HK40007646B - Asgpr antibodies and uses thereof - Google Patents

Description

ASGPR antibodies and uses thereof

The application is a divisional application of Chinese patent application 201380041685.8, the application date of the original application is 8/6 in 2013, and the invention name is ASGPR antibody and application thereof.

Technical Field

The present invention relates generally to antibodies specific for asialoglycoprotein receptor (ASGPR) and their use for the selective delivery of effector moieties that affect cellular activity. In addition, the invention relates to polynucleotides encoding such antibodies and vectors and host cells comprising such polynucleotides. The invention also relates to methods for producing the antibodies of the invention, and to methods of using them in the treatment of disease.

Background

Asialoglycoprotein receptor (ASGPR) is a transmembrane receptor composed of two subunits, H1 and H2. It is believed that these subunits oligomerize through their extracellular stem regions. ASGPR is a member of the C-type lectin family (calcium ion-dependent lectins) and mediates endocytosis and degradation of a wide variety of desialylated glycoproteins. ASGPR is selectively expressed on liver parenchymal cells (hepatocytes), which makes it an attractive target for liver-specific therapies. Many liver diseases, such as hepatitis, cirrhosis or hepatocellular carcinoma (HCC), may be caused directly or indirectly by viral infections such as Hepatitis B Virus (HBV) or Hepatitis C Virus (HCV) infection. Chronic infection with HCV is one of the major causes of cirrhosis and HCC. Similarly, chronic HBV infection accounts for 5-10% of chronic liver disease and cirrhosis in the united states. Approved therapies for HBV and HCV infection include Interferons (IFNs), such as interferon alpha. However, in many cases, side effects have hampered the development and widespread use of these therapies. It is believed that such IFN-related side effects are due in part to genes that induce interferon stimulation in peripheral blood cells (ISGs) following systemic exposure to IFN. Therefore, in order to minimize the side effects associated with IFN therapy for liver diseases and also to enhance the antiviral effects of conventional interferons, it is desirable to selectively deliver IFN to the liver. ASGPR has been considered as a potential target molecule on hepatocytes for such selective delivery. For example, WO92/22310 describes a protocol for targeting interferon to the liver by conjugation of recombinant IFN to asialoglycoprotein. In a similar scheme, the interferon molecule itself has been modified to produce asialo-interferon that binds to ASGPR (Eto and Takahashi, Nat Med 5,577-581 (1999)). More recently, a protocol based on anti-ASGPR single variable domain (dAb) antibodies has been described (WO 2011/086143).

However, none of these approaches have shown clinical success to date, and there remains a need for improved targeting molecules for selective delivery of therapeutic molecules, such as interferons, to the liver. The antibodies of the invention combine several advantageous properties which make them particularly suitable for targeting effector moieties such as interferons to ASGPR expressing cells, for example for the treatment of liver disease.

Summary of The Invention

In one aspect, the invention provides an antibody capable of specifically binding to asialoglycoprotein receptor (ASGPR), wherein the antibody comprises a) a heavy chain variable region sequence of SEQ ID NO:16 and a light chain variable region sequence of SEQ ID NO: 14; b) the heavy chain variable region sequence of SEQ ID NO 4 and the light chain variable region sequence of SEQ ID NO 2; c) the heavy chain variable region sequence of SEQ ID NO 8 and the light chain variable region sequence of SEQ ID NO 6; d) the heavy chain variable region sequence of SEQ ID NO 12 and the light chain variable region sequence of SEQ ID NO 10; e) the heavy chain variable region sequence of SEQ ID NO 20 and the light chain variable region sequence of SEQ ID NO 18; f) the heavy chain variable region sequence of SEQ ID NO 24 and the light chain variable region sequence of SEQ ID NO 22; g) the heavy chain variable region sequence of SEQ ID NO 28 and the light chain variable region sequence of SEQ ID NO 26; h) the heavy chain variable region sequence of SEQ ID NO 4 and the light chain variable region sequence of SEQ ID NO 30; i) the heavy chain variable region sequence of SEQ ID NO 4 and the light chain variable region sequence of SEQ ID NO 32; j) the heavy chain variable region sequence of SEQ ID NO 4 and the light chain variable region sequence of SEQ ID NO 34; or k) the heavy chain variable region sequence of SEQ ID NO. 24 and the light chain variable region sequence of SEQ ID NO. 22.

In one embodiment, the antibody comprises the heavy chain variable region sequence of SEQ ID NO 16 and the light chain variable region sequence of SEQ ID NO 14. In another specific embodiment, the antibody comprises the heavy chain variable region sequence of SEQ ID NO. 4 and the light chain variable region sequence of SEQ ID NO. 2.

In yet another aspect, the invention provides an antibody capable of specifically binding to ASGPR, wherein the antibody competes for binding to an epitope of ASGPR with an antibody comprising the heavy chain variable region sequence of SEQ ID No. 16 and the light chain variable region sequence of SEQ ID No. 14. In one embodiment, the antibody recognizes an epitope in the stem region of ASGPR. In one embodiment, the antibody is an affinity matured variant of an antibody comprising the heavy chain variable region sequence of SEQ ID NO 16 and the light chain variable region sequence of SEQ ID NO 14. In one embodiment, the antibody comprises a heavy chain variable region sequence that is at least about 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO 16 and a light chain variable region sequence that is at least about 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO 14. In one embodiment, the antibody comprises the light chain variable region sequence of SEQ ID No. 14 with one, two, three, four, five, six or seven, particularly two, three, four or five amino acid substitutions. In one embodiment, the antibody comprises the heavy chain variable region sequence of SEQ ID NO 16 with one, two, three, four, five, six or seven, particularly two, three, four or five amino acid substitutions.

In yet another aspect, the invention provides an antibody that specifically binds to ASGPR, wherein the antibody competes for binding to an epitope of ASGPR with an antibody comprising the heavy chain variable region sequence of SEQ ID No. 4 and the light chain variable region sequence of SEQ ID No. 2. In one embodiment, the antibody recognizes an epitope in the Carbohydrate Recognition Domain (CRD) of ASGPR. In one embodiment, the antibody is an affinity matured variant of an antibody comprising the heavy chain variable region sequence of SEQ ID NO. 4 and the light chain variable region sequence of SEQ ID NO. 2. In one embodiment, the antibody comprises a heavy chain variable region sequence that is at least about 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO. 4 and a light chain variable region sequence that is at least about 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO. 2. In one embodiment, the antibody comprises the light chain variable region sequence of SEQ ID No. 2 with one, two, three, four, five, six or seven, particularly two, three, four or five amino acid substitutions. In one embodiment, the antibody comprises the heavy chain variable region sequence of SEQ ID No. 4 with one, two, three, four, five, six or seven, particularly two, three, four or five amino acid substitutions. In one embodiment, the antibody comprises a) the heavy chain variable region sequence of SEQ ID NO 4 and the light chain variable region sequence of SEQ ID NO 36; b) the heavy chain variable region sequence of SEQ ID NO 4 and the light chain variable region sequence of SEQ ID NO 38; c) the heavy chain variable region sequence of SEQ ID NO 4 and the light chain variable region sequence of SEQ ID NO 40; d) the heavy chain variable region sequence of SEQ ID NO 4 and the light chain variable region sequence of SEQ ID NO 42; e) the heavy chain variable region sequence of SEQ ID NO 4 and the light chain variable region sequence of SEQ ID NO 44; f) the heavy chain variable region sequence of SEQ ID NO 4 and the light chain variable region sequence of SEQ ID NO 46; or g) the heavy chain variable region sequence of SEQ ID NO 4 and the light chain variable region sequence of SEQ ID NO 48.

In one embodiment, the antibody of the invention is capable of specifically binding to human ASGPR and cynomolgus monkey ASGPR. In one embodiment, the antibody has a dissociation constant (K) of less than 1 μ M, particularly less than 100nM, more particularly less than 1nM, as measured by Surface Plasmon Resonance (SPR) as a Fab fragment_D) Binding to human ASGPR. In one embodiment, the antibody has a K of less than 1 μ M, particularly less than 500nM, more particularly less than 100nM or even less than 10nM, when measured as IgG1 by fluorescence resonance energy transfer method (FRET)_DBinding to human ASGPR. In one embodiment, the antibody does not compete with the natural ligand of ASGPR for binding to ASGPR. In a specific embodiment, the natural ligand of ASGPR is asialofetuin. In one embodiment, the antibody does not detectably bind to CLEC10A, particularly human CLEC 10A. In one embodiment, the antibody does not specifically bind to cells, particularly human cells, more particularly human blood cells, lacking ASGPR expression. In one embodiment, once the antibody binds to ASGPR on the surface of an ASGPR-expressing cell, the antibody is internalized into the cell. In a particular embodiment, the antibody is recycled back to the surface of the cell at approximately the same rate as it is internalized into the cell. In one embodiment, once the antibody binds to ASGPR on the surface of the cell, the antibody does not significantly cause downregulation of ASGPR expression at the surface of the cell.

In one embodiment, the antibody of the invention is a human antibody. In one embodiment, the antibody comprises a human Fc region, particularly an IgG Fc region, more particularly an IgG Fc region₁An Fc region. In one embodiment, the antibody is a full length antibody. In one embodiment, the antibody is an IgG class antibody, particularly an IgG1 subclass antibody. In one embodiment, the antibody comprises an Fc regionModifications that reduce the binding affinity of an antibody to an Fc receptor, particularly an fey receptor. In a specific embodiment, the Fc receptor is an activating Fc receptor. In yet another specific embodiment, the Fc receptor is selected from the group consisting of Fc γ RIIIa (CD16a), Fc γ RI (CD64), Fc γ RIIa (CD32), and Fc α RI (CD 89). In a more specific embodiment, the Fc receptor is Fc γ RIIIa, particularly human Fc γ RIIIa. In one embodiment, the antibody comprises an amino acid substitution within the Fc region at a position selected from the group consisting of P329, L234 and L235(EU numbering). In one embodiment, the antibody comprises the amino acid substitutions P329G, L234A and L235A in the Fc region (EU numbering). In yet another embodiment, the antibody comprises a modification in the Fc region that promotes heterodimerization of two non-identical antibody heavy chains. In a specific embodiment, the modification is a knob-into-hole (knob-hole) modification, including a knob modification in one of the antibody heavy chains and a hole modification in the other of the two antibody heavy chains. In one embodiment, the antibody comprises a modification within the interface between two antibody heavy chains in the CH3 domain, wherein i) in the CH3 domain of one heavy chain, amino acid residues are replaced with amino acid residues having a larger side chain volume, thus creating a protuberance ("knob") within the interface in the CH3 domain of one heavy chain, which protuberance is positionable in a cavity ("pocket") within the interface in the CH3 domain of the other heavy chain, and ii) in the CH3 domain of the other heavy chain, amino acid residues are replaced with smaller amino acid residues having a side chain volume, thus creating a cavity ("pocket") within the interface in the second CH3 domain, within which cavity a protuberance ("knob") within the interface in the first CH3 domain is positionable. In one embodiment, the antibody comprises the amino acid substitution T366W and optionally amino acid substitution S354C in one of the antibody heavy chains and the amino acid substitutions T366S, L368A, Y407V and optionally Y349C in the other antibody heavy chain.

In one aspect, the invention provides an antibody capable of specifically binding to ASGPR according to any one of the above embodiments, wherein an effector moiety is linked to the antibody. In one embodiment, no more than one effector moiety is attached to the antibody. In one embodiment, the effector moiety is a cytokine molecule. In one embodiment, the cytokine molecule is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the antibody heavy chains, optionally via a peptide linker. In one embodiment, the cytokine molecule is a human cytokine. In one embodiment, the cytokine molecule is an interferon molecule. In a specific embodiment, the interferon molecule is interferon alpha, in particular human interferon alpha, more particularly human interferon alpha 2 (see SEQ ID NO:138) or human interferon alpha 2a (see SEQ ID NO: 139)). In one embodiment where the cytokine molecule is an interferon molecule, the antibody has antiviral activity in cells expressing ASGPR on the cell surface. In a specific embodiment, the antibody has no antiviral activity in cells that do not express significant levels of ASGPR on the cell surface. In yet another embodiment, the antiviral activity is selected from the group consisting of inhibiting viral infection, inhibiting viral replication, inhibiting cell killing, and inducing interferon stimulation.

The invention also provides polynucleotides encoding the antibodies of the invention. Also provided are vectors, particularly expression vectors, comprising a polynucleotide of the invention. In another aspect, the invention provides a host cell comprising a polynucleotide or vector of the invention. The invention also provides a method for producing an antibody of the invention comprising the steps of (i) culturing a host cell of the invention under conditions suitable for expression of the antibody, and (ii) recovering the antibody. Antibodies produced by the methods that are capable of specifically binding to ASGPR are also provided.

In one aspect, the invention provides a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable carrier. The antibodies or pharmaceutical compositions of the invention are also provided for use as a medicament and for the treatment or prevention of liver diseases, in particular viral infections, more particularly hepatitis virus infections, especially Hepatitis B Virus (HBV) infections. The antibodies or pharmaceutical compositions of the invention are also provided for the treatment or prevention of cancer, in particular liver cancer, more particularly hepatocellular carcinoma (HCC). Further provided are antibodies of the invention for use in the manufacture of a medicament for treating a disease in a subject in need thereof and methods of treating a disease in a subject comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody of the invention in a pharmaceutically acceptable form. In one embodiment, the disease is a liver disease. In a more specific embodiment, the liver disease is a viral infection. In an even more specific embodiment, the liver disease is a hepatitis virus infection, in particular an HBV infection. In another embodiment, the disease is cancer. In a more specific embodiment, the cancer is liver cancer. In an even more specific embodiment, the liver cancer is hepatocellular carcinoma (HCC). In one embodiment, the individual is a mammal, in particular a human. In yet another aspect, antibodies of the invention are provided for targeting cells expressing ASGPR in an individual. Also provided are methods for targeting cells expressing ASGPR in an individual comprising administering to the individual a composition comprising an antibody of the invention in a pharmaceutically acceptable form. In one embodiment, the cell is a liver cell, in particular a hepatocyte. In one embodiment, the individual is a mammal, in particular a human.

Brief Description of Drawings

FIG. 1 is a schematic representation of the resulting antigenic construct. Nucleotide sequences of total antigens and human-derived IgG₁The C-terminus of the Fc sequence is fused. ASGPR 1-derived and CLEC 10A-derived CRDs are fused to and co-expressed with sequences encoding Fc (hole) fragments, resulting in the display of a single dimer per Fc dimer. From left to right: Fc-CRD (ASGPR1), Fc-stem (ASGPR1), Fc-stem-CRD (ASGPR1), Fc-CRD (CLEC10A), Fc-stem (CLEC10A), Fc-stem-CRD (CLEC 10A). Thick bending line: (G)₄S)₃A joint; rough straight line: xa and IgAse cleavage sites.

FIG. 2. schematic diagram of the generation of a randomized generic Fab library within the CDR3 region of the heavy and light chains. In the first step, three PCR fragments were generated, which were subsequently fused by PCR (splicing by overlap extension; SOE). The final fragment was gel purified, digested with NcoI/NheI, while the acceptor phagemid was similarly treated, ligated and transformed into bacteria. PCR1(A, B, C): (1) LMB 3; (2) (A) VI _3_19_ L3r _ V/(B) VI _3_19_ L3r _ HV/(C) VI _3_19_ L3r _ HLV. PCR 2: (3) RJH 80; (4) DP47CDR3_ ba (mod). PCR3(A, B, C): (5) (A) DP47v44/(B) DP47v46/(C) DP47v 48; (6) fdseqlong.

FIG. 3 selection of anti-human ASGPR H1 specific clones as human IgG₁Binding analysis of antibodies to HepG2 cells. The antibody concentration was 30. mu.g/ml. Isotype control antibodies served as negative controls.

FIG. 4 FRET analysis of transiently transfected cells expressing terbium labeled transmembrane ASGPR H1-SNAP tag fusion protein. The analysis was performed by: antibodies were added at concentrations ranging from 50-0.39nM, followed by anti-human Fc-d2 (final 200nM per well) as the acceptor molecule. After 3 hours, the specific FRET signal was measured and the KD value (KD) was calculated_51A12＝200nM，KD_R7F12＝22nM，KD_R9E10＝6.2nM，KD_R5C2＝5.9nM，KD_4F3＝4.5nM)。

FIGS. 5 and 6 competition of asialofetuin (a natural ligand for ASGPR) and anti-ASGPR H1 antibody. HepG2 cells were pre-incubated with labeled asialofetuin prior to addition of the indicated antibodies to the cells in the dilution column. The binding of both components to cells was analyzed by FACS analysis. (A) Detecting an antibody; (B) and (3) detecting asialo fetuin.

FIG. 7. internalization assay of two anti-human ASGPR H1 antibodies, clones 51A12 and 4F3, as IgG. (A) Antibodies were incubated with HepG2 cells at 4 ℃ to prevent internalization and washed at 4 ℃, after which the cells were cultured in pre-warmed medium and incubated at 37 ℃ for up to 120 minutes. Samples were removed at the indicated time points, labeled with secondary antibody on ice and fixed using PFA. (B) The same procedure was performed as described under (a), except that the antibody was incubated with the cells at 37 ℃ to allow for ASGPR internalization. (C) The same procedure was carried out as described under (A) except that a direct FITC-labeled antibody was used. Cell surface bound antibodies were detected using PE conjugated anti-Fc antibodies. (D) The same experiment was performed as described under (C), but showing FITC signals representative of surface exposed and internalized antibodies.

FIG. 8 randomization strategy of LCDR3 region of clone 51A 12. Shows (a) the LCDR3 protein sequence of parental clone 51a12, (B) the LCDR3 protein sequence of the plasmid that serves as a library template without cysteine and glycosylation sequences, and (C) randomized positions in LCDR 3. During the generation of the library, the trinucleotide primers allow the exclusion of triplets encoding cysteine or amino acids that contribute to the formation of glycosylation sites.

FIG. 9. schematic diagram of generation of affinity maturation library randomized in LCDR3 of template 51A12(A82G, C112S, C113S, S116A) (SEQ ID NO: 33). In the first step, two PCR fragments were generated, which were subsequently fused by (SOE) PCR. The final fragment was gel purified, digested with NcoI/PstI, while the acceptor phagemid was similarly treated, ligated and transformed into bacteria. PCR1 (1) LMB3, (2) LCDR3 rev. PCR 2: (3) LCDR3rand, (4) fdseqlong.

FIG. 10 binding analysis of affinity matured 51A12 derived clones as human Fab fragments to HepG2 cells. Using Fab concentrations: 10. 3.3 and 1.1. mu.g/ml. Parental clones 51A12(SEQ ID NOS: 2 and 4), 51A12(S116A) (SEQ ID NOS: 4 and 30) (clones lacking glycosylation sequences) and 51A12(A82G, C112S, C113S, S116A) (SEQ ID NOS: 4 and 34) (template clones of affinity maturation libraries) served as controls.

FIG. 11 affinity matured 51A12 derived clone as human IgG₁Binding analysis of antibodies to HepG2 cells. Concentrations in the dilution series ranging from 0.01 to 20. mu.g/ml were used. The parental clone 51A12(SEQ ID NOS: 2 and 4) served as a control (A). Binding assay to Hela cells was performed using a negative control at a concentration of 10. mu.g/ml (B).

Figure 12 is a schematic of the antibody-cytokine conjugates produced. The gene encoding interferon- α 2a was fused to the C-terminus of the heavy chain of an ASGPR H1-specific antibody comprising a knob modification. While bivalent ASGPR binding of antibody-cytokine proteins was achieved by co-expressing the corresponding hole-modified ASGPR H1-specific heavy and light chains ((a), 2:1 valency), expression of the Fc (hole) fragment sequence resulted in a monomeric antibody-cytokine conjugate ((B), 1:1 valency) with only one ASGPR H1-specific binding site per molecule. Small black dots: modifications to prevent Fc γ R binding (e.g. L234A L235A P329G). Large black spots: modifications that promote heterodimerization (e.g., protuberance into a hole).

FIGS. 13-19. purification and analytical characterization of selected antibody-IFN α immunoconjugates (FIG. 13: 51A12kih IgG-IFN α; FIG. 14: 4F3kih IgG-IFN α; FIG. 15: 51A12(C7) kih IgG-IFN α; FIG. 16: 51A12(C1) kih IgG-IFN α; FIG. 17: 51A12(E7) kih IgG-IFN α; FIG. 18: isotype control kih IgG-IFN α; FIG. 19: monovalent 51A12kih IgG-IFN α). The purification process involved an affinity step (protein a) (a) followed by size exclusion chromatography (Superde x200, GE Healthcare) (B). The final product was analyzed and characterized by analytical size exclusion chromatography (Superde x200 column) (C) and microfluidic protein analysis (Caliper) or SDS-PAGE (D).

FIG. 20 binding selectivity of ASGPR specific IgG kih IFN α fusion constructs 51A12(A) and 4F3 (B). HepG2, human primary hepatocytes, Huh-7 cells, A549 cells, Hela cells and 293T cells were incubated with 1. mu.g/ml of 51A12-IFN α (A) or 4F3-IFN α (B) on ice for 45 minutes. After three washes, cells were stained with goat anti-human IgG secondary antibody on ice for 30 minutes and cells were washed 3 times before analysis using Calibur flow cytometry. Use ofThe R-phycoerythrin human IgG labeling kit binding to human PBMCs was performed by using 1 μ g/ml of directly labeled 51a12IgG kih IFN α (a) and 4F3IgG kih IFN α (B) according to the manufacturer's instructions, using isotypes IgG kih IFN α and CD81mAb as negative and positive controls, respectively.

FIG. 21 saturation curves of ASGPR mAb 4F3-IFN α binding on human primary hepatocytes and HepG2 cells. Saturation of binding of ASGPR mAb 4F3-IFN α on human hepatocytes (A-C) and HepG2 cells (D) derived from three different donors. Cells were incubated with 4F3IgG kih IFN α serially diluted for 45 minutes on ice. After three washes, cells were stained with goat anti-human IgG secondary antibody on ice for 30 minutes and washed 3 more times before analysis using Calibur flow cytometer.

Figure 22 analysis of ASGPR surface exposure levels over time in the presence of specific antibodies. HepG2 cells were incubated with ASGPR-specific clone 51a12IgG or the corresponding monovalent or bivalent antibody-cytokine conjugate. Cell samples were taken up to 5 hours later and the binding of the IgG construct to ASGPR was measured by detecting antibody (a) or cytokine (B). anti-CD 20 antibody (GA101) was used as a negative control.

Figure 23. rapid internalization of clone 51a12 antibody-cytokine conjugate. The Alexa 488-labeled 51a12IgG kih IFN α construct was incubated with HepG2 cells and ASGPR-mediated construct internalization was recorded by confocal microscopy in a10 stack range for 1 hour (z-level). Binding of antibody-cytokine conjugates on the cell surface occurs in a clustered fashion, rather than uniformly distributed (a). Once bound to the cell surface, the conjugate is very rapidly internalized in vesicles that are transported into the cell body (B, enclosed single cells). The vesicles are then recirculated back to the top surface of the cells (not shown).

Figure 24 antiviral activity of ASGPR mAb-IFN α molecules and other control IFN molecules in EMCV CPE (a) and HCV replicon (B) assays. (A) Hela cells were pretreated with serial dilutions of IFN molecules for 3 hours, after which EMCV virus was added. Cells were cultured for 24 hours and cell viability was measured by addition of CellTiter Glo. (B) Huh-72209 replicon cells were treated with serially diluted IFN molecules and luciferase activity was measured 3 days later.

FIG. 25 ISG was induced by 51A12-IFN α in various hepatocytes and non-hepatocytes. Hepatocytes (primary hepatocytes (B) and HepG2(a)) and non-hepatocytes (human pbmc (d) and hela (c)) were treated with multiple serial dilutions of IFN α molecules for 6 hours, total RNA was extracted and TaqMan RT-PCR was used to quantify ISG MX1(A, C) and Rsad2(B, D) gene expression. Data shown are from three or more experiments.

FIG. 26 ISG in human primary hepatocytes (PHH) (B) and Huh7(A) cells was persistently induced by 51A12-IFN α and 4F3-IFN α. Human primary hepatocytes (PHH) and Huh7 cells were treated with serially diluted IFN α molecules for 6 hours (left) and 72 hours (right), total RNA was extracted and TaqMan RT-PCR was used to quantify ISG MX1(a) and Rsad2(B) gene expression.

Figure 27 representative ISG expression in monkey liver samples. Monkey liver samples from the four dose groups taken at different time points were analyzed by microarray. The expression of 4 representative ISG genes is shown in 3D. 4 dose groups are indicated. For each dose group, bars represent the fold induction of ISG at day-5, day 2, day 4 and day 8 of 3 monkeys, from left to right.

FIG. 28 blood and liver gene expression heatmap (IFN Module M3.1). Blood PBMC samples and liver biopsy samples were subjected to mRNA microarray analysis and their IFN α responses were analyzed using gene modules determined from blood transcriptomics studies (Chaussabel et al, (2008), Immunity 29,150-64). In panel (a), fold change expression values of the genes of the interferon module M3.1 from baseline (see inset) were plotted as a heat map for blood and liver samples using the R-statistics software package (www.r-project. Unsupervised hierarchical clustering of hepatic interferon-induced genes revealed a subset (dashed rectangles) that were highly induced at a dose of 10 μ g/kg of 51a12, but not the isotype-IFN α compounds on days 1 and 3. (B) This gene set was plotted against liver and blood. Unsupervised hierarchical clustering of this subset revealed differential expression patterns between blood and liver, with the upper half of the heatmap showing 51a12 induced expression in the liver at 10 μ g/kg dose, but isoform-IFN α did not, and the lower half showing only isoform-IFN α induced expression in blood at high dose.

Detailed Description

Definition of

Unless defined otherwise below, terms are used herein as they are commonly used in the art.

"asialoglycoprotein receptor", abbreviated ASGPR, refers to any native ASGPR from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed ASGPR, as well as any form of ASGPR that results from processing in a cell. The term also encompasses naturally occurring variants of ASGPR, e.g., splice variants or allelic variants. In one embodiment, the antibody of the invention is capable of specifically binding to the extracellular domain of human ASGPR, particularly human ASGPR H1, more particularly human ASGPR H1.

The amino acid sequence of human ASGPR H1 (also known as CLEC4H1) is shown as UniProt (www.uniprot.org) accession number P07306 (version 131) or NCBI (www.ncbi.nlm.nih.gov /) RefSeq NP _ 001662.1. The extracellular domain (ECD) of human ASGPR H1 extends from amino acid position 62 to amino acid position 291. The nucleotide and amino acid sequences of human ASGPR H1ECD fused to a human Fc region are shown in SEQ ID NOS: 129 and 130, respectively. The ASGPR H1ECD comprises a stem region that extends from amino acid position 62 of the full sequence to about amino acid position 160(SEQ ID NOs: 123 and 124 show the nucleotide sequence and amino acid sequence of the stem region of human ASGPR H1 fused to a human Fc region), and a Carbohydrate Recognition Domain (CRD) that extends from amino acid position 161 of the full sequence to about amino acid position 278(SEQ ID NOs: 117 and 118 show the nucleotide sequence and amino acid sequence of the CRD region of human ASGPR H1 fused to a human Fc region).

In one embodiment, the antibody is also capable of binding to the extracellular domain of cynomolgus monkey ASGPR, in particular cynomolgus monkey ASGPR H1, more in particular cynomolgus monkey ASGPR H1. The sequence of cynomolgus monkey ASGPR H1 is shown at NCBI GenBank accession No. EHH 57654.1. 131 and 132 show the nucleotide and amino acid sequences, respectively, of cynomolgus monkey ASGPR H1ECD fused to a human Fc region.

By "human CLEC 10A" is meant the protein described by UniProt accession number Q8IUN9(86 edition), in particular the extracellular domain of said protein, which extends from amino acid position 61 to amino acid position 316 of the complete sequence. 133 and 134 show the nucleotide and amino acid sequences, respectively, of human CLEC10A ECD fused to a human Fc region.

As used herein, the term "conjugate" refers to a fusion polypeptide molecule comprising one effector moiety and another peptide molecule, particularly an antibody. The fusion protein of the antibody and the effector moiety is referred to as an "immunoconjugate". As referred to herein, an (immuno) conjugate is a fusion protein, i.e. the components of the (immuno) conjugate are linked to each other by peptide bonds, either directly or via a peptide linker.

An "epitope" is the region of an antigen to which an antibody binds. The term relates to the site on a polypeptide macromolecule where binding of an antibody to an antibody results in the formation of an antibody-antigen complex (e.g., a stretch of contiguous amino acids or a conformational configuration composed of discrete regions of amino acids).

An "antibody that competes with a reference antibody for binding to an epitope" refers to an antibody that blocks binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks binding of the antibody to its antigen by 50% or more in a competition assay. Exemplary competition assays are provided herein.

As used herein, the term "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to bind to a particular antigen can be measured by enzyme-linked immunosorbent assays (ELISAs) or other techniques familiar to those skilled in the art, such as Surface Plasmon Resonance (SPR) techniques (analysis on a BIAcore instrument) (Liljebelad et al, Glyco J17, 323-229 (2000)) and conventional binding assays (Heeley, Endocr Res 28,217-229 (2002)). In one embodiment, the degree of binding of the antibody to an unrelated protein is less than about 10% of the degree of binding of the antibody to the antigen as measured by SPR. In certain embodiments, the antibody that binds to the antigen has ≦ 1 μ M ≦ 100nM, ≦ 10nM, ≦ 1nM, ≦ 0.1nM, ≦ 0.01nM, or ≦ 0.001nM (e.g., 10 nM)^-8M or less, e.g. from 10^-8M to 10^-13M, e.g. from 10^-9M to 10^-13M) dissociation constant (K)_D)。

"affinity" or "binding affinity" refers to the sum of the strengths of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can be generally determined by the dissociation constant (K)_D) Typically, the dissociation constants are the dissociation and association rate constants (k, respectively)_offAnd k_on) The ratio of (a) to (b). Thus, equivalent affinities may comprise different rate constants, so long as the rates are constantThe ratio of numbers remains the same. Affinity can be measured by common methods known in the art, including those described herein. A specific method for measuring affinity is Surface Plasmon Resonance (SPR).

"reduced binding", e.g. reduced Fc receptor binding, refers to a decrease in affinity of the corresponding interaction, as measured by e.g. SPR. For clarity, the term also includes a reduction in affinity to zero (or below the detection limit of the analytical method), i.e., complete elimination of the interaction. Conversely, "increased binding" refers to an increase in binding affinity of the respective interaction.

By "internalization" is meant that the molecule moves from the surface of the cell into the intracellular space as a result of uptake of the molecule. One particular form of internalization is receptor-mediated endocytosis, which occurs through the inward bud-like bulging of plasma membrane vesicles containing the receptor and bound ligand or antibody upon binding of the ligand or antibody to a cell surface (transmembrane) receptor. Internalization can be assessed using techniques known in the art. Described in the examples herein is a method based on the determination of protein levels on cell surfaces by FACS.

As used herein, the term "recycle" refers to the re-appearance of a molecule on the surface of a cell after the molecule has previously internalized into the cell. Recycling suggests that the molecules are not degraded inside the cell when internalized. If recycling occurs at the same rate as internalization, a dynamic steady state is reached in which the number of molecules on the cell surface remains substantially constant. Recirculation can be detected by techniques well known in the art, for example by determining the level of protein on the cell surface by FACS or using (confocal) microscopy methods as described in the examples below.

By "downregulated" is meant a decrease in the copy number of a protein (e.g., a cell surface receptor) within or at the surface of a cell. Down-regulation as used herein refers in particular to a decrease in the copy number of a cell surface protein present at the cell surface, e.g. due to internalization and/or degradation or reduced expression. Downregulation of protein levels can be detected by a variety of methods established in the art, including, for example, western blotting (for total protein levels) or FACS (for surface protein levels).

As used herein, the term "effector moiety" refers to a molecule, particularly a polypeptide molecule (e.g., a protein or glycoprotein), that affects the activity of a cell (e.g., through signal transduction or other cellular pathways). Thus, the effector moiety may be associated with a receptor-mediated signaling process that propagates signals from outside the cell membrane to modulate a response in the cell carrying the one or more receptors of the effector moiety. In one embodiment, the effector moiety may elicit a cytotoxic response in cells carrying one or more receptors for the effector moiety. In another embodiment, the effector moiety may elicit a proliferative response in cells carrying one or more receptors for the effector moiety. In another embodiment, the effector moiety may stimulate differentiation in cells carrying the receptor for the effector moiety. In another embodiment, the effector moiety may alter (i.e., up-regulate or down-regulate) the expression of an endogenous cellular protein in a cell carrying the receptor for the effector moiety. Non-limiting examples of effector moieties include small molecules, cytokines, growth factors, hormones, enzymes, substrates, and cofactors. The effector moiety may associate with the antibody in a variety of configurations.

The term "linked" includes linkage by any kind of interaction, including chemical or peptide bonds.

"fused" refers to components joined by peptide bonds, either directly or via one or more peptide linkers.

As used herein, the term "cytokine" refers to a molecule that mediates and/or modulates a biological function or process or a cellular function or process (e.g., immunity, inflammation, and hematopoiesis). The term "cytokine" as used herein includes lymphokines, chemokines, monokines, and interleukins. Examples of cytokines include, but are not limited to, GM-CSF, IL-1 α, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IFN- α, IFN- β, IFN- γ, MIP-1 α, MIP-1 β, TGF- β, TNF- α, and TNF- β. A particular cytokine is an Interferon (IFN), in particular IFN- α. In a specific embodiment, the cytokine is a human cytokine. The sequences of human IFN α 2 and IFN α 2a as specific cytokines are shown in SEQ ID NO 138 and 139, respectively.

An "interferon-stimulated gene" (ISG) refers to a gene whose expression in a cell can be stimulated by contacting the cell with an interferon molecule, in particular an IFN α molecule. Generally, an ISG comprises one or more signaling molecules (e.g., STATs) to which interferon-activated signaling molecules can bind, thereby resulting in enhanced recognition sequences for ISG expression (e.g., interferon-stimulated response elements (ISREs)). Examples of ISGs include MX1 (myxovirus resistance 1, also known as interferon-induced protein p78), RSAD2 (containing the free radical S-adenosylmethionine domain 2, also known as cytomegalovirus-induced gene 5), HRASLS2 (HRAS-like repressor protein 2), IFIT1 (interferon-induced protein 1 with triangular tetrapeptide (tetratricopeptide) repeats) and IFITM2 (interferon-induced transmembrane protein 2).

As used herein, the term "single-chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In one embodiment, the effector moiety is a single chain peptide molecule. Non-limiting examples of single-chain effector moieties include cytokines, growth factors, hormones, enzymes, substrates, and cofactors. When the effector moiety is a cytokine and the cytokine of interest normally exists in nature as a multimer, then each subunit of the multimeric cytokine is in turn encoded by a single chain of effector moieties. Thus, non-limiting examples of useful single-chain effector moieties include GM-CSF, IL-1 α, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IFN- α, IFN- β, IFN- γ, MIP-1 α, MIP-1 β, TGF- β, TNF- α, and TNF- β.

As used herein, the term "effector moiety receptor" refers to a polypeptide molecule capable of specifically binding to an effector moiety. In the case where an effector moiety specifically binds to more than one receptor, all of the receptors specifically binding to the effector moiety are "effector moiety receptors" for that effector moiety. For example, where IFN α is an effector moiety, the effector moiety receptor that binds to an IFN α molecule (e.g., an antibody fused to IFN α) is IFN α receptor 1 or 2 (see UniProt accession No. P17181(121 edition) and NCBI RefSeq NP _000620.2 of human IFN α receptor 1, and UniProt accession No. P48551(131 edition) and NCBI RefSeq NP _997467.1 and NP _997468.1 of human IFN α receptor 2).

The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody constructs, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

An "antibody fragment" refers to a molecule other than an intact antibody, which molecule includes a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab '-SH, F (ab')₂Diabodies, linear antibodies, single chain antibody molecules (e.g., scFv), and single domain antibodies. For a review of certain antibody fragments, see Hudson et al, Nat Med 9, 129-. For reviews of scFv fragments, see, for example, Plouckthun, from The Pharmacology of Monoclonal Antibodies, Vol.113, Rosenburg and Moore, Springer-Verlag, New York, p.269-315 (1994); see also WO 93/16185; and U.S. patent nos. 5,571,894 and 5,587,458. Methods for making Fab and F (ab') fragments comprising rescue receptor (salvaging receptor) binding epitope residues and having increased in vivo half-life₂See U.S. Pat. No. 5,869,046 for a discussion of fragments. Diabodies are antibody fragments with two antigen binding sites, which may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; hudson et al, Nat Med 9, 129-; and Hollinger et al, Proc Natl Acad Sci USA 90, 6444-. Tri-and tetrad antibodies are also described in Hudson et al, Nat Med 9,129-134 (2003). Single domain antibodies are antibody fragments that comprise all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of the antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516B 1). Antibody fragments can be produced by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and from heavyThe recombinant host cells are produced (e.g., e.coli or phage), as described herein.

The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain containing an Fc region as defined herein.

"native antibody" refers to a naturally occurring immunoglobulin molecule with a varying structure. For example, a natural IgG class antibody is an heterotetrameric glycoprotein of about 150,000 daltons consisting of two light and two heavy chains bound by disulfide bonds. From N-terminus to C-terminus, each heavy chain has one variable region (VH), also known as variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also known as heavy chain constant regions. Similarly, from N-terminus to C-terminus, each light chain has one variable region (VL), also known as a variable light domain or light chain variable domain, followed by one light chain constant domain (CL), also known as a light chain constant region. Antibody heavy chains can be assigned to one of 5 classes, called α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), some of which can be divided into subclasses, e.g. γ₁(IgG1)、γ₂(IgG2)、γ₃(IgG₃)、γ₄(IgG₄)、α₁(IgA₁) And alpha₂(IgA₂). The light chains of antibodies can be divided into one of two types, called kappa and lambda, based on the amino acid sequences of their constant domains. An IgG class antibody essentially consists of two Fab fragments and one Fc domain connected by an immunoglobulin hinge region.

As used herein, a "Fab fragment" refers to an antibody fragment comprising a light chain fragment comprising the VL domain and constant domain of a light Chain (CL) and the VH domain and first constant domain of a heavy chain (CH 1).

The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region that a heavy chain possesses. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes may be further divided into subclasses (isotypes), e.g., IgG₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂. The heavy chain constant domains corresponding to different classes of antibodies or immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The term "variable region" or "variable domain" refers to a domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, each domain comprising four conserved Framework Regions (FR) and three hypervariable regions (HVRs). See, e.g., Kindt et al, Kuby Immunology, 6 th edition, w.h.freeman and co., page 91 (2007). A single VH domain or VL domain may be sufficient to confer antigen binding specificity.

As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain which is highly variable in sequence and/or forms structurally defined loops ("hypervariable loops"). Typically, a native 4 chain antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and/or from Complementarity Determining Regions (CDRs) which have the highest sequence variability and/or are involved in antigen recognition. Exemplary hypervariable loops are present at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2) and 96-101(H3) (Chothia and Lesk, J.mol.biol.196,901-917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) are present at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3 (Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The exception is CDR1 in VH, which usually comprises amino acid residues that form hypervariable loops. CDRs also contain "specificity determining residues" or "SDRs," which are residues that contact the antigen. SDR is contained within a CDR region, referred to as the abbreviated-CDR or a-CDR. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) are present at amino acid residues 31-34 of L1, amino acid residues 50-55 of L2, amino acid residues 89-96 of L3, amino acid residues 31-35B of H1, amino acid residues 50-58 of H2, and amino acid residues 95-102 of H3 (see Almagro and Fransson, front. biosci.13,1619-1633 (2008)). Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al, supra (referred to as "Kabat numbering").

"framework" or "FR" refers to variable domain residues other than the hypervariable region (HVR) residues. The FRs of the variable domain typically consist of the following 4 FR domains: FR1, FR2, FR3 and FR 4. Thus, HVR and FR sequences are typically present in the VH (or VL) as follows: FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.

An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs), wherein such alterations result in an improvement in the affinity of the antibody for an antigen compared to a parent antibody that does not possess the alterations.

The term "parent antibody" refers herein to an antibody that serves as a starting point or basis for making antibody variants. In one embodiment, the parent antibody is a humanized or human antibody.

A "human antibody" is an antibody that possesses an amino acid sequence corresponding to an antibody produced by a human or human cell or is derived from a non-human source using a human antibody library or other sequences encoding human antibodies. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or occurring during the production of a monoclonal antibody preparation), which are typically present in minute amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the invention can be produced by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods for producing monoclonal antibodies being described herein.

The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. The IgG Fc region comprises an IgG CH2 domain and an IgG CH3 domain. The "CH 2 domain" of the human IgG Fc region typically extends from an amino acid residue at about position 231 to an amino acid residue at about position 340. In one embodiment, the sugar chain is linked to a CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain. The "CH 3 domain" comprises a stretch of residues at the C-terminus of the CH2 domain in the Fc region (i.e., extending from an amino acid residue at about position 341 to an amino acid residue at about position 447 of the IgG). The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having an introduced "overhang" ("knob") in one strand thereof and a corresponding "hole" ("hole") in the other strand thereof; see U.S. Pat. No. 5,821,333, which is expressly incorporated herein by reference). Such variant CH3 domains can be used to promote heterodimerization of two non-identical antibody heavy chains as described herein. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system as described in Kabat et al, Sequences of Proteins of Immunological Interes, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD,1991, also known as the EU index.

The term "effector function" refers to those biological activities attributed to the Fc region of an antibody that vary with antibody isotype. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC), Fc receptor binding, antibody dependent cell mediated cytotoxicity (ADCC), Antibody Dependent Cellular Phagocytosis (ADCP), cytokine secretion, immune complex mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B cell receptors) and B cell activation.

An "activating Fc receptor" is an Fc receptor that, upon engagement by an antibody Fc region, initiates a signaling event that stimulates the receptor-bearing cell to perform effector functions. Activating Fc receptors include Fc γ RIIIa (CD16a), Fc γ RI (CD64), Fc γ RIIa (CD32), and Fc α RI (CD 89). A specific activating Fc receptor is human Fc γ RIIIa (see UniProt accession No. P08637(141 th)).

The term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides include, for example, (G)₄S)_n、(SG₄)_nOr G₄(SG₄)_nA peptide linker. "n" is generally a number between 1 and 10, typically between 2 and 4.

A "modification that promotes heterodimerization" is manipulation of the peptide backbone or post-translational modification of a polypeptide, such as an antibody heavy chain, that reduces or prevents association of the polypeptide with the same polypeptide to form a homodimer. Modifications that promote heterodimerization as used herein specifically include independent modifications made to each of the two polypeptides required to form a dimer, where the modifications are complementary to each other to promote association of the two polypeptides. For example, modifications that promote heterodimerization can alter the structure or charge of one or both of the two polypeptides required to form the dimer, thereby making their association sterically or electrostatically favorable, respectively. Heterodimerization occurs between two non-identical polypeptides, such as two antibody heavy chains, where the other components (e.g., effector moieties) attached to each heavy chain are not identical. In the antibodies of the invention, the modification that promotes heterodimerization is in the Fc domain, particularly in the CH3 domain. In some embodiments, the modification that promotes heterodimerization comprises an amino acid mutation, particularly an amino acid substitution. In a specific embodiment, the modification promoting heterodimerization comprises a separate amino acid mutation, in particular an amino acid substitution, in each of the two antibody heavy chains.

"knob-into-pocket modification" refers to a modification within the interface between two antibody heavy chains in the CH3 domain, wherein i) in the CH3 domain of one heavy chain, amino acid residues are replaced with amino acid residues having a larger side chain volume, thus creating a knob ("knob") within the interface in the CH3 domain of one heavy chain, which can fit into a cavity ("pocket") within the interface in the CH3 domain of the other heavy chain, and ii) in the CH3 domain of the other heavy chain, amino acid residues are replaced with smaller amino acid residues having a side chain volume, thus creating a cavity ("pocket") within the interface in the second CH3 domain, inside which a knob ("knob") within the interface in the first CH3 domain can fit. In one embodiment, a "protuberance-into-hole modification" comprises the amino acid substitution T366W and optionally the amino acid substitution S354C in one of the antibody heavy chains and the amino acid substitutions T366S, L368A, Y407V and optionally Y349C in the other antibody heavy chain.

Amino acid "substitution" refers to the replacement of one amino acid for another in a polypeptide. In one embodiment, an amino acid is replaced with another amino acid having similar structural and/or chemical properties, e.g., a conservative amino acid substitution. "conservative" amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine and histidine; and the positively charged (acidic) amino acids include aspartic acid and glutamic acid. Non-conservative substitutions will swap a member of one of these classes for a member of the other class. For example, an amino acid substitution can also result in the replacement of one amino acid with another amino acid having a different structural and/or chemical property, e.g.,an amino acid from one group (e.g., polar) is replaced with another amino acid from a different group (e.g., basic). Amino acid substitutions can be made using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, and the like. It is contemplated that methods of altering amino acid side chain groups by methods other than genetic engineering, such as chemical modification, may also be used. Various names may be used herein to indicate the same amino acid substitution. For example, substitutions from proline to glycine at position 329 of the Fc domain can be shown as 329G, G329, G₃₂₉P329G or Pro329 Gly.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, as necessary, to achieve the maximum percent sequence homology and not considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full-length sequences being compared. However, for purposes herein, the sequence comparison computer program ALIGN-2 was used to generate% amino acid sequence identity values. The ALIGN-2 sequence comparison computer program was authorized by Genentech, inc, and the source code had been submitted with the user document to the U.S. copyright office in washington, d.c., where it was registered with U.S. copyright registration number TXU 510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif. or may be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system (including digital UNIX V4.0D). All sequence comparison parameters are set by the ALIGN-2 program and are not changed. In the case of amino acid sequence comparison using ALIGN-2, the% amino acid sequence identity of a given amino acid sequence a with, or against a given amino acid sequence B (which may alternatively be described as a given amino acid sequence a having or comprising a certain% amino acid sequence identity with, or against a given amino acid sequence B) is calculated as follows:

100 times a fraction X/Y

Where X is the number of amino acid residues that are assessed by the sequence alignment program ALIGN-2 as identical matches in the A and B alignments of that program, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A relative to B will not be equal to the% amino acid sequence identity of B relative to A. Unless specifically stated otherwise, all% amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

"polynucleotide" or "nucleic acid" as used interchangeably herein refers to a polymer of nucleotides of any length, and includes DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogs thereof, or any substrate that can be incorporated into the polymer by a DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and analogs thereof. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may comprise modifications made post-synthetically, such as conjugation to a label.

The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid linked thereto. The term includes vectors which are self-replicating nucleic acid structures as well as vectors which are incorporated into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may instead contain a mutation. Included herein are mutant progeny that have the same function or biological activity as the progeny screened or selected for in the originally transformed cell. The host cell is any type of cell system that can be used to produce the antibodies of the invention. Host cells include cultured cells, e.g., cultured mammalian cells, such as CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, per.c6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, to mention only a few host cells; host cells also include cells contained within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

An "effective amount" of an agent refers to the amount required to produce a physiological change in the cell or tissue to which the drug is administered.

A "therapeutically effective amount" of an agent (e.g., a pharmaceutical composition) refers to an amount effective to achieve a desired therapeutic or prophylactic result at a desired dosage and for a desired period of time. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the adverse effects of the disease.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

The term "pharmaceutical composition" refers to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional components that are unacceptably toxic to a subject to whom the preparation will be administered.

"pharmaceutically acceptable carrier" refers to an ingredient of a pharmaceutical composition that is not toxic to a subject other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

As used herein, "treatment" (and grammatical variations thereof such as "treating" or "treating") refers to clinical intervention intended to alter the natural course of a disease in the individual being treated, and may be performed for prophylaxis or during the course of clinical pathology. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating a disease state, and ameliorating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay disease progression or to slow the progression of disease.

Antibodies of the invention

The present invention provides novel antibodies, particularly monoclonal antibodies, that bind to asialoglycoprotein receptor (ASGPR).

In a first aspect, the present invention provides an antibody capable of specifically binding to asialoglycoprotein receptor (ASGPR), wherein the antibody comprises a) a heavy chain variable region sequence of SEQ ID NO:16 and a light chain variable region sequence of SEQ ID NO: 14; b) the heavy chain variable region sequence of SEQ ID NO 4 and the light chain variable region sequence of SEQ ID NO 2; c) the heavy chain variable region sequence of SEQ ID NO 8 and the light chain variable region sequence of SEQ ID NO 6; d) the heavy chain variable region sequence of SEQ ID NO 12 and the light chain variable region sequence of SEQ ID NO 10; e) the heavy chain variable region sequence of SEQ ID NO 20 and the light chain variable region sequence of SEQ ID NO 18; f) the heavy chain variable region sequence of SEQ ID NO 24 and the light chain variable region sequence of SEQ ID NO 22; g) the heavy chain variable region sequence of SEQ ID NO 28 and the light chain variable region sequence of SEQ ID NO 26; h) the heavy chain variable region sequence of SEQ ID NO 4 and the light chain variable region sequence of SEQ ID NO 30; i) the heavy chain variable region sequence of SEQ ID NO 4 and the light chain variable region sequence of SEQ ID NO 32; j) the heavy chain variable region sequence of SEQ ID NO 4 and the light chain variable region sequence of SEQ ID NO 34; or k) the heavy chain variable region sequence of SEQ ID NO. 24 and the light chain variable region sequence of SEQ ID NO. 22.

One specific antibody of the invention is based on a heavy chain variable region sequence comprising SEQ ID NO 16 and a light chain variable region sequence comprising SEQ ID NO 14. This antibody clone was designated 4F 3. Another specific antibody of the present invention is based on a heavy chain variable region sequence comprising SEQ ID NO. 4 and a light chain variable region sequence comprising SEQ ID NO. 2. This antibody was designated 51a 12.

The present invention provides antibodies capable of specifically binding to ASGPR and competing with antibody 4F3 for binding to an epitope of ASGPR. In one embodiment, such an antibody binds to the same epitope as 4F 3. The 4F3 antibody recognizes an epitope in the stem region of ASGPR. Thus, in one embodiment, such an antibody recognizes an epitope in the stem region of ASGPR. Affinity matured variants of the 4F3 antibody are also contemplated by the invention. In one embodiment, such an antibody comprises a heavy chain variable region sequence that is at least about 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO 16 and a light chain variable region sequence that is at least about 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO 14. In one embodiment, such an antibody comprises the light chain variable region sequence of SEQ ID No. 14 with one, two, three, four, five, six or seven, particularly two, three, four or five amino acid substitutions. In one embodiment, such an antibody comprises the heavy chain variable region sequence of SEQ ID No. 16 with one, two, three, four, five, six or seven, particularly two, three, four or five amino acid substitutions. The variant of the 4F3 antibody may also comprise the same heavy chain variable region as the heavy chain variable region of 4F3, as well as a variant light chain variable region, or vice versa.

The invention also provides antibodies capable of specifically binding to ASGPR and competing with antibody 51a12 for binding to an epitope of ASGPR. In one embodiment, such an antibody binds to the same epitope as 51a 12. The 51a12 antibody recognizes an epitope in the Carbohydrate Recognition Domain (CRD) of ASGPR. Thus, in one embodiment, such an antibody recognizes an epitope in the CRD of ASGPR. The present invention also contemplates affinity matured variants of the 51a12 antibody, particularly the 51a12 antibody obtained by randomization of the light chain CDRs 3 or 51a 12. In one embodiment, such an antibody comprises a heavy chain variable region sequence that is at least about 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO. 4 and a light chain variable region sequence that is at least about 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO. 2. In one embodiment, such an antibody comprises the light chain variable region sequence of SEQ ID No. 2 with one, two, three, four, five, six or seven, particularly two, three, four or five amino acid substitutions. In one embodiment, such an antibody comprises the heavy chain variable region sequence of SEQ ID No. 4 with one, two, three, four, five, six or seven, particularly two, three, four or five amino acid substitutions. The variant of 51a12 antibody may also comprise the same heavy chain variable region as the heavy chain variable region of 51a12, and a variant light chain variable region, or vice versa. In one embodiment, such an antibody comprises a) the heavy chain variable region sequence of SEQ ID NO. 4 and the light chain variable region sequence of SEQ ID NO. 36; b) the heavy chain variable region sequence of SEQ ID NO 4 and the light chain variable region sequence of SEQ ID NO 38; c) the heavy chain variable region sequence of SEQ ID NO 4 and the light chain variable region sequence of SEQ ID NO 40; d) the heavy chain variable region sequence of SEQ ID NO 4 and the light chain variable region sequence of SEQ ID NO 42; e) the heavy chain variable region sequence of SEQ ID NO 4 and the light chain variable region sequence of SEQ ID NO 44; f) the heavy chain variable region sequence of SEQ ID NO 4 and the light chain variable region sequence of SEQ ID NO 46; or g) the heavy chain variable region sequence of SEQ ID NO 4 and the light chain variable region sequence of SEQ ID NO 48.

Preferably, the antibodies of the invention are human antibodies, i.e., the antibodies comprise human variable and constant regions. In one embodiment, the antibody comprises a human Fc region, particularly a human IgG Fc region, more particularly a human IgG Fc region₁An Fc region. Particular antibodies of the invention are full length antibodies, in particular full length IgG class antibodies, more particularly full length IgG class antibodies₁Subclass antibody. Alternatively, the antibody may be an antibody fragment. In one embodiment, the antibody is a Fab fragment or scFv fragment. In some embodiments, the antibody comprises an immunoglobulin hinge region, particularly a human IgG hinge region, more particularly a human IgG hinge region₁Hinge region linked Fab fragment and Fc region, particularly human IgG Fc region, more particularly human IgG₁An Fc region. In particular, an antibody may comprise a Fab fragment and an Fc region connected by an immunoglobulin hinge region, wherein no other Fab fragment is present. In such embodiments, the antibody is substantially a full-length antibody lacking one Fab fragment.

The Fc region included in the antibodies of the invention may comprise a plurality of modifications as compared to the native Fc region.

Although the Fc domain confers advantageous pharmacokinetic properties to the antibody, including promoting good accumulation in the target tissue and a long serum half-life for favorable tissue-blood distribution rates, it may at the same time result in the antibody inadvertently targeting Fc receptor-expressing cells rather than the preferred antigen-bearing cells. In addition, co-activation of the Fc receptor signaling pathway can lead to cytokine release, especially in antibodies with effector moieties (e.g., cytokines) attached, which leads to over-activation of cytokine receptors and serious side effects upon systemic administration. Thus, in some embodiments, the antibody comprises a modification within the Fc region that reduces the binding affinity of the antibody to an Fc receptor, particularly an fey receptor, as compared to a corresponding antibody comprising an unmodified Fc region. Binding to Fc receptors can be readily determined using standard instruments such as BIAcore instruments (GE Healthcare) and as Fc receptors which can be obtained by recombinant expression, for example by ELISA or by Surface Plasmon Resonance (SPR). Specific illustrative and exemplary embodiments for measuring binding affinity are described below. According to one embodiment, the ligand (Fc receptor) is immobilized on a CM5 chip at 25 deg.C usingT100 instrument (GE Healthcare), the binding affinity of Fc receptors was measured by surface plasmon resonance. Briefly, carboxymethylated dextran biosensor chips (CM5, GE Healthcare) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The reconstituted pool was diluted with 10mM sodium acetate, pH 5.5 to 0.5-30. mu.g/ml, after which time the conjugate protein was injected at a flow rate of 10. mu.l/min to achieve approximately 100-. After ligand loading, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, 3-fold to 5-fold serial dilutions (ranging between about 0.01nM and 300 nM) of the antibody were made at 25 ℃ in HBS-EP + (GE Healthcare, 10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20, pH 7.4) at a flow rate of about 30-50. mu.l/minAnd (4) loading. Using a simple one-to-one Langmuir binding model (T100 evaluation software version 1.1.1) association rates (k) were calculated by simultaneous fitting of association and dissociation sensorgrams (sensorgram)_on) And dissociation rate (k)_off). Will equilibrate the dissociation constant (K)_D) Is calculated as k_off/k_onAnd (4) the ratio. See, e.g., Chen et al, J Mol Biol 293, 865-. Alternatively, the binding affinity of an antibody to an Fc receptor can be assessed using a cell line known to express a particular Fc receptor, such as an NK cell expressing an Fc γ IIIa receptor.

The modification comprises one or more amino acid mutations that reduce the binding affinity of the antibody for the Fc receptor. Typically, the same amino acid mutation or mutations are present in each of the two antibody heavy chains in the Fc domain. In one embodiment, the amino acid mutation reduces the binding affinity of the antibody to the Fc receptor by at least 2 fold, at least 5 fold, or at least 10 fold. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of the antibody to the Fc receptor, the combination of these amino acid mutations can reduce the binding affinity of the antibody to the Fc receptor by at least 10 fold, at least 20 fold, or even at least 50 fold. In one embodiment, the antibody exhibits less than 20%, particularly less than 10%, more particularly less than 5% Fc receptor binding affinity compared to an antibody comprising an unmodified Fc domain.

In one embodiment, the Fc receptor is an activating Fc receptor. In a particular embodiment, the Fc receptor is an Fc γ receptor, more particularly an Fc γ RIIIa, Fc γ RI or Fc γ RIIa receptor. Preferably, binding to each of these receptors is reduced. In some embodiments, the binding affinity for complement components, particularly for C1q, is also reduced. In one embodiment, the binding affinity for neonatal Fc receptor (FcRn) is not reduced. Substantially similar FcRn binding is achieved when the antibody exhibits greater than about 70% of the binding affinity of the unmodified form of the antibody to FcRn, i.e., retains the binding affinity of the antibody for the receptor. Antibodies of the invention may exhibit such affinities of greater than about 80% and even greater than about 90%. In one embodiment, the amino acid mutation is an amino acid substitution. In one embodiment, the antibody comprises an amino acid substitution at position P329 (EU numbering) within the Fc region. In a more specific embodiment, the amino acid substitution is P329A or P329G, in particular P329G. In one embodiment, the antibody comprises a further amino acid substitution in the Fc region at a position selected from the group consisting of S228, E233, L234, L235, N297, and P331. In a more specific embodiment, the further amino acid substitution is S228P, E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a specific embodiment, the antibody comprises amino acid substitutions at positions P329, L234 and L235 within the Fc region. In a more specific embodiment, the antibody comprises the amino acid mutations L234A, L235A, and P329G (LALA P329G). This combination of amino acid substitutions almost completely abolished the Fc γ receptor binding effect of human IgG antibodies as described in PCT patent application No. PCT/EP2012/055393, which is herein incorporated in its entirety by reference. PCT patent application No. PCT/EP2012/055393 also describes methods for making such modified antibodies and methods for determining properties such as Fc receptor binding or effector function.

Antibodies comprising modifications in the Fc region can be made by amino acid deletion, substitution, insertion, or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of DNA coding sequences, PCR, gene synthesis, and the like. The correct nucleotide change can be verified, for example, by sequencing.

Antibodies comprising modifications that reduce Fc receptor binding typically have reduced effector function, particularly reduced ADCC, as compared to the unmodified corresponding antibody. In some embodiments, the antibody has reduced ADCC. In a particular embodiment, the reduced ADCC is less than 20% of the ADCC by a corresponding antibody comprising an unmodified Fc region. The effector function of an antibody can be measured by methods known in the art. Examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. nos. 5,500,362; hellstrom et al, Proc Natl Acad Sci USA 83,7059-6) And Hellstrom et al, Proc Natl Acad Sci USA 82, 1499-; U.S. Pat. nos. 5,821,337; bruggemann et al, J Exp Med 166, 1351-. Alternatively, non-radioactive analysis methods can be used (see, e.g., ACTI for flow cytometry)^TMNon-radioactive cytotoxicity assay (CellTechnology, inc. mountain View, CA); and CytoToxNon-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells for use in such assays include Peripheral Blood Mononuclear Cells (PBMCs) and Natural Killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al, Proc Natl Acad Sci USA 95, 652-. In some embodiments, the binding effect of the antibody to complement components, particularly to C1q, is also reduced. Thus, Complement Dependent Cytotoxicity (CDC) may also be reduced. A C1q binding assay may be performed to determine whether an antibody is capable of binding C1q and thus has CDC activity. See, e.g., WO 2006/029879 and WO 2005/100402 for C1q and C3C binding ELISAs. To assess complement activation, CDC assays may be performed (see, e.g., Gazzano-Santoro et al, J Immunol Methods 202,163 (1996); Cragg et al, Blood 101,1045-1052 (2003); and Cragg and Glennie, Blood 103,2738-2743 (2004)).

In addition to the antibodies described above and in PCT patent application No. PCT/EP2012/055393, antibodies with reduced Fc receptor binding and/or effector function also include those with substitutions to one or more of residues 238, 265, 269, 270, 297, 327 and 329 of the Fc region (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including so-called "DANA" Fc mutants having substitutions of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

And IgG₁Antibody versus IgG₄Antibodies exhibit reduced Fc receptor binding affinity and reduced effector function. Thus, is atIn some embodiments, the antibodies of the invention are IgG₄Subclass antibodies, in particular human IgG₄Subclass antibody. In one embodiment, the IgG is₄The antibody comprises an amino acid substitution at position S228 in the Fc region, in particular amino acid substitution S228P. To further reduce its Fc receptor binding affinity and/or its effector function, in one embodiment, IgG₄The antibody comprises an amino acid substitution at position L235, in particular the amino acid substitution L235E. In another embodiment, the IgG is₄The antibody comprises an amino acid substitution at position P329, in particular the amino acid substitution P329G. In a specific embodiment, the IgG₄The antibodies comprise amino acid substitutions at positions S228, L235 and P329, in particular amino acid substitutions S228P, L235E and P329G. Such modified IgG₄Antibodies and their Fc γ receptor binding properties are described in PCT patent application No. PCT/EP2012/055393, which is incorporated herein by reference in its entirety.

The antibodies of the invention may have effector moieties such as linked cytokines. In particular embodiments, the antibodies comprise only one single effector moiety fused to one of the two antibody heavy chains, and thus the antibodies comprise two non-identical polypeptide chains. Similarly, an antibody of the invention may be a full-length antibody, lacking one of the Fab fragments, and thus comprise a complete antibody heavy chain and an antibody heavy chain that lacks a VH domain and a CH1 domain. Recombinant co-expression and subsequent dimerization of these polypeptides yields several possible combinations of the two polypeptides, of which heterodimers of only two non-identical polypeptides are useful. In order to improve the yield and purity of such antibodies in recombinant production, it may therefore be advantageous to introduce in the Fc region of the antibody a modification that hinders the formation of homodimers of two identical polypeptides (e.g. two polypeptides comprising an effector moiety, or two polypeptides lacking an effector moiety) and/or promotes the formation of heterodimers of a polypeptide comprising an effector moiety and a polypeptide lacking an effector moiety. Thus, in some embodiments, an antibody of the invention comprises a modification in the Fc region that promotes heterodimerization of two non-identical antibody heavy chains. The site of most widespread protein-protein interaction between the two heavy chains of a human IgG antibody is in the CH3 domain of the Fc region. Thus, in one embodiment, the modification is in the CH3 domain of the Fc region. In a specific embodiment, the modification is a knob-into-hole (knob-hole) modification, comprising a knob modification in one of the antibody heavy chains and a hole modification in the other of the two antibody heavy chains. The lune-in-cave technique is described, for example, in US5,731,168; US7,695,936; ridgway et al, Prot Eng9,617-621(1996) and Carter, J Immunol Meth 248,7-15 (2001). In general, the method involves introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide such that the protuberance can be positioned in the cavity, thereby promoting heterodimer formation and hindering homodimer formation. The overhang is constructed by replacing the side chain of a small amino acid derived from the first polypeptide interface with a larger side chain (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller side chains (e.g., alanine or threonine), complementary "holes" of the same or similar size as the projections are created in the interface of the second polypeptide. Thus, in one embodiment, the antibody comprises a modification within the interface between two antibody heavy chains in the CH3 domain, wherein i) in the CH3 domain of one heavy chain, amino acid residues are replaced with amino acid residues having a larger side chain volume, thus creating a protuberance ("knob") within the interface in the CH3 domain of one heavy chain, which protuberance can be positioned in a cavity ("pocket") within the interface in the CH3 domain of the other heavy chain, and ii) in the CH3 domain of the other heavy chain, amino acid residues are replaced with amino acid residues having a smaller side chain volume, thus creating a cavity ("pocket") within the interface in the second CH3 domain, inside which cavity a protuberance ("knob") within the interface in the first CH3 domain can be positioned. The projections and cavities can be created by altering the nucleic acid encoding the polypeptide, for example by site-specific mutagenesis or by peptide synthesis. In a specific embodiment, the knob modification comprises the amino acid substitution T366W (EU numbering) in one of the two antibody heavy chains and the hole modification comprises the amino acid substitutions T366S, L368A and Y407V (EU numbering) in the other heavy chain of the two antibody heavy chains. In yet another specific embodiment, the antibody heavy chain comprising a knob modification additionally comprises the amino acid substitution S354C, and the antibody heavy chain comprising a hole modification additionally comprises the amino acid substitution Y349C. The introduction of these two cysteine residues results in the formation of disulfide bonds between the two antibody heavy chains within the Fc region, further stabilizing the dimer (Carter, J Immunol Methods 248,7-15 (2001)). In an alternative embodiment, the modification that promotes heterodimerization of two non-identical antibody heavy chains comprises a modification that mediates electrostatic steering effects, for example as described in PCT publication WO 2009/089004. Typically, such methods involve replacing one or more amino acid residues at the interface of two antibody heavy chains with charged amino acid residues, such that homodimer formation becomes electrostatically unfavorable and heterodimerization is electrostatically favorable. In one embodiment, wherein the antibody has an effector moiety attached thereto, the effector moiety is fused to an amino-terminal or carboxy-terminal amino acid of the heavy chain of the antibody comprising the knob modification. Without wishing to be bound by theory, fusion of the effector moiety to the knob-containing heavy chain will further minimize the production of homodimeric antibody comprising two effector moieties (steric hindrance of the two knob-containing polypeptides). Similarly, in embodiments where the antibody comprises only a single Fab fragment fused to an Fc region, the Fab fragment is preferably fused to a heavy chain comprising a knob modified Fc region.

The antibodies of the invention combine a number of properties that are particularly advantageous, for example in therapeutic applications. For example, antibodies cross-react with humans and cynomolgus monkeys, which makes possible in vivo studies, e.g., in cynomolgus monkeys, prior to human use. Thus, in one embodiment, the antibodies of the invention are capable of specifically binding to human ASGPR and cynomolgus monkey ASGPR. In addition, the antibodies of the invention bind ASGPR with particularly strong affinity and/or avidity. In one embodiment, the antibody has a dissociation constant (K) of less than 1 μ M, particularly less than 100nM, more particularly less than 1nM, as measured by Surface Plasmon Resonance (SPR) as a Fab fragment_D) Binding to human ASGPR. Described herein is a method of measuring binding affinity by SPR. Specifically, the measurement was performed at a temperature of 25 ℃. In one embodiment, a ProteOn XPR36 instrument (B) is used at 25 ℃iorad), measuring the affinity of the antibody as Fab fragment by SPR (K.K.sub.Dp) using biotinylated monovalent- (avi-Fc-human ASGPR H1CRD, SEQ ID NO:118) or bivalent (avi-Fc-human ASGPR H1 stem-CRD, SEQ ID NO:130) ASGPR H1 antigen immobilized on NLC chip by neutral avidin capture (K.sub.Dp)_D). In an exemplary method, the antigen used for immobilization was diluted to 10 μ g/ml with PBST (10mM phosphate, 150mM NaCl pH 7.4, 0.005% tween 20) and loaded at 30 μ l/min at different contact times to achieve a fixed level of 200, 400 or 800 Response Units (RU) in the vertical direction. Subsequently, the analyte (antibody) is loaded. For one-shot kinetic measurements, the loading direction was changed to horizontal and two-fold dilution series of purified Fab fragments (concentrations ranging between 100nM and 6.25 nM) were loaded simultaneously at 50, 60 or 100 μ Ι/min along independent channels 1-5 with an association time of 150 seconds or 200 seconds and a dissociation time of 240 seconds or 600 seconds. Buffer (PBST) was loaded along the sixth channel to provide an "in-line" blank for reference. Simple one-to-one Langmuir binding model in v3.1 software was managed with ProteOn, and the association rate constant (k) was calculated by fitting the association sensorgram and dissociation sensorgram simultaneously_on) And dissociation rate constant (k)_off). Will balance the dissociation constant (K)_D) Is calculated as k_off/k_onAnd (4) the ratio. Regeneration was performed horizontally using 10mM glycine, pH1.5 at a flow rate of 100. mu.l/min for a contact time of 30 seconds. In another embodiment, the antibody has a K of less than 1 μ M, particularly less than 500nM, more particularly less than 100nM or even less than 10nM, as measured by Fluorescence Resonance Energy Transfer (FRET) as IgG1_DBinding to human ASGPR. Described herein is a method for measuring binding affinity (or avidity) by FRET. In one embodiment, the measurement is performed by: cells expressing the FRET donor molecule labeled full-length ASGPR protein are contacted with an antibody and the bound antibody is detected by a second antibody labeled with a suitable FRET acceptor molecule. In an exemplary method, a DNA sequence encoding a SNAP tag (plasmid purchased from Cisbio) was amplified by PCR and ligated into an expression vector containing the full-length human ASGPR H1 sequence (Origene). The resulting fusion protein comprises the full length with the C-terminal SNAP tagASGPR H1. HEK293 cells were transfected with 10 μ g DNA using lipofectamine 2000 as transfection reagent. After 20 hours incubation time, cells were washed with PBS and incubated in LabMed buffer (Cisbio) containing 100nM SNAP-Lumi4Tb (Cibsio) for 1 hour at 37 ℃ resulting in specifically labeling the SNAP tag. Subsequently, the cells were washed 4 times with LabMed buffer to remove unbound dye. The efficiency of labeling was determined by measuring terbium emission at 615nm compared to the buffer. The cells can then be stored frozen at-80 ℃ for 6 months. Affinity was measured by: ASGPR-specific antibody was added to labeled cells (100 cells/well) at a concentration of 50-0.39nM, followed by anti-human Fc-d2(Cisbio, final concentration 200 nM/well) as an acceptor molecule for FRET. After 3 hours incubation time at room temperature, the emission of the acceptor dye (665nm) and the emission of the donor dye (615nm) were determined using a fluorescence reader (Victor3, Perkin Elmer). The ratio of acceptor to donor emission was calculated and subtracted from the background control (cells with anti-human Fc-d 2). Curves can be analyzed in GraphPad Prism5 software and K calculated_DThe value is obtained. Yet another advantage of the antibodies of the invention is that they do not compete with the natural ligand of the receptor (asialoglycoprotein, e.g. asialoglycoprotein) for binding to ASGPR, i.e. antibody binding is not affected by the presence of ASGPR ligand and does not interfere with the natural function of ASGPR. In one embodiment, the antibody does not compete with the natural ligand of ASGPR for binding to ASGPR. In a specific embodiment, the natural ligand of ASGPR is asialofetuin. Competition can be measured by methods well known in the art. In one embodiment, competition with the natural ligand for ASGPR is measured by FACS, e.g., using a cell line expressing ASGPR, a fluorescently labeled ligand and detecting bound antibody with a second antibody having a different fluorescent label. In an exemplary method, the hepatocellular carcinoma cell line HepG2 was used. 0.2mio cells/well in 96-well round bottom plates were incubated with 40. mu.l Alexa 488-labeled asialofetuin (from fetal bovine serum, Sigma Aldrich # A4781, final concentration 100. mu.g/ml) for 30 minutes at 4 ℃. The binding process is carried out in the presence of calcium, since the binding of ligand to ASGPR is calcium dependent. Unbound material was removed by washing the cells 1 time with HBSS containing 0.1% BSAA protein. Subsequently 40. mu.l of anti-ASGPR antibody (final concentration 30, 6 and 1.25. mu.g/ml) was added to the cells in the presence of 100. mu.g/ml asialofetuin. Cells were incubated at 4 ℃ for 30 minutes and unbound protein was removed by washing the cells 1 time. Fc gamma fragment specific second F (ab') goat anti-human IgG using APC conjugated AffiniPure₂The fragment (Jackson ImmunoResearch # 109-136-170; 1:50 working solution in HBSS containing 0.1% BSA) was used as the secondary antibody. After incubation at 4 ℃ for 30 minutes, unbound secondary antibody was removed by washing. Cells were fixed with 1% PFA and analyzed for ligand binding as well as antibody binding using BD FACS cantonii (software BD DIVA). The main advantage of the antibodies of the invention is their high specificity for ASGPR. For example, despite their strong binding to human ASGPR H1, the antibodies did not detectably bind CLEC10A, which was identified as the closest homolog of human ASGPR H1. In one embodiment, the antibody does not detectably bind CLEC10A, particularly human CLEC 10A. In particular, where binding is measured by SPR (as described herein), the antibody does not detectably bind CLEC 10A. In addition, the antibody binds to cells that do not express ASGPR only to a similar extent as the corresponding non-targeting antibody (isotype control). Thus, in one embodiment, the antibody does not specifically bind to cells lacking ASGPR expression, more particularly human cells, more particularly human blood cells. Exemplary cells lacking ASGPR expression include Hela cells (a human cell line derived from cervical cancer), a459 human non-small cell lung cancer cells, Human Embryonic Kidney (HEK) cells, and (human) PBMCs. Binding to specific cells or lack of binding can be readily determined, for example, by FACS. Such methods are well established in the art and are also described in the examples herein. An important feature of anti-ASGPR antibodies is their internalization profile. For example, if the antibody is to be used to target an effector moiety to cells expressing ASGPR, it is desirable that the antibody be present on the cell surface for a sufficient time to activate the effector moiety receptor. Once bound to ASGPR, the antibodies of the invention are internalized into ASGPR-expressing cells, however, they are recycled back to the cell surface without degradation inside the cell. Thus, in one embodiment, once the antibody is associated with the epitopeASGPR binding on the surface of ASGPR-reaching cells into which the antibody is internalized. In a particular embodiment, the antibody is recycled back to the surface of the cell at approximately the same rate as it is internalized into the cell. Internalization and recycling of cell surface proteins or antibodies bound thereto can be readily measured by established methods such as FACS or (confocal) microscopy. In one embodiment, internalization and/or recycling is measured by FACS. For an antibody to have sustained effect, it is important that its target antigen is present at a substantially constant level. Often, antibodies that bind to the target antigen cause the latter to be down-regulated, resulting in a reduction in the potency of the antibody. However, the antibodies of the present invention do not have this effect. In one embodiment, the antibody does not significantly cause downregulation of ASGPR expression at the cell surface once the antibody binds to ASGPR on the cell surface. The level of antigen expression at the cell surface can be readily determined by established methods such as FACS.

Antibodies with effector moieties attached thereto

Particularly useful antibodies of the invention are antibodies having an effector moiety (e.g., a cytokine) attached thereto. Antibodies fused to effector moieties such as cytokines are also referred to herein as immunoconjugates. The antibody having an effector moiety attached thereto may incorporate any of the features described above with respect to the antibodies of the invention, alone or in combination.

Accordingly, in one aspect, the invention provides an antibody capable of specifically binding to ASGPR according to any one of the above embodiments, wherein an effector moiety is linked to the antibody. In one embodiment, no more than one effector moiety is attached to the antibody. The absence of other effector moieties may reduce targeting of the antibody to the site where the corresponding effector moiety receptor is presented, thus improving the accumulation of ASGPR, the site where the actual target antigen of the antibody is presented, and where it is targeted. In addition, the absence of avidity effects of the receptors for the respective effector moieties may reduce activation of effector moiety receptor positive cells in peripheral blood upon intravenous administration of the antibody. The effector moiety for use in the present invention is typically a polypeptide that affects cellular activity (e.g., via a signal transduction pathway). Therefore, those useful in the present inventionThe effector moiety may be associated with receptor-mediated signaling that propagates signals from outside the cell membrane to modulate responses inside the cell. For example, the effector moiety may be a cytokine. In particular embodiments, the effector moiety is human. In particular embodiments, the effector moiety is a peptide molecule and is fused to the antibody by a peptide bond (i.e., the antibody and effector moiety form a fusion protein). In one embodiment, the effector moiety is a single chain peptide molecule. In yet another embodiment, the effector moiety is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the antibody heavy chains, optionally via a peptide linker. Suitable non-immunogenic peptide linkers include, for example, (G)₄S)_n、(SG₄)_nOr G₄(SG₄)_nA peptide linker. "n" is generally a number between 1 and 10, typically between 2 and 4. In embodiments in which the antibody comprises a knob-into-hole modification in the Fc region as described above, it is preferred that the effector moiety is fused to the heavy chain of the antibody comprising the knob modification.

In one embodiment, the effector moiety is a cytokine molecule. Examples of useful cytokines include, but are not limited to, GM-CSF, IL-1 α, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-21, IFN- β 0, IFN- β, IFN- γ, MIP-1 β 1, MIP-1 β, TGF- β, TNF- β 2, and TNF- β. In one embodiment, the cytokine molecule is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the antibody heavy chains, optionally via a peptide linker. In one embodiment, the cytokine molecule is a human cytokine. In one embodiment, the cytokine molecule is an interferon molecule. In a specific embodiment, the interferon molecule is interferon beta 3, in particular human interferon alpha, more particularly human interferon alpha 2 (see SEQ ID NO:138) or human interferon alpha 2a (see SEQ ID NO: 139). Interferon alpha is known to have antiviral activity. Thus, the attachment of interferon molecules to the antibodies of the invention is particularly useful for targeting virally infected ASGPR-expressing cells. In one embodiment where the cytokine molecule is an interferon moleculeThe antibodies have antiviral activity in cells expressing ASGPR on the cell surface. In one embodiment, the cell is a liver cell, particularly a hepatocyte, more particularly a human hepatocyte. In one embodiment, the antiviral activity is selective. In a specific embodiment, the antibody has no antiviral activity in cells that do not express significant levels of ASGPR on the cell surface. In one embodiment, the cell is a blood cell, in particular a human blood cell. In one embodiment, the antiviral activity. This selectivity of the interferon molecules linked to the anti-ASGPR antibodies of the present invention is in contrast to non-targeted interferon molecules, which do not distinguish between any desired target cells (e.g. hepatocytes) and cells that should not be affected (e.g. blood cells) and are critical for possible therapeutic use without major toxicity problems. In yet another specific embodiment, the antiviral activity is selected from the group consisting of inhibiting viral infection, inhibiting viral replication, inhibiting cell killing, and inducing one or more interferon-stimulated genes. In a specific embodiment, the one or more interferon-stimulated genes are selected from MX1 (myxovirus resistance 1, also known as interferon-induced protein p78), RSAD2 (containing the free radical S-adenosylmethionine domain 2, also known as cytomegalovirus-induced gene 5), HRASLS2 (HRAS-like repressor protein 2), IFIT1 (interferon-induced protein 1 with triangular tetrapeptide repeats), and IFITM2 (interferon-induced transmembrane protein 2). In one embodiment, the induction of one or more interferon-stimulated genes is at least 1.5-fold, particularly at least 2-fold, more particularly at least 5-fold induction at the mRNA level compared to the induction by a corresponding antibody not linked to the interferon molecule. Gene induction at the mRNA level can be measured by well established methods in the art, including quantitative Reverse Transcription (RT) PCR or microarray analysis, as described herein. Inhibition of cell killing can be determined, for example, by a viro-protection assay in which cells are pre-incubated with a test compound, followed by addition of virus and quantification of viable cells after incubation. This exemplary assay is described in the examples. Madin-Darby bovine kidney (MDBK) cells with antibodies and controlsPreincubation for 1-4 hours. The vesicular stomatitis virus is then added for an additional 16-24 hours. At the end of this incubation step, live cells were stained with crystal violet staining solution (0.5%) and quantified using a microplate reader at 550-. Exemplary assays for assessing viral replication are also provided in the examples. This assay uses a Huh 7-derived hepatoma cell line stably transfected with a bicistronic Hepatitis C Virus (HCV) replicon, where the first open reading frame of the replicon, driven by the HCV IRES, contains the renilla luciferase gene fused to the neomycin phosphotransferase gene (NPTII), and the second open reading frame, driven by the EMCV IRES, contains the HCV nonstructural genes NS3, NS4a, NS4b, NS5A, and NS5B derived from the NK5.1 replicon backbone. Cells were incubated at 37 ℃ with 5% CO₂Under a humid atmosphere supplemented with Glutamax^TMAnd 100mg/ml sodium pyruvate in DMEM. The medium was further supplemented with 10% (v/v) FBS, 1% (v/v) penicillin/streptomycin and 1% (v/v) geneticin. Cells in DMEM containing 5% (v/v) FBS were plated in 96-well plates at 5000 cells/well in a volume of 90 μ l. 24 hours after plating, the antibody (or the medium as a control) was added to the cells at a volume of 10. mu.l at a 3-fold dilution (0.01-2000pM) in a range of 12 wells, so that the final volume after antibody addition was 100. mu.l. At 72 hours after antibody addition, the Renilla luciferase reporter signal was read using the Renilla luciferase assay System (Promega, # E2820). EC (EC)₅₀Values were calculated as the concentration of compound at which a 50% reduction in renilla luciferase reporter levels was observed compared to control samples (in the absence of antibody). Dose-response curves and ECs were obtained by using the XLfit4 program (ID Business Solutions ltd., Surrey, UK)₅₀The value is obtained. In a specific embodiment, the antibody of the invention is a full-length human IgG comprising the heavy chain variable region sequence of SEQ ID NO 16 and the light chain variable region sequence of SEQ ID NO 14₁An antibody comprising within the Fc region a modification that reduces the binding affinity of the antibody to Fc γ RIIIa and a knob-into-hole modification comprising a knob modification in one of the antibody heavy chains and a hole modification in the other antibody heavy chain, andthe antibody has an IFN α 2 molecule fused at the N-terminal amino acid to the C-terminal amino acid of one of the antibody heavy chains via a peptide linker. In a specific embodiment, the modification that reduces the binding affinity of an antibody to fcyriiia comprises the amino acid substitutions L234A, L235A and P329G (EU numbering) in each antibody heavy chain. In yet another specific embodiment, the protuberance modification comprises the amino acid substitution T366W and the hole modification comprises the amino acid substitutions T366S, L368A and Y407V. In still another embodiment, the IFN α 2 molecule is fused to an antibody heavy chain comprising a knob modification. In an even more specific embodiment, the antibody comprises the polypeptide sequences SEQ ID NO 68, SEQ ID NO 70 and SEQ ID NO 72 or variants thereof that retain functionality.

In another embodiment, the antibody of the invention is a full-length human IgG comprising the heavy chain variable region sequence of SEQ ID NO. 4 and the light chain variable region sequence of SEQ ID NO. 2₁An antibody comprising within the Fc region a modification that reduces the binding affinity of the antibody to Fc γ RIIIa and a knob-into-hole modification comprising a knob modification in one of the antibody heavy chains and a hole modification in the other antibody heavy chain, and having an IFN α 2 molecule fused at the N-terminal amino acid to the C-terminal amino acid of one of the antibody heavy chains via a peptide linker. In a specific embodiment, the modification that reduces the binding affinity of an antibody to fcyriiia comprises the amino acid substitutions L234A, L235A and P329G (EU numbering) in each antibody heavy chain. In yet another specific embodiment, the knob modification comprises the amino acid substitution T366W and the hole modification comprises the amino acid substitutions T366S, L368A and Y407V. In still another embodiment, the IFN α 2 molecule is fused to an antibody heavy chain comprising a knob modification. In an even more specific embodiment, the antibody comprises the polypeptide sequences SEQ ID NO 50, SEQ ID NO 52 and SEQ ID NO 54 or functional retaining variants thereof.

In yet another specific embodiment, the antibody of the invention is a heavy chain variable region sequence comprising SEQ ID NO 4 and a light chain selected from the group consisting of SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46 and SEQ ID NO 48Full-length human IgG of variable region sequences₁An antibody comprising within the Fc region a modification that reduces the binding affinity of the antibody to Fc γ RIIIa and a knob-into-hole modification comprising a knob modification in one of the antibody heavy chains and a hole modification in the other antibody heavy chain, and having an IFN α 2 molecule fused at the N-terminal amino acid to the C-terminal amino acid of one of the antibody heavy chains via a peptide linker. In a specific embodiment, the modification that reduces the binding affinity of an antibody to fcyriiia comprises the amino acid substitutions L234A, L235A and P329G (EU numbering) in each antibody heavy chain. In yet another specific embodiment, the knob modification comprises the amino acid substitution T366W and the hole modification comprises the amino acid substitutions T366S, L368A and Y407V. In still another embodiment, the IFN α 2 molecule is fused to an antibody heavy chain comprising a knob modification. In an even more specific embodiment, the antibody comprises the polypeptide sequences SEQ ID NO 52, SEQ ID NO 54 and a polypeptide sequence selected from the group consisting of SEQ ID NO 96, SEQ ID NO 98, SEQ ID NO 100, SEQ ID NO 102, SEQ ID NO 104, SEQ ID NO 106, SEQ ID NO 108 or a functional retaining variant thereof.

In yet another embodiment, the antibody of the invention comprises the polypeptide sequences SEQ ID NO 92, SEQ ID NO 52 and SEQ ID NO 54 or a functional retaining variant thereof. In another embodiment, the antibody of the invention comprises the polypeptide sequences SEQ ID NO 94, SEQ ID NO 52 and SEQ ID NO 54 or functional retaining variants thereof. In yet another embodiment, the antibody of the invention comprises the polypeptide sequences SEQ ID NO 56, SEQ ID NO 58 and SEQ ID NO 60 or functional retaining variants thereof. In yet another embodiment, the antibody of the invention comprises the polypeptide sequences SEQ ID NO 62, SEQ ID NO 64 and SEQ ID NO 66 or a functional retaining variant thereof. In yet another embodiment, the antibody of the invention comprises the polypeptide sequences SEQ ID NO 74, SEQ ID NO 76 and SEQ ID NO 78 or variants thereof that retain functionality. In yet another embodiment, the antibody of the invention comprises the polypeptide sequences SEQ ID NO 80, SEQ ID NO 82 and SEQ ID NO 84 or functional retaining variants thereof. In yet another embodiment, the antibody of the invention comprises the polypeptide sequences SEQ ID NO 86, SEQ ID NO 88 and SEQ ID NO 90 or variants thereof that retain functionality.

In an alternative embodiment, the antibody of the invention is a full length human IgG lacking one of the two Fab fragments and comprising the heavy chain variable region sequence of SEQ ID NO 16 and the light chain variable region sequence of SEQ ID NO 14₁An antibody comprising within the Fc region a modification that reduces the binding affinity of the antibody to Fc γ RIIIa and a knob-into-hole modification comprising a knob modification in one of the antibody heavy chains and a hole modification in the other antibody heavy chain, and having an IFN α 2 molecule fused at the N-terminal amino acid to the C-terminal amino acid of one of the antibody heavy chains via a peptide linker. In a specific embodiment, the modification that reduces the binding affinity of an antibody to fcyriiia comprises the amino acid substitutions L234A, L235A and P329G (EU numbering) in each antibody heavy chain. In yet another specific embodiment, the knob modification comprises the amino acid substitution T366W and the hole modification comprises the amino acid substitutions T366S, L368A and Y407V. In still another embodiment, the IFN α 2 molecule is fused to an antibody heavy chain comprising a knob modification. In an even more specific embodiment, the antibody comprises the polypeptide sequences SEQ ID NO 68, SEQ ID NO 70 and SEQ ID NO 116 or variants thereof that retain functionality.

In another alternative embodiment, the antibody of the invention is a full length human IgG lacking one of the two Fab fragments and comprising the heavy chain variable region sequence of SEQ ID NO 4 and the light chain variable region sequence of SEQ ID NO 2₁An antibody comprising within the Fc region a modification that reduces the binding affinity of the antibody to Fc γ RIIIa and a knob-into-hole modification comprising a knob modification in one of the antibody heavy chains and a hole modification in the other antibody heavy chain, and having an IFN α 2 molecule fused at the N-terminal amino acid to the C-terminal amino acid of one of the antibody heavy chains via a peptide linker. In a specific embodiment, the modification that reduces the binding affinity of an antibody to fcyriiia comprises the amino acid substitutions L234A, L235A and P329G (EU numbering) in each antibody heavy chain. In yet another specific embodiment, said knob modification comprises the amino acid substitution T366W and is a hole modificationThe decorations contain the amino acid substitutions T366S, L368A and Y407V. In still another embodiment, the IFN α 2 molecule is fused to an antibody heavy chain comprising a knob modification. In an even more specific embodiment, the antibody comprises the polypeptide sequences SEQ ID NO 50, SEQ ID NO 52 and SEQ ID NO 116 or variants thereof which retain functionality.

In yet another alternative embodiment, the antibody of the invention is a full length human IgG lacking one of the two Fab fragments and comprising the heavy chain variable region sequence of SEQ ID NO 4 and the light chain variable region sequence selected from the group consisting of SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46 and SEQ ID NO 48₁An antibody comprising within the Fc region a modification that reduces the binding affinity of the antibody to Fc γ RIIIa and a knob-into-hole modification comprising a knob modification in one of the antibody heavy chains and a hole modification in the other antibody heavy chain, and having an IFN α 2 molecule fused at the N-terminal amino acid to the C-terminal amino acid of one of the antibody heavy chains via a peptide linker. In a specific embodiment, the modification that reduces the binding affinity of an antibody to fcyriiia comprises the amino acid substitutions L234A, L235A and P329G (EU numbering) in each antibody heavy chain. In yet another specific embodiment, the knob modification comprises the amino acid substitution T366W and the hole modification comprises the amino acid substitutions T366S, L368A and Y407V. In still another embodiment, the IFN α 2 molecule is fused to an antibody heavy chain comprising a knob modification. In an even more specific embodiment, the antibody comprises the polypeptide sequences SEQ ID NO 52, SEQ ID NO 116 and a polypeptide sequence selected from the group consisting of SEQ ID NO 96, SEQ ID NO 98, SEQ ID NO 100, SEQ ID NO 102, SEQ ID NO 104, SEQ ID NO 106, SEQ ID NO 108 or a functional retaining variant thereof.

In yet another embodiment, the antibody of the invention comprises the polypeptide sequences SEQ ID NO 92, SEQ ID NO 52 and SEQ ID NO 116 or variants thereof which retain functionality. In another embodiment, the antibody of the invention comprises the polypeptide sequences SEQ ID NO 94, SEQ ID NO 52 and SEQ ID NO 116 or a functional retaining variant thereof. In yet another embodiment, the antibody of the invention comprises the polypeptide sequences SEQ ID NO 56, SEQ ID NO 58 and SEQ ID NO 116 or functional retaining variants thereof. In yet another embodiment, the antibody of the invention comprises the polypeptide sequences SEQ ID NO 62, SEQ ID NO 64 and SEQ ID NO 116 or a functional retaining variant thereof. In yet another embodiment, the antibody of the invention comprises the polypeptide sequences SEQ ID NO 74, SEQ ID NO 76 and SEQ ID NO 116 or a functional retaining variant thereof. In yet another embodiment, the antibody of the invention comprises the polypeptide sequences SEQ ID NO 80, SEQ ID NO 82 and SEQ ID NO 116 or a functional retaining variant thereof. In yet another embodiment, the antibody of the invention comprises the polypeptide sequences SEQ ID NO 86, SEQ ID NO 88 and SEQ ID NO 116 or variants thereof which retain functionality.

Antibodies of the invention include those having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NOs 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 and 116, including functional fragments or variants thereof. Antibodies comprising these sequences with conservative amino acid substitutions are also encompassed by the present invention.

Polynucleotide

The invention also provides polynucleotides encoding the antibodies, or antigen-binding portions thereof, as described herein.

Polynucleotides of the invention include those that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequences set forth in SEQ ID NOs 1,3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109 and 117, including functional fragments or variants thereof.

The polynucleotides encoding the antibodies of the invention can be expressed as a single polynucleotide encoding an intact antibody or as multiple (e.g., two or more) polynucleotides that are co-expressed. Polypeptides encoded by the co-expressed polynucleotides may associate, for example, via disulfide bonds or other means, to form functional antibodies. For example, the light chain portion of an antibody may be encoded by a separate polynucleotide from the heavy chain portion of the antibody. When co-expressed, the heavy chain polypeptide will associate with the light chain polypeptide to form an antibody. In another example, the heavy chain portion of an antibody comprising an effector moiety may be encoded by a separate polynucleotide from another heavy chain portion of the antibody. When co-expressed, the heavy chain polypeptide will associate (with the light chain polypeptide) to form a functional antibody.

In one embodiment, the invention relates to a polynucleotide encoding an antibody or antigen-binding portion thereof, wherein the polynucleotide comprises a sequence encoding a variable region sequence as set forth in SEQ ID NOs 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48. In another embodiment, the invention relates to a polynucleotide encoding an antibody or antigen-binding portion thereof, wherein the polynucleotide comprises a sequence encoding a polypeptide sequence as set forth in SEQ ID NOs 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 116. In another embodiment, the invention also relates to a polynucleotide encoding an antibody or antigen binding portion thereof, wherein the polynucleotide comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence set forth in SEQ ID NO 1,3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, or 117. In another embodiment, the invention relates to a polynucleotide encoding an antibody or antigen binding portion thereof, wherein the polynucleotide comprises the nucleic acid sequence set forth in seq id No. 1,3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, or 117. In another embodiment, the invention relates to a polynucleotide encoding an antibody, or antigen binding portion thereof, wherein the polynucleotide comprises a sequence encoding a variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48. In another embodiment, the invention relates to a polynucleotide encoding an antibody or antigen-binding portion thereof, wherein the polynucleotide comprises a sequence encoding a polypeptide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 116. The invention encompasses polynucleotides encoding an antibody or antigen-binding portion thereof, wherein the polynucleotides comprise a sequence encoding a variable region sequence of SEQ ID NO 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 or 48 with conservative amino acid substitutions. The invention also encompasses polynucleotides encoding the antibodies of the invention or antigen binding portions thereof, wherein the polynucleotides comprise a sequence encoding the polypeptide sequence of SEQ ID NO 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 or 116 with conservative amino acid substitutions.

In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the invention is RNA, e.g., in the form of messenger RNA (mrna). The RNA of the present invention may be single-stranded or double-stranded.

Recombination method

The antibodies of the invention may be obtained, for example, by solid state peptide synthesis (e.g., Merrifield solid phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding the antibody (fragment) (e.g., as described above) are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such polynucleotides can be readily isolated and sequenced using conventional methods. In one embodiment, a vector, preferably an expression vector, comprising one or more polynucleotides of the invention is provided. Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence for the antibody (fragment) and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, e.g., techniques described in Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1989), and Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y. (1989). The expression vector may be part of a plasmid, virus, or may be a nucleic acid fragment. Expression vectors include expression cassettes in which polynucleotides encoding the antibody (fragment), i.e., the coding region, are cloned in operable linkage with a promoter and/or other transcriptional or translational control elements. As used herein, a "coding region" is a portion of a nucleic acid that consists of codons that are translated into amino acids. Although the "stop codon" (TAG, TGA or TAA) is not translated into an amino acid, it can be considered part of the coding region if present, but any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, 5 'and 3' untranslated regions, etc., are not part of the coding region. The two or more coding regions may be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (distinct) vectors. In addition, any vector may contain a single coding region, or may contain two or more coding regions, e.g., a vector of the invention may encode one or more polypeptides that are separated post-translationally or co-translationally into the final protein by proteolytic cleavage. Furthermore, the vectors, polynucleotides or nucleic acids of the invention may encode heterologous coding regions, which may or may not be fused to polynucleotides or variants encoding the antibodies (fragments) of the invention or derivatives thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs such as secretory signal peptides or heterologous functional domains. An operable linkage is a situation in which the coding region for a gene product (e.g., a polypeptide) is linked to one or more regulatory sequences in such a way that expression of the gene product is placed under the influence or control of the regulatory sequences. Two DNA fragments (e.g., a polypeptide coding region and a promoter linked thereto) are "operably linked" if induction of promoter function results in transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct expression of the gene product or with the ability of the DNA template to be transcribed. Thus, a promoter region is operably linked to a nucleic acid encoding a polypeptide if the promoter is capable of effecting transcription of the nucleic acid. The promoter may be a cell-specific promoter that directs the transcription of DNA only in predetermined cells in large quantities. Other transcriptional control elements besides promoters, such as enhancers, operators, repressors, and transcriptional termination signals, may be operably linked to the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein. Various transcriptional control regions are known to those skilled in the art. These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (e.g., immediate early promoter, along with intron-a), monkey virus 40 (e.g., early promoter), and retroviruses (e.g., rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes such as actin, heat shock proteins, bovine growth hormone, and rabbit alpha-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers and inducible promoters (e.g., tetracycline-inducible promoters). Similarly, a variety of translational control elements are known to those of ordinary skill in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (in particular, internal ribosome entry sites, or IRES, also known as CITE sequences). The expression cassette may also include other features such as an origin of replication, and/or a chromosomal integration element such as a retroviral Long Terminal Repeat (LTR) or an adeno-associated virus (AAV) Inverted Terminal Repeat (ITR).

The polynucleotide and nucleic acid coding regions of the present invention may be linked to additional coding regions encoding a secretion peptide or signal peptide that directs secretion of the polypeptide encoded by the polynucleotide of the present invention. For example, if secretion of the antibody is desired, DNA encoding a signal sequence can be placed upstream of the nucleic acid encoding the antibody or antigen-binding portion thereof of the invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence cleaved from the mature protein once the export of the growing protein chain across the rough endoplasmic reticulum has been initiated. One of ordinary skill in the art knows that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to yield the secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide, such as an immunoglobulin heavy or light chain signal peptide, or a functional derivative of such a sequence that retains the ability to direct secretion of a polypeptide to which such sequence is operably linked, is used. Alternatively, a heterologous mammalian signal peptide or functional derivative thereof may be used. For example, the wild-type leader sequence may be replaced with the leader sequence of human Tissue Plasminogen Activator (TPA) or mouse β -glucuronidase. Exemplary amino acid sequences of secretion signal peptides are shown in SEQ ID NO: 135-137.

DNA encoding short protein sequences that can be used to facilitate later purification (e.g., histidine tag) or to aid in labeling the antibody can be included within or at the end of the polynucleotide encoding the antibody (fragment).

In yet another embodiment, there is provided a pharmaceutical composition comprising one or more of the polynucleotides of the inventionA host cell. In certain embodiments, host cells comprising one or more vectors of the invention are provided. The polynucleotide and vector may incorporate any of the features described herein, individually or in combination, with respect to the polynucleotide and vector, respectively. In one such embodiment, the host cell comprises (e.g., has been transformed or transfected with) a vector comprising a polynucleotide encoding (part of) an antibody of the invention. As used herein, the term "host cell" refers to any kind of cellular system that can be engineered to produce an antibody or fragment thereof of the invention. Host cells suitable for replicating and supporting antibody expression are well known in the art. Such cells can be transfected or transduced with specific expression vectors, as desired, and large numbers of vector-containing cells can be grown for inoculation into large-scale fermentors to obtain sufficient quantities of antibody for clinical use. Suitable host cells include prokaryotic microorganisms such as E.coli, or a variety of eukaryotic cells such as Chinese hamster ovary Cells (CHO), insect cells, and the like. For example, the polypeptide may be produced in bacteria, particularly when glycosylation is not required. After expression, the polypeptide can be isolated from the bacterial cell paste in the soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning hosts or expression hosts for vectors encoding polypeptides, including fungi and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of polypeptides with partially or fully human glycosylation patterns. See Gerngross, Nat Biotech 22, 1409-. Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous strains of baculovirus have been identified that can be used with insect cells, particularly for transfecting Spodoptera frugiperda (Spodoptera frugiperda) cells. Plant cell cultures may also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing antibody-producing PLANTIBODIIES in transgenic plants^TMA technique). Vertebrate cells can also be used as hosts.For example, mammalian cell lines adapted for suspension culture may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lines (293 or 293T cells, as described in Graham et al, J Gen Virol 36,59 (1977)), baby hamster kidney cells (BHK), mouse support cells (TM4 cells as described in e.g.Mather, Biol Reprod 23,243-251 (1980)), monkey kidney cells (CV1), African Green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), Bufarro rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT060562), TRI cells (as described in e.g.Mather et al, Annals N.Y. Acad Sci 383,44-68 (1982)), MRC5 cells and 4 cells. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including dhfr^-CHO cells (Urlaub et al, Proc Natl Acad Sci USA 77,4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63, and Sp 2/0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol.248 (B.K.C.Lo., Humana Press, Totowa, NJ), pp.255-268 (2003). Host cells include cultured cells, e.g., cultured mammalian cells, yeast cells, insect cells, bacterial cells, and plant cells, to name just a few; host cells also include cells contained within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a Human Embryonic Kidney (HEK) cell, or a lymphoid cell (e.g., Y0, NS0, Sp20 cell).

Standard techniques for expressing foreign genes in these systems are known in the art. Cells expressing a polypeptide comprising an antibody heavy chain or light chain may be engineered so as to also express other antibody chains, so that the expression product is an antibody having a heavy chain and a light chain.

In one embodiment, a method of producing an antibody of the invention is provided, wherein the method comprises culturing a host cell comprising a polynucleotide encoding the antibody as provided above under conditions suitable for expression of the antibody, and recovering the antibody from the host cell (or host cell culture).

In the case of fusion of an antibody with an effector moiety, these components are genetically fused to each other. Antibodies can be designed such that their components are fused to each other directly or indirectly through linker sequences. The composition and length of the linker can be determined according to methods well known in the art and can be tested for efficacy. Additional sequences may also be included to incorporate cleavage sites to separate the various components of the fusion, such as endopeptidase recognition sequences, as desired.

In certain embodiments, an antibody of the invention comprises at least an antibody variable region capable of binding to ASGPR. The variable region may form part of and be derived from naturally or non-naturally occurring antibodies and fragments thereof. Methods for producing polyclonal and monoclonal Antibodies are well known in the art (see, e.g., Harlow and Lane, "Antibodies, a Laboratory", Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be recombinantly produced (e.g., as described in U.S. patent No. 4,186,567), or can be obtained, for example, by screening combinatorial libraries comprising variable heavy and variable light chains (see, e.g., U.S. patent No. 5,969,108 to McCafferty).

Antibodies of any animal species may be used in the present invention. Non-limiting antibodies useful in the present invention may be of murine, primate, or human origin. If the antibody is intended for use in humans, a chimeric form of the antibody may be used in which the constant region of the antibody is from a human. Humanized or fully human forms of antibodies may also be prepared according to methods well known in the art (see, e.g., U.S. Pat. No. 5,565,332 to Winter). Humanization can be achieved by a variety of methods including, but not limited to, (a) grafting non-human (e.g., donor antibody) CDRs onto human (e.g., acceptor antibody) frameworks and constant regions with or without retention of critical framework residues (e.g., those residues important for retaining good antigen binding affinity or antibody function), (b) grafting only non-human specificity determining regions (SDRs or a-CDRs; residues critical for antibody-antigen interaction) onto human frameworks and constant regions, or (c) grafting entire non-human variable domains, but "masking" them with human-like moieties by replacing surface residues. Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, Front Biosci 13,1619-1633(2008), and also, for example, in Riechmann et al, Nature 332,323-329 (1988); queen et al, Proc Natl Acad Sci USA 86, 10029-; U.S. Pat. nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; jones et al, Nature 321,522-525 (1986); morrison et al, Proc Natl Acad Sci 81,6851-6855 (1984); morrison and Oi, Adv Immunol 44,65-92 (1988); verhoeyen et al, Science 239,1534-1536 (1988); padlan, Molec Immun 31(3),169-217 (1994); kashmiri et al, Methods 36,25-34(2005) (description of SDR (a-CDR) grafting); padlan, Mol Immunol 28,489-498(1991) (description "resurfacing"); dall' Acqua et al, Methods 36,43-60(2005) (description "FR shuffling"); and Osbourn et al, Methods 36,61-68(2005) and Klimka et al, Br J Cancer 83, 252-. Particular antibodies of the invention are human antibodies. Human antibodies and human variable regions can be generated using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr Opin Pharmacol 5,368-74(2001) and Lonberg, Curr Opin Immunol 20, 450-. The human variable region may form part of, and be derived from, human Monoclonal antibodies produced by hybridoma methods (see, e.g., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Human antibodies and Human variable regions can also be prepared by administering an immunogen to a transgenic animal that has been modified to produce either a fully Human antibody or a fully antibody with a Human variable region in response to antigen challenge (see, e.g., Lonberg, Nat Biotech 23,1117-1125 (2005). Human antibodies and Human variable regions can also be generated by isolating Fv clone variable domain sequences selected from a Human-derived phage display library (see, e.g., Hoogenboom et al, reference Methods in Molecular Biology 178, 1-37(O' Brien et al, Human Press, Totowa, NJ, 2001); and McCafferty et al, Nature 348, 552-554; clackson et al, Nature 352,624-628(1991)), phage display antibody fragments, generally, as single chain fv (scFv) fragments or as Fab fragments, a detailed description of the production of antibodies by phage display can be found in the examples.

In certain embodiments, the antibodies of the invention are engineered to have enhanced binding affinity according to methods disclosed, for example, in PCT publication WO 2011/020783 (see examples relating to affinity maturation) or U.S. patent application publication No. 2004/0132066, the entire contents of which are hereby incorporated by reference. The ability of an antibody of the invention to bind to a particular epitope can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (Liljeblad et al, Glyco J17, 323-. Competition assays can be used to identify antibodies that compete with a reference antibody for binding to a particular antigen, e.g., antibodies that compete with the 51a12 antibody for binding to ASGPR. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear epitope or a conformational epitope) that the reference antibody binds. A detailed exemplary method for Mapping epitopes bound to antibodies is provided in Morris (1996) "Epitope Mapping Protocols", incorporated by reference in Methods in Molecular Biology volume 66 (Humana Press, Totowa, NJ). In an exemplary competition assay, an immobilized antigen (e.g., ASGPR) is incubated in a solution comprising a first labeled antibody (e.g., 51a12 antibody) that binds to the antigen and a second unlabeled antibody that is being tested for the ability to compete with the first antibody for binding to the antigen. The second antibody may be present in the hybridoma supernatant. As a control, the immobilized antigen was incubated in a solution containing the first labeled antibody but no second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to the antigen, excess unbound antibody is removed and the amount of label bound to the immobilized antigen is measured. If the amount of label bound to the immobilized antigen is substantially reduced in the test sample relative to the control sample, this indicates that the second antibody is competing with the first antibody for binding to the antigen. See Harlow and Lane (1988) Antibodies, Chapter 14 of A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Antibodies prepared as described herein can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend in part on a number of factors, such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors or antigens that bind to the antibody may be used. For example, for affinity chromatography purification of the antibody of the present invention, a matrix having protein a or protein G may be used. Protein a or G affinity chromatography and size exclusion chromatography in sequence may be used to separate antibodies, substantially as described in the examples. The purity of the antibody can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, and the like. For example, heavy chain fusion proteins expressed as described in the examples were shown to be intact and correctly assembled as demonstrated by reducing SDS-PAGE (see, e.g., fig. 13-19).

Compositions, formulations and routes of administration

In yet another aspect, the invention provides a pharmaceutical composition comprising any of the antibodies provided herein, e.g., for use in any of the methods of treatment described below. In one embodiment, the pharmaceutical composition comprises any of the antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition comprises any of the antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

Further provided is a method of producing an antibody of the invention in a form suitable for in vivo administration, the method comprising (a) obtaining an antibody of the invention, and (b) formulating the antibody with at least one pharmaceutically acceptable carrier, thereby formulating an antibody preparation for in vivo administration.

The pharmaceutical compositions of the invention comprise a therapeutically effective amount of one or more antibodies dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e., do not produce an adverse, allergic, or other untoward reaction when administered to an animal (e.g., to a human, as desired). In accordance with the present disclosure, the preparation of Pharmaceutical compositions containing at least one antibody and optionally additional active ingredients is known to those skilled in the art, as exemplified by Remington's Pharmaceutical Sciences, 18 th edition, Mack Printing Company,1990, which is incorporated herein by reference. In addition, for animal (e.g., human) administration, it is understood that the preparation should meet sterility, pyrogenicity, overall safety and purity standards as required by the FDA office of biological standards or corresponding authority in other countries. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, such similar materials, and combinations thereof, as known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18 th edition, Mack Printing Company,1990, 1289-1329, which is incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, use of the carrier in therapeutic or pharmaceutical compositions is contemplated.

The composition may contain different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form and whether it needs to be sterile for such routes of administration as an injection. The antibodies of the invention (and any additional therapeutic agent) can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrasplenically, intrarenally, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneous, subconjunctival, intracapsular, mucosal, intrapericardial, intraumbilical, intraventricular, oral, topical, localized, administration by inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, direct localized perfusion bathing of target cells, by catheter, by lavage, in cream, in liquid compositions (e.g., liposomes), or by other methods or any combination of the foregoing methods, as known to one of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18 th edition, Mack Printing Company,1990, incorporated herein by reference). Parenteral administration, especially intravenous injection, is most commonly used for administration of polypeptide molecules, such as the antibodies of the invention.

Parenteral compositions include those designed to be administered by injection, for example, subcutaneous, intradermal, intralesional, intravenous, intraarterial intramuscular, intrathecal, or intraperitoneal injection. For injections, the antibodies of the invention may be formulated in aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, ringer's solution or physiological saline. The solution may contain formulating agents such as suspending, solubilizing, stabilizing and/or dispersing agents. Alternatively, the antibody may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use. Sterile injectable solutions are prepared by: the antibodies of the invention are incorporated in the required amounts, as required, in a suitable solvent, along with various other ingredients enumerated below. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsions, the preferred methods of preparation are vacuum drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a liquid medium which has been previously sterile-filtered. The liquid medium should be suitably buffered and the liquid diluent first rendered isotonic with sufficient saline or glucose prior to injection, as desired. The composition must be stable under the conditions of manufacture and storage; and is protected from the contaminating action of microorganisms such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept to a minimum at safe levels, for example, less than 0.5ng/mg protein. Suitable pharmaceutically acceptable carriers include, but are not limited to: buffers such as phosphate, citric acid and other organic acids; antioxidants (including ascorbic acid and methionine); preservatives (e.g. octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzalkonium bromide; phenol, butanol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other sugars including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannose, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes) and/or nonionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, or the like. Optionally, the suspension may also contain suitable stabilizers or materials that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes.

The active ingredient may be embedded in microcapsules (e.g., hydroxymethylcellulose microcapsules or gelatin microcapsules and poly (methylmethacylate) microcapsules), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or macroemulsions, for example, prepared by coacervation techniques or interfacial polymerization, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18 th edition, Mack Printing Company, 1990). Sustained release articles can be prepared. Suitable examples of sustained-release articles include solid hydrophobic polymeric semipermeable matrices containing the polypeptide, which matrices are in the form of shaped articles, e.g., films, or microcapsules. In particular embodiments, prolonged absorption of the compositions can be brought about by the use in the injectable compositions of agents delaying absorption, for example, aluminum monostearate, gelatin or combinations thereof.

In addition to the compositions described previously, the antibodies may also be formulated as depot preparations. Such long acting formulations may be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the antibody may be formulated with suitable polymeric materials and hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins or as sparingly soluble derivatives, e.g., a sparingly soluble salt.

Pharmaceutical compositions comprising the antibodies of the invention may be manufactured by conventional mixing, dispersing, emulsifying, encapsulating, embedding or lyophilizing processes. The pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations which can be used pharmaceutically. Suitable formulations depend on the route of administration chosen.

The antibody may be formulated into the composition as a free acid or base, neutral or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These salts include acid addition salts, for example those formed with the free amino groups of the protein composition or with inorganic acids such as hydrochloric or phosphoric acids or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts with free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxide or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutically acceptable salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms.

Therapeutic methods and compositions

Any of the antibodies provided herein can be used in a method of treatment.

For use in a method of treatment, the antibodies of the invention will be formulated, administered and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of drug delivery, the method of administration, the administration schedule and other factors known to the medical practitioner.

In one aspect, an antibody of the invention is provided for use as a medicament. In other aspects, antibodies of the invention are provided for use in treating diseases. In certain embodiments, an antibody of the invention is provided for use in a method of treatment. In one embodiment, the invention provides an antibody as described herein for use in treating a disease in an individual in need thereof. In certain embodiments, the invention provides an antibody for use in a method of treating an individual having a disease, the method comprising administering to the individual a therapeutically effective amount of the antibody. In certain embodiments, the disease to be treated is a liver disease. Exemplary liver diseases include hepatitis, cirrhosis or liver cancer such as hepatocellular carcinoma. In a particular embodiment, the disease is a viral infection, in particular a hepatitis virus infection, more particularly an HBV infection. In another embodiment, the disease is cancer, particularly liver cancer, more particularly hepatocellular carcinoma (HCC). In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an antiviral agent if the disease to be treated is a viral infection, or an anticancer agent if the disease to be treated is a cancer. An "individual" according to any of the above embodiments is a mammal, preferably a human.

In a further aspect, the invention provides the use of an antibody of the invention in the manufacture or preparation of a medicament for the treatment of a disease in an individual in need thereof. In one embodiment, the medicament is for use in a method of treating a disease, the method comprising administering to an individual having the disease a therapeutically effective amount of the medicament. In certain embodiments, the disease to be treated is a liver disease. In a particular embodiment, the disease is a viral infection, in particular a hepatitis virus infection, more particularly an HBV infection. In other embodiments, the disease to be treated is cancer. In a specific embodiment, the disease is liver cancer, particularly hepatocellular carcinoma (HCC). In one embodiment, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an antiviral agent if the disease to be treated is a viral infection, or an anticancer agent if the disease to be treated is a cancer. An "individual" according to any of the above embodiments is a mammal, preferably a human.

In yet another aspect, the invention provides a method for treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of an antibody of the invention. In one embodiment, a composition comprising an antibody of the invention in a pharmaceutically acceptable form is administered to the individual. In certain embodiments, the disease to be treated is a liver disease. In a particular embodiment, the disease is a viral infection, in particular a hepatitis virus infection, more particularly an HBV infection. In other embodiments, the disease to be treated is cancer. In a specific embodiment, the disease is liver cancer, particularly hepatocellular carcinoma (HCC). In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an antiviral agent if the disease to be treated is a viral infection, or an anticancer agent if the disease to be treated is a cancer. An "individual" according to any of the above embodiments is a mammal, preferably a human.

The antibodies of the invention are also useful as diagnostic reagents. Binding of the antibody to the epitope can be readily detected by a label attached to the antibody or by using a labeled secondary antibody specific for the antibody of the invention.

In some embodiments, an effective amount of an antibody of the invention is administered to a cell. In other embodiments, a therapeutically effective amount of an antibody of the invention is administered to an individual to treat a disease.

For the prevention or treatment of disease, the appropriate dosage of an antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the patient's weight, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous or concomitant therapeutic intervention, the patient's clinical history and response to the antibody, and the discretion of the attending physician. In any event, the practitioner responsible for administration will determine the concentration of the active ingredient and the appropriate dosage in the composition for an individual subject. A variety of administration regimens are contemplated herein, including but not limited to single or multiple administrations at multiple time points, bolus administrations, and pulse infusions.

The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μ g/kg to 15mg/kg (e.g., 0.1mg/kg-10mg/kg) of antibody may be an initial candidate dose for administration to the patient, whether administered, for example, by one or more separate administrations or by continuous infusion. Depending on the factors mentioned above, a common daily dose may be from about 1. mu.g/kg to 100mg/kg or more. For repeated administration over a period of several days or longer, depending on the disease, treatment will generally continue until the desired suppression of disease symptoms occurs. An exemplary dose of the antibody is in the range of about 0.005mg/kg to about 10 mg/kg. In other non-limiting examples, the dosage may further include about 1 μ g/kg body weight, about 5 μ g/kg body weight, about 10 μ g/kg body weight, about 50 μ g/kg body weight, about 100 μ g/kg body weight, about 200 μ g/kg body weight, about 350 μ g/kg body weight, about 500 μ g/kg body weight, about 1mg/kg body weight, about 5mg/kg body weight, about 10mg/kg body weight, about 50mg/kg body weight, about 100mg/kg body weight, about 200mg/kg body weight, about 350mg/kg body weight, about 500mg/kg body weight to about 1000mg/kg body weight or more per administration; and any range derivable therein. In non-limiting examples of ranges derivable from the values listed herein, based on the values described above, ranges of about 5mg/kg body weight to about 100mg/kg body weight, about 5 μ g/kg body weight to about 500mg/kg body weight, and the like, may be administered. Thus, one or more doses (or any combination thereof) of about 0.5mg/kg, 2.0mg/kg, 5.0mg/kg, or 10mg/kg may be administered to the patient. Such doses may be administered intermittently, e.g., weekly or every 3 weeks (e.g., such that the patient receives from about 2 to about 20 or, e.g., about 6 doses of antibody). A higher initial loading dose may be administered followed by one or more lower doses. However, other dosage regimens may be used. The progress of such therapy is readily monitored by conventional techniques and assays.

The antibodies of the invention will generally be used in an amount effective to achieve the intended purpose. For use in treating or preventing a disease state, an antibody of the invention or a pharmaceutical composition thereof is administered or administered in a therapeutically effective amount. Determining a therapeutically effective amount is well within the ability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For systemic administration, the therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. A dose can be formulated in animal models to achieve inclusion of IC₅₀As determined in cell culture. This information can be used to more accurately determine the dosage for use in humans.

Initial doses can also be estimated from in vivo data (e.g., animal models) using techniques well known in the art. Administration to humans can be readily optimized by one of ordinary skill in the art based on animal data.

The dose number and interval may be adjusted individually to provide plasma levels of antibody sufficient to maintain therapeutic effect. A typical dose for patients administered by injection is about 0.1 to 50 mg/kg/day, typically about 0.5 to 1 mg/kg/day. Therapeutically effective plasma levels can be achieved by administering multiple doses per day. The level in plasma may be measured, for example, by HPLC.

In the case of topical administration or selective ingestion, the effective local concentration of the antibody may not be related to the plasma concentration. One skilled in the art will be able to optimize therapeutically effective local dosages without undue experimentation.

A therapeutically effective dose of the antibodies described herein will generally provide therapeutic benefit without causing substantial toxicity. Toxicity and therapeutic efficacy of the antibodies can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Cell culture assays and assaysThe study can be used to determine LD₅₀(dose lethal to 50% of the population) and ED₅₀(therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as LD₅₀/ED₅₀And (4) the ratio. Antibodies that exhibit large therapeutic indices are preferred. In one embodiment, the antibodies of the invention exhibit a high therapeutic index. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage suitable for use in humans. The dose is preferably positioned at a low or no toxicity level including ED₅₀In the circulating concentration range of (2). The dosage may vary within this range depending on a variety of factors, e.g., the dosage form used, the route of administration used, the condition of the subject, etc. The exact formulation, route of administration and dosage may be selected by The individual physician according to The condition of The patient (see, e.g., Fingl et al, 1975, cited in The Pharmacological Basis of Therapeutics, Chapter 1, page 1, which is incorporated herein by reference in its entirety).

The attending physician of a patient being treated with an antibody of the invention will know how and when to terminate, discontinue or adjust administration due to resulting toxicity, organ dysfunction, etc. Conversely, if the clinical response is inadequate (not including toxicity), the attending physician will also know to adjust the treatment to higher levels. The size of the dose administered in treating the condition of interest will vary with the severity of the condition to be treated, with the route of administration, and the like. The severity of the condition can be assessed, for example, in part, by standard prognostic assessment methods. In addition, the dosage and perhaps frequency of administration will also vary according to the age, weight and response of the individual patient.

Other Agents and treatments

The antibodies of the invention may be administered in combination with one or more other agents in therapy. For example, an antibody of the invention can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent administered for the treatment of a symptom or disease in an individual in need of such treatment. Such additional therapeutic agents may include any active ingredients suitable for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is an antiviral agent. In other embodiments, the additional therapeutic agent is an anti-cancer agent.

Such other agents are suitably present in an amount effective for the intended purpose. The effective amount of such other drugs will depend on the amount of antibody used, the type of disease or therapy, and other factors discussed above. These antibodies are generally used at the same dosages and using the same routes of administration as described herein, or at about 1% to 99% of the dosages described herein, or at any dosage and any route empirically/clinically determined to be appropriate.

Such combination therapies indicated above encompass combined administration (where two or more therapeutic agents are included in the same or separate compositions), and separate administration, in which case administration of the antibody of the invention may occur prior to, concurrently with, and/or after administration of additional therapeutic agents and/or adjuvants.

Article of manufacture

In another aspect of the invention, there is provided an article of manufacture comprising a substance as described above which is useful in the treatment, prevention and/or diagnosis of a disease. The article of manufacture comprises a container and or a label or package insert on or associated with the container. Suitable containers include, for example, vials, syringes, intravenous bags, and the like. The container may be formed from a variety of materials such as glass or plastic. The container contains a composition that is effective, by itself or in combination with another composition, in the treatment, prevention and/or diagnosis of a condition and may have a sterile access port (e.g., the container may be an intravenous bag or a vial having a stopper penetrable by a hypodermic injection needle). At least one active substance in the composition is an antibody of the invention. The label or package insert indicates that the composition is for use in treating a selected condition. Additionally, an article of manufacture can comprise (a) a first container having a composition therein, wherein the composition comprises an antibody of the invention; and (b) a second container having a composition therein, wherein the composition comprises a further therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the composition may be used to treat a particular disease. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution and dextrose solution. It may also include other materials that are popular from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

Examples

The following are examples of the methods and compositions of the present invention. It should be understood that various other embodiments may be implemented in view of the general description provided above.

Recombinant DNA technology

Such as Sambrook, J.et al, Molecular cloning: A laboratory manual; standard methods described in Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York,1989 were used to manipulate DNA. Molecular biological reagents were used according to the manufacturer's instructions.

General information on the nucleotide Sequences of human immunoglobulin light and heavy chains is given in Kabat, E.A. et al, (1991) Sequences of Proteins of Immunological Interest, 5 th edition, NIH published No. 91-3242.

DNA sequencing

The DNA sequence was determined by double-strand sequencing.

Gene synthesis

If desired, the gene segments of interest are generated by PCR using appropriate templates or synthesized by automated gene synthesis from synthetic oligonucleotides and PCR products in Geneart AG (Raugesberg, Germany). In the case where a precise gene sequence is not available, oligonucleotide primers are designed based on sequences from the closest homologues and the gene is isolated by RT-PCR from RNA derived from appropriate tissues. The gene segment flanked by a single restriction endonuclease cleavage site was cloned into a standard cloning/sequencing vector. Plasmid DNA was purified from the transformed bacteria and the concentration was determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. Gene segments are designed with appropriate restriction sites to allow subcloning into the corresponding expression vector. All constructs were designed with a 5' terminal DNA sequence encoding a leader peptide that directs protein secretion in eukaryotic cells. Exemplary leader peptides are given in SEQ ID NO 135-137.

Cloning of antigen expression vectors

The amplified DNA fragment encoding the antigen of interest was inserted in-frame into a mammalian recipient vector, downstream of the human IgG1Fc encoding fragment, which served as a lytic and purification tag (fig. 1). Expression of antigen-Fc fusions with wild-type Fc sequences (SEQ ID NOS: 123, 125, 127, 129, 131, 133) produced homodimeric molecules (avi-Fc-human ASGPR H1 stem: SEQ ID NO:124, avi-Fc-cynomolgus monkey ASGPR H1 stem: SEQ ID NO:126, avi-Fc-human ASGPR H1 stem CRD: SEQ ID NO:130, avi-Fc-cynomolgus monkey ASGPR H1 stem CRD: SEQ ID NO: 132). Protein CLEC10A was identified as the closest homolog of ASGPR H1 and the constructs avi-Fc-human CLEC10A stem (SEQ ID NO:128) and avi-Fc-human CLEC10A stem CRD (SEQ ID NO:134) were expressed to test the specificity of the selected binders. To express the antigen in the unimer state, the DNA fragment was fused to the Fc portion containing the "hole mutation (SEQ ID NO:117, 119) and co-expressed with the Fc-" knob "(SEQ ID NO:121) counterpart (Fc-human ASGPR H1 CRD: SEQ ID NO:118 and 122, avi-Fc-human CLEC10A CRD: SEQ ID NO:120 and 122). Antigen expression is typically driven by the MPSV promoter and transcription is terminated by a synthetic polyadenylation signal sequence located downstream of the CDS. In addition, all constructs contained an N-terminal Avi tag that allowed specific biotinylation during co-expression with Bir a biotin ligase. In addition to the expression cassette, each vector also contains an EBV oriP sequence for autonomous replication in EBV-EBNA expressing cell lines.

Antigen and antibody production and purification

Antigens and antibodies were transiently transfected into HEK293 cells stably expressing the EBV-derived protein EBNA. Simultaneously co-transfected plasmids encoding biotin ligase Bir a allowed Avi tag specific biotinylation in vivo. The protein was subsequently purified using a protein a column followed by gel filtration.

Generation of Attribute-like Lambda Fab libraries

Based on peopleGermline genes generate Fab-style generic lambda antibody libraries using the following V domain pairings: vl3_ 19. lamda. light chain and VH3_23 heavy chain, resulting in a DP 47-Lambda library. The library was randomized in CDR3 of the light chain (L3) and CDR3 of the heavy chain (H3) and assembled from the three fragments by "overlap extension splicing" (SOE) PCR. Fragment 1 contained the 5 'end of the antibody gene including randomized L3, fragment 2 was a central constant fragment encompassing the end of L3 to the beginning of H3, and fragment 3 contained the 3' portions of the randomized H3 and Fab fragments. The following primer combinations were used to generate library fragments for the library: fragment 1(LMB3(SEQ ID NO:146) -Vl 3_19_ L3r primer (SEQ ID NO:143-145)), fragment 2(RJH80(SEQ ID NO:148) -DP47CDR3_ ba (mod) (SEQ ID NO:149)), fragment 3(DP47-v4 primer (SEQ ID NO:140-142) -fdsolong (SEQ ID NO:147) (Table 1.) the PCR parameters used to generate the library fragments were initial denaturation at 94 ℃ for 5 minutes, 25 cycles of initial denaturation at 60 seconds 94 ℃, 60 seconds 55 ℃, 60 seconds 72 ℃ and final extension at 72 ℃ for 10 minutes using equal molar proportions of 3 fragments as templates, for assembly PCR the parameters were initial denaturation at 94 ℃ for 3 minutes and 5 cycles of 60 seconds 94 ℃, 60 seconds 55 ℃, 72 seconds 120 seconds, at which stage the outer primers were added and an additional 20 cycles were performed, followed by final extension at 72 ℃ for 10 min (FIG. 2). After assembling a sufficient amount of full-length randomized Fab fragments, they were digested with NcoI/NheI while the acceptor phagemid vector was similarly treated. Mu.g of the Fab library insert were ligated with 13.3. mu.g of phagemid vector. The purified ligation was used for 60 transformations yielding 1.5x10⁹And (4) a transformant. Phagemid particles displaying the Fab library were rescued and purified by PEG/NaCl purification for selection.

TABLE 1 sequences of primers used to generate class Attribute Lambda libraries

Selection of anti-human ASGPR H1 conjugates from a generic lambda Fab library

Human IgG expressed using HEK293₁Antibody Fc-portion (SEQ ID NO:118, 120, 124, 126, 128, 130, 132, 134) fused monomeric or dimeric human ASGPR protein fragments, the complete extracellular domain or extracellular domain (ECD) fragment of human ASGPR H1 was selected. Although ASGPR H1CRD and CLEC10A CRD were expressed as monomeric Fc fusions (only one Fc carrying a C-terminal fused CRD) using the Fc "hump-in-cave" format, all stem fragments and the entire ECD were expressed as homodimeric Fc fusion proteins (fig. 1). The antigen was enzymatically biotinylated by co-expressing biotin ligase Bir a via an N-terminal avi tag. The elutriation cycle was performed in solution according to the following pattern: (1) human IgG coated onto NUNC maxisorp plates at 10. mu.g/ml was used₁About 10 of prepurification¹²Individual phagemid particles to avoid Fc-binding, (2) non-Fc bound phagemid particles from the pre-wash supernatant were bound to 100nM biotinylated antigen protein in a total volume of 1ml for 0.5 h, (3) by addition of 5.4x 10⁷Streptavidin-coated magnetic beads for 10 min, capture biotinylated antigens and linked specifically bound phage, (4) wash the beads using 5x 1ml PBS/tween 20 and 5x 1ml PBS, (5) elute the phage particles by addition of 1ml 100mM Triethylamine (TEA) for 10 min and neutralize by addition of 500 μ Ι 1M Tris/HCl pH 7.4, (6) re-infect log phase e.coli TG1 cells with phage particles in the supernatant, infect with helper phage VCSM13 and then PEG/NaCl precipitate the phage particles for subsequent rounds of selection. Using constant or decreasing (10)^-7M to 2x 10^-9M) antigen concentration 3-5 rounds of selection were performed. In round 2, the antigen-phage complexes were captured using neutravidin plates instead of using streptavidin beads. Specific binders were identified by ELISA as follows: mu.l of 50nM biotinylated human Fc-stem-CRD, Fc-CRD or Fc-stem/well were coated on neutravidin plates. Fab containing bacterial supernatant was added and treated by using anti-Flag/HRP secondaryThe antibodies detect the bound Fab by means of their Flag-tag. Clones showing significantly higher signal than background were submitted for sequencing (SEQ ID NOs: 1,3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27) and further analysis.

Purification of Fab

Fab from bacterial cultures (protein sequences of the variable domains are listed as SEQ ID NO:2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28) were purified for exact analysis of kinetic parameters. For each clone, 500ml of the culture was inoculated with bacteria carrying the corresponding phagemid and at OD₆₀₀0.9 Induction with 1mM IPTG. Thereafter, the culture was incubated at 25 ℃ overnight and harvested by centrifugation. After incubating the resuspended pellet in 25ml of PPB buffer (30mM Tris-HCl pH 8, 1mM EDTA, 20% sucrose) for 20 minutes, the bacteria were again centrifuged and the supernatant harvested. Using 25ml of 5mM MgSO₄This incubation step was repeated 1 time with the solution. The supernatants from both incubation steps were combined, filtered and loaded onto an IMAC column (His gravitrap, GE Healthcare). Subsequently, the column was washed with 40ml of washing buffer (500mM NaCl, 20mM imidazole, 20mM NaH)₂PO₄pH 7.4). In elution (500mM NaCl, 500mM imidazole, 20mM NaH)₂PO₄pH 7.4), the eluate was rebuffered using a PD10 column (GE Healthcare). The kinetic parameters of the purified fabs were subsequently studied by SPR analysis (Proteon XPR36, Biorad) in a dilution series ranging from 200nM to 6.25 nM.

Determination of affinity by SPR

The affinity (K.sub.p.sub.m) of selected Fab clones was measured by surface plasmon resonance using a ProteOn XPR36 instrument (Biorad) at 25 ℃ with biotinylated monovalent- (avi-Fc-human ASGPR H1CRD, SEQ ID NO:118) or bivalent (avi-Fc-human ASGPR H1 stem-CRD, SEQ ID NO:130) ASGPR H1 antigens immobilized on NLC chip by neutral avidin capture_D). Immobilization of recombinant antigen (ligand): antigen was diluted to 10 μ g/ml with PBST (10mM phosphate, 150mM NaCl pH 7.4, 0.005% tween 20) and subsequently loaded at 30 μ l/min at different contact times to achieve a fixed level of 200, 400 or 800 Response Units (RU) in the vertical direction. Loading an analyte: for one shotKinetic measurements, changing the loading direction to horizontal, two-fold dilution series of purified Fab (concentrations ranging between 100nM and 6.25 nM) were loaded simultaneously at 50, 60 or 100 μ l/min along independent channels 1-5 with association time of 150 or 200 seconds and dissociation time of 240 or 600 seconds. Buffer (PBST) was loaded along the sixth channel to provide an "in-line" blank for reference. Association Rate constants (k) were calculated by fitting both association and dissociation sensorgrams with a simple one-to-one Langmuir binding model in ProteOn management v3.1 software_on) And dissociation rate constant (k)_off). Will balance the dissociation constant (K)_D) Is calculated as k_off/k_onAnd (4) the ratio. Regeneration was performed horizontally using 10mM glycine, pH1.5 at a flow rate of 100. mu.l/min for a contact time of 30 seconds. Two clones 51A12(SEQ ID NOS: 002 and 004) and 52C4(SEQ ID NOS: 006 and 008) were found to be specific for ASGPR H1 CRD. Notably, clone 51a12 revealed an affinity in the subnanomolar range. Clones 5A4(SEQ ID NOS: 010 and 012), 4F3(SEQ ID NOS: 014 and 016), R5C2(SEQ ID NOS: 018 and 020), R9E10(SEQ ID NOS: 022 and 024) and R9E10(SEQ ID NOS: 026 and 028) were generated against the stem region of ASGPR H1 or the interface between the stem and CRD. Affinity for their corresponding human and cynomolgus monkey epitopes was similar. In contrast, NO binding was detected to avi-Fc-human CLEC10A stem CRD (SEQ ID NO:134), demonstrating the high specificity of these binders. Interestingly, clone 5a4 showed strong binding to stem antigen rather than stem-CRD. All measured kinetic and thermodynamic data are summarized in table 2.

Table 2 kinetic and thermodynamic parameters of anti-ASGPR H1 Fab.

Cloning variable antibody domains into expression vectors

All Fab's shown to be specifically bound to their respective antigens by SPR were converted to IgG₁A/lambda antibody. Thus, PCR-amplified DNA fragments containing the heavy and light chain v domains were inserted in-frame with human IgG₁A constant heavy chain or a human constant lambda light chain. Antibody expression is driven by the MPSV promoter and transcription is terminated by a synthetic polyadenylation signal sequence located downstream of the CDS. In addition to the expression cassette, each vector also contains an EBV oriP sequence for autonomous replication in EBV-EBNA expressing cell lines.

Binding assay of antibodies to HepG2 cells

Measurement of human IgG by FACS₁Binding of anti-ASGPR antibody to hepatocellular carcinoma cell line HepG 2. Briefly, 0.2mio cells/well in 96-well round bottom plates were incubated at 300. mu.l at 4 ℃ for 30 minutes with anti-ASGPR antibody at a concentration of 30. mu.g/ml. Unbound antibody was removed by washing the cells with PBS containing 0.1% BSA. Second F (ab') specific to goat anti-human IgG Fc gamma fragment using FITC conjugated AffiniPure₂The fragment (Jackson ImmunoResearch # 109-096-098; 1:20 working solution in PBS, 0.1% BSA) detects the bound antibody. After incubation at 4 ℃ for 30 min, unbound antibody was removed by washing and the cells were fixed with 1% PFA. Cells were analyzed using a BD FACS cantonii (Software BD DIVA) (fig. 3). All antibodies showed strong binding to HepG2 cells.

Fluorescence resonance energy transfer assay

The affinity of IgG to its epitopes on ASGPR-expressing cells was determined by Fluorescence Resonance Energy Transfer (FRET) analysis. For this analysis, the DNA sequence encoding the SNAP tag (plasmid purchased from Cisbio) was amplified by PCR and ligated into an expression vector containing the full-length human ASGPR H1 sequence (Origene). The resulting fusion protein contained full-length ASGPR H1 with a C-terminal SNAP tag. HEK293 cells were transfected with 10 μ g DNA using lipofectamine 2000 as transfection reagent. After 20 hours incubation time, cells were washed with PBS and incubated in LabMed buffer (Cisbio) containing 100nM SNAP-Lumi4Tb (Cibsio) for 1 hour at 37 ℃ resulting in specifically labeling the SNAP tag. Subsequently, the cells were washed 4 times with LabMed buffer to remove the unknotA synthetic dye. The labeling efficiency was determined by measuring terbium emission at 615nm compared to the buffer. The cells were then stored frozen at-80 ℃ for up to 6 months. Affinity was measured by: ASGPR-specific antibody was added to labeled cells (100 cells/well) at a concentration of 50-0.39nM, followed by addition of anti-human Fc-d2 (final concentration 200 nM/well) as a FRET acceptor molecule. After 3 hours incubation time at room temperature, the emission of the acceptor dye (665nm) and the emission of the donor dye (615nm) were determined using a fluorescence reader (Victor3, Perkin Elmer). The ratio of acceptor to donor emission was calculated and subtracted from the background control (cells with anti-huFc-d 2). Curves were analyzed in GraphPad Prism5 and K was calculated_DValues (fig. 4). Although clone 4F3 showed the lowest affinity as measured by SPR for ASGPR H1 stem-CRD (table 2), the strength of binding to the cell surface as IgG was driven by strong avidity, so that 4F3 was the clone with the strongest binding strength at low concentrations. In contrast, clone 51a12 bound to CRD showed significantly weaker binding strength to cells at low antibody concentrations than the purified antigen in SPR studies.

Binding competition with native ASGPR ligand

The competition of ASGPR antibodies with desialylated glycoproteins (e.g. asialofetuin) as natural ligands for ASGPR was analyzed using the hepatocyte cancer cell line HepG 2. 0.2mio cells/well in 96-well round bottom plates were incubated with 40. mu.l Alexa 488-labeled asialofetuin (from fetal bovine serum, Sigma Aldrich # A4781, final concentration 100. mu.g/ml) for 30 minutes at 4 ℃. The binding process is carried out in the presence of calcium, since the binding of ligand to ASGPR is calcium dependent. Unbound protein was removed by washing the cells 1 time with HBSS containing 0.1% BSA. Subsequently 40. mu.l of anti-ASGPR antibody (final concentration 30, 6 and 1.25. mu.g/ml) was added to the cells in the presence of 100. mu.g/ml asialofetuin. Cells were incubated at 4 ℃ for 30 minutes and unbound protein was removed by washing the cells 1 time. Second F (ab') specific to goat anti-human IgG Fc gamma fragment using APC conjugated AffiniPure₂The fragment (Jackson ImmunoResearch # 109-136-170; 1:50 working solution in HBSS containing 0.1% BSA) was used as the secondary antibody. After incubation at 4 ℃ for 30 minutes, none was removed by washingA conjugated secondary antibody. Cells were fixed with 1% PFA and analyzed using BD FACS cantonii (software BD DIVA). Analysis of both CRD-specific and stem-CRD-specific antibodies revealed that the antibodies bound to ASGPR H1 independent of the presence of asialofetuin and vice versa, no binding competition occurred. (FIGS. 5 and 6).

Internalization study

It is known that the uptake of desialylated glycoproteins into liver cells occurs very rapidly upon binding to ASGPR. During this receptor-mediated endocytosis, the endosomal lumen becomes acidic, causing dissociation of the receptor-ligand complex. Although the ligand was targeted for degradation in lysosomes, ASGPR recycling was shown back to the cell surface. To analyze the residence time of the antibody on the cell surface, the internalization of ASGPR-antibody complexes was analyzed using the hepatocellular carcinoma cell line HepG 2. ASGPR positive HepG2 cells were transformed to 4 ℃ in cell culture medium to inhibit internalization. After incubation with antibody (30 μ g/ml) for 45 minutes at 4 ℃ on a shaker, unbound antibody was removed by washing 2 times with cold PBS, and cells were resuspended and cultured in pre-warmed media at 37 ℃ to reactivate cell metabolism, including receptor-mediated endocytosis. An aliquot was taken immediately and stored on ice, which represents time point zero. The remaining cells were incubated at 37 ℃ and after 5, 15, 30 and 120 minutes, additional samples were taken and washed with cold PBS to stop further internalization. Fc gamma specific second F (ab') against human IgG using PE conjugated AffiniPure goat₂Antibody fragments (Jackson ImmunoResearch #109-116-170, 1:50 working solution) detect cell surface bound antibodies. After incubation at 4 ℃ for 30 min, unbound antibody was removed by washing with PBS containing 0.1% BSA. Cells were fixed with 1% PFA and analyzed using BD FACS cantonii (software BD DIVA). Figure 7A shows exemplary cell surface exposed antibody levels of clones 4F3 and 51a 12. Interestingly, extracellular antibody signal decreased significantly during the first 30 minutes (to a 60% signal drop), but the subsequent drop was delayed over the remaining time course. This result indicates that the antibody internalizes very efficiently, but then eventually recycles back to the cell surface, resulting in a dynamic steady state of constant internalization and recyclingStatus. To support this hypothesis, the same experiment was performed, but the incubation of the cells with the antibody was performed at 37 ℃ for 45 minutes in cell culture medium. These conditions allow the receptor-antibody complex to form and internalize during the entire incubation time, ultimately leading to constant endocytosis and recycling homeostasis. After this time, unbound antibody was removed by washing 2 times with warm PBS and the cells were resuspended in warm medium. A sample was taken immediately and stored on ice, which represents time point zero. The remaining cells were incubated at 37 ℃ and after 5, 15, 30 and 120 minutes, additional samples were taken and washed with cold PBS to stop further internalization. Detection of surface exposed antibodies was performed as described above. FACS analysis revealed that the decrease in signal intensity was less pronounced during the experimental time course after incubation at 37 ℃ rather than 4 ℃, showing a balance of internalization and recycling of the antibody-receptor complex produced by incubation at 37 ℃ (fig. 7B). To further compliment the constant internalization and recycling hypothesis, the internalization of ASGPR H1-specific antibodies was further analyzed using a battery of FITC-directly labeled antibodies. Labeled antibodies were incubated with HepG2 cells at 4 ℃ allowing binding but not internalization of the antibody to ASGPR H1, as previously described. After incubation with antibody (30 μ g/ml) for 45 minutes at 4 ℃ on a shaker, unbound antibody was removed by washing 2 times with cold PBS, and cells were resuspended and cultured in pre-warmed media at 37 ℃ to reactivate cell metabolism, including receptor-mediated endocytosis. Cell aliquots were taken after 0,5, 15, 30 and 120 minutes and washed with cold PBS to stop further internalization. Fc gamma specific second F (ab') against human IgG using PE conjugated AffiniPure goat₂Antibody fragments (Jackson ImmunoResearch #109-116-170, 1:50 working solution) detect cell surface bound antibodies. As seen before, the detection level of surface exposed antibody decreased significantly during the first 30 minutes, after which it stabilized (fig. 7C). However, detection of IgG by FITC signal representing surface exposed and internalized antibodies revealed that total antibody amounts remained constant over time (fig. 7D). This result strongly supports the following findings: the antibody is in a dynamic steady state of constant internalization and recycling.

Generation of an L3 affinity library based on clone 51A12

Analysis of the antibody sequence revealed two hot spots in the CDR3 region of the 51a12 light chain, two adjacent cysteines and one glycosylation site (fig. 8). To generate 51a 12-derived cysteine-and glycosylation-free clones, a mature library randomized in LCDR3 was generated. The sequence of clone 51A12(A82G, C112S, C113S, S116A) (SEQ ID NO:33) was used as a randomized template. The triplet encoding position "RDISSNRAVRN" was randomized over the entire segment. To generate the library, the DNA portion resulting from the two fragment overlapping PCR products was cloned into a phage vector. To generate fragment 1, primer combinations LCDR3rand (SEQ ID NO:151) and fdseqlong (SEQ ID NO:147) (Table 1 and Table 3) were used, using clone 51A12(A82G, C112S, C113S, S116A) as template. The amplification conditions included an initial incubation step at 94 ℃ for 5 minutes followed by 25 cycles, each cycle consisting of 30 seconds of denaturation at 94 ℃, 30 seconds of renaturation at 60 ℃ and 90 seconds of extension at 72 ℃, followed by a final extension step at 72 ℃ for 10 minutes. The resulting fragments were purified on an agarose gel. Fragment 2 was generated using primer combinations LCDR3rev (SEQ ID NO:150) and LMB3(SEQ ID NO:146) (tables 1 and 3). The amplification conditions included an initial incubation step at 94 ℃ for 5 minutes followed by 25 cycles, each cycle consisting of a 30 second denaturation at 94 ℃, a 30 second renaturation at 60 ℃ and a 30 second extension step at 72 ℃, followed by a10 minute final extension step at 72 ℃. For assembly of the two fragments, equimolar amounts of fragment 1 and fragment 2 were used. The amplification conditions included an initial incubation step at 94 ℃ for 5 minutes followed by 5 cycles without primers, each cycle consisting of a denaturation step at 94 ℃ for 1 minute, a renaturation step at 60 ℃ for 1 minute and an extension step at 72 ℃ for 120 seconds. After addition of the outer primers LMB3 and fdseqlong, 20 additional cycles were performed using the same parameters. Finally, a final incubation step at 72 ℃ was performed for 10 minutes. The resulting gel-purified DNA fragment and clone 51A12(A82G, C112S, C113S, S116A) (SEQ ID NO:33) were both digested with NcoI/PstI (FIG. 9). To generate the library, ligation was performed with 10. mu.g insert and 30. mu.g vector. Transformation of purified ligation products into TG1 bacteria by electroporation yielded 3X 10⁹And (4) a transformant. Phagemid particles displaying the Fab library were rescued and purified by PEG/NaCl purification for selection.

TABLE 3 sequences of primers used to generate the L3 affinity maturation library.

Selection of 51A 12-derived affinity matured clones without cysteine and glycosylation sites

The generation of 51A 12-derived affinity matured Fab without cysteine and glycosylation sites inside LCDR3 was performed by phage display using standard protocols (Silacci et al, (2005), Proteomics 5,2340-50). In the first panning round, selection was performed in solution according to the following procedure: (1) about 10 in a total volume of 1ml¹²Individual phagemid particles bound to 10nM biotinylated Fc-CRD for 0.5 h, (2) by addition of 5.4 × 10⁷Streptavidin-coated magnetic beads for 10 min, capture biotinylated Fc-CRD and specifically bound phage particles, (3) wash the beads using 5x 1ml PBS/tween 20 and 5x 1ml PBS, (4) elute the phage particles by adding 1ml 100mM TEA for 10 min and neutralize by adding 500 μ Ι 1M Tris/HCl pH 7.4, (5) reinfect the log-grown e.coli TG1 bacteria, and (6) infect with helper phage VCSM13 and then PEG/NaCl precipitate the phage particles to be used in subsequent rounds of selection. Use (from 10x10^-9M to 0.5x10^-9M) reduced antigen concentration 3 rounds of selection were performed. In rounds 2 and 3, the antigen-phage complexes were captured using neutravidin plates instead of streptavidin beads. In addition, the neutral avidin plate in 2L PBS washing 3 hours. Specific binders were identified by ELISA as follows: mu.l 50nM biotinylated Fc-CRD/well were coated on neutravidin plates. Bacterial supernatants containing Fab were added and bound Fab was detected by their Flag-tag using anti-Flag/HRP secondary antibodies. ELISA positive clones were expressed as soluble Fab fragments in 96-well format with bacteria and supernatants were subjected to kinetic screening experiments by SPR analysis using Proteon XPR 36. Clones expressing the Fab with the highest affinity constant were identified and the light chain of the corresponding phagemid was sequenced (51A12_ C1, SEQ ID NO: 35; 51A12_ C7, SEQ ID NO: 37; 51A12_ E7, SEQ ID NO: 37; SEQ ID NO: 2; SEQ ID NO: 35; SEQ ID NO: 37; SEQ ID NO: 51A12_ E7; SEQ ID NO: 2; SEQ ID NO: 35-NO: 32; SEQ ID NO: 35; SEQ ID NO: 37; SEQ ID NO: 35-NO: 2; SEQ ID NO: 2),39 in SEQ ID NO; 51A12_ H3, SEQ ID NO: 41; 51A12_ A6, SEQ ID NO 43; 51A12_ D1, SEQ ID NO 45; 51A12_ H6, SEQ ID NO: 47). All clones lacked any of the critical amino acids in the light chain CDR3 region.

Determination of affinity of 51A 12-based affinity matured clones by SPR

The affinity (K.sub.p.sub.p.sub.m) of the purified 51A 12-derived Fab fragments was measured by surface plasmon resonance at 25 ℃ using a ProteOn XPR36 instrument (Biorad) with biotinylated monovalent (avi-Fc-human ASGPR H1CRD, SEQ ID NO:118) or divalent (avi-Fc-human ASGPR H1 stem-CRD, SEQ ID NO:130) ASGPR H1 antigen immobilized on NLC chip by neutral avidin capture (avid)_D) The Fab fragment consists of the parent heavy chain (SEQ ID NO:4) and an affinity mature light chain (51A12_ C1, SEQ ID NO: 36; 51A12_ C7, SEQ ID NO: 38; 51A12_ E7, SEQ ID NO: 40; 51A12_ H3, SEQ ID NO: 42; 51A12_ A6, SEQ ID NO: 44; 51A12_ D1, SEQ ID NO: 46; 51A12_ H6, SEQ ID NO: 48). Immobilization of recombinant antigen (ligand): antigen was diluted to 10 μ g/ml with PBST (10mM phosphate, 150mM NaCl pH 7.4, 0.005% tween 20) and subsequently loaded at 30 μ l/min at different contact times to achieve a fixed level of 200, 400 or 800 Response Units (RU) in the vertical direction. Loading an analyte: for one-shot kinetic measurements, the loading direction was changed to horizontal, and two-fold dilution series of purified Fab (concentrations ranging between 12.5nM and 0.78 nM) were loaded simultaneously at 100 μ Ι/min along independent channels 1-5 with association time of 150 or 200 seconds and dissociation time of 3600 seconds. Buffer (PBST) was loaded along the sixth channel to provide an "in-line" blank for reference. Simple one-to-one Langmuir binding model in v3.1 software was managed with ProteOn, and the association rate constant (k) was calculated by fitting the association sensorgram and dissociation sensorgram simultaneously_on) And dissociation rate constant (k)_off). Will equilibrate the dissociation constant (K)_D) Is calculated as k_off/k_onAnd (4) the ratio. Regeneration was performed horizontally using 10mM glycine, pH1.5 at a flow rate of 100. mu.l/min for a contact time of 30 seconds. Although most of the selected clones showed similar affinities to the parental clone, clone 51A12_ A6(SEQ ID NO:44) showed significantly improved affinities (Table 4).

TABLE 4 kinetic and thermodynamic parameters of affinity matured anti-ASGPR 1 Fab.

Binding assay of affinity matured 51A12 derivatives to HepG2 cells

Selected affinity matured 51a12 derivatives were measured for binding to ASGPR positive hepatocellular carcinoma cell line HepG2 by FACS. As a negative control, ASGPR negative cell line Hela was used. 0.2mio cells/well in 96-well round bottom plates were incubated with purified Fab fragments (1.1, 3.3 and 10. mu.g/ml) or human IgG at 4 ℃ in 300. mu.l₁The converted antibodies (0.01, 0.04, 0.1, 0.4, 1.1, 3.3 and 10. mu.g/ml) were incubated for 30 minutes. Unbound molecules were removed by washing the cells with PBS containing 0.1% BSA. Goat anti-human F (ab') with FITC conjugated AffiniPure₂Fragment-specific second F (ab')₂Fragment (Jackson Immuno Research Lab #109-096-097) or FITC conjugated AffiniP goat anti-human IgG Fc gamma fragment specific second F (ab')₂The fragment (Jackson ImmunoResearch #109-096-098, 1:20 working solution in PBS, 0.1% BSA) detects the bound molecules. After incubation at 4 ℃ for 30 min, unbound antibody was removed by washing and the cells were fixed with 1% PFA. Cells were analyzed using a BD FACS CantoII (Software BD DIVA). Analysis of Fab binding to HepG2 cells revealed strong binding of all clones (fig. 10). Variant 51A12_ A6(SEQ ID NO:44) was the most potent binder in both SPR analysis and cell binding studies. For all clones, as IgG₁Binding assays of the clonal variants of the transition antibody to HepG2 cells yielded binding patterns with similar intensity (fig. 11A), while binding to Hela cells was very weak or undetectable at the highest antibody concentration (fig. 11B), highlighting the specificity of these clonal variants.

Generation of IgG-IFN alpha DNA constructs

ASGPR H1-based antibodies 51A12, 51A12(S116A), 51A12(A82G, S116A), 52C4, 5A4, 4F3, R5C2, R9E10, R7E12, 51A12_ C1, 51A12_ C7, 51A12_ E7, 51A12_ H3, 51A12_ A6, 51A12_ D1, and 51A12_ H6 produced DNA sequences encoding IgG-IFN α fusion proteins targeting ASGPR H1, in which an interferon- α 2A (IFN α) is fused to the C-terminus of a heterodimeric heavy chain as shown in FIG. 12A. Targeting of hepatocytes in which ASGPR H1 is selectively expressed is achieved by a bivalent antibody Fab region (affinity effect). Heterodimerization leading to the presence of a single IFN α was achieved by applying the technique of protuberance-into-hole (kih). To minimize the production of homodimeric IgG-cytokine fusions by (G)₄S)₃The linker fuses the cytokine to the C-terminus of the knob-containing IgG heavy chain (C-terminal Lys residue deletion). The antibody-cytokine fusion has IgG-like properties. To reduce fcyr binding/effector function and prevent FcR co-activation, a P329G L234A L235A (LALA) mutation was introduced in the Fc domain. However, FcRn binding is not impaired. The DNA sequences encoding these immunoconjugates are set forth in SEQ ID NOS 49, 51 and 53(51A12), SEQ ID NOS 55, 57 and 59(52C4), SEQ ID NOS 93, 51 and 53(51A12A82G, S116A), SEQ ID NOS 91, 51 and 53(51A12, S116A), SEQ ID NOS 61, 63 and 65(5A4), SEQ ID NOS 67, 69 and 71(4F3), SEQ ID NOS 73, 75 and 77(R5C2), SEQ ID NOS 79, 81 and 83(R9E19), SEQ ID NOS 85, 87 and 89(R7E12), SEQ ID NOS 95, 51 and 53(51A12_ C1), SEQ ID NOS 97, 51 and 53(51A12_ C5), SEQ ID NOS 99, 51 and 53(51A12_ E42), SEQ ID NOS 101, 5953, SEQ ID NOS 6353, SEQ ID NOS 51 and 69551A 5951, SEQ ID NOS 6955, SEQ ID NOS 6351 and 69551A 9_ 8227, SEQ ID NOS 99, SEQ ID NOS 103, SEQ ID NO 6353, SEQ ID NOS 103 and 6955, 51 and 53(51A12_ D1), SEQ ID NOs: 107, 51 and 53(51A12_ H6). In addition, an alternative pocket-heavy chain was generated in which both the VH domain and the CH1 domain were deleted (SEQ ID NO: 115). The resulting Fc fragment was able to heterodimerize with the full-length knob heavy chain, resulting in a monovalent antibody with a single cytokine fusion (fig. 12B). As a negative control for the functional assay, a corresponding DNA construct was generated encoding a control DP47GS/DPL16 non-targeted IgG-IFNa protein in which IgG did not bind to a specific target. The DNA sequences of immunoconjugates of this isotype are given in SEQ ID NOS 109, 111 and 113.

Expression and purification of antibody-cytokine constructs

Exponential growth by cotransfection with mammalian expression vectors using calcium phosphate transfectionThe HEK293-EBNA cells of (a) produce an immunoconjugate. Alternatively, HEK293-EBNA cells grown in suspension were transfected with the corresponding expression vectors by Polyethylenimine (PEI). Subsequently, the IgG-cytokine fusion protein was purified from the supernatant by a method consisting of one affinity step (protein a) followed by size exclusion chromatography (Superdex200, GE Healthcare). Protein A column (HiTrap ProtA, GE Healthcare) was equilibrated in 20mM sodium phosphate, 20mM sodium citrate pH 7.5. After loading the supernatant, the column was washed first with 20mM sodium phosphate, 20mM sodium citrate, pH 7.5 and then with 13.3mM sodium phosphate, 20mM sodium citrate, 500mM sodium chloride, pH 5.45. IgG-cytokine fusion proteins were eluted with 20mM sodium citrate, 100mM sodium chloride, 100mM glycine, pH 3. Fractions were neutralized, pooled and purified by size exclusion chromatography (HiLoad16/60Superdex200, GE Healthcare) in final formulation buffer (25mM potassium phosphate, 125mM sodium chloride, 100mM glycine pH6.7 or 20mM histidine, 140mM NaCl, pH 6.0). The protein concentration of the purified protein sample was determined by measuring the Optical Density (OD) at 280nm using the molar extinction coefficient calculated based on the amino acid sequence. The purity and molecular weight of the immunoconjugates were analyzed by SDS-PAGE or Caliper in the presence and absence of reducing agent (5mM 1, 4-dithiothreitol). Used according to the manufacturer's instructionsPre-formed gel systems (Invitrogen) (4-20% Tris-glycine gel or 3-12% Bis-Tris). Superdex 20010/300 GL size exclusion column (GE Healthcare) in 2mM MOPS, 150mM NaCl, 0.02% NaN at 25 deg.C₃Samples of the immunoconjugate were analyzed for aggregate content in running buffer pH 7.3. Analytical data for selected clones are shown in FIG. 13(51A12kih IgG IFN α, SEQ ID NO:50, 52, 54), FIG. 14(4F3kih IgG IFN α, SEQ ID NO:68, 70, 72), FIG. 15(51A12_ C1kih IgG IFN α, SEQ ID NO:96, 52, 54), FIG. 16(51A12_ E7kih IgG IFN α, SEQ ID NO:100, 52, 54), FIG. 17(51A12_ C7kih IgG IFN α, SEQ ID NO:98, 52, 54), FIG. 18 (non-targeted kih IgG IFN α, SEQ ID NO:110, 112, 114) and FIG. 19 (monovalent 51A12kih IgG IFN α, SEQ ID NO:50, 52, 116)And (6) summarizing.

Determination of the affinity of IgG-IFN alpha immunoconjugates for ASGPR H1 by SPR

ASGPR H1 binding activity of clone 51a12 and clone 52C4 used as exemplary IgG-IFN α immunoconjugates was determined by Surface Plasmon Resonance (SPR) on a ProteOn XPR36 instrument (Biorad) and compared to the corresponding unmodified IgG antibodies. Biotinylated avi-Fc human ASGPR H1CRD antigen was immobilized on NLC chip via neutravidin capture. Immobilization of recombinant antigen (ligand): antigen was diluted to 10 μ g/ml with PBST (10mM phosphate, 150mM sodium chloride pH 7.4, 0.005% tween 20) and subsequently loaded at 30 μ l/min at different contact times to achieve a fixed level of 400 Response Units (RU) in the vertical direction. Analyte loading: for one-shot kinetic measurements, the loading direction was changed to horizontal, and two-fold dilution series of purified IgG, monovalent antibody-cytokine fusions, and bivalent antibody-cytokine fusions (ranging in concentration between 50nM and 3.25 nM) were loaded simultaneously at 50 μ Ι/min along independent channels 1-5, with an association time of 120 or 200 seconds and an dissociation time of 300 seconds. Buffer (PBST) was loaded along the sixth channel to provide an "in-line" blank for reference. Simple one-to-one Langmuir binding model in v3.1 software was managed with ProteOn, and the association rate constant (k) was calculated by fitting the association sensorgram and dissociation sensorgram simultaneously_on) And dissociation rate constant (k)_off). Will equilibrate the dissociation constant (K)_D) Is calculated as k_off/k_onAnd (4) a ratio. Regeneration was performed in the horizontal direction using 10mM glycine, pH1.5 at a flow rate of 100. mu.l/min for a contact time of 30 seconds. The data show-within the error of the method-that both the clone 51a 12-based immunoconjugate (SEQ ID NOs: 50, 52, 54) and clone 52C 4-based immunoconjugate (SEQ ID NOs: 56, 58, 60) retained affinity (monovalent display) and avidity (dimer display) for human ASGPR H1 (table 5).

TABLE 5 kinetic and thermodynamic parameters of the monovalent and divalent binding patterns of clone 51A12 and clone 52C4 to ASGPR H1.

Binding of IgG-IFN alpha immunoconjugates to ASGPR positive and negative cells

To characterize the specificity of the antibody conjugates, antibody-cytokine conjugates were incubated with ASGPR positive and negative cells and specific binding was measured by FACS analysis. For this purpose, human primary hepatocytes (from 3 donors; purchased from Celsis In Vitro Technologies (Baltimore, Md)), Huh-7 cells, HepG2 cells, A549 cells, Hela cells and 293T cells (1X 10 each)⁵Individual cells) were incubated with 1 μ g ASGPR H1-specific IgG kih IFN α samples for 45 minutes on ice. After washing, cells were incubated with labeled goat anti-human IgG secondary antibody (BD Biosciences, San Diego, CA) for 30 minutes on ice. After three washes, stained cells were analyzed by FACS analysis using a Calibur flow cytometer. In all FACS assays, isotype control conjugates (non-targeted kih IgG IFN. alpha., SEQ ID NO:110, 112, 114) were used to determine the background, which was subtracted from the MFI value of the test antibody. By using directly labeled antibody conjugates according to the manufacturer's instructions (R-phycoerythrin human IgG labeling kit, Life Technologies), binding assays of human Peripheral Blood Mononuclear Cells (PBMCs) were performed. Binding analysis revealed that clones 51A12IgG kih IFN α (SEQ ID NOS: 50, 52, 54) and 4F3IgG kih IFN α (SEQ ID NOS: 68, 70, 72) showed highly specific binding to ASGPR positive cells, while the signal on ASGPR negative cells was comparable to isotype control conjugates (FIG. 20). In addition, clone 4F3IgG kih IFN α was analyzed for binding saturation curves. For this purpose, antibody-IFN α conjugates were incubated with human primary hepatocytes (from 3 donors) in dilution columns ranging from 0.0001 to 6.7 μ g/ml and binding strength was recorded by FACS analysis. As shown in figure 21, saturation of binding was achieved on human primary hepatocytes as well as on the control cell line HepG2 at antibody concentrations of 0.25-0.74 μ g/ml, and higher antibody concentrations did not further significantly increase the binding signal.

Analysis of surface exposed ASGPR levels on HepG2 cells over time

The uptake of desialylated glycoproteins into liver cells is known to occur very rapidly upon binding to ASGPR. During this receptor-mediated endocytosis, the endosomal lumen becomes acidic, causing dissociation of the receptor-ligand complex. Although the ligand was targeted for degradation in lysosomes, ASGPR recycling was shown back to the cell surface. Since several receptors were shown to trigger down-regulation of receptor expression upon receptor binding followed by internalization, the level of sub-surface exposed ASGPR in the presence of anti-ASGPR H1 antibody was measured over time. For this experiment, HepG2 cells were incubated with clone 51a 12-derived antibody as IgG or as a monovalent or bivalent antibody-IFN α fusion protein for up to 5 hours. As a negative control, an irrelevant antibody (GA101) that did not specifically bind HepG2 cells was used (all at 30. mu.g/ml). During incubation at 37 ℃, samples were removed after 30, 60, 120, 180 and 300 minutes and washed with cold PBS. Fcg fragment specific F (ab')₂The fragment (Jackson Immuno Research Lab, 1:50 working solution) detects the cell surface bound antibody. After incubation at 4 ℃ for 30 min, unbound antibody was removed by washing with PBS containing 0.1% BSA. Cells were fixed with 1% PFA and analyzed using BD FACS cantonii (software BD DIVA). To verify the integrity of the antibody-cytokine fusion, the presence of IFN α was also detected. Cells were incubated at 4 ℃ with mouse monoclonal antibodies (MMHA-1, #21105-1, R) against human interferon alpha&D Systems, 5. mu.g/ml) for 30 minutes. Unbound antibody was removed by washing with PBS containing 0.1% BSA and FITC-conjugated anti-mouse F (ab')₂A fragment (Serotec, STAR 105F; 1:50 working fluid) was used as the second antibody. Cells were fixed with 1% PFA and analyzed using BD FACS cantonii (software BD DIVA). The results shown in figure 22 indicate that constant levels of surface exposed antibody bound to ASGPR without any binding-induced receptor downregulation over the measured period of time. Notably, the monomeric IgG-IFN α construct produces the strongest signal, likely due to the fact that: each ASGPR complex can be combined with 2 times of singleA bulk IgG-IFN α molecule (FIG. 22A).

Confocal microscopy

Three-dimensional and time-resolved analysis of ASGPR-mediated internalization of antibody-cytokine fusion constructs was performed by confocal microscopy. For this analysis, HepG2 cells were grown to 50-60% confluence on glass-bottom plates (Nunc) in a cell culture incubator. The plate was then rinsed 2 times with pre-warmed PBS (37 ℃) to replace the medium with PBS and quickly placed on the microscope stage (5% CO at 37 ℃)₂). For this experiment, clone 51a12kih IgG IFN α was directly labeled with Alexa 488. The labeled construct (20. mu.g/ml) was added directly to HepG2 cells at the microscope stage. Detection was started 5 minutes after antibody addition using a spinning disc confocal microscope. Data were collected every 3 seconds for 1 hour (100x magnification) on 10 stacks (z-level) covering the entire cell thickness. The binding of antibody-cytokine constructs to surface exposed ASGPR was not equally distributed but found to be clustered (fig. 23A). The clusters spread over the entire surface. Time resolved analysis of this experiment clearly revealed immediate internalization of the antibody-cytokine fusion construct within minutes (fig. 23B). After internalization of the IgG-cytokine construct in the blebs, the protein was subsequently transported back to the surface on the top surface of the cells (data not shown).

Determination of the affinity of IgG-IFN alpha immunoconjugates for interferon-alpha receptor 2 by SPR

The binding activity of IgG-IFN α immunoconjugates to the high affinity interferon- α receptor 2(IFNAR2) was determined by Surface Plasmon Resonance (SPR) on a ProteOn XPR36 instrument (Biorad) and compared to Roferon (Roferon). A commercially available IFNAR2-Fc fusion protein (R) was prepared by standard amine coupling&D Systems) are fixed in a vertical orientation on the sensor surface. For one-shot kinetic measurements, the loading direction was changed to horizontal, and two-fold dilution series of purified antibody-cytokine fusions (varying concentrations between 50nM and 3.25 nM) were loaded simultaneously at 50 μ Ι/min along independent channels 1-5 with association times of 120 or 200 seconds and dissociation times of 300 seconds. Buffer (PBST) was loaded along the sixth channel to provide an "in-line" blank for reference. By usingProteOn manages a simple one-to-one Langmuir binding model in v3.1 software, and calculates the association rate constant (k) by fitting the association sensorgram and dissociation sensorgram simultaneously_on) And dissociation rate constant (k)_off). Will equilibrate the dissociation constant (K)_D) Is calculated as k_off/k_onAnd (4) the ratio. Regeneration was performed horizontally using 10mM glycine, pH1.5 at a flow rate of 100. mu.l/min for a contact time of 30 seconds. The measured affinity of the antibody-cytokine fusion protein was about (k)_on1.57x10⁶ 1/Ms；k_off6.15x10^-31/s；K_D4nM) and thus comparable to the published affinities of the recombinantly produced protein rovolam, which indicates that the C-terminal fusion of IFN α with IgG does not affect the binding affinity to IFNAR 2.

Determination of antiviral Activity of ASGPR mAb-IFN alpha

To analyze the functional activity of IFN α as a component of an IgG-cytokine fusion and to compare it to roffern, the biological activity of the IFN α fusion constructs was tested in a viral protection assay. For this study, MDBK cells were preincubated with Rofern or antibody-cytokine fusion for 1-4 hours. Followed by the addition of vesicular stomatitis virus for an additional incubation of 16-24 hours. At the end of this incubation step, live cells were stained with crystal violet staining solution (0.5%) and quantified using a microplate reader at 550-. The biological activity of all IgG-cytokine constructs was determined against standardized rofiran solutions in a full dose-response curve analysis using a 4-parameter log fit function. As shown in table 6, the antibody-IFN α fusion constructs showed activity corresponding to about 5% of the activity of roffmann, regardless of the binding valency of the antibody. Since IFN α fusion to the C-terminal end of IgG does not affect binding affinity to IFNAR2 (shown above), interaction with low affinity interferon- α receptor 1(IFNAR1) may be sterically impaired, ultimately leading to reduced signaling of the IFNAR holocomplex.

Table 6 functional IFN α activity of antibody-cytokine conjugates compared to roffmam.

Antiviral activity of ASGPR-specific IgG-IFN alpha conjugates in HCV replicon assays and EMCV CPE assays

To characterize the functional activity of the IgG-IFN α fusion proteins and compare with commercially available Roferun and Pegasys (PEGylated interferon-. alpha.2a), antiviral activity was studied using ASGPR positive cells (Huh7-2209-3) and ASGPR negative cells (Hela).

To analyze the antiviral activity of compounds on ASGPR negative cells, Hela cells were seeded at 15,000 cells/well in 96-well opaque wall plates. After overnight incubation, wells were evacuated and 50 μ Ι of antibody-cytokine conjugate diluted in EMEM (10% FBS) was added. Hela cells were pretreated with the IgG-IFN α construct for 3 hours at 37 ℃ and 50. mu.l of EMCV (VR-1762, ATCC) was added to each well (2,000 TCID in EMEM)₅₀Hole/bore). Viable cells were measured 24 hours after infection using the CellTiter-Glo kit (G7572, Promega). Add 100. mu.l CellTiter-Glo reagent to each well and incubate for 10 min at room temperature with gentle shaking. Luminescence signals were then recorded using a Berthold Mithras luminometer (Berthold Technologies). Results represent the percentage of surviving cells (FIG. 24A), and total EC₅₀The values and the number of experimental replicates are summarized in table 7. EC of Roferun on ASGPR negative Hela cells₅₀The values were 75 times less than other compounds such as 4F3IgG kih IFN α, indicating that this compound is much more functionally active than the other compounds tested. In contrast, the activity of Pegasys was comparable to those of ASGPR specific IgG kih IFN α conjugates.

ASGPR positive Huh7 derived hepatoma cell lines 2209-23 were formed by stable transfection of a bicistronic HCV replicon, where the first open reading frame of the replicon, driven by the HCV IRES, contained the renilla luciferase gene fused to the neomycin phosphotransferase gene (NPTII) and the second open reading frame, driven by the EMCV IRES, contained the HCV nonstructural genes NS3, NS4a, NS4b, NS5A and NS5B derived from the NK5.1 replicon backbone. Cells were incubated at 37 ℃ with 5% CO₂Under a humid atmosphere of (a) supplemented with Glutamax^TMAnd 100mg/ml sodium pyruvate (#10569-010) in DMEM. The medium was further supplemented with 10% (v/v) FBS (#10082-139), 1% (v/v) penicillin/streptomycin (#15140-122) and 1% (v/v) geneticin. All reagents were obtained from Invitrogen.

Huh 72209-23 cells in DMEM containing 5% (v/v) fetal bovine serum were plated in 96-well plates at 5000 cells/well in a volume of 90. mu.l. At 24 hours after plating, the antibody-cytokine conjugate (or culture medium as a control) was added to the cells at a volume of 10 μ l at a dilution 3-fold (0.01-2000pM) in a range of 12 wells. After addition of compound, the final volume was 100. mu.l. At 72 hours after compound addition, the Renilla luciferase reporter signal was read using the Renilla luciferase assay System (Promega, # E2820). EC (EC)₅₀Values were calculated as the concentration of compound at which a 50% reduction in renilla luciferase reporter levels was observed compared to control samples (in the absence of compound). Dose-response curves and ECs were obtained by using the XLfit4 program (ID Business Solutions ltd., Surrey, UK)₅₀The value is obtained. Although the antiviral activity of the antibody-cytokine construct was reduced when exposed to ASGPR negative cells (table 6 and fig. 24A), clones 51a12IgG kih IFN α and 4F3IgG kih IFN α were more potent than Rofero in protecting cells from viral infection and proliferation when incubated with ASGPR positive cell line Huh 72209-23 (fig. 24B and table 7). In contrast, the potency of the isotype control (non-targeted IgG kih IFN α) was significantly lower, highlighting the positive results of targeting the IgG kih IFN α conjugate to ASGPR H1.

Table 7 summary of antiviral activity of various ASGPR H1-specific antibody-IFN α conjugates.

IFN alpha activity of ASGPR-specific IgG-IFN alpha conjugates in hepatocytes and non-hepatocytes

IFN α exerts its antiviral activity by inducing several hundred IFN-stimulated genes (ISGs). To validate antiviral activity and to further confirm that ASGPR targeting mediates enhanced IFN α activity, we measured ISG expression in hepatocytes and non-hepatocytes. Hepatocytes (primary hepatocytes and HepG2) and non-hepatocytes (human PBMC and Hela) were treated with multiple serial dilutions of IFN α molecules for 6 hours and total RNA was extracted from the cells using a 5PRIME RNA extraction kit (# FP2302530, 5PRIME, Gaithersburg, MD).

The TaqMan (real-time PCR) assay designed custom for the ISG genes MX1 and RSAD 2. The selected assay is located within the Affymetrix probe sequence of interest or within the 3' coding sequence of the reference mRNA of interest.

In ABI7900 Total Gene expression analysis was performed on the HT sequence detection System (Applied Biosystems). PCR mix for each reaction consisted of 10. mu.lqPCR FastMix、ROX^TM(Quanta), 1. mu.l TaqMan or 0.06. mu.l IDT test and 2. mu.l DEPC treated water (Ambion, Applied Biosystems). The cDNA samples were diluted to 10 ng/. mu.l in RNase-free water (Ambion, Applied Biosystems) and 7. mu.l was added to 384-well optical plates (Applied Biosystems) containing 13. mu.l of predistributed PCR test mix. All samples were examined with one test for one target Gene or one test for the endogenous control genes 18S, GAPDH (rhesus monkey), ACTB (rhesus monkey) and GUSB (rhesus monkey) (TaqMan Gene Expression Assays, Applied Biosystems). Each measurement was performed in triplicate. The following PCR conditions were used: 45 ℃ for 2 minutes, followed by 95 ℃ for 3 minutes, followed by 40 cycles as follows: 95 ℃ for 15 seconds and 60 ℃ for 45 seconds.

Expression levels of target genes were normalized to the geometric mean of 18S, ACTB, GAPDH and GUSB and expressed as relative expression (E), E ═ 2^(ΔCt)Wherein Δ Ct is the difference in amplification between the reference gene cycle and the target gene cycle that exceeds an arbitrary threshold. As shown in fig. 25, isotype IgG kih IFN α control showed reduced ISG induction compared to roffern in all cells, similar to Pegasys (PEG-IFN α) activity. In ASGPR negative Hela cells and PBMIn C cells, clone 51a12IgG kih IFN α also showed reduced activity, similar to isotype control. However, in ASGPR positive hepatocytes HepG2 and human primary hepatocytes, clone 51a12IgG kih IFN α showed enhanced IFN α activity compared to isotype control, similar to the level of roffern. This result confirms the above-described enhanced antiviral activity of the 51a12 antibody-IFN α conjugate.

To understand whether these ASGPR-targeted IFN α molecules have persistent IFN α activity, we monitored ISG expression in Huh-7 and primary hepatocytes for up to 72 hours. As shown in figure 26, both ASGPR-targeted IFN α molecules 51a12IgG kih IFN α and 4F3IgG kih IFN α showed persistent ISG induction at 72 hours post-treatment, while Pegasys and rofferun showed significantly reduced ISG induction at 72 hours.

Single administration PK/PD study in cynomolgus monkeys

Were inspired by the following in vitro results: based on the targeting of ASGPR antibodies IFN α molecules showing reduced IFN α activity in non-hepatocytes and enhanced IFN α activity in hepatocytes (liver-targeted IFN α effect), cynomolgus studies were designed to demonstrate liver-targeted IFN α effect in vivo. Since ASGPR specific clone 51a12 binds human ASGPR and monkey ASGPR with the same affinity and human IFN α has similar activity in monkeys, monkey could be used as a liver-targeted IFN α concept to validate the studied PK/PD model. In monkey studies, we directly compared 51a12IgG kih IFN α with isotype IgG kih IFN α control. Both molecules were injected subcutaneously at 1 μ g/kg or 10 μ g/kg dose levels, monkey blood samples and liver biopsy samples were collected before and after administration, and their PK (pharmacokinetics) and PD (pharmacodynamics) were monitored. The dose groups are listed in table 8.

Table 8. 12 cynomolgus monkeys were divided into 4 groups as shown below.

Sample collection, transport and storage

Blood (approximately 1ml) was collected from each animal 5 days prior to administration and at 2,6, 12, 24, 48, 72, 96, 168 and 336 hours post-injection. Samples for pharmacokinetics were collected into tubes without anticoagulants. The blood for pharmacokinetics is collected before the blood for pharmacodynamics is collected.

For gene expression studies, blood (approximately 2.5ml) was collected from each animal 5 days prior to administration and 2,6, 12, 24, 48, 72, 96, 168 and 336 hours after injection. Samples were collected into PAXgene^TMBlood RNA was collected in tubes and the tubes were inverted 8-10 times for mixing. PAXgene^TMThe blood RNA collection tube was the last tube withdrawn in the phlebotomy series (i.e. after clinical pathology and pharmacokinetic collection).

Liver tissue (at least 25 mg/sample) was collected laparoscopically from two discrete locations in the liver of each animal on day-5 and on days 2, 4 and 8. Each tissue sample was excised and immediately placed in a pre-weighed and labeled independent cryovial and snap frozen in liquid nitrogen. Since immediate freezing is required, the sample vial is not weighed after the liver sample is taken. For days-5 and 2, a first liver tissue aliquot was taken from the left lobe of the liver for each animal and a second liver tissue aliquot was taken from the right lobe of the liver for each animal. For days 4 and 8, a first liver tissue aliquot was taken from the left middle lobe of the liver for each animal and a second liver tissue aliquot was taken from the right middle lobe of the liver for each animal. Liver biopsy samples were snap frozen in 2ml tubes. RNAlater-ICE (P/N7031, Ambion) precooled on dry ICE was added to the frozen tissue and stored at-80 ℃. Blood samples were received in PAXgene tubes according to the study protocol and stored at-80 ℃ prior to processing.

Measurement of clone 51a12IgG kih IFN α in monkey serum samples and isotype IgG kih IFN α control aliquots of cynomolgus monkey sera were analyzed for the administered compounds using a sandwich ELISA assay using anti-IFN α antibodies (lot numbers 34495-28, Roche Nutley, NJ, usa) as capture reagents and HRP-labeled anti-human Fc antibodies (lot numbers Wbr72_ MM _090602, Roche Diagnostics, Penzberg, germany) as detection reagents. After coating the plates with anti-IFN α antibody for 1 hour at room temperature, the plates were treated with 2% BSA blocking buffer for 1 hour. After washing, HRP-labeled anti-human Fc antibody was added to each well and incubated for 1 hour with gentle shaking. After washing, 100. mu.l/well of TMB substrate solution (# 11484281001, Roche Diagnostics, Penzberg, Germany) was added and the reaction was allowed to proceed for about 20 minutes. The reaction was then stopped by the addition of 50. mu.l/well 2N HCl. The plate was read at 450nm with a reference wavelength of 650nm over 2 minutes. The lower limit of quantitation (LLOQ) for this method is 10 ng/ml. The accuracy (% CV) and accuracy (% relative error) of the assay meet acceptance criteria. The test performance, which was monitored by analyzing QC samples analyzed along with the samples, is shown in table 9. Serum concentrations are shown in tables 10-13. A single injection of the isotype IgG kih IFN α at 10 μ g/kg resulted in a significant exposure in the blood that peaked at about 100ng/ml for 1 week. In contrast, at the same dose level, 51a12IgG kih IFN α was below the quantitative level at any time point. At a dose level of 1. mu.g/kg, no two molecules could be detected in the blood. PK parameters are summarized in table 14.

TABLE 9 analytical Performance of clone 51A12IgG kih IFN α quality control samples in cynomolgus monkey serum.

TABLE 10.1 serum concentration (ng/ml) of isotype IgG kih IFN α at μ g/kg

Table 11: 1 u g/kg 51A12IgG kih IFN alpha serum concentration (ng/ml)

TABLE 12.10 μ g/kg 51A12IgG kih IFN α serum concentration (ng/ml)

TABLE 13.10 μ g/kg serum concentration of isotype IgG kih IFN α (ng/ml)

TABLE 14.10 μ g/kg isotype-IFN α cynomolgus monkey serum PK parameters

RNA extraction

Total RNA was extracted from 96 liver biopsies (48 animals x2 biopsies/sample) using Qiagen RNeasy mini kit (P/N74104) and quantified on Nanodrop 8000. Liver samples were evaluated for total RNA quality on a Caliper LabChip GX. RNA from all samples was of sufficient quantity and quality to perform qPCR and microarray-based gene expression measurements.

Total RNA isolation was isolated and quantified from 120 blood samples at Expression Analysis (Raleigh, NC). Total RNA quality of blood samples was evaluated on an Agilent Bioanalyzer 2100. RNA from all samples was of sufficient quantity and quality to perform qPCR and microarray-based gene expression measurements.

Microarray analysis

Two independent protocols were used to convert total RNA to cDNA: affymetrix GeneChip HT 3' IVT Express (P/N901225) and NuGEN Ovation RNA amplification System V2 (P/N3100-60).

Blood, blood-enriching agent and method for producing the same

100ng of total RNA from liver biopsies was converted to double stranded cDNA and amplified RNA (aRNA) using GeneChip HT 3'IVT expression kit according to the manufacturer's protocol. The hybridization mixture contained 12.5. mu.g aRNA, 2 Xhybridization mixture (P/N900720), DMSO, 20 Xhybridization control, and oligo B2 control.

Liver disease

Following the manufacturer's protocol, 50ng of total RNA from a blood sample was converted to single stranded cDNA using the NuGEN Ovation RNA amplification System V2 kit. The hybridization mixture contained 3 μ g SPIA amplified cDNA, 2 Xhybridization mixture (P/N900720), DMSO, 20 Xhybridization control, and oligo B2 control.

Hybridization mixtures of liver and blood samples were hybridized to Affymetrix GeneChip human genome U133Plus 2.0 arrays. The dyeing step and the washing step were carried out as recommended by the manufacturer (Affymetrix). Each hybridized Affymetrix GeneChip array was scanned using a GeneChip scanner 30007G (Agilent/Affymetrix). Image analysis was performed using Affymetrix GCOS software. The resulting cel file was evaluated using a standardized quality control measure method. Data were normalized and expression value calculations/differential expression were determined using standard data analysis packages.

In fig. 27, 4 representative ISGs in monkey liver samples: expression of HRASLS2, ITI44, IFIT1 and IFITM 2. There was a more robust induction of these 4 ISG expression in monkeys administered 51a12IgG kih IFN α compared to monkeys administered the isotype IgG kih IFN α.

IFN Gene expression analysis (M3.1 heatmap)

To more broadly analyze the gene expression of IFN α stimulation by IFN α molecules, the IFN α response was analyzed using an IFN gene module determined from blood transcriptomic studies (Chaussabel et al, (2008), Immunity 29,150-64). As shown in figure 28A, fold change expression values from baseline for the genes of interferon module M3.1 were plotted as a heat map for blood and liver samples using the R statistics software package (www.r-project. Unsupervised hierarchical clustering of hepatic interferon-induced genes revealed highly induced subsets at the 10 μ g/kg dose of 51a12 (dashed rectangles), but not at day 1 and day 3 of the isotype IFN α compounds. Unsupervised hierarchical clustering of this subset revealed a differential expression pattern between blood and liver, with some genes being more significantly induced by 51a12 but not by isoform IFN α in liver at 10 μ g/kg dose, and other genes being more significantly induced by isoform IFN α in blood at high dose (fig. 28B).

In summary, the ASGPR-targeted IFN α molecule 51a12IgG kih IFN α showed undetectable exposure in blood and lower IFN α activity in blood (ISG expression stimulation effect) but higher IFN α activity in monkey liver compared to isotype IFN α control.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, these illustrations and examples should not be construed to limit the scope of the invention. The disclosures of all patent and scientific documents cited herein are expressly incorporated by reference in their entirety.

Sequence listing

<110> Rochellicat Corp

<120> ASGPR antibody and use thereof

<130> 31129

<150> US 61/681239

<151> 2012-08-09

<160> 139

<170> PatentIn version 3.5

<210> 1

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12 VL

<400> 1

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctgcct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgt gatatttgtt gtaatcgatc tgtgcgtaat 300

ttcggcggag ggaccaagct gaccgtccta 330

<210> 2

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12 VL

<400> 2

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Ala Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ile Cys Cys Asn Arg

85 90 95

Ser Val Arg Asn Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 3

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12 VH

<400> 3

gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcagatga acagcctgag agccgaggac acggccgtat attactgtgc gaaaccgtac 300

ctgggttaca ctattgacta ctggggccaa ggaaccctgg tcaccgtctc gagt 354

<210> 4

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12 VH

<400> 4

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Pro Tyr Leu Gly Tyr Thr Ile Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 5

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> 52C4 VL

<400> 5

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactccggt gctagtagcg gtaatcagtt ggtattcggc 300

ggagggacca agctgaccgt ccta 324

<210> 6

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> 52C4 VL

<400> 6

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Gly Ala Ser Ser Gly Asn Gln

85 90 95

Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 7

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> 52C4 VH

<400> 7

gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcagatga acagcctgag agtcgaggac acggccgtat attactgtgc gaaaccggct 300

ggttactctt acggttactt cgactactgg ggccaaggaa ccctggtcac cgtctcgagt 360

<210> 8

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> 52C4 VH

<400> 8

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Pro Ala Gly Tyr Ser Tyr Gly Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 9

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> 5A4 VL

<400> 9

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgt ttgaggagcg ggaagatggt ggtattcggc 300

ggagggacca agctgaccgt ccta 324

<210> 10

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> 5A4 VL

<400> 10

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Leu Arg Ser Gly Lys Met

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 11

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> 5A4 VH

<400> 11

cattcggagg tgcaattgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60

agactctcct gtgcagcctc cggattcacc tttagcagtt atgccatgag ctgggtccgc 120

caggctccag ggaaggggct ggagtgggtc tcagctatta gtggtagtgg tggtagcaca 180

tactacgcag actccgtgaa gggccggttc accatctcca gagacaattc caagaacacg 240

ctgtatctgc agatgaacag cctgagagcc gaggacacgg ccgtatatta ctgtgcgaaa 300

agttggtacc tgccgggtcg tggtttcgac tactggggcc aaggaaccct ggtcaccgtc 360

tcgagt 366

<210> 12

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> 5A4 VH

<400> 12

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Ser Trp Tyr Leu Pro Gly Arg Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 13

<211> 327

<212> DNA

<213> Artificial sequence

<220>

<223> 4F3 VL

<400> 13

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactccctg gagaggatcg ggtatctttc ttatgtattc 300

ggcggaggga ccaagctgac cgtccta 327

<210> 14

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> 4F3 VL

<400> 14

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Leu Glu Arg Ile Gly Tyr Leu

85 90 95

Ser Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 15

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> 4F3 VH

<400> 15

gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcagatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagacttc 300

tcttctcgtc gttggtacct ggaatactgg ggccaaggaa ccctggtcac cgtctcgagt 360

<210> 16

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> 4F3 VH

<400> 16

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Phe Ser Ser Arg Arg Trp Tyr Leu Glu Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 17

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> R5C2 VL

<400> 17

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgg gatcgtagag gttattcggt attcggcgga 300

gggaccaagc tgaccgtcct a 321

<210> 18

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> R5C2 VL

<400> 18

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Arg Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Arg Arg Gly Tyr Ser

85 90 95

Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 19

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> R5C2 VH

<400> 19

gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcgg cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcagatga acagcctgag agccgaggac acggccgtat attactgtgc gaaatcttct 300

ttctcttacc tgcgtgcttt cgactactgg ggccaaggaa ccctggtcac cgtctcgagt 360

<210> 20

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> R5C2 VH

<400> 20

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Ser Ser Phe Ser Tyr Leu Arg Ala Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 21

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> R9E10 VL

<400> 21

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgg aatgttcgcg gtaagctcgt attcggcgga 300

gggaccaagc tgaccgtcct a 321

<210> 22

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> R9E10 VL

<400> 22

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asn Val Arg Gly Lys Leu

85 90 95

Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 23

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> R9E10 VH

<400> 23

gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcagatga acagcctgag agccgaggac acggccgtat attactgtgc gaaaaactct 300

tacacttacg gtcgtgctct ggactactgg ggccaaggaa ccctggtcac cgtctcgagt 360

<210> 24

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> R9E10 VH

<400> 24

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asn Ser Tyr Thr Tyr Gly Arg Ala Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 25

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> R7E12 VL

<400> 25

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgt aagagtagct cgaagaatgt tgtgttcggc 300

ggagggacca agctgaccgt ccta 324

<210> 26

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> R7E12 VL

<400> 26

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Lys Ser Ser Ser Lys Asn

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 27

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> R7E12 VH

<400> 27

gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcagatga acagcctgag agccgaggac acggccgtat attactgtgc gaaatcttct 300

ttcactttcg gtcgttactt cgactactgg ggccaaggaa ccctggtcac cgtctcgagt 360

<210> 28

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> R7E12 VH

<400> 28

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Ser Ser Phe Thr Phe Gly Arg Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 29

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12 S116A VL

<400> 29

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctgcct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgt gatatttgtt gtaatcgagc tgtgcgtaat 300

ttcggcggag ggaccaagct gaccgtccta 330

<210> 30

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12 S116A VL

<400> 30

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Ala Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ile Cys Cys Asn Arg

85 90 95

Ala Val Arg Asn Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 31

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12 A82G S116A VL

<400> 31

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgt gatatttgtt gtaatcgagc tgtgcgtaat 300

ttcggcggag ggaccaagct gaccgtccta 330

<210> 32

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12 A82G S116A VL

<400> 32

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ile Cys Cys Asn Arg

85 90 95

Ala Val Arg Asn Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 33

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12 A82G C112S C113S S116A VL

<400> 33

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgt gatattagta gtaatcgagc tgtgcgtaat 300

ttcggcggag ggaccaagct gaccgtccta 330

<210> 34

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12 A82G C112S C113S S116A VL

<400> 34

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ile Ser Ser Asn Arg

85 90 95

Ala Val Arg Asn Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 35

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12_C1 VL

<400> 35

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgt tcttacgctt tcaaccgtgt tgttcgtaac 300

ttcggcggag ggaccaagct gaccgtccta 330

<210> 36

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12_C1 VL

<400> 36

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Ser Tyr Ala Phe Asn Arg

85 90 95

Val Val Arg Asn Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 37

<211> 327

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12_C7 VL

<400> 37

tctgagctga ctcaggaccc tgctgtgtct gtggccttgg gacagacagt caggatcaca 60

tgccaaggag acagcctcag aagttattat gcaagctggt accagcagaa gccaggacag 120

gcccctgtac ttgtcatcta tggtaaaaac aaccggccct cagggatccc agaccgattc 180

tctggctcca gctcaggaaa cacagcttcc ttgaccatca ctggggctca ggcggaagat 240

gaggctgact attactgtaa ctcccgtgac atccgttaca accgtgttgt tcgtccgttc 300

ggcggaggga ccaagctgac cgtccta 327

<210> 38

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12_C7 VL

<400> 38

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ile Arg Tyr Asn Arg

85 90 95

Val Val Arg Pro Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 39

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12_E7 VL

<400> 39

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgt gactaccgtt acaaccgtgc tgttcgtccg 300

ttcggcggag ggaccaagct gaccgtccta 330

<210> 40

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12_E7 VL

<400> 40

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Tyr Arg Tyr Asn Arg

85 90 95

Ala Val Arg Pro Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 41

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12_H3 VL

<400> 41

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgt gactacaaat tcaaccgtgt tgttcgtaac 300

ttcggcggag ggaccaagct gaccgtccta 330

<210> 42

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12_H3 VL

<400> 42

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Tyr Lys Phe Asn Arg

85 90 95

Val Val Arg Asn Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 43

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12_A6 VL

<400> 43

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgt acttactctt acaaccgtgc tgttcgtaac 300

ttcggcggag ggaccaagct gaccgtccta 330

<210> 44

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12_A6 VL

<400> 44

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Thr Tyr Ser Tyr Asn Arg

85 90 95

Ala Val Arg Asn Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 45

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12_D1 VL

<400> 45

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgt acttactctt acaaccgtgc tgttcgtccg 300

ttcggcggag ggaccaagct gaccgtccta 330

<210> 46

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12_D1 VL

<400> 46

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Thr Tyr Ser Tyr Asn Arg

85 90 95

Ala Val Arg Pro Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 47

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12_H6 VL

<400> 47

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgc gactacaaat ggaaccgtgt tgttcgtcat 300

ttcggcggag ggaccaagct gaccgtccta 330

<210> 48

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12_H6 VL

<400> 48

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Tyr Lys Trp Asn Arg

85 90 95

Val Val Arg His Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 49

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12 LC

<400> 49

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctgcct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgt gatatttgtt gtaatcgatc tgtgcgtaat 300

ttcggcggag ggaccaagct gaccgtccta 330

<210> 50

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12 LC

<400> 50

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Ala Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ile Cys Cys Asn Arg

85 90 95

Ser Val Arg Asn Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 51

<211> 1881

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12 HC (carina)

<400> 51

gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcagatga acagcctgag agccgaggac acggccgtat attactgtgc gaaaccgtac 300

ctgggttaca ctattgacta ctggggccaa ggaaccctgg tcaccgtctc gagtgctagc 360

accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 420

gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480

tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540

tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600

tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 660

tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aagctgcagg gggaccgtca 720

gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 780

acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840

gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900

taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960

aagtgcaagg tctccaacaa agccctcggc gcccccatcg agaaaaccat ctccaaagcc 1020

aaagggcagc cccgagaacc acaggtgtac accctgcccc catgccggga tgagctgacc 1080

aagaaccagg tcagcctgtg gtgcctggtc aaaggcttct atcccagcga catcgccgtg 1140

gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200

tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 1260

gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1320

agcctctccc tgtctccggg tggcggcgga ggctccggag gcggaggatc tggcggcgga 1380

ggcagctgtg acctgcctca gacacacagc ctgggcagcc ggcggaccct gatgctgctg 1440

gcccagatgc ggaagatcag cctgttcagc tgcctgaagg accggcacga cttcggcttc 1500

cctcaggaag agttcggcaa ccagttccag aaggccgaga caatccccgt gctgcacgag 1560

atgatccagc agattttcaa cctgttcagc accaaggaca gcagcgccgc ctgggacgag 1620

acactgctgg acaagttcta caccgagctg taccagcagc tgaacgacct ggaagcctgc 1680

gtgatccagg gcgtgggcgt gaccgagaca cccctgatga aggaagatag catcctggcc 1740

gtgcggaagt atttccagcg gatcaccctg tacctgaaag agaagaagta cagcccctgc 1800

gcctgggagg tcgtgcgggc cgagatcatg cggagcttca gcctgagcac caacctgcag 1860

gaaagcctgc ggagcaaaga g 1881

<210> 52

<211> 627

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12 HC (carina)

<400> 52

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Pro Tyr Leu Gly Tyr Thr Ile Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Asp

450 455 460

Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu

465 470 475 480

Ala Gln Met Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg His

485 490 495

Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala

500 505 510

Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe Asn Leu

515 520 525

Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp

530 535 540

Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys

545 550 555 560

Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp

565 570 575

Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu

580 585 590

Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu

595 600 605

Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu Arg

610 615 620

Ser Lys Glu

625

<210> 53

<211> 1344

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12 HC (cave)

<400> 53

gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcagatga acagcctgag agccgaggac acggccgtat attactgtgc gaaaccgtac 300

ctgggttaca ctattgacta ctggggccaa ggaaccctgg tcaccgtctc gagtgctagc 360

accaagggcc cctccgtgtt ccccctggcc cccagcagca agagcaccag cggcggcaca 420

gccgctctgg gctgcctggt caaggactac ttccccgagc ccgtgaccgt gtcctggaac 480

agcggagccc tgacctccgg cgtgcacacc ttccccgccg tgctgcagag ttctggcctg 540

tatagcctga gcagcgtggt caccgtgcct tctagcagcc tgggcaccca gacctacatc 600

tgcaacgtga accacaagcc cagcaacacc aaggtggaca agaaggtgga gcccaagagc 660

tgcgacaaaa ctcacacatg cccaccgtgc ccagcacctg aagctgcagg gggaccgtca 720

gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 780

acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840

gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900

taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960

aagtgcaagg tctccaacaa agccctcggc gcccccatcg agaaaaccat ctccaaagcc 1020

aaagggcagc cccgagaacc acaggtgtgc accctgcccc catcccggga tgagctgacc 1080

aagaaccagg tcagcctctc gtgcgcagtc aaaggcttct atcccagcga catcgccgtg 1140

gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200

tccgacggct ccttcttcct cgtgagcaag ctcaccgtgg acaagagcag gtggcagcag 1260

gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1320

agcctctccc tgtctccggg taaa 1344

<210> 54

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12 HC (Point)

<400> 54

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Pro Tyr Leu Gly Tyr Thr Ile Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys

355 360 365

Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 55

<211> 642

<212> DNA

<213> Artificial sequence

<220>

<223> 52C4 LC

<400> 55

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactccggt gctagtagcg gtaatcagtt ggtattcggc 300

ggagggacca agctgaccgt cctaggtcaa cccaaggctg cccccagcgt gaccctgttc 360

ccccccagca gcgaggaact gcaggccaac aaggccaccc tggtctgcct gatcagcgac 420

ttctacccag gcgccgtgac cgtggcctgg aaggccgaca gcagccccgt gaaggccggc 480

gtggagacca ccacccccag caagcagagc aacaacaagt acgccgccag cagctacctg 540

agcctgaccc ccgagcagtg gaagagccac aggtcctaca gctgccaggt gacccacgag 600

ggcagcaccg tggagaaaac cgtggccccc accgagtgca gc 642

<210> 56

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> 52C4 LC

<400> 56

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Gly Ala Ser Ser Gly Asn Gln

85 90 95

Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys

100 105 110

Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln

115 120 125

Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly

130 135 140

Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly

145 150 155 160

Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala

165 170 175

Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser

180 185 190

Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val

195 200 205

Ala Pro Thr Glu Cys Ser

210

<210> 57

<211> 1887

<212> DNA

<213> Artificial sequence

<220>

<223> 52C4 HC (knob)

<400> 57

gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcagatga acagcctgag agtcgaggac acggccgtat attactgtgc gaaaccggct 300

ggttactctt acggttactt cgactactgg ggccaaggaa ccctggtcac cgtctcgagt 360

gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaagc tgcaggggga 720

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960

gagtacaagt gcaaggtctc caacaaagcc ctcggcgccc ccatcgagaa aaccatctcc 1020

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatg ccgggatgag 1080

ctgaccaaga accaggtcag cctgtggtgc ctggtcaaag gcttctatcc cagcgacatc 1140

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1260

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320

cagaagagcc tctccctgtc tccgggtggc ggcggaggct ccggaggcgg aggatctggc 1380

ggcggaggca gctgtgacct gcctcagaca cacagcctgg gcagccggcg gaccctgatg 1440

ctgctggccc agatgcggaa gatcagcctg ttcagctgcc tgaaggaccg gcacgacttc 1500

ggcttccctc aggaagagtt cggcaaccag ttccagaagg ccgagacaat ccccgtgctg 1560

cacgagatga tccagcagat tttcaacctg ttcagcacca aggacagcag cgccgcctgg 1620

gacgagacac tgctggacaa gttctacacc gagctgtacc agcagctgaa cgacctggaa 1680

gcctgcgtga tccagggcgt gggcgtgacc gagacacccc tgatgaagga agatagcatc 1740

ctggccgtgc ggaagtattt ccagcggatc accctgtacc tgaaagagaa gaagtacagc 1800

ccctgcgcct gggaggtcgt gcgggccgag atcatgcgga gcttcagcct gagcaccaac 1860

ctgcaggaaa gcctgcggag caaagag 1887

<210> 58

<211> 629

<212> PRT

<213> Artificial sequence

<220>

<223> 52C4 HC (carina)

<400> 58

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Pro Ala Gly Tyr Ser Tyr Gly Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met

465 470 475 480

Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp

485 490 495

Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln

500 505 510

Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe

515 520 525

Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu

530 535 540

Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu

545 550 555 560

Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys

565 570 575

Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu

580 585 590

Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg

595 600 605

Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser

610 615 620

Leu Arg Ser Lys Glu

625

<210> 59

<211> 1350

<212> DNA

<213> Artificial sequence

<220>

<223> 52C4 HC (Point)

<400> 59

gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcagatga acagcctgag agtcgaggac acggccgtat attactgtgc gaaaccggct 300

ggttactctt acggttactt cgactactgg ggccaaggaa ccctggtcac cgtctcgagt 360

gctagcacca agggcccctc cgtgttcccc ctggccccca gcagcaagag caccagcggc 420

ggcacagccg ctctgggctg cctggtcaag gactacttcc ccgagcccgt gaccgtgtcc 480

tggaacagcg gagccctgac ctccggcgtg cacaccttcc ccgccgtgct gcagagttct 540

ggcctgtata gcctgagcag cgtggtcacc gtgccttcta gcagcctggg cacccagacc 600

tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaagaa ggtggagccc 660

aagagctgcg acaaaactca cacatgccca ccgtgcccag cacctgaagc tgcaggggga 720

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960

gagtacaagt gcaaggtctc caacaaagcc ctcggcgccc ccatcgagaa aaccatctcc 1020

aaagccaaag ggcagccccg agaaccacag gtgtgcaccc tgcccccatc ccgggatgag 1080

ctgaccaaga accaggtcag cctctcgtgc gcagtcaaag gcttctatcc cagcgacatc 1140

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200

ctggactccg acggctcctt cttcctcgtg agcaagctca ccgtggacaa gagcaggtgg 1260

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320

cagaagagcc tctccctgtc tccgggtaaa 1350

<210> 60

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> 52C4 HC (cave)

<400> 60

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Pro Ala Gly Tyr Ser Tyr Gly Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 61

<211> 642

<212> DNA

<213> Artificial sequence

<220>

<223> 5A4 LC

<400> 61

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgt ttgaggagcg ggaagatggt ggtattcggc 300

ggagggacca agctgaccgt cctaggtcaa cccaaggctg cccccagcgt gaccctgttc 360

ccccccagca gcgaggaact gcaggccaac aaggccaccc tggtctgcct gatcagcgac 420

ttctacccag gcgccgtgac cgtggcctgg aaggccgaca gcagccccgt gaaggccggc 480

gtggagacca ccacccccag caagcagagc aacaacaagt acgccgccag cagctacctg 540

agcctgaccc ccgagcagtg gaagagccac aggtcctaca gctgccaggt gacccacgag 600

ggcagcaccg tggagaaaac cgtggccccc accgagtgca gc 642

<210> 62

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> 5A4 LC

<400> 62

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Leu Arg Ser Gly Lys Met

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys

100 105 110

Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln

115 120 125

Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly

130 135 140

Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly

145 150 155 160

Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala

165 170 175

Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser

180 185 190

Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val

195 200 205

Ala Pro Thr Glu Cys Ser

210

<210> 63

<211> 1892

<212> DNA

<213> Artificial sequence

<220>

<223> 5A4 HC (knob)

<400> 63

cattcggagg tgcaattgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60

agactctcct gtgcagcctc cggattcacc tttagcagtt atgccatgag ctgggtccgc 120

caggctccag ggaaggggct ggagtgggtc tcagctatta gtggtagtgg tggtagcaca 180

tactacgcag actccgtgaa gggccggttc accatctcca gagacaattc caagaacacg 240

ctgtatctgc agatgaacag cctgagagcc gaggacacgg ccgtatatta ctgtgcgaaa 300

agttggtacc tgccgggtcg tggtttcgac tactggggcc aaggaaccct ggtcaccgtc 360

tcgagtgcta gcaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc 420

tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg 480

gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 540

tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 600

cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagaaagtt 660

gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaagctgca 720

gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 780

acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 840

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 900

tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 960

ggcaaggagt acaagtgcaa ggtctccaac aaagccctcg gcgcccccat cgagaaaacc 1020

atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatgccgg 1080

gatgagctga ccaagaacca ggtcagcctg tggtgcctgg tcaaaggctt ctatcccagc 1140

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1200

cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1260

aggtggagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg cacaaccact 1320

acacgcagaa gagcctctcc ctgtctccgg gtggcggcgg aggctccgga ggcggaggat 1380

ctggcggcgg aggcagctgt gacctgcctc agacacacag cctgggcagc cggcggaccc 1440

tgatgctgct ggcccagatg cggaagatca gcctgttcag ctgcctgaag gaccggcacg 1500

acttcggctt ccctcaggaa gagttcggca accagttcca gaaggccgag acaatccccg 1560

tgctgcacga gatgatccag cagattttca acctgttcag caccaaggac agcagcgccg 1620

cctgggacga gacactgctg gacaagttct acaccgagct gtaccagcag ctgaacgacc 1680

tggaagcctg cgtgatccag ggcgtgggcg tgaccgagac acccctgatg aaggaagata 1740

gcatcctggc cgtgcggaag tatttccagc ggatcaccct gtacctgaaa gagaagaagt 1800

acagcccctg cgcctgggag gtcgtgcggg ccgagatcat gcggagcttc agcctgagca 1860

ccaacctgca ggaaagcctg cggagcaaag ag 1892

<210> 64

<211> 629

<212> PRT

<213> Artificial sequence

<220>

<223> 5A4 HC (knob)

<400> 64

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Ser Trp Tyr Leu Pro Gly Arg Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met

465 470 475 480

Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp

485 490 495

Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln

500 505 510

Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe

515 520 525

Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu

530 535 540

Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu

545 550 555 560

Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys

565 570 575

Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu

580 585 590

Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg

595 600 605

Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser

610 615 620

Leu Arg Ser Lys Glu

625

<210> 65

<211> 1350

<212> DNA

<213> Artificial sequence

<220>

<223> 5A4 HC (Point)

<400> 65

gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcagatga acagcctgag agccgaggac acggccgtat attactgtgc gaaaagttgg 300

tacctgccgg gtcgtggttt cgactactgg ggccaaggaa ccctggtcac cgtctcgagt 360

gctagcacca agggcccctc cgtgttcccc ctggccccca gcagcaagag caccagcggc 420

ggcacagccg ctctgggctg cctggtcaag gactacttcc ccgagcccgt gaccgtgtcc 480

tggaacagcg gagccctgac ctccggcgtg cacaccttcc ccgccgtgct gcagagttct 540

ggcctgtata gcctgagcag cgtggtcacc gtgccttcta gcagcctggg cacccagacc 600

tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaagaa ggtggagccc 660

aagagctgcg acaaaactca cacatgccca ccgtgcccag cacctgaagc tgcaggggga 720

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960

gagtacaagt gcaaggtctc caacaaagcc ctcggcgccc ccatcgagaa aaccatctcc 1020

aaagccaaag ggcagccccg agaaccacag gtgtgcaccc tgcccccatc ccgggatgag 1080

ctgaccaaga accaggtcag cctctcgtgc gcagtcaaag gcttctatcc cagcgacatc 1140

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200

ctggactccg acggctcctt cttcctcgtg agcaagctca ccgtggacaa gagcaggtgg 1260

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320

cagaagagcc tctccctgtc tccgggtaaa 1350

<210> 66

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> 5A4 HC (cave)

<400> 66

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Ser Trp Tyr Leu Pro Gly Arg Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 67

<211> 645

<212> DNA

<213> Artificial sequence

<220>

<223> 4F3 LC

<400> 67

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactccctg gagaggatcg ggtatctttc ttatgtattc 300

ggcggaggga ccaagctgac cgtcctaggt caacccaagg ctgcccccag cgtgaccctg 360

ttccccccca gcagcgagga actgcaggcc aacaaggcca ccctggtctg cctgatcagc 420

gacttctacc caggcgccgt gaccgtggcc tggaaggccg acagcagccc cgtgaaggcc 480

ggcgtggaga ccaccacccc cagcaagcag agcaacaaca agtacgccgc cagcagctac 540

ctgagcctga cccccgagca gtggaagagc cacaggtcct acagctgcca ggtgacccac 600

gagggcagca ccgtggagaa aaccgtggcc cccaccgagt gcagc 645

<210> 68

<211> 215

<212> PRT

<213> Artificial sequence

<220>

<223> 4F3 LC

<400> 68

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Leu Glu Arg Ile Gly Tyr Leu

85 90 95

Ser Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

100 105 110

Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu

115 120 125

Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro

130 135 140

Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala

145 150 155 160

Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala

165 170 175

Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg

180 185 190

Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr

195 200 205

Val Ala Pro Thr Glu Cys Ser

210 215

<210> 69

<211> 1887

<212> DNA

<213> Artificial sequence

<220>

<223> 4F3 HC (knob)

<400> 69

gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcagatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagacttc 300

tcttctcgtc gttggtacct ggaatactgg ggccaaggaa ccctggtcac cgtctcgagt 360

gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaagc tgcaggggga 720

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960

gagtacaagt gcaaggtctc caacaaagcc ctcggcgccc ccatcgagaa aaccatctcc 1020

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatg ccgggatgag 1080

ctgaccaaga accaggtcag cctgtggtgc ctggtcaaag gcttctatcc cagcgacatc 1140

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1260

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320

cagaagagcc tctccctgtc tccgggtggc ggcggaggct ccggaggcgg aggatctggc 1380

ggcggaggca gctgtgacct gcctcagaca cacagcctgg gcagccggcg gaccctgatg 1440

ctgctggccc agatgcggaa gatcagcctg ttcagctgcc tgaaggaccg gcacgacttc 1500

ggcttccctc aggaagagtt cggcaaccag ttccagaagg ccgagacaat ccccgtgctg 1560

cacgagatga tccagcagat tttcaacctg ttcagcacca aggacagcag cgccgcctgg 1620

gacgagacac tgctggacaa gttctacacc gagctgtacc agcagctgaa cgacctggaa 1680

gcctgcgtga tccagggcgt gggcgtgacc gagacacccc tgatgaagga agatagcatc 1740

ctggccgtgc ggaagtattt ccagcggatc accctgtacc tgaaagagaa gaagtacagc 1800

ccctgcgcct gggaggtcgt gcgggccgag atcatgcgga gcttcagcct gagcaccaac 1860

ctgcaggaaa gcctgcggag caaagag 1887

<210> 70

<211> 629

<212> PRT

<213> Artificial sequence

<220>

<223> 4F3 HC (knob)

<400> 70

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Phe Ser Ser Arg Arg Trp Tyr Leu Glu Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met

465 470 475 480

Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp

485 490 495

Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln

500 505 510

Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe

515 520 525

Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu

530 535 540

Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu

545 550 555 560

Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys

565 570 575

Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu

580 585 590

Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg

595 600 605

Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser

610 615 620

Leu Arg Ser Lys Glu

625

<210> 71

<211> 1350

<212> DNA

<213> Artificial sequence

<220>

<223> 4F3 HC (Point)

<400> 71

gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcagatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagacttc 300

tcttctcgtc gttggtacct ggaatactgg ggccaaggaa ccctggtcac cgtctcgagt 360

gctagcacca agggcccctc cgtgttcccc ctggccccca gcagcaagag caccagcggc 420

ggcacagccg ctctgggctg cctggtcaag gactacttcc ccgagcccgt gaccgtgtcc 480

tggaacagcg gagccctgac ctccggcgtg cacaccttcc ccgccgtgct gcagagttct 540

ggcctgtata gcctgagcag cgtggtcacc gtgccttcta gcagcctggg cacccagacc 600

tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaagaa ggtggagccc 660

aagagctgcg acaaaactca cacatgccca ccgtgcccag cacctgaagc tgcaggggga 720

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960

gagtacaagt gcaaggtctc caacaaagcc ctcggcgccc ccatcgagaa aaccatctcc 1020

aaagccaaag ggcagccccg agaaccacag gtgtgcaccc tgcccccatc ccgggatgag 1080

ctgaccaaga accaggtcag cctctcgtgc gcagtcaaag gcttctatcc cagcgacatc 1140

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200

ctggactccg acggctcctt cttcctcgtg agcaagctca ccgtggacaa gagcaggtgg 1260

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320

cagaagagcc tctccctgtc tccgggtaaa 1350

<210> 72

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> 4F3 HC (Point)

<400> 72

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Phe Ser Ser Arg Arg Trp Tyr Leu Glu Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 73

<211> 639

<212> DNA

<213> Artificial sequence

<220>

<223> R5C2 LC

<400> 73

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgg gatcgtagag gttattcggt attcggcgga 300

gggaccaagc tgaccgtcct aggtcaaccc aaggctgccc ccagcgtgac cctgttcccc 360

cccagcagcg aggaactgca ggccaacaag gccaccctgg tctgcctgat cagcgacttc 420

tacccaggcg ccgtgaccgt ggcctggaag gccgacagca gccccgtgaa ggccggcgtg 480

gagaccacca cccccagcaa gcagagcaac aacaagtacg ccgccagcag ctacctgagc 540

ctgacccccg agcagtggaa gagccacagg tcctacagct gccaggtgac ccacgagggc 600

agcaccgtgg agaaaaccgt ggcccccacc gagtgcagc 639

<210> 74

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> R5C2 LC

<400> 74

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Arg Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Arg Arg Gly Tyr Ser

85 90 95

Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala

100 105 110

Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala

115 120 125

Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala

130 135 140

Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val

145 150 155 160

Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser

165 170 175

Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr

180 185 190

Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala

195 200 205

Pro Thr Glu Cys Ser

210

<210> 75

<211> 1887

<212> DNA

<213> Artificial sequence

<220>

<223> R5C2 HC (knob)

<400> 75

gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcgg cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcagatga acagcctgag agccgaggac acggccgtat attactgtgc gaaatcttct 300

ttctcttacc tgcgtgcttt cgactactgg ggccaaggaa ccctggtcac cgtctcgagt 360

gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaagc tgcaggggga 720

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960

gagtacaagt gcaaggtctc caacaaagcc ctcggcgccc ccatcgagaa aaccatctcc 1020

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatg ccgggatgag 1080

ctgaccaaga accaggtcag cctgtggtgc ctggtcaaag gcttctatcc cagcgacatc 1140

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1260

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320

cagaagagcc tctccctgtc tccgggtggc ggcggaggct ccggaggcgg aggatctggc 1380

ggcggaggca gctgtgacct gcctcagaca cacagcctgg gcagccggcg gaccctgatg 1440

ctgctggccc agatgcggaa gatcagcctg ttcagctgcc tgaaggaccg gcacgacttc 1500

ggcttccctc aggaagagtt cggcaaccag ttccagaagg ccgagacaat ccccgtgctg 1560

cacgagatga tccagcagat tttcaacctg ttcagcacca aggacagcag cgccgcctgg 1620

gacgagacac tgctggacaa gttctacacc gagctgtacc agcagctgaa cgacctggaa 1680

gcctgcgtga tccagggcgt gggcgtgacc gagacacccc tgatgaagga agatagcatc 1740

ctggccgtgc ggaagtattt ccagcggatc accctgtacc tgaaagagaa gaagtacagc 1800

ccctgcgcct gggaggtcgt gcgggccgag atcatgcgga gcttcagcct gagcaccaac 1860

ctgcaggaaa gcctgcggag caaagag 1887

<210> 76

<211> 629

<212> PRT

<213> Artificial sequence

<220>

<223> R5C2 HC (carina)

<400> 76

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Ser Ser Phe Ser Tyr Leu Arg Ala Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met

465 470 475 480

Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp

485 490 495

Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln

500 505 510

Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe

515 520 525

Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu

530 535 540

Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu

545 550 555 560

Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys

565 570 575

Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu

580 585 590

Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg

595 600 605

Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser

610 615 620

Leu Arg Ser Lys Glu

625

<210> 77

<211> 1350

<212> DNA

<213> Artificial sequence

<220>

<223> R5C2 HC (Point)

<400> 77

gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcgg cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcagatga acagcctgag agccgaggac acggccgtat attactgtgc gaaatcttct 300

ttctcttacc tgcgtgcttt cgactactgg ggccaaggaa ccctggtcac cgtctcgagt 360

gctagcacca agggcccctc cgtgttcccc ctggccccca gcagcaagag caccagcggc 420

ggcacagccg ctctgggctg cctggtcaag gactacttcc ccgagcccgt gaccgtgtcc 480

tggaacagcg gagccctgac ctccggcgtg cacaccttcc ccgccgtgct gcagagttct 540

ggcctgtata gcctgagcag cgtggtcacc gtgccttcta gcagcctggg cacccagacc 600

tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaagaa ggtggagccc 660

aagagctgcg acaaaactca cacatgccca ccgtgcccag cacctgaagc tgcaggggga 720

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960

gagtacaagt gcaaggtctc caacaaagcc ctcggcgccc ccatcgagaa aaccatctcc 1020

aaagccaaag ggcagccccg agaaccacag gtgtgcaccc tgcccccatc ccgggatgag 1080

ctgaccaaga accaggtcag cctctcgtgc gcagtcaaag gcttctatcc cagcgacatc 1140

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200

ctggactccg acggctcctt cttcctcgtg agcaagctca ccgtggacaa gagcaggtgg 1260

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320

cagaagagcc tctccctgtc tccgggtaaa 1350

<210> 78

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> R5C2 HC (Point)

<400> 78

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Ser Ser Phe Ser Tyr Leu Arg Ala Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 79

<211> 639

<212> DNA

<213> Artificial sequence

<220>

<223> R9E10 LC

<400> 79

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgg aatgttcgcg gtaagctcgt attcggcgga 300

gggaccaagc tgaccgtcct aggtcaaccc aaggctgccc ccagcgtgac cctgttcccc 360

cccagcagcg aggaactgca ggccaacaag gccaccctgg tctgcctgat cagcgacttc 420

tacccaggcg ccgtgaccgt ggcctggaag gccgacagca gccccgtgaa ggccggcgtg 480

gagaccacca cccccagcaa gcagagcaac aacaagtacg ccgccagcag ctacctgagc 540

ctgacccccg agcagtggaa gagccacagg tcctacagct gccaggtgac ccacgagggc 600

agcaccgtgg agaaaaccgt ggcccccacc gagtgcagc 639

<210> 80

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> R9E10 LC

<400> 80

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asn Val Arg Gly Lys Leu

85 90 95

Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala

100 105 110

Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala

115 120 125

Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala

130 135 140

Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val

145 150 155 160

Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser

165 170 175

Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr

180 185 190

Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala

195 200 205

Pro Thr Glu Cys Ser

210

<210> 81

<211> 1887

<212> DNA

<213> Artificial sequence

<220>

<223> R9E10 HC (carina)

<400> 81

gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcagatga acagcctgag agccgaggac acggccgtat attactgtgc gaaaaactct 300

tacacttacg gtcgtgctct ggactactgg ggccaaggaa ccctggtcac cgtctcgagt 360

gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaagc tgcaggggga 720

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960

gagtacaagt gcaaggtctc caacaaagcc ctcggcgccc ccatcgagaa aaccatctcc 1020

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatg ccgggatgag 1080

ctgaccaaga accaggtcag cctgtggtgc ctggtcaaag gcttctatcc cagcgacatc 1140

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1260

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320

cagaagagcc tctccctgtc tccgggtggc ggcggaggct ccggaggcgg aggatctggc 1380

ggcggaggca gctgtgacct gcctcagaca cacagcctgg gcagccggcg gaccctgatg 1440

ctgctggccc agatgcggaa gatcagcctg ttcagctgcc tgaaggaccg gcacgacttc 1500

ggcttccctc aggaagagtt cggcaaccag ttccagaagg ccgagacaat ccccgtgctg 1560

cacgagatga tccagcagat tttcaacctg ttcagcacca aggacagcag cgccgcctgg 1620

gacgagacac tgctggacaa gttctacacc gagctgtacc agcagctgaa cgacctggaa 1680

gcctgcgtga tccagggcgt gggcgtgacc gagacacccc tgatgaagga agatagcatc 1740

ctggccgtgc ggaagtattt ccagcggatc accctgtacc tgaaagagaa gaagtacagc 1800

ccctgcgcct gggaggtcgt gcgggccgag atcatgcgga gcttcagcct gagcaccaac 1860

ctgcaggaaa gcctgcggag caaagag 1887

<210> 82

<211> 629

<212> PRT

<213> Artificial sequence

<220>

<223> R9E10 HC (knob)

<400> 82

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asn Ser Tyr Thr Tyr Gly Arg Ala Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met

465 470 475 480

Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp

485 490 495

Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln

500 505 510

Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe

515 520 525

Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu

530 535 540

Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu

545 550 555 560

Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys

565 570 575

Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu

580 585 590

Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg

595 600 605

Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser

610 615 620

Leu Arg Ser Lys Glu

625

<210> 83

<211> 1350

<212> DNA

<213> Artificial sequence

<220>

<223> R9E10 HC (Point)

<400> 83

gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcagatga acagcctgag agccgaggac acggccgtat attactgtgc gaaaaactct 300

tacacttacg gtcgtgctct ggactactgg ggccaaggaa ccctggtcac cgtctcgagt 360

gctagcacca agggcccctc cgtgttcccc ctggccccca gcagcaagag caccagcggc 420

ggcacagccg ctctgggctg cctggtcaag gactacttcc ccgagcccgt gaccgtgtcc 480

tggaacagcg gagccctgac ctccggcgtg cacaccttcc ccgccgtgct gcagagttct 540

ggcctgtata gcctgagcag cgtggtcacc gtgccttcta gcagcctggg cacccagacc 600

tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaagaa ggtggagccc 660

aagagctgcg acaaaactca cacatgccca ccgtgcccag cacctgaagc tgcaggggga 720

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960

gagtacaagt gcaaggtctc caacaaagcc ctcggcgccc ccatcgagaa aaccatctcc 1020

aaagccaaag ggcagccccg agaaccacag gtgtgcaccc tgcccccatc ccgggatgag 1080

ctgaccaaga accaggtcag cctctcgtgc gcagtcaaag gcttctatcc cagcgacatc 1140

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200

ctggactccg acggctcctt cttcctcgtg agcaagctca ccgtggacaa gagcaggtgg 1260

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320

cagaagagcc tctccctgtc tccgggtaaa 1350

<210> 84

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> R9E10 HC (cave)

<400> 84

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asn Ser Tyr Thr Tyr Gly Arg Ala Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 85

<211> 642

<212> DNA

<213> Artificial sequence

<220>

<223> R7E12 LC

<400> 85

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgt aagagtagct cgaagaatgt tgtgttcggc 300

ggagggacca agctgaccgt cctaggtcaa cccaaggctg cccccagcgt gaccctgttc 360

ccccccagca gcgaggaact gcaggccaac aaggccaccc tggtctgcct gatcagcgac 420

ttctacccag gcgccgtgac cgtggcctgg aaggccgaca gcagccccgt gaaggccggc 480

gtggagacca ccacccccag caagcagagc aacaacaagt acgccgccag cagctacctg 540

agcctgaccc ccgagcagtg gaagagccac aggtcctaca gctgccaggt gacccacgag 600

ggcagcaccg tggagaaaac cgtggccccc accgagtgca gc 642

<210> 86

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> R7E12 LC

<400> 86

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Lys Ser Ser Ser Lys Asn

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys

100 105 110

Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln

115 120 125

Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly

130 135 140

Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly

145 150 155 160

Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala

165 170 175

Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser

180 185 190

Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val

195 200 205

Ala Pro Thr Glu Cys Ser

210

<210> 87

<211> 1887

<212> DNA

<213> Artificial sequence

<220>

<223> R7E12 HC (carina)

<400> 87

gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcagatga acagcctgag agccgaggac acggccgtat attactgtgc gaaatcttct 300

ttcactttcg gtcgttactt cgactactgg ggccaaggaa ccctggtcac cgtctcgagt 360

gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaagc tgcaggggga 720

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960

gagtacaagt gcaaggtctc caacaaagcc ctcggcgccc ccatcgagaa aaccatctcc 1020

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatg ccgggatgag 1080

ctgaccaaga accaggtcag cctgtggtgc ctggtcaaag gcttctatcc cagcgacatc 1140

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1260

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320

cagaagagcc tctccctgtc tccgggtggc ggcggaggct ccggaggcgg aggatctggc 1380

ggcggaggca gctgtgacct gcctcagaca cacagcctgg gcagccggcg gaccctgatg 1440

ctgctggccc agatgcggaa gatcagcctg ttcagctgcc tgaaggaccg gcacgacttc 1500

ggcttccctc aggaagagtt cggcaaccag ttccagaagg ccgagacaat ccccgtgctg 1560

cacgagatga tccagcagat tttcaacctg ttcagcacca aggacagcag cgccgcctgg 1620

gacgagacac tgctggacaa gttctacacc gagctgtacc agcagctgaa cgacctggaa 1680

gcctgcgtga tccagggcgt gggcgtgacc gagacacccc tgatgaagga agatagcatc 1740

ctggccgtgc ggaagtattt ccagcggatc accctgtacc tgaaagagaa gaagtacagc 1800

ccctgcgcct gggaggtcgt gcgggccgag atcatgcgga gcttcagcct gagcaccaac 1860

ctgcaggaaa gcctgcggag caaagag 1887

<210> 88

<211> 629

<212> PRT

<213> Artificial sequence

<220>

<223> R7E12 HC (knob)

<400> 88

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Ser Ser Phe Thr Phe Gly Arg Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met

465 470 475 480

Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp

485 490 495

Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln

500 505 510

Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe

515 520 525

Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu

530 535 540

Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu

545 550 555 560

Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys

565 570 575

Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu

580 585 590

Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg

595 600 605

Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser

610 615 620

Leu Arg Ser Lys Glu

625

<210> 89

<211> 1350

<212> DNA

<213> Artificial sequence

<220>

<223> R7E12 HC (Point)

<400> 89

gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcagatga acagcctgag agccgaggac acggccgtat attactgtgc gaaatcttct 300

ttcactttcg gtcgttactt cgactactgg ggccaaggaa ccctggtcac cgtctcgagt 360

gctagcacca agggcccctc cgtgttcccc ctggccccca gcagcaagag caccagcggc 420

ggcacagccg ctctgggctg cctggtcaag gactacttcc ccgagcccgt gaccgtgtcc 480

tggaacagcg gagccctgac ctccggcgtg cacaccttcc ccgccgtgct gcagagttct 540

ggcctgtata gcctgagcag cgtggtcacc gtgccttcta gcagcctggg cacccagacc 600

tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaagaa ggtggagccc 660

aagagctgcg acaaaactca cacatgccca ccgtgcccag cacctgaagc tgcaggggga 720

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960

gagtacaagt gcaaggtctc caacaaagcc ctcggcgccc ccatcgagaa aaccatctcc 1020

aaagccaaag ggcagccccg agaaccacag gtgtgcaccc tgcccccatc ccgggatgag 1080

ctgaccaaga accaggtcag cctctcgtgc gcagtcaaag gcttctatcc cagcgacatc 1140

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200

ctggactccg acggctcctt cttcctcgtg agcaagctca ccgtggacaa gagcaggtgg 1260

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320

cagaagagcc tctccctgtc tccgggtaaa 1350

<210> 90

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> R7E12 HC (Point)

<400> 90

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Ser Ser Phe Thr Phe Gly Arg Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 91

<211> 648

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12 S116A LC

<400> 91

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctgcct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgt gatatttgtt gtaatcgagc tgtgcgtaat 300

ttcggcggag ggaccaagct gaccgtccta ggtcaaccca aggctgcccc cagcgtgacc 360

ctgttccccc ccagcagcga ggaactgcag gccaacaagg ccaccctggt ctgcctgatc 420

agcgacttct acccaggcgc cgtgaccgtg gcctggaagg ccgacagcag ccccgtgaag 480

gccggcgtgg agaccaccac ccccagcaag cagagcaaca acaagtacgc cgccagcagc 540

tacctgagcc tgacccccga gcagtggaag agccacaggt cctacagctg ccaggtgacc 600

cacgagggca gcaccgtgga gaaaaccgtg gcccccaccg agtgcagc 648

<210> 92

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12 S116A LC

<400> 92

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Ala Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ile Cys Cys Asn Arg

85 90 95

Ala Val Arg Asn Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 93

<211> 648

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12 A82G S116A LC

<400> 93

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgt gatatttgtt gtaatcgagc tgtgcgtaat 300

ttcggcggag ggaccaagct gaccgtccta ggtcaaccca aggctgcccc cagcgtgacc 360

ctgttccccc ccagcagcga ggaactgcag gccaacaagg ccaccctggt ctgcctgatc 420

agcgacttct acccaggcgc cgtgaccgtg gcctggaagg ccgacagcag ccccgtgaag 480

gccggcgtgg agaccaccac ccccagcaag cagagcaaca acaagtacgc cgccagcagc 540

tacctgagcc tgacccccga gcagtggaag agccacaggt cctacagctg ccaggtgacc 600

cacgagggca gcaccgtgga gaaaaccgtg gcccccaccg agtgcagc 648

<210> 94

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12 A82G S116A LC

<400> 94

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ile Cys Cys Asn Arg

85 90 95

Ala Val Arg Asn Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 95

<211> 649

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12_C1 LC

<400> 95

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgt tcttacgctt tcaaccgtgt tgttcgtaac 300

ttcggcggag ggaccaagct gaccgtccta ggtcaaccca aggctgcccc cagcgtgacc 360

ctgttccccc ccagcagcga ggaactgcag gccaacaagg ccaccctggt ctgcctgatc 420

agcgacttct acccaggcgc cgtgaccgtg gcctggaagg ccgacagcag ccccgtgaag 480

gccggcgtgg agaccaccac ccccagcaag cagagcaaca acaagtacgc cgccagcagc 540

tacctgagcc tgacccccga gcagtggaag agccacaggt cctacagctg ccaggtgacc 600

cacgagggca gcaccgtgga gaaaaccgtg gcccccaccg agtgcagct 649

<210> 96

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12_C1 LC

<400> 96

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Ser Tyr Ala Phe Asn Arg

85 90 95

Val Val Arg Asn Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 97

<211> 649

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12_C7 LC

<400> 97

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgt gacatccgtt acaaccgtgt tgttcgtccg 300

ttcggcggag ggaccaagct gaccgtccta ggtcaaccca aggctgcccc cagcgtgacc 360

ctgttccccc ccagcagcga ggaactgcag gccaacaagg ccaccctggt ctgcctgatc 420

agcgacttct acccaggcgc cgtgaccgtg gcctggaagg ccgacagcag ccccgtgaag 480

gccggcgtgg agaccaccac ccccagcaag cagagcaaca acaagtacgc cgccagcagc 540

tacctgagcc tgacccccga gcagtggaag agccacaggt cctacagctg ccaggtgacc 600

cacgagggca gcaccgtgga gaaaaccgtg gcccccaccg agtgcagct 649

<210> 98

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12_C7 LC

<400> 98

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ile Arg Tyr Asn Arg

85 90 95

Val Val Arg Pro Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 99

<211> 649

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12_E7 LC

<400> 99

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgt gactaccgtt acaaccgtgc tgttcgtccg 300

ttcggcggag ggaccaagct gaccgtccta ggtcaaccca aggctgcccc cagcgtgacc 360

ctgttccccc ccagcagcga ggaactgcag gccaacaagg ccaccctggt ctgcctgatc 420

agcgacttct acccaggcgc cgtgaccgtg gcctggaagg ccgacagcag ccccgtgaag 480

gccggcgtgg agaccaccac ccccagcaag cagagcaaca acaagtacgc cgccagcagc 540

tacctgagcc tgacccccga gcagtggaag agccacaggt cctacagctg ccaggtgacc 600

cacgagggca gcaccgtgga gaaaaccgtg gcccccaccg agtgcagct 649

<210> 100

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12_E7 LC

<400> 100

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Tyr Arg Tyr Asn Arg

85 90 95

Ala Val Arg Pro Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 101

<211> 649

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12_H3 LC

<400> 101

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgt gactacaaat tcaaccgtgt tgttcgtaac 300

ttcggcggag ggaccaagct gaccgtccta ggtcaaccca aggctgcccc cagcgtgacc 360

ctgttccccc ccagcagcga ggaactgcag gccaacaagg ccaccctggt ctgcctgatc 420

agcgacttct acccaggcgc cgtgaccgtg gcctggaagg ccgacagcag ccccgtgaag 480

gccggcgtgg agaccaccac ccccagcaag cagagcaaca acaagtacgc cgccagcagc 540

tacctgagcc tgacccccga gcagtggaag agccacaggt cctacagctg ccaggtgacc 600

cacgagggca gcaccgtgga gaaaaccgtg gcccccaccg agtgcagct 649

<210> 102

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12_H3 LC

<400> 102

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Tyr Lys Phe Asn Arg

85 90 95

Val Val Arg Asn Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 103

<211> 649

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12_A6 LC

<400> 103

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgt acttactctt acaaccgtgc tgttcgtaac 300

ttcggcggag ggaccaagct gaccgtccta ggtcaaccca aggctgcccc cagcgtgacc 360

ctgttccccc ccagcagcga ggaactgcag gccaacaagg ccaccctggt ctgcctgatc 420

agcgacttct acccaggcgc cgtgaccgtg gcctggaagg ccgacagcag ccccgtgaag 480

gccggcgtgg agaccaccac ccccagcaag cagagcaaca acaagtacgc cgccagcagc 540

tacctgagcc tgacccccga gcagtggaag agccacaggt cctacagctg ccaggtgacc 600

cacgagggca gcaccgtgga gaaaaccgtg gcccccaccg agtgcagct 649

<210> 104

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12_A6 LC

<400> 104

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Thr Tyr Ser Tyr Asn Arg

85 90 95

Ala Val Arg Asn Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 105

<211> 649

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12_D1 LC

<400> 105

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgt acttactctt acaaccgtgc tgttcgtccg 300

ttcggcggag ggaccaagct gaccgtccta ggtcaaccca aggctgcccc cagcgtgacc 360

ctgttccccc ccagcagcga ggaactgcag gccaacaagg ccaccctggt ctgcctgatc 420

agcgacttct acccaggcgc cgtgaccgtg gcctggaagg ccgacagcag ccccgtgaag 480

gccggcgtgg agaccaccac ccccagcaag cagagcaaca acaagtacgc cgccagcagc 540

tacctgagcc tgacccccga gcagtggaag agccacaggt cctacagctg ccaggtgacc 600

cacgagggca gcaccgtgga gaaaaccgtg gcccccaccg agtgcagct 649

<210> 106

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12_D1 LC

<400> 106

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Thr Tyr Ser Tyr Asn Arg

85 90 95

Ala Val Arg Pro Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 107

<211> 649

<212> DNA

<213> Artificial sequence

<220>

<223> 51A12_H6 LC

<400> 107

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgc gactacaaat ggaaccgtgt tgttcgtcat 300

ttcggcggag ggaccaagct gaccgtccta ggtcaaccca aggctgcccc cagcgtgacc 360

ctgttccccc ccagcagcga ggaactgcag gccaacaagg ccaccctggt ctgcctgatc 420

agcgacttct acccaggcgc cgtgaccgtg gcctggaagg ccgacagcag ccccgtgaag 480

gccggcgtgg agaccaccac ccccagcaag cagagcaaca acaagtacgc cgccagcagc 540

tacctgagcc tgacccccga gcagtggaag agccacaggt cctacagctg ccaggtgacc 600

cacgagggca gcaccgtgga gaaaaccgtg gcccccaccg agtgcagct 649

<210> 108

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> 51A12_H6 LC

<400> 108

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Tyr Lys Trp Asn Arg

85 90 95

Val Val Arg His Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 109

<211> 643

<212> DNA

<213> Artificial sequence

<220>

<223> DPL16 LC

<400> 109

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180

ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg taactcccgt gatagtagcg gtaatcatgt ggtattcggc 300

ggagggacca agctgaccgt cctaggtcaa cccaaggctg cccccagcgt gaccctgttc 360

ccccccagca gcgaggaact gcaggccaac aaggccaccc tggtctgcct gatcagcgac 420

ttctacccag gcgccgtgac cgtggcctgg aaggccgaca gcagccccgt gaaggccggc 480

gtggagacca ccacccccag caagcagagc aacaacaagt acgccgccag cagctacctg 540

agcctgaccc ccgagcagtg gaagagccac aggtcctaca gctgccaggt gacccacgag 600

ggcagcaccg tggagaaaac cgtggccccc accgagtgca gct 643

<210> 110

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> DPL16 LC

<400> 110

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys

100 105 110

Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln

115 120 125

Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly

130 135 140

Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly

145 150 155 160

Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala

165 170 175

Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser

180 185 190

Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val

195 200 205

Ala Pro Thr Glu Cys Ser

210

<210> 111

<211> 1872

<212> DNA

<213> Artificial sequence

<220>

<223> DP47GS HC (knob)

<400> 111

gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcagatga acagcctgag agccgaggac acggccgtat attactgtgc gaaaggcagc 300

ggatttgact actggggcca aggaaccctg gtcaccgtct cgagtgctag caccaagggc 360

ccatcggtct tccccctggc accctcctcc aagagcacct ctgggggcac agcggccctg 420

ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 480

ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc 540

agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc agacctacat ctgcaacgtg 600

aatcacaagc ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa 660

actcacacat gcccaccgtg cccagcacct gaagctgcag ggggaccgtc agtcttcctc 720

ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 780

gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 840

gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 900

gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 960

gtctccaaca aagccctcgg cgcccccatc gagaaaacca tctccaaagc caaagggcag 1020

ccccgagaac cacaggtgta caccctgccc ccatgccggg atgagctgac caagaaccag 1080

gtcagcctgt ggtgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1140

agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 1200

tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1260

ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320

ctgtctccgg gtggcggcgg aggctccgga ggcggaggat ctggcggcgg aggcagctgt 1380

gacctgcctc agacacacag cctgggcagc cggcggaccc tgatgctgct ggcccagatg 1440

cggaagatca gcctgttcag ctgcctgaag gaccggcacg acttcggctt ccctcaggaa 1500

gagttcggca accagttcca gaaggccgag acaatccccg tgctgcacga gatgatccag 1560

cagattttca acctgttcag caccaaggac agcagcgccg cctgggacga gacactgctg 1620

gacaagttct acaccgagct gtaccagcag ctgaacgacc tggaagcctg cgtgatccag 1680

ggcgtgggcg tgaccgagac acccctgatg aaggaagata gcatcctggc cgtgcggaag 1740

tatttccagc ggatcaccct gtacctgaaa gagaagaagt acagcccctg cgcctgggag 1800

gtcgtgcggg ccgagatcat gcggagcttc agcctgagca ccaacctgca ggaaagcctg 1860

cggagcaaag ag 1872

<210> 112

<211> 624

<212> PRT

<213> Artificial sequence

<220>

<223> DP47GS HC (carina)

<400> 112

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Ser Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly

435 440 445

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Asp Leu Pro Gln

450 455 460

Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met

465 470 475 480

Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly

485 490 495

Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile

500 505 510

Pro Val Leu His Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr

515 520 525

Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr

530 535 540

Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln

545 550 555 560

Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu

565 570 575

Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys

580 585 590

Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg

595 600 605

Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu

610 615 620

<210> 113

<211> 1335

<212> DNA

<213> Artificial sequence

<220>

<223> DP47GS HC (Point)

<400> 113

gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcagatga acagcctgag agccgaggac acggccgtat attactgtgc gaaaggcagc 300

ggatttgact actggggcca aggaaccctg gtcaccgtct cgagtgctag caccaagggc 360

ccctccgtgt tccccctggc ccccagcagc aagagcacca gcggcggcac agccgctctg 420

ggctgcctgg tcaaggacta cttccccgag cccgtgaccg tgtcctggaa cagcggagcc 480

ctgacctccg gcgtgcacac cttccccgcc gtgctgcaga gttctggcct gtatagcctg 540

agcagcgtgg tcaccgtgcc ttctagcagc ctgggcaccc agacctacat ctgcaacgtg 600

aaccacaagc ccagcaacac caaggtggac aagaaggtgg agcccaagag ctgcgacaaa 660

actcacacat gcccaccgtg cccagcacct gaagctgcag ggggaccgtc agtcttcctc 720

ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 780

gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 840

gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 900

gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 960

gtctccaaca aagccctcgg cgcccccatc gagaaaacca tctccaaagc caaagggcag 1020

ccccgagaac cacaggtgtg caccctgccc ccatcccggg atgagctgac caagaaccag 1080

gtcagcctct cgtgcgcagt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1140

agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 1200

tccttcttcc tcgtgagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1260

ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320

ctgtctccgg gtaaa 1335

<210> 114

<211> 445

<212> PRT

<213> Artificial sequence

<220>

<223> DP47GS HC (Point)

<400> 114

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Ser Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 115

<211> 681

<212> DNA

<213> Artificial sequence

<220>

<223> Fc (Point)

<400> 115

gacaaaactc acacatgccc accgtgccca gcacctgaag ctgcaggggg accgtcagtc 60

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300

tgcaaggtct ccaacaaagc cctcggcgcc cccatcgaga aaaccatctc caaagccaaa 360

gggcagcccc gagaaccaca ggtgtgcacc ctgcccccat cccgggatga gctgaccaag 420

aaccaggtca gcctctcgtg cgcagtcaaa ggcttctatc ccagcgacat cgccgtggag 480

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540

gacggctcct tcttcctcgt gagcaagctc accgtggaca agagcaggtg gcagcagggg 600

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660

ctctccctgt ctccgggtaa a 681

<210> 116

<211> 227

<212> PRT

<213> Artificial sequence

<220>

<223> Fc (Point)

<400> 116

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 117

<211> 1206

<212> DNA

<213> Artificial sequence

<220>

<223> avi-Fc-huasrGPR H1CRD (cave)

<400> 117

ggcctgaacg atatttttga agcccagaaa atcgaatggc atgaggacaa aactcacaca 60

tgcccaccgt gcccagcacc tgaactcctg gggggaccgt cagtcttcct cttcccccca 120

aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac 180

gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 240

aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc 300

ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac 360

aaagccctcc cagcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa 420

ccacaggtgt gcaccctgcc cccatcccgg gatgagctga ccaagaacca ggtcagcctc 480

tcgtgcgcag tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg 540

cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 600

ctcgtgagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 660

tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 720

ggtggatccg gcggtggtag tccgacacct ccgacacccg ggggtggtag caatggtagc 780

gaacgtacct gttgtccggt taattgggtt gaacatgaac gtagctgcta ttggtttagc 840

cgtagcggta aagcatgggc agatgcagat aattattgtc gtctggaaga tgcacatctg 900

gttgttgtga ccagctggga agaacagaaa tttgttcagc atcatattgg tccggtgaat 960

acctggatgg gtctgcatga tcagaatggt ccgtggaaat gggttgatgg caccgattat 1020

gaaaccggtt ttaaaaattg gcgtccggaa cagccggatg attggtatgg tcatggtctg 1080

ggtggtggtg aagattgtgc acattttacc gatgatggtc gttggaatga tgatgtttgt 1140

cagcgtccgt atcgttgggt ttgtgaaacc gaactggata aagcaagcca ggaaccgccg 1200

ctgctg 1206

<210> 118

<211> 402

<212> PRT

<213> Artificial sequence

<220>

<223> avi-Fc-huasprg H1CRD (Point)

<400> 118

Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Asp

1 5 10 15

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

20 25 30

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

35 40 45

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

50 55 60

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

65 70 75 80

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

85 90 95

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

100 105 110

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

115 120 125

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

130 135 140

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

145 150 155 160

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

165 170 175

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

180 185 190

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

195 200 205

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

210 215 220

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

225 230 235 240

Gly Gly Ser Gly Gly Gly Ser Pro Thr Pro Pro Thr Pro Gly Gly Gly

245 250 255

Ser Asn Gly Ser Glu Arg Thr Cys Cys Pro Val Asn Trp Val Glu His

260 265 270

Glu Arg Ser Cys Tyr Trp Phe Ser Arg Ser Gly Lys Ala Trp Ala Asp

275 280 285

Ala Asp Asn Tyr Cys Arg Leu Glu Asp Ala His Leu Val Val Val Thr

290 295 300

Ser Trp Glu Glu Gln Lys Phe Val Gln His His Ile Gly Pro Val Asn

305 310 315 320

Thr Trp Met Gly Leu His Asp Gln Asn Gly Pro Trp Lys Trp Val Asp

325 330 335

Gly Thr Asp Tyr Glu Thr Gly Phe Lys Asn Trp Arg Pro Glu Gln Pro

340 345 350

Asp Asp Trp Tyr Gly His Gly Leu Gly Gly Gly Glu Asp Cys Ala His

355 360 365

Phe Thr Asp Asp Gly Arg Trp Asn Asp Asp Val Cys Gln Arg Pro Tyr

370 375 380

Arg Trp Val Cys Glu Thr Glu Leu Asp Lys Ala Ser Gln Glu Pro Pro

385 390 395 400

Leu Leu

<210> 119

<211> 1179

<212> DNA

<213> Artificial sequence

<220>

<223> avi-Fc-huCLEC10A CRD (Point)

<400> 119

ggcctgaacg atatttttga agcccagaaa atcgaatggc atgaggacaa aactcacaca 60

tgcccaccgt gcccagcacc tgaactcctg gggggaccgt cagtcttcct cttcccccca 120

aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac 180

gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 240

aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc 300

ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac 360

aaagccctcc cagcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa 420

ccacaggtgt gcaccctgcc cccatcccgg gatgagctga ccaagaacca ggtcagcctc 480

tcgtgcgcag tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg 540

cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 600

ctcgtgagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 660

tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 720

ggtggatccg gcggtggtag tccgacacct ccgacacccg ggggtggtag ctgtccggtt 780

aattgggttg aacatcagga tagctgctat tggtttagcc atagcggtat gagctgggca 840

gaagcagaaa aatattgcca gctgaaaaat gcccatctgg ttgttattaa tagccgtgaa 900

gaacagaatt ttgtgcagaa atatctgggt agcgcatata cctggatggg tctgagcgat 960

ccggaaggtg catggaaatg ggttgatggc accgattatg caaccggttt tcagaattgg 1020

aaaccgggtc agccggatga ttggcagggt catggtctgg gtggtggtga agattgtgca 1080

cattttcatc cggatggtcg ttggaatgat gatgtttgtc agcgtccgta tcattgggtt 1140

tgtgaagcag gtctgggtca gaccagccag gaaagccat 1179

<210> 120

<211> 402

<212> PRT

<213> Artificial sequence

<220>

<223> avi-Fc-huCLEC10A CRD (Point)

<400> 120

Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Asp

1 5 10 15

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

20 25 30

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

35 40 45

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

50 55 60

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

65 70 75 80

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

85 90 95

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

100 105 110

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

115 120 125

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

130 135 140

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

145 150 155 160

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

165 170 175

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

180 185 190

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

195 200 205

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

210 215 220

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

225 230 235 240

Gly Gly Ser Gly Gly Gly Ser Pro Thr Pro Pro Thr Pro Gly Gly Gly

245 250 255

Ser Asn Gly Ser Glu Arg Thr Cys Cys Pro Val Asn Trp Val Glu His

260 265 270

Glu Arg Ser Cys Tyr Trp Phe Ser Arg Ser Gly Lys Ala Trp Ala Asp

275 280 285

Ala Asp Asn Tyr Cys Arg Leu Glu Asp Ala His Leu Val Val Val Thr

290 295 300

Ser Trp Glu Glu Gln Lys Phe Val Gln His His Ile Gly Pro Val Asn

305 310 315 320

Thr Trp Met Gly Leu His Asp Gln Asn Gly Pro Trp Lys Trp Val Asp

325 330 335

Gly Thr Asp Tyr Glu Thr Gly Phe Lys Asn Trp Arg Pro Glu Gln Pro

340 345 350

Asp Asp Trp Tyr Gly His Gly Leu Gly Gly Gly Glu Asp Cys Ala His

355 360 365

Phe Thr Asp Asp Gly Arg Trp Asn Asp Asp Val Cys Gln Arg Pro Tyr

370 375 380

Arg Trp Val Cys Glu Thr Glu Leu Asp Lys Ala Ser Gln Glu Pro Pro

385 390 395 400

Leu Leu

<210> 121

<211> 726

<212> DNA

<213> Artificial sequence

<220>

<223> avi-Fc (Point)

<400> 121

ggcctgaacg atatttttga agcccagaaa atcgaatggc atgaggacaa aactcacaca 60

tgcccaccgt gcccagcacc tgaactcctg gggggaccgt cagtcttcct cttcccccca 120

aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac 180

gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 240

aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc 300

ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac 360

aaagccctcc cagcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa 420

ccacaggtgt acaccctgcc cccatgccgg gatgagctga ccaagaacca ggtcagcctg 480

tggtgcctgg tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg 540

cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 600

ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 660

tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 720

ggtaaa 726

<210> 122

<211> 242

<212> PRT

<213> Artificial sequence

<220>

<223> avi-Fc (Point)

<400> 122

Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Asp

1 5 10 15

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

20 25 30

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

35 40 45

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

50 55 60

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

65 70 75 80

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

85 90 95

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

100 105 110

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

115 120 125

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

130 135 140

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

145 150 155 160

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

165 170 175

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

180 185 190

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

195 200 205

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

210 215 220

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

225 230 235 240

Gly Lys

<210> 123

<211> 1056

<212> DNA

<213> Artificial sequence

<220>

<223> avi-Fc-huasger H1 Stem

<400> 123

ggcctgaacg atatttttga agcccagaaa atcgaatggc atgaggacaa aactcacaca 60

tgcccaccgt gcccagcacc tgaactcctg gggggaccgt cagtcttcct cttcccccca 120

aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac 180

gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 240

aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc 300

ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac 360

aaagccctcg gcgcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa 420

ccacaggtgt acaccctgcc cccatcccgg gatgagctga ccaagaacca ggtcagcctg 480

acctgcctgg tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg 540

cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 600

ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 660

tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 720

ggtggcggag ggggatctgg aggtggcggc tccggaggcg gaggatctgg cggaggcgga 780

tcccagaata gccagctgca agaggaactg cgtggtctgc gtgaaacctt tagcaatttc 840

accgcaagca ccgaagcaca ggttaaaggt ctgagcaccc agggtggtaa tgttggtcgt 900

aaaatgaaaa gcctggaaag ccagctggaa aaacagcaga aagatctgag cgaagatcat 960

tcatcactgc tgctgcatgt taaacagttt gttagcgatc tgcgtagcct gagctgtcag 1020

atggcagcac tgcagggtaa tggtagcgaa cgtacc 1056

<210> 124

<211> 352

<212> PRT

<213> Artificial sequence

<220>

<223> avi-Fc-huasrGPR H1 Stem

<400> 124

Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Asp

1 5 10 15

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

20 25 30

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

35 40 45

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

50 55 60

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

65 70 75 80

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

85 90 95

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

100 105 110

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

115 120 125

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

130 135 140

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

145 150 155 160

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

165 170 175

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

180 185 190

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

195 200 205

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

210 215 220

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

225 230 235 240

Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

245 250 255

Gly Gly Gly Gly Ser Gln Asn Ser Gln Leu Gln Glu Glu Leu Arg Gly

260 265 270

Leu Arg Glu Thr Phe Ser Asn Phe Thr Ala Ser Thr Glu Ala Gln Val

275 280 285

Lys Gly Leu Ser Thr Gln Gly Gly Asn Val Gly Arg Lys Met Lys Ser

290 295 300

Leu Glu Ser Gln Leu Glu Lys Gln Gln Lys Asp Leu Ser Glu Asp His

305 310 315 320

Ser Ser Leu Leu Leu His Val Lys Gln Phe Val Ser Asp Leu Arg Ser

325 330 335

Leu Ser Cys Gln Met Ala Ala Leu Gln Gly Asn Gly Ser Glu Arg Thr

340 345 350

<210> 125

<211> 1056

<212> DNA

<213> Artificial sequence

<220>

<223> avi-Fc-cyASGPR H1 Stem

<400> 125

ggcctgaacg atatttttga agcccagaaa atcgaatggc atgaggacaa aactcacaca 60

tgcccaccgt gcccagcacc tgaactcctg gggggaccgt cagtcttcct cttcccccca 120

aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac 180

gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 240

aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc 300

ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac 360

aaagccctcg gcgcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa 420

ccacaggtgt acaccctgcc cccatcccgg gatgagctga ccaagaacca ggtcagcctg 480

acctgcctgg tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg 540

cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 600

ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 660

tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 720

ggtggcggag ggggatctgg aggtggcggc tccggaggcg gaggatctgg cggaggcgga 780

tcccaaaacg cccagctgca gcgggagctg cggggcctga gagagacgct cagcaacttc 840

acagcgagca ccgaggccca ggtcaagggc ttgagcaccc agggaggcaa tgtgggaaga 900

aagatgaagt cgctggagtc ccagctggag aaacagcaga aggacttgag tgaagatcac 960

tccagcctgc tgctccacgt gaagcagttc gtgtctgacc tgcggagcct gagctgtcag 1020

atggcggcgc tccagggcaa tggctcggaa agggcc 1056

<210> 126

<211> 352

<212> PRT

<213> Artificial sequence

<220>

<223> stems of avi-Fc-cyASGPR H1

<400> 126

Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Asp

1 5 10 15

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

20 25 30

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

35 40 45

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

50 55 60

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

65 70 75 80

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

85 90 95

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

100 105 110

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

115 120 125

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

130 135 140

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

145 150 155 160

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

165 170 175

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

180 185 190

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

195 200 205

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

210 215 220

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

225 230 235 240

Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

245 250 255

Gly Gly Gly Gly Ser Gln Asn Ala Gln Leu Gln Arg Glu Leu Arg Gly

260 265 270

Leu Arg Glu Thr Leu Ser Asn Phe Thr Ala Ser Thr Glu Ala Gln Val

275 280 285

Lys Gly Leu Ser Thr Gln Gly Gly Asn Val Gly Arg Lys Met Lys Ser

290 295 300

Leu Glu Ser Gln Leu Glu Lys Gln Gln Lys Asp Leu Ser Glu Asp His

305 310 315 320

Ser Ser Leu Leu Leu His Val Lys Gln Phe Val Ser Asp Leu Arg Ser

325 330 335

Leu Ser Cys Gln Met Ala Ala Leu Gln Gly Asn Gly Ser Glu Arg Ala

340 345 350

<210> 127

<211> 1023

<212> DNA

<213> Artificial sequence

<220>

<223> avi-Fc-huCLEC10A Stem

<400> 127

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300

tgcaaggtct ccaacaaagc cctcggcgcc cccatcgaga aaaccatctc caaagccaaa 360

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 420

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660

ctctccctgt ctccgggtgg cggaggggga tctggaggtg gcggctccgg aggcggagga 720

tctggcggag gcggatccca gaacagcaag ttccagcggg acctggtcac cctgcggacc 780

gacttcagca acttcaccag caacaccgtg gccgagatcc aggccctgac cagccagggc 840

agcagcctgg aagagacaat cgccagcctg aaggccgagg tggaaggctt caagcaggaa 900

cggcaggccg tccacagcga gatgctgctg cgggtgcagc agctggtgca ggacctgaag 960

aaactgacct gccaggtggc caccctgaac aacaacggcg aggaagctag cactgaaggg 1020

acc 1023

<210> 128

<211> 356

<212> PRT

<213> Artificial sequence

<220>

<223> avi-Fc-huCLEC10A Stem

<400> 128

Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Asp

1 5 10 15

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

20 25 30

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

35 40 45

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

50 55 60

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

65 70 75 80

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

85 90 95

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

100 105 110

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

115 120 125

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

130 135 140

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

145 150 155 160

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

165 170 175

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

180 185 190

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

195 200 205

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

210 215 220

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

225 230 235 240

Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

245 250 255

Gly Gly Gly Gly Ser Gln Asn Ser Lys Phe Gln Arg Asp Leu Val Thr

260 265 270

Leu Arg Thr Asp Phe Ser Asn Phe Thr Ser Asn Thr Val Ala Glu Ile

275 280 285

Gln Ala Leu Thr Ser Gln Gly Ser Ser Leu Glu Glu Thr Ile Ala Ser

290 295 300

Leu Lys Ala Glu Val Glu Gly Phe Lys Gln Glu Arg Gln Ala Val His

305 310 315 320

Ser Glu Met Leu Leu Arg Val Gln Gln Leu Val Gln Asp Leu Lys Lys

325 330 335

Leu Thr Cys Gln Val Ala Thr Leu Asn Asn Asn Gly Glu Glu Ala Ser

340 345 350

Thr Glu Gly Thr

355

<210> 129

<211> 1473

<212> DNA

<213> Artificial sequence

<220>

<223> avi-Fc-huasrGPR H1 Stem CRD

<400> 129

ggcctgaacg atatttttga agcccagaaa atcgaatggc atgaggacaa aactcacaca 60

tgcccaccgt gcccagcacc tgaactcctg gggggaccgt cagtcttcct cttcccccca 120

aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac 180

gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 240

aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc 300

ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac 360

aaagccctcg gcgcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa 420

ccacaggtgt acaccctgcc cccatcccgg gatgagctga ccaagaacca ggtcagcctg 480

acctgcctgg tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg 540

cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 600

ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 660

tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 720

ggtggcggag ggggatctgg aggtggcggc tccggaggcg gaggatctgg cggaggcgga 780

tcccagaata gccagctgca agaggaactg cgtggtctgc gtgaaacctt tagcaatttc 840

accgcaagca ccgaagcaca ggttaaaggt ctgagcaccc agggtggtaa tgttggtcgt 900

aaaatgaaaa gcctggaaag ccagctggaa aaacagcaga aagatctgag cgaagatcat 960

tcatcactgc tgctgcatgt taaacagttt gttagcgatc tgcgtagcct gagctgtcag 1020

atggcagcac tgcagggtaa tggtagcgaa cgtacctgtt gtccggttaa ttgggttgaa 1080

catgaacgta gctgctattg gtttagccgt agcggtaaag catgggcaga tgcagataat 1140

tattgtcgtc tggaagatgc acatctggtt gttgtgacca gctgggaaga acagaaattt 1200

gttcagcatc atattggtcc ggtgaatacc tggatgggtc tgcatgatca gaatggtccg 1260

tggaaatggg ttgatggcac cgattatgaa accggtttta aaaattggcg tccggaacag 1320

ccggatgatt ggtatggtca tggtctgggt ggtggtgaag attgtgcaca ttttaccgat 1380

gatggtcgtt ggaatgatga tgtttgtcag cgtccgtatc gttgggtttg tgaaaccgaa 1440

ctggataaag caagccagga accgccgctg ctg 1473

<210> 130

<211> 491

<212> PRT

<213> Artificial sequence

<220>

<223> avi-Fc-huasgrg H1 Stem CRD

<400> 130

Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Asp

1 5 10 15

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

20 25 30

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

35 40 45

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

50 55 60

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

65 70 75 80

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

85 90 95

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

100 105 110

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

115 120 125

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

130 135 140

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

145 150 155 160

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

165 170 175

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

180 185 190

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

195 200 205

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

210 215 220

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

225 230 235 240

Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

245 250 255

Gly Gly Gly Gly Ser Gln Asn Ser Gln Leu Gln Glu Glu Leu Arg Gly

260 265 270

Leu Arg Glu Thr Phe Ser Asn Phe Thr Ala Ser Thr Glu Ala Gln Val

275 280 285

Lys Gly Leu Ser Thr Gln Gly Gly Asn Val Gly Arg Lys Met Lys Ser

290 295 300

Leu Glu Ser Gln Leu Glu Lys Gln Gln Lys Asp Leu Ser Glu Asp His

305 310 315 320

Ser Ser Leu Leu Leu His Val Lys Gln Phe Val Ser Asp Leu Arg Ser

325 330 335

Leu Ser Cys Gln Met Ala Ala Leu Gln Gly Asn Gly Ser Glu Arg Thr

340 345 350

Cys Cys Pro Val Asn Trp Val Glu His Glu Arg Ser Cys Tyr Trp Phe

355 360 365

Ser Arg Ser Gly Lys Ala Trp Ala Asp Ala Asp Asn Tyr Cys Arg Leu

370 375 380

Glu Asp Ala His Leu Val Val Val Thr Ser Trp Glu Glu Gln Lys Phe

385 390 395 400

Val Gln His His Ile Gly Pro Val Asn Thr Trp Met Gly Leu His Asp

405 410 415

Gln Asn Gly Pro Trp Lys Trp Val Asp Gly Thr Asp Tyr Glu Thr Gly

420 425 430

Phe Lys Asn Trp Arg Pro Glu Gln Pro Asp Asp Trp Tyr Gly His Gly

435 440 445

Leu Gly Gly Gly Glu Asp Cys Ala His Phe Thr Asp Asp Gly Arg Trp

450 455 460

Asn Asp Asp Val Cys Gln Arg Pro Tyr Arg Trp Val Cys Glu Thr Glu

465 470 475 480

Leu Asp Lys Ala Ser Gln Glu Pro Pro Leu Leu

485 490

<210> 131

<211> 1473

<212> DNA

<213> Artificial sequence

<220>

<223> avi-Fc-cyASGPR H1 Stem CRD

<400> 131

ggcctgaacg atatttttga agcccagaaa atcgaatggc atgaggacaa aactcacaca 60

tgcccaccgt gcccagcacc tgaactcctg gggggaccgt cagtcttcct cttcccccca 120

aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac 180

gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 240

aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc 300

ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac 360

aaagccctcg gcgcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa 420

ccacaggtgt acaccctgcc cccatcccgg gatgagctga ccaagaacca ggtcagcctg 480

acctgcctgg tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg 540

cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 600

ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 660

tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 720

ggtggcggag ggggatctgg aggtggcggc tccggaggcg gaggatctgg cggaggcgga 780

tcccaaaacg cccagctgca gcgggagctg cggggcctga gagagacgct cagcaacttc 840

acagcgagca ccgaggccca ggtcaagggc ttgagcaccc agggaggcaa tgtgggaaga 900

aagatgaagt cgctggagtc ccagctggag aaacagcaga aggacttgag tgaagatcac 960

tccagcctgc tgctccacgt gaagcagttc gtgtctgacc tgcggagcct gagctgtcag 1020

atggcggcgc tccagggcaa tggctcggaa agggcctgct gcccagtcaa ctgggtggag 1080

cacgagcgca gctgctactg gttctctcgc tccgggaagg cctgggccga cgccgacaac 1140

tactgccggc tggaggacgc gcacctggtg gtggtcacgt cctgggagga gcagaaattt 1200

gtccagcacc acataggtcc tgtgaacacc tggatgggcc tccacgacca aaacgggccc 1260

tggaagtggg tggacgggac ggactacgag acgggcttca agaactggag accggagcag 1320

ccggacgact ggtacggcca cgggctcggg ggaggggagg actgtgccca cttcaccgac 1380

gacggccgct ggaacgacga cgtctgccag aggccctacc gctgggtctg cgagacagag 1440

ctggacaagg ccagccagga gccacctctc ctt 1473

<210> 132

<211> 491

<212> PRT

<213> Artificial sequence

<220>

<223> avi-Fc-cyASGPR H1 Stem CRD

<400> 132

Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Asp

1 5 10 15

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

20 25 30

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

35 40 45

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

50 55 60

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

65 70 75 80

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

85 90 95

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

100 105 110

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

115 120 125

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

130 135 140

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

145 150 155 160

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

165 170 175

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

180 185 190

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

195 200 205

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

210 215 220

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

225 230 235 240

Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

245 250 255

Gly Gly Gly Gly Ser Gln Asn Ala Gln Leu Gln Arg Glu Leu Arg Gly

260 265 270

Leu Arg Glu Thr Leu Ser Asn Phe Thr Ala Ser Thr Glu Ala Gln Val

275 280 285

Lys Gly Leu Ser Thr Gln Gly Gly Asn Val Gly Arg Lys Met Lys Ser

290 295 300

Leu Glu Ser Gln Leu Glu Lys Gln Gln Lys Asp Leu Ser Glu Asp His

305 310 315 320

Ser Ser Leu Leu Leu His Val Lys Gln Phe Val Ser Asp Leu Arg Ser

325 330 335

Leu Ser Cys Gln Met Ala Ala Leu Gln Gly Asn Gly Ser Glu Arg Ala

340 345 350

Cys Cys Pro Val Asn Trp Val Glu His Glu Arg Ser Cys Tyr Trp Phe

355 360 365

Ser Arg Ser Gly Lys Ala Trp Ala Asp Ala Asp Asn Tyr Cys Arg Leu

370 375 380

Glu Asp Ala His Leu Val Val Val Thr Ser Trp Glu Glu Gln Lys Phe

385 390 395 400

Val Gln His His Ile Gly Pro Val Asn Thr Trp Met Gly Leu His Asp

405 410 415

Gln Asn Gly Pro Trp Lys Trp Val Asp Gly Thr Asp Tyr Glu Thr Gly

420 425 430

Phe Lys Asn Trp Arg Pro Glu Gln Pro Asp Asp Trp Tyr Gly His Gly

435 440 445

Leu Gly Gly Gly Glu Asp Cys Ala His Phe Thr Asp Asp Gly Arg Trp

450 455 460

Asn Asp Asp Val Cys Gln Arg Pro Tyr Arg Trp Val Cys Glu Thr Glu

465 470 475 480

Leu Asp Lys Ala Ser Gln Glu Pro Pro Leu Leu

485 490

<210> 133

<211> 1434

<212> DNA

<213> Artificial sequence

<220>

<223> avi-Fc-huCLEC10A Stem CRD

<400> 133

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300

tgcaaggtct ccaacaaagc cctcggcgcc cccatcgaga aaaccatctc caaagccaaa 360

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 420

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660

ctctccctgt ctccgggtgg cggaggggga tctggaggtg gcggctccgg aggcggagga 720

tctggcggag gcggatccca gaacagcaag ttccagcggg acctggtcac cctgcggacc 780

gacttcagca acttcaccag caacaccgtg gccgagatcc aggccctgac cagccagggc 840

agcagcctgg aagagacaat cgccagcctg aaggccgagg tggaaggctt caagcaggaa 900

cggcaggccg tccacagcga gatgctgctg cgggtgcagc agctggtgca ggacctgaag 960

aaactgacct gccaggtggc caccctgaac aacaacggcg aggaagctag cactgaaggg 1020

acctgctgtc cggttaattg ggttgaacat caggatagct gctattggtt tagccatagc 1080

ggtatgagct gggcagaagc agaaaaatat tgccagctga aaaatgccca tctggttgtt 1140

attaatagcc gtgaagaaca gaattttgtg cagaaatatc tgggtagcgc atatacctgg 1200

atgggtctga gcgatccgga aggtgcatgg aaatgggttg atggcaccga ttatgcaacc 1260

ggttttcaga attggaaacc gggtcagccg gatgattggc agggtcatgg tctgggtggt 1320

ggtgaagatt gtgcacattt tcatccggat ggtcgttgga atgatgatgt ttgtcagcgt 1380

ccgtatcatt gggtttgtga agcaggtctg ggtcagacca gccaggaaag ccat 1434

<210> 134

<211> 493

<212> PRT

<213> Artificial sequence

<220>

<223> avi-Fc-huCLEC10A Stem CRD

<400> 134

Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Asp

1 5 10 15

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

20 25 30

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

35 40 45

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

50 55 60

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

65 70 75 80

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

85 90 95

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

100 105 110

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

115 120 125

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

130 135 140

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

145 150 155 160

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

165 170 175

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

180 185 190

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

195 200 205

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

210 215 220

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

225 230 235 240

Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

245 250 255

Gly Gly Gly Gly Ser Gln Asn Ser Lys Phe Gln Arg Asp Leu Val Thr

260 265 270

Leu Arg Thr Asp Phe Ser Asn Phe Thr Ser Asn Thr Val Ala Glu Ile

275 280 285

Gln Ala Leu Thr Ser Gln Gly Ser Ser Leu Glu Glu Thr Ile Ala Ser

290 295 300

Leu Lys Ala Glu Val Glu Gly Phe Lys Gln Glu Arg Gln Ala Val His

305 310 315 320

Ser Glu Met Leu Leu Arg Val Gln Gln Leu Val Gln Asp Leu Lys Lys

325 330 335

Leu Thr Cys Gln Val Ala Thr Leu Asn Asn Asn Gly Glu Glu Ala Ser

340 345 350

Thr Glu Gly Thr Cys Cys Pro Val Asn Trp Val Glu His Gln Asp Ser

355 360 365

Cys Tyr Trp Phe Ser His Ser Gly Met Ser Trp Ala Glu Ala Glu Lys

370 375 380

Tyr Cys Gln Leu Lys Asn Ala His Leu Val Val Ile Asn Ser Arg Glu

385 390 395 400

Glu Gln Asn Phe Val Gln Lys Tyr Leu Gly Ser Ala Tyr Thr Trp Met

405 410 415

Gly Leu Ser Asp Pro Glu Gly Ala Trp Lys Trp Val Asp Gly Thr Asp

420 425 430

Tyr Ala Thr Gly Phe Gln Asn Trp Lys Pro Gly Gln Pro Asp Asp Trp

435 440 445

Gln Gly His Gly Leu Gly Gly Gly Glu Asp Cys Ala His Phe His Pro

450 455 460

Asp Gly Arg Trp Asn Asp Asp Val Cys Gln Arg Pro Tyr His Trp Val

465 470 475 480

Cys Glu Ala Gly Leu Gly Gln Thr Ser Gln Glu Ser His

485 490

<210> 135

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> leader sequence 1

<400> 135

Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly

1 5 10 15

Ala His Ser

<210> 136

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> leader sequence 2

<400> 136

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ala Arg Cys

20

<210> 137

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> leader sequence 3

<400> 137

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser

<210> 138

<211> 165

<212> PRT

<213> human (Homo sapiens)

<400> 138

Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met

1 5 10 15

Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp

20 25 30

Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln

35 40 45

Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe

50 55 60

Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu

65 70 75 80

Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu

85 90 95

Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys

100 105 110

Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu

115 120 125

Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg

130 135 140

Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser

145 150 155 160

Leu Arg Ser Lys Glu

165

<210> 139

<211> 165

<212> PRT

<213> Artificial sequence

<220>

<223> IFNa2a

<400> 139

Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met

1 5 10 15

Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp

20 25 30

Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln

35 40 45

Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe

50 55 60

Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu

65 70 75 80

Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu

85 90 95

Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys

100 105 110

Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu

115 120 125

Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg

130 135 140

Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser

145 150 155 160

Leu Arg Ser Lys Glu

165

Claims (66)

1. An antibody capable of specifically binding to an asialoglycoprotein receptor, abbreviated ASGPR, wherein the antibody comprises the heavy chain variable region sequence of SEQ ID No. 16 and the light chain variable region sequence of SEQ ID No. 14.

2. The antibody of claim 1, wherein the antibody is capable of specifically binding to human and cynomolgus monkey ASGPR.

3. The antibody of claim 1, wherein the antibody has a dissociation constant, K, of less than 1 μ M as measured by surface plasmon resonance as a Fab fragment_DBinding to human ASGPR.

4. The antibody of claim 3, wherein the antibody has a dissociation constant K of less than 100nM, as measured by surface plasmon resonance as Fab fragment_DBinding to human ASGPR.

5. The antibody of claim 1, wherein the antibody has a K of less than 1 μ M as measured by fluorescence resonance energy transfer as IgG1_DBinding to human ASGPR.

6. The antibody of claim 5, wherein the antibody has a molecular mass of less than 500n as measured by fluorescence resonance energy transfer as IgG1K of M_DBinding to human ASGPR.

7. The antibody of claim 6, wherein the antibody has a K of less than 100nM as IgG1 measured by fluorescence resonance energy transfer method_DBinding to human ASGPR.

8. The antibody of claim 7, wherein the antibody has a K of less than 10nM as measured by fluorescence resonance energy transfer as IgG1_DBinding to human ASGPR.

9. The antibody of claim 1, wherein the antibody does not compete with the natural ligand of ASGPR for binding to ASGPR.

10. The antibody of claim 9, wherein the natural ligand of ASGPR is asialofetuin.

11. The antibody of claim 1 wherein the antibody does not bind to CLEC 10A.

12. The antibody of claim 11 wherein the antibody does not bind to human CLEC 10A.

13. The antibody of claim 1, wherein the antibody does not specifically bind to cells lacking ASGPR expression.

14. The antibody of claim 13, wherein the antibody does not specifically bind to human cells lacking ASGPR expression.

15. The antibody of claim 14, wherein the antibody does not specifically bind to human blood cells lacking ASGPR expression.

16. The antibody of claim 1, wherein when the antibody binds to ASGPR on the surface of a cell expressing ASGPR, the antibody is internalized into the cell.

17. The antibody of claim 16, wherein antibody is recycled back to the surface of the cell at the same rate as it is internalized into the cell.

18. The antibody of claim 1, wherein when the antibody binds to ASGPR on the surface of a cell, the antibody does not induce down-regulation of ASGPR expression at the surface of the cell.

19. The antibody of claim 1, wherein the antibody is a human antibody.

20. The antibody of claim 1, wherein the antibody comprises a human Fc region.

21. The antibody of claim 19, wherein the antibody comprises an IgG Fc region.

22. The antibody of claim 21, wherein the antibody comprises an IgG₁An Fc region.

23. The antibody of claim 1, wherein the antibody is a full length antibody.

24. The antibody of claim 1, wherein the antibody is an IgG class antibody.

25. The antibody of claim 24, wherein the antibody is an IgG₁Subclass antibody.

26. The antibody of claim 20, wherein the antibody comprises a modification in the Fc region that reduces the binding affinity of the antibody to an Fc receptor.

27. The antibody of claim 26, wherein the antibody comprises a modification in the Fc region that reduces the binding affinity of the antibody to an fey receptor.

28. The antibody of claim 26, wherein the Fc receptor is an activated Fc receptor.

29. The antibody of claim 26, wherein the Fc receptor is selected from the group consisting of fcyriiia (CD16a), fcyri (CD64), fcyriia (CD32), and fcyri (CD 89).

30. The antibody of claim 29, wherein the Fc receptor is fcyriiia.

31. The antibody of claim 30, wherein the Fc receptor is human fcyriiia.

32. The antibody of claim 26, wherein the antibody comprises an amino acid substitution within the Fc region at a position selected from the group consisting of P329, L234 and L235 according to EU numbering.

33. The antibody of claim 32, wherein the antibody comprises the amino acid substitutions P329G, L234A, and L235A in the Fc region according to EU numbering.

34. The antibody of claim 20, wherein the antibody comprises a modification in the Fc region that promotes heterodimerization of two non-identical antibody heavy chains.

35. The antibody of claim 34, wherein the modification is a protuberance-into-hole modification comprising a protuberance modification in one of the antibody heavy chains and a hole modification in the other of the two antibody heavy chains.

36. The antibody of claim 20, wherein the antibody comprises a modification within the interface between two antibody heavy chains in the CH3 domain, wherein i) in the CH3 domain of one heavy chain, amino acid residues are replaced with amino acid residues having a larger side chain volume, thus creating a protuberance ("knob") within the interface in the CH3 domain of one heavy chain that can fit into a cavity ("hole") within the interface in the CH3 domain of the other heavy chain, and ii) in the CH3 domain of the other heavy chain, amino acid residues are replaced with amino acid residues having a smaller side chain volume, thus creating a cavity ("hole") within the interface in the second CH3 domain inside which a protuberance ("knob") within the interface in the first CH3 domain can fit.

37. The antibody of claim 20, wherein the antibody comprises the amino acid substitution T366W and optionally amino acid substitution S354C in one of the antibody heavy chains and the amino acid substitutions T366S, L368A, Y407V and optionally Y349C in the other antibody heavy chain.

38. The antibody of any one of claims 1-37, wherein an effector moiety is linked to the antibody.

39. The antibody of claim 38, wherein no more than one effector moiety is attached to the antibody.

40. The antibody of claim 38, wherein the effector moiety is a cytokine molecule.

41. The antibody of claim 40, wherein the cytokine molecule is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the antibody heavy chains, optionally via a peptide linker.

42. The antibody of claim 40, wherein the cytokine molecule is a human cytokine.

43. The antibody of claim 40, wherein the cytokine molecule is an interferon molecule.

44. The antibody of claim 43, wherein the interferon molecule is interferon alpha.

45. The antibody of claim 44, wherein the interferon molecule is human interferon alpha.

46. The antibody of claim 45, wherein the interferon molecule is human interferon alpha 2.

47. The antibody of claim 46, wherein human interferon alpha 2 is human interferon alpha 2 a.

48. The antibody of claim 43, wherein the antibody has antiviral activity in cells expressing ASGPR on the cell surface.

49. The antibody of claim 43, wherein the antibody has no antiviral activity in cells that do not express ASGPR on the cell surface.

50. The antibody of claim 48 or 49, wherein the antiviral activity is selected from the group consisting of inhibiting viral infection, inhibiting viral replication, inhibiting cell killing, and inducing interferon stimulation.

51. A polynucleotide encoding the antibody or antigen binding portion thereof of any one of claims 1-50.

52. A vector comprising the polynucleotide of claim 51.

53. The vector of claim 52, which is an expression vector.

54. A host cell comprising the polynucleotide of claim 51.

55. A host cell comprising the vector of claim 52.

56. A method for producing the antibody of any one of claims 1-50, comprising the steps of (i) culturing the host cell of claim 54 or 55 under conditions suitable for expression of the antibody, and (ii) recovering the antibody.

57. An antibody capable of specifically binding to ASGPR produced by the method of claim 56.

58. A pharmaceutical composition comprising the antibody of any one of claims 1-50 or 57 and a pharmaceutically acceptable carrier.

59. Use of an antibody according to any one of claims 38-50 for the preparation of a medicament for the treatment of liver disease in an individual in need thereof.

60. The use according to claim 59, wherein the liver disease is a viral infection.

61. The use according to claim 60, wherein the liver disease is a hepatitis virus infection.

62. The use of claim 61, wherein the hepatitis virus infection is an HBV infection.

63. The use according to claim 59, wherein the liver disease is liver cancer.

64. The use of claim 63, wherein the liver cancer is hepatocellular carcinoma (HCC).

65. The use of any one of claims 59-64, wherein the individual is a mammal.

66. The use of claim 65, wherein the mammal is a human.