_IGHD1-7*01>1′_IGHJ2*01	1311	gnl|Fabrus|O12_IGKJ1*01	1101
  1632	VH3-23_IGHD1-7*01>3′_IGHJ2*01	1312	gnl|Fabrus|O12_IGKJ1*01	1101
  1633	VH3-23_IGHD1-14*01>1′_IGHJ2*01	1313	gnl|Fabrus|O12_IGKJ1*01	1101
  1634	VH3-23_IGHD1-14*01>2′_IGHJ2*01	1314	gnl|Fabrus|O12_IGKJ1*01	1101
  1635	VH3-23_IGHD1-14*01>3′_IGHJ2*01	1315	gnl|Fabrus|O12_IGKJ1*01	1101
  1636	VH3-23_IGHD1-20*01>1′_IGHJ2*01	1316	gnl|Fabrus|O12_IGKJ1*01	1101
  1637	VH3-23_IGHD1-20*01>2′_IGHJ2*01	1317	gnl|Fabrus|O12_IGKJ1*01	1101
  1638	VH3-23_IGHD1-20*01>3′_IGHJ2*01	1318	gnl|Fabrus|O12_IGKJ1*01	1101
  1639	VH3-23_IGHD1-26*01>1′_IGHJ2*01	1319	gnl|Fabrus|O12_IGKJ1*01	1101
  1640	VH3-23_IGHD1-26*01>1_IGHJ2*01_B	1320	gnl|Fabrus|O12_IGKJ1*01	1101
  1641	VH3-23_IGHD2-2*01>1′_IGHJ2*01	1321	gnl|Fabrus|O12_IGKJ1*01	1101
  1642	VH3-23_IGHD2-2*01>3′_IGHJ2*01	1322	gnl|Fabrus|O12_IGKJ1*01	1101
  1643	VH3-23_IGHD2-8*01>1′_IGHJ2*01	1323	gnl|Fabrus|O12_IGKJ1*01	1101
  1644	VH3-23_IGHD2-15*01>1′_IGHJ2*01	1324	gnl|Fabrus|O12_IGKJ1*01	1101
  1645	VH3-23_IGHD2-15*01>3′_IGHJ2*01	1325	gnl|Fabrus|O12_IGKJ1*01	1101
  1646	VH3-23_IGHD2-21*01>1′_IGHJ2*01	1326	gnl|Fabrus|O12_IGKJ1*01	1101
  1647	VH3-23_IGHD2-21*01>3′_IGHJ2*01	1327	gnl|Fabrus|O12_IGKJ1*01	1101
  1648	VH3-23_IGHD3-3*01>1′_IGHJ2*01	1328	gnl|Fabrus|O12_IGKJ1*01	1101
  1649	VH3-23_IGHD3-3*01>3′_IGHJ2*01	1329	gnl|Fabrus|O12_IGKJ1*01	1101
  1650	VH3-23_IGHD3-9*01>1′_IGHJ2*01	1330	gnl|Fabrus|O12_IGKJ1*01	1101
  1651	VH3-23_IGHD3-9*01>3′_IGHJ2*01	1331	gnl|Fabrus|O12_IGKJ1*01	1101
  1652	VH3-23_IGHD3-10*01>1′_IGHJ2*01	1332	gnl|Fabrus|O12_IGKJ1*01	1101
  1653	VH3-23_IGHD3-10*01>3′_IGHJ2*01	1333	gnl|Fabrus|O12_IGKJ1*01	1101
  1654	VH3-23_IGHD3-16*01>1′_IGHJ2*01	1334	gnl|Fabrus|O12_IGKJ1*01	1101
  1655	VH3-23_IGHD3-16*01>3′_IGHJ2*01	1335	gnl|Fabrus|O12_IGKJ1*01	1101
  1656	VH3-23_IGHD3-22*01>1′_IGHJ2*01	1336	gnl|Fabrus|O12_IGKJ1*01	1101
  1657	VH3-23_IGHD4-4*01 (1) >1′_IGHJ2*01	1337	gnl|Fabrus|O12_IGKJ1*01	1101
  1658	VH3-23_IGHD4-4*01 (1) >3′_IGHJ2*01	1338	gnl|Fabrus|O12_IGKJ1*01	1101
  1659	VH3-23_IGHD4-11*01 (1) >1′_IGHJ2*01	1339	gnl|Fabrus|O12_IGKJ1*01	1101
  1660	VH3-23_IGHD4-11*01 (1) >3′_IGHJ2*01	1340	gnl|Fabrus|O12_IGKJ1*01	1101
  1661	VH3-23_IGHD4-17*01>1′_IGHJ2*01	1341	gnl|Fabrus|O12_IGKJ1*01	1101
  1662	VH3-23_IGHD4-17*01>3′_IGHJ2*01	1342	gnl|Fabrus|O12_IGKJ1*01	1101
  1663	VH3-23_IGHD4-23*01>1′_IGHJ2*01	1343	gnl|Fabrus|O12_IGKJ1*01	1101
  1664	VH3-23_IGHD4-23*01>3′_IGHJ2*01	1344	gnl|Fabrus|O12_IGKJ1*01	1101
  1665	VH3-23_IGHD5-5*01 (2) >1′_IGHJ2*01	1345	gnl|Fabrus|O12_IGKJ1*01	1101
  1666	VH3-23_IGHD5-5*01 (2) >3′_IGHJ2*01	1346	gnl|Fabrus|O12_IGKJ1*01	1101
  1667	VH3-23_IGHD5-12*01>1′_IGHJ2*01	1347	gnl|Fabrus|O12_IGKJ1*01	1101
  1668	VH3-23_IGHD5-12*01>3′_IGHJ2*01	1348	gnl|Fabrus|O12_IGKJ1*01	1101
  1669	VH3-23_IGHD5-18*01 (2) >1′_IGHJ2*01	1349	gnl|Fabrus|O12_IGKJ1*01	1101
  1670	VH3-23_IGHD5-18*01 (2) >3′_IGHJ2*01	1350	gnl|Fabrus|O12_IGKJ1*01	1101
  1671	VH3-23_IGHD5-24*01>1′_IGHJ2*01	1351	gnl|Fabrus|O12_IGKJ1*01	1101
  1672	VH3-23_IGHD5-24*01>3′_IGHJ2*01	1352	gnl|Fabrus|O12_IGKJ1*01	1101
  1673	VH3-23_IGHD6-6*01>1′_IGHJ2*01	1353	gnl|Fabrus|O12_IGKJ1*01	1101
  1674	VH3-23_IGHD6-6*01>2′_IGHJ2*01	1354	gnl|Fabrus|O12_IGKJ1*01	1101
  1675	VH3-23_IGHD6-6*01>3′_IGHJ2*01	1355	gnl|Fabrus|O12_IGKJ1*01	1101
  1676	VH3-23_IGHD6-6*01>2_IGHJ2*01	1299	gnl|Fabrus|O12_IGKJ1*01	1101
  1677	VH3-23_IGHD6-13*01>1_IGHJ2*01	1300	gnl|Fabrus|O12_IGKJ1*01	1101
  1678	VH3-23_IGHD6-13*01>2_IGHJ2*01	1301	gnl|Fabrus|O12_IGKJ1*01	1101
  1679	VH3-23_IGHD6-19*01>1_IGHJ2*01	1302	gnl|Fabrus|O12_IGKJ1*01	1101
  1680	VH3-23_IGHD6-19*01>2_IGHJ2*01	1303	gnl|Fabrus|O12_IGKJ1*01	1101
  1681	VH3-23_IGHD6-25*01>1_IGHJ2*01	1304	gnl|Fabrus|O12_IGKJ1*01	1101
  1682	VH3-23_IGHD6-25*01>2_IGHJ2*01	1305	gnl|Fabrus|O12_IGKJ1*01	1101
  1683	VH3-23_IGHD7-27*01>1_IGHJ2*01	1306	gnl|Fabrus|O12_IGKJ1*01	1101
  1684	VH3-23_IGHD7-27*01>3_IGHJ2*01	1307	gnl|Fabrus|O12_IGKJ1*01	1101
  1685	VH3-23_IGHD6-13*01>1′_IGHJ2*01	1356	gnl|Fabrus|O12_IGKJ1*01	1101
  1686	VH3-23_IGHD6-13*01>2′_IGHJ2*01	1357	gnl|Fabrus|O12_IGKJ1*01	1101
  1687	VH3-23_IGHD6-13*01>2_IGHJ2*01_B	1358	gnl|Fabrus|O12_IGKJ1*01	1101
  1688	VH3-23_IGHD6-19*01>1′_IGHJ2*01	1359	gnl|Fabrus|O12_IGKJ1*01	1101
  1689	VH3-23_IGHD6-19*01>2′_IGHJ2*01	1360	gnl|Fabrus|O12_IGKJ1*01	1101
  1690	VH3-23_IGHD6-19*01>2_IGHJ2*01_B	1361	gnl|Fabrus|O12_IGKJ1*01	1101
  1691	VH3-23_IGHD6-25*01>1′_IGHJ2*01	1362	gnl|Fabrus|O12_IGKJ1*01	1101
  1692	VH3-23_IGHD6-25*01>3′_IGHJ2*01	1363	gnl|Fabrus|O12_IGKJ1*01	1101
  1693	VH3-23_IGHD7-27*01>1′_IGHJ2*01	1364	gnl|Fabrus|O12_IGKJ1*01	1101
  1694	VH3-23_IGHD7-27*01>2′_IGHJ2*01	1365	gnl|Fabrus|O12_IGKJ1*01	1101
  1695	VH3-23_IGHD1-1*01>1_IGHJ3*01	1366	gnl|Fabrus|O12_IGKJ1*01	1101
  1696	VH3-23_IGHD1-1*01>2_IGHJ3*01	1367	gnl|Fabrus|O12_IGKJ1*01	1101
  1697	VH3-23_IGHD1-1*01>3_IGHJ3*01	1368	gnl|Fabrus|O12_IGKJ1*01	1101
  1698	VH3-23_IGHD1-7*01>1_IGHJ3*01	1369	gnl|Fabrus|O12_IGKJ1*01	1101
  1699	VH3-23_IGHD1-7*01>3_IGHJ3*01	1370	gnl|Fabrus|O12_IGKJ1*01	1101
  1700	VH3-23_IGHD1-14*01>1_IGHJ3*01	1371	gnl|Fabrus|O12_IGKJ1*01	1101
  1701	VH3-23_IGHD1-14*01>3_IGHJ3*01	1372	gnl|Fabrus|O12_IGKJ1*01	1101
  1702	VH3-23_IGHD1-20*01>1_IGHJ3*01	1373	gnl|Fabrus|O12_IGKJ1*01	1101
  1703	VH3-23_IGHD1-20*01>3_IGHJ3*01	1374	gnl|Fabrus|O12_IGKJ1*01	1101
  1704	VH3-23_IGHD1-26*01>1_IGHJ3*01	1375	gnl|Fabrus|O12_IGKJ1*01	1101
  1705	VH3-23_IGHD1-26*01>3_IGHJ3*01	1376	gnl|Fabrus|O12_IGKJ1*01	1101
  1706	VH3-23_IGHD2-2*01>2_IGHJ3*01	1377	gnl|Fabrus|O12_IGKJ1*01	1101
  1707	VH3-23_IGHD2-2*01>3_IGHJ3*01	1378	gnl|Fabrus|O12_IGKJ1*01	1101
  1708	VH3-23_IGHD2-8*01>2_IGHJ3*01	1379	gnl|Fabrus|O12_IGKJ1*01	1101
  1709	VH3-23_IGHD2-8*01>3_IGHJ3*01	1380	gnl|Fabrus|O12_IGKJ1*01	1101
  1710	VH3-23_IGHD2-15*01>2_IGHJ3*01	1381	gnl|Fabrus|O12_IGKJ1*01	1101
  1711	VH3-23_IGHD2-15*01>3_IGHJ3*01	1382	gnl|Fabrus|O12_IGKJ1*01	1101
  1712	VH3-23_IGHD2-21*01>2_IGHJ3*01	1383	gnl|Fabrus|O12_IGKJ1*01	1101
  1713	VH3-23_IGHD2-21*01>3_IGHJ3*01	1384	gnl|Fabrus|O12_IGKJ1*01	1101
  1714	VH3-23_IGHD3-3*01>1_IGHJ3*01	1385	gnl|Fabrus|O12_IGKJ1*01	1101
  1715	VH3-23_IGHD3-3*01>2_IGHJ3*01	1386	gnl|Fabrus|O12_IGKJ1*01	1101
  1716	VH3-23_IGHD3-3*01>3_IGHJ3*01	1387	gnl|Fabrus|O12_IGKJ1*01	1101
  1717	VH3-23_IGHD3-9*01>2_IGHJ3*01	1388	gnl|Fabrus|O12_IGKJ1*01	1101
  1718	VH3-23_IGHD3-10*01>2_IGHJ3*01	1389	gnl|Fabrus|O12_IGKJ1*01	1101
  1719	VH3-23_IGHD3-10*01>3_IGHJ3*01	1390	gnl|Fabrus|O12_IGKJ1*01	1101
  1720	VH3-23_IGHD3-16*01>2_IGHJ3*01	1391	gnl|Fabrus|O12_IGKJ1*01	1101
  1721	VH3-23_IGHD3-16*01>3_IGHJ3*01	1392	gnl|Fabrus|O12_IGKJ1*01	1101
  1722	VH3-23_IGHD3-22*01>2_IGHJ3*01	1393	gnl|Fabrus|O12_IGKJ1*01	1101
  1723	VH3-23_IGHD3-22*01>3_IGHJ3*01	1394	gnl|Fabrus|O12_IGKJ1*01	1101
  1724	VH3-23_IGHD4-4*01 (1) >2_IGHJ3*01	1395	gnl|Fabrus|O12_IGKJ1*01	1101
  1725	VH3-23_IGHD4-4*01 (1) >3_IGHJ3*01	1396	gnl|Fabrus|O12_IGKJ1*01	1101
  1726	VH3-23_IGHD4-11*01 (1) >2_IGHJ3*01	1397	gnl|Fabrus|O12_IGKJ1*01	1101
  1727	VH3-23_IGHD4-11*01 (1) >3_IGHJ3*01	1398	gnl|Fabrus|O12_IGKJ1*01	1101
  1728	VH3-23_IGHD4-17*01>2_IGHJ3*01	1399	gnl|Fabrus|O12_IGKJ1*01	1101
  1729	VH3-23_IGHD4-17*01>3_IGHJ3*01	1400	gnl|Fabrus|O12_IGKJ1*01	1101
  1730	VH3-23_IGHD4-23*01>2_IGHJ3*01	1401	gnl|Fabrus|O12_IGKJ1*01	1101
  1731	VH3-23_IGHD4-23*01>3_IGHJ3*01	1402	gnl|Fabrus|O12_IGKJ1*01	1101
  1732	VH3-23_IGHD5-5*01 (2) >1_IGHJ3*01	1403	gnl|Fabrus|O12_IGKJ1*01	1101
  1733	VH3-23_IGHD5-5*01 (2) >2_IGHJ3*01	1404	gnl|Fabrus|O12_IGKJ1*01	1101
  1734	VH3-23_IGHD5-5*01 (2) >3_IGHJ3*01	1405	gnl|Fabrus|O12_IGKJ1*01	1101
  1735	VH3-23_IGHD5-12*01>1_IGHJ3*01	1406	gnl|Fabrus|O12_IGKJ1*01	1101
  1736	VH3-23_IGHD5-12*01>3_IGHJ3*01	1407	gnl|Fabrus|O12_IGKJ1*01	1101
  1737	VH3-23_IGHD5-18*01 (2) >1_IGHJ3*01	1408	gnl|Fabrus|O12_IGKJ1*01	1101
  1738	VH3-23_IGHD5-18*01 (2) >2_IGHJ3*01	1409	gnl|Fabrus|O12_IGKJ1*01	1101
  1739	VH3-23_IGHD5-18*01 (2) >3_IGHJ3*01	1410	gnl|Fabrus|O12_IGKJ1*01	1101
  1740	VH3-23_IGHD5-24*01>1_IGHJ3*01	1411	gnl|Fabrus|O12_IGKJ1*01	1101
  1741	VH3-23_IGHD5-24*01>3_IGHJ3*01	1412	gnl|Fabrus|O12_IGKJ1*01	1101
  1742	VH3-23_IGHD6-6*01>1_IGHJ3*01	1413	gnl|Fabrus|O12_IGKJ1*01	1101
  1743	VH3-23_IGHD1-1*01>1′_IGHJ3*01	1423	gnl|Fabrus|O12_IGKJ1*01	1101
  1744	VH3-23_IGHD1-1*01>2′_IGHJ3*01	1424	gnl|Fabrus|O12_IGKJ1*01	1101
  1745	VH3-23_IGHD1-1*01>3′_IGHJ3*01	1425	gnl|Fabrus|O12_IGKJ1*01	1101
  1746	VH3-23_IGHD1-7*01>1′_IGHJ3*01	1426	gnl|Fabrus|O12_IGKJ1*01	1101
  1747	VH3-23_IGHD1-7*01>3′_IGHJ3*01	1427	gnl|Fabrus|O12_IGKJ1*01	1101
  1748	VH3-23_IGHD1-14*01>1′_IGHJ3*01	1428	gnl|Fabrus|O12_IGKJ1*01	1101
  1749	VH3-23_IGHD1-14*01>2′_IGHJ3*01	1429	gnl|Fabrus|O12_IGKJ1*01	1101
  1750	VH3-23_IGHD1-14*01>3′_IGHJ3*01	1430	gnl|Fabrus|O12_IGKJ1*01	1101
  1751	VH3-23_IGHD1-20*01>1′_IGHJ3*01	1431	gnl|Fabrus|O12_IGKJ1*01	1101
  1752	VH3-23_IGHD1-20*01>2′_IGHJ3*01	1432	gnl|Fabrus|O12_IGKJ1*01	1101
  1753	VH3-23_IGHD1-20*01>3′_IGHJ3*01	1433	gnl|Fabrus|O12_IGKJ1*01	1101
  1754	VH3-23_IGHD1-26*01>1′_IGHJ3*01	1434	gnl|Fabrus|O12_IGKJ1*01	1101
  1755	VH3-23_IGHD1-26*01>3′_IGHJ3*01	1435	gnl|Fabrus|O12_IGKJ1*01	1101
  1756	VH3-23_IGHD2-2*01>1′_IGHJ3*01	1436	gnl|Fabrus|O12_IGKJ1*01	1101
  1757	VH3-23_IGHD2-2*01>3′_IGHJ3*01	1437	gnl|Fabrus|O12_IGKJ1*01	1101
  1758	VH3-23_IGHD2-8*01>1′_IGHJ3*01	1438	gnl|Fabrus|O12_IGKJ1*01	1101
  1759	VH3-23_IGHD2-15*01>1′_IGHJ3*01	1439	gnl|Fabrus|O12_IGKJ1*01	1101
  1760	VH3-23_IGHD2-15*01>3′_IGHJ3*01	1440	gnl|Fabrus|O12_IGKJ1*01	1101
  1761	VH3-23_IGHD2-21*01>1′_IGHJ3*01	1441	gnl|Fabrus|O12_IGKJ1*01	1101
  1762	VH3-23_IGHD2-21*01>3′_IGHJ3*01	1442	gnl|Fabrus|O12_IGKJ1*01	1101
  1763	VH3-23_IGHD3-3*01>1′_IGHJ3*01	1443	gnl|Fabrus|O12_IGKJ1*01	1101
  1764	VH3-23_IGHD3-3*01>3′_IGHJ3*01	1444	gnl|Fabrus|O12_IGKJ1*01	1101
  1765	VH3-23_IGHD3-9*01>1′_IGHJ3*01	1445	gnl|Fabrus|O12_IGKJ1*01	1101
  1766	VH3-23_IGHD3-9*01>3′_IGHJ3*01	1446	gnl|Fabrus|O12_IGKJ1*01	1101
  1767	VH3-23_IGHD3-10*01>1′_IGHJ3*01	1447	gnl|Fabrus|O12_IGKJ1*01	1101
  1768	VH3-23_IGHD3-10*01>3′_IGHJ3*01	1448	gnl|Fabrus|O12_IGKJ1*01	1101
  1769	VH3-23_IGHD3-16*01>1′_IGHJ3*01	1449	gnl|Fabrus|O12_IGKJ1*01	1101
  1770	VH3-23_IGHD3-16*01>3′_IGHJ3*01	1450	gnl|Fabrus|O12_IGKJ1*01	1101
  1771	VH3-23_IGHD3-22*01>1′_IGHJ3*01	1451	gnl|Fabrus|O12_IGKJ1*01	1101
  1772	VH3-23_IGHD4-4*01 (1) >1′_IGHJ3*01	1452	gnl|Fabrus|O12_IGKJ1*01	1101
  1773	VH3-23_IGHD4-4*01 (1) >3′_IGHJ3*01	1453	gnl|Fabrus|O12_IGKJ1*01	1101
  1774	VH3-23_IGHD4-11*01 (1) >1′_IGHJ3*01	1454	gnl|Fabrus|O12_IGKJ1*01	1101
  1775	VH3-23_IGHD4-11*01 (1) >3′_IGHJ3*01	1455	gnl|Fabrus|O12_IGKJ1*01	1101
  1776	VH3-23_IGHD4-17*01>1′_IGHJ3*01	1456	gnl|Fabrus|O12_IGKJ1*01	1101
  1777	VH3-23_IGHD4-17*01>3′_IGHJ3*01	1457	gnl|Fabrus|O12_IGKJ1*01	1101
  1778	VH3-23_IGHD4-23*01>1′_IGHJ3*01	1458	gnl|Fabrus|O12_IGKJ1*01	1101
  1779	VH3-23_IGHD4-23*01>3′_IGHJ3*01	1459	gnl|Fabrus|O12_IGKJ1*01	1101
  1780	VH3-23_IGHD5-5*01 (2) >1′_IGHJ3*01	1460	gnl|Fabrus|O12_IGKJ1*01	1101
  1781	VH3-23_IGHD5-5*01 (2) >3′_IGHJ3*01	1461	gnl|Fabrus|O12_IGKJ1*01	1101
  1782	VH3-23_IGHD5-12*01>1′_IGHJ3*01	1462	gnl|Fabrus|O12_IGKJ1*01	1101
  1783	VH3-23_IGHD5-12*01>3′_IGHJ3*01	1463	gnl|Fabrus|O12_IGKJ1*01	1101
  1784	VH3-23_IGHD5-18*01 (2) >1′_IGHJ3*01	1464	gnl|Fabrus|O12_IGKJ1*01	1101
  1785	VH3-23_IGHD5-18*01 (2) >3′_IGHJ3*01	1465	gnl|Fabrus|O12_IGKJ1*01	1101
  1786	VH3-23_IGHD5-24*01>1′_IGHJ3*01	1466	gnl|Fabrus|O12_IGKJ1*01	1101
  1787	VH3-23_IGHD5-24*01>3′_IGHJ3*01	1467	gnl|Fabrus|O12_IGKJ1*01	1101
  1788	VH3-23_IGHD6-6*01>1′_IGHJ3*01	1468	gnl|Fabrus|O12_IGKJ1*01	1101
  1789	VH3-23_IGHD6-6*01>2′_IGHJ3*01	1469	gnl|Fabrus|O12_IGKJ1*01	1101
  1790	VH3-23_IGHD6-6*01>3′_IGHJ3*01	1470	gnl|Fabrus|O12_IGKJ1*01	1101
  1791	VH3-23_IGHD6-6*01>2_IGHJ3*01	1414	gnl|Fabrus|O12_IGKJ1*01	1101
  1792	VH3-23_IGHD6-13*01>1_IGHJ3*01	1415	gnl|Fabrus|O12_IGKJ1*01	1101
  1793	VH3-23_IGHD6-13*01>2_IGHJ3*01	1416	gnl|Fabrus|O12_IGKJ1*01	1101
  1794	VH3-23_IGHD6-19*01>1_IGHJ3*01	1417	gnl|Fabrus|O12_IGKJ1*01	1101
  1795	VH3-23_IGHD6-19*01>2_IGHJ3*01	1418	gnl|Fabrus|O12_IGKJ1*01	1101
  1796	VH3-23_IGHD6-25*01>1_IGHJ3*01	1419	gnl|Fabrus|O12_IGKJ1*01	1101
  1797	VH3-23_IGHD6-25*01>2_IGHJ3*01	1420	gnl|Fabrus|O12_IGKJ1*01	1101
  1798	VH3-23_IGHD7-27*01>1_IGHJ3*01	1421	gnl|Fabrus|O12_IGKJ1*01	1101
  1799	VH3-23_IGHD7-27*01>3_IGHJ3*01	1422	gnl|Fabrus|O12_IGKJ1*01	1101
  1800	VH3-23_IGHD6-13*01>1′_IGHJ3*01	1471	gnl|Fabrus|O12_IGKJ1*01	1101
  1801	VH3-23_IGHD6-13*01>2′_IGHJ3*01	1472	gnl|Fabrus|O12_IGKJ1*01	1101
  1802	VH3-23_IGHD6-13*01>1_IGHJ6*01	1473	gnl|Fabrus|O12_IGKJ1*01	1101
  1803	VH3-23_IGHD6-19*01>1′_IGHJ3*01	1474	gnl|Fabrus|O12_IGKJ1*01	1101
  1804	VH3-23_IGHD6-19*01>2′_IGHJ3*01	1475	gnl|Fabrus|O12_IGKJ1*01	1101
  1805	VH3-23_IGHD6-19*01>3′_IGHJ3*01	1476	gnl|Fabrus|O12_IGKJ1*01	1101
  1806	VH3-23_IGHD6-25*01>1′_IGHJ3*01	1477	gnl|Fabrus|O12_IGKJ1*01	1101
  1807	VH3-23_IGHD6-25*01>3′_IGHJ3*01	1478	gnl|Fabrus|O12_IGKJ1*01	1101
  1808	VH3-23_IGHD7-27*01>1′_IGHJ3*01	1479	gnl|Fabrus|O12_IGKJ1*01	1101
  1809	VH3-23_IGHD7-27*01>2′_IGHJ3*01	1480	gnl|Fabrus|O12_IGKJ1*01	1101
  1810	VH3-23_IGHD1-1*01>1_IGHJ4*01	1481	gnl|Fabrus|O12_IGKJ1*01	1101
  1811	VH3-23_IGHD1-1*01>2_IGHJ4*01	1482	gnl|Fabrus|O12_IGKJ1*01	1101
  1812	VH3-23_IGHD1-1*01>3_IGHJ4*01	1483	gnl|Fabrus|O12_IGKJ1*01	1101
  1813	VH3-23_IGHD1-7*01>1_IGHJ4*01	1484	gnl|Fabrus|O12_IGKJ1*01	1101
  1814	VH3-23_IGHD1-7*01>3_IGHJ4*01	1485	gnl|Fabrus|O12_IGKJ1*01	1101
  1815	VH3-23_IGHD1-14*01>1_IGHJ4*01	1486	gnl|Fabrus|O12_IGKJ1*01	1101
  1816	VH3-23_IGHD1-14*01>3_IGHJ4*01	1487	gnl|Fabrus|O12_IGKJ1*01	1101
  1817	VH3-23_IGHD1-20*01>1_IGHJ4*01	1488	gnl|Fabrus|O12_IGKJ1*01	1101
  1818	VH3-23_IGHD1-20*01>3_IGHJ4*01	1489	gnl|Fabrus|O12_IGKJ1*01	1101
  1819	VH3-23_IGHD1-26*01>1_IGHJ4*01	1490	gnl|Fabrus|O12_IGKJ1*01	1101
  1820	VH3-23_IGHD1-26*01>3_IGHJ4*01	1491	gnl|Fabrus|O12_IGKJ1*01	1101
  1821	VH3-23_IGHD2-2*01>2_IGHJ4*01	1492	gnl|Fabrus|O12_IGKJ1*01	1101
  1822	VH3-23_IGHD2-2*01>3_IGHJ4*01	1493	gnl|Fabrus|O12_IGKJ1*01	1101
  1823	VH3-23_IGHD2-8*01>2_IGHJ4*01	1494	gnl|Fabrus|O12_IGKJ1*01	1101
  1824	VH3-23_IGHD2-8*01>3_IGHJ4*01	1495	gnl|Fabrus|O12_IGKJ1*01	1101
  1825	VH3-23_IGHD2-15*01>2_IGHJ4*01	1496	gnl|Fabrus|O12_IGKJ1*01	1101
  1826	VH3-23_IGHD2-15*01>3_IGHJ4*01	1497	gnl|Fabrus|O12_IGKJ1*01	1101
  1827	VH3-23_IGHD2-21*01>2_IGHJ4*01	1498	gnl|Fabrus|O12_IGKJ1*01	1101
  1828	VH3-23_IGHD2-21*01>3_IGHJ4*01	1499	gnl|Fabrus|O12_IGKJ1*01	1101
  1829	VH3-23_IGHD3-3*01>1_IGHJ4*01	1500	gnl|Fabrus|O12_IGKJ1*01	1101
  1830	VH3-23_IGHD3-3*01>2_IGHJ4*01	1501	gnl|Fabrus|O12_IGKJ1*01	1101
  1831	VH3-23_IGHD3-3*01>3_IGHJ4*01	1502	gnl|Fabrus|O12_IGKJ1*01	1101
  1832	VH3-23_IGHD3-9*01>2_IGHJ4*01	1503	gnl|Fabrus|O12_IGKJ1*01	1101
  1833	VH3-23_IGHD3-10*01>2_IGHJ4*01	1504	gnl|Fabrus|O12_IGKJ1*01	1101
  1834	VH3-23_IGHD3-10*01>3_IGHJ4*01	1505	gnl|Fabrus|O12_IGKJ1*01	1101
  1835	VH3-23_IGHD3-16*01>2_IGHJ4*01	1506	gnl|Fabrus|O12_IGKJ1*01	1101
  1836	VH3-23_IGHD3-16*01>3_IGHJ4*01	1507	gnl|Fabrus|O12_IGKJ1*01	1101
  1837	VH3-23_IGHD3-22*01>2_IGHJ4*01	1508	gnl|Fabrus|O12_IGKJ1*01	1101
  1838	VH3-23_IGHD3-22*01>3_IGHJ4*01	1509	gnl|Fabrus|O12_IGKJ1*01	1101
  1839	VH3-23_IGHD4-4*01 (1) >2_IGHJ4*01	1510	gnl|Fabrus|O12_IGKJ1*01	1101
  1840	VH3-23_IGHD4-4*01 (1) >3_IGHJ4*01	1511	gnl|Fabrus|O12_IGKJ1*01	1101
  1841	VH3-23_IGHD4-11*01 (1) >2_IGHJ4*01	1512	gnl|Fabrus|O12_IGKJ1*01	1101
  1842	VH3-23_IGHD4-11*01 (1) >3_IGHJ4*01	1513	gnl|Fabrus|O12_IGKJ1*01	1101
  1843	VH3-23_IGHD4-17*01>2_IGHJ4*01	1514	gnl|Fabrus|O12_IGKJ1*01	1101
  1844	VH3-23_IGHD4-17*01>3_IGHJ4*01	1515	gnl|Fabrus|O12_IGKJ1*01	1101
  1845	VH3-23_IGHD4-23*01>2_IGHJ4*01	1516	gnl|Fabrus|O12_IGKJ1*01	1101
  1846	VH3-23_IGHD4-23*01>3_IGHJ4*01	1517	gnl|Fabrus|O12_IGKJ1*01	1101
  1847	VH3-23_IGHD5-5*01 (2) >1_IGHJ4*01	1518	gnl|Fabrus|O12_IGKJ1*01	1101
  1848	VH3-23_IGHD5-5*01 (2) >2_IGHJ4*01	1519	gnl|Fabrus|O12_IGKJ1*01	1101
  1849	VH3-23_IGHD5-5*01 (2) >3_IGHJ4*01	1520	gnl|Fabrus|O12_IGKJ1*01	1101
  1850	VH3-23_IGHD5-12*01>1_IGHJ4*01	1521	gnl|Fabrus|O12_IGKJ1*01	1101
  1851	VH3-23_IGHD5-12*01>3_IGHJ4*01	1522	gnl|Fabrus|O12_IGKJ1*01	1101
  1852	VH3-23_IGHD5-18*01 (2) >1_IGHJ4*01	1523	gnl|Fabrus|O12_IGKJ1*01	1101
  1853	VH3-23_IGHD5-18*01 (2) >2_IGHJ4*01	1524	gnl|Fabrus|O12_IGKJ1*01	1101
  1854	VH3-23_IGHD5-18*01 (2) >3_IGHJ4*01	1525	gnl|Fabrus|O12_IGKJ1*01	1101
  1855	VH3-23_IGHD5-24*01>1_IGHJ4*01	1526	gnl|Fabrus|O12_IGKJ1*01	1101
  1856	VH3-23_IGHD5-24*01>3_IGHJ4*01	1527	gnl|Fabrus|O12_IGKJ1*01	1101
  1857	VH3-23_IGHD6-6*01>1_IGHJ4*01	1528	gnl|Fabrus|O12_IGKJ1*01	1101
  1858	VH3-23_IGHD1-1*01>1′_IGHJ4*01	1538	gnl|Fabrus|O12_IGKJ1*01	1101
  1859	VH3-23_IGHD1-1*01>2′_IGHJ4*01	1539	gnl|Fabrus|O12_IGKJ1*01	1101
  1860	VH3-23_IGHD1-1*01>3′_IGHJ4*01	1540	gnl|Fabrus|O12_IGKJ1*01	1101
  1861	VH3-23_IGHD1-7*01>1′_IGHJ4*01	1541	gnl|Fabrus|O12_IGKJ1*01	1101
  1862	VH3-23_IGHD1-7*01>3′_IGHJ4*01	1542	gnl|Fabrus|O12_IGKJ1*01	1101
  1863	VH3-23_IGHD1-14*01>1′_IGHJ4*01	1543	gnl|Fabrus|O12_IGKJ1*01	1101
  1864	VH3-23_IGHD1-14*01>2′_IGHJ4*01	1544	gnl|Fabrus|O12_IGKJ1*01	1101
  1865	VH3-23_IGHD1-14*01>3′_IGHJ4*01	1545	gnl|Fabrus|O12_IGKJ1*01	1101
  1866	VH3-23_IGHD1-20*01>1′_IGHJ4*01	1546	gnl|Fabrus|O12_IGKJ1*01	1101
  1867	VH3-23_IGHD1-20*01>2′_IGHJ4*01	1547	gnl|Fabrus|O12_IGKJ1*01	1101
  1868	VH3-23_IGHD1-20*01>3′_IGHJ4*01	1548	gnl|Fabrus|O12_IGKJ1*01	1101
  1869	VH3-23_IGHD1-26*01>1′_IGHJ4*01	1549	gnl|Fabrus|O12_IGKJ1*01	1101
  1870	VH3-23_IGHD1-26*01>1_IGHJ4*01_B	1550	gnl|Fabrus|O12_IGKJ1*01	1101
  1871	VH3-23_IGHD2-2*01>1′_IGHJ4*01	1551	gnl|Fabrus|O12_IGKJ1*01	1101
  1872	VH3-23_IGHD2-2*01>3′_IGHJ4*01	1552	gnl|Fabrus|O12_IGKJ1*01	1101
  1873	VH3-23_IGHD2-8*01>1′_IGHJ4*01	1553	gnl|Fabrus|O12_IGKJ1*01	1101
  1874	VH3-23_IGHD2-15*01>1′_IGHJ4*01	1554	gnl|Fabrus|O12_IGKJ1*01	1101
  1875	VH3-23_IGHD2-15*01>3′_IGHJ4*01	1555	gnl|Fabrus|O12_IGKJ1*01	1101
  1876	VH3-23_IGHD2-21*01>1′_IGHJ4*01	1556	gnl|Fabrus|O12_IGKJ1*01	1101
  1877	VH3-23_IGHD2-21*01>3′_IGHJ4*01	1557	gnl|Fabrus|O12_IGKJ1*01	1101
  1878	VH3-23_IGHD3-3*01>1′_IGHJ4*01	1558	gnl|Fabrus|O12_IGKJ1*01	1101
  1879	VH3-23_IGHD3-3*01>3′_IGHJ4*01	1559	gnl|Fabrus|O12_IGKJ1*01	1101
  1880	VH3-23_IGHD3-9*01>1′_IGHJ4*01	1560	gnl|Fabrus|O12_IGKJ1*01	1101
  1881	VH3-23_IGHD3-9*01>3′_IGHJ4*01	1561	gnl|Fabrus|O12_IGKJ1*01	1101
  1882	VH3-23_IGHD3-10*01>1′_IGHJ4*01	1562	gnl|Fabrus|O12_IGKJ1*01	1101
  1883	VH3-23_IGHD3-10*01>3′_IGHJ4*01	1563	gnl|Fabrus|O12_IGKJ1*01	1101
  1884	VH3-23_IGHD3-16*01>1′_IGHJ4*01	1564	gnl|Fabrus|O12_IGKJ1*01	1101
  1885	VH3-23_IGHD3-16*01>3′_IGHJ4*01	1565	gnl|Fabrus|O12_IGKJ1*01	1101
  1886	VH3-23_IGHD3-22*01>1′_IGHJ4*01	1566	gnl|Fabrus|O12_IGKJ1*01	1101
  1887	VH3-23_IGHD4-4*01 (1) >1′_IGHJ4*01	1567	gnl|Fabrus|O12_IGKJ1*01	1101
  1888	VH3-23_IGHD4-4*01 (1) >3′_IGHJ4*01	1568	gnl|Fabrus|O12_IGKJ1*01	1101
  1889	VH3-23_IGHD4-11*01 (1) >1′_IGHJ4*01	1569	gnl|Fabrus|O12_IGKJ1*01	1101
  1890	VH3-23_IGHD4-11*01 (1) >3′_IGHJ4*01	1570	gnl|Fabrus|O12_IGKJ1*01	1101
  1891	VH3-23_IGHD4-17*01>1′_IGHJ4*01	1571	gnl|Fabrus|O12_IGKJ1*01	1101
  1892	VH3-23_IGHD4-17*01>3′_IGHJ4*01	1572	gnl|Fabrus|O12_IGKJ1*01	1101
  1893	VH3-23_IGHD4-23*01>1′_IGHJ4*01	1573	gnl|Fabrus|O12_IGKJ1*01	1101
  1894	VH3-23_IGHD4-23*01>3′_IGHJ4*01	1574	gnl|Fabrus|O12_IGKJ1*01	1101
  1895	VH3-23_IGHD5-5*01 (2) >1′_IGHJ4*01	1575	gnl|Fabrus|O12_IGKJ1*01	1101
  1896	VH3-23_IGHD5-5*01 (2) >3′_IGHJ4*01	1576	gnl|Fabrus|O12_IGKJ1*01	1101
  1897	VH3-23_IGHD5-12*01>1′_IGHJ4*01	1577	gnl|Fabrus|O12_IGKJ1*01	1101
  1898	VH3-23_IGHD5-12*01>3′_IGHJ4*01	1578	gnl|Fabrus|O12_IGKJ1*01	1101
  1899	VH3-23_IGHD5-18*01 (2) >1′_IGHJ4*01	1579	gnl|Fabrus|O12_IGKJ1*01	1101
  1900	VH3-23_IGHD5-18*01 (2) >3′_IGHJ4*01	1580	gnl|Fabrus|O12_IGKJ1*01	1101
  1901	VH3-23_IGHD5-24*01>1′_IGHJ4*01	1581	gnl|Fabrus|O12_IGKJ1*01	1101
  1902	VH3-23_IGHD5-24*01>3′_IGHJ4*01	1582	gnl|Fabrus|O12_IGKJ1*01	1101
  1903	VH3-23_IGHD6-6*01>1′_IGHJ4*01	1583	gnl|Fabrus|O12_IGKJ1*01	1101
  1904	VH3-23_IGHD6-6*01>2′_IGHJ4*01	1584	gnl|Fabrus|O12_IGKJ1*01	1101
  1905	VH3-23_IGHD6-6*01>3′_IGHJ4*01	1585	gnl|Fabrus|O12_IGKJ1*01	1101
  1906	VH3-23_IGHD6-6*01>2_IGHJ4*01	1529	gnl|Fabrus|O12_IGKJ1*01	1101
  1907	VH3-23_IGHD6-13*01>1_IGHJ4*01	1530	gnl|Fabrus|O12_IGKJ1*01	1101
  1908	VH3-23_IGHD6-13*01>2_IGHJ4*01	1531	gnl|Fabrus|O12_IGKJ1*01	1101
  1909	VH3-23_IGHD6-19*01>1_IGHJ4*01	1532	gnl|Fabrus|O12_IGKJ1*01	1101
  1910	VH3-23_IGHD6-19*01>2_IGHJ4*01	1533	gnl|Fabrus|O12_IGKJ1*01	1101
  1911	VH3-23_IGHD6-25*01>1_IGHJ4*01	1534	gnl|Fabrus|O12_IGKJ1*01	1101
  1912	VH3-23_IGHD6-25*01>2_IGHJ4*01	1535	gnl|Fabrus|O12_IGKJ1*01	1101
  1913	VH3-23_IGHD7-27*01>1_IGHJ4*01	1536	gnl|Fabrus|O12_IGKJ1*01	1101
  1914	VH3-23_IGHD7-27*01>3_IGHJ4*01	1537	gnl|Fabrus|O12_IGKJ1*01	1101
  1915	VH3-23_IGHD6-13*01>1′_IGHJ4*01	1586	gnl|Fabrus|O12_IGKJ1*01	1101
  1916	VH3-23_IGHD6-13*01>2′_IGHJ4*01	1587	gnl|Fabrus|O12_IGKJ1*01	1101
  1917	VH3-23_IGHD6-13*01>2_IGHJ4*01_B	1588	gnl|Fabrus|O12_IGKJ1*01	1101
  1918	VH3-23_IGHD6-19*01>1′_IGHJ4*01	1589	gnl|Fabrus|O12_IGKJ1*01	1101
  1919	VH3-23_IGHD6-19*01>2′_IGHJ4*01	1590	gnl|Fabrus|O12_IGKJ1*01	1101
  1920	VH3-23_IGHD6-19*01>2_IGHJ4*01_B	1591	gnl|Fabrus|O12_IGKJ1*01	1101
  1921	VH3-23_IGHD6-25*01>1′_IGHJ4*01	1592	gnl|Fabrus|O12_IGKJ1*01	1101
  1922	VH3-23_IGHD6-25*01>3′_IGHJ4*01	1593	gnl|Fabrus|O12_IGKJ1*01	1101
  1923	VH3-23_IGHD7-27*01>1′_IGHJ4*01	1594	gnl|Fabrus|O12_IGKJ1*01	1101
  1924	VH3-23_IGHD7-27*01>2′_IGHJ4*01	1595	gnl|Fabrus|O12_IGKJ1*01	1101
  1925	VH3-23_IGHD1-1*01>1_IGHJ5*01	1596	gnl|Fabrus|O12_IGKJ1*01	1101
  1926	VH3-23_IGHD1-1*01>2_IGHJ5*01	1597	gnl|Fabrus|O12_IGKJ1*01	1101
  1927	VH3-23_IGHD1-1*01>3_IGHJ5*01	1598	gnl|Fabrus|O12_IGKJ1*01	1101
  1928	VH3-23_IGHD1-7*01>1_IGHJ5*01	1599	gnl|Fabrus|O12_IGKJ1*01	1101
  1929	VH3-23_IGHD1-7*01>3_IGHJ5*01	1600	gnl|Fabrus|O12_IGKJ1*01	1101
  1930	VH3-23_IGHD1-14*01>1_IGHJ5*01	1601	gnl|Fabrus|O12_IGKJ1*01	1101
  1931	VH3-23_IGHD1-14*01>3_IGHJ5*01	1602	gnl|Fabrus|O12_IGKJ1*01	1101
  1932	VH3-23_IGHD1-20*01>1_IGHJ5*01	1603	gnl|Fabrus|O12_IGKJ1*01	1101
  1933	VH3-23_IGHD1-20*01>3_IGHJ5*01	1604	gnl|Fabrus|O12_IGKJ1*01	1101
  1934	VH3-23_IGHD1-26*01>1_IGHJ5*01	1605	gnl|Fabrus|O12_IGKJ1*01	1101
  1935	VH3-23_IGHD1-26*01>3_IGHJ5*01	1606	gnl|Fabrus|O12_IGKJ1*01	1101
  1936	VH3-23_IGHD2-2*01>2_IGHJ5*01	1607	gnl|Fabrus|O12_IGKJ1*01	1101
  1937	VH3-23_IGHD2-2*01>3_IGHJ5*01	1608	gnl|Fabrus|O12_IGKJ1*01	1101
  1938	VH3-23_IGHD2-8*01>2_IGHJ5*01	1609	gnl|Fabrus|O12_IGKJ1*01	1101
  1939	VH3-23_IGHD2-8*01>3_IGHJ5*01	1610	gnl|Fabrus|O12_IGKJ1*01	1101
  1940	VH3-23_IGHD2-15*01>2_IGHJ5*01	1611	gnl|Fabrus|O12_IGKJ1*01	1101
  1941	VH3-23_IGHD2-15*01>3_IGHJ5*01	1612	gnl|Fabrus|O12_IGKJ1*01	1101
  1942	VH3-23_IGHD2-21*01>2_IGHJ5*01	1613	gnl|Fabrus|O12_IGKJ1*01	1101
  1943	VH3-23_IGHD2-21*01>3_IGHJ5*01	1614	gnl|Fabrus|O12_IGKJ1*01	1101
  1944	VH3-23_IGHD3-3*01>1_IGHJ5*01	1615	gnl|Fabrus|O12_IGKJ1*01	1101
  1945	VH3-23_IGHD3-3*01>2_IGHJ5*01	1616	gnl|Fabrus|O12_IGKJ1*01	1101
  1946	VH3-23_IGHD3-3*01>3_IGHJ5*01	1617	gnl|Fabrus|O12_IGKJ1*01	1101
  1947	VH3-23_IGHD3-9*01>2_IGHJ5*01	1618	gnl|Fabrus|O12_IGKJ1*01	1101
  1948	VH3-23_IGHD3-10*01>2_IGHJ5*01	1619	gnl|Fabrus|O12_IGKJ1*01	1101
  1949	VH3-23_IGHD3-10*01>3_IGHJ5*01	1620	gnl|Fabrus|O12_IGKJ1*01	1101
  1950	VH3-23_IGHD3-16*01>2_IGHJ5*01	1621	gnl|Fabrus|O12_IGKJ1*01	1101
  1951	VH3-23_IGHD3-16*01>3_IGHJ5*01	1622	gnl|Fabrus|O12_IGKJ1*01	1101
  1952	VH3-23_IGHD3-22*01>2_IGHJ5*01	1623	gnl|Fabrus|O12_IGKJ1*01	1101
  1953	VH3-23_IGHD3-22*01>3_IGHJ5*01	1624	gnl|Fabrus|O12_IGKJ1*01	1101
  1954	VH3-23_IGHD4-4*01 (1) >2_IGHJ5*01	1625	gnl|Fabrus|O12_IGKJ1*01	1101
  1955	VH3-23_IGHD4-4*01 (1) >3_IGHJ5*01	1626	gnl|Fabrus|O12_IGKJ1*01	1101
  1956	VH3-23_IGHD4-11*01 (1) >2_IGHJ5*01	1627	gnl|Fabrus|O12_IGKJ1*01	1101
  1957	VH3-23_IGHD4-11*01 (1) >3_IGHJ5*01	1628	gnl|Fabrus|O12_IGKJ1*01	1101
  1958	VH3-23_IGHD4-17*01>2_IGHJ5*01	1629	gnl|Fabrus|O12_IGKJ1*01	1101
  1959	VH3-23_IGHD4-17*01>3_IGHJ5*01	1630	gnl|Fabrus|O12_IGKJ1*01	1101
  1960	VH3-23_IGHD4-23*01>2_IGHJ5*01	1631	gnl|Fabrus|O12_IGKJ1*01	1101
  1961	VH3-23_IGHD4-23*01>3_IGHJ5*01	1632	gnl|Fabrus|O12_IGKJ1*01	1101
  1962	VH3-23_IGHD5-5*01 (2) >1_IGHJ5*01	1633	gnl|Fabrus|O12_IGKJ1*01	1101
  1963	VH3-23_IGHD5-5*01 (2) >2_IGHJ5*01	1634	gnl|Fabrus|O12_IGKJ1*01	1101
  1964	VH3-23_IGHD5-5*01 (2) >3_IGHJ5*01	1635	gnl|Fabrus|O12_IGKJ1*01	1101
  1965	VH3-23_IGHD5-12*01>1_IGHJ5*01	1636	gnl|Fabrus|O12_IGKJ1*01	1101
  1966	VH3-23_IGHD5-12*01>3_IGHJ5*01	1637	gnl|Fabrus|O12_IGKJ1*01	1101
  1967	VH3-23_IGHD5-18*01 (2) >1_IGHJ5*01	1638	gnl|Fabrus|O12_IGKJ1*01	1101
  1968	VH3-23_IGHD5-18*01 (2) >2_IGHJ5*01	1639	gnl|Fabrus|O12_IGKJ1*01	1101
  1969	VH3-23_IGHD5-18*01 (2) >3_IGHJ5*01	1640	gnl|Fabrus|O12_IGKJ1*01	1101
  1970	VH3-23_IGHD5-24*01>1_IGHJ5*01	1641	gnl|Fabrus|O12_IGKJ1*01	1101
  1971	VH3-23_IGHD5-24*01>3_IGHJ5*01	1642	gnl|Fabrus|O12_IGKJ1*01	1101
  1972	VH3-23_IGHD6-6*01>1_IGHJ5*01	1643	gnl|Fabrus|O12_IGKJ1*01	1101
  1973	VH3-23_IGHD1-1*01>1′_IGHJ5*01	1653	gnl|Fabrus|O12_IGKJ1*01	1101
  1974	VH3-23_IGHD1-1*01>2′_IGHJ5*01	1654	gnl|Fabrus|O12_IGKJ1*01	1101
  1975	VH3-23_IGHD1-1*01>3′_IGHJ5*01	1655	gnl|Fabrus|O12_IGKJ1*01	1101
  1976	VH3-23_IGHD1-7*01>1′_IGHJ5*01	1656	gnl|Fabrus|O12_IGKJ1*01	1101
  1977	VH3-23_IGHD1-7*01>3′_IGHJ5*01	1657	gnl|Fabrus|O12_IGKJ1*01	1101
  1978	VH3-23_IGHD1-14*01>1′_IGHJ5*01	1658	gnl|Fabrus|O12_IGKJ1*01	1101
  1979	VH3-23_IGHD1-14*01>2′_IGHJ5*01	1659	gnl|Fabrus|O12_IGKJ1*01	1101
  1980	VH3-23_IGHD1-14*01>3′_IGHJ5*01	1660	gnl|Fabrus|O12_IGKJ1*01	1101
  1981	VH3-23_IGHD1-20*01>1′_IGHJ5*01	1661	gnl|Fabrus|O12_IGKJ1*01	1101
  1982	VH3-23_IGHD1-20*01>2′_IGHJ5*01	1662	gnl|Fabrus|O12_IGKJ1*01	1101
  1983	VH3-23_IGHD1-20*01>3′_IGHJ5*01	1663	gnl|Fabrus|O12_IGKJ1*01	1101
  1984	VH3-23_IGHD1-26*01>1′_IGHJ5*01	1664	gnl|Fabrus|O12_IGKJ1*01	1101
  1985	VH3-23_IGHD1-26*01>3′_IGHJ5*01	1665	gnl|Fabrus|O12_IGKJ1*01	1101
  1986	VH3-23_IGHD2-2*01>1′_IGHJ5*01	1666	gnl|Fabrus|O12_IGKJ1*01	1101
  1987	VH3-23_IGHD2-2*01>3′_IGHJ5*01	1667	gnl|Fabrus|O12_IGKJ1*01	1101
  1988	VH3-23_IGHD2-8*01>1′_IGHJ5*01	1668	gnl|Fabrus|O12_IGKJ1*01	1101
  1989	VH3-23_IGHD2-15*01>1′_IGHJ5*01	1669	gnl|Fabrus|O12_IGKJ1*01	1101
  1990	VH3-23_IGHD2-15*01>3′_IGHJ5*01	1670	gnl|Fabrus|O12_IGKJ1*01	1101
  1991	VH3-23_IGHD2-21*01>1′_IGHJ5*01	1671	gnl|Fabrus|O12_IGKJ1*01	1101
  1992	VH3-23_IGHD2-21*01>3′_IGHJ5*01	1672	gnl|Fabrus|O12_IGKJ1*01	1101
  1993	VH3-23_IGHD3-3*01>1′_IGHJ5*01	1673	gnl|Fabrus|O12_IGKJ1*01	1101
  1994	VH3-23_IGHD3-3*01>3′_IGHJ5*01	1674	gnl|Fabrus|O12_IGKJ1*01	1101
  1995	VH3-23_IGHD3-9*01>1′_IGHJ5*01	1675	gnl|Fabrus|O12_IGKJ1*01	1101
  1996	VH3-23_IGHD3-9*01>3′_IGHJ5*01	1676	gnl|Fabrus|O12_IGKJ1*01	1101
  1997	VH3-23_IGHD3-10*01>1′_IGHJ5*01	1677	gnl|Fabrus|O12_IGKJ1*01	1101
  1998	VH3-23_IGHD3-10*01>3′_IGHJ5*01	1678	gnl|Fabrus|O12_IGKJ1*01	1101
  1999	VH3-23_IGHD3-16*01>1′_IGHJ5*01	1679	gnl|Fabrus|O12_IGKJ1*01	1101
  2000	VH3-23_IGHD3-16*01>3′_IGHJ5*01	1680	gnl|Fabrus|O12_IGKJ1*01	1101
  2001	VH3-23_IGHD3-22*01>1′_IGHJ5*01	1681	gnl|Fabrus|O12_IGKJ1*01	1101
  2002	VH3-23_IGHD4-4*01 (1) >1′_IGHJ5*01	1682	gnl|Fabrus|O12_IGKJ1*01	1101
  2003	VH3-23_IGHD4-4*01 (1) >3′_IGHJ5*01	1683	gnl|Fabrus|O12_IGKJ1*01	1101
  2004	VH3-23_IGHD4-11*01 (1) >1′_IGHJ5*01	1684	gnl|Fabrus|O12_IGKJ1*01	1101
  2005	VH3-23_IGHD4-11*01 (1) >3′_IGHJ5*01	1685	gnl|Fabrus|O12_IGKJ1*01	1101
  2006	VH3-23_IGHD4-17*01>1′_IGHJ5*01	1686	gnl|Fabrus|O12_IGKJ1*01	1101
  2007	VH3-23_IGHD4-17*01>3′_IGHJ5*01	1687	gnl|Fabrus|O12_IGKJ1*01	1101
  2008	VH3-23_IGHD4-23*01>1′_IGHJ5*01	1688	gnl|Fabrus|O12_IGKJ1*01	1101
  2009	VH3-23_IGHD4-23*01>3′_IGHJ5*01	1689	gnl|Fabrus|O12_IGKJ1*01	1101
  2010	VH3-23_IGHD5-5*01 (2) >1′_IGHJ5*01	1690	gnl|Fabrus|O12_IGKJ1*01	1101
  2011	VH3-23_IGHD5-5*01 (2) >3′_IGHJ5*01	1691	gnl|Fabrus|O12_IGKJ1*01	1101
  2012	VH3-23_IGHD5-12*01>1′_IGHJ5*01	1692	gnl|Fabrus|O12_IGKJ1*01	1101
  2013	VH3-23_IGHD5-12*01>3′_IGHJ5*01	1693	gnl|Fabrus|O12_IGKJ1*01	1101
  2014	VH3-23_IGHD5-18*01 (2) >1′_IGHJ5*01	1694	gnl|Fabrus|O12_IGKJ1*01	1101
  2015	VH3-23_IGHD5-18*01 (2) >3′_IGHJ5*01	1695	gnl|Fabrus|O12_IGKJ1*01	1101
  2016	VH3-23_IGHD5-24*01>1′_IGHJ5*01	1696	gnl|Fabrus|O12_IGKJ1*01	1101
  2017	VH3-23_IGHD5-24*01>3′_IGHJ5*01	1697	gnl|Fabrus|O12_IGKJ1*01	1101
  2018	VH3-23_IGHD6-6*01>1′_IGHJ5*01	1698	gnl|Fabrus|O12_IGKJ1*01	1101
  2019	VH3-23_IGHD6-6*01>2′_IGHJ5*01	1699	gnl|Fabrus|O12_IGKJ1*01	1101
  2020	VH3-23_IGHD6-6*01>3′_IGHJ5*01	1700	gnl|Fabrus|O12_IGKJ1*01	1101
  2021	VH3-23_IGHD6-6*01>2_IGHJ5*01	1644	gnl|Fabrus|O12_IGKJ1*01	1101
  2022	VH3-23_IGHD6-13*01>1_IGHJ5*01	1645	gnl|Fabrus|O12_IGKJ1*01	1101
  2023	VH3-23_IGHD6-13*01>2_IGHJ5*01	1646	gnl|Fabrus|O12_IGKJ1*01	1101
  2024	VH3-23_IGHD6-19*01>1_IGHJ5*01	1647	gnl|Fabrus|O12_IGKJ1*01	1101
  2025	VH3-23_IGHD6-19*01>2_IGHJ5*01	1648	gnl|Fabrus|O12_IGKJ1*01	1101
  2026	VH3-23_IGHD6-25*01>1_IGHJ5*01	1649	gnl|Fabrus|O12_IGKJ1*01	1101
  2027	VH3-23_IGHD6-25*01>2_IGHJ5*01	1650	gnl|Fabrus|O12_IGKJ1*01	1101
  2028	VH3-23_IGHD7-27*01>1_IGHJ5*01	1651	gnl|Fabrus|O12_IGKJ1*01	1101
  2029	VH3-23_IGHD7-27*01>3_IGHJ5*01	1652	gnl|Fabrus|O12_IGKJ1*01	1101
  2030	VH3-23_IGHD6-13*01>1′_IGHJ5*01	1701	gnl|Fabrus|O12_IGKJ1*01	1101
  2031	VH3-23_IGHD6-13*01>2′_IGHJ5*01	1702	gnl|Fabrus|O12_IGKJ1*01	1101
  2032	VH3-23_IGHD6-13*01>3′_IGHJ5*01	1703	gnl|Fabrus|O12_IGKJ1*01	1101
  2033	VH3-23_IGHD6-19*01>1′_IGHJ5*01	1704	gnl|Fabrus|O12_IGKJ1*01	1101
  2034	VH3-23_IGHD6-19*01>2′_IGHJ5*01	1705	gnl|Fabrus|O12_IGKJ1*01	1101
  2035	VH3-23_IGHD6-19*01>2_IGHJ5*01_B	1706	gnl|Fabrus|O12_IGKJ1*01	1101
  2036	VH3-23_IGHD6-25*01>1′_IGHJ5*01	1707	gnl|Fabrus|O12_IGKJ1*01	1101
  2037	VH3-23_IGHD6-25*01>3′_IGHJ5*01	1708	gnl|Fabrus|O12_IGKJ1*01	1101
  2038	VH3-23_IGHD7-27*01>1′_IGHJ5*01	1709	gnl|Fabrus|O12_IGKJ1*01	1101
  2039	VH3-23_IGHD7-27*01>2′_IGHJ5*01	1710	gnl|Fabrus|O12_IGKJ1*01	1101
  2040	VH3-23_IGHD1-1*01>1_IGHJ6*01	1711	gnl|Fabrus|O12_IGKJ1*01	1101
  2041	VH3-23_IGHD1-1*01>2_IGHJ6*01	1712	gnl|Fabrus|O12_IGKJ1*01	1101
  2042	VH3-23_IGHD1-1*01>3_IGHJ6*01	1713	gnl|Fabrus|O12_IGKJ1*01	1101
  2043	VH3-23_IGHD1-7*01>1_IGHJ6*01	1714	gnl|Fabrus|O12_IGKJ1*01	1101
  2044	VH3-23_IGHD1-7*01>3_IGHJ6*01	1715	gnl|Fabrus|O12_IGKJ1*01	1101
  2045	VH3-23_IGHD1-14*01>1_IGHJ6*01	1716	gnl|Fabrus|O12_IGKJ1*01	1101
  2046	VH3-23_IGHD1-14*01>3_IGHJ6*01	1717	gnl|Fabrus|O12_IGKJ1*01	1101
  2047	VH3-23_IGHD1-20*01>1_IGHJ6*01	1718	gnl|Fabrus|O12_IGKJ1*01	1101
  2048	VH3-23_IGHD1-20*01>3_IGHJ6*01	1719	gnl|Fabrus|O12_IGKJ1*01	1101
  2049	VH3-23_IGHD1-26*01>1_IGHJ6*01	1720	gnl|Fabrus|O12_IGKJ1*01	1101
  2050	VH3-23_IGHD1-26*01>3_IGHJ6*01	1721	gnl|Fabrus|O12_IGKJ1*01	1101
  2051	VH3-23_IGHD2-2*01>2_IGHJ6*01	1722	gnl|Fabrus|O12_IGKJ1*01	1101
  2052	VH3-23_IGHD2-2*01>3_IGHJ6*01	1723	gnl|Fabrus|O12_IGKJ1*01	1101
  2053	VH3-23_IGHD2-8*01>2_IGHJ6*01	1724	gnl|Fabrus|O12_IGKJ1*01	1101
  2054	VH3-23_IGHD2-8*01>3_IGHJ6*01	1725	gnl|Fabrus|O12_IGKJ1*01	1101
  2055	VH3-23_IGHD2-15*01>2_IGHJ6*01	1726	gnl|Fabrus|O12_IGKJ1*01	1101
  2056	VH3-23_IGHD2-15*01>3_IGHJ6*01	1727	gnl|Fabrus|O12_IGKJ1*01	1101
  2057	VH3-23_IGHD2-21*01>2_IGHJ6*01	1728	gnl|Fabrus|O12_IGKJ1*01	1101
  2058	VH3-23_IGHD2-21*01>3_IGHJ6*01	1729	gnl|Fabrus|O12_IGKJ1*01	1101
  2059	VH3-23_IGHD3-3*01>1_IGHJ6*01	1730	gnl|Fabrus|O12_IGKJ1*01	1101
  2060	VH3-23_IGHD3-3*01>2_IGHJ6*01	1731	gnl|Fabrus|O12_IGKJ1*01	1101
  2061	VH3-23_IGHD3-3*01>3_IGHJ6*01	1732	gnl|Fabrus|O12_IGKJ1*01	1101
  2062	VH3-23_IGHD3-9*01>2_IGHJ6*01	1733	gnl|Fabrus|O12_IGKJ1*01	1101
  2063	VH3-23_IGHD3-10*01>2_IGHJ6*01	1734	gnl|Fabrus|O12_IGKJ1*01	1101
  2064	VH3-23_IGHD3-10*01>3_IGHJ6*01	1735	gnl|Fabrus|O12_IGKJ1*01	1101
  2065	VH3-23_IGHD3-16*01>2_IGHJ6*01	1736	gnl|Fabrus|O12_IGKJ1*01	1101
  2066	VH3-23_IGHD3-16*01>3_IGHJ6*01	1737	gnl|Fabrus|O12_IGKJ1*01	1101
  2067	VH3-23_IGHD3-22*01>2_IGHJ6*01	1738	gnl|Fabrus|O12_IGKJ1*01	1101
  2068	VH3-23_IGHD3-22*01>3_IGHJ6*01	1739	gnl|Fabrus|O12_IGKJ1*01	1101
  2069	VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01	1740	gnl|Fabrus|O12_IGKJ1*01	1101
  2070	VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01	1741	gnl|Fabrus|O12_IGKJ1*01	1101
  2071	VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01	1742	gnl|Fabrus|O12_IGKJ1*01	1101
  2072	VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01	1743	gnl|Fabrus|O12_IGKJ1*01	1101
  2073	VH3-23_IGHD4-17*01>2_IGHJ6*01	1744	gnl|Fabrus|O12_IGKJ1*01	1101
  2074	VH3-23_IGHD4-17*01>3_IGHJ6*01	1745	gnl|Fabrus|O12_IGKJ1*01	1101
  2075	VH3-23_IGHD4-23*01>2_IGHJ6*01	1746	gnl|Fabrus|O12_IGKJ1*01	1101
  2076	VH3-23_IGHD4-23*01>3_IGHJ6*01	1747	gnl|Fabrus|O12_IGKJ1*01	1101
  2077	VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01	1748	gnl|Fabrus|O12_IGKJ1*01	1101
  2078	VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01	1749	gnl|Fabrus|O12_IGKJ1*01	1101
  2079	VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01	1750	gnl|Fabrus|O12_IGKJ1*01	1101
  2080	VH3-23_IGHD5-12*01>1_IGHJ6*01	1751	gnl|Fabrus|O12_IGKJ1*01	1101
  2081	VH3-23_IGHD5-12*01>3_IGHJ6*01	1752	gnl|Fabrus|O12_IGKJ1*01	1101
  2082	VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01	1753	gnl|Fabrus|O12_IGKJ1*01	1101
  2083	VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01	1754	gnl|Fabrus|O12_IGKJ1*01	1101
  2084	VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01	1755	gnl|Fabrus|O12_IGKJ1*01	1101
  2085	VH3-23_IGHD5-24*01>1_IGHJ6*01	1756	gnl|Fabrus|O12_IGKJ1*01	1101
  2086	VH3-23_IGHD5-24*01>3_IGHJ6*01	1757	gnl|Fabrus|O12_IGKJ1*01	1101
  2087	VH3-23_IGHD6-6*01>1_IGHJ6*01	1758	gnl|Fabrus|O12_IGKJ1*01	1101
  2088	VH3-23_IGHD6-6*01>2_IGHJ6*01	1759	gnl|Fabrus|O12_IGKJ1*01	1101
  2089	VH3-23_IGHD5-12*01>3′_IGHJ6*01	1815	gnl|Fabrus|O12_IGKJ1*01	1101
  2090	VH3-23_IGHD5-18*01(2)>1′_IGHJ6*01	1809	gnl|Fabrus|O12_IGKJ1*01	1101
  2091	VH3-23_IGHD5-18*01(2)>3′_IGHJ6*01	1810	gnl|Fabrus|O12_IGKJ1*01	1101
  2092	VH3-23_IGHD5-24*01>1′_IGHJ6*01	1811	gnl|Fabrus|O12_IGKJ1*01	1101
  2093	VH3-23_IGHD5-24*01>3′_IGHJ6*01	1812	gnl|Fabrus|O12_IGKJ1*01	1101
  2094	VH3-23_IGHD6-6*01>1′_IGHJ6*01	1813	gnl|Fabrus|O12_IGKJ1*01	1101
  2095	VH3-23_IGHD6-6*01>2′_IGHJ6*01	1814	gnl|Fabrus|O12_IGKJ1*01	1101
  2096	VH3-23_IGHD6-6*01>3′_IGHJ6*01	1815	gnl|Fabrus|O12_IGKJ1*01	1101
  2097	VH3-23_IGHD1-1*01>1′_IGHJ6*01	1768	gnl|Fabrus|O12_IGKJ1*01	1101
  2098	VH3-23_IGHD1-1*01>2′_IGHJ6*01	1769	gnl|Fabrus|O12_IGKJ1*01	1101
  2099	VH3-23_IGHD1-1*01>3′_IGHJ6*01	1770	gnl|Fabrus|O12_IGKJ1*01	1101
  2100	VH3-23_IGHD1-7*01>1′_IGHJ6*01	1771	gnl|Fabrus|O12_IGKJ1*01	1101
  2101	VH3-23_IGHD1-7*01>3′_IGHJ6*01	1772	gnl|Fabrus|O12_IGKJ1*01	1101
  2102	VH3-23_IGHD1-14*01>1′_IGHJ6*01	1773	gnl|Fabrus|O12_IGKJ1*01	1101
  2103	VH3-23_IGHD1-14*01>2′_IGHJ6*01	1774	gnl|Fabrus|O12_IGKJ1*01	1101
  2104	VH3-23_IGHD1-14*01>3′_IGHJ6*01	1775	gnl|Fabrus|O12_IGKJ1*01	1101
  2105	VH3-23_IGHD1-20*01>1′_IGHJ6*01	1776	gnl|Fabrus|O12_IGKJ1*01	1101
  2106	VH3-23_IGHD1-20*01>2′_IGHJ6*01	1777	gnl|Fabrus|O12_IGKJ1*01	1101
  2107	VH3-23_IGHD1-20*01>3′_IGHJ6*01	1778	gnl|Fabrus|O12_IGKJ1*01	1101
  2108	VH3-23_IGHD1-26*01>1′_IGHJ6*01	1779	gnl|Fabrus|O12_IGKJ1*01	1101
  2109	VH3-23_IGHD1-26*01>1_IGHJ6*01_B	1780	gnl|Fabrus|O12_IGKJ1*01	1101
  2110	VH3-23_IGHD2-2*01>2_IGHJ6*01_B	1781	gnl|Fabrus|O12_IGKJ1*01	1101
  2111	VH3-23_IGHD2-2*01>3′_IGHJ6*01	1782	gnl|Fabrus|O12_IGKJ1*01	1101
  2112	VH3-23_IGHD2-8*01>1′_IGHJ6*01	1783	gnl|Fabrus|O12_IGKJ1*01	1101
  2113	VH3-23_IGHD2-15*01>1′_IGHJ6*01	1784	gnl|Fabrus|O12_IGKJ1*01	1101
  2114	VH3-23_IGHD2-15*01>3′_IGHJ6*01	1785	gnl|Fabrus|O12_IGKJ1*01	1101
  2115	VH3-23_IGHD2-21*01>1′_IGHJ6*01	1786	gnl|Fabrus|O12_IGKJ1*01	1101
  2116	VH3-23_IGHD2-21*01>3′_IGHJ6*01	1787	gnl|Fabrus|O12_IGKJ1*01	1101
  2117	VH3-23_IGHD3-3*01>1′_IGHJ6*01	1788	gnl|Fabrus|O12_IGKJ1*01	1101
  2118	VH3-23_IGHD3-3*01>3′_IGHJ6*01	1789	gnl|Fabrus|O12_IGKJ1*01	1101
  2119	VH3-23_IGHD3-9*01>1′_IGHJ6*01	1790	gnl|Fabrus|O12_IGKJ1*01	1101
  2120	VH3-23_IGHD3-9*01>3′_IGHJ6*01	1791	gnl|Fabrus|O12_IGKJ1*01	1101
  2121	VH3-23_IGHD3-10*01>1′_IGHJ6*01	1792	gnl|Fabrus|O12_IGKJ1*01	1101
  2122	VH3-23_IGHD3-10*01>3′_IGHJ6*01	1793	gnl|Fabrus|O12_IGKJ1*01	1101
  2123	VH3-23_IGHD3-16*01>1′_IGHJ6*01	1794	gnl|Fabrus|O12_IGKJ1*01	1101
  2124	VH3-23_IGHD3-16*01>3′_IGHJ6*01	1795	gnl|Fabrus|O12_IGKJ1*01	1101
  2125	VH3-23_IGHD3-22*01>1′_IGHJ6*01	1796	gnl|Fabrus|O12_IGKJ1*01	1101
  2126	VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01	1797	gnl|Fabrus|O12_IGKJ1*01	1101
  2127	VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01	1798	gnl|Fabrus|O12_IGKJ1*01	1101
  2128	VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01	1799	gnl|Fabrus|O12_IGKJ1*01	1101
  2129	VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01	1800	gnl|Fabrus|O12_IGKJ1*01	1101
  2130	VH3-23_IGHD4-17*01>1′_IGHJ6*01	1801	gnl|Fabrus|O12_IGKJ1*01	1101
  2131	VH3-23_IGHD4-17*01>3′_IGHJ6*01	1802	gnl|Fabrus|O12_IGKJ1*01	1101
  2132	VH3-23_IGHD4-23*01>1′_IGHJ6*01	1803	gnl|Fabrus|O12_IGKJ1*01	1101
  2133	VH3-23_IGHD4-23*01>3′_IGHJ6*01	1804	gnl|Fabrus|O12_IGKJ1*01	1101
  2134	VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01	1805	gnl|Fabrus|O12_IGKJ1*01	1101
  2135	VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01	1806	gnl|Fabrus|O12_IGKJ1*01	1101
  2136	VH3-23_IGHD5-12*01>1′_IGHJ6*01	1807	gnl|Fabrus|O12_IGKJ1*01	1101
  2137	VH3-23_IGHD5-12*01>3′_IGHJ6*01	1808	gnl|Fabrus|O12_IGKJ1*01	1101
  2138	VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01	1809	gnl|Fabrus|O12_IGKJ1*01	1101
  2139	VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01	1810	gnl|Fabrus|O12_IGKJ1*01	1101
  2140	VH3-23_IGHD5-24*01>1′_IGHJ6*01	1811	gnl|Fabrus|O12_IGKJ1*01	1101
  2141	VH3-23_IGHD5-24*01>3′_IGHJ6*01	1812	gnl|Fabrus|O12_IGKJ1*01	1101
  2142	VH3-23_IGHD6-6*01>1′_IGHJ6*01	1813	gnl|Fabrus|O12_IGKJ1*01	1101
  2143	VH3-23_IGHD6-6*01>2′_IGHJ6*01	1814	gnl|Fabrus|O12_IGKJ1*01	1101
  2144	VH3-23_IGHD6-6*01>3′_IGHJ6*01	1815	gnl|Fabrus|O12_IGKJ1*01	1101
  2145	VH3-23_IGHD6-13*01>1′_IGHJ6*01	1816	gnl|Fabrus|O12_IGKJ1*01	1101
  2146	VH3-23_IGHD6-13*01>2′_IGHJ6*01	1817	gnl|Fabrus|O12_IGKJ1*01	1101
  2147	VH3-23_IGHD6-13*01>3′_IGHJ6*01	1818	gnl|Fabrus|O12_IGKJ1*01	1101
  2148	VH3-23_IGHD6-19*01>1′_IGHJ6*01	1819	gnl|Fabrus|O12_IGKJ1*01	1101
  2149	VH3-23_IGHD6-19*01>2′_IGHJ6*01	1820	gnl|Fabrus|O12_IGKJ1*01	1101
  2150	VH3-23_IGHD6-19*01>3′_IGHJ6*01	1821	gnl|Fabrus|O12_IGKJ1*01	1101
  2151	VH3-23_IGHD6-25*01>1′_IGHJ6*01	1822	gnl|Fabrus|O12_IGKJ1*01	1101
  2152	VH3-23_IGHD6-25*01>3′_IGHJ6*01	1823	gnl|Fabrus|O12_IGKJ1*01	1101
  2153	VH3-23_IGHD7-27*01>1′_IGHJ6*01	1824	gnl|Fabrus|O12_IGKJ1*01	1101
  2154	VH3-23_IGHD7-27*01>2′_IGHJ6*01	1825	gnl|Fabrus|O12_IGKJ1*01	1101
  2155	VH3-23_IGHD1-1*01>1_IGHJ1*01	1136	gnl|Fabrus|O18_IGKJ1*01	1102
  2156	VH3-23_IGHD1-1*01>2_IGHJ1*01	1137	gnl|Fabrus|O18_IGKJ1*01	1102
  2157	VH3-23_IGHD1-1*01>3_IGHJ1*01	1138	gnl|Fabrus|O18_IGKJ1*01	1102
  2158	VH3-23_IGHD1-7*01>1_IGHJ1*01	1139	gnl|Fabrus|O18_IGKJ1*01	1102
  2159	VH3-23_IGHD1-7*01>3_IGHJ1*01	1140	gnl|Fabrus|O18_IGKJ1*01	1102
  2160	VH3-23_IGHD1-14*01>1_IGHJ1*01	1141	gnl|Fabrus|O18_IGKJ1*01	1102
  2161	VH3-23_IGHD1-14*01>3_IGHJ1*01	1142	gnl|Fabrus|O18_IGKJ1*01	1102
  2162	VH3-23_IGHD1-20*01>1_IGHJ1*01	1143	gnl|Fabrus|O18_IGKJ1*01	1102
  2163	VH3-23_IGHD1-20*01>3_IGHJ1*01	1144	gnl|Fabrus|O18_IGKJ1*01	1102
  2164	VH3-23_IGHD1-26*01>1_IGHJ1*01	1145	gnl|Fabrus|O18_IGKJ1*01	1102
  2165	VH3-23_IGHD1-26*01>3_IGHJ1*01	1146	gnl|Fabrus|O18_IGKJ1*01	1102
  2166	VH3-23_IGHD2-2*01>2_IGHJ1*01	1147	gnl|Fabrus|O18_IGKJ1*01	1102
  2167	VH3-23_IGHD2-2*01>3_IGHJ1*01	1148	gnl|Fabrus|O18_IGKJ1*01	1102
  2168	VH3-23_IGHD2-8*01>2_IGHJ1*01	1149	gnl|Fabrus|O18_IGKJ1*01	1102
  2169	VH3-23_IGHD2-8*01>3_IGHJ1*01	1150	gnl|Fabrus|O18_IGKJ1*01	1102
  2170	VH3-23_IGHD2-15*01>2_IGHJ1*01	1151	gnl|Fabrus|O18_IGKJ1*01	1102
  2171	VH3-23_IGHD2-15*01>3_IGHJ1*01	1152	gnl|Fabrus|O18_IGKJ1*01	1102
  2172	VH3-23_IGHD2-21*01>2_IGHJ1*01	1153	gnl|Fabrus|O18_IGKJ1*01	1102
  2173	VH3-23_IGHD2-21*01>3_IGHJ1*01	1154	gnl|Fabrus|O18_IGKJ1*01	1102
  2174	VH3-23_IGHD3-3*01>1_IGHJ1*01	1155	gnl|Fabrus|O18_IGKJ1*01	1102
  2175	VH3-23_IGHD3-3*01>2_IGHJ1*01	1156	gnl|Fabrus|O18_IGKJ1*01	1102
  2176	VH3-23_IGHD3-3*01>3_IGHJ1*01	1157	gnl|Fabrus|O18_IGKJ1*01	1102
  2177	VH3-23_IGHD3-9*01>2_IGHJ1*01	1158	gnl|Fabrus|O18_IGKJ1*01	1102
  2178	VH3-23_IGHD3-10*01>2_IGHJ1*01	1159	gnl|Fabrus|O18_IGKJ1*01	1102
  2179	VH3-23_IGHD3-10*01>3_IGHJ1*01	1160	gnl|Fabrus|O18_IGKJ1*01	1102
  2180	VH3-23_IGHD3-16*01>2_IGHJ1*01	1161	gnl|Fabrus|O18_IGKJ1*01	1102
  2181	VH3-23_IGHD3-16*01>3_IGHJ1*01	1162	gnl|Fabrus|O18_IGKJ1*01	1102
  2182	VH3-23_IGHD3-22*01>2_IGHJ1*01	1163	gnl|Fabrus|O18_IGKJ1*01	1102
  2183	VH3-23_IGHD3-22*01>3_IGHJ1*01	1164	gnl|Fabrus|O18_IGKJ1*01	1102
  2184	VH3-23_IGHD4-4*01 (1) >2_IGHJ1*01	1165	gnl|Fabrus|O18_IGKJ1*01	1102
  2185	VH3-23_IGHD4-4*01 (1) >3_IGHJ1*01	1166	gnl|Fabrus|O18_IGKJ1*01	1102
  2186	VH3-23_IGHD4-11*01 (1) >2_IGHJ1*01	1167	gnl|Fabrus|O18_IGKJ1*01	1102
  2187	VH3-23_IGHD4-11*01 (1) >3_IGHJ1*01	1168	gnl|Fabrus|O18_IGKJ1*01	1102
  2188	VH3-23_IGHD4-17*01>2_IGHJ1*01	1169	gnl|Fabrus|O18_IGKJ1*01	1102
  2189	VH3-23_IGHD4-17*01>3_IGHJ1*01	1170	gnl|Fabrus|O18_IGKJ1*01	1102
  2190	VH3-23_IGHD4-23*01>2_IGHJ1*01	1171	gnl|Fabrus|O18_IGKJ1*01	1102
  2191	VH3-23_IGHD4-23*01>3_IGHJ1*01	1172	gnl|Fabrus|O18_IGKJ1*01	1102
  2192	VH3-23_IGHD5-5*01 (2) >1_IGHJ1*01	1173	gnl|Fabrus|O18_IGKJ1*01	1102
  2193	VH3-23_IGHD5-5*01 (2) >2_IGHJ1*01	1174	gnl|Fabrus|O18_IGKJ1*01	1102
  2194	VH3-23_IGHD5-5*01 (2) >3_IGHJ1*01	1175	gnl|Fabrus|O18_IGKJ1*01	1102
  2195	VH3-23_IGHD5-12*01>1_IGHJ1*01	1176	gnl|Fabrus|O18_IGKJ1*01	1102
  2196	VH3-23_IGHD5-12*01>3_IGHJ1*01	1177	gnl|Fabrus|O18_IGKJ1*01	1102
  2197	VH3-23_IGHD5-18*01 (2) >1_IGHJ1*01	1178	gnl|Fabrus|O18_IGKJ1*01	1102
  2198	VH3-23_IGHD5-18*01 (2) >2_IGHJ1*01	1179	gnl|Fabrus|O18_IGKJ1*01	1102
  2199	VH3-23_IGHD5-18*01 (2) >3_IGHJ1*01	1180	gnl|Fabrus|O18_IGKJ1*01	1102
  2200	VH3-23_IGHD5-24*01>1_IGHJ1*01	1181	gnl|Fabrus|O18_IGKJ1*01	1102
  2201	VH3-23_IGHD5-24*01>3_IGHJ1*01	1182	gnl|Fabrus|O18_IGKJ1*01	1102
  2202	VH3-23_IGHD6-6*01>1_IGHJ1*01	1183	gnl|Fabrus|O18_IGKJ1*01	1102
  2203	VH3-23_IGHD1-1*01>1′_IGHJ1*01	1193	gnl|Fabrus|O18_IGKJ1*01	1102
  2204	VH3-23_IGHD1-1*01>2′_IGHJ1*01	1194	gnl|Fabrus|O18_IGKJ1*01	1102
  2205	VH3-23_IGHD1-1*01>3′_IGHJ1*01	1195	gnl|Fabrus|O18_IGKJ1*01	1102
  2206	VH3-23_IGHD1-7*01>1′_IGHJ1*01	1196	gnl|Fabrus|O18_IGKJ1*01	1102
  2207	VH3-23_IGHD1-7*01>3′_IGHJ1*01	1197	gnl|Fabrus|O18_IGKJ1*01	1102
  2208	VH3-23_IGHD1-14*01>1′_IGHJ1*01	1198	gnl|Fabrus|O18_IGKJ1*01	1102
  2209	VH3-23_IGHD1-14*01>2′_IGHJ1*01	1199	gnl|Fabrus|O18_IGKJ1*01	1102
  2210	VH3-23_IGHD1-14*01>3′_IGHJ1*01	1200	gnl|Fabrus|O18_IGKJ1*01	1102
  2211	VH3-23_IGHD1-20*01>1′_IGHJ1*01	1201	gnl|Fabrus|O18_IGKJ1*01	1102
  2212	VH3-23_IGHD1-20*01>2′_IGHJ1*01	1202	gnl|Fabrus|O18_IGKJ1*01	1102
  2213	VH3-23_IGHD1-20*01>3′_IGHJ1*01	1203	gnl|Fabrus|O18_IGKJ1*01	1102
  2214	VH3-23_IGHD1-26*01>1′_IGHJ1*01	1204	gnl|Fabrus|O18_IGKJ1*01	1102
  2215	VH3-23_IGHD1-26*01>3′_IGHJ1*01	1205	gnl|Fabrus|O18_IGKJ1*01	1102
  2216	VH3-23_IGHD2-2*01>1′_IGHJ1*01	1206	gnl|Fabrus|O18_IGKJ1*01	1102
  2217	VH3-23_IGHD2-2*01>3′_IGHJ1*01	1207	gnl|Fabrus|O18_IGKJ1*01	1102
  2218	VH3-23_IGHD2-8*01>1′_IGHJ1*01	1208	gnl|Fabrus|O18_IGKJ1*01	1102
  2219	VH3-23_IGHD2-15*01>1′_IGHJ1*01	1209	gnl|Fabrus|O18_IGKJ1*01	1102
  2220	VH3-23_IGHD2-15*01>3′_IGHJ1*01	1210	gnl|Fabrus|O18_IGKJ1*01	1102
  2221	VH3-23_IGHD2-21*01>1′_IGHJ1*01	1211	gnl|Fabrus|O18_IGKJ1*01	1102
  2222	VH3-23_IGHD2-21*01>3′_IGHJ1*01	1212	gnl|Fabrus|O18_IGKJ1*01	1102
  2223	VH3-23_IGHD3-3*01>1′_IGHJ1*01	1213	gnl|Fabrus|O18_IGKJ1*01	1102
  2224	VH3-23_IGHD3-3*01>3′_IGHJ1*01	1214	gnl|Fabrus|O18_IGKJ1*01	1102
  2225	VH3-23_IGHD3-9*01>1′_IGHJ1*01	1215	gnl|Fabrus|O18_IGKJ1*01	1102
  2226	VH3-23_IGHD3-9*01>3′_IGHJ1*01	1216	gnl|Fabrus|O18_IGKJ1*01	1102
  2227	VH3-23_IGHD3-10*01>1′_IGHJ1*01	1217	gnl|Fabrus|O18_IGKJ1*01	1102
  2228	VH3-23_IGHD3-10*01>3′_IGHJ1*01	1218	gnl|Fabrus|O18_IGKJ1*01	1102
  2229	VH3-23_IGHD3-16*01>1′_IGHJ1*01	1219	gnl|Fabrus|O18_IGKJ1*01	1102
  2230	VH3-23_IGHD3-16*01>3′_IGHJ1*01	1220	gnl|Fabrus|O18_IGKJ1*01	1102
  2231	VH3-23_IGHD3-22*01>1′_IGHJ1*01	1221	gnl|Fabrus|O18_IGKJ1*01	1102
  2232	VH3-23_IGHD4-4*01 (1) >1′_IGHJ1*01	1222	gnl|Fabrus|O18_IGKJ1*01	1102
  2233	VH3-23_IGHD4-4*01 (1) >3′_IGHJ1*01	1223	gnl|Fabrus|O18_IGKJ1*01	1102
  2234	VH3-23_IGHD4-11*01 (1) >1′_IGHJ1*01	1224	gnl|Fabrus|O18_IGKJ1*01	1102
  2235	VH3-23_IGHD4-11*01 (1) >3′_IGHJ1*01	1225	gnl|Fabrus|O18_IGKJ1*01	1102
  2236	VH3-23_IGHD4-17*01>1′_IGHJ1*01	1226	gnl|Fabrus|O18_IGKJ1*01	1102
  2237	VH3-23_IGHD4-17*01>3′_IGHJ1*01	1227	gnl|Fabrus|O18_IGKJ1*01	1102
  2238	VH3-23_IGHD4-23*01>1′_IGHJ1*01	1228	gnl|Fabrus|O18_IGKJ1*01	1102
  2239	VH3-23_IGHD4-23*01>3′_IGHJ1*01	1229	gnl|Fabrus|O18_IGKJ1*01	1102
  2240	VH3-23_IGHD5-5*01 (2) >1′_IGHJ1*01	1230	gnl|Fabrus|O18_IGKJ1*01	1102
  2241	VH3-23_IGHD5-5*01 (2) >3′_IGHJ1*01	1231	gnl|Fabrus|O18_IGKJ1*01	1102
  2242	VH3-23_IGHD5-12*01>1′_IGHJ1*01	1232	gnl|Fabrus|O18_IGKJ1*01	1102
  2243	VH3-23_IGHD5-12*01>3′_IGHJ1*01	1233	gnl|Fabrus|O18_IGKJ1*01	1102
  2244	VH3-23_IGHD5-18*01 (2) >1′_IGHJ1*01	1234	gnl|Fabrus|O18_IGKJ1*01	1102
  2245	VH3-23_IGHD5-18*01 (2) >3′_IGHJ1*01	1235	gnl|Fabrus|O18_IGKJ1*01	1102
  2246	VH3-23_IGHD5-24*01>1′_IGHJ1*01	1236	gnl|Fabrus|O18_IGKJ1*01	1102
  2247	VH3-23_IGHD5-24*01>3′_IGHJ1*01	1237	gnl|Fabrus|O18_IGKJ1*01	1102
  2248	VH3-23_IGHD6-6*01>1′_IGHJ1*01	1238	gnl|Fabrus|O18_IGKJ1*01	1102
  2249	VH3-23_IGHD6-6*01>2′_IGHJ1*01	1239	gnl|Fabrus|O18_IGKJ1*01	1102
  2250	VH3-23_IGHD6-6*01>3′_IGHJ1*01	1240	gnl|Fabrus|O18_IGKJ1*01	1102
  2251	VH3-23_IGHD7-27*01>1′_IGHJ6*01	1824	gnl|Fabrus|O18_IGKJ1*01	1102
  2252	VH3-23_IGHD6-13*01>2_IGHJ6*01	1761	gnl|Fabrus|O18_IGKJ1*01	1102
  2253	VH3-23_IGHD6-19*01>1_IGHJ6*01	1762	gnl|Fabrus|O18_IGKJ1*01	1102
  2254	VH3-23_IGHD6-19*01>2_IGHJ6*01	1763	gnl|Fabrus|O18_IGKJ1*01	1102
  2255	VH3-23_IGHD6-25*01>1_IGHJ6*01	1764	gnl|Fabrus|O18_IGKJ1*01	1102
  2256	VH3-23_IGHD6-25*01>2_IGHJ6*01	1765	gnl|Fabrus|O18_IGKJ1*01	1102
  2257	VH3-23_IGHD7-27*01>1_IGHJ6*01	1766	gnl|Fabrus|O18_IGKJ1*01	1102
  2258	VH3-23_IGHD7-27*01>3_IGHJ6*01	1767	gnl|Fabrus|O18_IGKJ1*01	1102
  2259	VH3-23_IGHD6-13*01>1′_IGHJ6*01	1816	gnl|Fabrus|O18_IGKJ1*01	1102
  2260	VH3-23_IGHD6-13*01>2′_IGHJ6*01	1817	gnl|Fabrus|O18_IGKJ1*01	1102
  2261	VH3-23_IGHD6-13*01>2_IGHJ6*01_B	1761	gnl|Fabrus|O18_IGKJ1*01	1102
  2262	VH3-23_IGHD6-19*01>1′_IGHJ6*01	1819	gnl|Fabrus|O18_IGKJ1*01	1102
  2263	VH3-23_IGHD6-19*01>2′_IGHJ6*01	1820	gnl|Fabrus|O18_IGKJ1*01	1102
  2264	VH3-23_IGHD6-25*01>1_IGHJ6*01_B	1764	gnl|Fabrus|O18_IGKJ1*01	1102
  2265	VH3-23_IGHD6-25*01>1′_IGHJ6*01	1822	gnl|Fabrus|O18_IGKJ1*01	1102
  2266	VH3-23_IGHD6-25*01>3′_IGHJ6*01	1823	gnl|Fabrus|O18_IGKJ1*01	1102
  2267	VH3-23_IGHD7-27*01>1′_IGHJ6*01	1824	gnl|Fabrus|O18_IGKJ1*01	1102
  2268	VH3-23_IGHD7-27*01>2′_IGHJ6*01	1825	gnl|Fabrus|O18_IGKJ1*01	1102
  2269	VH3-23_IGHD7-27*01>1′_IGHJ6*01	1824	gnl|Fabrus|A20_IGKJ1*01	1077
  2270	VH3-23_IGHD6-13*01>2_IGHJ6*01	1761	gnl|Fabrus|A20_IGKJ1*01	1077
  2271	VH3-23_IGHD6-19*01>1_IGHJ6*01	1762	gnl|Fabrus|A20_IGKJ1*01	1077
  2272	VH3-23_IGHD6-19*01>2_IGHJ6*01	1763	gnl|Fabrus|A20_IGKJ1*01	1077
  2273	VH3-23_IGHD6-25*01>1_IGHJ6*01	1764	gnl|Fabrus|A20_IGKJ1*01	1077
  2274	VH3-23_IGHD6-25*01>2_IGHJ6*01	1765	gnl|Fabrus|A20_IGKJ1*01	1077
  2275	VH3-23_IGHD7-27*01>1_IGHJ6*01	1766	gnl|Fabrus|A20_IGKJ1*01	1077
  2276	VH3-23_IGHD7-27*01>3_IGHJ6*01	1767	gnl|Fabrus|A20_IGKJ1*01	1077
  2277	VH3-23_IGHD6-13*01>1′_IGHJ6*01	1816	gnl|Fabrus|A20_IGKJ1*01	1077
  2278	VH3-23_IGHD6-13*01>2′_IGHJ6*01	1817	gnl|Fabrus|A20_IGKJ1*01	1077
  2279	VH3-23_IGHD6-13*01>2_IGHJ6*01_B	1761	gnl|Fabrus|A20_IGKJ1*01	1077
  2280	VH3-23_IGHD6-19*01>1′_IGHJ6*01	1819	gnl|Fabrus|A20_IGKJ1*01	1077
  2281	VH3-23_IGHD6-19*01>2′_IGHJ6*01	1820	gnl|Fabrus|A20_IGKJ1*01	1077
  2282	VH3-23_IGHD6-25*01>1_IGHJ6*01_B	1764	gnl|Fabrus|A20_IGKJ1*01	1077
  2283	VH3-23_IGHD6-25*01>1′_IGHJ6*01	1822	gnl|Fabrus|A20_IGKJ1*01	1077
  2284	VH3-23_IGHD6-25*01>3′_IGHJ6*01	1823	gnl|Fabrus|A20_IGKJ1*01	1077
  2285	VH3-23_IGHD7-27*01>1′_IGHJ6*01	1824	gnl|Fabrus|A20_IGKJ1*01	1077
  2286	VH3-23_IGHD7-27*01>2′_IGHJ6*01	1825	gnl|Fabrus|A20_IGKJ1*01	1077
  2287	VH3-23_IGHD1-1*01>1_IGHJ6*01	1711	gnl|Fabrus|L11_IGKJ1*01	1087
  2288	VH3-23_IGHD1-1*01>2_IGHJ6*01	1712	gnl|Fabrus|L11_IGKJ1*01	1087
  2289	VH3-23_IGHD1-1*01>3_IGHJ6*01	1713	gnl|Fabrus|L11_IGKJ1*01	1087
  2290	VH3-23_IGHD1-7*01>1_IGHJ6*01	1714	gnl|Fabrus|L11_IGKJ1*01	1087
  2291	VH3-23_IGHD1-7*01>3_IGHJ6*01	1715	gnl|Fabrus|L11_IGKJ1*01	1087
  2292	VH3-23_IGHD1-14*01>1_IGHJ6*01	1716	gnl|Fabrus|L11_IGKJ1*01	1087
  2293	VH3-23_IGHD1-14*01>3_IGHJ6*01	1717	gnl|Fabrus|L11_IGKJ1*01	1087
  2294	VH3-23_IGHD1-20*01>1_IGHJ6*01	1718	gnl|Fabrus|L11_IGKJ1*01	1087
  2295	VH3-23_IGHD1-20*01>3_IGHJ6*01	1719	gnl|Fabrus|L11_IGKJ1*01	1087
  2296	VH3-23_IGHD1-26*01>1_IGHJ6*01	1720	gnl|Fabrus|L11_IGKJ1*01	1087
  2297	VH3-23_IGHD1-26*01>3_IGHJ6*01	1721	gnl|Fabrus|L11_IGKJ1*01	1087
  2298	VH3-23_IGHD2-2*01>2_IGHJ6*01	1722	gnl|Fabrus|L11_IGKJ1*01	1087
  2299	VH3-23_IGHD2-2*01>3_IGHJ6*01	1723	gnl|Fabrus|L11_IGKJ1*01	1087
  2300	VH3-23_IGHD2-8*01>2_IGHJ6*01	1724	gnl|Fabrus|L11_IGKJ1*01	1087
  2301	VH3-23_IGHD2-8*01>3_IGHJ6*01	1725	gnl|Fabrus|L11_IGKJ1*01	1087
  2302	VH3-23_IGHD2-15*01>2_IGHJ6*01	1726	gnl|Fabrus|L11_IGKJ1*01	1087
  2303	VH3-23_IGHD2-15*01>3_IGHJ6*01	1727	gnl|Fabrus|L11_IGKJ1*01	1087
  2304	VH3-23_IGHD2-21*01>2_IGHJ6*01	1728	gnl|Fabrus|L11_IGKJ1*01	1087
  2305	VH3-23_IGHD2-21*01>3_IGHJ6*01	1729	gnl|Fabrus|L11_IGKJ1*01	1087
  2306	VH3-23_IGHD3-3*01>1_IGHJ6*01	1730	gnl|Fabrus|L11_IGKJ1*01	1087
  2307	VH3-23_IGHD3-3*01>2_IGHJ6*01	1731	gnl|Fabrus|L11_IGKJ1*01	1087
  2308	VH3-23_IGHD3-3*01>3_IGHJ6*01	1732	gnl|Fabrus|L11_IGKJ1*01	1087
  2309	VH3-23_IGHD3-9*01>2_IGHJ6*01	1733	gnl|Fabrus|L11_IGKJ1*01	1087
  2310	VH3-23_IGHD3-10*01>2_IGHJ6*01	1734	gnl|Fabrus|L11_IGKJ1*01	1087
  2311	VH3-23_IGHD3-10*01>3_IGHJ6*01	1735	gnl|Fabrus|L11_IGKJ1*01	1087
  2312	VH3-23_IGHD3-16*01>2_IGHJ6*01	1736	gnl|Fabrus|L11_IGKJ1*01	1087
  2313	VH3-23_IGHD3-16*01>3_IGHJ6*01	1737	gnl|Fabrus|L11_IGKJ1*01	1087
  2314	VH3-23_IGHD3-22*01>2_IGHJ6*01	1738	gnl|Fabrus|L11_IGKJ1*01	1087
  2315	VH3-23_IGHD3-22*01>3_IGHJ6*01	1739	gnl|Fabrus|L11_IGKJ1*01	1087
  2316	VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01	1740	gnl|Fabrus|L11_IGKJ1*01	1087
  2317	VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01	1741	gnl|Fabrus|L11_IGKJ1*01	1087
  2318	VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01	1742	gnl|Fabrus|L11_IGKJ1*01	1087
  2319	VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01	1743	gnl|Fabrus|L11_IGKJ1*01	1087
  2320	VH3-23_IGHD4-17*01>2_IGHJ6*01	1744	gnl|Fabrus|L11_IGKJ1*01	1087
  2321	VH3-23_IGHD4-17*01>3_IGHJ6*01	1745	gnl|Fabrus|L11_IGKJ1*01	1087
  2322	VH3-23_IGHD4-23*01>2_IGHJ6*01	1746	gnl|Fabrus|L11_IGKJ1*01	1087
  2323	VH3-23_IGHD4-23*01>3_IGHJ6*01	1747	gnl|Fabrus|L11_IGKJ1*01	1087
  2324	VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01	1748	gnl|Fabrus|L11_IGKJ1*01	1087
  2325	VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01	1749	gnl|Fabrus|L11_IGKJ1*01	1087
  2326	VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01	1750	gnl|Fabrus|L11_IGKJ1*01	1087
  2327	VH3-23_IGHD5-12*01>1_IGHJ6*01	1751	gnl|Fabrus|L11_IGKJ1*01	1087
  2328	VH3-23_IGHD5-12*01>3_IGHJ6*01	1752	gnl|Fabrus|L11_IGKJ1*01	1087
  2329	VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01	1753	gnl|Fabrus|L11_IGKJ1*01	1087
  2330	VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01	1754	gnl|Fabrus|L11_IGKJ1*01	1087
  2331	VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01	1755	gnl|Fabrus|L11_IGKJ1*01	1087
  2332	VH3-23_IGHD5-24*01>1_IGHJ6*01	1756	gnl|Fabrus|L11_IGKJ1*01	1087
  2333	VH3-23_IGHD5-24*01>3_IGHJ6*01	1757	gnl|Fabrus|L11_IGKJ1*01	1087
  2334	VH3-23_IGHD6-6*01>1_IGHJ6*01	1758	gnl|Fabrus|L11_IGKJ1*01	1087
  2335	VH3-23_IGHD1-1*01>1′_IGHJ6*01	1768	gnl|Fabrus|L11_IGKJ1*01	1087
  2336	VH3-23_IGHD1-1*01>2′_IGHJ6*01	1769	gnl|Fabrus|L11_IGKJ1*01	1087
  2337	VH3-23_IGHD1-1*01>3′_IGHJ6*01	1770	gnl|Fabrus|L11_IGKJ1*01	1087
  2338	VH3-23_IGHD1-7*01>1′_IGHJ6*01	1771	gnl|Fabrus|L11_IGKJ1*01	1087
  2339	VH3-23_IGHD1-7*01>3′_IGHJ6*01	1772	gnl|Fabrus|L11_IGKJ1*01	1087
  2340	VH3-23_IGHD1-14*01>1′_IGHJ6*01	1773	gnl|Fabrus|L11_IGKJ1*01	1087
  2341	VH3-23_IGHD1-14*01>2′_IGHJ6*01	1774	gnl|Fabrus|L11_IGKJ1*01	1087
  2342	VH3-23_IGHD1-14*01>3′_IGHJ6*01	1775	gnl|Fabrus|L11_IGKJ1*01	1087
  2343	VH3-23_IGHD1-20*01>1′_IGHJ6*01	1776	gnl|Fabrus|L11_IGKJ1*01	1087
  2344	VH3-23_IGHD1-20*01>2′_IGHJ6*01	1777	gnl|Fabrus|L11_IGKJ1*01	1087
  2345	VH3-23_IGHD1-20*01>3′_IGHJ6*01	1778	gnl|Fabrus|L11_IGKJ1*01	1087
  2346	VH3-23_IGHD1-26*01>1′_IGHJ6*01	1779	gnl|Fabrus|L11_IGKJ1*01	1087
  2347	VH3-23_IGHD1-26*01>1_IGHJ6*01_B	1780	gnl|Fabrus|L11_IGKJ1*01	1087
  2348	VH3-23_IGHD2-2*01>2_IGHJ6*01_B	1781	gnl|Fabrus|L11_IGKJ1*01	1087
  2349	VH3-23_IGHD2-2*01>3′_IGHJ6*01	1782	gnl|Fabrus|L11_IGKJ1*01	1087
  2350	VH3-23_IGHD2-8*01>1′_IGHJ6*01	1783	gnl|Fabrus|L11_IGKJ1*01	1087
  2351	VH3-23_IGHD2-15*01>1′_IGHJ6*01	1784	gnl|Fabrus|L11_IGKJ1*01	1087
  2352	VH3-23_IGHD2-15*01>3′_IGHJ6*01	1785	gnl|Fabrus|L11_IGKJ1*01	1087
  2353	VH3-23_IGHD2-21*01>1′_IGHJ6*01	1786	gnl|Fabrus|L11_IGKJ1*01	1087
  2354	VH3-23_IGHD2-21*01>3′_IGHJ6*01	1787	gnl|Fabrus|L11_IGKJ1*01	1087
  2355	VH3-23_IGHD3-3*01>1′_IGHJ6*01	1788	gnl|Fabrus|L11_IGKJ1*01	1087
  2356	VH3-23_IGHD3-3*01>3′_IGHJ6*01	1789	gnl|Fabrus|L11_IGKJ1*01	1087
  2357	VH3-23_IGHD3-9*01>1′_IGHJ6*01	1790	gnl|Fabrus|L11_IGKJ1*01	1087
  2358	VH3-23_IGHD3-9*01>3′_IGHJ6*01	1791	gnl|Fabrus|L11_IGKJ1*01	1087
  2359	VH3-23_IGHD3-10*01>1′_IGHJ6*01	1792	gnl|Fabrus|L11_IGKJ1*01	1087
  2360	VH3-23_IGHD3-10*01>3′_IGHJ6*01	1793	gnl|Fabrus|L11_IGKJ1*01	1087
  2361	VH3-23_IGHD3-16*01>1′_IGHJ6*01	1794	gnl|Fabrus|L11_IGKJ1*01	1087
  2362	VH3-23_IGHD3-16*01>3′_IGHJ6*01	1795	gnl|Fabrus|L11_IGKJ1*01	1087
  2363	VH3-23_IGHD3-22*01>1′_IGHJ6*01	1796	gnl|Fabrus|L11_IGKJ1*01	1087
  2364	VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01	1797	gnl|Fabrus|L11_IGKJ1*01	1087
  2365	VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01	1798	gnl|Fabrus|L11_IGKJ1*01	1087
  2366	VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01	1799	gnl|Fabrus|L11_IGKJ1*01	1087
  2367	VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01	1800	gnl|Fabrus|L11_IGKJ1*01	1087
  2368	VH3-23_IGHD4-17*01>1′_IGHJ6*01	1801	gnl|Fabrus|L11_IGKJ1*01	1087
  2369	VH3-23_IGHD4-17*01>3′_IGHJ6*01	1802	gnl|Fabrus|L11_IGKJ1*01	1087
  2370	VH3-23_IGHD4-23*01>1′_IGHJ6*01	1803	gnl|Fabrus|L11_IGKJ1*01	1087
  2371	VH3-23_IGHD4-23*01>3′_IGHJ6*01	1804	gnl|Fabrus|L11_IGKJ1*01	1087
  2372	VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01	1805	gnl|Fabrus|L11_IGKJ1*01	1087
  2373	VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01	1806	gnl|Fabrus|L11_IGKJ1*01	1087
  2374	VH3-23_IGHD5-12*01>1′_IGHJ6*01	1807	gnl|Fabrus|L11_IGKJ1*01	1087
  2375	VH3-23_IGHD5-12*01>3′_IGHJ6*01	1808	gnl|Fabrus|L11_IGKJ1*01	1087
  2376	VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01	1809	gnl|Fabrus|L11_IGKJ1*01	1087
  2377	VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01	1810	gnl|Fabrus|L11_IGKJ1*01	1087
  2378	VH3-23_IGHD5-24*01>1′_IGHJ6*01	1811	gnl|Fabrus|L11_IGKJ1*01	1087
  2379	VH3-23_IGHD5-24*01>3′_IGHJ6*01	1812	gnl|Fabrus|L11_IGKJ1*01	1087
  2380	VH3-23_IGHD6-6*01>1′_IGHJ6*01	1813	gnl|Fabrus|L11_IGKJ1*01	1087
  2381	VH3-23_IGHD6-6*01>2′_IGHJ6*01	1814	gnl|Fabrus|L11_IGKJ1*01	1087
  2382	VH3-23_IGHD6-6*01>3′_IGHJ6*01	1815	gnl|Fabrus|L11_IGKJ1*01	1087
  2383	VH3-23_IGHD1-1*01>1_IGHJ6*01	1711	gnl|Fabrus|L12_IGKJ1*01	1088
  2384	VH3-23_IGHD1-1*01>2_IGHJ6*01	1712	gnl|Fabrus|L12_IGKJ1*01	1088
  2385	VH3-23_IGHD1-1*01>3_IGHJ6*01	1713	gnl|Fabrus|L12_IGKJ1*01	1088
  2386	VH3-23_IGHD1-7*01>1_IGHJ6*01	1714	gnl|Fabrus|L12_IGKJ1*01	1088
  2387	VH3-23_IGHD1-7*01>3_IGHJ6*01	1715	gnl|Fabrus|L12_IGKJ1*01	1088
  2388	VH3-23_IGHD1-14*01>1_IGHJ6*01	1716	gnl|Fabrus|L12_IGKJ1*01	1088
  2389	VH3-23_IGHD1-14*01>3_IGHJ6*01	1717	gnl|Fabrus|L12_IGKJ1*01	1088
  2390	VH3-23_IGHD1-20*01>1_IGHJ6*01	1718	gnl|Fabrus|L12_IGKJ1*01	1088
  2391	VH3-23_IGHD1-20*01>3_IGHJ6*01	1719	gnl|Fabrus|L12_IGKJ1*01	1088
  2392	VH3-23_IGHD1-26*01>1_IGHJ6*01	1720	gnl|Fabrus|L12_IGKJ1*01	1088
  2393	VH3-23_IGHD1-26*01>3_IGHJ6*01	1721	gnl|Fabrus|L12_IGKJ1*01	1088
  2394	VH3-23_IGHD2-2*01>2_IGHJ6*01	1722	gnl|Fabrus|L12_IGKJ1*01	1088
  2395	VH3-23_IGHD2-2*01>3_IGHJ6*01	1723	gnl|Fabrus|L12_IGKJ1*01	1088
  2396	VH3-23_IGHD2-8*01>2_IGHJ6*01	1724	gnl|Fabrus|L12_IGKJ1*01	1088
  2397	VH3-23_IGHD2-8*01>3_IGHJ6*01	1725	gnl|Fabrus|L12_IGKJ1*01	1088
  2398	VH3-23_IGHD2-15*01>2_IGHJ6*01	1726	gnl|Fabrus|L12_IGKJ1*01	1088
  2399	VH3-23_IGHD2-15*01>3_IGHJ6*01	1727	gnl|Fabrus|L12_IGKJ1*01	1088
  2400	VH3-23_IGHD2-21*01>2_IGHJ6*01	1728	gnl|Fabrus|L12_IGKJ1*01	1088
  2401	VH3-23_IGHD2-21*01>3_IGHJ6*01	1729	gnl|Fabrus|L12_IGKJ1*01	1088
  2402	VH3-23_IGHD3-3*01>1_IGHJ6*01	1730	gnl|Fabrus|L12_IGKJ1*01	1088
  2403	VH3-23_IGHD3-3*01>2_IGHJ6*01	1731	gnl|Fabrus|L12_IGKJ1*01	1088
  2404	VH3-23_IGHD3-3*01>3_IGHJ6*01	1732	gnl|Fabrus|L12_IGKJ1*01	1088
  2405	VH3-23_IGHD3-9*01>2_IGHJ6*01	1733	gnl|Fabrus|L12_IGKJ1*01	1088
  2406	VH3-23_IGHD3-10*01>2_IGHJ6*01	1734	gnl|Fabrus|L12_IGKJ1*01	1088
  2407	VH3-23_IGHD3-10*01>3_IGHJ6*01	1735	gnl|Fabrus|L12_IGKJ1*01	1088
  2408	VH3-23_IGHD3-16*01>2_IGHJ6*01	1736	gnl|Fabrus|L12_IGKJ1*01	1088
  2409	VH3-23_IGHD3-16*01>3_IGHJ6*01	1737	gnl|Fabrus|L12_IGKJ1*01	1088
  2410	VH3-23_IGHD3-22*01>2_IGHJ6*01	1738	gnl|Fabrus|L12_IGKJ1*01	1088
  2411	VH3-23_IGHD3-22*01>3_IGHJ6*01	1739	gnl|Fabrus|L12_IGKJ1*01	1088
  2412	VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01	1740	gnl|Fabrus|L12_IGKJ1*01	1088
  2413	VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01	1741	gnl|Fabrus|L12_IGKJ1*01	1088
  2414	VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01	1742	gnl|Fabrus|L12_IGKJ1*01	1088
  2415	VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01	1743	gnl|Fabrus|L12_IGKJ1*01	1088
  2416	VH3-23_IGHD4-17*01>2_IGHJ6*01	1744	gnl|Fabrus|L12_IGKJ1*01	1088
  2417	VH3-23_IGHD4-17*01>3_IGHJ6*01	1745	gnl|Fabrus|L12_IGKJ1*01	1088
  2418	VH3-23_IGHD4-23*01>2_IGHJ6*01	1746	gnl|Fabrus|L12_IGKJ1*01	1088
  2419	VH3-23_IGHD4-23*01>3_IGHJ6*01	1747	gnl|Fabrus|L12_IGKJ1*01	1088
  2420	VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01	1748	gnl|Fabrus|L12_IGKJ1*01	1088
  2421	VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01	1749	gnl|Fabrus|L12_IGKJ1*01	1088
  2422	VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01	1750	gnl|Fabrus|L12_IGKJ1*01	1088
  2423	VH3-23_IGHD5-12*01>1_IGHJ6*01	1751	gnl|Fabrus|L12_IGKJ1*01	1088
  2424	VH3-23_IGHD5-12*01>3_IGHJ6*01	1752	gnl|Fabrus|L12_IGKJ1*01	1088
  2425	VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01	1753	gnl|Fabrus|L12_IGKJ1*01	1088
  2426	VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01	1754	gnl|Fabrus|L12_IGKJ1*01	1088
  2427	VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01	1755	gnl|Fabrus|L12_IGKJ1*01	1088
  2428	VH3-23_IGHD5-24*01>1_IGHJ6*01	1756	gnl|Fabrus|L12_IGKJ1*01	1088
  2429	VH3-23_IGHD5-24*01>3_IGHJ6*01	1757	gnl|Fabrus|L12_IGKJ1*01	1088
  2430	VH3-23_IGHD6-6*01>1_IGHJ6*01	1758	gnl|Fabrus|L12_IGKJ1*01	1088
  2431	VH3-23_IGHD1-1*01>1′_IGHJ6*01	1768	gnl|Fabrus|L12_IGKJ1*01	1088
  2432	VH3-23_IGHD1-1*01>2′_IGHJ6*01	1769	gnl|Fabrus|L12_IGKJ1*01	1088
  2433	VH3-23_IGHD1-1*01>3′_IGHJ6*01	1770	gnl|Fabrus|L12_IGKJ1*01	1088
  2434	VH3-23_IGHD1-7*01>1′_IGHJ6*01	1771	gnl|Fabrus|L12_IGKJ1*01	1088
  2435	VH3-23_IGHD1-7*01>3′_IGHJ6*01	1772	gnl|Fabrus|L12_IGKJ1*01	1088
  2436	VH3-23_IGHD1-14*01>1′_IGHJ6*01	1773	gnl|Fabrus|L12_IGKJ1*01	1088
  2437	VH3-23_IGHD1-14*01>2′_IGHJ6*01	1774	gnl|Fabrus|L12_IGKJ1*01	1088
  2438	VH3-23_IGHD1-14*01>3′_IGHJ6*01	1775	gnl|Fabrus|L12_IGKJ1*01	1088
  2439	VH3-23_IGHD1-20*01>1′_IGHJ6*01	1776	gnl|Fabrus|L12_IGKJ1*01	1088
  2440	VH3-23_IGHD1-20*01>2′_IGHJ6*01	1777	gnl|Fabrus|L12_IGKJ1*01	1088
  2441	VH3-23_IGHD1-20*01>3′_IGHJ6*01	1778	gnl|Fabrus|L12_IGKJ1*01	1088
  2442	VH3-23_IGHD1-26*01>1′_IGHJ6*01	1779	gnl|Fabrus|L12_IGKJ1*01	1088
  2443	VH3-23_IGHD1-26*01>1_IGHJ6*01_B	1780	gnl|Fabrus|L12_IGKJ1*01	1088
  2444	VH3-23_IGHD2-2*01>2_IGHJ6*01_B	1781	gnl|Fabrus|L12_IGKJ1*01	1088
  2445	VH3-23_IGHD2-2*01>3′_IGHJ6*01	1782	gnl|Fabrus|L12_IGKJ1*01	1088
  2446	VH3-23_IGHD2-8*01>1′_IGHJ6*01	1783	gnl|Fabrus|L12_IGKJ1*01	1088
  2447	VH3-23_IGHD2-15*01>1′_IGHJ6*01	1784	gnl|Fabrus|L12_IGKJ1*01	1088
  2448	VH3-23_IGHD2-15*01>3′_IGHJ6*01	1785	gnl|Fabrus|L12_IGKJ1*01	1088
  2449	VH3-23_IGHD2-21*01>1′_IGHJ6*01	1786	gnl|Fabrus|L12_IGKJ1*01	1088
  2450	VH3-23_IGHD2-21*01>3′_IGHJ6*01	1787	gnl|Fabrus|L12_IGKJ1*01	1088
  2451	VH3-23_IGHD3-3*01>1′_IGHJ6*01	1788	gnl|Fabrus|L12_IGKJ1*01	1088
  2452	VH3-23_IGHD3-3*01>3′_IGHJ6*01	1789	gnl|Fabrus|L12_IGKJ1*01	1088
  2453	VH3-23_IGHD3-9*01>1′_IGHJ6*01	1790	gnl|Fabrus|L12_IGKJ1*01	1088
  2454	VH3-23_IGHD3-9*01>3′_IGHJ6*01	1791	gnl|Fabrus|L12_IGKJ1*01	1088
  2455	VH3-23_IGHD3-10*01>1′_IGHJ6*01	1792	gnl|Fabrus|L12_IGKJ1*01	1088
  2456	VH3-23_IGHD3-10*01>3′_IGHJ6*01	1793	gnl|Fabrus|L12_IGKJ1*01	1088
  2457	VH3-23_IGHD3-16*01>1′_IGHJ6*01	1794	gnl|Fabrus|L12_IGKJ1*01	1088
  2458	VH3-23_IGHD3-16*01>3′_IGHJ6*01	1795	gnl|Fabrus|L12_IGKJ1*01	1088
  2459	VH3-23_IGHD3-22*01>1′_IGHJ6*01	1796	gnl|Fabrus|L12_IGKJ1*01	1088
  2460	VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01	1797	gnl|Fabrus|L12_IGKJ1*01	1088
  2461	VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01	1798	gnl|Fabrus|L12_IGKJ1*01	1088
  2462	VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01	1799	gnl|Fabrus|L12_IGKJ1*01	1088
  2463	VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01	1800	gnl|Fabrus|L12_IGKJ1*01	1088
  2464	VH3-23_IGHD4-17*01>1′_IGHI6*01	1801	gnl|Fabrus|L12_IGKJ1*01	1088
  2465	VH3-23_IGHD4-17*01>3′_IGHJ6*01	1802	gnl|Fabrus|L12_IGKJ1*01	1088
  2466	VH3-23_IGHD4-23*01>1′_IGHJ6*01	1803	gnl|Fabrus|L12_IGKJ1*01	1088
  2467	VH3-23_IGHD4-23*01>3′_IGHJ6*01	1804	gnl|Fabrus|L12_IGKJ1*01	1088
  2468	VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01	1805	gnl|Fabrus|L12_IGKJ1*01	1088
  2469	VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01	1806	gnl|Fabrus|L12_IGKJ1*01	1088
  2470	VH3-23_IGHD5-12*01>1′_IGHJ6*01	1807	gnl|Fabrus|L12_IGKJ1*01	1088
  2471	VH3-23_IGHD5-12*01>3′_IGHJ6*01	1808	gnl|Fabrus|L12_IGKJ1*01	1088
  2472	VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01	1809	gnl|Fabrus|L12_IGKJ1*01	1088
  2473	VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01	1810	gnl|Fabrus|L12_IGKJ1*01	1088
  2474	VH3-23_IGHD5-24*01>1′_IGHJ6*01	1811	gnl|Fabrus|L12_IGKJ1*01	1088
  2475	VH3-23_IGHD5-24*01>3′_IGHI6*01	1812	gnl|Fabrus|L12_IGKJ1*01	1088
  2476	VH3-23_IGHD6-6*01>1′_IGHJ6*01	1813	gnl|Fabrus|L12_IGKJ1*01	1088
  2477	VH3-23_IGHD6-6*01>2′_IGHJ6*01	1814	gnl|Fabrus|L12_IGKJ1*01	1088
  2478	VH3-23_IGHD6-6*01>3′_IGHJ6*01	1815	gnl|Fabrus|L12_IGKJ1*01	1088
  2479	VH3-23_IGHD1-1*01>1_IGHJ6*01	1711	gnl|Fabrus|O1_IGKJ1*01	1100
  2480	VH3-23_IGHD1-1*01>2_IGHJ6*01	1712	gnl|Fabrus|O1_IGKJ1*01	1100
  2481	VH3-23_IGHD1-1*01>3_IGHJ6*01	1713	gnl|Fabrus|O1_IGKJ1*01	1100
  2482	VH3-23_IGHD1-7*01>1_IGHJ6*01	1714	gnl|Fabrus|O1_IGKJ1*01	1100
  2483	VH3-23_IGHD1-7*01>3_IGHJ6*01	1715	gnl|Fabrus|O1_IGKJ1*01	1100
  2484	VH3-23_IGHD1-14*01>1_IGHJ6*01	1716	gnl|Fabrus|O1_IGKJ1*01	1100
  2485	VH3-23_IGHD1-14*01>3_IGHJ6*01	1717	gnl|Fabrus|O1_IGKJ1*01	1100
  2486	VH3-23_IGHD1-20*01>1_IGHJ6*01	1718	gnl|Fabrus|O1_IGKJ1*01	1100
  2487	VH3-23_IGHD1-20*01>3_IGHJ6*01	1719	gnl|Fabrus|O1_IGKJ1*01	1100
  2488	VH3-23_IGHD1-26*01>1_IGHJ6*01	1720	gnl|Fabrus|O1_IGKJ1*01	1100
  2489	VH3-23_IGHD1-26*01>3_IGHJ6*01	1721	gnl|Fabrus|O1_IGKJ1*01	1100
  2490	VH3-23_IGHD2-2*01>2_IGHJ6*01	1722	gnl|Fabrus|O1_IGKJ1*01	1100
  2491	VH3-23_IGHD2-2*01>3_IGHJ6*01	1723	gnl|Fabrus|O1_IGKJ1*01	1100
  2492	VH3-23_IGHD2-8*01>2_IGHJ6*01	1724	gnl|Fabrus|O1_IGKJ1*01	1100
  2493	VH3-23_IGHD2-8*01>3_IGHJ6*01	1725	gnl|Fabrus|O1_IGKJ1*01	1100
  2494	VH3-23_IGHD2-15*01>2_IGHJ6*01	1726	gnl|Fabrus|O1_IGKJ1*01	1100
  2495	VH3-23_IGHD2-15*01>3_IGHJ6*01	1727	gnl|Fabrus|O1_IGKJ1*01	1100
  2496	VH3-23_IGHD2-21*01>2_IGHJ6*01	1728	gnl|Fabrus|O1_IGKJ1*01	1100
  2497	VH3-23_IGHD2-21*01>3_IGHJ6*01	1729	gnl|Fabrus|O1_IGKJ1*01	1100
  2498	VH3-23_IGHD3-3*01>1_IGHJ6*01	1730	gnl|Fabrus|O1_IGKJ1*01	1100
  2499	VH3-23_IGHD3-3*01>2_IGHJ6*01	1731	gnl|Fabrus|O1_IGKJ1*01	1100
  2500	VH3-23_IGHD3-3*01>3_IGHJ6*01	1732	gnl|Fabrus|O1_IGKJ1*01	1100
  2501	VH3-23_IGHD3-9*01>2_IGHJ6*01	1733	gnl|Fabrus|O1_IGKJ1*01	1100
  2502	VH3-23_IGHD3-10*01>2_IGHJ6*01	1734	gnl|Fabrus|O1_IGKJ1*01	1100
  2503	VH3-23_IGHD3-10*01>3_IGHJ6*01	1735	gnl|Fabrus|O1_IGKJ1*01	1100
  2504	VH3-23_IGHD3-16*01>2_IGHJ6*01	1736	gnl|Fabrus|O1_IGKJ1*01	1100
  2505	VH3-23_IGHD3-16*01>3_IGHJ6*01	1737	gnl|Fabrus|O1_IGKJ1*01	1100
  2506	VH3-23_IGHD3-22*01>2_IGHJ6*01	1738	gnl|Fabrus|O1_IGKJ1*01	1100
  2507	VH3-23_IGHD3-22*01>3_IGHJ6*01	1739	gnl|Fabrus|O1_IGKJ1*01	1100
  2508	VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01	1740	gnl|Fabrus|O1_IGKJ1*01	1100
  2509	VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01	1741	gnl|Fabrus|O1_IGKJ1*01	1100
  2510	VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01	1742	gnl|Fabrus|O1_IGKJ1*01	1100
  2511	VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01	1743	gnl|Fabrus|O1_IGKJ1*01	1100
  2512	VH3-23_IGHD4-17*01>2_IGHJ6*01	1744	gnl|Fabrus|O1_IGKJ1*01	1100
  2513	VH3-23_IGHD4-17*01>3_IGHJ6*01	1745	gnl|Fabrus|O1_IGKJ1*01	1100
  2514	VH3-23_IGHD4-23*01>2_IGHJ6*01	1746	gnl|Fabrus|O1_IGKJ1*01	1100
  2515	VH3-23_IGHD4-23*01>3_IGHJ6*01	1747	gnl|Fabrus|O1_IGKJ1*01	1100
  2516	VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01	1748	gnl|Fabrus|O1_IGKJ1*01	1100
  2517	VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01	1749	gnl|Fabrus|O1_IGKJ1*01	1100
  2518	VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01	1750	gnl|Fabrus|O1_IGKJ1*01	1100
  2519	VH3-23_IGHD5-12*01>1_IGHJ6*01	1751	gnl|Fabrus|O1_IGKJ1*01	1100
  2520	VH3-23_IGHD5-12*01>3_IGHJ6*01	1752	gnl|Fabrus|O1_IGKJ1*01	1100
  2521	VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01	1753	gnl|Fabrus|O1_IGKJ1*01	1100
  2522	VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01	1754	gnl|Fabrus|O1_IGKJ1*01	1100
  2523	VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01	1755	gnl|Fabrus|O1_IGKJ1*01	1100
  2524	VH3-23_IGHD5-24*01>1_IGHJ6*01	1756	gnl|Fabrus|O1_IGKJ1*01	1100
  2525	VH3-23_IGHD5-24*01>3_IGHJ6*01	1757	gnl|Fabrus|O1_IGKJ1*01	1100
  2526	VH3-23_IGHD6-6*01>1_IGHJ6*01	1758	gnl|Fabrus|O1_IGKJ1*01	1100
  2527	VH3-23_IGHD1-1*01>1′_IGHJ6*01	1768	gnl|Fabrus|O1_IGKJ1*01	1100
  2528	VH3-23_IGHD1-1*01>2′_IGHJ6*01	1769	gnl|Fabrus|O1_IGKJ1*01	1100
  2529	VH3-23_IGHD1-1*01>3′_IGHJ6*01	1770	gnl|Fabrus|O1_IGKJ1*01	1100
  2530	VH3-23_IGHD1-7*01>1′_IGHJ6*01	1771	gnl|Fabrus|O1_IGKJ1*01	1100
  2531	VH3-23_IGHD1-7*01>3′_IGHJ6*01	1772	gnl|Fabrus|O1_IGKJ1*01	1100
  2532	VH3-23_IGHD1-14*01>1′_IGHJ6*01	1773	gnl|Fabrus|O1_IGKJ1*01	1100
  2533	VH3-23_IGHD1-14*01>2′_IGHJ6*01	1774	gnl|Fabrus|O1_IGKJ1*01	1100
  2534	VH3-23_IGHD1-14*01>3′_IGHJ6*01	1775	gnl|Fabrus|O1_IGKJ1*01	1100
  2535	VH3-23_IGHD1-20*01>1′_IGHJ6*01	1776	gnl|Fabrus|O1_IGKJ1*01	1100
  2536	VH3-23_IGHD1-20*01>2′_IGHJ6*01	1777	gnl|Fabrus|O1_IGKJ1*01	1100
  2537	VH3-23_IGHD1-20*01>3′_IGHJ6*01	1778	gnl|Fabrus|O1_IGKJ1*01	1100
  2538	VH3-23_IGHD1-26*01>1′_IGHJ6*01	1779	gnl|Fabrus|O1_IGKJ1*01	1100
  2539	VH3-23_IGHD1-26*01>1_IGHJ6*01_B	1780	gnl|Fabrus|O1_IGKJ1*01	1100
  2540	VH3-23_IGHD2-2*01>2_IGHJ6*01_B	1781	gnl|Fabrus|O1_IGKJ1*01	1100
  2541	VH3-23_IGHD2-2*01>3′_IGHJ6*01	1782	gnl|Fabrus|O1_IGKJ1*01	1100
  2542	VH3-23_IGHD2-8*01>1′_IGHJ6*01	1783	gnl|Fabrus|O1_IGKJ1*01	1100
  2543	VH3-23_IGHD2-15*01>1′_IGHJ6*01	1784	gnl|Fabrus|O1_IGKJ1*01	1100
  2544	VH3-23_IGHD2-15*01>3′_IGHJ6*01	1785	gnl|Fabrus|O1_IGKJ1*01	1100
  2545	VH3-23_IGHD2-21*01>1′_IGHJ6*01	1786	gnl|Fabrus|O1_IGKJ1*01	1100
  2546	VH3-23_IGHD2-21*01>3′_IGHJ6*01	1787	gnl|Fabrus|O1_IGKJ1*01	1100
  2547	VH3-23_IGHD3-3*01>1′_IGHJ6*01	1788	gnl|Fabrus|O1_IGKJ1*01	1100
  2548	VH3-23_IGHD3-3*01>3′_IGHJ6*01	1789	gnl|Fabrus|O1_IGKJ1*01	1100
  2549	VH3-23_IGHD3-9*01>1′_IGHJ6*01	1790	gnl|Fabrus|O1_IGKJ1*01	1100
  2550	VH3-23_IGHD3-9*01>3′_IGHJ6*01	1791	gnl|Fabrus|O1_IGKJ1*01	1100
  2551	VH3-23_IGHD3-10*01>1′_IGHJ6*01	1792	gnl|Fabrus|O1_IGKJ1*01	1100
  2552	VH3-23_IGHD3-10*01>3′_IGHJ6*01	1793	gnl|Fabrus|O1_IGKJ1*01	1100
  2553	VH3-23_IGHD3-16*01>1′_IGHJ6*01	1794	gnl|Fabrus|O1_IGKJ1*01	1100
  2554	VH3-23_IGHD3-16*01>3′_IGHJ6*01	1795	gnl|Fabrus|O1_IGKJ1*01	1100
  2555	VH3-23_IGHD3-22*01>1′_1GHJ6*01	1796	gnl|Fabrus|O1_IGKJ1*01	1100
  2556	VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01	1797	gnl|Fabrus|O1_IGKJ1*01	1100
  2557	VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01	1798	gnl|Fabrus|O1_IGKJ1*01	1100
  2558	VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01	1799	gnl|Fabrus|O1_IGKJ1*01	1100
  2559	VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01	1800	gnl|Fabrus|O1_IGKJ1*01	1100
  2560	VH3-23_IGHD4-17*01>1′_IGHJ6*01	1801	gnl|Fabrus|O1_IGKJ1*01	1100
  2561	VH3-23_IGHD4-17*01>3′_IGHJ6*01	1802	gnl|Fabrus|O1_IGKJ1*01	1100
  2562	VH3-23_IGHD4-23*01>1′_IGHJ6*01	1803	gnl|Fabrus|O1_IGKJ1*01	1100
  2563	VH3-23_IGHD4-23*01>3′_IGHJ6*01	1804	gnl|Fabrus|O1_IGKJ1*01	1100
  2564	VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01	1805	gnl|Fabrus|O1_IGKJ1*01	1100
  2565	VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01	1806	gnl|Fabrus|O1_IGKJ1*01	1100
  2566	VH3-23_IGHD5-12*01>1′_IGHJ6*01	1807	gnl|Fabrus|O1_IGKJ1*01	1100
  2567	VH3-23_IGHD5-12*01>3′_IGHJ6*01	1808	gnl|Fabrus|O1_IGKJ1*01	1100
  2568	VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01	1809	gnl|Fabrus|O1_IGKJ1*01	1100
  2569	VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01	1810	gnl|Fabrus|O1_IGKJ1*01	1100
  2570	VH3-23_IGHD5-24*01>1′_IGHJ6*01	1811	gnl|Fabrus|O1_IGKJ1*01	1100
  2571	VH3-23_IGHD5-24*01>3′_IGHJ6*01	1812	gnl|Fabrus|O1_IGKJ1*01	1100
  2572	VH3-23_IGHD6-6*01>1′_IGHJ6*01	1813	gnl|Fabrus|O1_IGKJ1*01	1100
  2573	VH3-23_IGHD6-6*01>2′_IGHJ6*01	1814	gnl|Fabrus|O1_IGKJ1*01	1100
  2574	VH3-23_IGHD6-6*01>3′_IGHJ6*01	1815	gnl|Fabrus|O1_IGKJ1*01	1100
  2575	VH3-23_IGHD1-1*01>1_IGHJ5*01	1596	gnl|Fabrus|A2_IGKJ1*01	1076
  2576	VH3-23_IGHD1-1*01>2_IGHJ5*01	1597	gnl|Fabrus|A2_IGKJ1*01	1076
  2577	VH3-23_IGHD1-1*01>3_IGHJ5*01	1598	gnl|Fabrus|A2_IGKJ1*01	1076
  2578	VH3-23_IGHD1-7*01>1_IGHJ5*01	1599	gnl|Fabrus|A2_IGKJ1*01	1076
  2579	VH3-23_IGHD1-7*01>3_IGHJ5*01	1600	gnl|Fabrus|A2_IGKJ1*01	1076
  2580	VH3-23_IGHD1-14*01>1_IGHJ5*01	1601	gnl|Fabrus|A2_IGKJ1*01	1076
  2581	VH3-23_IGHD1-14*01>3_IGHJ5*01	1602	gnl|Fabrus|A2_IGKJ1*01	1076
  2582	VH3-23_IGHD1-20*01>1_IGHJ5*01	1603	gnl|Fabrus|A2_IGKJ1*01	1076
  2583	VH3-23_IGHD1-20*01>3_IGHJ5*01	1604	gnl|Fabrus|A2_IGKJ1*01	1076
  2584	VH3-23_IGHD1-26*01>1_IGHJ5*01	1605	gnl|Fabrus|A2_IGKJ1*01	1076
  2585	VH3-23_IGHD1-26*01>3_IGHJ5*01	1606	gnl|Fabrus|A2_IGKJ1*01	1076
  2586	VH3-23_IGHD2-2*01>2_IGHJ5*01	1607	gnl|Fabrus|A2_IGKJ1*01	1076
  2587	VH3-23_IGHD2-2*01>3_IGHJ5*01	1608	gnl|Fabrus|A2_IGKJ1*01	1076
  2588	VH3-23_IGHD2-8*01>2_IGHJ5*01	1609	gnl|Fabrus|A2_IGKJ1*01	1076
  2589	VH3-23_IGHD2-8*01>3_IGHJ5*01	1610	gnl|Fabrus|A2_IGKJ1*01	1076
  2590	VH3-23_IGHD2-15*01>2_IGHJ5*01	1611	gnl|Fabrus|A2_IGKJ1*01	1076
  2591	VH3-23_IGHD2-15*01>3_IGHJ5*01	1612	gnl|Fabrus|A2_IGKJ1*01	1076
  2592	VH3-23_IGHD2-21*01>2_IGHJ5*01	1613	gnl|Fabrus|A2_IGKJ1*01	1076
  2593	VH3-23_IGHD2-21*01>3_IGHJ5*01	1614	gnl|Fabrus|A2_IGKJ1*01	1076
  2594	VH3-23_IGHD3-3*01>1_IGHJ5*01	1615	gnl|Fabrus|A2_IGKJ1*01	1076
  2595	VH3-23_IGHD3-3*01>2_IGHJ5*01	1616	gnl|Fabrus|A2_IGKJ1*01	1076
  2596	VH3-23_IGHD3-3*01>3_IGHJ5*01	1617	gnl|Fabrus|A2_IGKJ1*01	1076
  2597	VH3-23_IGHD3-9*01>2_IGHJ5*01	1618	gnl|Fabrus|A2_IGKJ1*01	1076
  2598	VH3-23_IGHD3-10*01>2_IGHJ5*01	1619	gnl|Fabrus|A2_IGKJ1*01	1076
  2599	VH3-23_IGHD3-10*01>3_IGHJ5*01	1620	gnl|Fabrus|A2_IGKJ1*01	1076
  2600	VH3-23_IGHD3-16*01>2_IGHJ5*01	1621	gnl|Fabrus|A2_IGKJ1*01	1076
  2601	VH3-23_IGHD3-16*01>3_IGHJ5*01	1622	gnl|Fabrus|A2_IGKJ1*01	1076
  2602	VH3-23_IGHD3-22*01>2_IGHJ5*01	1623	gnl|Fabrus|A2_IGKJ1*01	1076
  2603	VH3-23_IGHD3-22*01>3_IGHJ5*01	1624	gnl|Fabrus|A2_IGKJ1*01	1076
  2604	VH3-23_IGHD4-4*01 (1) >2_IGHJ5*01	1625	gnl|Fabrus|A2_IGKJ1*01	1076
  2605	VH3-23_IGHD4-4*01 (1) >3_IGHJ5*01	1626	gnl|Fabrus|A2_IGKJ1*01	1076
  2606	VH3-23_IGHD4-11*01 (1) >2_IGHJ5*01	1627	gnl|Fabrus|A2_IGKJ1*01	1076
  2607	VH3-23_IGHD4-11*01 (1) >3_IGHJ5*01	1628	gnl|Fabrus|A2_IGKJ1*01	1076
  2608	VH3-23_IGHD4-17*01>2_IGHJ5*01	1629	gnl|Fabrus|A2_IGKJ1*01	1076
  2609	VH3-23_IGHD4-17*01>3_IGHJ5*01	1630	gnl|Fabrus|A2_IGKJ1*01	1076
  2610	VH3-23_IGHD4-23*01>2_IGHJ5*01	1631	gnl|Fabrus|A2_IGKJ1*01	1076
  2611	VH3-23_IGHD4-23*01>3_IGHJ5*01	1632	gnl|Fabrus|A2_IGKJ1*01	1076
  2612	VH3-23_IGHD5-5*01 (2) >1_IGHJ5*01	1633	gnl|Fabrus|A2_IGKJ1*01	1076
  2613	VH3-23_IGHD5-5*01 (2) >2_IGHJ5*01	1634	gnl|Fabrus|A2_IGKJ1*01	1076
  2614	VH3-23_IGHD5-5*01 (2) >3_IGHJ5*01	1635	gnl|Fabrus|A2_IGKJ1*01	1076
  2615	VH3-23_IGHD5-12*01>1_IGHJ5*01	1636	gnl|Fabrus|A2_IGKJ1*01	1076
  2616	VH3-23_IGHD5-12*01>3_IGHJ5*01	1637	gnl|Fabrus|A2_IGKJ1*01	1076
  2617	VH3-23_IGHD5-18*01 (2) >1_IGHJ5*01	1638	gnl|Fabrus|A2_IGKJ1*01	1076
  2618	VH3-23_IGHD5-18*01 (2) >2_IGHJ5*01	1639	gnl|Fabrus|A2_IGKJ1*01	1076
  2619	VH3-23_IGHD5-18*01 (2) >3_IGHJ5*01	1640	gnl|Fabrus|A2_IGKJ1*01	1076
  2620	VH3-23_IGHD5-24*01>1_IGHJ5*01	1641	gnl|Fabrus|A2_IGKJ1*01	1076
  2621	VH3-23_IGHD5-24*01>3_IGHJ5*01	1642	gnl|Fabrus|A2_IGKJ1*01	1076
  2622	VH3-23_IGHD6-6*01>1_IGHJ5*01	1643	gnl|Fabrus|A2_IGKJ1*01	1076
  2623	VH3-23_IGHD1-1*01>1′_IGHJ5*01	1653	gnl|Fabrus|A2_IGKJ1*01	1076
  2624	VH3-23_IGHD1-1*01>2′_IGHJ5*01	1654	gnl|Fabrus|A2_IGKJ1*01	1076
  2625	VH3-23_IGHD1-1*01>3′_IGHJ5*01	1655	gnl|Fabrus|A2_IGKJ1*01	1076
  2626	VH3-23_IGHD1-7*01>1′_IGHJ5*01	1656	gnl|Fabrus|A2_IGKJ1*01	1076
  2627	VH3-23_IGHD1-7*01>3′_IGHJ5*01	1657	gnl|Fabrus|A2_IGKJ1*01	1076
  2628	VH3-23_IGHD1-14*01>1′_IGHJ5*01	1658	gnl|Fabrus|A2_IGKJ1*01	1076
  2629	VH3-23_IGHD1-14*01>2′_IGHJ5*01	1659	gnl|Fabrus|A2_IGKJ1*01	1076
  2630	VH3-23_IGHD1-14*01>3′_IGHJ5*01	1660	gnl|Fabrus|A2_IGKJ1*01	1076
  2631	VH3-23_IGHD1-20*01>1′_IGHJ5*01	1661	gnl|Fabrus|A2_IGKJ1*01	1076
  2632	VH3-23_IGHD1-20*01>2′_IGHJ5*01	1662	gnl|Fabrus|A2_IGKJ1*01	1076
  2633	VH3-23_IGHD1-20*01>3′_IGHJ5*01	1663	gnl|Fabrus|A2_IGKJ1*01	1076
  2634	VH3-23_IGHD1-26*01>1′_IGHJ5*01	1664	gnl|Fabrus|A2_IGKJ1*01	1076
  2635	VH3-23_IGHD1-26*01>3′_IGHJ5*01	1665	gnl|Fabrus|A2_IGKJ1*01	1076
  2636	VH3-23_IGHD2-2*01>1′_IGHJ5*01	1666	gnl|Fabrus|A2_IGKJ1*01	1076
  2637	VH3-23_IGHD2-2*01>3′_IGHJ5*01	1667	gnl|Fabrus|A2_IGKJ1*01	1076
  2638	VH3-23_IGHD2-8*01>1′_IGHJ5*01	1668	gnl|Fabrus|A2_IGKJ1*01	1076
  2639	VH3-23_IGHD2-15*01>1′_IGHJ5*01	1669	gnl|Fabrus|A2_IGKJ1*01	1076
  2640	VH3-23_IGHD2-15*01>3′_IGHJ5*01	1670	gnl|Fabrus|A2_IGKJ1*01	1076
  2641	VH3-23_IGHD2-21*01>1′_IGHJ5*01	1671	gnl|Fabrus|A2_IGKJ1*01	1076
  2642	VH3-23_IGHD2-21*01>3′_IGHJ5*01	1672	gnl|Fabrus|A2_IGKJ1*01	1076
  2643	VH3-23_IGHD3-3*01>1′_IGHJ5*01	1673	gnl|Fabrus|A2_IGKJ1*01	1076
  2644	VH3-23_IGHD3-3*01>3′_IGHJ5*01	1674	gnl|Fabrus|A2_IGKJ1*01	1076
  2645	VH3-23_IGHD3-9*01>1′_IGHJ5*01	1675	gnl|Fabrus|A2_IGKJ1*01	1076
  2646	VH3-23_IGHD3-9*01>3′_IGHJ5*01	1676	gnl|Fabrus|A2_IGKJ1*01	1076
  2647	VH3-23_IGHD3-10*01>1′_IGHJ5*01	1677	gnl|Fabrus|A2_IGKJ1*01	1076
  2648	VH3-23_IGHD3-10*01>3′_IGHJ5*01	1678	gnl|Fabrus|A2_IGKJ1*01	1076
  2649	VH3-23_IGHD3-16*01>1′_IGHJ5*01	1679	gnl|Fabrus|A2_IGKJ1*01	1076
  2650	VH3-23_IGHD3-16*01>3′_IGHJ5*01	1680	gnl|Fabrus|A2_IGKJ1*01	1076
  2651	VH3-23_IGHD3-22*01>1′_IGHJ5*01	1681	gnl|Fabrus|A2_IGKJ1*01	1076
  2652	VH3-23_IGHD4-4*01 (1) >1′_IGHJ5*01	1682	gnl|Fabrus|A2_IGKJ1*01	1076
  2653	VH3-23_IGHD4-4*01 (1) >3′_IGHJ5*01	1683	gnl|Fabrus|A2_IGKJ1*01	1076
  2654	VH3-23_IGHD4-11*01 (1) >1′_IGHJ5*01	1684	gnl|Fabrus|A2_IGKJ1*01	1076
  2655	VH3-23_IGHD4-11*01 (1) >3′_IGHJ5*01	1685	gnl|Fabrus|A2_IGKJ1*01	1076
  2656	VH3-23_IGHD4-17*01>1′_IGHJ5*01	1686	gnl|Fabrus|A2_IGKJ1*01	1076
  2657	VH3-23_IGHD4-17*01>3′_IGHJ5*01	1687	gnl|Fabrus|A2_IGKJ1*01	1076
  2658	VH3-23_IGHD4-23*01>1′_IGHJ5*01	1688	gnl|Fabrus|A2_IGKJ1*01	1076
  2659	VH3-23_IGHD4-23*01>3′_IGHJ5*01	1689	gnl|Fabrus|A2_IGKJ1*01	1076
  2660	VH3-23_IGHD5-5*01 (2) >1′_IGHJ5*01	1690	gnl|Fabrus|A2_IGKJ1*01	1076
  2661	VH3-23_IGHD5-5*01 (2) >3′_IGHJ5*01	1691	gnl|Fabrus|A2_IGKJ1*01	1076
  2662	VH3-23_IGHD5-12*01>1′_IGHJ5*01	1692	gnl|Fabrus|A2_IGKJ1*01	1076
  2663	VH3-23_IGHD5-12*01>3′_IGHJ5*01	1693	gnl|Fabrus|A2_IGKJ1*01	1076
  2664	VH3-23_IGHD5-18*01 (2) >1′_IGHJ5*01	1694	gnl|Fabrus|A2_IGKJ1*01	1076
  2665	VH3-23_IGHD5-18*01 (2) >3′_IGHJ5*01	1695	gnl|Fabrus|A2_IGKJ1*01	1076
  2666	VH3-23_IGHD5-24*01>1′_IGHJ5*01	1696	gnl|Fabrus|A2_IGKJ1*01	1076
  2667	VH3-23_IGHD5-24*01>3′_IGHJ5*01	1697	gnl|Fabrus|A2_IGKJ1*01	1076
  2668	VH3-23_IGHD6-6*01>1′_IGHJ5*01	1698	gnl|Fabrus|A2_IGKJ1*01	1076
  2669	VH3-23_IGHD6-6*01>2′_IGHJ5*01	1699	gnl|Fabrus|A2_IGKJ1*01	1076
  2670	VH3-23_IGHD6-6*01>3′_IGHJ5*01	1700	gnl|Fabrus|A2_IGKJ1*01	1076
  2671	VH3-23_IGHD1-1*01>1_IGHJ6*01	1711	gnl|Fabrus|L2_IGKJ1*01	1090
  2672	VH3-23_IGHD1-1*01>2_IGHJ6*01	1712	gnl|Fabrus|L2_IGKJ1*01	1090
  2673	VH3-23_IGHD1-1*01>3_IGHJ6*01	1713	gnl|Fabrus|L2_IGKJ1*01	1090
  2674	VH3-23_IGHD1-7*01>1_IGHJ6*01	1714	gnl|Fabrus|L2_IGKJ1*01	1090
  2675	VH3-23_IGHD1-7*01>3_IGHJ6*01	1715	gnl|Fabrus|L2_IGKJ1*01	1090
  2676	VH3-23_IGHD1-14*01>1_IGHJ6*01	1716	gnl|Fabrus|L2_IGKJ1*01	1090
  2677	VH3-23_IGHD1-14*01>3_IGHJ6*01	1717	gnl|Fabrus|L2_IGKJ1*01	1090
  2678	VH3-23_IGHD1-20*01>1_IGHJ6*01	1718	gnl|Fabrus|L2_IGKJ1*01	1090
  2679	VH3-23_IGHD1-20*01>3_IGHJ6*01	1719	gnl|Fabrus|L2_IGKJ1*01	1090
  2680	VH3-23_IGHD1-26*01>1_IGHJ6*01	1720	gnl|Fabrus|L2_IGKJ1*01	1090
  2681	VH3-23_IGHD1-26*01>3_IGHJ6*01	1721	gnl|Fabrus|L2_IGKJ1*01	1090
  2682	VH3-23_IGHD2-2*01>2_IGHJ6*01	1722	gnl|Fabrus|L2_IGKJ1*01	1090
  2683	VH3-23_IGHD2-2*01>3_IGHJ6*01	1723	gnl|Fabrus|L2_IGKJ1*01	1090
  2684	VH3-23_IGHD2-8*01>2_IGHJ6*01	1724	gnl|Fabrus|L2_IGKJ1*01	1090
  2685	VH3-23_IGHD2-8*01>3_IGHJ6*01	1725	gnl|Fabrus|L2_IGKJ1*01	1090
  2686	VH3-23_IGHD2-15*01>2_IGHJ6*01	1726	gnl|Fabrus|L2_IGKJ1*01	1090
  2687	VH3-23_IGHD2-15*01>3_IGHJ6*01	1727	gnl|Fabrus|L2_IGKJ1*01	1090
  2688	VH3-23_IGHD2-21*01>2_IGHJ6*01	1728	gnl|Fabrus|L2_IGKJ1*01	1090
  2689	VH3-23_IGHD2-21*01>3_IGHJ6*01	1729	gnl|Fabrus|L2_IGKJ1*01	1090
  2690	VH3-23_IGHD3-3*01>1_IGHJ6*01	1730	gnl|Fabrus|L2_IGKJ1*01	1090
  2691	VH3-23_IGHD3-3*01>2_IGHJ6*01	1731	gnl|Fabrus|L2_IGKJ1*01	1090
  2692	VH3-23_IGHD3-3*01>3_IGHJ6*01	1732	gnl|Fabrus|L2_IGKJ1*01	1090
  2693	VH3-23_IGHD3-9*01>2_IGHJ6*01	1733	gnl|Fabrus|L2_IGKJ1*01	1090
  2694	VH3-23_IGHD3-10*01>2_IGHJ6*01	1734	gnl|Fabrus|L2_IGKJ1*01	1090
  2695	VH3-23_IGHD3-10*01>3_IGHJ6*01	1735	gnl|Fabrus|L2_IGKJ1*01	1090
  2696	VH3-23_IGHD3-16*01>2_IGHJ6*01	1736	gnl|Fabrus|L2_IGKJ1*01	1090
  2697	VH3-23_IGHD3-16*01>3_IGHJ6*01	1737	gnl|Fabrus|L2_IGKJ1*01	1090
  2698	VH3-23_IGHD3-22*01>2_IGHJ6*01	1738	gnl|Fabrus|L2_IGKJ1*01	1090
  2699	VH3-23_IGHD3-22*01>3_IGHJ6*01	1739	gnl|Fabrus|L2_IGKJ1*01	1090
  2700	VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01	1740	gnl|Fabrus|L2_IGKJ1*01	1090
  2701	VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01	1741	gnl|Fabrus|L2_IGKJ1*01	1090
  2702	VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01	1742	gnl|Fabrus|L2_IGKJ1*01	1090
  2703	VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01	1743	gnl|Fabrus|L2_IGKJ1*01	1090
  2704	VH3-23_IGHD4-17*01>2_IGHJ6*01	1744	gnl|Fabrus|L2_IGKJ1*01	1090
  2705	VH3-23_IGHD4-17*01>3_IGHJ6*01	1745	gnl|Fabrus|L2_IGKJ1*01	1090
  2706	VH3-23_IGHD4-23*01>2_IGHJ6*01	1746	gnl|Fabrus|L2_IGKJ1*01	1090
  2707	VH3-23_IGHD4-23*01>3_IGHJ6*01	1747	gnl|Fabrus|L2_IGKJ1*01	1090
  2708	VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01	1748	gnl|Fabrus|L2_IGKJ1*01	1090
  2709	VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01	1749	gnl|Fabrus|L2_IGKJ1*01	1090
  2710	VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01	1750	gnl|Fabrus|L2_IGKJ1*01	1090
  2711	VH3-23_IGHD5-12*01>1_IGHJ6*01	1751	gnl|Fabrus|L2_IGKJ1*01	1090
  2712	VH3-23_IGHD5-12*01>3_IGHJ6*01	1752	gnl|Fabrus|L2_IGKJ1*01	1090
  2713	VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01	1753	gnl|Fabrus|L2_IGKJ1*01	1090
  2714	VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01	1754	gnl|Fabrus|L2_IGKJ1*01	1090
  2715	VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01	1755	gnl|Fabrus|L2_IGKJ1*01	1090
  2716	VH3-23_IGHD5-24*01>1_IGHJ6*01	1756	gnl|Fabrus|L2_IGKJ1*01	1090
  2717	VH3-23_IGHD5-24*01>3_IGHJ6*01	1757	gnl|Fabrus|L2_IGKJ1*01	1090
  2718	VH3-23_IGHD6-6*01>1_IGHJ6*01	1758	gnl|Fabrus|L2_IGKJ1*01	1090
  2719	VH3-23_IGHD1-1*01>1′_IGHJ6*01	1768	gnl|Fabrus|L2_IGKJ1*01	1090
  2720	VH3-23_IGHD1-1*01>2′_IGHJ6*01	1769	gnl|Fabrus|L2_IGKJ1*01	1090
  2721	VH3-23_IGHD1-1*01>3′_IGHJ6*01	1770	gnl|Fabrus|L2_IGKJ1*01	1090
  2722	VH3-23_IGHD1-7*01>1′_IGHJ6*01	1771	gnl|Fabrus|L2_IGKJ1*01	1090
  2723	VH3-23_IGHD1-7*01>3′_IGHJ6*01	1772	gnl|Fabrus|L2_IGKJ1*01	1090
  2724	VH3-23_IGHD1-14*01>1′_IGHJ6*01	1773	gnl|Fabrus|L2_IGKJ1*01	1090
  2725	VH3-23_IGHD1-14*01>2′_IGHJ6*01	1774	gnl|Fabrus|L2_IGKJ1*01	1090
  2726	VH3-23_IGHD1-14*01>3′_IGHJ6*01	1775	gnl|Fabrus|L2_IGKJ1*01	1090
  2727	VH3-23_IGHD1-20*01>1′_IGHJ6*01	1776	gnl|Fabrus|L2_IGKJ1*01	1090
  2728	VH3-23_IGHD1-20*01>2′_IGHJ6*01	1777	gnl|Fabrus|L2_IGKJ1*01	1090
  2729	VH3-23_IGHD1-20*01>3′_IGHJ6*01	1778	gnl|Fabrus|L2_IGKJ1*01	1090
  2730	VH3-23_IGHD1-26*01>1′_IGHJ6*01	1779	gnl|Fabrus|L2_IGKJ1*01	1090
  2731	VH3-23_IGHD1-26*01>1_IGHJ6*01_B	1780	gnl|Fabrus|L2_IGKJ1*01	1090
  2732	VH3-23_IGHD2-2*01>2_IGHJ6*01_B	1781	gnl|Fabrus|L2_IGKJ1*01	1090
  2733	VH3-23_IGHD2-2*01>3′_IGHJ6*01	1782	gnl|Fabrus|L2_IGKJ1*01	1090
  2734	VH3-23_IGHD2-8*01>1′_IGHJ6*01	1783	gnl|Fabrus|L2_IGKJ1*01	1090
  2735	VH3-23_IGHD2-15*01>1′_IGHJ6*01	1784	gnl|Fabrus|L2_IGKJ1*01	1090
  2736	VH3-23_IGHD2-15*01>3′_IGHJ6*01	1785	gnl|Fabrus|L2_IGKJ1*01	1090
  2737	VH3-23_IGHD2-21*01>1′_IGHJ6*01	1786	gnl|Fabrus|L2_IGKJ1*01	1090
  2738	VH3-23_IGHD2-21*01>3′_IGHJ6*01	1787	gnl|Fabrus|L2_IGKJ1*01	1090
  2739	VH3-23_IGHD3-3*01>1′_IGHJ6*01	1788	gnl|Fabrus|L2_IGKJ1*01	1090
  2740	VH3-23_IGHD3-3*01>3′_IGHJ6*01	1789	gnl|Fabrus|L2_IGKJ1*01	1090
  2741	VH3-23_IGHD3-9*01>1′_IGHJ6*01	1790	gnl|Fabrus|L2_IGKJ1*01	1090
  2742	VH3-23_IGHD3-9*01>3′_IGHJ6*01	1791	gnl|Fabrus|L2_IGKJ1*01	1090
  2743	VH3-23_IGHD3-10*01>1′_IGHJ6*01	1792	gnl|Fabrus|L2_IGKJ1*01	1090
  2744	VH3-23_IGHD3-10*01>3′_IGHJ6*01	1793	gnl|Fabrus|L2_IGKJ1*01	1090
  2745	VH3-23_IGHD3-16*01>1′_IGHJ6*01	1794	gnl|Fabrus|L2_IGKJ1*01	1090
  2746	VH3-23_IGHD3-16*01>3′_IGHJ6*01	1795	gnl|Fabrus|L2_IGKJ1*01	1090
  2747	VH3-23_IGHD3-22*01>1′_IGHJ6*01	1796	gnl|Fabrus|L2_IGKJ1*01	1090
  2748	VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01	1797	gnl|Fabrus|L2_IGKJ1*01	1090
  2749	VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01	1798	gnl|Fabrus|L2_IGKJ1*01	1090
  2750	VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01	1799	gnl|Fabrus|L2_IGKJ1*01	1090
  2751	VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01	1800	gnl|Fabrus|L2_IGKJ1*01	1090
  2752	VH3-23_IGHD4-17*01>1′_IGHJ6*01	1801	gnl|Fabrus|L2_IGKJ1*01	1090
  2753	VH3-23_IGHD4-17*01>3′_IGHJ6*01	1802	gnl|Fabrus|L2_IGKJ1*01	1090
  2754	VH3-23_IGHD4-23*01>1′_IGHJ6*01	1803	gnl|Fabrus|L2_IGKJ1*01	1090
  2755	VH3-23_IGHD4-23*01>3′_IGHJ6*01	1804	gnl|Fabrus|L2_IGKJ1*01	1090
  2756	VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01	1805	gnl|Fabrus|L2_IGKJ1*01	1090
  2757	VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01	1806	gnl|Fabrus|L2_IGKJ1*01	1090
  2758	VH3-23_IGHD5-12*01>1′_IGHJ6*01	1807	gnl|Fabrus|L2_IGKJ1*01	1090
  2759	VH3-23_IGHD5-12*01>3′_IGHJ6*01	1808	gnl|Fabrus|L2_IGKJ1*01	1090
  2760	VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01	1809	gnl|Fabrus|L2_IGKJ1*01	1090
  2761	VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01	1810	gnl|Fabrus|L2_IGKJ1*01	1090
  2762	VH3-23_IGHD5-24*01>1′_IGHJ6*01	1811	gnl|Fabrus|L2_IGKJ1*01	1090
  2763	VH3-23_IGHD5-24*01>3′_IGHJ6*01	1812	gnl|Fabrus|L2_IGKJ1*01	1090
  2764	VH3-23_IGHD6-6*01>1′_IGHJ6*01	1813	gnl|Fabrus|L2_IGKJ1*01	1090
  2765	VH3-23_IGHD6-6*01>2′_IGHJ6*01	1814	gnl|Fabrus|L2_IGKJ1*01	1090
  2766	VH3-23_IGHD6-6*01>3′_IGHJ6*01	1815	gnl|Fabrus|L2_IGKJ1*01	1090
  2767	VH3-23_IGHD1-1*01>1_IGHJ6*01	1711	gnl|Fabrus|L6_IGKJ1*01	1097
  2768	VH3-23_IGHD1-1*01>2_IGHJ6*01	1712	gnl|Fabrus|L6_IGKJ1*01	1097
  2769	VH3-23_IGHD1-1*01>3_IGHJ6*01	1713	gnl|Fabrus|L6_IGKJ1*01	1097
  2770	VH3-23_IGHD1-7*01>1_IGHJ6*01	1714	gnl|Fabrus|L6_IGKJ1*01	1097
  2771	VH3-23_IGHD1-7*01>3_IGHJ6*01	1715	gnl|Fabrus|L6_IGKJ1*01	1097
  2772	VH3-23_IGHD1-14*01>1_IGHJ6*01	1716	gnl|Fabrus|L6_IGKJ1*01	1097
  2773	VH3-23_IGHD1-14*01>3_IGHJ6*01	1717	gnl|Fabrus|L6_IGKJ1*01	1097
  2774	VH3-23_IGHD1-20*01>1_IGHJ6*01	1718	gnl|Fabrus|L6_IGKJ1*01	1097
  2775	VH3-23_IGHD1-20*01>3_IGHJ6*01	1719	gnl|Fabrus|L6_IGKJ1*01	1097
  2776	VH3-23_IGHD1-26*01>1_IGHJ6*01	1720	gnl|Fabrus|L6_IGKJ1*01	1097
  2777	VH3-23_IGHD1-26*01>3_IGHJ6*01	1721	gnl|Fabrus|L6_IGKJ1*01	1097
  2778	VH3-23_IGHD2-2*01>2_IGHJ6*01	1722	gnl|Fabrus|L6_IGKJ1*01	1097
  2779	VH3-23_IGHD2-2*01>3_IGHJ6*01	1723	gnl|Fabrus|L6_IGKJ1*01	1097
  2780	VH3-23_IGHD2-8*01>2_IGHJ6*01	1724	gnl|Fabrus|L6_IGKJ1*01	1097
  2781	VH3-23_IGHD2-8*01>3_IGHJ6*01	1725	gnl|Fabrus|L6_IGKJ1*01	1097
  2782	VH3-23_IGHD2-15*01>2_IGHJ6*01	1726	gnl|Fabrus|L6_IGKJ1*01	1097
  2783	VH3-23_IGHD2-15*01>3_IGHJ6*01	1727	gnl|Fabrus|L6_IGKJ1*01	1097
  2784	VH3-23_IGHD2-21*01>2_IGHJ6*01	1728	gnl|Fabrus|L6_IGKJ1*01	1097
  2785	VH3-23_IGHD2-21*01>3_IGHJ6*01	1729	gnl|Fabrus|L6_IGKJ1*01	1097
  2786	VH3-23_IGHD3-3*01>1_IGHJ6*01	1730	gnl|Fabrus|L6_IGKJ1*01	1097
  2787	VH3-23_IGHD3-3*01>2_IGHJ6*01	1731	gnl|Fabrus|L6_IGKJ1*01	1097
  2788	VH3-23_IGHD3-3*01>3_IGHJ6*01	1732	gnl|Fabrus|L6_IGKJ1*01	1097
  2789	VH3-23_IGHD3-9*01>2_IGHJ6*01	1733	gnl|Fabrus|L6_IGKJ1*01	1097
  2790	VH3-23_IGHD3-10*01>2_IGHJ6*01	1734	gnl|Fabrus|L6_IGKJ1*01	1097
  2791	VH3-23_IGHD3-10*01>3_IGHJ6*01	1735	gnl|Fabrus|L6_IGKJ1*01	1097
  2792	VH3-23_IGHD3-16*01>2_IGHJ6*01	1736	gnl|Fabrus|L6_IGKJ1*01	1097
  2793	VH3-23_IGHD3-16*01>3_IGHJ6*01	1737	gnl|Fabrus|L6_IGKJ1*01	1097
  2794	VH3-23_IGHD3-22*01>2_IGHJ6*01	1738	gnl|Fabrus|L6_IGKJ1*01	1097
  2795	VH3-23_IGHD3-22*01>3_IGHJ6*01	1739	gnl|Fabrus|L6_IGKJ1*01	1097
  2796	VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01	1740	gnl|Fabrus|L6_IGKJ1*01	1097
  2797	VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01	1741	gnl|Fabrus|L6_IGKJ1*01	1097
  2798	VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01	1742	gnl|Fabrus|L6_IGKJ1*01	1097
  2799	VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01	1743	gnl|Fabrus|L6_IGKJ1*01	1097
  2800	VH3-23_IGHD4-17*01>2_IGHJ6*01	1744	gnl|Fabrus|L6_IGKJ1*01	1097
  2801	VH3-23_IGHD4-17*01>3_IGHJ6*01	1745	gnl|Fabrus|L6_IGKJ1*01	1097
  2802	VH3-23_IGHD4-23*01>2_IGHJ6*01	1746	gnl|Fabrus|L6_IGKJ1*01	1097
  2803	VH3-23_IGHD4-23*01>3_IGHJ6*01	1747	gnl|Fabrus|L6_IGKJ1*01	1097
  2804	VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01	1748	gnl|Fabrus|L6_IGKJ1*01	1097
  2805	VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01	1749	gnl|Fabrus|L6_IGKJ1*01	1097
  2806	VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01	1750	gnl|Fabrus|L6_IGKJ1*01	1097
  2807	VH3-23_IGHD5-12*01>1_IGHJ6*01	1751	gnl|Fabrus|L6_IGKJ1*01	1097
  2808	VH3-23_IGHD5-12*01>3_IGHJ6*01	1752	gnl|Fabrus|L6_IGKJ1*01	1097
  2809	VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01	1753	gnl|Fabrus|L6_IGKJ1*01	1097
  2810	VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01	1754	gnl|Fabrus|L6_IGKJ1*01	1097
  2811	VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01	1755	gnl|Fabrus|L6_IGKJ1*01	1097
  2812	VH3-23_IGHD5-24*01>1_IGHJ6*01	1756	gnl|Fabrus|L6_IGKJ1*01	1097
  2813	VH3-23_IGHD5-24*01>3_IGHJ6*01	1757	gnl|Fabrus|L6_IGKJ1*01	1097
  2814	VH3-23_IGHD6-6*01>1_IGHJ6*01	1758	gnl|Fabrus|L6_IGKJ1*01	1097
  2815	VH3-23_IGHD1-1*01>1′_IGHJ6*01	1768	gnl|Fabrus|L6_IGKJ1*01	1097
  2816	VH3-23_IGHD1-1*01>2′_IGHJ6*01	1769	gnl|Fabrus|L6_IGKJ1*01	1097
  2817	VH3-23_IGHD1-1*01>3′_IGHJ6*01	1770	gnl|Fabrus|L6_IGKJ1*01	1097
  2818	VH3-23_IGHD1-7*01>1′_IGHJ6*01	1771	gnl|Fabrus|L6_IGKJ1*01	1097
  2819	VH3-23_IGHD1-7*01>3′_IGHJ6*01	1772	gnl|Fabrus|L6_IGKJ1*01	1097
  2820	VH3-23_IGHD1-14*01>1′_IGHJ6*01	1773	gnl|Fabrus|L6_IGKJ1*01	1097
  2821	VH3-23_IGHD1-14*01>2′_IGHJ6*01	1774	gnl|Fabrus|L6_IGKJ1*01	1097
  2822	VH3-23_IGHD1-14*01>3′_IGHJ6*01	1775	gnl|Fabrus|L6_IGKJ1*01	1097
  2823	VH3-23_IGHD1-20*01>1′_IGHJ6*01	1776	gnl|Fabrus|L6_IGKJ1*01	1097
  2824	VH3-23_IGHD1-20*01>2′_IGHJ6*01	1777	gnl|Fabrus|L6_IGKJ1*01	1097
  2825	VH3-23_IGHD1-20*01>3′_IGHJ6*01	1778	gnl|Fabrus|L6_IGKJ1*01	1097
  2826	VH3-23_IGHD1-26*01>1′_IGHJ6*01	1779	gnl|Fabrus|L6_IGKJ1*01	1097
  2827	VH3-23_IGHD1-26*01>1_IGHJ6*01_B	1780	gnl|Fabrus|L6_IGKJ1*01	1097
  2828	VH3-23_IGHD2-2*01>2_IGHJ6*01_B	1781	gnl|Fabrus|L6_IGKJ1*01	1097
  2829	VH3-23_IGHD2-2*01>3′_IGHJ6*01	1782	gnl|Fabrus|L6_IGKJ1*01	1097
  2830	VH3-23_IGHD2-8*01>1′_IGHJ6*01	1783	gnl|Fabrus|L6_IGKJ1*01	1097
  2831	VH3-23_IGHD2-15*01>1′_IGHJ6*01	1784	gnl|Fabrus|L6_IGKJ1*01	1097
  2832	VH3-23_IGHD2-15*01>3′_IGHJ6*01	1785	gnl|Fabrus|L6_IGKJ1*01	1097
  2833	VH3-23_IGHD2-21*01>1′_IGHJ6*01	1786	gnl|Fabrus|L6_IGKJ1*01	1097
  2834	VH3-23_IGHD2-21*01>3′_IGHJ6*01	1787	gnl|Fabrus|L6_IGKJ1*01	1097
  2835	VH3-23_IGHD3-3*01>1′_IGHJ6*01	1788	gnl|Fabrus|L6_IGKJ1*01	1097
  2836	VH3-23_IGHD3-3*01>3′_IGHJ6*01	1789	gnl|Fabrus|L6_IGKJ1*01	1097
  2837	VH3-23_IGHD3-9*01>1′_IGHJ6*01	1790	gnl|Fabrus|L6_IGKJ1*01	1097
  2838	VH3-23_IGHD3-9*01>3′_IGHJ6*01	1791	gnl|Fabrus|L6_IGKJ1*01	1097
  2839	VH3-23_IGHD3-10*01>1′_IGHJ6*01	1792	gnl|Fabrus|L6_IGKJ1*01	1097
  2840	VH3-23_IGHD3-10*01>3′_IGHJ6*01	1793	gnl|Fabrus|L6_IGKJ1*01	1097
  2841	VH3-23_IGHD3-16*01>1′_IGHJ6*01	1794	gnl|Fabrus|L6_IGKJ1*01	1097
  2842	VH3-23_IGHD3-16*01>3′_IGHJ6*01	1795	gnl|Fabrus|L6_IGKJ1*01	1097
  2843	VH3-23_IGHD3-22*01>1′_IGHJ6*01	1796	gnl|Fabrus|L6_IGKJ1*01	1097
  2844	VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01	1797	gnl|Fabrus|L6_IGKJ1*01	1097
  2845	VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01	1798	gnl|Fabrus|L6_IGKJ1*01	1097
  2846	VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01	1799	gnl|Fabrus|L6_IGKJ1*01	1097
  2847	VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01	1800	gnl|Fabrus|L6_IGKJ1*01	1097
  2848	VH3-23_IGHD4-17*01>1′_IGHJ6*01	1801	gnl|Fabrus|L6_IGKJ1*01	1097
  2849	VH3-23_IGHD4-17*01>3′_IGHJ6*01	1802	gnl|Fabrus|L6_IGKJ1*01	1097
  2850	VH3-23_IGHD4-23*01>1′_IGHJ6*01	1803	gnl|Fabrus|L6_IGKJ1*01	1097
  2851	VH3-23_IGHD4-23*01>3′_IGHJ6*01	1804	gnl|Fabrus|L6_IGKJ1*01	1097
  2852	VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01	1805	gnl|Fabrus|L6_IGKJ1*01	1097
  2853	VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01	1806	gnl|Fabrus|L6_IGKJ1*01	1097
  2854	VH3-23_IGHD5-12*01>1′_IGHJ6*01	1807	gnl|Fabrus|L6_IGKJ1*01	1097
  2855	VH3-23_IGHD5-12*01>3′_IGHJ6*01	1808	gnl|Fabrus|L6_IGKJ1*01	1097
  2856	VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01	1809	gnl|Fabrus|L6_IGKJ1*01	1097
  2857	VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01	1810	gnl|Fabrus|L6_IGKJ1*01	1097
  2858	VH3-23_IGHD5-24*01>1′_IGHJ6*01	1811	gnl|Fabrus|L6_IGKJ1*01	1097
  2859	VH3-23_IGHD5-24*01>3′_IGHJ6*01	1812	gnl|Fabrus|L6_IGKJ1*01	1097
  2860	VH3-23_IGHD6-6*01>1′_IGHJ6*01	1813	gnl|Fabrus|L6_IGKJ1*01	1097
  2861	VH3-23_IGHD6-6*01>2′_IGHJ6*01	1814	gnl|Fabrus|L6_IGKJ1*01	1097
  2862	VH3-23_IGHD6-6*01>3′_IGHJ6*01	1815	gnl|Fabrus|L6_IGKJ1*01	1097
  2863	VH3-23_IGHD1-1*01>1_IGHJ5*01	1596	gnl|Fabrus|L25_IGKJ1*01	1093
  2864	VH3-23_IGHD1-1*01>2_IGHJ5*01	1597	gnl|Fabrus|L25_IGKJ1*01	1093
  2865	VH3-23_IGHD1-1*01>3_IGHJ5*01	1598	gnl|Fabrus|L25_IGKJ1*01	1093
  2866	VH3-23_IGHD1-7*01>1_IGHJ5*01	1599	gnl|Fabrus|L25_IGKJ1*01	1093
  2867	VH3-23_IGHD1-7*01>3_IGHJ5*01	1600	gnl|Fabrus|L25_IGKJ1*01	1093
  2868	VH3-23_IGHD1-14*01>1_IGHJ5*01	1601	gnl|Fabrus|L25_IGKJ1*01	1093
  2869	VH3-23_IGHD1-14*01>3_IGHJ5*01	1602	gnl|Fabrus|L25_IGKJ1*01	1093
  2870	VH3-23_IGHD1-20*01>1_IGHJ5*01	1603	gnl|Fabrus|L25_IGKJ1*01	1093
  2871	VH3-23_IGHD1-20*01>3_IGHJ5*01	1604	gnl|Fabrus|L25_IGKJ1*01	1093
  2872	VH3-23_IGHD1-26*01>1_IGHJ5*01	1605	gnl|Fabrus|L25_IGKJ1*01	1093
  2873	VH3-23_IGHD1-26*01>3_IGHJ5*01	1606	gnl|Fabrus|L25_IGKJ1*01	1093
  2874	VH3-23_IGHD2-2*01>2_IGHJ5*01	1607	gnl|Fabrus|L25_IGKJ1*01	1093
  2875	VH3-23_IGHD2-2*01>3_IGHJ5*01	1608	gnl|Fabrus|L25_IGKJ1*01	1093
  2876	VH3-23_IGHD2-8*01>2_IGHJ5*01	1609	gnl|Fabrus|L25_IGKJ1*01	1093
  2877	VH3-23_IGHD2-8*01>3_IGHJ5*01	1610	gnl|Fabrus|L25_IGKJ1*01	1093
  2878	VH3-23_IGHD2-15*01>2_IGHJ5*01	1611	gnl|Fabrus|L25_IGKJ1*01	1093
  2879	VH3-23_IGHD2-15*01>3_IGHJ5*01	1612	gnl|Fabrus|L25_IGKJ1*01	1093
  2880	VH3-23_IGHD2-21*01>2_IGHJ5*01	1613	gnl|Fabrus|L25_IGKJ1*01	1093
  2881	VH3-23_IGHD2-21*01>3_IGHJ5*01	1614	gnl|Fabrus|L25_IGKJ1*01	1093
  2882	VH3-23_IGHD3-3*01>1_IGHJ5*01	1615	gnl|Fabrus|L25_IGKJ1*01	1093
  2883	VH3-23_IGHD3-3*01>2_IGHJ5*01	1616	gnl|Fabrus|L25_IGKJ1*01	1093
  2884	VH3-23_IGHD3-3*01>3_IGHJ5*01	1617	gnl|Fabrus|L25_IGKJ1*01	1093
  2885	VH3-23_IGHD3-9*01>2_IGHJ5*01	1618	gnl|Fabrus|L25_IGKJ1*01	1093
  2886	VH3-23_IGHD3-10*01>2_IGHJ5*01	1619	gnl|Fabrus|L25_IGKJ1*01	1093
  2887	VH3-23_IGHD3-10*01>3_IGHJ5*01	1620	gnl|Fabrus|L25_IGKJ1*01	1093
  2888	VH3-23_IGHD3-16*01>2_IGHJ5*01	1621	gnl|Fabrus|L25_IGKJ1*01	1093
  2889	VH3-23_IGHD3-16*01>3_IGHJ5*01	1622	gnl|Fabrus|L25_IGKJ1*01	1093
  2890	VH3-23_IGHD3-22*01>2_IGHJ5*01	1623	gnl|Fabrus|L25_IGKJ1*01	1093
  2891	VH3-23_IGHD3-22*01>3_IGHJ5*01	1624	gnl|Fabrus|L25_IGKJ1*01	1093
  2892	VH3-23_IGHD4-4*01 (1) >2_IGHJ5*01	1625	gnl|Fabrus|L25_IGKJ1*01	1093
  2893	VH3-23_IGHD4-4*01 (1) >3_IGHJ5*01	1626	gnl|Fabrus|L25_IGKJ1*01	1093
  2894	VH3-23_IGHD4-11*01 (1) >2_IGHJ5*01	1627	gnl|Fabrus|L25_IGKJ1*01	1093
  2895	VH3-23_IGHD4-11*01 (1) >3_IGHJ5*01	1628	gnl|Fabrus|L25_IGKJ1*01	1093
  2896	VH3-23_IGHD4-17*01>2_IGHJ5*01	1629	gnl|Fabrus|L25_IGKJ1*01	1093
  2897	VH3-23_IGHD4-17*01>3_IGHJ5*01	1630	gnl|Fabrus|L25_IGKJ1*01	1093
  2898	VH3-23_IGHD4-23*01>2_IGHJ5*01	1631	gnl|Fabrus|L25_IGKJ1*01	1093
  2899	VH3-23_IGHD4-23*01>3_IGHJ5*01	1632	gnl|Fabrus|L25_IGKJ1*01	1093
  2900	VH3-23_IGHD5-5*01 (2) >1_IGHJ5*01	1633	gnl|Fabrus|L25_IGKJ1*01	1093
  2901	VH3-23_IGHD5-5*01 (2) >2_IGHJ5*01	1634	gnl|Fabrus|L25_IGKJ1*01	1093
  2902	VH3-23_IGHD5-5*01 (2) >3_IGHJ5*01	1635	gnl|Fabrus|L25_IGKJ1*01	1093
  2903	VH3-23_IGHD5-12*01>1_IGHJ5*01	1636	gnl|Fabrus|L25_IGKJ1*01	1093
  2904	VH3-23_IGHD5-12*01>3_IGHJ5*01	1637	gnl|Fabrus|L25_IGKJ1*01	1093
  2905	VH3-23_IGHD5-18*01 (2) >1_IGHJ5*01	1638	gnl|Fabrus|L25_IGKJ1*01	1093
  2906	VH3-23_IGHD5-18*01 (2) >2_IGHJ5*01	1639	gnl|Fabrus|L25_IGKJ1*01	1093
  2907	VH3-23_IGHD5-18*01 (2) >3_IGHJ5*01	1640	gnl|Fabrus|L25_IGKJ1*01	1093
  2908	VH3-23_IGHD5-24*01>1_IGHJ5*01	1641	gnl|Fabrus|L25_IGKJ1*01	1093
  2909	VH3-23_IGHD5-24*01>3_IGHJ5*01	1642	gnl|Fabrus|L25_IGKJ1*01	1093
  2910	VH3-23_IGHD6-6*01>1_IGHJ5*01	1643	gnl|Fabrus|L25_IGKJ1*01	1093
  2911	VH3-23_IGHD1-1*01>1′_IGHJ5*01	1653	gnl|Fabrus|L25_IGKJ1*01	1093
  2912	VH3-23_IGHD1-1*01>2′_IGHJ5*01	1654	gnl|Fabrus|L25_IGKJ1*01	1093
  2913	VH3-23_IGHD1-1*01>3′_IGHJ5*01	1655	gnl|Fabrus|L25_IGKJ1*01	1093
  2914	VH3-23_IGHD1-7*01>1′_IGHJ5*01	1656	gnl|Fabrus|L25_IGKJ1*01	1093
  2915	VH3-23_IGHD1-7*01>3′_IGHJ5*01	1657	gnl|Fabrus|L25_IGKJ1*01	1093
  2916	VH3-23_IGHD1-14*01>1′_IGHJ5*01	1658	gnl|Fabrus|L25_IGKJ1*01	1093
  2917	VH3-23_IGHD1-14*01>2′_IGHJ5*01	1659	gnl|Fabrus|L25_IGKJ1*01	1093
  2918	VH3-23_IGHD1-14*01>3′_IGHJ5*01	1660	gnl|Fabrus|L25_IGKJ1*01	1093
  2919	VH3-23_IGHD1-20*01>1′_IGHJ5*01	1661	gnl|Fabrus|L25_IGKJ1*01	1093
  2920	VH3-23_IGHD1-20*01>2′_IGHJ5*01	1662	gnl|Fabrus|L25_IGKJ1*01	1093
  2921	VH3-23_IGHD1-20*01>3′_IGHJ5*01	1663	gnl|Fabrus|L25_IGKJ1*01	1093
  2922	VH3-23_IGHD1-26*01>1′_IGHJ5*01	1664	gnl|Fabrus|L25_IGKJ1*01	1093
  2923	VH3-23_IGHD1-26*01>3′_IGHJ5*01	1665	gnl|Fabrus|L25_IGKJ1*01	1093
  2924	VH3-23_IGHD2-2*01>1′_IGHJ5*01	1666	gnl|Fabrus|L25_IGKJ1*01	1093
  2925	VH3-23_IGHD2-2*01>3′_IGHJ5*01	1667	gnl|Fabrus|L25_IGKJ1*01	1093
  2926	VH3-23_IGHD2-8*01>1′_IGHJ5*01	1668	gnl|Fabrus|L25_IGKJ1*01	1093
  2927	VH3-23_IGHD2-15*01>1′_IGHJ5*01	1669	gnl|Fabrus|L25_IGKJ1*01	1093
  2928	VH3-23_IGHD2-15*01>3′_IGHJ5*01	1670	gnl|Fabrus|L25_IGKJ1*01	1093
  2929	VH3-23_IGHD2-21*01>1′_IGHJ5*01	1671	gnl|Fabrus|L25_IGKJ1*01	1093
  2930	VH3-23_IGHD2-21*01>3′_IGHJ5*01	1672	gnl|Fabrus|L25_IGKJ1*01	1093
  2931	VH3-23_IGHD3-3*01>1′_IGHJ5*01	1673	gnl|Fabrus|L25_IGKJ1*01	1093
  2932	VH3-23_IGHD3-3*01>3′_IGHJ5*0 1	1674	gnl|Fabrus|L25_IGKJ1*01	1093
  2933	VH3-23_IGHD3-9*01>1′_IGHJ5*01	1675	gnl|Fabrus|L25_IGKJ1*01	1093
  2934	VH3-23_IGHD3-9*01>3′_IGHJ5*01	1676	gnl|Fabrus|L25_IGKJ1*01	1093
  2935	VH3-23_IGHD3-10*01>1′_IGHJ5*01	1677	gnl|Fabrus|L25_IGKJ1*01	1093
  2936	VH3-23_IGHD3-10*01>3′_IGHJ5*01	1678	gnl|Fabrus|L25_IGKJ1*01	1093
  2937	VH3-23_IGHD3-16*01>1′_IGHJ5*01	1679	gnl|Fabrus|L25_IGKJ1*01	1093
  2938	VH3-23_IGHD3-16*01>3′_IGHJ5*01	1680	gnl|Fabrus|L25_IGKJ1*01	1093
  2939	VH3-23_IGHD3-22*01>1′_IGHJ5*01	1681	gnl|Fabrus|L25_IGKJ1*01	1093
  2940	VH3-23_IGHD4-4*01 (1) >1′_IGHJ5*01	1682	gnl|Fabrus|L25_IGKJ1*01	1093
  2941	VH3-23_IGHD4-4*01 (1) >3′_IGHJ5*01	1683	gnl|Fabrus|L25_IGKJ1*01	1093
  2942	VH3-23_IGHD4-11*01 (1) >1′_IGHJ5*01	1684	gnl|Fabrus|L25_IGKJ1*01	1093
  2943	VH3-23_IGHD4-11*01 (1) >3′_IGHJ5*01	1685	gnl|Fabrus|L25_IGKJ1*01	1093
  2944	VH3-23_IGHD4-17*01>1′_IGHJ5*01	1686	gnl|Fabrus|L25_IGKJ1*01	1093
  2945	VH3-23_IGHD4-17*01>3′_IGHJ5*01	1687	gnl|Fabrus|L25_IGKJ1*01	1093
  2946	VH3-23_IGHD4-23*01>1′_IGHJ5*01	1688	gnl|Fabrus|L25_IGKJ1*01	1093
  2947	VH3-23_IGHD4-23*01>3′_IGHJ5*01	1689	gnl|Fabrus|L25_IGKJ1*01	1093
  2948	VH3-23_IGHD5-5*01 (2) >1′_IGHJ5*01	1690	gnl|Fabrus|L25_IGKJ1*01	1093
  2949	VH3-23_IGHD5-5*01 (2) >3′_IGHJ5*01	1691	gnl|Fabrus|L25_IGKJ1*01	1093
  2950	VH3-23_IGHD5-12*01>1′_IGHJ5*01	1692	gnl|Fabrus|L25_IGKJ1*01	1093
  2951	VH3-23_IGHD5-12*01>3′_IGHJ5*01	1693	gnl|Fabrus|L25_IGKJ1*01	1093
  2952	VH3-23_IGHD5-18*01 (2) >1′_IGHJ5*01	1694	gnl|Fabrus|L25_IGKJ1*01	1093
  2953	VH3-23_IGHD5-18*01 (2) >3′_IGHJ5*01	1695	gnl|Fabrus|L25_IGKJ1*01	1093
  2954	VH3-23_IGHD5-24*01>1′_IGHJ5*01	1696	gnl|Fabrus|L25_IGKJ1*01	1093
  2955	VH3-23_IGHD5-24*01>3′_IGHJ5*01	1697	gnl|Fabrus|L25_IGKJ1*01	1093
  2956	VH3-23_IGHD6-6*01>1′_IGHJ5*01	1698	gnl|Fabrus|L25_IGKJ1*01	1093
  2957	VH3-23_IGHD6-6*01>2′_IGHJ5*01	1699	gnl|Fabrus|L25_IGKJ1*01	1093
  2958	VH3-23_IGHD6-6*01>3′_IGHJ5*01	1700	gnl|Fabrus|L25_IGKJ1*01	1093
  2959	VH3-23_IGHD1-1*01>1_IGHJ5*01	1596	gnl|Fabrus|B3_IGKJ1*01	1085
  2960	VH3-23_IGHD1-1*01>2_IGHJ5*01	1597	gnl|Fabrus|B3_IGKJ1*01	1085
  2961	VH3-23_IGHD1-1*01>3_IGHJ5*01	1598	gnl|Fabrus|B3_IGKJ1*01	1085
  2962	VH3-23_IGHD1-7*01>1_IGHJ5*01	1599	gnl|Fabrus|B3_IGKJ1*01	1085
  2963	VH3-23_IGHD1-7*01>3_IGHJ5*01	1600	gnl|Fabrus|B3_IGKJ1*01	1085
  2964	VH3-23_IGHD1-14*01>1_IGHJ5*01	1601	gnl|Fabrus|B3_IGKJ1*01	1085
  2965	VH3-23_IGHD1-14*01>3_IGHJ5*01	1602	gnl|Fabrus|B3_IGKJ1*01	1085
  2966	VH3-23_IGHD1-20*01>1_IGHJ5*01	1603	gnl|Fabrus|B3_IGKJ1*01	1085
  2967	VH3-23_IGHD1-20*01>3_IGHJ5*01	1604	gnl|Fabrus|B3_IGKJ1*01	1085
  2968	VH3-23_IGHD1-26*01>1_IGHJ5*01	1605	gnl|Fabrus|B3_IGKJ1*01	1085
  2969	VH3-23_IGHD1-26*01>3_IGHJ5*01	1606	gnl|Fabrus|B3_IGKJ1*01	1085
  2970	VH3-23_IGHD2-2*01>2_IGHJ5*01	1607	gnl|Fabrus|B3_IGKJ1*01	1085
  2971	VH3-23_IGHD2-2*01>3_IGHJ5*01	1608	gnl|Fabrus|B3_IGKJ1*01	1085
  2972	VH3-23_IGHD2-8*01>2_IGHJ5*01	1609	gnl|Fabrus|B3_IGKJ1*01	1085
  2973	VH3-23_IGHD2-8*01>3_IGHJ5*01	1610	gnl|Fabrus|B3_IGKJ1*01	1085
  2974	VH3-23_IGHD2-15*01>2_IGHJ5*01	1611	gnl|Fabrus|B3_IGKJ1*01	1085
  2975	VH3-23_IGHD2-15*01>3_IGHJ5*01	1612	gnl|Fabrus|B3_IGKJ1*01	1085
  2976	VH3-23_IGHD2-21*01>2_IGHJ5*01	1613	gnl|Fabrus|B3_IGKJ1*01	1085
  2977	VH3-23_IGHD2-21*01>3_IGHJ5*01	1614	gnl|Fabrus|B3_IGKJ1*01	1085
  2978	VH3-23_IGHD3-3*01>1_IGHJ5*01	1615	gnl|Fabrus|B3_IGKJ1*01	1085
  2979	VH3-23_IGHD3-3*01>2_IGHJ5*01	1616	gnl|Fabrus|B3_IGKJ1*01	1085
  2980	VH3-23_IGHD3-3*01>3_IGHJ5*01	1617	gnl|Fabrus|B3_IGKJ1*01	1085
  2981	VH3-23_IGHD3-9*01>2_IGHJ5*01	1618	gnl|Fabrus|B3_IGKJ1*01	1085
  2982	VH3-23_IGHD3-10*01>2_IGHJ5*01	1619	gnl|Fabrus|B3_IGKJ1*01	1085
  2983	VH3-23_IGHD3-10*01>3_IGHJ5*01	1620	gnl|Fabrus|B3_IGKJ1*01	1085
  2984	VH3-23_IGHD3-16*01>2_IGHJ5*01	1621	gnl|Fabrus|B3_IGKJ1*01	1085
  2985	VH3-23_IGHD3-16*01>3_IGHJ5*01	1622	gnl|Fabrus|B3_IGKJ1*01	1085
  2986	VH3-23_IGHD3-22*01>2_IGHJ5*01	1623	gnl|Fabrus|B3_IGKJ1*01	1085
  2987	VH3-23_IGHD3-22*01>3_IGHJ5*01	1624	gnl|Fabrus|B3_IGKJ1*01	1085
  2988	VH3-23_IGHD4-4*01 (1) >2_IGHJ5*01	1625	gnl|Fabrus|B3_IGKJ1*01	1085
  2989	VH3-23_IGHD4-4*01 (1) >3_IGHJ5*01	1626	gnl|Fabrus|B3_IGKJ1*01	1085
  2990	VH3-23_IGHD4-11*01 (1) >2_IGHJ5*01	1627	gnl|Fabrus|B3_IGKJ1*01	1085
  2991	VH3-23_IGHD4-11*01 (1) >3_IGHJ5*01	1628	gnl|Fabrus|B3_IGKJ1*01	1085
  2992	VH3-23_IGHD4-17*01>2_IGHJ5*01	1629	gnl|Fabrus|B3_IGKJ1*01	1085
  2993	VH3-23_IGHD4-17*01>3_IGHJ5*01	1630	gnl|Fabrus|B3_IGKJ1*01	1085
  2994	VH3-23_IGHD4-23*01>2_IGHJ5*01	1631	gnl|Fabrus|B3_IGKJ1*01	1085
  2995	VH3-23_IGHD4-23*01>3_IGHJ5*01	1632	gnl|Fabrus|B3_IGKJ1*01	1085
  2996	VH3-23_IGHD5-5*01 (2) >1_IGHJ5*01	1633	gnl|Fabrus|B3_IGKJ1*01	1085
  2997	VH3-23_IGHD5-5*01 (2) >2_IGHJ5*01	1634	gnl|Fabrus|B3_IGKJ1*01	1085
  2998	VH3-23_IGHD5-5*01 (2) >3_IGHJ5*01	1635	gnl|Fabrus|B3_IGKJ1*01	1085
  2999	VH3-23_IGHD5-12*01>1_IGHJ5*01	1636	gnl|Fabrus|B3_IGKJ1*01	1085
  3000	VH3-23_IGHD5-12*01>3_IGHJ5*01	1637	gnl|Fabrus|B3_IGKJ1*01	1085
  3001	VH3-23_IGHD5-18*01 (2) >1_IGHJ5*01	1638	gnl|Fabrus|B3_IGKJ1*01	1085
  3002	VH3-23_IGHD5-18*01 (2) >2_IGHJ5*01	1639	gnl|Fabrus|B3_IGKJ1*01	1085
  3003	VH3-23_IGHD5-18*01 (2) >3_IGHJ5*01	1640	gnl|Fabrus|B3_IGKJ1*01	1085
  3004	VH3-23_IGHD5-24*01>1_IGHJ5*01	1641	gnl|Fabrus|B3_IGKJ1*01	1085
  3005	VH3-23_IGHD5-24*01>3_IGHJ5*01	1642	gnl|Fabrus|B3_IGKJ1*01	1085
  3006	VH3-23_IGHD6-6*01>1_IGHJ5*01	1643	gnl|Fabrus|B3_IGKJ1*01	1085
  3007	VH3-23_IGHD1-1*01>1′_IGHJ5*01	1653	gnl|Fabrus|B3_IGKJ1*01	1085
  3008	VH3-23_IGHD1-1*01>2′_IGHJ5*01	1654	gnl|Fabrus|B3_IGKJ1*01	1085
  3009	VH3-23_IGHD1-1*01>3′_IGHJ5*01	1655	gnl|Fabrus|B3_IGKJ1*01	1085
  3010	VH3-23_IGHD1-7*01>1′_IGHJ5*01	1656	gnl|Fabrus|B3_IGKJ1*01	1085
  3011	VH3-23_IGHD1-7*01>3′_IGHJ5*01	1657	gnl|Fabrus|B3_IGKJ1*01	1085
  3012	VH3-23_IGHD1-14*01>1′_IGHJ5*01	1658	gnl|Fabrus|B3_IGKJ1*01	1085
  3013	VH3-23_IGHD1-14*01>2′_IGHJ5*01	1659	gnl|Fabrus|B3_IGKJ1*01	1085
  3014	VH3-23_IGHD1-14*01>3′_IGHJ5*01	1660	gnl|Fabrus|B3_IGKJ1*01	1085
  3015	VH3-23_IGHD1-20*01>1′_IGHJ5*01	1661	gnl|Fabrus|B3_IGKJ1*01	1085
  3016	VH3-23_IGHD1-20*01>2′_IGHJ5*01	1662	gnl|Fabrus|B3_IGKJ1*01	1085
  3017	VH3-23_IGHD1-20*01>3′_IGHJ5*01	1663	gnl|Fabrus|B3_IGKJ1*01	1085
  3018	VH3-23_IGHD1-26*01>1′_IGHJ5*01	1664	gnl|Fabrus|B3_IGKJ1*01	1085
  3019	VH3-23_IGHD1-26*01>3′_IGHJ5*01	1665	gnl|Fabrus|B3_IGKJ1*01	1085
  3020	VH3-23_IGHD2-2*01>1′_IGHJ5*01	1666	gnl|Fabrus|B3_IGKJ1*01	1085
  3021	VH3-23_IGHD2-2*01>3′_IGHJ5*01	1667	gnl|Fabrus|B3_IGKJ1*01	1085
  3022	VH3-23_IGHD2-8*01>1′_IGHJ5*01	1668	gnl|Fabrus|B3_IGKJ1*01	1085
  3023	VH3-23_IGHD2-15*01>1′_IGHJ5*01	1669	gnl|Fabrus|B3_IGKJ1*01	1085
  3024	VH3-23_IGHD2-15*01>3′_IGHJ5*01	1670	gnl|Fabrus|B3_IGKJ1*01	1085
  3025	VH3-23_IGHD2-21*01>1′_IGHJ5*01	1671	gnl|Fabrus|B3_IGKJ1*01	1085
  3026	VH3-23_IGHD2-21*01>3′_IGHJ5*01	1672	gnl|Fabrus|B3_IGKJ1*01	1085
  3027	VH3-23_IGHD3-3*01>1′_IGHJ5*01	1673	gnl|Fabrus|B3_IGKJ1*01	1085
  3028	VH3-23_IGHD3-3*01>3′_IGHJ5*01	1674	gnl|Fabrus|B3_IGKJ1*01	1085
  3029	VH3-23_IGHD3-9*01>1′_IGHJ5*01	1675	gnl|Fabrus|B3_IGKJ1*01	1085
  3030	VH3-23_IGHD3-9*01>3′_IGHJ5*01	1676	gnl|Fabrus|B3_IGKJ1*01	1085
  3031	VH3-23_IGHD3-10*01>1′_IGHJ5*01	1677	gnl|Fabrus|B3_IGKJ1*01	1085
  3032	VH3-23_IGHD3-10*01>3′_IGHJ5*01	1678	gnl|Fabrus|B3_IGKJ1*01	1085
  3033	VH3-23_IGHD3-16*01>1′_IGHJ5*01	1679	gnl|Fabrus|B3_IGKJ1*01	1085
  3034	VH3-23_IGHD3-16*01>3′_IGHJ5*01	1680	gnl|Fabrus|B3_IGKJ1*01	1085
  3035	VH3-23_IGHD3-22*01>1′_IGHJ5*01	1681	gnl|Fabrus|B3_IGKJ1*01	1085
  3036	VH3-23_IGHD4-4*01 (1) >1′_IGHJ5*01	1682	gnl|Fabrus|B3_IGKJ1*01	1085
  3037	VH3-23_IGHD4-4*01 (1) >3′_IGHJ5*01	1683	gnl|Fabrus|B3_IGKJ1*01	1085
  3038	VH3-23_IGHD4-11*01 (1) >1′_IGHJ5*01	1684	gnl|Fabrus|B3_IGKJ1*01	1085
  3039	VH3-23_IGHD4-11*01 (1) >3′_IGHJ5*01	1685	gnl|Fabrus|B3_IGKJ1*01	1085
  3040	VH3-23_IGHD4-17*01>1′_IGHJ5*01	1686	gnl|Fabrus|B3_IGKJ1*01	1085
  3041	VH3-23_IGHD4-17*01>3′_IGHJ5*01	1687	gnl|Fabrus|B3_IGKJ1*01	1085
  3042	VH3-23_IGHD4-23*01>1′_IGHJ5*01	1688	gnl|Fabrus|B3_IGKJ1*01	1085
  3043	VH3-23_IGHD4-23*01>3′_IGHJ5*01	1689	gnl|Fabrus|B3_IGKJ1*01	1085
  3044	VH3-23_IGHD5-5*01 (2) >1′_IGHJ5*01	1690	gnl|Fabrus|B3_IGKJ1*01	1085
  3045	VH3-23_IGHD5-5*01 (2) >3′_IGHJ5*01	1691	gnl|Fabrus|B3_IGKJ1*01	1085
  3046	VH3-23_IGHD5-12*01>1′_IGHJ5*01	1692	gnl|Fabrus|B3_IGKJ1*01	1085
  3047	VH3-23_IGHD5-12*01>3′_IGHJ5*01	1693	gnl|Fabrus|B3_IGKJ1*01	1085
  3048	VH3-23_IGHD5-18*01 (2) >1′_IGHJ5*01	1694	gnl|Fabrus|B3_IGKJ1*01	1085
  3049	VH3-23_IGHD5-18*01 (2) >3′_IGHJ5*01	1695	gnl|Fabrus|B3_IGKJ1*01	1085
  3050	VH3-23_IGHD5-24*01>1′_IGHJ5*01	1696	gnl|Fabrus|B3_IGKJ1*01	1085
  3051	VH3-23_IGHD5-24*01>3′_IGHJ5*01	1697	gnl|Fabrus|B3_IGKJ1*01	1085
  3052	VH3-23_IGHD6-6*01>1′_IGHJ5*01	1698	gnl|Fabrus|B3_IGKJ1*01	1085
  3053	VH3-23_IGHD6-6*01>2′_IGHJ5*01	1699	gnl|Fabrus|B3_IGKJ1*01	1085
  3054	VH3-23_IGHD6-6*01>3′_IGHJ5*01	1700	gnl|Fabrus|B3_IGKJ1*01	1085
  3055	VH3-23_IGHD1-1*01>1_IGHJ5*01	1596	gnl|Fabrus|A26_IGKJ1*01	1079
  3056	VH3-23_IGHD1-1*01>2_IGHJ5*01	1597	gnl|Fabrus|A26_IGKJ1*01	1079
  3057	VH3-23_IGHD1-1*01>3_IGHJ5*01	1598	gnl|Fabrus|A26_IGKJ1*01	1079
  3058	VH3-23_IGHD1-7*01>1_IGHJ5*01	1599	gnl|Fabrus|A26_IGKJ1*01	1079
  3059	VH3-23_IGHD1-7*01>3_IGHJ5*01	1600	gnl|Fabrus|A26_IGKJ1*01	1079
  3060	VH3-23_IGHD1-14*01>1_IGHJ5*01	1601	gnl|Fabrus|A26_IGKJ1*01	1079
  3061	VH3-23_IGHD1-14*01>3_IGHJ5*01	1602	gnl|Fabrus|A26_IGKJ1*01	1079
  3062	VH3-23_IGHD1-20*01>1_IGHJ5*01	1603	gnl|Fabrus|A26_IGKJ1*01	1079
  3063	VH3-23_IGHD1-20*01>3_IGHJ5*01	1604	gnl|Fabrus|A26_IGKJ1*01	1079
  3064	VH3-23_IGHD1-26*01>1_IGHJ5*01	1605	gnl|Fabrus|A26_IGKJ1*01	1079
  3065	VH3-23_IGHD1-26*01>3_IGHJ5*01	1606	gnl|Fabrus|A26_IGKJ1*01	1079
  3066	VH3-23_IGHD2-2*01>2_IGHJ5*01	1607	gnl|Fabrus|A26_IGKJ1*01	1079
  3067	VH3-23_IGHD2-2*01>3_IGHJ5*01	1608	gnl|Fabrus|A26_IGKJ1*01	1079
  3068	VH3-23_IGHD2-8*01>2_IGHJ5*01	1609	gnl|Fabrus|A26_IGKJ1*01	1079
  3069	VH3-23_IGHD2-8*01>3_IGHJ5*01	1610	gnl|Fabrus|A26_IGKJ1*01	1079
  3070	VH3-23_IGHD2-15*01>2_IGHJ5*01	1611	gnl|Fabrus|A26_IGKJ1*01	1079
  3071	VH3-23_IGHD2-15*01>3_IGHJ5*01	1612	gnl|Fabrus|A26_IGKJ1*01	1079
  3072	VH3-23_IGHD2-21*01>2_IGHJ5*01	1613	gnl|Fabrus|A26_IGKJ1*01	1079
  3073	VH3-23_IGHD2-21*01>3_IGHJ5*01	1614	gnl|Fabrus|A26_IGKJ1*01	1079
  3074	VH3-23_IGHD3-3*01>1_IGHJ5*01	1615	gnl|Fabrus|A26_IGKJ1*01	1079
  3075	VH3-23_IGHD3-3*01>2_IGHJ5*01	1616	gnl|Fabrus|A26_IGKJ1*01	1079
  3076	VH3-23_IGHD3-3*01>3_IGHJ5*01	1617	gnl|Fabrus|A26_IGKJ1*01	1079
  3077	VH3-23_IGHD3-9*01>2_IGHJ5*01	1618	gnl|Fabrus|A26_IGKJ1*01	1079
  3078	VH3-23_IGHD3-10*01>2_IGHJ5*01	1619	gnl|Fabrus|A26_IGKJ1*01	1079
  3079	VH3-23_IGHD3-10*01>3_IGHJ5*01	1620	gnl|Fabrus|A26_IGKJ1*01	1079
  3080	VH3-23_IGHD3-16*01>2_IGHJ5*01	1621	gnl|Fabrus|A26_IGKJ1*01	1079
  3081	VH3-23_IGHD3-16*01>3_IGHJ5*01	1622	gnl|Fabrus|A26_IGKJ1*01	1079
  3082	VH3-23_IGHD3-22*01>2_IGHJ5*01	1623	gnl|Fabrus|A26_IGKJ1*01	1079
  3083	VH3-23_IGHD3-22*01>3_IGHJ5*01	1624	gnl|Fabrus|A26_IGKJ1*01	1079
  3084	VH3-23_IGHD4-4*01 (1) >2_IGHJ5*01	1625	gnl|Fabrus|A26_IGKJ1*01	1079
  3085	VH3-23_IGHD4-4*01 (1) >3_IGHJ5*01	1626	gnl|Fabrus|A26_IGKJ1*01	1079
  3086	VH3-23_IGHD4-11*01 (1) >2_IGHJ5*01	1627	gnl|Fabrus|A26_IGKJ1*01	1079
  3087	VH3-23_IGHD4-11*01 (1) >3_IGHJ5*01	1628	gnl|Fabrus|A26_IGKJ1*01	1079
  3088	VH3-23_IGHD4-17*01>2_IGHJ5*01	1629	gnl|Fabrus|A26_IGKJ1*01	1079
  3089	VH3-23_IGHD4-17*01>3_IGHJ5*01	1630	gnl|Fabrus|A26_IGKJ1*01	1079
  3090	VH3-23_IGHD4-23*01>2_IGHJ5*01	1631	gnl|Fabrus|A26_IGKJ1*01	1079
  3091	VH3-23_IGHD4-23*01>3_IGHJ5*01	1632	gnl|Fabrus|A26_IGKJ1*01	1079
  3092	VH3-23_IGHD5-5*01 (2) >1_IGHJ5*01	1633	gnl|Fabrus|A26_IGKJ1*01	1079
  3093	VH3-23_IGHD5-5*01 (2) >2_IGHJ5*01	1634	gnl|Fabrus|A26_IGKJ1*01	1079
  3094	VH3-23_IGHD5-5*01 (2) >3_IGHJ5*01	1635	gnl|Fabrus|A26_IGKJ1*01	1079
  3095	VH3-23_IGHD5-12*01>1_IGHJ5*01	1636	gnl|Fabrus|A26_IGKJ1*01	1079
  3096	VH3-23_IGHD5-12*01>3_IGHJ5*01	1637	gnl|Fabrus|A26_IGKJ1*01	1079
  3097	VH3-23_IGHD5-18*01 (2) >1_IGHJ5*01	1638	gnl|Fabrus|A26_IGKJ1*01	1079
  3098	VH3-23_IGHD5-18*01 (2) >2_IGHJ5*01	1639	gnl|Fabrus|A26_IGKJ1*01	1079
  3099	VH3-23_IGHD5-18*01 (2) >3_IGHJ5*01	1640	gnl|Fabrus|A26_IGKJ1*01	1079
  3100	VH3-23_IGHD5-24*01>1_IGHJ5*01	1641	gnl|Fabrus|A26_IGKJ1*01	1079
  3101	VH3-23_IGHD5-24*01>3_IGHJ5*01	1642	gnl|Fabrus|A26_IGKJ1*01	1079
  3102	VH3-23_IGHD6-6*01>1_IGHJ5*01	1643	gnl|Fabrus|A26_IGKJ1*01	1079
  3103	VH3-23_IGHD1-1*01>1′_IGHJ5*01	1653	gnl|Fabrus|A26_IGKJ1*01	1079
  3104	VH3-23_IGHD1-1*01>2′_IGHJ5*01	1654	gnl|Fabrus|A26_IGKJ1*01	1079
  3105	VH3-23_IGHD1-1*01>3′_IGHJ5*01	1655	gnl|Fabrus|A26_IGKJ1*01	1079
  3106	VH3-23_IGHD1-7*01>1′_IGHJ5*01	1656	gnl|Fabrus|A26_IGKJ1*01	1079
  3107	VH3-23_IGHD1-7*01>3′_IGHJ5*01	1657	gnl|Fabrus|A26_IGKJ1*01	1079
  3108	VH3-23_IGHD1-14*01>1′_IGHJ5*01	1658	gnl|Fabrus|A26_IGKJ1*01	1079
  3109	VH3-23_IGHD1-14*01>2′_IGHJ5*01	1659	gnl|Fabrus|A26_IGKJ1*01	1079
  3110	VH3-23_IGHD1-14*01>3′_IGHJ5*01	1660	gnl|Fabrus|A26_IGKJ1*01	1079
  3111	VH3-23_IGHD1-20*01>1′_IGHJ5*01	1661	gnl|Fabrus|A26_IGKJ1*01	1079
  3112	VH3-23_IGHD1-20*01>2′_IGHJ5*01	1662	gnl|Fabrus|A26_IGKJ1*01	1079
  3113	VH3-23_IGHD1-20*01>3′_IGHJ5*01	1663	gnl|Fabrus|A26_IGKJ1*01	1079
  3114	VH3-23_IGHD1-26*01>1′_IGHJ5*01	1664	gnl|Fabrus|A26_IGKJ1*01	1079
  3115	VH3-23_IGHD1-26*01>3′_IGHJ5*01	1665	gnl|Fabrus|A26_IGKJ1*01	1079
  3116	VH3-23_IGHD2-2*01>1′_IGHJ5*01	1666	gnl|Fabrus|A26_IGKJ1*01	1079
  3117	VH3-23_IGHD2-2*01>3′_IGHJ5*01	1667	gnl|Fabrus|A26_IGKJ1*01	1079
  3118	VH3-23_IGHD2-8*01>1′_IGHJ5*01	1668	gnl|Fabrus|A26_IGKJ1*01	1079
  3119	VH3-23_IGHD2-15*01>1′_IGHJ5*01	1669	gnl|Fabrus|A26_IGKJ1*01	1079
  3120	VH3-23_IGHD2-15*01>3′_IGHJ5*01	1670	gnl|Fabrus|A26_IGKJ1*01	1079
  3121	VH3-23_IGHD2-21*01>1′_IGHJ5*01	1671	gnl|Fabrus|A26_IGKJ1*01	1079
  3122	VH3-23_IGHD2-21*01>3′_IGHJ5*01	1672	gnl|Fabrus|A26_IGKJ1*01	1079
  3123	VH3-23_IGHD3-3*01>1′_IGHJ5*01	1673	gnl|Fabrus|A26_IGKJ1*01	1079
  3124	VH3-23_IGHD3-3*01>3′_IGHJ5*01	1674	gnl|Fabrus|A26_IGKJ1*01	1079
  3125	VH3-23_IGHD3-9*01>1′_IGHJ5*01	1675	gnl|Fabrus|A26_IGKJ1*01	1079
  3126	VH3-23_IGHD3-9*01>3′_IGHJ5*01	1676	gnl|Fabrus|A26_IGKJ1*01	1079
  3127	VH3-23_IGHD3-10*01>1′_IGHJ5*01	1677	gnl|Fabrus|A26_IGKJ1*01	1079
  3128	VH3-23_IGHD3-10*01>3′_IGHJ5*01	1678	gnl|Fabrus|A26_IGKJ1*01	1079
  3129	VH3-23_IGHD3-16*01>1′_IGHJ5*01	1679	gnl|Fabrus|A26_IGKJ1*01	1079
  3130	VH3-23_IGHD3-16*01>3′_IGHJ5*01	1680	gnl|Fabrus|A26_IGKJ1*01	1079
  3131	VH3-23_IGHD3-22*01>1′_IGHJ5*01	1681	gnl|Fabrus|A26_IGKJ1*01	1079
  3132	VH3-23_IGHD4-4*01 (1) >1′_IGHJ5*01	1682	gnl|Fabrus|A26_IGKJ1*01	1079
  3133	VH3-23_IGHD4-4*01 (1) >3′_IGHJ5*01	1683	gnl|Fabrus|A26_IGKJ1*01	1079
  3134	VH3-23_IGHD4-11*01 (1) >1′_IGHJ5*01	1684	gnl|Fabrus|A26_IGKJ1*01	1079
  3135	VH3-23_IGHD4-11*01 (1) >3′_IGHJ5*01	1685	gnl|Fabrus|A26_IGKJ1*01	1079
  3136	VH3-23_IGHD4-17*01>1′_IGHJ5*01	1686	gnl|Fabrus|A26_IGKJ1*01	1079
  3137	VH3-23_IGHD4-17*01>3′_IGHJ5*01	1687	gnl|Fabrus|A26_IGKJ1*01	1079
  3138	VH3-23_IGHD4-23*01>1′_IGHJ5*01	1688	gnl|Fabrus|A26_IGKJ1*01	1079
  3139	VH3-23_IGHD4-23*01>3′_IGHJ5*01	1689	gnl|Fabrus|A26_IGKJ1*01	1079
  3140	VH3-23_IGHD5-5*01 (2) >1′_IGHJ5*01	1690	gnl|Fabrus|A26_IGKJ1*01	1079
  3141	VH3-23_IGHD5-5*01 (2) >3′_IGHJ5*01	1691	gnl|Fabrus|A26_IGKJ1*01	1079
  3142	VH3-23_IGHD5-12*01>1′_IGHJ5*01	1692	gnl|Fabrus|A26_IGKJ1*01	1079
  3143	VH3-23_IGHD5-12*01>3′_IGHJ5*01	1693	gnl|Fabrus|A26_IGKJ1*01	1079
  3144	VH3-23_IGHD5-18*01 (2) >1′_IGHJ5*01	1694	gnl|Fabrus|A26_IGKJ1*01	1079
  3145	VH3-23_IGHD5-18*01 (2) >3′_IGHJ5*01	1695	gnl|Fabrus|A26_IGKJ1*01	1079
  3146	VH3-23_IGHD5-24*01>1′_IGHJ5*01	1696	gnl|Fabrus|A26_IGKJ1*01	1079
  3147	VH3-23_IGHD5-24*01>3′_IGHJ5*01	1697	gnl|Fabrus|A26_IGKJ1*01	1079
  3148	VH3-23_IGHD6-6*01>1′_IGHJ5*01	1698	gnl|Fabrus|A26_IGKJ1*01	1079
  3149	VH3-23_IGHD6-6*01>2′_IGHJ5*01	1699	gnl|Fabrus|A26_IGKJ1*01	1079
  3150	VH3-23_IGHD6-6*01>3′_IGHJ5*01	1700	gnl|Fabrus|A26_IGKJ1*01	1079
  3151	VH3-23_IGHD1-1*01>1_IGHJ5*01	1596	gnl|Fabrus|A14_IGKJ1*01	1074
  3152	VH3-23_IGHD1-1*01>2_IGHJ5*01	1597	gnl|Fabrus|A14_IGKJ1*01	1074
  3153	VH3-23_IGHD1-1*01>3_IGHJ5*01	1598	gnl|Fabrus|A14_IGKJ1*01	1074
  3154	VH3-23_IGHD1-7*01>1_IGHJ5*01	1599	gnl|Fabrus|A14_IGKJ1*01	1074
  3155	VH3-23_IGHD1-7*01>3_IGHJ5*01	1600	gnl|Fabrus|A14_IGKJ1*01	1074
  3156	VH3-23_IGHD1-14*01>1_IGHJ5*01	1601	gnl|Fabrus|A14_IGKJ1*01	1074
  3157	VH3-23_IGHD1-14*01>3_IGHJ5*01	1602	gnl|Fabrus|A14_IGKJ1*01	1074
  3158	VH3-23_IGHD1-20*01>1_IGHJ5*01	1603	gnl|Fabrus|A14_IGKJ1*01	1074
  3159	VH3-23_IGHD1-20*01>3_IGHJ5*01	1604	gnl|Fabrus|A14_IGKJ1*01	1074
  3160	VH3-23_IGHD1-26*01>1_IGHJ5*01	1605	gnl|Fabrus|A14_IGKJ1*01	1074
  3161	VH3-23_IGHD1-26*01>3_IGHJ5*01	1606	gnl|Fabrus|A14_IGKJ1*01	1074
  3162	VH3-23_IGHD2-2*01>2_IGHJ5*01	1607	gnl|Fabrus|A14_IGKJ1*01	1074
  3163	VH3-23_IGHD2-2*01>3_IGHJ5*01	1608	gnl|Fabrus|A14_IGKJ1*01	1074
  3164	VH3-23_IGHD2-8*01>2_IGHJ5*01	1609	gnl|Fabrus|A14_IGKJ1*01	1074
  3165	VH3-23_IGHD2-8*01>3_IGHJ5*01	1610	gnl|Fabrus|A14_IGKJ1*01	1074
  3166	VH3-23_IGHD2-15*01>2_IGHJ5*01	1611	gnl|Fabrus|A14_IGKJ1*01	1074
  3167	VH3-23_IGHD2-15*01>3_IGHJ5*01	1612	gnl|Fabrus|A14_IGKJ1*01	1074
  3168	VH3-23_IGHD2-21*01>2_IGHJ5*01	1613	gnl|Fabrus|A14_IGKJ1*01	1074
  3169	VH3-23_IGHD2-21*01>3_IGHJ5*01	1614	gnl|Fabrus|A14_IGKJ1*01	1074
  3170	VH3-23_IGHD3-3*01>1_IGHJ5*01	1615	gnl|Fabrus|A14_IGKJ1*01	1074
  3171	VH3-23_IGHD3-3*01>2_IGHJ5*01	1616	gnl|Fabrus|A14_IGKJ1*01	1074
  3172	VH3-23_IGHD3-3*01>3_IGHJ5*01	1617	gnl|Fabrus|A14_IGKJ1*01	1074
  3173	VH3-23_IGHD3-9*01>2_IGHJ5*01	1618	gnl|Fabrus|A14_IGKJ1*01	1074
  3174	VH3-23_IGHD3-10*01>2_IGHJ5*01	1619	gnl|Fabrus|A14_IGKJ1*01	1074
  3175	VH3-23_IGHD3-10*01>3_IGHJ5*01	1620	gnl|Fabrus|A14_IGKJ1*01	1074
  3176	VH3-23_IGHD3-16*01>2_IGHJ5*01	1621	gnl|Fabrus|A14_IGKJ1*01	1074
  3177	VH3-23_IGHD3-16*01>3_IGHJ5*01	1622	gnl|Fabrus|A14_IGKJ1*01	1074
  3178	VH3-23_IGHD3-22*01>2_IGHJ5*01	1623	gnl|Fabrus|A14_IGKJ1*01	1074
  3179	VH3-23_IGHD3-22*01>3_IGHJ5*01	1624	gnl|Fabrus|A14_IGKJ1*01	1074
  3180	VH3-23_IGHD4-4*01 (1) >2_IGHJ5*01	1625	gnl|Fabrus|A14_IGKJ1*01	1074
  3181	VH3-23_IGHD4-4*01 (1) >3_IGHJ5*01	1626	gnl|Fabrus|A14_IGKJ1*01	1074
  3182	VH3-23_IGHD4-11*01 (1) >2_IGHJ5*01	1627	gnl|Fabrus|A14_IGKJ1*01	1074
  3183	VH3-23_IGHD4-11*01 (1) >3_IGHJ5*01	1628	gnl|Fabrus|A14_IGKJ1*01	1074
  3184	VH3-23_IGHD4-17*01>2_IGHJ5*01	1629	gnl|Fabrus|A14_IGKJ1*01	1074
  3185	VH3-23_IGHD4-17*01>3_IGHJ5*01	1630	gnl|Fabrus|A14_IGKJ1*01	1074
  3186	VH3-23_IGHD4-23*01>2_IGHJ5*01	1631	gnl|Fabrus|A14_IGKJ1*01	1074
  3187	VH3-23_IGHD4-23*01>3_IGHJ5*01	1632	gnl|Fabrus|A14_IGKJ1*01	1074
  3188	VH3-23_IGHD5-5*01 (2) >1_IGHJ5*01	1633	gnl|Fabrus|A14_IGKJ1*01	1074
  3189	VH3-23_IGHD5-5*01 (2) >2_IGHJ5*01	1634	gnl|Fabrus|A14_IGKJ1*01	1074
  3190	VH3-23_IGHD5-5*01 (2) >3_IGHJ5*01	1635	gnl|Fabrus|A14_IGKJ1*01	1074
  3191	VH3-23_IGHD5-12*01>1_IGHJ5*01	1636	gnl|Fabrus|A14_IGKJ1*01	1074
  3192	VH3-23_IGHD5-12*01>3_IGHJ5*01	1637	gnl|Fabrus|A14_IGKJ1*01	1074
  3193	VH3-23_IGHD5-18*01 (2) >1_IGHJ5*01	1638	gnl|Fabrus|A14_IGKJ1*01	1074
  3194	VH3-23_IGHD5-18*01 (2) >2_IGHJ5*01	1639	gnl|Fabrus|A14_IGKJ1*01	1074
  3195	VH3-23_IGHD5-18*01 (2) >3_IGHJ5*01	1640	gnl|Fabrus|A14_IGKJ1*01	1074
  3196	VH3-23_IGHD5-24*01>1_IGHJ5*01	1641	gnl|Fabrus|A14_IGKJ1*01	1074
  3197	VH3-23_IGHD5-24*01>3_IGHJ5*01	1642	gnl|Fabrus|A14_IGKJ1*01	1074
  3198	VH3-23_IGHD6-6*01>1_IGHJ5*01	1643	gnl|Fabrus|A14_IGKJ1*01	1074
  3199	VH3-23_IGHD1-1*01>1′_IGHJ5*01	1653	gnl|Fabrus|A14_IGKJ1*01	1074
  3200	VH3-23_IGHD1-1*01>2′_IGHJ5*01	1654	gnl|Fabrus|A14_IGKJ1*01	1074
  3201	VH3-23_IGHD1-1*01>3′_IGHJ5*01	1655	gnl|Fabrus|A14_IGKJ1*01	1074
  3202	VH3-23_IGHD1-7*01>1′_IGHJ5*01	1656	gnl|Fabrus|A14_IGKJ1*01	1074
  3203	VH3-23_IGHD1-7*01>3′_IGHJ5*01	1657	gnl|Fabrus|A14_IGKJ1*01	1074
  3204	VH3-23_IGHD1-14*01>1′_IGHJ5*01	1658	gnl|Fabrus|A14_IGKJ1*01	1074
  3205	VH3-23_IGHD1-14*01>2′_IGHJ5*01	1659	gnl|Fabrus|A14_IGKJ1*01	1074
  3206	VH3-23_IGHD1-14*01>3′_IGHJ5*01	1660	gnl|Fabrus|A14_IGKJ1*01	1074
  3207	VH3-23_IGHD1-20*01>1′_IGHJ5*01	1661	gnl|Fabrus|A14_IGKJ1*01	1074
  3208	VH3-23_IGHD1-20*01>2′_IGHJ5*01	1662	gnl|Fabrus|A14_IGKJ1*01	1074
  3209	VH3-23_IGHD1-20*01>3′_IGHJ5*01	1663	gnl|Fabrus|A14_IGKJ1*01	1074
  3210	VH3-23_IGHD1-26*01>1′_IGHJ5*01	1664	gnl|Fabrus|A14_IGKJ1*01	1074
  3211	VH3-23_IGHD1-26*01>3′_IGHJ5*01	1665	gnl|Fabrus|A14_IGKJ1*01	1074
  3212	VH3-23_IGHD2-2*01>1′_IGHJ5*01	1666	gnl|Fabrus|A14_IGKJ1*01	1074
  3213	VH3-23_IGHD2-2*01>3′_IGHJ5*01	1667	gnl|Fabrus|A14_IGKJ1*01	1074
  3214	VH3-23_IGHD2-8*01>1′_IGHJ5*01	1668	gnl|Fabrus|A14_IGKJ1*01	1074
  3215	VH3-23_IGHD2-15*01>1′_IGHJ5*01	1669	gnl|Fabrus|A14_IGKJ1*01	1074
  3216	VH3-23_IGHD2-15*01>3′_IGHJ5*01	1670	gnl|Fabrus|A14_IGKJ1*01	1074
  3217	VH3-23_IGHD2-21*01>1′_IGHJ5*01	1671	gnl|Fabrus|A14_IGKJ1*01	1074
  3218	VH3-23_IGHD2-21*01>3′_IGHJ5*01	1672	gnl|Fabrus|A14_IGKJ1*01	1074
  3219	VH3-23_IGHD3-3*01>1′_IGHJ5*01	1673	gnl|Fabrus|A14_IGKJ1*01	1074
  3220	VH3-23_IGHD3-3*01>3′_IGHJ5*01	1674	gnl|Fabrus|A14_IGKJ1*01	1074
  3221	VH3-23_IGHD3-9*01>1′_IGHJ5*01	1675	gnl|Fabrus|A14_IGKJ1*01	1074
  3222	VH3-23_IGHD3-9*01>3′_IGHJ5*01	1676	gnl|Fabrus|A14_IGKJ1*01	1074
  3223	VH3-23_IGHD3-10*01>1′_IGHJ5*01	1677	gnl|Fabrus|A14_IGKJ1*01	1074
  3224	VH3-23_IGHD3-10*01>3′_IGHJ5*01	1678	gnl|Fabrus|A14_IGKJ1*01	1074
  3225	VH3-23_IGHD3-16*01>1′_IGHJ5*01	1679	gnl|Fabrus|A14_IGKJ1*01	1074
  3226	VH3-23_IGHD3-16*01>3′_IGHJ5*01	1680	gnl|Fabrus|A14_IGKJ1*01	1074
  3227	VH3-23_IGHD3-22*01>1′_IGHJ5*01	1681	gnl|Fabrus|A14_IGKJ1*01	1074
  3228	VH3-23_IGHD4-4*01 (1) >1′_IGHJ5*01	1682	gnl|Fabrus|A14_IGKJ1*01	1074
  3229	VH3-23_IGHD4-4*01 (1) >3′_IGHJ5*01	1683	gnl|Fabrus|A14_IGKJ1*01	1074
  3230	VH3-23_IGHD4-11*01 (1) >1′_IGHJ5*01	1684	gnl|Fabrus|A14_IGKJ1*01	1074
  3231	VH3-23_IGHD4-11*01 (1) >3′_IGHJ5*01	1685	gnl|Fabrus|A14_IGKJ1*01	1074
  3232	VH3-23_IGHD4-17*01>1′_IGHJ5*01	1686	gnl|Fabrus|A14_IGKJ1*01	1074
  3233	VH3-23_IGHD4-17*01>3′_IGHJ5*01	1687	gnl|Fabrus|A14_IGKJ1*01	1074
  3234	VH3-23_IGHD4-23*01>1′_IGHJ5*01	1688	gnl|Fabrus|A14_IGKJ1*01	1074
  3235	VH3-23_IGHD4-23*01>3′_IGHJ5*01	1689	gnl|Fabrus|A14_IGKJ1*01	1074
  3236	VH3-23_IGHD5-5*01 (2) >1′_IGHJ5*01	1690	gnl|Fabrus|A14_IGKJ1*01	1074
  3237	VH3-23_IGHD5-5*01 (2) >3′_IGHJ5*01	1691	gnl|Fabrus|A14_IGKJ1*01	1074
  3238	VH3-23_IGHD5-12*01>1′_IGHJ5*01	1692	gnl|Fabrus|A14_IGKJ1*01	1074
  3239	VH3-23_IGHD5-12*01>3′_IGHJ5*01	1693	gnl|Fabrus|A14_IGKJ1*01	1074
  3240	VH3-23_IGHD5-18*01 (2) >1′_IGHJ5*01	1694	gnl|Fabrus|A14_IGKJ1*01	1074
  3241	VH3-23_IGHD5-18*01 (2) >3′_IGHJ5*01	1695	gnl|Fabrus|A14_IGKJ1*01	1074
  3242	VH3-23_IGHD5-24*01>1′_IGHJ5*01	1696	gnl|Fabrus|A14_IGKJ1*01	1074
  3243	VH3-23_IGHD5-24*01>3′_IGHJ5*01	1697	gnl|Fabrus|A14_IGKJ1*01	1074
  3244	VH3-23_IGHD6-6*01>1′_IGHJ5*01	1698	gnl|Fabrus|A14_IGKJ1*01	1074
  3245	VH3-23_IGHD6-6*01>2′_IGHJ5*01	1699	gnl|Fabrus|A14_IGKJ1*01	1074
  3246	VH3-23_IGHD6-6*01>3′_IGHJ5*01	1700	gnl|Fabrus|A14_IGKJ1*01	1074
  3247	VH3-23_IGHD1-1*01>1_IGHJ5*01	1596	gnl|Fabrus|A27_IGKJ1*01	1080
  3248	VH3-23_IGHD1-1*01>2_IGHJ5*01	1597	gnl|Fabrus|A27_IGKJ1*01	1080
  3249	VH3-23_IGHD1-1*01>3_IGHJ5*01	1598	gnl|Fabrus|A27_IGKJ1*01	1080
  3250	VH3-23_IGHD1-7*01>1_IGHJ5*01	1599	gnl|Fabrus|A27_IGKJ1*01	1080
  3251	VH3-23_IGHD1-7*01>3_IGHJ5*01	1600	gnl|Fabrus|A27_IGKJ1*01	1080
  3252	VH3-23_IGHD1-14*01>1_IGHJ5*01	1601	gnl|Fabrus|A27_IGKJ1*01	1080
  3253	VH3-23_IGHD1-14*01>3_IGHJ5*01	1602	gnl|Fabrus|A27_IGKJ1*01	1080
  3254	VH3-23_IGHD1-20*01>1_IGHJ5*01	1603	gnl|Fabrus|A27_IGKJ1*01	1080
  3255	VH3-23_IGHD1-20*01>3_IGHJ5*01	1604	gnl|Fabrus|A27_IGKJ1*01	1080
  3256	VH3-23_IGHD1-26*01>1_IGHJ5*01	1605	gnl|Fabrus|A27_IGKJ1*01	1080
  3257	VH3-23_IGHD1-26*01>3_IGHJ5*01	1606	gnl|Fabrus|A27_IGKJ1*01	1080
  3258	VH3-23_IGHD2-2*01>2_IGHJ5*01	1607	gnl|Fabrus|A27_IGKJ1*01	1080
  3259	VH3-23_IGHD2-2*01>3_IGHJ5*01	1608	gnl|Fabrus|A27_IGKJ1*01	1080
  3260	VH3-23_IGHD2-8*01>2_IGHJ5*01	1609	gnl|Fabrus|A27_IGKJ1*01	1080
  3261	VH3-23_IGHD2-8*01>3_IGHJ5*01	1610	gnl|Fabrus|A27_IGKJ1*01	1080
  3262	VH3-23_IGHD2-15*01>2_IGHJ5*01	1611	gnl|Fabrus|A27_IGKJ1*01	1080
  3263	VH3-23_IGHD2-15*01>3_IGHJ5*01	1612	gnl|Fabrus|A27_IGKJ1*01	1080
  3264	VH3-23_IGHD2-21*01>2_IGHJ5*01	1613	gnl|Fabrus|A27_IGKJ1*01	1080
  3265	VH3-23_IGHD2-21*01>3_IGHJ5*01	1614	gnl|Fabrus|A27_IGKJ1*01	1080
  3266	VH3-23_IGHD3-3*01>1_IGHJ5*01	1615	gnl|Fabrus|A27_IGKJ1*01	1080
  3267	VH3-23_IGHD3-3*01>2_IGHJ5*01	1616	gnl|Fabrus|A27_IGKJ1*01	1080
  3268	VH3-23_IGHD3-3*01>3_IGHJ5*01	1617	gnl|Fabrus|A27_IGKJ1*01	1080
  3269	VH3-23_IGHD3-9*01>2_IGHJ5*01	1618	gnl|Fabrus|A27_IGKJ1*01	1080
  3270	VH3-23_IGHD3-10*01>2_IGHJ5*01	1619	gnl|Fabrus|A27_IGKJ1*01	1080
  3271	VH3-23_IGHD3-10*01>3_IGHJ5*01	1620	gnl|Fabrus|A27_IGKJ1*01	1080
  3272	VH3-23_IGHD3-16*01>2_IGHJ5*01	1621	gnl|Fabrus|A27_IGKJ1*01	1080
  3273	VH3-23_IGHD3-16*01>3_IGHJ5*01	1622	gnl|Fabrus|A27_IGKJ1*01	1080
  3274	VH3-23_IGHD3-22*01>2_IGHJ5*01	1623	gnl|Fabrus|A27_IGKJ1*01	1080
  3275	VH3-23_IGHD3-22*01>3_IGHJ5*01	1624	gnl|Fabrus|A27_IGKJ1*01	1080
  3276	VH3-23_IGHD4-4*01 (1) >2_IGHJ5*01	1625	gnl|Fabrus|A27_IGKJ1*01	1080
  3277	VH3-23_IGHD4-4*01 (1) >3_IGHJ5*01	1626	gnl|Fabrus|A27_IGKJ1*01	1080
  3278	VH3-23_IGHD4-11*01 (1) >2_IGHJ5*01	1627	gnl|Fabrus|A27_IGKJ1*01	1080
  3279	VH3-23_IGHD4-11*01 (1) >3_IGHJ5*01	1628	gnl|Fabrus|A27_IGKJ1*01	1080
  3280	VH3-23_IGHD4-17*01>2_IGHJ5*01	1629	gnl|Fabrus|A27_IGKJ1*01	1080
  3281	VH3-23_IGHD4-17*01>3_IGHJ5*01	1630	gnl|Fabrus|A27_IGKJ1*01	1080
  3282	VH3-23_IGHD4-23*01>2_IGHJ5*01	1631	gnl|Fabrus|A27_IGKJ1*01	1080
  3283	VH3-23_IGHD4-23*01>3_IGHJ5*01	1632	gnl|Fabrus|A27_IGKJ1*01	1080
  3284	VH3-23_IGHD5-5*01 (2) >1_IGHJ5*01	1633	gnl|Fabrus|A27_IGKJ1*01	1080
  3285	VH3-23_IGHD5-5*01 (2) >2_IGHJ5*01	1634	gnl|Fabrus|A27_IGKJ1*01	1080
  3286	VH3-23_IGHD5-5*01 (2) >3_IGHJ5*01	1635	gnl|Fabrus|A27_IGKJ1*01	1080
  3287	VH3-23_IGHD5-12*01>1_IGHJ5*01	1636	gnl|Fabrus|A27_IGKJ1*01	1080
  3288	VH3-23_IGHD5-12*01>3_IGHJ5*01	1637	gnl|Fabrus|A27_IGKJ1*01	1080
  3289	VH3-23_IGHD5-18*01 (2) >1_IGHJ5*01	1638	gnl|Fabrus|A27_IGKJ1*01	1080
  3290	VH3-23_IGHD5-18*01 (2) >2_IGHJ5*01	1639	gnl|Fabrus|A27_IGKJ1*01	1080
  3291	VH3-23_IGHD5-18*01 (2) >3_IGHJ5*01	1640	gnl|Fabrus|A27_IGKJ1*01	1080
  3292	VH3-23_IGHD5-24*01>1_IGHJ5*01	1641	gnl|Fabrus|A27_IGKJ1*01	1080
  3293	VH3-23_IGHD5-24*01>3_IGHJ5*01	1642	gnl|Fabrus|A27_IGKJ1*01	1080
  3294	VH3-23_IGHD6-6*01>1_IGHJ5*01	1643	gnl|Fabrus|A27_IGKJ1*01	1080
  3295	VH3-23_IGHD1-1*01>1′_IGHJ5*01	1653	gnl|Fabrus|A27_IGKJ1*01	1080
  3296	VH3-23_IGHD1-1*01>2′_IGHJ5*01	1654	gnl|Fabrus|A27_IGKJ1*01	1080
  3297	VH3-23_IGHD1-1*01>3′_IGHJ5*01	1655	gnl|Fabrus|A27_IGKJ1*01	1080
  3298	VH3-23_IGHD1-7*01>1′_IGHJ5*01	1656	gnl|Fabrus|A27_IGKJ1*01	1080
  3299	VH3-23_IGHD1-7*01>3′_IGHJ5*01	1657	gnl|Fabrus|A27_IGKJ1*01	1080
  3300	VH3-23_IGHD1-14*01>1′_IGHJ5*01	1658	gnl|Fabrus|A27_IGKJ1*01	1080
  3301	VH3-23_IGHD1-14*01>2′_IGHJ5*01	1659	gnl|Fabrus|A27_IGKJ1*01	1080
  3302	VH3-23_IGHD1-14*01>3′_IGHJ5*01	1660	gnl|Fabrus|A27_IGKJ1*01	1080
  3303	VH3-23_IGHD1-20*01>1′_IGHJ5*01	1661	gnl|Fabrus|A27_IGKJ1*01	1080
  3304	VH3-23_IGHD1-20*01>2′_IGHJ5*01	1662	gnl|Fabrus|A27_IGKJ1*01	1080
  3305	VH3-23_IGHD1-20*01>3′_IGHJ5*01	1663	gnl|Fabrus|A27_IGKJ1*01	1080
  3306	VH3-23_IGHD1-26*01>1′_IGHJ5*01	1664	gnl|Fabrus|A27_IGKJ1*01	1080
  3307	VH3-23_IGHD1-26*01>3′_IGHJ5*01	1665	gnl|Fabrus|A27_IGKJ1*01	1080
  3308	VH3-23_IGHD2-2*01>1′_IGHJ5*01	1666	gnl|Fabrus|A27_IGKJ1*01	1080
  3309	VH3-23_IGHD2-2*01>3′_IGHJ5*01	1667	gnl|Fabrus|A27_IGKJ1*01	1080
  3310	VH3-23_IGHD2-8*01>1′_IGHJ5*01	1668	gnl|Fabrus|A27_IGKJ1*01	1080
  3311	VH3-23_IGHD2-15*01>1′_IGHJ5*01	1669	gnl|Fabrus|A27_IGKJ1*01	1080
  3312	VH3-23_IGHD2-15*01>3′_IGHJ5*01	1670	gnl|Fabrus|A27_IGKJ1*01	1080
  3313	VH3-23_IGHD2-21*01>1′_IGHJ5*01	1671	gnl|Fabrus|A27_IGKJ1*01	1080
  3314	VH3-23_IGHD2-21*01>3′_IGHJ5*01	1672	gnl|Fabrus|A27_IGKJ1*01	1080
  3315	VH3-23_IGHD3-3*01>1′_IGHJ5*01	1673	gnl|Fabrus|A27_IGKJ1*01	1080
  3316	VH3-23_IGHD3-3*01>3′_IGHJ5*01	1674	gnl|Fabrus|A27_IGKJ1*01	1080
  3317	VH3-23_IGHD3-9*01>1′_IGHJ5*01	1675	gnl|Fabrus|A27_IGKJ1*01	1080
  3318	VH3-23_IGHD3-9*01>3′_IGHJ5*01	1676	gnl|Fabrus|A27_IGKJ1*01	1080
  3319	VH3-23_IGHD3-10*01>1′_IGHJ5*01	1677	gnl|Fabrus|A27_IGKJ1*01	1080
  3320	VH3-23_IGHD3-10*01>3′_IGHJ5*01	1678	gnl|Fabrus|A27_IGKJ1*01	1080
  3321	VH3-23_IGHD3-16*01>1′_IGHJ5*01	1679	gnl|Fabrus|A27_IGKJ1*01	1080
  3322	VH3-23_IGHD3-16*01>3′_IGHJ5*01	1680	gnl|Fabrus|A27_IGKJ1*01	1080
  3323	VH3-23_IGHD3-22*01>1′_IGHJ5*01	1681	gnl|Fabrus|A27_IGKJ1*01	1080
  3324	VH3-23_IGHD4-4*01 (1) >1′_IGHJ5*01	1682	gnl|Fabrus|A27_IGKJ1*01	1080
  3325	VH3-23_IGHD4-4*01 (1) >3′_IGHJ5*01	1683	gnl|Fabrus|A27_IGKJ1*01	1080
  3326	VH3-23_IGHD4-11*01 (1) >1′_IGHJ5*01	1684	gnl|Fabrus|A27_IGKJ1*01	1080
  3327	VH3-23_IGHD4-11*01 (1) >3′_IGHJ5*01	1685	gnl|Fabrus|A27_IGKJ1*01	1080
  3328	VH3-23_IGHD4-17*01>1′_IGHJ5*01	1686	gnl|Fabrus|A27_IGKJ1*01	1080
  3329	VH3-23_IGHD4-17*01>3′_IGHJ5*01	1687	gnl|Fabrus|A27_IGKJ1*01	1080
  3330	VH3-23_IGHD4-23*01>1′_IGHJ5*01	1688	gnl|Fabrus|A27_IGKJ1*01	1080
  3331	VH3-23_IGHD4-23*01>3′_IGHJ5*01	1689	gnl|Fabrus|A27_IGKJ1*01	1080
  3332	VH3-23_IGHD5-5*01 (2) >1′_IGHJ5*01	1690	gnl|Fabrus|A27_IGKJ1*01	1080
  3333	VH3-23_IGHD5-5*01 (2) >3′_IGHJ5*01	1691	gnl|Fabrus|A27_IGKJ1*01	1080
  3334	VH3-23_IGHD5-12*01>1′_IGHJ5*01	1692	gnl|Fabrus|A27_IGKJ1*01	1080
  3335	VH3-23_IGHD5-12*01>3′_IGHJ5*01	1693	gnl|Fabrus|A27_IGKJ1*01	1080
  3336	VH3-23_IGHD5-18*01 (2) >1′_IGHJ5*01	1694	gnl|Fabrus|A27_IGKJ1*01	1080
  3337	VH3-23_IGHD5-18*01 (2) >3′_IGHJ5*01	1695	gnl|Fabrus|A27_IGKJ1*01	1080
  3338	VH3-23_IGHD5-24*01>1′_IGHJ5*01	1696	gnl|Fabrus|A27_IGKJ1*01	1080
  3339	VH3-23_IGHD5-24*01>3′_IGHJ5*01	1697	gnl|Fabrus|A27_IGKJ1*01	1080
  3340	VH3-23_IGHD6-6*01>1′_IGHJ5*01	1698	gnl|Fabrus|A27_IGKJ1*01	1080
  3341	VH3-23_IGHD6-6*01>2′_IGHJ5*01	1699	gnl|Fabrus|A27_IGKJ1*01	1080
  3342	VH3-23_IGHD6-6*01>3′_IGHJ5*01	1700	gnl|Fabrus|A27_IGKJ1*01	1080
  3343	VH3-23_IGHD6-6*01>2_IGHJ1*01	1184	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3344	VH3-23_IGHD6-13*01>1_IGHJ1*01	1185	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3345	VH3-23_IGHD6-13*01>2_IGHJ1*01	1186	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3346	VH3-23_IGHD6-19*01>1_IGHJ1*01	1187	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3347	VH3-23_IGHD6-19*01>2_IGHJ1*01	1188	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3348	VH3-23_IGHD6-25*01>1_IGHJ1*01	1189	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3349	VH3-23_IGHD6-25*01>2_IGHJ1*01	1190	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3350	VH3-23_IGHD7-27*01>1_IGHJ1*01	1191	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3351	VH3-23_IGHD7-27*01>3_IGHJ1*01	1192	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3352	VH3-23_IGHD6-13*01>1′_IGHJ1*01	1241	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3353	VH3-23_IGHD6-13*01>2′_IGHJ1*01	1242	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3354	VH3-23_IGHD6-13*01>2_IGHJ1*01_B	1243	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3355	VH3-23_IGHD6-19*01>1′_IGHJ1*01	1244	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3356	VH3-23_IGHD6-19*01>2′_IGHJ1*01	1245	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3357	VH3-23_IGHD6-19*01>2_IGHJ1*01_B	1246	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3358	VH3-23_IGHD6-25*01>1′_IGHJ1*01	1247	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3359	VH3-23_IGHD6-25*01>3′_IGHJ1*01	1248	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3360	VH3-23_IGHD7-27*01>1′_IGHJ1*01_B	1249	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3361	VH3-23_IGHD7-27*01>2′_IGHJ1*01	1250	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3362	VH3-23_IGHD6-6*01>2_IGHJ2*01	1299	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3363	VH3-23_IGHD6-13*01>1_IGHJ2*01	1300	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3364	VH3-23_IGHD6-13*01>2_IGHJ2*01	1301	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3365	VH3-23_IGHD6-19*01>1_IGHJ2*01	1302	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3366	VH3-23_IGHD6-19*01>2_IGHJ2*01	1303	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3367	VH3-23_IGHD6-25*01>1_IGHJ2*01	1304	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3368	VH3-23_IGHD6-25*01>2_IGHJ2*01	1305	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3369	VH3-23_IGHD7-27*01>1_IGHJ2*01	1306	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3370	VH3-23_IGHD7-27*01>3_IGHJ2*01	1307	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3371	VH3-23_IGHD6-13*01>1′_IGHJ2*01	1356	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3372	VH3-23_IGHD6-13*01>2′_IGHJ2*01	1357	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3373	VH3-23_IGHD6-13*01>2_IGHJ2*01_B	1358	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3374	VH3-23_IGHD6-19*01>1′_IGHJ2*01	1359	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3375	VH3-23_IGHD6-19*01>2′_IGHJ2*01	1360	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3376	VH3-23_IGHD6-19*01>2_IGHJ2*01_B	1361	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3377	VH3-23_IGHD6-25*01>1′_IGHJ2*01	1362	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3378	VH3-23_IGHD6-25*01>3′_IGHJ2*01	1363	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3379	VH3-23_IGHD7-27*01>1′_IGHJ2*01	1364	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3380	VH3-23_IGHD7-27*01>2′_IGHJ2*01	1365	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3381	VH3-23_IGHD6-6*01>2_IGHJ3*01	1414	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3382	VH3-23_IGHD6-13*01>1_IGHJ3*01	1415	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3383	VH3-23_IGHD6-13*01>2_IGHJ3*01	1416	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3384	VH3-23_IGHD6-19*01>1_IGHJ3*01	1417	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3385	VH3-23_IGHD6-19*01>2_IGHJ3*01	1418	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3386	VH3-23_IGHD6-25*01>1_IGHJ3*01	1419	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3387	VH3-23_IGHD6-25*01>2_IGHJ3*01	1420	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3388	VH3-23_IGHD7-27*01>1_IGHJ3*01	1421	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3389	VH3-23_IGHD7-27*01>3_IGHJ3*01	1422	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3390	VH3-23_IGHD6-13*01>1′_IGHJ3*01	1471	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3391	VH3-23_IGHD6-13*01>2′_IGHJ3*01	1472	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3392	VH3-23_IGHD6-13*01>3′_IGHJ6*01	1818	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3393	VH3-23_IGHD6-19*01>1′_IGHJ3*01	1474	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3394	VH3-23_IGHD6-19*01>2′_IGHJ3*01	1475	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3395	VH3-23_IGHD6-19*01>3′_IGHJ3*01	1476	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3396	VH3-23_IGHD6-25*01>1′_IGHJ3*01	1477	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3397	VH3-23_IGHD6-25*01>3′_IGHJ3*01	1478	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3398	VH3-23_IGHD7-27*01>1′_IGHJ3*01	1479	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3399	VH3-23_IGHD7-27*01>2′_IGHJ3*01	1480	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3400	VH3-23_IGHD6-6*01>2_IGHJ4*01	1529	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3401	VH3-23_IGHD6-13*01>1_IGHJ4*01	1530	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3402	VH3-23_IGHD6-13*01>2_IGHJ4*01	1531	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3403	VH3-23_IGHD6-19*01>1_IGHJ4*01	1532	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3404	VH3-23_IGHD6-19*01>2_IGHJ4*01	1533	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3405	VH3-23_IGHD6-25*01>1_IGHJ4*01	1534	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3406	VH3-23_IGHD6-25*01>2_IGHJ4*01	1535	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3407	VH3-23_IGHD7-27*01>1_IGHJ4*01	1536	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3408	VH3-23_IGHD7-27*01>3_IGHJ4*01	1537	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3409	VH3-23_IGHD6-13*01>1′_IGHJ4*01	1586	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3410	VH3-23_IGHD6-13*01>2′_IGHJ4*01	1587	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3411	VH3-23_IGHD6-13*01>2_IGHJ4*01_B	1588	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3412	VH3-23_IGHD6-19*01>1′_IGHJ4*01	1589	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3413	VH3-23_IGHD6-19*01>2′_IGHJ4*01	1590	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3414	VH3-23_IGHD6-19*01>2_IGHJ4*01_B	1591	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3415	VH3-23_IGHD6-25*01>1′_IGHJ4*01	1592	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3416	VH3-23_IGHD6-25*01>3′_IGHJ4*01	1593	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3417	VH3-23_IGHD7-27*01>1′_IGHJ4*01	1594	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3418	VH3-23_IGHD7-27*01>2′_IGHJ4*01	1595	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3419	VH3-23_IGHD6-6*01>2_IGHJ5*01	1644	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3420	VH3-23_IGHD6-13*01>1_IGHJ5*01	1645	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3421	VH3-23_IGHD6-13*01>2_IGHJ5*01	1646	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3422	VH3-23_IGHD6-19*01>1_IGHJ5*01	1647	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3423	VH3-23_IGHD6-19*01>2_IGHJ5*01	1648	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3424	VH3-23_IGHD6-25*01>1_IGHJ5*01	1649	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3425	VH3-23_IGHD6-25*01>2_IGHJ5*01	1650	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3426	VH3-23_IGHD7-27*01>1_IGHJ5*01	1651	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3427	VH3-23_IGHD7-27*01>3_IGHJ5*01	1652	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3428	VH3-23_IGHD6-13*01>1′_IGHJ5*01	1701	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3429	VH3-23_IGHD6-13*01>2′_IGHJ5*01	1702	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3430	VH3-23_IGHD6-13*01>3′_IGHJ5*01	1703	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3431	VH3-23_IGHD6-19*01>1′_IGHJ5*01	1704	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3432	VH3-23_IGHD6-19*01>2′_IGHJ5*01	1705	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3433	VH3-23_IGHD6-19*01>2_IGHJ5*01_B	1706	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3434	VH3-23_IGHD6-25*01>1′_IGHJ5*01	1707	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3435	VH3-23_IGHD6-25*01>3′_IGHJ5*01	1708	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3436	VH3-23_IGHD7-27*01>1′_IGHJ5*01	1709	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3437	VH3-23_IGHD7-27*01>2′_IGHJ5*01	1710	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3438	VH3-23_IGHD6-6*01>2_IGHJ6*01	1759	gnl|Fabrus|V1-11_IGLJ2*01	1104
  3439	VH3-23_IGHD6-6*01>2_IGHJ1*01	1184	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3440	VH3-23_IGHD6-13*01>1_IGHJ1*01	1185	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3441	VH3-23_IGHD6-13*01>2_IGHJ1*01	1186	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3442	VH3-23_IGHD6-19*01>1_IGHJ1*01	1187	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3443	VH3-23_IGHD6-19*01>2_IGHJ1*01	1188	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3444	VH3-23_IGHD6-25*01>1_IGHJ1*01	1189	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3445	VH3-23_IGHD6-25*01>2_IGHJ1*01	1190	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3446	VH3-23_IGHD7-27*01>1_IGHJ1*01	1191	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3447	VH3-23_IGHD7-27*01>3_IGHJ1*01	1192	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3448	VH3-23_IGHD6-13*01>1′_IGHJ1*01	1241	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3449	VH3-23_IGHD6-13*01>2′_IGHJ1*01	1242	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3450	VH3-23_IGHD6-13*01>2_IGHJ1*01_B	1243	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3451	VH3-23_IGHD6-19*01>1′_IGHJ1*01	1244	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3452	VH3-23_IGHD6-19*01>2′_IGHJ1*01	1245	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3453	VH3-23_IGHD6-19*01>2_IGHJ1*01_B	1246	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3454	VH3-23_IGHD6-25*01>1′_IGHJ1*01	1247	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3455	VH3-23_IGHD6-25*01>3′_IGHJ1*01	1248	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3456	VH3-23_IGHD7-27*01>1′_IGHJ1*01_B	1249	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3457	VH3-23_IGHD7-27*01>2′_IGHJ1*01	1250	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3458	VH3-23_IGHD6-6*01>2_IGHJ2*01	1299	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3459	VH3-23_IGHD6-13*01>1_IGHJ2*01	1300	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3460	VH3-23_IGHD6-13*01>2_IGHJ2*01	1301	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3461	VH3-23_IGHD6-19*01>1_IGHJ2*01	1302	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3462	VH3-23_IGHD6-19*01>2_IGHJ2*01	1303	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3463	VH3-23_IGHD6-25*01>1_IGHJ2*01	1304	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3464	VH3-23_IGHD6-25*01>2_IGHJ2*01	1305	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3465	VH3-23_IGHD7-27*01>1_IGHJ2*01	1306	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3466	VH3-23_IGHD7-27*01>3_IGHJ2*01	1307	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3467	VH3-23_IGHD6-13*01>1′_IGHJ2*01	1356	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3468	VH3-23_IGHD6-13*01>2′_IGHJ2*01	1357	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3469	VH3-23_IGHD6-13*01>2_IGHJ2*01_B	1358	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3470	VH3-23_IGHD6-19*01>1′_IGHJ2*01	1359	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3471	VH3-23_IGHD6-19*01>2′_IGHJ2*01	1360	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3472	VH3-23_IGHD6-19*01>2_IGHJ2*01_B	1361	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3473	VH3-23_IGHD6-25*01>1′_IGHJ2*01	1362	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3474	VH3-23_IGHD6-25*01>3′_IGHJ2*01	1363	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3475	VH3-23_IGHD7-27*01>1′_IGHJ2*01	1364	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3476	VH3-23_IGHD7-27*01>2′_IGHJ2*01	1365	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3477	VH3-23_IGHD6-6*01>2_IGHJ3*01	1414	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3478	VH3-23_IGHD6-13*01>1_IGHJ3*01	1415	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3479	VH3-23_IGHD6-13*01>2_IGHJ3*01	1416	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3480	VH3-23_IGHD6-19*01>1_IGHJ3*01	1417	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3481	VH3-23_IGHD6-19*01>2_IGHJ3*01	1418	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3482	VH3-23_IGHD6-25*01>1_IGHJ3*01	1419	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3483	VH3-23_IGHD6-25*01>2_IGHJ3*01	1420	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3484	VH3-23_IGHD7-27*01>1_IGHJ3*01	1421	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3485	VH3-23_IGHD7-27*01>3_IGHJ3*01	1422	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3486	VH3-23_IGHD6-13*01>1′_IGHJ3*01	1471	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3487	VH3-23_IGHD6-13*01>2′_IGHJ3*01	1472	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3488	VH3-23_IGHD6-13*01>1_IGHJ6*01	1818	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3489	VH3-23_IGHD6-19*01>1′_IGHJ3*01	1474	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3490	VH3-23_IGHD6-19*01>2′_IGHJ3*01	1475	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3491	VH3-23_IGHD6-19*01>3′_IGHJ3*01	1476	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3492	VH3-23_IGHD6-25*01>1′_IGHJ3*01	1477	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3493	VH3-23_IGHD6-25*01>3′_IGHJ3*01	1478	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3494	VH3-23_IGHD7-27*01>1′_IGHJ3*01	1479	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3495	VH3-23_IGHD7-27*01>2′_IGHJ3*01	1480	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3496	VH3-23_IGHD6-6*01>2_IGHJ4*01	1529	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3497	VH3-23_IGHD6-13*01>1_IGHJ4*01	1530	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3498	VH3-23_IGHD6-13*01>2_IGHJ4*01	1531	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3499	VH3-23_IGHD6-19*01>1_IGHJ4*01	1532	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3500	VH3-23_IGHD6-19*01>2_IGHJ4*01	1533	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3501	VH3-23_IGHD6-25*01>1_IGHJ4*01	1534	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3502	VH3-23_IGHD6-25*01>2_IGHJ4*01	1535	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3503	VH3-23_IGHD7-27*01>1_IGHJ4*01	1536	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3504	VH3-23_IGHD7-27*01>3_IGHJ4*01	1537	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3505	VH3-23_IGHD6-13*01>1′_IGHJ4*01	1586	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3506	VH3-23_IGHD6-13*01>2′_IGHJ4*01	1587	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3507	VH3-23_IGHD6-13*01>2_IGHJ4*01_B	1588	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3508	VH3-23_IGHD6-19*01>1′_IGHJ4*01	1589	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3509	VH3-23_IGHD6-19*01>2′_IGHJ4*01	1590	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3510	VH3-23_IGHD6-19*01>2_IGHJ4*01_B	1591	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3511	VH3-23_IGHD6-25*01>1′_IGHJ4*01	1592	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3512	VH3-23_IGHD6-25*01>3′_IGHJ4*01	1593	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3513	VH3-23_IGHD7-27*01>1′_IGHJ4*01	1594	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3514	VH3-23_IGHD7-27*01>2′_IGHJ4*01	1595	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3515	VH3-23_IGHD6-6*01>2_IGHJ5*01	1644	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3516	VH3-23_IGHD6-13*01>1_IGHJ5*01	1645	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3517	VH3-23_IGHD6-13*01>2_IGHJ5*01	1646	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3518	VH3-23_IGHD6-19*01>1_IGHJ5*01	1647	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3519	VH3-23_IGHD6-19*01>2_IGHJ5*01	1648	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3520	VH3-23_IGHD6-25*01>1_IGHJ5*01	1649	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3521	VH3-23_IGHD6-25*01>2_IGHJ5*01	1650	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3522	VH3-23_IGHD7-27*01>1_IGHJ5*01	1651	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3523	VH3-23_IGHD7-27*01>3_IGHJ5*01	1652	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3524	VH3-23_IGHD6-13*01>1′_IGHJ5*01	1701	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3525	VH3-23_IGHD6-13*01>2′_IGHJ5*01	1702	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3526	VH3-23_IGHD6-13*01>3′_IGHJ5*01	1703	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3527	VH3-23_IGHD6-19*01>1′_IGHJ5*01	1704	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3528	VH3-23_IGHD6-19*01>2′_IGHJ5*01	1705	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3529	VH3-23_IGHD6-19*01>2_IGHJ5*01_B	1706	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3530	VH3-23_IGHD6-25*01>1′_IGHJ5*01	1707	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3531	VH3-23_IGHD6-25*01>3′_IGHJ5*01	1708	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3532	VH3-23_IGHD7-27*01>1′_IGHJ5*01	1709	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3533	VH3-23_IGHD7-27*01>2′_IGHJ5*01	1710	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3534	VH3-23_IGHD6-6*01>2_IGHJ6*01	1759	gnl|Fabrus|V1-13_IGLJ5*01	1105
  3535	VH3-23_IGHD6-6*01>2_IGHJ1*01	1184	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3536	VH3-23_IGHD6-13*01>1_IGHJ1*01	1185	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3537	VH3-23_IGHD6-13*01>2_IGHJ1*01	1186	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3538	VH3-23_IGHD6-19*01>1_IGHJ1*01	1187	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3539	VH3-23_IGHD6-19*01>2_IGHJ1*01	1188	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3540	VH3-23_IGHD6-25*01>1_IGHJ1*01	1189	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3541	VH3-23_IGHD6-25*01>2_IGHJ1*01	1190	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3542	VH3-23_IGHD7-27*01>1_IGHJ1*01	1191	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3543	VH3-23_IGHD7-27*01>3_IGHJ1*01	1192	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3544	VH3-23_IGHD6-13*01>1′_IGHJ1*01	1241	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3545	VH3-23_IGHD6-13*01>2′_IGHJ1*01	1242	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3546	VH3-23_IGHD6-13*01>2_IGHJ1*01_B	1243	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3547	VH3-23_IGHD6-19*01>1′_IGHJ1*01	1244	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3548	VH3-23_IGHD6-19*01>2′_IGHJ1*01	1245	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3549	VH3-23_IGHD6-19*01>2_IGHJ1*01_B	1246	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3550	VH3-23_IGHD6-25*01>1′_IGHJ1*01	1247	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3551	VH3-23_IGHD6-25*01>3′_IGHJ1*01	1248	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3552	VH3-23_IGHD7-27*01>1′_IGHJ1*01_B	1249	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3553	VH3-23_IGHD7-27*01>2′_IGHJ1*01	1250	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3554	VH3-23_IGHD6-6*01>2_IGHJ2*01	1299	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3555	VH3-23_IGHD6-13*01>1_IGHJ2*01	1300	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3556	VH3-23_IGHD6-13*01>2_IGHJ2*01	1301	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3557	VH3-23_IGHD6-19*01>1_IGHJ2*01	1302	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3558	VH3-23_IGHD6-19*01>2_IGHJ2*01	1303	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3559	VH3-23_IGHD6-25*01>1_IGHJ2*01	1304	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3560	VH3-23_IGHD6-25*01>2_IGHJ2*01	1305	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3561	VH3-23_IGHD7-27*01>1_IGHJ2*01	1306	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3562	VH3-23_IGHD7-27*01>3_IGHJ2*01	1307	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3563	VH3-23_IGHD6-13*01>1′_IGHJ2*01	1356	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3564	VH3-23_IGHD6-13*01>2′_IGHJ2*01	1357	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3565	VH3-23_IGHD6-13*01>2_IGHJ2*01_B	1358	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3566	VH3-23_IGHD6-19*01>1′_IGHJ2*01	1359	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3567	VH3-23_IGHD6-19*01>2′_IGHJ2*01	1360	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3568	VH3-23_IGHD6-19*01>2_IGHJ2*01_B	1361	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3569	VH3-23_IGHD6-25*01>1′_IGHJ2*01	1362	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3570	VH3-23_IGHD6-25*01>3′_IGHJ2*01	1363	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3571	VH3-23_IGHD7-27*01>1′_IGHJ2*01	1364	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3572	VH3-23_IGHD7-27*01>2′_IGHJ2*01	1365	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3573	VH3-23_IGHD6-6*01>2_IGHJ3*01	1414	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3574	VH3-23_IGHD6-13*01>1_IGHJ3*01	1415	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3575	VH3-23_IGHD6-13*01>2_IGHJ3*01	1416	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3576	VH3-23_IGHD6-19*01>1_IGHJ3*01	1417	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3577	VH3-23_IGHD6-19*01>2_IGHJ3*01	1418	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3578	VH3-23_IGHD6-25*01>1_IGHJ3*01	1419	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3579	VH3-23_IGHD6-25*01>2_IGHJ3*01	1420	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3580	VH3-23_IGHD7-27*01>1_IGHJ3*01	1421	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3581	VH3-23_IGHD7-27*01>3_IGHJ3*01	1422	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3582	VH3-23_IGHD6-13*01>1′_IGHJ3*01	1471	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3583	VH3-23_IGHD6-13*01>2′_IGHJ3*01	1472	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3584	VH3-23_IGHD6-13*01>1_IGHJ6*01	1818	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3585	VH3-23_IGHD6-19*01>1′_IGHJ3*01	1474	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3586	VH3-23_IGHD6-19*01>2′_IGHJ3*01	1475	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3587	VH3-23_IGHD6-19*01>3′_IGHJ3*01	1476	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3588	VH3-23_IGHD6-25*01>1′_IGHJ3*01	1477	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3589	VH3-23_IGHD6-25*01>3′_IGHJ3*01	1478	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3590	VH3-23_IGHD7-27*01>1′_IGHJ3*01	1479	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3591	VH3-23_IGHD7-27*01>2′_IGHJ3*01	1480	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3592	VH3-23_IGHD6-6*01>2_IGHJ4*01	1529	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3593	VH3-23_IGHD6-13*01>1_IGHJ4*01	1530	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3594	VH3-23_IGHD6-13*01>2_IGHJ4*01	1531	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3595	VH3-23_IGHD6-19*01>1_IGHJ4*01	1532	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3596	VH3-23_IGHD6-19*01>2_IGHJ4*01	1533	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3597	VH3-23_IGHD6-25*01>1_IGHJ4*01	1534	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3598	VH3-23_IGHD6-25*01>2_IGHJ4*01	1535	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3599	VH3-23_IGHD7-27*01>1_IGHJ4*01	1536	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3600	VH3-23_IGHD7-27*01>3_IGHJ4*01	1537	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3601	VH3-23_IGHD6-13*01>1′_IGHJ4*01	1586	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3602	VH3-23_IGHD6-13*01>2′_IGHJ4*01	1587	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3603	VH3-23_IGHD6-13*01>2_IGHJ4*01_B	1588	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3604	VH3-23_IGHD6-19*01>1′_IGHJ4*01	1589	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3605	VH3-23_IGHD6-19*01>2′_IGHJ4*01	1590	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3606	VH3-23_IGHD6-19*01>2_IGHJ4*01_B	1591	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3607	VH3-23_IGHD6-25*01>1′_IGHJ4*01	1592	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3608	VH3-23_IGHD6-25*01>3′_IGHJ4*01	1593	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3609	VH3-23_IGHD7-27*01>1′_IGHJ4*01	1594	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3610	VH3-23_IGHD7-27*01>2′_IGHJ4*01	1595	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3611	VH3-23_IGHD6-6*01>2_IGHJ5*01	1644	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3612	VH3-23_IGHD6-13*01>1_IGHJ5*01	1645	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3613	VH3-23_IGHD6-13*01>2_IGHJ5*01	1646	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3614	VH3-23_IGHD6-19*01>1_IGHJ5*01	1647	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3615	VH3-23_IGHD6-19*01>2_IGHJ5*01	1648	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3616	VH3-23_IGHD6-25*01>1_IGHJ5*01	1649	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3617	VH3-23_IGHD6-25*01>2_IGHJ5*01	1650	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3618	VH3-23_IGHD7-27*01>1_IGHJ5*01	1651	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3619	VH3-23_IGHD7-27*01>3_IGHJ5*01	1652	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3620	VH3-23_IGHD6-13*01>1′_IGHJ5*01	1701	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3621	VH3-23_IGHD6-13*01>2′_IGHJ5*01	1702	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3622	VH3-23_IGHD6-13*01>3′_IGHJ5*01	1703	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3623	VH3-23_IGHD6-19*01>1′_IGHJ5*01	1704	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3624	VH3-23_IGHD6-19*01>2′_IGHJ5*01	1705	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3625	VH3-23_IGHD6-19*01>2_IGHJ5*01_B	1706	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3626	VH3-23_IGHD6-25*01>1′_IGHJ5*01	1707	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3627	VH3-23_IGHD6-25*01>3′_IGHJ5*01	1708	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3628	VH3-23_IGHD7-27*01>1′_IGHJ5*01	1709	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3629	VH3-23_IGHD7-27*01>2′_IGHJ5*01	1710	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3630	VH3-23_IGHD6-6*01>2_IGHJ6*01	1759	gnl|Fabrus|V1-16_IGLJ6*01	1106
  3631	VH3-23_IGHD6-6*01>2_IGHJ1*01	1184	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3632	VH3-23_IGHD6-13*01>1_IGHJ1*01	1185	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3633	VH3-23_IGHD6-13*01>2_IGHJ1*01	1186	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3634	VH3-23_IGHD6-19*01>1_IGHJ1*01	1187	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3635	VH3-23_IGHD6-19*01>2_IGHJ1*01	1188	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3636	VH3-23_IGHD6-25*01>1_IGHJ1*01	1189	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3637	VH3-23_IGHD6-25*01>2_IGHJ1*01	1190	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3638	VH3-23_IGHD7-27*01>1_IGHJ1*01	1191	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3639	VH3-23_IGHD7-27*01>3_IGHJ1*01	1192	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3640	VH3-23_IGHD6-13*01>1′_IGHJ1*01	1241	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3641	VH3-23_IGHD6-13*01>2′_IGHJ1*01	1242	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3642	VH3-23_IGHD6-13*01>2_IGHJ1*01_B	1243	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3643	VH3-23_IGHD6-19*01>1′_IGHJ1*01	1244	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3644	VH3-23_IGHD6-19*01>2′_IGHJ1*01	1245	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3645	VH3-23_IGHD6-19*01>2_IGHJ1*01_B	1246	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3646	VH3-23_IGHD6-25*01>1′_IGHJ1*01	1247	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3647	VH3-23_IGHD6-25*01>3′_IGHJ1*01	1248	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3648	VH3-23_IGHD7-27*01>1′_IGHJ1*01_B	1249	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3649	VH3-23_IGHD7-27*01>2′_IGHJ1*01	1250	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3650	VH3-23_IGHD6-6*01>2_IGHJ2*01	1299	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3651	VH3-23_IGHD6-13*01>1_IGHJ2*01	1300	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3652	VH3-23_IGHD6-13*01>2_IGHJ2*01	1301	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3653	VH3-23_IGHD6-19*01>1_IGHJ2*01	1302	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3654	VH3-23_IGHD6-19*01>2_IGHJ2*01	1303	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3655	VH3-23_IGHD6-25*01>1_IGHJ2*01	1304	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3656	VH3-23_IGHD6-25*01>2_IGHJ2*01	1305	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3657	VH3-23_IGHD7-27*01>1_IGHJ2*01	1306	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3658	VH3-23_IGHD7-27*01>3_IGHJ2*01	1307	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3659	VH3-23_IGHD6-13*01>1′_IGHJ2*01	1356	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3660	VH3-23_IGHD6-13*01>2′_IGHJ2*01	1357	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3661	VH3-23_IGHD6-13*01>2_IGHJ2*01_B	1358	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3662	VH3-23_IGHD6-19*01>1′_IGHJ2*01	1359	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3663	VH3-23_IGHD6-19*01>2′_IGHJ2*01	1360	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3664	VH3-23_IGHD6-19*01>2_IGHJ2*01_B	1361	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3665	VH3-23_IGHD6-25*01>1′_IGHJ2*01	1362	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3666	VH3-23_IGHD6-25*01>3′_IGHJ2*01	1363	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3667	VH3-23_IGHD7-27*01>1′_IGHJ2*01	1364	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3668	VH3-23_IGHD7-27*01>2′_IGHJ2*01	1365	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3669	VH3-23_IGHD6-6*01>2_IGHJ3*01	1414	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3670	VH3-23_IGHD6-13*01>1_IGHJ3*01	1415	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3671	VH3-23_IGHD6-13*01>2_IGHJ3*01	1416	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3672	VH3-23_IGHD6-19*01>1_IGHJ3*01	1417	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3673	VH3-23_IGHD6-19*01>2_IGHJ3*01	1418	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3674	VH3-23_IGHD6-25*01>1_IGHJ3*01	1419	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3675	VH3-23_IGHD6-25*01>2_IGHJ3*01	1420	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3676	VH3-23_IGHD7-27*01>1_IGHJ3*01	1421	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3677	VH3-23_IGHD7-27*01>3_IGHJ3*01	1422	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3678	VH3-23_IGHD6-13*01>1′_IGHJ3*01	1471	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3679	VH3-23_IGHD6-13*01>2′_IGHJ3*01	1472	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3680	VH3-23_IGHD6-13*01>1_IGHJ6*01	1818	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3681	VH3-23_IGHD6-19*01>1′_IGHJ3*01	1474	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3682	VH3-23_IGHD6-19*01>2′_IGHJ3*01	1475	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3683	VH3-23_IGHD6-19*01>3′_IGHJ3*01	1476	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3684	VH3-23_IGHD6-25*01>1′_IGHJ3*01	1477	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3685	VH3-23_IGHD6-25*01>3′_IGHJ3*01	1478	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3686	VH3-23_IGHD7-27*01>1′_IGHJ3*01	1479	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3687	VH3-23_IGHD7-27*01>2′_IGHJ3*01	1480	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3688	VH3-23_IGHD6-6*01>2_IGHJ4*01	1529	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3689	VH3-23_IGHD6-13*01>1_IGHJ4*01	1530	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3690	VH3-23_IGHD6-13*01>2_IGHJ4*01	1531	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3691	VH3-23_IGHD6-19*01>1_IGHJ4*01	1532	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3692	VH3-23_IGHD6-19*01>2_IGHJ4*01	1533	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3693	VH3-23_IGHD6-25*01>1_IGHJ4*01	1534	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3694	VH3-23_IGHD6-25*01>2_IGHJ4*01	1535	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3695	VH3-23_IGHD7-27*01>1_IGHJ4*01	1536	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3696	VH3-23_IGHD7-27*01>3_IGHJ4*01	1537	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3697	VH3-23_IGHD6-13*01>1′_IGHJ4*01	1586	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3698	VH3-23_IGHD6-13*01>2′_IGHJ4*01	1587	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3699	VH3-23_IGHD6-13*01>2_IGHJ4*01_B	1588	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3700	VH3-23_IGHD6-19*01>1′_IGHJ4*01	1589	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3701	VH3-23_IGHD6-19*01>2′_IGHJ4*01	1590	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3702	VH3-23_IGHD6-19*01>2_IGHJ4*01_B	1591	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3703	VH3-23_IGHD6-25*01>1′_IGHJ4*01	1592	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3704	VH3-23_IGHD6-25*01>3′_IGHJ4*01	1593	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3705	VH3-23_IGHD7-27*01>1′_IGHJ4*01	1594	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3706	VH3-23_IGHD7-27*01>2′_IGHJ4*01	1595	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3707	VH3-23_IGHD6-6*01>2_IGHJ5*01	1644	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3708	VH3-23_IGHD6-13*01>1_IGHJ5*01	1645	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3709	VH3-23_IGHD6-13*01>2_IGHJ5*01	1646	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3710	VH3-23_IGHD6-19*01>1_IGHJ5*01	1647	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3711	VH3-23_IGHD6-19*01>2_IGHJ5*01	1648	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3712	VH3-23_IGHD6-25*01>1_IGHJ5*01	1649	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3713	VH3-23_IGHD6-25*01>2_IGHJ5*01	1650	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3714	VH3-23_IGHD7-27*01>1_IGHJ5*01	1651	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3715	VH3-23_IGHD7-27*01>3_IGHJ5*01	1652	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3716	VH3-23_IGHD6-13*01>1′_IGHJ5*01	1701	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3717	VH3-23_IGHD6-13*01>2′_IGHJ5*01	1702	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3718	VH3-23_IGHD6-13*01>3′_IGHJ5*01	1703	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3719	VH3-23_IGHD6-19*01>1′_IGHJ5*01	1704	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3720	VH3-23_IGHD6-19*01>2′_IGHJ5*01	1705	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3721	VH3-23_IGHD6-19*01>2_IGHJ5*01_B	1706	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3722	VH3-23_IGHD6-25*01>1′_IGHJ5*01	1707	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3723	VH3-23_IGHD6-25*01>3′_IGHJ5*01	1708	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3724	VH3-23_IGHD7-27*01>1′_IGHJ5*01	1709	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3725	VH3-23_IGHD7-27*01>2′_IGHJ5*01	1710	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3726	VH3-23_IGHD6-6*01>2_IGHJ6*01	1759	gnl|Fabrus|V1-2_IGLJ7*01	1108
  3727	VH3-23_IGHD6-6*01>2_IGHJ1*01	1184	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3728	VH3-23_IGHD6-13*01>1_IGHJ1*01	1185	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3729	VH3-23_IGHD6-13*01>2_IGHJ1*01	1186	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3730	VH3-23_IGHD6-19*01>1_IGHJ1*01	1187	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3731	VH3-23_IGHD6-19*01>2_IGHJ1*01	1188	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3732	VH3-23_IGHD6-25*01>1_IGHJ1*01	1189	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3733	VH3-23_IGHD6-25*01>2_IGHJ1*01	1190	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3734	VH3-23_IGHD7-27*01>1_IGHJ1*01	1191	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3735	VH3-23_IGHD7-27*01>3_IGHJ1*01	1192	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3736	VH3-23_IGHD6-13*01>1′_IGHJ1*01	1241	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3737	VH3-23_IGHD6-13*01>2′_IGHJ1*01	1242	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3738	VH3-23_IGHD6-13*01>2_IGHJ1*01_B	1243	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3739	VH3-23_IGHD6-19*01>1′_IGHJ1*01	1244	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3740	VH3-23_IGHD6-19*01>2′_IGHJ1*01	1245	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3741	VH3-23_IGHD6-19*01>2_IGHJ1*01_B	1246	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3742	VH3-23_IGHD6-25*01>1′_IGHJ1*01	1247	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3743	VH3-23_IGHD6-25*01>3′_IGHJ1*01	1248	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3744	VH3-23_IGHD7-27*01>1′_IGHJ1*01_B	1249	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3745	VH3-23_IGHD7-27*01>2′_IGHJ1*01	1250	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3746	VH3-23_IGHD6-6*01>2_IGHJ2*01	1299	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3747	VH3-23_IGHD6-13*01>1_IGHJ2*01	1300	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3748	VH3-23_IGHD6-13*01>2_IGHJ2*01	1301	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3749	VH3-23_IGHD6-19*01>1_IGHJ2*01	1302	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3750	VH3-23_IGHD6-19*01>2_IGHJ2*01	1303	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3751	VH3-23_IGHD6-25*01>1_IGHJ2*01	1304	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3752	VH3-23_IGHD6-25*01>2_IGHJ2*01	1305	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3753	VH3-23_IGHD7-27*01>1_IGHJ2*01	1306	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3754	VH3-23_IGHD7-27*01>3_IGHJ2*01	1307	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3755	VH3-23_IGHD6-13*01>1′_IGHJ2*01	1356	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3756	VH3-23_IGHD6-13*01>2′_IGHJ2*01	1357	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3757	VH3-23_IGHD6-13*01>2_IGHJ2*01_B	1358	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3758	VH3-23_IGHD6-19*01>1′_IGHJ2*01	1359	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3759	VH3-23_IGHD6-19*01>2′_IGHJ2*01	1360	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3760	VH3-23_IGHD6-19*01>2_IGHJ2*01_B	1361	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3761	VH3-23_IGHD6-25*01>1′_IGHJ2*01	1362	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3762	VH3-23_IGHD6-25*01>3′_IGHJ2*01	1363	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3763	VH3-23_IGHD7-27*01>1′_IGHJ2*01	1364	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3764	VH3-23_IGHD7-27*01>2′_IGHJ2*01	1365	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3765	VH3-23_IGHD6-6*01>2_IGHJ3*01	1414	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3766	VH3-23_IGHD6-13*01>1_IGHJ3*01	1415	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3767	VH3-23_IGHD6-13*01>2_IGHJ3*01	1416	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3768	VH3-23_IGHD6-19*01>1_IGHJ3*01	1417	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3769	VH3-23_IGHD6-19*01>2_IGHJ3*01	1418	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3770	VH3-23_IGHD6-25*01>1_IGHJ3*01	1419	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3771	VH3-23_IGHD6-25*01>2_IGHJ3*01	1420	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3772	VH3-23_IGHD7-27*01>1_IGHJ3*01	1421	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3773	VH3-23_IGHD7-27*01>3_IGHJ3*01	1422	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3774	VH3-23_IGHD6-13*01>1′_IGHJ3*01	1471	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3775	VH3-23_IGHD6-13*01>2′_IGHJ3*01	1472	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3776	VH3-23_IGHD6-13*01>1_IGHJ6*01	1818	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3777	VH3-23_IGHD6-19*01>1′_IGHJ3*01	1474	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3778	VH3-23_IGHD6-19*01>2′_IGHJ3*01	1475	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3779	VH3-23_IGHD6-19*01>3′_IGHJ3*01	1476	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3780	VH3-23_IGHD6-25*01>1′_IGHJ3*01	1477	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3781	VH3-23_IGHD6-25*01>3′_IGHJ3*01	1478	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3782	VH3-23_IGHD7-27*01>1′_IGHJ3*01	1479	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3783	VH3-23_IGHD7-27*01>2′_IGHJ3*01	1480	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3784	VH3-23_IGHD6-6*01>2_IGHJ4*01	1529	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3785	VH3-23_IGHD6-13*01>1_IGHJ4*01	1530	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3786	VH3-23_IGHD6-13*01>2_IGHJ4*01	1531	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3787	VH3-23_IGHD6-19*01>1_IGHJ4*01	1532	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3788	VH3-23_IGHD6-19*01>2_IGHJ4*01	1533	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3789	VH3-23_IGHD6-25*01>1_IGHJ4*01	1534	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3790	VH3-23_IGHD6-25*01>2_IGHJ4*01	1535	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3791	VH3-23_IGHD7-27*01>1_IGHJ4*01	1536	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3792	VH3-23_IGHD7-27*01>3_IGHJ4*01	1537	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3793	VH3-23_IGHD6-13*01>1′_IGHJ4*01	1586	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3794	VH3-23_IGHD6-13*01>2′_IGHJ4*01	1587	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3795	VH3-23_IGHD6-13*01>2_IGHJ4*01_B	1588	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3796	VH3-23_IGHD6-19*01>1′_IGHJ4*01	1589	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3797	VH3-23_IGHD6-19*01>2′_IGHJ4*01	1590	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3798	VH3-23_IGHD6-19*01>2_IGHJ4*01_B	1591	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3799	VH3-23_IGHD6-25*01>1′_IGHJ4*01	1592	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3800	VH3-23_IGHD6-25*01>3′_IGHJ4*01	1593	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3801	VH3-23_IGHD7-27*01>1′_IGHJ4*01	1594	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3802	VH3-23_IGHD7-27*01>2′_IGHJ4*01	1595	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3803	VH3-23_IGHD6-6*01>2_IGHJ5*01	1644	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3804	VH3-23_IGHD6-13*01>1_IGHJ5*01	1645	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3805	VH3-23_IGHD6-13*01>2_IGHJ5*01	1646	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3806	VH3-23_IGHD6-19*01>1_IGHJ5*01	1647	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3807	VH3-23_IGHD6-19*01>2_IGHJ5*01	1648	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3808	VH3-23_IGHD6-25*01>1_IGHJ5*01	1649	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3809	VH3-23_IGHD6-25*01>2_IGHJ5*01	1650	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3810	VH3-23_IGHD7-27*01>1_IGHJ5*01	1651	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3811	VH3-23_IGHD7-27*01>3_IGHJ5*01	1652	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3812	VH3-23_IGHD6-13*01>1′_IGHJ5*01	1701	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3813	VH3-23_IGHD6-13*01>2′_IGHJ5*01	1702	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3814	VH3-23_IGHD6-13*01>3′_IGHJ5*01	1703	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3815	VH3-23_IGHD6-19*01>1′_IGHJ5*01	1704	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3816	VH3-23_IGHD6-19*01>2′_IGHJ5*01	1705	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3817	VH3-23_IGHD6-19*01>2_IGHJ5*01_B	1706	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3818	VH3-23_IGHD6-25*01>1′_IGHJ5*01	1707	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3819	VH3-23_IGHD6-25*01>3′_IGHJ5*01	1708	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3820	VH3-23_IGHD7-27*01>1′_IGHJ5*01	1709	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3821	VH3-23_IGHD7-27*01>2′_IGHJ5*01	1710	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3822	VH3-23_IGHD6-6*01>2_IGHJ6*01	1759	gnl|Fabrus|V1-20_IGLJ6*01	1109
  3823	VH3-23_IGHD6-6*01>2_IGHJ1*01	1184	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3824	VH3-23_IGHD6-13*01>1_IGHJ1*01	1185	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3825	VH3-23_IGHD6-13*01>2_IGHJ1*01	1186	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3826	VH3-23_IGHD6-19*01>1_IGHJ1*01	1187	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3827	VH3-23_IGHD6-19*01>2_IGHJ1*01	1188	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3828	VH3-23_IGHD6-25*01>1_IGHJ1*01	1189	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3829	VH3-23_IGHD6-25*01>2_IGHJ1*01	1190	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3830	VH3-23_IGHD7-27*01>1_IGHJ1*01	1191	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3831	VH3-23_IGHD7-27*01>3_IGHJ1*01	1192	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3832	VH3-23_IGHD6-13*01>1′_IGHJ1*01	1241	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3833	VH3-23_IGHD6-13*01>2′_IGHJ1*01	1242	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3834	VH3-23_IGHD6-13*01>2_IGHJ1*01_B	1243	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3835	VH3-23_IGHD6-19*01>1′_IGHJ1*01	1244	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3836	VH3-23_IGHD6-19*01>2′_IGHJ1*01	1245	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3837	VH3-23_IGHD6-19*01>2_IGHJ1*01_B	1246	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3838	VH3-23_IGHD6-25*01>1′_IGHJ1*01	1247	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3839	VH3-23_IGHD6-25*01>3′_IGHJ1*01	1248	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3840	VH3-23_IGHD7-27*01>1′_IGHJ1*01_B	1249	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3841	VH3-23_IGHD7-27*01>2′_IGHJ1*01	1250	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3842	VH3-23_IGHD6-6*01>2_IGHJ2*01	1299	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3843	VH3-23_IGHD6-13*01>1_IGHJ2*01	1300	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3844	VH3-23_IGHD6-13*01>2_IGHJ2*01	1301	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3845	VH3-23_IGHD6-19*01>1_IGHJ2*01	1302	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3846	VH3-23_IGHD6-19*01>2_IGHJ2*01	1303	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3847	VH3-23_IGHD6-25*01>1_IGHJ2*01	1304	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3848	VH3-23_IGHD6-25*01>2_IGHJ2*01	1305	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3849	VH3-23_IGHD7-27*01>1_IGHJ2*01	1306	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3850	VH3-23_IGHD7-27*01>3_IGHJ2*01	1307	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3851	VH3-23_IGHD6-13*01>1′_IGHJ2*01	1356	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3852	VH3-23_IGHD6-13*01>2′_IGHJ2*01	1357	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3853	VH3-23_IGHD6-13*01>2_IGHJ2*01_B	1358	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3854	VH3-23_IGHD6-19*01>1′_IGHJ2*01	1359	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3855	VH3-23_IGHD6-19*01>2′_IGHJ2*01	1360	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3856	VH3-23_IGHD6-19*01>2_IGHJ2*01_B	1361	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3857	VH3-23_IGHD6-25*01>1′_IGHJ2*01	1362	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3858	VH3-23_IGHD6-25*01>3′_IGHJ2*01	1363	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3859	VH3-23_IGHD7-27*01>1′_IGHJ2*01	1364	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3860	VH3-23_IGHD7-27*01>2′_IGHJ2*01	1365	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3861	VH3-23_IGHD6-6*01>2_IGHJ3*01	1414	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3862	VH3-23_IGHD6-13*01>1_IGHJ3*01	1415	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3863	VH3-23_IGHD6-13*01>2_IGHJ3*01	1416	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3864	VH3-23_IGHD6-19*01>1_IGHJ3*01	1417	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3865	VH3-23_IGHD6-19*01>2_IGHJ3*01	1418	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3866	VH3-23_IGHD6-25*01>1_IGHJ3*01	1419	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3867	VH3-23_IGHD6-25*01>2_IGHJ3*01	1420	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3868	VH3-23_IGHD7-27*01>1_IGHJ3*01	1421	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3869	VH3-23_IGHD7-27*01>3_IGHJ3*01	1422	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3870	VH3-23_IGHD6-13*01>1′_IGHJ3*01	1471	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3871	VH3-23_IGHD6-13*01>2′_IGHJ3*01	1472	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3872	VH3-23_IGHD6-13*01>1_IGHJ6*01	1818	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3873	VH3-23_IGHD6-19*01>1′_IGHJ3*01	1474	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3874	VH3-23_IGHD6-19*01>2′_IGHJ3*01	1475	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3875	VH3-23_IGHD6-19*01>3′_IGHJ3*01	1476	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3876	VH3-23_IGHD6-25*01>1′_IGHJ3*01	1477	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3877	VH3-23_IGHD6-25*01>3′_IGHJ3*01	1478	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3878	VH3-23_IGHD7-27*01>1′_IGHJ3*01	1479	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3879	VH3-23_IGHD7-27*01>2′_IGHJ3*01	1480	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3880	VH3-23_IGHD6-6*01>2_IGHJ4*01	1529	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3881	VH3-23_IGHD6-13*01>1_IGHJ4*01	1530	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3882	VH3-23_IGHD6-13*01>2_IGHJ4*01	1531	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3883	VH3-23_IGHD6-19*01>1_IGHJ4*01	1532	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3884	VH3-23_IGHD6-19*01>2_IGHJ4*01	1533	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3885	VH3-23_IGHD6-25*01>1_IGHJ4*01	1534	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3886	VH3-23_IGHD6-25*01>2_IGHJ4*01	1535	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3887	VH3-23_IGHD7-27*01>1_IGHJ4*01	1536	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3888	VH3-23_IGHD7-27*01>3_IGHJ4*01	1537	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3889	VH3-23_IGHD6-13*01>1′_IGHJ4*01	1586	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3890	VH3-23_IGHD6-13*01>2′_IGHJ4*01	1587	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3891	VH3-23_IGHD6-13*01>2_IGHJ4*01_B	1588	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3892	VH3-23_IGHD6-19*01>1′_IGHJ4*01	1589	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3893	VH3-23_IGHD6-19*01>2′_IGHJ4*01	1590	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3894	VH3-23_IGHD6-19*01>2_IGHJ4*01_B	1591	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3895	VH3-23_IGHD6-25*01>1′_IGHJ4*01	1592	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3896	VH3-23_IGHD6-25*01>3′_IGHJ4*01	1593	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3897	VH3-23_IGHD7-27*01>1′_IGHJ4*01	1594	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3898	VH3-23_IGHD7-27*01>2′_IGHJ4*01	1595	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3899	VH3-23_IGHD6-6*01>2_IGHJ5*01	1644	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3900	VH3-23_IGHD6-13*01>1_IGHJ5*01	1645	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3901	VH3-23_IGHD6-13*01>2_IGHJ5*01	1646	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3902	VH3-23_IGHD6-19*01>1_IGHJ5*01	1647	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3903	VH3-23_IGHD6-19*01>2_IGHJ5*01	1648	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3904	VH3-23_IGHD6-25*01>1_IGHJ5*01	1649	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3905	VH3-23_IGHD6-25*01>2_IGHJ5*01	1650	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3906	VH3-23_IGHD7-27*01>1_IGHJ5*01	1651	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3907	VH3-23_IGHD7-27*01>3_IGHJ5*01	1652	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3908	VH3-23_IGHD6-13*01>1′_IGHJ5*01	1701	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3909	VH3-23_IGHD6-13*01>2′_IGHJ5*01	1702	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3910	VH3-23_IGHD6-13*01>3′_IGHJ5*01	1703	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3911	VH3-23_IGHD6-19*01>1′_IGHJ5*01	1704	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3912	VH3-23_IGHD6-19*01>2′_IGHJ5*01	1705	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3913	VH3-23_IGHD6-19*01>2_IGHJ5*01_B	1706	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3914	VH3-23_IGHD6-25*01>1′_IGHJ5*01	1707	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3915	VH3-23_IGHD6-25*01>3′_IGHJ5*01	1708	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3916	VH3-23_IGHD7-27*01>1′_IGHJ5*01	1709	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3917	VH3-23_IGHD7-27*01>2′_IGHJ5*01	1710	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3918	VH3-23_IGHD6-6*01>2_IGHJ6*01	1759	gnl|Fabrus|V1-3_IGLJ1*01	1110
  3919	VH3-23_IGHD1-1*01>1_IGHJ6*01	1711	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3920	VH3-23_IGHD1-1*01>2_IGHJ6*01	1712	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3921	VH3-23_IGHD1-1*01>3_IGHJ6*01	1713	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3922	VH3-23_IGHD1-7*01>1_IGHJ6*01	1714	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3923	VH3-23_IGHD1-7*01>3_IGHJ6*01	1715	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3924	VH3-23_IGHD1-14*01>1_IGHJ6*01	1716	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3925	VH3-23_IGHD1-14*01>3_IGHJ6*01	1717	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3926	VH3-23_IGHD1-20*01>1_IGHJ6*01	1718	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3927	VH3-23_IGHD1-20*01>3_IGHJ6*01	1719	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3928	VH3-23_IGHD1-26*01>1_IGHJ6*01	1720	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3929	VH3-23_IGHD1-26*01>3_IGHJ6*01	1721	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3930	VH3-23_IGHD2-2*01>2_IGHJ6*01	1722	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3931	VH3-23_IGHD2-2*01>3_IGHJ6*01	1723	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3932	VH3-23_IGHD2-8*01>2_IGHJ6*01	1724	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3933	VH3-23_IGHD2-8*01>3_IGHJ6*01	1725	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3934	VH3-23_IGHD2-15*01>2_IGHJ6*01	1726	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3935	VH3-23_IGHD2-15*01>3_IGHJ6*01	1727	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3936	VH3-23_IGHD2-21*01>2_IGHJ6*01	1728	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3937	VH3-23_IGHD2-21*01>3_IGHJ6*01	1729	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3938	VH3-23_IGHD3-3*01>1_IGHJ6*01	1730	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3939	VH3-23_IGHD3-3*01>2_IGHJ6*01	1731	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3940	VH3-23_IGHD3-3*01>3_IGHJ6*01	1732	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3941	VH3-23_IGHD3-9*01>2_IGHJ6*01	1733	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3942	VH3-23_IGHD3-10*01>2_IGHJ6*01	1734	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3943	VH3-23_IGHD3-10*01>3_IGHJ6*01	1735	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3944	VH3-23_IGHD3-16*01>2_IGHJ6*01	1736	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3945	VH3-23_IGHD3-16*01>3_IGHJ6*01	1737	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3946	VH3-23_IGHD3-22*01>2_IGHJ6*01	1738	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3947	VH3-23_IGHD3-22*01>3_IGHJ6*01	1739	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3948	VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01	1740	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3949	VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01	1741	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3950	VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01	1742	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3951	VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01	1743	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3952	VH3-23_IGHD4-17*01>2_IGHJ6*01	1744	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3953	VH3-23_IGHD4-17*01>3_IGHJ6*01	1745	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3954	VH3-23_IGHD4-23*01>2_IGHJ6*01	1746	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3955	VH3-23_IGHD4-23*01>3_IGHJ6*01	1747	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3956	VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01	1748	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3957	VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01	1749	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3958	VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01	1750	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3959	VH3-23_IGHD5-12*01>1_IGHJ6*01	1751	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3960	VH3-23_IGHD5-12*01>3_IGHJ6*01	1752	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3961	VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01	1753	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3962	VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01	1754	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3963	VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01	1755	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3964	VH3-23_IGHD5-24*01>1_IGHJ6*01	1756	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3965	VH3-23_IGHD5-24*01>3_IGHJ6*01	1757	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3966	VH3-23_IGHD6-6*01>1_IGHJ6*01	1758	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3967	VH3-23_IGHD1-1*01>1′_IGHJ6*01	1768	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3968	VH3-23_IGHD1-1*01>2′_IGHJ6*01	1769	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3969	VH3-23_IGHD1-1*01>3′_IGHJ6*01	1770	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3970	VH3-23_IGHD1-7*01>1′_IGHJ6*01	1771	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3971	VH3-23_IGHD1-7*01>3′_IGHJ6*01	1772	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3972	VH3-23_IGHD1-14*01>1′_IGHJ6*01	1773	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3973	VH3-23_IGHD1-14*01>2′_IGHJ6*01	1774	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3974	VH3-23_IGHD1-14*01>3′_IGHJ6*01	1775	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3975	VH3-23_IGHD1-20*01>1′_IGHJ6*01	1776	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3976	VH3-23_IGHD1-20*01>2′_IGHJ6*01	1777	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3977	VH3-23_IGHD1-20*01>3′_IGHJ6*01	1778	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3978	VH3-23_IGHD1-26*01>1′_IGHJ6*01	1779	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3979	VH3-23_IGHD1-26*01>1_IGHJ6*01_B	1780	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3980	VH3-23_IGHD2-2*01>2_IGHJ6*01_B	1781	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3981	VH3-23_IGHD2-2*01>3′_IGHJ6*01	1782	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3982	VH3-23_IGHD2-8*01>1′_IGHJ6*01	1783	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3983	VH3-23_IGHD2-15*01>1′_IGHJ6*01	1784	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3984	VH3-23_IGHD2-15*01>3′_IGHJ6*01	1785	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3985	VH3-23_IGHD2-21*01>1′_IGHJ6*01	1786	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3986	VH3-23_IGHD2-21*01>3′_IGHJ6*01	1787	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3987	VH3-23_IGHD3-3*01>1′_IGHJ6*01	1788	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3988	VH3-23_IGHD3-3*01>3′_IGHJ6*01	1789	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3989	VH3-23_IGHD3-9*01>1′_IGHJ6*01	1790	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3990	VH3-23_IGHD3-9*01>3′_IGHJ6*01	1791	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3991	VH3-23_IGHD3-10*01>1′_IGHJ6*01	1792	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3992	VH3-23_IGHD3-10*01>3′_IGHJ6*01	1793	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3993	VH3-23_IGHD3-16*01>1′_IGHJ6*01	1794	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3994	VH3-23_IGHD3-16*01>3′_IGHJ6*01	1795	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3995	VH3-23_IGHD3-22*01>1′_IGHJ6*01	1796	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3996	VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01	1797	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3997	VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01	1798	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3998	VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01	1799	gnl|Fabrus|V2-13_IGLJ2*01	1117
  3999	VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01	1800	gnl|Fabrus|V2-13_IGLJ2*01	1117
  4000	VH3-23_IGHD4-17*01>1′_IGHJ6*01	1801	gnl|Fabrus|V2-13_IGLJ2*01	1117
  4001	VH3-23_IGHD4-17*01>3′_IGHJ6*01	1802	gnl|Fabrus|V2-13_IGLJ2*01	1117
  4002	VH3-23_IGHD4-23*01>1′_IGHJ6*01	1803	gnl|Fabrus|V2-13_IGLJ2*01	1117
  4003	VH3-23_IGHD4-23*01>3′_IGHJ6*01	1804	gnl|Fabrus|V2-13_IGLJ2*01	1117
  4004	VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01	1805	gnl|Fabrus|V2-13_IGLJ2*01	1117
  4005	VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01	1806	gnl|Fabrus|V2-13_IGLJ2*01	1117
  4006	VH3-23_IGHD5-12*01>1′_IGHJ6*01	1807	gnl|Fabrus|V2-13_IGLJ2*01	1117
  4007	VH3-23_IGHD5-12*01>3′_IGHJ6*01	1808	gnl|Fabrus|V2-13_IGLJ2*01	1117
  4008	VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01	1809	gnl|Fabrus|V2-13_IGLJ2*01	1117
  4009	VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01	1810	gnl|Fabrus|V2-13_IGLJ2*01	1117
  4010	VH3-23_IGHD5-24*01>1′_IGHJ6*01	1811	gnl|Fabrus|V2-13_IGLJ2*01	1117
  4011	VH3-23_IGHD5-24*01>3′_IGHJ6*01	1812	gnl|Fabrus|V2-13_IGLJ2*01	1117
  4012	VH3-23_IGHD6-6*01>1′_IGHJ6*01	1813	gnl|Fabrus|V2-13_IGLJ2*01	1117
  4013	VH3-23_IGHD6-6*01>2′_IGHJ6*01	1814	gnl|Fabrus|V2-13_IGLJ2*01	1117
  4014	VH3-23_IGHD6-6*01>3′_IGHJ6*01	1815	gnl|Fabrus|V2-13_IGLJ2*01	1117
  4015	VH3-23_IGHD1-1*01>1_IGHJ6*01	1711	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4016	VH3-23_IGHD1-1*01>2_IGHJ6*01	1712	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4017	VH3-23_IGHD1-1*01>3_IGHJ6*01	1713	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4018	VH3-23_IGHD1-7*01>1_IGHJ6*01	1714	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4019	VH3-23_IGHD1-7*01>3_IGHJ6*01	1715	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4020	VH3-23_IGHD1-14*01>1_IGHJ6*01	1716	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4021	VH3-23_IGHD1-14*01>3_IGHJ6*01	1717	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4022	VH3-23_IGHD1-20*01>1_IGHJ6*01	1718	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4023	VH3-23_IGHD1-20*01>3_IGHJ6*01	1719	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4024	VH3-23_IGHD1-26*01>1_IGHJ6*01	1720	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4025	VH3-23_IGHD1-26*01>3_IGHJ6*01	1721	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4026	VH3-23_IGHD2-2*01>2_IGHJ6*01	1722	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4027	VH3-23_IGHD2-2*01>3_IGHJ6*01	1723	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4028	VH3-23_IGHD2-8*01>2_IGHJ6*01	1724	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4029	VH3-23_IGHD2-8*01>3_IGHJ6*01	1725	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4030	VH3-23_IGHD2-15*01>2_IGHJ6*01	1726	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4031	VH3-23_IGHD2-15*01>3_IGHJ6*01	1727	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4032	VH3-23_IGHD2-21*01>2_IGHJ6*01	1728	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4033	VH3-23_IGHD2-21*01>3_IGHJ6*01	1729	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4034	VH3-23_IGHD3-3*01>1_IGHJ6*01	1730	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4035	VH3-23_IGHD3-3*01>2_IGHJ6*01	1731	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4036	VH3-23_IGHD3-3*01>3_IGHJ6*01	1732	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4037	VH3-23_IGHD3-9*01>2_IGHJ6*01	1733	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4038	VH3-23_IGHD3-10*01>2_IGHJ6*01	1734	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4039	VH3-23_IGHD3-10*01>3_IGHJ6*01	1735	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4040	VH3-23_IGHD3-16*01>2_IGHJ6*01	1736	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4041	VH3-23_IGHD3-16*01>3_IGHJ6*01	1737	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4042	VH3-23_IGHD3-22*01>2_IGHJ6*01	1738	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4043	VH3-23_IGHD3-22*01>3_IGHJ6*01	1739	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4044	VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01	1740	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4045	VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01	1741	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4046	VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01	1742	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4047	VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01	1743	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4048	VH3-23_IGHD4-17*01>2_IGHJ6*01	1744	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4049	VH3-23_IGHD4-17*01>3_IGHJ6*01	1745	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4050	VH3-23_IGHD4-23*01>2_IGHJ6*01	1746	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4051	VH3-23_IGHD4-23*01>3_IGHJ6*01	1747	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4052	VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01	1748	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4053	VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01	1749	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4054	VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01	1750	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4055	VH3-23_IGHD5-12*01>1_IGHJ6*01	1751	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4056	VH3-23_IGHD5-12*01>3_IGHJ6*01	1752	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4057	VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01	1753	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4058	VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01	1754	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4059	VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01	1755	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4060	VH3-23_IGHD5-24*01>1_IGHJ6*01	1756	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4061	VH3-23_IGHD5-24*01>3_IGHJ6*01	1757	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4062	VH3-23_IGHD6-6*01>1_IGHJ6*01	1758	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4063	VH3-23_IGHD1-1*01>1′_IGHJ6*01	1768	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4064	VH3-23_IGHD1-1*01>2′_IGHJ6*01	1769	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4065	VH3-23_IGHD1-1*01>3′_IGHJ6*01	1770	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4066	VH3-23_IGHD1-7*01>1′_IGHJ6*01	1771	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4067	VH3-23_IGHD1-7*01>3′_IGHJ6*01	1772	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4068	VH3-23_IGHD1-14*01>1′_IGHJ6*01	1773	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4069	VH3-23_IGHD1-14*01>2′_IGHJ6*01	1774	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4070	VH3-23_IGHD1-14*01>3′_IGHJ6*01	1775	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4071	VH3-23_IGHD1-20*01>1′_IGHJ6*01	1776	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4072	VH3-23_IGHD1-20*01>2′_IGHJ6*01	1777	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4073	VH3-23_IGHD1-20*01>3′_IGHJ6*01	1778	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4074	VH3-23_IGHD1-26*01>1′_IGHJ6*01	1779	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4075	VH3-23_IGHD1-26*01>1_IGHJ6*01_B	1780	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4076	VH3-23_IGHD2-2*01>2_IGHJ6*01_B	1781	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4077	VH3-23_IGHD2-2*01>3′_IGHJ6*01	1782	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4078	VH3-23_IGHD2-8*01>1′_IGHJ6*01	1783	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4079	VH3-23_IGHD2-15*01>1′_IGHJ6*01	1784	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4080	VH3-23_IGHD2-15*01>3′_IGHJ6*01	1785	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4081	VH3-23_IGHD2-21*01>1′_IGHJ6*01	1786	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4082	VH3-23_IGHD2-21*01>3′_IGHJ6*01	1787	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4083	VH3-23_IGHD3-3*01>1′_IGHJ6*01	1788	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4084	VH3-23_IGHD3-3*01>3′_IGHJ6*01	1789	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4085	VH3-23_IGHD3-9*01>1′_IGHJ6*01	1790	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4086	VH3-23_IGHD3-9*01>3′_IGHJ6*01	1791	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4087	VH3-23_IGHD3-10*01>1′_IGHJ6*01	1792	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4088	VH3-23_IGHD3-10*01>3′_IGHJ6*01	1793	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4089	VH3-23_IGHD3-16*01>1′_IGHJ6*01	1794	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4090	VH3-23_IGHD3-16*01>3′_IGHJ6*01	1795	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4091	VH3-23_IGHD3-22*01>1′_IGHJ6*01	1796	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4092	VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01	1797	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4093	VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01	1798	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4094	VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01	1799	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4095	VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01	1800	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4096	VH3-23_IGHD4-17*01>1′_IGHJ6*01	1801	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4097	VH3-23_IGHD4-17*01>3′_IGHJ6*01	1802	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4098	VH3-23_IGHD4-23*01>1′_IGHJ6*01	1803	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4099	VH3-23_IGHD4-23*01>3′_IGHJ6*01	1804	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4100	VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01	1805	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4101	VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01	1806	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4102	VH3-23_IGHD5-12*01>1′_IGHJ6*01	1807	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4103	VH3-23_IGHD5-12*01>3′_IGHJ6*01	1808	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4104	VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01	1809	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4105	VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01	1810	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4106	VH3-23_IGHD5-24*01>1′_IGHJ6*01	1811	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4107	VH3-23_IGHD5-24*01>3′_IGHJ6*01	1812	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4108	VH3-23_IGHD6-6*01>1′_IGHJ6*01	1813	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4109	VH3-23_IGHD6-6*01>2′_IGHJ6*01	1814	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4110	VH3-23_IGHD6-6*01>3′_IGHJ6*01	1815	gnl|Fabrus|V2-14_IGLJ4*01	1118
  4111	VH3-23_IGHD1-1*01>1_IGHJ6*01	1711	gnl|Fabrus|V2-15_IGLJ7*01	1118
  4112	VH3-23_IGHD1-1*01>2_IGHJ6*01	1712	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4113	VH3-23_IGHD1-1*01>3_IGHJ6*01	1713	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4114	VH3-23_IGHD1-7*01>1_IGHJ6*01	1714	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4115	VH3-23_IGHD1-7*01>3_IGHJ6*01	1715	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4116	VH3-23_IGHD1-14*01>1_IGHJ6*01	1716	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4117	VH3-23_IGHD1-14*01>3_IGHJ6*01	1717	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4118	VH3-23_IGHD1-20*01>1_IGHJ6*01	1718	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4119	VH3-23_IGHD1-20*01>3_IGHJ6*01	1719	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4120	VH3-23_IGHD1-26*01>1_IGHJ6*01	1720	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4121	VH3-23_IGHD1-26*01>3_IGHJ6*01	1721	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4122	VH3-23_IGHD2-2*01>2_IGHJ6*01	1722	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4123	VH3-23_IGHD2-2*01>3_IGHJ6*01	1723	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4124	VH3-23_IGHD2-8*01>2_IGHJ6*01	1724	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4125	VH3-23_IGHD2-8*01>3_IGHJ6*01	1725	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4126	VH3-23_IGHD2-15*01>2_IGHJ6*01	1726	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4127	VH3-23_IGHD2-15*01>3_IGHJ6*01	1727	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4128	VH3-23_IGHD2-21*01>2_IGHJ6*01	1728	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4129	VH3-23_IGHD2-21*01>3_IGHJ6*01	1729	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4130	VH3-23_IGHD3-3*01>1_IGHJ6*01	1730	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4131	VH3-23_IGHD3-3*01>2_IGHJ6*01	1731	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4132	VH3-23_IGHD3-3*01>3_IGHJ6*01	1732	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4133	VH3-23_IGHD3-9*01>2_IGHJ6*01	1733	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4134	VH3-23_IGHD3-10*01>2_IGHJ6*01	1734	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4135	VH3-23_IGHD3-10*01>3_IGHJ6*01	1735	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4136	VH3-23_IGHD3-16*01>2_IGHJ6*01	1736	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4137	VH3-23_IGHD3-16*01>3_IGHJ6*01	1737	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4138	VH3-23_IGHD3-22*01>2_IGHJ6*01	1738	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4139	VH3-23_IGHD3-22*01>3_IGHJ6*01	1739	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4140	VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01	1740	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4141	VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01	1741	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4142	VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01	1742	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4143	VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01	1743	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4144	VH3-23_IGHD4-17*01>2_IGHJ6*01	1744	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4145	VH3-23_IGHD4-17*01>3_IGHJ6*01	1745	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4146	VH3-23_IGHD4-23*01>2_IGHJ6*01	1746	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4147	VH3-23_IGHD4-23*01>3_IGHJ6*01	1747	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4148	VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01	1748	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4149	VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01	1749	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4150	VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01	1750	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4151	VH3-23_IGHD5-12*01>1_IGHJ6*01	1751	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4152	VH3-23_IGHD5-12*01>3_IGHJ6*01	1752	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4153	VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01	1753	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4154	VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01	1754	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4155	VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01	1755	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4156	VH3-23_IGHD5-24*01>1_IGHJ6*01	1756	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4157	VH3-23_IGHD5-24*01>3_IGHJ6*01	1757	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4158	VH3-23_IGHD6-6*01>1_IGHJ6*01	1758	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4159	VH3-23_IGHD1-1*01>1′_IGHJ6*01	1768	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4160	VH3-23_IGHD1-1*01>2′_IGHJ6*01	1769	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4161	VH3-23_IGHD1-1*01>3′_IGHJ6*01	1770	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4162	VH3-23_IGHD1-7*01>1′_IGHJ6*01	1771	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4163	VH3-23_IGHD1-7*01>3′_IGHJ6*01	1772	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4164	VH3-23_IGHD1-14*01>1′_IGHJ6*01	1773	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4165	VH3-23_IGHD1-14*01>2′_IGHJ6*01	1774	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4166	VH3-23_IGHD1-14*01>3′_IGHJ6*01	1775	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4167	VH3-23_IGHD1-20*01>1′_IGHJ6*01	1776	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4168	VH3-23_IGHD1-20*01>2′_IGHJ6*01	1777	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4169	VH3-23_IGHD1-20*01>3′_IGHJ6*01	1778	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4170	VH3-23_IGHD1-26*01>1′_IGHJ6*01	1779	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4171	VH3-23_IGHD1-26*01>1_IGHJ6*01_B	1780	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4172	VH3-23_IGHD2-2*01>2_IGHJ6*01_B	1781	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4173	VH3-23_IGHD2-2*01>3′_IGHJ6*01	1782	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4174	VH3-23_IGHD2-8*01>1′_IGHJ6*01	1783	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4175	VH3-23_IGHD2-15*01>1′_IGHJ6*01	1784	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4176	VH3-23_IGHD2-15*01>3′_IGHJ6*01	1785	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4177	VH3-23_IGHD2-21*01>1′_IGHJ6*01	1786	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4178	VH3-23_IGHD2-21*01>3′_IGHJ6*01	1787	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4179	VH3-23_IGHD3-3*01>1′_IGHJ6*01	1788	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4180	VH3-23_IGHD3-3*01>3′_IGHJ6*01	1789	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4181	VH3-23_IGHD3-9*01>1′_IGHJ6*01	1790	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4182	VH3-23_IGHD3-9*01>3′_IGHJ6*01	1791	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4183	VH3-23_IGHD3-10*01>1′_IGHJ6*01	1792	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4184	VH3-23_IGHD3-10*01>3′_IGHJ6*01	1793	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4185	VH3-23_IGHD3-16*01>1′_IGHJ6*01	1794	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4186	VH3-23_IGHD3-16*01>3′_IGHJ6*01	1795	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4187	VH3-23_IGHD3-22*01>1′_IGHJ6*01	1796	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4188	VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01	1797	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4189	VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01	1798	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4190	VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01	1799	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4191	VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01	1800	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4192	VH3-23_IGHD4-17*01>1′_IGHJ6*01	1801	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4193	VH3-23_IGHD4-17*01>3′_IGHJ6*01	1802	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4194	VH3-23_IGHD4-23*01>1′_IGHJ6*01	1803	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4195	VH3-23_IGHD4-23*01>3′_IGHJ6*01	1804	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4196	VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01	1805	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4197	VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01	1806	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4198	VH3-23_IGHD5-12*01>1′_IGHJ6*01	1807	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4199	VH3-23_IGHD5-12*01>3′_IGHJ6*01	1808	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4200	VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01	1809	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4201	VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01	1810	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4202	VH3-23_IGHD5-24*01>1′_IGHJ6*01	1811	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4203	VH3-23_IGHD5-24*01>3′_IGHJ6*01	1812	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4204	VH3-23_IGHD6-6*01>1′_IGHJ6*01	1813	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4205	VH3-23_IGHD6-6*01>2′_IGHJ6*01	1814	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4206	VH3-23_IGHD6-6*01>3′_IGHJ6*01	1815	gnl|Fabrus|V2-15_IGLJ7*01	1119
  4207	VH3-23_IGHD1-1*01>1_IGHJ6*01	1711	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4208	VH3-23_IGHD1-1*01>2_IGHJ6*01	1712	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4209	VH3-23_IGHD1-1*01>3_IGHJ6*01	1713	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4210	VH3-23_IGHD1-7*01>1_IGHJ6*01	1714	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4211	VH3-23_IGHD1-7*01>3_IGHJ6*01	1715	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4212	VH3-23_IGHD1-14*01>1_IGHJ6*01	1716	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4213	VH3-23_IGHD1-14*01>3_IGHJ6*01	1717	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4214	VH3-23_IGHD1-20*01>1_IGHJ6*01	1718	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4215	VH3-23_IGHD1-20*01>3_IGHJ6*01	1719	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4216	VH3-23_IGHD1-26*01>1_IGHJ6*01	1720	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4217	VH3-23_IGHD1-26*01>3_IGHJ6*01	1721	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4218	VH3-23_IGHD2-2*01>2_IGHJ6*01	1722	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4219	VH3-23_IGHD2-2*01>3_IGHJ6*01	1723	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4220	VH3-23_IGHD2-8*01>2_IGHJ6*01	1724	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4221	VH3-23_IGHD2-8*01>3_IGHJ6*01	1725	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4222	VH3-23_IGHD2-15*01>2_IGHJ6*01	1726	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4223	VH3-23_IGHD2-15*01>3_IGHJ6*01	1727	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4224	VH3-23_IGHD2-21*01>2_IGHJ6*01	1728	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4225	VH3-23_IGHD2-21*01>3_IGHJ6*01	1729	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4226	VH3-23_IGHD3-3*01>1_IGHJ6*01	1730	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4227	VH3-23_IGHD3-3*01>2_IGHJ6*01	1731	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4228	VH3-23_IGHD3-3*01>3_IGHJ6*01	1732	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4229	VH3-23_IGHD3-9*01>2_IGHJ6*01	1733	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4230	VH3-23_IGHD3-10*01>2_IGHJ6*01	1734	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4231	VH3-23_IGHD3-10*01>3_IGHJ6*01	1735	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4232	VH3-23_IGHD3-16*01>2_IGHJ6*01	1736	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4233	VH3-23_IGHD3-16*01>3_IGHJ6*01	1737	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4234	VH3-23_IGHD3-22*01>2_IGHJ6*01	1738	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4235	VH3-23_IGHD3-22*01>3_IGHJ6*01	1739	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4236	VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01	1740	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4237	VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01	1741	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4238	VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01	1742	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4239	VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01	1743	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4240	VH3-23_IGHD4-17*01>2_IGHJ6*01	1744	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4241	VH3-23_IGHD4-17*01>3_IGHJ6*01	1745	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4242	VH3-23_IGHD4-23*01>2_IGHJ6*01	1746	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4243	VH3-23_IGHD4-23*01>3_IGHJ6*01	1747	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4244	VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01	1748	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4245	VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01	1749	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4246	VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01	1750	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4247	VH3-23_IGHD5-12*01>1_IGHJ6*01	1751	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4248	VH3-23_IGHD5-12*01>3_IGHJ6*01	1752	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4249	VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01	1753	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4250	VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01	1754	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4251	VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01	1755	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4252	VH3-23_IGHD5-24*01>1_IGHJ6*01	1756	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4253	VH3-23_IGHD5-24*01>3_IGHJ6*01	1757	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4254	VH3-23_IGHD6-6*01>1_IGHJ6*01	1758	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4255	VH3-23_IGHD1-1*01>1′_IGHJ6*01	1768	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4256	VH3-23_IGHD1-1*01>2′_IGHJ6*01	1769	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4257	VH3-23_IGHD1-1*01>3′_IGHJ6*01	1770	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4258	VH3-23_IGHD1-7*01>1′_IGHJ6*01	1771	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4259	VH3-23_IGHD1-7*01>3′_IGHJ6*01	1772	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4260	VH3-23_IGHD1-14*01>1′_IGHJ6*01	1773	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4261	VH3-23_IGHD1-14*01>2′_IGHJ6*01	1774	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4262	VH3-23_IGHD1-14*01>3′_IGHJ6*01	1775	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4263	VH3-23_IGHD1-20*01>1′_IGHJ6*01	1776	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4264	VH3-23_IGHD1-20*01>2′_IGHJ6*01	1777	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4265	VH3-23_IGHD1-20*01>3′_IGHJ6*01	1778	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4266	VH3-23_IGHD1-26*01>1′_IGHJ6*01	1779	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4267	VH3-23_IGHD1-26*01>1_IGHJ6*01_B	1780	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4268	VH3-23_IGHD2-2*01>2_IGHJ6*01_B	1781	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4269	VH3-23_IGHD2-2*01>3′_IGHJ6*01	1782	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4270	VH3-23_IGHD2-8*01>1′_IGHJ6*01	1783	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4271	VH3-23_IGHD2-15*01>1′_IGHJ6*01	1784	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4272	VH3-23_IGHD2-15*01>3′_IGHJ6*01	1785	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4273	VH3-23_IGHD2-21*01>1′_IGHJ6*01	1786	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4274	VH3-23_IGHD2-21*01>3′_IGHJ6*01	1787	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4275	VH3-23_IGHD3-3*01>1′_IGHJ6*01	1788	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4276	VH3-23_IGHD3-3*01>3′_IGHJ6*01	1789	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4277	VH3-23_IGHD3-9*01>1′_IGHJ6*01	1790	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4278	VH3-23_IGHD3-9*01>3′_IGHJ6*01	1791	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4279	VH3-23_IGHD3-10*01>1′_IGHJ6*01	1792	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4280	VH3-23_IGHD3-10*01>3′_IGHJ6*01	1793	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4281	VH3-23_IGHD3-16*01>1′_IGHJ6*01	1794	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4282	VH3-23_IGHD3-16*01>3′_IGHJ6*01	1795	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4283	VH3-23_IGHD3-22*01>1′_IGHJ6*01	1796	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4284	VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01	1797	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4285	VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01	1798	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4286	VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01	1799	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4287	VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01	1800	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4288	VH3-23_IGHD4-17*01>1′_IGHJ6*01	1801	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4289	VH3-23_IGHD4-17*01>3′_IGHJ6*01	1802	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4290	VH3-23_IGHD4-23*01>1′_IGHJ6*01	1803	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4291	VH3-23_IGHD4-23*01>3′_IGHJ6*01	1804	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4292	VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01	1805	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4293	VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01	1806	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4294	VH3-23_IGHD5-12*01>1′_IGHJ6*01	1807	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4295	VH3-23_IGHD5-12*01>3′_IGHJ6*01	1808	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4296	VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01	1809	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4297	VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01	1810	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4298	VH3-23_IGHD5-24*01>1′_IGHJ6*01	1811	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4299	VH3-23_IGHD5-24*01>3′_IGHJ6*01	1812	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4300	VH3-23_IGHD6-6*01>1′_IGHJ6*01	1813	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4301	VH3-23_IGHD6-6*01>2′_IGHJ6*01	1814	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4302	VH3-23_IGHD6-6*01>3′_IGHJ6*01	1815	gnl|Fabrus|V2-17_IGLJ2*01	1120
  4303	VH3-23_IGHD1-1*01>1_IGHJ6*01	1711	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4304	VH3-23_IGHD1-1*01>2_IGHJ6*01	1712	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4305	VH3-23_IGHD1-1*01>3_IGHJ6*01	1713	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4306	VH3-23_IGHD1-7*01>1_IGHJ6*01	1714	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4307	VH3-23_IGHD1-7*01>3_IGHJ6*01	1715	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4308	VH3-23_IGHD1-14*01>1_IGHJ6*01	1716	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4309	VH3-23_IGHD1-14*01>3_IGHJ6*01	1717	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4310	VH3-23_IGHD1-20*01>1_IGHJ6*01	1718	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4311	VH3-23_IGHD1-20*01>3_IGHJ6*01	1719	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4312	VH3-23_IGHD1-26*01>1_IGHJ6*01	1720	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4313	VH3-23_IGHD1-26*01>3_IGHJ6*01	1721	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4314	VH3-23_IGHD2-2*01>2_IGHJ6*01	1722	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4315	VH3-23_IGHD2-2*01>3_IGHJ6*01	1723	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4316	VH3-23_IGHD2-8*01>2_IGHJ6*01	1724	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4317	VH3-23_IGHD2-8*01>3_IGHJ6*01	1725	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4318	VH3-23_IGHD2-15*01>2_IGHJ6*01	1726	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4319	VH3-23_IGHD2-15*01>3_IGHJ6*01	1727	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4320	VH3-23_IGHD2-21*01>2_IGHJ6*01	1728	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4321	VH3-23_IGHD2-21*01>3_IGHJ6*01	1729	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4322	VH3-23_IGHD3-3*01>1_IGHJ6*01	1730	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4323	VH3-23_IGHD3-3*01>2_IGHJ6*01	1731	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4324	VH3-23_IGHD3-3*01>3_IGHJ6*01	1732	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4325	VH3-23_IGHD3-9*01>2_IGHJ6*01	1733	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4326	VH3-23_IGHD3-10*01>2_IGHJ6*01	1734	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4327	VH3-23_IGHD3-10*01>3_IGHJ6*01	1735	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4328	VH3-23_IGHD3-16*01>2_IGHJ6*01	1736	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4329	VH3-23_IGHD3-16*01>3_IGHJ6*01	1737	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4330	VH3-23_IGHD3-22*01>2_IGHJ6*01	1738	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4331	VH3-23_IGHD3-22*01>3_IGHJ6*01	1739	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4332	VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01	1740	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4333	VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01	1741	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4334	VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01	1742	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4335	VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01	1743	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4336	VH3-23_IGHD4-17*01>2_IGHJ6*01	1744	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4337	VH3-23_IGHD4-17*01>3_IGHJ6*01	1745	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4338	VH3-23_IGHD4-23*01>2_IGHJ6*01	1746	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4339	VH3-23_IGHD4-23*01>3_IGHJ6*01	1747	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4340	VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01	1748	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4341	VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01	1749	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4342	VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01	1750	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4343	VH3-23_IGHD5-12*01>1_IGHJ6*01	1751	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4344	VH3-23_IGHD5-12*01>3_IGHJ6*01	1752	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4345	VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01	1753	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4346	VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01	1754	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4347	VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01	1755	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4348	VH3-23_IGHD5-24*01>1_IGHJ6*01	1756	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4349	VH3-23_IGHD5-24*01>3_IGHJ6*01	1757	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4350	VH3-23_IGHD6-6*01>1_IGHJ6*01	1758	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4351	VH3-23_IGHD1-1*01>1′_IGHJ6*01	1768	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4352	VH3-23_IGHD1-1*01>2′_IGHJ6*01	1769	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4353	VH3-23_IGHD1-1*01>3′_IGHJ6*01	1770	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4354	VH3-23_IGHD1-7*01>1′_IGHJ6*01	1771	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4355	VH3-23_IGHD1-7*01>3′_IGHJ6*01	1772	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4356	VH3-23_IGHD1-14*01>1′_IGHJ6*01	1773	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4357	VH3-23_IGHD1-14*01>2′_IGHJ6*01	1774	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4358	VH3-23_IGHD1-14*01>3′_IGHJ6*01	1775	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4359	VH3-23_IGHD1-20*01>1′_IGHJ6*01	1776	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4360	VH3-23_IGHD1-20*01>2′_IGHJ6*01	1777	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4361	VH3-23_IGHD1-20*01>3′_IGHJ6*01	1778	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4362	VH3-23_IGHD1-26*01>1′_IGHJ6*01	1779	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4363	VH3-23_IGHD1-26*01>1_IGHJ6*01_B	1780	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4364	VH3-23_IGHD2-2*01>2_IGHJ6*01_B	1781	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4365	VH3-23_IGHD2-2*01>3′_IGHJ6*01	1782	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4366	VH3-23_IGHD2-8*01>1′_IGHJ6*01	1783	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4367	VH3-23_IGHD2-15*01>1′_IGHJ6*01	1784	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4368	VH3-23_IGHD2-15*01>3′_IGHJ6*01	1785	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4369	VH3-23_IGHD2-21*01>1′_IGHJ6*01	1786	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4370	VH3-23_IGHD2-21*01>3′_IGHJ6*01	1787	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4371	VH3-23_IGHD3-3*01>1′_IGHJ6*01	1788	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4372	VH3-23_IGHD3-3*01>3′_IGHJ6*01	1789	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4373	VH3-23_IGHD3-9*01>1′_IGHJ6*01	1790	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4374	VH3-23_IGHD3-9*01>3′_IGHJ6*01	1791	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4375	VH3-23_IGHD3-10*01>1′_IGHJ6*01	1792	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4376	VH3-23_IGHD3-10*01>3′_IGHJ6*01	1793	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4377	VH3-23_IGHD3-16*01>1′_IGHJ6*01	1794	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4378	VH3-23_IGHD3-16*01>3′_IGHJ6*01	1795	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4379	VH3-23_IGHD3-22*01>1′_IGHJ6*01	1796	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4380	VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01	1797	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4381	VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01	1798	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4382	VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01	1799	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4383	VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01	1800	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4384	VH3-23_IGHD4-17*01>1′_IGHJ6*01	1801	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4385	VH3-23_IGHD4-17*01>3′_IGHJ6*01	1802	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4386	VH3-23_IGHD4-23*01>1′_IGHJ6*01	1803	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4387	VH3-23_IGHD4-23*01>3′_IGHJ6*01	1804	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4388	VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01	1805	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4389	VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01	1806	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4390	VH3-23_IGHD5-12*01>1′_IGHJ6*01	1807	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4391	VH3-23_IGHD5-12*01>3′_IGHJ6*01	1808	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4392	VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01	1809	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4393	VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01	1810	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4394	VH3-23_IGHD5-24*01>1′_IGHJ6*01	1811	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4395	VH3-23_IGHD5-24*01>3′_IGHJ6*01	1812	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4396	VH3-23_IGHD6-6*01>1′_IGHJ6*01	1813	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4397	VH3-23_IGHD6-6*01>2′_IGHJ6*01	1814	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4398	VH3-23_IGHD6-6*01>3′_IGHJ6*01	1815	gnl|Fabrus|V2-6_IGLJ4*01	1122
  4399	VH3-23_IGHD1-1*01>1_IGHJ6*01	1711	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4400	VH3-23_IGHD1-1*01>2_IGHJ6*01	1712	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4401	VH3-23_IGHD1-1*01>3_IGHJ6*01	1713	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4402	VH3-23_IGHD1-7*01>1_IGHJ6*01	1714	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4403	VH3-23_IGHD1-7*01>3_IGHJ6*01	1715	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4404	VH3-23_IGHD1-14*01>1_IGHJ6*01	1716	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4405	VH3-23_IGHD1-14*01>3_IGHJ6*01	1717	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4406	VH3-23_IGHD1-20*01>1_IGHJ6*01	1718	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4407	VH3-23_IGHD1-20*01>3_IGHJ6*01	1719	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4408	VH3-23_IGHD1-26*01>1_IGHJ6*01	1720	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4409	VH3-23_IGHD1-26*01>3_IGHJ6*01	1721	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4410	VH3-23_IGHD2-2*01>2_IGHJ6*01	1722	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4411	VH3-23_IGHD2-2*01>3_IGHJ6*01	1723	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4412	VH3-23_IGHD2-8*01>2_IGHJ6*01	1724	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4413	VH3-23_IGHD2-8*01>3_IGHJ6*01	1725	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4414	VH3-23_IGHD2-15*01>2_IGHJ6*01	1726	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4415	VH3-23_IGHD2-15*01>3_IGHJ6*01	1727	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4416	VH3-23_IGHD2-21*01>2_IGHJ6*01	1728	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4417	VH3-23_IGHD2-21*01>3_IGHJ6*01	1729	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4418	VH3-23_IGHD3-3*01>1_IGHJ6*01	1730	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4419	VH3-23_IGHD3-3*01>2_IGHJ6*01	1731	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4420	VH3-23_IGHD3-3*01>3_IGHJ6*01	1732	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4421	VH3-23_IGHD3-9*01>2_IGHJ6*01	1733	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4422	VH3-23_IGHD3-10*01>2_IGHJ6*01	1734	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4423	VH3-23_IGHD3-10*01>3_IGHJ6*01	1735	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4424	VH3-23_IGHD3-16*01>2_IGHJ6*01	1736	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4425	VH3-23_IGHD3-16*01>3_IGHJ6*01	1737	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4426	VH3-23_IGHD3-22*01>2_IGHJ6*01	1738	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4427	VH3-23_IGHD3-22*01>3_IGHJ6*01	1739	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4428	VH3-23_IGHD4-4*01 (1) >2_IGHJ6*01	1740	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4429	VH3-23_IGHD4-4*01 (1) >3_IGHJ6*01	1741	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4430	VH3-23_IGHD4-11*01 (1) >2_IGHJ6*01	1742	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4431	VH3-23_IGHD4-11*01 (1) >3_IGHJ6*01	1743	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4432	VH3-23_IGHD4-17*01>2_IGHJ6*01	1744	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4433	VH3-23_IGHD4-17*01>3_IGHJ6*01	1745	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4434	VH3-23_IGHD4-23*01>2_IGHJ6*01	1746	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4435	VH3-23_IGHD4-23*01>3_IGHJ6*01	1747	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4436	VH3-23_IGHD5-5*01 (2) >1_IGHJ6*01	1748	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4437	VH3-23_IGHD5-5*01 (2) >2_IGHJ6*01	1749	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4438	VH3-23_IGHD5-5*01 (2) >3_IGHJ6*01	1750	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4439	VH3-23_IGHD5-12*01>1_IGHJ6*01	1751	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4440	VH3-23_IGHD5-12*01>3_IGHJ6*01	1752	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4441	VH3-23_IGHD5-18*01 (2) >1_IGHJ6*01	1753	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4442	VH3-23_IGHD5-18*01 (2) >2_IGHJ6*01	1754	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4443	VH3-23_IGHD5-18*01 (2) >3_IGHJ6*01	1755	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4444	VH3-23_IGHD5-24*01>1_IGHJ6*01	1756	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4445	VH3-23_IGHD5-24*01>3_IGHJ6*01	1757	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4446	VH3-23_IGHD6-6*01>1_IGHJ6*01	1758	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4447	VH3-23_IGHD1-1*01>1′_IGHJ6*01	1768	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4448	VH3-23_IGHD1-1*01>2′_IGHJ6*01	1769	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4449	VH3-23_IGHD1-1*01>3′_IGHJ6*01	1770	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4450	VH3-23_IGHD1-7*01>1′_IGHJ6*01	1771	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4451	VH3-23_IGHD1-7*01>3′_IGHJ6*01	1772	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4452	VH3-23_IGHD1-14*01>1′_IGHJ6*01	1773	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4453	VH3-23_IGHD1-14*01>2′_IGHJ6*01	1774	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4454	VH3-23_IGHD1-14*01>3′_IGHJ6*01	1775	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4455	VH3-23_IGHD1-20*01>1′_IGHJ6*01	1776	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4456	VH3-23_IGHD1-20*01>2′_IGHJ6*01	1777	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4457	VH3-23_IGHD1-20*01>3′_IGHJ6*01	1778	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4458	VH3-23_IGHD1-26*01>1′_IGHJ6*01	1779	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4459	VH3-23_IGHD1-26*01>1_IGHJ6*01_B	1780	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4460	VH3-23_IGHD2-2*01>2_IGHJ6*01_B	1781	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4461	VH3-23_IGHD2-2*01>3′_IGHJ6*01	1782	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4462	VH3-23_IGHD2-8*01>1′_IGHJ6*01	1783	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4463	VH3-23_IGHD2-15*01>1′_IGHJ6*01	1784	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4464	VH3-23_IGHD2-15*01>3′_IGHJ6*01	1785	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4465	VH3-23_IGHD2-21*01>1′_IGHJ6*01	1786	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4466	VH3-23_IGHD2-21*01>3′_IGHJ6*01	1787	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4467	VH3-23_IGHD3-3*01>1′_IGHJ6*01	1788	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4468	VH3-23_IGHD3-3*01>3′_IGHJ6*01	1789	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4469	VH3-23_IGHD3-9*01>1′_IGHJ6*01	1790	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4470	VH3-23_IGHD3-9*01>3′_IGHJ6*01	1791	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4471	VH3-23_IGHD3-10*01>1′_IGHJ6*01	1792	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4472	VH3-23_IGHD3-10*01>3′_IGHJ6*01	1793	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4473	VH3-23_IGHD3-16*01>1′_IGHJ6*01	1794	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4474	VH3-23_IGHD3-16*01>3′_IGHJ6*01	1795	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4475	VH3-23_IGHD3-22*01>1′_IGHJ6*01	1796	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4476	VH3-23_IGHD4-4*01 (1) >1′_IGHJ6*01	1797	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4477	VH3-23_IGHD4-4*01 (1) >3′_IGHJ6*01	1798	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4478	VH3-23_IGHD4-11*01 (1) >1′_IGHJ6*01	1799	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4479	VH3-23_IGHD4-11*01 (1) >3′_IGHJ6*01	1800	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4480	VH3-23_IGHD4-17*01>1′_IGHJ6*01	1801	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4481	VH3-23_IGHD4-17*01>3′_IGHJ6*01	1802	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4482	VH3-23_IGHD4-23*01>1′_IGHJ6*01	1803	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4483	VH3-23_IGHD4-23*01>3′_IGHJ6*01	1804	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4484	VH3-23_IGHD5-5*01 (2) >1′_IGHJ6*01	1805	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4485	VH3-23_IGHD5-5*01 (2) >3′_IGHJ6*01	1806	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4486	VH3-23_IGHD5-12*01>1′_IGHJ6*01	1807	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4487	VH3-23_IGHD5-12*01>3′_IGHJ6*01	1808	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4488	VH3-23_IGHD5-18*01 (2) >1′_IGHJ6*01	1809	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4489	VH3-23_IGHD5-18*01 (2) >3′_IGHJ6*01	1810	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4490	VH3-23_IGHD5-24*01>1′_IGHJ6*01	1811	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4491	VH3-23_IGHD5-24*01>3′_IGHJ6*01	1812	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4492	VH3-23_IGHD6-6*01>1′_IGHJ6*01	1813	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4493	VH3-23_IGHD6-6*01>2′_IGHJ6*01	1814	gnl|Fabrus|V2-7_IGLJ2*01	1123
  4494	VH3-23_IGHD6-6*01>3′_IGHJ6*01	1815	gnl|Fabrus|V2-7_IGLJ2*01	1123

• Typically, the addressable combinatorial germline libraries are spatially arrayed in a multiwell plate, such as a 96-well plate, wherein each well of the plate corresponds to one antibody that is different from the antibodies in all the other wells. The antibodies can be immobilized to the surface of the wells of the plate or can be present in solution. Alternatively, the antibodies are attached to a solid support, such as for example, a filter, chip, slide, bead or cellulose. The antibodies can also be identifiably labeled, such as for example, with a colored, chromogenic, luminescent, chemical, fluorescent or electronic label. The combinatorial addressable germline antibody libraries can be screened for binding or activity against a target protein to identify antibodies or portions thereof that bind to a target protein and/or modulate an activity of a target protein. By virtue of the fact that these libaries are arrayed, the identity of each individual member in the collection is known during screening thereby allowing facile identification of a “Hit” antibody. Screening for binding or a functional activity can be by any method known to one of skill in the art, for example, any described in Section E.1.
• For example, as described in the Examples, an addressable antibody library is exemplified to screen for “Hits” against a target antigen using an MSD electrochemiluminescence binding assay or by ELISA. Since the library was addressable, the sequence of the identified “Hit” was immediately known. A similar assay is exemplified to identify a related antibody as discussed further below.
• b. Identification of a Related Antibody
• In the method provided herein, comparison to a related antibody that has reduced or less activity for the target antigen than the first antibody provides information of SAR that can be used for affinity maturation herein. In the method, residues to mutagenize in the antibody to be affinity matured are identified by comparison of the amino acid sequence of the variable heavy or light chain of the first antibody (e.g.“Hit”) with the corresponding amino acid sequence of the variable heavy or light chain of a related antibody. For purposes of practice of the method herein, a related antibody has sequence similarity or identity to the “Hit” antibody across the entire sequence of the antibody (heavy and light chain), but is not itself identical in sequence to the “Hit” antibody. In addition, the related antibody exhibits less activity (e.g. binding or binding affinity) for the target antigen than the first antibody.
• In the method herein, once a first antibody is chosen for affinity maturation herein as set forth above, one or more related antibodies are selected. It is not necessary that the first antibody and related antibodies are identified from the same library or even using the same screening method. All that is necessary is that the related antibody has less activity to a target antigen than the first antibody and that the related antibody exhibits sequence similarity to the antibody that is being affinity matured. For convenience, a related antibody is typically identified using the same screening method and assay system used for identification of the first antibody. Hence, any of the methods of generating an antibody, including any of the antibody libraries, described in Section C.1 above can be used for identification of a related antibody. Exemplary of an antibody library is an addressable combinatorial antibody library described above and herein in the Examples. As previously mentioned, the addressable combinatorial antibody library has the benefit of immediate knowledge of the structure-activity relationship of all members of the library for binding to a target antigen. Hence, like a “Hit” antibody, the sequence and activity of a related antibody is immediately known. Accordingly, assessment of sequence similarity between a “Hit” and related antibody can be determined almost instantaneously upon completion of a screening assay for a target antigen.
• Generally, the related antibody is an antibody that exhibits 80% of less of the activity of the first antibody, generally 5% to 80% of the activity, and in particular 5% to 50% of the activity, such as 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less the activity towards the target antigen compared to the first antibody. For example, the related antibody can be an antibody that does not bind or that shows negligible binding to the target antigen for which the “Hit” antibody binds (e.g. a level of binding that is the same or similar to binding of a negative control used in the assay). Thus, a related antibody can be initially identified because it does not specifically bind to the target antigen for which the chosen first antibody specifically binds. For example, a related antibody can exhibit a binding affinity that is 10⁻⁴ M or higher, for example, 10⁻⁴ M, 10⁻³ M, 10⁻² M, or higher. In comparing an activity (e.g. binding and/or binding affinity) of first antibody to a related antibody, the same target antigen is used and activity is assessed in the same or similar assay. In addition, corresponding forms of the antibodies are compared such that the structure of the antibody also is the same (e.g. full-length antibody or fragment thereof such as a Fab).
• A related antibody that is chosen for practice of the method is related to the first antibody because it exhibits sequence similarity or identity to a first antibody across its entire sequence (heavy and light chain) or across its variable heavy or variable light chain. For example, the amino acid sequence of the variable heavy chain and/or variable light chain of the related antibody is at least 50% identical in amino acid sequence to the first antibody, generally at least 75% identical in sequence, for example it is or is about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical in amino acid sequence to the first antibody, typically at least at or about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% similar in sequence. The related antibody is not identical to the first antibody in both the variable heavy and light chain, but can be identical to the first antibody in one of the variable heavy or light chains and exhibit less than 100% sequence similarity in the other chain. Thus, it is understood that for practice of the method, the variable portion of the related antibody used in the method is less than 100% identical to the identified “Hit” antibody. For example, in many instances, a related antibody might exhibit 100% sequence identity to the first antibody in the variable light chain sequence, but less than 100% sequence similarity to the first antibody in the variable heavy chain sequence, while still exhibiting a requisite sequence similarity. In that instance, only the variable heavy chain sequence of the related antibody is used in the practice of the method as described herein. Any method for determining sequence similarity known to one of skill in the art can be used as described elsewhere herein, including, but not limited to, manual methods or the use of available programs such as BLAST.
• For example, a related antibody can contain a variable heavy chain that is identical to the variable heavy chain of the first antibody, and a variable light chain that exhibits sequence similarity to the first antibody. In other examples, neither the variable heavy or variable light chain of the related antibody are identical to the amino acid sequence of the first antibody, but both exhibit sequence similarity to the first antibody. Thus, in some instances, a related antibody used in the method of affinity maturing the variable heavy chain of the first antibody is different from a related antibody used in the method of affinity maturing the variable light chain of the first antibody. Accordingly, more than one related antibody can be selected for practice of the method herein. For example, as exemplified in the examples, three related antibodies are selected for affinity maturation of the variable light chain. In either case, a variable chain (heavy or light) of a related antibody that exhibits sequence similarity to the corresponding heavy or light chain of the first antibody is used in the method to identify a region or regions in the first antibody that differ and thus are responsible for the differing binding abilities of the “first antibody and related antibodies. Such region or regions are targeted for affinity maturation and mutagenesis in the method herein as described further below.
• Generally, the variable heavy and/or light chain of a first antibody and a related antibody are derived from the same or related, such as from the same gene family, antibody variable region germline segments. For example, a related antibody is encoded by a sequence of nucleic acids that contains one or more variable heavy chain V_H, D_H and/or J_H germline segments or variable light chain V_κ and J_κ or V_λ and J_λ germline segments that is not identical to, but is of the same gene family, as contained in the nucleic acid sequence encoding the first antibody. Typically, a related antibody is encoded by a sequence of nucleic acids that contains identical germline segments to the nucleic acid sequence encoding the first antibody, except that 1, 2, 3, 4, or 5 of the germline segments are different or related. For example, a related antibody is encoded by a nucleic acid sequence encoding the VH or VL chain that contains the same variable heavy chain V_H, and D_H germline segments, or the same variable light chain V_κ or V_λ germline segments, but different or related J_H, and J_κ or J_λ germline segments. As exemplified in the Examples, the variable heavy chain of a related antibody was chosen for practice of the method herein because it was encoded by a sequence of nucleic acids that contained identical variable heavy chain V_H and J_H germline segments (i.e., VH5-51 and IGHJ4*01) but had a different D_H germline segment (i.e., IGHD5-51*01>3 versus IGHD6-25*01) compared to the sequence of nucleic acids encoding the variable heavy chain sequence of the chosen “Hit”. The sequence of the variable heavy chain of the related antibody exhibits 98% sequence similarity to the first antibody. In another example, the variable heavy chain of a related antibody was chosen for practice of the method herein because it was encoded by a sequence of nucleic acids that contained identical V_H germline segments (i.e., VH1-46), but different J_H germline segments (i.e., IGHJ4*01 versus IGHJ1*01), and related D_H germline segments (i.e., IGHD6-13*01 versus IGHD6-6*01, sharing the same gene family IGHD6) compared to the sequence of nucleic acids encoding the variable heavy chain sequence of the chosen first antibody. The sequence of the variable heavy chain of the related antibody exhibits 95% sequence similarity to the first antibody.
• One of skill in the art knows and is familiar with germline segment sequences of antibodies, and can identify the germline segment sequences encoding an antibody heavy or light chain. Exemplary antibody germline sources include but are not limited to databases at the National Center for Biotechnology Information (NCBI), the international ImMunoGeneTics information System® (IMGT), the Kabat database and the Tomlinson's VBase database (Lefranc (2003) Nucleic Acids Res., 31:307-310; Martin et al., Bioinformatics Tools for Antibody Engineering in Handbook of Therapeutic Antibodies, Wiley-VCH (2007), pp. 104-107). Germline segments also are known for non-humans. For example, an exemplary mouse germline databases is ABG database available at ibt.unam.mx/vir/v_mice.html. Germline segment sequences are known by various nomenclatures, including for example, IMGT gene names and definitions approved by the Human Genome Organization (HUGO) nomenclature committee, Lefranc, M.-P. Exp Clin Immunogenet, 18:100-116 (2001), Zachau, H. G. Immunologist, 4:49-54 (1996), Lefranc, M.-P. Exp Clin Immunogenet, 18:161-174 (2000), Kawasaki et al, Genome Res, 7:250-261 (1997), Lefranc, M.-P. Exp Clin Immunogenet, 18:242-254 (2001). Any desired naming convention can be used to identify antibody germline segments. One of skill in the art can identify a nucleic acid sequence using any desired naming convention. For example, for IMGT nomenclature, the first three letters indicate the locus (IGH, IGK or IGL), the fourth letter represents the gene (e.g., V for V-gene, D for D-gene, J for J-gene), the fifth position indicates the number of the subgroup, followed by a hyphen indicating the gene number classification. For alleles, the IMGT name is followed by an asterisk and a two figure number. U.S. Provisional Application Nos. 61/198,764 and 61/211,204 set forth exemplary human heavy chain and light chain (kappa and lambda) germline segment sequences.
• c. Comparison of the Amino Acid Sequences of the First Antibody and Related Antibodies
• Once a first antibody is chosen and a related antibody or antibodies are identified that have a related variable heavy chain and/or variable light chain, sequence comparison of the antibodies is effected. Comparison of the amino acid sequence of the variable heavy chain and/or the variable light chain of the parent or first antibody and the related antibody permits identification of regions that differ between the first antibody and the related antibody. Such region or regions are targeted for affinity maturation and mutagenesis.
• In the method, the amino acid sequence of the VH chain and/or the VL chain of the parent first antibody is aligned to the respective VH chain or VL chain of at least one related antibody to identify regions of the polypeptide that differ, or vary, between the first antibody and related antibodies. The amino acid sequences of the antibodies can be aligned by any method commonly known in the art. The methods include manual alignment, computer assisted sequence alignment, and combinations thereof. A number of algorithms (which are generally computer implemented) for performing sequence alignment are widely available, or can be produced by one of skill. These methods include, e.g., the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482; the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443; the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (USA) 85:2444; and/or by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.).
• For example, software for performing sequence identity (and sequence similarity) analysis using the BLAST algorithm is described in Altschul et al., (1990) J. Mol. Biol. 215:403-410. This software is publicly available, e.g., through the National Center for Biotechnology Information on the world wide web at ncbi.nlm.nih.gov. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP (BLAST Protein) program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).
• Additionally, the BLAST algorithm performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences occurs by chance. For example, a nucleic acid is considered similar to a reference sequence (and, therefore, in this context, homologous) if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, or less than about 0.01, and or even less than about 0.001.
• An additional example of an algorithm that is suitable for multiple DNA, or amino acid, sequence alignments is the CLUSTALW program (Thompson, J. D. et al., (1994) Nucl. Acids. Res. 22: 4673-4680). CLUSTALW performs multiple pairwise comparisons between groups of sequences and assembles them into a multiple alignment based on homology. Gap open and Gap extension penalties can be, e.g., 10 and 0.05 respectively. For amino acid alignments, the BLOSUM algorithm can be used as a protein weight matrix. See, e.g., Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919.
• By aligning the amino acid sequences of the antibodies, one skilled in the art can identify regions that differ between the amino acid sequence of the first antibody and the related antibodies. A region that differs between the antibodies can occur along any portion of the VH chain and/or VL chain. Typically, a region that differs or varies occurs at a CDR or framework (FR) region, for example, CDR1, CDR2, CDR3, FR1, FR2, FR3 and/or FR4, and in particular in a CDR, for example CDR3. One of skill in the art knows and can identify the CDRs and FR based on Kabat or Chothia numbering (see e.g., Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). For example, based on Kabat numbering, CDR-L1 corresponds to residues L24-L34; CDR-L2 corresponds to residues L50-L56; CDR-L3 corresponds to residues L89-L97; CDR-H1 corresponds to residues H31-H35, 35a or 35b depending on the length; CDR-H2 corresponds to residues H50-H65; and CDR-H3 corresponds to residues H95-H102. For example, based on Kabat numbering, FR-L1 corresponds to residues L1-L23; FR-L2 corresponds to residues L35-L49; FR-L3 corresponds to residues L57-L88; FR-L4 corresponds to residues L98-L109; FR-H1 corresponds to residues H1-H30; FR-H2 corresponds to residues H36-H49; FR-H3 corresponds to residues H66-H94; and FR-H4 corresponds to residues H103-H113.
• A region(s) that differs is identified as a target region because it contains at least one acid differences or variation at corresponding amino acid positions in the variable heavy chain and/or variable light chain amino acid sequence of a first antibody and a related antibody. A variant position includes an amino acid deletion, addition or substitution in the first antibody polypeptide as compared to the related antibody polypeptide. For purposes herein, an identified region contains one or more, typically two or more, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more variant amino acid positions in at least one region of a variable chain of the first antibody compared to a related antibody. In some examples, more then one region, for example, 1, 2, 3, 4 or more regions can be identified that contain at least one variant amino acid positions between a first antibody and a related antibody. Any one or more of the regions can be targeted for affinity maturation by mutagenesis. Generally, a CDR is targeted for mutagenesis.
• d. Mutagenesis of an Identified Region
• In the method, mutagenesis is performed on target residues within the identified target region. For example, some or up to all amino acid residues of the selected target region in the heavy chain and/or light chain of the first antibody are mutated, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acid residues. Each target amino acid residue selected for mutagenesis can be mutated to all 19 other amino acid residues, or to a restricted subset thereof.
• In one example, all amino acid residues in the identified target region, e.g. CDR3, can be subject to mutagenesis. In another example, a subset of amino acid residues in the selected target region can be subject to mutagenesis. For example, only the amino acid residues at positions that differ between the first antibody and related antibody are subject to mutagenesis. In another example, only the amino acid residues at positions that are the same between the first antibody and a related antibody are subject to mutagenesis. In an additional example, scanning mutagenesis is optionally performed to identify residues that increase binding to the target antigen. In such examples, only those residues that are identified as “UP” mutants as discussed below are subject to further saturation mutagenesis.
• For example, typically, a CDR can contain 3 to 25 amino acid residues. All or subset of the amino acids within a CDR can be targeted for mutagenesis, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid residues can be targeted for mutagenesis. In some examples, all amino acids within a CDR are selected for mutagenesis. In other examples, only a subset of amino acids within a CDR are selected for mutagenesis. In some instances, only one amino acid residue within a CDR is selected for mutagenesis. In other instances, two or more amino acids are selected for mutagenesis.
• The amino acid residues that are selected for further mutagenesis can be modified by any method known to one of skill in the art. The amino acid residues can be modified rationally or can be modified by random mutagenesis. This can be accomplished by modifying the encoding DNA. One of skill in the art is familiar with mutagenesis methods. Mutagenesis methods include, but are not limited to, site-mediated mutagenesis, PCR mutagenesis, cassette mutagenesis, site-directed mutagenesis, random point mutagenesis, mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA, point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, double-strand break repair, and many others known to persons of skill. See, e.g., Arnold (1993) Current Opinion in Biotechnology 4:450-455; Bass et al., (1988) Science 242:240-245; Botstein and Shortie (1985) Science 229:1193-1201; Carter et al., (1985) Nucl. Acids Res. 13: 4431-4443; Carter (1986) Biochem. J. 237:1-7; Carter (1987) Methods in Enzymol. 154: 382-403; Dale et al., (1996) Methods Mol. Biol. 57:369-374; Eghtedarzadeh and Henikoff (1986) Nucl. Acids Res. 14: 5115; Fritz et al., (1988) Nucl. Acids Res. 16: 6987-6999; Grundstrom et al., (1985) Nucl. Acids Res. 13: 3305-3316; Kunkel (1987) “The efficiency of oligonucleotide directed mutagenesis” in Nucleic Acids and Molecular Biology (Eckstein, F. and Lilley, D. M. J. eds., Springer Verlag, Berlin); Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al., (1987) Methods in Enzymol. 154, 367-382; Kramer et al., (1984) Nucl. Acids Res. 12: 9441-9456; Kramer and Fritz (1987) Methods in Enzymol. 154:350-367; Kramer et al., (1984) Cell 38:879-887; Kramer et al., (1988) Nucl. Acids Res. 16: 7207; Ling et al., (1997) Anal Biochem. 254(2): 157-178; Lorimer and Pastan (1995) Nucleic Acids Res. 23, 3067-8; Mandecki (1986) Proc. Natl. Acad. Sci. USA 83:7177-7181; Nakamaye and Eckstein (1986) Nucl. Acids Res. 14: 9679-9698; Nambiar et al., (1984) Science 223: 1299-1301; Sakamar and Khorana (1988) Nucl. Acids Res. 14: 6361-6372; Sayers et al., (1988) Nucl. Acids Res. 16:791-802; Sayers et al., (1988) Nucl. Acids Res. 16:803-814; Sieber et al., (2001) Nature Biotechnology 19:456-460; Smith (1985) Ann. Rev. Genet. 19:423-462; Stemmer (1994) Nature 370, 389-91; Taylor et al., (1985) Nucl. Acids Res. 13: 8749-8764; Taylor et al., (1985) Nucl. Acids Res. 13: 8765-8787; Wells et al., (1986) Phil. Trans. R. Soc. Load. A 317: 415-423; Wells et al. (1985) Gene 34:315-323; Zoller and Smith (1982) Nucleic Acids Res. 10:6487-6500; Zoller and Smith (1983) Methods in Enzymol. 100:468-500; and Zoller and Smith (1987) Methods in Enzymol. 154:329-350. In some examples, the amino acid residues are modified by NNK mutagenesis. In other examples, the amino acid residues are modified by cassette mutagenesis.
• In some examples, selected target amino acid residues can be mutagenized individually such that each mutagenesis is performed by the replacement of a single amino acid residue at only one target position, such that each individual mutant generated is the single product of each single mutagenesis reaction. The single amino acid replacement mutagenesis reactions can be repeated for each of the replacing amino acids selected at each of the target positions in the selected region. Thus, a plurality of mutant protein molecules are produced, whereby each mutant protein contains a single amino acid replacement at only one of the target positions. The mutagenesis can be effected in an addressable array such that the identity of each mutant protein is known. For example, site-directed mutagenesis methods can be used to individually generate mutant proteins.
• In other examples, a mutagenized antibody can be generated that has random amino acids at specific target positions in the variable heavy or light chain Generally, selected target amino acid residues can be mutagenized simultaneously, i.e., one or more amino acid residues are mutagenized at the same time. For example, random mutagenesis methodology can be used such that target amino acids are replaced by all (or a group) of the 20 amino acids. Either single or multiple replacements at different amino acid positions are generated on the same molecule, at the same time. In this approach neither the amino acid position nor the amino acid type are restricted; and every possible mutation is generated and tested. Multiple replacements can randomly happen at the same time on the same molecule. The resulting collection of mutant molecules can be assessed for activity as described below, and those that exhibit binding are identified and sequenced.
• In random mutagenesis methods, it is contemplated that any known method of introducing randomization into a sequence can be utilized. For example, error prone PCR can introduce random mutations into nucleic acid sequences encoding the polypeptide of interest (see, e.g., Hawkins et al., J. Mol. Biol., (1992) 226(3): 889-96). Briefly, PCR is run under conditions which compromise the fidelity of replication, thus introducing random mutations in sequences as those skilled in the art can accomplish.
• Exemplary of a method of introducing randomization into one or more target amino acid positions is the use of a deoxyribonucleotide “doping strategy,” which can cover the introduction of all 20 amino acids while minimizing the number of encoded stop codons. For example, NNK mutagenesis can be employed whereby N can be A, C, G, or T (nominally equimolar) and K is G or T (nominally equimolar). In other examples, NNS mutagenesis can be employed whereby S can be G or C. Thus, NNK or NNS (i) code for all the amino acids, (ii) code for only one stop codon, and (iii) reduce the range of codon bias from 6:1 to 3:1. There are 32 possible codons resulting from the NNK motif: 1 for each of 12 amino acids, 2 for each of 5 amino acids, 3 for each of 3 amino acids, and only one of the three stop codons. Other alternatives include, but are not limited to: NNN which can provide all possible amino acids and all stops; NNY which eliminates all stops and still cover 14 of 20 amino acids; and NNR which covers 14 of 20 amino acids. The third nucleotide position in the codon can be custom engineered using any of the known degenerate mixtures. However, the group NNK, NNN, NNY, NNR, NNS covers the most commonly used doping strategies and the ones used herein.
• Mutagenized proteins are expressed and assessed for activity to the target antigen. Any method known to one of skill in the art to assess activity, for example, as described further herein below in Section E.1, can be used. For example, exemplary binding assays include, but are not limited to immunoassays such as competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, Meso Scale Discovery electrochemiluminescence assays (MSD, Gaithersburg, Md.), immunoprecipitation assays, ELISPOT, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such assays are routine and well known in the art (see, e.g., Ausubel et al., cds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). For example, in the methods provided herein, binding of an antibody to a target antigen is determined using an ECL binding assay. In another example, binding is determined by ELISA.
• Identified mutant antibodies that exhibit improved or increased binding to the target antigen compared to the parent first antibody are identified. The amino acid mutations in the variable heavy or light chain in the identified mutant antibody can be determined. As discussed below, further mutagenesis and iterative screening can be effected on an identified mutant antibody to further optimize the activity for a target antigen. For example, the mutations of all mutant antibodies of a parent first antibody that were identified as exhibiting improved binding for a target antigen can be determined. All or a subset of the identified amino acid mutations can be combined to generate a combination mutant antibody.
• 2. SAR by Scanning Mutagenesis
• Scanning mutagenesis is a simple and widely used technique in the determination of the functional role of protein residues. Scanning mutagenesis can be used in methods of affinity maturation herein to determine SAR of a first antibody. Scanning mutagenesis can be performed on a first antibody without comparison to a related antibody. In other examples, scanning mutagenesis is optionally performed prior to mutagenesis of a target region above in order to more rationally identify amino acif residues to mutate.
• In the scanning mutagenesis methods herein, every residue across the full-length of the variable heavy chain and/or variable light chain of the antibody is replaced by a scanning amino acid. Alternatively, every residue in a region of the variable heavy chain or variable light chain is replaced by a scanning amino acid. For example, at least one CDR (e.g. a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 or CDRL3) is selected for scanning. The scanning amino acid can be any amino acid, but is generally an alanine, theronine, proline or glycine Amino acid substitution is typically effected by site-directed mutagenesis Alanine is generally the substitution residue of choice since it eliminates the side chain beyond the [beta] carbon and yet does not alter the main-chain conformation (as can glycine or proline), nor does it impose extreme electrostatic or steric effects. Generally, all amino acid residues selected for mutageneis are scanned (e.g. mutated to) the same amino acid residue. Often, it is necessary to use other scanning amino acid residues. For example, if the target amino acid residue already is an alanine, then another amino acid residue such as threonine, proline or glycine can be used.
• When performing scanning mutagenesis, all or a subset of amino acids across the full-length polypeptide or in a selected region are targeted for scanning mutagenesis, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues are subjected to scanning mutagenesis. In examples where scanning mutagenesis is performed in addition to comparison to a related antibody, all amino acid residues in a target region, or a subset or amino acid residues in a target region, are scanned. In one example, only the amino acid residues that differ between the first antibody and a related antibody are targeted for scanning mutagenesis. Generally, all amino acid residues in a target region are subjected to scanning mutagenesis. Mutagenized proteins are expressed and assessed for activity to the target antigen as described above and in Section E below.
• Following scanning, scanned (e.g. mutated) antibodies are screened for an activity to identify amino acid residues for further mutation. Generally, most prior art scanning mutagenesis methods involve or are limited to identification of scanned positions that knock down or decrease the activity of the protein of interest. The rationale is that these residues are critical for activity in some way. For purposes of practice of the method herein, however, residues that are “Up” mutants are selected for further mutagenesis following scanning. These are antibodies that exhibit retained or increased activity when mutated to contain a scanned amino acid compared to the parent antibody. Further, only residues with scanned substitutions that are in contact-making CDRs are selected. Thus in an exemplary embodiment, only residues with scanned substitutions that are in contact-making CDRs and that do not affect activity or confer an improvement are selected herein to further mutate individually to other amino acids.
• A benefit of this approach is that generally antibodies that are selected for affinity maturation herein exhibit a micromolar or high nanomolar affinity. Such affinities mean that the antibodies exhibit a low interaction for the target antigen. This is in contrast to many proteins that are typically affinity matured that already are highly evolved for their functional activity. Thus, for antibodies selected for affinity maturation that exhibit a weaker activity for a target antigen, there is more opportunity to improve or optimize weak interactions. Thus, in practicing the method herein, scanned residues that result in an increased or retained activity of the antibody are selected for further mutagenesis. This, allows new interactions to take place, for example, creating new contact residues, that did not exist prior to affinity maturation.
• Thus, in scanning mutagenesis methods herein, selected amino acids are subjected to scanning mutagenesis to identify those amino acid residues that are “Up” mutants (i.e. exhibit retained or increased activity). Further mutagenesis is performed only at scanned amino acid positions that exhibit a retained or an increase in activity to the target antigen compared to the parent antibody. An antibody that retains an activity to a target antigen can exhibit some increase or decrease in binding, but generally exhibits the same binding as the first antibody not containing the scanned mutation, for example, exhibits at least 75% of the binding activity, such as 75% to 120% of the binding, for example, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110% or 115% of the binding. An antibody that exhibits increased activity to a target antigen generally exhibits greater than 115% of the activity, such as greater than 115%, 120%, 130%, 140%, 150%, 200% or more activity than the first antibody not containing the mutation. Thus, scanning mutagenesis can be employed to restrict the subset of target amino acid residues in the target region that are further mutagenized. Once identified, mutagenesis is performed on all or a subset of the amino acid residues as described in Section C.4 above. The further mutagenized antibodies are expressed and assessed for activity to the target antigen as described above and in Section E below. Antibodies that exhibit an improved or optimized activity compared to the first antibody are selected.
• 3. Further Optimization
• The affinity maturation methods provided herein can be performed iteratively to further optimize antibodies. Additionally or alternatively, all or a subset of the amino acid modifications within a variable heavy or light chain that result in improved or increased activity to the target antigen can be selected and combined and further assessed for activity. These intermediate antibodies also can be used as templates for further mutagenesis using the affinity maturation methods herein. In some examples, variable heavy or light chains with one or more amino acid modification(s) incorporated can be used as templates for further mutagenesis and optimization of activity. In addition, further regions of an antibody can be mutagenized.
• The method further provides for optimization of regions of the variable heavy or light chain that were not initially selected for mutagenesis based on the amino acid sequence comparison of the first antibody and related antibodies. An additional region selected for further mutagenesis can occur along any portion of the variable heavy or light chain. For example, a further region can include a CDR or a framework region. Typically, a CDR, for example, CDR1, CDR2 and/or CDR3, is selected and targeted. Any one or more of the regions can be targeted for affinity maturation by mutagenesis. As exemplified in Examples 9 and 12 below, CDRH1 and CDRH2 are selected for additional mutagenesis.
• Additional regions of the variable heavy or light chain can be subjected to further mutagenesis at the same time, or alternatively, they can be mutagenized iteratively. For example, mutations in one region that optimize an activity of the antibody can first be identified by further mutagenesis herein, followed by optimization of a second region. The selection of amino acid residues to mutagenize within a selected target region can be determined by the person practicing the method. In some examples, all amino acids in that region are targeted for mutagenesis. In other examples, only a subset of amino acids in that region are targeted for mutagenesis. In an additional example, scanning mutagenesis is performed to identify residues that increase or retain activity to the target antigen. In such examples, only residues that increase or do not affect binding affinity are further mutagenized to identify mutations that increase binding affinity to the target antigen. Typically, mutagenesis is performed for one or both of the heavy and/or light chain(s) independently of the other. The amino acid residues that are selected for further mutagenesis can be modified by any method known to one of skill in the art. Mutagenized proteins are expressed and assessed for binding to the target antigen. Exemplary binding assays are described in Section E.1 below.
• The amino acid residues in a region that are selected for further mutagenesis can be modified by any method known to one of skill in the art, as described in Sections C.4 and C.5 above. In some examples, the selected amino acids are subjected to scanning mutagenesis to identify “Up” mutants for further mutagenesis. In other examples, the selected amino acids are randomly mutagenized, for example, the amino acid residues are modified by saturation mutagenesis and/or cassette mutagenesis. Mutagenized proteins are expressed and assessed for activity to the target antigen, as described in Sections F and E. Antibodies containing amino acid mutations that increase activity to the target antigen are identified.
• Combination mutants also can be generated. In the methods provided herein, amino acid mutations that result in increased activity of the antibody towards the target antigen can be combined to generate a variable heavy or light chain with multiple amino acid modifications. Typically, combination mutants have 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations per variable heavy and/or light chain. In some examples, combination mutants contain two amino acid modifications. In other examples, combination mutants contain three or more amino acid modifications. As exemplified in Example 9 below, a variable heavy chain is generated containing 4 amino acid mutations.
• In addition, intermediate antibodies containing multiple amino acid modifications within the variable heavy or light chain can be generated at any step in the method. A variable heavy and/or light chain of an intermediate antibody, i.e., one containing multiple previously identified amino acid modifications, can be used as a “template” for further mutagenesis and affinity maturation.
• Further, the method herein provides for pairing of any modified heavy chains with any modified light chains thereby generating intermediate or affinity matured antibodies in which both the heavy and light chains contain mutations. Mutated heavy and light chains can be paired at any step in the method, expressed and assessed for binding to the target antigen. Thus, further optimization of an antibody can be achieved.
• At any step in of further optimization in the methods herein, the affinity matured antibodies can be further evaluated for activity as described in Section E.
• a. Complementarity Determining Regions
• In some examples, a region is selected for further mutagenesis. Generally, a region is a CDR, for example, CDR1, CDR2 and/or CDR3 of the variable heavy or light chain. The amino acid residues within a variable heavy or light chain CDR can be identified by one of skill in the art. CDRs can be identified by any standard definition, including those of Kabat (see, e.g., Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91-3242.); Chothia (see, e.g. Chothia & Lesk, (1987) J Mol Biol. 196(4):901-17; Al-Lazikani et al., (1997) J Mol Biol. 273(4):927-48); Abm (see, e.g., Martin et al., (1989) Proc Natl Acad Sci USA 86:9268-9272); or contact residues based on crystal structure data (see, e.g., MacCalllum et al., (1996) J. Mol. Biol. 262, 732-745). Amino acids contained within heavy and light chain CDRs, as defined based on Kabat numbering, are described in Section C.3. above.
• Typically, a CDR contains 3 to 25 residues, all or part of which can be targeted for further mutagenesis. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid residues can be targeted for mutagenesis. As exemplified in Example 9, only selected residues of CDRH1 were mutagenized whereas in Example 10, all residues within CDRL2 were mutagenized.
• Selected amino acids are subjected to mutagenesis and the antibodies are expressed and assayed for activity to the target antigen as described in sections C.4 above and E. and F. below.
• b. Framework Regions
• In some examples, a region selected for further mutagenesis is part of a framework region, for example, FR1, FR2, FR3 and/or FR4, of the variable heavy or light chain. As is the case for CDRs, framework regions can be identified by any standard definition, according to the numbering of Kabat, Chothia, Abm or contact residues Amino acids that make up the framework regions within the heavy and light chain variable regions as defined based on Kabat numbering are described in Section C.3. above. Typically, a framework region contains 11 to 32 amino acids. All or part of a framework region can be targeted for mutagenesis, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 amino acids can be subjected to full or partial saturation mutagenesis. A selected region with a framework region can include one or more amino acid residues. In some examples, only one amino acid residue is mutagenized. In other examples, two or more amino acid residues are mutagenized. Selected amino acid residues can be mutagenized individually, or alternatively, selected amino acid residues can be mutagenized simultaneously, i.e., one or more amino acid residues are mutagenized at the same time. For example, double mutants are generated and assayed for their ability to bind to the target antigen.
• Selected amino acids are subjected to mutagenesis and the antibodies are expressed and assayed for activity to the target antigen as described in sections C.4 above and E. and F below.
• c. Germline Swapping
• In some examples, a region selected for further mutagenesis is a germline segment, i.e., a variable heavy chain V, D or J segment, or a variable kappa or lambda light chain V or J segment, e.g., V_H, D_H, J_H, V_κ, V_λ, J_κ, and J_λ. In a variable heavy chain, germline segment V_H contains amino acids within CDR1 and CDR2 while germline segments D_H and J_H contain amino acid residues within CDR3. In a variable light chain, V germline segments (e.g., V_κ or V_λ) contain amino acid residues within CDR1, CDR2 and the 5′ end of CDR3 while J germline segments (e.g., V_λ and J_κ) contain amino acid residues at the 3′ end of CDR3. When a germline segment is targeted for mutagenesis, amino acid modifications are introduced into a variable heavy or light chain by swapping, or replacing, an entire germline segment with another germline segment of the same type. For example, a J_H germline segment, e.g., IGHJ1*01, is replaced with a different J_H germline segment, e.g., IGHJ2*01, or any other IGHJ germline segment. As exemplified in Example 13A and FIG. 4A, swapping of IGHJ1*01 allows for simultaneous mutation of 6 amino acid residues within heavy chain CDR3 and a seventh residue within framework region 4. One germline segment is swapped, such as, for example, J_H, or alternatively, two germline segments can be swapped, for example, both D_H and J_H can be swapped within one variable heavy chain.
• Typically, a D or J germline segment is selected for mutagenesis since these germline segments encode for CDR3 of both the heavy and light chain. More specifically, germline segments D_H, J_H, J_κ, and/or J_λ are selected. As exemplified in Example 13B, swapping of both D_H and J_H segments leads to an almost complete scan of heavy chain CDR3. As shown in FIG. 4B, germline segment J_H is swapped with three different J_H segments serving to mutate 6 amino acids at the 3′ end of CDRH3 and as shown in FIG. 4C, 5 amino acids within the middle of CDRH3 are modified.
• Germline swapped antibodies are expressed and assayed for activity to the target antigen as described in section E. and F below. Antibodies containing swapped germline segments that increase activity to the target antigen can be used as intermediate antibodies for further modifications, as described in this section herein.
D. METHOD OF ANTIBODY CONVERSION
• Provided herein is a method of antibody conversion. The method is based on the elucidation that antibodies with varying affinities, while maintaining their specificity to a target antigen, can exhibit a range of activities ranging from agonist or activator-modulator activity to antagonist activity for the same target antigen. As described herein, the pharmacologic activity of antibodies is dependent on their affinity, with qualitatively different activities (activations vs. inhibition) occurring in antibodies recognizing the same epitope but with disparate affinities. It is contemplated herein that activation of an activity is due to the enhancement of signaling through receptor clustering and rapid on/off kinetics of the low affinity variant. In contrast, high affinity binders grab on to their ligand and do not let go, thereby preventing transmission of a signal. Thus, an antibody can have a therapeutic benefit as a low affinity agonist or activator-modulator or as a high affinity antagonist of the same target antigen.
• Nearly all antibodies in clinical use are high-affinity antagonists, despite the fact that multiple mechanisms of action are typically seen for several classes of small molecule drugs. For example, small molecule drugs have several mechanisms of action, including acting as antagonists, agonists, partial agonists or antagonists and modulators. In contrast, most antibody therapeutics act as antagonists. The discovery selection mechanisms in hybridoma and display-based systems drive affinity and dominant epitope binding. Thus, most methods of antibody engineering exhibit affinity-based bias. This is because most existing display-based libraries select antibodies based on the ability to rapidly identify high-affinity binders. For example, most methods rely on competitive selection based on target affinity. Thus, most existing methods, for example, traditional display-based methods that rely on competitive affinity screens can miss potential therapeutics simply because they are incompatible with high affinity.
• Thus, provided herein are methods of antibody conversion, whereby antibodies are converted from antagonists to partial agonists, antagonists or activators-modulators, or can be converted from agonists or activators-modulators to antagonists or partial antagonists. The method is based on converting antibodies by modulating or altering the binding affinity of an antibody for the same target antigen in order to get a range of activities from antagonism, partial antagonism or activation-modulation. The methods combine mutagenesis approaches of a starting antibody with endpoint analysis for binding affinity and functional activity assessment of resulting activities. By employing random or rational mutagenesis strategies, libraries can be generated that can be screened through a wide dynamic range of affinities to identify antibodies with antagonist, partial antagonist or activator/modulator activities. In some examples, the libraries are in arrayed formats such that the identity of each member in the library is known. In another example, a structure/activity relationship (SAR) mutagensis strategy can be employed similar to the affinity maturation method described in Section C.
• 1. Choosing the Starting or Reference Antibody
• In the method, a starting or reference antibody, or portion thereof, to be converted is chosen. The antibody that is chosen is one that 1) exhibits a known activity against a particular target antigen (e.g. antagonist or agonist), and 2) for which there would be a potential therapeutic benefit if the activity of the antibody was inversed or partially inversed. For example, an antibody that exhibits an antagonist or partial antagonist activity can be chosen, whereby an antibody exhibiting the inverse agonist, partial agonist or activator-modulator activity towards the same target antigen also is desired. In another example, an antibody that exhibits an agonist, partial agonist or activator-modulator activity towards a target antigen can be chosen, whereby an antibody exhibiting the inverse antagonist or partial antagonist activity towards the same target antigen also is desired.
• The first or starting antibody is an antibody that is known or that is identified as having an activity to a target antigen. The target antigen can be a polypeptide, carbohydrate, lipid, nucleic acid or a small molecule (e.g. neurotransmitter). The antibody can exhibit activity for the antigen expressed on the surface of a virus, bacterial, tumor or other cell, or exhibits an activity (e.g. binding) for the purified antigen. Generally, the target antigen is a protein that is a target for a therapeutic intervention. Exemplary target antigens include, but are not limited to, targets involved in cell proliferation and differentiation, cell migration, apoptosis and angiogenesis. Such targets include, but are not limited to, growth factors, cytokines, lymphocytic antigens, other cellular activators and receptors thereof. Exemplary of such targets include, membrane bound receptors, such as cell surface receptors, including, but are not limited to, a VEGFR-1, VEGFR-2, VEGFR-3 (vascular endothelial growth factor receptors 1, 2, and 3), a epidermal growth factor receptor (EGFR), ErbB-2, ErbB-b3, IGF-R1, C-Met (also known as hepatocyte growth factor receptor; HGFR), DLL4, DDR1 (discoidin domain receptor), KIT (receptor for c-kit), FGFR1, FGFR2, FGFR4 (fibroblast growth factor receptors 1, 2, and 4), RON (recepteur d'origine nantais; also known as macrophage stimulating 1 receptor), TEK (endothelial-specific receptor tyrosine kinase), TIE (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains receptor), CSF1R (colony stimulating factor 1 receptor), PDGFRB (platelet-derived growth factor receptor B), EPHA1, EPHA2, EPHB1 (erythropoietin-producing hepatocellular receptor A1, A2 and B1), TNF-R1, TNF-R2, HVEM, LT-βR, CD20, CD3, CD25, NOTCH, G-CSF-R, GM-CSF-R and EPO-R. Other targets include membrane-bound proteins such as selected from among a cadherin, integrin, CD52 or CD44. Exemplary ligands that can be targets, include, but are not limited to, VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF, EGF, HGF, TNF-α, LIGHT, BTLA, lymphotoxin (LT), IgE, G-CSF, GM-CSF and EPO.
• The first or starting antibody that has activity for the target antigen is known in the art or is identified as having a particular activity for a target antigen or antigens. For example, any method for identifying or selecting antibodies against particular target antigens can be used to choose or select a starting antibody including, but not limited to, immunization and hybridoma screening approaches, display library screening methods (e.g. antibody phage display libraries), or addressable combinatorial antibody libraries. For example, methods of identifying antibodies with particular activities or affinities is described in Section B.2 herein. Further, it is understood that the description of the methods for choosing or selecting a first or starting antibody described for the affinity maturation method herein in Section C.1, and in particular in section C.1.ai and ii, can also be used choose or select a first antibody to be converted in the antibody conversion method herein. In addition, any antibody that has been affinity matured, and which, typically, exhibits antagonist activity, can be selected as the starting or first antibody. As discussed elsewhere herein, affinity maturation methods are known in the art (see e.g. Section B.3). Also, the affinity maturation method described in Section C also can be used to identify an antibody, generally one with high affinity, that can be subsequently used in the conversion method herein.
• If not known, the activity of a first or starting antibody can be determined. The binding affinity and/or functional activity (e.g. as an agonist, antagonist or activator-modulator) can be determined. Exemplary assays are described herein in Section E and in the Examples. The particular assay chosen depends on the target antigen and/or its requirements for activity. For example, DLL4 is a cell-surface ligand that activates the Notch1 receptor, also expressed on the cell surface. Thus, typically, cell-based assays are employed to assess activity. Exemplary of cell-based assays are reporter assays as described herein and in the Examples. Based on the descriptions herein, it is within the level of one of skill in the art to determine and or optimize a particular assay for each antibody.
• 2. Mutagenesis
• Once a first or starting antibody is chosen, amino acid residues in the variable heavy chain and/or variable light chain are subjected to mutagenesis. Generally, amino acid residues in a CDR or CDRs are mutated, for example, residues in CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and/or CDRH3 of the antibody are mutated. For example, typically, a CDR can contain 3 to 25 amino acid residues. All or subset of the amino acids within a CDR can be targeted for mutagenesis, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid residues can be targeted for mutagenesis.
• The amino acid residues that are selected for further mutagenesis can be modified by any method known to one of skill in the art. The amino acid residues can be modified rationally or can be modified by random mutagenesis. This can be accomplished by modifying the encoding DNA. One of skill in the art is familiar with mutagenesis methods. For example, any of the mutagenesis methods described in Section C.1.d can be used. In one example, if residues in the first or starting antibody are known that are involved in binding, those residues can be rationally targeted by any of a variety of mutagenesis strategies. In another example, random mutagenesis methods can be employed. Exemplary of such mutagenesis strategies introduce randomization into a sequence using methods know in the art, including but not limited to, error prone PCR or doping strategies. Mutagenized proteins are expressed as described in Section F. Libraries or collections of variant antibodies can be generated and screened for conversion as described herein below. In some examples, the libraries are addressable libraries.
• 3. Selecting for a Converted Antibody
• Mutagenized proteins are expressed and assessed for their binding affinity to the target antigen and/or for effects on modulation of a functional activity towards the target antigen. Converted antibodies are selected for that have a binding affinity and activity that is inversed (e.g. higher or lower; antagonist vs. agonist/activator-modulator) compared to the starting of first antibody.
• a. Binding
• In the first step of selection of a converted antibody, binding affinity is assessed. Any method known to one of skill in the art to assess activity, for example, as described further herein below in Section E.1, can be used. For example, exemplary binding assays include, but are not limited to immunoassays such as competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, Meso Scale Discovery electrochemiluminescence assays (MSD, Gaithersburg, Md.), immunoprecipitation assays, ELISPOT, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). For example, in the methods provided herein, binding of an antibody to a target antigen is determined using an ECL binding assay. In another example, binding is determined by ELISA. As discussed elsewhere herein, comparison of binding affinities between a first antibody and a mutagenized antibody are typically made between antibodies that have the same structure, e.g. Fab compared to Fab of IgG compated to IgG.
• For example, if an antagonist antibody is chosen as the first or starting antibody, an agonist, partial agonist or activator-modulator is selected by first testing the antibody for its binding affinity. Antibodies that exhibit a decreased binding affinity (e.g. higher binding affinity) than the first or starting antibody are selected. For example, antibodies are selected that exhibit a binding affinity that is decreased by 2-fold to 5000-fold, for example, 10-fold to 5000-fold, such as 100-fold to 1000-fold. For example, if the binding affinity of the first or starting antibody is 10⁻⁹ M, and antibody exhibiting a binding affinity of 10⁻⁷ M exhibits a 1000-fold decreased binding affinity.
• In another example, if an agonist, partial agonist, or an activator-modulator antibody is chosen as the first or starting antibody, an antagonist or partial antagonist antibody is selected by first testing the antibody for its binding affinity. Antibodies that exhibit an enhanced or increased binding affinity (e.g. lower binding affinity) then the first or starting antibody are selected. For example, antibodies are selected that exhibit a binding affinity that is enhanced or increased by 10-fold to 10,000 fold, for example, 100-fold to 5000-fold, such as about 500-fold to 2500-fold. For example, if the binding affinity of the first or starting antibody is 10⁻⁷ M, an antibody exhibiting a binding affinity of 10⁻⁹ M is selected as exhibiting a 1000-fold increased or enhanced binding affinity.
• b. Functional Activity
• Mutagenized antibodies initially selected based on binding affinity are then selected for the inversed modulation of a functional activity. Assays to assess the functional activities are well known to those of skill in the art and can be empirically determined depending on the particular target protein. Typically, the assay is a cell-based assay. Exemplary assays, including exemplary cell lines, are described herein in Section E. The cells to be assayed express the particular target protein of interest. Control cells not expressing the protein also can be used to assess specificity. The assay that is employed is one that is capable of providing a read-out that that provides a quantitative assessment of activity, which can be readily assessed. For example, exemplary functional assays include reporter assays, whereby upon activation of a cell-surface receptor, for example by an exogenously added ligand, a reporter signal is induced that can be measured. In the presence of an antagonist or partial antagonist antibody to the cell-surface receptor or ligand, the measured read-out is decreased consistent with the inhibitory effect of the antibody. In contrast, in the presence of an agonist, partial agonist or activator-modulator, the measured read-out is increased consistent with an activating effect of the antibody.
• For example, if the starting or first antibody is an antagonist of a target protein, mutant antibodies of the first antibody that are initially selected as having decreased binding affinity in a) above (e.g. higher binding affinity), are further tested for activity as an agonist, partial agonist and/or activator-modulator for the same target protein. Antibodies selected as being converted are those that exhibit an activating activity on the target protein. Thus, the presence of the antibody results in increased activity of the target protein, or on the end-point activity of the target protein, compared to the activity that is exhibited under the same activating conditions without the antibody present. For example, if a target protein is normally activated in the presence of a ligand, a set measured activity is achieved; in the additional presence of an agonist, partial agonist or activator-modulator antibody, the measured activity is increased. In another example, if the target protein is a ligand that normally activates a receptor, the ligand-receptor interaction results in a set measured activity; in the additional presence of an antibody to the ligand the measured activity is increased. For example, activity of the target protein is increased by 1.2 to 2-fold, 2-fold to 1000-fold, for example, is increased 5-fold to 500-fold, such as 10-fold to 200-fold, for example, is increased 1.2-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold or more compared to the activity of the target protein under the same activating conditions without the antibody present.
• In another example, if the starting or first antibody is an agonist, partial agonist or activator-modulator of a target protein, mutant antibodies of the first antibody that are initially selected as having increased or enhanced binding affinity in a) above (e.g. lower binding affinity), are further tested for activity as an antagonist or partial antagonist for the same target protein. Antibodies selected as being converted are those that exhibit an inhibitory activity on the target protein. Thus, the presence of the antibody results in decreased activity of the target protein, or on the end-point activity of the target protein, compared to the activity that is exhibited under the same activating conditions without the antibody present. For example, if a target protein is normally activated in the presence of a ligand, a set measured activity is achieved; in the additional presence of an antagonist or partial antagonist antibody, the measured activity is decreased. In another example, if the target protein is a ligand that normally activates a receptor, the ligand-receptor interaction results in a set measured activity; in the additional presence of an antibody to the ligand the measured activity is decreased. For example, activity of the target protein is decreased by 1.2 to 2-fold, 2-fold to 1000-fold, for example, is decreased by 5-fold to 500-fold, such as 10-fold to 200-fold, for example, is deceased 1.2-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold or more compared to the activity of the target protein under the same activating conditions without the antibody present.
• In some examples of the antibody conversion method herein, the initial step of selecting an antibody based on an increased or decreased binding affinity is not performed. Hence, the method of antibody conversion herein can be effected directly by choosing a first or starting antibody as described herein, mutagenizing it as described herein, and directly testing the collection of mutant antibodies for an inverse functional activity of the first or starting antibody. Converted antibodies are selected that exhibit the inverse activity.
• In practicing the method provided herein, typically only the variable heavy chain and/or variable light chain of the antibody is subjected to mutagenesis. The ultimate antibody that is selected typically at least contains a variable heavy chain and a variable light chain, or portion thereof sufficient to form an antigen binding site. It is understood, however, that the antibody also can include all or a portion of the constant heavy chain (e.g. one or more CH domains, such as CH1, CH2, CH3 and CH4, and/or a constant light chain (CL)). Hence, the antibody can include those that are full-length antibodies, and also include fragments or portions thereof including, for example, Fab, Fab′, F(ab′)₂, single-chain Fvs (scFv), Fv, dsFv, diabody, Fd and Fd′ fragments, Fab fragments, scFv fragments, and scFab fragments. It also is understood that once the antibody is converted as provided herein, the resulting antibody can be produced as a full-length antibody or a fragment thereof, such as a Fab, Fab′, F(ab′)₂, single-chain Fvs (scFv), Fv, dsFv, diabody, Fd and Fd′ fragments, Fab fragments, scFv fragments, and scFab fragments. Further, the constant region of any isotype can be used in the generation of full or partial antibody fragments, including IgG, IgM, IgA, IgD and IgE constant regions. Such constant regions can be obtained from any human or animal species. It is understood that activities and binding affinities can differ depending on the structure of an antibody, although it is not expected that an activity as, for example an agonist or antagonist, will substantially change. For example, generally a bivalent antibody, for example a bivalent F(ab′)₂ fragment or full-length IgG, has a better binding affinity then a monovalent Fab antibody. As a result, where a Fab has a specified binding affinity for a particular target, it is excepted that the binding affinity is even greater for a full-length IgG that is bivalent.
• The resulting converted antibodies are candidate therapeutics. Exemplary of practice of the method is described herein in the Examples. For example, Example 19 shows that two different anti-DLL4 germline antibodies, having low affinity for DLL4, exhibited agonist activity. Mutagenesis of each of the antibodies by the affinity maturation method described herein resulted in conversion of the antibodies to antagonist antibodies with higher affinity for the same target antigen.
E. ASSAYS
• Antibodies produced in the methods herein can be assessed for their activity towards the target antigen. Antibodies can be screened to identify mutant or modified antibodies that have improved binding affinity or that alter or modulate (increase or decrease) an activity of a target. Typically, the methods herein includes screening or testing antibodies for their binding to a target antigen. Other activities also can be assayed for, including but not limited to cytotoxicity, differentiation or proliferation of cells, cell migration, apoptosis, angiogenesis and alteration of gene expression.
• 1. Binding Assays
• The antibodies provided herein can be screened for their ability to bind a selected target by any method known to one of skill in the art. Exemplary target antigens are described in Section C.1. Binding assays can be performed in solution, suspension or on a solid support. For example, target antigens can be immobilized to a solid support (e.g. a carbon or plastic surface or chip) and contacted with antibody. Unbound antibody or target protein can be washed away and bound complexes can then be detected. Binding assays can be performed under conditions to reduce nonspecific binding, such as by using a high ionic strength buffer (e.g. 0.3-0.4 M NaCl) with nonionic detergent (e.g. 0.1% Triton X-100 or Tween 20) and/or blocking proteins (e.g. bovine serum albumin or gelatin). Negative controls also can be including in such assays as a measure of background binding. Binding affinities can be determined using Scatchard analysis (Munson et al., Anal. Biochem., 107:220 (1980)), BIACore or other methods known to one of skill in the art.
• Exemplary binding assays include, but are not limited to immunoassays such as competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, Meso Scale Discovery (MSD, Gaithersburg, Md.), immunoprecipitation assays, ELISPOT, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Other assay formats include liposome immunoassays (LIA), which use liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques (see Monroe et al., (1986) Amer. Clin. Prod. Rev. 5:34-41).
• Generally, binding is detected using a detectable moiety or label (e.g. an enzyme, a radionuclide, a fluorescent probe, electrochemiluminescent label, or a color dye) typically attached to the target or, if desired, directly to the antibody members in the library. Alternatively, binding can be detected by a further third reagent that itself is labeled or detectable. For example, detection of an antibody bound to a target protein can be achieved using a labeled capture molecule in a sandwich assay format. Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G also can be used as the label agent. These proteins exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, e.g., Kronval et al., (1973) J. Immunol. 111:1401-1406; Akerstrom et al., (1985) J. Immunol. 135:2589-2542). The detection agent can be modified with a detectable moiety, such as biotin, to which another molecule can specifically bind, such as streptavidin. A variety of detectable moieties are well known to those skilled in the art.
• The choice of label or detectable group used in the assay is not critical, as long as it does not significantly interfere with the specific binding of the antibody used in the assay. Generally, the choice depends on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions. One of skill in the art is familiar with labels and can identify a detectable label suitable for and compatible with the assay employed.
• The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field of immunoassays and, in general, most any label useful in such methods can be applied herein. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels include magnetic beads (e.g., DYNABEADS™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3H, 125I, 35S, 14C, or 32P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), chemiluminescent labels (luciferin and 2,3-dihydrophtahlazinediones, e.g., luminol), and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.). For a review of various labeling or signal producing systems that can be used, see e.g. U.S. Pat. No. 4,391,904.
• Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it can be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence can be detected visually, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels can be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Finally simple colorimetric labels can be detected simply by observing the color associated with the label.
• Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies. In this case, antigen-coated particles are agglutinated by samples containing the target antibodies. In this format, none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.
• Alternatively, the antibodies provided herein can be screened for their ability to bind to cells, using whole cell panning, with or without subtractive panning. Screening can be done against live cells or against intact, mildly fixed target cells. Methods for whole cell panning have been described previously (see e.g. Siegel et al. (1997) J. Immunol. Methods 206:73-85 incorporated herein by reference). Other techniques for screening which can be applied include fluorescent activated cell sorting (FACS).
• For high-throughput screening, assays can be multiplexed. Thus, the binding affinities of antibodies to a number of different target proteins can be determined at once. In one example, different target proteins can be separately labeled with different detectable moieities. For example, different antigens can be coupled to color-coded beads (Schwenk et al. (2007) Mol. Cell. Prot., 6:125-132). In another example, multi-spot plates can be used that permit assay multiplexing by absorption of up to 100 proteins in a locus of the plate (e.g. using Multi-Array or Multi-Spot plates from Meso Scale Discovery; MSD, Gaithersburg, Md.). In such an example, antibodies can be screened by addition of a different antibody to each well of a multi-spot plate. The assay readily permits the screening of thousands of antibodies at once against numerous target proteins.
• In the methods of screening herein, antibodies generally are identified that specifically bind to a target antigen, and that have an increased binding affinity compared to a first antibody. The increase in affinity, measured as decrease in Kd, can be achieved either through an increase in association rate (k_on), or a reduction in dissociation rate k_off; or both. For example, the binding affinity of the antibodies is determined to identify or select antibodies that have high affinity for a target protein. For example, the affinity matured antibodies generated by practice of the method can have a binding affinity for a target antigen that is 1×10⁻⁹ M or less, generally 1×10⁻⁹ M to 1×10⁻¹¹ M, for example that is or is about 1×10⁻⁹ M, 2×10⁻⁹ M, 3×10⁻⁹ M, 4×10⁻⁹ M, 5×10⁻⁹ M, 6×10⁻⁹ M, 7×10⁻⁹ M, 8×10⁻⁹ M, 9×10⁻⁹ M, 1×10⁻¹⁰ M, 2×10⁻¹⁰ M, 3×10⁻¹⁰ M, 4×10⁻¹⁰ M, 5×10⁻¹⁰ M, 6×10⁻¹⁰ M, 7×10⁻¹⁰ M, 8×10⁻¹⁰ M, 9×10⁻¹⁰ M or less.
• Any method known to one of skill in the art can be used to measure the binding affinity of an antibody. For example, the binding properties of an antibody can be assessed by performing a saturation binding assay, for example, a saturation ELISA, whereby binding to a target protein is assessed with increasing amounts of antibody. In such experiments, it is possible to assess whether the binding is dose-dependent and/or saturable. In addition, the binding affinity can be extrapolated from the 50% binding signal. Typically, apparent binding affinity is measured in terms of its association constant (Ka) or dissociation constant (Kd) and determined using Scatchard analysis (Munson et al., Anal. Biochem., 107:220 (1980). For example, binding affinity to a target protein can be assessed in a competition binding assay in where increasing concentrations of unlabeled protein is added, such as by radioimmunoassay (RIA) or ELISA. Binding affinity also can be analyzed using BIAcore kinetic analysis. This involves analyzing the binding and dissociation of an antibody member from chips containing immobilized target proteins on their surface. The Biacore evaluation software generates the values of Ka and Kd by fitting the data to interaction models. It is understood that the binding affinity of an antibody can vary depending on the assay and conditions employed, although all assays for binding affinity provide a rough approximation. By performing various assays under various conditions it is possible to estimate the binding affinity of an antibody.
• In addition, binding affinities can differ depending on the structure of an antibody. For example, generally a bivalent antibody, for example a bivalent F(ab′)2 fragment or full-length IgG, has a better binding affinity then a monovalent Fab antibody. Hence, it is understood that where a Fab has a specified binding affinity for a particular target, it is excepted that the binding affinity is even greater for a full-length IgG that is bivalent.
• 2. Functional Activity
• The antibodies generated by the method herein can be screened for their ability to modulate the functional activity of a target by any method known to one of skill in the art. Assays can be designed to identify antibodies capable of binding and/or modulating cell surface receptors. Such antibodies can either be agonists, mimicking the normal effects of receptor binding, or antagonists, inhibiting the normal effects of receptor binding. Of particular interest is the identification of agents which bind to the receptors and modulate intracellular signaling.
• In some example, such assays are cell-based assays. Generally, assays are performed using cell lines known to express the target of interest. Such cells are known to one of skill in the art. For example, one can consult the ATCC Catalog (atcc.org) to identify cell lines. Also, if a particular cell type is desired, the means for obtaining such cells, and/or their instantly available source is known to those in the art. An analysis of the scientific literature can readily reveal appropriate choice of cells expressing any desired target. Table 5 lists exemplary cells lines that express targets of interest that can be screened in functional activities herein against antibody libraries provided herein.

• TABLE 5
  Cell lines expressing targets

  Target	Cell Lines	References
  GP IIb/IIIa	MEG-01 chronic	Ogura et al. Establishment of a novel human
  	myelogenous leukemia	megakaryoblastic leukemia cell line, MEG-01, with positive
  	megakaryoblast cells	Philadelphia chromosome. Blood 66: 1384-1392, 1985;
  	(ATCC CRL-2021);	Komatsu et al. Establishment and Characterization of a
  	UT-7 human leukemia	Human Leukemic Cell Line with Megakaryocytic Features:
  	cell ine	Dependency on Granulocyte-Macrophage Colony-
  		stimulating Factor, Interleukin 3, or Erythropoietin for
  		Growth and Survival. Cancer Research 51: 341-348 (1991)
  GM-CSF-R	VA-ES-BJ epitheloid	Int J Oncol 1995; 7: 51-56; Ali Habib et al. A urokinase-
  	sarcoma cells (ATCC	activated recombinant diphtheria toxin targeting the
  	CRL-2138);	granulocyte-macrophage colony-stimulating factor receptor
  	TF1-HaRas;	is selectively cytotoxic to human acute myeloid leukemia
  	TF1-vRaf;	blasts. Blood 104(7): 2143-2148 (2004); Kiser et al.
  	TF1-vSrc;	Oncogene-dependent engraftment of human myeloid
  	HL-60 (ATCC CCL-	leukemia cells in immunosuppressed mice. Leukemia
  	240);	15(5): 814-818 (2001)
  	U-937 (ATCC CRL-
  	1593.2);
  	ML-2
  VEGFA	Human A673	Gerber et al. Complete inhibition of rhabdomyosarcoma
  	rhabdomyosarcoma cells	xenograft growth and neovascularization requires blockade
  	(ATCC CRL-1598);	of both tumor and host vascular endothelial growth factor.
  	Breast carcinoma MDA-	Cancer Res. 60(22): 6253-8 (2000); Presta et al.
  	MB-435 cells (ATCC);	Humanization of an anti-vascular endothelial growth factor
  	Bovine adrenal cortex-	monoclonal antibody for the therapy of solid tumors and
  	derived capillary	other disorders. Cancer Research, 57(20): 4593-4599 (1997)
  	endothelial cells
  CD3	Jurkat E6.1 Human	Buhler et al. A bispecific diabody directed against prostate-
  	leukemic T cell	specific membrane antigen and CD3 induces T-cell
  	lymphoblast (Sigma	mediated lysis of prostate cancer cells. Cancer Immunol
  	Aldrich 88042803)	Immunother. 57(1): 43-52 (2008)
  EGFR	DiFi human colorectal	Olive et al. Characterization of the DiFi rectal carcinoma
  	carcinoma cells;	cell line derived from a familial adenomatous polyposis
  	A431 cells (ATCC CRL-	patient. In Vitro Cell Dev Biol. 29A(3 Pt 1): 239-248 (1993);
  	1555);	Wu et al. Apoptosis induced by an anti-epidermal growth
  	Caco-2 colorectal	factor receptor monoclonal antibody in a human colorectal
  	adenocarcinoma cells	carcinoma cell line and its delay by insulin. Clin. Invest.
  	(ATCC HTB-37);	95(4): 1897-1905 (1995)
  	HRT-18 colorectal
  	adenocarcinoma cells
  	(ATCC CCL-244);
  	HT-29 colorectal
  	adenocarcinoma cells
  	(ATCC HTB-38)
  EPO	A2780 ovarian cancer	Jeong et al. Characterization of erythropoietin receptor and
  receptor	cells;	erythropoietin expression and function in human ovarian
  	UT-7 human leukemia	cancer cells. Int J Cancer. 122(2): 274-280 (2008); Elliott et
  	cell ine	al. Activation of the Erythropoietin (EPO) Receptor by
  		Bivalent Anti-EPO Receptor Antibodies. J Biol Chem.
  		271(40): 24691-24697 (1996)
  Her2/Neu	BT-474 ductal	Le et al. Roles of human epidermal growth factor receptor 2,
  receptor	carcinoma breast cancer	c-jun NH2-terminal kinase, phosphoinositide 3-kinase, and
  	cell (ATCC HTB-20);	p70 S6 kinase pathways in regulation of cyclin G2
  	SK-BR-3	expression in human breast cancer cells. Mol Cancer Ther.
  	adenocarcinoma breast	6(11): 2843-2857 (2007)
  	cancer cell (ATCC HTB-
  	30);
  	MDA-MB-453
  	metastatic carcinoma cell
  	line (ATCC HTB-131)
  cMet	H1993 lung	Ma et al. Functional expression and mutations of c-Met and
  	adenocarcinoma cells	its therapeutic inhibition with SU11274 and small interfering
  	(ATCC CRL-5909);	RNA in non-small cell lung cancer. Cancer Res. 65(4): 1479-1488
  	H1838 lung	(2005);
  	adenocarcinoma cells	Ma et al. A selective small molecule c-MET Inhibitor,
  	(ATCC CRL-5899);	PHA665752, cooperates with rapamycin. Clin Cancer Res
  	SW 900 lung squamous	11(6): 2312-2319 (2005)
  	cell carcinoma cells
  	(ATCC HTB-59);
  	H358 lung
  	bronchioalveolar
  	carcinoma cells (ATCC
  	CRL-5807);
  	SK-Lu-1 lung
  	adenocarcinoma cells
  	(ATCC HTB-57);
  	H441 Non-small cell
  	lung cancer cells (ATCC
  	HTB-174)
  CD20	Ramos Burkitt's	Jazirehi et al. Rituximab (anti-CD20) selectively modifies
  	lymphoma B cells	Bcl-xL and apoptosis protease activating factor-1 (Apaf-1)
  	(ATCC CRL-1596);	expression and sensitizes human non-Hodgkin's lymphoma
  	Raji Burkitt's lymphoma	B cell lines to paclitaxel-induced apoptosis. Mol Cancer
  	B cells (ATCC CCL-86):	Ther. 2(11): 1183-1193 (2003)
  	Daudi Burkitt's
  	lymphoma B cells
  	(ATCC CCL-213);
  	2F7 Burkitt's lymphoma
  	B cells

• In addition, cells lines expressing a target of interest can be generated by transient or stable transfection with an expression vector expressing a target of interest. Methods of transfection and expression are known to those of skill in the art (see e.g., Kaufman R. J. (1990) Methods in Enzymology 185:537-566; Kaufman et al. (1990) Methods in Enzymology 185:537-566). In addition, any primary cell or cell line can be assessed for expression of a particular target (e.g. cell surface marker). Cell surface markers can be assayed using fluorescently labeled antibodies and FACS. Suitable cell lines include A549 (lung), HeLa, Jurkat, BJAB, Colo205, H1299, MCF7, MDA-MB-231, PC3, HUMEC, HUVEC, and PrEC.
• Any suitable functional effect can be measured, as described herein. For example, cellular morphology (e.g., cell volume, nuclear volume, cell perimeter, and nuclear perimeter), ligand binding, substrate binding, nuclease activity, apoptosis, chemotaxis or cell migrations, cell surface marker expression, cellular proliferation, GFP positivity and dye dilution assays (e.g., cell tracker assays with dyes that bind to cell membranes), DNA synthesis assays (e.g., 3H-thymidine and fluorescent DNA-binding dyes such as BrdU or Hoechst dye with FACS analysis) and nuclear foci assays, are all suitable assays to identify potential modulators using a cell based system. Other functional activities that can be measured include, but are not limited to, ligand binding, substrate binding, endonuclease and/or exonuclease activity, transcriptional changes to both known and uncharacterized genetic markers (e.g., northern blots), changes in cell metabolism, changes related to cellular proliferation, cell surface marker expression, DNA synthesis, marker and dye dilution assays (e.g., GFP and cell tracker assays), contact inhibition, tumor growth in nude mice, and others.
• For example, antibodies generated by the method provided herein can be assessed for their modulation of one or more phenotypes of a cell known to express a target protein. Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to screen antibody libraries. Representative phenotypic assays, which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assays including enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, Calif.; Amersham Biosciences, Piscataway, N.J.).
• Cells determined to be appropriate for a particular phenotypic assay (i.e., A549, HeLa, Jurkat, BJAB, Colo205, H1299, MCF7, MDA-MB-231, PC3, HUMEC, HUVEC, and PrEC and any others known to express the target of interest) are treated with antibodies as well as control compounds. If necessary, a ligand for the receptor target is included so that activation of the receptor is effected. At the end of the treatment period, treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.
• Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest.
• The assays can be performed to assess the direct effects of an antibody on a target protein. For example, if the target protein is a cell surface receptor, an antibody can be added to assess whether the target protein directly modulates, such as by stimulation, the activity or function of the receptor. In such instances, the antibody is deemed an agonist antibody. In other examples, if the target protein is a cell surface receptor, the activity of the receptor can be stimulated in the presence of a ligand or other stimulating agent in the presence or absence of the antibody to determine if the antibody modulates (e.g. inhibits) the actions of the antibody. For example, the antibody can act by blocking the ability of the ligand to interact with the receptor and/or otherwise induce a negative stimulatory signal. In such instances, the antibody is deemed to be an antagonist of the receptor. Thus, the methods of screening herein by functional activity permits identification of agonist and antagonist antibodies.
• a. Differentiation
• Cellular differentiation can be analyzed using any assay that allows a detection of a physical, chemical or phenotypic change. Various assays are used to quantitatively determine cellular proliferation and activation in response to an external stimuli. Cell proliferation assays are used to quantitatively determine cellular proliferation by incorporating a reagent into the DNA of newly synthesized cells upon cell division. Such reagents include, but are not limited to ³H-thymidine, 5-bromo-2′-deoxyuridine (BrdU) and fluorescent Hoechst dyes. Cell viability assays are used to determine the number of healthy cells in a sample by staining cells with a dye and measuring how many cells uptake the dye based on the fact that living cells will exclude the dye. Such dyes include but are not limited to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MIT), 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT), and 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1). Uptake of the reagent is measured either colorimetrically using a spectrophotometer or by measuring radiation with a scintillation counter. Details of these methods are well-known to one skilled in the art.
• Fluorescent dyes are commonly used for the detection of live cells and key functional activities in a variety of cell-based assays. There are several non-radioactive, fluorescence-based assays that are not dependent on cellular metabolism. The fluorescent dye binds nucleic acids and the fluorescence can then be measured quantitatively or qualitatively. Such dyes include, but are not limited to, propidium iodide and Hoechst 33342. The cell number can then be quantitated based on the fluorescence. DNA content can also be quantitated using the tools available in the imaging instruments. Details of these methods are well known to one skilled in the art.
• The degree of invasiveness into Matrigel or some other extracellular matrix constituent can be used as an assay to identify antibodies that are capable of inhibiting abnormal cell proliferation and tumor growth. Tumor cells exhibit a good correlation between malignancy and invasiveness of cells into Matrigel or some other extracellular matrix constituent. In this assay, tumorigenic cells are typically used as host cells. Therefore, antibodies can be identified by measuring changes in the level of invasiveness between the host cells before and after the introduction of potential modulators.
• Briefly, the level of invasion of host cells can be measured by using filters coated with Matrigel or some other extracellular matrix constituent. Penetration into the gel, or through to the distal side of the filter, is rated as invasiveness, and rated histologically by number of cells and distance moved, or by prelabeling the cells with 125I and counting the radioactivity on the distal side of the filter or bottom of the dish. (see, e.g., Freshney, Culture of Animal Cells a Manual of Basic Technique, 3rd ed., Wiley-Liss, New York (1994), herein incorporated by reference).
• b. Alteration of Gene Expression
• Detection of binding and/or modulation of a target by an antibody can be accomplished by detecting a biological response, such as, for example, measuring Ca²⁺ ion flux, cAMP, IP3, PIP3 or transcription of reporter genes. Analysis of the genotype of the cell (measurement of the expression of one or more of the genes of the cell using a reporter gene assay) after treatment is also used as an indicator of the efficacy or potency of the antibody. Hallmark genes, or those genes suspected to be associated with a signal transduction pathway are measured in both treated and untreated cells.
• Assays can be performed that measure the activation of a reporter gene. Suitable reporter genes include endogenous genes as well as exogenous genes that are introduced into a cell by any of the standard methods familiar to the skilled artisan, such as transfection, electroporation, lipofection and viral infection. For example, cells expressing a recombinant receptor can be transfected with a reporter gene (e.g., chloramphenicol acetyltransferase, firefly luciferase, bacterial luciferase, β-galactosidase and alkaline phosphatase) operably linked to a response element. The cells are then incubated with antibodies and the expression of the reporter gene is compared to expression in control cells that do not express the recombinant receptor but that are essentially identical in other respects. A statistically significant change in reporter gene expression in the receptor-expressing cells is indicative of a test compound that interacts with the receptor. Furthermore, the protein of interest can be used as an indirect reporter via attachment to a second reporter such as red or green fluorescent protein (see, e.g., Mistili & Spector, (1997) Nature Biotechnology 15:961-964).
• The reporter construct is typically transfected into a cell. After treatment with a potential modulator, the amount of reporter gene transcription, translation, or activity is measured according to standard techniques known to those of skill in the art. The use of a reporter gene assay using luciferase to measure activiation of STATS directly or by induction of cyclin-D promoter is exemplified in Example 12.
• c. Cytotoxicity Activity
• Antibodies can be screened for their ability to directly induce apoptosis or programmed cell death or to indirectly induce apoptosis by blocking growth factor receptors, thereby effectively arresting proliferation. Antibodies also bind complement, leading to direct cell toxicity, known as complement dependent cytotoxicity (CDC). Thus, assays can be performed to assess complement-dependent cytotoxicity.
• A variety of assays to assess apoptosis are known to one of skill in the art. For example, apoptosis assays include those that assay for the activation of a caspase, which are enzymes involved in apoptosis. Caspase assays are based on the measurement of zymogen processing to an active enzyme and proteolytic activity. A number of commercial kits and reagents are available to assess apoptosis based on caspase function including, but not limited to, PhiPhiLux (OncoImmunin, Inc.), Caspase 3 activity assay (Roche Applied science), Homogenous Caspase assay (Roche Applied Science), Caspase-Glo Assays (Promega), Apo-ONE Homogeneous Caspase-3/7 Assay (Promega), CaspACE Assay System Colorimetric or Fluormetric (Promega), EnzChek Caspase-3 Assay Kit (Invitrogen), Imag-iT LIVE green Caspase-3 and 7 Detection Kit (Invitrogen), Active Caspase-3 Detection Kits (Stratagene), Caspase-mediated Apoptosis Products (BioVision) and CasPASE Apoptosis Assay Kit (Genotech).
• Assays for apoptosis include TUNEL and DNA fragmentation assays that measure the activation of nucleases and subsequent cleavage of DNA into 180 to 200 base pair increments. Such assays and kits are commercially available and include, but are not limited to, Apoptotic DNA Ladder Kit (Roche Applied Science), Cellular DNA Fragmentation ELISA (Roche Applied Science), Cell Death Detection ELISAPLUS (Roche Applied Science), In Situ Cell Death Detection Kit (Roche Applied Science), DeadEnd Fluorometirc or Colorimetric TUNEL System (Promega), APO-BrdU TUNEL Assay Kit (Invitrogen), and TUNEL Apoptosis Detection Kit (Upstate).
• Other assays to assess apoptosis include, for example, cell permeability assays that evaluate the loss of membrane integrity. For example, to determine whether the antibody is able to induce cell death, loss of membrane integrity as evaluated by uptake of propidium iodide (PI), trypan blue, or 7-aminoactinomycin D (7AAD) can be assessed relative to untreated cells. In addition, commercial kits such as APOPercentage Assay (Biocolor Assays) can be used to measure apoptosis. Annexin V assays also can be employed. Annexin V binds to phosphatidylserine, which is normally found on the inner surface of the cytoplasmic membrane. During apoptosis, phosphatidylserine is translocated to the outer surface and can be detected by Annexin V. For example, standard binding assays using a fluorescent labeled Annexin V can be used (e.g. Annexin V, Alex Fluor 350 Conjugate from Invitrogen). Apoptosis also can be measured by assessing the presence of other markers of apoptosis, assessing protein cleavage, and/or by mitochondrial and ATP/ADP assays. Such assays are routine and known to one of skill in the art.
• For example, apoptosis analysis can be used as an assay to identify functional antibodies using cell lines, such as RKO or HCT116, or other cells expressing a target protein of interest. The cells can be co-transfected with a construct containing a marker gene, such as a gene that encodes green fluorescent protein, or a cell tracker dye. The apoptotic change can be determined using methods known in the art, such as DAPI staining and TUNEL assay using fluorescent microscope. For TUNEL assay, commercially available kit can be used (e.g., Fluorescein FragEL DNA Fragmentation Detection Kit (Oncogene Research Products, Cat.# QIA39) and Tetramethyl-rhodamine-5-dUTP (Roche, Cat. #1534 378)). Cells contacted with an antibody exhibit, e.g., an increased apoptosis compared to control.
• Cell death in vitro can be determined in the absence of complement and immune effector cells to distinguish cell death induced by antibody dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). Thus, the assay for cell death can be performed using heat inactivated serum (i.e. in the absence of complement) and in the absence of immune effector cells.
• 3. In Vivo Assays
• Once an affinity matured antibody or converted antibody is generated by the methods herein, it can be assessed in vivo assays associated with aberrant activity of the target. In general, the method involves administering an antibody to a subject, generally a non-human animal model for a disease or condition and determining the effect of the antibody on the on the disease or condition of the model animal. In vivo assays include controls, where suitable controls include a sample in the absence of the antibody. Generally a plurality of assay mixtures is run in parallel with different antibody concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.
• Non-human animals models include those induced to have a disease such as by injection with disease and/or phenotype-inducing substances prior to administration of the antibodies to monitor the effects on disease progression. Genetic models also are useful. Animals, such as mice, can be generated which mimic a disease or condition by the overexpression, underexpression or knock-out of one or more genes. Such animals can be generated by transgenic animal production techniques well-known in the art or using naturally-occurring or induced mutant strains. One of skill in the art is familiar with various animal models associated with particular targets.
• Such animal model systems include, but are not limited to, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e.g. baboons, chimpanzees and monkey. Any animal system well-known in the art can be used. Several aspects of the procedure can vary; said aspects include, but are not limited to, the temporal regime of administering the antibodies (e.g., prophylactic and/or therapeutic agents), whether such antibodies are administered separately or as an admixture, and the frequency of administration of the antibodies.
• Recombinant (transgenic) animal models can be engineered by introducing the coding portion of the genes identified herein into the genome of animals of interest, using standard techniques for producing transgenic animals Animals that can serve as a target for transgenic manipulation include, without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e.g. baboons, chimpanzees and monkeys. Techniques known in the art to introduce a transgene into such animals include pronucleic microinjection (U.S. Pat. No. 4,873,191); retrovirus-mediated gene transfer into germ lines (e.g., Van der Putten et al., (1985) Proc. Natl. Acad. Sci. USA 82:6148-615); gene targeting in embryonic stem cells (Thompson et al., (1989) Cell 56:313-321); electroporation of embryos (Lo, (1983) Mol. Cel. Biol. 3:1803-1814); sperm-mediated gene transfer (Lavitrano et al., (1989) Cell 57:717-73). For review, see, for example, U.S. Pat. No. 4,736,866.
• Animal models can be used to assess the efficacy of an antibody, a composition, or a combination therapy provided herein. Examples of animal models for lung cancer include, but are not limited to, lung cancer animal models (see e.g. Zhang et al., (1994) In Vivo 8(5):755-69) and a transgenic mouse model with disrupted p53 function (see, e.g., Morris et al., (1998) J La State Med Soc 150(4):179-85). An example of an animal model for breast cancer includes, but is not limited to, a transgenic mouse that overexpresses cyclin D1 (see, e.g., Hosokawa et al., (2001) Transgenic Res 10(5):471-8). An example of an animal model for colon cancer includes, but is not limited to, a TCR b and p53 double knockout mouse (see, e.g., Kado et al., (2001), Cancer Res 61(6):2395-8). Examples of animal models for pancreatic cancer include, but are not limited to, a metastatic model of Panc02 murine pancreatic adenocarcinoma (see, e.g., Wang et al., (2001) Int J Pancreatol 29(1):37-46) and nu-nu mice generated in subcutaneous pancreatic tumors (see, e.g., Ghaneh et al., (2001) Gene Ther 8(3):199-208). Examples of animal models for non-Hodgkin's lymphoma include, but are not limited to, a severe combined immunodeficiency (“SCID”) mouse (see, e.g., Bryant et al., (2000) Lab Invest 80(4):553-73) and an IgHmu-HOX11 transgenic mouse (see, e.g., Hough et al., (1998) Proc Natl Acad Sci USA 95(23):13853-8). An example of an animal model for esophageal cancer includes, but is not limited to, a mouse transgenic for the human papillomavirus type 16 E7 oncogene (see, e.g., Herber et al., (1996) J Virol 70(3):1873-81). Examples of animal models for colorectal carcinomas include, but are not limited to, Apc mouse models (see, e.g., Fodde & Smits, (2001) Trends Mol Med 7(8):369-73 and Kuraguchi et al., (2000) Oncogene 19(50):5755-63).
• Animal models for arthritis include, but are not limited to, rheumatoid arthritis rats (see e.g. Pearson, (1956) Proc. Soc. Exp. Biol. Med., 91:95-101) and collagen induced arthritis in mice and rats (see e.g. Current Protocols in Immunology, Eds. J. E. Cologan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, John Wiley & Sons, Inc., 1994). An example of an animal model for asthma, includes but is not limited to, a mouse model of pulmonary hypersensitivity (see e.g. Riese et al. (1998) J. Clin. Invest. 101:2351-2363 and Shi, et al. (1999) Immunity 10:197-206). Animal models for allogenic rejection include, but are not limited to, rat allogeneic heart transplant models (see e.g. Tanabe et al. (1994) Transplantation 58:23-27 and Tinubu et al. (1994) J. Immunol. 153:4330-4338) and rat heterocardiac allograft rejection (Jae-Hyuck Sim et al. (2002) Proc Natl Acad Sci 99(16):10617-10622). Steel mice are used as a model of human aplastic anemia (see e.g. Jones, (1983) Exp. Hematol., 11:571-580). An example of an animal model for anemia, includes but is not limited to, hemolytic anemia guinea pigs (see e.g. Schreiber, et al. (1972) J. Clin. Invest. 51:575). An example of an animal model for neutropenia, includes but is not limited to, neutropenia neutropenic CD rats (see, e.g. Nohynek et al. (1997) Cancer Chemother. Pharmacol. 39:259-266).
F. METHODS OF PRODUCTION OF ANTIBODIES
• Nucleic acid molecules and antibodies generated by the methods provided herein can be made by any method known to one of skill in the art. Such procedures are routine and are well known to the skill artisan. They include routine molecular biology techniques including gene synthesis, PCR, ligation, cloning, transfection and purification techniques. A description of such procedures is provided below.
• For example, nucleic acid sequences can be constructed using gene synthesis techniques as discussed herein above. Gene synthesis or routine molecular biology techniques also can be used to effect insertion, deletion, addition or replacement of nucleotides. For example, additional nucleotide sequences can be joined to a nucleic acid sequence. In one example linker sequences can be added, such as sequences containing restriction endonuclease sites for the purpose of cloning the synthetic gene into a vector, for example, a protein expression vector or a vector designed for the amplification of the antibody constant region coding DNA sequences. Furthermore, additional nucleotide sequences specifying functional DNA elements can be operatively linked to a recombined germline encoding nucleic acid molecule. Examples of such sequences include, but are not limited to, promoter sequences designed to facilitate intracellular protein expression, and leader peptide sequences designed to facilitate protein secretion. Additional nucleotide sequences such as sequences specifying protein binding regions also can be linked to nucleic acid sequences. Such regions include, but are not limited to, sequences to facilitate uptake of recombined antibodies or fragments thereof into specific target cells, or otherwise enhance the pharmacokinetics of the synthetic gene.
• Nucleic acid sequences can be further engineered as described herein, such as by mutagenesis, to generate mutant antibodies. Mutagenesis can be effected entirely through gene synthesis. For example, nucleic acid molecules can be designed manually or in silico for synthesis to encode mutant antibodies. The benefit of using gene synthesis methods is that the mutations can be effected so that the resulting nucleic acid molecules are in-frame and are “productive” as discussed herein above. Other methods of synthesis exist where randomization can be achieved during the gene synthesis. For example, a protocol has been developed by which synthesis of an oligonucleotide is “doped” with non-native phosphoramidites, resulting in randomization of the gene section targeted for random mutagenesis (Wang and Hoover (1997) J. Bacteriol., 179:5812-9). This method allows control of position selection while retaining a random substitution rate. Alternatively, mutagenesis can be effected through other molecular biology techniques. Generally, site-directed mutagenesis strategies can be employed.
• Other current methods can be used to create mutant antibodies include, but are not limited to, error-prone polymerase chain reaction (Caldwell and Joyce (1992); Gram et al. (1992) Proc. Natl. Acad. Sci., 89:3576-80); cassette mutagenesis in which the specific region to be optimized is replaced with a synthetically mutagenized oligonucleotide (Stemmer and Morris (1992) Biotechniques, 13:214-20); Arkin and Youvan (1992) Proc. Natl. Acad. Sci., 89:7811-7815; Oliphant et al. (1986) Gene, 44:177-83; Hermes et al. (1990) Proc. Natl. Acad. Sci, 87:696-700); the use of mutator strains of hosts cells to add mutational frequency (Greener et al. (1997) Mol. Biotechnol., 7:189-95); DNA shuffling (Crameri et al. (1998) Nature, 391:288-291; U.S. Pat. No. 6,177,263; U.S. Pat. No. 5,965,408; Ostermeier et al. (1999) Nat. Biotechnol., 17:1205-1209); and other random mutagenesis methods.
• Antibodies provided herein can be generated or expressed as full-length antibodies or as antibodies that are less than full length, including, but not limited to Fabs, Fab hinge fragment, scFv fragment, scFv tandem fragment and scFv hinge and scFv hinge (ΔE) fragments. Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see e.g. Morimoto et al. (1992) Journal of Biochemical and Biophysical Methods, 24:107-117; Brennance et al. (1985) Science, 229:81). Fragments also can be produced directly by recombinant host cells. Fab, Fv and scFv antibody fragments can all be expressed in and secreted from host cells, such as E. coli, thus allowing the facile production of large amounts of these fragments. Also, Fab′-SH fragments can be chemically coupled to form F(ab′)₂ fragments (Carter et al. (1992) Bio/Technology, 10:163-167). According to another approach, F(ab′)₂ fragments can be isolated directly from recombinant host cell culture. In other examples, the antibody of choice is a single chain Fv fragment (scFv) (see e.g. WO93/16185; U.S. Pat. No. 5,571,894 and U.S. Pat. No. 5,587,458. Fv and sFv are the only species with intact combining sites that are devoid of constant regions; thus, they are suitable for reduced nonspecific binding during in vivo use. sFv fusion proteins can be constructed to yield fusion of an effector protein at either the amino or the carboxy terminius of an sFv. The antibody fragment can also be a linear antibody (see e.g. U.S. Pat. No. 5,641,870). Such linear antibody fragments can be monospecific or bispecific. Other techniques for the production of antibody fragments or antibody multimers are known to one of skill in the art.
• For example, upon expression, antibody heavy and light chains pair by disulfide bond to form a full-length antibody or fragments thereof. For example, for expression of a full-length Ig, sequences encoding the V_H-C_H1-hinge-C_H2-C _H3 can be cloned into a first expression vector and sequences encoding the V_L-C_L domains can be cloned into a second expression vector. Upon co-expression with the second expression vector encoding the V_L-C_L domains, a full-length antibody is expressed. In another example, to generate a Fab, sequences encoding the V_H-C_H1 can be cloned into a first expression vector and sequences encoding the V_L-C_L domains can be cloned into a second expression vector. The heavy chain pairs with a light chain and a Fab monomer is generated. In this example, exemplary vectors include Plasmids A, C, D and E as described elsewhere herein. Sequences of C_H1, hinge, C_H2 and/or C _H3 of various IgG sub-types are known to one of skill in the art (see e.g. U.S. Published Application No. 20080248028; see also SEQ ID NO: 2922). Similarly, sequences of CL, lambda or kappa, also is known (see e.g. U.S. Published Application No. 20080248028; see also SEQ ID NOS: 2923-2924).
• 1. Vectors
• Provided herein are vectors for expression of nucleic acid encoding variable heavy chain or a variable light chain. The nucleic acids encoding antibody polypeptides are typically cloned into a intermediate vector before transformation into prokaryotic or eukaryotic cells. Choice of vector can depend on the desired application. For example, after insertion of the nucleic acid, the vectors typically are used to transform host cells, for example, to amplify the antibody genes for replication and/or expression thereof. In such examples, a vector suitable for high level expression is used. In other cases, a vector is chosen that is compatible with display of the expressed polypeptide on the surface of the cell.
• The nucleic acids encoding antibody polypeptides are typically cloned into a vector before transformation into prokaryotic or eukaryotic cells. Choice of vector can depend on the desired application. For example, after insertion of the nucleic acid, the vectors typically are used to transform host cells, for example, to amplify the antibody genes for replication and/or expression thereof. In such examples, a vector suitable for high level expression is used. Expression can be in any cell expression system known to one of skill in the art. Exemplary cells for expression include, but are not limited to, 293FS cells, HEK293-6E cells or CHO cells. Other expression vectors and host cells are described below.
• Generally, nucleic acid encoding the heavy chain of an antibody is cloned into a vector and the nucleic acid encoding the light chain of an antibody is cloned into the vector. The genes can be cloned into a single vector for dual expression thereof, or into separate vectors. If desired, the vectors also can contain further sequences encoding additional constant region(s) or hinge regions to generate other antibody forms.
• Many expression vectors are available and known to those of skill in the art for the expression of antibodies or portions thereof. The choice of an expression vector is influenced by the choice of host expression system. Such selection is well within the level of skill of the skilled artisan. In general, expression vectors can include transcriptional promoters and optionally enhancers, translational signals, and transcriptional and translational termination signals. Expression vectors that are used for stable transformation typically have a selectable marker which allows selection and maintenance of the transformed cells. In some cases, an origin of replication can be used to amplify the copy number of the vectors in the cells. Vectors also generally can contain additional nucleotide sequences operably linked to the ligated nucleic acid molecule (e.g. His tag, Flag tag). For purposes herein, vectors generally include sequences encoding the constant region. Thus, recombined antibodies or portions thereof also can be expressed as protein fusions. For example, a fusion can be generated to add additional functionality to a polypeptide. Examples of fusion proteins include, but are not limited to, fusions of a signal sequence, an epitope tag such as for localization, e.g. a his₆ tag or a myc tag, or a tag for purification, for example, a GST fusion, and a sequence for directing protein secretion and/or membrane association.
• For example, expression of the proteins can be controlled by any promoter/enhancer known in the art. Suitable bacterial promoters are well known in the art and described herein below. Other suitable promoters for mammalian cells, yeast cells and insect cells are well known in the art and some are exemplified below. Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application. Promoters which can be used include but are not limited to eukaryotic expression vectors containing the SV40 early promoter (Bernoist and Chambon, Nature 290:304-310 (1981)), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al. Cell 22:787-797 (1980)), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)), the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296:39-42 (1982)); prokaryotic expression vectors such as the β-lactamase promoter (Jay et al., (1981) Proc. Natl. Acad. Sci. USA 78:5543) or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25 (1983)); see also “Useful Proteins from Recombinant Bacteria”: in Scientific American 242:79-94 (1980)); plant expression vectors containing the nopaline synthetase promoter (Herrara-Estrella et al., Nature 303:209-213 (1984)) or the cauliflower mosaic virus 35S RNA promoter (Gardner et al., Nucleic Acids Res. 9:2871 (1981)), and the promoter of the photosynthetic enzyme ribulose bisphosphate carboxylase (Herrera-Estrella et al., Nature 310:115-120 (1984)); promoter elements from yeast and other fungi such as the Gal4 promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinase promoter, the alkaline phosphatase promoter, and the following animal transcriptional control regions that exhibit tissue specificity and have been used in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., Cell 38:639-646 (1984); Ornitz et al., Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald, Hepatology 7:425-515 (1987)); insulin gene control region which is active in pancreatic beta cells (Hanahan et al., Nature 315:115-122 (1985)), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., Cell 38:647-658 (1984); Adams et al., Nature 318:533-538 (1985); Alexander et al., Mol. Cell Biol. 7:1436-1444 (1987)), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., Cell 45:485-495 (1986)), albumin gene control region which is active in liver (Pinckert et al., Genes and Devel. 1:268-276 (1987)), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., Mol. Cell. Biol. 5:1639-1648 (1985); Hammer et al., Science 235:53-58 1987)), alpha-1 antitrypsin gene control region which is active in liver (Kelsey et al., Genes and Devel. 1:161-171 (1987)), beta globin gene control region which is active in myeloid cells (Magram et al., Nature 315:338-340 (1985); Kollias et al., Cell 46:89-94 (1986)), myelin basic protein gene control region which is active in oligodendrocyte cells of the brain (Readhead et al., Cell 48:703-712 (1987)), myosin light chain-2 gene control region which is active in skeletal muscle (Shani, Nature 314:283-286 (1985)), and gonadotrophic releasing hormone gene control region which is active in gonadotrophs of the hypothalamus (Mason et al., Science 234:1372-1378 (1986)).
• In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the antibody, or portion thereof, in host cells. A typical expression cassette contains a promoter operably linked to the nucleic acid sequence encoding the germline antibody chain and signals required for efficient polyadenylation of the transcript, ribosome binding sites and translation termination. Additional elements of the cassette can include enhancers. In addition, the cassette typically contains a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region can be obtained from the same gene as the promoter sequence or can be obtained from different genes.
• Some expression systems have markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase. Alternatively, high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a nucleic acid sequence encoding a germline antibody chain under the direction of the polyhedron promoter or other strong baculovirus promoter.
• Exemplary expression vectors include any mammalian expression vector such as, for example, pCMV. For bacterial expression, such vectors include pBR322, pUC, pSKF, pET23D, and fusion vectors such as MBP, GST and LacZ. Exemplary of such a vector are bacterial expression vectors such as, for example, plasmid A, plasmid C, plasmid D and plasmid E, described herein. Other eukaryotic vectors, for example any containing regulatory elements from eukaryotic viruses can be used as eukaryotic expression vectors. These include, for example, SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Bar virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMT010/A+, pMAMneo-5, baculovirus pDSCE, and any other vector allowing expression of proteins under the direction of the CMV promoter, SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedron promoter, or other promoters shown effective for expression in eukaryotes.
• Vectors can be provided that contain a sequence of nucleotides that encodes a constant region of an antibody operably linked to the nucleic acid sequence encoding the variable region of the antibody. The vector can include the sequence for one or all of a CH1, CH2, CH3 or CH4 and/or CL. Generally, such as for expression of Fabs, the vector contains the sequence for a CH1 or CL. In one example, nucleic acid encoding the heavy chain of an antibody, is ligated into a first expression vector and nucleic acid encoding the light chain of an antibody, is ligated into a second expression vector. The expression vectors can be the same or different, although generally they are sufficiently compatible to allow comparable expression of proteins (heavy and light chain) therefrom. The first and second expression vectors are generally co-transfected into host cells, typically at a 1:1 ratio. Exemplary of vectors include, but are not limited to, pγ1HC and pκLC (Tiller et al. (2008) J Immunol. Methods, 329:112-24). Other expression vectors include the light chain expression vector pAG4622 and the heavy chain expression vector pAH4604 (Coloma et al. (1992) J Immunol. Methods, 152:89-104). The pAG4622 vector contains the genomic sequence encoding the C-region domain of the human κL chain and the gpt selectable marker. The pAH4604 vectors contain the hisD selectable marker and sequences encoding the human H chain γ1 C-region domain. In another example, the heavy and light chain can be cloned into a single vector that has expression cassettes for both the heavy and light chain. Other exemplary expression vectors include Plasmids A, C, D and E, described elsewhere herein.
• For purposes herein, vectors are provided that contain a sequence of nucleotides that encodes a constant region of an antibody operably linked to the nucleic acid sequence encoding the recombined variable region of the antibody. The vector can include the sequence for one or all of a CH1, CH2, hinge, CH3 or CH4 and/or CL. Generally, such as for expression of Fabs, the vector contains the sequence for a CH1 (amino acids 1-103 of SEQ ID NO:2922) or CL (for kappa light chains, see SEQ ID NO:2923; for lambda light chains, see SEQ ID NO:2924). The sequences of constant regions or hinge regions are known to one of skill in the art (see e.g. U.S. Published Application No. 20080248028 and SEQ ID NOS:2922-2924, including CH1 (amino acids 1-103 of SEQ ID NO:2922), IgG1 hinge region (amino acids 104-119 of SEQ ID NO:2922), IgG1 CH2 (amino acids 120-223 of SEQ ID NO:2922), IgG1 CH3 (amino acids 224-330 of SEQ ID NO:2922), CL kappa (SEQ ID NO:2923) and CL lambda (SEQ ID NO:2924). Exemplary of such vectors containing a heavy chain constant region gene (e.g. CH1) are plasmids A and D, described herein. Exemplary of such vectors containing a light chain constant region genes are plasmids C and E, described herein.
• Exemplary plasmid vectors for transformation of E. coli cells, include, for example, the ColE1 replication vectors described herein. Several features common to all these vectors include (a) a pBAD inducible promoter; (b) an AraC gene, which controls the pBAD promoter; (c) a synthetic ribosomal binding site (RBS) for efficient translation; (d) a ColE1 origin of replication, allowing for high copy expression; (e) a STII leader sequence, allowing for expressed proteins to be translocated to the periplasm; (f) a f1 origin of replication; and (g) a gene for conferring antibiotic resistance. Such plasmids include plasmid A (SEQ ID NO:84), plasmid C (SEQ ID NO:86), plasmid D (SEQ ID NO:85) and plasmid E (SEQ ID NO:87). Plasmid A and Plasmid D are utilized for expression of heavy chain antibody genes in as they contain a gene for the heavy chain constant region (CH1) operably linked to the inserted gene for the heavy chain variable region. The vectors contain NheI and NcoI restriction sites to allow for cloning of the recombined antibody genes described herein. Both vectors contain a pUC origin of replication, a ColE1 type origin of replication, and an aminoglycoside phosphotransferase gene conferring kanamycin resistance. Plasmid A contains a (His)₆ Tag and a Flag Tag for protein purification. Plasmid D contains both a (His)₆ Tag and a Flag Tag, and an additional LPETG tag, which allows for covalent attachment of the resulting protein using a sortase. Plasmid C and Plasmid E are utilized for expression of light chain antibody genes in as they contain a gene for the light chain constant region (CL) operably linked to the inserted gene for the light chain variable region. Plasmid C is specific for kappa light chains and contains BseWI and NcoI restriction sites to allow for cloning of the recombined antibody genes described herein. Plasmid E is specific for lambda light chains and contains AcrII and NcoI restriction sites to allow for cloning of the recombined antibody genes described herein. Both vectors contain a 3.3 origin of replication, a ColE1 type origin of replication, and a gene conferring chloramphenicol resistance. The vectors described above are designed to be utilized in a dual vector system, in which a light chain vector and a heavy chain vector are co-transformed. Thus, they contain two different but compatible ColE1 origins of replication utilized, one for heavy chains and one light chain. This allows for efficient expression of both chains of the antibody when the vectors are co-transformed and expressed.
• Any methods known to those of skill in the art for the insertion of DNA fragments into a vector can be used to construct expression vectors containing a nucleic acid encoding an antibody chain. These methods can include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. If the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules can be enzymatically modified. Alternatively, any site desired can be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers can contain specific chemically synthesized nucleic acids encoding restriction endonuclease recognition sequences.
• 2. Cells and Expression Systems
• Cells containing the vectors also are provided. Generally, any cell type that can be engineered to express heterologous DNA and has a secretory pathway is suitable. Expression hosts include prokaryotic and eukaryotic organisms such as bacterial cells (e.g. E. coli), yeast cells, fungal cells, Archea, plant cells, insect cells and animal cells including human cells. Expression hosts can differ in their protein production levels as well as the types of post-translational modifications that are present on the expressed proteins. Further, the choice of expression host is often related to the choice of vector and transcription and translation elements used. For example, the choice of expression host is often, but not always, dependent on the choice of precursor sequence utilized. For example, many heterologous signal sequences can only be expressed in a host cell of the same species (i.e., an insect cell signal sequence is optimally expressed in an insect cell). In contrast, other signal sequences can be used in heterologous hosts such as, for example, the human serum albumin (hHSA) signal sequence which works well in yeast, insect, or mammalian host cells and the tissue plasminogen activator pre/pro sequence which has been demonstrated to be functional in insect and mammalian cells (Tan et al., (2002) Protein Eng. 15:337). The choice of expression host can be made based on these and other factors, such as regulatory and safety considerations, production costs and the need and methods for purification. Thus, the vector system must be compatible with the host cell used.
• Expression in eukaryotic hosts can include expression in yeasts such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as Drosophila cells and lepidopteran cells, plants and plant cells such as tobacco, corn, rice, algae, and lemna. Eukaryotic cells for expression also include mammalian cells lines such as Chinese hamster ovary (CHO) cells or baby hamster kidney (BHK) cells. Eukaryotic expression hosts also include production in transgenic animals, for example, including production in serum, milk and eggs.
• Recombinant molecules can be introduced into host cells via, for example, transformation, transfection, infection, electroporation and sonoporation, so that many copies of the gene sequence are generated. Generally, standard transfection methods are used to produce bacterial, mammalian, yeast, or insect cell lines that express large quantity of antibody chains, which is then purified using standard techniques (see e.g., Colley et al. (1989) J. Biol. Chem., 264:17619-17622; Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed.), 1990). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison (1977) J. Bact. 132:349-351; Clark-Curtiss and Curtiss (1983) Methods in Enzymology, 101, 347-362). For example, any of the well-known procedures for introducing foreign nucleotide sequences into host cells can be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistics, liposomes, microinjection, plasma vectors, viral vectors and any other the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell. Generally, for purposes herein, host cells are transfected with a first vector encoding at least a VH chain and a second vector encoding at least a VL chain. Thus, it is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least both genes into the host cell capable of expressing germline, or modified form thereof, antibody polypeptide.
• Transformation of host cells with recombinant DNA molecules that incorporate the isolated recombined variable region gene, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene. Thus, the gene can be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA. Generally, After the expression vector is introduced into the cells, the transfected cells are cultured under conditions favoring expression of the germline chain, which is recovered from the culture using standard purification techniques identified below.
• Antibodies and portions thereof can be produced using a high throughput approach by any methods known in the art for protein production including in vitro and in vivo methods such as, for example, the introduction of nucleic acid molecules encoding antibodies or portions thereof into a host cell or host animal and expression from nucleic acid molecules encoding antibodies in vitro. Prokaryotes, especially E. coli, provide a system for producing large amounts of antibodies or portions thereof, and are particularly desired in applications of high-throughput expression and purification of proteins. Transformation of E. coli is a simple and rapid technique well known to those of skill in the art. E. coli host strains for high throughput expression include, but are not limited to, BL21 (EMD Biosciences) and LMG194 (ATCC). Exemplary of such an E. coli host strain is BL21. Vectors for high throughput expression include, but are not limited to, pBR322 and pUC vectors. Exemplary of such vectors are the vectors described herein, including plasmid A, plasmid C, plasmid D and plasmid E. Automation of expression and purification can facilitate high-throughput expression. For example, use of a Piccolo™ system (Wollerton et al. (2006) JALA, 11:291-303), a fully automatic system that combines cell culture with automated harvesting, lysing and purification units, or other similar robotic system can be employed.
• a. Prokaryotic Expression
• Prokaryotes, especially E. coli, provide a system for producing large amounts of antibodies or portions thereof. Transformation of E. coli is a simple and rapid technique well known to those of skill in the art. Expression vectors for E. coli can contain inducible promoters that are useful for inducing high levels of protein expression and for expressing proteins that exhibit some toxicity to the host cells. Examples of inducible promoters include the lac promoter, the trp promoter, the hybrid tac promoter, the T7 and SP6 RNA promoters and the temperature regulated λP_L promoter.
• Antibodies or portions thereof can be expressed in the cytoplasmic environment of E. coli. The cytoplasm is a reducing environment and for some molecules, this can result in the formation of insoluble inclusion bodies. Reducing agents such as dithiothreitol and (3-mercaptoethanol and denaturants (e.g., such as guanidine-HCl and urea) can be used to resolubilize the proteins. An exemplary alternative approach is the expression of antibodies or fragments thereof in the periplasmic space of bacteria which provides an oxidizing environment and chaperonin-like and disulfide isomerases leading to the production of soluble protein. Typically, a leader sequence is fused to the protein to be expressed which directs the protein to the periplasm. The leader is then removed by signal peptidases inside the periplasm. There are three major pathways to translocate expressed proteins into the periplasm, namely the Sec pathway, the SRP pathway and the TAT pathway. Examples of periplasmic-targeting leader sequences include the pelB leader from the pectate lyase gene, the StII leader sequence, and the DsbA leader sequence. An exemplary leader sequence is a DsbA leader sequence. In some cases, periplasmic expression allows leakage of the expressed protein into the culture medium. The secretion of proteins allows quick and simple purification from the culture supernatant. Proteins that are not secreted can be obtained from the periplasm by osmotic lysis. Similar to cytoplasmic expression, in some cases proteins can become insoluble and denaturants and reducing agents can be used to facilitate solubilization and refolding. Temperature of induction and growth also can influence expression levels and solubility. Typically, temperatures between 25° C. and 37° C. are used. Mutations also can be used to increase solubility of expressed proteins. Typically, bacteria produce aglycosylated proteins. Thus, if proteins require glycosylation for function, glycosylation can be added in vitro after purification from host cells.
• b. Yeast
• Yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Kluyveromyces lactis, and Pichia pastoris are useful expression hosts for recombined antibodies or portions thereof. Yeast can be transformed with episomal replicating vectors or by stable chromosomal integration by homologous recombination. Typically, inducible promoters are used to regulate gene expression. Examples of such promoters include AOX1, GAL1, GAL7, and GAL5 and metallothionein promoters such as CUP1. Expression vectors often include a selectable marker such as LEU2, TRP1, HIS3, and URA3 for selection and maintenance of the transformed DNA. Proteins expressed in yeast are often soluble. Co-expression with chaperonins such as Bip and protein disulfide isomerase can improve expression levels and solubility. Additionally, proteins expressed in yeast can be directed for secretion using secretion signal peptide fusions such as the yeast mating type alpha-factor secretion signal from Saccharomyces cerevisae and fusions with yeast cell surface proteins such as the Aga2p mating adhesion receptor or the Arxula adeninivorans glucoamylase. A protease cleavage site such as for the Kex-2 protease, can be engineered to remove the fused sequences from the expressed polypeptides as they exit the secretion pathway. Yeast also is capable of glycosylation at Asn-X-Ser/Thr motifs.
• c. Insects
• Insect cells, particularly using baculovirus expression, are useful for expressing antibodies or portions thereof. Insect cells express high levels of protein and are capable of most of the post-translational modifications used by higher eukaryotes. Baculovirus have a restrictive host range which improves the safety and reduces regulatory concerns of eukaryotic expression. Typical expression vectors use a promoter for high level expression such as the polyhedrin promoter and p10 promoter of baculovirus. Commonly used baculovirus systems include the baculoviruses such as Autographa californica nuclear polyhedrosis virus (AcNPV), and the Bombyx mori nuclear polyhedrosis virus (BmNPV) and an insect cell line such as Sf9 derived from Spodoptera frugiperda and TN derived from Trichoplusia ni. For high-level expression, the nucleotide sequence of the molecule to be expressed is fused immediately downstream of the polyhedrin initiation codon of the virus. To generate baculovirus recombinants capable of expressing human antibodies, a dual-expression transfer, such as pAcUW51 (PharMingen) is utilized. Mammalian secretion signals are accurately processed in insect cells and can be used to secrete the expressed protein into the culture medium
• An alternative expression system in insect cells is the use of stably transformed cells. Cell lines such as Sf9 derived cells from Spodoptera frugiperda and TN derived cells from Trichoplusia ni can be used for expression. The baculovirus immediate early gene promoter IE1 can be used to induce consistent levels of expression. Typical expression vectors include the pIE1-3 and pI31-4 transfer vectors (Novagen). Expression vectors are typically maintained by the use of selectable markers such as neomycin and hygromycin.
• d. Mammalian Cells
• Mammalian expression systems can be used to express antibodies or portions thereof. Expression constructs can be transferred to mammalian cells by viral infection such as adenovirus or by direct DNA transfer such as liposomes, calcium phosphate, DEAE-dextran and by physical means such as electroporation and microinjection. Expression vectors for mammalian cells typically include an mRNA cap site, a TATA box, a translational initiation sequence (Kozak consensus sequence) and polyadenylation elements. Such vectors often include transcriptional promoter-enhancers for high-level expression, for example the SV40 promoter-enhancer, the human cytomegalovirus (CMV) promoter and the long terminal repeat of Rous sarcoma virus (RSV). These promoter-enhancers are active in many cell types. Tissue and cell-type promoters and enhancer regions also can be used for expression. Exemplary promoter/enhancer regions include, but are not limited to, those from genes such as elastase I, insulin, immunoglobulin, mouse mammary tumor virus, albumin, alpha fetoprotein, alpha 1 antitrypsin, beta globin, myelin basic protein, myosin light chain 2, and gonadotropic releasing hormone gene control. Selectable markers can be used to select for and maintain cells with the expression construct. Examples of selectable marker genes include, but are not limited to, hygromycin B phosphotransferase, adenosine deaminase, xanthine-guanine phosphoribosyl transferase, aminoglycoside phosphotransferase, dihydrofolate reductase and thymidine kinase. Antibodies are typically produced using a NEO^R/G418 system, a dihydrofolate reductase (DHFR) system or a glutamine synthetase (GS) system. The GS system uses joint expression vectors, such as pEE12/pEE6, to express both heavy chain and light chain. Fusion with cell surface signaling molecules such as TCR-ζ and Fc_εRI-γ can direct expression of the proteins in an active state on the cell surface.
• Many cell lines are available for mammalian expression including mouse, rat human, monkey, chicken and hamster cells. Exemplary cell lines include but are not limited to CHO, Balb/3T3, HeLa, MT2, mouse NS0 (nonsecreting) and other myeloma cell lines, hybridoma and heterohybridoma cell lines, lymphocytes, fibroblasts, Sp2/0, COS, NIH3T3, HEK293, 293S, 2B8, and HKB cells. Cell lines also are available adapted to serum-free media which facilitates purification of secreted proteins from the cell culture media. One such example is the serum free EBNA-1 cell line (Pham et al., (2003) Biotechnol. Bioeng. 84:332-42.)
• e. Plants
• Transgenic plant cells and plants can be used to express proteins such as any antibody or portion thereof described herein. Expression constructs are typically transferred to plants using direct DNA transfer such as microprojectile bombardment and PEG-mediated transfer into protoplasts, and with agrobacterium-mediated transformation. Expression vectors can include promoter and enhancer sequences, transcriptional termination elements and translational control elements. Expression vectors and transformation techniques are usually divided between dicot hosts, such as Arabidopsis and tobacco, and monocot hosts, such as corn and rice. Examples of plant promoters used for expression include the cauliflower mosaic virus CaMV 35S promoter, the nopaline synthase promoter, the ribose bisphosphate carboxylase promoter and the maize ubiquitin-1 (ubi-1) promoter promoters. Selectable markers such as hygromycin, phosphomannose isomerase and neomycin phosphotransferase are often used to facilitate selection and maintenance of transformed cells. Transformed plant cells can be maintained in culture as cells, aggregates (callus tissue) or regenerated into whole plants. Transgenic plant cells also can include algae engineered to produce proteases or modified proteases (see for example, Mayfield et al. (2003) PNAS 100:438-442). Because plants have different glycosylation patterns than mammalian cells, this can influence the choice of protein produced in these hosts.
• 3. Purification
• Antibodies and portions thereof are purified by any procedure known to one of skill in the art. The antibodies generated or used by the methods herein can be purified to substantial purity using standard protein purification techniques known in the art including but not limited to, SDS-PAGE, size fraction and size exclusion chromatography, ammonium sulfate precipitation, chelate chromatography, ionic exchange chromatography or column chromatography. For example, antibodies can be purified by column chromatography. Exemplary of a method to purify antibodies is by using column chromatography, wherein a solid support column material is linked to Protein G, a cell surface-associated protein from Streptococcus, that binds immunoglobulins with high affinity. The antibodies can be purified to 60%, 70%, 80% purity and typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% purity. Purity can be assessed by standard methods such as by SDS-PAGE and coomassie staining.
• Methods for purification of antibodies or portions thereof from host cells depend on the chosen host cells and expression systems. For secreted molecules, proteins are generally purified from the culture media after removing the cells. For intracellular expression, cells can be lysed and the proteins purified from the extract. When transgenic organisms such as transgenic plants and animals are used for expression, tissues or organs can be used as starting material to make a lysed cell extract. Additionally, transgenic animal production can include the production of polypeptides in milk or eggs, which can be collected, and if necessary further the proteins can be extracted and further purified using standard methods in the art.
• When antibodies are expressed by transformed bacteria in large amounts, typically after promoter induction, although expression can be constitutive, the polypeptides can form insoluble aggregates. There are several protocols that are suitable for purification of polypeptide inclusion bodies known to one of skill in the art. Numerous variations will be apparent to those of skill in the art.
• For example, in one method, the cell suspension is generally centrifuged and the pellet containing the inclusion bodies resuspended in buffer which does not dissolve but washes the inclusion bodies, e.g., 20 mM Tris-HCL (pH 7.2), 1 mM EDTA, 150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It can be necessary to repeat the wash step to remove as much cellular debris as possible. The remaining pellet of inclusion bodies can be resuspended in an appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mM NaCl). Other appropriate buffers are apparent to those of skill in the art.
• Alternatively, antibodies can be purified from bacteria periplasm. Where the polypeptide is exported into the periplasm of the bacteria, the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to those of skill in the art. For example, in one method, to isolate recombinant polypeptides from the periplasm, the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20% sucrose. To lyse the cells, the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO₄ and kept in an ice bath for approximately 10 minutes. The cell suspension is centrifuged and the supernatant decanted and saved. The recombinant polypeptides present in the supernatant can be separated from the host proteins by standard separation techniques well known to those of skill in the art. These methods include, but are not limited to, the following steps: solubility fractionation, size differential filtration, and column chromatography.
G. ANTI-DLL4 ACTIVATOR/MODULATOR ANTIBODIES AND USES THEREOF
• Provided herein are anti-DLL4 multimer antibodies that specifically bind to human Delta-like ligand 4 (DLL4) DLL4 and that are activator/modulators of DLL4 activity. Thus, the multimer antibodies can be used as antiangiogenic therapeutics to treat diseases or disorders characterized by excessive or aberrant angiogenesis, such as for example, cancer or macular degeneration.
• 1. DLL4
• a. Structure
• DLL4 (set forth in SEQ ID NO:2904; and encoded by a sequence of nucleotides set forth in SEQ ID NO:2905) is a transmembrane protein ligand for Notch transmembrane receptors. The extracellular region contains 8 EGF-like repeats, as well as a DSL domain that is conserved among all Notch ligands and is necessary for receptor binding. The protein also contains a transmembrane region, and a cytoplasmic tail lacking any catalytic motifs. Human DLL4 is a 685 amino acid protein and contains the following domains corresponding to amino acids set forth in SEQ ID NO:2904: signal peptide (amino acids 1-25); MNNL (amino acids 26-92); DSL (amino acids 155-217); EGF-Like 1 (EGF1; amino acids 221-251); EGF-Like 2 (EGF2; amino acids 252-282); EGF-Like 3 (EGF3; amino acids 284-322); EGF-Like 4 (EGF4; amino acids 324-360); EGF-Like 5 (EGF5; amino acids 366-400); EGF-Like 6 (EGF6; amino acids 402-438); EGF-Like 7 (EGF7; amino acids 440-476); EGF-Like 8 (EGFR; amino acids 480-518); transmembrane (amino acids 529-551); and cytoplasmic domain (amino acids 553-685).
• b. Expression
• DLL4 is expressed widely in a variety of tissues, but its expression is predominantly localized to the vasculature. It is required for normal vascular development and is expressed on tumor vessels. It is upregulated in blood vessels during tumor angiogenesis and expression is dependent on VEGF signaling. DLL4 also is expressed on activated macrophages exposed to proinflammatory stimuli such as lipopolysaccharide, interleukin-1β, Toll-like receptor 4 ligands and other proinflammatory stimuli and it's signaling through the Notch pathway plays a role in inflammatory states characterized by macrophage activation (Fung et al. (2007) Circulation, 115: 2948-2956).
• c. Function
• DLL4 binds to Notch receptors. The evolutionary conserved Notch pathway is a key regulator of many developmental processes as well as postnatal self-renewing organ systems. From invertebrates to mammals, Notch signaling guides cells through a myriad of cell fate decisions and incluences proliferation, differentiation and apoptosis (Miele and Osborne (1999) J Cell Physiol., 181:393-409). The Notch family is made up of structurally conserved cell surface receptors that are activated by membrane bound ligands of the DSL gene family (named for Delta and Serrate from Drosophila and Lag-2 from C. elegans). Mammals have four receptors (Notch 1, Notch 2, Notch 3 and Notch 4) and five ligands (Jag 1, Jag 2, DLL1, DLL3, and DLL4). Upon activation by ligands presented on neighboring cells, Notch receptors undergo successive proteolytic cleavages; an extracellular cleavage mediated by an ADAM protease and a cleavage within the trnamembrane domain mediated by gamma secretase. This leads to the release of the Notch Intra-Cellular Domain (NICD), which translocates into the nucleus and forms a transcriptional complex with the DNA binding protein, RBP-Jk (also known as CSL for CBF1/Su(H)/Lag-1) and other transcriptional cofactors. The primary target genes of Notch activation include the HES (Hairy/Enhance of Split) gene family and HES-related genes (Hey, CHF, HRT, HESR), which in turn regulate the downstream transcriptional effectors in a tissue and cell-type specific manner (Iso et al. (2003) J Cell Physiol., 194:237-255; L1 and Harris (2005) Cancer Cell, 8:1-3).
• Signaling by Notch receptors implicate a variety of cellular processes including, but not limited to, the normal maintenance and leukemic transformation of hematopoietic stem cells (HSCs; Kopper & Hajdu (2004) Pathol. Oncol. Res., 10:69-73); maintenance of neural stem cells including in their normal maintenance as well as in brain cancers (Kopper & Hajdu (2004) Pathol. Oncol. Res., 10:69-73; Purow et al. (2005) Cancer Res. 65:2353-63; Hallahan et al., (2004) Cancer Res. 64:7794-800); generation of a number of human cancers including in lymphoblastic leukemia/lymphoma (Ellisen et al. (1991) Cell, 66:649-61; Robey et al. (1996) Cell, 87:483-92; Pear et al. (1996) J. Exp. Med. 183:2283-91; Yan et al. (2001) Blood 98:3793-9; Bellavia et al. (2000) EMBO J. 19:3337-48; Pear & Aster (2004) Curr. Opin. Hematol., 11:416-33); breast cancer (Gallahan & Callahan (1987) J. Virol., 61:66-74; Brennan & Brown (2003) Breast Cancer Res., 5:69; Politi et al. (2004) Semin. Cancer Biol., 14:341-7; Weijzen et al. (2002) Nat. Med., 8:979-86; Parr et al. (2004) Int. J. Mol. Med., 14:779-86); cervical cancer (Zagouras et al. (1995) PNAS, 92:6414-8); renal cell carcinomas (Rae et al (2000) Int. J. Cancer, 88:726-32); head and neck squamous cell carcinomas (Leethanakul et al (2000) Oncogene, 19:3220-4); endometrial cancers (Suzuki et al. (2000) Int. J. Oncol., 17:1131-9); and neuroblastomas (van Limpt et al. (2000) Med. Pediatr. Oncol., 35:554-8). The Notch pathway also is involved in multiple aspects of vascular development including proliferation, migration, smooth muscle differentiation, angiogenesis and arterial-venous differentiation (Iso et al. (2003) Arterioscler. Thromb. Vasc. Biol. 23: 543).
• The Notch ligand DLL4, which interacts with Notch-1 (Uniprot accession No. P46531; SEQ ID NO:2906) and Notch-4 receptors (Uniprot accession No. Q99466; SEQ ID NO:2907), is expressed predominantly in the vasculature. Studies assessing the effects of overexpression of DLL4 have shown that DLL4 is a negative regulator of angiogenesis, endothelial cell proliferation, migration and vessel branching (see e.g. Trindade et al. (2008) Blood 1:112). One explanation for the antiangiogenic activity of DLL4 is that it is a VEGF responsive gene and acts as a negative regulator of VEGF signaling, which is a proangiogenic factor. Thus, targeting the activation of DLL4 promotes the antiangiogenic activity of DLL4.
• In contrast, blocking DLL4 is associated with nonproductive angiogensis. Although DLL4 increases angiogenesis characterized by sprouting and branching of blood vessels, it also is associated with a decrease in vessel function, thereby resulting in decreased tumor growth (Ridgway et al. (2006) Nature, 444:1083; Noguera-Troise et al. (2006) Nature, 444:1032). Accordingly, DLL4 function is associated with deregulated angiogenesis by uncoupling of tumor growth from tumor vascular density. Thus, blocking DLL4 signaling effectively reduces tumor growth by disrupting productive angiogenesis. Accordingly, targeting the inhibition of DLL4 also can be used to treat tumors undergoing angiogenesis (see e.g. International PCT application No. WO2009/085209).
• 2. Activator/Modulator Anti-DLL4 Multimer Antibodies
• Provided herein are antibodies or antibody fragments thereof that are activator/modulators of DLL4 activity. The antibodies activate or increase the activity of DLL4, and thereby act as anti-angiogenic agents. For example, the antibody multimers provided herein increase the activity of DLL4-mediated receptor activation, for example activation of DLL4-mediated Notch-1 or Notch-4 signaling, compared to activation in the absence of the antibody multimer. DLL4-mediated activity is increased at least 1.1-fold, for example, between or about 1.2-fold to 5-fold, such as 1.1-fold, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2.0-, 2.5-, 3.0-, 3.5-, 4.0-, 4.5-, 5-fold or more in the presence of the antibody multimer compared to activation in its absence. Thus, the antibodies can be used to treat angiogenic diseases or disorders. In some examples, the antibodies provided herein are agonists. In other examples, the antibodies provided herein are activator/modulators of DLL4 by activating Notch signaling.
• The antibody multimers provided herein exhibit rapid on/off kinetics for their binding site on DLL4. In particular, the antibody exhibits a fast k_off. For example, when assessed as a monomeric Ig fragment, antibodies provided herein have a k_off that is or is about between 1 s⁻¹ to 5×10⁻² s⁻¹, for example, 0.5 s⁻¹ to 0.01 s⁻¹, such as for example, at or about 0.1 s⁻¹. For example, the k_off of antibodies provided herein, when assessed in Fab form, is at or about 5×10⁻² s⁻¹, 4×10⁻² s⁻¹, 3×10⁻² s⁻¹, 2×10⁻² s⁻¹, 1×10⁻² s⁻¹, 0.02 s⁻¹, 0.03 s⁻¹, 0.04 s⁻¹, 0.05 s⁻¹, 0.06 s⁻¹, 0.07 s⁻¹, 0.08 s⁻¹, 0.09 s⁻¹, 0.1 s⁻¹, 0.2 s⁻¹, 0.3 s⁻¹, 0.4 s⁻¹, 0.5 s⁻¹, 0.6 s⁻¹, 0.7 s⁻¹, 0.8 s⁻¹, 0.9 s⁻¹, 1 s⁻¹ or faster, so long as the antibody multimer specifically binds to DLL4. In some examples, the antibodies provided herein exhibit a dissociation half-life (t_1/2), when assessed as a monomeric Ig fragment, that is between 0.5 seconds (s) to 150 s, for example, 1 s to 100 s, 5 s to 50 s or 5 s to 10 s. For example, the t_1/2 of antibodies provided herein, when assessed as a monomeric Ig fragment, is or is about 1 s, 2 s, 3 s, 4 s, 5 s, 6 s, 7 s, 8 s, 9 s, 10 s, 20 s, 30 s, 40 s, 50 s, 60 s, 70 s, 80 s, 90 s, 100 s, 110 s, 120 s, 130 s, 140 s or 150 s. Methods to determine kinetic rate constants of antibodies are known to one of skill in the art. For example, surface plasmon resonance using Biacore™ instrument can be used (BiaCore Life Science; GE Healthcare). Services offering Biacore instrumentation and other instrumentations are available (Biosensor Tools; Salt Lake City, Utah; biosensortools.com/index.php).
• Typically, antibody multimers provided herein exhibit a generally low binding affinity. For example, when assessed as a monomeric Ig fragment, antibodies provided herein exhibit a binding affinity that is 10⁻⁸ M or lower binding affinity. For example, the binding affinity is between 10⁻⁶ M to 10⁻⁸ M, such as between 4×10⁻⁶ M to 10⁻⁸ M, for example between 1×10⁻⁷ M to 10⁻⁸ M. For example, the binding affinity of antibodies provided herein, as a monomeric Ig fragment, is at or about 1×10⁻⁶ M, 2×10⁻⁶ M, 3×10⁻⁶ M, 4×10⁻⁶ M, 5×10⁻⁶ M, 6×10⁻⁶ M, 7×10⁻⁶ M, 8×10⁻⁶ M, 9×10⁻⁶ M, 1×10⁻⁷ M, 2×10⁻⁷ M, 3×10⁻⁷ M, 4×10⁻⁷ M, 5×10⁻⁷ M, 6×10⁻⁷ M, 7×10⁻⁷ M, 8×10⁻⁷ M, 9×10⁻⁷ M or 1×10⁻⁸ M. Methods to assess binding affinity are known to one of skill in the art and are described elsewhere herein in Section E.
• The antibodies provided herein are multimers, such that they contain at least two antigen-binding sites. Generally, the antibodies provided herein contain at least two variable heavy chain, or a sufficient portion thereof to bind antigen; and two variable light chains, or a sufficient portion thereof to bind antigen that are associated by a multimerization domain. The multimers can be dimers, trimers or higher-order multimers of monomeric immunoglobulin molecules. The multimers include those that are bivalent, trivalent, tetravalent, pentavalent, hexavalent, heptavalent, or greater valency (i.e., containing 2, 3, 4, 5, 6, 7 or more antigen-binding sites). For example, dimers of whole immunoglobulin molecules or of F(ab′)2 fragments are tetravalent, whereas dimers of Fab fragments or scFv molecules are bivalent.
• Individual antibodies within a multimer can have the same or different binding specificites. Typically, the multimers are monospecific, containing two or more antigen-binding domains that immunospecifically bind to the same epitope on DLL4. In some examples, antibody multimers can be generated that are multispecific, containing two or more antigen-binding domains that immunospecifically bind to two of more different epitopes. The epitopes can be DLL4 epitopes. In some examples, the antibody multimers bind an epitope in DLL4 and also bind an epitope in another different target antigen.
• Techniques for engineering antibody multimers are known in the art, and include, for example, linkage of two or more variable heavy chains and variable light chains via covalent, non-covalent, or chemical linkage. Multimerization of antibodies can be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. Thus, multimerization between two antibody polypeptide chains or antigen-binding fragments can be spontaneous, or can occur due to forced linkage of two or more polypeptides. In one example, antibody multimers can be generated by disulfide bonds formed between cysteine residues on different polypeptide chains. In another example, antibody multimers are generated by joining polypeptides via covalent or non-covalent interactions. In some examples, multimers can be generated form peptides such as peptide linkers (spacers), or peptides that have the property of promoting multimerization. In some examples, antibody multimers can be formed through chemical linkage, such as for example, by using heterobifunctional linkers.
• For example, antibody multimers include antibodies that contain a light chain containing a VL-CL and a heavy chain containing a VH-CH1-hinge and a sufficient portion of CH2-CH3 (or CH4 if of an IgE or IgM class) to permit association of heavy chains. Upon purification, such antibodies (e.g. full length IgG1) spontaneously form aggregates containing antibody homodimers, and other higher-order antibody multimers. Exemplary of a constant region can include a constant region portion of an immunoglobulin molecule, such as from IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgM, and IgE. Sequences of antibody regions are known and can be used to recombinantly generate antibody multimers (see e.g. US20080248028). For example, a light chain amino acid sequence can include the CL domain, kappa (set forth in SEQ ID NO:2923) or lambda (SEQ ID NO:2924). A heavy chain amino acid sequence can include one or more of a CH1, hinge, CH2, CH3 or CH4 from an IgG1 (SEQ ID NO:2922), IgG2 (SEQ ID NO: 2937), IgG3 (SEQ ID NO:2925), IgA (SEQ ID NO:2926 or 2927) or IgM (SEQ ID NO:2928 or 2929) subclass. In particular, antibody multimers provided herein are full-length antibodies that contain a light chain containing a VL-CL and a heavy chain containing a VH-CH1-hinge-CH2-CH3. For example, in such an antibody multimer, the resulting antibody molecule is at least a four chain molecule where each heavy chain is linked to a light chain by a disulfide bond, and the two heavy chains are linked to each other by disulfide bonds. Linkage of the heavy chains also is mediated by a flexible region of the heavy chain, known as the hinge region.
• Alternatively, antibody homodimers can be formed through chemical linkage techniques known in the art. For example, heterobifunctional crosslinking agents, including, but not limited to, SMCC [succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate] and SATA [N-succinimidyl S-acethylthio-acetate] (available, for example, from Pierce Biotechnology, Inc. (Rockford, Ill.)) can be used to form antibody multimers. An exemplary protocol for the formation of antibody homodimers is given in Ghetie et al., Proceedings of the National Academy of Sciences USA (1997) 94:7509-7514. Antibody homodimers can be converted to Fab′2 homodimers through digestion with pepsin. Another way to form antibody homodimers is through the use of the autophilic T15 peptide described in Zhao and Kohler, The Journal of Immunology (2002) 25:396-404.
• ScFv dimers can also be formed through recombinant techniques known in the art. For example, such an antibody multimer contains a variable heavy chain connected to a variable light chain on the same polypeptide chain (V_H-V_L) connected by a peptide linker that is too short to allow pairing between the two domains on the same chain. This forces pairing with the complementary domains of another chain and promotes the assembly of a dimeric molecule with two functional antigen binding sites. An example of the construction of scFv dimers is given in Goel et al., (2000) Cancer Research 60:6964-6971.
• Alternatively, antibodies can be made to multimerize through recombinant DNA techniques. IgM and IgA naturally form antibody multimers through the interaction with the mature J chain polypeptide (e.g., SEQ ID NO:2930). Non-IgA or non-IgM molecules, such as IgG molecules, can be engineered to contain the J chain interaction domain of IgA or IgM, thereby conferring the ability to form higher order multimers on the non-IgA or non-IgM molecules. (see, for example, Chintalacharuvu et al., (2001) Clinical Immunology 101:21-31. and Frigerio et al., (2000) Plant Physiology 123:1483-94). IgA dimers are naturally secreted into the lumen of mucosa-lined organs. This secretion is mediated through interaction of the J chain with the polymeric IgA receptor (pIgR) on epithelial cells. If secretion of an IgA form of an antibody (or of an antibody engineered to contain a J chain interaction domain) is not desired, it can be greatly reduced by expressing the antibody molecule in association with a mutant J chain that does not interact well with pIgR (e.g., SEQ ID NOS:2931-2933; Johansen et al., The Journal of Immunology (2001) 167:5185-5192). SEQ ID NO:2931 is a mutant form of a human mature J chain with C134S mutation compared to the mature form of human J chain (SEQ ID NO:2930). SEQ ID NO:2932 is a mutant form of a human mature J chain with amino acids 113-137 deleted compared to the mature form of human J chain (SEQ ID NO:2930). SEQ ID NO:2933 shows a mutant form of human mature J chain with C109S and C134S mutation compared to the mature form of human J chain (SEQ ID NO:2930). Expression of an antibody with one of these mutant J chains will reduce its ability to bind to the polymeric IgA receptor on epithelial cells, thereby reducing transport of the antibody across the epithelial cell and its resultant secretion into the lumen of mucosa lined organs.
• Antibody multimers may be purified using any suitable method known in the art, including, but not limited to, size exclusion chromatography. Exemplary methods for purifying antibodies are described elsewhere herein.
• Exemplary Antibodies
• An exemplary antibody multimer provided herein contains a variable heavy chain that contains a CDRH1 (corresponding to amino acid positions 26-35 based on kabat numbering) that has a sequence of amino acids of SYYMH (SEQ ID NO:2920), such as GYTFTSYYMH (SEQ ID NO: 2908), a CDRH2 (corresponding to amino acid positions 50-65 based on kabat numbering) that has a sequence of amino acids of IINPSGGSTSYAQKFQG (SEQ ID NO:2909), and a CDRH3 (corresponding to amino acid positions 95-102) that has a sequence of amino acids of EEYSSSSAEYFQH (SEQ ID NO:2910); and contains a variable light chain that contains a CDRL1 (corresponding to amino acid positions 24 to 33 or 34 based on kabat numbering) that has a sequence of amino acids of RASQSVSSYLA (SEQ ID NO: 2911), a CDRL2 (corresponding to amino acid positions 50-56 based on kabat numbering) that has a sequence of amino acids of amino acids of DASNRAT (SEQ ID NO:2912), and a CDRL3 (corresponding to amino acid positions 89-97 based on kabat numbering) that has a sequence of amino acids of QQRSNWPPWT (SEQ ID NO:2913). Also provided are antibody multimers that have a variable heavy chain containing a CDRH1, CDRH2 and CDRH3 that is at least 70% identical to any of SEQ ID NOS:2908-2910, respectively and a variable light chain containing a CDRL1, CDRL2, and CDRL3 that is at least 70% identical to any of SEQ ID NOS:2911-2913, respectively, whereby the antibody multimer binds to DLL4 and is an activator of DLL4. For example, sequence identity can be at or about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or more. For example, the antibody multimer is an antibody that at least contains a variable heavy chain set forth in SEQ ID NO:88 and a variable light chain set forth in SEQ ED NO:107, or a variable heavy chain or variable light chain that is at least 60% identical to SEQ ID NO:88 and/or 107, respectively. The antibody can be multimerized as described herein above. For example, provided herein is an antibody multimer that has a heavy chain containing a variable heavy chain region set forth in SEQ ID NO:88, and a CH1-hinge-CH2-CH3 set forth in SEQ ID NO: 2922, and contains a light chain containing a variable light chain set forth in SEQ ID NO:107 and a kappa or lambda CL chain set forth in SEQ ID NO:2923 or 2924.
• In another example, an exemplary antibody multimer provided herein contains a variable heavy chain that contains a CDRH1 (corresponding to amino acid positions 26-35 based on kabat numbering) that has a sequence of amino acids of SYWIG (SEQ ID NO: 2921), such as GYSFTSYWIG (SEQ ID NO:2914), a CDRH2 (corresponding to amino acid positions 50-65 based on kabat numbering) that has a sequence of amino acids of IIYPGDSDTRYSPSFQG (SEQ ID NO:2915), and a CDRH3 (corresponding to amino acid positions 95-102) that has a sequence of amino acids of RGYSYGYDYFDY (SEQ ID NO:2916); a contains a variable light chain that contains CDRL1 (corresponding to amino acid positions 24 to 33 or 34 based on kabat numbering) that has a sequence of amino acids of GLSSGSVSTSYYPS (SEQ ID NO:2917); a CDRL2 (corresponding to amino acid positions 50-56 based on kabat numbering) that has a sequence of amino acids of amino acids of STNTRSS (SEQ ID NO: 2918); and a CDRL3 (corresponding to amino acid positions 89-97 based on kabat numbering) that has a sequence of amino acids of VLYMGSGISYV (SEQ ID NO:2919). Also provided are antibody multimers that have a variable heavy chain containing a CDRH1, CDRH2 and CDRH3 that is at least 70% identical to any of SEQ ID NOS:2914-2916, respectively and a variable light chain containing a CDRL1, CDRL2, and CDRL3 that is at least 70% identical to any of SEQ ID NOS:2917-2919, respectively, whereby the antibody multimer binds to DLL4 and is an activator of DLL4. For example, sequence identity can be at or about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or more. For example, the antibody multimer is an antibody that at least contains a variable heavy chain set forth in SEQ ID NO:89 and a variable light chain set forth in SEQ ID NO:108, or a variable heavy chain or variable light chain that is at least 60% identical to SEQ ID NO:89 and/or 108, respectively. The antibody can be multimerized as described herein above. For example, provided herein is an antibody multimer that has a heavy chain containing a variable heavy chain region set forth in SEQ ID NO:89, and a CH1-hinge-CH2-CH3 set forth in SEQ ID NO: 2922, and contains a light chain containing a variable light chain set forth in SEQ ID NO:108 and a kappa or lambda CL chain set forth in SEQ ID NO:2923 or 2924.
• In some examples, that anti-DLL4 antibody multimers provided herein include activator/modulators of DLL4 activity, with the proviso that the antibody is not an antibody that has a heavy chain containing a variable heavy chain set forth in SEQ ID NO:88 and a variable light chain set forth in SEQ ID NO:107; or is not an antibody that has a heavy chain containing a variable heavy chain set forth in SEQ ID NO:89 and a variable light chain set forth in SEQ ID NO:108.
• 3. Modifications
• The anti-DLL4 antibody multimers provided herein can be further modified so long as the antibody retains binding to DLL4 and is an activator of DLL4 activity. Modification of an anti-DLL4 antibody multimer provided herein can improve one or more properties of the antibody, including, but not limited to, decreasing the immunogenicity of the antibody; improving the half-life of the antibody, such as reducing the susceptibility to proteolysis and/or reducing susceptibility to oxidation; altering or improving of the binding properties of the antibody; and/or modulating the effector functions of the antibody. Exemplary modifications include modification of the primary sequence of the antibody and/or alteration of the post-translational modification of an antibody. Exemplary post-translational modifications include, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with protecting/blocking group, proteolytic cleavage, and linkage to a cellular ligand or other protein. Other exemplary modifications include attachment of one or more heterologous peptides to the antibody to alter or improve one or more properties of the antibody.
• Generally, the modifications do not result in increased immunogenicity of the antibody or antigen-binding fragment thereof or significantly negatively affect the binding of the antibody to DLL4 or its activity as an activator. Methods of assessing the binding of the modified antibodies to DLL4 are provided herein and are known in the art. For example, modified antibodies can be assayed for binding to DLL4 by methods such as, but not limited to, ELISA or FACS binding assays. Methods to assess activating activity of the antibody also are known to one of skill in the art and described elsewhere herein, for examples, in the Examples. For example, activity can be determined using a reporter assay for activity of a Notch receptor.
• Modification of the anti-DLL4 antibodies produced herein can include one or more amino acid substitutions, deletions or additions, compared to the parent antibody from which it was derived. Methods for modification of polypeptides, such as antibodies, are known in the art and can be employed for the modification of any antibody or antigen-binding fragment provided herein. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide molecule encoding an antibody or an antigen-binding fragment provided herein in order to produce a polypeptide with one or more amino acid substitutions. Exemplary techniques for introducing mutations include, but are not limited to, site-directed mutagenesis and PCR-mediated mutagenesis.
• The antibodies can be recombinantly fused to a heterologous polypeptide at the N-terminus or C-terminus or chemically conjugated, including covalent and non-covalent conjugation, to a heterologous polypeptide or other composition. The fusion does not necessarily need to be direct, but can occur through a linker peptide. In some examples, the linker peptide contains a protease cleavage site which allows for removal of the purification peptide following purification by cleavage with a protease that specifically recognizes the protease cleavage site.
• For example, the anti-DLL4 antibodies provided herein can be modified by the attachment of a heterologous peptide to facilitate purification. Generally such peptides are expressed as a fusion protein containing the antibody fused to the peptide at the C- or N-terminus of the antibody. Exemplary peptides commonly used for purification include, but are not limited to, hexa-histidine peptides, hemagglutinin (HA) peptides, and flag tag peptides (see e.g., Wilson et al. (1984) Cell 37:767; Witzgall et al. (1994) Anal Biochem 223:2, 291-8). In another example, the anti-DLL4 antibodies provided herein can be modified by the covalent attachment of any type of molecule, such as a diagnostic or therapeutic molecule. Exemplary diagnostic and therapeutic moieties include, but are not limited to, drugs, radionucleotides, toxins, fluorescent molecules (see, e.g. International PCT Publication Nos. WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387). Diagnostic polypeptides or diagnostic moieties can be used, for example, as labels for in vivo or in vitro detection. In a further example, anti-DLL4 antibody multimers provided herein can be modified by attachment to other molecules or moieties, such as any that increase the half-life, stability, immunogenicity or that affect or alter the targeting of the antibody in vivo.
• Exemplary modifications are described herein below. It is within the level of one of skill in the art to modify any of the antibodies provided herein depending on the particular application of the antibody.
• a. Modifications to Reduce Immunogenicity
• In some examples, the antibodies provided herein can be modified to reduce the immunogenicity in a subject, such as a human subject. For example, one or more amino acids in the antibody can be modified to alter potential epitopes for human T-cells in order to eliminate or reduce the immunogenicity of the antibody when exposed to the immune system of the subject. Exemplary modifications include substitutions, deletions and insertion of one or more amino acids, which eliminate or reduce the immunogenicity of the antibody. Generally, such modifications do not alter the binding specificity of the antibody for its respective antigen. Reducing the immunogenicity of the antibody can improve one or more properties of the antibody, such as, for example, improving the therapeutic efficacy of the antibody and/or increasing the half-life of the antibody in vivo.
• b. Glycosylation
• The anti-DLL4 antibodies provided herein can be modified by either N-linked or O-linked glycosylation. N-linked glycosylation includes the attachment of a carbohydrate moiety to the side chain of an asparagine residue within the tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation includes the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used. The anti-DLL4 antibodies can be further modified to incorporate additional glycosylation sites by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration can also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites). Where the antibody comprises an Fc region, the carbohydrate attached thereto can be altered (see, e.g., U.S. Patent Pub. Nos. 2003/0157108, 2005/0123546 and US 2004/0093621; International Patent Pub. Nos. WO 2003/011878, WO 1997/30087, WO 1998/58964, WO 1999/22764; and U.S. Pat. No. 6,602,684).
• For example, a glycosylation variantion is in the Fc region of the antibody, wherein a carbohydrate structure attached to the Fc region lacks fucose. Such variants have improved ADCC function. Optionally, the Fc region further contains one or more amino acid substitutions therein which further improve ADCC, for example, substitutions at positions 298, 333, and/or 334 of the Fc region (Eu numbering of residues) (see, e.g., US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)). Examples of cell lines producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al., especially at Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)).
• c. Fc Modifications
• The anti-DLL4 antibody multimers provided herein can contain wild-type or modified Fc region. The antibodies provided herein can be engineered to contain modified Fc regions. In some examples, the Fc region can be modified to alter one or more properties of the Fc polypeptide. For example, the Fc region can be modified to alter (i.e. increase or decrease) effector functions compared to the effector function of an Fc region of a wild-type immunoglobulin heavy chain. Thus, a modified Fc domain can have altered affinity, including but not limited to, increased or low or no affinity for the Fc receptor. Altering the affinity of an Fc region for a receptor can modulate the effector functions induced by the Fc domain.
• In one example, an Fc region is used that is modified for optimized binding to certain FcγRs to better mediate effector functions, such as for example, antibody-dependent cellular cytotoxicity, ADCC. Such modified Fc regions can contain modifications at one or more of amino acid residues (according to the Kabat numbering scheme, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services), including, but not limited to, amino acid positions 249, 252, 259, 262, 268, 271, 273, 277, 280, 281, 285, 287, 296, 300, 317, 323, 343, 345, 346, 349, 351, 352, 353, and 424. For example, modifications in an Fc region can be made corresponding to any one or more of G119S, G119A, S122D, S122E, S122N, S122Q, S122T, K129H, K129Y, D132Y, R138Y, E141Y, T143H, V147I, S150E, H151D, E155Y, E155I, E155H, K157E, G164D, E166L, E166H, S181A, S181D, S187T, S207G, S2071, K209T, K209E, K209D, A210D, A213Y, A213L, A213I, I215D, I215E, I215N, I215Q, E216Y, E216A, K217T, K217F, K217A, and P279L of the exemplary Fc sequence set forth in SEQ ID NO:2922, or combinations thereof. A modified Fc containing these mutations can have enhanced binding to an FcR such as, for example, the activating receptor FcγIIIa and/or can have reduced binding to the inhibitory receptor FcγRIIb (see e.g., US 2006/0024298). Fc regions modified to have increased binding to FcRs can be more effective in facilitating the destruction of the fungal cells in patients.
• In some examples, the antibodies or antigen-binding fragments provided herein can be further modified to improve the interaction of the antibody with the FcRn receptor in order to increase the in vivo half-life and pharmacokinetics of the antibody (see, e.g. U.S. Pat. No. 7,217,797; and U.S. Pat. Pub. Nos. 2006/0198840 and 2008/0287657). FcRn is the neonatal FcR, the binding of which recycles endocytosed antibody from the endosomes back to the bloodstream. This process, coupled with preclusion of kidney filtration due to the large size of the full length molecule, results in favorable antibody serum half-lives ranging from one to three weeks. Binding of Fc to FcRn also plays a role in antibody transport.
• Exemplary modifications of the Fc region include but are not limited to, mutation of the Fc described in U.S. Pat. No. 7,217,797; U.S. Pat. Pub. Nos. 2006/0198840, 2006/0024298 and 2008/0287657; and International Patent Pub. No. WO 2005/063816, such as mutations at one or more of amino acid residues (Kabat numbering, Kabat et al. (1991)) 251-256, 285-90, 308-314, in the C_H2 domain and/or amino acids residues 385-389, and 428-436 in the C _H3 domain of the Fc heavy chain constant region, where the modification alters Fc receptor binding affinity and/or serum half-life relative to unmodified antibody. In some examples, the Fc region is modified at one or more of amino acid positions 250, 251, 252, 254, 255, 256, 263, 308, 309, 311, 312 and 314 in the C_H2 domain and/or amino acid positions 385, 386, 387, 389, 428, 433, 434, 436, and 459 in the C _H3 domain of the Fc heavy chain constant region. Such modifications correspond to amino acids Gly120, Pro121, Ser122, Phe124 Leu125, Phe126, Thr133, Pro174, Arg175, Glu177, Gln178, and Asn180 in the C_H2 domain and amino acids Gln245, Val246, Ser247, Thr249, Ser283, Gly285, Ser286, Phe288, and Met311 in the C _H3 domain in an exemplary Fc sequence set forth in SEQ ID NO:2922 In some examples, the modification is at one or more surface-exposed residues, and the modification is a substitution with a residue of similar charge, polarity or hydrophobicity to the residue being substituted.
• In particular examples, a Fc heavy chain constant region is modified at one or more of amino acid positions 251, 252, 254, 255, and 256 (Kabat numbering), where position 251 is substituted with Leu or Arg, position 252 is substituted with Tyr, Phe, Ser, Trp or Thr, position 254 is substituted with Thr or Ser, position 255 is substituted with Leu, Gly, Ile or Arg, and/or position 256 is substituted with Ser, Arg, Gln, Glu, Asp, Ala, Asp or Thr. In some examples, a Fc heavy chain constant region is modified at one or more of amino acid positions 308, 309, 311, 312, and 314 (Kabat numbering), where position 308 is substituted with Thr or Ile, position 309 is substituted with Pro, position 311 is substituted with serine or Glu, position 312 is substituted with Asp, and/or position 314 is substituted with Leu. In some examples, a Fc heavy chain constant region is modified at one or more of amino acid positions 428, 433, 434, and 436 (Kabat numbering), where position 428 is substituted with Met, Thr, Leu, Phe, or Ser, position 433 is substituted with Lys, Arg, Ser, Ile, Pro, Gln, or His, position 434 is substituted with Phe, Tyr, or His, and/or position 436 is substituted with His, Asn, Asp, Thr, Lys, Met, or Thr. In some examples, a Fc heavy chain constant region is modified at one or more of amino acid positions 263 and 459 (Kabat numbering), where position 263 is substituted with Gln or Glu and/or position 459 is substituted with Leu or Phe.
• In some examples, a Fc heavy chain constant region can be modified to enhance binding to the complement protein C1q. In addition to interacting with FcRs, Fc also interact with the complement protein C1q to mediate complement dependent cytotoxicity (CDC). C1q forms a complex with the serine proteases C1r and C1s to form the C1 complex. C1q is capable of binding six antibodies, although binding to two IgGs is sufficient to activate the complement cascade. Similar to Fc interaction with FcRs, different IgG subclasses have different affinity for C1q, with IgG1 and IgG3 typically binding substantially better than IgG2 and IgG4. Thus, a modified Fc having increased binding to C1q can mediate enhanced CDC, and can enhance destruction of fungal cells. Exemplary modifications in an Fc region that increase binding to C1q include, but are not limited to, amino acid modifications at positions 345 and 253 (Kabat numbering). Exemplary modifications are include those corresponding to K209W, K209Y, and E216S in an exemplary Fc sequence set forth in SEQ ID NO:2922.
• In another example, a variety of Fc mutants with substitutions to reduce or ablate binding with FcγRs also are known. Such muteins are useful in instances where there is a need for reduced or eliminated effector function mediated by Fc. This is often the case where antagonism, but not killing of the cells bearing a target antigen is desired. Exemplary of such an Fc is an Fc mutein described in U.S. Pat. No. 5,457,035, which is modified at amino acid positions 248, 249 and 251 (Kabat numbering). In an exemplary Fc sequence set forth in amino acids 100-330 of SEQ ID NO:2922, amino acid 118 is modified from Leu to Ala, amino acid 119 is modified from Leu to Glu, and amino acid 121 is modified from Gly to Ala. Similar mutations can be made in any Fc sequence such as, for example, the exemplary Fc sequence. This mutein exhibits reduced affinity for Fc receptors.
• d. Pegylation
• The anti-DLL4 antibody multimers provided herein can be conjugated to polymer molecules, or water soluble polymers, such as high molecular weight polyethylene glycol (PEG) to increase half-life and/or improve their pharmacokinetic profiles. Water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde can have advantages in manufacturing due to its stability in water. The polymer can be of any molecular weight, and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, and whether the antibody derivative will be used in a therapy under defined conditions.
• Conjugation can be carried out by techniques known to those skilled in the art. Conjugation of therapeutic antibodies with PEG has been shown to enhance pharmacodynamics while not interfering with function (see, e.g., Deckert et al., Int. J. Cancer 87: 382-390, 2000; Knight et al., Platelets 15: 409-418, 2004; Leong et al., Cytokine 16: 106-119, 2001; and Yang et al., Protein Eng. 16: 761-770, 2003). PEG can be attached to the antibodies or antigen-binding fragments with or without a multifunctional linker either through site-specific conjugation of the PEG to the N- or C-terminus of the antibodies or antigen-binding fragments or via epsilon-amino groups present on lysine residues. Linear or branched polymer derivatization that results in minimal loss of biological activity can be used. The degree of conjugation can be monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibodies. Unreacted PEG can be separated from antibody-PEG conjugates by, e.g., size exclusion or ion-exchange chromatography. PEG-derivatized antibodies can be tested for binding activity to DLL4 as well as for in vivo efficacy using methods known to those skilled in the art, for example, by functional assays described herein.
• 4. Compositions, Formulations, Administration and Articles of Manufacture/Kits
• a. Compositions and Formulations
• The antibody multimers provided herein can be provided as a formulation for administration. While it is possible for the active ingredient to be administered alone, generally it is present as a pharmaceutical formulation. Compositions or formulations contain at least one active ingredient, together with one or more acceptable carriers thereof. Each carrier must be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient. Formulations include those suitable for oral, rectal, nasal, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations can conveniently be presented in unit dosage form and can be prepared by methods well known in the art of pharmacy. See, e.g., Gilman, et al. (eds. 1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co., Easton, Pa.; Avis, et al. (eds. 1993) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, NY; Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms Tablets Dekker, NY; and Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms Disperse Systems Dekker, NY.
• The route of antibody administration is in accord with known methods, e.g., injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, subcutaneous, intraocular, intraarterial, intrathecal, inhalation or intralesional routes, topical or by sustained release systems as noted below. The antibody is typically administered continuously by infusion or by bolus injection. One can administer the antibodies in a local or systemic manner.
• The antibody multimers provided herein can be prepared in a mixture with a pharmaceutically acceptable carrier. Techniques for formulation and administration of the compounds are known to one of skill in the art (see e.g. “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.). This therapeutic composition can be administered intravenously or through the nose or lung, preferably as a liquid or powder aerosol (lyophilized). The composition also can be administered parenterally or subcutaneously as desired. When administered systematically, the therapeutic composition should be sterile, pyrogen-free and in a parenterally acceptable solution having due regard for pH, isotonicity, and stability. These conditions are known to those skilled in the art.
• Therapeutic formulations can be administered in many conventional dosage formulations. Briefly, dosage formulations of the antibodies provided herein are prepared for storage or administration by mixing the compound having the desired degree of purity with physiologically acceptable carriers, excipients, or stabilizers. Such materials are non-toxic to the recipients at the dosages and concentrations employed, and can include buffers such as TRIS HCl, phosphate, citrate, acetate and other organic acid salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) peptides such as polyarginine, proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidinone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium and/or nonionic surfactants such as TWEEN, PLURONICS or polyethyleneglycol.
• When used for in vivo administration, the antibody multimer formulation should be sterile and can be formulated according to conventional pharmaceutical practice. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. The antibody ordinarily will be stored in lyophilized foam or in solution. Other vehicles such as naturally occurring vegetable oil like sesame, peanut, or cottonseed oil or a synthetic fatty vehicle like ethyl oleate or the like may be desired. Buffers, preservatives, antioxidants and the like can be incorporated according to accepted pharmaceutical practice.
• Pharmaceutical compositions suitable for use include compositions wherein one or more antibody multimers are contained in an amount effective to achieve their intended purpose. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. Therapeutically effective dosages can be determined by using in vitro and in vivo methods.
• An effective amount of antibody to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient. In addition, the attending physician takes into consideration various factors known to modify the action of drugs including severity and type of disease, body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. Typically, the clinician will administer antibody until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays.
• For any antibody containing a peptide, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the EC50 as determined in cell culture (e.g., the concentration of the test molecule which promotes or inhibits cellular proliferation or differentiation). Such information can be used to more accurately determine useful doses in humans.
• Toxicity and therapeutic efficacy of the antibody multimers described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. Molecules which exhibit high therapeutic indices can be used. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such molecules lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1, p. 1).
• Dosage amount and interval can be adjusted individually to provide plasma levels of the antibody which are sufficient to promote or inhibit cellular proliferation or differentiation or minimal effective concentration (MEC). The MEC will vary for each antibody, but can be estimated from in vitro data using described assays. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.
• Dosage intervals can also be determined using MEC value. Antibody molecules should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.
• In cases of local administration or selective uptake, the effective local concentration of the antibody may not be related to plasma concentration.
• A typical daily dosage might range of antibody multimers provided herein is from about 1 μ/kg to up to 1000 mg/kg or more, depending on the factors mentioned above. Typically, the clinician will administer the molecule until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays.
• Depending on the type and severity of the disease, from about 0.001 mg/kg to abut 1000 mg/kg, such as about 0.01 mg to 100 mg/kg, for example about 0.010 to 20 mg/kg of the antibody multimer, is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs or the desired improvement in the patient's condition is achieved. However, other dosage regimes also are contemplated.
• b. Articles of Manufacture and Kits
• Pharmaceutical compounds of selected antibodies or nucleic acids encoding selected antibodies, or a derivative or a biologically active portion thereof can be packaged as articles of manufacture containing packaging material, a pharmaceutical composition which is effective for treating the disease or disorder, and a label that indicates that selected antibody or nucleic acid molecule is to be used for treating the disease or disorder.
• The articles of manufacture provided herein contain packaging materials. Packaging materials for use in packaging pharmaceutical products are well known to those of skill in the art. See, for example, U.S. Pat. Nos. 5,323,907, 5,052,558 and 5,033,252, each of which is incorporated herein in its entirety. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment. A wide array of formulations of the compounds and compositions provided herein are contemplated as are a variety of treatments for any EPO-mediated disease or disorder or therapeutic polypeptide-mediated disease or disorder.
• Antibodies and nucleic acid molecules encoding the antibodies thereof also can be provided as kits. Kits can include a pharmaceutical composition described herein and an item for administration. For example, a selected antibody can be supplied with a device for administration, such as a syringe, an inhaler, a dosage cup, a dropper, or an applicator. The kit can, optionally, include instructions for application including dosages, dosing regimens and instructions for modes of administration. Kits also can include a pharmaceutical composition described herein and an item for diagnosis. For example, such kits can include an item for measuring the concentration, amount or activity of the antibody in a subject.
• 5. Methods of Treatment and Uses
• Provided herein are methods of treatment or uses of anti-DLL4 antibody multimers to treat diseases that manifest aberrant angiogenesis or neovascularization. Angiogenesis is a process by which new blood vessels are formed. It occurs for example, in a healthy body for healing wounds and for restoring blood flow to tissues after injury or insult. In females, angiogenesis also occurs during the monthly reproductive cycle to rebuild the uterus lining, to mature the egg during ovulation and during pregnancy to build the placenta. In some situations ‘too much’ angiogenesis can be detrimental, such as angiogenesis that supplies blood to tumor foci, in inflammatory responses and other aberrant angiogenic-related conditions. The growth of tumors, or sites of proliferation in chronic inflammation, generally requires the recruitment of neighboring blood vessels and vascular endothelial cells to support their metabolic requirements. This is because the diffusion is limited for oxygen in tissues. Exemplary conditions associated with angiogenesis include, but are not limited to solid tumors and hematologic malignancies such as lymphomas, acute leukemia, and multiple myeloma, where increased numbers of blood vessels are observed in the pathologic bone marrow.
• Hence, angiogenesis is implicated in the pathogenesis of a variety of disorders. These include solid tumors and metastasis, atherosclerosis, retrolental fibroplasia, hemangiomas, chronic inflammation, intraocular neovascular diseases such as proliferative retinopathies, e.g., diabetic retinopathy, age-related macular degeneration (AMD), neovascular glaucoma, immune rejection of transplanted corneal tissue and other tissues, rheumatoid arthritis, and psoriasis. Folkman et al., J. Biol. Chem. 267:10931-34 (1992); Klagsbrun et al., Annu. Rev. Physiol. 53:217-39 (1991); and Garner A., “Vascular diseases,” In: Pathobiology of Ocular Disease. A Dynamic Approach, Garner A., Klintworth G K, eds., 2nd Edition (Marcel Dekker, NY, 1994), pp 1625-1710.
• In the case of tumor growth, angiogenesis appears to be crucial for the transition from hyperplasia to neoplasia, and for providing nourishment for the growth and metastasis of the tumor. Folkman et al., Nature 339:58 (1989). The neovascularization allows the tumor cells to acquire a growth advantage and proliferative autonomy compared to the normal cells. A tumor usually begins as a single aberrant cell which can proliferate only to a size of a few cubic millimeters due to the distance from available capillary beds, and it can stay ‘dormant’ without further growth and dissemination for a long period of time. Some tumor cells then switch to the angiogenic phenotype to activate endothelial cells, which proliferate and mature into new capillary blood vessels. These newly formed blood vessels not only allow for continued growth of the primary tumor, but also for the dissemination and recolonization of metastatic tumor cells. Accordingly, a correlation has been observed between density of microvessels in tumor sections and patient survival in breast cancer as well as in several other tumors. Weidner et al., N Engl. J. Med. 324:1-6 (1991); Horak et al., Lancet 340:1120-24 (1992); Macchiarini et al., Lancet 340:145-46 (1992). The precise mechanisms that control the angiogenic switch is not well understood, but it is believed that neovascularization of tumor mass results from the net balance of a multitude of angiogenesis stimulators and inhibitors (Folkman, Nat. Med. 1(1):27-31 (1995)).
• Angiogenesis also play a role in inflammatory diseases. These diseases have a proliferative component, similar to a tumor focus. In rheumatoid arthritis, one component of this is characterized by aberrant proliferation of synovial fibroblasts, resulting in pannus formation. The pannus is composed of synovial fibroblasts which have some phenotypic characteristics with transformed cells. As a pannus grows within the joint it expresses many proangiogenic signals, and experiences many of the same neo-angiogenic requirements as a tumor. The need for additional blood supply, neoangiogenesis, is critical. Similarly, many chronic inflammatory conditions also have a proliferative component in which some of the cells composing it may have characteristics usually attributed to transformed cells.
• Another example of a condition involving excess angiogenesis is diabetic retinopathy (Lip et al. Br J Ophthalmology 88: 1543, 2004)). Diabetic retinopathy has angiogenic, inflammatory and proliferative components; overexpression of VEGF, and angiopoietin-2 are common. This overexpression is likely required for disease-associated remodeling and branching of blood vessels, which then supports the proliferative component of the disease.
• Hence, provided herein are methods of treatment with anti-DLL4 antibody multimers for angiogenic diseases and conditions. Such diseases or conditions include, but are not limited to, inflammatory diseases, immune diseases, cancers, and other diseases that manifest aberrant angiogenesis and abnormal vascularization. Cancers include breast, lung, colon, gastric cancers, pancreatic cancers and others. Inflammatory diseases, include, for example, diabetic retinopathies and/or neuropathies and other inflammatory vascular complications of diabetes, autoimmune diseases, including autoimmune diabetes, atherosclerosis, Crohn's disease, diabetic kidney disease, cystic fibrosis, endometriosis, diabetes-induced vascular injury, inflammatory bowel disease, Alzheimers disease and other neurodegenerative diseases. Treatment can be effected by administering by suitable route formulations of the antibody multimers, which can be provided in compositions as polypeptides. In some examples, the antibody multimers can be linked to targeting agents, for targeted delivery or encapsulated in delivery vehicles, such as liposomes.
• For example, treatments using the anti-DLL4 multimers provided herein, include, but are not limited to treatment of diabetes-related diseases and conditions including periodontal, autoimmune, vascular, and tubulointerstitial diseases. Treatments using the anti-DLL4 antibody multimers also include treatment of ocular disease including macular degeneration, cardiovascular disease, neurodegenerative disease including Alzheimer's disease, inflammatory diseases and conditions including rhematoid arthritis, and diseases and conditions associated with cell proliferation including cancers. One of skill in the art can assess based on the type of disease to be treated, the severity and course of the disease, whether the molecule is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to therapy, and the discretion of the attending physician appropriate dosage of a molecule to administer.
• Combination Therapy
• Anti-DLL4 antibody multimers provided herein can be administered in combination with another therapy. For example, anti-DLL4 antibody multimers are used in combinations with anti-cancer therapeutics or anti-neovascularization therapeutics to treat various neoplastic or non-neoplastic conditions. In one embodiment, the neoplastic or non-neoplastic condition is characterized by pathological disorder associated with aberrant or undesired angiogenesis. Exemplary combination therapies also include any set forth in U.S. Published application No. 20090246199. The anti-DLL4 antibody multimer can be administered serially or in combination with another agent that is effective for those purposes, either in the same composition or as separate compositions. The anti-DLL4 antibody multimers can be administered sequentially, simultaneously or intermittently with a therapeutic agent. Alternatively, or additionally, multiple inhibitors of DLL4 can be administered.
• The administration of the anti-DLL4 antibody multimer can be done simultaneously, e.g., as a single composition or as two or more distinct compositions using the same or different administration routes. Alternatively, or additionally, the administration can be done sequentially, in any order. In certain embodiments, intervals ranging from minutes to days, to weeks to months, can be present between the administrations of the two or more compositions. For example, the anti-cancer agent can be administered first, followed by the DLL4 antibody multimer. Simultaneous administration or administration of the anti-DLL4 antibody multimer first also is contemplated.
• The effective amounts of therapeutic agents administered in combination with an anti-DLL4 antibody multimer will be at the physician's or veterinarian's discretion. Dosage administration and adjustment is done to achieve maximal management of the conditions to be treated. The dose will additionally depend on such factors as the type of therapeutic agent to be used and the specific patient being treated. Suitable dosages for the anti-cancer agent are those presently used and can be lowered due to the combined action (synergy) of the anti-cancer agent and the anti-DLL4 antibody multimer.
• Typically, the anti-DLL4 antibody multimer and anti-cancer agents are suitable for the same or similar diseases to block or reduce a pathological disorder such as tumor growth or growth of a cancer cell. In one embodiment the anti-cancer agent is an anti-angiogenesis agent. Antiangiogenic therapy in relationship to cancer is a cancer treatment strategy aimed at inhibiting the development of tumor blood vessels required for providing nutrients to support tumor growth. Because angiogenesis is involved in both primary tumor growth and metastasis, the antiangiogenic treatment is generally capable of inhibiting the neoplastic growth of tumor at the primary site as well as preventing metastasis of tumors at the secondary sites, therefore allowing attack of the tumors by other therapeutics.
• Many anti-angiogenic agents have been identified and are known in the arts, including those listed herein, e.g., listed under Definitions, and by, e.g., Carmeliet and Jain, Nature 407:249-257 (2000); Ferrara et al., Nature Reviews. Drug Discovery, 3:391-400 (2004); and Sato Int. J. Clin. Oncol., 8:200-206 (2003). See also, US Patent Application US20030055006. In one embodiment, an anti-DLL4 antibody multimer is used in combination with an anti-VEGF neutralizing antibody (or fragment) and/or another VEGF antagonist or a VEGF receptor antagonist including, but not limited to, for example, soluble VEGF receptor (e.g., VEGFR-1, VEGFR-2, VEGFR-3, neuropillins (e.g., NRP1, NRP2)) fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, low molecule weight inhibitors of VEGFR tyrosine kinases (RTK), antisense strategies for VEGF, ribozymes against VEGF or VEGF receptors, antagonist variants of VEGF; and any combinations thereof. Alternatively, or additionally, two or more angiogenesis inhibitors can optionally be co-administered to the patient in addition to VEGF antagonist and other agent. In certain embodiment, one or more additional therapeutic agents, e.g., anti-cancer agents, can be administered in combination with anti-DLL4 antibody multimer, the VEGF antagonist, and an anti-angiogenesis agent.
• In certain aspects, other therapeutic agents useful for combination angiogenic or tumor therapy with a anti-DLL4 antibody mulitmer include other cancer therapies, (e.g., surgery, radiological treatments (e.g., involving irradiation or administration of radioactive substances), chemotherapy, treatment with anti-cancer agents listed herein and known in the art, or combinations thereof). Alternatively, or additionally, two or more antibodies binding the same or two or more different antigens disclosed herein can be co-administered to the patient. Sometimes, it can be beneficial to also administer one or more cytokines to the patient.
H. EXAMPLES
• The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1 Generation of Mutant Fab Antibodies
• In this Example, mutant Fab antibodies were generated by alanine-scanning, NNK mutagenesis, and ligation of oligo pairs into BsaI modified plasmids that allow cloning of any modified CDR region in a high-throughput manner
A. Alanine Scanning Mutagenesis
• Alanine mutants were generated by overlapping PCR using the parent heavy or light chain DNA as a template. Forward and reverse primers that specifically generate the desired mutation at the target codon were used to amplify the parent DNA in the appropriate plasmid.
• In the first round of PCR, two separate PCR reactions with different primer pairs were used to amplify two segments of the gene. The first reaction used the specific reverse primer with an EcoRI forward primer and amplified the first half of the gene. The second reaction used the specific forward primer with an FLXhoI reverse primer and amplified the second half of the gene. The gene segments were generated using 20 cycles of PCR with the following conditions: 94° C. for 30 sec; 50° C. for 30 sec; and 72° C. for 90 sec. The PCR products were isolated and purified from 1% agarose gel and mixed together as a template for the second round of PCR. In the second round of PCR, EcoRI forward and FLXhoI reverse primers were used to amplify the full length gene product. The gene product was generated using 20 cycles of PCR with the following conditions: 94° C. for 30 sec; 55° C. for 30 sec; and 72° C. for 90 sec.
• The PCR product was isolated and subsequently digested with EcoRI and XhoI (New England Biolabs) and ligated into the similarly digested plasmid. After transformation of the ligation product in E. coli DH5α and plating, individual colonies were selected and grown in a 96-well block containing 1.5 ml of Terrific Broth (EMD, San Diego, Calif.) supplemented with 50 μg/ml Kanamycin, and 0.4% glucose, and grown at 37° C. overnight. The DNA was isolated using a mini-prep kit (Qiagen) and alanine mutations were confirmed by DNA sequencing.
• As an example, Table 6 sets forth primer pairs used to generate the mutant VH5-51_IGHD5-18*01>3_IGHJ4*01 R99A and VH1-46_IGHD6-6*01_IGHJ1*01 E100A. Primers R99A_F and R99A_R were utilized to specifically amplify the R99 to alanine mutation. Primers E100A_F and E100A_R were utilized to specifically amplify the E100 to alanine mutation. Primers EcoRI_F and FLXhoI_R were utilized to amplify the remaining segments of the gene.

• TABLE 6
  Example primer pairs for alanine scanning mutagenesis

  Primer	Sequence	SEQ ID NO

  VH5-51_IGHD5-18*01>3_IGHJ4*01

  R99A_F	GCCATGTATTACTGTGCGAGAGCCGGATACAGCTATGGTTACGAC	1
  R99A_R	GTCGTAACCATAGCTGTATCCGGCTCTCGCACAGTAATACATGGC	2

  VH1-46_IGHD6-6*01_IGHJ1*01

  E100A_F	GTGTATTACTGTGCGAGAGAGGCCTATAGCAGCTCGTCCGCTG		3
  E100A_R	CAGCGGACGAGCTGCTATAGGCCTCTCTCGCACAGTAATACAC	4

  Plasmid A and D

  EcoRI_F	TTGGGCGAATTCCCTAGATAATTAATTAGGAGG	5
  FLXhoI_R	TTAAACCTCGAGCCGCGGTTCATTAAAG	6

B. NNK Mutagenesis by Overlapping PCR
• NNK mutagenesis by overlapping PCR was carried out as described above for alanine scanning mutagenesis, with initial primers that generate the desired NNK mutations. Therefore, in the first round of PCR, specific primer pairs were used in which the target codon was replaced with NNK (forward) and MNN (reverse). For example, Table 7 below sets forth forward and reverse primers used to generate VH5-51_IGHD5-18*01>3_IGHJ4*01 G100 NNK mutants and VH1-46_IGHD6-6*01_IGHJ1*01 S102 NNK mutants.
• Individual clones were subjected to DNA sequencing (by BATJ, Inc., San Diego, Calif.) to identify the amino acid substitution. Depending on the number of colonies picked per NNK mutation reaction, mutation rate varies—as low as 4 to 5 amino acid changes, and as high as 18 to 19 amino acid changes per mutation were observed.

• TABLE 7
  Example primer pairs for NNK mutagenesis

  		SEQ
  		ID
  Primer	Sequence	NO

  VH5-51_IGHD5-18*01>3_IGHJ4*01

  G100_NNK_F	GTATTACTGTGCGAGACGTNNKTACAGCTATGGTT	7
  	ACGAC
  G100_NNK_R	GTCGTAACCATAGCTGTAMNNACGTCTCGCACAGT	8
  	AATAC

  VH1-46_IGHD6-6*01_IGHJ1*01

  S102_NNK_F	TGCGAGAGAGGGGTATNNKAGCAGCTGGTACGACT	9
  S102_NNK_R	AGTCGTACCAGCTGCTMNNATACCCCTCTCTCGCA	10

C. Cassette Mutagenesis Using Type II Restriction Enzyme Based Digestion and Ligation of Oligo Pairs
• In this example, Fab mutants were generated in a in a high-throughput manner by cloning of specific synthetic CDR1, CDR2 and/or CDR3 sequences into plasmids previously modified to contain BsaI cloning sites. Specifically, for each heavy or light chain, three vectors each were generated whereby a BsaI restriction site was incorporated at both the 5′ and 3′ end of each CDR region. To generate Fab mutants, forward and reverse primers encoding a CDR with specific mutations and additionally BsaI overlapping ends were synthesized and annealed. These cassettes, or mutated CDR regions, were then ligated into the corresponding BsaI digested vector, thereby generating a plasmid containing a specifically modified CDR region.
• For example, specific primers were synthesized (IDT, see Table 8 below) and used to generate three vectors each for heavy chains VH1-46_IGHD6-6*01_IGHJ1*01 and VH5-51_IGHD5-18*01>3_IGHJ4*01 and light chains L6_IGKJ1*01 and V3-4_IGLJ1*01, to incorporate a BsaI site at the beginning and end of CDR1, CDR2 and CDR3. The vectors were generated as described above using the specific forward and reverse primers in the first round of PCR and the parent heavy or light chain DNA as a template. Individual clones were subjected to DNA sequencing (by BATJ, Inc., San Diego, Calif.) to confirm the incorporation of two BsaI sites in each CDR.
• Subsequently, each BsaI containing plasmid was digested with BsaI (New England Biolabs) and the DNA was gel purified. Specific primers were synthesized (IDT) to generate desired mutants. Briefly, 1 μl of each forward and reverse primer were annealed by heating to 95° C. in TE for 2 min, followed by slow cooling to room temperature. 1 μl of the annealed primers were then ligated with 2 ng of the BsaI digested vector and transformed into E. coli DH5a cell. Mutations were confirmed by DNA sequencing. The ligation reactions can be carried out in a 96-well plate thereby allowing for high-throughput mutagenesis.
• For example, Table 8-9 below sets forth primers to generate VH1-46_IGHD6-6*01_IGHJ1*01_APFF CDR2 mutants.

• TABLE 8
  BsaI restriction enzyme mutagenesis primers

  		SEQ ID
  Primer	Sequence	NO
  VH1-46_C DR1_F	gagacctactatggttcgggtctctgggtgcgacaggcc	11
  VH1-46_C DR2_F	gagacctactatggttcgggtctcaagttccagggcagagtcac	12
  VH1-46_C DR3_F	gagacctactatggttcgggtctctggggccagggcac	13
  VH5-51_C DR1_F	gagacctactatggttcgggtctctgggtgcgccagatg	14
  VH5-51_C DR2_F	gagacctactatggttcgggtctccaggtcaccatctcagccg	15
  VH5-51_C DR3_F	gagacctactatggttcgggtctctggggccaaggaaccc	16
  L6_CDR1_F	gagacctactatggttcgggtctctggtaccaacagaaacctggc	17
  L6_CDR2_F	gagacctactatggttcgggtctcggcatcccagccagg	18
  L6_CDR3_F	gagacctactatggttcgggtctcttcggccaagggacca	19
  V3-4 CDR1_F	gagacctactatggttcgggtctctggtaccagcagacccca	20
  V3-4 CDR2_F	gagacctactatggttcgggtctcggggtccctgatcgcttc	21
  V3-4 CDR3_F	gagacctactatggttcgggtctcttcggaactgggaccaag	22
  Lambda_BSA_F	gagtggagacgaccacaccc	23
  VH1-46_C DR1_R	GAGACCCGAACCATAGTAGGTCTCAGATGCCTTGCAGGAAACC	24
  VH1-46_C DR2_R	GAGACCCGAACCATAGTAGGTCTCTCCCATCCACTCAAGCCC	25
  VH1-46_C DR3_R	GAGACCCGAACCATAGTAGGTCTCTCTCGCACAGTAATACACGG	26
  	C
  VH5-51_C DR1_R	GAGACCCGAACCATAGTAGGTCTCAGAACCCTTACAGGAGATCT	27
  	TCA
  VH5-51_C DR2_R	GAGACCCGAACCATAGTAGGTCTCCCCCATCCACTCCAGGC	28
  VH5-51_C DR3_R	GAGACCCGAACCATAGTAGGTCTCTCTCGCACAGTAATACATGG	29
  	C
  L6_CDR1_R	GAGACCCGAACCATAGTAGGTCTCGCAGGAGAGGGTGGCTC	30
  L6_CDR2_R	GAGACCCGAACCATAGTAGGTCTCATAGATGAGGAGCCTGGGA	31
  	G
  L6_CDR3_R	GAGACCCGAACCATAGTAGGTCTCACAGTAATAAACTGCAAAAT	32
  	CTTCAG
  V3-4_CDR1_R	GAGACCCGAACCATAGTAGGTCTCACAAGTGAGTGTGACTGTCC	33
  	CT
  V3-4_CDR2_R	GAGACCCGAACCATAGTAGGTCTCGTAGATGAGCGTGCGTGG	34
  V3-4_CDR3_R	GAGACCCGAACCATAGTAGGTCTCACAGTAATAATCAGATTCAT	35
  	CATCTGC

• TABLE 9
  VH1-46_IGHD6-6*01_IGHJ1*01_APFF_CDR2 BsaI mutagenesis primers

  		SEQ ID
  Primer	Sequence	NO
  A_ILPTH_F	tgggaataattctccctactggtcatagcacaagctacgcacaga	36
  A_VLPTH_F	tgggaatagtgctccctactggtcatagcacaagctacgcacaga	37
  A_ALPTH_F	tgggaatagctctccctactggtcatagcacaagctacgcacaga	38
  A_GLPTH_F	tgggaataggcctccctactggtcatagcacaagctacgcacaga	39
  A_TLPTH_F	tgggaataaccctccctactggtcatagcacaagctacgcacaga	40
  A_SLPTH_F	tgggaatatccctccctactggtcatagcacaagctacgcacaga	41
  A_YLPTH_F	tgggaatatacctccctactggtcatagcacaagctacgcacaga	42
  A_WLPTH_F	tgggaatatggctccctactggtcatagcacaagctacgcacaga	43
  A_HLPTH_F	tgggaatacacctccctactggtcatagcacaagctacgcacaga	44
  A_RLPTH_F	tgggaatacgcctccctactggtcatagcacaagctacgcacaga	45
  A_ELPTH_F	tgggaatagaactccctactggtcatagcacaagctacgcacaga	46
  A_NLPTH_F	tgggaataaacctccctactggtcatagcacaagctacgcacaga	47
  A_TLVTH_F	tgggaataaccctcgtgactggtcatagcacaagctacgcacaga	48
  A_TLATH_F	tgggaataaccctcgctactggtcatagcacaagctacgcacaga	49
  A_TLGTH_F	tgggaataaccctcggcactggtcatagcacaagctacgcacaga	50
  A_TLTTH_F	tgggaataaccctcaccactggtcatagcacaagctacgcacaga	51
  A_TLSTH_F	tgggaataaccctctccactggtcatagcacaagctacgcacaga	52
  A_TLYTH_F	tgggaataaccctctacactggtcatagcacaagctacgcacaga	53
  A_TLWTH_F	tgggaataaccctctggactggtcatagcacaagctacgcacaga	54
  A_TLHTH_F	tgggaataaccctccacactggtcatagcacaagctacgcacaga	55
  A_TLRTH_F	tgggaataaccctccgcactggtcatagcacaagctacgcacaga	56
  A_TLETH_F	tgggaataaccctcgaaactggtcatagcacaagctacgcacaga	57
  A_TLNTH_F	tgggaataaccctcggcactggtcatagcacaagctacgcacaga	58
  A_TLMTH_F	tgggaataaccctcatgactggtcatagcacaagctacgcacaga	59
  A_ILPTH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTAGGGAGAATTATT		60
  A_VLPTH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTAGGGAGCACTATT	61
  A_ALPTH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTAGGGAGAGCTATT	62
  A_GLPTH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTAGGGAGGCCTATT	63
  A_TLPTH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTAGGGAGGGTTATT	64
  A_SLPTH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTAGGGAGGGATATT	65
  A_YLPTH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTAGGGAGGTATATT	66
  A_WLPTH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTAGGGAGCCATATT	67
  A_HLPTH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTAGGGAGGTGTATT	68
  A_RLPTH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTAGGGAGGCGTATT	69
  A_ELPTH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTAGGGAGTTCTATT	70
  A_NLPTH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTAGGGAGGTTTATT	71
  A_TLVTH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTCACGAGGGTTATT	72
  A_TLATH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTAGCGAGGGTTATT	73
  A_TLGTH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTGCCGAGGGTTATT	74
  A_TLTTH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTGGTGAGGGTTATT	75
  A_TLSTH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTGGAGAGGGTTATT	76
  A_TLYTH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTGTAGAGGGTTATT	77
  A_TLWTH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTCCAGAGGGTTATT	78
  A_TLHTH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTGTGGAGGGTTATT	79
  A_TLRTH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTGCGGAGGGTTATT	80
  A_TLETH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTTTCGAGGGTTATT	81
  A_TLNTH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTGCCGAGGGTTATT	82
  A_TLMTH_R	AACTTCTGTGCGTAGCTTGTGCTATGACCAGTCATGAGGGTTATT	83

Example 2 Cloning and High Throughput Growth and Purification of Fab Libraries
• In this Example, Fab antibodies were generated by cloning heavy or light chain variable region DNA into their respective plasmids followed by co-transformation and high throughput protein growth/purification.
A. Cloning and Co-Transformation of Variable Heavy and Light Chains
• DNA encoding a heavy or light chain variable region was cloned into plasmids containing constant heavy or light chains as appropriate for co-transformation and expression of combinatorial Fabs. Plasmid A (SEQ ID NO:84) and plasmid D (SEQ ID NO:85) contain heavy chain constant regions sequences. Plasmid C (SEQ ID NO:86) contains a kappa light chain constant region sequence and Plasmid E (SEQ ID NO:87) contains a lambda light chain constant region sequence.
• DNA encoding a variable heavy chain was digested with Nhe I and Nco I and ligated into Plasmid A with a StII leader sequence using standard molecular techniques. DNA encoding a variable kappa light chain was digested with NcoI and BsiWI and DNA encoding a variable lambda chain was digested with NcoI and AvrII, and were ligated into Plasmid C or Plasmid E, respectively, with a StII leader sequence, using standard molecular biology techniques.
• Plasmid A and one of either Plasmid C or Plasmid E, each containing various combinations of variable heavy and light chains, were co-transformed into E. coli. The process was repeated for all combinations of heavy and light chains. Briefly, plasmid A (encoding a Fab heavy chain) and plasmid C or Plasmid E (encoding a Fab light chain) were resuspended separately in TE buffer to a final concentration of 1 ng/μl. One (1) μL of heavy chain plasmid and 1 μL of light chain plasmid were combined in a PCR tube or a PCR plate and were mixed with 20 μL ice cold LMG194 competent cells. The transformation reaction was incubated on ice for 10 minutes followed by heat shock in a preheated PCR block at 42° C. for 45 seconds. The tube was then placed on ice for an additional 2 minutes followed by addition of 200 μL SOC medium. The cells were allowed to recover for 1.5 hours at 37° C. A 100 μL aliquot of the transformation culture was used to inoculate 0.9 mL LB (Luria-Bertani Broth) containing 0.4% (w/v) glucose, 17 μg/mL kanamycin (Sigma Aldrich) and 34 chloramphenicol (Sigma Aldrich). The culture was grown at 30° C. with vigorous shaking for 20 hours. The transformation culture was grown and purified using the Piccolo™ system as described below.
B. High Throughput Growth and Purification of Fab Antibodies
• Following transformation, the cells were grown overnight in 2 ml deep well 96-well plates (VWR) block covered with breathable tape. The overnight culture was used directly for inoculation in Piccolo™ (Wollerton et al. (2006) JALA, 11:291-303.)
• High throughput, parallel expression and purification of Fab antibodies was performed using Piccolo™ (The Automation Partnership (TAP)), which automates protein expression and purification. The expression and purification parameters for Piccolo™ were prepared using Run Composer software (TAP). A ‘Strain File’ was generated mapping the location of each clone in the seed culture plate. This was submitted to the Run Composer software and the basic machine settings were set as follows: Pre-induction Incubator set at 30° C.; Expression Incubator 1 set at 16° C.; Centrifuge set at 6° C. and 5000×g; Media Pump 1 primed with TB (Terrific Broth; per liter contains 12 g tryptone, 24 g yeast extract, 9.4 g potassium phosphate, dibasic, and 2.2 g potassium phosphate, monobasic) (EMD Biosciences; catalog No. 71754), 50 μg/mL kanamycin (Sigma Aldrich), 35 μg/mL chloramphenicol (Sigma Aldrich), 0.4% (w/v) glucose (Sigma Aldrich) and 0.015% (v/v) Antifoam 204 (Sigma Aldrich); Inducer Pump 1 primed with 0.2% (w/v) arabinose (EMD Biosciences); Incubator Gassing Rate set at 2 sec with 51% oxygen, 0.1 mL inoculation volume; Induction Statistic Mean set w/o Outliers (i.e. block mean OD₆₀₀ determined after excluding the 3 highest and 3 lowest values); culture vessel blocks (CVB) pre-induction delay set at 1 hr 20 min and Expression Incubator Acclimatization set at 30 min.
• The seed cultures were prepared and loaded into Piccolo™ along with the necessary labware: 24-well culture vessel blocks (CVBs; The Automation Partnership), 24-well Filter Plates (The Automation Partnership), 24-well Output Plates (Seahorse Bioscience) and Pipette Tip Boxes (MBP) as specified by the manufacturer. The TB media supplemented as described above, arabinose inducer and associated pumps were prepared under sterile conditions and attached to the machine. The centrifuge counterbalance weight was set and placed inside the centrifuge. Lastly, purification reagents were prepared and attached to the system pumps (lysis buffer, resin, wash buffer and elution buffer as described below). Once this was complete, the machine was started and processing began.
• Before inoculation, the inocula were mapped to specific wells of 24-well CVB, and expression and induction conditions were set as described below. Each well of the CVBs was filled with 10 mL of TB media supplemented as described above prior to inoculation from the seed plate. Each well of each CVB was inoculated with 0.1 mL seed culture and then returned to the storage carousel to await scheduled admission to pre-induction incubation. Once a CVB was queued to begin pre-induction incubation it was removed from the storage carousel and coupled to an aeration assembly (which provides agitation, well sealing and a means for controlled administration of oxygen/air) and then placed in the pre-induction incubator set at 30° C. OD₆₀₀ readings were taken upon commencement of incubation and approximately every 30 minutes thereafter. Piccolo operation control software monitors the OD₆₀₀ measurements to predict when each CVB will reach the 1.0 OD₆₀₀ set point. Approximately 30 minutes prior to the CVB reaching the OD₆₀₀ set point the assembly was moved to the expression incubator to equilibrate to the expression temperature of 20° C., and then the cultures in the CVB were induced by addition of 0.032% arabinose inducer followed by 45 hours of expression.
• Following culture inoculation and growth induction of cultures, the cells were harvested and lysed for purification of Fabs. Piccolo™ was used for purification of the expressed Fab proteins using an automated expression and purification ‘Lifecycle’ of a whole culture purification. After controlled expression, CVBs were chilled for 30 minutes at 6° C. in the storage carousel prior to lysis. The CVB was moved to the liquid handling bed and lysis buffer (2.5 mL of Popculture with 1:1000 Lysonase (EMD Biosciences)) was added to each well with thorough mixing. The lysis proceeded for 10 minutes and then the CVB was centrifuged for 10 minutes at 5000×g to pellet cell debris. During centrifugation, a Filter Plate was placed in the filter bed and resin (2 mL of a 50% slurry of Ni-charged His-Bind resin (EMD Biosciences)) was added to each well. Soluble lysate was added to the corresponding wells of the filter plate containing resin and allowed to bind for 10 minutes prior to draining to waste. Wash buffer (12 mL, of wash buffer (50 mM Sodium Phosphate, 300 mM NaCl, 30 mM Imidazole, pH 8.0)) was added in two steps to each well and allowed to drain to waste. Finally, an Output Plate was placed under the Filter Plate in the filter bed and IMAC elution buffer (50 mM Sodium Phosphate, 300 mM NaCl, 500 mM Imidazole) was added in two steps draining into the output plate. The output plate was returned to the storage carousel as was all other labware. Once this process was complete for each CVB in the designed run, the machine was unloaded.
Example 3 Orthogonal Secondary Purification of Fab Antibodies
• To rapidly further purify partially pure Fabs generated after the Piccolo™ process, an orthogonal method of purification was developed. Fabs were expressed and purified as described above in Example 2 using the Piccolo™ machine.
• Two different affinity resins were used depending on the light chain classes. Fabs with a kappa light chain were further purified on Protein G column (GE Healthcare), and Fabs with a lambda light chain were further purified on CaptureSelect Fab Lambda affinity column (BAC, Netherlands). First, the protein samples were transferred to a deep well 96-well block (VWR). Approximately 1.8 mL of the IMAC elution per Fab sample was purified on either a 1 mL Hi-Trap Protein G column or a 0.5 mL CaptureSelect Fab Lambda affinity column at 4° C. using the Akta purifier (GE Healthcare) and A-905 autosampler (GE Healthcare) according to the manufacturer's protocol. Protein concentration was determined by measuring absorbance at A280 on a Molecular Dynamic plate reader and calculated from the exctinction coefficient of the corresponding Fab. Extinction coefficients are calculated based on the total numbers of Tyrosine+Tryptophan+Phenylalanine in the Fab heavy and light chains. Following purification using the Piccolo™ system, expressed protein was generally less than 20% pure. After orthogonal purification with protein G, Fab purity was greater than 95% pure as indicated by SDS-PAGE.
Example 4 Electrochemiluminescence Binding Assay
• In this example, an electrochemiluminescence (ECL) binding assay was used to screen a Fab library (e.g. see Table 4) for antibodies capable of binding to one of nine different antigens, including the human epidermal growth factor 2 receptor (ErbB2), epidermal growth factor receptor (EGF R), hepatocyte growth factor receptor (HGF R/c-Met), Notch-1, CD44, insulin-like growth factor-1 soluble receptor (IGF-1 sR), P-cadherin, erythropoietin receptor (Epo R) and delta-like protein 4 (DLL4). In an ECL assay, an antigen-antibody interaction is detected by addition of a detection antibody labeled with ruthenium tri-bispyridine-(4-methysulfone) (Ru(bpy)₂ ²⁺). Upon application of an electric current, the Ru(bpy)₂ ²⁺-label undergoes an oxidation-reduction cycle in the presence of a co-reactant and light is emitted. A signal is only generated when the Ru(bpy)₂ ²⁺-label is in close proximity to the electrode, eliminating the need for washing. Detected light intensity is proportional to the amount of captured protein.
• Recombinant human proteins were obtained from R&D Systems and included: rHuman ErbB2/Fc Chimera, CF (Cat#1129-ER); rHuman EGF R/Fc Chimera, CF (Cat#344-ER); rHuman HGF R/c-MET/Fc Chimera, CF (Cat#358-MT/CF); rHuman Notch-1/Fc Chimera, CF (Cat#3647-TK); rHuman CD44/Fc Chimera, CF (Cat#3660-CD); rHuman IGF-1 sR, (IGF-1 sR), CF (Cat#391-GR); rHuman P-Cadherin/Fc Chimera, CF (Cat#861-PC); rHuman Erythropoietin R/Fc Chimera, CF (Cat#963-ER); and Recombinant Human DLL4 (Cat#1506-D4/CF).
A. Multispot ECL Assay for Binding to Multiple Antigens
• Each of the antigens listed above were immobilized onto each well of 10 plates by spotting 50 nanoliters (nl) of each protein (of a 60 μg/mL antigen) on the surface of a 96-well Multi-Spot 10 Highbind plate (Meso Scale Discovery; Gaithersburg Md.). Spot 10 was left blank as a control.
• An 150 μl aliquot of 1% Bovine Serum Albumin (BSA) in Tris-buffered Saline Tween (TBST) was added to each well and allowed to incubate for 30 min at 20° C. followed by washing and tap drying to completely remove any residual solution. Subsequently, a 12.5 μl aliquot of 1% BSA TBST was added to each well followed by the addition of a 12.5 μl aliquot of a purified Fab. The plate was sealed and incubated for 1 hour at 20° C. with shaking
• Detection antibodies were prepared by individually conjugating both goat anti-human Kappa light chain polyclonal antibody (K3502-1MG, Sigma-Aldrich) and goat anti-human Lambda light chain polyclonal antibody (L1645-1ML, Sigma-Aldrich) with Ruthenium (II) tris-bipyridine-(4-methylsulfone)-N-hydroxysuccinimide (SULFO-TAG NHS-ester, Meso. Scale Discovery) according to the manufacturer's instructions. TAG-detection antibody at 25 μl was added to each well and allowed to incubate for 1 hour at 20° C. with shaking. Finally, 15 μl of Read Buffer P with Surfactant (Cat # R92PC-1, Meso Scale Discovery) was added to each well. The electrochemiluminescence was measured using a Sector Imager 2400 (Meso Scale Discovery). Data was analyzed by comparing the ECL signals for an antigen to the blank of each well. A signal to blank ratio of 4 or more was considered a “Hit” Fab.
• Using the Multispot ECL assay antibodies were identified that bind to the selected antigens. Table 10, below, lists the Fabs (including the heavy chain and light chain) that were identified as “hits” using the Multispot ECL assay and the target(s) of the identified Fab “hit.” Several Fabs were identified that bind to multiple targets. For example, VH1-46_IGHD6-13*01_IGH41*01 & B3_IGKJ1*01, shows affinity for both Human ErbB2/Fc and Human Erythropoietin R/Fc chimeras; Fab VH1-46_IGHD2-15*01_IGHJ2*01 & L12_IGKJ1*01 binds to EGF R, Epo R and DLL4 and Fab VH1-46_IGHD3-10*01_IGHJ4*01 & L12_IGKJ1*01 binds to Notch-1, P-cadherin and DLL4.

• TABLE 10
  IDENTIFIED FAB “HITS”

  		SEQ		SEQ ID
  Target	Heavy Chain	ID NO	Light Chain	NO

  rHuman DLL4	VH1-46_IGHD6-	88	L6_IGKJ1*01	107
  	6*01_IGHJ1*01
  rHuman DLL4	VH5-51_IGHD5-	89	V3-	108
  	18*01>3_IGHJ4*01		4_IGLJ1*01
  rHuman DLL4	VH6-1_IGHD3-	90	V4-	109
  	3*01_IGHJ4*01		3_IGLJ4*01
  rHuman ErbB2/Fc chimera	VH4-31_IGHD1-	91	A27_IGKJ1*01	110
  	26*01_IGHJ2*01
  rHuman Epo R/Fc chimera	VH1-46_IGHD3-	92	B3_IGKJ1*01	111
  	10*01_IGHJ4*01
  rHuman ErbB2/Fc chimera and	VH1-46_IGHD6-	93	B3_IGKJ1*01	111
  rHuman Epo R/Fc chimera	13*01_IGHJ4*01
  Epo R/Fc chimera	VH4-28_IGHD7-	94	L2_IGKJ1*01	112
  	27*01_IGHJ1*01
  Epo R/Fc chimera	VH4-31_IGHD7-	95	L2_IGKJ1*01	112
  	27*01_IGHJ5*01
  ErbB2/Fc chimera	VH2-5_IGHD7-	96	L2_IGKJ1*01	112
  	27*01_IGHJ2*01
  Epo R/Fc chimera	VH1-46_IGHD7-	97	A27_IGKJ1*01	110
  	27*01_IGHJ2*01
  ErbB2/Fc chimera	VH1-69_IGHD1-	98	A17_IGKJ1*01	113
  	1*01_IGHJ6*01
  Epo R/Fc chimera and EGF R/Fc	VH1-46_IGHD2-	99	L2_IGKJ1*01	112
  chimera	15*01_IGHJ2*01
  EGF R/Fc chimera, Notch-1/Fc	VH1-46_IGHD6-	93	L2_IGKJ1*01	112
  chimera, P-cadherin/Fc chimera,	13*01_IGHJ4*01
  Epo R/Fc chimera and DLL4
  DLL4	VH4-34_IGHD7-	100	L5_IGKJ1*01	114
  	27*01_IGHJ4*01
  Notch-1/Fc chimera, P-	VH1-46_IGHD6-	93	A27_IGKJ1*01	110
  cadherin/Fc chimera, Epo R/Fc	13*01_IGHJ4*01
  chimera and DLL4
  P-cadherin/Fc chimera	VH1-46_IGHD7-	97	L6_IGKJ1*01	107
  	27*01_IGHJ2*01
  DLL4	VH1-3_IGHD4-	101	L12_IGKJ1*01	115
  	23*01_IGHJ4*01
  EGF R/Fc chimera, Epo R/Fc	VH1-46_IGHD2-	99	L12_IGKJ1*01	115
  chimera and DLL4	15*01_IGHJ2*01
  Notch-1/Fc chimera, P-	VH1-46_IGHD3-	92	L12_IGKJ1*01	115
  cadherin/Fc chimera and DLL4	10*01_IGHJ4*01
  DLL4	VH1-8_IGHD2-	102	L12_IGKJ1*01	115
  	2*01_IGHJ6*01
  Epo R/Fc chimera	VH1-46_IGHD3-	92	O1_IGKJ1*01	116
  	10*01_IGHJ4*01
  Epo R/Fc chimera and DLL4	VH1-46_IGHD6-	93	O1_IGKJ1*01	116
  	13*01_IGHJ4*01
  DLL4	VH4-34_IGHD7-	100	V1-	117
  	27*01_IGHJ4*01		4_IGLJ4*01
  DLL4	VH4-31_IGHD2-	103	V1-	117
  	15*01_IGHJ2*01		4_IGLJ4*01
  DLL4	VH4-34_IGHD7-	100	V4-	118
  	27*01_IGHJ4*01		6_IGLJ4*01
  P-cadherin/Fc chimera and Epo	VH3-23_IGHD3-	104	O12_IGKJ1*01	119
  R/Fc chimera	10*01>3_IGHJ6*01
  P-cadherin/Fc chimera	VH3-23_IGHD3-	105	O12_IGKJ1*01	119
  	10*01>1′_IGHJ3*01

• To confirm a “Hit” from the initial Multispot ECL screening, a Fab concentration dependent titration was carried out to determine the Fab-antigen binding affinity. The Multispot ECL assay procedure was the same as described above, except that the concentration of Fab antibody was varied between wells from 0.1 nM to 2.4 μM as indicated in the Tables below depending on each Fab tested. The data are set forth in Tables 11-33 below.

• TABLE 11
  Binding affinity of Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01

  Fab[nM]	2383	595.8	148.9	37.2	9.3	2.3	0.6	0.1

  ErbB2/Fc	454	321	247	384	354	291	215	306
  EGF R/Fc	621	403	290	228	424	289	309	311
  HGF R/Fc	762	353	205	207	324	253	256	286
  Notch-1/Fc	690	306	375	402	492	333	337	378
  CD44/Fc	559	372	348	356	396	317	238	323
  IGF-1 sR	527	335	322	295	315	231	313	241
  P-Cadherin/Fc	728	617	687	649	452	401	321	235
  EPO R/Fc	658	378	373	315	306	429	337	373
  DLL4	11794	17203	16253	16717	13210	3055	508	317
  Blank	344	285	218	199	287	234	226	201

• TABLE 12
  Binding affinity of Fab VH5-51_IGHD5-
  18*01>3_IGHJ4*01 & V3-4_IGLJ1*01

  	Fab[nM]	154	51	17	6

  	ErbB2/Fc	1593	1248	1033	873
  	EGF R/Fc	1398	816	805	742
  	HGF R/Fc	1520	1044	914	831
  	Notch-1/Fc	929	685	558	464
  	CD44/Fc	960	651	518	547
  	IGF-1 sR	1396	1051	872	854
  	P-Cadherin/Fc	1733	854	542	358
  	EPO R/Fc	1195	750	620	548
  	DLL4	40392	17025	7158	1946
  	Blank	447	335	143	191

• TABLE 13
  Binding affinity of Fab VH6-
  1_IGHD3-3*01_IGHJ4*01 & V4-3_IGLJ4*01

  Fab[nM]	480	240	120	60	30	15	7.5	3.8

  ErbB2/Fc	965	833	822	777	726	713	695	714
  EGF R/Fc	877	690	658	679	585	584	582	511
  HGF R/Fc	951	834	785	623	640	694	558	519
  Notch-1/Fc	545	368	472	415	425	508	392	383
  CD44/Fc	541	470	442	434	484	454	444	419
  IGF-1 sR	741	625	813	654	697	705	642	463
  P-Cadherin/Fc	596	383	450	372	440	351	352	281
  EPO R/Fc	621	478	431	423	325	397	443	407
  DLL4	1532	1273	938	875	736	690	598	462
  Blank	362	316	363	237	213	261	217	198

• TABLE 14
  Binding affinity of Fab VH4-
  31_IGHD1-26*01_IGHJ2*01 & A27_IGKJ1*01

  Fab[nM]	410	205	102.5	51.3	25.6	12.8	6.4	3.2

  ErbB2/Fc	5422	5260	4355	3588	2992	2255	1796	868
  EGF R/Fc	734	595	455	379	373	320	249	254
  HGF R/Fc	753	735	425	456	382	258	234	294
  Notch-1/Fc	804	722	607	408	270	249	279	275
  CD44/Fc	767	613	461	409	332	273	240	295
  IGF-1 sR	600	565	443	316	311	323	209	313
  P-Cadherin/Fc	814	769	714	424	323	245	197	206
  EPO R/Fc	797	595	587	498	409	338	264	233
  DLL4	859	599	550	474	384	268	256	242
  Blank	637	430	437	337	345	227	133	172

• TABLE 15
  Binding affinity of Fab VH1-
  46_IGHD3-10*01_IGHJ4*01 & B3_IGKJ1*01

  Fab[nM]	1410	705	352.5	176.3	88.1	44.1	22	11

  ErbB2/Fc	932	671	514	448	200	347	363	216
  EGF R/Fc	1071	692	769	428	376	428	312	201
  HGF R/Fc	903	839	606	418	392	336	203	268
  Notch-1/Fc	1034	958	715	664	440	331	389	404
  CD44/Fc	885	693	556	376	340	302	317	296
  IGF-1 sR	426	630	528	393	273	309	347	289
  P-Cadherin/Fc	1059	827	649	532	278	343	215	270
  EPO R/Fc	4314	4894	4105	3519	3368	2387	2241	1824
  DLL4	1265	981	660	460	434	388	342	254
  Blank	709	483	494	346	301	200	289	212

• TABLE 16
  Binding affinity of Fab VH1-46_IGHD6-13*01_IGHJ4*01 & B3_IGKJ1*01

  Fab[nM]	1000	500	250	125	62.5	31.3	15.6	7.8

  ErbB2/Fc	8731	10241	11026	12956	13124	13911	14791	13220
  EGF R/Fc	2236	1468	1138	860	602	447	346	379
  HGF R/Fc	2109	1371	1221	778	578	299	293	282
  Notch-1/Fc	2267	1975	1241	802	536	563	418	486
  CD44/Fc	1966	1685	1175	764	591	439	473	409
  IGF-1 sR	1667	1334	993	654	491	385	349	353
  P-Cadherin/Fc	4495	3447	2784	1481	1173	1105	971	695
  EPO R/Fc	8594	10305	8535	9237	7749	7878	8357	6765
  DLL4	2785	2319	1560	912	715	528	525	407
  Blank	1133	680	590	403	268	250	294	316

• TABLE 17
  Binding affinity of Fab VH4-
  28_IGHD7-27*01_IGHJ1*01 & L2_IGKJ1*01

  	Fab[nM]	360	36

  	ErbB2/Fc	647	600
  	EGF R/Fc	957	711
  	HGF R/Fc	581	613
  	Notch-1/Fc	1026	773
  	CD44/Fc	740	679
  	IGF-1 sR	535	486
  	P-Cadherin/Fc	636	693
  	EPO R/Fc	4715	2977
  	DLL4	866	799
  	Blank	462	413

• TABLE 18
  Binding affinity of Fab VH1-46_IGHD2-15*01_IGHJ2*01 &
  L2_IGKJ1*01

  	Fab[μM]	0.25	0.0625	0.01563	0.00391

  	ErbB2/Fc	29608	9033	4495	1667
  	EGF R/Fc	116674	94778	70836	35936
  	HGF R/Fc	13427	4108	1998	913
  	Notch-1/Fc	21447	5848	2800	1282
  	CD44/Fc	23015	6746	3182	1295
  	IGF-1 sR	11050	3150	1742	822
  	P-Cadherin/Fc	25459	7739	4945	1962
  	EPO R/Fc	49177	21136	11342	5022
  	DLL4	27691	8051	4015	1551
  	Blank	6344	1738	906	576

• TABLE 19
  Binding affinity of Fab VH1-46_IGHD6-13*01_IGHJ4*01 &
  L2_IGKJ1*01

  	Fab[μM]	1.19	0.2975	0.07438	0.01859

  	ErbB2/Fc	38410	15111	7551	5531
  	EGF R/Fc	62454	42213	16605	11750
  	HGF R/Fc	45494	17396	6611	4566
  	Notch-1/Fc	72018	37503	21990	17565
  	CD44/Fc	47145	28601	10922	7322
  	IGF-1 sR	35187	17389	5804	3779
  	P-Cadherin/Fc	69710	26043	14807	11672
  	EPO R/Fc	192967	167064	153692	188065
  	DLL4	74900	34726	20719	18888
  	Blank	24999	5019	2504	1776

• TABLE 20
  Binding affinity of Fab VH4-34_IGHD7-27*01_IGHJ4*01 &
  L5_IGKJ1*01

  	Fab[μM]	0.51	0.1275	0.03188	0.00797

  	ErbB2/Fc	1532	857	584	493
  	EGF R/Fc	2363	1061	694	530
  	HGF R/Fc	1989	853	693	419
  	Notch-1/Fc	2773	1497	849	654
  	CD44/Fc	2012	926	653	490
  	IGF-1 sR	2236	1045	765	564
  	P-Cadherin/Fc	2389	957	775	502
  	EPO R/Fc	2624	1067	789	566
  	DLL4	5183	2382	1282	872
  	Blank	1096	530	536	364

• TABLE 21
  Binding affinity of Fab VH1-46_IGHD6-
  13*01_IGHJ4*01 & A27_IGKJ1*01

  	Fab[μM]	0.48	0.096	0.0192

  	ErbB2/Fc	11287	3365	2313
  	EGF R/Fc	14638	4509	3115
  	HGF R/Fc	8002	2328	1582
  	Notch-1/Fc	15931	4802	3041
  	CD44/Fc	13445	4320	2915
  	IGF-1 sR	8927	2449	1826
  	P-Cadherin/Fc	15595	6654	5040
  	EPO R/Fc	70938	57356	62037
  	DLL4	16065	5586	3555
  	Blank	2945	917	751

• TABLE 22
  Binding affinity of Fab VH1-46_IGHD7-
  27*01_IGHJ2*01 & L6_IGKJ1*01

  	Fab[μM]	1.56	0.312	0.0624

  	ErbB2/Fc	7577	3659	2146
  	EGF R/Fc	7832	4328	2415
  	HGF R/Fc	10267	4691	2453
  	Notch-1/Fc	9447	4462	2352
  	CD44/Fc	7595	4171	2110
  	IGF-1 sR	6913	3508	2034
  	P-Cadherin/Fc	15016	7098	4226
  	EPO R/Fc	9480	5020	2678
  	DLL4	10897	5484	2585
  	Blank	4357	1977	960

• TABLE 23
  Binding affinity of Fab VH1-3_IGHD4-23*01_IGHJ4*01 &
  L12_IGKJ1*01

  	Fab[nM]	60	15	3.75	0.9375

  	ErbB2/Fc	2155	740	291	268
  	EGF R/Fc	2563	842	371	224
  	HGF R/Fc	2298	743	394	243
  	Notch-1/Fc	2886	1058	375	348
  	CD44/Fc	2355	748	307	251
  	IGF-1 sR	2666	859	314	204
  	P-Cadherin/Fc	2662	837	331	191
  	EPO R/Fc	3214	970	358	238
  	DLL4	17270	7728	1569	453
  	Blank	1433	536	191	153

• TABLE 24
  Binding affinity of Fab VH1-46_IGHD2-15*01_IGHJ2*01 &
  L12_IGKJ1*01

  	Fab[nM]	280	70	17.5	4.375

  	ErbB2/Fc	3953	1358	541	384
  	EGF R/Fc	6667	2574	1305	542
  	HGF R/Fc	3564	1289	565	193
  	Notch-1/Fc	4382	1492	680	480
  	CD44/Fc	4069	1370	664	424
  	IGF-1 sR	3533	1319	626	369
  	P-Cadherin/Fc	5400	1817	949	469
  	EPO R/Fc	8496	2485	1262	594
  	DLL4	8111	2747	1219	558
  	Blank	1691	635	304	305

• TABLE 25
  Binding affinity of Fab VH1-46_IGHD3-10*01_IGHJ4*01 &
  L12_IGKJ1*01

  	Fab[nM]	920	230	57.5	14.375

  	ErbB2/Fc	10924	4078	2447	1594
  	EGF R/Fc	13406	5723	3858	2672
  	HGF R/Fc	10708	3934	2297	1600
  	Notch-1/Fc	20086	9737	5886	4206
  	CD44/Fc	9698	3817	2313	1488
  	IGF-1 sR	10246	4764	2833	1746
  	P-Cadherin/Fc	16666	6484	4110	2318
  	EPO R/Fc	16429	6949	4038	2718
  	DLL4	73638	119436	144126	125422
  	Blank	4082	1656	954	738

• TABLE 26
  Binding affinity of Fab VH1-8_IGHD2-2*01_IGHJ6*01 &
  L12_IGKJ1*01

  	Fab[nM]	130	32.5	8.1	2.0

  	ErbB2/Fc	1533	556	557	382
  	EGF R/Fc	1746	645	560	424
  	HGF R/Fc	1882	525	551	356
  	Notch-1/Fc	1759	706	612	539
  	CD44/Fc	1754	573	528	447
  	IGF-1 sR	1973	561	518	367
  	P-Cadherin/Fc	1845	556	573	250
  	EPO R/Fc	2151	673	660	433
  	DLL4	7738	2989	1548	605
  	Blank	1153	473	435	316

• TABLE 27
  Binding affinity of Fab FabVH1-46_IGHD3-10*01_IGHJ4*01
  & O1_IGKJ1*01

  	Fab[nM]	1570	392.5	98.1	24.5

  	ErbB2/Fc	1263	539	247	241
  	EGF R/Fc	2481	744	4386	317
  	HGF R/Fc	1638	581	335	211
  	Notch-1/Fc	1639	749	313	434
  	CD44/Fc	1381	498	265	267
  	IGF-1 sR	1428	466	309	239
  	P-Cadherin/Fc	1793	459	347	257
  	EPO R/Fc	6121	5863	5628	4531
  	DLL4	2701	735	402	339
  	Blank	866	338	210	149

• TABLE 28
  Binding affinity of Fab VH1-46_IGHD6-13*01_IGHJ4*01 &
  O1_IGKJ1*01

  	Fab[nM]	930	232.5	58.1	14.5

  	ErbB2/Fc	2225	779	322	274
  	EGF R/Fc	3110	803	444	357
  	HGF R/Fc	2344	790	432	373
  	Notch-1/Fc	2206	778	388	317
  	CD44/Fc	1917	607	375	212
  	IGF-1 sR	1915	569	343	234
  	P-Cadherin/Fc	2438	655	478	277
  	EPO R/Fc	3009	1472	829	660
  	DLL4	8162	3586	1876	1149
  	Blank	1206	460	225	117

• TABLE 29
  Binding affinity of Fab VH4-34_IGHD7-27*01_IGHJ4*01 &
  V1-4_IGLJ4*01

  	Fab[nM]	580	145	36.3	9.1

  	ErbB2/Fc	1712	1123	1029	987
  	EGF R/Fc	1631	856	831	800
  	HGF R/Fc	2341	1173	1065	894
  	Notch-1/Fc	1585	860	633	754
  	CD44/Fc	1228	692	629	607
  	IGF-1 sR	1364	794	799	788
  	P-Cadherin/Fc	2240	850	684	589
  	EPO R/Fc	1579	845	722	697
  	DLL4	4420	2140	1399	1030
  	Blank	679	357	314	276

• TABLE 30
  Binding affinity of Fab VH4-31_IGHD2-15*01_IGHJ2*01 &
  V1-4_IGLJ4*01

  	Fab [nM]	210	52.5	13.1	3.3

  	ErbB2/Fc	1977	1511	930	1031
  	EGF R/Fc	1617	1109	824	847
  	HGF R/Fc	2060	1286	981	849
  	Notch-1/Fc	1972	1323	669	726
  	CD44/Fc	1395	897	708	621
  	IGF-1 sR	1431	911	814	743
  	P-Cadherin/Fc	4410	2161	1062	678
  	EPO R/Fc	2123	1319	776	695
  	DLL4	4108	1951	1107	922
  	Blank	833	467	376	359

• TABLE 31
  Binding affinity of Fab VH4-34_IGHD7-27*01_IGHJ4*01 &
  V4-6_IGLJ4*01

  	Fab [nM]	340	170	85.0	42.5

  	ErbB2/Fc	1226	964	844	866
  	EGF R/Fc	1208	826	1001	528
  	HGF R/Fc	1238	757	998	607
  	Notch-1/Fc	1209	816	780	649
  	CD44/Fc	959	660	693	522
  	IGF-1 sR	1042	832	891	646
  	P-Cadherin/Fc	1160	744	709	421
  	EPO R/Fc	1255	790	817	494
  	DLL4	2332	1462	1311	877
  	Blank	554	262	292	162

• TABLE 32
  Binding affinity of Fab VH3-23_IGHD3-10*01>3_IGHJ6*01 &
  O12_IGKJ1*01

  	Fab [nM]	120	12	1.2	0.12

  	ErbB2/Fc	17294	4358	677	287
  	EGF R/Fc	14925	1984	464	272
  	HGF R/Fc	15917	2703	412	287
  	Notch-1/Fc	14382	2582	660	218
  	CD44/Fc	13519	1321	341	291
  	IGF-1 sR	13265	1135	181	175
  	P-Cadherin/Fc	61714	28490	1684	318
  	EPO R/Fc	33268	10966	1014	260
  	DLL4	20627	2510	319	210
  	Blank	6749	573	227	264

• TABLE 33
  Binding affinity of Fab VH3-23_IGHD3-10*01>1′_IGHJ3*01 &
  O12_IGKJ1*01

  	Fab [nM]	421.12	42.112

  	ErbB2/Fc	868	524
  	EGF R/Fc	765	422
  	HGF R/Fc	1202	565
  	Notch-1/Fc	1061	437
  	CD44/Fc	903	360
  	IGF-1 sR	1065	364
  	P-Cadherin/Fc	2949	1546
  	EPO R/Fc	1299	759
  	DLL4	1090	404
  	Blank	639	323

B. 96-Well Plate ECL Assay for Binding to DLL4
• A similar ECL assay was performed as above, except only one antigen was immobilized to a single-spot per well plate for testing. Recombinant Human DLL4 (Cat#1506-D4/CF) was immobilized onto a 96-well plate by adding 5 μL (of 10 μg/ml DLL4 in PBS+0.03% Triton-X-100) to each well and incubating overnight at 20° C. One well was left blank as a control. The protein was removed and an 150 μl aliquot of 1% BSA in TBST was added to each well and allowed to incubate for 1 hour at 20° C. followed by washing 2 times with 150 μl TBST and tap drying to completely remove any residual solution. Subsequently, 25 μl aliquot of each Fab (with 1% BSA with TBST) was added to each well. The plate was sealed and incubated for 1 hour at 20° C. with shaking. As described in Examples 7 and 12, two different combinations of antigen and Fab concentrations were utilized. In one experiment, 5 μL of 30 μg/mL antigen was used to coat the plate and each Fab was tested at a concentration of 0.02 μM. In the other experiment, 5 μL of 15 μg/mL antigen was used to coat the plate and each Fab was tested at a concentration of 0.004 μM.
• The Fab was subsequently removed and 25 μl anti-human Kappa Ruthenium antibody or anti-human Lambda Ruthenium antibody (1 μg/ml in 1% BSA with TBST) was added to each well and allowed to incubate for 1 hour at 20° C. with shaking Finally, 15 μl of Read Buffer P with Surfactant (Cat # R92PC-1, Meso Scale Discovery) was added to each well. The electrochemiluminescence was measured using a Sector Imager 2400 (Meso Scale Discovery). Data was analyzed by comparing the ECL signals for an antigen to the blank of each well. A signal to blank ratio of 4 or more was considered a “Hit” Fab. The results are depicted in Examples 7-15 below.
Example 5 Surface Plasmon Resonance
• In this example, the binding affinities of selected Fabs to recombinant human DLL4 (R&D Systems) were analyzed using Surface Plasmon Resonance (SPR) (Biosensor Tools, Salt Lake City, Utah). The Fabs include germline antibodies identified in the initial ECL screen as binding to DLL4 (as shown in Example 4).
• The results are shown in Table 34 below. Table 34 sets forth the Fab, the k_a (M⁻¹s⁻¹), the k_d (s⁻¹), and the K_D (nM) and the standard deviation (in parentheses). Germline Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01 has an average K_D of 4.8 μM. Germline Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 binds DLL4 with an average K_D of 730 nM. Germline Fab VH6-1_IGHD3-3*01_IGHJ4*01 & V4-3_IGLJ4*01 has an average binding affinity of 38 μM while germline Fab VH1-46_IGHD3-10*01_IGHJ4*01 & L12_IGKJ1*01 has an average K_D of 500 nM.

• TABLE 34
  Binding affinity of DLL4 Fabs

  	SEQ		SEQ
  Heavy Chain	ID NO	Light Chain	ID NO	ka (M−1s−1)	kd (s−1)	KD (nM)
  VH5-51_IGHD5-	89	V3-4_IGLJ1*01	108	n/a	n/a	4800(200)
  18*01 > 3_IGHJ4*01
  VH1-46_IGHD6-	88	L6_IGKJ1*01	107	1.63(3)e5	0.101(2)	730(130)
  6*01_IGHJ1*01
  VH6-1_IGHD3-	90	V4-3_IGLJ4*01	109	n/a	n/a	38000(4000)
  3*01_IGHJ4*01
  VH1-46_IGHD3-	92	L12_IGKJ1*01	115	5(1)e5	0.29(2)	500(100)
  10*01_IGHJ4*01

Example 6 ELISA Binding Assay
• In this example, an ELISA binding assay was used to determine the binding of Fab antibodies to DLL4.
A. 96-Well Plate
• Briefly, 50 μl of a 0.5 μg/ml solution of DLL4 in 100 mM NaHCO₃, pH 9 was added to each well of a 96-well Costar plate (Cat #3370, Corning Inc.) and allowed to incubate for 1 hour at room temperature. The plate was blocked by adding 1% BSA in Tris-buffered Saline Tween (TBST) and incubating for 1 hour at room temperature followed by washing 2 times with 150 μl TBST. A Fab antibody was serially diluted in 1% BSA in TBST, starting at a concentration of 1000 nM. A 50 μl aliquot of each serial dilution was added, in triplicate, to each well and the plate was incubated for 1 hour at room temperature followed by washing 2 times with TBST. 50 μl of goat anti-DDDDK tag HRP conjugated polyclonal antibody diluted 1:1000 in 1% BSA TBST (Cat # AB1238-200, Abeam), was added to each well and the plate was incubated for 30 minutes at room temperature followed by washing 3 times with 200 μl TBST. Finally, 100 μl TMB one-component reagent (Cat # TMBW-1000-01, BioFax) was added and allowed to develop for 2 minutes at room temperature. The reaction was immediately halted by the addition of 100 μl 0.5 M H₂SO₄ and the absorbance at 450 nm was measured using an ELISA plate reader. Results using this assay are depicted in Examples 9 and 10.
B. 384-Well Plate
• Briefly, 10 μl of a 0.5 μg/ml solution of DLL4 in 100 mM NaHCO₃, pH 9 was added to each well of a 384-well Nunc Maxisorp plate (Cat #464718, Nalgene Nunc International) and allowed to incubate for 90 minutes at room temperature. The plate was blocked by adding 1% BSA in Tris-buffered Saline Tween (TBST) and incubating for 1 hour at room temperature followed by washing 2 times with 100 μl TBST. Fab antibody was serially diluted in 1% BSA in TBST, starting at a concentration of 1000 nM. A 20 μl aliquot of each serial dilution was added, in triplicate, to each well and the plate was incubated for 1 hour at room temperature followed by washing 2 times with 100 μl TBST. Depending on the light chain, 20 μl of goat anti-kappa HRP conjugated polyclonal antibody, diluted 1:1000 in 1% BSA TBST (Cat # A7164-1mL, Sigma-Aldrich) or goat anti-lambda HRP conjugated polyclonal antibody, diluted 1:1000 in 1% BSA TBST (Cat # L1645-1ml, Sigma-Aldrich) was added to each well and the plate was incubated for 1 hour at room temperature followed by washing 4 times with 100 μl TBST. Finally, 25 μl TMB one-component reagent (Cat # TMBW-1000-01, BioFax) was added and allowed to develop for 1-5 minutes at room temperature. The reaction was immediately halted by the addition of 25 μl 0.5 M H₂SO₄ and the absorbance at 450 nm was measured using an ELISA plate reader. Results using this assay are depicted in Examples 9 and 10.
Example 7 Affinity Maturation of the Heavy Chain of Anti-DLL4 “Hit” VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01
• a. Summary
• The heavy and light chain amino acid sequence of Fab “Hit” VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 (SEQ ID NOS:88 and 107) against DLL4, identified in Example 4 using the Multispot ECL binding assay, was aligned with the heavy and light chain amino acid sequence of a related “non-Hit” Fab antibody that had a related heavy or light chain but did not bind to DLL4. Based on the alignment, amino acid residues that differed between the “Hit” and “non-Hit” antibodies were identified in each of the heavy and light chain as potential amino acids involved in binding for subsequent affinity maturation. Affinity maturation of the heavy chain is described in Examples 7-9. Affinity maturation of the light chain is described in Example 10.
• Briefly, the identified amino acid residues were subjected to alanine-scanning mutagenesis and resultant mutant Fabs tested to assess the affect of the mutation on binding of the antibody to DLL4. Mutated residues that did not affect binding of the antibody to DLL4 were identified and subjected to further mutagenesis using overlapping PCR with NNK mutagenesis. Mutant antibodies were assessed for DLL4 binding, and mutations that improved binding to DLL4 were identified. Combinations mutants were generated containing each of the identified single mutants; combination mutants were further assayed for binding to DLL4. Further optimization was performed by mutating other regions of the antibody. By this method, anti-DLL4 antibodies were generated with significantly improved binding affinity for DLL4 compared to the parent “Hit” VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 Fab antibody.
• b. Affinity Maturation of Heavy Chain
• i. Identification of the CDR Potential Binding Site
• The amino acid sequence of the heavy chain (SEQ ID NO:88) for the parent “Hit” VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 was aligned with the amino acid sequence of a related heavy chain (SEQ ID NO:93) of a non-Hit that was identified as not binding to DLL4, i.e. VH1-46_IGHD6-13*01_IGHJ4*01 & L6_IGKJ1*01. “Hit” Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 had an ECL signal/blank ratio of 23.1 while that of the non-Hit Fab VH1-46_IGHD6-13*01_IGHJ4*01 & L6_IGKJ1*01 was only 2.4. These two Fabs are related because they share the same V_H germline segment. Further, the D_H germline segment is of the same gene family (i.e. IGHD6). The sequence alignment is set forth in FIG. 1. Based on the alignment, amino acid residues were identified that differed between the “Hit” and “non-Hit,” thus accounting for the differences in binding of the “Hit” and “non-Hit” anti-DLL4 antibodies. The identified amino acid residues were located in CDR3, which was identified as the region of the heavy chain that is important for binding affinity.
• ii. Alanine Scanning of CDR3
• Alanine scanning mutagenesis was performed on amino acid residues in the CDR3 of the heavy chain sequence of parent Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 to identify amino acid residues that do not appear to be involved in DLL4 binding. Alanine-scanning of the CDR3 region of the heavy chain sequence of parent Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 was performed by mutating every residue of the CDR3 region to an alanine, except amino acid residues A106, Y108, and F109. The mutant Fab antibodies were expressed and purified as described in Example 2 above.
• Purified Fab alanine mutants were tested for binding to DLL4 using the ECL 96-well plate assay as described in Example 4B. 5 μL of 10 μg/mL recombinant Human DLL4 antigen was coated to a 96-well plate, and tested Fab mutants were added at a concentration of 0.04 μM. As a control, background binding of the Fab to a blank well of the 96-well plate also was determined. The data were depicted as a Signal/Noise ratio of the ECL signal, which is the ratio of the ECL signal for binding to DLL4 divided by the ECL signal for residual binding to the plate. Table 35 sets forth the mutant Fabs tested and the Signal/Noise ratio observed for binding to DLL4. The results show that mutation of E100, Y101, 5105, E107 or Q110 with alanine caused a reduction in the ECL signal and therefore decreased binding affinity to DLL4. These residues, therefore, appeared to be involved in the DLL4 binding and were not further mutagenized. In contrast, mutation of S102, S103, S104 or H111 with alanine resulted in either an increased ECL signal or no difference in ECL signal compared to the parent and thus either improved binding affinity or did not affect binding affinity to DLL4. Accordingly, these residues were identified as residues for further mutagenesis.
• The ECL binding experiments above were repeated, except with varying concentrations of mutant Fab and DLL4 protein. Table 36 sets forth the mutant Fab, the ECL signal, and the Signal/Noise ratio for two different concentrations of DLL4 antigen and mutant Fab. The results are consistent for both assays and confirm the initial results above. Substitution of E100, Y101, 5105, E107 or Q110 with alanine caused a reduction in ECL signal for binding to DLL4 while substitution of S102, S103, S104 or H111 with alanine either improved the ECL signal for binding or did not affect the ECL signal for binding to DLL4.

• TABLE 35
  Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 alanine mutant
  binding data

  Fab	Signal/Noise

  Heavy Chain	SEQ ID NO	Light Chain	SEQ ID NO	(0.04 μM)

  E100A	129	L6_IGKJ1*01	107	0.9
  Y101A	130	L6_IGKJ1*01	107	0.8
  S102A	124	L6_IGKJ1*01	107	5.6
  S103A	131	L6_IGKJ1*01	107	3.5
  S104A	122	L6_IGKJ1*01	107	1.3
  S105A	132	L6_IGKJ1*01	107	0.8
  E107A	133	L6_IGKJ1*01	107	0.7
  Q110A	134	L6_IGKJ1*01	107	0.9
  H111A	135	L6_IGKJ1*01	107	2.4
  parental	88	L6_IGKJ1*01	107	3.1

• TABLE 36
  Fab VH1-46_IGHD6-6*01_IGHJ1*01 &
  L6_IGKJ1*01 alanine mutant binding data

  Fab

  	0.02 μM Fab	0.004 μM Fab
  	30 μg/mL DLL4	15 μg/mL DLL4

  	SEQ	Light Chain	ECL	Signal/	ECL	Signal/
  Heavy Chain	ID NO	(SEQ ID NO: 107)	Signal	Noise	Signal	Noise

  VH1-46_IGHD6-	88	L6_IGKJ1*01	8714	23.0	4261	29.2
  6*01_IGHJ1*01
  E100A	129	L6_IGKJ1*01	1296	3.4	536	3.7
  Y101A	130	L6_IGKJ1*01	237	0.6	340	2.3
  S102A	124	L6_IGKJ1*01	19056	50.3	10338	70.8
  S103A	131	L6_IGKJ1*01	11553	30.5	5150	35.3
  S104A	122	L6_IGKJ1*01	163452	431.3	3614	24.8
  S105A	132	L6_IGKJ1*01	1103	2.9	181	1.2
  E107A	133	L6_IGKJ1*01	338	0.9	146	1.0
  Q110A	134	L6_IGKJ1*01	257	0.7	128	0.9
  H111A	135	L6_IGKJ1*01	11582	30.6	5023	34.4

• iii. NNK Mutagenesis of Heavy Chain Amino Acid Residues S102, S103, S104
• Following alanine scanning mutagenesis of CDR3, heavy chain amino acid residues S102, S103 and S104 were selected for further mutation using overlapping PCR with NNK mutagenesis as described in Example 1 using parent Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 as a template.
• The binding affinity of each generated Fab mutant for DLL4 was determined using the 96-well plate ECL assay described in Example 4 with varying concentrations of Fab and DLL4 protein. Table 37 sets forth the Signal/Noise ratio for each of the S102, S103 and S104 NNK mutants. Fab NNK mutants were selected at random prior to sequencing and therefore several mutants, such as S103L, were purified and tested multiple times giving consistent results. Three mutations in Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 were identified that resulted in a Fab with an increased signal/noise ratio and therefore improved binding affinity to DLL4. Two Fab mutants, S102A and S103P, each had an signal/noise ratio for DLL4 approximately 3-fold greater than parent Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01. A third mutant, heavy chain Fab mutant S104F, had a signal/noise ratio for binding to DLL4 at least 4-fold greater than that of parent Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01. Two additional mutations were identified that resulted in a slight increase in the signal/noise ratio for binding to DLL4, namely Fab heavy chain mutants S103A and S104H.

• TABLE 37
  NNK mutagenesis of Fab VH1-46_IGHD6-6*01_IGHJ1*01 and
  L6_IGKJ1*01 at amino acid residues S102, S103 and S104

  	0.02 μM	0.004 μM
  Fab	Fab	Fab

  	SEQ	Light Chain	30 μg/mL	15 μg/mL
  	ID	(SEQ ID	DLL4	DLL4
  Heavy Chain	NO	NO: 107)	Signal/Noise	Signal/Noise

  VH1-46_IGHD6-	88	L6_IGKJ1*01	19.5	25.0
  6*01_IGHJ1*01
  VH1-46_IGHD6-	88	L6_IGKJ1*01	24.8	19.5
  6*01_IGHJ1*01
  VH1-46_IGHD6-	88	L6_IGKJ1*01	20.3	28.3
  6*01_IGHJ1*01
  S102Q	136	L6_IGKJ1*01	40.6	31.9
  S102V	137	L6_IGKJ1*01	35.9	36.5
  S102I	138	L6_IGKJ1*01	35.3	34.5
  S102A	124	L6_IGKJ1*01	51.7	69.8
  S102G	139	L6_IGKJ1*01	5.1	5.2
  S103stop	234	L6_IGKJ1*01	0.8	1.1
  S103L	140	L6_IGKJ1*01	25.8	36.6
  S103W	141	L6_IGKJ1*01	16.3	25.0
  S103L	140	L6_IGKJ1*01	27.0	36.8
  S103L	140	L6_IGKJ1*01	39.8	44.9
  S103F	142	L6_IGKJ1*01	16.4	20.7
  S103L	140	L6_IGKJ1*01	22.5	30.7
  S103L	140	L6_IGKJ1*01	18.7	28.1
  S103N	143	L6_IGKJ1*01	18.8	23.8
  S103H	144	L6_IGKJ1*01	21.7	31.7
  S103C	145	L6_IGKJ1*01	27.1	27.4
  S103L	140	L6_IGKJ1*01	22.1	36.3
  S103L	140	L6_IGKJ1*01	24.0	40.4
  S103A	131	L6_IGKJ1*01	30.9	44.5
  S103A	131	L6_IGKJ1*01	29.1	32.9
  S103L	140	L6_IGKJ1*01	26.6	30.7
  S103G	146	L6_IGKJ1*01	9.1	8.3
  S103W	141	L6_IGKJ1*01	25.8	38.8
  S103F	142	L6_IGKJ1*01	21.9	21.2
  S103P	123	L6_IGKJ1*01	59.7	82.4
  S103N	143	L6_IGKJ1*01	13.4	22.4
  S104G	147	L6_IGKJ1*01	23.4	20.0
  S104C	148	L6_IGKJ1*01	9.9	8.4
  S104H	149	L6_IGKJ1*01	24.9	79.2
  S104L	150	L6_IGKJ1*01	23.5	43.8
  S104R	151	L6_IGKJ1*01	23.4	28.6
  S104G	147	L6_IGKJ1*01	45.5	67.8
  S104F	121	L6_IGKJ1*01	76.5	134.2
  S104L	150	L6_IGKJ1*01	24.8	25.6

• The Fab heavy chain mutants, S102A, S103A, S103P, S104H and S104F, each containing a mutation in the heavy chain parent Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01, were subsequently re-assayed using the ECL multispot assay as describe in Example 4A to confirm the observed increased binding affinity for DLL4. Each Fab mutant was tested against a panel of antigens at two different Fab concentrations. The results are set forth in Tables 38-39 below. Table 29 sets forth the results for the ECL signal and signal/noise ratio of each mutant for binding to DLL4. Table 38 sets forth the signal/noise ratio for binding to all of the tested antigens. The results show that the heavy chain mutants S102A, S103P, S104H and S104F all have increased signals for binding to DLL4 as compared to parent Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01, and additionally these mutants bind in a dose-dependent and antigen specific manner. Further, the results show that the signal for binding of heavy chain mutant S103A to DLL4 is about the same as binding of parent Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01.

• TABLE 38
  Binding affinity of Fab VH1-46_IGHD6-6*01_IGHJ1*01 and L6_IGKJ1*01
  heavy chain mutants S102A, S103A, S103P, S104H, and S104F for DLL4

  	SEQ	Light Chain	Fab			Signal/
  Heavy Chain	ID NO	(SEQ ID NO: 107)	[μM]	Signal	Blank	Noise

  S103A

  	131	L6_IGKJ1*01	0.1	7108	225	31.6
  S103A	131	L6_IGKJ1*01	0.01	1192	265	4.5
  S103P	123	L6_IGKJ1*01	0.1	19284	139	138.7
  S103P	123	L6_IGKJ1*01	0.01	4095	179	22.9
  S104H	149	L6_IGKJ1*01	0.1	20053	227	88.3
  S104H	149	L6_IGKJ1*01	0.01	4159	154	27.0
  S104F	121	L6_IGKJ1*01	0.1	27072	139	194.8
  S104F	121	L6_IGKJ1*01	0.01	4283	280	15.3
  Parent	88	L6_IGKJ1*01	0.1	7002	171	40.9
  Parent	88	L6_IGKJ1*01	0.01	1030	210	4.9
  S102A	124	L6_IGKJ1*01	0.1	15754	220	71.6
  S102A	124	L6_IGKJ1*01	0.01	2598	259	10.0

• TABLE 39
  Binding affinity and specificity of Fab VH1-46_IGHD6-6*01_IGHJ1*01 and
  L6_IGKJ1*01 heavy chain mutants S102A, S103A, S103P, S104H, and S104F

  	Fab
  	[μM]	ErbB2	EGF R	HGF R	Notch-1	CD44	IGF-1	P-Cad	EPO R	DLL4

  S103A	0.1	1.0	1.2	1.0	1.2	1.4	1.2	1.4	1.4	31.6
  S103A	0.01	0.9	1.3	1.2	1.4	1.3	1.1	1.3	1.1	4.5
  S103P	0.1	2.2	2.3	1.9	2.6	2.1	2.0	1.4	2.4	138.7
  S103P	0.01	2.0	1.8	1.2	1.8	1.5	1.0	1.1	1.8	22.9
  S104H	0.1	1.0	0.6	0.8	0.8	1.1	1.0	0.8	1.0	88.3
  S104H	0.01	1.0	1.0	1.0	1.4	1.4	1.5	1.0	1.2	27.0
  S104F	0.1	1.8	2.0	1.6	2.6	1.9	1.7	1.4	2.1	194.8
  S104F	0.01	0.7	0.8	0.6	0.8	0.8	0.6	0.4	0.7	15.3
  Parent	0.1	1.2	1.2	1.3	1.7	1.8	1.6	1.6	1.2	40.9
  Parent	0.01	1.0	0.9	0.9	0.5	1.1	0.8	1.0	1.0	4.9
  S102A	0.1	0.8	0.9	0.5	1.4	1.3	1.3	1.0	1.3	71.6
  S102A	0.01	1.0	0.8	0.5	0.6	0.9	0.5	0.2	0.8	10.0

• iv. Combination Mutants Based on NNK Mutagenesis Results
• Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 heavy chain mutants S102A, S103P and S104F, identified as contributing to increased binding to DLL4, were combined to generate a triple mutant. The triple mutant is designated as Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F & L6_IGKJ1*01 (H:APF & L:wt). The binding affinity and specificity of the Fab APF triple mutant was determined using both the ECL multispot assay and ELISA.
• The ECL multispot assay described in Example 4A was used to compare the specificity and binding affinity of the APF triple mutant and the parent antibody for binding to DLL4 and other antigens at various concentrations of antibody. Table 40 sets forth the signal/noise ratio for binding of the parent and APF triple mutant against the tested antigens. The results show that the heavy chain APF triple mutant binds DLL4 with 10-fold greater binding affinity than the parent antibody. Additionally, the APF triple mutant specifically binds DLL4, since no detectable signal was observed for binding to any other tested antigen.
• The binding of the APF triple mutant to DLL4 was further analyzed by ELISA as described in Example 6 at Fab concentrations of 125 nm to 1000 nm antibody. The results are set forth in Table 41 below. At the tested concentrations, the parent Fab antibody did not show a detectable signal for binding to DLL4. In contrast, the APF triple mutant had a detectable signal evidencing DLL4 binding in a concentration dependent manner. These results confirm that the ECL assay is more sensitive then the ELISA assay.

• TABLE 40
  Binding affinity and specificity of triple mutant Fab VH1-46_IGHD6-6*01_IGHJ1*01
  S102A/S103P/S104F (APF) & L6_IGKJ1*01 (SEQ ID NOS: 125 and 107) as compared to parent Fab
  VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 (SEQ ID NOS: 88 and 107)

  	Fab
  	[μM]	ErbB2	EGF R	HGF R	Notch-1	CD44	IGF-1	P-Cad	EPO R	DLL4

  Wt	500.00	0.3	0.7	0.3	0.8	0.4	0.4	0.2	0.8	16.2
  	50.00	0.6	0.9	0.6	0.6	0.3	0.5	0.7	0.9	33.5
  	5.00	1.0	1.0	0.9	0.8	1.3	1.1	0.9	1.1	32.5
  	0.50	1.0	1.4	0.6	2.0	1.0	1.2	1.3	0.9	2.9
  S102A,	500.00	1.7	5.5	2.2	4.2	2.4	1.5	3.4	10.4	181.4
  S103P,	50.00	0.7	1.0	0.7	1.1	0.7	0.5	0.9	1.6	274.5
  S104F	5.00	1.1	1.1	0.8	0.9	1.3	1.1	1.0	1.8	482.1
  	0.50	1.0	1.1	0.8	1.4	1.0	1.3	0.8	0.9	34.5

• TABLE 41
  Binding affinity of triple mutant Fab VH1-46_IGHD6-6*01_IGHJ1*01
  S102A/S103P/S104F (APF) & L6_IGKJ1*01 (SEQ ID NOS: 125 and
  107) as compared to parent Fab VH1-46_IGHD6-6*01_IGHJ1*01
  & L6_IGKJ1*01 (SEQ ID NOS: 88 and 107)

  			S102A, S103P,
  Fab [nM]	Wildtype	Blank	S104F	Blank

  1000	0.071	0.060	0.463	0.080
  500	0.070	0.069	0.307	0.074
  250	0.069	0.064	0.231	0.071
  125	0.070	0.066	0.173	0.075

Example 8 Further Optimization of the Heavy Chain of Anti-DLL4 APF Triple Mutant for Binding to DLL4
• In this example, the heavy chain of the APF triple mutant described and generated in Example 7 was further optimized to improve its binding affinity for DLL4. The APF triple mutant Fab was used as a template for further mutagenesis of heavy chain amino acid residues in the remaining CDR regions of the antibody heavy chain Amino acid residue G55 of CDR2 and amino acid residues E100, A106, Y108, F109, and H111 of CDR3 were subjected to mutagenesis using overlapping PCR with NNK mutagenesis, as described above in Example 1.
• The Fab APF triple mutant containing further mutations at amino acid residues E100, A106, Y108, F109, and H111 were tested for binding to DLL4 and other antigens using the ECL Multispot Assay at a concentration of 10 nM Fab. The results are set forth in Tables 42-43 below. The Signal/Noise ratio of each mutant Fab tested for binding to DLL4 is set forth in Table 42. Table 43 sets forth the ECL signal and blank (background binding to control well containing no antigen) for the binding of each mutant Fab to various tested antigens. Amino acid mutations designated with X (for any amino acid) did not show appreciable binding and therefore were not sequenced to identify the exact mutation. The results show that mutation of amino acid residues G55, E100, A106, Y108, or F109 with any other amino acid generally caused a reduction in binding affinity to DLL4 as evidenced by a reduction in ECL signal while substitution of H111 either improved binding affinity or did not affect binding affinity to DLL4 as evidenced by an increased ECL signal or no change in ECL signal. In particular, Fab heavy chain mutant VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F & L6_IGKJ1*01 (H:APFF & L:wt) had a 2 to 4-fold better signal/noise ratio for binding to DLL4 than the Fab APF triple mutant. Additionally, none of the mutants showed any appreciable binding to any of the other tested antigens (see Table 43 below.)

• TABLE 42
  NNK mutagenesis of Fab VH1-46_IGHD6-6*01_IGHJ1*01
  S102A/S103P/S104F (APF) & L6_IGKJ1*01 at amino acid residues
  G55, E100, A106, Y108, F109 and H111

  Fab [10 nM]

  		Light (SEQ	Signal/
  Heavy	SEQ ID NO	ID NO: 107)	Noise

  S102A/S103P/S104F	125	L6_IGKJ1*01	12.8
  S102A/S103P/S104F	125	L6_IGKJ1*01	10.4
  S102A/S103P/S104F G55W	152	L6_IGKJ1*01	8.0
  S102A/S103P/S104F G55X	235	L6_IGKJ1*01	1.4
  S102A/S103P/S104F G55X	235	L6_IGKJ1*01	1.1
  S102A/S103P/S104F G55X	235	L6_IGKJ1*01	1.0
  S102A/S103P/S104F G55X	235	L6_IGKJ1*01	0.8
  S102A/S103P/S104F G55X	235	L6_IGKJ1*01	0.6
  S102A/S103P/S104F G55D	153	L6_IGKJ1*01	1.2
  S102A/S103P/S104F	125	L6_IGKJ1*01	11.1
  S102A/S103P/S104F G55X	235	L6_IGKJ1*01	1.3
  S102A/S103P/S104F E100X	236	L6_IGKJ1*01	1.2
  S102A/S103P/S104F E100X	236	L6_IGKJ1*01	1.0
  S102A/S103P/S104F	125	L6_IGKJ1*01	20.4
  S102A/S103P/S104F E100X	236	L6_IGKJ1*01	1.1
  S102A/S103P/S104F E100X	236	L6_IGKJ1*01	1.0
  S102A/S103P/S104F E100X	236	L6_IGKJ1*01	1.7
  S102A/S103P/S104F E100X	236	L6_IGKJ1*01	1.2
  S102A/S103P/S104F E100X	236	L6_IGKJ1*01	1.5
  S102A/S103P/S104F	125	L6_IGKJ1*01	14.9
  S102A/S103P/S104F A106X	237	L6_IGKJ1*01	0.7
  S102A/S103P/S104F A106X	237	L6_IGKJ1*01	0.9
  S102A/S103P/S104F A106X	237	L6_IGKJ1*01	1.2
  S102A/S103P/S104F A106X	237	L6_IGKJ1*01	1.7
  S102A/S103P/S104F A106X	237	L6_IGKJ1*01	1.1
  S102A/S103P/S104F A106X	237	L6_IGKJ1*01	1.5
  S102A/S103P/S104F A106X	237	L6_IGKJ1*01	1.9
  S102A/S103P/S104F	125	L6_IGKJ1*01	16.0
  S102A/S103P/S104F	125	L6_IGKJ1*01	13.8
  S102A/S103P/S104F A106X	237	L6_IGKJ1*01	1.1
  S102A/S103P/S104F Y108X	238	L6_IGKJ1*01	0.9
  S102A/S103P/S104F Y108X	238	L6_IGKJ1*01	1.6
  S102A/S103P/S104F Y108X	238	L6_IGKJ1*01	11.7
  S102A/S103P/S104F Y108X	238	L6_IGKJ1*01	1.2
  S102A/S103P/S104F	125	L6_IGKJ1*01	17.6
  S102A/S103P/S104F Y108X	238	L6_IGKJ1*01	6.2
  S102A/S103P/S104F	125	L6_IGKJ1*01	18.0
  S102A/S103P/S104F A106E	154	L6_IGKJ1*01	4.3
  S102A/S103P/S104F Y108X	238	L6_IGKJ1*01	8.0
  S102A/S103P/S104F Y108X	238	L6_IGKJ1*01	0.8
  S102A/S103P/S104F F109X	239	L6_IGKJ1*01	1.1
  S102A/S103P/S104F F109X	239	L6_IGKJ1*01	1.2
  S102A/S103P/S104F	125	L6_IGKJ1*01	9.9
  S102A/S103P/S104F F109X	239	L6_IGKJ1*01	4.5
  S102A/S103P/S104F F109X	239	L6_IGKJ1*01	0.9
  S102A/S103P/S104F	125	L6_IGKJ1*01	12.0
  S102A/S103P/S104F F109X	239	L6_IGKJ1*01	1.0
  S102A/S103P/S104F F109X	239	L6_IGKJ1*01	1.3
  S102A/S103P/S104F F109X	239	L6_IGKJ1*01	26.4
  S102A/S103P/S104F	125	L6_IGKJ1*01	1.8
  S102A/S103P/S104F H111X	240	L6_IGKJ1*01	1.1
  S102A/S103P/S104F H111F	126	L6_IGKJ1*01	42.5
  S102A/S103P/S104F	125	L6_IGKJ1*01	14.5
  S102A/S103P/S104F	125	L6_IGKJ1*01	13.7
  S102A/S103P/S104F H111X	240	L6_IGKJ1*01	2.4
  S102A/S103P/S104F	125	L6_IGKJ1*01	12.3
  S102A/S103P/S104F H111X	240	L6_IGKJ1*01	12.4
  S102A/S103P/S104F H111X	240	L6_IGKJ1*01	6.2
  S102A/S103P/S104F	125	L6_IGKJ1*01	24.7
  S102A/S103P/S104F H111S	155	L6_IGKJ1*01	24.0

• TABLE 43
  NNK mutagenesis of Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F
  (APF) & L6_IGKJ1*01 at amino acid residues G55, E100, A106, Y108, F109 and H111

  Heavy Chain
  [10 nM Fab]	ErbB2	EGF R	HGF R	Notch-1	CD44	IGF-1	P-Cad	EPO R	Blank

  APF	330	272	306	257	189	241	297	304	271
  APF	157	237	272	334	96	197	208	329	204
  APF G55W	334	312	365	327	159	250	391	296	271
  APF G55X	284	190	331	333	165	330	234	275	317
  APF G55X	189	280	182	208	256	202	190	235	301
  APF G55X	145	207	277	436	298	228	301	339	314
  APF G55X	323	307	301	334	257	247	357	261	324
  APF G55X	113	192	254	182	172	192	128	279	235
  APF G55D	302	272	268	302	173	191	243	329	248
  APF	340	216	171	130	236	174	256	285	239
  APF G55X	305	352	377	383	234	248	440	343	245
  APF E100X	273	273	322	265	291	309	271	304	222
  APF E100X	358	287	318	358	304	249	226	284	297
  APF	91	159	212	181	127	238	59	159	95
  APF E100X	314	365	451	418	262	177	430	327	326
  APF E100X	357	267	379	171	257	241	205	222	229
  APF E100X	172	158	188	142	197	169	206	140	132
  APF E100X	229	285	306	144	159	177	249	324	273
  APF E100X	279	267	395	293	295	355	436	302	220
  APF	314	241	388	304	188	291	396	303	243
  APF A106X	200	170	441	336	158	241	267	309	366
  APF A106X	288	244	319	153	276	221	235	248	283
  APF A106X	306	428	452	268	268	320	336	398	390
  APF A106X	349	350	324	270	239	215	367	239	157
  APF A106X	24	253	177	319	297	248	368	258	232
  APF A106X	393	406	380	434	339	404	506	333	237
  APF A106X	174	238	122	63	296	246	159	161	247
  APF	202	138	190	189	199	190	152	179	214
  APF	378	277	317	370	262	207	422	312	306
  APF A106X	273	324	240	331	242	229	251	308	249
  APF Y108X	270	300	294	315	169	285	285	384	385
  APF Y108X	283	272	236	306	321	258	313	334	167
  APF Y108X	322	253	314	314	295	240	189	345	219
  APF Y108X	405	355	438	464	376	334	340	399	321
  APF	413	324	269	390	385	270	301	421	320
  APF Y108X	336	320	276	297	208	343	246	178	211
  APF	200	255	258	336	214	230	280	228	198
  APF A106E	189	226	212	156	192	312	308	204	219
  APF Y108X	239	261	277	292	325	337	333	271	368
  APF Y108X	388	355	423	348	248	380	469	276	336
  APF F109X	378	397	429	362	440	400	509	479	428
  APF F109X	405	444	462	544	324	442	503	441	402
  APF	513	460	339	433	298	318	338	252	372
  APF F109X	294	442	433	382	350	272	379	440	387
  APF F109X	417	334	371	446	235	320	416	463	438
  APF	356	371	434	363	417	293	293	389	344
  APF F109X	304	241	246	369	392	320	351	340	347
  APF F109X	350	399	340	217	338	407	314	376	331
  APF F109X	147	158	298	249	334	260	206	241	148
  APF	221	296	319	251	221	344	449	222	182
  APF H111X	410	414	382	427	362	488	607	430	476
  APF H111F	370	409	493	356	360	345	461	343	290
  APF	381	206	379	450	363	453	384	326	487
  APF	391	428	426	299	400	434	433	480	472
  APF H111X	395	315	298	380	322	387	392	443	454
  APF	525	467	422	376	345	361	305	494	363
  APF H111X	91	292	134	297	164	158	143	291	186
  APF H111X	207	188	256	177	192	142	223	181	185
  APF	302	394	200	283	340	213	118	343	204
  APF H111S	314	286	235	272	244	136	178	277	203

• The APF triple mutant and APFF mutant were further compared for binding to DLL4 using the ECL Multispot Assay. The Fab antibodies were assayed at various concentrations to assess the dose dependence for binding to DLL4. The Fab antibodies also were assayed against various antigens to assess the specificity. The APFF mutant was tested in duplicate. Table 44 sets forth the signal/noise ratio for binding to DLL4. The results show that the H:APFF & L:wt mutant exhibits slightly increased affinity (70 nM) for DLL4 as compared to the H:APF & L:wt mutant (122 nM). Additionally, the results in Table 45, which depict the ECL signal observed in the assay, confirm that both Fab mutants specifically bind to DLL4 compared to other antigens tested.

• TABLE 44
  Binding affinity of Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/
  S103P/S104F (APF) & L6_IGKJ1*01 versus Fab VH1-46_IGHD6-
  6*01_IGHJ1*01 S102A/S103P/S104F/H111F (APFF) & L6_IGKJ1*01

  Heavy Chain

  	S102A/S103P/	S102A/S103P/	S102A/S103P/
  	S104F	S104F/H111F	S104F/H111F
  	(SEQ ID NO: 125)	(SEQ ID NO: 126)	(SEQ ID NO: 126)

  Light Chain

  	L6_IGKJ1*01	L6_IGKJ1*01	L6_IGKJ1*01
  Fab	(SEQ ID NO: 107)	(SEQ ID NO: 107)	(SEQ ID NO: 107)

  [nM]	Signal/Noise

  500.00	65.9	47.3	54.8
  50.00	207.3	239.1	355.7
  5.00	260.4	747.6	282.9
  0.50	46.9	87.6	36.6

• TABLE 45
  Binding affinity and specificity of Fab VH1-46_IGHD6-6*01_IGHJ1*01
  S102A/S103P/S104F (APF) & L6_IGKJ1*01 versus Fab VH1-46_IGHD6-6*01_IGHJ1*01
  S102A/S103P/S104F/H111F (APFF) & L6_IGKJ1*01

  	Fab
  	[μM]	ErbB2	EGF R	HGF R	Notch-1	CD44	IGF-1	P-Cad	EPO R	DLL4	Blank

  S102A	500.00	1578	1477	760	785	874	613	1008	1213	28930	439
  S103P	50.00	672	585	525	509	557	558	652	768	80005	386
  S104F	5.00	401	356	309	338	343	300	547	423	54938	211
  	0.50	199	152	182	230	207	190	161	235	6666	142
  S102A	500.00	908	2409	945	1607	1282	722	1011	4722	26937	570
  S103P	50.00	394	452	368	559	449	283	349	736	79372	332
  S104F	5.00	225	229	208	260	168	232	290	294	76254	102
  H111F	0.50	130	137	104	158	129	94	106	122	8322	95
  S102A	500.00	712	2895	723	1333	1143	736	785	4966	27150	495
  S103P	50.00	503	552	380	470	550	485	453	879	79326	223
  S104F	5.00	286	303	258	304	313	323	280	423	75810	268
  H111F	0.50	222	266	215	265	279	184	201	298	7539	206

Example 9 Further Optimization of the Heavy Chain of Anti-DLL4 APFF Mutant for Binding to DLL4
• In this Example, the heavy chain amino acid sequence of the APFF mutant that was affinity matured for binding to DLL4 as described in Examples 7 and 8, was used as a template for further mutations of other CDR regions of the antibody polypeptide. Mutant Fabs were expressed and assayed for binding to DLL4.
• i. Alanine Scanning of CDR1
• Heavy chain APFF mutant was used as a template for alanine scanning mutagenesis of amino acid residues in CDR1 (amino acids 26-35) to determine residues involved in antibody binding to DLL4. Alanine scanning was performed by mutating only residues T28, F29, T30, S31 and Y33 of CDR1 to an alanine. The mutant Fab antibodies were expressed and purified as described in Example 2 above.
• Purified Fab alanine mutants were tested at a concentration of 10 nM for binding to DLL4 and other antigens using the ECL multispot binding assay. The results for the ECL assay are set forth in Tables 46 and 47. Table 46 sets forth the mutant Fabs and the Signal/Noise ratio for binding to DLL4. The results show that mutation of amino acid residues F29 and Y33 with alanine caused a reduction in the signal/noise ratio for binding to DLL4. Thus, these residues were not selected for further mutagenesis. Mutation of amino acid residues T28, T30 or S31 with alanine resulted in a slight increase in the signal/noise ratio for binding to DLL4 compared to the parent heavy chain APFF mutant. Table 47, which sets forth the ECL signal for binding to various antigens and to a blank well containing no antigen, shows that all antibodies tested exhibited specificity for DLL4. Table 46 also depicts the results of an ELISA assay performed as described in Example 6 using 100 nM of Fab mutant. The results of the ELISA also show that amino acid residue Y33 is involved in DLL4 binding. The differing results observed in the ECL assay compared to the ELISA are likely due to the fact that the ELISA assay selects for long off-rates whereas the ECL assay detects equilibrium binding. Therefore a mutant with a reduced on-rate but improved off rate can exhibit strong binding by ELISA, but it will not necessarily correlate to a strong ECL signal. In contrast, a mutant with an improved on-rate but reduced off rate can exhibit weak binding by ELISA.
• A further experiment was performed to confirm binding of the alanine mutants to DLL4 using an ECL Assay. Table 48 sets forth the ECL signal for DLL4 antigen and blank and signal/ration of each mutant Fab for binding to DLL4. Table 40 sets forth the ECL signals of each mutant Fab for binding to all tested antigens. The results in Tables 48 and 49 confirm the ECL results observed in Tables 46 and 47, respectively.

• TABLE 46
  Binding of Fab heavy chain VH1-46_IGHD6-6*01_IGHJ1*01
  S102A/S103P/S104F/H111F (APFF) & L6_IGKJ1 CDR1 alanine mutants to DLL4

  Fab

  	SEQ	Light Chain	ECL
  Heavy Chain	ID	(SEQ ID	Signal/Noise	ELISA
  VH1-46_IGHD6-6*01_IGHJ1*01	NO	NO: 107)	[10 nM Fab]	(Signal-Noise)
  S102A/S103P/S104F/H111F	126	L6_IGKJ1*01	202.2	0.78
  S102A/S103P/S104F/H111F T28A	156	L6_IGKJ1*01	334.0	0.77
  S102A/S103P/S104F/H111F F29A	157	L6_IGKJ1*01	189.8	0.67
  S102A/S103P/S104F/H111F T30A	158	L6_IGKJ1*01	456.9	0.64
  S102A/S103P/S104F/H111F S31A	159	L6_IGKJ1*01	453.3	0.47
  S102A/S103P/S104F/H111F Y33A	160	L6_IGKJ1*01	136.3	0.09

• TABLE 47
  Binding and specificity of Fab heavy chain VH1-46_IGHD6-6*01_IGHJ1*01
  S102A/S103P/S104F/H111F (APFF) & L6_IGKJ1 CDR1 alanine mutants

  Heavy Chain	ErbB2	EGF R	HGF R	Notch-1	CD44	IGF-1	P-Cad	EPO R	DLL4	Blank

  APFF	354	383	347	369	404	397	347	438	78437	388
  APFF T28A	411	389	427	432	471	408	381	480	140295	420
  APFF F29A	244	293	404	374	414	315	276	466	80652	425
  APFF T30A	272	427	413	270	439	356	275	428	140273	307
  APFF S31A	207	394	398	333	379	405	255	454	137810	304
  APFF Y33A	394	372	345	244	294	308	383	373	26978	198

• TABLE 48
  Binding of Fab heavy chain VH1-46_IGHD6-6*01_IGHJ1*01
  S102A/S103P/S104F/H111F (APFF) & L6_IGKJ1*01 CDR1
  alanine mutants to DLL4

  Heavy Chain	Light Chain
  VH1-46_IGHD6-	(SEQ ID			Signal/
  6*01_IGHJ1*01	NO: 107)	Signal	Blank	Noise

  S102A/S103P/S104F/H111F	L6_IGKJ1*01	181427	449	404.1
  T28A
  S102A/S103P/S104F/H111F	L6_IGKJ1*01	109225	459	238.0
  F29A
  S102A/S103P/S104F/H111F	L6_IGKJ1*01	177678	353	503.3
  T30A
  S102A/S103P/S104F/H111F	L6_IGKJ1*01	176308	333	529.5
  S31A
  APFF	L6_IGKJ1*01	196536	283	694.5
  S102A/S103P/S104F/H111F	L6_IGKJ1*01	59547	265	224.7
  Y33A

• TABLE 49
  Binding and specificity of Fab heavy chain VH1-46_IGHD6-6*01_IGHJ1*01
  S102A/S103P/S104F/H111F (APFF) & L6_IGKJ1*01 CDR1 alanine mutants

  Heavy Chain	ErbB2	EGF R	HGF R	Notch-1	CD44	IGF-1	P-Cad	EPO R	DLL4	Blank

  APFF T28A	316	329	353	478	497	377	477	477	181427	449
  APFF F29A	1292	537	512	6089	978	439	508	1055	109225	459
  APFF T30A	408	351	353	368	396	343	337	479	177678	353
  APFF S31A	253	377	358	427	235	268	262	507	176308	333
  APFF	263	279	252	389	425	342	318	536	196536	283
  APFF Y33A	298	281	248	334	290	227	178	430	59547	265

• ii. Alanine Scanning of CDR2
• Heavy chain APFF mutant was used as a template for alanine scanning mutagenesis of amino acid residues in CDR2 (amino acids 50-66) to determine residues involved in antibody binding to DLL4 Amino acid residues Y60 to G66 were not mutated. The mutant Fab antibodies were expressed and purified as described in Example 2 above.
• Purified Fab alanine mutants were tested at a concentration of 10 nM for binding to DLL4 using the ECL multispot binding assay. The results for the ECL assay are set forth in Tables 50 and 51. Table 50 sets forth the mutant Fabs and the Signal/Noise ratio for binding to DLL4. The results show that mutation of amino acid residues 150, G55, S57, T58, or S59 with alanine caused a reduction in the signal/noise ratio for binding to DLL4, and thus these residues were not further mutagenized. In contrast, mutation of amino acid residues I51, N52, P53, S54 or G56 with alanine improved the signal/noise ratio for binding to DLL4 2- to 4-fold over the parent heavy chain APFF mutant, and thus these residues were identified as residues for further mutagenesis. Table 51, which sets forth the ECL signals for binding various antigens and to a blank well containing no antigen, shows that all antibodies tested exhibited specificity for DLL4. Table 50 also depicts the results of an ELISA assay performed as described in Example 6 using 100 nM of Fab mutant. The results of the ELISA generally confirmed the results observed by the ECL assay. Mutation of amino acid residues 150, G55, S57, T58 and S59 exhibited decreased binding to DLL4 compared to the parent APFF mutant as observed by ELISA.

• TABLE 50
  Binding of Fab heavy chain VH1-46_IGHD6-6*01_IGHJ1*01
  S102A/S103P/S104F/H111F CDR1 and CDR2 alanine mutants to DLL4

  Fab

  		Light Chain	Signal/Noise	ELISA
  Heavy Chain		(SEQ ID	[10 nM	(Signal-
  VH1-46_IGHD6-6*01_IGHJ1*01	SEQ ID NO	NO: 107)	Fab]	Noise)

  APFF	126	L6_IGKJ1*01	202.2	0.78
  S102A/S103P/S104F/H111F I50A	161	L6_IGKJ1*01	9.8	0.01
  S102A/S103P/S104F/H111F I51A	162	L6_IGKJ1*01	637.2	0.49
  S102A/S103P/S104F/H111F N52A	163	L6_IGKJ1*01	721.1	0.60
  S102A/S103P/S104F/H111F P53A	164	L6_IGKJ1*01	462.3	0.41
  S102A/S103P/S104F/H111F G55A	166	L6_IGKJ1*01	44.2	0.02
  S102A/S103P/S104F/H111F G56A	167	L6_IGKJ1*01	441.5	1.60
  S102A/S103P/S104F/H111F S57A	168	L6_IGKJ1*01	293.1	0.39
  S102A/S103P/S104F/H111F T58A	169	L6_IGKJ1*01	142.4	0.14
  S102A/S103P/S104F/H111F S59A	170	L6_IGKJ1*01	17.1	0.02
  S102A/S103P/S104F/H111F S54A	165	L6_IGKJ1*01	122.1	0.255
  APFF	126	L6_IGKJ1*01	71.1	0.123

• TABLE 51
  Binding and specificity of Fab heavy chain VH1-46_IGHD6-6*01_IGHJ1*01
  S102A/S103P/S104F/H111F CDR1 and CDR2 alanine mutants

  Heavy Chain	ErbB2	EGF R	HGF R	Notch-1	CD44	IGF-1	P-Cad	EPO R	DLL4	Blank

  APFF	354	383	347	369	404	397	347	438	78437	388
  I50A	369	402	301	297	326	247	313	252	2668	271
  I51A	344	373	440	312	391	383	144	380	159290	250
  N52A	378	340	369	383	362	362	353	468	168745	234
  P53A	203	439	337	393	378	374	390	427	151173	327
  G55A	474	217	221	381	365	392	426	305	14500	328
  G56A	279	355	313	331	330	422	214	466	189405	429
  S57A	304	302	388	365	439	417	232	477	112266	383
  T58A	320	384	304	289	318	271	294	329	47422	333
  S59A	312	358	280	333	346	273	339	382	4502	264

• iii. NNK Mutagenesis of CDR2 Residues N52, S54 and G56
• The Fab heavy chain APFF mutant was subsequently used as a template for further mutagenesis of amino acid residues N52, S54, G56 using NNK mutagenesis, as described above.
• Fab heavy chain mutants containing mutations of amino acid residues N52, S65 and G56 in the parent APFF mutant template I-LAPFF & L:wt were tested for binding to DLL4 using the 96-well plate DLL4 ECL binding assay described in Example 4B and the ELISA assay described in Example 6. Table 52 depicts the ECL and ELISA signal for binding to DLL4 for the various mutants tested. Double mutants, such as I51T/N52V, were inadvertently generated during the PCR reaction. Several Fab mutants that contained a combination of two mutations at a specific amino acid position are designated as such. For example, G56E/D indicates the tested antibody was a mixture of two Fabs, one containing the mutation G56E and the other containing the mutation G56D. Both the ECL and ELISA results show that several Fab heavy chain mutants containing mutations in the Fab APFF mutant, including N52L, N52W, S54T, G56H and G56W, all bind DLL4 with greater affinity than the parent Fab APFF mutant.

• TABLE 52
  Binding of Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F
  (APFF) & L6_IGKJ1*01 NNK heavy chain mutants to DLL4

  Fab

  ECL

  ELISA

  Heavy Chain	SEQ	Light Chain	Signal	Signal
  VH1-46_IGHD6-6*01_IGHJ1*01	ID	(SEQ ID	[10 nM	[100 nM
  Mutant	NO	NO: 107)	Fab]	Fab]

  S102A/S103P/S104F/H111F I51T/N52V	171	L6_IGKJ1*01	71708	1.23
  S102A/S103P/S104F/H111F N52G	172	L6_IGKJ1*01	55584	0.47
  S102A/S103P/S104F/H111F N52T	173	L6_IGKJ1*01	66771	0.61
  S102A/S103P/S104F/H111F N52P	174	L6_IGKJ1*01	44756	0.18
  APFF	126	L6_IGKJ1*01	42782	0.18
  S102A/S103P/S104F/H111F N52L	175	L6_IGKJ1*01	75452	1.06
  S102A/S103P/S104F/H111F N52W	176	L6_IGKJ1*01	87011	0.42
  S102A/S103P/S104F/H111F N52Y	177	L6_IGKJ1*01	24501	0.01
  S102A/S103P/S104F/H111F N52R	183	L6_IGKJ1*01	21642	0.01
  S102A/S103P/S104F/H111F N52V	178	L6_IGKJ1*01	64665	0.24
  S102A/S103P/S104F/H111F N52S	179	L6_IGKJ1*01	62211	0.28
  S102A/S103P/S104F/H111F N52Q	180	L6_IGKJ1*01	60646	0.10
  S102A/S103P/S104F/H111F N52K	181	L6_IGKJ1*01	67116	0.45
  S102A/S103P/S104F/H111F N52A	163	L6_IGKJ1*01	52534	0.12
  S102A/S103P/S104F/H111F G56V	182	L6_IGKJ1*01	68585	0.23
  S102A/S103P/S104F/H111F G56E/G	241	L6_IGKJ1*01	61039	0.21
  S102A/S103P/S104F/H111F G56V/N	242	L6_IGKJ1*01	68876	0.25
  S102A/S103P/S104F/H111F G56S	184	L6_IGKJ1*01	65728	0.18
  S102A/S103P/S104F/H111F G56K	185	L6_IGKJ1*01	66152	0.19
  S102A/S103P/S104F/H111F G56E/D	243	L6_IGKJ1*01	70474	0.24
  S102A/S103P/S104F/H111F G56T	186	L6_IGKJ1*01	60689	0.20
  S102A/S103P/S104F/H111F G56L	187	L6_IGKJ1*01	64709	0.12
  S102A/S103P/S104F/H111F G56A	167	L6_IGKJ1*01	63058	0.24
  APFF	126	L6_IGKJ1*01	51792	0.09
  S102A/S103P/S104F/H111F G56R	188	L6_IGKJ1*01	64277	0.20
  S102A/S103P/S104F/H111F G56H	189	L6_IGKJ1*01	68804	0.65
  S102A/S103P/S104F/H111F G56I	190	L6_IGKJ1*01	76973	0.23
  S102A/S103P/S104F/H111F G56L	187	L6_IGKJ1*01	63372	0.19
  S102A/S103P/S104F/H111F G56W	191	L6_IGKJ1*01	69571	0.54
  S102A/S103P/S104F/H111F G56A	167	L6_IGKJ1*01	65124	0.26
  S102A/S103P/S104F/H111F S54I	192	L6_IGKJ1*01	18450	0.03
  APFF	126	L6_IGKJ1*01	46641	0.07
  S102A/S103P/S104F/H111F S54E	193	L6_IGKJ1*01	36826	0.04
  S102A/S103P/S104F/H111F S54R	194	L6_IGKJ1*01	26284	0.02
  S102A/S103P/S104F/H111F S54G	195	L6_IGKJ1*01	47033	0.06
  S102A/S103P/S104F/H111F S54T	196	L6_IGKJ1*01	57232	0.08
  S102A/S103P/S104F/H111F S54L	197	L6_IGKJ1*01	28172	0.02
  S102A/S103P/S104F/H111F S54V	198	L6_IGKJ1*01	22155	0.00
  S102A/S103P/S104F/H111F S54Q	264	L6_IGKJ1*01	41757	0.07
  S102A/S103P/S104F/H111F S54A	165	L6_IGKJ1*01	32598	0.02
  S102A/S103P/S104F/H111F S54N	199	L6_IGKJ1*01	31710	0.02
  S102A/S103P/S104F/H111F S54P	200	L6_IGKJ1*01	10059	0.00
  S102A/S103P/S104F/H111F I50T/S54P	201	L6_IGKJ1*01	229	0.00
  S102A/S103P/S104F/H111F S54A	165	L6_IGKJ1*01	35277	0.02
  S102A/S103P/S104F/H111F S54A/S59N	202	L6_IGKJ1*01	17305	0.100
  APFF	126	L6_IGKJ1*01	42886	0.06

• iv. Further Mutagenesis of CDR2 Amino Acid Residue I51
• A Fab mutant containing N52L, S54T and G56H was generated. Thus, the resulting Fab mutant contains seven mutations in the heavy chain of the antibody: S102A/S103P/S104F/H111F N52L/S54T/G56H, and is designated Fab mutant VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F N52L/S54T/G56H & L6_IGKJ1*01 (H:APFF LTH & L:wt). The H:APFF LTH mutant was used as a template for further NNK mutagenesis of CDR2 amino acid residue I51. The I51 mutants were tested for binding to DLL4 using the 96-well plate ECL binding assay described in Example 4B and ELISA described in Example 6. The results are depicted in Table 53, which sets forth the ECL and ELISA signals. The results show that mutation of amino acid residue I51 to valine (I51V) in the H:APFF LTH parent backbone caused a further increase in binding affinity to DLL4 compared to the H:APFF LTH parent.

• TABLE 53
  Binding of Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F
  (APFF) & L6_IGKJ1*01 I51 NNK heavy chain mutants to DLL4

  Fab

  ECL

  ELISA

  Heavy Chain	SEQ	Light Chain	Signal	Signal
  VH1-46_IGHD6-6*01_IGHJ1*01	ID	(SEQ ID	[10 nM	[100 nM
  Mutant	NO	NO: 107)	Fab]	Fab]

  S102A/S103P/S104F/H111F/	203	L6_IGKJ1*01	165312	1.101
  N52L/S54T/G56H (APFF LTH)
  S102A/S103P/S104F/H111F/	204	L6_IGKJ1*01	142542	0.620
  I51A/N52L/S54T/G56H (APFF ALTH)
  S102A/S103P/S104F/H111F/	205	L6_IGKJ1*01	123199	0.641
  I51T/N52L/S54T/G56H (APFF TLTH)
  S102A/S103P/S104F/H111F/	206	L6_IGKJ1*01	154612	0.513
  I51Y/N52L/S54T/G56H (APFF YLTH)
  S102A/S103P/S104F/H111F/	207	L6_IGKJ1*01	155073	0.647
  I51H/N52L/S54T/G56H (APFF HLTH)
  S102A/S103P/S104F/H111F/	208	L6_IGKJ1*01	166549	0.995
  I51E/N52L/S54T/G56H (APFF ELTH)
  S102A/S103P/S104F/H111F/	209	L6_IGKJ1*01	192273	1.105
  I51V/N52L/S54T/G56H (APFF VLTH)
  S102A/S103P/S104F/H111F/	210	L6_IGKJ1*01	130722	0.407
  I51G/N52L/S54T/G56H (APFF GLTH)
  S102A/S103P/S104F/H111F/	211	L6_IGKJ1*01	134860	0.786
  I51S/N52L/S54T/G56H (APFF SLTH)
  S102A/S103P/S104F/H111F/	212	L6_IGKJ1*01	126271	0.088
  I51W/N52L/S54T/G56H (APFF WLTH)
  S102A/S103P/S104F/H111F/	213	L6_IGKJ1*01	92415	0.512
  I51R/N52L/S54T/G56H (APFF RLTH)
  S102A/S103P/S104F/H111F/	214	L6_IGKJ1*01	125869	1.091
  I51N/N52L/S54T/G56H (APFF NLTH)

• v. NNK Mutagenesis of CDR2 Amino Acid Residue P53
• Fab mutant VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F I51T/N52L/S54T/G56H & L6_IGKJ1*01 (H:APFF TLTH) was used as a template for NNK mutagenesis of CDR2 amino acid residue P53. The P53 mutants were tested for binding to DLL4 using the 96-well plate ECL binding assay described in Example 4B and ELISA assay described in Example 6. Table 54 sets forth the ECL and ELISA signals. The results show that mutation of Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F I51T/N52L/S54T/G56H(H:APFF TLTH) & L6_IGKJ1*01 heavy chain residue P53 to alanine (P53A) causes an increase in binding affinity to DLL4 compared to the H:APFF TLTH mutant.

• TABLE 54
  Binding of Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F
  (APFF) & L6_IGKJ1*01 P53 NNK heavy chain mutants to DLL4

  Fab

  ECL

  ELISA

  	SEQ	Light Chain	Signal	Signal
  Heavy Chain	ID	(SEQ ID	[10 nM	[100 nM
  VH1-46_IGHD6-6*01_IGHJ1*01 Mutant	NO	NO: 107)	Fab]	Fab]

  S102A/S103P/S104F/H111F/	205	L6_IGKJ1*01	123199	0.641
  I51T/N52L/S54T/G56H (APFF TLTH)
  S102A/S103P/S104F/H111F/	215	L6_IGKJ1*01	91483	0.035
  I51T/N52L/P53V/S54T/G56H (APFF TLVTH)
  S102A/S103P/S104F/H111F/	216	L6_IGKJ1*01	103398	0.018
  I51T/N52L/P53G/S54T/G56H (APFF TLGTH)
  S102A/S103P/S104F/H111F/	217	L6_IGKJ1*01	135290	0.076
  I51T/N52L/P53S/S54T/G56H (APFF TLSTH)
  S102A/S103P/S104F/H111F/	218	L6_IGKJ1*01	126454	0.433
  I51T/N52L/P53W/S54T/G56H
  (APFF TLWTH)
  S102A/S103P/S104F/H111F/	219	L6_IGKJ1*01	63200	0.070
  I51T/N52L/P53R/S54T/G56H (APFF TLRTH)
  S102A/S103P/S104F/H111F/	220	L6_IGKJ1*01	113788	0.021
  I51T/N52L/P53N/S54T/G56H (APFF TLNTH)
  S102A/S103P/S104F/H111F/	221	L6_IGKJ1*01	163025	0.330
  I51T/N52L/P53A/S54T/G56H (APFF
  TLATH)
  S102A/S103P/S104F/H111F/	222	L6_IGKJ1*01	124867	0.219
  I51T/N52L/P53T/S54T/G56H (APFF TLTTH)
  S102A/S103P/S104F/H111F/	223	L6_IGKJ1*01	99517	0.274
  I51T/N52L/P53Y/S54T/G56H (APFF TLYTH)
  S102A/S103P/S104F/H111F/	224	L6_IGKJ1*01	107908	0.287
  I51T/N52L/P53H/S54T/G56H (APFF TLHTH)
  S102A/S103P/S104F/H111F/	225	L6_IGKJ1*01	91504	0.017
  I51T/N52L/P53E/S54T/G56H (APFF TLETH)
  S102A/S103P/S104F/H111F/	226	L6_IGKJ1*01	105485	0.341
  I51T/N52L/P53M/S54T/G56H (APFF
  TLMTH)

• Heavy chain mutants APFF LTH (SEQ ID NO:203), APFF ELTH (SEQ ID NO: 208), APPF VLTH (SEQ ID NO: 209), APFF NLTH (SEQ ID NO: 214), APFF TLATH (SEQ ID NO: 221) and APFF I51T/N52V (SEQ ID NO: 171) were each paired with parent light chain L6_IGKJ1*01 (SEQ ID NO:107) and further analyzed for binding to DLL4 by ELISA using 2-fold serial dilutions of Fab, starting at a concentration of 20 nM. The results are set forth in Table 55 below. The results show that Fabs containing heavy chain mutants APFF LTH (SEQ ID NO:206), APFF ELTH (SEQ ID NO: 208), APPF VLTH (SEQ ID NO: 209) and APFF NLTH (SEQ ID NO: 214) bind DLL4 with a Kd of approximately between 1 nM and 10 nM. Fabs containing heavy chain mutants APFF TLATH (SEQ ID NO: 221) and APFF I51T/N52V (SEQ ID NO:171) have lower affinity for DLL4 as compared to the other tested Fabs. Heavy chain mutant APFF TLATH has an approximate Kd greater than 100 nM and heavy chain mutant APFF I51T/N52V has a Kd between 10 and 100 nM.

• TABLE 55
  Heavy chain Fab mutant VH1-46_IGHD6-6*01_IGHJ1*01
  S102A/S103P/S104F/H111F (APFF) & L6_IGKJ1*01 (SEQ
  ID NO: 107) binding to DLL4 by ELISA

  Fab	APFF	APFF	APFF	APFF	APFF	APFF
  [nM]	LTH	ELTH	VLTH	NLTH	TLATH	I51T/N52V

  20	2.402	2.290	2.052	1.627	1.109	0.648
  10	2.345	2.168	1.854	1.362	0.875	0.506
  5	2.477	2.333	2.198	1.751	1.272	0.724
  2.5	2.151	1.982	1.656	1.165	0.592	0.358
  1.3	0.653	0.402	0.252	0.143	0.078	0.055
  0.63	1.367	1.010	0.785	0.419	0.227	0.115
  0.31	2.402	2.290	2.052	1.627	1.109	0.648
  0.16	2.345	2.168	1.854	1.362	0.875	0.506

• vi. NNK Mutagenesis of Framework Amino Acid Residue S84
• Fab heavy chain APFF mutant was used as a template for further mutagenesis of amino acid residue S84 in the framework region of the heavy chain using overlapping PCR with NNK mutagenesis, as described above. The resulting mutants were tested for binding to DLL4 and other antigens using the ECL Multispot binding assay as described in Example 4A and ELISA as described in Example 6. The results for the ECL and ELISA are set forth in Tables 56. Table 56 sets forth mutant Fabs and the Signal/Noise ratio for binding to DLL4 by the ECL method or the ELISA assay. Table 57 sets forth the ECL signals of each mutant Fab for binding to all tested antigens. In general, the results show that Fab heavy chain VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F S84 mutants showed no increase in binding to DLL4 by either ECL or ELISA. One mutant, Fab heavy chain VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F S84T (SEQ ID NO:233), showed greater binding to DLL4 by the ECL MSD assay but had the same binding by ELISA.

• TABLE 56
  Binding of Fab heavy chain VH1-46_IGHD6-6*01_IGHJ1*01
  S102A/S103P/S104F/H111F S84 NNK mutants to DLL4

  Fab

  ELISA

  Heavy Chain	SEQ ID	Light Chain	Signal/Blank	(Signal-
  VH1-46_IGHD6-6*01_IGHJ1*01	NO	(SEQ ID NO: 107)	[10 nM Fab]	Noise)

  S102A/S103P/S104F/H111F S84G	227	L6_IGKJ1*01	346.1	0.53
  S102A/S103P/S104F/H111F S84Q	228	L6_IGKJ1*01	413.1	0.39
  S102A/S103P/S104F/H111F S84N	229	L6_IGKJ1*01	497.4	0.47
  S102A/S103P/S104F/H111F S84H	230	L6_IGKJ1*01	457.0	0.41
  S102A/S103P/S104F/H111F S84R	231	L6_IGKJ1*01	432.9	0.26
  S102A/S103P/S104F/H111F S84K	232	L6_IGKJ1*01	447.6	0.29
  S102A/S103P/S104F/H111F S84T	233	L6_IGKJ1*01	1079.0	0.40
  S102A/S103P/S104F/H111F	126	L6_IGKJ1*01	441.3	0.57
  S102A/S103P/S104F/H111F	126	L6_IGKJ1*01	309.9	0.24
  S102A/S103P/S104F/H111F	126	L6_IGKJ1*01	584.6	0.26
  S102A/S103P/S104F/H111F	126	L6_IGKJ1*01	718.7	0.37

• TABLE 57
  Binding and specificity of Fab heavy chain VH1-46_IGHD6-6*01_IGHJ1*01
  S102A/S103P/S104F/H111F S84 NNK mutants

  Heavy Chain	ErbB2	EGF R	HGF R	Notch-1	CD44	IGF-1	P-Cad	EPO R	DLL4	Blank

  APFF S84G	299	435	419	473	457	395	434	429	130821	378
  APFF S84Q	311	347	255	416	372	373	357	288	122273	296
  APFF S84N	307	337	375	309	251	324	167	415	134783	271
  APFF S84H	301	306	374	331	382	353	319	318	138028	302
  APFF S84R	372	435	392	377	335	395	310	393	139388	322
  APFF S84K	354	301	317	400	386	405	517	528	164261	367
  APFF S84T	297	293	274	372	352	281	180	328	162923	151
  APFF	379	425	332	429	470	468	399	437	149144	338
  APFF	292	329	237	377	326	357	277	449	126118	407
  APFF	351	209	176	359	332	306	138	414	148493	254
  APFF	322	409	263	417	316	173	240	328	132249	184

Example 10 Affinity Maturation of the Light Chain of Identified “Hit” Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 Against DLL4
• In this Example, the light chain of parent “Hit” Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 against DLL4 was subjected to affinity maturation similar to the affinity maturation of the heavy chain as described in Examples 7-9 above.
• i. Identification of the CDR Potential Binding Site
• The amino acid sequence of the light chain (SEQ ID NO:107) for the “Hit” VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 was aligned with the amino acid sequence of related light chains of three “non-Hits” that were identified as not binding to DLL4 (see Table 58 below) These four Fabs are related because they share the same J_L germline segment. Further, the V_L germline segment is of the same subgroup (i.e. IGKV3). The sequence alignment is set forth in FIG. 2. Based on the alignment, amino acid residues were identified that differed between the “Hit” and “non-Hits,” thus accounting for the differences in binding affinity of the “Hit” and “non-Hits.” The identified amino acid residues were located in CDR3, which was identified as the region of the light chain that is important for binding affinity.

• TABLE 58
  “Hit” and “non-Hit” Antibodies for Light Chain Sequence Alignment

  	SEQ		SEQ	ECL
  	ID		ID	signal/
  Heavy Chain	NO	Light Chain	NO	blank

  VH1-	88	L6_IGKJ1*01	107	23.1
  46_IGHD6-6*01_IGHJ1*01
  VH1-	88	A27_IGKJ1*01	110	1.3
  46_IGHD6-6*01_IGHJ1*01
  VH1-	88	L25_IGKJ1*01	120	1.4
  46_IGHD6-6*01_IGHJ1*01
  VH1-	88	L2_IGKJ1*01	112	1.4
  46_IGHD6-6*01_IGHJ1*01

• NNK Mutagenesis of CDR3
• Amino acid residues R91, S92, N93, and W94 of CDR3 of the light chain L6_IGKJ1*01 were mutated by NNK mutagenesis using overlapping PCR to further identify amino acid residues that are in binding to DLL4. CDR3 amino acid residues Q89, Q90, P95, P96, W97 and T98 were conserved among the four aligned light chains (see FIG. 2), and therefore were not subjected to NNK mutagenesis. Heavy chain triple mutant APF (see e.g. Example 7; Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F (H:APF) & L6_IGKJ1*01) was used as a parent template for NNK mutagenesis of amino acid residues R91 and S92. Heavy chain quadruple mutant APFF (see e.g., Example 9; Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F (H:APFF) & L6_IGKJ1*01) was used as a parent template for NNK mutagenesis of amino acid residues S92, N93 and W94. Amino acid mutations designated with X (for any amino acid) did not show appreciable binding and therefore were not sequenced to identify the exact mutation. The resulting mutants were assayed using the ECL multispot assay as described in Example 4A. The results are set forth in Tables 59 and 60 below. Amino acid mutations designated with X (for any amino acid) did not show appreciable binding and therefore were not sequenced to identify the exact mutation. The results show that mutagenesis of amino acid residues R91, S92, N93 and W94 caused a reduction in ECL signal for binding to DLL4 compared either the APF or APFF parent template antibody, and therefore these residues were not further mutagenized.

• TABLE 59
  NNK mutagenesis of Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F
  (APF) (SEQ ID NO: 125) & L6_IGKJ1*01 or Fab VH1-46_IGHD6-6*01_IGHJ1*01
  S102A/S103P/S104F/H111F (APFF) (SEQ ID NO: 126) & L6_IGKJ1*01 at light chain
  amino acid residues R91, S92, N93 and W94

  Fab

  Heavy Chain
  VH1-46_IGHD6-	Light Chain				Signal/
  6*01_IGHJ1*01	L6_IGKJ1*01	SEQ ID NO	Signal	Blank	Blank

  S102A/S103P/S104F	R91P	247	1280	271	4.7
  S102A/S103P/S104F	R91L	248	375	273	1.4
  S102A/S103P/S104F	parent	107	2585	229	11.3
  S102A/S103P/S104F	R91G	249	292	209	1.4
  S102A/S103P/S104F	R91X	361	1673	262	6.4
  S102A/S103P/S104F	parent	107	2442	287	8.5
  S102A/S103P/S104F	R91Q	250	817	261	3.1
  S102A/S103P/S104F	R91X	361	248	296	0.8
  S102A/S103P/S104F	S92X	362	180	259	0.7
  S102A/S103P/S104F	S92X	362	255	395	0.6
  S102A/S103P/S104F	S92X	362	2911	244	11.9
  S102A/S103P/S104F	parent	107	2832	224	12.6
  S102A/S103P/S104F	S92N	251	2092	271	7.7
  S102A/S103P/S104F	S92X	362	701	140	5.0
  S102A/S103P/S104F	S92X	362	2204	342	6.4
  S102A/S103P/S104F	S92C	252	401	338	1.2
  S102A/S103P/S104F	parent	107	3482	271	12.8
  S102A/S103P/S104F	parent	107	2123	204	10.4
  S102A/S103P/S104F/H111F	N93Y	253	1385	270	5.1
  S102A/S103P/S104F/H111F	N93S	254	6436	206	31.2
  S102A/S103P/S104F/H111F	N93H	255	14711	331	44.4
  S102A/S103P/S104F/H111F	N93Q	256	704	239	2.9
  S102A/S103P/S104F/H111F	W94R	257	75771	256	296.0
  S102A/S103P/S104F/H111F	W94S	258	108653	479	226.8
  S102A/S103P/S104F/H111F	W94T	259	23228	438	53.0
  S102A/S103P/S104F/H111F	W94L	260	11613	200	58.1
  S102A/S103P/S104F/H111F	W94P	261	332	169	2.0
  S102A/S103P/S104F/H111F	W94M	262	33801	241	140.3
  S102A/S103P/S104F/H111F	S92P	263	2412	292	8.3
  S102A/S103P/S104F/H111F	S92P	263	446	166	2.7
  S102A/S103P/S104F/H111F	S92A/X	363	1755	265	6.6
  S102A/S103P/S104F/H111F	S92Q	265	348	255	1.4
  S102A/S103P/S104F/H111F	S92V	266	327	317	1.0
  S102A/S103P/S104F/H111F	parent	107	164982	282	585.0
  S102A/S103P/S104F/H111F	parent	107	164992	277	595.6
  S102A/S103P/S104F/H111F	parent	107	164224	274	599.4
  S102A/S103P/S104F/H111F	S92T	267	54083	278	194.5
  S102A/S103P/S104F/H111F	S92C	252	1343	348	3.9
  S102A/S103P/S104F/H111F	S92C	252	1263	504	2.5
  S102A/S103P/S104F/H111F	S92C	252	1229	428	2.9
  S102A/S103P/S104F/H111F	S92R	252	418	252	1.7
  S102A/S103P/S104F/H111F	S92G	269	89202	254	351.2
  S102A/S103P/S104F/H111F	S92V	266	405	225	1.8
  S102A/S103P/S104F/H111F	S92M	271	390	201	1.9
  S102A/S103P/S104F/H111F	S92N	251	824	224	3.7
  S102A/S103P/S104F/H111F	S92G	269	80151	294	272.6
  S102A/S103P/S104F/H111F	S92G	269	80671	208	387.8
  S102A/S103P/S104F/H111F	parent	107	188914	309	611.4
  S102A/S103P/S104F/H111F	S92R	268	587	219	2.7
  S102A/S103P/S104F/H111F	S92P	263	484	220	2.2
  S102A/S103P/S104F/H111F	S92P	263	4751	296	16.1
  S102A/S103P/S104F/H111F	S92G	269	91432	325	281.3

• TABLE 60
  NNK mutagenesis of Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F (APF) (SEQ
  ID NO: 125) & L6_IGKJ1*01 or Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F
  (APFF) (SEQ ID NO: 126) & L6_IGKJ1*01 at light chain amino acid residues R91, S92, N93 and W94

  Heavy	Light
  Chain	Chain	ErbB2	EGF R	HGF R	Notch-1	CD44	IGF-1	P-Cad	EPO R	DLL4	Blank

  APF	R91P	333	216	273	228	252	199	296	275	1280	271
  APF	R91L	526	367	255	383	236	382	437	459	375	273
  APF	parent	331	363	307	398	223	223	189	252	2585	229
  APF	R91G	236	271	239	170	163	260	235	306	292	209
  APF	R91X	268	329	279	297	254	282	180	193	1673	262
  APF	parent	317	226	344	358	205	162	250	319	2442	287
  APF	R91Q	234	290	325	229	268	210	314	263	817	261
  APF	R91X	219	210	341	138	191	269	324	193	248	296
  APF	S92X	262	163	260	82	228	208	176	208	180	259
  APF	S92X	258	209	267	354	257	264	323	327	255	395
  APF	S92X	257	306	334	272	270	216	326	220	2911	244
  APF	parent	149	279	275	171	197	168	171	0	2832	224
  APF	S92N	293	346	405	193	316	211	240	304	2092	271
  APF	S92X	298	228	131	135	99	200	290	227	701	140
  APF	S92X	248	300	333	243	279	247	266	309	2204	342
  APF	S92C	295	143	335	125	156	303	265	302	401	338
  APF	parent	330	272	306	257	189	241	297	304	3482	271
  APF	parent	157	237	272	334	96	197	208	329	2123	204
  APFF	N93Y	369	464	380	453	333	318	499	541	1385	270
  APFF	N93S	351	364	328	345	346	238	321	420	6436	206
  APFF	N93H	307	347	307	342	345	268	293	425	14711	331
  APFF	N93Q	240	337	309	310	452	256	304	477	704	239
  APFF	W94R	283	325	293	375	443	303	364	546	75771	256
  APFF	W94S	351	419	453	486	469	450	466	506	108653	479
  APFF	W94T	396	414	377	418	453	387	481	432	23228	438
  APFF	W94L	274	257	187	369	309	263	296	333	11613	200
  APFF	W94P	299	267	275	228	241	187	268	292	332	169
  APFF	W94M	244	302	302	321	327	340	346	435	33801	241
  APFF	S92P	219	345	242	346	282	236	354	391	2412	292
  APFF	S92P	268	317	256	328	292	280	307	385	446	166
  APFF	S92A/X	212	268	252	242	228	193	325	262	1755	265
  APFF	S92Q	282	332	373	351	312	246	340	330	348	255
  APFF	S92V	188	319	230	262	248	244	373	371	327	317
  APFF	parent	259	290	321	380	346	249	302	1062	164982	282
  APFF	parent	311	307	267	266	351	221	299	467	164992	277
  APFF	parent	236	266	339	279	367	305	283	473	164224	274
  APFF	S92T	237	295	290	231	290	308	387	424	54083	278
  APFF	S92C	425	452	472	439	458	471	786	601	1343	348
  APFF	S92C	573	638	616	611	646	666	930	845	1263	504
  APFF	S92C	526	588	589	642	554	642	805	742	1229	428
  APFF	S92R	272	292	265	386	365	248	387	318	418	252
  APFF	S92G	274	273	238	296	263	229	213	405	89202	254
  APFF	S92V	246	305	288	347	331	237	390	368	405	225
  APFF	S92M	301	367	346	385	304	271	328	340	390	201
  APFF	S92N	242	293	243	407	336	312	271	314	824	224
  APFF	S92G	384	347	296	280	306	257	294	428	80151	294
  APFF	S92G	228	160	314	203	284	297	238	418	80671	208
  APFF	parent	289	326	185	310	211	336	295	433	188914	309
  APFF	S92R	266	322	315	437	358	256	410	395	587	219
  APFF	S92P	240	332	281	399	367	282	321	378	484	220
  APFF	S92P	299	315	222	397	393	296	288	495	4751	296
  APFF	S92G	377	420	287	541	413	323	402	543	91432	325

• iii. NNK Mutagenesis of CDR1
• Amino acid residues S28, S30, S31, and Y32 of CDR1 of the light chain L6_IGKJ1*01 were mutated by NNK mutagenesis using overlapping PCR to further identify amino acid residues that are important for binding to DLL4. The APF triple mutant (see e.g. Example 7; Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F (H:APF) & L6_IGKJ1*01) was used as a template for NNK mutagenesis of S30 and Y32. The APFF heavy chain quadruple mutant (see e.g. Example 9; Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F (H:APFF) & L6_IGKJ1*01) was used as a template for NNK mutagenesis of S28, S30 and S31. The resulting mutants were assayed using the ECL multispot assay as described in Example 4A above. The results are set forth in Tables 61 and 62 below. Double mutants, such as R24G/Q27L, were inadvertently generated during the PCR reaction. Amino acid mutations designated with X (for any amino acid) did not show appreciable binding and therefore were not sequenced to identify the exact mutation. The results show that mutagenesis of amino acid residue Y32 caused a reduction in binding affinity to DLL4 compared to the APF parent template, and therefore this residue was not further mutagenized. Mutagenesis of amino acid residue S28, S30 and S31 either improved binding affinity or did not affect binding affinity to DLL4 compared to the APF or APFF parent templates, and thus these residues were identified as residues for further mutagenesis. Three light chain mutants, namely L6_IGKJ1*01 S28D, S30N, and S31H, slightly increased antibody binding affinity to DLL4.

• TABLE 61
  NNK mutagenesis of Fab VH1-46_IGHD6-6*01_IGHJ1*01
  S102A/S103P/S104F (APF) (SEQ ID NO: 125) & L6_IGKJ1*01 or Fab
  VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F (APFF) (SEQ ID NO:
  126) & L6_IGKJ1*01 at light chain amino acid residues S28, S30, S31 and Y32

  Heavy Chain
  VH1-46_IGHD6-	Light Chain				Signal/
  6*01_IGHJ1*01	L6_IGKJ1*01	SEQ ID NO	Signal	Blank	Blank

  S102A/S103P/S104F	S30W	300	791	186	4.3
  S102A/S103P/S104F	parent	107	803	125	6.4
  S102A/S103P/S104F	S30X	364	101	112	0.9
  S102A/S103P/S104F	S30R	298	745	95	7.8
  S102A/S103P/S104F	S30X	364	593	204	2.9
  S102A/S103P/S104F	S30T	297	1016	206	4.9
  S102A/S103P/S104F	S30X	364	1374	204	6.7
  S102A/S103P/S104F	S30X	364	1299	210	6.2
  S102A/S103P/S104F	S30L	296	1627	235	6.9
  S102A/S103P/S104F	Y32X	365	648	196	3.3
  S102A/S103P/S104F	Y32X	365	817	193	4.2
  S102A/S103P/S104F	Y32X	365	1753	261	6.7
  S102A/S103P/S104F	Y32X	365	1209	155	7.8
  S102A/S103P/S104F	R24G/Q27L	276	197	87	2.3
  S102A/S103P/S104F	Y32V	277	427	164	2.6
  S102A/S103P/S104F	Y32S	278	1031	210	4.9
  S102A/S103P/S104F	parent	107	4266	256	16.7
  S102A/S103P/S104F	Y32X	365	293	253	1.2
  S102A/S103P/S104F	parent	107	3052	242	12.6
  S102A/S103P/S104F/H111F	S28G	279	182961	343	533.4
  S102A/S103P/S104F/H111F	S28K	280	124246	395	314.5
  S102A/S103P/S104F/H111F	S28V	281	83083	237	350.6
  S102A/S103P/S104F/H111F	S28F	282	133659	249	536.8
  S102A/S103P/S104F/H111F	parent	107	182026	400	455.1
  S102A/S103P/S104F/H111F	S28P	244	178227	393	453.5
  S102A/S103P/S104F/H111F	S28T	283	159288	305	522.3
  S102A/S103P/S104F/H111F	S28L	284	72299	329	219.8
  S102A/S103P/S104F/H111F	S28Q	285	133486	353	378.1
  S102A/S103P/S104F/H111F	S28A	286	156761	332	472.2
  S102A/S103P/S104F/H111F	S28N	287	203926	262	778.3
  S102A/S103P/S104F/H111F	S28H	288	209433	344	608.8
  S102A/S103P/S104F/H111F	S28I	289	106041	343	309.2
  S102A/S103P/S104F/H111F	S28R	290	110363	449	245.8
  S102A/S103P/S104F/H111F	S28W	291	165026	303	544.6
  S102A/S103P/S104F/H111F	S28M	292	108166	322	335.9
  S102A/S103P/S104F/H111F	S28E	293	184227	420	438.6
  S102A/S103P/S104F/H111F	S30C	294	128661	915	140.6
  S102A/S103P/S104F/H111F	S30D	295	225396	397	567.7
  S102A/S103P/S104F/H111F	S30L	296	198641	379	524.1
  S102A/S103P/S104F/H111F	S30T	297	122207	407	300.3
  S102A/S103P/S104F/H111F	S30R	298	145575	416	349.9
  S102A/S103P/S104F/H111F	S30P	299	1143	262	4.4
  S102A/S103P/S104F/H111F	parent	107	207955	306	679.6
  S102A/S103P/S104F/H111F	S30W	300	190872	289	660.5
  S102A/S103P/S104F/H111F	S30Y/S	366	143412	294	487.8
  S102A/S103P/S104F/H111F	S30Q	302	202637	198	1023.4
  S102A/S103P/S104F/H111F	S30A	303	183649	356	515.9
  S102A/S103P/S104F/H111F	S30G	304	180489	272	663.6
  S102A/S103P/S104F/H111F	S30N	245	174926	352	496.9
  S102A/S103P/S104F/H111F	S30P	299	1262	302	4.2
  S102A/S103P/S104F/H111F	S30G	304	177646	351	506.1
  S102A/S103P/S104F/H111F	S30A	303	186732	184	1014.8
  S102A/S103P/S104F/H111F	S30T	297	136426	392	348.0
  S102A/S103P/S104F/H111F	S30V	305	141111	284	496.9
  S102A/S103P/S104F/H111F	S30R	298	189471	278	681.6
  S102A/S103P/S104F/H111F	S30Q	302	196711	327	601.6
  S102A/S103P/S104F/H111F	S31T	306	191253	332	576.1
  S102A/S103P/S104F/H111F	S31N	307	177897	294	605.1
  S102A/S103P/S104F/H111F	S31K	246	179257	511	350.8
  S102A/S103P/S104F/H111F	parent	107	171775	442	388.6
  S102A/S103P/S104F/H111F	S31L	308	155112	416	372.9
  S102A/S103P/S104F/H111F	S31M	309	167080	442	378.0
  S102A/S103P/S104F/H111F	S31F	310	188723	411	459.2
  S102A/S103P/S104F/H111F	S31I	311	173649	321	541.0
  S102A/S103P/S104F/H111F	S31V	312	176358	345	511.2
  S102A/S103P/S104F/H111F	S31H	313	221327	264	838.4
  S102A/S103P/S104F/H111F	S31A	314	192365	218	882.4
  S102A/S103P/S104F/H111F	S31P	315	53282	341	156.3
  S102A/S103P/S104F/H111F	S31D	316	154331	493	313.0
  S102A/S103P/S104F/H111F	S31R	317	166188	298	557.7
  S102A/S103P/S104F/H111F	S31Y	318	187896	284	661.6
  S102A/S103P/S104F/H111F	S31Q	319	165030	407	405.5
  S102A/S103P/S104F/H111F	S31E	320	171114	331	517.0
  S102A/S103P/S104F/H111F	S31G	321	65521	231	283.6

• TABLE 62
  NNK mutagenesis of Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F (APF) (SEQ
  ID NO: 125) & L6_IGKJ1*01 or Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F
  (APFF) (SEQ ID NO: 126) & L6_IGKJ1*01 at light chain amino acid residues S28, S30, S31 and Y32

  Heavy	Light
  Chain	Chain	ErbB2	EGF R	HGF R	Notch-1	CD44	IGF-1	P-Cad	EPO R	DLL4	Blank

  APF	S30W	73	132	62	105	186	157	39	30	791	186
  APF	parent	61	161	86	135	66	217	117	105	803	125
  APF	S30X	119	67	75	45	6	56	83	93	101	112
  APF	S30R	35	140	108	155	89	86	39	87	745	95
  APF	S30X	319	99	122	231	239	144	224	227	593	204
  APF	S30T	243	274	297	127	229	204	195	207	1016	206
  APF	S30X	213	188	337	247	223	176	233	267	1374	204
  APF	S30X	210	218	311	79	156	207	262	211	1299	210
  APF	S30L	244	288	250	296	240	193	260	259	1627	235
  APF	Y32X	240	223	259	241	203	170	199	248	648	196
  APF	Y32X	155	93	176	148	147	142	38	190	817	193
  APF	Y32X	125	240	299	168	236	247	260	214	1753	261
  APF	Y32X	124	256	167	255	147	139	148	170	1209	155
  APF	R24G/Q27L	225	252	185	177	119	49	236	191	197	87
  APF	Y32V	156	57	283	56	120	151	186	144	427	164
  APF	Y32S	154	208	222	137	162	175	51	230	1031	210
  APF	parent	223	268	205	344	200	332	285	366	4266	256
  APF	Y32X	275	266	358	306	206	304	382	374	293	253
  APF	parent	383	296	265	107	273	132	366	254	3052	242
  APFF	S28G	334	360	333	324	436	360	491	494	182961	343
  APFF	S28K	270	386	355	395	464	348	443	477	124246	395
  APFF	S28V	231	327	338	289	380	284	344	446	83083	237
  APFF	S28F	242	283	223	367	402	275	336	413	133659	249
  APFF	parent	333	406	432	350	451	386	368	539	182026	400
  APFF	S28P	427	370	318	416	365	392	605	492	178227	393
  APFF	S28T	271	321	371	249	368	355	676	380	159288	305
  APFF	S28L	222	378	317	392	365	346	418	404	72299	329
  APFF	S28Q	345	517	380	331	420	404	809	437	133486	353
  APFF	S28A	348	351	377	440	502	378	521	424	156761	332
  APFF	S28N	363	325	406	243	399	331	447	440	203926	262
  APFF	S28H	381	435	346	482	513	355	447	517	209433	344
  APFF	S28I	265	386	369	442	412	353	416	450	106041	343
  APFF	S28R	318	403	378	425	378	437	395	542	110363	449
  APFF	S28W	316	283	414	349	404	489	385	489	165026	303
  APFF	S28M	271	320	305	382	313	341	410	360	108166	322
  APFF	S28E	389	396	401	433	461	361	393	513	184227	420
  APFF	S30C	1007	1187	1229	1472	1081	1027	1686	1792	128661	915
  APFF	S30D	284	325	312	415	434	357	543	496	225396	397
  APFF	S30L	270	406	315	389	295	332	351	540	198641	379
  APFF	S30T	332	360	375	413	423	410	370	497	122207	407
  APFF	S30R	434	456	458	576	455	404	465	571	145575	416
  APFF	S30P	391	394	328	544	334	356	348	520	1143	262
  APFF	parent	412	386	349	565	411	409	466	540	207955	306
  APFF	S30W	289	398	399	372	500	471	342	542	190872	289
  APFF	S30Y/S	319	299	345	306	346	283	429	520	143412	294
  APFF	S30Q	262	353	339	243	400	342	298	423	202637	198
  APFF	S30A	251	322	414	380	390	400	454	561	183649	356
  APFF	S30G	404	387	355	382	427	393	369	485	180489	272
  APFF	S30N	241	400	297	296	437	362	396	525	174926	352
  APFF	S30P	358	385	383	346	411	312	413	418	1262	302
  APFF	S30G	260	298	263	346	343	304	397	480	177646	351
  APFF	S30A	295	337	311	364	451	342	317	475	186732	184
  APFF	S30T	269	383	320	375	521	401	418	470	136426	392
  APFF	S30V	279	412	394	294	375	365	333	536	141111	284
  APFF	S30R	404	395	452	313	472	422	442	525	189471	278
  APFF	S30Q	340	381	344	326	411	354	393	376	196711	327
  APFF	S31T	285	351	432	261	384	303	332	423	191253	332
  APFF	S31N	197	246	300	267	384	379	342	363	177897	294
  APFF	S31K	262	355	221	334	370	505	471	522	179257	511
  APFF	parent	312	370	347	367	457	433	450	438	171775	442
  APFF	S31L	288	375	319	365	371	405	346	427	155112	416
  APFF	S31M	352	380	293	474	488	445	510	573	167080	442
  APFF	S31F	295	342	280	349	256	267	369	599	188723	411
  APFF	S31I	222	363	303	421	506	365	444	500	173649	321
  APFF	S31V	300	363	288	374	384	335	360	509	176358	345
  APFF	S31H	307	373	352	421	426	350	480	504	221327	264
  APFF	S31A	383	415	309	424	406	334	361	461	192365	218
  APFF	S31P	372	488	431	461	466	404	493	594	53282	341
  APFF	S31D	479	438	429	510	471	407	451	596	154331	493
  APFF	S31R	313	331	261	358	423	374	270	465	166188	298
  APFF	S31Y	236	320	197	351	445	293	361	604	187896	284
  APFF	S31Q	392	390	329	383	438	415	379	548	165030	407
  APFF	S31E	313	297	324	460	390	367	273	441	171114	331
  APFF	S31G	311	391	378	426	381	301	384	414	65521	231

• Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F (H:APFF) & L6_IGKJ1*01 light chain mutants S28D, S281I, S30N and S31H were subsequently re-assayed for binding to DLL4 by ELISA. The results are set forth in Table 63 below. The results show that Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F (H:APFF) & L6_IGKJ1*01 light chain mutants S28N, and S31H slightly increase binding affinity to DLL4 compared to the H:APFF parental template antibody. By ELISA at the concentrations tested, the Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F (H:APFF) & L6_IGKJ1*01 light chain mutant S28H and S30D did not increase binding affinity to DLL4 compared to the APFF parental template antibody.

• TABLE 63
  Binding affinity of VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F
  (H: APFF) & L6_IGKJ1*01 Fab mutants to DLL4

  	Heavy Chain
  	S102A/S103P/S104F/H111F (SEQ ID NO: 126)
  	Light Chain

  	L6_IGKJ1*01	S28N	S28H	S30D	L6_IGKJ1*01	S31H
  	(SEQ ID	(SEQ ID	(SEQ ID	(SEQ ID	(SEQ ID	(SEQ ID
  	NO: 107)	NO: 287)	NO: 288)	NO: 295)	NO: 107)	NO: 313)

  400 nM	0.13	0.19	0.13	0.13	0.13	0.20
  200 nM	0.10	0.17	0.14	0.11	0.08	0.11
  100 nM	0.07	0.13	0.09	0.09	0.07	0.09
  50 nM	0.06	0.07	0.05	0.06	0.04	0.05
  25 nM	0.02	0.04	0.03	0.03	0.02	0.03
  25 nM	0.03	0.05	0.03	0.03	0.02	0.03
  0	0.00	0.00	0.01	0.00	0.00	0.00
  0	0.00	0.00	0.00	0.01	0.01	0.00

• iv. Combination Mutants Based on NNK Mutagenesis of CDR1
• Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F (H:APFF) & L6_IGKJ1*01 light chain mutants S28D, S30N and S31H were combined into one triple mutant, designated as Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F (H:APFF) & L6_IGKJ1*01 S28D/S30N/S31H (L:NDH) (H:APFF & L:NDH). The binding affinity of the H:APFF & L:NDH mutant to DLL4 was assayed using both ELISA and the 96-well plate ECL assay. Additionally, the light chain triple mutant L6_IGKJ1*01 S28D/S30N/S31H (L:NDH) was assayed in combination with heavy chain mutants VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F/G56A (H:APFF G56A) and VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F/S54A (H:APFF S54A).
• The results are set forth in Tables 64 and 65 below. The results show the antibody mutant APFF-NDH binds DLL4 with 4-fold increased affinity as compared to parent antibody APFF mutant. The antibody Fab H:APFF G56A & L:NDH resulted in 8-fold greater affinity for binding to DLL4 as compared to the H:APFF & L:wt parental antibody mutant, and also exhibited increased binding affinity compared to the other antibodies tested. The antibody Fab H:APFF S54A & L:NDH resulted in a slight decrease in binding affinity compared to the H:APFF & L:NDH antibody mutant. Table 65 provides a comparison of binding affinity of antibodies containing the triple light chain mutant and various mutated heavy chain mutants. The results in Tables 64 and 65 show that the H:APFF G56A & L:NDH, containing 5 mutations in the heavy chain and three mutations in the light chain, exhibited the highest binding affinity of the antibodies tested.

• TABLE 64
  Binding affinity of VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 Fab mutants

  Heavy Chain

  			APFF G56A	APFF G56A	APFF S54A
  	APFF (SEQ ID	APFF (SEQ ID	(SEQ ID	(SEQ ID	(SEQ ID
  	NO: 126)	NO: 126)	NO: 167)	NO: 167)	NO: 165)

  Light Chain

  	Parent	S28N/S30D/	Parent	S28N/S30D/	S28N/S30D/
  	(SEQ ID	S31H (SEQ ID	(SEQ ID	S31H (SEQ ID	S31H (SEQ ID
  	NO: 107)	NO: 323)	NO: 107)	NO: 323)	NO: 323)

  100 nM	0.072	0.259	0.338	0.453	0.213
  75 nM	0.072	0.268	0.399	0.543	0.212
  50 nM	0.060	0.202	0.301	0.366	0.154
  0	0.006	0.002	0.002	0.002	0.000

• TABLE 65
  Binding affinity of VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 Fab mutants

  Fab

  ELISA

  	SEQ		SEQ	ECL	Signal
  Heavy Chain	ID	Light Chain	ID	Signal	[100 nM
  VH1-46_IGHD6-6*01_IGHJ1*01	NO	L6_IGKJ1*01	NO	[10 nM Fab]	Fab]
  S102A/S103P/S104F/H111F	126	S28N/S30D/S31H	323	48997	0.08
  S102A/S103P/S104F/H111F/G56A	167	S28N/S30D/S31H	323	71603	0.20
  S102A/S103P/S104F/H111F/S54A	165	S28N/S30D/S31H	323	46700	0.08

• v. Alanine Scanning of CDR2
• Amino acid residues D50, A51, S52, N53, R54, A55 and T56 of CDR2 of the light chain L6_IGKJ1*01 were mutated by alanine scanning mutagenesis to further identify amino acid residues that are important for binding to DLL4. Amino acid residues A51 and A55 were mutated to threonine. The APFF heavy chain quadruple mutant (see e.g. Example 9; Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F (H:APFF) & L6_IGKJ1*01) was used as a template.
• The results are set forth in Table 66 below. The results show that mutation of amino acid residues D50, R54 and T56 with alanine and substitution of amino acid residue A51 with threonine caused a reduction in ECL signal for binding to DLL4 and therefore these residues were not further mutagenized. Mutation of amino acid residues S52 and N53 with alanine and mutation of amino acid residue A55 with threonine either improved the ECL signal or did not affect the ECL signal for binding to DLL4 and therefore these residues were identified as amino acid residues for further mutagenesis.

• TABLE 66
  Binding affinity of Fab VH1-46_IGHD6-6*01_IGHJ1*01
  S102A/S103P/S104F/H111F (APFF) & L6_IGKJ1*01 CDR2 alanine mutants

  Heavy Chain VH1-46_IGHD6-	SEQ ID	Light Chain	SEQ ID
  6*01_IGHJ1*01	NO	L6_IGKJ1*01	NO	Signal [10 nM Fab]

  S102A/S103P/S104F/H111F	126	wildtype	107	13516
  S102A/S103P/S104F/H111F	126	D50A	324	4231
  S102A/S103P/S104F/H111F	126	A51T	325	2849
  S102A/S103P/S104F/H111F	126	S52A	326	19311
  S102A/S103P/S104F/H111F	126	N53A	327	14166
  S102A/S103P/S104F/H111F	126	R54A	328	11626
  S102A/S103P/S104F/H111F	126	A55T	329	13228
  S102A/S103P/S104F/H111F	126	T56A	330	7260

• vi. NNK Mutagenesis of CDR2 Residues S52, N53, and A55
• Fab mutant H:APFF & L:NDH (see Example 10 above; VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F (H:APFF) & L6_IGKJ1*01 S28D/S30N/S31H (L:NDH)) was used as a template for NNK mutagenesis of CDR2 amino acid residues S52, N53 and A55. The Fab mutants were tested for binding to DLL4 using the 96-well plate ECL binding assay and ELISA. Table 67 sets forth the ECL and ELISA signals. Amino acid mutations designated with X (for any amino acid) did not show appreciable binding and therefore were not sequenced to identify the exact mutation. The results show that various mutants of H:APFF & L:NDH exhibited greater ECL and ELISA signals for binding to DLL4 as compared to the parental H:APFF & L:NDH, including those having further mutations S52T, S52L, N53H, A55S and A55G in the light chain.
• Light chain mutants H:APFF & L:NDH S52T, H:APFF & L:NDH S52L, H:APFF & L:NDH S52T/S, H:APFF & L:NDH S52X, H:APFF & L:NDH N53H, H:APFF & L:NDH A55S and H:APFF & L:NDH A55G were further analyzed for binding to DLL4 by ELISA using 2-fold serial dilutions of Fab, starting at a concentration of 100 nM. The results are set forth in Table 68 below. Antibody mutants H:APFF & L:NDH S52L, H:APFF & L:NDH A55S and H:APFF & L:NDH A55G had a slightly increased affinity for binding to DLL4 as compared to the parental H:APFF & L:NDH mutant. All of the Fab light chain mutants bind DLL4 within the same range of affinity as the parental H:APFF & L:NDH mutant.

• TABLE 67
  Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F (H:APFF) &
  L6_IGKJ1*01 S28N/S30D/S31H (L:NDH) light chain CDR2 NNK mutant binding data

  Fab

  Heavy Chain				ELISA
  VH1-46_IGHD6-		SEQ		(Avgerage
  6*01_IGHJ1*01 (SEQ ID		ID	ECL	signal-
  NO: 126)	Light Chain	NO	Signal	noise)

  S102A/S103P/S104F/H111F	S28N/S30D/S31H S52L	331	17810	0.285
  S102A/S103P/S104F/H111F	S28N/S30D/S31H S52G/V	367	17589	0.233
  S102A/S103P/S104F/H111F	S28N/S30D/S31H S52T/S	368	17769	0.261
  S102A/S103P/S104F/H111F	S28N/S30D/S31H S52R	333	20009	0.244
  S102A/S103P/S104F/H111F	S28N/S30D/S31H S52S/Y	369	15572	0.218
  S102A/S103P/S104F/H111F	S28N/S30D/S31H S52X	370	2757	0.077
  S102A/S103P/S104F/H111F	S28N/S30D/S31H S52X	370	15250	0.232
  S102A/S103P/S104F/H111F	S28N/S30D/S31H S52X	370	16779	0.299
  S102A/S103P/S104F/H111F	S28N/S30D/S31H S52X	370	16012	0.303
  S102A/S103P/S104F/H111F	S28N/S30D/S31H S52X	370	15424	0.272
  S102A/S103P/S104F/H111F	S28N/S30D/S31H S52X	370	16839	0.366
  S102A/S103P/S104F/H111F	S28N/S30D/S31H S52X	370	15263	0.273
  S102A/S103P/S104F/H111F	S28N/S30D/S31H S52W	334	16341	0.177
  S102A/S103P/S104F/H111F	S28N/S30D/S31H S52R	333	20497	0.179
  S102A/S103P/S104F/H111F	NDH	323	18697	0.165
  S102A/S103P/S104F/H111F	S28N/S30D/S31H S52N/X	371	20512	0.221
  S102A/S103P/S104F/H111F	S28N/S30D/S31H S52R	333	20573	0.243
  S102A/S103P/S104F/H111F	S28N/S30D/S31H S52P/X	372	19361	0.233
  S102A/S103P/S104F/H111F	S28N/S30D/S31H S52T	332	20097	0.263
  S102A/S103P/S104F/H111F	S28N/S30D/S31H S52M	337	19458	0.185
  S102A/S103P/S104F/H111F	S28N/S30D/S31H N53X	373	12235	0.106
  S102A/S103P/S104F/H111F	S28N/S30D/S31H N53E	338	17553	0.204
  S102A/S103P/S104F/H111F	S28N/S30D/S31H N53X	373	200	0.000
  S102A/S103P/S104F/H111F	S28N/S30D/S31H N53X	373	9412	0.110
  S102A/S103P/S104F/H111F	S28N/S30D/S31H N53G	339	20572	0.163
  S102A/S103P/S104F/H111F	S28N/S30D/S31H N53X	373	15916	0.132
  S102A/S103P/S104F/H111F	S28N/S30D/S31H N53X	373	3627	−0.001
  S102A/S103P/S104F/H111F	S28N/S30D/S31H N53M	340	17793	0.162
  S102A/S103P/S104F/H111F	S28N/S30D/S31H N53X	373	13341	0.161
  S102A/S103P/S104F/H111F	S28N/S30D/S31H N53C/F	374	18046	0.266
  S102A/S103P/S104F/H111F	S28N/S30D/S31H N53H	342	20061	0.230
  S102A/S103P/S104F/H111F	S28N/S30D/S31H N53X	373	14078	0.139
  S102A/S103P/S104F/H111F	S28N/S30D/S31H N53X	373	456	0.060
  S102A/S103P/S104F/H111F	S28N/S30D/S31H	375	16809	0.166
  	N53M/L
  S102A/S103P/S104F/H111F	S28N/S30D/S31H N53P	343	18132	0.120
  S102A/S103P/S104F/H111F	S28N/S30D/S31H N53X	373	203	0.015
  S102A/S103P/S104F/H111F	S28N/S30D/S31H N53A	344	14213	0.151
  S102A/S103P/S104F/H111F	S28N/S30D/S31H N53X	373	14322	0.127
  S102A/S103P/S104F/H111F	S28N/S30D/S31H N53X	373	260	−0.001
  S102A/S103P/S104F/H111F	S28N/S30D/S31H A55R	345	9031	0.106
  S102A/S103P/S104F/H111F	S28N/S30D/S31H A55C	346	8226	0.146
  S102A/S103P/S104F/H111F	S28N/S30D/S31H A55X	376	14187	0.202
  S102A/S103P/S104F/H111F	S28N/S30D/S31H A55S	347	20047	0.383
  S102A/S103P/S104F/H111F	S28N/S30D/S31H A55X	376	899	0.019
  S102A/S103P/S104F/H111F	S28N/S30D/S31H A55G	348	21381	0.323
  S102A/S103P/S104F/H111F	S28N/S30D/S31H A55X	376	8799	0.092
  S102A/S103P/S104F/H111F	S28N/S30D/S31H A55X	376	5320	0.068
  S102A/S103P/S104F/H111F	NDH	323	17201	0.214
  S102A/S103P/S104F/H111F	S28N/S30D/S31H A55X	376	13643	0.116
  S102A/S103P/S104F/H111F	S28N/S30D/S31H A55X	376	275	0.016
  S102A/S103P/S104F/H111F	S28N/S30D/S31H A55X	376	1370	0.010
  S102A/S103P/S104F/H111F	S28N/S30D/S31H A55X	376	13611	0.151
  S102A/S103P/S104F/H111F	S28N/S30D/S31H A55X	376	167	0.007
  S102A/S103P/S104F/H111F	S28N/S30D/S31H A55G	348	18042	0.301
  S102A/S103P/S104F/H111F	S28N/S30D/S31H A55X	376	296	0.023
  S102A/S103P/S104F/H111F	S28N/S30D/S31H A55G	348	19264	0.298
  S102A/S103P/S104F/H111F	S28N/S30D/S31H A55X	376	5246	0.068

• TABLE 68
  Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F (APFF) (SEQ ID NO: 126) &
  L6_IGKJ1*01 S28N/S30D/S31H (NDH) light chain S52, N53 and A55 mutant binding to DLL4 by ELISA

  H

  APFF

  APFF

  APFF

  APFF

  APFF

  APFF

  APFF

  APFF

  Fab

  L

  [nM]	NDH/S52L	NDH/S52T/S	NDH/S52X	NDH/S52T	NDH/N53H	NDH/A55S	NDH/A55G	NDH

  100	0.791	0.696	0.686	0.653	0.608	0.858	0.814	0.686
  50	0.546	0.500	0.508	0.490	0.416	0.588	0.510	0.507
  25	0.335	0.297	0.309	0.323	0.238	0.407	0.316	0.310
  12.5	0.215	0.186	0.192	0.215	0.167	0.258	0.198	0.192
  6.25	0.142	0.115	0.125	0.130	0.109	0.154	0.125	0.125
  3.125	0.095	0.088	0.096	0.099	0.089	0.108	0.093	0.096

• vii. NNK Mutagenesis of Framework 3 Residues S76 and F62
• Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F (H:APFF) and L6_IGKJ1*01 was used as template for further mutagenesis of amino acid residues S76 and F62 in the framework 3 region of the light chain. These residues were mutated using overlapping PCR with NNK mutagenesis, as described above. Binding to DLL4 was assayed using an ECL Multispot assay as described in Example 4A or in an ELISA assay as described in Example 6. The results are set forth in Tables 69-71, below. The results show that mutation of amino acid residues S76 and F62 caused a decrease in the ECL and ELISA signals for binding to DLL4.

• TABLE 69
  Binding affinity of Fab VH1-46_IGHD6-6*01_IGHJ1*01
  S102A/S103P/S104F/H111F (APFF) & L6_IGKJ1*01 S76 and F62 Mutants

  Heavy Chain VH1-46_IGHD6-	SEQ ID	Light Chain	SEQ ID
  6*01_IGHJ1*01	NO	L6_IGKJ1*01	NO	Signal [10 nM Fab]

  S102A/S103P/S104F/H111F	126	S76L	351	13688
  S102A/S103P/S104F/H111F	126	S76T	352	15747
  S102A/S103P/S104F/H111F	126	S76G	353	13404
  S102A/S103P/S104F/H111F	126	wildtype	107	13516
  S102A/S103P/S104F/H111F	126	S76A/K	377	16525
  S102A/S103P/S104F/H111F	126	S76Y	355	14825
  S102A/S103P/S104F/H111F	126	F62L	356	261

• TABLE 70
  Binding affinity of Fab VH1-46_IGHD6-6*01_IGHJ1*01
  S102A/S103P/S104F/H111F (H:APFF) & L6_IGKJ1*01 S76 and F62 Mutants

  Heavy Chain		Light Chain			ELISA
  VH1-46_IGHD6-	SEQ ID	L6_IGKJ1*	SEQ ID	ECL	(Signal-
  6*01_IGHJ1*01	NO	01	NO	Signal/Noise	Noise)
  S102A/S103P/S104F/H111F	126	S76E	357	217.5	0.36
  S102A/S103P/S104F/H111F	126	S76Q	358	187.3	0.32
  S102A/S103P/S104F/H111F	126	S76P	359	100.0	0.29
  S102A/S103P/S104F/H111F	126	S76N	360	118.2	0.28
  S102A/S103P/S104F/H111F	126	wildtype	107	441.3	0.57
  S102A/S103P/S104F/H111F	126	wildtype	107	309.9	0.24
  S102A/S103P/S104F/H111F	126	wildtype	107	584.6	0.26
  S102A/S103P/S104F/H111F	126	wildtype	107	718.7	0.37

• TABLE 71
  Binding affinity and specificity of Fab VH1-46_IGHD6-6*01_IGHJ1*01
  S102A/S103P/S104F/H111F (H: APFF) (SEQ ID NO: 126) & L6_IGKJ1*01 S76 and F62 Mutants

  Light	ErbB2	EGF R	HGF R	Notch-1	CD44	IGF-1	P-Cad	EPO R	DLL4	Blank

  S76E	277	266	228	313	439	336	338	440	51555	237
  S76Q	264	324	386	255	287	188	364	430	48330	258
  S76P	260	331	394	402	313	347	271	371	29787	298
  S76N	436	385	429	298	369	378	329	384	51989	440
  wildtype	379	425	332	429	470	468	399	437	149144	338
  wildtype	292	329	237	377	326	357	277	449	126118	407
  wildtype	351	209	176	359	332	306	138	414	148493	254
  wildtype	322	409	263	417	316	173	240	328	132249	184

Example 11 Heavy Chain and Light Chain Fab Combination Mutants
• Heavy chain and light chain mutants that were identified in Examples 7-10 as contributing to binding to DLL4 were paired into various combination mutants. Heavy chain mutants included VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F N52L/S54T/G56H(H:APFF LTH), VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F I51A/N52L/S54T/G56H(H:APFF ALTH), and VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F I51V/N52L/S54T/G56H(H:APFF VLTH). Light chain mutants included L6_IGKJ1*01 S28D/S30N/S31H S52L/A55S (L:NDH LS) and L6_IGKJ1*01 S28D/S30N/S31H S52L/A55G (L:NDH LG).
• Table 72 below sets forth the Fabs and the ECL signal for binding to DLL4. In general, Fabs with H:APFF LTH and H:APFF VLTH heavy chains had an increased ECL signal for binding to DLL4 as compared to a Fab with a heavy chain H:APFF ALTH. Depending on the antibody tested, the particular light chain mutants also further affected binding to DLL4. Similar results were obtained by ELISA (Table 73). The mutants were further analyzed for binding to DLL4 by ELISA using 3-fold serial dilutions of Fab, starting at a concentration of 20 nM. The results are set forth in Table 73 below. Antibodies containing the H:APFF LTH and APFF H:VLTH heavy chain mutations had approximately 10-fold increased binding affinity to DLL4 compared to the antibody mutants containing the heavy chain mutant H:APFF ALTH.

• TABLE 72
  Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F
  (H:APFF) & L6_IGKJ1*01 S28N/S30D/S31H (L:NDH) CDR2 combination mutants

  Fab

  Heavy Chain
  VH1-46_IGHD6-		Light Chain
  6*01_IGHJ1*01		L6_IGKJ1*01	SEQ
  S102A/S103P/S104F/H111F	SEQ ID	S28N/S30D/S31H	ID	ECL
  (APFF)	NO	(NDH)	NO	Signal

  N52L/S54T/G56H (LTH)	203	(NDH)	323	6023
  N52L/S54T/G56H (LTH)	203	S52L/A55G (NDH LG)	349	9007
  N52L/S54T/G56H (LTH)	203	S52L/A55S (NDH LS)	350	11493
  I51A/N52L/S54T/G56H (ALTH)	204	(NDH)	323	1840
  I51A/N52L/S54T/G56H (ALTH)	204	S52L/A55G (NDH LG)	349	1759
  I51A/N52L/S54T/G56H (ALTH)	204	S52L/A55S (NDH LS)	350	3720
  I51V/N52L/S54T/G56H (VLTH)	209	(NDH)	323	9789
  I51V/N52L/S54T/G56H (VLTH)	209	S52L/A55G (NDH LG)	349	12246
  I51V/N52L/S54T/G56H (VLTH)	209	S52L/A55S (NDH LS)	350	8000

• TABLE 73
  Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ*01 mutant binding to DLL4
  by ELISA

  Heavy Chain	Light Chain
  VH1-46_IGHD6-6*01_IGHJ1*01	L6_IGKJ1*01
  S102A/S103P/S104F/H111F	S28N/S30D/S31H
  (APFF)	(NDH)	20	6.67	2.22	0.74
  N52L/S54T/G56H (LTH)	(NDH)	0.863	0.739	0.463	0.270
  N52L/S54T/G56H (LTH)	S52L/A55G (NDH LG)	1.008	0.880	0.594	0.368
  N52L/S54T/G56H (LTH)	S52L/A55S (NDH LS)	1.054	0.916	0.557	0.398
  I51A/N52L/S54T/G56H (ALTH)	(NDH)	0.391	0.232	0.069	0.024
  I51A/N52L/S54T/G56H (ALTH)	S52L/A55G (NDH LG)	0.390	0.212	0.069	0.028
  I51A/N52L/S54T/G56H (ALTH)	S52L/A55S (NDH LS)	0.458	0.282	0.040	0.046
  I51V/N52L/S54T/G56H (VLTH)	(NDH)	0.979	0.776	0.608	0.288
  I51V/N52L/S54T/G56H (VLTH)	S52L/A55G (NDH LG)	1.057	0.916	0.755	0.397
  I51V/N52L/S54T/G56H (VLTH)	S52L/A55S (NDH LS)	0.910	0.747	0.523	0.263

Summary
• As a result of affinity maturation, the affinity of parental Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 for binding to DLL4 was increased 430-fold. Table 75 below sets for the binding affinity of the various affinity matured antibodies for DLL4, as determined by SPR (see Example 5). Parent Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 binds DLL4 with a K_D of 730 nM. Mutation of four heavy chain amino acids, namely S102A/S103P/S104F/H111F (H:APFF), resulted in a Fab with 10-fold increased affinity for DLL4 (K_D=70.6 nM). Affinity matured heavy and light chain mutant Fab H:APFF VLTH & L:NDH LS has a K_D of 1.7 nM, a 430-fold increase in binding affinity for DLL4.

• TABLE 75
  Surface Plasmon Resonance Binding affinity of DLL4 Fabs

  		ka (×105)	kd	KD
  Heavy Chain	Light Chain	(M−1s−1)	(s−1)	(nM)
  VH1-46_IGHD6-6*01_IGHJ1*01	L6_IGKJ1*01	1.63	0.101	730
  (parental)	(parental)	(±3)	(±2)	(±130)
  VH1-46_IGHD6-6*01_IGHJ1*01	L6_IGKJ1*01	5.0	0.19	380
  S104F		(±0.8)	(±0.01)	(±60)
  VH1-46_IGHD6-6*01_IGHJ1*01	L6_IGKJ1*01	4.05	0.0492	122
  S102A/S103P/S104F (APF)		(±0.05)	(±0.0004)	(±1)
  VH1-46_IGHD6-6*01_IGHJ1*01	L6_IGKJ1*01	4.25	0.0300	70.6
  S102A/S103P/S104F/H111F		(±0.04)	(±0.0002)	(±0.7)
  (APFF)
  VH1-46_IGHD6-6*01_IGHJ1*01	L6_IGKJ1*01	3.40	0.0317	93.1
  S102A/S103P/S104F/H111Y		(±0.03)	(±0.0002)	(±0.9)
  (APFY)
  VH1-46_IGHD6-6*01_IGHJ1*01	L6_IGKJ1*01 S31K	3.50	0.0392	112
  S102A/S103P/S104F (APF)		(±0.05)	(0.0004)	(±2)
  VH1-46_IGHD6-6*01_IGHJ1*01	L6_IGKJ1*01	3.51	0.0101	32.7
  S102A/S103P/S104F/H111F		(±1.84)	(±0.000716)	(±11.6)
  G56H (APFF G56H)
  VH1-46_IGHD6-6*01_IGHJ1*01	L6_IGKJ1*01	4.44	0.0689	*155.2
  S102A/S103P/S104F/H111F	S28N/S30D/S31H			and 14
  (APFF)	(NDH)
  VH1-46_IGHD6-6*01_IGHJ1*01	L6_IGKJ1*01	4.30	0.00113	2.7
  S102A/S103P/S104F/H111F	S28N/S30D/S31H	(±1.45)	(±0.000138)	(±0.6)
  I51V/N52L/S54T/G56H	(NDH)
  (APFF VLTH)
  VH1-46_IGHD6-6*01_IGHJ1*01	L6_IGKJ1*01	6.84	0.00109	1.7
  S102A/S103P/S104F/H111F	S28N/S30D/S31H	(±2.51)	(±0.000106)	(±0.5)
  I51V/N52L/S54T/G56H	S52L/A55S
  (APFF VLTH)	(NDH LS)
  *Fab Fab VH1-46_IGHD6-6*01_IGHJ1*01 S102A/S103P/S104F/H111F & L6_IGKJ1*01 S28N/S30D/S31H displays 2-site binding: 89% with Kd of 155.2 nM and 10% with Kd of 14 nM.

Example 12 Affinity Maturation of Identified Parent “Hit” Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01 Against DLL4
• The parent “Hit” Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01 (SEQ ID NOS:89 and 108) against DLL4, identified in Example 4 using the electroluminescence Meso Scale Discovery (MSD) multispot binding assay, was subjected to affinity maturation as described above in Examples 7-11. By this method, an anti-DLL4 antibody was generated with significantly improved binding affinity for DLL4 compared to the parent “Hit” VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01 Fab antibody.
A. Heavy Chain
• 1. Identification of the CDR Potential Binding Site
• The amino acid sequence of the heavy chain (SEQ ID NO:89) for the parent “Hit” VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01 was aligned with the amino acid sequence of a related heavy chain (SEQ ID NO:106) of a non-Hit that was identified as not binding to DLL4, i.e. VH5-51_IGHD6-25*01_IGHJ4*01. These two Fabs are related because they share the same V_H and J_H germline segments. The sequence alignment is set forth in FIG. 3. Based on the alignment, amino acid residues were identified that differed between the “Hit” and “non-Hit,” thus accounting for the differences in binding of the “Hit” and “non-Hit” antibody for DLL4. The identified amino acid residues were located in CDR3, which was identified as the region of the heavy chain that is important for binding affinity.
• 2. Alanine Scanning of CDR3
• Alanine scanning mutagenesis was performed on amino acid residues in the CDR3 of the heavy chain sequence of parent Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01 to identify amino acid residues that do not appear to be involved in DLL4 binding. Alanine-scanning of the CDR3 region of the heavy chain was performed by mutating every residue of the CDR3 region to an alanine, except amino acid residues Y107, F108, D109, and Y110. Purified Fab alanine mutants were tested for binding to DLL4. The results are set forth in Table 76. Mutation of R99, Y101, S102, Y103, Y105, or D106 with alanine caused a reduction in the ECL signal for binding to DLL4, and therefore these residues were not further mutagenized. In contrast, mutation of G100 or G104 with alanine either resulted in an increased ECL signal or did not affect the ECL signal for binding to DLL4, and thus these residues were identified as residues for further mutagenesis. The results were confirmed in a repeat experiment using varying concentrations of mutant Fab and DLL4 protein (see Table 77).

• TABLE 76
  Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01 alanine
  mutant binding data

  Heavy Chain				Signal/
  VH5-51_IGHD5-	SEQ ID		SEQ ID	Noise
  18*01_IGHJ4*01	NO	Light Chain	NO	(0.04 μM)

  wildtype	89	V3-4_IGLJ1*01	108	14.7
  R99A	382	V3-4_IGLJ1*01	108	1.3
  G100A	383	V3-4_IGLJ1*01	108	30.4
  Y101A	384	V3-4_IGLJ1*01	108	1.2
  S102A	385	V3-4_IGLJ1*01	108	2
  Y103A	386	V3-4_IGLJI*01	108	1.2
  G104A	387	V3-4_IGLJ1*01	108	15.5
  Y105A	388	V3-4_IGLJ1*01	108	9.6
  D106A	389	V3-4_IGLJ1*01	108	1.2
  wildtype	89	V3-4_IGLJ1*01	108	15.5

• TABLE 77
  Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01 alanine
  mutant binding data

  	0.1 μM	0.02 μM
  	Fab	Fab
  Fab	30 μg/mL	15 μg/mL

  Heavy Chain		Light Chain	DLL4	DLL4
  VH5-51_IGHD5-	SEQ ID	(SEQ ID	Signal/	Signal/
  18*01_IGHJ4*01	NO	NO: 108)	Noise	Noise

  wildtype	89	V3-4_IGLJ1*01	24.0	15.2
  R99A	382	V3-4_IGLJ1*01	1.1	1.0
  G100A	383	V3-4_IGLJ1*01	53.3	24.2
  Y101A	384	V3-4_IGLJ1*01	1.1	1.3
  S102A	385	V3-4_IGLJ1*01	4.7	1.8
  Y103A	386	V3-4_IGLJ1*01	4.0	1.5
  G104A	387	V3-4_IGLJ1*01	41.5	12.5
  Y105A	388	V3-4_IGLJ1*01	1.0	1.0
  D106A	389	V3-4_IGLJ1*01	1.3	1.0

• 3. NNK Mutagenesis of Heavy Chain Amino Acid Residues G100 and G104
• Following alanine scanning mutagenesis of CDR3, heavy chain amino acid residues G100 and G104 were selected for further mutation using overlapping PCR with NNK mutagenesis using wildtype Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01 as a template, similar to the experiment described in Example 7.b.iii above. The results are set forth in Table 78 below. Amino acid mutations designated with X (for any amino acid) did not show appreciable binding and therefore were not sequenced to identify the exact mutation. Two mutations, G100K and G104T, in the heavy chain were identified that resulted in a Fab with an improved ECL signal for binding to DLL4. Each mutant exhibited an ECL signal for binding to DLL4 approximately 2-fold greater than parent Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01.

• TABLE 78
  NNK mutagenesis of parent Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 &
  V3-4_IGLJ1*01 at amino acid residues G100 and G104

  	0.1 μM Fab	0.02 μM Fab
  Fab	30 μg/mL	15 μg/mL

  		Light Chain	DLL4	DLL4
  Heavy Chain	SEQ	(SEQ ID	Signal/	Signal/
  VH5-51_IGHD5-18*01_IGHJ4*01	ID NO	NO: 108)	Noise	Noise

  G100L	390	V3-4_IGLJ1*01	27.2	13.0
  G104stop	436	V3-4_IGLJ1*01	1.0	1.1
  G100L	390	V3-4_IGLJ1*01	66.2	32.5
  G100D	391	V3-4_IGLJ1*01	5.8	2.0
  G100T	392	V3-4_IGLJ1*01	26.0	11.0
  G100K	378	V3-4_IGLJ1*01	133.9	72.6
  G100R	379	V3-4_IGLJ1*01	90.6	39.9
  G100L	390	V3-4_IGLJ1*01	40.2	15.6
  G100L	390	V3-4_IGLJ1*01	59.0	28.7
  G104D	393	V3-4_IGLJ1*01	42.5	23.2
  G104A	387	V3-4_IGLJ1*01	6.7	2.6
  G104L	394	V3-4_IGLJ1*01	28.4	9.3
  G104P	395	V3-4_IGLJ1*01	1.0	1.0
  wildtype	89	V3-4_IGLJ1*01	31.4	13.2
  G104R	396	V3-4_IGLJ1*01	23.2	9.1
  G104T	380	V3-4_IGLJ1*01	45.4	20.2
  G104X	437	V3-4_IGLJ1*01	44.5	22.5
  G104T	380	V3-4_IGLJ1*01	63.2	29.0
  G104stop	436	V3-4_IGLJ1*01	1.2	0.9
  G104M	397	V3-4_IGLJ1*01	29.1	12.3
  wildtype	89	V3-4_IGLJ1*01	32.6	15.6
  G104L	394	V3-4_IGLJ1*01	23.4	10.8
  G104stop	436	V3-4_IGLJ1*01	1.0	1.0
  G104K	398	V3-4_IGLJ1*01	17.6	9.1
  wildtype	89	V3-4_IGLJ1*01	42.4	17.6
  G104R	396	V3-4_IGLJ1*01	20.4	7.8
  G104S	399	V3-4_IGLJ1*01	47.8	25.6
  G104R/Y101H	400	V3-4_IGLJ1*01	1.2	1.0
  G104T	380	V3-4_IGLJ1*01	67.8	35.8

• 4. Combination Mutant Based on NNK Mutagenesis of CDR3
• Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01 heavy chain mutants G100K and G104T, identified as having increased binding affinity to DLL4, were combined to generate a double mutant, designated as Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 G100K/G104T & V3-4_IGLJ1*01 (H:KT). The binding of the KT double mutant to DLL4 was compared to the binding of the parent Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01 to DLL4 by assaying various concentrations each antibody. The results are set forth in Tables 79-80 below. The results show that the KT double mutant exhibits an increased ECL signal for binding to DLL4 as compared to the parent Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01. Both Fabs exhibit specific binding to DLL4 as compared to the various other tested antigens (see Table 80).

• TABLE 79
  Binding affinity of double mutant Fab VH5-51_IGHD5-
  18*01>3_IGHJ4*01 G100K/G104T & V3-4_IGLJ1*01
  (SEQ ID NO: 108) as compared to wildtype Fab VH5-
  51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01

  	Wildtype	G100K, G104T		G100K, G104T
  Heavy	(SEQ ID	(SEQ ID	Wildtype (SEQ	(SEQ ID
  Chain	NO: 89)	NO: 381)	ID NO: 89)	NO: 381)
  Fab [μM]	Signal	Signal	Signal/Noise	Signal/Noise

  200.00	4750	69079	36.3	76.9
  20.00	2199	45123	21.1	157.2
  2.00	443	5379	2.2	72.7
  0.20	348	350	3.0	3.0

• TABLE 80
  Binding affinity and specificity of double mutant Fab VH5-51_IGHD5-18*01 >
  3_IGHJ4*01 G100K/G104T & V3-4_IGLJ1*01 as compared to wildtype Fab
  VH5-51_IGHD5-18*01 > 3_IGHJ4*01 & V3-4_IGLJ1*01

  	Fab
  Heavy Chain	[μM]	ErbB2	EGF R	HGF R	Notch-1	CD44	IGF-1	P-Cad	EPO R	DLL4

  Wt	200.00	3.1	2.9	3.5	1.3	1.6	2.8	1.5	2.2	36.3
  	20.00	4.4	2.5	4.0	1.6	2.6	1.8	0.9	2.0	21.1
  	2.00	1.8	1.1	1.8	1.5	1.1	1.6	1.1	1.2	2.2
  	0.20	2.6	3.4	3.1	1.7	1.4	2.9	1.5	2.5	3.0
  G100K,	200.00	1.7	1.6	1.7	1.7	1.4	1.5	2.0	1.7	76.9
  G104T	20.00	1.2	1.1	0.9	1.2	1.1	1.1	1.1	1.1	157.2
  	2.00	3.2	3.8	6.0	1.9	3.5	3.7	4.3	4.1	72.7
  	0.20	2.5	1.5	2.4	1.9	1.3	1.9	1.7	1.7	3.0

B. Further Optimization of the Heavy Chain
• 1. Summary
• The heavy chain of the KT double mutant described and generated above was further optimized to improve its binding for DLL4. The heavy chain mutant KT double mutant was used as a template for further mutagenesis of heavy chain amino acid residues in the CDR1 (amino acids 26-35), CDR2 (amino acid residues 50-66) and framework region of the heavy chain by alanine scanning mutagenesis.
• 2. Alaninie Scanning of Residues in CDR1
• Alanine scanning was performed by mutating every amino acid residue of CDR1, except G26. Three additional flanking amino acid residues, namely G24, I34, and G35 were also mutated to alanine. Purified Fab alanine mutants were tested for binding to DLL4 using the ECL multispot binding assay. The results are set forth in Tables 81-83 below. Mutation of amino acid residues Y27, F29, T30, S31, Y32, W33, or 134 with alanine caused a reduction in the ECL and ELISA signals for binding to DLL4, and thus these residues were not further mutagenized. Mutation of amino acid residues G24, S28, or G35 with alanine either improved the ECL signal or did not affect the ECL signal for binding to DLL4, and thus these residues were identified as residues for further mutagenesis. ELISA experiments also were performed, but little or no detectable signal was observed in the ELISA experiments (Table 81). Table 83 shows that the tested antibodies exhibit specificity for DLL4 compared to other tested antigens.

• TABLE 81
  Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 G100K/G104T (KT)
  & V3-4_IGLJ1*01 CDR1 alanine mutant binding data

  Fab

  Heavy Chain			ECL	ELISA
  VH5-51_IGHD5-	SEQ	Light Chain	Signal/	(Signal-
  18*01_IGHJ4*01	ID NO	(SEQ ID NO: 108)	Blank	Noise)

  G100K/G104T G24A	401	V3-4_IGLJ1*01	122.1	0.02
  G100K/G104T I34A	402	V3-4_IGLJ1*01	2.6	0.01
  G100K/G104T G35A	403	V3-4_IGLJ1*01	180.5	0.02
  G100K/G104T S28A	404	V3-4_IGLJ1*01	112.1	0.01
  G100K/G104T	381	V3-4_IGLJ1*01	85.9	0.00
  G100K/G104T F29A	405	V3-4_IGLJ1*01	67.9	0.02
  G100K/G104T T30A	406	V3-4_IGLJ1*01	69.4	0.00
  G100K/G104T	381	V3-4_IGLJ1*01	188.0	0.00
  G100K/G104T W33A	407	V3-4_IGLJ1*01	3.0	0.02
  G100K/G104T	381	V3-4_IGLJ1*01	153.3	0.01

• TABLE 82
  Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 G100K/G104T (KT)
  & V3-4_IGLJ1*01 CDR1 alanine mutant binding data

  Fab

  Heavy Chain			ECL
  VH5-51_IGHD5-	SEQ	Light Chain	Signal/
  18*01_IGHJ4*01	ID NO	(SEQ ID NO: 108)	Blank

  G100K/G104T	381	V3-4_IGLJ1*01	49.2
  G100K/G104T Y27A	2899	V3-4_IGLJ1*01	9.1
  G100K/G104T S31A	2900	V3-4_IGLJ1*01	3.0
  G100K/G104T Y32A	2901	V3-4_IGLJ1*01	2.7

• TABLE 83
  Fab VH5-51_IGHD5-18*01 > 3_IGHJ4*01 G100K/G104T (KT)
  & V3-4_IGLJ1*01 CDR1 alanine mutant binding data

  Heavy Chain	ErbB2	EGF R	HGF R	Notch-1	CD44	IGF-1	P-Cad	EPO R	DLL4	Blank

  KT G24A	869	757	803	493	547	879	212	551	45546	373
  KT I34A	1149	883	1084	564	608	923	349	505	772	300
  KT G35A	911	760	939	400	624	899	305	506	53618	297
  KT S28A	1072	839	1040	432	497	924	317	586	35439	316
  KT	1095	852	838	543	579	877	319	554	36440	424
  KT F29A	1040	887	985	601	621	945	502	586	22867	337
  KT T30A	1071	853	868	539	698	968	438	553	24346	351
  KT	1068	915	936	507	633	964	346	497	45120	240
  KT W33A	921	761	735	561	513	788	302	424	731	240
  KT	1098	768	867	437	540	781	226	421	32658	213

• 3. NNK Mutagenesis of Amino Acid Residues G24, S28 and G35
• Following alanine scanning mutagenesis of CDR1, heavy chain amino acid residues G24, S28 and G35 were selected for further mutation using overlapping PCR with NNK mutagenesis using the heavy chain KT double mutant as a template. The results are set forth in Table 84 below. Amino acid mutations designated with X (for any amino acid) did not show appreciable binding and therefore were not sequenced to identify the exact mutation. Several Fab mutants that contained a combination of two mutations at a specific amino acid position are designated as such. For example, G24S/T indicates the tested antibody was a mixture of two Fabs, one containing the mutation G24S and the other containing the mutation G24T. The results show that mutation of additional amino acids (G24L, S28R, S28K and G35V) in the heavy chain of the KT double mutant result in increase the ECL signal for binding to DLL4 compared to the parental KT double mutant template.

• TABLE 84
  Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 G100K/G104T (KT) &
  V3-4_IGLJ1*01 CDR1 NNK mutant binding data

  Fab

  Heavy Chain	SEQ			ELISA
  VH5-51_IGHD5-	ID	Light Chain	ECL	(Signal-
  18*01_IGHJ4*01	NO	(SEQ ID NO: 108)	Signal	Noise)

  G100K/G104T G24L	408	V3-4_IGLJ1*01	19617	0.09
  G100K/G104T G24X	438	V3-4_IGLJ1*01	291	0.03
  G100K/G104T G24X	438	V3-4_IGLJ1*01	13304	0.06
  G100K/G104T G24X	438	V3-4_IGLJ1*01	250	0.03
  G100K/G104T G24X	438	V3-4_IGLJ1*01	10339	0.06
  G100K/G104T G24X	438	V3-4_IGLJ1*01	7395	0.05
  G100K/G104T G24X	438	V3-4_IGLJ1*01	1294	0.03
  G100K/G104T G24X	438	V3-4_IGLJ1*01	4299	0.04
  G100K/G104T G24X	438	V3-4_IGLJ1*01	319	0.02
  G100K/G104T G24S/T	439	V3-4_IGLJ1*01	22221	0.09
  G100K/G104T G24X	438	V3-4_IGLJ1*01	9771	0.06
  G100K/G104T G24X	438	V3-4_IGLJ1*01	7554	0.05
  G100K/G104T G24L/G	440	V3-4_IGLJ1*01	7970	0.05
  G100K/G104T G24X	438	V3-4_IGLJ1*01	517	0.04
  G100K/G104T G24X	438	V3-4_IGLJ1*01	1267	0.04
  G100K/G104T G24X	438	V3-4_IGLJ1*01	12665	0.05
  G100K/G104T G24X	438	V3-4_IGLJ1*01	12614	0.06
  G100K/G104T G24X	438	V3-4_IGLJ1*01	8746	0.05
  G100K/G104T G24X	438	V3-4_IGLJ1*01	2330	0.04
  G100K/G104T G24X	438	V3-4_IGLJ1*01	7003	0.05
  G100K/G104T S28R	411	V3-4_IGLJ1*01	36903	0.25
  G100K/G104T S28X	441	V3-4_IGLJ1*01	1882	0.06
  G100K/G104T S28K	412	V3-4_IGLJ1*01	32324	0.28
  G100K/G104T S28X	441	V3-4_IGLJ1*01	5811	0.06
  G100K/G104T G24R	410	V3-4_IGLJ1*01	4203	0.06
  G100K/G104T S28X	441	V3-4_IGLJ1*01	6855	0.05
  G100K/G104T S28X	441	V3-4_IGLJ1*01	356	0.03
  G100K/G104T S28X	441	V3-4_IGLJ1*01	8482	0.05
  G100K/G104T S28R	411	V3-4_IGLJ1*01	64124	0.49
  G100K/G104T S28X	441	V3-4_IGLJ1*01	14585	0.10
  G100K/G104T S28X	441	V3-4_IGLJ1*01	10205	0.07
  G100K/G104T S28X	441	V3-4_IGLJ1*01	834	0.04
  G100K/G104T S28X	441	V3-4_IGLJ1*01	4605	0.04
  G100K/G104T S28X	441	V3-4_IGLJ1*01	344	0.03
  G100K/G104T S28X	441	V3-4_IGLJ1*01	8017	0.05
  G100K/G104T S28X	441	V3-4_IGLJ1*01	9895	0.05
  G100K/G104T S28R	411	V3-4_IGLJ1*01	51418	0.29
  G100K/G104T S28N	413	V3-4_IGLJ1*01	17255	0.09
  G100K/G104T S28X	441	V3-4_IGLJ1*01	7681	0.05
  G100K/G104T G35X	442	V3-4_IGLJ1*01	6027	0.05
  G100K/G104T G35X	442	V3-4_IGLJ1*01	302	0.02
  G100K/G104T G35T	414	V3-4_IGLJ1*01	14452	0.07
  G100K/G104T G35X	442	V3-4_IGLJ1*01	937	0.04
  G100K/G104T G35X	442	V3-4_IGLJ1*01	4954	0.05
  G100K/G104T G35X	442	V3-4_IGLJ1*01	812	0.03
  G100K/G104T G35X	442	V3-4_IGLJ1*01	1088	0.04
  G100K/G104T G35X	442	V3-4_IGLJ1*01	1231	0.03
  G100K/G104T G35X	442	V3-4_IGLJ1*01	5067	0.04
  G100K/G104T G35A	403	V3-4_IGLJ1*01	19695	0.06
  G100K/G104T G35V	416	V3-4_IGLJ1*01	21169	0.09
  G100K/G104T G35X	442	V3-4_IGLJ1*01	2122	0.04
  G100K/G104T G35X	442	V3-4_IGLJ1*01	1426	0.04
  G100K/G104T G35X	442	V3-4_IGLJ1*01	326	0.03
  G100K/G104T G35X	442	V3-4_IGLJ1*01	3106	0.03
  G100K/G104T G35X	442	V3-4_IGLJ1*01	1373	0.03
  G100K/G104T G35X	442	V3-4_IGLJ1*01	5986	0.06
  G100K/G104T G35X	442	V3-4_IGLJ1*01	3787	0.04
  G100K/G104T G35X	442	V3-4_IGLJ1*01	4871	0.04
  G100K/G104T G35X	442	V3-4_IGLJ1*01	370	0.03
  G100K/G104T G35X	442	V3-4_IGLJ1*01	841	0.04

• 4. Combination Mutants of G24, S28 and G35
• Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 G100K/G104T (KT) & V3-4_IGLJ1*01 heavy chain mutants G24L, G24T, G24A, S28R and G35V were combined to generate antibodies containing three to five mutations in the heavy chain. The mutants generated are set forth in Table 85. The mutants were assessed for binding to DLL4 using an ECL assay. All combination mutants exhibited greater ECL signals for binding to DLL4 compared to the KT double mutant. The results show that the mutant Fab H:KT TRV & L:wt had the greatest affinity towards binding to DLL4.

• TABLE 85
  Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 G100K/G104T (KT) &
  V3-4_IGLJ1*01 CDR1 combination mutants

  Fab

  Heavy Chain	SEQ ID		SEQ ID	ECL
  VH5-51_IGHD5-18*01_IGHJ4*01	NO	Light Chain	NO	Signal

  G100K/G104T (KT)	381	V3-4_IGLJ1*01	108	588
  G100K/G104T/S28R (KT S28R)	411	V3-4_IGLJ1*01	108	6423
  G100K/G104T/G24L/S28R/G35V	417	V3-4_IGLJ1*01	108	15333
  (KT LRV)
  G100K/G104T/G24T/S28R/G35V	430	V3-4 _IGLJ1* 01	108	26072
  (KT TRV)
  G100K/G104T/G24A/S28R/G35V	431	V3-4_IGLJ1*01	108	17357
  (KT ARV)

• 5. Alanine Scanning of CDR2
• The KT double mutant was used as a template for alanine scanning mutagenesis of CDR2 (amino acids 50-58) to determine residues important for antibody binding to DLL4. Purified Fab alanine mutants were tested for binding to DLL4 using the ECL multispot binding assay. The results are set forth in Tables 86-88 below. Mutation of amino acid residues 150, 151, Y52, P53, G54, D55, or D57 with alanine caused a reduction in the ECL signal for binding to DLL4, and thus these residues were not targeted for further mutagenesis. Substitution of amino acid residues S56 or T58 with alanine either improved the ECL signal or did not affect the ECL signal for binding to DLL4, and thus these residues were subjected to further mutagenesis. Similar experiments also were performed by ELISA, although little to no detectable signal was observed. Table 88 shows that all antibodies exhibit specificity for DLL4.

• TABLE 86
  Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 G100K/G104T (KT) &
  V3-4_IGLJ1*01 CDR2 alanine mutant binding data

  Fab

  Heavy Chain			ECL	ELISA
  VH5-51_IGHD5-	SEQ	Light Chain	Signal/	(Signal-
  18*01_IGHJ4*01	ID NO	(SEQ ID NO: 108)	Blank	Noise)

  G100K/G104T D57A	418	V3-4_IGLJ1*01	2.8	0.01
  G100K/G104T	381	V3-4_IGLJ1*01	85.9	0.00
  G100K/G104T	381	V3-4_IGLJ1*01	188.0	0.00
  G100K/G104T	381	V3-4_IGLJ1*01	153.3	0.01
  G100K/G104T I50A	419	V3-4_IGLJ1*01	40.9	0.02
  G100K/G104T I51A	420	V3-4_IGLJ1*01	30.6	0.01
  G100K/G104T Y52A	421	V3-4_IGLJ1*01	2.7	0.04
  G100K/G104T P53A	422	V3-4_IGLJ1*01	57.7	0.00
  G100K/G104T D55A	423	V3-4_IGLJ1*01	2.5	0.00

• TABLE 87
  Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 G100K/G104T (KT)
  & V3-4_IGLJ1*01 CDR2 alanine mutant binding data

  Fab

  Heavy Chain			ECL
  VH5-51_IGHD5-	SEQ	Light Chain	Signal/
  18*01_IGHJ4*01	ID NO	(SEQ ID NO: 108)	Blank

  G100K/G104T	381	V3-4_IGLJ1*01	49.2
  G100K/G104T G54A	2902	V3-4_IGLJ1*01	4.1
  G100K/G104T S56A	2903	V3-4_IGLJ1*01	55
  G100K/G104T T58A	425	V3-4_IGLJ1*01	101.9

• TABLE 88
  Fab VH5-51_IGHD5-18*01 > 3_IGHJ4*01 G100K/G104T (KT) &
  V3-4_IGLJ1*01 CDR1 and CDR2 alanine mutant binding data

  Heavy Chain	ErbB2	EGF R	HGF R	Notch-1	CD44	IGF-1	P-Cad	EPO R	DLL4	Blank

  KT D57A	1203	915	1126	523	600	982	365	456	888	321
  KT	1095	852	838	543	579	877	319	554	36440	424
  KT	1068	915	936	507	633	964	346	497	45120	240
  KT	1098	768	867	437	540	781	226	421	32658	213
  KT I50A	925	794	822	443	632	785	343	523	9682	237
  KT I51A	1092	803	875	612	517	828	432	497	6578	215
  KT Y52A	989	745	803	566	591	827	334	584	735	277
  KT P53A	1145	976	1000	536	556	943	424	563	20135	349
  KT D55A	1028	729	856	683	606	898	310	479	761	306

• 6. NNK Mutagenesis of Amino Acid Residues T58 and S56
• Following alanine scanning mutagenesis of CDR2, heavy chain amino acid residues T58 and S56 were selected for further mutation using overlapping PCR with NNK mutagenesis using the H:KT & L:wt double mutant as a template. The results are set forth in Table 89 below Amino acid mutations designated with X (for any amino acid) did not show appreciable binding and therefore were not sequenced to identify the exact mutation. Mutation of heavy chain KT amino acid residue T58 to alanine (T58A) and aspartic acid (T58D) resulted in an increase in ECL signal for binding to DLL4.

• TABLE 89
  Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 G100K/G104T (H:KT) & V3-
  4_IGLJ1*01 CDR1 and CDR2 T58 and S56 NNK mutant binding data

  Fab

  Heavy Chain	SEQ			ELISA
  VH5-51_IGHD5-	ID	Light Chain	ECL	(Signal-
  18*01_IGHJ4*01	NO	(SEQ ID NO: 108)	Signal	Noise)

  G100K/G104T T58D/K	443	V3-4_IGLJ1*01	823	0.03
  G100K/G104T/T58X	444	V3-4_IGLJ1*01	5040	0.03
  G100K/G104T/T58X	444	V3-4_IGLJ1*01	765	0.03
  G100K/G104T/T58X	444	V3-4_IGLJ1*01	520	0.02
  G100K/G104T/T58A	425	V3-4_IGLJ1*01	12938	0.07
  G100K/G104T/T58X	444	V3-4_IGLJ1*01	2272	0.03
  G100K/G104T/T58X	444	V3-4_IGLJ1*01	1059	0.03
  G100K/G104T/T58X	444	V3-4_IGLJ1*01	619	0.03
  G100K/G104T/T58X	444	V3-4_IGLJ1*01	2994	0.04
  G100K/G104T/T58X	444	V3-4_IGLJ1*01	7341	0.05
  G100K/G104T/T58X	444	V3-4_IGLJ1*01	1422	0.03
  G100K/G104T/T58X	444	V3-4_IGLJ1*01	5119	0.05
  G100K/G104T/T58D	424	V3-4_IGLJ1*01	11468	0.07
  G100K/G104T/T58D	424	V3-4_IGLJ1*01	10459	0.06
  G100K/G104T/T58X	444	V3-4_IGLJ1*01	476	0.03
  G100K/G104T/T58X	444	V3-4_IGLJ1*01	1421	0.03
  G100K/G104T/T58X	444	V3-4_IGLJ1*01	658	0.03
  G100K/G104T/T58X	444	V3-4_IGLJ1*01	4278	0.03
  G100K/G104T/S56X	445	V3-4_IGLJ1*01	1436	0.04
  G100K/G104T/S56X	445	V3-4_IGLJ1*01	1553	0.03
  G100K/G104T/S56X	445	V3-4_IGLJ1*01	1372	0.04
  G100K/G104T/S56X	445	V3-4_IGLJ1*01	585	0.03
  G100K/G104T/S56X	445	V3-4_IGLJ1*01	1165	0.03
  G100K/G104T/S56X	445	V3-4_IGLJ1*01	335	0.03
  G100K/G104T/S56X	445	V3-4_IGLJ1*01	1139	0.04
  G100K/G104T/S56X	445	V3-4_IGLJ1*01	3206	0.04
  G100K/G104T/S56X	445	V3-4_IGLJ1*01	3239	0.03
  G100K/G104T/S56G	426	V3-4_IGLJ1*01	8433	0.05
  G100K/G104T/S56X	445	V3-4_IGLJ1*01	1125	0.03
  G100K/G104T/S56X	445	V3-4_IGLJ1*01	1927	0.04
  G100K/G104T/S56X	445	V3-4_IGLJ1*01	502	0.04
  G100K/G104T/S56X	445	V3-4_IGLJ1*01	1509	0.04
  G100K/G104T/S56X	445	V3-4_IGLJ1*01	1951	0.03
  G100K/G104T/S56X	445	V3-4_IGLJ1*01	4317	0.04
  G100K/G104T/S56X	445	V3-4_IGLJ1*01	2065	0.04
  G100K/G104T/S56X	445	V3-4_IGLJ1*01	1486	0.02

• 7. Mutagenesis of Amino Acid Residues S84 and D109
• The heavy chain KT double mutant was used as a template for mutagenesis of amino acid residues S84 and D109. These amino acid residues were mutated using overlapping PCR with NNK mutagenesis or by alanine scanning. The results are shown in Tables 90-92 below, which depict ECL and ELISA results for binding to DLL4 or various antigens. Mutation of heavy chain residues S84 and D109 caused a reduction in ECL signal for binding to DLL4 as compared to heavy chain mutant Fab KT & V3-4_IGLJ*01.

• TABLE 90
  Binding of Fab heavy chain VH5-51_IGHD5-18*01>3_IGHJ4*01
  G100K/G104T (H:KT) & V3-4_IGLJ1*01 S84 and
  D109A mutants to DLL4

  Fab

  Heavy Chain				ELISA
  VH5-51_IGHD5-	SEQ	Light Chain	ECL	(Signal-
  18*01_IGHJ4*01	ID NO	(SEQ ID NO: 108)	Signal	Noise)

  G100K/G104T S84V	427	V3-4_IGLJ1*01	37.7	0.02
  G100K/G104T S84L	428	V3-4_IGLJ1*01	3.2	0.00
  G100K/G104T D109A	429	V3-4_IGLJ1*01	76.8	0.00
  G100K/G104T	381	V3-4_IGLJ1*01	85.9	0.00

• TABLE 91
  Binding and specificity of Fab heavy chain VH5-51_IGHD5-18*01 >
  3_IGHJ4*01 G100K/G104T (H: KT) & V3-4_IGLJ1*01 S84 and D109A mutants to DLL4

  Heavy Chain	ErbB2	EGF R	HGF R	Notch-1	CD44	IGF-1	P-Cad	EPO R	DLL4	Blank

  S84V	1042	805	811	505	577	914	362	484	8889	236
  S84L	1092	864	933	410	545	908	320	458	713	223
  D109A	1099	791	846	443	538	967	406	612	21807	284
  G100K/G104T	1095	852	838	543	579	877	319	554	36440	424

• TABLE 92
  Binding of Fab heavy chain VH5-51_IGHD5-18*01>3_IGHJ4*01
  G100K/G104T (H:KT) & V3-4_IGLJ1*01 S84I to DLL4

  	SEQ	Light Chain
  Heavy Chain	ID	(SEQ ID	ECL
  VH5-51_IGHD5-18*01_IGHJ4*01	NO	NO: 108)	Signal
  G100K/G104T	381	V3-4_IGLJ1*01	9355
  G100K/G104T S84I	409	V3-4_IGLJ1*01	7937

C. Light Chain
• 1. Alanine Scanning of CDR3
• Alanine scanning mutagenesis was performed on amino acid residues in the CDR3 of the light chain of parent Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 G100K/G104T (H:KT) & V3-4_IGLJ*01 to identify amino acid residues that do not appear to be involved in DLL4 binding. Alanine scanning mutagenesis was performed by mutation of every residue of CDR3. Purified Fab alanine mutants were tested at a concentration of 0.04 μM for binding to DLL4 using the ECL multispot assay. The results are set forth in Tables 93-94 below. The results show that mutation of amino acid residues L92, Y93, G95, G97, I98, or S99 with alanine resulted in reduced binding to DLL4, and therefore these residues were not further mutagenized. Substitution of V91, M94, or S96 with alanine either improved binding or did not affect binding to DLL4 and thus these residues were identified as residues for further mutagenesis.

• TABLE 93
  Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 G100K/G104T
  (H:KT) & V3-4_IGLJ1*01 alanine mutant binding data

  Fab

  Heavy Chain
  VH5-51_IGHD5-	SEQ ID	Light Chain	SEQ ID	ECL Signal/
  18*01_IGHJ4*01	NO	V3-4_IGLJ1*01	NO	Noise
  G100K/G104T	381	Parental	108	49.2
  G100K/G104T	381	V91A	446	48.5
  G100K/G104T	381	L92A	447	30.3
  G100K/G104T	381	Y93A	448	21.3
  G100K/G104T	381	M94A	449	53.1
  G100K/G104T	381	G95A	450	34.4
  G100K/G104T	381	G97A	451	24.7
  G100K/G104T	381	S96A	452	57.9
  G100K/G104T	381	I98A	453	32.6
  G100K/G104T	381	S99A	454	41.0

• TABLE 94
  Fab VH5-51_IGHD5-18*01 > 3_IGHJ4*01 G100K/G104T (H:
  KT) & V3-4_IGLJ1*01 CDR3 alanine mutant binding data

  Light Chain	ErbB2	EGF R	HGF R	Notch-1	CD44	IGF-1	P-Cad	EPO R	DLL4	Blank

  V91A	1118	833	1107	682	632	1031	484	675	33377	372
  L92A	1374	1012	1172	693	695	959	326	582	11698	328
  Y93A	1404	918	1130	725	700	1049	497	602	8107	388
  M94A	1203	1126	1151	574	633	1094	472	614	35311	388
  G95A	1250	995	999	707	657	1091	345	637	10445	341
  G97A	1292	1059	1112	660	642	1034	474	528	14892	248
  S96A	1275	1004	1115	715	678	927	491	684	32312	321
  I98A	1375	1054	1227	700	708	1098	359	584	15096	1623
  S99A	1323	956	909	674	670	943	500	693	18191	394

• 2. NNK Mutagenesis of CDR3 Amino Acid Residues V91, M94 and S96
• Following alanine scanning mutagenesis of CDR3, light chain amino acid residues V91, M94 and S96 were selected for further mutation using overlapping PCR with NNK mutagenesis using Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 G100K/G104T & V3-4_IGLJ*01 as a template. The resulting mutants were assayed using the ECL multispot assay as described in Example 4 or by ELISA as described in Example 6. The results are set forth in Table 95. The ECL results show that V3-4_IGLJ*01 amino acid mutants M94R, S96M and S96E exhibited increased binding to DLL4. No detectable signal was observed by ELISA for any of the mutants tested.

• TABLE 95
  Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 G100K/G104T (H:KT) &
  V3-4_IGLJ1*01 V91, M94 and S96 NNK mutant binding data

  Fab

  Heavy Chain			ECL	ELISA
  VH5-51_IGHD5-	Light Chain	SEQ	Signal	Signal
  18*01>3_IGHJ4*01	V3-	ID	[10 nM	[100 nM
  (SEQ ID NO: 381)	4_IGLJ1*01	NO	Fab]	Fab]

  G100K/G104T	V91P	455	920	0.06
  G100K/G104T	V91T	456	32717	0.01
  G100K/G104T	V91S	457	32077	0.01
  G100K/G104T	V91L	458	41576	0.02
  G100K/G104T	V91R	459	13432	0.00
  G100K/G104T	V91A	446	35576	0.01
  G100K/G104T	parent	108	42851	0.01
  G100K/G104T	V91C	460	38330	0.02
  G100K/G104T	V91E	461	22524	0.00
  G100K/G104T	V91W	462	12523	0.00
  G100K/G104T	V91N	463	46674	0.00
  G100K/G104T	V91I	464	51236	0.01
  G100K/G104T	V91G	465	45254	0.01
  G100K/G104T	V91H	466	27123	0.01
  G100K/G104T	V91A	446	33817	0.02
  G100K/G104T	M94E	467	32481	0.01
  G100K/G104T	M94S	468	49579	0.02
  G100K/G104T	M94G	469	20338	0.01
  G100K/G104T	M94L	470	46770	0.02
  G100K/G104T	M94P	471	39930	0.01
  G100K/G104T	M94V	472	47326	0.02
  G100K/G104T	M94D	473	52677	0.01
  G100K/G104T	M94R	474	77777	0.01
  G100K/G104T	M94N	475	51284	0.01
  G100K/G104T	M94T	476	43017	0.02
  G100K/G104T	M94F	477	26330	0.01
  G100K/G104T	M94A	449	33484	0.01
  G100K/G104T	M94A	449	37962	0.00
  G100K/G104T	S96W	478	52299	0.02
  G100K/G104T	S96G	479	40377	0.01
  G100K/G104T	S96P	480	53997	0.03
  G100K/G104T	S96A/E	579	43247	0.02
  G100K/G104T	S96R	481	54259	0.02
  G100K/G104T	S96L	482	39950	0.02
  G100K/G104T	S96M	483	61737	0.02
  G100K/G104T	S96E	484	57030	0.02
  G100K/G104T	parent	108	36614	0.01
  G100K/G104T	S96V	485	42293	0.01
  G100K/G104T	S96A	452	1128	0.00

• 3. Combination Mutants of M94 and S96
• V3-4_IGLJ1*01 light chain mutants M94R and S96M, identified as contributing to increased binding to DLL4, were combined to generate a double mutant. The double mutant is designated as V3-4_IGLJ1*01 M94R/S96M (L:RM). The binding affinity of the L:RM double mutant, as paired with various heavy chain mutants including H:KT, H:KT S28R, H:KT LRV, H:KT TRV, and H:KT ARV, was determined by ECL assay as described in Example 4. The results are set forth in Table 96 below. Fab H:KT TRV & L:RM exhibited the greatest ECL signal for binding to DLL4 compared to other Fab antibodies tested.
• The mutant Fabs above were further analyzed for binding to DLL4 by ELISA as described in Example 6 using 3-fold serial dilutions of Fab, starting at a concentration of 20 nM. The results are set forth in Table 97 below. Similar to the ECL results, Fab H:KT TRV & L:RM exhibited the greatest ELISA signal for binding to DLL4 compared to other mutant Fab antibodies tested.

• TABLE 96
  Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 G100K/G104T (KT)
  & V3-4_IGLJ1*01 CDR3 combination mutants

  Fab

  Heavy Chain	SEQ ID	Light Chain	SEQ ID	ECL
  VH5-51_IGHD5-18*01_IGHJ4*01	NO	V3-4_IGLJ1*01	NO	Signal

  G100K/G104T	381	M94R/S96M	486	564
  G100K/G104T S28R	411	M94R/S96M	486	530
  G100K/G104T G24L/S28R/G35V	417	M94R/S96M	486	889
  G100K/G104T G24T/S28R/G35V	430	M94R/S96M	486	17277
  G100K/G104T G24A/S28R/G35V	431	M94R/S96M	486	1202

• TABLE 97
  Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ*01
  mutant binding to DLL4 by ELISA

  Heavy Chain
  VH5-51_IGHD5-	Light Chain
  18*01>3_IGHJ4*01	V3-4_IGLJ*01	20	6.67	2.22	0.74

  G100K/G104T	parent	0.018	0.042	0.014	0.019
  G100K/G104T S28R	parent	0.009	0.003	0.000	0.000
  G100K/G104T	parent	0.027	0.005	0.000	0.006
  G24L/S28R/G35V
  G100K/G104T	parent	0.054	0.023	0.000	0.002
  G24T/S28R/G35V
  G100K/G104T	parent	0.054	0.025	0.002	0.008
  G24A/S28R/G35V
  G100K/G104T	M94R/S96M	0.087	0.023	0.007	0.000
  G100K/G104T	0.011	0.001	0.003	0.000
  S28R	M94R/S96M
  G100K/G104T	M94R/S96M	0.003	0.000	0.000	0.000
  G24L/S28R/G35V
  G100K/G104T	M94R/S96M	0.122	0.062	0.028	0.006
  G24T/S28R/G35V
  G100K/G104T	M94R/S96M	0.006	0.034	0.000	0.000
  G24A/S28R/G35V

• 4. Alanine Scanning of CDR1 of Light Chain
• Heavy chain KT double mutant (Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 G100K/G104T & V3-4_IGLJ*01) was used as a template for alanine scanning mutagenesis of CDR1 (amino acids 23-33) of the light chain to determine residues important for antibody binding to DLL4.
• Purified Fab alanine mutants were tested for at a concentration of 100 nM for binding to DLL4 using the ECL multispot binding assay as described in Example 4A. The results are set forth in Table 98 below. Mutation of amino acid residues Y33, Y34 and P35 with alanine resulted in reduced binding to DLL4 as evidenced by the reduced ECL signal. Mutation of amino acid residues G23, L24, S25, S26, G27, S28, V29, S30, T31, and S32 with alanine either improved binding or did not affect binding to DLL4 as evidenced by an increased ECL signal or no change in ECL signal compared to the parent KT double mutant having no mutations in the light chain.

• TABLE 98
  Binding affinity of Fab VH5-51_IGHD5-18*01>3_IGHJ4*01
  G100K/G104T (H:KT) & V3-4_IGLJ*01 light chain CDR1 and
  CDR2 alanine mutants

  Heavy Chain
  VH5-51_IGHD5-
  18*03_IGHJ4*01	Light Chain	SEQ ID
  (SEQ ID NO: 381)	V3-4_IGLJ1*01	NO	ECL Signal

  G100K/G104T	wildtype		108	9355
  G100K/G104T	L24A	487	9631
  G100K/G104T	S26A	488	11673
  G100K/G104T	G27A	489	10680
  G100K/G104T	S28A	490	11488
  G100K/G104T	V29A	491	9323
  G100K/G104T	S30A	492	10342
  G100K/G104T	T31A	493	13507
  G100K/G104T	S32A	494	10377
  G100K/G104T	Y33A	495	7705
  G100K/G104T	Y34A	496	2198
  G100K/G104T	P35A	497	8255
  G100K/G104T	S36A	498	9690
  G100K/G104T	G23A	499	13487
  G100K/G104T	S25A	500	10150

• 5. NNK Mutagenesis of Amino Acid Residue G23
• Following alanine scanning mutagenesis of CDR1, the light chain amino acid residue G23 was selected for further NNK mutagenesis using the Fab H:KT & L:wt double mutant as a template. The ECL and ELISA signals are set forth in Table 99 below. Amino acid mutations designated with X (for any amino acid) did not show appreciable binding and therefore were not sequenced to identify the exact mutation.

• TABLE 99
  Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 G100K/G104T (H:KT) &
  V3-4_IGLJ*01 CDR1 G23 NNK mutant binding data

  Fab

  Heavy Chain
  VH5-51_IGHD5-
  18*01>3_IGHJ4*01
  G100K/G104T		SEQ ID	ECL	ELISA
  (SEQ ID NO: 381)	Light Chain	NO	Signal	Signal

  G100K/G104T	G23R	501	68243	0.11
  G100K/G104T	G23X	580	61919	0.10
  G100K/G104T	G23X	580	327	0.09
  G100K/G104T	G23X	580	68201	0.12
  G100K/G104T	G23X	580	384	0.09
  G100K/G104T	G23X	580	67230	0.11
  G100K/G104T	G23X	580	70515	0.09
  G100K/G104T	G23X	580	56769	0.10
  G100K/G104T	G23X	580	322	0.09
  G100K/G104T	G23L	502	67320	0.10
  G100K/G104T	G23L	502	67618	0.10
  G100K/G104T	G23X	580	66603	0.12
  G100K/G104T	G23X	580	62101	0.10
  G100K/G104T	G23X	580	50904	0.10
  G100K/G104T	G23X	580	61718	0.11
  G100K/G104T	G23X	580	67917	0.11
  G100K/G104T	G23X	580	414	0.09
  G100K/G104T	G23X	580	52864	0.10
  G100K/G104T	G23X	580	53493	0.10

• 6. Alanine Scanning of CDR2
• Heavy chain KT double mutant (Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 G100K/G104T & V3-4_IGLJ*01) was used as a template for alanine scanning mutagenesis of CDR2 (amino acids 52-58) to determine residues important for antibody binding to DLL4.
• Purified Fab alanine mutants were tested for binding to DLL4 using the ECL multispot binding assay as described in Example 4. The results are set forth in Table 100 below. Mutation of amino acid residues S52, T53, N54, T55, R56, S57 and S58 with alanine either improved binding or did not affect binding to DLL4 as evidenced by an increased ECL signal or no change in ECL signal compared to the parent KT double mutant having no mutations in the light chain.

• TABLE 100
  Binding affinity of Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 G100K/
  G104T (H:KT) & V3-4_IGLJ*01 light chain CDR2 alanine mutants

  Heavy Chain
  VH5-51_IGHD5-
  18*03_IGHJ4*01	Light Chain	SEQ ID
  (SEQ ID NO: 381)	V3-4_IGLJ1*01	NO	ECL Signal

  G100K/G104T	wildtype		108	9355
  G100K/G104T	S52A	503	15240
  G100K/G104T	T53A	504	13197
  G100K/G104T	N54A	505	12936
  G100K/G104T	T55A	506	12717
  G100K/G104T	R56A	507	16833
  G100K/G104T	S57A	508	12612
  G100K/G104T	S58A	509	12557
  G100K/G104T	R56A	507	13609

• 7. NNK Mutagenesis of Amino Acid Residues S52 and R56
• Following alanine scanning mutagenesis of CDR2, light chain amino acid residues S52 and R56 were selected for further NNK mutagenesis using the heavy chain KT double mutant as a template. The ECL and ELISA signals are set forth in Table 101 below. Amino acid mutations designated with X (for any amino acid) did not show appreciable binding and therefore were not sequenced to identify the exact mutation. Light chain mutants S52G, R56Y/S, R56A and R56G exhibited increased binding to DLL4 as assessed by both ECL and ELISA.
• Various Fabs, containing various combinations of mutations of the heavy chain and light chain, were further analyzed for binding to DLL4 by ELISA using 2-fold serial dilutions of Fab, starting at a concentration of 100 nM. The results are set forth in Table 102 below. Fab H:KT S28R & L:wt exhibited the greatest binding to DLL4 as evidenced by the ELISA signal compared to other Fab mutants tested.

• TABLE 101
  Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 G100K/G104T (KT) & V3-
  4_IGLJ*01 CDR1 S52 and R56 NNK mutant binding data

  Fab

  Heavy Chain
  VH5-51_IGHD5-
  18*01>3_IGHJ4*01
  G100K/G104T	Light Chain	SEQ ID	ECL	ELISA
  (SEQ ID NO: 381)	V3-4_IGLJ*01	NO	Signal	Signal

  G100K/G104T	S52X	581	64794	0.10
  G100K/G104T	S52X	581	58732	0.10
  G100K/G104T	S52C	511	64255	0.10
  G100K/G104T	S52X	581	84622	0.13
  G100K/G104T	S52X	581	78239	0.14
  G100K/G104T	S52X	581	62099	0.11
  G100K/G104T	S52X	581	76278	0.14
  G100K/G104T	S52X	581	84797	0.15
  G100K/G104T	S52G	510	85929	0.21
  G100K/G104T	S52G	510	86660	0.18
  G100K/G104T	S52X	581	81950	0.13
  G100K/G104T	S52X	581	79552	0.11
  G100K/G104T	S52X	581	84470	0.14
  G100K/G104T	S52X	581	356	0.09
  G100K/G104T	S52R	512	85879	0.15
  G100K/G104T	S52X	581	84017	0.16
  G100K/G104T	S52X	581	67861	0.14
  G100K/G104T	S52X	581	100221	0.17
  G100K/G104T	S52X	581	61304	0.12
  G100K/G104T	R56X	582	69586	0.13
  G100K/G104T	R56X	582	75844	0.15
  G100K/G104T	R56X	582	93607	0.13
  G100K/G104T	R56X	582	58626	0.11
  G100K/G104T	R56X	582	82996	0.14
  G100K/G104T	R56X	582	71685	0.12
  G100K/G104T	R56X	582	73639	0.11
  G100K/G104T	R56I	513	94265	0.13
  G100K/G104T	R56Y/S	583	95103	0.28
  G100K/G104T	R56X	582	367	0.09
  G100K/G104T	R56X	582	82747	0.26
  G100K/G104T	R56X	582	80011	0.16
  G100K/G104T	R56D	515	87363	0.19
  G100K/G104T	R56G	516	93708	0.19
  G100K/G104T	R56A	507	83853	0.27
  G100K/G104T	R56X	582	91910	0.15
  G100K/G104T	R56X	582	58466	0.11
  G100K/G104T	R56X	582	45685	0.11
  G100K/G104T	R56X	582	55229	0.12

• TABLE 102
  Fab VH5-51_IGHD5-18*01 > 3_IGHJ4*01 & V3-
  4_IGLJ*01 mutant binding to DLL4 by ELISA

  H

  	KT	KT	KT	KT	KT
  	G24L	S28R	G35V	T58A	T58D	KT	KT	KT

  L

  Fab	par-	par-	par-	par-	par-
  [nM]	ent	ent	ent	ent	ent	S52G	R56Y	R56A

  100	0.298	0.529	0.271	0.253	0.219	0.209	0.231	0.251
  50	0.245	0.456	0.232	0.209	0.230	0.194	0.211	0.239
  25	0.221	0.365	0.232	0.220	0.218	0.227	0.205	0.227
  12.5	0.233	0.309	0.244	0.230	0.223	0.215	0.184	0.212
  6.25	0.278	0.303	0.245	0.249	0.224	0.207	0.182	0.200
  3.125	0.257	0.246	0.251	0.244	0.252	0.216	0.180	0.213
  H—heavy chain
  L—Light Chain

• 8. Mutagenesis of Framework 3 Amino Acid Residue T78
• The KT heavy chain double mutant (Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 G100K/G104T (H:KT) & V3-4_IGLJ*01) was used as a template for further mutagenesis of amino acid residue T78 in the framework 3 region of the light chain. This residue was mutated using overlapping PCR with NNK mutagenesis. Table 103 sets forth the ECL signal for binding to DLL4. Mutation of amino acid residue T78 either improved binding or did not affect binding to DLL4 as evidenced by an increased ECL signal or no change in ECL signal compared to the parent KT double mutant having no mutations in the light chain. Two additional light chain double mutants G23A/N175K (in the constant region) and S52A/A116T (in the framework 4 region) also were generated and they exhibited improved binding for DLL4 compared to the KT double mutant template antibody as evidenced by an increased ECL signal.

• TABLE 103
  Binding affinity of Fab VH5-51_IGHD5-18*01>3_IGHJ4*01
  G100K/G104T (H:KT) & V3-4_IGLJ*01 light chain mutants

  Heavy Chain
  VH5-51_IGHD5-
  18*03_IGHJ4*01	Light Chain	SEQ ID
  (SEQ ID NO: 381)	V3-4_IGLJ1*01	NO	ECL Signal

  G100K/G104T	wildtype		108	9355
  G100K/G104T	T78S	518	7554
  G100K/G104T	T78E	519	10559
  G100K/G104T	T78Y/M	584	12364
  G100K/G104T	T78L	522	9554
  G100K/G104T	T78K	523	9620
  G100K/G104T	T78V	524	9833
  G100K/G104T	G23A, N175K	525	17828
  G100K/G104T	S25A, A116T	526	12178

• 9. Paired Mutants of Heavy Chain KT TRV
• The SPR data (see Example 5 and Table 108) for Fabs H:KT TRV & V3-4_IGLJ1*01 and H:KT TRV & L:RM indicated that these Fabs have a short off-rate. Thus, in order to increase binding affinity of these antibodies, heavy chain H:KT TRV was paired with various V3-4_IGLJ1*01 light chain mutants and the binding affinity towards DLL4 was assayed by ELISA since the ELISA assay selects for long off-rates whereas the ECL assay detects equilibrium binding.
• Purified Fab mutants were tested for binding to DLL4 using ELISA performed as described in Example 6 at a concentration of 100 nM Fab. The results for the ELISA assay are set forth in Table 104. Fabs containing light chain mutants V91A, T31A, S52A, T53A, S57A, V91L, S96G and S96P exhibited increased binding to DLL4 as compared to a Fab with parental light chain V3-4_IGLJ1*01 as evidenced by a greater ELISA signal-blank.

• TABLE 104
  Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 G100K/G104T G24T/S28R/G35V (H:KT
  TRV) & V3-4_IGLJ1*01 light chain mutant binding data

  Fab	ELISA Signal-

  Heavy Chain	SEQ ID	Light Chain	SEQ ID	blank
  VH5-51_IGHD5-18*01_IGHJ4*01	NO	V3-4_IGLJ1*01	NO	(100 nM Fab)
  G100K/G104T G24T/S28R/G35V	430	V91A	446	0.606
  G100K/G104T G24T/S28R/G35V	430	L92A	447	0.186
  G100K/G104T G24T/S28R/G35V	430	Y93A	448	0.185
  G100K/G104T G24T/S28R/G35V	430	M94A	449	0.277
  G100K/G104T G24T/S28R/G35V	430	G95A	450	0.216
  G100K/G104T G24T/S28R/G35V	430	S96A	452	0.436
  G100K/G104T G24T/S28R/G35V	430	G97A	451	0.129
  G100K/G104T G24T/S28R/G35V	430	I98A	453	0.162
  G100K/G104T G24T/S28R/G35V	430	S99A	454	0.300
  G100K/G104T G24T/S28R/G35V	430	T78S	518	0.093
  G100K/G104T G24T/S28R/G35V	430	T78E	519	0.217
  G100K/G104T G24T/S28R/G35V	430	T78Y/M	584	0.459
  G100K/G104T G24T/S28R/G35V	430	T78L	522	0.347
  G100K/G104T G24T/S28R/G35V	430	T78K	523	0.480
  G100K/G104T G24T/S28R/G35V	430	T78V	524	0.340
  G100K/G104T G24T/S28R/G35V	430	G23A	499	0.405
  G100K/G104T G24T/S28R/G35V	430	L24A	487	0.244
  G100K/G104T G24T/S28R/G35V	430	S25A	500	0.483
  G100K/G104T G24T/S28R/G35V	430	S26A	488	0.395
  G100K/G104T G24T/S28R/G35V	430	G27A	489	0.398
  G100K/G104T G24T/S28R/G35V	430	S28A	490	0.478
  G100K/G104T G24T/S28R/G35V	430	V29A	491	0.394
  G100K/G104T G24T/S28R/G35V	430	S30A	492	0.344
  G100K/G104T G24T/S28R/G35V	430	T31A	493	0.552
  G100K/G104T G24T/S28R/G35V	430	S32A	494	0.502
  G100K/G104T G24T/S28R/G35V	430	Y33A	495	0.301
  G100K/G104T G24T/S28R/G35V	430	Y34A	496	0.085
  G100K/G104T G24T/S28R/G35V	430	P35A	497	0.236
  G100K/G104T G24T/S28R/G35V	430	S36A	498	0.380
  G100K/G104T G24T/S28R/G35V	430	S52A	503	0.574
  G100K/G104T G24T/S28R/G35V	430	T53A	504	0.532
  G100K/G104T G24T/S28R/G35V	430	N54A	505	0.318
  G100K/G104T G24T/S28R/G35V	430	T55A	506	0.382
  G100K/G104T G24T/S28R/G35V	430	R56A	507	0.442
  G100K/G104T G24T/S28R/G35V	430	S57A	508	0.598
  G100K/G104T G24T/S28R/G35V	430	S58A	509	0.451
  G100K/G104T G24T/S28R/G35V	430	V91L	458	0.734
  G100K/G104T G24T/S28R/G35V	430	V91P	455	0.078
  G100K/G104T G24T/S28R/G35V	430	V91T	456	0.197
  G100K/G104T G24T/S28R/G35V	430	V91S	457	0.264
  G100K/G104T G24T/S28R/G35V	430	V91R	459	0.025
  G100K/G104T G24T/S28R/G35V	430	V91A	446	0.529
  G100K/G104T G24T/S28R/G35V	430	Parent	108	0.393
  G100K/G104T G24T/S28R/G35V	430	V91C	460	0.625
  G100K/G104T G24T/S28R/G35V	430	V91E	461	0.152
  G100K/G104T G24T/S28R/G35V	430	V91W	462	0.080
  G100K/G104T G24T/S28R/G35V	430	V91N	463	0.203
  G100K/G104T G24T/S28R/G35V	430	V91I	464	0.336
  G100K/G104T G24T/S28R/G35V	430	V91G	465	0.248
  G100K/G104T G24T/S28R/G35V	430	V91H	466	0.127
  G100K/G104T G24T/S28R/G35V	430	M94T	476	0.395
  G100K/G104T G24T/S28R/G35V	430	M94E	467	0.171
  G100K/G104T G24T/S28R/G35V	430	M94S	468	0.195
  G100K/G104T G24T/S28R/G35V	430	M94G	469	0.199
  G100K/G104T G24T/S28R/G35V	430	M94L	470	0.388
  G100K/G104T G24T/S28R/G35V	430	M94P	471	0.256
  G100K/G104T G24T/S28R/G35V	430	M94V	472	0.315
  G100K/G104T G24T/S28R/G35V	430	M94D	473	0.070
  G100K/G104T G24T/S28R/G35V	430	M94R	474	0.197
  G100K/G104T G24T/S28R/G35V	430	M94N	475	0.205
  G100K/G104T G24T/S28R/G35V	430	M94F	477	0.317
  G100K/G104T G24T/S28R/G35V	430	M94A	449	0.216
  G100K/G104T G24T/S28R/G35V	430	S96W	478	0.261
  G100K/G104T G24T/S28R/G35V	430	S96G	479	0.562
  G100K/G104T G24T/S28R/G35V	430	S96P	480	0.813
  G100K/G104T G24T/S28R/G35V	430	S96A/E	579	0.538
  G100K/G104T G24T/S28R/G35V	430	S96R	481	0.499
  G100K/G104T G24T/S28R/G35V	430	S96L	482	0.355
  G100K/G104T G24T/S28R/G35V	430	S96M	483	0.358
  G100K/G104T G24T/S28R/G35V	430	S96E	484	0.439
  G100K/G104T G24T/S28R/G35V	430	Parent	108	0.437
  G100K/G104T G24T/S28R/G35V	430	S96V	485	0.452
  G100K/G104T G24T/S28R/G35V	430	Parent	108	0.455
  G100K/G104T G24T/S28R/G35V	430	Parent	108	0.430

• 10. Cassette Mutagenesis Using Type II Restriction Enzyme Ligatioin of Amino Acid Residues S52, T53 and S57
• Following analysis of paired Fab mutants of heavy chain H:KT TRV, light chain double mutant V3-4_IGLJ1*01 V91L/S96P (L:LP) was generated. Three additional light chain amino acid residues (S52, T53 and S57) that exhibited increased binding to DLL4 by ELISA (see Table 103 above) were selected for further mutagenesis using type II restriction enzyme ligation using Fab H: KT TRV & L:LP as a template. The ELISA signals are set forth in Table 105 below. Light chain mutants L:LP S52G, L:LP S52 M, L:LP S52N and L:LP S52H exhibited increased binding to DLL4 as assessed by ELISA.
• Four Fabs, containing various combinations of mutations of the heavy chain and light chain, were further analyzed for binding to DLL4 by ELISA using 3-fold serial dilutions of Fab, starting at a concentration of 100 nM. The results are set forth in Table 106 below. Fab H:KT TRV & L:LP S52G exhibited the greatest binding to DLL4 as evidenced by the ELISA signal compared to other Fab mutants tested.

• TABLE 105
  Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 G100K/G104T G24T/S28R/G35V (H:KT
  TRV) & V3-4_IGLJ1*01 V91L/S96P (L:LP) light chain mutant binding data

  Fab	ELISA Signal-

  Heavy Chain	SEQ ID	Light Chain	SEQ ID	blank
  VH5-51_IGHD5-18*01_IGHJ4*01	NO	V3-4_IGLJ1*01	NO	(100 nM Fab)
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S52F	527	0.33
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S52L	528	0.40
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S52I	529	0.42
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S52M	530	0.46
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S52V	531	0.44
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S52P	532	0.32
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S52T	533	0.34
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S52Y	534	0.41
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S52H	535	0.44
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S52Q	536	0.39
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S52N	537	0.45
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S52K	538	0.32
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S52D	539	0.39
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S52E	540	0.38
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S52W	541	0.29
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S52G	543	0.53
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P	544	0.39
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P T53F	545	0.15
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P T53L	546	0.18
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P T53I	547	0.30
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P T53M	548	0.01
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P T53V	549	0.29
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P T53S	550	0.18
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P T53P	551	0.39
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P T53Y	552	0.22
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P T53H	553	0.14
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P T53Q	554	0.11
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P T53N	555	0.15
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P T53K	556	0.12
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P T53D	557	0.16
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P T53E	558	0.09
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P T53W	559	0.06
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P T53R	560	0.05
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P T53G	561	0.08
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P	544	0.30
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S57F	562	0.10
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S57L	563	0.30
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S57I	564	0.24
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S57M	565	0.30
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S57V	566	0.34
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S57P	567	0.36
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S57T	568	0.30
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S57Y	569	0.28
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S57H	570	0.21
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S57Q	571	0.21
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S57N	572	0.24
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S57K	573	0.17
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S57D	574	0.17
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S57E	575	0.20
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S57W	576	0.12
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S57R	577	0.18
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P S57G	578	0.23
  G100K/G104T G24T/S28R/G35V	430	V91L/S96P	544	0.29

• TABLE 106
  Binding affinity of Fab VH5-51_IGHD5-18*01>3_IGHJ4*01
  G100K/G104T G24T/S28R/G35V (H:KT TRV) & V3-4_IGLJ1*01
  light chain mutants

  			V91L/S96P	V91L/S96P
  	Wildtype	V91L/S96P	S52M	S52G
  	(SEQ ID	(SEQ ID	(SEQ ID	(SEQ ID
  Light Chain	NO: 108)	NO: 544)	NO: 530)	NO: 543)
  Fab [μM]	Signal	Signal	Signal	Signal

  100	0.16	0.34	0.24	0.69
  33.33	0.08	0.19	0.12	0.35
  11.11	0.04	0.07	0.06	0.17
  3.70	0.03	0.03	0.03	0.06
  1.23	0.01	0.03	0.03	0.03
  0.41	0.01	0.02	0.03	0.01
  0.14	0.00	0.03	0.02	0.02
  0.05	0.01	0.02	0.02	0.02

• 11. Paired Fab Mutants
• Twenty four mutant Fabs, containing various combinations of mutations of the heavy chain and light chain, were further analyzed for binding to DLL4 by ELISA using 2-fold serial dilutions of Fab, starting at a concentration of 100 nM. The results are set forth in Table 107 below. Fabs H:KT TRV & L:LP S52K and H:KT TRV & L:LP S52G exhibited the greatest binding affinity to DLL4 as evidenced by the ELISA signal compared to other Fab mutants tested. Fabs H:KT TRV & L:LP S52H and H:KT TRV & L:LP S52N had slightly reduced binding affinity to DLL4 as compared to Fabs H:KT TRV & L:LP S52K and H:KT TRV & L:LP S52G.

• TABLE 107
  Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ*01 mutant binding to DLL4
  by ELISA

  Heavy Chain	Light Chain
  VH5-51_IGHD5-18*01>3_IGHJ4*01	V3-4_IGLJ*01	100	50	25	12.5
  G100K/G104T G24L/S28R/G35V Y105H	Wildtype	0.23	0.20	0.19	0.21
  (SEQ ID NO: 432)
  G100K/G104T G24T/S28R/G35V Y105N	Wildtype	0.25	0.18	0.19	0.21
  (SEQ ID NO: 433)
  G100K/G104T G24A/S28R/G35V Y107F	Wildtype	0.28	0.24	0.20	0.21
  (SEQ ID NO: 434)
  G100K/G104T G24L/S28R/G35V D109Q	Wildtype	0.30	0.25	0.22	0.24
  (SEQ ID NO: 435)
  G100K/G104T G24T/S28R/G35V	V91L/S96P	1.00	0.81	0.58	0.45
  G100K	Wildtype	0.20	0.19	0.18	0.19
  Wildtype	Wildtype	0.17	0.16	0.18	0.17
  G104T	Wildtype	0.17	0.17	0.18	0.19
  G100K/G104T	Wildtype	0.18	0.18	0.16	0.18
  G100K/G104T G24T/S28R/G35V	Wildtype	0.45	0.32	0.26	0.23
  G100K/G104T S28R	Wildtype	0.26	0.23	0.20	0.18
  G100K/G104T G24A/S28R/G35V	V91L/S96P S52V	0.95	0.74	0.60	0.43
  G100K/G104T G24L/S28R/G35V	V91L/S96P S52F	0.99	0.69	0.49	0.42
  G100K/G104T G24T/S28R/G35V	V91L/S96P S52L	1.02	0.78	0.58	0.43
  G100K/G104T G24A/S28R/G35V	V91L/S96P S52I	1.04	0.82	0.60	0.40
  G100K/G104T G24L/S28R/G35V	V91L/S96P S52M	1.01	0.80	0.59	0.41
  G100K/G104T G24T/S28R/G35V	V91L/S96P S52G	1.14	1.02	0.90	0.63
  G100K/G104T G24A/S28R/G35V	V91L/S96P S52P	1.00	0.79	0.59	0.43
  G100K/G104T G24L/S28R/G35V	V91L/S96P S52T	0.99	0.79	0.62	0.41
  G100K/G104T G24T/S28R/G35V	V91L/S96P S52Y	0.90	0.72	0.56	0.41
  G100K/G104T G24A/S28R/G35V	V91L/S96P S52H	1.09	0.91	0.73	0.50
  G100K/G104T G24L/S28R/G35V	V91L/S96P S52Q	0.96	0.81	0.67	0.47
  G100K/G104T G24T/S28R/G35V	V91L/S96P S52N	1.05	0.90	0.86	0.65
  G100K/G104T G24T/S28R/G35V	V91L/S96P S52K	1.23	1.03	0.79	0.56

Summary
• As a result of affinity maturation, the affinity of parental Hit Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01 for binding to DLL4 was increased 130-fold (see SPR data in Table 108 below). Parental Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01 binds DLL4 with a K_D of 4.8 μM. Heavy chain mutant Fab H:KT & L:wt has 13-fold increased affinity for DLL4 (K_D=355 nM). Affinity matured heavy and light chain mutant Fab H:KT TRV & L:wt has a K_D of 36.2 nM, a 130-fold increase in binding affinity for DLL4. Affinity matured heavy and light chain mutant Fabs H:KT TRV & L:LP and H:KT TRV & L:LP S52G have a K_D of 3.3 and 5.0 nM, respectively, a 1000-fold increase in binding affinity for DLL4.

• TABLE 108
  Binding affinity of VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01 DLL4
  mutant Fabs by Surface Plasmon Resonance

  		ka (×105)	kd (×10−3)	KD
  Heavy Chain	Light Chain	(M−1s−1)	(s−1)	(nM)
  VH5-51_IGHD5-18*01>3_IGHJ4*01	V3-4_IGLJ1*01	n/a	n/a	4800
  (parental)	(parental)			(±200)
  VH5-51_IGHD5-18*01>3_IGHJ4*01	V3-4_IGLJ1*01	0.645	0.023	355
  G100K/G104T (KT)		(±0.092)	(±0.004)	(±7)
  VH5-51_IGHD5-18*01>3_IGHJ4*01	V3-4_IGLJ1*01	7.4	0.0845	114
  G100K/G104T S28R (KT S28R)		(±0.6)	(±0.0050)	(±6)
  VH5-51_IGHD5-18*01>3_IGHJ4*01	V3-4_IGLJ1*01	20.90	0.0717	36.2
  G100K/G104T G24T/S28R/G35V		(±6.24)	(±0.00351)	(±8.5)
  (KT TRV)
  VH5-51_IGHD5-18*01>3_IGHJ4*01	V3-4_IGLJ1*01	25.30	0.101	40.3
  G100K/G104T G24T/S28R/G35V	M94R/S96M (RM)	(±4.16)	(±0.0153)	(±9.3)
  (KT TRV)
  VH5-51_IGHD5-18*01>3_IGHJ4*01	V3-4_IGLJ1*01	110	36	3.3
  G100K/G104T G24T/S28R/G35V	V91L/S96P
  (KT TRV)	(LP)
  VH5-51_IGHD5-18*01>3_IGHJ4*01	V3-4_IGLJ1*01	29.6	14.7	5.0
  G100K/G104T G24T/S28R/G35V	V91L/S96P S52G
  (KT TRV)	(LP S52G)

Example 13 Germline Segment Swapping
• In this example, two antibody “Hit” Fabs against DLL4, identified in Example 4 using the Multispot ECL binding assay, were subjected to mutagenesis by J-swapping or D-swapping of the J_H or D_H germline segments, respectively. J-swapping involves substitution of the parent “Hit” Fab J_H germline segment with a different J_H germline segment. D-swapping involves substitution of the parent “Hit” D_H germline segment with a different D_H germline segment. Since the D_H germline segment constitutes the 5′ end of the heavy chain CDR3 and J_H segment constitutes the 3′ end of the heavy chain CDR3, D-swapping and J-swapping allow for facile mutagenesis of this important antibody binding region.
A. Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01
• For Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01, J-swapping of IGHJ1*01 with IGHJ2*01, IGHJ4*01, and IGHJ5*01 allowed analysis of the 3′ end of CDR3 from amino acid residues A106 to H111 (see FIG. 4A). Purified Fab J-swapped mutants were tested for binding to DLL4 using the ECL assay as described in Example 4. The results are set forth in Tables 109-110 below. The results show that swapping of IGHJ1*01 with either IGHJ2*01, IGHJ4*01, or IGHJ5*01 reduced binding of the antibody to DLL4 as assessed by a decreased ECL signal compared to the parent template antibody containing the IGHJ1*01 J_H germline segment.

• TABLE 109
  Fab VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 J-swap binding
  data

  				Sig-
  				nal/
  	SEQ		SEQ	Noise
  	ID		ID	(0.04
  Heavy Chain	NO	Light Chain	NO	μM)

  VH1-46_IGHD6-6*01_IGHJ2*01	585	L6_IGKJ1*01	107	0.8
  wildtype	88	L6_IGKJ1*01	107	1.7
  VH1-46_IGHD6-6*01_IGHJ4*01	586	L6_IGKJ1*01	107	0.8
  VH1-46_IGHD6-6*01_IGHJ5*01	587	L6_IGKJ1*01	107	0.8

• TABLE 110
  Fab VH1-46 _IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 J-swap mutant
  binding data

  	0.02 μM Fab	0.004 μM Fab
  	30 μg/mL DLL4	15 μg/mL DLL4

  Fab

  ECL

  Signal/

  ECL

  Signal/

  Heavy Chain	Light Chain	Signal	Noise	Signal	Noise

  IGHJ2*01	L6_IGKJ1*01	232	0.6	185	1.3
  wildtype	L6_IGKJ1*01	8714	23.0	4261	29.2
  IGHJ4*01	L6_IGKJ1*01	203	0.5	178	1.2
  IGHJ5*01	L6_IGKJ1*01	244	0.6	137	0.9

B. Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ*01
• For Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ*01, J-swapping of IGHJ4*01 with IGHJ1*01, IGHJ3*01, and IGHJ5*01 allowed analysis of the 3′ end of CDR3 from amino acid residues 106-110 (see FIG. 4B). D-swapping of IGHD5-18*01 with IGHD5-12*01 and IGHD5-24*01 allowed analysis of the 5′ end of CDR3 from amino acid residues 100-104 (see FIG. 4C). Purified J-swapped and D-swapped mutants were tested for binding to DLL4 using the ECL assay as described in Example 4. The ECL results for binding to DLL4 are set forth in Tables 111-112 below. The results show that swapping of IGHJ4*01 with either IGHJ1*01, IGHJ3*01, or IGHJ5*01 reduced binding of the antibody to DLL4 as assessed by a decreased ECL signal compared to the parent template antibody containing the IGHJ4*01 J_H germline segment. Additionally, swapping of IGHD5-18*01 with IGHD5-12*01 or IGHD5-24*01 reduced binding of the antibody to DLL4 as assessed by a decreased ECL signal compared to the parent template antibody containing the IGHD5-18*01 D_H germline segment.

• TABLE 111
  Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ*01
  D-swap and J-swap mutant binding data

  Heavy Chain	SEQ		SEQ	Signal/
  VH5-51_IGHD5-	ID		ID	Noise
  18*01>3_IGHJ4*01	NO	Light Chain	NO	(0.04 μM)

  IGHJ1*01	588	V3-4_IGLJ1*01	108	1.2
  wildtype	89	V3-4_IGLJ1*01	108	14.7
  IGHJ3*01	589	V3-4_IGLJ1*01	108	3.1
  IGHJ5*01	590	V3-4_IGLJ1*01	108	1.2
  IGHD5-12*01	591	V3-4_IGLJ1*01	108	1.2
  IGHD5-24*01	592	V3-4_IGLJ1*01	108	1.3
  wildtype	89	V3-4_IGLJ1*01	108	15.5

• TABLE 112
  Fab VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ*01
  D-swap and J-swap mutant binding data

  	0.1 μM	0.02 μM
  	Fab	Fab
  Fab	30 μg/mL	15 μg/mL

  Heavy Chain	SEQ	Light Chain	DLL4	DLL4
  VH5-51_IGHD5-	ID	(SEQ ID	Signal/	Signal/
  18*01_IGHJ4*01	NO	NO: 108)	Noise	Noise

  IGHJ1*01	588	V3-4_IGLJ1*01	1.0	1.1
  wildtype	89	V3-4_IGLJ1*01	24.0	15.2
  IGHJ3*01	589	V3-4_IGLJ1*01	7.9	3.5
  IGHJ5*01	590	V3-4_IGLJ1*01	1.0	0.9
  IGHD5-12*01	591	V3-4_IGLJ1*01	1.1	1.2
  IGHD5-24*01	592	V3-4_IGLJ1*01	1.7	1.0

Example 14 Affinity Maturation of Fab VH3-23_IGHD2-21*01>3_IGHJ6*01 & V2-13_IGLJ2*01 Against Hepatocyte Growth Factor Receptor
• Fab VH3-23_IGHD2-21*01>3_IGHJ6*01 & V2-13_IGLJ2*01 (SEQ ID NOS:2803 and 594) against hepatocyte growth factor receptor (HGFR; C-Met) identified using the electroluminescence Meso Scale Discovery (MSD) multispot binding assay, was subjected to affinity maturation as described above in Examples 7-9. Mutations of amino acid residues were carried out by ligation of oligo pairs using method described in Example 1C.
• i. Identification of the CDR Potential Binding Site
• The amino acid sequence of the heavy chain (SEQ ID NO:2803) for the parent “Hit” VH3-23_IGHD2-21*01>3_IGHJ6*01 & V2-13_IGLJ2*01 was aligned with the amino acid sequences of three heavy chains (SEQ ID NOS:2797, 2799 and 2801) of three related “Hits” that also bind HGFR, albeit with slightly reduced affinity. These four Fabs share the same V_H and J_H germline segments. The sequence alignment is set forth in FIG. 5. Based on the alignment, amino acid residues were identified that differed between the “Hit” and the related “Hits”, thus accounting for differences in binding of the “Hit” and related “Hits” for HGFR. The identified amino acid residues were located in CDR3, which was identified as the region of the heavy chain that is important for binding affinity.
• ii. Alanine Scanning of Heavy Chain CDR3
• CDR3 of the heavy chain sequence of parent Fab VH3-23_IGHD2-21*01>3_IGHJ6*01 & V2-13_IGLJ2*01 (SEQ ID NOS:2803 and 594) was subjected to alanine scanning mutagenesis and analyzed using the ECL multispot assay using 100 nM Fab. The results are set forth in Table 113 below. Mutation of amino acid residues E99, V102, V103, V104, and I105 with alanine and A106 with threonine caused a significant reduction in binding to HGFR as assessed by a decreased ECL signal. Mutation of H100, I101, I107, and S108 with alanine slightly reduced binding to HGFR as assessed by a decreased ECL signal.

• TABLE 113
  Binding of Fab VH3-23_IGHD2-21*01 > 3_IGHJ6*01
  & V2-13_IGLJ2*01 CDR3 alanine mutants to HGFR

  	SEQ
  	ID NO	ErbB2	EGF R	HGF R	Notch-1	CD44	IGF-1	P-Cad	EPO R	DLL4

  Wt	2803	2.4	2.8	18.5	1.7	1.6	0.9	12.7	16.5	1.3
  E99A	595	2.5	2.3	5.8	1.9	1.4	1.1	9.7	11.6	1.4
  H100A	596	1.3	1.8	14.1	1.0	1.0	1.0	4.8	7.2	2.2
  I101A	597	2.8	3.0	14.8	1.7	1.2	1.1	23.2	26.6	1.5
  V102A	598	1.4	1.4	5.3	1.0	1.0	1.0	4.9	8.3	1.4
  V103A	599	0.9	1.1	2.2	0.8	0.7	0.9	3.9	6.2	1.0
  V104A	600	1.3	1.4	2.3	1.3	1.1	1.1	2.6	5.3	1.4
  I105A	601	1.0	1.1	1.1	1.2	0.9	1.1	1.2	5.5	1.1
  A106T	602	1.3	1.4	6.9	1.5	1.3	1.4	2.3	3.2	1.9
  I107A	603	4.8	4.3	13.7	2.7	1.5	1.1	19.6	43.6	3.6
  S108A	604	1.9	2.0	12.9	1.5	1.3	1.2	4.8	9.5	2.3

• iii. NNK Mutagenesis of Y113
• Amino acid residue Y113 of the heavy chain sequence of Fab VH3-23_IGHD2-21*01>3_IGHJ6*01 H100E/S108P(H:EP) & V2-13_IGLJ2*01 (SEQ ID NOS:593 and 594) was subjected to NNK mutagenesis and analyzed using the ECL multispot assay using 20 nM Fab. The results are set forth in Table 114 below. EP mutants Y113G, Y113I, Y113S, Y113T, Y113N, Y113N and Y113W had increased binding to HGFR as compared to heavy chain EP as evidenced by an increase in ECL signal.

• TABLE 114
  Binding of Fab VH3-23_IGHD2-21*01 > 3_IGHJ6*01 H100E/S108P
  (EP) & V2-13_IGLJ2*01 mutants to HGFR

  	SEQ
  	ID NO	ErbB2	EGF R	HGF R	Notch-1	CD44	IGF-1	P-Cad	EPO R	DLL4

  Parent	593	4.1	4.2	33.4	2.1	1.8	1.6	20.6	39.4	2.3
  Y113G	605	11.7	8.4	104.6	2.3	1.7	1.7	27.5	126.1	2.3
  Y113I	606	40.7	17.8	178.9	5.5	3.7	3.3	58.6	116.5	5.0
  Y113S	607	19.1	9.2	133.1	3.3	2.3	1.8	41.0	142.2	3.0
  Y113P	608	1.6	1.4	13.0	1.4	1.1	1.4	2.3	2.1	1.6
  Y113T	609	35.4	18.9	185.0	6.1	4.1	3.1	65.9	174.4	5.5
  Y113H	610	6.3	3.6	107.1	1.7	1.4	1.5	16.0	55.9	2.0
  Y113N	611	28.4	11.0	122.6	4.3	2.4	1.6	38.5	114.2	3.1
  Y113E	612	50.6	20.0	48.6	7.3	3.9	3.4	41.8	142.0	5.3
  Y113W	613	21.8	11.7	130.8	4.2	3.9	1.9	44.3	169.8	3.3
  Y113R	614	48.4	19.3	76.4	9.4	6.5	3.4	56.3	183.2	4.6

• iv. NNK Mutagenesis of Y109, Y110, Y111, Y112 and Y114
• Amino acid residues Y109, Y110, Y111, Y112 and Y114 of the heavy chain sequence of Fab VH3-23_IGHD2-21*01>3_IGHJ6*01 H100E/S108P/Y113G (EPG) & V2-13_IGLJ2*01 (SEQ ID NOS:605 and 594) were subjected to NNK mutagenesis and analyzed using the ECL multispot assay using 20 nM Fab. The results are set forth in Table 115 below. Mutation of EPG heavy chain residue Y110 to isoleucine resulted in increased binding to HGFR as evidenced by an increased ECL signal as compared to heavy chain EPG. EPG mutants Y109W, Y112, Y112T and Y112W had slightly increased binding to HGFR as compared to heavy chain EPG as evidenced by a slight increase in ECL signal.

• TABLE 115
  Binding of Fab VH3-23_IGHD2-21*01 > 3_IGHJ6*01 H100E/S108P/Y113G
  (EPG) & V2-13_IGLJ2*01 Y109, Y110, Y111, Y112, and Y114 mutants to HGFR

  	SEQ
  	ID NO	ErbB2	EGF R	HGF R	Notch-1	CD44	IGF-1	P-Cad	EPO R	DLL4

  Parent	2803	4.1	4.2	33.4	2.1	1.8	1.6	20.6	39.4	2.3
  EPG	605	11.7	8.4	104.6	2.3	1.7	1.7	27.5	126.1	2.3
  Y109L	615	2.1	2.2	52.9	1.9	1.6	1.7	4.0	22.1	2.0
  Y109P	616	1.5	1.6	1.8	1.1	1.1	1.4	1.9	1.8	1.6
  Y109T	617	1.7	1.4	16.0	1.7	1.1	1.3	2.1	6.0	1.5
  Y109H	618	1.7	1.4	27.7	1.2	1.1	1.2	3.9	18.3	1.4
  Y109Q	619	1.3	1.7	14.7	1.3	1.3	1.1	2.2	3.1	1.2
  Y109D	620	1.3	1.4	2.9	1.3	1.0	1.4	1.8	2.0	1.4
  Y109W	621	16.1	11.1	125.3	4.4	2.4	1.5	32.0	168.8	4.3
  Y109R	622	2.0	1.8	39.6	1.4	1.0	1.3	7.4	30.1	1.5
  Y109G	623	1.4	2.0	8.7	1.5	1.5	1.6	2.7	11.1	1.9
  Y110I	624	11.4	8.6	163.2	2.5	1.7	1.4	39.0	73.7	2.0
  Y110S	625	1.0	1.3	13.1	0.8	1.0	1.1	1.9	4.5	1.2
  Y110P	626	0.9	1.1	4.8	1.1	1.2	1.2	1.7	2.9	1.4
  Y110T	627	0.8	1.8	21.1	2.3	1.6	1.8	1.0	3.5	1.8
  Y110H	628	2.2	1.7	8.8	1.4	1.4	1.3	2.9	3.7	1.9
  Y110N	629	1.2	0.9	2.3	1.3	0.8	0.9	1.2	1.6	1.2
  Y110E	630	1.7	1.6	1.8	1.5	1.3	1.4	2.0	2.2	1.8
  Y110W	631	16.5	7.6	110.2	3.2	2.1	2.3	38.8	116.8	3.9
  Y110R	632	2.1	1.6	3.9	1.6	1.3	1.4	3.3	4.8	1.8
  Y110G	633	1.3	1.5	1.0	1.6	1.0	1.4	0.8	2.0	1.3
  Y111I	634	1.7	1.9	10.2	1.8	1.3	1.0	1.9	6.9	1.5
  Y111S	635	2.1	1.8	23.9	1.9	1.2	1.3	5.0	30.1	1.7
  Y111P	636	1.6	1.5	1.7	1.6	1.3	1.2	1.3	1.9	1.4
  Y111T	637	2.6	2.6	42.0	2.0	1.8	1.2	6.2	38.8	2.2
  Y111H	638	3.0	2.9	37.5	1.5	1.3	1.2	7.7	49.8	1.6
  Y111N	639	1.5	1.4	17.0	1.3	0.9	0.8	2.9	9.3	1.1
  Y111E	640	1.5	1.4	2.2	1.5	1.1	1.4	2.3	2.9	1.5
  Y111W	641	26.5	16.3	121.4	5.3	3.4	1.4	49.2	195.3	2.8
  Y111R	642	3.3	2.6	24.3	2.3	1.4	1.3	15.7	22.6	1.4
  Y111G	643	2.2	1.5	18.8	1.9	1.3	1.1	5.0	10.0	1.7
  Y112I	644	25.0	21.5	126.2	10.4	6.5	2.1	43.1	81.7	3.7
  Y112S	645	3.5	2.3	67.9	2.3	1.5	1.3	7.1	31.0	1.7
  Y112P	646	2.3	1.8	41.8	1.4	1.1	1.1	5.0	32.2	1.5
  Y112T	647	8.8	8.4	137.6	2.1	1.8	1.2	25.5	90.9	1.7
  Y112H	648	3.4	2.7	86.6	1.8	1.4	1.7	9.7	40.6	1.8
  Y112N	649	1.2	1.3	29.5	0.8	0.9	1.1	1.9	4.2	1.3
  Y112E	650	1.4	1.5	7.3	1.2	1.1	1.2	2.0	4.7	1.3
  Y112W	651	25.5	18.7	127.0	9.2	5.8	2.1	50.5	156.8	3.2
  Y112R	652	5.9	3.7	120.5	2.7	1.6	1.5	30.0	85.1	2.6
  Y112G	653	1.4	1.7	10.0	2.1	1.2	1.0	2.3	7.9	1.3
  Y114I	654	11.4	7.1	82.2	2.6	1.8	1.4	22.6	161.8	2.4
  Y114S	655	8.7	5.0	48.9	2.9	1.4	1.3	15.8	68.5	2.2
  Y114P	656	1.4	1.2	2.7	1.4	1.1	0.9	1.3	2.3	1.1
  Y114T	657	1.4	1.3	1.8	1.8	1.1	1.1	1.7	2.0	1.6
  Y114H	658	12.5	8.7	67.5	3.3	1.8	1.4	27.0	119.7	2.3
  Y114N	659	3.5	2.6	23.1	2.0	1.2	1.2	5.9	35.0	1.6
  Y114E	660	7.4	6.8	18.2	3.3	1.5	1.5	13.9	69.2	2.2
  Y114W	661	9.3	6.6	56.7	2.2	1.6	1.1	16.7	51.5	1.9
  Y114R	662	6.4	4.3	70.4	2.0	1.4	1.1	15.6	61.8	1.9
  Y114G	663	3.2	2.1	14.7	1.6	1.2	1.2	6.4	15.8	1.7

• v. Alanine Scanning of Heavy Chain CDR1
• CDR1 of the heavy chain sequence of Fab VH3-23_IGHD2-21*01>3_IGHJ6*01 H100E/S108P/Y113G (H:EPG) & V2-13_IGLJ2*01 (SEQ ID NOS:605 and 594) was subjected to alanine scanning mutagenesis and analyzed using the ECL multispot assay using 20 nM Fab. The results are set forth in Table 116 below. Mutation of amino acid residues F27 and A33 with alanine resulted in reduced binding to HGFR as evidenced by a reduced ECL signal. Mutation of amino acid residues G26, T28, F29, S30, S31, Y32, M34, and S35 with alanine either improved binding or did not affect binding to HGFR as evidenced by an increased ECL signal or no change in ECL signal compared to the EPG triple mutant having no mutations in the light chain.

• TABLE 116
  Binding of Fab VH3-23_IGHD2-21*01 > 3_IGHJ6*01 H100E/S108P/Y113G
  (H: EPG) & V2-13_IGLJ2*01 CDR1 alanine mutants to HGFR

  	SEQ
  	ID NO	ErbB2	EGF R	HGF R	Notch-1	CD44	IGF-1	P-Cad	EPO R	DLL4

  G26A	664	12.1	7.5	110.9	2.3	2.0	1.6	29.4	161.3	2.7
  F27A	665	6.1	3.6	87.3	1.4	1.3	1.1	14.8	64.2	1.7
  T28A	666	13.3	8.7	140.3	2.2	1.8	1.3	32.6	180.5	2.2
  F29A	667	11.6	8.1	120.5	2.4	1.5	1.4	32.7	157.6	2.5
  S30A	668	11.4	8.9	118.3	2.4	1.7	1.3	26.8	153.7	2.0
  S31A	669	12.4	9.1	121.2	2.1	1.7	1.3	32.4	143.5	4.3
  Y32A	670	5.8	4.1	104.7	1.9	1.4	1.6	14.7	65.8	2.3
  A33T	671	6.3	5.3	35.7	1.7	1.2	1.1	25.9	114.3	1.8
  M34A	672	12.0	9.8	129.2	2.5	1.9	1.3	32.2	197.6	2.6
  S35A	673	12.0	8.3	108.5	2.6	1.8	1.3	32.4	184.4	2.4
  Parent	2803	4.1	4.2	33.4	2.1	1.8	1.6	20.6	39.4	2.3
  EPG	605	11.7	8.4	104.6	2.3	1.7	1.7	27.5	126.1	2.3

• vi. Alanine Scanning of Heavy Chain CDR2
• CDR2 of the heavy chain sequence of Fab VH3-23_IGHD2-21*01>3_IGHJ6*01 H100E/S108P/Y113G (H:EPG) & V2-13_IGLJ2*01 (SEQ ID NOS:605 and 594) was subjected to alanine scanning mutagenesis and analyzed using the ECL multispot assay using 20 nM Fab. The results are set forth in Table 117 below. Mutation of amino acid residues I51, G56, Y59, and A61 with alanine resulted in reduced binding to HGFR as evidenced by a reduced ECL signal. Double mutant S46A/G47A had reduced binding to HGFR as evidenced by a reduced ECL signal. Mutation of amino acid residues G53, S54 G55, S57, T58, Y60, D62, V64 and K65 with alanine either improved binding or did not affect binding to HGFR as evidenced by an increased ECL signal or no change in ECL signal compared to the H:EPG triple mutant having no mutations in the light chain.

• TABLE 117
  Binding of Fab VH3-23_IGHD2-21*01 > 3_IGHJ6*01 H100E/S108P (H:
  EP) or H100E/S108P/Y113G (H: EPG) & V2-13_IGLJ2*01 CDR2 alanine mutants to HGFR

  	SEQ
  	ID NO	ErbB2	EGF R	HGF R	Notch-1	CD44	IGF-1	P-Cad	EPO R	DLL4

  Parent	2803	4.1	4.2	33.4	2.1	1.8	1.6	20.6	39.4	2.3
  EPG	605	11.7	8.4	104.6	2.3	1.7	1.7	27.5	126.1	2.3
  I51A	674	9.4	5.3	77.4	2.8	1.6	1.3	20.3	112.6	2.2
  S52A/G53A	675	8.3	5.2	85.0	2.2	1.5	1.4	16.7	75.6	2.3
  G53A	676	16.7	10.9	159.2	3.9	2.5	1.8	36.5	222.6	3.1
  S54A	677	15.1	8.9	115.2	2.8	1.9	1.3	33.4	160.7	2.4
  G55A	678	11.1	7.7	111.3	2.5	1.7	1.3	26.9	143.0	2.1
  G56A	679	9.5	6.8	79.4	2.7	1.6	1.4	23.6	100.0	2.3
  S57A	680	12.9	8.7	124.0	3.4	1.8	1.7	33.0	150.8	2.5
  T58A	681	15.9	9.6	167.0	3.1	1.5	1.2	36.9	158.1	2.3
  Y59A	682	1.6	1.4	3.3	2.1	1.4	1.3	2.4	2.5	2.8
  Y60A	683	11.5	6.2	112.7	2.6	1.5	1.2	25.3	109.8	2.3
  A61T	684	11.2	7.1	81.4	2.9	2.0	1.6	20.9	146.7	2.6
  D62A	685	21.7	11.6	154.4	3.5	2.0	1.4	45.8	244.1	2.4
  EP V64A	686	16.5	9.1	100.9	3.0	2.2	1.2	30.6	172.9	2.7
  EP K65A	687	12.1	7.1	95.8	3.0	1.7	1.4	21.6	120.4	2.5

Example 15 Affinity Maturation of Fab VH3-23_IGHD3-10*01>3_IGHJ6*01 & O12_IGKJ1*01 Against P-Cadherin and Epo
• Fab VH3-23_IGHD3-10*01>3_IGHJ6*01 & V2-13_IGLJ2*01 (SEQ ID NOS:688 and 594) against P-cadherin and EPO, identified as described in Example 4 using the electroluminescence Meso Scale Discovery (MSD) multispot binding assay, was subjected to affinity maturation as described above in Examples 7-9.
• vii. NNK Mutagenesis of CDR3 Amino Acid Residues R104, Y110, Y112, Y113, and Y114
• CDR3 amino acid residues R104, Y110, Y112, Y113, and Y114 were mutagenized using NNK mutagenesis and tested for their ability to bind P-cadherin and EPO by ECL multispot assay. The results are set forth in Table 118 below. Mutant-3Y is a deletion mutant in which tyrosines 110, 111 and 112 were deleted. Mutation of amino acid residue Y115 to proline (Y115P) and Y110 to valine (Y110V) resulted an increased binding to both P-cadherin and EPO as compared to the wildtype template antibody as evidenced by an increase in ECL binding signal. Mutation of amino acid residue Y111 to arginine (Y111R) resulted in an increase in binding to P-cadherin as compared to wildtype as evidenced by an increase in ECL binding signal. Additionally, as set forth in Table 116 below, mutants Y115P, Y110V and Y111R all bind P-cadherin as evidenced by ELISA binding results.

• TABLE 118
  Binding of Fab VH3-23_IGHD3-10*01 > 3_IGHJ6*01 & O12_IGKJ1*01 NNK mutants

  	SEQ
  	ID NO	ErbB2	EGF R	HGF R	Notch-1	CD44	IGF-1	P-Cad	EPO R	DLL4

  Y114N	689	1.1	1.0	1.2	1.2	1.2	1.0	1.7	1.3	1.2
  Y114T	690	0.8	0.8	1.2	0.7	0.9	0.7	1.3	1.2	0.9
  Y114I	691	1.2	1.4	1.5	1.1	1.2	0.7	1.1	1.4	1.2
  Y115P	692	3.4	2.7	4.3	1.7	1.3	1.7	27.2	37.0	4.2
  Y115R	693	1.9	1.7	2.0	1.7	1.5	1.4	4.7	3.9	1.9
  Y115G	694	1.9	2.0	2.1	1.7	1.5	1.9	9.7	17.0	2.7
  Y115E	695	1.3	1.1	1.3	0.8	1.4	1.0	1.3	1.4	1.1
  R104A	696	1.8	1.4	2.3	2.0	1.3	1.1	9.3	8.5	2.7
  -3Y	697	1.2	1.4	1.1	0.7	0.9	1.0	1.1	1.7	1.3
  Y110V	698	1.5	1.2	2.0	1.3	1.1	1.1	17.2	10.8	2.0
  Y110S	699	1.6	1.2	1.4	1.4	1.4	1.1	1.5	1.4	1.4
  Y110P	700	1.3	1.6	1.5	1.6	1.4	1.2	1.1	1.7	1.5
  Y110G	701	1.3	1.2	0.9	1.4	0.9	1.2	1.0	1.4	1.3
  Y110R	702	2.5	2.1	3.0	2.8	1.4	2.5	11.3	9.2	3.0
  Y111S	703	1.2	1.3	1.3	1.3	1.0	0.9	1.4	1.5	1.2
  Y111D	704	1.2	0.9	1.1	2.0	1.4	1.4	1.1	1.1	1.1
  Y111R	705	2.5	2.4	3.2	3.0	1.5	1.9	11.9	7.3	2.9
  Y112A	706	1.3	1.5	0.8	1.1	1.5	1.4	1.1	1.6	1.2
  Y112G	707	2.9	2.1	2.3	3.3	2.5	2.3	1.4	1.6	2.2
  Y112Q	708	1.5	1.2	1.4	1.7	1.4	1.6	3.0	2.4	1.8
  Y112P	709	0.9	1.0	1.1	1.1	1.1	0.8	1.4	0.9	1.0
  Y112V	710	1.6	1.2	1.3	1.4	1.0	0.8	9.8	4.3	2.0
  Y113H	711	1.4	1.4	1.6	1.0	1.0	1.2	7.3	5.1	1.8
  Y113L	712	0.8	1.6	1.0	1.5	1.2	1.4	1.4	1.7	1.4
  Y113W	713	1.8	1.5	2.0	1.4	1.4	1.2	5.6	4.0	1.8
  Y113E	714	1.1	1.1	1.2	1.3	1.3	1.0	1.2	1.6	1.4
  Y113P	715	1.3	1.4	1.4	1.4	0.8	1.2	2.0	2.0	1.3
  Y113K	716	0.9	1.1	1.2	1.2	1.0	0.8	1.2	1.3	1.3
  Y114K	717	0.8	0.9	0.9	1.0	0.8	0.6	1.0	1.2	1.1
  Y114F	718	1.1	1.1	1.4	1.0	1.0	1.1	2.0	2.1	1.2
  Y114R	719	2.0	2.0	2.4	2.4	1.6	1.5	2.9	2.3	2.4
  wt	688	1.8	1.4	1.6	1.2	0.9	1.1	9.2	7.9	1.6
  wt	688	1.6	1.5	2.0	1.4	1.3	1.4	9.5	9.3	1.7

Example 16 Binding to DLL4 Expressed on the Surface of CHO Cells
• In this example, Fabs H:APFF VLTH & L:NDH LS (SEQ ID NOS:209 and 350; identified as exhibiting about 1.7 nM affinity as shown in Table 75) and H:KT TRV & L:LP S52G (SEQ ID NOS:430 and 543; identified as exhibiting about 5 nM affinity as shown in Table 108) were tested for their ability to bind to DLL4 expressed on the surface of CHO cells as detected by flow cytometry.
• To generate a DLL4 expression construct, human DLL4 cDNA (SEQ ID NO:2905, Accession No. BC106950; and encoding amino acids set forth in SEQ ID NO:2904, Accession No. AAI06951) in pCR-BluntII-TOPO (SEQ ID NO:2934) as a glycerol stock was obtained from Open Biosystems (Clone ID#40034887). The stock was streaked on kanamycin agar plates and a colony picked for purification of the DNA. DNA was obtained with Purelink™ Quick Plasmid Miniprep Kit (Invitrogen, Catalog #K210010).
• Full-length DLL4 was digested out from the OpenBiosystems vector and ligated into pcDNA5/FRT (SEQ ID NO:2935; Invitrogen Catalog #K601001) between NheI and NotI. Ligation was performed with Rapid DNA Ligation Kit (Roche, Catalog #11 635 379 001) and cells transformed using heat shock into One Shot® Max Efficiency® DH5α™-T1® Competent Cells (Invitrogen, Catalog #12297016). Cells were selected on carbenicillin plates. Colonies were picked and inoculated overnight in luria broth (LB) containing 1:1000 100 mg/mL, carbenicillin. Plasmid DNA was extracted by miniprep (Invitrogen; Catalog #K210011).
• Using Invitrogen's Lipofectamine™ Transfection Reagent, pcDNA5/FRT containing full-length DLL4 and pOG44 recombinase vector (SEQ ID NO:2936; Invitrogen Catalog #K601001) were transfected into Invitrogen's Flp-In™-CHO Cell Line (Cat. No. R75807) according to Flp-In™ System protocol. Cells were approximately 90% confluent in a 12-well plate. Transfected cells were selected with 400 μg/ml Hygromycin after a couple days. Colonies were picked about 5 days after and transferred into a 10 cm² tissue culture dish. These cell lines were maintained with hygromycin selection
• CHO cells expressing full-length DLL4 and control CHO cells were detached from tissue culture plates (BD Falcon 10 cm²) using Accutase™ Enzyme Cell Detachment Medium (Cat#00-4555-56, eBioscience). After washing the cells in 2% Bovine Serum Albumin in Phosphate Buffered Saline (2% BSA/PBS), 10 nM to 50 nM Fab in 2% BSA/PBS was added and incubated at on ice for 30 minutes. The cells were washed one time with 2% BSA/PBS and mouse anti-human kappa-PE antibody (diluted 1:100, Cat# MH10514, Invitrogen) or mouse anti-human lambda-PE antibody (diluted 1:100, Cat# MH10614, Invitrogen) was added and incubated on ice for 10 minutes. Secondary antibody mouse anti-human kappa-PE alone (without Fab) was used as a control for DLL4-expressing CHO cells. The cells were then washed twice in 2% BSA/PBS and analyzed by flow cytometry on a BD FACSAria. The results show that the tested Fabs bind DLL4 expressed on the surface of CHO cells. Neither Fab showed significant binding to CHO cells without DLL4 over-expression.
Example 17 Inhibition of DLL4-Notch Interaction by Flow Cytometry
• In this example, three DLL4 binding Fabs were functionally screened for their ability to block the binding of Notch-Fc to DLL4. In this assay, DLL4-expressing CHO cells were incubated in the presence of both Fab and biotinylated-Notch-Fc. Streptavidin-PE was used as a detection molecule. If Notch-Fc binds to DLL4-expressing CHO cells, these cells will be detected by a PE signal at 578 nm. Alternatively, if the Fab blocks the binding of Notch-Fc to DLL4, the DLL4-expressing CHO cells will not be labeled or detected. The tested Fabs included H:APFF VLTH & L:NDH LS (SEQ ID NOS:209 and 350), H:KT TRV & V3-4_IGLJ1*01 (SEQ ID NOS:430 and 108) and H:KT TRV & L:LP S52G (SEQ ID NOS:430 and 543).
• In short, CHO cells expressing full-length DLL4 (CHO-DLL4) as described in Example 16 were detached from tissue culture plates using Accutase™ Enzyme Cell Detachment Medium (Cat#00-4555-56, eBioscience). Fab was 5-fold serially diluted in 2% BSA/PBS from a starting concentration of 50 nM. Notch-FC (cat#3647-TK-050, R&D Systems) was biotinylated following using EZ-Link NHS-Biotin Reagent (cat#20217. Pierce) according to the manufacturers instructions. Detached cells were treated with 250 nM biotinylated Notch-FC in 2% BSA/PBS and 30 μL Fab for 30 minutes on ice. PE-labeled streptavidin (Cat#21627, Pierce-Thermo Scientific) was then added to a final dilution of 1:5 followed by incubation for 10 minutes at room temperature. The cells were then washed twice in 2% BSA/PBS and analyzed by flow cytometry on a BD FACSAria.
• The results are set forth in Table 119 below. All three Fabs effectively block Notch-Fc binding to CHO-DLL4. Fab H:APFF VLTH & L:NDH LS completely blocks the binding of Notch to DLL4 by 80% at a Fab concentration of 2 nM. Fab H:KT TRV & V3-4_IGLJ1*01 blocks the binding of Notch to DLL4 by 50% at a concentration of 50 nM Fab. Fab H:KT TRV & L:LP S52G blocks the binding of Notch to DLL4 by 80% at a concentration of 50 nM Fab.

• TABLE 119
  Inhibition of DLL4-Notch interaction

  	Fab	H:APFF VLTH &	H:KT TRV &	H:KT TRV &
  	[nM]	L:NDH LS	L:wt	L:LP S52G

  	50	30	141	105
  	10	30	244	190
  	2	117	448	250
  	0.4	277	Not tested	324
  	0	531	531	531

Example 18 IgG Cloning and Expression
• In this example, Fab antibodies that bind to DLL4 were converted into IgGs by cloning into the pFUSE vectors. Briefly, sequences encoding heavy and light chains were cloned separately into the pFUSE family of vectors (pFUSE-hIgG2-Fc2, Cat# pfuse-hfc2, InvivoGen; SEQ ID NO:2938)) behind the included IL-2 signal sequence. These two vectors were then co-transformed into 293F cells and the protein was expressed and purified.
Light Chain:
• The Sequence encoding the Fab light chain (excluding the N-terminal E. coli sorting signal Met Ala) was amplified by PCR with primers containing EcoRI and NheI ends. The amplified Fab light chain was subcloned into pFUSE-hIgG2-Fc2, previously digested with EcoRI and NheI. The Fab light chain immediately follows the IL-2 signal sequence, and completely replaces the Fc sequence in pFUSE-hIgG2-Fc2.
Heavy Chain:
• A full-length IgG1 heavy chain sequence (SEQ ID NO:2922) also including a NheI site between VH and CH1-CH2-CH3 was synthesized by Genscript, amplified by PCR with primers containing EcoRI and XbaI ends, and subcloned into pFUSE-hIgG2-Fc2, previously digested with EcoRI and NheI. Ligation of the XbaI and NheI compatible cohesive ends eliminates both sties at this position, making the NheI site between VH and CH1-CH2-CH3 of the IgG1 heavy chain sequence unique. The sequence encoding Fab heavy chain (excluding the N-terminal E. coli sorting signal Met Ala) was amplified by PCR with EcoRI and NheI ends. The vector containing the full length IgG1 heavy chain was then digested with EcoRI and NheI, which removed the VH sequence, and the amplified Fab heavy chain was subcloned into the digested vector. Thus the Fab Heavy chain was subcloned between IL-2 and the IgG1 heavy chain.
Protein Expression and Purification:
• To produce IgG, the heavy and light chain plasmids were co-transfected into 293F cells (Cat# R790-07, Invitrogen) using 293fectin (Cat#12347, Invitrogen) per manufacturer's instructions. Cells grown in serum-free 293Freestyle media (Cat#12338026, Invitrogen) were transfected at 1×106 cells/ml in 50 ml spinner flask. Cell culture media were harvested 3 and 6 days after transfection and pooled together for purification by column chromatography using Protein-G Sepharose (GE Healthcare). IgG elution fractions were pooled and dialysed into PBS.
Example 19 Activity of Antibodies by DLL4-Notch Interaction by a Reporter Assay
• In this example, two DLL4 binding antibodies were assayed for their ability to inhibit DLL4-dependent Notch 1 signaling using a luciferase reporter assay. Reporter cells were generated by stably transfecting human glioma T98G cells, known for the presence of Notch 1 on their cell surface (see Purow et al. (2005) Cancer Res., 65:2353-63), with a Notch reporter plasmid (p6xCBF) containing six C promoter binding factor-1 (CBF-1) responsive elements (set forth in SEQ ID NO:2939; see Nefedova et al. (2004), Blood. 103(9):3503-10). Subsequent addition of DLL4-CHO cells (see Example 16 above) to the reporter T98G cells results in expression of firefly luciferase due to the Notch1-DLL4 interaction. Disruption of the Notch1-DLL4 by a DLL4 binding antibody therefore causes a decrease in luciferase expression.
A. Notch Reporter Plasmids
• A reporter construct containing six C promoter binding factor-1 (CBF-1) response elements (set forth in SEQ ID NO:2939; CBF Notch-response elements are indicated by bold; ggtacctgagctcgctagcgatctggtgtaaacacgccgtgggaaaaaatttatggatctggtgtaaacacgccgtgggaaaaaattta tggagctcgctagcgatctggtgtaaacacgccgtgggaaaaaatttatggatctggtgtaaacacgccgtgggaaaaaatttatgac gaggatctggtgtaaacacgccgtgggaaaaaatttatggatctggtgtaaacacgccgtgggaaaaaatttatgaagctt;) was digested with KpnI and HindIII. The digested product was then into the luciferase reporter vectors pGL4.26 (SEQ ID NO:2940; Promega, Catalog #E8441)) at the KpnI and HindIII sites. The pGL4.26 vector allows for hygromycin selection, which facilitates the production of a cell line with a stably-integrated copy of the reporter. Also, the use of pGL4.26 eliminates the need to transiently transfect the reporter and normalize for variable transfection efficiency.
B. Assay
• T98G cells from ATCC (No. CRL-1690™) were plated onto a 96-well tissue culture plate at 20,000 cells per well in Eagle's Minimum Essential Media (EMEM, Invitrogen) supplemented with 10% Fetal Bovine Serum (BSA, Invitrogen) and 1× penicillin/streptomycin/glutamine (P/S/G, Invitrogen).
• The following day, T98G cells were transfected with the Notch reporter construct expressing Firefly luciferase (p6xCBF) and stable integrants were selected with 200 ug/ml Hygromycin B (Invitrogen). CHO cells expressing DLL4 or control CHO cells were propagated in F12 media (Invitrogen) supplemented with 10% FBS and P/S/G. Separately, T98G Notch reporter cells (2×10⁵ cells/well) in EMEM with 10% FBS and P/S/G were plated onto 96-well tissue culture plates. Notch-expressing T98G cells were stimulated by CHO-DLL4 or control CHO cells (1×10⁵ cells/well). Media on T98G cells was replaced by 100 μl of serum free F12 media supplemented with P/S/G. Fabs H:APFF VLTH & L:NDH LS (SEQ ID NOS:209 and 350) and H:KT TRV & L:LP S52G (SEQ ID NOS:430 and 543) and their corresponding IgGs, and control Fab (that does not bind DLL4; VH6-1_IGHD6-13*01_IGHJ4*01 and V2-17_IGLJ2*01 set forth in SEQ ID NOS: 2152 and 2941, respectively) were added at 100, 20, 4 and 0.8 nM. In addition, the non-affinity matured germline parent Fabs also were tested to determine their Notch reporter response. For this, corresponding IgGs of VH5-51_IGHD5-18*01>3_IGHJ4*01 & V3-4_IGLJ1*01 (set forth in SEQ ID NOS: 89 and 108; the parent germline Fab of H:KT TRV & L:LP S52G) and VH1-46_IGHD6-6*01_IGHJ1*01 & L6_IGKJ1*01 (set forth in SEQ ID NOS:88 and 107; the parent germline Fab of H:APFF VLTH & L:NDH LS) were control IgG was added at 200, 100 and 20 nM.
• After 24 hours, luciferase-reporter expression was measured with Bright-Glo luciferase assay reagent (Cat# E2620, Promega). Luminenscence was read using a Wallac Victor II model 1420 plate reader. Each condition was performed in quadruplicate.
• The results are depicted in Tables 120 below. The results in Table 120 show that incubation of the T98G reporter cells with CHO-DLL4 resulted in 8- to 9-fold increase in Notch1 reporter levels compared to those incubated with CHO cells alone. The Notch1 activation remained constant in the presence of the control Fab that does not bind to DLL4. The activation was reduced in the presence of increasing concentration of anti-DLL4 antibody Fabs H:APFF VLTH & L:NDH LS and H:KT TRV & L:LP S52G. The reduction was even more pronounced with an IgG version of H:APFF VLTH & L:NDH LS (IC₅₀˜6 nM), which was almost 10-fold more efficient than the corresponding Fab. The IgG version of H:KT TRV & L:LP S52G was also more effective than the corresponding Fab, displaying about 30% reduction in Notch1 activation at 0.8 nM. Neither Fab nor IgG form of H:KT TRV & L:LP S52G showed complete suppression of Notch1 activation at higher concentrations (>100 nM). The results show that the IgG H:APFF VLTH & L:NDH LS is a complete inhibitor, whereas IgG H:KT TRV & L:LP S52G is a partial antagonist of the DLL4-Notch activation.

• TABLE 120
  Cell type	treatment	Conc [nM]	1	2	3	4	Avg ± SE

  CHO-DLL4	VH6-1 IGH36-	0.8	4482	4541	3908	4221	4288 ± 144
  	13*01	4	4809	4921	4187	4520	4609 ± 164
  	IGHJ4*01 and	20	5402	4988	4323	4546	4815 ± 240
  	V2-	100	4821	4813	4034	4473	4535 ± 186
  	17_IGLJ2*01
  	(control Fab)
  	H: KT TRV &	0.8	4878	4716	4078	4278	4488 ± 186
  	L: LP S52G	4	4792	4771	4321	4469	4588 ± 116
  	(Fab)	20	4245	4371	4148	4075	4210 ± 64
  		100	3321	3483	3012	3083	3225 ± 109
  	H: KT TRV &	0.8	3711	3485	3092	3292	3395 ± 132
  	L: LP S52G	4	3276	3339	3091	2911	3154 ± 97
  	(IgG)	20	3020	2904	2598	2652	2794 ± 101
  		100	2811	2545	2276	2519	2538 ± 109
  	H: APFF	0.8	4739	4886	3818	4076	4380 ± 257
  	VLTH &	4	4837	4877	4251	4667	4658 ± 143
  	L: NDH LS	20	4376	4482	3960	3993	4203 ± 133
  	(Fab)	100	2397	2285	2148	2169	2250 ± 58
  	H: APFF	0.8	4445	4521	3899	3985	4213 ± 158
  	VLTH &	4	4261	3862	3949	3765	3959 ± 107
  	L: NDH LS	20	1250	1269	1174	1191	1221 ± 23
  	(IgG)	100	757	807	678	688	733 ± 30
  CHO	VH6-1 IGH36-	0.8	572	569	555	583	570 ± 6
  	13*01	4	557	547	539	450	523 ± 25
  	IGHJ4*01 and	20	508	532	550	476	517 ± 16
  	V2-	100	488	487	491	464	483 ± 6
  	17_IGLJ2*01
  	(control Fab)

• Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.

Claims (15)

1-97. (canceled)

98. A method of affinity maturation of a first antibody, or antigen-binding portion thereof, for a target antigen, comprising:

a) identifying a related antibody, or antigen-binding portion thereof, that exhibits a reduced activity for the target antigen than the corresponding form of a first antibody, wherein the related antibody, or antigen-binding portion thereof, contains a related variable heavy chain or a related variable light chain that is either:

one in which the corresponding variable heavy chain or variable light chain of the related antibody, or antigen-binding portion thereof, exhibits at least 75% amino acid sequence identity to the variable heavy chain or variable light chain of the first antibody, or antigen-binding portion thereof, but does not exhibit 100% sequence identity therewith; or

one in which at least one of the V_H, D_H, and J_H germline segments of a nucleic acid molecule encoding the variable heavy chain of the related antibody, or antigen-binding portion thereof, is identical to one of the V_H, D_H, and J_H germline segments of the nucleic acid molecule encoding the variable heavy chain of the first antibody, or antigen-binding portion thereof, and/or at least one of the V_κ and J_κ or at least one of the V_λ and J_λ germline segments of the nucleic acid molecule encoding the variable light chain is identical to one of the V_κ and J_κ or V_λ and J_λ germline segments of the nucleic acid molecule encoding the variable light chain of the first antibody, or antigen-binding portion thereof; and

b) comparing the amino acid sequence of the variable heavy chain or variable light chain of the first antibody, or antigen-binding portion thereof, to the amino acid sequence of the corresponding related variable heavy chain or variable light chain of the related antibody, or antigen-binding portion thereof;

c) identifying a target region within the variable heavy chain or variable light chain of a first antibody, or antigen-binding portion thereof, wherein the target region exhibits at least one amino acid difference compared to the same region in the related antibody, or antigen-binding portion thereof;

d) producing a plurality of modified antibodies, or antigen-binding portions thereof, each comprising a variable heavy chain and a variable light chain, or a portion thereof, wherein at least one of the variable heavy chain or variable light chain is modified in its target region by replacement of a single amino acid residue, whereby the target region in each of the plurality of antibodies, or antigen-binding portions thereof, contains replacement of an amino acid to a different amino acid compared to the first antibody, or antigen-binding portion thereof;

e) screening each of the plurality of modified antibodies, or antigen-binding portions thereof, for an activity to the target antigen; and

f) selecting those modified antibodies, or antigen-binding portions thereof, that exhibit increased activity for the target antigen compared to the first antibody, or antigen-binding portion thereof.

99. A method according to claim 98 that further includes at least one of the following:

a) wherein the plurality of modified antibodies, or antigen-binding portions thereof, in part (d) are produced by producing a plurality of nucleic acid molecules that encode modified forms of a variable heavy chain or a variable light chain of the first antibody, or antigen-binding portion thereof, wherein the nucleic acid molecules contain one codon encoding an amino acid in the target region that encodes a different amino acid as compared to the unmodified variable heavy or variable light chain, whereby each nucleic acid molecule of the plurality encodes a variable heavy chain or variable light chain that is modified in its target region by replacement of a single amino acid residue;

b) wherein the target region in the first antibody, or antigen-binding portion thereof, exhibits 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid differences compared to the corresponding region in the related antibody, or antigen-binding portion thereof;

c) wherein the related antibody, or antigen-binding portion thereof, is 1, 2, 3, 4, or 5 related antibodies, or antigen-binding portions thereof;

d) wherein the activity is selected from the group consisting of:

i. binding, optionally binding as assessed by a method selected from the group consisting of an immunoassay, optionally an immunoassay selected from the group consisting of a radioimmunoassay, an enzyme linked immunosorbent assay (ELISA), and an electrochemiluminescence assay, wherein the electrochemiluminescence assay optionally is meso-scale discovery (MSD); whole cell panning; and surface plasmon resonance (SPR);

ii. signal transduction;

iii. differentiation;

iv. alteration of gene expression;

v. cellular proliferation;

vi. apoptosis;

vii. chemotaxis;

viii. cytotoxicity;

ix. cancer cell invasion;

x. endothelial cell proliferation; and

xi. tube formation;

e) wherein the first antibody, or antigen-binding portion thereof, binds to the target antigen when in a Fab form with a binding affinity that is about 10⁻⁴ M or lower, about 10⁻⁴ M to about 10⁻⁸ M, or at or about 10⁻⁴ M, 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, or lower;

f) wherein the related antibody, or antigen-binding portion thereof, exhibits a binding affinity that is less than the binding affinity of the first antibody, or antigen-binding portion thereof, whereby the binding affinity of the related antibody, or antigen-binding portion thereof, in its Fab form is about 10⁻⁴ M or lower; about 10⁻⁴ M to about 10⁻⁸ M; or at or about 10⁻⁴ M, 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, or lower;

g) wherein the related antibody, or antigen-binding portion thereof, exhibits about 80% or less activity than the corresponding form of the first antibody, or antigen-binding portion thereof; about 5% to about 80% of the activity of the corresponding form of the first antibody; or less than or about 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or less activity than the corresponding form of the first antibody.

h) wherein the related antibody, or antigen-binding portion thereof, exhibits the same or similar level of activity to the target antigen compared to a negative control;

i) wherein the target region is identified within the variable heavy chain of the first antibody, or antigen-binding portion thereof, and steps d)-f) are performed therefrom;

j) wherein the target region is identified within the variable light chain of the first antibody, or antigen-binding portion thereof, and steps d)-f) are performed therefrom;

k) wherein:

a target region is identified within the variable heavy chain of the first antibody, or antigen-binding portion thereof, and steps d)-f) are performed therefrom; and

separately and independently a target region is identified within the variable light chain of the first antibody, or antigen-binding portion thereof, and steps d)-f) are performed therefrom;

l) wherein a related antibody, or antigen-binding portion thereof, that contains the related corresponding variable heavy chain is different than a related antibody, or antigen-binding portion thereof, that contains the related corresponding variable light chain;

m) wherein a related antibody, or antigen-binding portion thereof, that contains the related corresponding variable heavy chain is the same as a related antibody, or antigen-binding portion thereof, that contains the related corresponding variable light chain;

n) wherein the amino acid sequence of the variable heavy chain or variable light chain of the first antibody, or antigen-binding portion thereof, exhibits at least about 80% or more sequence identity with the corresponding amino acid sequence of the related variable heavy chain or variable light chain of the related antibody, or antigen-binding portion thereof; about 80% to about 99% of the sequence identity with the corresponding amino acid sequence of the related variable heavy chain or variable light chain of the related antibody, or antigen-binding portion thereof; or at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the corresponding amino acid sequence of the related variable heavy chain or variable light chain of the related antibody, or antigen-binding portion thereof;

o) wherein the variable heavy chain or variable light chain of the first antibody, or antigen-binding portion thereof, exhibits at least about 95% sequence identity with the corresponding amino acid sequence of the related variable heavy chain or variable light chain of the related antibody, or antigen-binding portion thereof;

p) wherein the related antibody, or antigen-binding portion thereof, contains a related variable heavy chain or variable light chain that is one in which at least one of the V_H, D_H, and J_H germline segments of the nucleic acid molecule encoding the variable heavy chain of the first antibody, or antigen-binding portion thereof, is identical to one of the V_H, D_H, and J_H germline segments of the nucleic acid molecule encoding the variable heavy chain of the related antibody, or antigen-binding portion thereof; and/or at least one of the V_κ and J_κ or at least one of the V_λ and J_λ germline segments of the nucleic acid molecule encoding the variable light chain of the first antibody, or antigen-binding portion thereof, is identical to one of the V_κ and J_κ or V_λ and J_λ germline segments of the nucleic acid molecule encoding the variable light chain of the related antibody, or antigen-binding portion thereof;

q) wherein the target antigen is selected from the group consisting of a polypeptide, carbohydrate, lipid, nucleic acid, and a small molecule;

r) wherein the target antigen is expressed on the surface of a virus, bacteria, tumor or other cell, or is a recombinant protein or peptide;

s) wherein the target antigen is a protein that is a target for therapeutic intervention, optionally selected from the group consisting of VEGFR-1, VEGFR-2, VEGFR-3 (vascular endothelial growth factor receptors 1, 2, and 3), a epidermal growth factor receptor (EGFR), ErbB-2, ErbB-3, IGF-R1, C-Met (also known as hepatocyte growth factor receptor; HGFR), DLL4, DDR1 (discoidin domain receptor), KIT (receptor for c-kit), FGFR1, FGFR2, FGFR4 (fibroblast growth factor receptors 1, 2, and 4), RON (recepteur d'origine nantais; also known as macrophage stimulating 1 receptor), TEK (endothelial-specific receptor tyrosine kinase), TIE (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains receptor), CSF1R (colony stimulating factor 1 receptor), PDGFRB (platelet-derived growth factor receptor B), EPHA1, EPHA2, EPHB1 (erythropoietin-producing hepatocellular receptor A1, A2 and B1), TNF-R1, TNF-R2, HVEM, LT-βR, CD20, CD3, CD25, NOTCH, G-CSF-R, GM-CSF-R, EPO-R., a cadherin, an integrin, CD52, CD44, VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF, EGF, HGF, TNF-α, LIGHT, BTLA, lymphotoxin (LT), IgE, G-CSF, GM-CSF and EPO;

t) wherein the target antigen is involved in cell proliferation and differentiation, cell migration, apoptosis or angiogenesis;

u) wherein a subset of the amino acid residues in the target region are modified by amino acid replacement;

v) wherein only the amino acid residues that differ between the first antibody and related antibody in the target region are modified by amino acid replacement;

w) wherein only the amino acid residues that are the same between the first antibody and the related antibody in the target region are modified by amino acid replacement;

x) wherein all of the amino acids residues in the target region are modified by amino acid replacement;

y) wherein each amino acid residue that is modified in the target region is modified to all 19 other amino acid residues, or a restricted subset thereof;

z) further comprising determining the amino acid modifications that are altered in the modified antibody compared to the first antibody not containing the amino acid replacements;

aa) wherein the method is repeated iteratively, wherein a modified antibody, or antigen-binding portion thereof, is selected and used in step a) as the first antibody, or antigen-binding portion thereof, for subsequent affinity maturation thereof;

bb) wherein one or more amino acid replacements in the target region of one or more variable heavy chains or one or more variable light chains of selected modified antibodies, or antigen-binding portions thereof, are combined to generate a further modified antibody, or antigen-binding portion thereof, whereby the further modified antibody(ies), or antigen-binding portion(s) thereof, are screened for an activity to the target antigen to identify a further modified antibody, or antigen-binding portion thereof, that exhibits an increased activity for the target antigen compared to the first antibody, or antigen-binding portion thereof, and to the selected modified antibody(ies), or antigen-binding portion(s) thereof; and

cc) wherein the antibody, or antigen-binding portion thereof, comprising a variable heavy chain and a variable light chain, or a portion thereof, is selected from the group consisting of a Fab, Fab′, F(ab′)₂, single-chain Fv (scFv), scFab, Fv, dsFv, diabody, Fd, Fd′, Fab fragment, Fd fragment, Fd′ fragment, scFv fragment, and scFab fragment.

100. A method according to claim 98, wherein the related antibody, or antigen-binding portion thereof, contains a related variable heavy chain or variable light that is one in which at least one of the V_H, D_H, and J_H germline segments of the nucleic acid molecule encoding the variable heavy chain of the first antibody, or antigen-binding portion thereof, is from the same gene family as one of the V_H, D_H, and J_H germline segments of the nucleic acid molecule encoding the variable heavy chain of the related antibody, or antigen-binding portion thereof; and/or at least one of the V_κ and J_κ or at least one of the V_λ and J_λ germline segments of the nucleic acid molecule encoding the variable light chain of the first antibody, or antigen-binding portion thereof, is from the same gene family as one of the V_κ and J_κ or V_λ and J_λ germline segments of the nucleic acid molecule encoding the variable light chain of the related antibody, or antigen-binding portion thereof.

101. A method according to claim 98, wherein the variable heavy chain or variable light chain of the first antibody, or antigen binding portion thereof, exhibits at least 60% or more sequence identity with the corresponding related variable heavy chain or variable light chain of the related antibody, or antigen binding portion thereof; 60% to 99% of the sequence identity with the corresponding related variable heavy chain or variable light chain of the related antibody, or antigen binding portion thereof; or at least or about 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the corresponding related variable heavy chain or variable light chain of the related antibody, or antigen binding portion thereof.

102. A method according to claim 98, wherein the target region is selected from the group consisting of a CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4.

103. A method according to claim 98, wherein:

a) the first antibody, or antigen-binding portion thereof, is identified by screening a combinatorial antibody library or combinatorial antigen-binding antibody fragment library;

b) the combinatorial library is produced by a method comprising:

i) combining a V_H, a D_H, and a J_H human germline segment or portion thereof in frame to generate a sequence of a nucleic acid molecule encoding a VH chain or a portion thereof;

ii) combining a V_κ and a J_κ human germline segment or portion thereof, or a V_λ and a J_λ germline segment or portion thereof in frame to generate a sequence of a nucleic acid molecule encoding a VL chain or a portion thereof, wherein:

in steps i) and ii), each of the portions of the V_H, D_H, J_H, V_κ, J_κ, V_λ, or J_λ are sufficient to produce an antibody or antigen-binding portion thereof containing a VH or VL or portion thereof that forms a sufficient antigen binding site;

iii) repeating steps i) and ii) a plurality of times to generate sequences of a plurality of different nucleic acid molecules;

iv) synthesizing the nucleic acid molecules to produce two libraries, wherein:

the first library comprises nucleic acid molecules encoding a VH chain or a portion thereof; and

the second library comprises nucleic acid molecules encoding a VL chain or a portion thereof;

v) introducing a nucleic acid molecule from the first library and from the second library into a cell and repeating this a plurality of times to produce a library of cells, wherein each cell contains nucleic acid molecules encoding a different combination of VH and VL from at least some of the other cells in the library of cells; and

vi) growing the cells to express the antibodies, or antigen-binding portions thereof, thereby producing a plurality of antibodies, or antigen-binding portion thereof, wherein the different antibodies, or antigen-binding portions thereof, in the library each comprise a different combination of a VH and a VL chain or a sufficient portion thereof to form an antigen binding site; and

c) screening of the library is effected by:

i) contacting an antibody, or antigen-binding portion thereof, in the library with a target protein;

ii) assessing binding of the antibody, or antigen-binding portion thereof, with the target protein and/or whether the antibody, or antigen-binding portion thereof, modulates a functional activity of the target protein; and

iii) identifying an antibody, or antigen-binding portion thereof, that exhibits an activity for the target protein, wherein the identified antibody, or antigen-binding portion thereof, is a first antibody.

104. A method according to claim 103 that further includes at least one of the following:

a) the related antibody also is identified by screening a combinatorial antibody library by steps a)-c), whereby the related antibody exhibits reduced activity for the target antigen compared to the first antibody;

b) the library is an addressable library, whereby:

in step iv), the synthesized nucleic acid sequences are individually addressed, thereby generating a first addressed nucleic acid library and a second addressed nucleic acid library;

in step v), the cells are addressed, wherein each locus comprises a cell that contains nucleic acid molecules encoding a different combination of a VH and a VL from every other cell in the addressed library of cells; and

in step vi) the plurality of antibodies or portions thereof are addressed, wherein:

the antibodies or portions thereof at each locus in the library are the same antibody and are different from those at each and every other locus; and

the identity of the antibody or portion thereof is known by its address, wherein optionally the antibodies in the addressable library are arranged in a spatial array, optionally a multiwell plate, wherein each individual locus of the array corresponds to a different antibody member;

c) wherein the antibodies are in an addressable library, wherein optionally the antibodies in the addressable library are arranged in a spatial array, optionally a multiwell plate, wherein optionally each individual locus of the array corresponds to a different antibody member are attached to a solid support selected from the group consisting of a filter, chip, slide, bead or cellulose, and the different antibody members are immobilized to the surface thereof;

d) wherein the plurality of nucleic acid molecules are generated by a method selected from the group consisting of PCR mutagenesis, cassette mutagenesis, site-directed mutagenesis, random point mutagenesis, mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA, point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, and double-strand break repair; and

e) wherein the plurality of nucleic acid molecules are generated by a method selected from the group consisting of NNK, NNS, NNN, NNY or NNR mutagenesis.

105. A method according to claim 98, further comprising before step d),

g) performing scanning mutagenesis of the first antibody, or antigen-binding portion thereof, comprising producing a plurality of modified antibodies, or antigen-binding portions thereof, comprising a variable heavy chain and a variable light chain, or a portion thereof, wherein at least one of the variable heavy chain or variable light chain, or portion thereof, is one that is modified by replacement of a single amino acid residue with a scanned amino acid residue in the target region, whereby each of the plurality of antibodies, or antigen-binding portion thereof, contains replacement of an amino acid in the target region compared to the first antibody, or antigen-binding portion thereof, wherein the scanned amino acid optionally is selected from the group consisting of alanine, threonine, proline, glycine, and a non-natural amino acid;

h) screening each of the plurality of modified antibodies, or antigen-binding portions thereof, for an activity to the target antigen; and

i) selecting a second antibody, or antigen-binding portion thereof, from among the modified antibodies, or antigen-binding portions thereof, that exhibits retained or increased activity for the target antigen compared to the first antibody, or antigen-binding portion thereof, not containing the amino acid replacement, whereby the second antibody, or antigen-binding portion thereof, is used in place of the first antibody, or antigen-binding portion thereof, in step b).

106. A method according to claim 105 that further includes at least one of the following:

a) wherein the plurality of modified antibodies, or antigen-binding portions thereof, in step g) are produced by producing a plurality of nucleic acid molecules that encode modified forms of a variable heavy chain or a variable light chain of the first antibody, or antigen-binding portion thereof, containing the target region, wherein the nucleic acid molecules contain one codon that encodes a scanned amino acid in the target region compared to the corresponding codon of the unmodified variable heavy or variable light chain that does not encode the scanned amino acid, whereby each nucleic acid molecule of the plurality encodes a variable heavy chain or variable light chain that is modified by replacement of a single amino acid residue to the same scanned amino acid residue in the target region;

b) wherein a second antibody is, or antigen-binding portion thereof, selected that exhibits an activity that is at least 75% or more of the activity of the corresponding form of the first antibody, or antigen-binding portion thereof; is at least 75% to 200% of the activity of the corresponding form of the first antibody, or antigen-binding portion thereof; or is at least or about 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 130%, 140%, 150%, 200% or more of the activity of the corresponding form of the first antibody, or antigen-binding portion thereof;

c) further comprising after step i) determining the amino acid residue position that is modified in the second antibody, or antigen-binding portion thereof, to contain a neutral amino acid compared to the first antibody not containing the amino acid replacement;

d) wherein a subset of the amino acid residues in the target region are modified by amino acid replacement to a scanned amino acid;

e) wherein only the amino acid residues that differ between the first antibody, or antigen-binding portion thereof, and related antibody, or antigen-binding portion thereof, in the target region are modified by amino acid replacement to a scanned amino acid;

f) wherein all of the amino acids in the target region are modified by amino acid replacement to a scanned amino acid;

g) wherein the selected modified antibody, or antigen-binding portion thereof, exhibits about 2-fold, 5-fold, 10-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 10000-fold, or more improved activity for the target antigen compared to the first antibody, or antigen-binding portion thereof; and

h) wherein the modified antibody, or antigen-binding portion thereof, exhibits a binding affinity that is greater than the binding affinity of the first antibody, or antigen-binding portion thereof, and is about 1×10⁻⁹ M or less; 1×10⁻⁹ M to 1×10⁻¹¹ M; or is or is about 1×10⁻⁹ M, 2×10⁻⁹ M, 3×10⁻⁹ M, 4×10⁻⁹ M, 5×10⁻⁹ M, 6×10⁻⁹ M, 7×10⁻⁹ M, 8×10⁻⁹ M, 9×10⁻⁹ M, 1×10⁻¹⁰ M, 2×10⁻¹⁰ M, 3×10⁻¹⁰ M, 4×10⁻¹⁰ M, 5×10⁻¹⁰ M, 6×10⁻¹⁰ M, 7×10⁻¹⁰ M, 8×10⁻¹⁰ M, 9×10⁻¹⁰ M, or less

107. A method according to claim 98, comprising:

performing steps a)-f) on the variable heavy chain of the first antibody, or antigen-binding portion thereof, and selecting first modified antibodies, or antigen-binding portions thereof, each containing an amino acid replacement in the target region;

performing steps a)-f) independently and separately on the variable light chain of the first antibody and selecting second modified antibodies, or antigen-binding portions thereof, each containing an amino acid replacement in the target region;

combining the variable heavy chain of a first modified antibody, or antigen-binding portion thereof, with the variable light chain of a second modified antibody, or antigen-binding portion thereof, to generate a plurality of different third modified antibodies, or antigen-binding portions thereof, each comprising an amino acid replacement in the target region of the variable heavy chain and variable light chain; and

screening each of the plurality of third modified antibodies, or antigen-binding portions thereof, for binding to the target antigen; and

selecting those third modified antibodies, or antigen-binding portions thereof, that exhibit an increased activity for the target antigen compared to the first and second modified antibodies.

108. A method according to claim 98, further comprising after selecting a first modified antibody, or antigen-binding portion thereof, in step f):

j) selecting another different region within the variable heavy chain or variable light chain of the first modified antibody, or antigen-binding portion thereof, for further mutagenesis, wherein optionally the further different region is selected from the group consisting of a CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4;

k) producing a plurality of nucleic acid molecules that encode modified forms of the variable heavy chain or variable light chain of the first modified antibody, or antigen-binding portion thereof, wherein the nucleic acid molecules contain one codon encoding an amino acid in the selected region that encodes a different amino acid from the first modified variable heavy or variable light chain, whereby each nucleic acid molecule of the plurality encodes a variable heavy chain or variable light chain that is modified in the selected region by replacement of a single amino acid residue;

l) producing a plurality of further modified antibodies, or antigen-binding portions thereof, each comprising a variable heavy chain and a variable light chain, or a portion thereof, wherein at least one of the variable heavy chain or variable light chain is one produced in step k), whereby the selected region in each of the plurality of antibodies, or antigen-binding portions thereof, contains replacement of an amino acid to a different amino acid compared to the first modified antibody, or antigen-binding portion thereof;

m) screening each of the plurality of further modified antibodies, or antigen-binding portions thereof, for binding to the target antigen; and

n) selecting those further modified antibodies, or antigen-binding portions thereof, that exhibit increased activity for the target antigen compared to the first modified antibody, or antigen-binding portion thereof.

109. A method of affinity maturation of an antibody, or antigen-binding portion thereof, for a target antigen, comprising:

a) performing scanning mutagenesis of a first antibody, or antigen-binding portion thereof, comprising producing a plurality of nucleic acid molecules that encode modified forms of a variable heavy chain or a variable light chain of a first antibody, or antigen-binding portion thereof, wherein the nucleic acid molecules contain one codon that encodes another amino acid compared to the corresponding codon of the unmodified variable heavy or variable light chain, or portion thereof, that does not encode the other amino acid, whereby each nucleic acid molecule of the plurality encodes a variable heavy chain or variable light chain, or portion thereof, that is modified by replacement of a single amino acid residue to another amino acid such that every position across the full-length of the encoded variable heavy or light chain, or portion thereof, is replaced or every position in a selected region of the encoded variable heavy or variable light chain, or portion thereof, is replaced, whereby each replacement is to the same amino acid residue;

b) producing a plurality of modified antibodies, or antigen-binding portions thereof, each comprising a variable heavy chain and a variable light chain, or a portion thereof, wherein at least one of the variable heavy chain or variable light chain, or portion thereof, is one produced in step a), whereby each of the plurality of antibodies, or antigen-binding portions thereof, contains replacement of an amino acid position with another amino acid compared to the first antibody, or antigen-binding portion thereof;

c) screening each of the plurality of modified antibodies, or antigen-binding portions thereof, for an activity to the target antigen;

d) selecting a second antibody, or antigen-binding portion thereof, from among the modified antibodies, or antigen-binding portions thereof, that exhibits retained or increased activity for the target antigen compared to the first antibody, or antigen-binding portion thereof, not containing the amino acid replacement;

e) performing further mutagenesis of the second antibody, or antigen-binding portion thereof, comprising producing a plurality of nucleic acid molecules that encode modified forms of a variable heavy chain or a variable light chain, or portion thereof, of the second antibody, or antigen-binding portion thereof, wherein the nucleic acid molecules contain one codon encoding an amino acid at the scanned amino acid position that encodes a different amino acid than the scanned amino acid in the second antibody, or antigen-binding portion thereof, whereby each nucleic acid molecule of the plurality encodes a variable heavy chain or variable light chain, or portion thereof, that is modified at the scanned amino acid position by a single amino acid residue; and

f) producing a plurality of further modified antibodies, or antigen-binding portions thereof, each comprising a variable heavy chain and a variable light chain, or a portion thereof, wherein at least one of the variable heavy chain or variable light chain, or portion thereof, is one produced in step e), whereby the scanned amino acid position contains replacement to a different amino acid compared to the second antibody, or antigen-binding portion thereof;

g) screening each of the plurality of further modified antibodies, or antigen-binding portions thereof, for an activity to the target antigen; and

h) selecting a third antibody, or antigen-binding portion thereof, that exhibits increased activity for the target antigen compared to the first and/or second antibody, or antigen-binding portion(s) thereof.

110. A method according to claim 109 that further includes at least one of the following:

a) wherein in step a) every position in a region of the encoded variable heavy or variable light chain is replaced;

b) wherein the selected region is a complementary determining region in the variable heavy chain or variable light chain selected from the group consisting of a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3;

c) wherein a second antibody, or antigen-binding portion thereof, is selected that exhibits an activity that is at least 75% or more of the activity of the corresponding form of the first antibody; is 75% to 200% of the activity of the corresponding form of the first antibody, or antigen-binding portion thereof; or is at least or about 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 130%, 140%, 150%, 200% or more of the activity of the corresponding form of the first antibody, or antigen-binding portion thereof;

d) a method further comprising after step d) determining the amino acid residue position that is modified in the second antibody, or antigen-binding portion thereof, to contain a scanned amino acid compared to the first antibody, or antigen-binding portion thereof, not containing the amino acid replacement;

e) wherein the other amino acid is selected from the group consisting of alanine, threonine, proline, glycine, and non-natural amino acid;

f) wherein each of the plurality of nucleic acid molecules encodes a variable heavy chain or variable light chain, or portion thereof, that is modified by replacement of a single amino acid residue to the same scanned amino acid;

g) wherein an activity is selected from the group consisting of binding, signal transduction, differentiation, alteration of gene expression, cellular proliferation, apoptosis, chemotaxis, cytotoxicity, cancer cell invasion, endothelial cell proliferation and tube formation;

h) wherein the activity is selected from the group consisting of:

i. binding, optionally binding as assessed by a method selected from the group consisting of an immunoassay, optionally an immunoassay selected from the group consisting of a radioimmunoassay, an enzyme linked immunosorbent assay (ELISA), and an electrochemiluminescence assay, wherein the electrochemiluminescence assay optionally is meso-scale discovery (MSD); whole cell panning; and surface plasmon resonance (SPR);

ii. signal transduction;

iii. differentiation;

iv. alteration of gene expression;

v. cellular proliferation;

vi. apoptosis;

vii. chemotaxis;

viii. cytotoxicity;

ix. cancer cell invasion;

x. endothelial cell proliferation; and

xi. tube formation;

i) wherein the plurality of nucleic acid molecules produced in step e) are generated by a method selected from the group consisting of PCR mutagenesis, cassette mutagenesis, site-directed mutagenesis, random point mutagenesis, mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA, point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, and double-strand break repair;

j) wherein the plurality of nucleic acid molecules produced in step e) are generated by a method selected from the group consisting of NNK, NNS, NNN, NNY or NNR mutagenesis

k) wherein in step e), the scanned amino acid position is modified by amino acid replacement to all other amino acid residues, or to a restricted subset thereof, wherein optionally the modification does not include amino acid replacement to the scanned amino acid or to the original amino acid at that position in the first antibody, or antigen-binding portion thereof;

l) wherein the target antigen is selected from the group consisting of a polypeptide, carbohydrate, lipid, nucleic acid, and a small molecule;

m) wherein the target antigen is expressed on the surface of a virus, bacteria, tumor or other cell, or is a recombinant protein or peptide;

n) wherein the target antigen is a protein that is a target for therapeutic intervention;

o) wherein the target antigen is involved in cell proliferation and differentiation, cell migration, apoptosis or angiogenesis;

p) wherein the target antigen is selected from the group consisting of a VEGFR-1, VEGFR-2, VEGFR-3 (vascular endothelial growth factor receptors 1, 2, and 3), a epidermal growth factor receptor (EGFR), ErbB-2, ErbB-3, IGF-R1, C-Met (also known as hepatocyte growth factor receptor; HGFR), DLL4, DDR1 (discoidin domain receptor), KIT (receptor for c-kit), FGFR1, FGFR2, FGFR4 (fibroblast growth factor receptors 1, 2, and 4), RON (recepteur d'origine nantais; also known as macrophage stimulating 1 receptor), TEK (endothelial-specific receptor tyrosine kinase), TIE (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains receptor), CSF1R (colony stimulating factor 1 receptor), PDGFRB (platelet-derived growth factor receptor B), EPHA1, EPHA2, EPHB1 (erythropoietin-producing hepatocellular receptor A1, A2 and B1), TNF-R1, TNF-R2, HVEM, LT-βR, CD20, CD3, CD25, NOTCH, G-CSF-R, GM-CSF-R, EPO-R., a cadherin, an integrin, CD52, CD44, VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF, EGF, HGF, TNF-α, LIGHT, BTLA, lymphotoxin (LT), IgE, G-CSF, GM-CSF and EPO;

q) wherein the first antibody, or antigen-binding portion thereof, binds to the target antigen with a binding affinity when the antibody, or antigen-binding portion thereof, is in a Fab form that is about 10⁻⁴ M or lower; about 10⁻⁴ M to about 10⁻⁸ M; or that is at or about 10⁻⁴ M, 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, or lower;

r) wherein scanning mutagenesis is performed within the variable heavy chain of the first antibody, or antigen-binding portion thereof, and steps a)-h) are performed therefrom;

s) wherein scanning mutagenesis is performed within the variable light chain of the first antibody, or antigen-binding portion thereof, and steps a)-h) are performed therefrom

t) wherein scanning mutagenesis is performed within the variable heavy chain of the first antibody and steps a)-h) are performed therefrom, and separately and independently, scanning mutagenesis is performed within the variable light chain of the first antibody, and steps a)-h) are performed therefrom;

u) wherein the third antibody, or antigen-binding portion thereof, exhibits at least 2-fold improved activity for the target antigen compared to the first and/or second antibody(ies), or antigen-binding portion(s) thereof; 2-fold to 10000-fold or 2-fold to 1000-fold improved activity for the target antigen compared to the first and/or second antibody(ies), or antigen-binding portion(s) thereof; or at least 2-fold, 5-fold, 10-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 10000-fold or more improved activity for the target antigen compared to the first and/or second antibody(ies), or antigen-binding portion(s) thereof;

v) wherein the third antibody, or antigen-binding portion thereof, exhibits a binding affinity that is greater than the binding affinity of the first antibody, or antigen-binding portion thereof, and is about 1×10⁻⁹ M or less; is 1×10⁻⁹ M to 1×10⁻¹¹ M; or is or is about 1×10⁻⁹ M, 2×10⁻⁹ M, 3×10⁻⁹ M, 4×10⁻⁹ M, 5×10⁻⁹ M, 6×10⁻⁹ M, 7×10⁻⁹ M, 8×10⁻⁹ M, 9×10⁻⁹ M, 1×10⁻¹⁰ M, 2×10⁻¹⁰ M, 3×10⁻¹⁰ M, 4×10⁻¹⁰ M, 5×10⁻¹⁰ M, 6×10⁻¹⁰ M, 7×10⁻¹⁰ M, 8×10⁻¹⁰ M, 9×10⁻¹⁰ M, or less;

w) further comprising determining the amino acid modifications that are altered in the third antibody, or antigen-binding portion thereof, compared to the first antibody, or antigen-binding portion thereof, not containing the amino acid replacements;

x) wherein the method is repeated iteratively, and wherein the third antibody, or antigen-binding portion thereof, identified in step h) is selected and used in step a) as the first antibody, or antigen-binding portion thereof, for subsequent maturation thereof, whereby the amino acid residue that is modified is not further modified in subsequent iterations of the method;

y) wherein one or more amino acid replacement in one or more variable heavy chains or one or more variable light chains, or portions thereof, of selected third antibodies, or antigen-binding portions thereof, are combined to generate a further modified antibody, or antigen-binding portion thereof, whereby the further modified antibodies, or antigen-binding portions thereof, are screened for an activity to the target antigen to identify a further modified antibody, or antigen-binding portion thereof, that exhibits an increased activity for the target antigen compared to the first, second, and/or third antibodies, or antigen-binding portions thereof; and

z) wherein the method comprises performing steps a)-h) on the variable heavy chain, or portion thereof, of the first antibody, or antigen-binding portion thereof, and selecting third antibodies, or antigen-binding portion thereof, each containing an amino acid replacement in the variable heavy chain, or portion thereof, compared to the corresponding variable heavy chain, or portion thereof, of the first antibody, or antigen-binding portion thereof;

performing steps a)-h) independently and separately on the variable light chain, or portion thereof, of the first antibody, or antigen-binding portion thereof, and selecting different third modified antibodies, or antigen-binding portions thereof, each containing an amino replacement in the variable light chain, or portion thereof, compared to the corresponding variable light chain, or portion thereof, of the first antibody;

combining the variable heavy chain, or portion thereof, of a third antibody, or antigen-binding portion thereof, with the variable light chain, or portion thereof, of a different third antibody, or antigen-binding portion thereof, to generate a plurality of different further modified antibodies, or antigen-binding portions thereof, each comprising an amino acid replacement of the variable heavy chain, or portion thereof, and variable light chain, or portion thereof, compared to the corresponding variable heavy or light chains, or portion(s) thereof, of the first antibody, or antigen-binding portion thereof;

screening each of the plurality of further modified antibodies, or antigen-binding portions thereof, for binding to the target antigen; and

selecting those fourth antibodies, or antigen-binding portions thereof, that exhibit an increased activity for the target antigen compared to the first, second, and/or third antibodies, or antigen-binding portions thereof; and

aa) wherein the antibody, or antigen-binding portion thereof, comprising a variable heavy chain and a variable light chain, or a portion thereof, is selected from the group consisting of a Fab, Fab′, F(ab′)₂, single-chain Fv (scFv), scFab, Fv, dsFv, diabody, Fd, Fd′, Fab fragment, Fd fragment, Fd′ fragment, scFv fragment, and scFab fragment.

111. A method according to claim 109, further comprising after selecting a third antibody in step h),

i) selecting another different region within the variable heavy chain or variable light chain of the third antibody, or antigen-binding portion thereof, optionally a different region selected from the group consisting of a CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4, for further mutagenesis;

j) producing a plurality of nucleic acid molecules that encode modified forms of the variable heavy chain or variable light chain of the third antibody, or antigen-binding portion thereof, wherein the nucleic acids molecules contain one codon encoding an amino acid in the selected region that encodes a different amino acid from the first modified variable heavy or variable light chain, whereby each nucleic acid molecule of the plurality encodes a variable heavy chain or variable light chain that is modified in the selected region by replacement of a single amino acid residue;

k) producing a plurality of further modified antibodies each comprising a variable heavy chain and a variable light chain, or a portion thereof, wherein at least one of the variable heavy chain or variable light chain is one produced in step j), whereby the selected region in each of the plurality of antibodies, or antigen-binding portions thereof, contains replacement of an amino acid to a different amino acid compared to the third antibody, or antigen-binding portion thereof;

l) screening each of the plurality of further modified antibodies, or antigen-binding portions thereof, for binding to the target antigen; and

m) selecting those further modified antibodies, or antigen-binding portions thereof, that exhibit increased activity for the target antigen compared to the third antibody, or antigen-binding portion thereof.

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