RU2810777C2 - Antibodies to il-6 and their fusions and conjugates - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: following is proposed: fusion proteins for binding VEGF and IL-6, each of which contains an antagonistic antibody that binds to IL-6 and a VEGF trap. The VEGF trap contains domain 2 of VEGFR1 and domain 3 of VEGFR2. Conjugates of these fusion proteins are also proposed. Besides, the invention relates to nucleic acids encoding fusion proteins, expression vectors, cells to produce fusion proteins, methods of obtaining and using the said fusion proteins.

EFFECT: invention may find its further application in therapy.

73 cl, 77 dwg, 35 tbl, 27 ex

Description

[0001] The contents of any and all applications for which foreign or domestic priority is claimed in the application information sheet filed with this application are incorporated by reference in accordance with 37 CFR §1.57.

List of sequences

[0002] This application is filed together with a sequence listing in electronic form. This sequence listing is provided as a file called "KDIAK044.txt", created on March 1, 2019, which is 395,430 bytes in size. The complete information contained in the electronic sequence listing is incorporated herein by reference.

REGULATIONS ON THE DEPOSIT OF BIOLOGICAL SAMPLES

[0003] Some embodiments provide anti-IL-6 antibodies, fusions and conjugates thereof, which are deposited in the American Type Culture Collection (ATCC) under the Treaty of Budapest under numbers ____ (date) _____.

[0004] These deposits are made in accordance with the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the instructions thereto (Budapest Treaty). This ensures that the deposited sample is preserved for 30 years from the date of deposit. The deposited design will be made available to the ATCC in accordance with the terms of the Treaty of Budapest and by agreement between the applicant and the ATCC, which will ensure the continued and unrestricted availability to the public of the deposited design upon the issuance of the applicable U.S. patent or upon publication of any U.S. patent application or foreign patent application, whichever , whichever occurs first, and ensures the availability of the deposited sample to persons determined by the United States Patent and Trademark Commissioner to be entitled thereto under 35 USC § 122 and related rules of the Commissioner (including 37 CFR US Acts) § 1.14). The availability of deposited biological material shall not be construed as permission to carry out an invention in violation of rights granted by the competent authority of any government under its patent laws.

TECHNICAL FIELD

[0005] The present invention generally relates to fusion constructs that bind to IL-6 and/or VEGF.

BACKGROUND OF THE ART

[0006] Vascular endothelial growth factor-A (VEGF-A) is a signaling protein that mediates proangiogenic functions such as endothelial cell survival, proliferation, migration, and intercellular permeability. Its activity has been shown to promote the progression of retinal diseases such as choroidal neovascularization (CNV) in age-related macular degeneration (AMD) and diabetic macular edema (DMA). Inhibition of VEGF-A signaling has been shown to be an effective way to stop the progression of retinal neovascular diseases (Ferrara et al., Retina, 2006). Various therapeutic molecules have been developed to inhibit VEGF function. Among these molecules, monoclonal anti-VEGF antibodies such as ranibizumab and bevacizumab have been shown to be safe and effective treatments for pathological angiogenesis. Recently, recombinantly produced VEGFR (vascular endothelial growth factor receptor - VEGF) fusion proteins, such as aflibercept (Eylea) and conbercept (China), which act as decoys for VEGF, are more effective and have a longer lasting effect than their competitors are antibodies.

[0007] Inflammation has been implicated in the pathogenesis of retinal diseases, and anti-inflammatory therapies such as steroids are effective in the treatment of uveitis and diabetic macular edema (DMA). Careful studies of inflammation and infection in the eye have shown that the levels of the proinflammatory cytokine, interleukin-6 (IL-6), are significantly elevated in the aqueous fluid of patients with refractory/chronic uveitis, and inhibition of IL-6 in animal models prevents the occurrence of uveitis. High levels of IL-6 in the intraocular fluid are also found in patients with PVD and retinal vein occlusion. Moreover, chronic inflammatory cells were observed on the surface of Bruch's membrane in eyes with neovascular VJP, and patients with VJP reportedly exhibited elevated serum levels of IL-6. Interestingly, IL-6 was also observed to stimulate impaired angiogenesis. In addition to autoimmune disorders such as rheumatoid arthritis, treatments targeting IL-6 have been shown to effectively treat uveitis and macular edema in uveitis.

BRIEF DESCRIPTION OF THE INVENTION

[0008] In accordance with some aspects, an isolated antagonist antibody that specifically binds IL-6 conjugated to a polymer is provided.

[0009] In accordance with some aspects, an isolated antagonist antibody is provided. The antibody contains the amino acid sequence of the heavy chain variable region constituting the heavy chain in Table 1; and the amino acid sequence of the light chain variable region constituting the light chain in Table 2.

[0010] In accordance with some aspects, an isolated antagonist antibody is provided. The antibody contains: a heavy chain variable region (VH) containing complementarity determining region (CDR) 1 (CDR1) VH, CDR2 VH and CDR3 VH, having an amino acid sequence selected from the group consisting of CDRs in table 1; and a light chain variable region (VL) comprising CDR1 VL, CDR2 VL and CDR3 VL having an amino acid sequence selected from the group consisting of CDRs in Table 2.

[0011] In accordance with some aspects, an isolated antagonist antibody is provided that binds to IL-6. The antibody contains at least one of the following mutations according to EU numbering: L234A, L235A and G237A.

[0012] In accordance with some aspects, an isolated antagonist antibody is provided that binds to IL-6. The specified antibody contains CDR _H 1, which is CDR _H 1 in table 1; CDR _H 2, which is CDR _H 2 in Table 1; CDR _H 3, which is CDR _H 3 in Table 1; CDR _L 1, which is CDR _L 1 in Table 2; CDR _L 2, which is CDR _L 2 in Table 2; CDR _L 3, which is CDR _L 3 in Table 2; at least one of the following mutations (EU numbering): L234A, L235A and G237A; and at least one of the following mutations (EU numbering): Q347C or L443C.

[0013] In accordance with some aspects, there is provided a fusion protein comprising an isolated antagonist antibody; and a fragment of VEGF-binding protein.

[0014] In accordance with some aspects, a conjugate is provided comprising any of the isolated antagonistic antibodies of the present invention and a polymer. The polymer is covalently attached to the antibody.

[0015] In accordance with certain aspects, a conjugate is provided. Said conjugate contains a fusion protein of the present invention and a polymer, said polymer being covalently attached to the fusion protein.

[0016] In accordance with some aspects, an isolated cell line is provided that produces an isolated antagonist antibody or fusion protein.

[0017] In accordance with some aspects, an isolated nucleic acid encoding an isolated antagonist antibody or fusion protein is provided.

[0018] In accordance with certain aspects, a recombinant expression vector is provided. The specified vector contains a nucleic acid encoding any of these constructs proposed according to the present invention.

[0019] In accordance with some aspects, a host cell is provided containing an expression vector of the present invention.

[0020] In accordance with some aspects, a method for producing an IL-6 antagonist antibody or a fusion protein thereof is provided. The method includes: culturing a cell line that recombinantly produces an isolated antagonistic antibody or fusion protein under conditions in which the antibody is produced; and antibody recovery.

[0021] In accordance with certain aspects, a pharmaceutical composition is provided. It contains an isolated antagonistic antibody or fusion protein and/or conjugate according to the present invention and a pharmaceutically acceptable carrier.

[0022] In accordance with some aspects, a method of treating or preventing a disease in a patient in need thereof is provided. The method involves administering to a patient an isolated antagonist antibody or fusion protein and/or conjugate of the present invention.

[0023] In accordance with some aspects, a method of treating or preventing a disease in a patient in need thereof is provided. The method includes identifying a patient with hyperactivity of IL-6 and/or VEGF; and administering to said patient an isolated antagonist antibody or fusion protein and/or conjugate of the present invention.

[0024] In accordance with some aspects, a fusion protein is provided comprising IL-6 VH, IL-6 VL, IL-6 Fc, and a VEGF trap, wherein said VEGF trap is fused to IL-6 in one of the following ways: 1) with N -the end of the heavy chain containing IL-6 VH; or 2) between the hinge region and after the CH1 domain of the heavy chain containing IL-6 VH.

[0025] In accordance with some aspects, an isolated anti-IL-6 antagonist antibody is provided comprising a heavy chain variable region that comprises a heavy chain that has the sequence of at least one of SEQ ID NOs: 7-13, 19-27, 89. 90, 256-262; and a light chain variable region that contains a light chain that has the sequence of at least one of SEQ ID NOs: 91-93, 28-30.

[0026] In accordance with some aspects, an isolated anti-IL-6 antagonist antibody is provided comprising: a heavy chain variable region (VH) comprising 3 complementarity determining regions: CDR1 VH, CDR2 VH and CDR3 VH, having the amino acid sequence of the CDRs listed in SEQ ID NO: 256; and a light chain variable region (VL) comprising CDR1 VL, CDR2 VL and CDR3 VL having an amino acid sequence selected from the group of CDRs listed in SEQ ID NOs: 91-93.

[0027] In accordance with some aspects, an isolated antagonist antibody is provided that binds to IL-6. The antibody contains at least one of the following mutations according to EU numbering: L234A, L235A and G237A.

[0028] In some embodiments, an isolated antagonist antibody that binds to IL-6 is provided and comprises: CDR _H 1, which is CDR _H 1 in SEQ ID NO: 172; CDR _H 2, which is CDR _H 2 in SEQ ID NO: 173; CDR _H 3, which is CDR _H 3 in SEQ ID NO: 174; CDR _L 1, which is CDR _L 1 in SEQ ID NO: 199; CDR _L 2, which is CDR _L 2 in SEQ ID NO: 200; CDR _L 3, which is CDR _L 3 in SEQ ID NO: 201; at least one of the following mutations (EU numbering): L234A, L235A and G237A; and at least one of the following mutations (EU numbering): Q347C or L443C.

[0029] In accordance with some aspects, there is provided a fusion protein comprising: an isolated antagonist antibody from any of those provided by the present invention (including fragments thereof), and a VEGF trap.

[0030] In accordance with some aspects, a conjugate is provided comprising: any of the isolated antagonistic antibodies (including fragments) provided by the present invention; and a polymer, wherein said polymer is covalently attached to the antibody.

[0031] In accordance with some aspects, there is provided a conjugate comprising a fusion protein of the present invention; and a polymer, wherein said polymer is covalently attached to the fusion protein.

[0032] In accordance with some aspects, a dual VEGFR-anti-IL-6 inhibitor is provided. Said dual VEGFR-anti-IL-6 inhibitor comprises a decoy-antibody fusion construct of an anti-IL6 antibody and an anti-VEGF decoy (VEGFR1/2), wherein the dual inhibitor contains at least one point mutation in the VEGFR sequence to reduce protein degradation VEGFR.

[0033] In accordance with some aspects, a protein construct is provided comprising: at least 3 heavy chain CDRs; at least 3 light chain CDRs; VEGF trap sequence; and a linker sequence, each of said sequences being selected from the corresponding sequence in FIGS. 18-25 or from the corresponding sequences in the tables herein (including, for example, 1, 2, 6, 7, 8 and 9).

[0034] In accordance with some aspects, there is provided a fusion protein comprising IL-6 VH; IL-6 VL; IL-6 Fc; and a VEGF trap, wherein the fusion protein modifies the proliferation of human umbilical vein endothelial cells (HUEC). In some embodiments, altering ECHF proliferation inhibits VEGF/IL6-mediated proliferation.

[0035] In accordance with some aspects, a fusion protein is provided comprising a sequence that is at least 80% identical to SEQ ID NO: 263 and at least 80% identical to SEQ ID NO: 117, wherein the fusion protein is further conjugated to polymer.

[0036] In accordance with some aspects, an isolated antagonist antibody is provided that binds to IL-6. The antibody contains at least one of the following mutations according to EU numbering: L234A, L235A and G237A.

[0037] In accordance with some aspects, an isolated antagonist antibody is provided that binds to IL-6, wherein the antibody comprises: CDR _H 1, which is CDR _H 1 in SEQ ID NO: 172; CDR _H 2, which is CDR _H 2 in SEQ ID NO: 173; CDR _H 3, which is CDR _H 3 in SEQ ID NO: 174; CDR _L 1, which is CDR _L 1 in SEQ ID NO: 199; CDR _L 2, which is CDR _L 2 in SEQ ID NO: 200; CDR _L 3, which is CDR _L 3 in SEQ ID NO: 201; at least one of the following mutations (EU numbering): L234A, L235A and G237A; and at least one of the following mutations (EU numbering): Q347C or L443C.

[0038] In some aspects, the dual anti-IL-6 VEGFR inhibitor comprises anti-IL-6 heavy chain variable region sequences selected from SEQ ID NOs: 7-13, 89, 90 and/or 256-262.

[0039] In some aspects, the dual anti-IL-6 VEGFR inhibitor comprises VEGF decoy sequences selected from at least one of SEQ ID NO: 145, 15, 16, or 17.

[0040] In some aspects, the linker sequence is SEQ ID NO: 18.

[0041] In accordance with some aspects, there is provided a heavy chain sequence for an anti-IL-6 molecule selected from at least one of SEQ ID NOs: 19-27 or comprising at least a sequence in one of SEQ ID NOs: 89, 90 , 256-262.

[0042] In accordance with some aspects, a light chain sequence for an anti-IL-6 molecule is provided comprising at least 1, 2 or 3 light chain CDRs from at least one of SEQ ID NOs: 76-84.

[0043] In accordance with some aspects, a heavy chain sequence for an anti-IL-6 molecule is provided comprising at least 1, 2, or 3 heavy chain CDRs from at least one of SEQ ID NOs: 49-75.

[0044] In accordance with some aspects, a dual VEGFR-anti-IL-6 inhibitor is provided comprising a VEGFR-Fc sequence from at least one of SEQ ID NOs: 85-88.

[0045] In accordance with some aspects, there is provided a dual VEGFR-anti-IL-6 inhibitor comprising one or more sequences in any one or more of SEQ ID NO: 7-13, 145, 15-17, 18-84.

[0046] Some embodiments provide a dual anti-IL-6 VEGFR inhibitor comprising an IL-6 VH; IL-6 VL; IL-6 Fc; VEGF trap; and a linker. In some embodiments, the IL-6 VH comprises a sequence from the IL6 VH sequence in any of SEQ ID NOs: 19-27, 31-39, 89, 90, or 256-262. In some embodiments, IL-6 VL comprises a sequence from the IL6 VL sequence in any of SEQ ID NOs: 28-30 or 91-93. In some embodiments, the Fc comprises a sequence from the Fc sequence in any of SEQ ID NOs: 40-48. In some embodiments, the VEGF trap comprises a sequence from the VEGF trap sequence in any of SEQ ID NO: 145, 15, 16, or 17.

[0047] In accordance with some aspects, there is provided a fusion protein comprising IL-6 VH; IL-6 VL; IL-6 Fc; and a VEGF trap, wherein the fusion protein alters the proliferation of ECHRVs. In some embodiments, altering ECHF proliferation inhibits VEGF/IL6-mediated proliferation.

[0048] In accordance with some aspects, a fusion protein is provided comprising a sequence that is at least 80% identical to SEQ ID NO: 263 and at least 80% identical to SEQ ID NO: 117. The fusion protein is further conjugated to a polymer. In some embodiments, the fusion protein is at least 95% identical to SEQ ID NO: 263 and at least 95% identical to SEQ ID NO: 117. In some embodiments, the protein comprises at least a) SEQ ID NO: 172, 173 , 174, 199, 200 and 201 or b) substitution of 1, 2 or 3 amino acids in SEQ ID NO: 172, 173, 174, 199, 200 and/or 201, wherein said substitution is a conservative substitution.

[0049] In accordance with some aspects, an antibody of any one provided herein comprises a) an amino acid sequence of SEQ ID NOs: 169 and 170, or b) an amino acid sequence that is at least 80% identical to SEQ ID NO: 169 and at least 80% identical to SEQ ID NO: 170.

[0050] In accordance with some aspects, a fusion protein as provided herein comprises a) an amino acid sequence of SEQ ID NOs: 169 and 170, or b) an amino acid sequence that is at least 80% identical to SEQ ID NO: 169 and at least at least 80% identical to SEQ ID NO: 170.

[0051] In certain aspects, a conjugate of any of those provided herein comprises a) an amino acid sequence of SEQ ID NOs: 169 and 170, or b) an amino acid sequence that is at least 80% identical to SEQ ID NO: 169 and at least 80% identical to SEQ ID NO: 170.

[0052] In accordance with some aspects, a dual VEGFR-anti-IL-6 inhibitor as provided herein comprises a) an amino acid sequence of SEQ ID NOs: 169 and 170, or b) an amino acid sequence that is at least 80% identical to SEQ ID NO: 169 and is at least 80% identical to SEQ ID NO: 170.

[0053] In accordance with some aspects, a protein as provided herein comprises a) an amino acid sequence of SEQ ID NOs: 169 and 170, or b) an amino acid sequence that is at least 80% identical to SEQ ID NO: 169 and at least 80% identical to SEQ ID NO: 170.

BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 shows the sequence of IL-6.

[0055] Figure 2A shows Connection L.

[0056] Figure 2B shows Connection K.

[0057] Figure 2C shows the synthesis of OG1802 from R3707.

[0058] Figure 2D shows OG1786.

[0059] Figure 2E shows the synthesis of OG1546 from OG1550.

[0060] Figure 2F shows the synthesis of OG1784 from OG1546 and OG1563.

[0061] Figure 2G shows the synthesis of OG1405 from OG1784.

[0062] Figure 2H shows the synthesis of OG1785 from OG1405.

[0063] Figure 2I shows the synthesis of OG1786 from OG1785.

[0064] Figure 2J shows OG1801.

[0065] FIG. 2K shows OG1802.

[0066] Figure 2L shows Compound E.

[0067] Figure 3 shows a flowchart for antibody selection and optimization.

[0068] Figure 4 presents ELISA data demonstrating that an anti-IL-6 monoclonal antibody (mAb) binds to IL-6 but not the IL-6/IL-6R complex, and that the anti-IL-6 mAb binds to IL-6. 6 inhibits the formation of the IL-6/IL-6R complex.

[0069] Figure 5 shows some embodiments of the variable regions of the heavy and light chain of an antibody (Ab, AT) to IL-6. CDR implementation options are shown in the boxed areas. These sequences can also be used in an anti-IL-6-VEGF decoy Ab fusion construct.

[0070] Figure 6 shows some embodiments of an IL-6-VEGF decoy fusion protein. The VEGF trap domains are located either at the N terminus immediately preceding the variable domain (left) or are located between the Fab region and the hinge region of the antibody (right).

[0071] Figure 7 provides sensorgrams and a table that demonstrate that the dual VEGFR inhibitor molecules anti-IL-6 and anti-IL-6-VEGFR bind with similar affinity to VEGF-A as the anti-VEGF antibody, OG1950, and Eylea . Thus, the position of the VEGF decoy does not change its affinity for the target.

[0072] Figure 8 shows sensorgrams of independent and combined binding of VEGF-A and IL-6 with dual inhibitors. IL-6 sensograms for mixed targets were compared with the theoretical curve (sum of individual IL-6 and VEGF-A sensograms). The results show an overlap of the theoretical and experimental curves, which qualitatively indicates that both targets can bind to the dual inhibitor molecule and do not affect each other's binding.

[0073] Figure 9 shows results at OD450 nm from various IL-6 ELISA assays. In the bridging ELISA (top), both dual inhibitors bridged btVEGF to IL-6, indicating that both configurations can bind to both targets. EC50 of VEGF trap-anti-IL-6 constructs = 0.079 nM and anti-IL-6-VEGF trap = 0.026 nM. Eylea and anti-IL-6 served as negative controls. Figure 9 also shows ELISA results for the IL-6/IL-6R complex (middle). Dual inhibitors and anti-IL-6 inhibited the formation of the IL-6/IL-6R complex to an equal extent. IC50 values: anti-IL-6=0.36 nM, VEGF-anti-IL-6 trap construct=0.47 nM and anti-IL-6-VEGF-trap construct=0.32 nM. Eylea served as a negative control. Figure 9 also shows the results of the competitive ELISA for VEGF/VEGFR (bottom). The dual inhibitors, Eylea and OG1950, inhibited VEGF binding to VEGFR to varying degrees. Eylea and the anti-IL-6-VEGF-trap construct were comparable (4.24 nM vs. 4.53 nM), while the VEGF-anti-IL-6 trap construct was ~2-fold more potent (1.74 nM) and OG1950 showed maximal inhibition. superior to other inhibitors (1.55 nM).

[0074] Figure 10 shows some embodiments of a method for producing an antibody conjugate (which can also be used for an AT-decoy or decoy-AT conjugate). Although depicted as an antibody, it will be apparent to one skilled in the art in the context of the present invention that the AT depiction in FIG. 10 can be replaced by a trap fusion construct (shown in FIG. 6). For simplicity, the generic antibody depicted in this figure represents both antibody and fusion variants in the context of a trap fusion construct (unless, of course, it is already depicted as a fusion construct).

[0075] Figures 11A-11B show sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) bands for SeeBlue®Plus2 standard, anti-IL-6, anti-IL-6-VEGFR construct, and VEGFR-anti- IL-6. 11C depicts a VEGFR-anti-IL6 conjugated construct, which is a fusion of anti-VEGF (VEGFR1/2) and anti-IL-6 conjugated to a phosphorylcholine polymer.

[0076] Figure 12 shows the results of transfer to a PVDF membrane, N-terminal Edman sequencing, which demonstrate that the digested products contain the same N-terminal sequence (LTHRQT), indicating that the cleavage site is located in the VEGF trap region.

[0077] Figures 13A-B show SDS-PAGE bands for the SeeBlue®Plus2 standard and 19 VEGF trap designs.

[0078] FIGS. 13C-13E provide sequence listings for: VEGF_trap_option_1, VEGF_trap_option_2, and VEGF_trap_option_3.

[0079] Figures 13F-13G show SDS-PAGE bands for the SeeBlue®Plus2 standard and 4 VEGF trap variants, including VEGFR_variant_3 containing the T94I and H95I double point mutations.

[0080] Figures 14A-14F present the results of the Biacore assay and VEGF-A affinity measurements.

[0081] Figure 15 shows a reporter assay of VEGF-stimulated VEGFR in a cellular system.

[0082] FIG. 16A presents an assay for inhibition of VEGF/IL6-mediated tube formation by human umbilical vein endothelial cells ("HVECs").

[0083] Figures 16B-16C show statistics from tube formation assays with various parameters.

[0084] FIG. 17 shows the results of the ECHR proliferation assay.

[0085] Figure 18 shows variant heavy chain variable region anti-IL-6 sequences. CDRs are underlined.

[0086] Figure 19 shows various variants of VEGF trap sequences. The region that varies among the specified sequences is highlighted in bold and underlined.

[0087] Figure 20 shows some variants of the linker (GS) sequence. The linker may be present as a Gly-Gly-Gly-Gly-Ser (GS) linker with a double repeat.

[0088] Figure 21 shows some heavy chain sequence variations for anti-IL-6 molecules. CDRs are underlined. FIG. 21 includes both FIG. 21A and FIG. 21B, which illustrate various CDR variants in the indicated sequences.

[0089] Figure 22 shows some variant light chain sequences for anti-IL-6 molecules. CDRs are underlined. FIG. 22 includes both FIG. 22A and FIG. 22B, which illustrate various CDR variants in the indicated sequences.

[0090] Figure 23 shows some variant heavy chain sequences for anti-IL-6 molecules. CDRs are underlined. FIG. 23 includes both FIG. 23A and FIG. 23B, which illustrate various CDR variants in the indicated sequences.

[0091] FIGS. 24A-24B show some variations of the CDR combinations of FIGS. 21-23.

[0092] Figure 25 shows some variants of the VEGFR-Fc sequence. The region that varies among the specified sequences is highlighted in bold and underlined.

[0093] Figures 26A-26C present binding affinity data.

[0094] Figure 27 shows the sequences of some variant VEGFR anti-IL6 sequences. CDRs (defined according to Kabat) are underlined. Areas colored gray represent VEGFR constructs. The linker region is highlighted in bold. Mutations L234A, L235A, G237A and L443C (EU numbering) are underlined with a double line. Each of these regions may be replaced by other appropriate regions presented herein (eg, alternative linkers or CDRs, etc.).

[0095] Figure 28 shows SDS-PAGE of the reduction reaction products of the VEGFR-antiIL6 construct (Cys - decapping). Lanes are as follows: 1. VEGFR-antiIL6; 2. VEGFR-antiIL6 - completely reduced (TCEP); 3. Novex sharp pre-stained protein standard (Novex pre-stained protein standard); 4. VEGFR-antiIL6+30x TCEP, starting point; 5. VEGFR-antiIL6+30x TCEP, after 30 minutes; 6. VEGFR-antiIL6+30x TCEP, after 60 minutes; 7. VEGFR-anti-IL6 treated with TCEP, buffer replaced; 8. VEGFR-antiIL6+15x dHAA, starting point; 9. VEGFR-antiIL6+15x dHAA, after 30 minutes; 10. VEGFR-antiIL6+15x dHAA, after 60 minutes; 11. VEGFR-antiIL6, unblocked; 12. VEGFR-antiIL6, unblocked, completely reduced (TCEP); Gel: NuPAGE Bis-Tris 4-12% Protein Amount: 4 µg/lane; 30x TCEP=30 times molar excess of TCEP, 15x dHAA=15 times molar excess of dHAA.

[0096] Figure 29 shows SDS-PAGE for cation exchange chromatography (CEX) of the VEGFR-anti-IL6-OG1802 conjugate. Non-reducing gel: Bis-Tris NuPAGE 4-12%. Buffer A: 20 mM sodium acetate, pH 5.5. Buffer B: 20 mM sodium acetate, pH 5.5, 500 mM NaCl. Lanes are as follows: 1. VEGFR-antiIL6; 2. VEGFR-antiIL6-OG1802 (download); 3. Novex sharp pre-stained protein standard (Novex pre-stained protein standard); 4. Flow fraction; 5. Chase; 6. 30% buffer B - aliquot 1; 7. 30% buffer B - aliquot 2; 8. 30% buffer B - aliquot 3; 9. 30% buffer B - aliquot; 10. 40% buffer B - aliquot 1; 11. 40% buffer B - aliquot 2; 12. 40% buffer B - aliquot 3; 13. 40% buffer B - aliquot 4; 14. 60% buffer B - aliquot 1; 15. 60% buffer B - aliquot 2; 16. 60% buffer B - aliquot 3; 17. 100% buffer B - strip. Lanes 6-13 show conjugated protein material, the conjugate cannot penetrate the gel due to its large size. Lanes 14-17 show a mixture of proteins, possibly containing aggregated, conjugated and unconjugated protein material.

[0097] Figure 30 shows SDS-PAGE of a protein-polymer conjugate relative to unconjugated protein material. Gel analysis shows a ratio of 57% heavy chain band intensity and 96% light chain band intensity when comparing the bioconjugate (lane 3) to the VEGFR-antiIL6 reference standard (lane 2), indicating the presence of one OG1802 polymer per VEGFR-antiIL6 molecule. The restorative gel is: Bis-Tris NuPAGE 4-12%. The restorative gel is: Bis-Tris NuPAGE 4-12%.

[0098] Figure 31 shows size exclusion chromatography with multi-angle light scattering (SEC-MALS) for the VEGFR-anti-IL6-OG1802 construct. The molecular weight of the VEGFR-anti-IL6 conjugate was determined using integrated size exclusion chromatography (Shodex-SB806M-HQ) and light scattering (MALS). Top graph. The chromatogram shows the presence of a single elution peak. The absence of additional peaks and shoulders suggests the absence of aggregates and decay material after conjugation and subsequent separation steps by CEX. Bottom graph. Selected peak protein conjugate analysis demonstrated an experimentally measured average molecular weight (Mw) of 983 kDa for the VEGFR-antiIL6-OG1802 bioconjugate. This value results from the conjugation of one VEGFR-antiIL6 molecule (MW ~ 189 kDa) and one OG1802 polymer (MW ~ 794 kDa).

[0099] Figure 32 shows the results of CEX chromatography of the VEGFR-anti-IL6 construct. Gel: is 8-16% Tris-Glycine Novex (reducing conditions), M=SeeBlue®Plus2 standard. Lanes D12-F11 correspond to samples aliquoted at different concentrations of buffer B, as shown in the chromatogram. Intact (I) and cleaved (C) heavy chains are shown. In a Porus XS column, buffer A is 20 mM sodium phosphate, pH 6, and buffer B is 20 mM sodium phosphate, pH 6, 1 M NaCl.

[0100] Figure 33 shows hydrophobic interaction chromatography (HIC) results for the VEGFR-anti-IL6 construct. Gel: is 8-16% Tris-Glycine Novex/reducing conditions, M=SeeBlue®Plus2 standard. L=VEGFR-antiIL6 construct, intact and digested mixture (loading). The remaining lanes correspond to samples aliquoted at different concentrations of buffer B, as shown in the chromatogram. Intact (I) and cleaved (C) heavy chains are shown. In the Hi Trap Butly HP column, buffer A is: 20 mM sodium phosphate pH 6, 1 M ammonium sulfate. Buffer B is: 20 mM sodium phosphate, pH 6.

[0101] Figure 34 shows the results of a competitive ELISA for VEGF/VEGFR.

[0102] Figure 35 shows ELISA results for the IL6/IL6R complex.

[0103] Figure 36 shows the results of a reporter assay for VEGF-stimulated VEGFR in a cellular system.

[0104] Figure 37 shows lipopolysaccharide-stimulated tube formation in ECHF.

[0105] Figure 38 shows lipopolysaccharide-stimulated tube formation in ECHF.

[0106] Figure 39 shows lipopolysaccharide-stimulated tube formation in ECHF.

[0107] FIG. 40A shows inhibition of VEGF/IL6-mediated proliferation by HEC.

[0108] FIG. 40B shows the inhibition of ECHF proliferation with increasing number of cells per well.

DETAILED DESCRIPTION OF THE INVENTION

[0109] To directly reduce the concomitant inflammation and impaired angiogenesis that drive the pathogenesis of retinal neovascular pathologies, the present invention provides engineered molecules that simultaneously block the functions of the proinflammatory cytokine IL-6 and the proangiogenic signaling protein VEGF. These molecules consist of (1) a monoclonal anti-IL-6 antibody fused to (2) two VEGF-binding domains of VEGF receptors (VEGFRs). The anti-IL-6 fragment specifically binds IL-6 and inhibits its interaction with the IL-6 receptor (IL-6R). The VEGF decoy fragment contains a fusion of two VEGF-binding domains (VEGFR1 domain 2, VEGFR2 domain 3) that act as a VEGF decoy and prevent VEGF from binding to VEGF receptors. In addition, in some embodiments, each of these dual inhibitor molecules is equipped with an unpaired cysteine at the C-terminus, which can be conjugated to a phosphorylcholine-based biopolymer to increase the half-life.

[0110] The various embodiments presented herein utilize, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology that are within the skill of the art. Such methods are fully described in the literature, for example, in Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney, ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995).

[0111] It should be understood that the following terms, unless otherwise indicated, have the following meanings: the term “isolated molecule” refers to a molecule (wherein said molecule is, for example, a polypeptide, polynucleotide or antibody) that, by virtue of its origin or source of production (1) is not associated with the naturally associated components that accompany it in its native state, (2) is substantially free from other molecules from the same source, e.g., species, cell in which it is expressed, library, etc. .d., (3) is expressed by a cell of another species, or (4) does not occur in nature. Thus, a molecule chemically synthesized or expressed in a cellular system different from the system from which it occurs in nature will be "separated" from its naturally associated components. The molecule can also be substantially freed from its naturally associated components by isolation using purification techniques well known in the art. Analysis of the purity or homogeneity of molecules can be accomplished by a number of methods well known in the art. For example, the purity of a polypeptide sample can be analyzed using polyacrylamide gel electrophoresis and gel staining to visualize the polypeptide using methods well known in the art. For some purposes, higher resolution can be achieved by using HPLC or other purification methods well known in the art.

[0112] As used herein, unless otherwise indicated, the term “IL-6” or “IL6” refers to human IL-6. In some embodiments, other forms of IL-6 are provided and will be designated by specific reference to other organisms, such as dog, cat, horse, and bovine. One representative human IL-6 has UniProt accession number P05231.

[0113] Anti-IL-6 antibodies or other biologics described herein are typically presented in isolated form. This means that the antibody is generally at least 50% by weight pure and free of undesirable proteins and other contaminants resulting from its preparation or purification, but does not exclude the possibility that the antibody is combined with a pharmaceutically acceptable excipient intended to facilitate its use. Sometimes the antibodies are at least 60, 70, 80, 90, 95 or 99 wt.% pure and free of unwanted proteins and contaminants as a result of preparation or purification. Often the antibody (or antibody conjugate) is the predominant macromolecule species that remains after its purification.

[0114] An “antibody” is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term includes not only intact polyclonal or monoclonal antibodies, but also, unless otherwise specified, any antigen binding portion thereof that competes with the intact antibody for specific binding, fusion proteins containing an antigen binding portion, and any other modified configuration of the immunoglobulin molecule , which contains the antigen recognition site. Antigen-binding moieties include, for example, Fab, Fab', F(ab') ₂ , Fd, Fv, domain antibodies (dAbs, e.g. shark and camelid antibodies), fragments containing complementarity determining regions (CDRs), antibodies, being single chain variable fragments (scFv), maxibodies, minibodies, intrabodies, diabodies, tribodies, tetrabodies, v-NAR and bis-scFvs, and polypeptides that contain at least a portion of an immunoglobulin sufficient to impart specific antigen binding properties to the polypeptide. An antibody includes an antibody of any class, such as IgG, IgA or IgM (or a subclass thereof), and the antibody need not be of any particular class. Depending on the amino acid sequence of the constant region of the heavy chains of antibodies, immunoglobulins can be classified into different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into subclasses (isotypes), for example, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . The heavy chain constant regions that correspond to different classes of immunoglobulins are designated alpha, delta, epsilon, gamma and mu, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known.

[0115] The term “variable region” of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. As is known in the art, each heavy and light chain variable region consists of four framework regions (FRs) connected by three complementarity determining regions (CDRs), also known as hypervariable regions, and contributes to the formation of the antigen binding site of antibodies. If variants of the subject variable region are desired, in particular those containing substitutions of amino acid residues outside the CDR region (i.e., in the framework region), the corresponding amino acid substitution, preferably a conservative amino acid substitution, can be identified by comparison of the subject variable region with the variable regions of other antibodies, which contain CDR1 and CDR2 sequences, in the same canonical class as the variable region in question (Chothia and Lesk, J Mol Biol 196(4): 901-917, 1987).

[0116] In some embodiments, precise determination of the CDR and identification of residues constituting the antibody binding site is accomplished by determining the structure of the antibody and/or determining the structure of the antibody-ligand complex. In some embodiments, this can be accomplished by any of various methods known to one skilled in the art, such as x-ray crystallography. In some implementations, various analysis techniques may be used to identify or approximate CDR regions. In some implementations, various analysis techniques may be used to identify or approximate CDR regions. Examples of such methods include, but are not limited to, the Kabat determination, the Chotia determination, the IMGT approach (Lefranc et al., 2003) Dev Comp Immunol. 27:55-77), computer programs such as Paratome (Kunik et al., 2012, Nucl Acids Res. W521-4), AbM determination and conformational determination.

[0117] The Kabat definition is a standard for numbering residues in an antibody and is typically used to identify CDR regions. See, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8. The Chotia definition is similar to the Kabat definition, but the Chotia definition takes into account the positions of certain structural loop regions. See, for example, Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83. AbM determination uses a comprehensive suite of computer programs developed by the Oxford Molecular Group that model the structure of the antibody. See, for example, Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; “AbM™, A Computer Program for Modeling Variable Regions of Antibodies,” Oxford, UK; Oxford Molecular, Ltd. The AbM definition allows the tertiary structure of an antibody to be modeled from a primary sequence using a combination of knowledge bases and ab initio methods such as those described by Samudrala et al., 1999, “Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach,” in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198. Contact determination is based on analysis of available complex crystal structures. See, for example, MacCallum et al., 1996, J. Mol. Biol., 5:732-45. According to another approach, referred to herein as "conformational determination" of CDRs, CDR positions can be defined as residues that make an enthalpic contribution to antigen binding. See, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166. Other definitions of CDR boundaries may not strictly follow one of the above approaches, but will nevertheless overlap with at least part of the CDR according to Kabat, although they may be shortened or lengthened due to prediction or experimental evidence that particular residues or groups of residues do not have a significant effect on antigen binding. As used herein, the term “CDR” may refer to CDR regions defined by any approach known in the art, including combinations of approaches. The methods used according to the present invention can use CDRs determined in accordance with any of these approaches. For any particular embodiment including more than one CDR, the CDRs may be defined according to any of the Kabat, Chotia, extended, IMGT, Paratome, AbM, and/or conformational definitions, or a combination of any of the above definitions.

[0118] As is known in the art, the term “constant region” of an antibody refers to the light chain constant region of an antibody or the heavy chain constant region of an antibody, either alone or in combination.

[0119] As used herein, the term “monoclonal antibody” refers to an antibody derived from a population of essentially homogeneous antibodies, i.e. the individual antibodies composing this population are identical except for possible naturally occurring mutations that may be present in minute quantities. Monoclonal antibodies are highly specific, being directed against a single antigenic determinant. Moreover, unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. The designation "monoclonal" indicates the nature of the antibody as an antibody obtained from an essentially homogeneous population of antibodies, and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies for use in the present invention can be produced by a hybridoma-based method first described by Kohler and Milstein, 1975, Nature 256:495, or can be produced by recombinant DNA-based methods such as those described in US Pat. No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries obtained using techniques described, for example, in McCafferty et al., 1990, Nature 348:552-554. As used herein, the term “humanized” antibody refers to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding antibody subsequences) that contain minimal sequence derived from non-human immunoglobulin. Preferably, the humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the CDR of the recipient antibody are replaced by residues from the CDR of a non-human species (donor antibody), such as mouse, rat or rabbit, having the desired specificity, affinity and potency . The humanized antibody may contain residues not found in either the recipient antibody or the input CDR or framework sequences, but are included to further improve and optimize the effectiveness of the antibody.

[0120] A “human antibody” is an antibody that contains an amino acid sequence that corresponds to the sequence of an antibody produced by a human and/or that is produced using any of the methods for producing human antibodies described herein. This definition of a human antibody specifically excludes a humanized antibody containing non-human antigen-binding moieties.

[0121] The term "chimeric antibody" refers to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from from a human antibody. The term "epitope" refers to that part of the molecule that can be recognized and bound by an antibody in one or more antigen-binding regions of the specified antibody. Epitopes often consist of a surface group of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structure characteristics as well as specific charge characteristics. In some embodiments, the epitope may be a protein epitope. Protein epitopes may be linear or conformational. In a linear epitope, all points of interaction between the protein and the interacting molecule (such as an antibody) are located linearly along the primary amino acid sequence of the protein. A "non-linear epitope" or "conformational epitope" contains non-contiguous polypeptides (or amino acids) within an antigenic protein to which an antibody specific for that epitope binds. As used herein, the term “antigenic epitope” is defined as that portion of an antigen to which an antibody can specifically bind, as determined by any method well known in the art, such as conventional immunoassays. Once the desired epitope on an antigen has been determined, antibodies to that epitope can be produced, for example, using the techniques described herein. Alternatively, during the discovery process, production and characterization of antibodies can provide information about desired epitopes. Based on this information, antibodies can then be competitively screened for binding to the same epitope. An approach to achieve this is to conduct competition and cross-competition studies to detect antibodies that compete or cross-compete with each other for binding to IL-6, for example, antibodies competing for binding to the specified antigen.

[0122] As used herein, the term “compete” when applied to an antibody means that a first antibody or antigen-binding portion thereof binds to an epitope in a manner sufficiently similar to binding of a second antibody or antigen-binding portion thereof such that the result of binding of the first antibody to its recognized epitope is detectable is reduced in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. Alternatively, there may also be a detectable decrease in the binding of the second antibody to the epitope in the presence of the first antibody. That is, the first antibody can inhibit the binding of the second antibody to an epitope without the second antibody inhibiting the binding of the first antibody to the corresponding epitope. However, when each antibody detectably inhibits the binding of another antibody to its recognized epitope or ligand, whether to the same, greater or lesser extent, the antibodies are said to "cross-compete" with each other for binding to the corresponding epitope(s). . The present invention includes both competitive and cross-competitive antibodies. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof), one skilled in the art will appreciate from the teachings presented herein that such competitive and/or or cross-competing antibodies are included and may be suitable for the methods described herein.

[0123] As used herein, an antibody “interacts with” IL-6 when the equilibrium dissociation constant is equal to or less than 20 nM, preferably less than about 6 nM, more preferably less than about 1 nM, most preferably less than about 0.75 nM. In some embodiments, the antibody has an affinity of 400 to 800 pM, such as 450-700 or 500-600 pM.

[0124] an IL-6 antagonist antibody includes antibodies that block, antagonize, inhibit or reduce (to any extent, including substantially) the biological activity of IL-6, such as binding to IL-6R, binding of the IL-6 complex 6/IL-6R with gp130, phosphorylation and activation of Stat3, cell proliferation and stimulation of IL-6-mediated inflammatory or proangiogenic pathways. For the purpose of the present invention, it is understood that the term "antagonistic antibody to IL-6" includes all previously defined terms, names and functional states and characteristics whereby IL-6 itself, the biological activity of IL-6 or the consequences of said biological activity are substantially eliminated , are reduced or neutralized to any significant extent. In some embodiments, the anti-IL-6 antagonist antibody binds IL-6. The present description provides examples of IL-6 antagonist antibodies.

[0125] An antibody that “preferentially binds” or “specifically binds” (interchangeable herein) to an epitope is a term well known in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or binds more often and/or faster and/or for a longer duration and/or with greater affinity to a particular cell or substance than to alternative cells or substances. substances. An antibody "specifically binds" or "preferentially binds" to a target if it binds with greater affinity and/or avidity and/or more readily and/or for a longer duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to an IL-6 epitope is an antibody that binds that epitope with greater affinity and/or avidity, and/or more readily, and/or for a longer duration than it binds other IL epitopes -6 or non-IL-6 epitopes. It is also clear from this definition that, for example, an antibody (or fragment or epitope) that specifically or preferentially binds to a first target may specifically or preferentially bind to a second target, or may not specifically or preferentially bind to a second target. Accordingly, “specific binding” or “preferential binding” does not necessarily require (although it may include) exclusive binding. Typically, but not necessarily, an indication of binding means a preferred binding.

[0126] As used herein, the term “substantially pure” refers to a substance that is at least 50% pure (i.e., free of contaminants), more preferably at least 90% pure, more preferably at least at least 95% pure, more preferably at least 98% pure, and most preferably at least 99% pure.

[0127] The term “host cell” includes a single cell or cell culture that can be or has been the recipient of the vector(s) for incorporating polynucleotide insertions. Host cells include the progeny of a single host cell, and these progeny may not necessarily be completely identical (in morphology or complementary genomic DNA sequence) to the original parent cell due to natural, accidental, or intentional mutation. The host cell includes cells transfected in vivo with the polynucleotide(s) of the present invention.

[0128] As is known in the art, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region of a human IgG heavy chain is generally defined to extend from the amino acid residue at position Cys226 or Pro230 to the carboxyl terminus. The numbering of residues in the Fc region is EU numbering, as in Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of immunoglobulins typically contains two constant domains, CH2 and CH3. As is known in the art, the Fc region may be present in the form of a dimer or monomer.

[0129] In the art, the terms "Fc receptor" and "FcR" describe a receptor that binds to the Fc region of an antibody. A preferred FcR is a human FcR with a native sequence. Moreover, a preferred FcR is an FcR that binds an IgG antibody (receptor gamma), and includes receptors of the FcγRI, FcγRII and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which contain similar amino acid sequences that differ mainly in their cytoplasmic domains. FcRs are reviewed in Ravetch and Kinet, 1991, Ann. Rev. Immunol., 9:457-92; Capel et al., 1994, Immunomethods, 4:25-34; and de Haas et al., 1995, J. Lab. Clin. Med., 126:330-41. The term "FcR" also includes the neonatal receptor, FcRn, responsible for the transfer of maternal IgG to the fetus (Guyer et al., 1976, J. Immunol., 117:587; and Kim et al., 1994, J. Immunol., 24:249 ).

[0130] A “functional Fc region” has at least one native sequence Fc region effector function. Typical “effector functions” include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity; phagocytosis; down-regulation of cell surface receptors (eg B-cell receptor), etc. Such effector functions typically require the combination of an Fc region with a binding domain (eg, an antibody variable domain) and can be assessed using various assays known in the art for assessing such antibody effector functions.

[0131] A “native sequence Fc region” contains an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. A “variant Fc region” contains an amino acid sequence that differs from the amino acid sequence of the native sequence Fc region by at least one amino acid modification, but nevertheless retains at least one effector function of the native sequence Fc region. Preferably, the variant Fc region contains at least one amino acid substitution relative to the native sequence Fc region or the Fc region of the parent polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in the Fc region with the native sequence or in the Fc region of the original polypeptide. The variant Fc region of the present invention preferably has at least about 80% sequence identity to the Fc region with the native sequence and/or the Fc region of the parent polypeptide, and most preferably at least about 90% sequence identity to it , more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity thereto.

[0132] As used herein, “treatment” is an approach to obtain beneficial or desired clinical results.

[0133] As used herein, “disorders associated with IL-6 and/or VEGF” include, for example, ocular disorders and systemic disorders. Eye disorders include eye disorders such as ocular inflammatory disease scleritis, nonproliferative diabetic retinopathy, proliferative diabetic retinopathy, diabetic macular edema, preventing diabetic macular edema, preventing proliferative diabetic retinopathy, wet age-related macular degeneration, preventing wet age-related degeneration macular degeneration, dry age-related macular degeneration, venous, arterial, or other occlusion of the blood vessels of the eye or retina with or without retinal edema, anterior and posterior uveitis, macular edema with uveitis, and intraocular tumors. IL-6-related disorders also include disorders in which increased levels of IL-6 activity are observed due to the interaction of IL-6 with IL-6R or soluble IL-6R (sIL-6R). In some embodiments, any one or more fusion proteins provided by the present invention and/or any one or more conjugates provided by the present invention can be used to treat or prevent any one or more disorders associated with IL-6 and/or VEGF. In some embodiments, the disorders include systemic diseases affecting the eye, such as Graves' disease or neuromyelitis optica, or systemic diseases not affecting the eye, such as multiple sclerosis, rheumatoid arthritis. In some embodiments, the disorders include post-CAR cytokine release syndrome. T or similar immuno-oncology drugs.

[0134] In addition, anti-IL-6 molecules suppress the induction of IL-6 expression observed after treatment with anti-PD-1/PD-L1 molecules (Tsukamoto et al, Cancer Res; 2018 78(17); 5011- 22). It has also been shown that blockade of VEGF signaling may improve the effectiveness of anti-PD-L1 treatment (Allen et al, Sci Transl Med 2017 April 12: 9(385)). Thus, these dual inhibitors can be used in combination with PD-1/PDL-1 modulators and/or other immune checkpoint inhibitors for synergistic cancer treatment. Other disorders may include cerebral edema in glioblastoma, in which anti-IL6 therapy may show beneficial effects in addition to anti-VEGF treatment. Other disorders include those associated with solid tumors.

[0135] As used herein, the term “improvement” means a reduction or alleviation of one or more symptoms compared to the absence of administration of an anti-IL-6 antibody, an anti-IL-6 antibody-VEGF decoy fusion, an anti-IL-6 antibody conjugate, and/or a conjugate anti-IL-6-VEGF decoy fusion construct. The term "improvement" also includes shortening or reducing the duration of a symptom.

[0136] As used herein, the term “VEGF decoy” or similar term refers to VEGF binding domains (VEGFR1 domain 2, VEGFR2 domain 3). This fragment allows the protein to act as a VEGF decoy and prevent VEGF from binding to VEGF receptors expressed by cells. An example of this sequence can be found in Table 10. In some embodiments, the VEGF trap contains only VEGFR1 domain 2, VEGFR2 domain 3. Various variants of decoy proteins are known in the art and can be found, for example, in US Publication No. 20150376271, the entire contents of which with respect to various variants of VEGF decoys (which are VEGFR proteins or fragments thereof) and fusion constructs thereof are included herein. via link. In some embodiments, the term “VEGF decoy” or similar term refers to the full-length extracellular region, or any portion thereof, or a combination of portions thereof, from various VEGF receptors that can antagonize signal transduction between at least one VEGF and VEGFR.

[0137] As used herein, the terms “anti-IL-6-VEGF Trap fusion”, “anti-IL-6-VEGF Trap”, “anti-IL-6-VEGF Trap”, “anti-IL-6-VEGF Trap” , "VEGFR-anti-IL6", "VEGFR-anti-IL-6", "VEGF trap-anti-IL6 antibody fusion (TAF)", "VEGF-IL6 trap", "VEGFR IL-6", "IL6-VEGFR" or a similar term, or vice versa terms (for example, “VEGF-trap anti-IL-6 antibody,” “VEGF-trap anti-IL-6 antibody fusion construct,” etc.) mean the fusion of an IL-6 antibody and a VEGF trap. The options are presented in Fig.6. When used in general, the order of the two terms can be reversed. When used in a specific case, the order of the two terms refers to the relative position of the components in the design. The term “TA-trap”, “IL-6 VEGF trap AT”, “IL-6-VEGF trap AT” or “IL-6 trap AT”, or “anti-IL-6 VEGF trap”, “trap” -AT”, “anti-IL-6-VEGFR”, “anti-IL6-VEGFR” or other similar term or terms to the contrary (e.g., “VEGF-Trap Anti-IL-6 Ab”, “VEGF-Trap Anti-IL-6 Antibody Fusion” etc.) means the arrangement of the AT fused to the appropriate domains of the VEGF-binding protein in such a way as to obtain a VEGF trap. As noted above, this region of the VEGF binding protein is a region that prevents VEGF from binding to VEGF receptors. As described herein, the arrangement (order) of the trap and antibody portions can be varied. Thus, unless expressly stated otherwise or the context indicates otherwise, terms used herein in relation to AT-trap (or IL-6/VEGF trap, etc.) fusion constructs mean all antibody arrangements described. and traps. Thus, unless otherwise noted, the term AT-trap (or Il-6/VEGF trap, etc.) means the left option in FIG. 6 and the right option in FIG. 6, and both options in FIG. 6. Thus, this general expression describes all three options for convenience. If a particular orientation is indicated, it may be indicated, for example, by indicating that the “location” may be one of: trap-AT, trap-IL-6 AT, VEGF-trap anti-IL-6 AT, VEGF-trap anti-IL6 AT. Likewise, it will be apparent that in the context of some of the examples herein, the particular orientation or arrangement of molecules indicated in the context of the examples is indicated. Both arrangements (alternative and combined) are explicitly provided for throughout the description of the fusion proteins proposed according to the present invention. In addition, due to the arrangement, it is apparent that the term "IL-6 Ab" when used in the context of a fusion protein includes both the option where the antibody is contiguous, Figure 6, left side, and the option where the decoy is located " within the AT (Fig. 6, right side). Again, the term "Ab" or "antibody" when used in the context of a fusion protein (or other similar term) includes all three options (left side of FIG. 6, right side of FIG. 6, and both) unless otherwise noted . In some embodiments, the VEGF decoy is fused to IL-6 in one of the following ways: to the N-terminus of a heavy chain containing IL-6 VH; or between the hinge region and after the CH1 domain of the heavy chain containing VH to IL-6. There is no distinction between the designations "Ab", "antibody", "anti" or other similar term when used in a name to refer to an antibody or fragment thereof. There is no distinction between the designations "Il-6" or "IL6" or "IL-6". As used herein, when referring to an IL-6 fusion construct, the terms “VEGF”, “VEGFR”, “VEGF Trap”, “VEGFR Trap” are used interchangeably. These terms may have different meanings when used apart from their location in the IL-6 fusion, which will depend on the context of the term in question.

[0138] As used herein, the term “biopolymer” means that the polymer is associated with a protein of interest. The term may also be defined as a “conjugated” form of the protein. This can be done for all proteins described herein. Thus, for all such anti-IL-6 Abs and anti-IL-6 antibody-VEGF decoy constructs provided by the present invention, biopolymers of the anti-IL-6 Ab and biopolymers of the anti-IL-6 antibody-VEGF decoy construct are provided. In addition to this, VEGF trap biopolymers are also proposed.

[0139] As used herein, the term “antagonistic antibody” means an antibody that blocks one or more functions or activities of a molecule to which the antibody binds.

[0140] As used herein, the term “effective dose” or “effective amount” of a drug, compound or pharmaceutical composition is an amount sufficient to provide any one or more beneficial or desired results. In more specific aspects, the effective amount prevents, alleviates or eliminates symptoms of a disease and/or increases the life expectancy of the subject being treated. When used prophylactically, beneficial or desired results include eliminating or reducing the risk, reducing the severity or delaying the onset of disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological manifestations present during the development of the disease. In the case of therapeutic use, beneficial or desired results include clinical results such as a reduction in one or more symptoms of a disease such as, for example, ADHD, including, for example, without limitation, the dry form of ADHD and the wet form of ADHD, a reduction in the dose of other drugs needed for the treatment of the specified disease, enhancing the effect of another drug and/or slowing down the progression of VAD in patients. An effective dose can be administered in one or more administrations. For purposes of the present invention, an effective dose of a drug, compound or pharmaceutical composition is an amount sufficient to provide prophylactic or therapeutic treatment, either directly or indirectly. As understood in a clinical context, an effective dose of a drug, compound or pharmaceutical composition may or may not be achieved in combination with another drug, compound or pharmaceutical composition. Thus, an “effective dose” may be considered in the context of administering one or more therapeutic agents, and may be considered administering a single agent in an effective amount if, in combination with one or more other agents, the desired result can be or is achieved.

[0141] Anti-IL-6 antibodies are administered in an effective regimen, meaning a dose, route of administration, and frequency of administration, that delays the onset, reduces the severity, prevents further deterioration, and/or eliminates at least one sign or symptom of the disorder. If the patient already suffers from a disorder, the regimen may be referred to as a therapeutically effective regimen. If a patient is at increased risk of experiencing a disorder relative to the general population but is not yet experiencing symptoms, the regimen may be referred to as a prophylactically effective regimen. In some cases, therapeutic or prophylactic efficacy can be observed in an individual patient relative to historical controls or past experience in the same patient. In other cases, therapeutic or prophylactic efficacy may be demonstrated in a preclinical or clinical study in a population of treated patients relative to a control population of untreated patients.

[0142] Anti-IL-6-VEGF Trap constructs are administered in an effective regimen, meaning a dose, route of administration, and frequency of administration, that delays the onset, reduces the severity, prevents further deterioration, and/or eliminates at least one sign or symptom of the disorder. If the patient already suffers from a disorder, the regimen may be referred to as a therapeutically effective regimen. If a patient is at increased risk of experiencing a disorder relative to the general population but is not yet experiencing symptoms, the regimen may be referred to as a prophylactically effective regimen. In some cases, therapeutic or prophylactic efficacy can be observed in an individual patient relative to historical controls or past experience in the same patient. In other cases, therapeutic or prophylactic efficacy may be demonstrated in a preclinical or clinical study in a population of treated patients relative to a control population of untreated patients.

[0143] The “biological half-life” of a substance is a pharmacokinetic parameter that refers to the time required for half of the substance to be eliminated from the body after administration of the substance to said body.

[0144] The term “preventing” or “preventing” refers to (a) preventing the occurrence of a disorder, (b) delaying the occurrence of a disorder or the onset of symptoms of a disorder, or (c) slowing the progression of an existing condition. Unless otherwise specified, the term “prevention” does not imply the complete prevention of the occurrence of a phenomenon.

[0145] The "individual" or "subject" is a mammal or bird, more preferably a human. Mammals also include, but are not limited to, farm animals (eg, cows, pigs, horses, chickens, etc.), sporting animals, pets, primates, horses, dogs, cats, mice and rats.

[0146] As used herein, the term “vector” means a construct capable of delivering and preferably expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, deproteinized DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors, encapsulated in liposomes, and some eukaryotic cells, such as producer cells.

[0147] As used herein, the term “expression control sequence” means a nucleic acid sequence that directs transcription of the nucleic acid. The expression control sequence may be a promoter, such as a constitutive or inducible promoter, or an enhancer. The expression control sequence is operably linked to the transcribed nucleic acid sequence.

[0148] As used herein, the term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any substance that, when combined with an active ingredient, allows said ingredient to retain biological activity and that does not react with the immune system of a subject. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as phosphate buffered saline, water, emulsions such as oil-in-water emulsion, various types of wetting agents, detergents such as polysorbate 20 to prevent aggregation and sugars , such as sucrose, as a cryoprotectant. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or isotonic (0.9%) saline. Compositions containing such carriers are prepared by well known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000).

[0149] As used herein, the term “k _on ” refers to the rate constant of association of an antibody (or bioconjugate) with an antigen. In particular, rate constants (k _on and k _off ) and equilibrium dissociation constants are measured using full-length antibodies and/or Fab antibody fragments (ie monovalent) and IL-6.

[0150] As used herein, the term " _koff " refers to the rate constant for the dissociation of an antibody (or bioconjugate) from an antibody/antigen complex.

[0151] As used herein, the term “K _D ” refers to the equilibrium dissociation constant of an antibody-antigen (or bioconjugate-antigen) interaction.

[0152] The term “about” when applied to a value or parameter as used herein includes (and describes) implementations that relate to that value or parameter per se. For example, a description referring to "about X" includes a description of "X". Numeric ranges include the numbers that define that range.

[0153] The term “patient” includes humans and other subjects (including mammals) who are receiving either prophylactic or therapeutic treatment.

[0154] For purposes of classifying amino acid substitutions as conservative or non-conservative, amino acids are grouped as follows: group I (hydrophobic side chains): met, ala, val, leu, ile; group II (neutral hydrophilic side chains): cys, ser, thr; group III (acid side chains): asp, glu; group IV (main side chains): asn, gln, his, lys, arg; group V (residues affecting chain orientation): gly, pro; and group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions include substitutions between amino acids in the same class. Non-conservative substitutions are the replacement of a representative of one of these classes with a representative of another.

[0155] Percent sequence identity is determined for antibody sequences maximally aligned according to the Kabat numbering system for the variable region or EU numbering for the constant region. After alignment, if a region of a subject antibody (e.g., the entire mature heavy or light chain variable region) is compared to the same region of a reference antibody, the percentage of sequence identity between the specified regions of the subject antibody and the reference antibody is the number of positions occupied by the same amino acid in the region of both the antibody in question and the reference antibody, divided by the total number of aligned positions of the two regions, excluding gaps, multiplied by 100 to convert to a percentage. Sequence identity for other sequences can be determined by sequence alignment using algorithms such as BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, version 7.0, Genetics Computer Group, 575 Science Dr., Madison, WI, using gap parameters by default, or visually, and the best alignment (i.e., providing the highest percentage of sequence similarity in the comparison window). Percent sequence identity is calculated by comparing two optimally aligned sequences in the comparison window, determining the number of positions at which identical residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., size window) and multiplying the result by 100 to obtain the percentage of sequence identity.

[0156] The term "antibody-dependent cellular cytotoxicity" or ADCC is a mechanism for inducing cell death that depends on the interaction of antibody-coated target cells (i.e., cells with bound antibody) with lytic immune cells (also called effector cells). cells). Such effector cells include natural killer cells, monocytes/macrophages and neutrophils. ADCC is triggered by interactions between the Fc region of a cell-bound antibody and Fcγ receptors, particularly FcγRI and FcγRIII, on immune effector cells such as neutrophils, macrophages, and natural killer cells. The target cell is eliminated by phagocytosis or lysis depending on the type of mediating effector cell. Death of the antibody-coated target cell occurs as a result of the activity of the effector cell.

[0157] A humanized antibody is a genetically engineered antibody in which CDRs from a non-human “donor” antibody are “grafted” into the sequence of a human “acceptor” antibody (see, for example, Queen, US 5530101 and 5585089; Winter, US 5225539, Carter , US 6407213, Adair, US 5859205 6881557, Foote, US 6881557). The acceptor antibody sequences may be, for example, a mature human antibody sequence, a composite of such sequences, a human antibody consensus sequence, or a germline sequence. Thus, a humanized antibody is an antibody containing some or all of the CDRs, wholly or substantially from a donor antibody, and the variable region framework sequences and constant regions, if present, wholly or substantially from human antibody sequences. Likewise, the humanized heavy chain contains at least one, two, and typically all three CDRs, all or substantially from the heavy chain of the donor antibody, and a heavy chain variable region framework sequence and a heavy chain constant region, if present, substantially from the variable heavy chain framework sequences. heavy chain regions and human constant region sequences. Likewise, the humanized light chain contains at least one, two, and typically all three CDRs, all or substantially from the light chain of the donor antibody, and a light chain variable region framework sequence and a light chain constant region, if present, substantially from the variable region framework sequences. light chain region and human constant region sequences. With the exception of nanobodies and dAbs, a humanized antibody contains a humanized heavy chain and a humanized light chain. A CDR in a humanized antibody is derived substantially from the corresponding CDR in a non-human antibody when at least 85%, 90%, 95%, or 100% of the corresponding residues (as determined by Kabat) are identical between the corresponding CDRs. The antibody variable chain framework sequences or the antibody constant region chain sequences are derived substantially from the human variable region framework sequence or the human constant region sequence, respectively, when at least 85%, 90%, 95% or 100% of the corresponding residues, as determined according to Kabat, are identical .

[0158] Although humanized antibodies often contain all six CDRs (preferably defined according to Kabat) from a mouse antibody, they can also be prepared with fewer than all CDRs (for example, at least 3, 4 or 5 CDRs from a mouse antibody) ( for example, Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al., Mol. Immunol. 36: 1079-1091, 1999 ; Tamura et al, Journal of Immunology, 164: 1432-1441, 2000).

[0159] A chimeric antibody is an antibody in which the mature light and heavy chain variable regions of a non-human (eg, mouse) antibody are combined with human light and heavy chain constant regions. Such antibodies retain substantially or completely the binding specificity of the murine antibody and are approximately two-thirds human.

[0160] A venated antibody is a type of humanized antibody that retains some, and typically all, of the CDRs and some variable region framework residues of a non-human antibody, but replaces other variable region framework residues that may contribute to binding to B or T cell epitopes, for example, replace exposed residues (Padlan, Mol. Immunol. 28:489, 1991) with residues from corresponding positions in the human antibody sequence. The result is an antibody in which the CDRs are derived wholly or substantially from an antibody of non-human origin, and the degree of humanization of the framework portions of the variable regions of the non-human antibody is increased by such substitutions. The human antibody may be isolated from a human or otherwise produced by expression of human immunoglobulin genes (eg, in a transgenic mouse, in vitro , or through phage display). Methods for producing human antibodies include the triome method according to Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, US Patent No. 4634664; and Engleman et al., US Pat. No. 4,634,666, use of transgenic mice containing human immunoglobulin genes (see, for example, Lonberg et al., W093/12227 (1993); US 5,877,397, US 5,874,299, US 5,814,318, US 5,789,650, US 5,770,429, US 5661016, US 5633425, US 5625126, US 5569825, US 5545806, Nature 148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91/10741 (1991) and phage display methods (see eg Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047, US 5877218, US 5871907, US 5858657, US 5837242, US 5733743 and US 5565332.

[0161] A “polymer” is a molecule composed of many repeating subunits. These subunits, also sometimes called "monomers", may be the same or different. There are both natural and synthetically produced polymers. Examples of natural polymers are DNA, protein and complex carbohydrates. Examples of synthetically produced polymers are polystyrene and polyacrylamide. A polymer consisting of repeating units of one monomer is called a homopolymer. A polymer made up of two or more monomers is called a copolymer or sometimes a heteropolymer. A copolymer in which certain types of monomers are grouped together is sometimes called a block copolymer. Polymers can be linear or branched. When a polymer is branched, the polymer chains having a common origin are sometimes called branch(s) of the polymer.

[0162] An "initiator" is a compound capable of serving as a substrate upon which one or more polymerizations can occur using the monomers or comonomers described herein. The polymerization may be a traditional free radical polymerization or preferably a controlled/living radical polymerization such as atom transfer radical polymerization (ATRP), reversible addition-fragmentation-termination polymerization (RAFT), or nitroxide-mediated polymerization (NMP). The polymerization may be “pseudo” controlled polymerization, such as degenerate transfer. Initiators suitable for ATRP contain one or more labile bonds that can be homolytically cleaved to produce an initiator moiety, I, which is a radical capable of initiating radical polymerization, and a radical scavenger, I', which reacts with a radical of the growing polymer chain to reversibly terminate polymerization . Said radical scavenger I' is typically a halogen, but may also be an organic moiety such as a nitrile. In some embodiments of the present invention, the initiator contains one or more 2-bromoisobutyrate groups as sites for ATRP polymerization.

[0163] The term "chemical linker" refers to a chemical moiety that links two groups, such as a half-life extending moiety and a protein. The linker may be cleavable or non-cleavable. Cleavable linkers can be hydrolyzable, enzymatically cleavable, pH-sensitive, photolabile, or disulfide linkers. Other linkers include homobifunctional and heterobifunctional linkers. A "linking group" is a functional group capable of forming a covalent bond, consisting of one or more bonds, with a bioactive agent. Non-limiting examples include those presented in Table 1 in WO2013059137 (the contents of which are incorporated by reference).

[0164] The term "reactive group" refers to a group that is capable of reacting with another chemical group to form a covalent bond, i.e. covalently reactive under suitable reaction conditions, and typically provides a point of attachment for another substance. A reactive group is a group, such as a maleimide or succinimidyl ester, that is capable of chemically reacting with a functional group on another moiety to form a covalent bond. Reactive groups typically include nucleophiles, electrophiles, and photoactivatable groups.

[0165] As used herein, the term "phosphorylcholine", also referred to as "PC", refers to the following:

[0166] where * indicates the attachment point. Phosphorylcholine is a zwitterionic group and includes salts (such as internal salts) and their protonated and deprotonated forms.

[0167] As used herein, a “phosphorylcholine-based polymer” is a polymer containing phosphorylcholine. The term "zwitterion-containing polymer" refers to a polymer that contains a zwitterion.

[0168] The term “poly(acryloyloxyethylphosphorylcholine)-containing polymer” refers to a polymer containing 2-(acryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate as a monomer.

[0169] The term “poly(methacryloyloxyethylphosphorylcholine)-containing polymer” refers to a polymer containing 2-(methacryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate as a monomer.

[0170] As used herein, “molecular weight” in the context of a polymer may be expressed as either a number average molecular weight, a weight average molecular weight, or a peak molecular weight. Unless otherwise indicated, all references to molecular weight in this specification refer to peak molecular weight. These determinations of molecular weight, number average (Mn), mass average (Mw) and peak (Mp), can be made using size exclusion chromatography or other liquid chromatography techniques. Other methods for measuring molecular weight values may also be used, such as using end group analysis or measuring colligative properties (such as freezing point depression, boiling point elevation, or osmotic pressure) to determine number-average molecular weight, or using light scattering, ultracentrifugation, or viscometry techniques to determine mass-average molecular weight. molecular weight. In a preferred embodiment of the present invention, molecular weight is measured by SEC-MALS (size exclusion chromatography - multi-angle light scattering). The polymer reagents of the present invention are typically polydisperse (ie, the number average molecular weight and weight average molecular weight of the polymers are not equal). The polydispersity index (PDI) provides a measure of the dispersity of polymers in a mixture. PDI is determined by the formula Mw/Mn. In this regard, a homogeneous protein will have a PDI of 1.0 (Mn is the same as Mw). The PDI for polymers will generally be higher than 1.0. The polymers of the present invention preferably have relatively low polydispersity values (PDI), for example, less than about 1.5, as assessed, for example, by SEC-MALS. In other embodiments, polydispersity values (PDI) are more preferably in the range of from about 1.4 to about 1.2, more preferably less than about 1.15, and more preferably less than about 1.10, more preferably less than about 1.05, and most preferably less than about 1.03.

[0171] As used herein, the terms “protected,” “protected form,” “protecting group,” and “protecting group” refer to the presence of a group (i.e., a protecting group) that prevents or blocks the reaction of a particular reactive functional group in the molecule under certain reaction conditions. Protecting groups vary depending on the type of reactive group being protected, as well as the reaction conditions used and the presence of additional reactive or protecting groups in the molecule, if any. Suitable protecting groups include those described in Greene et al., “Protective Groups In Organic Synthesis,” ^3rd Edition, John Wiley and Sons, Inc., New York, 1999.

[0172] As used herein, the term “alkyl” refers to a linear or branched saturated aliphatic radical containing the specified number of carbon atoms. For example, C ₁ -C ₆ alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, isopentyl, hexyl, etc. Other alkyl groups include, but are not limited to, heptyl, octyl, nonyl, decyl, etc. Alkyl can contain any number of carbon atoms, for example, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6 and 5-6 carbon atoms.

[0173] The term "lower", mentioned above and below in the present description in combination with organic radicals or compounds, respectively, defines a compound or radical that can be branched or unbranched and contain up to 7 inclusive, preferably up to 4 inclusive and (as unbranched) one or two carbon atoms.

[0174] As used herein, the term “alkylene” refers to an alkyl group, as defined above, linking at least two other groups, i.e. divalent hydrocarbon radical. These two alkylene-linked groups may be linked to the same alkylene atom or different alkylene atoms. For example, linear alkylene may be a divalent radical -(CH ₂ ) _n, where n is 1, 2, 3, 4, 5 or 6. Alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene , isobutylene, sec-butylene, pentylene and hexylene.

[0175] Substituents for alkyl, alkenyl, alkylene, heteroalkyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl and heterocycloalkenyl radicals may be one or more of various groups selected from, but not limited to: -OR',= O,=NR',=N-OR', -NR'R”, -SR', -halogen, -SiR'R”R”', -OC(O)R', -C(O)R', -CO ₂ R', -CONR'R”, -OC(O)NR'R”, -NR”C(O)R’, -NR’-C(O)NR”R”’, -NR”C (O) ₂ R',-NR-C(NR'R”R'”)=NR””, -NR-C(NR'R”)=NR'”, -S(O)R', -S (O) ₂ R', -S(O) ₂ NR'R”, -NRSO ₂ R', -CN and -NO ₂ , in amounts ranging from 1 to (2m'+1), where m' represents the total number of carbon atoms in such a radical. Each of R', R”, R”' and R”” independently refers to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, for example, aryl containing 1-3 halogen atoms as substituents, containing substituents or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. In the case where R' and R” are attached to the same nitrogen atom, they can form, together with the specified nitrogen atom, a 5-, 6- or 7-membered ring. For example, -NR'R” includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl.

[0176] As used herein, the term “alkoxy” refers to an alkyl group attached to an oxygen atom and forms the radical -OR, where R is alkyl. Alkoxy groups include, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 2-butoxy, isobutoxy, sec-butoxy, t-butoxy, pentoxy, hexoxy, etc. Alkoxy groups may additionally contain various substituents described herein. For example, alkoxy groups may contain halogen atoms as substituents to form a "haloalkoxy" group.

[0177] As used herein, the term “carboxyalkyl” means an alkyl group (as defined herein) substituented with a carboxy group. The term "carboxycycloalkyl" means a cycloalkyl group (as defined herein) substituted with a carboxy group. The term "alkoxyalkyl" means an alkyl group (as defined herein) substituted with an alkoxy group. As used herein, the term “carboxy” refers to carboxylic acids and their esters.

[0178] As used herein, the term “haloalkyl” refers to an alkyl as defined above in which some or all of the hydrogen atoms are replaced by halogen atoms. Halogen (halogen) is preferably chlorine or fluorine, but may also be bromine or iodine. For example, haloalkyl includes trifluoromethyl, fluoromethyl, 1,2,3,4,5-pentafluorophenyl, etc. The term "perfluoro" defines a compound or radical in which all available hydrogen atoms have been replaced by fluorine. For example, perfluorophenyl refers to 1,2,3,4,5-pentafluorophenyl, perfluoromethyl refers to 1,1,1-trifluoromethyl, and perfluoromethoxy refers to 1,1,1-trifluoromethoxy. Haloalkyl may also be referred to as halogen-substituted alkyl, such as fluorine-substituted alkyl.

[0179] As used herein, a “cytokine” as used herein is a member of a group of protein signaling molecules that may participate in intercellular communication in immune and inflammatory responses. Cytokines are, as a rule, small water-soluble glycoproteins, the mass of which is approximately 8-35 kDa.

[0180] As used herein, the term “cycloalkyl” refers to a saturated mono- or polycyclic aliphatic ring system that contains from about 3 to 12, 3 to 10, 3 to 7, or 3 to 6 carbon atoms. In the case where the cycloalkyl group consists of two or more rings, said rings can be connected to each other to form a fused ring or spiro ring structure. In the case where the cycloalkyl group consists of three or more rings, said rings may also be connected to each other to form a bridging ring structure. Monocyclic rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cyclooctyl. Bicyclic and polycyclic rings include, for example, bicyclo[1.1.1]pentane, bicyclo[2.1.1]heptane, norbornane, decahydronaphthalene and adamantane. For example, C _3-8 cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl and norbornane.

[0181] As used herein, the term “endocyclic” refers to an atom or group of atoms forming part of a cyclic ring structure.

[0182] As used herein, the term “exocyclic” refers to an atom or group of atoms that are attached to, but do not define, a cyclic ring structure.

[0183] As used herein, the term “cyclic alkyl ether” refers to a 4- or 5-membered cyclic alkyl group containing 3 or 4 endocyclic carbon atoms and 1 endocyclic oxygen or sulfur atom (eg, oxetane, thietane, tetrahydrofuran, tetrahydrothiophene); or a 6-7 membered cyclic alkyl group containing 1 or 2 endocyclic oxygen or sulfur atoms (for example, tetrahydropyran, 1,3-dioxane, 1,4-dioxane, tetrahydrothiopyran, 1,3-dithiane, 1,4-dithiane, 1,4-oxathian).

[0184] As used herein, the term “alkenyl” refers to either a linear or branched hydrocarbon of 2-6 carbon atoms containing at least one double bond. Examples of alkenyl groups include, but are not limited to, vinyl, propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl or 1,3,5-hexatrienyl. Alkenyl groups may also contain 2 to 3, 2 to 4, 2 to 5, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6 and 5 to 6 atoms carbon.

[0185] As used herein, the term “alkenylene” refers to an alkenyl group, as defined above, linking at least two other groups, i.e. divalent hydrocarbon radical. The two alkenylene-related groups may be bonded to the same alkenylene atom or to different alkenylene atoms. Alkenylene groups include, but are not limited to, ethenylene, propenylene, isopropenylene, butenylene, isobutenylene, sec-butenylene, pentenylene and hexenylene.

[0186] As used herein, the term “alkynyl” refers to either a linear or branched hydrocarbon of 2-6 carbon atoms containing at least one triple bond. Examples of alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadinyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadinyl, 1,4- pentadiinyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadinyl, 1,4-hexadinyl, 1,5-hexadinyl, 2,4-hexadinyl or 1,3,5-hexatriinyl. Alkynyl groups may also contain 2 to 3, 2 to 4, 2 to 5, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6 and 5 to 6 atoms carbon.

[0187] As used herein, the term “alkynylene” refers to an alkynyl group, as defined above, linking at least two other groups, i.e. divalent hydrocarbon radical. The two alkynylene-related groups may be bonded to the same alkynylene atom or to different alkynylene atoms. Alkynylene groups include, but are not limited to, ethynylene, propynylene, butynylene, sec-butynylene, pentynylene and hexynylene.

[0188] As used herein, the term “cycloalkylene” refers to a cycloalkyl group, as defined above, linking at least two other groups, i.e. divalent hydrocarbon radical. These two groups associated with cycloalkylene may be associated with the same atom or different atoms of cycloalkylene. Cycloalkylene groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene and cyclooctylene.

[0189] As used herein, the term “heterocycloalkyl” refers to a ring system containing from 3 ring atoms to about 20 ring atoms and from 1 to about 5 heteroatoms, such as N, O, and S. Additional heteroatoms may also be suitable, including, but not limited to, B, Al, Si and P. Heteroatoms may also be oxidized, for example, but not limited to, -S(O)- and -S(O) ₂ -. For example, the heterocycle includes, but is not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, morpholino, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, piperidinyl, indolinyl, quinuclidinyl and 1,4-dioxa-8-aza-spiro[4.5] dets-8-il.

[0190] As used herein, the term “heterocycloalkylene” refers to a heterocycloalkyl group, as defined above, linking at least two other groups. These two groups associated with heterocycloalkylene may be associated with the same atom or different atoms of the heterocycloalkylene.

[0191] As used herein, the term “aryl” refers to a monocyclic or polycyclic (eg, fused bicyclic, tricyclic, or more ring) aromatic ring system containing from 6 to 16 carbon atoms. For example, aryl may be phenyl, benzyl or naphthyl, and is preferably phenyl. Aryl groups can be mono-, di- or tri-substituted and contain as substituents one, two or three radicals selected from alkyl, alkoxy, aryl, hydroxy, halogen, cyano, amino, aminoalkyl, trifluoromethyl, alkylenedioxy and oxy-C ₂ - C ₃ -alkylene; all of which optionally also contain substituents such as those defined above; or 1- or 2-naphthyl; or 1- or 2-phenanthrenyl. Alkylenedioxy is a divalent substituent attached to two adjacent phenyl carbons, such as methylenedioxy or ethylenedioxy. Oxy-C ₂ -C ₃ -alkylene is also a divalent substituent attached to two adjacent phenyl carbon atoms, for example, oxyethylene or oxypropylene. An example of oxy-C ₂ -C ₃ -alkylenephenyl is 2,3-dihydrobenzofuran-5-yl.

[0192] Preferred aryl is naphthyl, phenyl or phenyl mono- or disubstituted with alkoxy, phenyl, halogen, alkyl or trifluoromethyl, especially phenyl or phenyl mono- or disubstituted with alkoxy, halogen or trifluoromethyl, and in particular phenyl.

[0193] Examples of substituted phenyl groups as R include, for example, 4-chlorophen-1-yl, 3,4-dichlorophen-1-yl, 4-methoxyphen-1-yl, 4-methylphen-1-yl, 4 -aminomethylphen-1-yl, 4-methoxyethylaminomethylphen-1-yl, 4-hydroxyethylaminomethylphen-1-yl, 4-hydroxyethyl-(methyl)-aminomethylphen-1-yl, 3-aminomethylphen-1-yl, 4-N-acetylaminomethylphen -1-yl, 4-aminophen-1-yl, 3-aminophen-1-yl, 2-aminophen-1-yl, 4-phenyl-phen-1-yl, 4-(imidazol-1-yl)-phenyl , 4-(imidazol-1-ylmethyl)-phen-1-yl, 4-(morpholin-1-yl)-phen-1-yl, 4-(morpholin-1-ylmethyl)-phen-1-yl, 4 -(2-methoxyethylaminomethyl)-phen-1-yl and 4-(pyrrolidin-1-ylmethyl)-phen-1-yl, 4-(thiophenyl)-phen-1-yl, 4-(3-thiophenyl)-phen -1-yl, 4-(4-methylpiperazin-1-yl)-phen-1-yl and 4-(piperidinyl)-phenyl, and 4-(pyridinyl)-phenyl, possibly containing substituents on the heterocyclic ring.

[0194] As used herein, the term “arylene” refers to an aryl group, as defined above, linking at least two other groups. The two arylene-related groups are bonded to different arylene atoms. Arylene groups include, but are not limited to, phenylene.

[0195] As used herein, the term “aryleneoxy” refers to an arylene group as defined above, wherein one of the groups associated with the arylene is linked through an oxygen atom. Aryleneoxy groups include, but are not limited to, phenyleneoxy.

[0196] Likewise, substituents for aryl and heteroaryl groups vary and are selected from: -halogen, -OR', -OC(O)R', -NR'R”, -SR', -R', -CN, - NO ₂ , -CO ₂ R', -CONR'R”, -C(O)R’, -OC(O)NR’R”, -NR”C(O)R’, -NR”C(O) ₂ R', -NR'-C(O)NR”R”', -NH-C(NH ₂ )=NH, -NR'C(NH ₂ )=NH, -NH-C(NH ₂ )=NR ', -S(O)R', -S(O) ₂ R', -S(O) ₂ NR'R”, -N ₃ , -CH(Ph) ₂ , perfluoro(C ₁ -C ₄ )alkoxy and perfluoro(C ₁ -C ₄ )alkyl in an amount ranging from zero to the total number of free valencies in the aromatic ring system; and wherein R', R” and R”' are independently selected from hydrogen, (C ₁ -C ₈ )alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C ₁ -C ₄ )alkyl and (unsubstituted aryl)oxy-(C ₁ -C ₄ )alkyl.

[0197] Two of these substituents on adjacent atoms of an aryl or heteroaryl ring may be replaced by a substituent of the formula -TC(O)-(CH ₂ ) _q -U-, where T and U are independently -NH-, -O-, -CH ₂ - or a single bond, and q is an integer from 0 to 2. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may be replaced by a substituent of the formula -A-(CH ₂ ) _r -B- where A and B are independently -CH ₂ -, -O-, -NH-, -S-, -S(O)-, -S(O) ₂ -, -S(O) ₂ NR'- or single bond, and r is an integer from 1 to 3. One of the single bonds of the new ring thus formed can be replaced by a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may be replaced by a substituent of the formula -(CH ₂ ) _s -X-(CH ₂ ) _t -, where s and t are independently integers from 0 to 3, and X is -O-, -NR'-, -S-, -S(O)-, -S(O) ₂ - or -S(O) ₂ NR'-. The R' substituent in -NR'- and -S(O) ₂ NR'- is selected from hydrogen or unsubstituted (C ₁ -C ₆ )alkyl.

[0198] As used herein, the term “heteroaryl” refers to a monocyclic or fused bicyclic or tricyclic aromatic ring system containing from 5 to 16 ring atoms, where each of the 1 to 4 ring atoms is a N, O, or S heteroatom. For example, heteroaryl includes pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl or any other radicals containing as substituents (especially mono - and disubstituted), for example, alkyl, nitro or halogen. Pyridyl is 2-, 3- or 4-pyridyl, preferably 2- or 3-pyridyl. Thienyl is 2- or 3-thienyl. Quinolinyl is preferably 2-, 3- or 4-quinolinyl. Isoquinolinyl is preferably 1-, 3- or 4-isoquinolinyl. Benzopyranyl, benzothiopyranyl are preferably 3-benzopyranyl or 3-benzothiopyranyl, respectively. The thiazolyl is preferably 2- or 4-thiazolyl and most preferably 4-thiazolyl. Triazolyl is preferably 1-, 2- or 5-(1,2,4-triazolyl). Tetrazolyl is preferably 5-tetrazolyl.

[0199] Preferably, heteroaryl is pyridyl, indolyl, quinolinyl, pyrrolyl, thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl, benzothienyl, oxazolyl, indazolyl, or any of the substituent-containing radicals, especially mono- or disubstituted.

[0200] The term “heteroalkyl” refers to an alkyl group containing 1 to 3 heteroatoms, such as N, O, and S. Additional heteroatoms may also be suitable, including, but not limited to, B, Al, Si, and P. Heteroatoms may also be oxidized, for example, but not limited to, -S(O)- and -S(O) ₂ -. For example, heteroalkyl may include ethers, thioethers, alkylamines and alkylthiols.

[0201] The term “heteroalkylene” refers to a heteroalkyl group, as defined above, linking at least two other groups. These two groups associated with heteroalkylene may be associated with the same atom or different atoms of the heteroalkylene.

[0202] As used herein, the term "electrophile" refers to an ion or atom or group of atoms, which may be ionic, containing an electrophilic center, i.e. an electron-withdrawing center capable of reacting with a nucleophile. An electrophile (or electrophilic reagent) is a reagent that forms a bond with its reaction partner (nucleophile) by accepting both bonding electrons from that reaction partner.

[0203] As used herein, the term “nucleophile” refers to an ion or atom or group of atoms, which may be ionic, containing a nucleophilic center, i.e. a center that “seeks” an electrophilic center or is capable of reacting with an electrophile. A nucleophile (or nucleophilic reagent) is a reagent that forms a bond with its reaction partner (electrophile) by donating both bonding electrons. The term "nucleophilic group" refers to a nucleophile after it has reacted with a reactive group. Non-limiting examples include amino, hydroxyl, alkoxy, haloalkoxy and the like.

[0204] As used herein, the term “maleimido” refers to a pyrrol-2,5-dione-1-yl group having the structure:

[0205] which, when reacted with a sulfhydryl (e.g., thioalkyl), forms an -S-maleimido group having the structure

[0206] where "•" indicates the point of attachment of the maleimido group, and " " indicates the point of attachment of the thiol sulfur atom to the remainder of the original sulfhydryl-containing group.

[0207] For the purpose of the present invention, "naturally occurring amino acids" found in proteins and polypeptides are L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamine, L-glutamic acid, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and /or L-valine. "Non-naturally occurring amino acids" found in proteins are any amino acid other than those listed as naturally occurring amino acids. Non-naturally occurring amino acids include, without limitation, D-isomers of naturally occurring amino acids and mixtures of D- and L-isomers of naturally occurring amino acids. Other amino acids such as 4-hydroxyproline, desmosine, isodesmosine, 5-hydroxylysine, epsilon-N-methyllysine, 3-methylhistidine, although found in naturally occurring proteins, are considered non-naturally occurring amino acids , found in proteins, for the purpose of the present invention, since they are typically introduced by means other than ribosomal translation of mRNA.

[0208] As used herein, the term “linear”, when applied to the geometry, structure, or overall structure of a polymer, refers to a polymer containing a single branch of the polymer.

[0209] As used herein, the term “branched” as applied to the geometry, structure, or overall structure of a polymer refers to a polymer containing 2 or more polymer branches extending from a core structure contained in an initiator. The initiator can be used in an atom transfer radical polymerization (ATRP) reaction. A branched polymer may contain 2 polymer chains (polymer branches), 3 polymer branches, 4 polymer branches, 5 polymer branches, 6 polymer branches, 7 polymer branches, 8 polymer branches, 9 polymer branches or more. Each polymer branch extends from the center of initiation of polymer synthesis. Each initiation center for polymer synthesis can be a center for the growth of a polymer chain through the addition of monomers. For example, but not by way of limitation, when using ATRP, the initiation site for polymer synthesis on the initiator is typically an organic halide undergoing a reversible redox process catalyzed by a transition metal compound such as a copper halide. Preferably, the halide is bromine.

[0210] As used herein, the term “pharmaceutically acceptable excipient” refers to an excipient that can be included in the compositions of the present invention and that does not cause significant adverse toxicological effects in a patient and is or may be approved by the FDA for therapeutic use, in particular , in people. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, saline, lactated Ringer's solution, standard sucrose solution, standard glucose solution, and the like.

[0211] As used herein, "OG1786" is a 9-arm initiator used for polymer synthesis, with the structure shown in Figure 2D, which represents the trifluoroacetic acid salt form of OG1786. OG1786 can be used in accordance with the present invention in the form of other salts or as a free base.

[0212] As used herein, "OG1801" is an approximately (+/- 15%) 750 kDa polymer (either Mn or Mp) prepared using OG1786 as an initiator for ATRP synthesis using the HEMA-PC monomer. The structure of OG1801 is shown in Figure 2J.

[0213] As used herein, "OG1802" is OG1801 with a maleimide functionality attached and has the structure shown in Figure 2K, wherein each of n ₁ , n ₂ , n 3 , n ₄ , n ₅ , _{n 6 ,} n ₇ , n ₈ and n ₉ is a positive integer (from 0 to about 3000), such that the total molecular weight of said polymer is (Mw) 750,000 ± 15% Dalton. When the term "OG1802" is used to modify the term protein (such as VEGF trap or anti-IL6 antibody), it means that the protein is a conjugated protein.

[0214] Unless otherwise defined, all technical and scientific terms used in this description have the same meanings as commonly understood by one skilled in the art to which the present invention relates. In the event of any conflict, this specification, including definitions, will control. Throughout this specification and claims, the term “comprise” or variations such as “comprises” or “comprising” means the inclusion of a specified integer or group of integers, but not the exclusion of any another integer or group of integers. Unless the context otherwise requires, terms in the singular include the plural and terms in the plural include the singular. Any example(s) following the term “for example” are not intended to be exhaustive or limiting.

[0215] A singular entity term refers to one or more of such entities; for example, the term "connection" refers to one or more connections or at least one connection. Thus, the singular terms “one or more” and “at least one” may be used interchangeably herein.

[0216] As used herein, the term “about” means the variation that may be observed across measurements made with different instruments, across different samples, and during sample production.

[0217] In the present description, the positions of the CDRs are in the order of their appearance in the variable domain when described. For example, positions in the heavy chain may be described as S35H or G66D. In this specification, Fc provisions follow EU numbering when describing or indicating that they are given according to EU numbering. For example, antibody mutations may be described as L234A or L235A, which corresponds to EU numbering. Provisions may also be defined in accordance with specified provisions in a particular SEQ ID or sequence presented in the present description.

[0218] Multi-angle light scattering ( MALS ) is a method for analyzing macromolecules in which laser light strikes a molecule and the light's oscillating electric field induces an oscillating dipole in it. This oscillating dipole will emit light back and can be measured using a MALS detector such as Wyatt's miniDawn TREOS. The intensity of the emitted light depends on the magnitude of the dipole induced in the macromolecule, which in turn is proportional to the polarizability of the macromolecule; the larger the induced dipole, the greater the intensity of the scattered light. Accordingly, to analyze the scattering of such macromolecules from a solution, it is necessary to know their polarizability relative to the environment (for example, a solvent). It can be determined by measuring the change, Δn , in the refractive index of a solution n with a change in molecular concentration, Δc , by measuring the dn/dc value (= Δn / Δc ) using an Optilab T-rEX Wyatt differential refractometer. The two molar mass parameters used in determining MALS are number average molecular weight (Mn) and mass average molecular weight (Mw), with the polydispersity index (PDI) being equal to Mw divided by Mn. SEC also allows the determination of another average molecular weight, the peak molecular weight Mp, which is defined as the molecular weight of the highest peak in the SEC.

[0219] PDI is used as a measure of the breadth of the molecular weight distribution for a polymer and a bioconjugate resulting from the conjugation of a single protein to a polydisperse biopolymer (eg, OG1802). For a protein sample, the polydispersity is close to 1.0 because it is a product of translation, in which each protein molecule in solution is expected to have almost the same length and molar mass. In contrast, due to the polydisperse nature of the biopolymer, where polymer chains of varying lengths are synthesized during the polymerization process, determining the PDI of a sample as one of its quality indicators for a narrow molecular weight distribution is very important.

[0220] Size exclusion chromatography ( SEC ) is a chromatography technique that separates molecules in solution based on their size. Typically, an aqueous solution is used to move the sample through a column filled with resins of varying pore sizes. The resin is expected to be inert to the analyte as it passes through the column, and the analytes will be separated from each other based on their unique size and the pore size characteristics of the selected column.

[0221] Combining SEC with MALS or SEC/MALS provides accurate molar mass and size distribution (rms radius) as opposed to relying on a set of SEC calibration standards. This type of device has many advantages over traditional column calibration methods. Since light scattering and concentration are measured for each eluting fraction, the molar mass and size can be determined regardless of the elution position. This is especially important for substances with non-globular macromolecules, such as biopolymers (OG1802) or bioconjugates; such substances generally do not elute in a manner that can be described by a set of column calibration standards.

[0222] In some embodiments, the SEC/MALS analysis includes a Waters HPLC system with an Alliance 2695 solvent injection module and a Waters 2996 photodiode array detector equipped with a Shodex SEC-HPLC column (7.8 x 300 mm). It is connected online to the miniDawn TREOS Wyatt and the Optilab T-rEX Wyatt differential refractometer. Waters' Empower software can be used to monitor a Waters HPLC system, and Wyatt's ASTRA V 6.1.7.16 software can be used to obtain MALS data from Wyatt's miniDawn TREOS, dn/dc data from the T-rEX detector and mass recovery data using the A280 absorbance signal from a Waters 2996 photodiode array detector. SEC can be carried out at a rate of 1 ml/min in 1x PBS pH 7.4, after sample injection the MALS and RI signals can be analyzed using ASTRA software to determine absolute molar mass (Mp, Mw , Mn ) and polydispersity index ( PDI ). In addition to this, the calculation also includes dn/dc input values for the polymer and protein, such as 0.142 and 0.183, respectively. For bioconjugates, the dn/dc value is calculated based on the determined molecular weight of the polymer and protein using the formula below and is approximately 0.148.

dn/dc of conjugate = 0.142 x [MW polymer / (MW polymer + MW protein )] + 0.183 x [MW protein / (MW polymer + MW protein )]

[0223] When the polymer MW for OG1802, measured by SEC-MALS, is approximately 800 kDa, and the protein MW for anti-IL-6, measured by SEC-MALS, is approximately 145 kDa, the expected total molecular weight of the bioconjugate, measured by SEC -MALS is approximately 1000 kDa. The protein MW for the anti-IL-6-VEGF trap or VEGF-anti-IL-6 trap construct is approximately 192 kDa, and the expected total molecular weight of the bioconjugate is 1000-1100 kDa.

[0224] Typical methods and substances are described herein, although methods and substances similar or equivalent to those described herein may also be used in the practice or testing of the present invention. The specified substances, methods and examples are provided for illustrative purposes only and are not limiting.

I. IL-6 ANTAGONIST ANTIBODIES, AB IL-6-VEGF TRAP CONSTRUCTS AND/OR THEIR CONJUGATES

[0225] The present invention provides anti-IL-6 antibodies that block, inhibit or reduce (including significantly reduce) the biological activity of IL-6, including downstream events mediated by IL-6. In some embodiments, an anti-IL-6 antagonist antibody will comprise one or more CDR sequences provided herein.

[0226] In some embodiments, the isolated antagonist antibody specifically binds to IL-6.

[0227] In some embodiments, the antibody preferably reacts with IL-6 in a manner that inhibits IL-6 signaling function. In some embodiments, the anti-IL-6 antagonist antibody specifically binds primate IL-6.

[0228] Antibodies suitable for the present invention may include monoclonal antibodies, polyclonal antibodies, antibody fragments (eg, Fab, Fab', F(ab') ₂ , Fv, Fc, etc.), chimeric antibodies, bispecific antibodies , heteroconjugate antibodies, single chain fragments (ScFv), mutants thereof, fusion proteins containing an antibody moiety (e.g., a domain antibody), humanized antibodies, and any other modified configuration of an immunoglobulin molecule that contains an antigen recognition site of the required specificity, including glycosylated variants antibodies, amino acid sequence variants of antibodies and covalently modified antibodies. The antibodies may be mouse, rat, human, or any other origin (including chimeric or humanized antibodies). In some embodiments, the anti-IL-6 antagonist antibody is a monoclonal antibody. In some embodiments, the antibody is a human antibody or a humanized antibody.

[0229] In some embodiments, the antibody comprises a heavy chain variable region amino acid sequence presented in Tables 1, 2, 6, 7, 8 and/or 9, or FIG. 5. In some embodiments, the isolated antagonist antibody comprises a heavy chain variable region (VH) comprising VH complementarity determining region 1 (CDR1), VH CDR2, and VH CDR3 having the amino acid sequence set forth in Tables 1, 2, 6, 7, 8 and/ or 9, and a light chain variable region (VL) containing CDR1 VL, CDR2 VL and CDR3 VL having the amino acid sequence shown in the table.

[0230] In some embodiments, an isolated anti-IL-6 antagonist antibody is provided. The antibody contains a heavy chain constant domain containing one or more mutations to reduce effector function. In some embodiments, the one or more mutations reduce the effector functions of the antibody associated with the complement system, for example, reduce activation of the complement system. In some embodiments, the reduction in effector function is at least about 50%.

[0231] Some embodiments provide an isolated antagonist antibody that specifically binds IL-6, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the antibody contains the mutations L234A, L235A, and G237A (according to EU numbering). In some embodiments, the isolated antagonist antibody contains the L234A, L235A, and G237A mutations. In some embodiments, the isolated antagonist antibody containing the mutations exhibits minimized binding to FC or C1q gamma receptors. In some embodiments, an isolated antagonist antibody comprising the L234A, L235A, and G237A mutations exhibits minimized binding to FC or C1q gamma receptors. In some embodiments, an isolated anti-IL-6 antagonist antibody is provided in which the mutation(s) is located at one or more of the following amino acid positions (EU numbering): E233, L234, L235, G236, G237, A327, A330, and P331. In some embodiments, an isolated anti-IL-6 antagonist antibody is provided in which the mutation(s) are selected from the group consisting of E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S.

[0232] In some embodiments, an isolated anti-IL-6 antagonist antibody is provided. The heavy chain constant domain additionally contains a cysteine residue introduced by recombinant DNA. In some embodiments, said cysteine residue is selected from the group consisting of Q347C and L443C (EU numbering). In some embodiments, the cysteine residue is L443C (EU numbering).

[0233] In some embodiments, the antibody contains all three of the following mutations (EU numbering): L234A, L235A, and G237A, and the antibody contains L443C (EU numbering). In some embodiments, the antibody is human IgG1, and the heavy chain constant domain of the antibody contains one or more mutations that reduce immune-mediated effector function.

[0234] In some embodiments, an isolated antagonist antibody is provided that binds an epitope on human IL-6 that is the same as or overlaps with an epitope recognized by an antibody comprising amino acid sequences in any one or more of Tables 1 and 2 and/or 6- 9. In some embodiments, an anti-IL-6 antibody is provided that contains cys and is linked through this cysteine to a polymer (as shown in Formula 17 herein).

[0235] In some embodiments, an isolated antagonist antibody is provided that binds to IL-6. In some embodiments, said isolated antagonist antibody that binds IL-6 comprises a heavy chain comprising the amino acid sequence shown in Tables 1, 2, 6, 7, 8 and/or 9, with or without a C-terminal lysine, and a light chain containing the amino acid sequence shown in Tables 1, 2, 6, 7, 8 and/or 9.

[0236] In some embodiments, an isolated antagonist antibody is provided that binds to IL-6. The specified antibody contains a VH containing the amino acid sequence presented in tables 1, 2, 6, 7, 8 and/or 9, or a sequence that is at least 90% identical to it, containing amino acid substitutions at residues not included in the CDR. In some embodiments, the antibody comprises one or more of: HCDR1: in Figure 5, HCDR2: in Figure 5, HCDR3: in Figure 5; LCDR1: in Fig.5, LCDR2: in Fig.5, LCDR3: in Fig.5, for example, 1, 2, 3, 4, 5 or all 6 CDRs.

[0237] In some embodiments, an antibody is provided that binds to IL-6, wherein the antibody comprises CDRH1, which is CDRH1 in Tables 1, 2, 6, 7, 8 and/or 9, CDRH2, which is CDRH2 in Tables 1, 2, 6, 7, 8 and/or 9, CDRH3, being CDRH3 in tables 1, 2, 6, 7, 8 and/or 9, CDRL1, being CDRL1 in tables 1, 2, 6, 7, 8 and/or 9, CDRL2, representing CDRL2 in tables 1, 2, 6, 7, 8 and/or 9, CDRL3, representing CDRL3 in tables 1, 2, 6, 7, 8 and/or 9; at least one of the following mutations: L234A, L235A and G237A according to EU numbering and at least one of the following mutations: Q347C or L443C according to EU numbering.

[0238] In some embodiments, an isolated anti-IL-6 antagonist antibody is provided. The heavy chain variable region of said antibody contains three complementarity determining regions (CDRs) containing the amino acid sequences presented in Table 1. In some embodiments, an isolated anti-IL-6 antagonist antibody is provided, wherein the light chain variable region of said antibody contains three complementarity determining regions (CDR) containing the amino acid sequences presented in Table 2. In some embodiments, the specified antibody is an antibody that contains one or more sequences defined in Fig. 5, for example, one or more CDRs (including 2, 3, 4, 5 or 6 framed CDRs) and/or entire heavy and light chain variable regions.

[0239] In some embodiments, the isolated anti-IL-6 antagonist antibody comprises a heavy chain variable region (VH) that contains three CDRs comprising the amino acid sequences presented in Table 1 and an antibody light chain variable region (VL) that contains three CDRs , containing the amino acid sequences presented in Table 2.

[0240] In some embodiments, an isolated anti-IL-6 antagonist antibody is provided. VH contains the amino acid sequences presented in Table 1, and the antibody light chain variable region contains three CDRs containing the amino acid sequences presented in Table 2.

[0241] In some embodiments, an isolated anti-IL-6 antagonist antibody is provided, wherein the antibody comprises a VL comprising the amino acid sequence shown in Table 2, or a variant thereof containing one amino acid substitution in the non-CDR amino acids. In some embodiments, an isolated anti-IL-6 antagonist antibody is provided, wherein the antibody comprises a VH containing the amino acid sequence shown in Table 1, or a variant thereof containing multiple amino acid substitutions in amino acids not included in the CDR.

[0242] In some embodiments, an isolated antagonist antibody is provided, wherein the antibody comprises a heavy chain comprising the amino acid sequence shown in Table 1 with a C-terminal lysine, and a light chain comprising the amino acid sequence shown in Table 2. In some embodiments provide an isolated antagonistic antibody, wherein the antibody comprises a heavy chain containing the amino acid sequence presented in Table 1, without the C-terminal lysine, and a light chain containing the amino acid sequence presented in Table 2.

[0243] IL-6 antagonist antibodies can be prepared by any method known in the art. General methods for producing human and mouse antibodies are known in the art and/or described herein.

[0244] IL-6 antagonist antibodies can be identified or characterized using methods known in the art that detect and/or measure the reduction, elimination, or neutralization of IL-6 biological activity. In some embodiments, an anti-IL-6 antagonist antibody is identified by incubating the candidate antibody with IL-6 and monitoring binding to IL-6R or IL-6/IL-6R binding to gp130, and/or concomitant reduction or neutralization of IL biological activity -6. The binding assay can be performed, for example, using purified IL-6 polypeptide(s) or cells naturally expressing (eg, different strains) or transfected to express IL-6 polypeptide(s). In one embodiment, the binding assay is a competitive binding assay that evaluates the ability of a candidate antibody to compete with a known IL-6 antagonist antibody for binding to IL-6. This assay can be performed in various formats, including ELISA format.

[0245] Following initial identification, the activity of a candidate IL-6 antagonist antibody can be further confirmed and refined by bioassays known to test target biological activity. In some embodiments, an in vitro cell-based assay is used to further characterize the candidate IL-6 antagonist antibody.

[0246] Characterization of IL-6 antagonist antibodies can be obtained using methods well known in the art. For example, one method is to identify the epitope to which it binds, or “epitope mapping.” There are many methods for mapping and determining the location of epitopes on proteins known in the art, including determination of the crystal structure of the antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays described, for example, in Chapter 11 in Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999. In an additional example, epitope mapping can be used to determine the sequence to which an IL-6 antagonist antibody binds. Epitope mapping for the IL-6 antagonist antibody is commercially available from various sources, for example, Pepscan Systems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). The epitope may be a linear epitope, i.e. contained in a single region of amino acids, or a conformational epitope formed as a result of the three-dimensional interaction of amino acids that may not necessarily be contained in a single region. Peptides of various lengths (eg, at least 4-6 amino acids in length) can be isolated or synthesized (eg, recombinantly) and used for IL-6 antagonist antibody binding assays. In another example, the epitope to which an anti-IL-6 antagonist antibody binds can be determined in a systematic screen by using overlapping peptides derived from the IL-6 sequence and determining binding by the IL-6 antagonist antibody. In gene fragment expression assays, the open reading frame encoding IL-6 is fragmented either randomly or through specific genetic constructs, and the reactivity of the expressed IL-6 fragments with the test antibody is determined. Gene fragments can be obtained, for example, by PCR, and then transcribed and translated into protein in vitro in the presence of radioactive amino acids. Antibody binding to radiolabeled IL-6 fragments is then determined by immunoprecipitation and gel electrophoresis. Some epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries) or yeast (yeast display). Alternatively, a defined library of overlapping peptide fragments can be tested for binding to a test antibody in simple binding assays. In a further example, antigen mutagenesis, domain swap experiments, and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and/or essential for epitope binding. For example, alanine scanning mutagenesis experiments can be performed using mutant IL-6 in which various residues in the IL-6 polypeptide are replaced with alanine. By assessing antibody binding to mutant IL-6, the importance of specific IL-6 residues for antibody binding can be assessed.

[0247] In some embodiments, an isolated anti-IL-6 antagonist antibody is provided, wherein the antibody binds human IL-6 with an affinity of from about 0.01 pM to about 10 nM. In some embodiments, an isolated anti-IL-6 antagonist antibody is provided, wherein the antibody binds human IL-6 with an affinity of from about 0.1 pM to about 2 nM. In some embodiments, an isolated anti-IL-6 antagonist antibody is provided, wherein the antibody binds human IL-6 with an affinity of about 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 15 , 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 pM.

[0248] The binding affinity (K _D ) of an IL-6 antagonist antibody for IL-6 can range from about 0.001 nM to about 200 nM. In some embodiments, the binding affinity is any of: about 200 nM, about 100 nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, about 60 pM, about 50 pM, about 20 pM , about 15 pM, about 10 pM, about 5 pM, about 2 pM, or about 1 pM. In some embodiments, the binding affinity is less than any of: about 250 nM, about 200 nM, about 100 nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, about 50 pM, about 20 pM , about 10 pM, about 5 pM, about 2 pM, about 1 pM, about 0.5 pM, about 0.1 pM, about 0.05 pM, about 0.01 pM, about 0.005 pM, or about 0.001 pM.

[0249] In some embodiments, an isolated anti-IL-6 antagonist antibody is provided, wherein the antibody binds human IL-6 with a koff of at least 5.0E-03 at 37 degrees. In some embodiments, koff is 5E-04. In some embodiments, an isolated anti-IL-6 antagonist antibody is provided, wherein the antibody binds human IL-6 to a koff that is greater than 5.0E-04 at 37 degrees.

[0250] In some embodiments, binding affinity may be defined in terms of one or more of an association constant ( _ka ), a dissociation constant (k _d ), and an analyte concentration that produces half-maximal binding capacity (KD). In some embodiments, k _a may range from about 0.50E+05 to about 5.00E+08. In some embodiments, k _d may range from about 0.50E-06 to about 5.00E-03. In some embodiments, K _D may range from about 0.50E-12 to about 0.50E-07.

[0251] In some embodiments, a pharmaceutical composition is provided comprising any of the antibodies described herein. In some embodiments, a pharmaceutical composition is provided containing any of the conjugates described herein. In some embodiments, said pharmaceutical composition contains one or more pharmaceutically acceptable carriers. In some embodiments, the pharmaceutical composition is a liquid. In some embodiments, the pharmaceutical composition has an endotoxin level of less than about 0.2 EU/ml. In some embodiments, the pharmaceutical composition is a liquid and has an endotoxin level of less than about 0.2 EU/ml. In some embodiments, the pharmaceutical composition is a liquid and has an endotoxin level of less than about 2.0, 1, 0.5, 0.2 EU/ml. In some embodiments, such as intravitreal injection, the endotoxin limit is 0.01-0.02 EU/injection/eye.

[0252] Some embodiments provide any of the following or compositions (including pharmaceutical compositions) comprising an antibody comprising a partial light chain sequence and a partial heavy chain sequence shown in Tables 1 and 2, or variants thereof. In Tables 1 and 2, the underlined sequences represent some variants of the CDR sequences presented herein.

Table 1. Anti-IL-6 heavy chain variable region sequences. CDRs are underlined.

Table 2. Anti-IL-6 light chain variable region sequences. CDRs are underlined.

ID Subsequence III DIQLTQSPSSLSASVGDRVTITC SASISVSYLY WYQQKPGKAPK LLIYDDSSLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQWSGYPYT FGQGTKVEIK(SEQ ID NO: 91) IV DIQLTQSPSSLSASVGDRVTITC SASISVSYLY WYQQKPGKAPK LLIYDASSLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQWSGYPYT FGQGTKVEIK (SEQ ID NO: 92) V DIQLTQSPSSLSASVGDRVTITC SASISVSYLY WYQQKPGKAPK LLIYDDSNLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQWSGYPYT FGQGTKVEIK (SEQ ID NO: 93)

[0253] In some embodiments, the antibody does not contain one or more (or none) of the following CDRs, Tables 3, 4, and/or 5.

Table 3

Subsequence Heavy chain CDR1 GFTFSPFAMS (SEQ ID NO: 94) CDR2 KISPGGSWTYYSDTVTG (SEQ ID NO: 95) CDR3 QLWGYYALDI (SEQ ID NO: 171) Light chain CDR1 SASISVSYMY (SEQ ID NO: 96) CDR2 DMSNLAS(SEQ ID NO: 97) CDR3 MQWSGYPYT (SEQ ID NO: 98)

Table 4

Subsequence Heavy chain CDR1 PFAMS (SEQ ID NO: 244) CDR2 KISPGGSWTYYSDTVTG (SEQ ID NO: 245) CDR3 QLWGYYALDI (SEQ ID NO: 246) Light chain CDR1 SASISVSYMY (SEQ ID NO: 247) CDR2 DMSNLAS (SEQ ID NO: 248) CDR3 MQWSGYPYT (SEQ ID NO: 249)

Table 5

Subsequence Heavy chain CDR1 GFTFSPFAMS (SEQ ID NO: 250) CDR2 WVAKISPGGSWTYYSDTVTG (SEQ ID NO: 251) CDR3 ARQLWGYYALDI (SEQ ID NO: 252) Light chain CDR1 SASISVSYMY (SEQ ID NO: 253) CDR2 LLIYDMSNLAS (SEQ ID NO: 254) CDR3 MQWSGYPYT (SEQ ID NO: 255)

[0254] In some embodiments, the composition described herein comprises an antibody comprising a partial or complete light chain sequence and a partial or complete heavy chain sequence from any of those set forth in Tables 1, 2, 6, 7, 8 and/or 9. or their variants. In some embodiments, the antibody (or binding fragment thereof) may contain any one or more of the CDRs presented in Tables 1, 2, 6, 7, 8 and/or 9. In some embodiments, the antibody (or binding fragment thereof) may contain any three or more CDRs presented in Tables 1, 2, 6, 7, 8 and/or 9. In some embodiments, an antibody (or a binding fragment thereof) may contain any or all of the six CDRs presented in Tables 1, 2, 6, 7, 8 and/or 9. In some embodiments, the heavy and/or light chain may be any one or more of the other antibody constructs provided by the present invention, including, for example, those presented in FIGS. 5, 18 and /or 21-24 and in tables 1, 3, 4, 5, 6, 7, 8 and/or 9.

[0255] In some embodiments, a composition described herein comprises an antibody comprising a partial or complete light chain CDR sequence and a partial or complete heavy chain CDR sequence from any of the options set forth in Tables 1, 2, 6, 7, 8, and /or 9.

[0256] In some embodiments, CDR portions of IL-6 antagonist antibodies are also provided. The definition of CDR regions is well known to one skilled in the art. It is understood that in some embodiments, the CDRs may be a combination of IMGT and Paratome CDRs (also referred to as “combined CDRs” or “extended CDRs”). The definition of CDR is well known to one skilled in the art. In some embodiments, the CDRs are IMGT CDRs. In some embodiments, the CDRs are Paratome CDRs. In other embodiments, the CDRs are extended CDRs, AbM, conformational CDRs, Kabat or Chotia CDRs. In embodiments with more than one CDR, the CDRs may be any of: IMGT CDR, Paratome, extended CDR, Kabat CDR, Chotia, AbM, conformational CDR, or combinations thereof. In some implementations, other CDR definitions may also be used. In some embodiments, only residues that are common to 2, 3, 4, 5, 6, or 7 definitions above are used (resulting in a shorter sequence). In some embodiments, any residue in any of 2, 3, 4, 5, 6, or 7 definitions above (resulting in a longer sequence) may be used.

[0257] In some embodiments, the anti-IL-6 antagonist antibody comprises three CDRs of any of the heavy chain variable regions presented in Tables 1, 2, 6, 7, 8 and/or 9. In some embodiments, the antibody contains three CDRs of any of light chain variable regions presented in Tables 1, 2, 6, 7, 8 and/or 9. In some embodiments, the antibody comprises three CDRs of any of the heavy chain variable regions presented in Table 1 and three CDRs of any of the light chain variable regions circuits shown in Table 2. In some embodiments, the CDRs are one or more of those listed in Tables 6 and/or 7 or 8 and/or 9 below:

Table 6. ANTI-IL-6 HEAVY CHAIN CDR SEQUENCES

ID CDR1 CDR2 CDR3 I GFTFSPFAIS (SEQ ID NO: 99) VAKISPGGSWTYYSDTVTD (SEQ ID NO: 100) ARQLWGYYALDV (SEQ ID NO: 101) II GFTFSPFAWS (SEQ ID NO: 102) VAKISPGGSWTYYSDTVTD (SEQ ID NO: 103) ARQLWGYYALDV (SEQ ID NO: 104) IIa GFTFSPFAMH (SEQ ID NO: 49) VAKISPGGSWTYYSDTVTD (SEQ ID NO: 50) ARQAWGYYALDI (SEQ ID NO: 51) IIb GFTFSPFAMH (SEQ ID NO:52) VAKISPGGSWTYYSDTVTD (SEQ ID NO:53) ARQSWGYYALDI (SEQ ID NO:54) IIc GFTFSPFAMH (SEQ ID NO:55) VAKISPGGSWTYYSDTVTD (SEQ ID NO: 56) ARQGWGYYALDI (SEQ ID NO: 57) IId GFTFSPFAMH (SEQ ID NO: 58) VAKISPGGSWTYYSDTVTD (SEQ ID NO: 59) ARQTWGYYALDI (SEQ ID NO: 60) IIe GFTFSPFAMH (SEQ ID NO: 61) VAKISPGGSWTYYSDTVTD (SEQ ID NO: 62) ARQVWGYYALDI (SEQ ID NO: 63) IIf GFTFSPFAMH (SEQ ID NO: 64) VAKISPGGSWTYYSDTVTD (SEQ ID NO: 65) ARQQWGYYALDI (SEQ ID NO: 66) IIg GFTFSPFAMH (SEQ ID NO: 67) VAKISPGGSWTYYSDTVTD (SEQ ID NO: 68) ARQKWGYYALDI (SEQ ID NO: 69)

Table 7. ANTI-IL-6 HEAVY CHAIN CDR SEQUENCES. Kabat

CDR1 CDR2 CDR3 PFAMH SEQ ID NO. 172) KISPGGSWTYYSDTVTD SEQ ID NO. 173) QAWGYYALDI SEQ ID NO. 174) PFAMH SEQ ID NO. 175) KISPGGSWTYYSDTVTD SEQ ID NO. 176) QSWGYYALDI SEQ ID NO. 177) PFAMH SEQ ID NO. 178) KISPGGSWTYYSDTVTD SEQ ID NO. 179) QGWGYYALDI SEQ ID NO. 180) PFAMH SEQ ID NO. 181) KISPGGSWTYYSDTVTD SEQ ID NO. 182) QTWGYYALDI SEQ ID NO. 183) PFAMH SEQ ID NO. 184) KISPGGSWTYYSDTVTD SEQ ID NO. 185) QVWGYYALDI SEQ ID NO. 186) PFAMH SEQ ID NO. 187) KISPGGSWTYYSDTVTD SEQ ID NO. 188) QQWGYYALDI SEQ ID NO. 189) PFAMH SEQ ID NO. 190) KISPGGSWTYYSDTVTD SEQ ID NO. 191) QKWGYYALDI SEQ ID NO. 192) PFAIS SEQ ID NO. 193) KISPGGSWTYYSDTVTD SEQ ID NO. 194) QLWGYYALDV SEQ ID NO. 195) PFAWS SEQ ID NO. 196) KISPGGSWTYYSDTVTD SEQ ID NO. 197) QLWGYYALDV SEQ ID NO. 198)

Table 8. CDR SEQUENCES OF ANTI-IL-6 LIGHT CHAIN

ID CDR1 CDR2 CDR3 III SASISVSYLY (SEQ ID NO: 105) LLIYDDSSLAS (SEQ ID NO: 106) QQWSGYPYT (SEQ ID NO: 107) IV SASISVSYLY (SEQ ID NO: 108) LLIYDASSLAS (SEQ ID NO: 109) QQWSGYPYT (SEQ ID NO: 110) V SASISVSYLY (SEQ ID NO: 111) LLIYDDSNLAS (SEQ ID NO: 112) QQWSGYPYT (SEQ ID NO: 113)

Table 9. ANTI-IL-6 LIGHT CHAIN CDR SEQUENCES

CDR1 CDR2 CDR3 SASISVSYLY SEQ ID NO. 199) DDSSLAS SEQ ID NO. 200) QQWSGYPYT SEQ ID NO. 201) SASISVSYLY SEQ ID NO. 202) DASSLAS SEQ ID NO. 203) QQWSGYPYT SEQ ID NO. 204) SASISVSYLY SEQ ID NO. 205) DDSNLAS SEQ ID NO. 206) QQWSGYPYT SEQ ID NO. 207)

[0258] In some embodiments, the antibody used to bind to IL-6 may be an antibody comprising one or more of the sequences in Tables 1, 2, 6, 7, 8, and/or 9. In some embodiments, the antibody used for binding to IL-6 may be an antibody comprising three or more sequences in Tables 1, 2, 6, 7, 8 and/or 9. In some embodiments, the antibody used to bind to IL-6 may be an antibody comprising the six sequences in any of Tables 1, 2, 6 and/or 7, 8 and/or 9. In some embodiments, the antibody that binds to IL-6 may be an antibody that competes for binding with an antibody containing 6 from the CDRs specified in any of Tables 1, 2, 6 and/or 7, 8 and/or 9.

[0259] In some embodiments, the antibody may be linked or fused to a VEGF trap sequence. In some embodiments, the trap sequence may be as shown in Table 10. In some embodiments, the sequence is at least 80% identical to the sequence shown in Table 10, for example, at least 80, 85, 90, 95 , 96, 97, 98, 99% identical to the sequence presented in Table 10. In some embodiments of the present invention, any of the VEGF decoy molecules from US Publication No. 20150376271 can be used. In some embodiments, the VEGF trap sequence is fused to IL-6 in one of the following ways: to the N terminus of the heavy chain containing IL-6 VH (Fig. 6 left), or between the hinge region and after the CH1 domain of the heavy chain containing IL -6 VH (Fig. 6 on the right). Unless otherwise noted, embodiments presented herein that involve an AT-trap fusion design provide for both alternatives and together. In some embodiments, the term “decoy” refers to the full-length extracellular region, or any portion or combination of portions thereof from various VEGF receptors that can antagonize signal transduction between at least one VEGF and VEGFR. Preferably, the extracellular segment of the trap contains at least one domain from one of VEGFR-1, -2 or -3, and more preferably at least two adjacent domains, such as D2 and D3. The extracellular domain optionally contains at least one domain from at least two different VEGFRs. The preferred extracellular domain contains or consists essentially of D2 from VEGFR-1 and D3 from VEGFR-2.

Table 10. Fusion sequence of VEGFR1 domain 2 and VEGFR2 domain 3

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK (SEQ ID NO: 114)

[0260] In some embodiments, the anti-IL-6 VEGF trap Ab construct may comprise any of the sequences presented in Table 11. In some embodiments, the construct may be at least identical to the sequences in Table 11, e.g., at 80, 85 , 90, 95, 96, 97, 98, 99% or more. In some embodiments, the fusion protein may meet these percentages except that the anti-IL-6 antibody domain does not contain one or more of the CDRs in Tables 3, 4, and/or 5. In some embodiments, the fusion protein is a fusion protein that contains one or more sequences defined in Figure 5, for example, one or more CDRs (including 2, 3, 4, 5 or 6 framed CDRs) and/or entire heavy and light chain variable regions together with a VEGF trap sequence ( for example, table 10). In some embodiments, these sequences may be directly fused to each other. In some embodiments, one or more flexible binding sequences or regions may be used. The binding sequence may be located between the AT sequence and the VEGF trap sequence. The length of these sequences can range from 5 to 30 amino acids. In some embodiments, the binding sequence may contain G and S in a ratio of about 4:1. In some embodiments, the linker contains the following sequence: GGGGSGGGGS (SEQ ID NO: 115). In some embodiments, any flexible linker may be used. In some embodiments, the Fc portion of the anti-Il-6 antibody is IgG1.

Table 11. Heavy and light chain sequences for dual inhibitor molecules. CDRs in the heavy and light chains are underlined, the VEGF trap sequence is in bold black, and the Gly-Ser linker is in italics

ID Heavy chain Light chain A SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK GGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAISWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 116) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDDSSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 117) B SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK GGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAISWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 118) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 119) C SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK GGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAISWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 120) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDDSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 121) D SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK GGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAWSWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 122) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDDSSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 123) E SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK GGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAWSWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 124) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 125) F SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK GGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAWSWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 126) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDDSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 127) G SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK GGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAMHWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQAWGYYALDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 263) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDDSSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 117) H SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK GGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAMHWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQAWGYYALDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 263) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 119) I SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK GGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAMHWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQAWGYYALDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 263) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDDSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 121) J EVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAISWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSD QGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 128) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDDSSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 129) K EVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAISWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSD QGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 130) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 131) L EVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAISWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSD QGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 132) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDDSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 133) M EVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAWSWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSD QGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 134) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDDSSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 135) N EVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAWSWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSD QGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 136) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 137) O EVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAWSWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSD QGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 138) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDDSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 139) P EVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAMHWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQAWGYYALDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQICNVNH KPSNTKVDKKVEPKSCGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQ GLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 266) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDDSSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 117) Q EVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAMHWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQAWGYYALDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQICNVNH KPSNTKVDKKVEPKSCGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQ GLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 267) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 119) R EVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAMHWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQAWGYYALDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQICNVNH KPSNTKVDKKVEPKSCGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQ GLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 268) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDDSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 121)

[0261] For the expression of anti-IL-6 antibodies and/or IL-6-VEGF trap constructs provided by the present invention, DNA fragments encoding the described VH and VL regions can first be obtained. Various modifications, such as mutations, deletions and/or additions, can also be introduced into the DNA sequence using standard methods known to one skilled in the art. For example, mutagenesis can be carried out using standard methods, such as PCR-mediated mutagenesis, in which mutated nucleotides are included in PCR primers, resulting in a PCR product containing the desired mutations, or site-directed mutagenesis.

[0262] The present invention includes modifications of the variable regions presented in the present description. For example, the present invention includes antibodies containing functionally equivalent variable regions and CDRs that do not significantly affect their properties, as well as variants having increased or decreased activity and/or affinity. For example, the amino acid sequence may be mutated to produce an antibody having the desired binding affinity for IL-6. Modification of polypeptides is routine practice in this art and need not be described in detail herein. Examples of modified polypeptides include polypeptides containing conservative substitutions of amino acid residues, one or more deletions, or additions of amino acids that do not significantly adversely affect or alter functional activity or that result in maturation (increase) of the polypeptide's affinity for its ligand, or the use of chemical analogs.

[0263] Amino acid sequence insertions include amino- and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as insertions of one or more amino acid residues within a sequence. Examples of terminal insertion include an antibody with an N-terminal methionyl residue or an antibody fused to an epitope tag. Other insertion options for an antibody molecule include fusion to the N- or C-terminus of the antibody with an enzyme or polypeptide that increases the half-life of the antibody from circulation.

[0264] In variants containing a substitution, at least one amino acid residue in the antibody molecule is removed and another residue is inserted in its place. Sites of greatest interest for substitution mutagenesis include hypervariable regions, but changes in the framework region are also contemplated. Conservative substitutions are shown in Table 12 under the heading “Conservative Substitutions.” If such substitutions result in a change in biological activity, then more significant changes, referred to as “typical substitutions” in Table 12 or further described below for amino acid classes, can be made and products screened.

Table 12. Amino acid substitutions

Original balance Conservative Substitutions Typical substitutions Ala (A) Val Val; Leu; Ile Arg(R) Lys Lys; Gln; Asn Asn(N) Gln Gln; His; Asp, Lys; Arg Asp (D) Glu Glu; Asn Cys(C) Ser Ser; Ala Gln (Q) Asn Asn; Glu Glu (E) Asp Asp; Gln Gly (G) Ala Ala His(H) Arg Asn; Gln; Lys; Arg Ile (I) Leu Leu; Val; Met; Ala; Ph; norleucine Leu (L) Ile Norleucine; Ile; Val; Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met(M) Leu Leu; Ph; Ile Phe (F) Tyr Leu; Val; Ile; Ala; Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp(W) Tyr Tyr; Phe Tyr (Y) Phe Trp; Ph; Thr; Ser Val(V) Leu Ile; Leu; Met; Ph; Ala; norleucine

[0265] Significant modifications to the biological properties of an antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the region of the substitution, such as a β-sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at target site or (c) side chain volume. Naturally occurring residues are divided into groups based on common side chain properties:

[0266] (1) Non-polar: norleucine, Met, Ala, Val, Leu, Ile;

[0267] (2) Polar uncharged: Cys, Ser, Thr, Asn, Gln;

[0268] (3) Acidic (negatively charged): Asp, Glu;

[0269] (4) Basic (positively charged): Lys, Arg;

[0270] (5) Residues affecting chain orientation: Gly, Pro; And

[0271] (6) Aromatic: Trp, Tyr, Phe, His.

[0272] Non-conservative substitutions are made by replacing a member of one of these classes with a member from another class.

[0273] One type of substitution, for example, that can be made is to replace one or more cysteine residues in the antibody, which may be reactive, with another residue, such as, but not limited to, alanine or serine. For example, a non-canonical cysteine can be replaced. The replacement may be in the CDR or framework region of the variable domain or in the constant region of the antibody. In some embodiments, cysteine is canonical. Any cysteine residue not involved in maintaining the proper conformation of the antibody can also be replaced, typically with serine, to increase the oxidative stability of the molecule and prevent cross-linking disruption. Conversely, cysteine linkage(s) may be introduced into the antibody to increase its stability, in particular when said antibody is an antibody fragment, such as an Fv fragment.

[0274] Antibodies can also be modified, for example, in the variable domains of the heavy and/or light chains, for example, to change the binding properties of the antibody. Changes in the variable region may alter binding affinity and/or specificity. In some embodiments, no more than one to five conservative amino acid substitutions are made within a CDR domain. In other embodiments, no more than one to three conservative amino acid substitutions are made within a CDR domain. For example, a mutation may be made in one or more CDRs. areas to increase or decrease the K _D of the antibody for IL-6, to increase or decrease the k _off , or to change the binding specificity of the antibody. Site-directed mutagenesis techniques are well known in the art. See, for example, Sambrook et al. and Ausubel et al., supra.

[0275] In one aspect, the IgG domain of an IL-6 antagonist antibody may be IgG1, IgG2, IgG3, or IgG4. In another aspect, the IgG domain may be a composite domain in which the constant regions are formed from more than one of the above isotypes (e.g., the CH ₁ region from IgG2 or IgG4, the hinge region, the CH ₂ and CH ₃ regions from IgG1). It is known in the art that, upon isotype selection, the human IgG1 and IgG3 isotypes have complement-mediated cytotoxicity, while the human IgG2 and IgG4 isotypes have little or no complement-mediated cytotoxicity. In some embodiments, the IL-6 antagonist antibody isotype is IgG1.

[0276] The light chain constant region can be either human lambda or kappa. In some embodiments, the anti-IL-6 antagonist antibody comprises a human kappa light chain constant region.

[0277] Human constant regions exhibit allotypic variation and isoallotypic variation across individuals, i.e. constant regions may vary between individuals at one or more polymorphic positions. Isoallotypes differ from allotypes in that the serum proteins that recognize an isoallotype bind to a non-polymorphic region of one or more other isotypes. A reference to a human constant region includes a constant region with any natural allotype or any permutation of residues occupying polymorphic positions in natural allotypes, or containing up to 3, 5 or 10 substitutions to reduce or increase effector function, as described below.

[0278] One or more amino acids at the amino or carboxy terminus of the light and/or heavy chains, such as the C-terminal lysine of the heavy chain, may be absent or derivatized in part or all of the molecules.

[0279] Substitutions can be made in constant regions to reduce or increase effector function, such as complement-mediated cytotoxicity (CMC), antibody-dependent cell-mediated cytotoxicity (ADCC) (see, for example, Winter et al., US patent No. 5624821; Tso et al., US Pat. No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to increase half-life in humans (see, for example, Hinton et al., J Biol Chem 279:6213, 2004).

[0280] In some embodiments, the IL-6 antagonist antibodies of the present invention contain one or more substitutions that reduce complement-mediated cytotoxicity. Reduced complement-mediated cytotoxicity can be achieved with or without decreased Fc receptor binding, depending on the nature of the mutation(s). Antibodies with reduced complement-mediated cytotoxicity, but little or no reduction in Fc receptor binding, provide the desired effect of Fc-mediated phagocytosis of iC3b without complement activation, which may contribute to side effects. Representative mutations in human constant regions known to reduce complement-mediated cytotoxicity include mutations at positions 241, 264, 265, 270, 296, 297, 322, 329 and 331 according to EU numbering. There is evidence that mutations at positions 318, 320 and 322 in mouse antibodies reduce complement activation. The preferred residue to occupy these positions in the mutated constant region is alanine. Some typical human antibody mutations that have been used include F241A, V264A, D265A, V296A, N297A, K322A, and P331S in human IgG3 and D270A or E, N297Q, K322A, P329A, and P331S in human IgGl (EU numbering).

[0281] Here, as elsewhere herein, the EU numbering scheme is used to number amino acids in the constant region of an antibody. When reference is made herein to a residue in a variable region (unless otherwise indicated), residue numbering corresponds to the variable domain (or, if indicated, SEQ ID NO). Substitution at any or all of positions 234, 235, 236 and/or 237 reduces affinity for Fcγ receptors, in particular the FcγRI receptor, and also reduces complement binding and activation ( see, for example , US 6624821 WO/2009/052439). Substitution to alanine at positions 234, 235 and 237 reduces effector functions, particularly in the context of human IgGl. Optionally, positions 234, 236 and/or 237 in human IgG2 are replaced by alanine, and position 235 by glutamine (see, for example, US 5624821) to reduce binding to the Fc receptor. Typical substitutions to increase half-life include Gln at position 250 and/or Leu at position 428. In accordance with one aspect of the present invention, when the provided anti-IL6 antibody is of the human IgG1 isotype, it is preferred that the antibody contains at least one mutation in the constant region. Preferably, the mutation reduces the fixation or activation of complement by the constant region. In accordance with the most preferred aspects of the present invention, the antibody contains one or more mutations at positions E233, L234, L235, G236, G237, A327, A330 and P331 according to the EU numbering. More preferably, said mutations are one or more of the following: E233P, L234V, L234A, L235A, G237A, A327G, A330S and P331S according to EU numbering. In the most preferred embodiments, human IgG1 contains the following mutations: L234A, L235A and G237A according to EU numbering.

Conjugates

[0282] The half-life of IL-6 antagonist antibodies and/or anti-IL-6-VEGF trap Ab constructs can be increased by attaching “half-life extending moieties” or “half-life extending moieties”; these terms are interchangeable herein and refer to one or more chemical groups attached to one or more amino acid side chain functional groups, such as -SH, -OH, -COOH, -CONH2, -NH2, or one or more N structures - and/or O-glycans, and which can increase the half-life of proteins/peptides from the blood in vivo when conjugated with these proteins/peptides. Examples of half-life increasing moieties include the polymers described herein, in particular polymers of zwitterionic monomers, e.g., HEMA-phosphorylcholine, PEG, biocompatible fatty acids and derivatives thereof, hydroxyalkyl starch (HAS), e.g., hydroxyethyl starch (HES), polyethylene glycol (PEG), poly(Gly _x -Ser _y ) (HAP), hyaluronic acid (HA), heparosan polymers (HEP), fleximers, dextran, polysialic acids (PSA), Fc domains, transferrin, 25 albumin, elastin-like peptides (ELP), XTEN polymers, proline-alanine-serine (PAS) polymers, polyamide (PA) polymers, albumin-binding peptides, CTP peptides, FcRn-binding peptides, and any combination thereof.

[0283] In some embodiments, the antibody is conjugated to a polymer containing phosphorylcholine. In some embodiments, the antibody is conjugated to a poly(acryloyloxyethylphosphorylcholine)-containing polymer, such as an acrylic acid polymer containing at least one acryloyloxyethylphosphorylcholine monomer, such as 2-methacryloyloxyethylphosphorylcholine (i.e., 2-methacryloyl-2'-trimethylammonium ethyl phosphate) .

[0284] In some embodiments, the antibody and/or VEGF trap antibody fusion construct is conjugated to a water-soluble polymer, which refers to a polymer that is soluble in water. The water-soluble polymer solution can transmit at least about 75%, more preferably at least about 95% of the light transmitted by the same solution after filtration. By weight, the water-soluble polymer or segment thereof may be at least about 35%, at least about 50%, about 70%, about 85%, about 95%, or 100% (by weight of dry polymer) soluble in water.

[0285] In one embodiment, the half-life extending moiety may be conjugated to IL-6 antagonist antibodies and/or an anti-IL-6 VEGF decoy Ab construct via the free amino groups of the protein using N-hydroxysuccinimide esters (NHS) . Amino conjugation reagents may randomly react with the ϵ-amino group of lysine residues, the α-amino group of N-terminal amino acids, and the δ-amino group of histidine residues.

[0286] However, the IL-6 antagonist antibodies of the present invention contain many amino groups available for conjugation to the polymer. Thus, conjugation of polymers with free amino groups may negatively affect the ability of the antibody to bind to the epitope.

[0287] In another embodiment, the half-life increasing moiety is coupled to one or more free SH groups using any suitable thiol-active chemistry, including, without limitation, maleimide chemistry or coupling of polymeric hydrazides or polymeric amines with carbohydrate moieties IL-6 antagonist antibodies and/or anti-IL-6 VEGF trap Ab constructs after pre-oxidation. The use of a maleimide combination is the most preferred embodiment of the present invention. The coupling preferably occurs at cysteine residues present naturally or introduced through genetic engineering.

[0288] In some embodiments, polymers are covalently attached to cysteine residues introduced into IL-6 antagonist antibodies and/or anti-IL-6 VEGF trap Ab constructs by site-directed mutagenesis. In some embodiments, cysteine residues in the Fc portion of an IL-6 antagonist antibody and/or an anti-IL-6 VEGF decoy Ab construct may be used. In some embodiments, sites for introducing cysteine residues into the Fc region are provided in WO 2013/093809, US 7521541, WO 2008/020827, US 8008453, US 8455622 and US2012/0213705, the contents of which are incorporated herein by reference for all purposes. In some embodiments, the cysteine mutations are Q347C and L443C, assigned to the human IgG heavy chain according to the Kabat EU index. In some embodiments, the cysteine added by site-directed mutagenesis for subsequent polymer attachment is L443C. In some embodiments, the stoichiometry of the IL-6 antagonist antibody and the polymer is 1:1; in other words, the conjugate consists essentially of molecules each containing one molecule of an IL-6 antagonist antibody and/or an anti-IL-6 VEGF trap Ab construct conjugated to one molecule of a polymer. In some embodiments, the coupling may occur at one or more lysine residues.

[0289] In some embodiments, the conjugate comprises an isolated antagonist antibody that specifically binds IL-6, or an anti-IL-6 VEGF decoy Ab construct conjugated to a polymer. In some embodiments, the polymer comprises a zwitterionic monomer. In some embodiments, said zwitterionic monomer is, without limitation, HEMA-phosphorylcholine, PEG, biocompatible fatty acids and derivatives thereof, hydroxyalkyl starch (HAS), e.g., hydroxyethyl starch (HES), polyethylene glycol (PEG), poly(Gly _x -Ser _y ) ( HAP), hyaluronic acid (HA), heparosan polymers (HEP), fleximers, dextran, polysialic acids (PSA), Fc domains, transferrin, 25 albumin, elastin-like peptides (ELP), XTEN polymers, PAS polymers, PA polymers, albumin binding peptides, CTP peptides or FcRn binding peptides. In some embodiments, the polymer containing the zwitterionic monomer is a half-life increasing moiety

[0290] In some embodiments, the anti-IL-6 antagonist antibody and/or the anti-IL-6 VEGF trap Ab construct may contain a half-life extending moiety attached. In some embodiments, the half-life extending moiety is a zwitterionic polymer, but alternatively, PEG or other half-life extending agents discussed below may be used. In some embodiments, the zwitterionic polymer is formed by monomers containing a phosphorylcholine group. In some embodiments, the monomer is 2-(acryloyloxyethyl)-2'-(trimethylammonium methyl)phosphate. In some embodiments, the monomer is 2-(methacryloyloxyethyl)-2'-(trimethylammonium methyl)phosphate (HEMA-PC).

[0291] In some embodiments, the polymer conjugated to an IL-6 antagonist antibody and/or an anti-IL-6 VEGF trap Ab construct contains at least 2 or 3 or more arms. Some polymers contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 branches. In some embodiments, the polymer contains 3, 6, or 9 branches. In some embodiments, the polymer contains 9 branches. In some embodiments, the peak molecular weight of the polymer is from 300,000 to 1,750,000 Da. In some embodiments, the polymer has a peak molecular weight of 500,000 to 1,000,000 Da. In some embodiments, the polymer has a peak molecular weight of 600,000 to 800,000 Da.

[0292] In some embodiments, a conjugate of an antagonistic anti-IL-6 antibody and/or an anti-IL-6 VEGF decoy Ab construct and a polymer is provided. In some embodiments, the polymer has a peak molecular weight of 300,000 to 1,750,000 Daltons, as measured by size exclusion chromatography-multiangle light scattering (hereinafter referred to herein as "SEC-MALS"). In some embodiments, the polymer has a peak molecular weight of 500,000 to 1,000,000 Daltons as measured by SEC-MALS. In some embodiments, the polymer has a peak molecular weight of 600,000 to 800,000 Daltons, as measured by SEC-MALS.

[0293] In some embodiments, the half-life extending moiety may be conjugated to a naturally occurring cysteine residue of an IL-6 antagonist antibody and/or an anti-IL-6-VEGF decoy Ab construct of the present invention. In some embodiments, the half-life extending moiety is conjugated to a cysteine added by site-directed mutagenesis. In some embodiments, a cysteine is added by using a recombinant DNA technique to attach an SGGGC or CAA peptide to the C-terminus of either the light or heavy chain. In some embodiments, the peptide is attached to a heavy chain. In some embodiments, the cysteine residue introduced by the recombinant DNA method is selected from the group consisting of (EU numbering) Q347C and L443C.

[0294] In accordance with another aspect of the present invention, there is provided a pharmaceutical composition comprising an anti-IL-6 antagonist antibody and/or an anti-IL-6 VEGF trap Ab construct, and a pharmaceutically acceptable excipient.

[0295] IL-6 antagonist antibodies and/or anti-IL-6 VEGF trap Ab constructs (or other constructs provided by the present invention) can be produced by recombinant expression, including (i) obtaining recombinant DNA through genetic engineering, ( ii) introducing the recombinant DNA into prokaryotic or eukaryotic cells, for example and without limitation, by transfection, electroporation or microinjection, (iii) culturing the transformed cells, (iv) expression of anti-IL-6 antibodies, for example, constitutively or by induction and (v ) isolating the anti-IL-6 antibody, for example, from a culture medium or by collecting transformed cells to (vi) obtain a purified anti-IL-6 antibody.

[0296] In some embodiments, a conjugate is provided comprising an anti-IL6 antibody and/or an anti-IL-6 VEGF decoy Ab construct and a polymer (as well as other constructs provided by the present invention, such as a bioconjugate). The antibody and/or AT trap construct is any of the antibodies and/or traps described herein. In some embodiments, the antibody is an IgG. In some embodiments, the antibody is IgA, IgE, IgD, or IgM. In some embodiments, the polymer is covalently linked to a sulfhydryl group. In some embodiments, the polymer is covalently linked to a sulfhydryl group of a cysteine residue. In some embodiments, the polymer is covalently linked to a sulfhydryl group of a cysteine residue on the heavy chain. In some embodiments, the polymer is covalently linked to the sulfhydryl group of a cysteine residue on the IgG heavy chain. In some embodiments, the antibody contains a cysteine residue at position 347 or 443 (EU numbering). In some embodiments, the polymer is covalently linked to the sulfhydryl group of a cysteine residue at position 347 or 443 (EU numbering).

[0297] IL-6 antagonist antibodies and/or an anti-IL-6 VEGF trap Ab construct can be produced by expression in a suitable prokaryotic or eukaryotic host system characterized by the production of a pharmacologically acceptable anti-IL-6 antibody molecule. Examples of eukaryotic cells are mammalian cells such as CHO, COS, HEK 293, BHK, SK-Hip and HepG2. Other suitable expression systems are prokaryotic cells (eg E. coli with the pET/BL21 expression system), yeast cells (Saccharomyces cerevisiae and/or Pichia pastoris systems) and insect cells. In some embodiments, an isolated cell line is provided that produces any of the antibodies and/or AT-trap constructs described herein. In some embodiments, said isolated cell line is selected from, without limitation, one or more of: CHO, k1SV, XCeed, CHOK1SV, GS-KO.

[0298] In some embodiments, an isolated nucleic acid encoding any of the antibodies and/or decoy constructs described herein is provided. In some embodiments, a recombinant expression vector containing an isolated nucleic acid is provided. In some embodiments, the host cell contains an expression vector.

[0299] A wide range of vectors can be used to produce IL-6 antagonist antibodies and/or anti-IL-6 VEGF trap Ab constructs and can be selected from eukaryotic and prokaryotic expression vectors. Examples of prokaryotic expression vectors include plasmids such as, but not limited to, preset, pet and pad, wherein promoters used in prokaryotic expression vectors include one or more of, but not limited to, lac, trc, trp, recA or araBAD. Examples of vectors for eukaryotic expression include, but are not limited to: (i) for expression in yeast: vectors such as but not limited to pAO, pPIC, pYES or pMET using promoters such as but not limited to AOX1, GAP, GAL1 or AUG1 ; (ii) for expression in insect cells: vectors such as but not limited to pMT, pAc5, pIB, pMIB or pBAC using promoters such as but not limited to PH, p10, MT, Ac5, OpIE2, gp64 or polh and (iii) for expression in mammalian cells: vectors such as, but not limited to, pSVL, pCMV, pRc/RSV, pcDNA3, or pBPV, and vectors derived in accordance with one aspect from viral systems, such as but not limited to , vaccinia virus, adeno-associated viruses, herpes viruses or retroviruses, using promoters such as, but not limited to, CMV, SV40, EF-1, UbC, RSV, ADV, BPV and beta-actin.

[0300] In some embodiments, a method of producing an IL-6 antagonist antibody and/or an anti-IL-6 VEGF decoy Ab construct is provided. In some embodiments, the method comprises culturing a cell line that recombinantly produces any of the antibodies and/or Ab decoy constructs described herein under conditions under which the antibody is produced and recovered. In some embodiments, a method for producing an antagonist antibody is provided. IL-6 and/or anti-IL-6 VEGF trap Ab constructs. In some embodiments, the method comprises culturing a cell line containing a nucleic acid encoding an antibody and/or an AT-trap construct comprising a heavy chain comprising the amino acid sequence set forth in Table 1 and a light chain comprising the amino acid sequence set forth in Table 2 , under conditions in which an anti-IL-6-VEGF trap antibody and/or Ab construct is produced and recovered. In some embodiments, the heavy and light chains of the anti-IL-6-VEGF Trap antibody and/or Ab constructs are encoded by separate vectors. In some embodiments, the antibody heavy and light chains and/or anti-IL-6-VEGF trap Ab constructs are encoded by a single vector.

[0301] In accordance with another aspect of the present invention, methods are provided for the synthesis of zwitterionic polymer-anti-IL-6 antagonist antibody and/or anti-IL-6 Ab-VEGF decoy conjugates, wherein the conjugate contains one or more functional agents and one or more branches polymer, wherein each of the polymer branches contains one or more monomer units, wherein at least one of these units contains a zwitterion. For example, such a method may include the steps:

a. providing an initiator containing one or more polymerization sites of a monomer and a first linker containing an amino group, said initiator being a salt of trifluoroacetic acid;

b. obtaining one or more monomers suitable for polymerization, wherein at least one of these monomers is zwitterionic;

c. reacting the monomers with an initiator to form one or more polymer branches, each corresponding to polymerization sites of the monomer, to produce an initiator-polymer conjugate containing a first amino group linker;

d. providing a second linker containing at least second and third reactive groups;

e. combining one of said second and third reactive groups of the second linker with an amino group of the first linker of the initiator-polymer conjugate to produce a linker-initiator-polymer conjugate containing one or more reactive groups not used in the coupling step; And

f. combining one or more functional agents with one or more unreacted reactive groups of a linker-initiator-polymer conjugate to form a polymer-functional agent conjugate.

[0302] In some embodiments, a conjugate is provided comprising an isolated antagonist antibody and/or an anti-IL-6-VEGF decoy Ab construct that specifically binds to IL-6, and a phosphorylcholine-containing polymer. In some embodiments, the polymer is covalently linked to the antibody and/or the AT-trap construct. In some embodiments, the polymer is non-covalently linked to the antibody and/or the AT-trap construct.

[0303] In some embodiments, a conjugate is provided comprising an anti-IL6 antibody and/or an anti-IL-6 VEGF decoy Ab construct and a phosphorylcholine-containing polymer. In some embodiments, the polymer is covalently linked to the antibody outside the variable region of the antibody. In some embodiments, a conjugate is provided comprising an anti-IL6 antibody and/or an anti-IL-6 VEGF decoy Ab construct and a phosphorylcholine-containing polymer. The polymer is covalently linked to the antibody at a cysteine residue outside the variable region of the antibody. In some embodiments, a conjugate is provided comprising an anti-IL6 antibody and/or an anti-IL-6 VEGF decoy Ab construct and a phosphorylcholine-containing polymer, wherein said polymer is covalently linked to the antibody at a cysteine residue outside the variable region of the antibody, wherein said cysteine was added by recombinant DNA. In some embodiments of the conjugate, the polymer contains 2(methacryloyloxy)ethyl (2-(trimethylammonium)ethyl)phosphate (MPC) monomers.

[0304] In accordance with another aspect of the present invention, there is provided a method in which, after synthesis of the polymer, a conjugation group (eg, maleimide) is added. It is sometimes called the "snap-on strategy" or the "universal polymer strategy." See, for example, US Patent Application No. 14/916180 (published as US Patent Application Publication No. 20160199501), the entire contents of which are incorporated herein by reference. In some embodiments, a single initiator moiety can be used for large-scale polymer synthesis. In this way, conditions can be developed to scale up optimal polymer synthesis. Such polymers can then be adapted to different types of functional agents by attaching different types of linkers. For example, if it is necessary to conjugate a larger functional agent to a polymer of the present invention, such as an antibody or even a Fab fragment, a longer linker sequence may be “locked” to said polymer. In contrast, smaller functional agents may require relatively shorter linker sequences.

[0305] In some embodiments of the methods, the initiator contains about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 polymer synthesis initiation sites. In some embodiments, the initiator contains about 3, about 6, or about 9 initiation sites for polymer synthesis.

[0306] In accordance with another aspect of the present invention, the second linker contains second, third, fourth, fifth and sixth reactive groups. More preferably, the second linker contains only the second and third reactive groups.

[0307] In accordance with one aspect of the present invention, each polymer branch contains from about 20 to about 2000 monomer units. Preferably, each branch contains from about 100 to 500 monomer units, or from about 500 to 1000 monomer units, or from about 1000 to 1500 monomer units, or from about 1500 to 2000 monomer units.

[0308] In accordance with one aspect of the present invention, the peak molecular weight of the polymer-functional agent conjugate is from about 100,000 to 1,500,000 Da. Preferably, the peak molecular weight of the polymer-functional agent conjugate is from about 200,000 to about 300,000 Daltons, from about 400,000 to about 600,000 Daltons, or from about 650,000 to about 850,000 Daltons.

[0309] In accordance with another aspect of the present invention, the first linker is preferably an alkyl substituted alkyl, alkylene, alkoxy, carboxyalkyl, haloalkyl, cycloalkyl, cyclic alkyl ether, alkenyl, alkenylene, alkynyl, alkynylene, cycloalkylene, heterocycloalkyl, heterocycloalkylene, aryl , arylene, arylenoxy, heteroaryl, amino, amido, or any combination thereof. More preferably, the first linker has the formula:

Formula 1)

where m is from 1 to 10. In some embodiments, the first linker has the formula above (Formula (1)), and m is 4.

[0310] In some embodiments, the initiator preferably comprises a structure selected from the group consisting of:

Formula (2)

Formula (3) and

Formula (4)

where X is selected from the group consisting of NCS, F, Cl, Br and I. More preferably, X in Formula (2), Formula (3) and/or Formula (4) is Br.

[0311] In some embodiments, the monomer is selected from the group consisting of:

Formula (5)

Formula (6)

Formula (7)

Formula (8) and

Formula (9)

where R7 is H or C1-6 alkyl, and t is from 1 to 6.

[0312] More preferably, the monomer is selected from the group consisting of 2-(methacryloyloxyethyl)-2'-(trimethylammonium methyl)phosphate (HEMA-PC) and 2-(acryloyloxyethyl)-2'-(trimethylammonium methyl)phosphate.

[0313] Most preferably, the monomer is 2-(methacryloyloxyethyl)-2'-(trimethylammonium methyl)phosphate.

[0314] The second linker moiety preferably comprises an activated ester having the structure

Formula (10)

where R8 is selected from the group consisting of:

And ,

and R9 represents

Formula (11)

where p is from 1 to 12.

[0315] In more preferred embodiments of the present invention, the polymer contains 9 branches, m is 2-4, R9 is

Formula (11)

where p is 4 to 15. More preferably, m is 4 and p is 12.

[0316] In some embodiments, the radically polymerizable monomer is

Formula (12)

where R1 is H or C1-6 alkyl, R2, R3, R4 are the same or different and represent H or C1-4 alkyl, and X and Y are the same or different and are integers 1-6. In some embodiments, R1, R2, R3, and R4 are each methyl, and X and Y are each equal to 2 in Formula (12).

[0317] In some embodiments, the radically polymerizable monomer is

Formula (13)

wherein R1 is H or C1-6 alkyl, R2 and R3 are the same or different and represent H or C1-4 alkyl, R4 is PO4-, SO3- or CO2-, and X and Y are the same or different and represent are integers 1-6. In some embodiments, R1, R2, and R3 are methyl, R4 is PO4-, and X and Y are each 2 in Formula (13).

[0318] In some embodiments, the monomer is

Formula (14)

where R1 is H or C1-6 alkyl, R2, R3 and R4 are the same or different and are H or C1-4 alkyl, R5 is PO4-, SO3- or CO2-, and X and Y are the same or different and represent integers 1-6. In some embodiments, R1, R2, R3, and R4 are methyl, R5 is PO4-, and X and Y are 2 in Formula (14).

[0319] When a polymer is conjugated via a cysteine (or other specified residue), the polymer may be linked to said residue directly or indirectly (eg, through an intermediate initiator and/or spacer, etc.).

[0320] In some embodiments, the phosphorylcholine-containing polymer contains 2-(methacryloyloxyethyl)-2'-(trimethylammonium)ethyl phosphate (MPC) monomers, as follows:

Formula (15),

such that said polymer contains the following repeating units:

where n is an integer from 1 to 3000 and the wavy lines indicate the points of attachment between monomer units in the polymer.

[0321] In some embodiments, the polymer contains three or more branches, or is synthesized using an initiator containing 3 or more polymer synthesis initiation sites. In some embodiments, the polymer contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 branches or is synthesized using an initiator containing 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11 or 12 initiation centers for polymer synthesis. More preferably, the polymer contains 3, 6 or 9 branches or is synthesized using an initiator containing 3, 6 or 9 polymer initiation sites. In some embodiments, the polymer contains 9 branches or is synthesized using an initiator containing 9 initiation sites for polymer synthesis.

[0322] In some embodiments, the attachable polymer has a molecular weight of from about 300,000 to about 1,750,000 Da (SEC-MALs). In some embodiments, the polymer has a molecular weight of from about 500,000 to about 1,000,000 Da. In some embodiments, the polymer has a molecular weight of from about 600,000 to about 900,000 Da. In some embodiments, the polymer has a molecular weight of from about 750,000 to about 850,000 Da. In some embodiments, the polymer has a molecular weight of from about 800,000 to about 850,000 Da. In some embodiments, the polymer has a molecular weight of from about 750,000 to about 800,000 Da.

[0323] In some embodiments, any of the antibodies and/or Ab decoy constructs described herein can be further conjugated to a polymer to form a bioconjugate. The molecular weight of said bioconjugate (total, SEC-MALs) may be from about 350,000 to 2,000,000 Dalton, for example, from about 450,000 to 1,900,000 Dalton, from about 550,000 to 1,800,000 Dalton, from about 650,000 to 1,700,000 Dalton, from about 75,000 0 to 1600000 Dalton, from about 850,000 to 1,500,000 Dalton, from about 900,000 to 1,400,000 Dalton, from about 950,000 to 1,300,000 Dalton, from about 900,000 to 1,000,000 Dalton, from about 1,000,000 to 1,300,000 Dalton, from about 850,000 to 13 00000 Dalton, from about 850000 to 1000000 Dalton and from approximately 1,000,000 to 1,200,000 Dalton. In some embodiments, the bioconjugate has a molecular weight of from about 350,000 to 1,900,000 Daltons.

[0324] In some embodiments, the antibody and/or AT-trap construct is purified. In some embodiments, the polymer in the conjugate aspect of the antibody and/or AT-trap construct is polydisperse, i.e. The PDI of the polymer is not 1.0. In some embodiments, the PDI is less than 1.5. In some embodiments, the PDI is less than 1.4. In some embodiments, the PDI is less than 1.3. In some embodiments, the PDI is less than 1.2. In some embodiments, the PDI is less than 1.1. In some embodiments, the PDI of the conjugate is equal to or less than 1.5.

[0325] In some embodiments, the antibody conjugate and/or Ab-trap construct comprises an anti-IL-6 immunoglobulin G (IgG) linked to a polymer, wherein the polymer comprises MPC monomers, wherein the anti-IL-6 heavy chain sequence is presented in Table 1, and the anti-IL-6 light chain sequence is presented in Table 2, and the antibody and/or AT-trap construct is bound to the polymer only at C442. In some embodiments, the polymer contains 9 branches and has a molecular weight of from about 600,000 to about 1,000,000 Da.

[0326] In some embodiments, the antibody conjugate and/or Ab-trap construct comprises an anti-IL-6 immunoglobulin G (IgG) linked to a polymer, wherein the polymer comprises MPC monomers, wherein the anti-IL-6 heavy chain sequence is contained in Table 1, and the anti-IL-6 light chain sequence is contained in Table 2, and the antibody is bound to the polymer only at C443 (EU numbering). In some embodiments, the polymer contains 9 branches and has a molecular weight of from about 600,000 to about 1,000,000 Da. In some embodiments, the conjugate comprises a 9-arm polymer, and the polymer has a molecular weight of from about 600,000 to about 900,000 Da.

[0327] In some embodiments, the antibody conjugate has the following structure:

where: each anti-IL-6 antibody heavy chain is designated by the letter H and each anti-IL-6 antibody light chain is designated by the letter L (which in some embodiments may also be associated with the AT-trap structure presented in FIG. 6);

the polymer is linked to the anti-IL-6 antibody (and/or decoy construct) via the sulfhydryl group C443 (EU numbering); the specified bond is depicted on one of the heavy chains; PC represents

,

where the wavy line shows the point of attachment to the rest of the polymer; where X=a) OR, where R=H, methyl, ethyl, propyl, isopropyl, b) H or c) any halide including Br; and n1, n2, n3, n4, n5, n6, n7, n8 and n9 are the same or different so that the sum of n1, n2, n3, n4, n5, n6, n6, n7, n8 and n9 is 2500 plus or minus 10%. In some embodiments, n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 3000. In some embodiments, n1, n2, n3, n4, n5, n6 , n7, n8, and n9 are the same or different and are integers from 0 to 500. In some embodiments, X=OR, wherein R is sugar, aminoalkyl, monosubstituted, polysubstituted, or unsubstituted variants of the following residues: saturated C ₁ -C ₂₄ alkyl, unsaturated C ₂ -C ₂₄ alkenyl or C ₂ -C ₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonyl acyl, amino, aminocarbonyl, aminocarboyl oxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano and halogenated alkyl, including polyhalogenated alkyl, --CO--O--R ₇ , carbonyl --CCO--R ₇ , --CO-- NR ₈ R ₉ , --(CH ₂ ) _n --COOR ₇ , --CO--(CH) _n --COOR ₇ , --(CH ₂ ) _n --NR ₈ R ₉ , ester, alkoxycarbonyl, aryloxycarbonyl, where n is an integer from 1 to 6, wherein each of R ₇ , R ₈ and R ₉ is separately selected from the group consisting of a hydrogen atom, a halogen atom, monosubstituted, polysubstituted or unsubstituted variants of the following residues: saturated C ₁ -C ₂₄ alkyl, unsaturated C ₂ -C ₂₄ alkenyl or C ₂ -C ₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacel, amino, aminocarbonyl, aminocar boyiloxy , nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano and halogenated alkyl, including polyhalogenated alkyl, 5-membered ring and 6-membered ring. In some embodiments, Formula 17 may be a portion of a fusion protein that will further comprise part or all of the sequence from Table 10 to thereby produce an anti-IL-6 Ab-VEGF trap fusion protein. In some embodiments, the fusion construct in FIG. 27 may be used in Formula 17, wherein the fusion construct replaces the antibody depicted.

[0328] In some embodiments, the antibody conjugate has the following structure:

Where:

each antibody heavy chain is designated by the letter H, and each anti-IL-6 antibody light chain is designated by the letter L (which in some embodiments may also be associated with the location of the AT-trap, as shown in FIG. 6);

the polymer is linked to the antibody through the sulfhydryl group C443 (EU numbering); the specified bond is depicted on one of the heavy chains;

PC represents

where the wavy line shows the point of attachment to the rest of the polymer, where X=a) OR, where R=H, methyl, ethyl, propyl, isopropyl, b) H or c) any halide including Br; And

n1, n2, n3, n4, n5, n6, n7, n8 and n9 are the same or different so that the sum of n1, n2, n3, n4, n5, n6, n6, n7, n8 and n9 is 2500 plus or minus 15 %. In some embodiments, the sum of n1, n2, n3, n4, n5, n6, n6, n7, n8, and n9 is from about 1500 to about 3500, plus or minus about 10% to about 20%. In some embodiments, the fusion construct of FIG. 27 may be used in Formula 17, wherein the fusion construct replaces the antibody shown.

[0329] In some embodiments, the antibody conjugate and/or the AT-trap construct is in a liquid formulation. In some embodiments, the antibody conjugate and/or the Ab-trap construct is combined with a pharmaceutically acceptable carrier.

[0330] In some embodiments, the heavy chain isotype of the anti-IL-6 antibody (and/or anti-IL-6 VEGF Trap Ab construct) is IgG1 and comprises a CH ₁ domain, a hinge domain, a CH ₂ - and a CH ₃ - domain. domains. In some embodiments, the light chain isotype is a kappa isotype.

[0331] In some embodiments, the IgG1 domain of an anti-IL-6 antibody (and/or anti-IL-6 VEGF trap Ab constructs) contains one or more mutations to modulate an effector function such as ADCC, antibody-dependent cellular phagocytosis (ADCP) and KOTS. In some embodiments, said IgG1 mutations reduce effector function. In some embodiments, the amino acids used for mutations for effector function include (EU numbering) E233X, L234X, L235X, G236X, G237X, G236X, D270X, K322X, A327X, P329X, A330X, A330X, P331X, and P331X, where X represents any natural or unnatural amino acid. In some embodiments, the mutations include one or more of the following: E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S (EU numbering). In some embodiments, the anti-IL-6 heavy chain contains the following mutations (EU numbering): L234A, L235A, and G237A. In some embodiments, the number of mutations for an effector function relative to a natural human IgG1 sequence is no more than 10. In some embodiments, the number of mutations for an effector function relative to a natural human IgG1 sequence is no more than 5, 4, 3, 2, or 1. In some embodiments, the antibody (and/or anti-IL-6 VEGF-trap Ab) exhibits a decrease in Fc-gamma binding and/or complement C1q binding, thereby reducing the ability of the antibody to mediate effector function. This may be especially beneficial for ophthalmic symptoms/disturbances.

[0332] In some embodiments, the anti-IL6 antibody (and/or anti-IL-6 VEGF trap Ab) contains one or more of the following amino acid mutations: L234A, L235A, G237A, and L443C (EU numbering, or 451A, 452A, 454A, and 660C in SEQ ID NO: 170, as indicated by the double underline in FIG. 27).

[0333] In some embodiments, the anti-IL6 antibody (and/or anti-IL-6 VEGF trap Ab) is or is a portion of human immunoglobulin G (IgG1).

[0334] In some embodiments, the anti-IL-6 antibody (and/or the anti-IL-6 VEGF trap Ab) comprises a heavy chain constant domain containing one or more mutations that reduce immune-mediated effector function.

[0335] In some embodiments, the anti-IL-6 heavy chain comprises a cysteine residue mutated by recombinant DNA that can be used to conjugate a half-life extending moiety. In some embodiments, the mutation is Q347C (EU numbering) and/or L443C (EU numbering). In some embodiments, the mutation is L443C (EU numbering). In some embodiments, the stoichiometry of antibody and polymer is 1:1; in other words, the conjugate contains one antibody molecule conjugated to one polymer molecule.

[0336] The half-life of anti-IL-6 antibodies can be increased by attaching "half-life extending moieties" or "half-life extending moieties". Moieties that increase half-life include peptides and proteins, which may be expressed in frame with the biological drug in question (or chemically conjugated as appropriate), and various polymers, which may be attached or conjugated to one or more pendant functional groups. amino acid chains or terminal functional groups such as -SH, -OH, -COOH, -CONH2, -NH2, or one or more N- and/or O-glycan structures. Half-life extending fragments generally act to increase the half-life of biological drugs from the blood in vivo .

[0337] Examples of peptide/protein fragments that increase half-life include Fc fusion (Capon DJ, Chamow SM, Mordenti J, et al. Designing CD4 immunoadhesions for AIDS therapy. Nature. 1989. 337:525-31), fusion with human serum albumin (HAS) (Yeh P, Landais D, Lemaitre M, et al. Design of yeast-secreted albumin derivatives for human therapy: biological and antiviral properties of a serum albumin-CD4 genetic conjugate. Proc Natl Acad Sci USA. 1992 . 89:1904-08), fusion with carboxy-terminal peptide (CTP) (Fares FA, Suganuma N. Nishimori K., et al. Design of a long-acting follitropin agonist by fusing the C-terminal sequence of the chorionic gonadotropin beta subunit to the follitropin beta subunit. Proc Natl Acad Sci USA. 1992. 89:4304-08), fusion with a loosely repeated peptide sequence by genetic engineering (XTEN) (Schellenberger V, Wang CW, Geething NC, et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat Biotechnol. 2009. 27:1186-90), elastin-like peptide (ELP) (ELP fusion) (MCpherson DT, Morrow C, Minehan DS, et al. Production and purification of a recombinant elastomeric polypeptide, G(VPGVG19-VPGV, from Escheriachia coli Biotechnol Prog. 1992. 8:347-52), fusion with human transferrin (Prior CP, Lai CH, Sadehghi H et al. Modified transferrin fusion proteins. patent WO2004/020405. 2004), proline-alanine-serine (PASylation) (Skerra A, Theobald I, Schlapsky M. Biological active proteins having increased in vivo and/or vitro stability. Patent WO2008/155134 A1. 2008), homoamino acid polymer (HAPylation) (Schlapschy M, Theobald I, Mack H, et al. Fusion of a recombinant antibody fragment with a homo-amino acid polymer: effects on biophysical properties and plasma prolonged half-life. Protein Eng Des Sel. 2007. 20:273-84) and fusion with gelatin-like protein (GLK) (Huang YS, Wen XF, Zaro JL, et al. Engineering a pharmacologically superior form of granulocyte-colony-stimulating-factor by fusion with gelatin-like protein polymer. Eur J. Pharm Biopharm. 2010. 72:435-41).

[0338] Examples of half-life extending polymer moieties include polyethylene glycol (PEG), branched PEG, PolyPEG® (Warwick Effect Polymers; Coventry, UK), polysialic acid (PSA), starch, hydroxyethyl starch (HES), hydroxyalkyl starch (HAS) ), carbohydrate, polysaccharides, pullulan, chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, dextran, carboxymethyldextran, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG), polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, polyphosphazene, polyoxazoline, ethylene-maleic anhydride copolymer, styrene-maleic anhydride copolymer, poly(1-hydroxymethylenehydroxymethylformal) (PHF), zwitterionic polymer, phosphorylcholine-containing polymer and MPC-containing polymer, poly(Gly _x -Ser _y ) , hyaluronic acid (HA), heparosan polymers (HEP), fleximers, dextran and polysialic acids (PSA).

[0339] In one embodiment, the half-life extending moiety can be conjugated to the antibody via free amino groups of the protein using N-hydroxysuccinimide (NHS) esters. Amino conjugation reagents may randomly react with the ε-amino group of lysine residues, the α-amino group of N-terminal amino acids, and the δ-amino group of histidine residues.

[0340] However, the anti-IL-6 antibodies (and/or anti-IL-6 VEGF trap Ab constructs) described herein may contain many amino groups available for conjugation to the polymer. Thus, conjugation of polymers with free amino groups may negatively affect the ability of antibody proteins to bind to IL-6 (and/or anti-IL-6 VEGF-trap Ab constructs).

[0341] In some embodiments, the half-life increasing moiety is coupled to one or more free SH groups using any suitable thiol-active chemistry, including, without limitation, maleimide chemistry or coupling of polymeric hydrazides or polymeric amines with carbohydrate moieties antibodies after preliminary oxidation. In some embodiments, a combination using maleimide is used. In some embodiments, the coupling occurs at cysteine residues that are naturally present or introduced through genetic engineering.

[0342] Some embodiments provide conjugates of an antibody and high molecular weight polymers that serve as half-life extending agents. In some embodiments, the conjugate comprises an antibody (and/or anti-IL-6-VEGF trap Ab construct) coupled to a zwitterionic polymer, wherein the polymer is formed from one or more monomeric units, and wherein at least one monomeric unit contains a zwitterionic group. In some embodiments, said zwitterionic group is phosphorylcholine.

[0343] In some embodiments, one of the monomer units is HEMA-PC. In some embodiments, the polymer is synthesized from a single monomer, which is HEMA-PC.

[0344] In some embodiments, certain antibody conjugates (and/or anti-IL-6-VEGF trap Ab constructs) contain 2, 3, or more polymer arms in which the monomer is HEMA-PC. In some embodiments, the conjugates contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 polymer arms in which the monomer is HEMA-PC. In some embodiments, the conjugates contain 3, 6, or 9 branches. In some embodiments, the conjugate contains 9 branches.

[0345] In some embodiments, the polymer-antibody (and/or polymer-anti-IL-6-VEGF trap Ab) conjugates contain a polymer moiety with a molecular weight of 100,000 to 1,500,000 Da. In some embodiments, the conjugate contains a polymer moiety with a molecular weight of 500,000 to 1,000,000 Da. In some embodiments, the conjugate contains a polymer moiety with a molecular weight of 600,000 to 800,000 Da. In some embodiments, the conjugate contains a polymer moiety with a molecular weight of 600,000 to 850,000 Da and contains 9 branches. When the molecular weight is given for an antibody conjugated to a polymer, the stated molecular weight is the sum of the molecular weight of the protein, including any associated carbohydrate moieties, and the molecular weight of the polymer.

[0346] In some embodiments, an anti-IL6 antibody (and/or a VEGF-trap anti-IL-6 Ab construct) is provided that comprises a HEMA-PC polymer having a molecular weight, measured as Mw, of from about 100 kDa to 1650 kDa . In some embodiments, the molecular weight of the polymer, measured as Mw, is from about 500 kDa to 1000 kDa. In some embodiments, the molecular weight of the polymer, measured as Mw, is from about 600 kDa to about 900 kDa. In some embodiments, the molecular weight of the polymer, measured as Mw, is 750 or 800 kDa plus or minus 15%.

[0347] In some embodiments, the polymer is prepared from an initiator suitable for ATRP containing one or more initiation sites for polymer synthesis. In some embodiments, the polymer synthesis initiation site comprises a 2-bromoisobutyrate center. In some embodiments, the initiator contains 3 or more initiation sites for polymer synthesis. In some embodiments of the methods, the initiator contains 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 initiation sites for polymer synthesis. In some embodiments, the initiator contains 3, 6, or 9 polymer synthesis initiation sites. In some embodiments, the initiator contains 9 polymer synthesis initiation sites. In some embodiments, the initiator is OG1786.

[0348] Anti-IL-6 antibodies (and/or anti-IL-6 VEGF-trap Ab constructs) can be produced by recombinant expression involving (i) producing recombinant DNA through genetic engineering, (ii) introducing recombinant DNA into prokaryotic or eukaryotic cells, for example and without limitation, by transfection, electroporation or microinjection, (iii) culturing the transformed cells, (iv) expressing the antibody, such as constitutively or upon induction, and (v) isolating the antibody, such as from culture medium or by collecting transformed cells to (vi) obtain purified antibody.

[0349] Anti-IL-6 antibodies (and/or anti-IL-6 VEGF trap Ab constructs) can be produced by expression in a suitable prokaryotic or eukaryotic host system characterized by the production of a pharmacologically acceptable antibody molecule. Examples of eukaryotic cells are mammalian cells such as CHO, COS, HEK 293, BHK, SK-Hip and HepG2. Other suitable expression systems are prokaryotic cells (eg E. coli with the pET/BL21 expression system), yeast cells (Saccharomyces cerevisiae and/or Pichia pastoris systems) and insect cells.

[0350] A wide variety of vectors can be used to produce the antibodies (and/or anti-IL-6-VEGF trap Ab constructs) described herein, and are selected from eukaryotic and prokaryotic expression vectors. Examples of prokaryotic expression vectors include plasmids such as, but not limited to, preset, pet and pad, wherein promoters used in prokaryotic expression vectors include one or more of, but not limited to, lac, trc, trp, recA or araBAD. Examples of eukaryotic expression vectors include: (i) for expression in yeast: vectors such as but not limited to pAO, pPIC, pYES or pMET using promoters such as but not limited to AOX1, GAP, GAL1 or AUG1; (ii) for expression in insect cells: vectors such as but not limited to pMT, pAc5, pIB, pMIB or pBAC using promoters such as but not limited to PH, p10, MT, Ac5, OpIE2, gp64 or polh and (iii) for expression in mammalian cells: vectors such as, but not limited to, pSVL, pCMV, pRc/RSV, pcDNA3, or pBPV, and vectors derived in accordance with one aspect from viral systems, such as but not limited to , vaccinia virus, adeno-associated viruses, herpes viruses or retroviruses, using promoters such as, but not limited to, CMV, SV40, EF-1, UbC, RSV, ADV, BPV and beta-actin.

[0351] In some embodiments, there is at least one polymer per protein. In some embodiments, the protein to polymer ratio is from 1:4 to 1:6. In some embodiments, the ratio of protein (Ab, AT) to polymer is a 1:5 molar ratio. In some embodiments, the amount of protein is 5, 4, 3, 2, or about 2.4 mg/ml.

[0352] In some embodiments, purification after combining the polymer and antibody also includes the use of a cation exchange column.

[0353] In some embodiments, the presence of a polymer attached to IL-6 antagonist antibodies and/or anti-IL-6 VEGF Trap Ab constructs may provide greater efficacy due to the presence of the polymer itself. In some embodiments, the presence of a polymer, when used in combination with a heparin binding domain (such as in VEGF), produces a conjugated molecule with higher potency. In some embodiments, the molecule has superior angiogenesis efficacy, as determined by the EHR data in the examples below. In some embodiments, the difference is in the nature of the conjugate molecule rather than simply in degree (eg, comparing a conjugate to unconjugated molecules). Moreover, as demonstrated in the ECHR proliferation assays in the examples below, in some embodiments, the presence of the polymer provides synergistic inhibition.

[0354] Additionally, in some embodiments, as the cell density increases (eg, from 4000 to 6000), the potency of the conjugate may also increase.

[0355] In some embodiments, the amount (by weight of protein) of the conjugate used is greater than 50 mg/ml. It may be, for example, 55, 60, 65, 70 mg/ml or more (based on the weight of the protein before conjugation).

Method for conjugating proteins with polymers

[0356] In some embodiments, a method is provided for preparing a therapeutic protein-half-life extending moiety conjugate, comprising the step of conjugating a therapeutic protein containing a recombinantly DNA-linked cysteine residue to a half-life extending moiety containing a sulfhydryl-specific reactive moiety selected from the group consisting of maleimide, vinyl sulfones, orthopyridyl disulfides and iodoacetamides to produce a therapeutic protein-fragment conjugate that increases the half-life.

[0357] In some embodiments, a method for producing an antibody conjugate (and/or anti-IL-6-VEGF decoy Ab construct) is provided. As shown in FIG. 10, the method involves reducing the protein using a 30x molar excess of the reducing agent TCEP (FIG. 10). After reduction, the antibody is oxidized to produce a deblocked antibody in which the disulfide bonds between and within the light and heavy chains naturally occurring in the antibody are formed, but the engineered cysteine at position L443C of the heavy chain (EU numbering) has yet to be deblocked (Figure 10). The antibody is then conjugated by adding an excipient and adding a 2-10x molar excess of maleimide biopolymer (FIG. 10). The specified biopolymer binds to the antibody by a covalent thiol ether bond (Fig. 10). After conjugation, the antibody conjugate is purified to remove both unconjugated antibody and polymer (Figure 10). In embodiments in which the protein is an AT-trap construct, the same position on the antibody can be used and the same approach can be used.

[0358] The protein and method described above can also be varied. Thus, in some embodiments, a method for producing a conjugated protein (which need not be an antibody or anti-IL6 antibody) is provided. The method involves reducing one or more cysteine residues in a protein to form a deprotected protein in solution. After reduction of said one or more cysteine residues, the deprotected protein is re-oxidized to reduce at least one disulfide bond in the reduced protein while retaining the engineered cysteine residue in the protein in the form of a free thiol to form a re-oxidized deprotected protein in solution. At least one excipient is then added to the solution. This excipient reduces polymer-induced protein precipitation. After adding the excipient, a polymer is added to the solution and conjugated to the re-oxidized deprotected protein at the engineered cysteine residue to form the conjugated protein.

[0359] In some embodiments, the molar excess of the reducing agent can be changed to any value that performs its function. In some embodiments, a 10, 20, 30, 40, 50, 60, 70, 80, 90x molar excess of reducing agent (which need not be TCEP in all embodiments) may be used. In some embodiments, the reducing agent may be trisodium 3,3',3''-phosphinetriyl tris(benzene-1-sulfonate) (TPPTS). In some embodiments, any antibody (therapeutic or otherwise) can be used. In some embodiments, a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15x molar excess of maleimide biopolymer may be used. In some embodiments, there is an excess of deprotected protein relative to the polymer. In some embodiments, the amount of protein recovered is less than the amount of polymer. In some embodiments, the amount of protein recovered is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1% of amount of polymer. In some embodiments, 10-15 times more polymer is used than protein. In some embodiments, the amount of reduced antibody is greater than the amount of polymer. In some embodiments, the amount of polymer is greater than the amount of reconstituted antibody (and/or anti-IL-6-VEGF-trap Ab construct).

[0360] In some implementations, the cleanup step is optional.

[0361] In some embodiments, a method of producing an antibody conjugate (and/or a decoy Ab construct) comprises conjugating an anti-IL-6 antibody (and/or an anti-IL-6 Ab decoy VEGF construct) to a phosphorylcholine-containing polymer. In some embodiments, the method includes the steps of conjugating an anti-IL-6 antibody to a polymer containing phosphorylcholine. Anti-IL6 antibody contains an amino acid residue attached by recombinant DNA. In some embodiments, said attached amino acid residue is a cysteine residue. In some embodiments, said cysteine residue is attached outside the variable region of the antibody. The cysteine residue can be attached to either the heavy chain or the light chain of the antibody.

[0362] In some embodiments, the polymer contains or consists of a polymer containing phosphorylcholine. In some embodiments, the phosphorylcholine-containing polymer contains a sulfhydryl-specific reactive group selected from the group consisting of maleimide, vinyl sulfone, orthopyridyl disulfide, and iodoacetamide. In some embodiments, said sulfhydryl-specific reactive group in the phosphorylcholine-containing polymer reacts with a cysteine residue in the anti-IL-6 antibody to form an antibody conjugate (and/or an anti-IL-6-VEGF decoy Ab construct).

[0363] In some embodiments, the conjugated protein may be an antibody, an antibody fusion protein (e.g., an anti-IL-6 VEGF Trap Ab construct), or a binding fragment thereof. In some embodiments, the protein is not an antibody, but rather an enzyme, ligand, receptor or other protein, or mutants or variants thereof. In some embodiments, the native protein contains at least one disulfide bond and at least one non-native cysteine.

[0364] In some embodiments, the excipient may be an acid or a base. In some embodiments, the excipient is a detergent, sugar, or charged amino acid. In some embodiments, the excipient helps keep the protein in solution during conjugation with the polymer. In some embodiments, the excipient is added to the solution containing the protein before adding the polymer to the solution containing the protein.

[0365] In some embodiments, the reaction is conducted under aqueous conditions, pH from about pH 5 to about pH 9. In some embodiments, the reaction is conducted from 6.0 to 8.5, 6.5 to 8.0, or 7. 0 to 7.5.

[0366] In some embodiments, the polymer is conjugated to the protein at 2-37 degrees Celsius. In some embodiments, conjugation is performed at 0-40 degrees Celsius, 5-35 degrees Celsius, 10-30 degrees Celsius, and 15-25 degrees Celsius.

[0367] In some embodiments, the conjugated proteins described herein may be contacted with an ion exchange or hydrophobic interaction chromatography or affinity chromatography medium for purification (to separate conjugated from unconjugated). In some embodiments, said ion exchange, hydrophobic interaction chromatography, and/or affinity chromatography media allows separation of the conjugated protein from the unconjugated free polymer and from the unconjugated re-oxidized released protein.

[0368] In some embodiments, the methods described herein and summarized in FIG. 10 include an excipient that can promote and/or maintain the solubility system. In some embodiments, the method ensures that the solution maintains the solubility of two interacting components. Solubility may include the solubility of the protein and the polymer, and then also the final conjugate. In some embodiments without an excipient approach, the problem may be that although the protein is soluble, when a biopolymer is added, the solubility in the solution (eg, the protein) decreases and it precipitates out of solution. Of course, once the protein precipitates, it cannot be effectively conjugated to the biopolymer. Thus, an excipient can be used to maintain protein solubility in the presence of the biopolymer so that the two can combine to form a protein conjugate (or, as shown in FIG. 10, an antibody conjugate). This also helps maintain the solubility of the conjugate.

[0369] In some embodiments, the polymers described herein may contain one or more of the following: a zwitterion, a phosphorylcholine, or a PEG linker bridging the center of the polymer's branch point to a maleimide functionality. In some embodiments, any of the polymers provided by the present invention may be attached to a protein by the methods provided by the present invention.

[0370] In some embodiments, any of the proteins provided by the present invention may be conjugated to any of the polymers provided by the present invention by one or more methods provided by the present invention.

[0371] In some embodiments, the method(s) provided by the present invention allows for a larger scale process for the production and purification of protein and/or antibody conjugates. In some embodiments, the volume used is at least 1 liter, such as 1, 10, 100, 1000, 5000, 10,000 liters or more. In some embodiments, the amount of the antibody conjugate produced and/or purified may be 0.1, 1, 10, 100, 1000, or more grams.

[0372] In some embodiments, the therapeutic protein may be any of the anti-IL-6 antibodies (and/or anti-IL-6 VEGF trap Ab constructs) described herein containing a recombinantly DNA-linked cysteine residue. In some embodiments, the anti-IL-6 antibody heavy chain contains the following CDRs: CDR _H 1: in Table 1, CDR _H 2: in Table 1, and CDR _H 3: in Table 1. The heavy chain may also contain arginine (R) in position 84 (according to sequential numbering). In some embodiments, the anti-IL-6 light chain comprises the following CDRs: CDR _L 1: in Table 2, CDR _L 2: in Table 2, and CDR _L 3: in Table 2.

[0373] In some embodiments, the anti-IL6 antibody (and/or anti-IL-6 VEGF trap Ab) comprises IgG1. In some embodiments, the heavy chain contains one or more mutations to modulate effector function. In some embodiments, the mutations are at one or more of the following amino acid positions (EU numbering): E233, L234, L235, G236, G237, A327, A330, and P331. In some embodiments, the mutations are selected from the group consisting of: E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S (EU numbering). In some embodiments, the mutations are (EU numbering) L234A, L235A, and G237A.

[0374] In some embodiments, the cysteine residue attached to the therapeutic protein by recombinant DNA should not be involved in the formation of Cys-Cys disulfide bonds. In this regard, therapeutic proteins may be dimeric. For example, an intact anti-IL6 antibody (and/or anti-IL-6 VEGF trap antibody) contains two light chains and two heavy chains. If a Cys residue is introduced into the heavy chain, for example, the intact antibody will contain two such introduced cysteine residues in identical positions, and it is likely that these cysteine residues will form intrachain disulfide bonds. If the introduced cysteine residues form Cys-Cys disulfide bonds or are prone to do so, the introduced Cys residue will not be suitable for conjugation. It is known in the art to avoid positions in the heavy and light chains that result in the formation of intrachain disulfide bonds. See, for example, US Patent Application No. 2015/0158952.

[0375] In some embodiments, the cysteine residue introduced by the recombinant DNA method is selected from the group consisting of (EU numbering) Q347C and L443C. In some embodiments, the cysteine residue is L443C (EU numbering). In some embodiments, the antibody heavy chain comprises the amino acid sequence shown in Table 1 and the light chain contains the amino acid sequence shown in Table 2.

[0376] In some embodiments, the sulfhydryl-specific reactive group is a maleimide.

[0377] In some embodiments, the half-life increasing moiety is selected from the group consisting of polyethylene glycol (PEG), branched PEG, PolyPEG ® (Warwick Effect Polymers; Coventry, UK), polysialic acid (PSA), starch, hydroxyethyl starch (HES ), hydroxyalkyl starch (HAS), carbohydrate, polysaccharides, pullulan, chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, dextran, carboxymethyldextran, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG), polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (P VA) , polycarboxylate, polyvinylpyrrolidone, polyphosphazene, polyoxazoline, ethylene-maleic anhydride copolymer, styrene-maleic anhydride copolymer, poly(1-hydroxymethylenehydroxymethylformal) (PHF), zwitterionic polymer, phosphorylcholine-containing polymer, and 2-methacryloyloxy-2-containing polymer '-ethyltrimethylammonium phosphate (MPC).

[0378] In some embodiments, the half-life increasing moiety is a zwitterionic polymer. In some embodiments, the zwitterion is phosphorylcholine, i.e. a polymer containing phosphorylcholine. In some embodiments, said polymer consists of MPC units.

[0379] In some embodiments, the MPC polymer contains three or more branches. In some embodiments, the MPC polymer contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 branches. In some embodiments, the MPC polymer contains 3, 6, or 9 branches. In some embodiments, the MPC polymer contains 9 branches. In some embodiments, the polymer is synthesized using an initiator containing 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more polymer synthesis initiation sites.

[0380] In some embodiments, the MPC polymer has a molecular weight of from about 300,000 to 1,750,000 Da. In some embodiments, the MPC polymer has a molecular weight of from about 500,000 to 1,000,000 Daltons, or from about 600,000 to 900,000 Daltons.

[0381] In some embodiments, a method of producing a therapeutic protein-fragment half-life extending conjugate includes the additional step of contacting the therapeutic protein with a thiol reducing agent under conditions to produce a reduced sulfhydryl group of the cysteine. As discussed above, it is preferable that the cysteine residues added by recombinant DNA be unpaired, i.e. were not involved in intrachain Cys-Cys disulfide bonds or were essentially not involved in the formation of such bonds. However, it is known that Cys residues that are not involved in the formation of such Cys-Cys disulfide bonds and are free for conjugation react with free cysteine in the culture medium to form disulfide adducts. See, for example, WO 2009/052249. Cysteine derivatized in this way will not be available for conjugation. To release the newly attached cysteine from the disulfide adduct, the protein after purification is treated with a reducing agent, for example, dithiothreitol. However, such treatment with a reducing agent will reduce all cysteine residues in the therapeutic protein, including native cysteine residues, many of which are involved in inter- and intrachain Cys-Cys disulfide bonds. Native Cys-Cys disulfides are typically critical to protein stability and activity and must be re-formed. In some embodiments, all native (eg, inter- and intrachain) Cys-Cys disulfides are re-formed.

[0382] In order to re-form native inter- and intrachain disulfide residues, after reduction to remove cysteine disulfide adducts, the therapeutic protein is exposed to oxidative conditions and/or oxidizing agents for a set period of time, for example, overnight. In some embodiments, overnight exposure to ambient air may be used to form re-native disulfide bonds. In some embodiments, an oxidizing agent is used to return native disulfide bonds. In some embodiments, said oxidizing agent is selected from the group consisting of aqueous CuSO4 and dehydroascorbic acid (DHAA). In some embodiments, the oxidizing agent is DHAA. In some embodiments, the DHAA used is in the range of 5-30 equivalents. In some embodiments, this range is 10-30 equivalents. In some embodiments, the range is 15 equivalents.

[0383] In some embodiments, the thiol reducing agent is selected from the group consisting of: 3,3',3''-phosphantriyltripropanoic acid (TCEP), dithiothreitol (DTT), dithioerythritol (DTE), sodium borohydride (NaBH ₄ ), sodium cyanoborohydride (NaCNBH3), β-mercaptoethanol (BME), cysteine hydrochloride, trisodium 3,3',3''-phosphinetriyltris(benzene-1-sulfonate) (TPPTS) and cysteine. In some embodiments, the thiol reducing agent is TCEP.

[0384] In some embodiments, the concentration of the thiol reducing agent is 1-100 times a molar excess relative to the concentration of the therapeutic protein. In some embodiments, the concentration of the thiol reducing agent is a 20- to 50-fold molar excess relative to the concentration of the therapeutic protein. In some embodiments, the thiol reducing agent is removed after incubation with the therapeutic protein prior to oxidation of the therapeutic protein.

[0385] In some embodiments, a method of conjugating a therapeutic protein to a half-life extending moiety includes the additional step of purifying the therapeutic protein conjugate after conjugation. In some embodiments, the therapeutic protein conjugate is purified using a method selected from the group consisting of ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, and affinity chromatography, or combinations thereof.

[0386] In some embodiments, the therapeutic protein conjugate retains at least 20% of the biological activity relative to the unconjugated therapeutic protein. In some embodiments, the therapeutic protein conjugate retains at least 50% of the biological activity relative to the unconjugated therapeutic protein. In some embodiments, the therapeutic protein conjugate retains at least 90% of the biological activity relative to the native therapeutic protein.

[0387] In some embodiments, the therapeutic protein conjugate exhibits an increased half-life relative to the unconjugated therapeutic protein. In some embodiments, the therapeutic protein conjugate exhibits at least a 1.5-fold increase in half-life relative to the unconjugated therapeutic protein. In some embodiments, the therapeutic protein conjugate exhibits at least a 1.5-, 2-, 2.5-, 3-, 3.5-, 4-, 4.5-, or 5-fold increase in half-life relative to the unconjugated therapeutic protein .

[0388] In some embodiments, the zwitterionic polymer in a method of conjugating a therapeutic protein to a half-life extending moiety is a radically polymerizable monomer containing a zwitterionic moiety, and the method includes the further step of polymerizing said free radically polymerizable zwitterionic monomer in a polymerization medium to produce the polymer , wherein said medium contains: a radically polymerizable zwitterionic monomer; transition metal catalyst M _t ^(q-1)+ , where M _t represents a transition metal, q represents a higher oxidation state of the metal, and q-1 represents a lower oxidation state of the metal, wherein the metal catalyst is represented as salts of the form Mt ^(q-1)+ X' ^(q-1) , where X' is a counterion or group, or a transition metal catalyst are prepared in situ by using an inactive metal salt in a higher oxidation state M _t ^q+ X' _q together with a reducing agent that is capable of reducing the transition metal from an oxidized inactive state to a reduced active state; ligand; and initiator.

[0389] To function as a transition metal catalyst for ATRP, the transition metal must have at least two readily accessible oxidation states separated by one electron, a higher oxidation state and a lower oxidation state. In ATRP, a reversible redox reaction causes the transition metal catalyst to cycle between a higher oxidation state and a lower oxidation state while the polymer chains cycle between the ends of the growing chain and the ends of the dormant chain. See, for example, US Patent No. 7893173.

[0390] In some embodiments, the radically polymerizable zwitterionic monomer is selected from the group consisting of:

Formula (18)

Formula (19)

Formula (20) and

Formula (21)

where R1 represents H or C1-6 alkyl, ZW represents a zwitterion, and n represents an integer from 1-6.

[0391] In some embodiments, the radically polymerizable monomer is

Formula (12)

where R1 is H or C1-6 alkyl, R2, R3, R4 are the same or different and represent H or C1-4 alkyl, and X and Y are the same or different and are integers 1-6. In some embodiments, R1, R2, R3, and R4 are each methyl, and X and Y are each equal to 2 in Formula (12).

[0392] In some embodiments, the radically polymerizable monomer is

Formula (13)

wherein R1 is H or C1-6 alkyl, R2 and R3 are the same or different and represent H or C1-4 alkyl, R4 is PO4-, SO3- or CO2-, and X and Y are the same or different and represent are integers 1-6. In some embodiments, R1, R2, and R3 are methyl, R4 is PO4-, and X and Y are each 2 in Formula (13).

[0393] In some embodiments, the monomer is

Formula (14)

where R1 is H or C1-6 alkyl, R2, R3 and R4 are the same or different and are H or C1-4 alkyl, R5 is PO4-, SO3- or CO2-, and X and Y are the same or different and represent integers 1-6. In some embodiments, R1, R2, R3, and R4 are methyl, R5 is PO4-, and X and Y are 2 in Formula (14).

[0394] In some embodiments, the transition metal Mt is selected from the group consisting of Cu, Fe, Ru, Cr, Mo, W, Mn, Rh, Re, Co, V, Zn, Au, and Ag. In some embodiments, the metal catalyst is in the form of a salt of the form Mt ^(q-1)+ X' _(q-1) . M _t ^(q-1)+ is selected from the group consisting of Cu ¹⁺ , Fe ²⁺ , Ru ²⁺ , Cr ²⁺ , Mo ²⁺ , W ²⁺ , Mn ³⁺ , Rh ³⁺ , Re ²⁺ , Co ⁺ , V ²⁺ , Zn ⁺ , Au ⁺ and Ag ⁺ , and X' is selected from the group consisting of halogen, C _1-6 alkoxy, (SO ₄ ) _1/2 , (PO ₄ ) _1/3 , ( R7PO ₄ ) _1/2 , (R7 ₂ PO ₄ ), triflate, hexaluorophosphate, methanesulfonate, arylsulfonate, CN and R7CO ₂ where R7 represents H or a linear or branched C _1-6 alkyl group, which may contain as substituents halogen from 1 to 5 times. In some embodiments, M _t ^(q-1)+ is Cu ¹⁺ and X' is Br.

[0395] In some embodiments, M _t ^(q-1)+ is produced in situ . In some embodiments, M _t ^q+ X _q is CuBr ₂ . In some embodiments, the reducing agent is an inorganic compound. In some embodiments, the reducing agent is selected from the group consisting of a low oxidation state sulfur compound, sodium hydrogen sulfite, an inorganic metal ion salt, a metal hydrazine hydrate, and derivatives of such compounds. In some embodiments, the reducing agent is a metal. In some embodiments, the reducing agent is Cu ⁰ .

[0396] In some embodiments, the reducing agent is an organic compound. In some embodiments, said organic compound is selected from the group consisting of alkylthiols, mercaptoethanol or carbonyl compounds that can be readily enolized, ascorbic acid, acetylacetonate, camphorsulfonic acid, hydroxyacetone, reducing sugars, monosaccharides, glucose, aldehydes, and derivatives of such organic compounds.

[0397] In some embodiments, the ligand is selected from the group consisting of 2,2'-bipyridine, 4,4'-di-5-nonyl-2,2'-bipyridine, 4,4-dinonyl-2,2'- dipyridyl, 4,4',4''-tris(5-nonyl)-2,2':6',2''-terpyridine, N,N,N',N',N''-pentamethyldiethylenetriamine, 1, 1,4,7,10,10-hexamethyltriethylenetetramine, tris(2-dimethylaminoethyl)amine, N,N-bis(2-pyridylmethyl)octadecylamine, N,N,N',N'-tetra[(2-pyridal)methyl ]ethylenediamine, tris[(2-pyridyl)methyl]amine, tris(2-aminoethyl)amine, tris(2-bis(3-butoxy-3-oxopropyl)aminoethyl)amine, tris(2-bis(3-(2 -ethylhexoxy)-3-oxopropyl)aminoethyl)amine and tris(2-bis(3-dodecoxy-3-oxopropyl)aminoethyl)amine. In some embodiments, the ligand is 2,2'-bipyridine.

[0398] In some implementations, the initiator has the structure:

Formula (22)

where R1 represents a nucleophilic reactive group, R2 contains a linker, and R3 contains a polymer synthesis initiator moiety having the structure

Formula (23)

where R4 and R5 are the same or different and are selected from the group consisting of alkyl, alkylene, alkoxy, carboxyalkyl, haloalkyl, cycloalkyl, cyclic alkyl ether, alkenyl, alkenylene, alkynyl, alkynylene, cycloalkylene, heterocycloalkyl, heterocycloalkylene, aryl , arylene, aryleneoxy, heteroaryl, amino, amido, or any combination thereof; Z represents halogen or CN; and s is an integer from 1 to 20.

[0399] In some embodiments, Z in Formula (23) is Br, and R4 and R5 are each methyl. In some embodiments, R1 in Formula (22) is selected from the group consisting of NH ₂ -, OH- and SH-.

[0400] In some embodiments, R2 in Formula (22) is alkyl, alkylene, alkoxy, carboxyalkyl, haloalkyl, cycloalkyl, cyclic alkyl ether, alkenyl, alkenylene, alkynyl, alkynylene, cycloalkylene, heterocycloalkyl, heterocycloalkylene, aryl , arylene, arylenoxy, heteroaryl, amino, amido, or any combination thereof. In some implementations, R2 in Formula (22) is

Formula (24)

where X and Y are the same or different and represent integers 1-20. In some implementations, X and Y are each 4.

[0401] In some embodiments, R3 in Formula (22) is

Formula (25)

where R6, R7 and R8 are the same or different and are selected from the group consisting of

Formula (26),

Formula (27) and

Formula (28)

where Z is NCS, F, Cl, Br, or I. In some embodiments, Z in Formula (26), Formula (27), and/or Formula (28) is Br, and each of R6, R7, and R8 in Formula (25) represents

Formula (29)

[0402] In some implementations, the initiator has the structure:

Formula (30)

where A and B are the same or different and are integers from 2 to 12, and Z is any halide, such as Br. In some embodiments, A and B are each equal to 4 in Formula (30).

[0403] In some embodiments, the method further includes the step of reacting the polymer with a maleimide reagent to produce a polymer containing a terminal maleimide. In some embodiments, said maleimide compound is

Formula (31).

[0404] A modification or mutation can also be made in the framework region or constant region to increase the half-life of the IL-6 antagonist antibody (and/or anti-IL-6 VEGF decoy Ab construct). See, for example, PCT publication No. WO 00/09560. It is also possible to mutate the framework region or constant region to alter the immunogenicity of an antibody, provide a site for covalent or non-covalent binding to another molecule, or to alter properties such as complement fixation, FcR binding, and antibody-dependent cell-mediated cytotoxicity. In some embodiments, no more than one to five conservative amino acid substitutions are made within the framework region or constant region. In other embodiments, no more than one to three conservative amino acid substitutions are made within the framework region or constant region. In accordance with the present invention, one antibody may contain mutations in any one or more CDR or framework regions of the variable domain, or constant region.

[0405] Modifications also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with various sugars, acetylation, and phosphorylation. Antibodies are glycosylated at conserved positions in constant regions (Jefferis and Lund, 1997, Chem. Immunol. 65:111-128; Wright and Morrison, 1997, TibTECH 15:26-32). The oligosaccharide side chains of immunoglobulins influence protein function (Boyd et al., 1996, Mol. Immunol. 32:1311-1318; Wittwe and Howard, 1990, Biochem. 29:4175-4180) and intramolecular interactions between glycoprotein moieties that can influence on the conformation and apparent three-dimensional surface of the glycoprotein (Jefferis and Lund supra; Wyss and Wagner, 1996, Current Opin. Biotech. 7:409-416). Oligosaccharides may also serve to target a particular glycoprotein to specific molecules based on structures for specific recognition. There is also evidence that antibody glycosylation influences antibody-dependent cellular cytotoxicity (ADCC). There is evidence that antibodies produced by CHO cells with tetracycline-regulated expression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase that catalyzes the formation of branching GlcNAc, have increased ADCC activity (Umana et al., 1999 , Nature Biotech 17:176–180).

[0406] Antibody glycosylation is typically either N-linked or O-linked. N-linking refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine, where X is any amino acid except proline, are recognition sequences for enzymatically attaching a carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a possible glycosylation site. O-linked glycosylation refers to the addition of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0407] The addition of glycosylation sites to an antibody (and/or AT decoy construct) is conveniently accomplished by altering the amino acid sequence to contain one or more of the tripeptide sequences described above (for N-linked glycosylation sites). This change can also be made by adding or substituting one or more serine or threonine residues in the sequence of the parent antibody (for O-linked glycosylation sites).

[0408] The glycosylation pattern of antibodies (and/or AT decoy constructs) can also be changed without changing the basic nucleotide sequence. Glycosylation is highly dependent on the host cell used to express the antibody. Because the cell type used to express recombinant glycoproteins, such as antibodies as potential therapeutics, is rarely the native cell, variation in the glycosylation pattern of antibodies can be expected (see, e.g., Hse et al., 1997, J. Biol. Chem 272:9062-9070).

[0409] In addition to the selection of host cells, factors affecting glycosylation during recombinant production of antibodies (and/or AT decoy constructs) include growth mode, media composition, culture density, oxygenation, pH, purification schemes, and the like. Various methods have been proposed to alter the glycosylation pattern carried out in a particular host organism, including introduction or overexpression of certain enzymes involved in the production of oligosaccharides (US Patent Nos. 5,047,335; 5,510,261 and 5,278,299). Glycosylation or some types of glycosylation can be enzymatically removed from the glycoprotein, for example, using endoglycosidase H (Endo H), N-glycosidase F, endoglycosidase F1, endoglycosidase F2, endoglycosidase F3. Moreover, a recombinant host cell can be genetically engineered to exhibit impaired processing of certain types of polysaccharides. These and similar techniques are well known in the art.

[0410] Certain embodiments disclosed herein may be used as a therapeutic agent for inflammatory retinal diseases and/or retinal vesicular diseases with an inflammatory component. In addition to angiogenesis, inflammation has been implicated in the pathogenesis of these retinal diseases. Anti-inflammatory therapies, such as steroids, are effective in treating both uveitis (a spectrum of diseases whose defining characteristic is intraocular inflammation) and diabetic macular edema (DMA). Similarly, genetically inherited IL-6 polymorphisms have been associated with a higher incidence of proliferative diabetic retinopathy (PDR) in patients with type 2 diabetes. Moreover, there is evidence that disease progression in cases of VJP, diabetic retinopathy (DR), and retinal vein occlusion (RVO) is associated with elevated serum and/or ocular IL-6 levels. In addition, chronic inflammatory cells were observed on the surface of Bruch's membrane in eyes with neovascular VJP. IL-6 has been implicated in resistance to anti-VEGF treatment in patients with PVD. This is believed to be partly an indirect result of IL-6-mediated up-regulation of VEGF expression [link] as well as more direct VEGF-independent angiogenic functions mediated by IL-6 signaling that occur in the presence of VEGF inhibitors. In some embodiments, any one or more of these conditions can be treated and/or prevented by one or more compositions provided by the present invention.

[0411] FIG. 11C illustrates some design options for a bioconjugate comprising a trap-antibody fusion (TAF) of a VEGF trap (VEGFR1/2) and an anti-IL-6 antibody conjugated to a phosphorylcholine biopolymer. In some embodiments, the anti-VEGF/anti-IL-6 bioconjugate may have a molecular weight of 1.0 MDa with a clinical dose of 6.0 mg in some embodiments. The equivalent molar dose of the anti-VEGF moiety in the bioconjugate may be 6 times greater than the clinical dose of ranibizumab, while the ocular half-life may be 3 to 5 times greater than that of ranibizumab. CH is a heavy chain constant region, CL is a light chain constant region, Fab is an antigen binding fragment, Fc is a crystallizable fragment, VEGFR is a vascular endothelial growth factor receptor, VH is a heavy chain variable region, VL is a light chain variable region chains, and * represent CDR regions. Equivalent values are given as fold changes relative to ranibizumab.

[0412] A different arrangement of sequences in the anti-IL6 antibody/VEGF trap (VEGFR1/2) construct may be used or the sequences may be presented in a different arrangement. FIG. 21 illustrates some possible anti-IL-6 heavy chain variable region sequences. CDRs are underlined.

[0413] Based on the results for the VEGF Trap variants and the improved anti-IL-6 paratopes, 216 molecules were designed and produced in two different configurations that contained combinations of the sequences shown in FIGS. 19, 20, 21, 22, and 23. first configuration (VEGFR-anti-IL-6), the VEGF trap, represented by sequences 1A-1D in Fig. 19, was located at the beginning of the protein, followed by a double repeat of the Gly-Gly-Gly-Gly-Ser (GS) linker, represented by sequence 2A on FIG. 20, which connects the decoy to the N-terminus of the anti-IL-6 heavy chain represented by sequences 3A-3I in FIG. 21. These constructs can be paired with the light chains listed as sequences 4A-4C in FIG. 22. In some embodiments, the VEGF trap (in Fig. 19) can be combined with any of the light chains and/or anti-IL-6 heavy chains shown in any of the figures or tables presented herein.

[0414] In some embodiments, any of the VEGF trap variants provided herein may be paired with any of: the anti-IL-6 antibody heavy and light chain sequences provided herein, and may be further modified by any of the polymers, presented in this description. In some embodiments, any of the VEGF trap variants provided herein may be paired with any of the anti-IL-6 antibody heavy and light chain variable region sequences provided herein and may be further modified by any of the polymers provided in the present description. In some embodiments, any of the VEGF trap variants provided herein may be paired with any of: The 3 heavy and light chain CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) of the anti-IL-6 antibodies provided herein description, and can be further modified by any of the polymers presented in the present description. In some embodiments, the IL-6-VEGF Trap fusion construct comprises the components of FIG. 27.

[0415] In some embodiments, the sequence of the anti-IL-6 VEGF decoy construct (or dual inhibitor, fusion protein, or conjugate thereof) comprises SEQ ID NO: 169 as a light chain, SEQ ID NO: 170 as a heavy chain subjected to fusion with the VEGFR trap via a linker. In some embodiments, the sequence of the anti-IL-6 VEGF decoy construct (or dual inhibitor or fusion protein or conjugate thereof) is at least 80% identical to the molecule comprising SEQ ID NOs: 169 and 170, including, for example, at least 85, 90, 95, 96, 97, 98, 99% or greater identity to the combination of SEQ ID NOs: 170 and 169. In some embodiments, the sequence of the anti-IL-6-VEGF trap construct (or a conjugate thereof) is at least is at least 80% identical to the molecule containing SEQ ID NO: 169, and at least 80% identical to the molecule containing SEQ ID NO: 170, including, for example, at least 85, 90, 95, 96, 97, 98, 99 % or greater identity of SEQ ID NO: 170, and at least 85, 90, 95, 96, 97, 98, 99% or greater identity of 169. In some embodiments, the CDRs remain as depicted in any of the CDRs presented in herein for an anti-IL-6 antibody (including those regions in SEQ ID NOs: 170 and 169), while the remaining sequences (heavy and light chain, heavy and light chain variable regions and/or complete fusion sequences (such as SEQ ID NOs 169 and 170)) can be varied such that the overall sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the original sequence. In some embodiments, the CDR may be varied by 1, 2, or 3 conservative changes, while the remaining sequences (heavy and light chain, heavy and light chain variable regions, and/or complete fusion sequences (such as SEQ ID NOs 169 and 170) ) can be varied such that the overall sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the original sequence. In some embodiments, the trap sequence (shown in gray in FIG. 27 or FIG. 19) may retain at least 80, 85, 90, 95, 96, 97, 98, 99 percent identity or more, and the CDR can be varied by 1, 2 or 3 conservative changes, while the remaining sequences (heavy and light chain, heavy and light chain variable regions and/or complete fusion sequences (such as SEQ ID NOs 169 and 170)) can be varied so that the entire the sequence was at least 80, 85, 90, 95, 96, 97, 98, 99% or more identical to the original sequence.

[0416] In some embodiments, the fusion protein (or dual inhibitor) or conjugate thereof contains 1, 2, 3, 4, 5, or all 6 CDRs provided herein. In some embodiments, the fusion protein or conjugate thereof contains at least SEQ ID NOs: 49, 50, and 51 (or a variant containing 1, 2, or 3 conservative substitutions). In some embodiments, the fusion protein (or dual inhibitor) or conjugate thereof contains at least SEQ ID NOs: 172, 173, and 174 (or a variant containing 1, 2, or 3 conservative substitutions). In some embodiments, the fusion protein or conjugate thereof contains at least SEQ ID NOs: 49, 50, and 51 (or a variant containing 1, 2, or 3 conservative substitutions) and at least SEQ ID NOs: 172, 173, and 174 (or variant containing 1, 2 or 3 conservative substitutions). In some embodiments, these 6 CDRs (or variants thereof) are contained in the framework region of a human antibody. In some embodiments, the CDRs are located in the framework region of any of the antibodies presented herein, or variants thereof, that are at least 80, 85, 90, 95, 96, 97, 98, 99 percent identical to the severe and /or light chain presented herein, including, without limitation, those shown in Tables 1 for the heavy chain and 2 for the light chain. In some embodiments, the fusion protein (or dual inhibitor) or conjugate thereof contains at least SEQ ID NO: 49, 50, and 51 (or a variant containing 1, 2, or 3 conservative substitutions) and at least SEQ ID NO: 172. 173 and 174 (or a variant containing 1, 2 or 3 conservative substitutions), and these CDRs (including variants) are replaced by CDRs in one or more sequences in Table 11 or in a variant in Table 11 (where excluding the CDRs in Table 11, the remaining sequence is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 percent or more identical to the heavy chain region and the light chain region in Table 11 ). Thus, in some embodiments, these 6 CDRs (and variants thereof) may be replaced not only by the CDR sequences listed in Tables 1 and 2, but also by variants thereof. In some embodiments, the CDRs presented in Tables 3, 4, 5 are excluded as possible regions in the candidate CDR options. In some embodiments, the antibody (including fragments thereof) is an antibody that is at least 80% identical to the antibody comprising the heavy and light chain variable regions presented in FIG. 27, while maintaining the specific CDRs in FIG. 27 (thus in In this embodiment, there is no variation in the CDR portion).

[0417] In some embodiments, an isolated anti-IL-6 antagonist antibody is provided. It may contain a heavy chain variable region, which contains a heavy chain that has the sequence of at least one of SEQ ID NO: 7-13, 19-27, 89, 90, 256-262; and a light chain variable region that contains a light chain that has the sequence of at least one of SEQ ID NOs: 91-93, 28-30.

[0418] Some embodiments provide an isolated anti-IL-6 antagonist antibody comprising a heavy chain variable region (VH) comprising 3 complementarity determining regions: CDR1 VH, CDR2 VH, and CDR3 VH, having the amino acid sequence of the CDRs listed in SEQ ID NO: 256; and a light chain variable region (VL) comprising CDR1 VL, CDR2 VL and CDR3 VL having an amino acid sequence selected from the group of CDRs listed in SEQ ID NOs: 91-93.

[0419] In some embodiments, an isolated antagonist antibody is provided that binds to IL-6. The antibody contains at least one of the following mutations according to EU numbering: L234A, L235A and G237A.

[0420] In some embodiments, an isolated antagonist antibody is provided that binds to IL-6, the antibody comprising: CDR _H 1 being CDR _H 1 in SEQ ID NO: 172; CDR _H 2, which is CDR _H 2 in SEQ ID NO: 173; CDR _H 3, which is CDR _H 3 in SEQ ID NO: 174; CDR _L 1, which is CDR _L 1 in SEQ ID NO: 199; CDR _L 2, which is CDR _L 2 in SEQ ID NO: 200; CDR _L 3, which is CDR _L 3 in SEQ ID NO: 201; at least one of the following mutations (EU numbering): L234A, L235A and G237A; and at least one of the following mutations (EU numbering): Q347C or L443C.

[0421] In some embodiments, the dual anti-IL-6 VEGFR inhibitor comprises anti-IL-6 heavy chain variable region sequences selected from SEQ ID NOs: 7-13, 89, 90 and/or 256-262.

[0422] In some embodiments, the dual anti-IL-6 VEGFR inhibitor comprises VEGF decoy sequences selected from at least one of SEQ ID NO: 145, 15, 16, or 17.

[0423] In some embodiments, the linker sequence is SEQ ID NO: 18.

[0424] In some embodiments, the heavy chain sequence for the anti-IL-6 molecules is selected from at least one of SEQ ID NOs: 19-27 or contains at least a sequence in one of SEQ ID NOs: 89, 90, 256- 262.

[0425] In some embodiments, the light chain sequence for an anti-IL-6 molecule comprises at least 1, 2, or 3 light chain CDRs from at least one of SEQ ID NOs: 76-84.

[0426] In some embodiments, the heavy chain sequence for the anti-IL-6 molecule comprises at least 1, 2, or 3 heavy chain CDRs from at least one of SEQ ID NOs: 49-75.

[0427] In some embodiments, the dual VEGFR-anti-IL-6 inhibitor comprises a VEGFR-Fc sequence from at least one of SEQ ID NOs: 85-88.

[0428] In some embodiments, a dual VEGFR-anti-IL-6 inhibitor comprises one or more sequences in any one or more of SEQ ID NOs: 7-13, 145, 15-17, 18-84.

[0429] In some embodiments, the dual anti-IL-6 VEGFR inhibitor comprises an IL-6 VH; IL-6 VL; IL-6 Fc; VEGF trap; and a linker. In some embodiments, the IL-6 VH comprises a sequence from the IL6 VH sequence in any of SEQ ID NOs: 19-27, 31-39, 89, 90, or 256-262. In some embodiments, the IL-6 VL comprises a sequence from the IL6 VL sequence in any of SEQ ID NOs: 28-30 or 91-93. In some embodiments, the Fc comprises a sequence from the Fc sequence in any of SEQ ID NOs: 40-48. In some embodiments, the VEGF trap comprises a sequence from the VEGF trap sequence in any of SEQ ID NO: 145, 15, 16, or 17.

[0430] In some embodiments, a fusion protein is provided comprising IL-6 VH; IL-6 VL; IL-6 Fc; and a VEGF trap, wherein the fusion protein alters the proliferation of ECHRVs. In some embodiments, altering ECHF proliferation inhibits VEGF/IL6-mediated proliferation.

[0431] In some embodiments, a fusion protein is provided comprising a sequence that is at least 80% identical to SEQ ID NO: 263 and at least 80% identical to SEQ ID NO: 117. The fusion protein is further conjugated to a polymer. In some embodiments, the fusion protein is at least 95% identical to SEQ ID NO: 263 and at least 95% identical to SEQ ID NO: 117. In some embodiments, the protein comprises at least a) SEQ ID NO: 172, 173 , 174, 199, 200 and 201 or b) substitution of 1, 2 or 3 amino acids in SEQ ID NO: 172, 173, 174, 199, 200 and/or 201, wherein said substitution is a conservative substitution.

[0432] In some embodiments, any of the constructs provided herein may contain one or more mutations at positions 94 and/or 95 of the VEGFR sequence. In some embodiments, said mutation(s) may be T94I and H95I. In some embodiments, it reduces any degradation of the VEGFR protein (as detailed in the examples below).

[0433] In some embodiments (in which the orientation is anti-IL-6-VEGFR), the heavy chain variable and constant domains represented by the heavy chain - Fab, sequences 5A-5I in Fig. 23 are coupled to a VEGF decoy represented by the sequences 1A-1D in FIG. 19, through the GS linker represented by sequence 2A in FIG. 20, and then the Fc domain represented by the heavy chain - Fc, sequences 5A-5I in FIG. 23, is fused to the C-terminal region of the VEGF trap. . Thus, the VEGF trap is sandwiched between the Fab and Fc regions of the antibody. In some embodiments, these constructs may be paired with the light chains listed as sequences 4A-4C in FIG. 22.

[0434] Figure 19 shows some variant VEGF trap sequences. Variations between specified sequences are underlined and in bold. In some embodiments, any of the fused structures presented in the present description may contain any of the bolded variations presented in this figure or in any of the figures in the present description.

[0435] Figure 20 shows some variants of the Gly-Gly-Gly-Gly-Ser double repeat linker sequence (GS). In some embodiments, more than one sequence unit may be used (eg, two may be used for a double repeat, three, or more). In some embodiments, other linker sequences may be used. In some embodiments, alternative linker sequences may be used.

[0436] Figure 21 shows the heavy chain sequences for some variants of anti-IL-6 molecules. CDRs are underlined. In some embodiments, any of the sequences presented herein may be used in place of other heavy chain sequences that are part of the fusion constructs herein.

[0437] Figure 22 shows some variant light chain sequences for anti-IL-6 molecules. CDRs are underlined. In some embodiments, any of the sequences presented herein may be used in place of other light chain sequences that are part of the fusion constructs herein.

[0438] Figure 23 shows some variant heavy chain sequences (divided into Fab and Fc) for anti-IL-6 molecules. CDRs are underlined. In some embodiments, any of the sequences presented herein may be used in place of other heavy chain sequences that are part of the fusion constructs herein.

[0439] FIGS. 24A-24B show the CDR combinations of FIGS. 21-23. FIG. 24A shows the heavy chain CDR sequences defined in FIGS. 21 and 23. FIG. 24B shows the light chain CDR sequences defined in FIG. 22. In some embodiments, instead of using the entire heavy and/or light chain variable region in the design, only the CDR regions from one or more of the figures presented herein may be used.

[0440] In some embodiments, the VEGFR-Fc fusion construct is part of an engineered IgG1 framework. In addition to the VEGFR-anti-IL-6 and anti-IL-6-VEGFR constructs, an anti-VEGF molecule (VEGFR-Fc) can be generated containing a VEGF trap with the cleavage-resistant variants described above and an engineered Fc domain IgG1, represented by sequences 6A-6D in Fig. 25. The specified Fc domain for these molecules may contain substitutions L234A, L235A and G237A, which minimize effector function, and L443C, which provides site-specific conjugation to a phosphorylcholine-based biopolymer that increases half-life (residue positions correspond to EU numbering). FIG. 25 shows some VEGFR-Fc sequence variants. Variation between specified sequences is underlined and in bold. It should be noted that these variations and/or combinations may provide a biotherapeutic that is further superior to and/or an alternative to other compounds currently used in therapy.

[0441] As shown in FIGS. 26A-26C, VEGFR-Fc and Eylea bind to VEGF with similar affinities. FIG. 26A illustrates Biacore assays for VEGFR-Fc. FIG. 26B illustrates Biacore analyzes for Eylea. Figure 26C shows the experimental results (Ka, Kd, KD and Rmax) for VEGFR-Fc and Eylea.

[0442] In some embodiments, any one or more sequences for a specified amino acid sequence in any one or more of FIGS. 18-23 and 25 may be replaced by the corresponding structure of any of the other embodiments presented herein, or replaced by any of the other sequences presented in the present description. For example, any of the sequences in FIGS. 13C-13E, 19, or 25 can be used with any of the IL-6 heavy chains in FIGS. 18, 21, or 23, with any of the linkers provided herein (e.g., FIG. 20) , with any of the light chains (Fig. 22). In some embodiments, the construct is a VEGFR-anti-IL-6 configuration and contains a combination of one of the sequences 1A-1D (FIG. 19) linked to a linker (e.g., FIG. 20, sequence 2A) linked to an IL heavy chain sequence -6 (eg, FIG. 21, sequence 3A or 3B) linked to a light chain sequence (eg, FIG. 22, sequence 4A). In some embodiments, any of sequences 1A-1D (FIG. 19) can be combined with a linker (FIG. 20) and an anti-IL-6 heavy chain sequence (FIG. 21, sequences 3A-3I) and an anti-IL sequence -6 light chain (Fig. 22, sequence 4A-4C). In some embodiments, any of the other corresponding sequences for any particular design unit presented herein may be replaced by or in place of any of these units.

[0443] In some embodiments, a dual inhibitor provides one or more synergistic results. In some embodiments, a dual inhibitor is synergistically superior to each site administered as monotherapy (eg, a combination of IL-6 therapy and VEGF therapy). In some embodiments, the VEGFR-IL-6 combination construct provides a synergistic result as demonstrated by proliferation assay results (e.g., while the dual inhibitor has an apparent effect on the proliferation of ECHRVs, an important component of angiogenesis, neither Eylea nor anti-IL-6 provide no change). Thus, in some embodiments, there is provided a method of controlling the proliferation of ECHRVs that utilizes the VEGFR-anti-IL-6 construct of the present invention.

[0444] In other embodiments, the fusion construct simply provides the additional benefit of achieving both the VEGF scavenger and anti-IL-6 results.

[0445] In some embodiments, any one or more components in any one or more tables presented herein may be combined to thereby produce a VEGFR-anti-IL-6 construct or to thereby produce an anti-IL-6 construct -VEGFR. In some embodiments, the construct will also comprise an Fc region. In some embodiments, said Fc region may be a human Fc region.

[0446] In some embodiments, CDRs alone (with or without any specific framework region) may be used as the antibody region. Thus, any one or more (eg, 3 or 6) CDRs in each chain or pair of chains (heavy and light) can be used from any of the tables or figures presented herein, in combination with any of the other components (VEGFR and /or linkers). The heavy chain CDRs and/or 1, 2 or 3 CDRs from any of the IL-6 constructs provided herein can be combined with any one or more VEGFR sequences provided herein. In some embodiments, any heavy and/or light chain variable region from any of the IL-6 constructs provided herein can be combined with any one or more VEGFR sequences provided herein. In some embodiments, any of the linkers may also be present in the final design.

[0447] In some embodiments, any nucleic acid sequence encoding any one or more of the amino acid sequences provided herein (eg, isolated, in a vector or in a cell, etc.) can also be obtained.

[0448] In some embodiments, any one or more polymers for conjugation to form a bioconjugate can be combined with any of the VEGFR-IL6 constructs provided herein.

[0449] In some embodiments, the VEGFR-anti-IL-6 combination is the sequence 1A-1D (FIG. 19) for VEGFR linked to a linker (2A in FIG. 20) linked to a heavy chain (3A or 3B in FIG. .21) associated with light chain 4A in FIG. 22.

[0450] In some embodiments, the VEGFR-anti-IL-6 combination is a 1A sequence (FIG. 19) for VEGFR linked to a linker (2A in FIG. 20) linked to a heavy chain (3A in FIG. 21), associated with light chain 4A in FIG. 22. In some embodiments, the VEGFR-anti-IL-6 combination is sequence 1B (FIG. 19) for VEGFR linked to a linker (2A in FIG. 20) linked to a heavy chain (3A in FIG. 21) linked to a light chain. circuit 4A in Fig.22. In some embodiments, the VEGFR-anti-IL-6 combination is a 1C sequence (FIG. 19) for VEGFR linked to a linker (2A in FIG. 20) linked to a heavy chain (3A in FIG. 21) linked to a light chain. circuit 4A in Fig.22. In some embodiments, the VEGFR-anti-IL-6 combination is a 1D sequence (FIG. 19) for VEGFR linked to a linker (2A in FIG. 20) linked to a heavy chain (3A in FIG. 21) linked to a light chain. circuit 4A in Fig.22.

[0451] In some embodiments, the VEGFR-anti-IL-6 combination is sequence 1A (Figure 19) for VEGFR linked to a linker (2A in Figure 20) linked to the heavy chain (3B in Figure 21), associated with light chain 4A in FIG. 22. In some embodiments, the VEGFR-anti-IL-6 combination is sequence 1B (FIG. 19) for VEGFR linked to a linker (2A in FIG. 20) linked to a heavy chain (3B in FIG. 21) linked to a light chain. circuit 4A in Fig.22. In some embodiments, the VEGFR-anti-IL-6 combination is sequence 1C (FIG. 19) for VEGFR linked to a linker (2A in FIG. 20) linked to a heavy chain (3B in FIG. 21) linked to a light chain. circuit 4A in Fig.22. In some embodiments, the VEGFR-anti-IL-6 combination is a 1D sequence (FIG. 19) for VEGFR linked to a linker (2A in FIG. 20) linked to a heavy chain (3B in FIG. 21) linked to a light chain. circuit 4A in Fig.22.

[0452] In some embodiments, any of the anti-IL-6 antibody sequences can be used with any of the VEGFR sequences provided herein. In some embodiments, any 1, 2, 3, 4, 5, or 6 CDR sequences (1, 2, or 3 light chain and 1, 2, or 3 heavy chain) of anti-IL-6 can be used with VEGFR in any of the locations.

[0453] In some embodiments, any anti-IL-6 antibody light and/or heavy chain containing any one or more point mutations presented in any one or more of Tables 30-33 can be used. In some embodiments, the fusion construct uses an anti-IL-6 construct comprising a point mutation at any one or more positions defined in any one or more of Tables 30-33. In some embodiments, the modified positions are those underlined and bolded in the respective figures as indicating the modified residues. In some embodiments, the construct is a construct comprising heavy chain changes including one or more of: S35H, G66D, L100A, or L100S, and light chain changes including one or more of: M32L, M50D, N52S, or M88Q. When describing the positions of the CDRs, they are given in the order of their appearance in the variable domain (eg, S35H, G66D...). When describing the position, Fc corresponds to the EU numbering (for example, L234A, L235A...). In some embodiments, the construct (e.g., an anti-IL-6 antibody and/or an IL-6-VEGF trap, and/or a bioconjugate (with one or more biopolymers provided herein) of an IL-6-VEGF trap construct) contains in a heavy and light chain 1, 2, 3, 4, 5, 6, 7 or 8 of the following: S35H, G66D, L100A and/or L100S in the heavy chain and M32L, M50D, N52S and/or M88Q in the light chain. In some embodiments, these point mutations can be used in any of the anti-Il-6 antibodies or anti-Il-6 antibody-containing constructs provided herein.

[0454] As will be apparent to one skilled in the art, the present invention provides a variety of designs. In general, these constructs can be divided into: anti-IL-6 antibodies and/or anti-IL-6-VEGF decoy constructs (fusion of an antibody and a VEGF decoy), and/or a bioconjugate of an IL-6-VEGF decoy construct (fusion of one or more biopolymers presented herein), and/or VEGF trap constructs, and/or VEGF trap biopolymers. Thus, any of the individual components presented herein (and variations thereof) can be used alone or in combination with each other. In some embodiments, a construct of any of these groups (an anti-IL-6 antibody and/or an anti-IL-6-VEGF trap construct (fusion of an antibody and a VEGF trap), and/or a bioconjugate of an IL-6-VEGF trap construct (fusion of one or more biopolymers presented herein), and/or VEGF trap constructs, and/or VEGF trap biopolymers) can be used for any of the methods or other compositions presented herein. For example, any of these options can be used for any of the treatments described with respect to IL-6-VEGF Trap construct therapy and/or IL-6 therapy and/or IL-6-VEGF Trap construct bioconjugates. As will be appreciated, the characteristics and properties of each construct may be different for each treatment in accordance with the properties of the components described in detail herein. Thus, the description herein with respect to, for example, cell lines, nucleic acids, therapies, and other embodiments is provided not only specifically for the anti-IL-6-VEGF trap construct and/or bioconjugates thereof, but also for antibodies ( and, for example, fragments thereof), antibody bioconjugates and VEGF trap constructs separate from the anti-IL-VEGF trap constructs and/or bioconjugates. For example, in some embodiments, any of the point mutations (or combinations thereof) for IL-6 can be used in an antibody (or a binding fragment thereof) for a treatment that is based only on the antibody rather than on the antibody and the VEGF decoy. Thus, point mutations for VEGF trap and anti-IL-6 antibodies are not limited to the context of fusion structures (although they are proposed), since the context also describes such constructs (and their use and method of preparation) separately from the fusion structures. It should be noted that, rather than repeating all such embodiments separately, any such description for one of the structures (an anti-IL-6 antibody and/or an anti-IL-6-VEGF trap construct (fusion of an antibody and a VEGF trap), and/or a bioconjugate construct IL-6-VEGF trap (fusion with one or more biopolymers presented herein), and/or VEGF trap constructs, and/or VEGF trap biopolymers) is applicable and defines options for all other such structures (anti-IL-6 and /or an anti-IL-6-VEGF Trap construct (fusion of an antibody and a VEGF Trap), and/or a bioconjugate of an IL-6-VEGF Trap construct (fusion with one or more biopolymers provided herein), and/or a VEGF Trap construct , and/or VEGF trap biopolymers).

[0455] In some embodiments, any of the VEGF trap constructs are provided for use as compositions, components, or therapies, including, for example, those in FIGS. 13C-13E, 19, and 25 of the embodiments presented herein.

[0456] In some embodiments, any of the anti-IL-6 antibody constructs are provided for use as compositions, components, or therapies, including, for example, those in Tables 1 and 3, 4, 5, and FIG. , 18, 21, 22, 23 and 24 of the embodiments presented herein.

[0457] In some embodiments, the fusion protein contains a mutation at position 94 in the VEGF trap sequence, at position 95 in the VEGF trap sequence, or at T94I and H95I in the VEGF trap sequence.

[0458] In some embodiments, a dual VEGFR-anti-IL-6 inhibitor is provided. The dual VEGFR-anti-IL-6 inhibitor comprises a decoy-antibody fusion construct of an anti-IL6 antibody and a VEGF decoy (VEGFR1/2), wherein the dual inhibitor contains at least one point mutation in the VEGFR sequence to reduce cleavage of the VEGFR sequence. In some embodiments, the dual VEGFR-anti-IL-6 inhibitor has a molecular weight of 1.0 MDa.

[0459] In some embodiments, a dual VEGR-anti-IL-6 inhibitor provides therapy for inflammatory diseases of the retina.

[0460] In some embodiments, a dual anti-IL-6 VEGFR inhibitor comprises a heavy chain constant region, a light chain constant region, an antigen binding fragment, a crystallizable fragment (Fc), a vascular endothelial growth factor receptor (VEGFR), a heavy chain variable region, light chain variable region and CDR region.

[0461] In some embodiments, the anti-IL-6 sequences may be selected from options VI, VII, VIII, IX, X, XI, or XII in FIG. 18.

[0462] In some embodiments, the VEGF trap sequence may be selected from at least one of options 1A, 1B, 1C, or 1D in FIG. 19.

[0463] In some embodiments, for a dual VEGR-anti-IL-6 inhibitor, the heavy chain sequence for the anti-IL-6 molecules may be selected from at least one of 3A, 3B, 3C, 3D, 3E, 3F, 3G , 3H or 3I in Fig. 21.

[0464] In some embodiments, for a dual VEGFR-anti-IL-6 inhibitor, the light chain sequence for the anti-IL-6 molecules comprises at least 1, 2, or 3 light chain CDRs from at least one of variants 4A, 4B, or 4C in Fig. 24B.

[0465] In some embodiments, for a dual VEGFR-anti-IL-6 inhibitor, the heavy chain sequence for the anti-IL-6 molecule comprises at least 1, 2, or 3 heavy chain CDRs from at least one of variants 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H or 5I in Fig. 24A.

[0466] In some embodiments, the dual VEGFR-anti-IL-6 inhibitor comprises a VEGFR-Fc sequence from at least one of options 6A, 6B, 6C, or 6D in FIG. 25.

[0467] In some embodiments, the dual VEGFR-anti-IL-6 inhibitor comprises one or more of the sequences of FIGS. 18-25.

[0468] In some embodiments, a dual VEGFR-anti-IL-6 inhibitor comprises IL-6 VH, IL-6 VL, IL-6 Fc, a VEGF decoy, and a linker. In some embodiments, the IL-6 VH comprises a sequence from the IL6 VH sequence in FIG. 21 or 23. In some embodiments, the IL-6 VL comprises a sequence from the IL6 VL sequence in FIG. 22. In some embodiments, the Fc comprises a sequence from the Fc sequence in FIG. 23. In some embodiments, the VEGF trap comprises a sequence from a VEGF trap sequence.

[0469] In some embodiments, a protein construct is provided comprising: at least 3 heavy chain CDRs; at least 3 light chain CDRs; VEGF trap sequence; and a linker sequence, each of these sequences being selected from the sequence in FIGS. 18-25.

[0470] In some embodiments, a fusion protein is provided comprising: IL-6 VH, IL-6 VL, IL-6 Fc, a VEGF trap, and wherein the fusion protein modifies the proliferation of ECHRVs. In some embodiments, each sequence is selected from the sequence in FIGS. 18-25.

Polynucleotides, vectors and host cells

[0471] The present invention also provides polynucleotides encoding any of the antibodies (and/or anti-IL-6-VEGF-trap Ab constructs), including antibody fragments and modified antibodies described herein. In accordance with another aspect of the present invention, a method for producing any of the polynucleotides described herein is provided. Polynucleotides can be prepared and expressed by methods known in the art. Accordingly, the present invention provides polynucleotides or compositions, including pharmaceutical compositions, containing polynucleotides encoding any of the IL-6 antagonist antibodies (and/or anti-IL-6 VEGF-trap Ab constructs) described herein.

[0472] The present invention also includes polynucleotides complementary to any such sequences. Polynucleotides can be single-stranded (coding or antisense) or double-stranded and can be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include heterogeneous nuclear RNA (hnRNA) molecules, which contain introns and correspond one-to-one to the DNA molecule, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but not necessarily, be present in the polynucleotide of the present invention, and the polynucleotide may not necessarily be associated with other molecules and/or a support.

[0473] Polynucleotides may contain a native sequence (ie, an endogenous sequence encoding an antibody or fragment thereof) or may contain a variant of such a sequence. Polynucleotide variants contain one or more substitutions, additions, deletions and/or insertions such that the immunoreactivity of the encoded polypeptide is not reduced relative to the native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide can generally be assessed as described herein. The variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity, more preferably at least about 90% identity, and most preferably at least about 95% identity to the polynucleotide sequence encoding the native antibody or fragment thereof.

[0474] Two polynucleotide or polypeptide sequences are said to be “identical” if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum matching, as described below. Comparison of two sequences is typically accomplished by comparing the specified sequences in a comparison window to identify and compare local regions of sequence similarity. As used herein, the term “comparison window” refers to a segment of at least about 20 consecutive positions, typically 30 to about 75 or 40 to about 50, in which the sequence can be compared to a reference sequence of the same number consecutive positions after the optimal alignment of the two sequences.

[0475] Optimal sequence alignment for comparison can be performed using the MegAlign ^® program in the Lasergene ^® bioinformatics software package (DNASTAR ^® , Inc., Madison, WI) using default parameters. This program includes several alignment schemes described in the following references: Dayhoff, MO, 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol.5, Suppl. 3, pp.345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp.626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM, 1989, CABIOS 5:151-153; Myers, E.W. and Muller W., 1988, CABIOS 4:11-17; Robinson, E.D., 1971, Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath, P.H.A. and Sokal, R.R., 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA 80:726-730.

[0476] Preferably, "percentage sequence identity" is determined by comparing two optimally aligned sequences within a comparison window of at least 20 positions, wherein a portion of the polynucleotide or polypeptide sequence within said comparison window may contain additions or deletions (i.e., gaps) 20 percent or less, typically 5 to 15 percent or 10 to 12 percent compared to reference sequences (which contain no additions or deletions), for optimal alignment of two sequences. The percentage is calculated by determining the number of positions at which identical nucleic acid bases or amino acid residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., window size), and multiplying the results by 100 to obtain percent sequence identity.

[0477] Variants may also, or alternatively, be substantially homologous to the native gene or a portion or set thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions with a naturally occurring DNA sequence encoding the native antibody (or complementary sequence).

[0478] Suitable "moderately stringent conditions" include pre-wash in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization at 50°C-65°C, 5 X SSC overnight; followed by washing twice at 65°C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0.1% SDS.

[0479] As used herein, “very stringent conditions” or “high stringency conditions” are conditions that: (1) use low ionic strength and high temperature for washing, for example, 0.015 M sodium chloride/0.0015 M sodium citrate /0.1% sodium dodecyl sulfate at 50°C; (2) a denaturing agent such as formamide is used during hybridization, e.g. 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium -phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) use 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated Salmon milt DNA (50 µg/ml), 0.1% SDS and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C followed by a high stringency wash consisting of 0.1 x SSC containing EDTA at 55°C. One skilled in the art will know how to adjust temperature, ionic strength, etc. if necessary, to accommodate factors such as probe length, etc.

[0480] It will be apparent to one skilled in the art that, due to the degeneracy of the genetic code, there are many nucleotide sequences encoding the polypeptide described herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. However, polynucleotides that vary due to differences in codon usage are particularly contemplated by the present invention. Moreover, alleles of genes containing polynucleotide sequences presented in the present description are included within the scope of the present invention. Alleles are endogenous genes that have been altered by one or more mutations, such as deletions, additions, and/or nucleotide substitutions. The resulting mRNA and protein may, but need not, have altered structure or function. Alleles can be identified using standard methods (such as hybridization, amplification and/or database sequence comparison).

[0481] The polynucleotides of the present invention can be produced using chemical synthesis, recombinant methods, or PCR. Methods for chemically synthesizing polynucleotides are well known in the art and do not require detailed description herein. One skilled in the art can use the sequences presented herein and a commercial DNA synthesizer to obtain the desired DNA sequence.

[0482] To produce polynucleotides using recombinant methods, a polynucleotide containing the desired sequence can be inserted into a suitable vector, and the vector in turn can be introduced into a suitable host cell for replication and amplification, as described below herein. Polynucleotides can be introduced into host cells by any methods known in the art. Cells are transformed by introducing an exogenous polynucleotide through direct uptake, endocytosis, transfection, F-pairing, or electroporation. Once introduced, the exogenous polynucleotide may be contained in the cell as a non-integrated vector (such as a plasmid) or may be integrated into the genome of the host cell. The polynucleotide thus amplified can be isolated from the host cell by methods well known in the art. See, for example, Sambrook et al., 1989.

[0483] Alternatively, PCR allows DNA sequences to be reproduced. PCR technology is well known in the art and is described in US Patent Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.

[0484] The RNA can be produced by using the isolated DNA in a suitable vector and introducing it into a suitable host cell. Once the cell has replicated and the DNA is transcribed into RNA, the RNA can then be isolated using methods well known to one of ordinary skill in the art, as described, for example, in Sambrook et al., 1989, supra.

[0485] Suitable cloning vectors can be constructed according to standard methods or can be selected from the large number of cloning vectors available in the art. Although the cloning vector chosen may vary depending on the host cell used, suitable cloning vectors generally have the ability to self-replicate, may have a single target for a particular restriction endonuclease, and/or may carry marker genes that can be used in the selection of clones containing vector. Suitable examples include plasmids and bacterial viruses, e.g. pUC18, pUC19, Bluescript (eg pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA and shuttle vectors such as pSA3 and pAT28 . These and many other cloning vectors are available from commercial suppliers such as BioRad, Strategene, and Invitrogen.

[0486] Expression vectors are also provided. Expression vectors are typically replicable polynucleotide constructs that contain a polynucleotide of the present invention. It is understood that the expression vector must replicate in host cells either as episomes or as part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and the expression vector(s) described in PCT Publication No. WO 87/04462. Vector components may generally include, but are not limited to, one or more of the following components: a signal sequence; replication origin point; one or more marker genes; suitable transcription control elements (such as promoters, enhancers and terminator). Expression (ie, translation) also typically requires one or more translation control elements, such as ribosome binding sites, translation initiation sites, and stop codons.

[0487] Vectors containing polynucleotides of interest can be introduced into a host cell by any of a number of suitable methods, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; microparticle bombardment; lipofection; and infection (eg, when the vector is an infectious agent such as vaccinia virus). The choice of vectors or polynucleotides to be introduced often depends on the characteristics of the host cell.

[0488] The present invention also provides host cells containing any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DNA can be used to isolate genes encoding an antibody, polypeptide or protein of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa and CHO cells. See also PCT Publication No. WO 87/04462. Suitable non-mammalian host cells include prokaryotes (such as E. coli or B. subtillis ) and yeasts (such as S. cerevisae , S. pombe , or K. lactis ). Preferably, the host cells express the cDNA at a level that is about 5 times higher, more preferably 10 times higher, more preferably 20 times higher than the level for the corresponding endogenous antibody or protein of interest, if present, in the host cells. Screening of host cells for specific binding to IL-6 is carried out by immunoassay or FACS. A cell overexpressing an antibody or protein of interest may be identified.

[0489] An expression vector can be used to direct expression of an IL-6 antagonist antibody (and/or an anti-IL-6 VEGF decoy Ab construct). One skilled in the art will be familiar with the introduction of expression vectors to achieve exogenous protein expression in vivo . See, for example, US patent No. 6436908; 6413942; and 6,376,471. Administration of expression vectors includes local or systemic administration, including injection, oral administration, gene gun or catheter administration, and topical administration. In another embodiment, the expression vector is introduced directly into the sympathetic trunk or ganglion, or into a coronary artery, atrium, ventricle or pericardium.

[0490] Targeted delivery of therapeutic compositions containing an expression vector or subgenomic polynucleotides can also be used. Methods for receptor-mediated DNA delivery are described, for example, in Findeis et al., Trends Biotechnol., 1993, 11:202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer, J. A. Wolff, ed., 1994; Wu et al., J. Biol. Chem., 1988, 263:621; Wu et al., J. Biol. Chem., 1994, 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA 1990, 87:3655; Wu et al., J. Biol. Chem., 1991, 266:338. Therapeutic compositions containing polynucleotide are administered in the range of from about 100 ng to about 200 mg of DNA when administered topically in a gene therapy protocol. Concentration ranges from about 500 ng to about 50 mg, from about 1 μg to about 2 mg, from about 5 μg to about 500 μg, and from about 20 μg to about 100 μg of DNA can also be used in a gene therapy protocol. Therapeutic polynucleotides and polypeptides can be delivered using gene delivery vehicles. Said gene delivery vehicle may be of viral or non-viral origin (in general, see Jolly, Cancer Gene Therapy, 1994, 1:51; Kimura, Human Gene Therapy, 1994, 5:845; Connelly, Human Gene Therapy, 1995, 1: 185; and Kaplitt, Nature Genetics, 1994, 6:148). Expression of such coding sequences can be induced using endogenous mammalian promoters or heterologous promoters. Expression of a coding sequence can be either constitutive or regulated.

[0491] Virus-based vectors for delivery of the desired polynucleotide and expression in the desired cell are well known in the art. Typical viral-based delivery vehicles include, but are not limited to, recombinant retroviruses (see, for example, PCT Publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; US Patent Nos. 5219740 and 4777127; UK Patent No. 2200651; and European Patent No. 0345242), alphavirus vectors (eg, Sindbis virus vectors, Semliki Forest virus (ATCC VR- 67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)) and adeno-associated virus (AAV) vectors (see, for example, PCT Publication Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). You can also use the introduction of DNA associated with inactivated adenovirus, as described in Curiel, Hum. Gene Ther., 1992, 3:147.

[0492] Non-viral delivery vehicles and methods may also be used, including, but not limited to, polycationic fused DNA bound or unbound only to inactivated adenovirus (see, e.g., Curiel, Hum. Gene Ther., 1992, 3:147) ; DNA bound to a ligand (see, for example, Wu, J. Biol. Chem., 1989, 264:16985); eukaryotic cell-based delivery vehicles (see, for example, US Pat. No. 5,814,482; PCT Publications No. WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and neutralizing the nucleic acid charge or fusion with cell membranes. Deproteinized DNA can also be used. Exemplary methods for introducing deproteinized DNA are described in PCT Publication No. WO 90/11092 and US Patent No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in US Pat. No. 5,422,120; PCT publications No. WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additional approaches are described in Philip, Mol. Cell Biol., 1994, 14:2411 and in Woffendin, Proc. Natl. Acad. Sci., 1994, 91:1581.

Compositions

[0493] The present invention also provides pharmaceutical compositions containing an effective amount of an IL-6 antagonist antibody conjugate (and/or an anti-IL-6-VEGF trap Ab construct) described herein. Examples of such compositions, as well as how to make them, are also described herein. In some embodiments, the composition contains one or more IL-6 antagonist antibodies (and/or anti-IL-6 VEGF Trap Ab constructs) and/or antibody conjugates (and/or Trap Ab constructs). In other embodiments, said anti-IL-6 antagonist antibody (and/or VEGF-trap anti-IL-6 antibody construct) recognizes human IL-6. In other embodiments, the anti-IL-6 antagonist antibody is a human antibody. In other embodiments, the anti-IL-6 antagonist antibody is a humanized antibody. In some embodiments, the anti-IL-6 antagonist antibody comprises a constant region that does not trigger an adverse or unwanted immune response, such as antibody-mediated lysis or ADCC. In other embodiments, the IL-6 antagonist (and/or the anti-IL-6 VEGF trap Ab construct) comprises one or more antibody CDRs (e.g., one, two, three, four, five, or in some embodiments, all six CDRs).

[0494] It is understood that the compositions may contain more than one IL-6 antagonist antibody (and/or anti-IL-6 VEGF trap Ab constructs) (e.g., a mixture of anti-IL-6 antibodies that recognize different epitopes of IL-6) . Other typical compositions contain more than one IL-6 antagonist antibody (and/or anti-IL-6 VEGF trap Ab constructs) recognizing the same epitope(s), or different types of IL-6 antagonist antibodies (and/or anti-IL-6-VEGF-trap Ab constructs) that bind to different epitopes of IL-6. In some embodiments, the compositions contain a mixture of IL-6 antagonist antibodies (and/or anti-IL-6 VEGF trap Ab constructs) recognizing different IL-6 variants. In addition, in some embodiments, the composition may also or alternatively contain more than one VEGF trap. These alternative VEGF trap portions may include different sequences for the VEGF trap portion as long as the portions at least exhibit the same or similar functionality as described herein.

[0495] The composition used in accordance with the present invention may further contain pharmaceutically acceptable carriers, excipients or stabilizers (Remington: The Science and practice of Pharmacy 20th Ed., 2000, Lippincott Williams and Wilkins, Ed. K. E. Hoover) in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations indicated, and may include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; 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 carbohydrates, including glucose, mannose or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as Polysorbate, PLURONIC™ or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are described below.

[0496] Pharmaceutical compositions for parenteral administration include an aqueous and non-aqueous sterile injection solution that may contain antioxidants, buffers, bacteriostatic agents and solutes that render the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. Excipients that can be used for injection solutions include water, alcohols, polyols, glycerin and vegetable oils, for example. The compositions may be presented in single-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use. Injection solutions and suspensions for immediate use can be prepared from sterile powders, granules and tablets. Pharmaceutical compositions may be substantially isotonic, meaning an osmolality of about 250-350 mOsm/kg water.

[0497] Pharmaceutical compositions may contain preservatives, solubilizing agents, stabilizers, wetting agents, emulsifiers, sweeteners, colors, flavors, salts (the substances of the present invention may themselves be in the form of a pharmaceutically acceptable salt), buffers, coating agents, or antioxidants. They may also contain therapeutically active agents in addition to the substance of the present invention. The pharmaceutical compositions of the present invention can be used in combination with one or more pharmaceutically acceptable excipients. Such excipients may include, but are not limited to, saline, buffered saline (such as phosphate buffered saline), dextrose, liposomes, water, glycerol, ethanol, and combinations thereof.

[0498] IL-6 antagonist antibodies (and/or anti-IL-6-VEGF trap Ab constructs), IL-6 antagonist antibody conjugates (and/or anti-IL-6-VEGF trap Ab constructs) and pharmaceutical compositions according to the present The invention can be used alone or in combination with other compounds, such as therapeutic compounds or molecules, for example, anti-inflammatory drugs, analgesics or antibiotics. Such administration together with other compounds may be simultaneous, separate or sequential. The components may be presented in the form of a kit, which may contain instructions if necessary.

Methods for preventing or treating ophthalmic disorders

[0499] In some embodiments, antibodies (and/or anti-IL-6-VEGF trap Ab constructs) and antibody conjugates (and/or anti-IL-6-Trap VEGF Ab constructs) are suitable for a variety of uses, including, but not limited to , methods of therapeutic treatment.

[0500] In some embodiments, a method is provided for treating an eye disease, such as, for example, ADHD. In some embodiments, a method of treating VAD in a subject comprises administering to the subject in need thereof an effective amount of a composition (e.g., a pharmaceutical composition) comprising any of the anti-IL-6 or anti-IL6-VEGF trap Ab antibody constructs described herein. As used herein, VJP includes the dry form of VJP and the wet form of VJP. In some embodiments, there is provided a method of treating VAD in a subject, comprising administering to the subject in need thereof an effective amount of a composition comprising IL-6 antagonist antibodies or anti-IL6-VEGF trap Ab constructs, or conjugates thereof, as described herein. In some embodiments, an anti-IL-6 antibody or anti-IL6 VEGF-trap Ab construct and/or conjugates thereof can be used to treat and/or prevent and/or reduce the risk of wet macular degeneration or wet neovascular macular degeneration age-related macular degeneration or neovascular age-related macular degeneration, dry form of age-related macular degeneration, venous, arterial or other occlusion of the blood vessels of the eye and/or retina with or without retinal edema, anterior and posterior uveitis, macular edema with uveitis, diabetic retinopathy, proliferative diabetic retinopathy, diabetic macular edema, non-proliferative diabetic macular edema and intraocular tumors. In some embodiments, an anti-IL6 VEGF trap antibody or Ab construct may be used for: (i) patients who are refractory to VEGF therapy, (ii) patients who are experiencing loss of effectiveness of anti-VEGF agents due to progression of additional underlying pathologies, such such as fibrosis, wound healing and atrophy, and/or (iii) if undertreated due to the need for frequent intravitreal injections.

[0501] In some embodiments, a method of treating an eye disease, such as a systemic disease, is provided. In some embodiments, systemic diseases affecting the eye, such as Graves' disease or neuromyelitis optica, or systemic diseases not affecting the eye, such as multiple sclerosis, rheumatoid arthritis, can be treated by the methods and compositions of the present invention.

[0502] In some embodiments, the compositions of the present invention can be used in combination with immunomodulators, such as PD-1/PDL-1, when indicated in the case of cancer.

[0503] In some embodiments, the methods described herein further include the step of treating a subject with one or more additional forms of therapy. In some embodiments, said adjunctive form of therapy is an adjunctive therapy for VAD, including, but not limited to, ^Visudin® , laser photocoagulation, or intravitreal injection, e.g., ^Lucentis® , ^Macugene® , ^Eylea® , Ozurdex® ^{, Iluvien®} , ^Triesens® ( Triesence ^® ) or Trivarisa ^® .

[0504] For all methods described herein, reference to IL-6 antagonist antibodies (and/or anti-IL6 VEGF trap Ab construct) also includes compositions containing one or more additional agents. These compositions may further contain suitable excipients, such as pharmaceutically acceptable excipients, including buffers, which are well known in the art. The present invention can be used alone or in combination with other treatment methods.

[0505] In some embodiments, a method is provided for treating or preventing a disease in a patient in need thereof. In some embodiments, the method comprises administering to a patient any of the isolated antagonist antibodies (and/or anti-IL-6-VEGF-trap Ab constructs) described herein. In some embodiments, the method includes administering to a patient any of the conjugates described herein. In some embodiments, the method includes administering to a patient any of the compositions described herein. In some embodiments, the method includes identifying a patient with IL-6 hyperactivity, and administering to said patient any of the isolated antagonist antibodies described herein. In some embodiments, the method includes identifying a patient with IL-6 hyperactivity, and administering to said patient any of the conjugates described herein. In some embodiments, the method includes identifying a patient with IL-6 hyperactivity, and administering to said patient any of the compositions described herein. In some embodiments, the patient exhibits increased VEGF activity instead of or in addition to increased IL-6 activity.

[0506] In some embodiments, the disorder is selected from the group consisting of wet age-related macular degeneration or neovascular macular degeneration, wet age-related macular degeneration or neovascular age-related macular degeneration, dry age-related macular degeneration, venous, arterial occlusion, or other occlusion of the blood vessels of the eye and/or retina with or without retinal edema, anterior and posterior uveitis, uveitis macular edema, diabetic retinopathy, proliferative diabetic retinopathy, diabetic macular edema, non-proliferative diabetic macular edema and intraocular tumors.

[0507] In some embodiments, the isolated antibody (and/or anti-IL-6-VEGF trap Ab construct), conjugate (and/or conjugate of the anti-IL-6-VEGF Trap Ab construct), composition, or combination thereof is administered no more than once per month. In some embodiments, the isolated antibody, conjugate, composition, or combination thereof is administered no more than once every two months. In some embodiments, the isolated antibody (and/or anti-IL-6-VEGF trap Ab construct), conjugate (and/or conjugate of the anti-IL-6-VEGF Trap Ab construct), composition, or combination thereof is administered no more than once every three months. . In some embodiments, the isolated construct (eg, AT or AT-trap with or without polymer) is administered at a frequency of once per year to once every two months. In some embodiments, the treatment may be a single application of the antibody. In some embodiments, the treatment may be as many applications of the antibody as needed or required to achieve a result.

[0508] An anti-IL-6 antagonist antibody (and/or a VEGF-trap anti-IL6 Ab construct) or a conjugate thereof can be administered to a subject by any suitable route. It will be apparent to one skilled in the art that the examples described herein are not limiting, but illustrative of the methods available. Accordingly, in some embodiments, an anti-IL-6 antagonist antibody (and/or a VEGF decoy anti-IL6 Ab construct) is administered to a subject according to known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time. period of time, intramuscular, intraperitoneal, intracerebrospinal, transdermal, subcutaneous, intraarticular, sublingual, intrasynovial, insufflation, intrathecal, oral, inhalation or topical route. Administration may be systemic, such as intravenous, or local. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are suitable for administration. Liquid formulations can be sprayed directly, and lyophilized powder can be sprayed after reconstitution. Alternatively, the anti-IL-6 antagonist antibody (and/or the anti-IL6 VEGF trap Ab construct) can be aerosolized using a fluorocarbon formulation and a metered dose inhaler or administered by inhalation as a lyophilized and ground powder.

[0509] In some embodiments, IL-6 antagonist antibodies (and/or anti-IL-6-VEGF-trap Ab constructs), IL-6 antagonist antibody conjugates (and/or anti-IL-6-VEGF-trap Ab constructs), and The pharmaceutical compositions described herein are used for the prevention or treatment of an eye disease or condition. The conjugates used in this manner are typically formulated for such administration and are administered by ocular, intraocular and/or intravitreal injection, and/or periscleral injection, and/or sub-Tenon injection, and/or suprachoroidal injection, and/or topical injection into in the form of eye drops and/or ointment. Such IL-6 antagonist antibodies (and/or anti-IL-6-VEGF trap Ab constructs), IL-6 antagonist antibody conjugates (and/or anti-IL-6-VEGF Trap Ab constructs) and compositions can be delivered in a variety of ways, for example, intravitreal in the form of a device and/or depot that provides slow release of the compound into the vitreous, including those described in such sources as Intraocular Drug Delivery, Jaffe, Jaffe, Ashton, and Pearson, editors, Taylor & Francis (March 2006 ). In one example, the device may take the form of a minimal and/or matrix and/or passive diffusion system and/or encapsulated cells that release the compound over an extended period of time (Intraocular Drug Delivery, Jaffe, Jaffe, Ashton, and Pearson, editors, Taylor & Francis (March 2006) In therapy or as a prophylactic agent, the active agent can be administered to an individual in the form of an injectable composition, for example, in the form of a sterile aqueous dispersion, preferably isotonic or substantially isotonic.

[0510] Formulations for ocular, intraocular or intravitreal administration can be prepared by methods and using ingredients known in the art. Suitable penetration into the eye is necessary for effective treatment. Unlike diseases of the anterior eye, where drugs can be delivered topically, retinal diseases require a more site-specific approach. Eye drops and ointments rarely penetrate the back of the eye, and the blood-ophthalmic barrier prevents systemically administered drugs from penetrating the ocular tissue. In some embodiments, a preferred method of drug delivery for the treatment of a retinal disease such as VAD and CNV is direct intravitreal injection. In some embodiments, intravitreal injections are repeated at intervals that depend on the patient's condition and the properties and half-life of the drug being delivered.

[0511] For administration to mammals and, in particular, humans, the dose of the active agent is expected to range from 0.01 mg/kg body weight to typically about 1 mg/kg if administered systemically. For ocular diseases that require local administration (eg, intravitreal, suprachoroidal, periocular, etc.), the dose is typically 0.1 mg/eye/dose to 10 mg/eye/dose or more. In some embodiments, the dose is 100 μl/dose/eye. In some embodiments, a needle may be used to administer said dose. The needle may be, for example, a 30 gauge ½" needle or a 27G or 29G ½" needle. The physician may determine the actual dosage most appropriate for the individual, which depends on factors including the age, body weight, sex and response of the individual, the disease or disorder being treated, and the age and condition of the individual being treated. The above dosages are typical for the average case. Of course, there may be times when higher or lower doses are needed.

[0512] This dose can be repeated at any desired frequency (eg, weekly, biweekly, monthly, quarterly). If side effects develop, the dosage amount and/or frequency may be reduced in accordance with normal clinical practice. In one embodiment, the pharmaceutical composition can be administered once every one to thirty days.

[0513] IL-6 antagonist antibodies (and/or anti-IL-6 VEGF trap Ab constructs) of the present invention can be used in accordance with the present invention by expressing such polypeptides in a patient in vivo , i.e. gene therapy. There are two main approaches to introducing a nucleic acid (possibly contained in a vector) into a patient's cells: in vivo and ex vivo . For in vivo delivery, the nucleic acid is administered by injection directly to the patient, typically at sites where the therapeutic protein is needed, i.e. where the biological activity of the therapeutic protein is required. For ex vivo treatment, cells from a patient are extracted, the isolated cells are injected with nucleic acid, and the modified cells are administered to the patient either directly or, for example, encapsulated in porous membranes that are implanted into the patient (see, for example, US Pat. Nos. 4,892,538 and 5,283,187). There are various methods for introducing nucleic acids into viable cells. These methods vary depending on whether the nucleic acid is transferred into cultured cells in vitro or transferred into cells of the intended host in vivo . Methods suitable for transferring nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, transduction, cell fusion, DEAE-dextran, calcium phosphate precipitation method, etc. Transduction involves the binding of a replication-defective recombinant viral (preferably retroviral) particle to a cellular receptor, followed by the introduction of nucleic acids contained in the said particle into the cell. The vector commonly used for ex vivo gene delivery is a retrovirus.

[0514] Currently, preferred methods for in vivo nucleic acid transfer include transfection with viral or non-viral vectors (e.g., adenovirus, lentivirus, herpes simplex virus I, or adeno-associated virus (AAV)) and lipid-based systems (lipids suitable for lipid-mediated gene transfer are, for example, DOTMA, DOPE and DC-Chol; see, for example, Tonkinison et al., Cancer Investigation, 14(1): 54-65 (1996)). The most preferred vectors for use in gene therapy are viruses, most preferably adenoviruses, AAVs, lentiviruses or retroviruses. A viral vector, such as a retroviral vector, contains at least one transcriptional promoter/enhancer or locus defining element(s), or other elements that control gene expression by other means, such as alternative splicing, nuclear export of RNA, or post-translational modification of the messenger. In addition, a viral vector, such as a retroviral vector, contains a nucleic acid molecule that, when transcribed in the presence of a gene encoding a therapeutic protein, is operably linked thereto and acts as a translation initiation sequence. Such vector constructs also contain a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate for the virus being used (if not already present in the viral vector). In addition, such a vector typically contains a signal sequence for secretion of the propolypeptide from the host cell into which it is placed. Preferably, said signal sequence for this purpose is a mammalian signal sequence, most preferably a native signal sequence for the therapeutic protein. Optionally, the vector construct may also contain a signal sequence that directs polyadenylation, as well as one or more restriction sites and a transcription termination sequence. For example, such vectors typically contain a 5' LTR, a tRNA binding site, a packaging signal, a start point for second-strand DNA synthesis, and a 3' LTR or part thereof. Other vectors that are non-viral can be used, such as cationic lipids, polylysine and dendrimers.

[0515] In some situations, it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell surface or target cell membrane protein, a ligand for a receptor on the target cell, etc. When using liposomes, proteins that bind to cell surface membrane proteins associated with endocytosis can be used to target and/or facilitate uptake, e.g. capsid proteins or fragments thereof that are cell type tropic, antibodies to proteins that are internalized during the cycle, and proteins that specifically affect intracellular localization and increase the intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, in Wu et al., J. Biol. Chem., 262: 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA, 87: 3410-3414(1990). For a review of currently known gene tagging and gene therapy protocols, see Anderson et al., Science, 256: 808-813 (1992). Also see WO 93/25673 and references therein.

[0516] Suitable gene therapy and methods for producing retroviral particles and structural proteins can be found, for example, in US patent No. 5681746.

[0517] In accordance with some aspects, a method of treating or preventing an eye disease in a mammal is provided, wherein a nucleic acid molecule encoding an anti-IL-6 antagonist antibody (and/or a VEGF decoy anti-IL-6 Ab construct) is administered.

[0518] In some embodiments, these dual IL6/VEGF inhibitor molecules can be used to treat ocular disorders such as ocular inflammatory disease scleritis, nonproliferative diabetic retinopathy, proliferative diabetic retinopathy (PDR), diabetic macular edema, diabetic macular edema prevention , prevention of proliferative diabetic retinopathy, wet age-related macular degeneration, prevention of wet age-related macular degeneration, uveitis and macular edema with uveitis.

[0519] VEGFR-anti-IL6 drugs can also be used to treat systemic diseases affecting the eye, such as Graves' disease or neuromyelitis optica, or systemic diseases not affecting the eye, such as multiple sclerosis, rheumatoid arthritis.

[0520] These molecules may also be used to treat cytokine release syndrome following CAR-T or similar immuno-oncology drugs. In addition, anti-IL-6 molecules suppress the induction of IL-6 expression observed after treatment with anti-PD-1/PD-L1 molecules (Tsukamoto et al, 2018). In addition, blockade of VEGF signaling may also improve the effectiveness of treatment with anti-PD-L1 agents (Allen et al., 2017). Thus, these dual inhibitors can be used in combination with immune checkpoint inhibitors (such as PD-1/PDL-1 modulators) to synergistically treat cancer. In addition, inhibition of VEGF has additional beneficial effects. Another use for these molecules is for cerebral edema in glioblastoma, where anti-IL6 therapy (anti-IL6 therapy) may provide additional beneficial effects to anti-VEGF treatment. The biopolymer may also provide increased tissue distribution, which is beneficial for penetration into solid tumors.

[0521] In some embodiments, the conjugate (or various proteins) is suitable for preventing any one or more of the disorders presented herein.

Compositions

[0522] Therapeutic compositions of IL-6 antagonist antibodies (and/or anti-IL-6-VEGF trap Ab constructs) and IL-6 antagonist antibody conjugates (and/or anti-IL-6-VEGF Trap Ab constructs) administered according to of the present invention are prepared by mixing an antibody exhibiting the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000) for storage in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the doses and concentrations used, and may include buffers such as phosphate, citrate and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; 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 carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as TWIN™, PLURONIC™ or polyethylene glycol (PEG).

[0523] Liposomes containing an anti-IL-6 antagonist antibody (and/or an anti-IL-6-VEGF decoy Ab construct) and/or an IL-6 antagonist antibody conjugate (and/or an anti-IL-6-VEGF decoy Ab construct) , are prepared by methods known in the art, such as those described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl Acad. Sci. USA 77:4030 (1980); and US Pat. Nos. 4,485,045 and 4,544,545. Extended circulation time liposomes are described in US Pat. No. 5,013,556. The most suitable liposomes can be prepared by reverse phase evaporation using a lipid composition containing phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of specific pore sizes to produce liposomes with the desired diameter.

[0524] The active ingredients can also be encapsulated in microcapsules obtained, for example, by coacervation methods or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems ( for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are described in Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[0525] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, said matrices being shaped into articles such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (eg, poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides (US Pat. No. 3,773,919), copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene vinyl acetate , degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate and poly-D-(-)-3-hydroxybutyric acid.

[0526] Compositions used for administrationin vivo, must be sterile. Sterilization is easily accomplished by, for example, filtration through sterile filtration membranes. Therapeutic compositions of IL-6 antagonist antibody (and/or anti-IL-6-VEGF trap Ab constructs) and/or antibody conjugate (and/or anti-IL-6-VEGF Trap Ab construct) are typically placed in a container containing sterile access port, such as a bag or bottle of intravenous solution with a stopper pierced by a hypodermal injection needle. In some embodiments, the antibody and/or antibody conjugate composition is placed into a syringe to form a pre-filled syringe.

[0527] In some embodiments, the composition is a pyrogen-free composition that is substantially free of endotoxins and/or related pyrogenic substances. Endotoxins include toxins that are trapped within a microorganism and are released only when those microorganisms are destroyed or killed. Pyrogens also include fever-inducing, heat-stable substances from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension, and shock when administered to humans. Because of possible harmful effects, even low levels of endotoxins must be removed from intravenous drug solutions. For systemic injection, such as intravenous or intraperitoneal, the US Food and Drug Administration ("FDA") has established an upper limit of 5 endotoxin units (EU) per dose per kilogram of body weight in one one-hour period for intravenous drug administration. funds (The United States Pharmacopeial Convention, Pharmacopeial Forum 26 (1):223 (2000)). When therapeutic proteins are administered at levels of several hundred or thousands of milligrams per kilogram of body weight, as may be the case for proteins of interest (eg antibodies), even trace amounts of harmful and dangerous endotoxin must be removed. In some embodiments, the levels of endotoxins and pyrogens in the composition are less than 10 EU/mg, or less than 5 EU/mg, or less than 1 EU/mg, or less than 0.1 EU/mg, or less than 0.01 EU/mg, or less than 0.001 EU/mg. In some embodiments, 0.1 EU/eye/injection is allowed in the compositions or methods provided herein. In some embodiments, 0.05 EU/eye/injection is allowed in the compositions or methods provided herein. In some embodiments, 0.02 EU/eye/injection is allowed in the compositions or methods provided herein. In some embodiments, 0.01 EU/eye/injection is allowed in the compositions or methods provided herein.

[0528] The compositions of the present invention may be presented in unit dosage forms, such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.

[0529] To prepare solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical carrier, for example, traditional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gum, and with other pharmaceutical diluents, for example, water, to form a solid preformulated composition containing a homogeneous mixture of a compound of the present invention or a non-toxic pharmaceutically acceptable salt thereof. When these preformulated compositions are referred to as homogeneous, it means that the active ingredient is uniformly dispersed throughout the composition so that said composition can be readily divided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation is then divided into unit dosage forms of the type described above, containing from about 0.1 to about 500 mg of the active ingredient of the present invention. Tablets or pills of the new composition may be coated or otherwise formulated to provide a dosage form that provides the benefit of extended release. For example, a tablet or pill may contain an internal dose component and an external dose component, the latter being in the form of a coating over the former. The two components may be separated by an enteric layer, which serves to prevent degradation in the stomach and allows the internal component to pass intact into the duodenum or to be released in a delayed manner. Various substances can be used for such enteric layers or coatings; such substances include a number of polymer acids and mixtures of polymer acids with substances such as shellac, cetyl alcohol and cellulose acetate.

[0530] Suitable surfactants include, but are not limited to, nonionic agents such as Polysorbate or polyoxyethylene sorbitans (e.g., Tween™ 20, 40, 60, 80, or 85) and other sorbitans (e.g., Span™ 20, 40, 60, 80 or 85). Surfactant compositions typically contain 0.01% to 5% surfactant and may contain 0.01 to 0.02% or 0.1 to 2.5% (polysorbate 20 or 80). It will be appreciated that other ingredients, such as mannitol, or other pharmaceutically acceptable carriers, may be added as necessary.

[0531] Suitable emulsions can be prepared using commercially available fat emulsions such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipifisan™. The active ingredient can either be dissolved in a premixed emulsion composition, or alternatively it can be dissolved in an oil (eg, soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and an emulsion formed by mixing with a phospholipid (eg egg phospholipids, soy phospholipids or soy lecithin) and water. It will be appreciated that other ingredients, such as glycerol or glucose, may be added to adjust the tonicity of the emulsion. Suitable emulsions typically contain up to 20% oil, for example 5 to 20%. The fat emulsion may contain small fat droplets of 0.1 to 1.0 µm, in particular 0.1 to 0.5 µm, and have a pH in the range of 5.5 to 8.0.

[0532] Emulsion compositions may be compositions prepared by mixing an IL-6 antagonist antibody (and/or an anti-IL-6 VEGF trap Ab construct) with Intralipid™ or its components (soybean oil, egg phospholipids, glycerol and water) .

[0533] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients listed above. In some embodiments, the compositions are administered by the oral or nasal route to provide local or systemic effects. The compositions in preferably sterile pharmaceutically acceptable solvents can be nebulized using gases. Nebulized solutions may be inhaled directly from the nebulizing device, or said nebulizing device may be connected to a face mask, tent, or intermittent positive pressure breathing apparatus. The compositions in the form of a solution, suspension or powder can be administered, preferably orally or nasally, from devices delivering the composition in a suitable manner.

Sets

[0534] The present invention also provides kits containing any or all of the antibodies described herein. The kits of the present invention contain one or more vials containing an anti-IL-6 antagonist antibody (and/or a VEGF-trap anti-IL-6 Ab construct) or a conjugate described herein and instructions for use in accordance with any of the methods according to the present invention described herein. Typically, these instructions contain a description of the administration of an IL-6 antagonist antibody (and/or an anti-IL-6 VEGF trap Ab construct) or a conjugate for the therapeutic treatment described above. In some embodiments, single dose kits are provided. In some embodiments, the kit may include both a first container containing a dried protein and a second container containing an aqueous formulation. In some embodiments, kits containing single and multi-chamber prefilled syringes (eg, liquid syringes and lyophilisate syringes) are included.

[0535] In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a monoclonal antibody. Instructions for the use of an IL-6 antagonist antibody (and/or anti-IL-6 VEGF decoy antibody construct) or conjugate generally contain information regarding the dose, dosage regimen, and route of administration for the intended treatment. Containers may be single doses, bulk packages (eg, multi-dose packages), or subunit doses. The instructions provided in the kits of the present invention are typically written instructions on a label or package insert (e.g., a piece of paper included in the kit), but machine-readable instructions (e.g., instructions contained on a magnetic disk) are also acceptable. or optical storage device).

[0536] Kits according to the present invention are provided in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (eg, sealed mylar or plastic bags), and the like. Packaging is also provided for use in combination with a specific device, such as a prefilled syringe, inhaler, a nasal delivery device (eg, a nebulizer) or an infusion device, such as a minipump. The kit may have a sterile access port (eg, the container may be a bag or vial of intravenous solution with a stopper pierced by a hypodermal injection needle). The container may also have a sterile access port (eg, the container may be a bag or vial of intravenous solution with a stopper pierced by a hypodermal injection needle). At least one active agent in the composition is an anti-IL-6 antagonist antibody or a conjugate thereof. The container may further contain a second pharmaceutically active agent.

[0537] Additional components such as buffers and explanatory information may be provided in the kits. In some embodiments, the kits may include an additional syringe and needle used to prime the dispensing syringe. Typically the kit contains a container and a label or leaflet(s) package insert on the container or attached to the container.

[0538] The following examples are offered for purposes of illustration only and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the present invention in addition to those presented and described herein will be apparent to one skilled in the art from the description given above and are intended to be within the scope of the appended claims.

EXAMPLES

[0539] Despite the therapeutic success of anti-vascular endothelial growth factor (VEGF) biologics, there remains an unmet need for the treatment of retinal neovascular diseases due to the presence of: (i) patients refractory to VEGF therapy ( ii) patients who experience loss of effectiveness of anti-VEGF agents due to progression of additional underlying pathologies such as fibrosis, wound healing and atrophy, and/or (iii) insufficient treatment due to the need for frequent intravitreal injections. The following are results related to new bispecific biological agents designed to inhibit both VEGF and IL-6.

[0540] Methods. Dual inhibitors were designed and their binding affinities for their respective targets were characterized by surface plasmon resonance (SPR). Functional aspects of the molecules were tested: (1) by ELISA assays aimed at competitively blocking each target for the corresponding receptor (2) VEGF- and/or IL-6-stimulated proliferation of primary retinal microvascular endothelial cells (HRMVEC) stimulated and (3) three-dimensional coculture assays of primary cells can be performed to assess inhibition of blood vessel sprouting.

[0541] Anti-IL-6 VEGF decoy Ab constructs bound with pM affinity to VEGF and IL-6. The binding kinetics of each target was independent of the presence of the other, indicating that both targets can bind simultaneously without interference. Binding of these molecules to each of their targets inhibited interactions with the corresponding cognate receptors.

[0542] When combined within a biopolymer conjugate, these dual inhibitor molecules provide an additional therapeutic agent for the next generation of treatment and prevention of retinal diseases.

Example 1 - production and screening of antibodies

Methods/results:

Anti-IL-6

[0543] The examples herein briefly describe the construction of a dual anti-IL-6 and anti-VEGF inhibitor molecule comprising an anti-IL-6 antibody in an engineered human IgG1 framework and an IgG1 Fc domain fused to an anti-VEGF decoy . The specified Fc domain for these molecules contains substitutions 234A, L235A and G237A, which minimize effector function, and L443C, which provides site-specific conjugation to a phosphorylcholine-based biopolymer that increases half-life (residue positions correspond to EU numbering).

[0544] An anti-IL-6 paratope in an engineered human IgG1 framework was subjected to affinity maturation using a combination of library scanning mutagenesis in E. coli followed by site-directed mutagenesis in a mammalian system. Construction of a dual expression plasmid system containing heavy and light chain Fabs with anti-IL-6 CDR sequences was carried out in E. coli. FIG. 3 is a diagram of this process that resulted in the heavy and light chain variable regions of the antibody of FIG. 5.

[0545] Fab libraries were constructed containing random single point mutations for a total of 71 CDR positions, excluding substitutions for Cys, Met, and Asn due to the chemical instability associated with these amino acids. Small-scale expression in a 96-well plate format was used to screen Fab libraries. E. coli colonies (TG1 cells, Zymo Research) containing random single point mutations were collected in 96-well plates (1.5 ml) and grown overnight at 30°C in LB Amp + 2% glucose. The next day, a separate 96-well plate was formed by removing 100 μl of culture and adding 100 μl of 30% glycerol, then stored at -80°C. The remaining culture was centrifuged for 20 min and then resuspended in 1.5 ml LB Amp containing 1 mM IPTG to induce protein expression at 30°C. After 5 hours, cells were centrifuged, resuspended in 500 μl of HBS-EP+ buffer, and lysed using a single freeze/thaw cycle. Lysates were centrifuged for 30 min and vacuum filtered (200 μl) into round-bottomed 96-well Biacore assay plates.

[0546] Anti-IL-6 Fab affinity was determined using Biacore T200 (GE Healthcare) at 25°C. CM5 chips were activated by EDC and NHS, recombinant human IL-6 (R&D Systems) was diluted in 10 mM sodium acetate, pH 5, to a concentration of 0.1 mg/ml and injected onto the activated chip. Three antigen density ranges were obtained (100-150 RU, 300-400 RU, 400-600 RU) by using different flow times for each channel of the chip, then blocked with ethanolamine. Filtered culture lysate supernatants from 96 wells were injected at a rate of 50 μl/min for 120 seconds. Fitting the dissociation curves (300 seconds) to a single exponential decay curve was used to determine _dissociation rates ( koff ). The chip surfaces were regenerated with 10 mM glycine pH 1.7 solution injected at a rate of 50 μL/min for 60 seconds after each sample binding and dissociation cycle. Clones exhibiting similar or improved k _off values compared to the wild type were subjected to DNA sequencing to identify the mutation.

[0547] Screening data showed that out of a total of 71 CDR residue positions, 15 residues were critical for IL-6 binding and could not be replaced by any other amino acid. Substitutions at valid positions resulted in koff values similar to those for _the parent molecule (Table 13). Most of these single substitutions were then introduced into full heavy and light chain genes cloned into mammalian expression vectors, which were cotransfected into 3 ml Expi293 cell cultures for protein production. On the fifth day, these cultures were centrifuged and the supernatants containing secreted antibodies were collected and diluted in HBS-EP+ running buffer (1:20 ratio). Point mutants are presented in tables 13-18.

Table 13

Heavy chain CDR1

[0548] The table provides koff (s-1) values for each valid substitution within a specific CDR. In general, the mutants exhibited a koff value similar to that of the parent molecule (4.94E-03±7.67E-04). Residues that are critical for IL-6 binding and do not allow substitutions are designated “critical.”

Table 14

Heavy chain CDR2

[0549] The table provides koff (s-1) values for each valid substitution within a specific CDR. In general, the mutants exhibited a koff value similar to that of the parent molecule (4.94E-03±7.67E-04). Residues that are critical for IL-6 binding and do not allow substitutions are designated “critical.”

Table 15

Heavy chain CDR3

[0550] The table provides koff (s-1) values for each valid substitution within a specific CDR. In general, the mutants exhibited a koff value similar to that of the parent molecule (4.94E-03±7.67E-04). Residues that are critical for IL-6 binding and do not allow substitutions are designated “critical.”

Table 16

Heavy chain CDR1

[0551] The table provides koff (s-1) values for each valid substitution within a specific CDR. In general, the mutants exhibited a koff value similar to that of the parent molecule (4.94E-03±7.67E-04). Residues that are critical for IL-6 binding and do not allow substitutions are designated “critical.”

Table 17

Heavy chain CDR2

[0552] The table provides koff (s-1) values for each valid substitution within a specific CDR. In general, the mutants exhibited a koff value similar to that of the parent molecule (4.94E-03±7.67E-04). Residues that are critical for IL-6 binding and do not allow substitutions are designated “critical.”

Table 18

Heavy chain CDR3

[0553] The table provides koff (s-1) values for each valid substitution within a specific CDR. In general, the mutants exhibited a koff value similar to that of the parent molecule (4.94E-03±7.67E-04). Residues that are critical for IL-6 binding and do not allow substitutions are designated “critical.”

[0554] The binding kinetics of these single mutation molecules to IL-6 was measured at 25°C using a Biacore T200. Antibodies were captured onto a protein A chip (GE) by injecting cell supernatant at a 1:20 dilution at a rate of 10 μL/min for 60 seconds. Captured antibodies were passed through five concentrations (0.56, 1.67, 5, 15, and 45 nM) of IL-6 over 60 seconds at a flow rate of 30 μL/min and dissociated for 180 seconds in the Biacore single-dose kinetics assay format. cycle. The sensor chip was regenerated by a 60-second injection of 10 mM glycine, pH 1.7, at a flow rate of 50 μL/min. A similar assay system was used to monitor interactions between purified antibody samples and IL-6 at 37°C, where applicable. For this system, 1 μg/mL of purified antibody was injected onto the Protein A chip at a rate of 10 μL/min for 25–28 seconds. Data analysis for supernatant and purified samples was performed using BIAevaluation (GE) software. Two reference signals were subtracted from all sensorgrams and fitted using a 1:1 Langmuir binding model.

[0555] Of all single point mutations tested (Table 19), the G66D mutation in the heavy chain CDR2 (sequential numbering of the antibody variable domain) resulted in a marked increase in affinity (~2-fold decrease in K _D compared to wild type: 6.95x10 ^-11 M relative to 1.58x10 ^-10 M respectively). G66D was retained, and the remaining heavy chain and light chain CDR residues that may be chemically unstable during production (Cys, Met, Asn) were mutagenized. Based on this position, two heavy chain variants containing triple mutations were obtained (Table 21): I) M34I/G66D/I108V, II) M34W/G66D/I108V, and three light chain variants containing triple and quadruple mutations (Table 22): III) M32L/M50D/N52S/M88Q, IV) M32L/M50A/N52S/M88Q, V) M32L/M50D/M88Q (residue positions correspond to the sequential numbering of the variable region of the antibody heavy chain). We assessed the binding of IL-6 to all possible combinations among these variants using the same Biacore assay system as described for single point mutations. The results obtained by the present inventors demonstrated that these mutations counteracted the effect of the single point mutation G66D while maintaining an affinity for IL-6 closer to the original CDR sequence (Table 20). Based on these results, these modified CDRs were incorporated into the development of anti-IL-6 and anti-IL-6-VEGF decoy Ab constructs.

Table 19

Sud./ 25°C Peeled / 37°C CDR Heavy chain - mutation Light chain - mutation Capture ka (1/Ms) kd (1/s) KD(M) Rmax (RU) Chi² (RU²) Capture ka (1/Ms) kd (1/s) KD(M) Rmax (RU) Chi² (RU²) DT DT 981.4 3.15E+05 1.26E-04 3.99E-10 223.2 0.108 138.1 8.80E+05 1.39E-04 1.58E-10 34.6 0.397 Heavy chain - CDR1 G26S DT 437.8 5.51E+05 1.15E-04 2.09E-10 96.7 0.0358 172.4 8.42E+05 3.90E-04 4.63E-10 39.8 0.0688 G26R DT 638.4 4.02E+05 1.21E-04 3.02E-10 141.5 0.0304 183.3 7.02E+05 3.26E-04 4.65E-10 42.8 0.185 F27G DT 764 8.56E+05 2.06E-04 2.40E-10 129.3 0.19 F27P DT 734.7 5.70E+05 1.96E-04 3.44E-10 125.9 0.0913 F27R DT 809.6 5.03E+05 2.09E-04 4.15E-10 137.7 0.0638 F27S DT 573.2 7.31E+05 2.03E-04 2.78E-10 93.5 0.12 F27V DT 699.4 4.70E+05 2.08E-04 4.42E-10 114 0.0548 T28Y DT 797.6 3.32E+05 1.37E-04 4.12E-10 165.6 0.229 T28K DT 773.6 3.86E+05 1.43E-04 3.70E-10 171 0.157 T28A DT 705.1 3.93E+05 1.39E-04 3.54E-10 157.5 0.0635 T28E DT 832.8 4.69E+05 1.63E-04 3.47E-10 192.9 0.112 F29H DT 653.3 4.95E+05 1.35E-04 2.73E-10 148.1 0.0271 156.2 8.50E+05 3.97E-04 4.66E-10 37.2 0.0579 F29Y DT 623.6 4.99E+05 1.33E-04 2.66E-10 139.3 0.0423 174.1 8.42E+05 3.74E-04 4.44E-10 40.2 0.078 S30W DT 619.7 4.17E+05 1.57E-04 3.76E-10 127.9 0.145 S30Y DT 545.2 3.65E+05 1.22E-04 3.34E-10 110.9 0.0853 S30G DT 686.2 3.98E+05 1.32E-04 3.33E-10 156 0.108 P31 DT CRITICAL F32G DT 328.5 4.18E+05 1.33E-04 3.18E-10 68.3 0.0332 155.1 6.50E+05 3.70E-04 5.69E-10 36.2 0.0601 A33 DT CRITICAL M34W DT 634.5 2.96E+05 1.15E-04 3.90E-10 119.6 0.0557 118.3 5.99E+05 2.36E-04 3.94E-10 24.2 0.226 S35H DT 942.1 3.48E+05 1.32E-04 3.79E-10 202.8 0.0305 S35Q DT 587.6 3.75E+05 1.30E-04 3.46E-10 128.9 0.0834 Heavy chain - CDR2 V48I DT 182.6 4.60E+05 1.59E-04 3.46E-10 42.2 0.0224 V48Q DT 49.5 6.54E+05 1.38E-04 2.10E-10 12.8 0.0418 A49T DT 410.6 4.48E+05 1.51E-04 3.37E-10 96.2 0.0495 K50 DT CRITICAL I51R DT 605.9 4.62E+05 7.60E-05 1.65E-10 132.1 0.168 126 6.79E+05 3.43E-04 5.05E-10 28.5 0.173 S52 DT CRITICAL P53 DT CRITICAL G54 DT CRITICAL G55 DT CRITICAL S56R DT 521.8 2.73E+05 1.55E-04 5.67E-10 97.4 0.0349 S56H DT 582.3 3.56E+05 1.61E-04 4.52E-10 128.4 0.0384 W57F DT 335.5 5.77E+05 1.28E-04 2.22E-10 78.1 0.0213 146.5 8.64E+05 4.53E-04 5.24E-10 37.2 0.0527 T58K DT 553.5 4.60E+05 1.79E-04 3.90E-10 121 0.0168 T58R DT 606.4 3.57E+05 1.52E-04 4.26E-10 131.5 0.0529 T58V DT 621.7 8.03E+05 1.81E-04 2.26E-10 144 0.0386 Y59 DT CRITICAL Y60 DT CRITICAL S61R DT 510.6 3.48E+05 1.62E-04 4.67E-10 119.1 0.0401 S61A DT 546.2 3.79E+05 1.49E-04 3.94E-10 123.6 0.0376 D62Q DT 1055.5 4.03E+05 1.48E-04 3.66E-10 237.3 0.0974 70.3 8.75E+05 6.01E-04 6.87E-10 17.5 0.156 D62F DT 279.2 4.20E+05 1.47E-04 3.50E-10 63.8 0.0214 T63D DT 547.4 4.10E+05 1.74E-04 4.23E-10 127.4 0.0677 T63E DT 622.5 3.80E+05 1.69E-04 4.45E-10 141.7 0.0419 T63K DT 882.7 5.46E+05 2.12E-04 3.88E-10 137.4 0.134 T63G DT 702.6 5.17E+05 2.09E-04 4.04E-10 134 0.0425 T63Q DT 900.9 3.09E+05 1.84E-04 5.94E-10 208.1 0.261 V64A DT 558.1 4.44E+05 1.78E-04 4.02E-10 130.3 0.0683 T65Y DT 559.1 749000 0.000206 2.75E-10 123.8 0.0642 T65G DT 3.2 597000 5.69E-05 9.54E-11 3 0.0498 G66E DT 743 1.02E+06 1.10E-04 1.07E-10 176.8 0.242 105.2 1.91E+06 1.71E-04 8.95E-11 31.3 0.522 G66D DT 783.5 1.30E+06 1.17E-04 9.02E-11 184.9 0.254 142.8 1.96E+06 1.36E-04 6.95E-11 39.9 0.513 Heavy Chain - CDR3 A97V DT 547.3 3.11E+05 1.79E-04 5.74E-10 128.2 0.0654 R98G DT 693.2 1.76E+05 2.51E-04 1.42E-09 148.9 0.344 Q99A DT 879.2 3.68E+05 7.37E-04 2.00E-09 183.2 0.0575 Q99H DT 1004.6 5.39E+05 1.05E-03 1.94E-09 213.9 0.094 L100Q DT 1264.1 3.98E+05 1.53E-04 3.84E-10 281.2 0.0485 121.4 8.23E+05 4.10E-04 4.99E-10 28.4 0.155 W101 DT CRITICAL G102D DT 777.7 4.52E+05 6.00E-04 1.33E-09 136 0.0832 Y103D DT 774.6 7.37E+05 2.19E-04 2.97E-10 140 0.0826 Y103S DT 1016.4 5.46E+05 2.31E-04 4.23E-10 177.8 0.125 Y103E DT 794.4 7.18E+05 1.65E-04 2.29E-10 192.4 0.0289 Y104V DT 276.8 5.25E+05 7.03E-04 1.34E-09 41 0.0745 A105Q DT 524.4 4.74E+05 4.93E-04 1.04E-09 94.8 0.107 A105H DT 520.8 4.04E+05 7.23E-04 1.79E-09 108 0.0869 L106F DT 1138.8 5.91E+05 1.85E-04 3.14E-10 252.2 0.0955 444.9 8.52E+05 5.41E-04 6.34E-10 100.5 0.17 L106I DT 268.1 4.79E+05 1.34E-04 2.79E-10 60 0.0889 D107E DT 900.1 5.09E+05 1.47E-04 2.90E-10 200.7 0.0643 344.2 7.62E+05 4.02E-04 5.27E-10 78.7 0.145 I108F DT 1177.7 3.65E+05 1.46E-04 3.99E-10 251.7 0.0728 193.5 6.83E+05 4.01E-04 5.87E-10 42.7 0.216 I108E DT 1135.2 5.71E+05 1.74E-04 3.05E-10 254.4 0.156 234.8 9.21E+05 3.72E-04 4.04E-10 53.8 0.255 I108S DT 815.4 5.54E+05 1.41E-04 2.56E-10 177.3 0.0421 89.5 8.58E+05 3.36E-04 3.91E-10 20.8 0.362 Light chain - CDR1 DT S24P 44.3 4.84E+05 2.67E-06 5.51E-12 11.2 0.0502 DT S24K 739 3.11E+05 1.32E-04 4.24E-10 167.3 0.025 DT S24V 787.2 3.40E+05 1.37E-04 4.03E-10 180.6 0.225 DT S24I 597.7 4.76E+05 1.35E-04 2.84E-10 129.6 0.0541 DT A25S 386.6 5.35E+05 1.05E-04 1.97E-10 90.6 0.0195 351.4 7.82E+05 3.42E-04 4.38E-10 86.1 0.21 DT A25T 513 5.08E+05 1.19E-04 2.35E-10 118 0.0222 77.5 8.89E+05 3.78E-04 4.25E-10 19.8 0.163 DT S26G 5.9 7.93E+05 2.23E-04 2.81E-10 3.6 0.15 DT S26H 525.5 4.15E+05 1.49E-04 3.60E-10 117.4 0.111 DT S26K 696.4 3.79E+05 1.64E-04 4.33E-10 155.4 0.144 DT I27L 878.5 4.37E+05 1.45E-04 3.32E-10 202 0.0356 166.2 8.11E+05 4.49E-04 5.53E-10 39.1 0.18 DT I27W 516.3 4.87E+05 1.61E-04 3.30E-10 113.3 0.129 151.7 1.06E+06 7.64E-04 7.19E-10 30.6 0.221 DT I27F 748.4 4.36E+05 1.93E-04 4.43E-10 168 0.0802 165.3 1.01E+06 6.90E-04 6.85E-10 38.5 0.186 DT S28E 673.8 4.20E+05 1.21E-04 2.89E-10 159.5 0.266 DT S28R 468 3.69E+05 1.71E-04 4.64E-10 110.9 0.0196 DT V29L 385.6 3.51E+05 1.71E-04 4.87E-10 83.4 0.171 DT S30G 84.5 5.48E+05 1.73E-04 3.16E-10 19.3 0.0863 DT S30A 760 2.55E+05 1.63E-04 6.39E-10 169 0.0653 DT Y31G 405.9 3.05E+05 2.03E-04 6.66E-10 92.8 0.0183 DT Y31F 745.1 2.92E+05 1.38E-04 4.72E-10 184.2 0.0274 DT M32L 563.2 5.91E+05 1.18E-04 1.99E-10 130 0.0192 188.7 8.85E+05 3.50E-04 3.95E-10 44.6 0.216 DT M32V 426.5 5.05E+05 1.16E-04 2.30E-10 98.8 0.0226 175.6 8.61E+05 3.69E-04 4.29E-10 43.4 0.188 DT Y33 CRITICAL Light chain - CDR2 DT L45 CRITICAL DT L46V 486.4 4.60E+05 2.03E-04 4.42E-10 96.3 0.0311 DT I47V 490 4.71E+05 1.95E-04 4.14E-10 93.3 0.0258 DT Y48W 500.3 3.45E+05 1.23E-04 3.56E-10 121.7 0.0146 DT D49E 376.2 4.59E+05 1.11E-04 2.41E-10 79.7 0.0725 164.3 6.68E+05 4.17E-04 6.24E-10 36.5 0.181 DT M50D 544.3 8.06E+05 1.12E-04 1.38E-10 129.3 0.0328 48.4 1.49E+06 4.64E-04 3.11E-10 13.7 0.188 DT S51K 817 3.96E+05 2.12E-04 5.35E-10 137.3 0.0479 DT S51R 419.5 4.99E+05 2.31E-04 4.63E-10 64.6 0.0401 DT S51D 552.6 7.29E+05 2.16E-04 2.96E-10 77.5 0.0487 DT N52S 671.2 4.30E+05 1.52E-04 3.53E-10 160.2 0.0246 DT L53H 420.5 5.44E+05 2.18E-04 4.01E-10 64.9 0.0396 DT L53W 10.2 1.19E+06 6.14E-06 5.18E-12 3.5 0.0526 DT L53R 445.3 4.16E+05 1.85E-04 4.46E-10 87.7 0.0331 DT A54G 302 4.75E+05 1.98E-04 4.16E-10 54.2 0.0241 DT A54Y 104.2 6.52E+05 1.09E-04 1.68E-10 18 0.0725 DT A54L 391.8 4.20E+05 2.10E-04 4.99E-10 68.2 0.0473 DT S55W 472.5 5.63E+05 2.26E-04 4.01E-10 69.3 0.0716 DT S55Y 615.7 4.36E+05 2.01E-04 4.61E-10 102.7 0.0384 DT S55R 17.8 9.87E+05 1.07E-04 1.09E-10 5.2 0.0981 DT S55L 661 6.68E+05 2.20E-04 3.29E-10 99.8 0.131 Light chain - CDR3 DT M88V 240.3 4.48E+05 1.07E-04 2.39E-10 49.4 0.0502 128.8 6.19E+05 4.41E-04 7.13E-10 29.8 0.176 DT M88A 328.8 4.51E+05 1.27E-04 2.82E-10 51.3 0.976 151.7 5.86E+05 1.32E-03 2.25E-09 27.5 0.141 DT Q89S 471.5 3.62E+05 2.72E-04 7.52E-10 61.2 0.0171 DT Q89A 186.6 5.43E+05 2.27E-04 4.19E-10 32.2 0.085 DT W90 CRITICAL DT S91A 345.3 3.41E+05 1.39E-04 4.07E-10 79 0.0325 DT G92 CRITICAL DT Y93 CRITICAL DT P94 CRITICAL DT Y95G 503.8 8.82E+05 3.38E-04 3.83E-10 51 0.126 DT Y95I 274.3 5.93E+05 4.44E-03 7.48E-09 42.2 0.107 DT T96L 399 5.20E+05 1.35E-04 2.59E-10 83.8 1.01 172.8 8.82E+05 7.95E-04 9.01E-10 37.1 0.234 DT T96V 523 4.54E+05 1.92E-04 4.23E-10 110.6 0.169 147.3 9.21E+05 6.39E-04 6.95E-10 34.8 0.202

[0556] Biacore kinetics for single anti-IL-6 point mutations. Data were obtained for samples before (supernatant (supernatant)) at 25°C and after purification at 37°C. Mutations selected for the final CDR combinations (Table 19) are in italics. CDR positions that did not allow mutations during bacterial screening are designated as critical.

Table 20

[0557] Biacore kinetics data for anti-IL-6 CDR variants. Kinetic data were obtained for cell culture supernatants (supernatants) at 25°C and purified samples at 37°C. Heavy and light chain CDR mutations are shown on the left and right sides, respectively (heavy/light). The table shows the uptake rates, kon (ka), koff (kd) and dissociation constants.

Table 21. Anti-IL-6 heavy chain variable region sequences. Some CDR options are underlined

ID Subsequence I EVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAISWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSS (SEQ ID NO: 140) II EVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAWSWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSS (SEQ ID NO: 141)

Table 22. Anti-IL-6 light chain variable region sequences. Some CDR options are underlined

ID Subsequence III DIQLTQSPSSLSASVGDRVTTITCSASISVSYLYWYQQKPGKAPKLLIYDDSSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIK (SEQ ID NO: 142) IV DIQLTQSPSSLSASVGDRVTTITCSASISVSYLYWYQQKPGKAPKLLIYDASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIK (SEQ ID NO: 143) V DIQLTQSPSSLSASVGDRVTTITCSASISVSYLYWYQQKPGKAPKLLIYDDSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIK (SEQ ID NO: 144)

Example 2

Development and construction of the ab anti-IL-6 VEGF trap molecule

[0558] The VEGF binding fragment in the anti-IL-6 VEGF Trap Ab construct consisted of a fusion of VEGFR1 domain 2 and VEGFR2 domain 3 (Table 23). This fusion acts as a decoy for VEGF and prevents VEGF from binding to cellular VEGF receptors.

Table 23. VEGF Trap Sequence

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK (SEQ ID NO: 145)

[0559] Twelve designs containing the VEGF trap in two different configurations were tested (Table 24). In the first configuration, the VEGF trap was positioned at the beginning of the protein, followed by a double Gly-Gly-Gly-Gly-Ser (GS) linker sequence connecting the VEGF trap to the N-terminus of the anti-IL-6 heavy chain (VEGF-anti-IL-trap). 6; Table 24, AF molecules). In the second configuration, the heavy chain variable and constant domains (Fab region) were connected to the VEGF trap via a GS linker, and then the Fc domain was fused to the C terminus of the VEGF trap. Thus, in this configuration, the VEGF trap was sandwiched between the Fab and Fc regions of the antibody (anti-IL-6 VEGF trap; Table 24, GL molecules).

Table 24. Heavy and light chain sequences for dual inhibitor molecules. Some CDR variants are underlined in the heavy and light chains, the VEGF trap sequence is in bold, the Gly-Ser linker is in italics (and bold)

ID Heavy chain Light chain A SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK GGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAISWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 269) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDDSSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 146) B SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK GGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAISWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 147) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 148) C SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK GGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAISWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 149) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDDSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 150) D SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK GGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAWSWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 151) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDDSSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 152) E SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK GGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAWSWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 153) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 154) F SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK GGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAWSWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 155) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDDSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 156) G EVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAISWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSD QGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 157) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDDSSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 158) H EVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAISWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSD QGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 159) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 160) I EVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAISWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSD QGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 161) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDDSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 162) J EVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAWSWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSD QGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 163) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDDSSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 164) K EVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAWSWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSD QGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 165) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 166) L EVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAWSWVRQAPGKGLEWVAKISPGGSWTYYSDTVTDRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARQLWGYYALDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSD QGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPGK (SEQ ID NO: 167) DIQLTQSPSSLSASVGDRVTITCSASISVSYLYWYQQKPGKAPKLLIYDDSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSGYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 168)

[0560] Kinetic data from Biacore (the system described in the previous analysis) showed that although the K _D values among all variants were comparable, there was a difference in the capture values for the supernatant samples (Table 25). This parameter is sensitive to the amount of antibody present in the solution, suggesting variation in the level of protein expression among these molecules due to different CDR compositions. On the other hand, the two VEGF trap positions tested did not show any significant effect on the kinetic data. Experiments monitoring expression levels (data not shown) showed that the IL-6-VEGF trap configuration provided approximately 20% higher expression levels compared with the VEGF-antiIL-6 trap configuration. Preliminary data from Biacore also indicate that the anti-IL-6 decoy configuration of VEGF binds more strongly to the VEGF homologue, placenta growth factor (PLGF) (data not shown).

Table 25

[0561] Biacore kinetics data for dual inhibitor AL variants binding to IL-6. Kinetic data were obtained for cell culture supernatant samples (supernatant) at 25°C and purified samples at 37°C. Capture rates, kon (ka), koff (kd) and dissociation constants are given.

Sud./ 25 °C Cleaned / 37 °C ID Capture ka (1/Ms) kd (1/s) KD(M) Capture ka (1/Ms) kd (1/s) KD(M) A 521.4 3.09E+05 1.82E-04 5.89E-10 100 1.11E+06 5.38E-04 4.84E-10 B 144.3 6.05E+05 1.85E-04 3.05E-10 106.6 1.06E+06 5.36E-04 5.07E-10 C 474.6 3.79E+05 1.50E-04 3.94E-10 92 1.01E+06 4.81E-04 4.77E-10 D 356.2 4.33E+05 1.09E-04 2.53E-10 105.4 1.23E+06 4.47E-04 3.65E-10 E 228 5.04E+05 1.51E-04 2.99E-10 97.9 1.08E+06 4.59E-04 4.24E-10 F 272.1 6.11E+05 1.37E-04 2.25E-10 93.9 1.31E+06 4.46E-04 3.4E-10 G 519 2.52E+05 1.49E-04 5.89E-10 109 1.26E+06 5.67E-04 4.51E-10 H 424.7 4.54E+05 1.62E-04 3.56E-10 97.3 1.34E+06 5.29E-04 3.96E-10 I 552.8 2.17E+05 1.47E-04 6.79E-10 108.9 1.15E+06 4.99E-04 4.35E-10 J 335 5.34E+05 1.49E-04 2.80E-10 88.9 1.42E+06 6.64E-04 4.68E-10 K 279.8 6.29E+05 1.78E-04 2.82E-10 93.9 1.28E+06 5.79E-04 4.52E-10 L 312.4 7.05E+05 1.69E-04 2.39E-10 91.9 1.49E+06 6.50E-04 4.36E-10

Example 3

Dual Inhibitor Binding to VEGF by Biacore Assay

[0562] To evaluate the binding kinetics of different dual inhibitor configurations to VEGF, a series of Biacore assays were performed to measure the affinity among these molecules. Binding kinetics were measured at 37°C in HBS-EP+ buffer containing 1 mg/ml BSA. Anti-VEGF agents (VEGF-antiIL-6 trap construct (5 µg/ml), anti-IL-6-VEGF trap construct (5 µg/ml), OG1950 (1 µg/ml) and Eylea (0.5 µg/ml) ) was captured onto the protein A chip (GE) at a rate of 10 μl/min for 25 seconds, five concentrations of VEGF-A165 (0.19, 0.56, 1.67, 5, 15 nM) were passed through the captured antibodies for 120 seconds at a rate of 30 μL/min and dissociated for 30 minutes. The surface of the sensor chip was regenerated by a 60-second injection of 10 mM glycine, pH 1.7, at a flow rate of 50 μL/min. Two reference signals were subtracted from all sensorgrams and fitted using a 1:1 Langmuir binding model.

[0563] Data indicate that the VEGF decoy component of the dual inhibitor molecule binds tightly to VEGF-A regardless of configuration. The anti-IL-6 component of the molecule did not appear to affect the binding of VEGF-A to the VEGF decoy, as the dual inhibitor molecules exhibit similar VEGF affinity to another VEGF decoy molecule (Eylea). (Fig.7).

Binding of IL-6 and VEGF-A to dual inhibitor molecules

[0564] To directly monitor the dual binding of IL-6 and VEGF-A, the Biacore assay was performed. Dual inhibitor molecules (2 μg/ml) were captured onto the protein A (GE) chip at a rate of 10 μl/min for 25 seconds, five concentrations of IL-6 and/or VEGF-A165 (0.19, 0.56, 1 ,67, 5, 15 nM) were passed individually or in mixture through the captured antibodies for 120 seconds at a rate of 30 μl/min and dissociated for 30 minutes. The surface of the sensor chip was regenerated by a 60-second injection of 10 mM glycine, pH 1.7, at a flow rate of 50 μL/min. Data from the two controls were subtracted from all data, and the sum of the individual IL-6 and VEGF-A sensorgrams was used to construct a theoretical curve, which was compared with the sensorgrams of the mixed samples.

[0565] The results showed that the theoretical and experimental curves are virtually identical when superimposed, which strongly supports a model in which both targets bind to the dual inhibitor molecules and do not affect their respective interactions (Figure 8).

ELISA assay for anti-IL-6 binding to the IL-6/IL-6-R complex

[0566] IL-6/IL-6R complexes can bind to the membrane-bound receptor gp130, and this trimeric complex stimulates IL-6 signaling such as proliferation and up-regulation of VEGF. Inhibition of either IL-6 binding to IL-6R or IL-6/IL-6R binding to gp130 blocks IL-6-mediated downstream signaling pathways. To determine whether anti-IL-6 antibodies block any of these binding processes, an ELISA was first performed to assess whether they could bind IL-6 when it was complexed with the IL-6 receptor.

[0567] Nunc MaxiSorp ELISA was coated with 100 μl of 1 μg/ml recombinant human IL-6R (R&D Systems) overnight. 10 nM IL-6 (R&D Systems) was mixed with a series of 3-fold dilutions of anti-IL-6 (final concentrations ranging from 30 nM to 10 pM). After blocking and washing, the mixture was added to IL-6R-coated plates. This assay was also performed by first incubating the IL-6R-coated plate with 10 nM IL-6, washing, and then adding anti-IL-6 antibody. For both methods, bound antibody/IL-6 complexes were detected with HRP anti-human IgG antibody and developed with TMB (KPL) for 10 minutes. Absorbance was measured at 450 nM on a SpectraMax Plus plate reader (Molecular Devices) and plotted against antibody concentration using GraphPad Prism software and fitted to a nonlinear regression curve.

[0568] Nunc MaxiSorp ELISA was coated with 100 μl of 1 μg/ml recombinant human IL-6 (R&D systems) overnight. A series of 3-fold dilutions of anti-IL-6 (R&D Systems; final concentrations from 30 nM to 10 pM) were carried out and the solutions were added to blocked IL-6-coated plates. Antibody/IL-6 complexes were detected with HRP anti-human IgG and developed with TMB (KPL) for 10 minutes. Absorbance was measured at 450 nM on a SpectraMax Plus plate reader (Molecular Devices) and plotted against antibody concentrations using GraphPad Prism software and resulted in a nonlinear regression curve.

[0569] Under the conditions tested, the anti-IL6 antibody described herein specifically binds to IL-6, but not when complexed with the IL-6 receptor, suggesting that the antibody and IL-6R share a common epitope on IL-6. The results are presented in Fig. 4.

IL-6/IL-6R Competitive ELISA Assay

[0570] An ELISA was then performed to determine whether the anti-IL6 antibody and dual IL-6/VEGF inhibitors blocked the binding of IL-6 to the IL-6R.

[0571] Nunc MaxiSorp ELISA was coated with 100 μl of 1 μg/ml antibody to the IL-6/IL-6R complex (R&D systems; IL-6/IL-6R DuoSet) overnight. Block the plate with 2% bovine serum albumin (BSA) in PBS-Tween solution (PBS-T) for 2 hours. During blocking, 6 nM IL-6 (R&D Systems), 1 nM IL-6R (R&D Systems), and a 3-fold dilution series to obtain 8 dilution points of dual inhibitors or one group of controls (starting at 30 nM) were incubated at room temperature. This mixture was then added to the ELISA plate after a washing step. After 1 hour, the plate was washed and co-incubated with biotinylated anti-IL-6/IL-6R complex antibody (R&D Duoset). IL-6/IL-6R complexes were detected using streptavidin-HRP (1:2000 dilution; R&D Systems) and developed using TMB reagent (KPL) for 10 minutes. Absorbance was measured at 450 nM on a SpectraMax Plus plate reader (Molecular Devices) and plotted against antibody concentration using GraphPad Prism software and fitted to a nonlinear regression curve.

[0572] Under these conditions, anti-IL6 antibody and dual IL-6/VEGF inhibitors effectively compete with IL-6R for binding to IL-6 and therefore inhibit IL-6/IL-6R interaction. The IC50 values for the dual inhibitor molecules are comparable (anti-IL-6=0.36 nM, VEGF-anti-IL-6 decoy construct=0.47 nM and anti-IL-6-VEGF decoy construct=0.32 nM), indicating that the configuration of the molecule does not affect IL-6 binding or inhibition. Eilea served as a negative control. (Fig.9).

VEGF/VEGFR Competitive ELISA Assay

[0573] To evaluate VEGF decoy function, the ability of dual IL-6/VEGF inhibitors to block VEGF binding to the receptor (VEGFR) was tested by competitive ELISA.

[0574] Nunc MaxiSorp ELISA was coated with 100 μl of 1 μg/ml recombinant human VEGFR-Fc (R&D systems) overnight. Block the plate with 2% bovine serum albumin (BSA) in PBS-Tween solution (PBS-T) for 2 hours. During blocking, 100 pM biotinylated VEGF (btVEGF; R&D Systems) was co-incubated with increasing concentrations of the anti-IL-6-VEGF trap construct, the VEGF-anti-IL-6 trap construct, or the divalent anti-VEGF antibody, OG1950. Bound bt-VEGF was detected using streptavidin-HRP (1:2000 dilution; R&D Systems) and developed using TMB reagent (KPL) for 10 minutes. Absorbance was measured on a SpectraMax Plus plate reader (Molecular Devices) at 450 nM and applied plotted against antibody concentration using GraphPad Prism software and resulted in a nonlinear regression curve.

[0575] Under these conditions, dual IL-6/VEGF inhibitors bind to VEGF and inhibit its binding to the receptor. When the VEGF trap is located at the N terminus of the anti-IL-6 antibody, the inhibition of VEGF is approximately 2.5 times greater than for a molecule in which the VEGF trap is located between the Fab and Fc domains of the anti-IL-6 antibody (IC50 1.74 nM vs. 4.53 nM, respectively), likely due to a change in valence from 2 binding sites to 1 binding site. The results are presented in Fig.9.

Example 4

IL-6-VEGF Bridge ELISA Assay

[0576] To confirm that the dual inhibitors could simultaneously bind to both IL-6 and VEGF, they were subjected to a bridge ELISA in which they were first reacted with plate-bound IL-6 and then co-incubated with biotinylated VEGF (btVEGF). Detection of btVEGF by streptavidin-HRP indicates that molecules associated with IL-6 are also able to bind btVEGF.

[0577] Nunc MaxiSorp ELISA was coated with 100 μl of 1 μg/ml recombinant human IL-6 (Tonbo Sciences) overnight. Block the plate with 2% bovine serum albumin (BSA) in PBS-Tween solution (PBS-T) for 2 hours. During blocking, 100 pM btVEGF was co-incubated with increasing concentrations of the anti-IL-6-VEGF trap construct, the VEGF-anti-IL-6 trap construct, or the divalent anti-VEGF antibody, OG1950, as a negative control. Bound btVEGF was detected using streptavidin-HRP (1:2000 dilution; R&D systems) and developed using TMB reagent (KPL) for 10 minutes. Absorbance was measured at 450 nM on a SpectraMax Plus plate reader (Molecular Devices) and plotted as a function of antibody concentration using GraphPad Prism software and resulted in a nonlinear regression curve.

[0578] This analysis confirmed that dual IL-6/VEGF inhibitors simultaneously bind IL-6 and btVEGF. The orientation of anti-IL-6 and VEGF trap shows slightly variable binding, with the orientation of anti-IL-6-VEGF trap being more suitable in these conditions (EC50 of VEGF-anti-IL-6 trap construct = 0.079 nM and anti-IL-6-trap construct VEGF=0.026 nM). Eylea and anti-IL-6 served as negative controls. The results are presented in Fig.9.

Example 5

Path 1. Synthesis of OG1802

[0579] The first route for the synthesis of OG1802 is as follows. First, a TFA/amine salt initiator (Compound L) having the structure shown in FIG. 2A was synthesized as follows.

[0580] First, Compound K having the structure shown in Figure 2B was synthesized as follows. Compound J (OG1563) (1.9 g, 2.67 mmol, 3.3 eq.) was placed in a 200 mL round bottom flask under nitrogen atmosphere.

CONNECTION J

and Compound E (0.525 g, 0.81 mmol, 1.0 eq.) (see Figure 2L), followed by dimethylformamide (10 mL), then diisopropylethylamine (2.5 mL, 14.6 mmol, 18 eq. ). The flask was cooled to 0°C using an ice bath. To this was added a solution of propylphosphonic anhydride (50 wt% in ethyl acetate, 2.5 mL, 4.04 mmol, 5 eq.) over ~6 minutes.

[0581] The reaction mixture was warmed to room temperature and stirred for 15 minutes. The reaction was quenched by adding water (20 ml), saturated aqueous sodium bicarbonate (20 ml) and ethyl acetate (100 ml). The organic phase was separated and the aqueous phase was extracted with ethyl acetate (75 ml). The combined organic phases were washed with saturated aqueous sodium bicarbonate (30 ml), 0.5 M aqueous citric acid (40 ml), water (25 ml) and saturated aqueous sodium chloride (40 ml), then dried (sodium sulfate), filtered and concentrated in a vacuum. The residue, which was used without further purification, provided 2.0 g (0.80 mmol, 99%) of Compound K.

¹ H NMR (400 MHz DMSO-d6): (=1.36 (s, 9H, OC CH3 ), 1.90 (s, 54H, CC (CH3)2 Br), 2.31 (t, J=7 .2 Hz, 6H, C CH2 CH2NH), 2.98 (d, J=5.6 Hz, 6H, C CH2 NH), 3.04 (q, J=6.0 Hz, 2H, OCH2 CH2 NH) , 3.18 (s, 2H, O CH2 C), 3.3-3.37 (m, 8H , CH2 ), 3.47-3.55 (m, 12H, CH2 ), 3.58 (s, 6H, O CH2 C), 3.87 (s, 6H, O=C CH2 O), 4.27 (s, 18H, C CH2 OC=O), 6.74 (br t, 1H, CH2 NH C= O), 7.69 (t, J=6.8 Hz, 3H, CH2 NH C=O), 7.84 (t, J=6.0 Hz, 3H, CH2 NH C=O).

LC-MS (ES, m/z): [(M+2H-boc)/2]+calc. for (C84H136Br9N7O33+2H-Boc)/2=1196.6; 1196.6 detected.

[0582] Compound L (Figure 2A) was then synthesized as follows: Compound K (2.0 g, 0.8 mmol), dichloromethane (10 mL), and then trifluoroacetic acid (5 ml). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was concentrated in vacuo. The reaction mixture was diluted with dichloromethane (10 ml) and concentrated in vacuo. The residue was dissolved using acetonitrile (10 ml), filtered through a syringe filter (Acrodisc CR25, PN 4225T) and loaded onto a preparative HPLC column, and eluted with 60% acetonitrile in water (with 0.1% trifluoroacetic acid) - 98% acetonitrile ( with 0.1% trifluoroacetic acid). Tubes containing the product were pooled, concentrated in vacuo, frozen and placed in a lyophilizer. The result was 990 mg (0.4 mmol, 50% in 2 steps) of Compound L as a white powder.

¹ H NMR (400 MHz DMSO-d6): (=1.90 (s, 54H, CC (CH3)2 Br), 2.31 (t, J=7.2 Hz, 6H, C CH2 CH2NH), 2 .97-3.0 (m, 8H, C CH2 NH and OCH2 CH2 NH), 3.17 (s, 2H, O CH2 C), 3.3 (q, 6H, CH2 CH2 NHC=O), 3. 4-3.59 (m, 20H, CH2 ), 3.87 (s, 6H, O=C CH2 O), 4.27 (s, 18H, C CH2 OC=O), 7.69-7.84 (m, 9H, both CH2 NH C=O and NH3 +).

LC-MS (ES, m/z): [(M+2H)/2]+calc. for (C84H136Br9N7O33+2H)/2=1196.6; found 1197.4.

[0583] Compound L (Figure 2A) was then used as an initiator for the synthesis of the MPC polymer. The initiator is typically prepared as a stock solution in DMF at approximately 100 mg/mL. The initiator and ligand (2,2'-bipyridyl) were introduced into a Schlenk vessel. The resulting solution was cooled to -78°C using dry ice/acetone and degassed in vacuo for 10 minutes. The flask was refilled under argon and the catalyst (CuBr unless otherwise noted) kept under argon was introduced into the Schlenk flask (molar ratio of bromine atom in initiator/catalyst (CuBr)/ligand was maintained at 1/1/2) . The solution immediately turned dark brown. The Schlenk flask was sealed and immediately purged using a short vacuum/argon cycle. A HEMA-PC solution was prepared by mixing a certain amount of monomer, prepared in a glove box under nitrogen, with degassed 200 proof ethanol. The monomer solution was added dropwise into the Schlenk vessel (via cannula) (and homogenized by gentle stirring). The temperature was maintained at -78°C. The reaction mixture was subjected to full vacuum for at least 10-15 minutes until no more bubbles erupted from solution. The vessel was then filled with argon again and heated to room temperature. The solution was stirred and as polymerization proceeded, the solution became viscous. After 3-8 hours or after simply leaving overnight, the reaction is quenched by direct exposure to air to oxidize Cu(I) to Cu(II), the mixture turns blue-green and is passed through a silica column to remove the copper catalyst . The collected solution was concentrated by rotary evaporation and the resulting mixture was either precipitated with tetrahydrofuran or dialyzed against water followed by freeze-drying to obtain a free-flowing white powder. Table 26 below presents data for a polymer in which Compound L was used as an initiator.

Table 26

Theor. MM
(kDa) Polymer ID No. Initiator Mn (kDa) Mp (kDa) PDI 500 130 L 490 530 1.1 750 150 L 645 750 1.1

[0584] The Maleimide ester Mal-PEG4-PFP (as shown in Figure 2C) was then “clipped” to the above 750 kDa polymer to produce OG1802. Polymer R3707 (750 kDa polymer prepared using compound L as initiator, 515 mg) was placed in a 20 ml vial and dissolved using ethanol (4.0 ml) after stirring for 40 minutes. 1% 4-methylmorpholine solution was added in acetonitrile (22 µl). In a separate vial, Mal-PEG4-PFP (1.97 mg) was dissolved in acetonitrile (1.0 ml), and this solution was added to the polymer solution for ~2 minutes at room temperature, and the resulting solution was stirred overnight. The reaction mixture was diluted with 0.1% aqueous trifluoroacetic acid (2 ml) (pH ~5), then with water (~12 ml), filtered through a syringe filter (Acrodisc Supor, PN 4612) and equally placed in 3 centrifuge tubes with an Amicon dialysis membrane (30,000 nominal molecular weight cutoff (mwco)). These tubes were diluted and mixed with water (~5 ml each), placed in a centrifuge (3200 rpm) for 25 minutes. The filtrate was recovered for analysis, and the retentate was diluted and mixed with water (~10 ml/tube). The centrifugation procedure was repeated 5 more times, after which the retentate was removed and placed in a vial. Amicon membrane tubes were washed with water (2 x ~2 ml each tube) and combined with the retentate. The retentate solution was filtered through a syringe filter (Acrodisc Supor, PN 4612), frozen and placed in a lyophilizer. The result was 485 mg of white powder.

Example 6: Synthesis of OG1786 initiator

[0585] OG1786 is a nine-arm initiator for polymer synthesis used as a precursor in the synthesis of OG1802. Each branch ends in 2-bromoisobutyrate, which is capable of initiating polymerization under ATRP conditions. OG1786 is a trifluoroacetic acid (TFA) salt shown in Figure 2D. OG1786 was prepared as follows. First, OG1550 was reacted with TFA (trifluoroacetic acid) to produce OG1546, as shown in Figure 2E.

[0586] To a 1 L round bottom flask equipped with a magnetic stirrer and dropping funnel, OG1550 (14.8 g), methyl tert-butyl ether (MTBE) (350 mL), and water (30 mL) were added. The mixture was stirred with dissolved OG1550, then cooled in an ice bath. A solution of trifluoroacetic acid (4.9 ml) in water (90 ml) was added dropwise to this mixture over 90 minutes. After addition was complete, the mixture was stirred for an additional 15 minutes, then removed from the ice bath and allowed to warm to room temperature. The mixture was stirred (after removing from the ice bath) for another 4-5 hours until thin layer chromatography (TLC) showed that ~5% of the starting material remained and the pH of the aqueous mixture was between 3 and 4 (pH test paper ).

[0587] The mixture was separated. The MTBE phase was washed with water (30 ml). The aqueous phases were combined, then extracted with MTBE (150 ml). The second phase of MTBE was washed with water (30 ml). The combined aqueous phases were washed with a third portion of MTBE (100 ml). The third phase of MBTE was washed with water (25 ml). The aqueous phases were combined again (~250 ml, pH ~4 using pH paper).

[0588] The product was collected by lyophilization. 11.5 g of a white solid were obtained. This substance is extremely hygroscopic, so manipulations with it are best carried out in a nitrogen atmosphere. The product was confirmed by LC-MS.

[0589] The resulting OG1546 was then reacted with OG1563 to produce OG1784 (as shown in Figure 2F).

[0590] OG1546 (hygroscopic, 9.0 g) followed by N,N-dimethylformamide (110 mL) was added to a 250 mL flask under nitrogen atmosphere equipped with a stirrer. The mixture was stirred at room temperature until all of the OG1546 had dissolved (approximately 15 minutes), then OG1563 (29.9 g) was added and the mixture was stirred for another 3 minutes until the OG1563 had dissolved. The resulting solution was cooled in an ice bath and N,N-diisopropylethylamine (37.6 ml) was added over 3 minutes, followed by propylphosphonic anhydride (T3P), 50% in ethyl acetate (34.5 ml) dropwise over 5 minutes (addition T3P is exothermic). Once the addition of T3P was complete, the flask was removed from the cooling bath and allowed to warm to room temperature. Samples were then taken for LC-MS analysis at 5-min intervals. The reaction mixture showed a very light yellow-brown color.

[0591] After 20 minutes, the reaction mixture was cooled again in an ice bath and 5 ml of water was added. The mixture was then removed from the cooling bath and an additional 50 ml portion of water was added, followed by 50 ml of 0.5 M citric acid, then isopropyl acetate (300 ml). The mixture was separated. The aqueous phase (~300 ml) was extracted with additional isopropyl acetate (150 ml). The aqueous phase was AQ1 for HPLC testing. The combined organic phases were washed with aqueous citric acid (115 ml, 65 mM, which was a mixture of 15 ml 0.5 M citric acid plus 100 ml water), and the aqueous phase was AQ2 (pH~3). The organic phase was washed with water/saturated sodium chloride (100 ml/25 ml), and the aqueous phase was AQ3 (pH~3). The organic phase was finally washed with saturated sodium chloride (100 ml) and the aqueous phase was AQ4. None of the aqueous (AQ) fractions contained any significant product (data not shown). The product was confirmed in the organic phase by LC-MS. The product was dried over sodium sulfate (80 g), filtered and washed with isopropyl acetate (75 ml), and concentrated on a rotary evaporator to give a tan oil (33.2 g). The crude product was stored overnight under a nitrogen atmosphere.

[0592] The next day, the crude product was allowed to reach room temperature, then dissolved in acetonitrile/water (46 ml/12 ml) and filtered using an HPLC filter disk (Cole-Parmer PTFE 0.2 µm, product number 02915-20) . The filtrate was divided into three equal parts and purified in three series.

[0593] The filtrate was loaded onto a C18 RediSep Rf Gold column (275 g, SN 69-2203-339, lot # 24126-611Y) equilibrated with 50% acetonitrile/water. The substance was eluted at 100 ml/min using the following gradient (solvent A: water, solvent B: acetonitrile). All relevant fractions were verified by HPLC. Fractions found to be sufficiently pure were pooled (from all three batches) and concentrated (bath temperature maintained at approximately 20°C) on a rotary evaporator, then partitioned between dichloromethane (100 ml) and water (5 ml)/saturated sodium chloride (25 ml). The aqueous phase was extracted two more times with dichloromethane (2 × 30 ml). The combined organic phases were dried over sodium sulfate (35 g), filtered, washed with dichloromethane (DCM) (30 ml) and concentrated. Product and purity were confirmed by LC-MS methods. Actual yield and purity of batches R5172 and R5228 are presented in Table 27.

Table 27

Batch OG1784 R5172 R5228 Used OG1546 5.3 g 9.0 g Used OG1563 17.6 g 29.9 g Actual output 53% 58% Purity (a/a 210 nm) 99.3% 100.0%

[0594] OG1405 was then prepared from OG1784 as shown in Figure 2G. To a 500 mL round bottom flask equipped with a magnetic stirrer, OG1784 (20.9 g) was added, followed by dichloromethane (50 mL), then trifluoroacetic acid (20 mL). The mixture was stirred at room temperature and HPLC analysis demonstrated complete deprotection after 23 minutes. The mixture was concentrated on a rotary evaporator, redissolved in dichloromethane (25 ml) and concentrated again, then redissolved in acetonitrile (25 ml) and concentrated again. The product was confirmed by LC-MS. The above material (OG1405, 34.5 g, estimated quantitative yield 21.0 g) was used as a crude oil in the next step. There was no need for cleaning.

[0595] OG1405 was then reacted with OG1402 to produce OG1785, as shown in Figure 2H. In a 500 ml flask under nitrogen atmosphere, equipped with a stirrer, was placed OG1402 (5.5 g), then acetonitrile (70 ml), then N,N-diisopropylethylamine (26.3 ml) and the T3P solution (see above) (7, 9 ml). The solution was stirred at room temperature for 30 minutes, then cooled in an ice water bath and a solution of OG1405 (crude oil from step above, 34.5 g) in acetonitrile (70 ml) was added. The mixture was warmed to room temperature. After 20 minutes, the reaction mixture was cooled in an ice water bath and quenched with water (5 ml). The mixture was then concentrated in vacuo using a rotary evaporator to half volume. Samples were taken for LC-MS.

[0596] More water (50 ml) was added, followed by 0.5 M citric acid (75 ml) and isopropyl acetate (175 ml). After 5 minutes the mixture was separated. The aqueous phase was extracted with additional isopropyl acetate (50 ml). The combined organic phases were washed with aqueous citric acid (0.13 M, 30 ml, consisting of 10 ml of 0.5 M citric acid and 20 ml of water). The organic phases were then washed with a mixture of saturated sodium chloride (25 ml) and water (25 ml), then finally washed with saturated sodium chloride (25 ml). They were then dried over sodium sulfate (124 g), filtered and washed with isopropyl acetate (30 ml), and concentrated on a rotary evaporator to give a tan oil (27.3 g). Samples were taken for LC-MS analysis.

[0597] The said oil was dissolved in acetonitrile/water (3:1, 15 ml/5 ml), filtered through an HPLC filter disk (Cole-Parmer PTFE membrane 0.2 μm, product number 02915-20) and divided into three equal portions, each of which was separately purified as follows.

[0598] Portions were loaded onto a C18 Redi-Sep Gold column (275 g, SN - 69-2203-339, lot 241234-611W) equilibrated with 50% solvent B (acetonitrile)/50% solvent A (water). The material was then purified by reverse phase HPLC using a solvent A:water/solvent B:acetonitrile gradient. The appropriate fractions were pooled and partitioned between dichloromethane (150 ml) and water (5 ml)/saturated sodium chloride (25 ml). The aqueous phase was extracted twice with dichloromethane (2 x 50 ml). The combined organic phases were dried over sodium sulfate (60 g), filtered and washed with dichloromethane (40 ml), and concentrated. Structure and purity were confirmed by various analytical methods, including LC-MS: OG1785 was isolated as a foamy solid (R5329, 19.0 g, 83% yield, 95.1% purity (a/a 210 nm), stored under nitrogen at 4°C.

[0599] The tert-butyloxycarbonyl protecting group on OG1785 was then removed using trifluoroacetic acid (TFA) to obtain OG1786, as shown in Figure 2I.

Example 7 Synthesis of OG1801 polymer

[0600] The OG1801 polymer was first prepared from the initiator OG1786. OG1801 contains an amino functional group, which is more stable (than maleimide) during polymer synthesis. To synthesize the polymer OG1801, a modified version of ATRP was used, in which the copper compound (Cu(I)) was prepared in situ by adding metallic copper to Cu(II). The starting materials and reagents required in this reaction were calculated based on the loading batch of monomer (HEMA-PC) OG47 as well as the target molecular weight (MW).

[0601] In a glove box, 50 g of OG47 monomer was weighed and 200 ml of degassed EtOH was added to dissolve the said monomer at room temperature; samples were taken to test monomer concentration. Cu(II), Bpy, Cu(0) were weighed and placed in a 500 ml flask; purged with argon while adding the monomer solution to the flask; the flask was stoppered and subjected to vacuum for 25 minutes until the bubbles disappeared. The reaction mixture gradually changed color from light green to dark green, then to light brown; ~200 mg of OG1786 initiator was weighed in a glove box and dissolved in ~2000 μL of DMF at room temperature to obtain a stock solution of 100 mg/mL; samples were taken to test initiator concentration and purity; The initiator solution was added to the flask in an argon atmosphere. Over time, the reaction solution turned dark brown and began to thicken; the system was closed and the reaction was allowed to proceed for 2 days.

[0602] OG1801 was then prepared for maleimide addition and catalyst (copper) removal as follows: A RediSep® Rf normal phase silica packed column was used to remove the catalyst. The column size was chosen based on the amount of copper in the reaction mixture. For example, for a 50 g batch of OG1801, a 330 g column was used (Cat. No. 69-2203-330, column size 330 g, column volume (CV)=443 ml). Teflon tubing was used for connection since the elution solvent was EtOH.

[0603] After copper removal, all fractions were transferred in batches to a round bottom flask and the EtOH was evaporated to dryness using a rotary evaporator at 45-50°C under reduced pressure. At this stage, the volume of EtOH collected from the condensation was monitored to ensure that EtOH removal was >90%. The polymer was dissolved in 250 ml of water for injection and filtered using a 0.2 μm filter. The result was a transparent, light yellow polymer solution at a concentration of ~150 mg/ml. This solution can be stored at 2-8°C for up to 3 months before use.

Example 8 Synthesis of polymer OG1802

[0604] The starting materials and reagents required in this reaction were calculated based on the loading batch of OG1801. The linker is an NHS ester of 3-maleimidopropionic acid. To 50 g of polymer solution (~150 mg/ml) was added 30 ml of 0.5 M sodium phosphate (in water for injection, pH 8). Leave to stir for 1 minute; The pH measured by pH indicator paper was 8.0. 204.8 mg of linker was weighed and dissolved in 4.1 ml of DMF to obtain a stock solution of 50 mg/ml. The linker solution was added dropwise to the polymer solution at a rate of 815 μL per minute with vigorous stirring. Adding 4095 μL of linker solution took 5 minutes. The reaction was carried out for 30 minutes at room temperature. The reaction was quenched with 20 ml of 5% acetic acid to achieve a final pH of 5. The solution was filtered using a 1 L vacuum filter (0.2 μm).

[0605] OG1802 (shown in Figure 2K) was then purified as follows: Millipore cross-flow filtration cassettes were used to purify the polymer in an aqueous system. We started by concentrating the polymer solution to 250 ml (~200 mg/ml). Fresh WFI was added from the reservoir and the flow rate of the fresh WFI feed was adjusted to the permeate level (~2 mL/min). UV/DF was set at 2–8°C overnight. Typically, 2.5 L of water for injection was used (10x volume ratio to polymer solution). A sample of the retentate was taken for purity testing. Target purity was >98%. The polymer solution was filtered using a 1 L filter bottle of 0.2 μM. The polymer solution can be stored at 2-8°C for up to 3 months before conjugation.

Example 9 Alternative Polymers Containing Phosphorylcholine

[0606] The HEA-PC polymer was synthesized as described below. HEA-PC (2-(acryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate), which is an acrylate, unlike the methacrylate HEMA-PC described above, has the following structure:

HEA-PC

[0607] HEA-PC was polymerized with the initiator shown in Example 5 as Compound L.

Table 28

Reagent Name Quantity MM Initiator Connection L (see above) 1.65 mg 2505.5 Monomer HEA-PC 0.461 g 281.24 Catalyst Cu(I) bromide 1.2 mg 143.45 Ligand Tris-[2-(dimethylamino)ethyl]amine
(Me6TREN) 2.73 mg 230.39 Solvent A N,N-dimethylformamide (DMF) 21.85 µl 73.09 Solvent B Water 0.7 ml 18.02 Solvent C Methanol 0.7 ml 32.04

[0608] A 200 mg/mL initiator stock solution was prepared by dissolving 2.2 mg of initiator in 11 μL of dry DMF and a 200 mg/mL ligand solution by dissolving 4.6 mg of Me6TREN in 23 μL of dry DMF. 8.25 μl of the initial initiator solution and 13.6 μl of the ligand were measured into the test tube. Degassed at -78°C for 5 minutes, then refilled with argon and added 1.2 mg CuBr. Degassed and filled again with argon. Added a stock solution of HEA-PC in methanol (weighed 0.461 g of HEA-PC and dissolved it in 0.5 ml of methanol) to the solution inside the reactor at -78°C. The vial was washed with 200 μl of methanol and added to the reactor at -78°C, followed by 0.5 ml of distilled water, then another 200 μl of water. Carefully degassed until no bubbles were released and all heterogeneity disappeared (dissolution or disappearance of solid particles). The mixture was again filled with 4 psi argon and the reaction was allowed to proceed at room temperature for an hour. The reaction mixture was already viscous. The reaction was allowed to proceed for approximately one hour. A solution of bipyrindine in methanol (5 mg in 0.5 μl) was added. Another 2-3 ml of methanol was added and the catalyst was left to oxidize overnight at 4°C. The conversion determined by 1H NMR was 94%.

[0609] The next day, the polymer was dialyzed and analyzed for SEC/MALS using an SB806M_HQ Shodex column (7.8 x 300 mm) in 1x PBS, pH 7.4 at 1 mL/min, yielding a PDI of 1.157, Mn equal to 723.5 kDa, Mp equal to 820.4 kDa, and Mw equal to 837.2 kDa (pre-dialysis PDI was 1.12, Mn=695 kDa, Mp=778 kDa). A maleimide functional group was then added to the polymer so that it could be conjugated to the protein.

[0610] The maleimide ester Mal-PEG4-PFP (see Example 20 above) was then “snapped” to the HEA-PC polymer as shown in Example 20. The resulting HEA-PC polymer containing the maleimide functionality can then be conjugated to sulfhydryl groups, as discussed herein for HEMA-PC polymers.

[0611] An acrylamide polymer containing PC was also prepared using 2-(acrylamyl)ethyl-2-(trimethylammonium)ethyl phosphate (Am-PC) monomer having the following structure:

[0612] Am-PC was used for polymerization using a 3-arm initiator (TFA salt) having the structure:

[0613] The Am-PC polymer was synthesized as follows:

Table 29

Reagent Name/feature Quantity MM Initiator Initiator with 3 branches (see above) 2.2 mg 885.35 Monomer Am-PC 0.5 g 280.26 Catalyst (I) Copper(I) bromide 1 mg 143.45 Catalyst (II) Copper(II) bromide 0.2 mg 223.35 Ligand Tris-[2-(dimethylamino)ethyl]amine
(Me6TREN) 3.94 mg 230.39 Solvent A N,N-dimethylformamide (DMF) 31.7 µl 73.09 Solvent B Water 1 ml 18.02 Solvent C Methanol 1 ml 32.04

[0614] A 200 mg/mL ligand stock solution was prepared by dissolving 9 mg of Me6TREN in 45 μL of dry DMF. Add 19.7 μl of stock solution to the reaction vessel. A stock initiator solution with a concentration of 200 mg/ml was prepared by dissolving 6.5 mg of the substance in 32.5 μl of dry DMF. 11 μl of the initiator stock solution was added to the ligand obtained above. Degassed for 5 minutes. 1 mg CuBr was added. A stock solution of CuBr ₂ with a concentration of 200 mg/ml was prepared by dissolving 4 mg of CuBr ₂ in 20 μl of dry DMF. Add 0.5 g of monomer (AmPC) to 1 mL of methanol (slow dissolution/viscous solution), followed by 1 μL of CuBr ₂ stock solution. The monomer solution was added dropwise to the reaction mixture obtained above. Wash with 1 ml of water. The reaction mixture was thoroughly degassed (freeze-thawing). The reaction was allowed to proceed for 24 hours.

[0615] The Am-PC polymer can then be dialyzed. The molecular weight of the polymer above was determined by SEC/MALS: Mn was 215 kDa, Mp: 250 kDa, PDI was 1.17. The conversion was determined by ¹ H NMR and was 94%. The maleimide functionality can be attached to the Am-PC polymer, as discussed above for HEMA-PC and HEA-PC. The Am-PC polymer containing the maleimide functionality can be conjugated to the protein as described above.

Example 10: Inverse Ellman Analysis to Calculate Free Maleimide in a Compound

[0616] After the maleimide functionality was attached to the OG1801 polymer to form OG1802 (see above), the Ellman assay was used to determine the amount of maleimide functionality, expressed as a percentage function (ie, conjugated), in the sample. Thiol converted Ellman's reagent (DTNB) to TNB-, then to TNB2- in water at neutral and alkaline pH, producing a yellow color (measured at 412 nm). A standard curve was constructed with cysteine. Since maleimide reacts with a thiol, this assay actually measured the remaining thiol (cysteine). Inhibition was calculated as molarity ratios (initial thiol - thiol remaining after addition of maleimide to the polymer)/(initial thiol) and expressed as a percentage, with the higher the percentage, the higher the maleimide functionalization.

[0617] Reagents used in the assay: A standard curve was prepared using cysteine from 62.5 μM to 2 μM. Initial polymer solutions were prepared by dissolving the powder in 1xPBS pH 7.4 (reaction buffer) and thoroughly mixing. Solutions of polymer and cysteine with the same molarity were mixed and left to react at 27°C for 30 minutes. A 150 μM DTNB solution was added to standard cysteine and polymer/cysteine reaction mixture solutions and color developed at 27°C for 5 min. OD at 412 nm was read on a Spectramax plate reader and percent inhibition was calculated using Softmax Pro software and a cysteine standard curve.

Example 11: Purification and release of anti-IL-6-VEGF decoy molecules

[0618] The heavy and light chains were cloned into expression plasmids and transfected into CHO cells. Cells were grown in appropriate media and harvested. The antibody (or alternatively the AT-trap construct) was purified using capture and elution on a protein A affinity chromatography column. The cysteine residue of the antibody (or alternatively the AT-trap construct) at position 443 (L443C ( EU numbering)) is typically “blocked” or oxidized by chemicals in cell culture media and is not available for conjugation. Therefore, the purified antibody (or, alternatively, the AT-trap construct) was subjected to a deblocking process to remove the blockage and allow conjugation of the free (i.e., not involved in Cys-Cys disulfide bonds) cysteine residue to the maleimide functional group of the polymer. Deblocking was accomplished by mixing the purified antibody protein (or alternatively an Ab decoy construct) with a 30x molar excess of a reducing agent such as TCEP (3,3',3''-phosphantriyltripropanoic acid), trisodium 3,3 ',3''-phosphinetriyltris(benzene-1-sulfonate) (TPPTS), etc., for 1 hour at ambient temperature. The reduction reaction was monitored by SDS-PAGE. After reconstitution, the antibody protein buffer (or alternatively the AT-trap construct) was replaced using a Pellicon XL Ultrafiltration Cassette with 20 mM Tris pH 7.5, 100 mM NaCl, 0.5 mM TCEP buffer for unlocking. The TCEP reagent was then removed in the same buffer exchange system with 20 mM Tris pH 7.5, 100 mM NaCl. The reconstituted antibody (or alternatively the AT-trap construct) was re-oxidized using a 15x molar excess of the oxidant DHAA (dehydroxyascorbic acid) for 1 hour at ambient temperature, which was again monitored by SDS-PAGE analysis. The DHAA reagent was then removed in the same buffer exchange system with 20 mM Tris pH 7.5, 100 mM NaCl.

Example 12A. Conjugation of an antibody (or alternatively an AT-trap construct) to an MPC polymer

[0619] The released antibody (or, alternatively, the AT-trap construct) was conjugated to the OG1802 polymer to produce a bioconjugate. An excess of OG1802 was used (3-20-fold molar excess). Conjugation was monitored by cation exchange HPLC chromatography and brought to near completion. The antibody conjugate (or alternatively the Ab-trap constructs) was purified by cation exchange chromatography and buffer exchanged to formulate by ultrafiltration/diafiltration (UF/DF). The antibody conjugate (or alternatively the Ab-trap construct) was purified by chromatography as described above.

Example 12B. Conjugation reaction of the VEGFR-antiIL6 construct with OG1802

[0620] The method for conjugating the VEGFR-anti-IL6 construct to OG1802 involved two steps: an initial reduction reaction to remove the cysteine S protecting groups (deprotection) followed by conjugation to the OG1802 biopolymer via maleimide-cysteine chemistry. Unblocking was performed first by reducing (30-60 minutes) the VEGFR-antiIL6 construct with a 30x molar excess of 3,3',3''-phosphantriyltripropanoic acid (TCEP), followed by buffer exchange to 20 mM Tris-HCl pH 7.5, 100 mM NaCl with cysteine unblocking and TCEP removed (FIG. 28). Alternatively, other reducing agents such as 3',3''-phosphantriyl tris(benzenesulfonic acid) trisodium salt (TPPTS) can also be used for the deblocking step.

[0621] The reconstituted VEGFR-antiIL6 construct was then oxidized (30-60 minutes) with a 15x molar excess of dehydroascorbic acid (dHAA), followed by a buffer exchange to 20 mM Tris pH 7.5, 100 mM NaCl to remove the oxidant (Figure 28). The overall yield for the deblocking process was 89% (TCEP reduction 94%, dHAA oxidation 95%).

[0622] The conjugation process was carried out by mixing the VEGFR-anti-IL6 construct at a final concentration of 2.4 mg/ml (or 12.5 μM) with a 5x molar excess of OG1802 biopolymer at pH 8.0. The conjugation process took place at 2-8°C for a period of 2 to 3 days. The VEGFR-anti-IL6 conjugated construct was purified from unconjugated material by cation exchange chromatography (CEX) with Poros XS resin using a step gradient of increasing salt concentrations (0-500 mM NaCl) in a buffer containing 20 mM sodium acetate, pH 5.5 (Fig. .29). Fractions containing the VEGFR-antiIL6-OG1802 bioconjugate were pooled and the buffer was replaced with phosphate buffer solution (PBS), pH 7.4. Conjugation efficiency was approximately 60% (determined by SDS-PAGE under non-reducing conditions and ImageJ analysis). The total yield from the starting protein substance to the bioconjugate was approximately 27%.

[0623] SDS-PAGE (FIG. 30) and SEC-MALS (FIG. 31) analysis demonstrates one molecule of VEGFR-anti-IL6 (~189 kDa) conjugated to one molecule of OG1802 polymer (~794 kDa), resulting in a bioconjugate of approximately ~983 kDa. A SEC-MALS experiment was performed by injecting 10 μg (protein mass) at a flow rate of 1 mL/min into a size exclusion chromatography column (Shodex-SB806M-HQ) pre-equilibrated with PBS buffer, along with light scattering (miniDAWN TREOS) and refractive index detectors (Optilab T-rEX) (Wyatt Technology). Data and protein conjugate analysis was performed in ASTRA 7 (Wyatt Technology).

Example 13 Detecting and Resolving VEGF Trap Cleavage

[0624] As shown in FIGS. 11A and 11B, SDS-PAGE of Protein A purified reconstituted VEGFR-anti-IL-6 and anti-IL-6-VEGFR products obtained from ExpiCHO-S cells exhibited unexpected bands, the size of which depended from the orientations of the specified structure. These bands indicated that some of the dual inhibitor molecules were cleaved during preparation. In this example, the VEGFR-antiIL6 construct corresponds to a heavy chain molecule containing the sequences 1A-2A-3H paired with a light chain containing the sequence 4A. The antiIL-6-VEGFR construct corresponds to a heavy chain containing the sequences 5H(Fab)-2A-1A-5H(Fc) paired with a light chain containing the 4A sequence.

[0625] As shown in Figure 12, these protein bands were transferred to a PVDF membrane and the first six residues of the protein bands were sequenced by N-terminal Edman sequencing (Procise 494HT device). The results showed that the cleaved products contained a common N-terminal sequence (93LTHRQT), which is located in domain 2 of VEGFR1 in the VEGF trap region.

[0626] Figures 13A and 13B show the results of mutations in the region flanking the cleavage site (K89TNY/LTHR96), which prevented protein cleavage while maintaining overall protein stability and VEGF binding properties. These mutations were selected based on sequence conservation, using Basic Local Alignment Search Tool (BLAST) to search for sequence similarity and alignment between VEGFR1 domain 2 sequence and protein homologs within the mammalian class. The top 500 results were selected based on their BLAST E-value. From this initial cluster of 500 sequences (cluster_1), the best performing sequence for each individual species was selected, reducing it to a set of 106 representative sequences (cluster_2). Based on the sequence variations in the region flanking the cleavage site observed in cluster_2, 16 mutants of the anti-IL-6-VEGFR construct with single point mutations were generated and assessed for protein cleavage. These mutations were initially introduced only into the anti-IL-6-VEGFR construct because it exhibits better size separation between cleaved and intact products due to differences in their molecular weight (∼35 kDa).

[0627] FIGS. 13A and 13B show SDS-PAGE results of mutants of the anti-IL-6-VEGFR construct, demonstrating that modifications to the cleavage site reduced the amount of cleaved product (indicated by arrows). The T94I and H95I mutations (residue numbering according to position in the VEGF trap sequence) markedly reduced VEGF trap cleavage. In particular, FIGS. 13A and 13B show that most of the mutations reduced protein degradation, with mutations T94I (VEGFR_variant_1), shown in FIG. 13C, and H95I (VEGFR_variant_2), shown in FIG. 13D, having a more significant effect. The single point mutation (T94I, H95I) and double point mutation (T94I/H95I, VEGFR_variant_3) shown in Fig. 13E were then introduced into the VEGFR-anti-IL-6 construct. 13C shows the position of T94I (underlined). 13D shows the position of H95I (underlined). 13E shows the position of T94I and H95I (underlined). As shown in FIGS. 13F and 13G, SDS-PAGE showed a similar reduction in protein degradation, and this effect was further enhanced when a double mutation was introduced.

Example 14 Binding of Dual VEGFR Inhibitor Variants to VEGF by Biacore Assay

[0628] Figures 14A-14F present the results of a series of Biacore assays used to determine the affinity between anti-VEGF and VEGF agents. Binding kinetics between molecules was measured using Biacore T200 (GE Healthcare) at 37°C in HBS-EP+ buffer containing 1 mg/ml BSA. Anti-VEGF agents (VEGFR-anti-IL-6 construct (5 µg/ml), VEGFR_variant_1-anti-IL-6 construct (5 µg/ml), VEGFR_variant_2-anti-IL-6 construct (5 µg/ml), construct VEGFR_variant_3-anti-IL-6 (5 μg/ml) and Eylea (3 μg/ml)) were captured on the chip with protein A (GE) at a rate of 10 μl/min for 25 seconds, five concentrations of VEGF-A165 (0 ,19, 0.56, 1.67, 5, 15 nM) were passed through the captured antibodies for 120 seconds at a rate of 30 μl/min and dissociated for 60 minutes. The surface of the sensor chip was regenerated by a 60-second injection of 10 mM glycine, pH 1.7, at a flow rate of 50 μL/min. Two reference signals were subtracted from all sensorgrams and fitted using a 1:1 Langmuir binding model. 14A-14F show that introduction of single point mutations T94I (VEGFR_variant_1-anti-IL-6 construct), H95I (VEGFR_variant_2-anti-IL-6 construct), and T94I/H95I double mutation (VEGFR_variant_3-anti-IL-6 construct) 6) minimally affects binding to VEGF. As seen in Figure 14F, the sensorgrams demonstrate that dual VEGFR anti-IL-6 inhibitor molecules and variants bind strongly to VEGF-A, as does Eylea. In this example, VEGFR-anti-IL6 molecules correspond to VEGFR variants (sequences 1A-1D) fused to an anti-IL-6 moiety via a GS linker (sequences 2A-3H) paired with a light chain containing sequence 4A.

Example 15 Reporter assay of VEGF-stimulated VEGFR in a cellular system

[0629] The results of the VEGF-stimulated VEGFR reporter assay in a cellular system are presented in FIG. 15. The dual VEGFR-anti-IL6 inhibitor, as above, blocked VEGF binding to VEGFR and stimulation of downstream signaling to the same extent as Eylea (IC50 0.18 nM vs 0.11 nM), whereas the anti-IL6 antibody had no effect influence.

[0630] For these experiments, thaw-ready HEK293 KDR/VEGF Promega cells (CS181405; Promega) were thawed and added to 4.6 ml of assay medium (10% FBS in DMEM). 25 μl of cells were transferred to each well of a 96-well plate. VEGF (final concentration 500 pM) was mixed with a series of 1:3 dilutions of inhibitors to obtain 10 dilution points starting at 100 nM (final concentration) and incubated for 30 minutes before adding 50 μl to the seeded cells. After 6 hours, 75 μl of prepared Bio-Glo reagent (Promega) was added to the cells and incubated for 3-10 minutes at room temperature. Luminescence was measured on an iD3 plate reader (Molecular Devices). Relative luminescence units resulted in the lowest inhibitor concentration for each curve and were plotted as the mean of replicates with standard error against the logarithm of antibody concentration using GraphPad Prism software and fitted to a nonlinear regression curve. In this example, the VEGFR-antiIL6 construct corresponds to a heavy chain molecule containing the sequences 1A-2A-3H paired with a light chain containing the sequence 4A.

Example 16. Analysis of the formation of ECHF tubes

[0631] FIGS. 16A-16C show the results of an analysis of the formation of EHCR tubules.

[0632] 2 or 3 subcultures of EHRV cells were cultured in vascular cell basal media supplemented with 1x Bovine Brain Extract endothelial cell growth kit (ATCC; culture media) in 5% CO2 at 37°C. Once the cells reached 70% confluency, the cells were separated from the plate using trypsin (0.05%; ATCC) and 0.75 x 10^6 cells were seeded into a T25 flask in culture media. The next day, the media were replaced with assay media (2% FBS in vascular cell basal medium). After 6 hours, the media were directly supplemented with 5 μg/ml Calcein AM and incubated for 30 minutes at 37°C. Simultaneously, 200 μl of pre-thawed Matrigel (growth factor-reduced; Corning) was added to each well of an 8-well glass slide and incubated at 37°C for 30 minutes. During incubation, 25 nM of each inhibitor (final concentrations) was mixed with 3 nM VEGF (R&D), 12.5 nM IL6 (Tonbo), and 25 nM IL6R (Tonbo) (all final concentrations). After a 30-minute incubation with Calcein AM, the cells were washed with 3x PBS and then treated with trypsin. The collected cells were centrifuged for 3 minutes at 1500×g and resuspended in assay medium at a density of 500,000 cells/ml. Then, 100 μl of cells were mixed with 100 μl of the prepared inhibitor/complex solutions and then transferred to Matrigel-coated glass slides and incubated overnight. The next day, the tubules were imaged using a 10× objective lens and a GFP filter on an EVOS fluorescence microscope (Life Technologies). For each condition, at least 2 representative images were acquired and then analyzed using the Angiogenesis Analyzer Plugin for ImageJ (NIH), which evaluates and quantifies 20 parameters of tubule network formation.

[0633] Figures 16A-16C present data as the mean with standard error from 4 independent experiments. Statistical significance was determined for all replicates of 4 independent experiments using an unpaired two-tailed Student's t test. As shown in Fig. 16C, the VEGFR-anti-IL6 construct significantly inhibited angiogenesis parameters: 17/20 parameters compared to control. In contrast, Eylea inhibited 4/20 parameters, and anti-IL-6 inhibited 7/20 parameters. The VEGFR-anti-IL6 construct was significantly more effective than Eylea and anti-IL-6 in 12/20 parameters, as indicated by asterisks in Fig. 16B. In addition, t-test statistics are presented in Fig. 16C. In this example, the VEGFR-anti-IL6 construct corresponds to a heavy chain molecule containing the sequences 1A-2A-3H paired with a light chain containing the sequence 4A.

[0634] In some embodiments, the VEGFR anti-IL6 construct provides synergistic mechanisms to improve efficacy and durability in specific at-risk populations. Populations at risk include those at risk for or diagnosed with diabetic retinopathy or one or more inflammatory diseases of the retina.

Example 17: EHPV Proliferation Assay

[0635] FIG. 17 shows the results of the ECHR proliferation assay. ECHV cells (2-5 passages) were cultured in basal media for vascular cells with the addition of 1x Bovine Brain Extract endothelial cell growth kit (ATCC; nutrient media) in 5% CO2 at 37 degrees. When cells reached 70% confluency, cells were detached from the plate using trypsin (0.05%; ATCC) and 3500 cells/well were seeded into the central 60 wells of a gelatin-coated 96-well plate in 100 μl assay media (basal media for vascular cells with the addition of l-glutamine, ascorbic acid, heparan sulfate, hydrocortisone and 1% FBS) (Corning). After 24 hours in minimal medium, cells were stimulated with VEGF (1.5 nM), IL-6 (5 nM), and IL6R (10 nM) mixed with a series of 1:3 dilutions of inhibitors to obtain 10 dilution points starting at 100 nM. These concentrations of VEGF and IL-6 induced proliferation at submaximal levels when added independently and exhibited a synergistic growth curve when combined. After 48 hours, cell viability was measured using CellTiterBlue reagent (Promega) according to the manufacturer's instructions on an iD3 plate reader (Molecular Devices). For uptake measurements, the A570/A605 ratio was determined and data normalized to control (no inhibitor) cells for each replicate. Fluorescence measurements (555ex/605em) resulted in control cells for each replicate.

[0636] As shown in Figure 17, the reported values were plotted as the mean of replicates with standard error versus antibody concentration using GraphPad Prism software and resulted in a nonlinear regression curve.

[0637] As seen in FIG. 17, the dual VEGFR-anti-IL6 inhibitor blocked the synergistic stimulation of IL-6 and VEGF on EHPV proliferation, whereas neither Eylea nor anti-Il-6 affected EHPV growth under these conditions.

Example 18: Preparation of Improved Anti-IL-6 Paratopes

[0638] The present example describes the preparation of improved anti-IL-6 paratopes. Biacore kinetic results are presented in Tables 30, 31, 32 and 33.

[0639] To obtain a better anti-IL-6 paratope in an engineered human IgG1 framework, the heavy chain mutant G66D (CDR2) was subjected to affinity maturation. First, complete G66D heavy chain libraries were constructed containing random single point mutations at positions S35, I51, T63, and T65, excluding substitutions at Cys, Met, and Asn due to the chemical instability associated with these amino acids. These four positions were chosen based on their tolerance for mutations observed in bacterial and mammalian screens. Mammalian expression vectors containing heavy chain mutations were cotransfected with the wild-type light chain and the M32L/M50D/N52S/M88Q variant in 3 ml of Expi293 cell culture and incubated for five days. These cultures were centrifuged and supernatants containing secreted antibodies were collected and diluted in HBS-EP+ running buffer (1:20 ratio).

[0640] The various designs and resulting kinetic properties are presented below in Tables 30-33.

Table 30

Biacore kinetics for anti-IL-6 heavy chain double mutants

(column and row names are as in table 1.7 above)

Table 31

Biacore kinetics of anti-IL-6 heavy chain mutagenized light chain double mutants

Table 32

Biacore kinetics for anti-IL-6 heavy chain triple mutants

Table 33

Biacore Kinetics for Anti-IL-6 Heavy Chain Triple Mutants Paired with a Mutagenesized Light Chain

[0641] The IL-6 binding kinetics of these mutant molecules was measured at 37°C using a Biacore T200. Antibodies were captured onto a protein A chip (GE) by injecting cell supernatant at 1:20 dilutions at a rate of 10 μl/min for 60 seconds. Captured antibodies were passed through five concentrations (0.56, 1.67, 5, 15, and 45 nM) of IL-6 over 60 seconds at a flow rate of 30 μL/min and dissociated for 180 seconds in the Biacore single-dose kinetics assay format. cycle. The sensor chip was regenerated by a 60-second injection of 10 mM glycine, pH 1.7, at a flow rate of 50 μL/min. A similar assay system was used to monitor interactions between purified antibody samples and IL-6 at 37°C, where applicable. For this system, 1 μg/mL of purified antibody was injected onto the protein A chip at a rate of 10 μL/min for 25 seconds. Captured antibodies were passed through five concentrations (0.56, 1.67, 5, 15, and 45 nM) of IL-6 over 60 seconds at a flow rate of 30 μL/min and dissociated for 1800 seconds in the Biacore single-dose kinetics assay format. cycle. The sensor chip was regenerated by a 60-second injection of 10 mM glycine, pH 1.7, at a flow rate of 50 μL/min. Data analysis for supernatant and purified samples was performed using BIAevaluation (GE) software. Two reference signals were subtracted from all sensorgrams and fitted using a 1:1 Langmuir binding model.

[0642] As shown in Tables 30 and 31, the heavy chain of the S35H G66D double mutant exhibited increased protein expression, as indicated by an approximately two-fold increase in uptake relative to the wild-type heavy chain. This mutant also showed a slight increase in affinity depending on the paired light chain (wild type: ∼2-fold, M32L/M50D/N52S/M88Q: ∼1.3-fold). Table 30 shows G66D optimization of wild-type Biacore light chain kinetics for anti-IL-6 heavy chain double mutants. Data were obtained for samples before (susp.) and after purification, in both cases at 37°C. Table 31 shows G66D optimization of Biacore light chain mutant kinetics for anti-IL-6 heavy chain double mutants paired with a mutagenized light chain. Data were obtained for samples before (susp.) and after purification, in both cases at 37°C.

[0643] Thus, this double mutant was chosen as the starting point for another step in which the substitution-permissible positions Q99 and L100 in CDR3 were randomly mutagenized, expressed, and assessed for IL-6 binding as described for the G66D screening step. As shown in Tables 32 and 33, substitutions of L100 with Ala, Ser, Gly, Thr, Gln, Lys showed better expression compared to the wild type, as indicated by uptake levels. Accordingly, these mutants were selected for purification and evaluation of IL-6 binding. Affinity measurements for the purified substance also demonstrated better binding of IL-6, as shown in Tables 32 and 33. Based on these results, these additional modified CDRs shown in FIG. 18 were included in the creation of dual anti-IL-6 and anti-IL-6 molecules. VEGF inhibitor.

[0644] Table 32 presents the Biacore kinetics for the G66D/S35H heavy chain modification paired with the wild-type light chain for anti-IL-6 heavy chain triple mutants. Data were obtained for samples before (susp.) and after purification, in both cases at 37°C. Table 33 shows the Biacore kinetics for the G66D/S35H heavy chain modification paired with a light chain mutant for anti-IL-6 heavy chain triple mutants paired with a mutagenized light chain. Data were obtained for samples before (susp.) and after purification, in both cases at 37°C.

Example 19 Dual Anti-IL-6 Anti-VEGF Inhibitor Options

[0645] Based on the results for the VEGF Trap variants and the improved anti-IL-6 paratopes, 216 molecules were designed in two different configurations that contained combinations of sequences as shown in FIGS. 19, 20, 21, 22, and 23. In the case of the first configuration (VEGFR-anti-IL-6), the VEGF trap, represented by sequences 1A-1D in Fig. 19, was located at the beginning of the protein, followed by a double repeat of the Gly-Gly-Gly-Gly-Ser (GS) linker, represented by sequence 2A in Fig. .20, which connects the trap to the N-terminus of the anti-IL-6 heavy chain represented by sequences 3A-3I in FIG. 21. These constructs can be paired with the light chains listed as sequences 4A-4C in FIG. 22. In the case of the second configuration (anti-IL-6-VEGFR), the heavy chain variable and constant domains, represented by the heavy chain - Fab, sequences 5A-5I in Fig. 23, were connected to the VEGF trap, represented by sequences 1A-1D in Fig. 19. through the GS linker, represented by sequence 2A in Fig. 20, and then the Fc domain, represented by the heavy chain - Fc, sequences 5A-5I in Fig. 23, was fused to the C-terminus of the VEGF trap. Thus, the VEGF trap was “sandwiched” between the Fab and Fc regions of the antibody.

[0646] Figure 19 shows VEGF trap sequences. Sequence variation is underlined and in bold. Figure 20 shows the linker sequence (GS) Gly-Gly-Gly-Gly-Ser with a double repeat. Figure 21 shows the heavy chain sequences for anti-IL-6 molecules. CDRs are underlined. Figure 22 shows light chain sequences for anti-IL-6 molecules. CDRs are underlined. Figure 23 shows the heavy chain sequences (divided into Fab and Fc) for anti-IL-6 molecules. CDRs are underlined.

[0647] In some embodiments, any of the embodiments of the VEGF trap, in which it is sandwiched between the Fab and Fc regions of the antibody, can be paired with the light chains listed as sequences 4A-4C in FIG. 22. Thus, in some embodiments, the constructs presented in FIG. 19 (VEGFR sequences) can be used for any of the VEGF trap locations presented herein, or alone.

Example 20 Binding kinetics between VEGFR-trap-Fc fusion and VEGF

[0648] Figures 26A-26C present the results of Biacore assays used to determine the affinity between anti-VEGF and VEGF agents. Binding kinetics were measured using Biacore T200 at 37°C in HBS-EP+ buffer containing 1 mg/ml BSA. VEGFR-Fc (1 μg/ml) and Eylea (1 μg/ml) were captured onto the protein A chip (GE) at a rate of 10 μl/min for 25 seconds, five concentrations of VEGF-A165 (0.19, 0.56 , 1.67, 5, 15 nM) were passed through the captured anti-VEGF molecules for 120 seconds at a rate of 30 μl/min and dissociated for 30 minutes. The surface of the sensor chip was regenerated by a 60-second injection of 10 mM glycine, pH 1.7, at a flow rate of 50 μL/min. Two reference signals were subtracted from all sensorgrams and fitted using a 1:1 Langmuir binding model. As shown in FIGS. 26A-26C, VEGFR-Fc and Eylea bind to VEGF with similar affinities. In this example, VEGFR-Fc contains the 6A sequence.

Example 21 Removal of Cleavage Product

[0649] As discussed in Example 13, the VEGFR-anti-IL6 construct exhibits a minor degree of cleavage in VEGFR1 domain 2 in the VEGF trap region. The present inventors have developed two orthogonal chromatography methods for separating intact protein from digested material after the protein A purification step: i) cation exchange chromatography (CEX) and ii) hydrophobic interaction chromatography (HIC).

[0650] For the CEX method, the pH of the VEGFR-antiIL6 Protein A column eluate solution was adjusted to 6, then injected into a Porous XS column pre-equilibrated with 20 mM sodium phosphate, pH 6. The intact protein eluted at a lower ionic strength than the digested material, and was almost completely separated along a linear gradient of increasing ionic strength (0-1 M sodium chloride) ( Figure 32 ). Fractions enriched in intact protein were pooled and the overall recovery after recovery was approximately 57%.

[0651] For the HIC method, the intact VEGFR-anti-IL6 protein mixture enriched with digest was injected onto a Butyl HP column pre-equilibrated with 20 mM sodium phosphate, pH 6, 500 mM-1 M ammonium sulfate. Intact and digested proteins were separated along a linear gradient of decreasing ionic strength (1-0 M ammonium sulfate), wherein the intact protein eluted at a lower ionic strength than the digest and could be partially or completely separated depending on the pooled fractions ( Fig. 33 ).

Example 22 Affinity of the VEGFR-anti-IL6 construct for IL6 and VEGF-A measured by KinExA

[0652] The Kinetic Exclusion Assay (KinExA®) 3200 system (Sapidyne Instruments Inc., Boise, Idaho) was used to measure the affinity and equilibrium binding kinetics between the VEGFR-anti-IL6 or VEGFR-anti-IL6-OG1802 construct (the protein component of which is shown in FIG. 27 ) and VEGF or IL6 in solution. Azlactone beads coated with VEGF or IL6 were used to capture the free construct fraction of VEGFR-antiIL6 or VEGFR-antiIL6-OG1802 from a counterbalanced sample of VEGFR-antiIL6 or VEGFR-antiIL6-OG1802 and VEGF or IL6. Captured dual inhibitor molecules were detected using a fluorescently labeled polyclonal anti-human immunoglobulin antibody (goat anti-human IgG antibody Alexa 647).

[0653] The affinity between the dual inhibitor molecules and VEGF was determined by 2-3 titration curves at increasing concentrations of the dual inhibitor and the corresponding serial titration dilutions of VEGF (indicated in parentheses) listed below: I) VEGFR-anti-IL6 construct: 1 nM (20 nM , 2-fold dilution, 12 dilutions), incubation 3.5 hours; 50 pM (500 pM, 2-fold dilution, 14 dilutions), incubation 2.5 hours; 10 pM (1 nM, 2-fold dilution, 13 dilutions), incubation 23 hours; ii) VEGFR-anti-IL6-OG1802 construct: 2 nM (30 nM, 2-fold dilution, 12 dilutions), incubation 45 minutes; 100 pM (15 nM, 2-fold dilution, 14 dilutions), incubation 6 hours; 7.5 pM (1 nM, 2-fold dilution, 14 dilutions), incubation 48 hours. Similar measurements were performed with IL6 under the following conditions: III) VEGFR-anti-IL6 construct: 1 nM (20 nM, 2-fold dilution, 13 dilutions), incubation 7 hours; 10 pM (1 nM, 2-fold dilution, 13 dilutions), incubation 42 hours; 100 pM (1 nM, 2-fold dilution, 13 dilutions), incubation 2.5 hours; IV) VEGFR-antiIL6-OG1802 construct: 1 nM (22.5 nM, 2-fold dilution, 13 dilutions), incubation 5.5 hours; 100 pM (22.5 nM, 2-fold dilution, 14 dilutions), incubation 12 hours.

[0654] Association rates ( k _on , M ^{-1 s -1} ) between dual inhibitor molecules and VEGF or IL6 were directly determined from kinetics experiments (1-2 curves for each analyzed pair), while dissociation rates ( k _off , s ^-1 ) was calculated based on the following equation: k _off =K _d x k _on . Measurements were performed on four preparations containing i) 100 pM VEGFR-antiIL6 and 86.4 pM VEGF, ii) 217 pM VEGFR-antiIL6-OG1802 and 200 pM VEGF, iii) 100 pM VEGFR-antiIL6 and 84.3 pM IL6, iv ) 1000 pM VEGFR-antiIL6 and 900 pM IL6.

[0655] All measurements were performed at 37°C using 1x PBS running buffer (pH 7.4) and samples prepared in 1x PBS (pH 7.4) with 1 mg/ml BSA. Data analysis was performed using KinExA Pro 4.3.11 software.

[0656] Under the conditions tested, the VEGFR-anti-IL6-OG1802 construct bound to VEGF and IL6 comparable to the VEGFR-anti-IL6 construct, with Kd values of 2.10 pM for the VEGFR-anti-IL6 and VEGF construct, 1.02 pM for the VEGFR-anti-IL6- construct OG1802 and VEGF, 21.10 pM for the VEGFR-anti-IL6 and IL6 construct, 12.10 pM for the VEGFR-anti-IL6-OG1802 and IL6 construct (see Tables 34 and 35).

Table 34. VEGF VEGFR-antiIL6 VEGFR-antiIL6-OG1802 Kd (pm) 2.10 1.02 95% confidence interval (pM) 1.34 - 2.91 0.46 - 1.80 Association rate (M ^{-1 s -1} ) 4.04 ×10 ⁷ 3.75 ×10 ⁶ confidence interval 95% 3.31 ×10 ⁷ - 4.92 ×10 ⁷ 2.90 ×10 ⁶ - 4.90 ×10 ⁶ Dissociation rate (s ^-1 ) 8.49 ×10 ^-5 3.83 ×10 ^-6

Table 35. IL-6

VEGFR-antiIL6 VEGFR-antiIL6-OG1802 Kd (pm) 21.10 12.10 95% confidence interval (pM) 14.30 - 30.80 8.47 - 16.30 Association rate (M ^{-1 s -1} ) 1.15 ×10 ⁷ 8.00 × 10 ⁶ confidence interval 95% 9.57 ×10 ⁶ - 1.37 ×10 ⁷ 6.44 ×10 ⁶ - 9.93 ×10 ⁶ Dissociation rate (s ^-1 ) 2.42 ×10 ^-4 9.66×10 ^-5

[0657] The measured affinity of the VEGFR-anti-IL6 and VEGFR-anti-IL6-OG1802 constructs was within error for both IL6 and VEGF; thus, these results demonstrate that the biopolymer did not affect the binding of dual inhibitors to their targets.

Example 23 VEGF/VEGFR Competitive ELISA Assay

[0658] This assay was performed as described in Example 3. In this assay, the VEGFR-anti-IL6, VEGFR-anti-IL6-OG1802 (as shown in FIG. 27), and Eylea constructs all inhibited the binding of VEGF to VEGFR to varying degrees ( FIG. 34 ). The VEGFR-anti-IL6-OG1802 construct showed greater inhibition (IC ₅₀ =0.99 nM) compared to the VEGFR-anti-IL6 construct (IC ₅₀ =1.98 nM) and Eylea (IC ₅₀ =1.38 nM). In addition, the VEGFR-anti-IL6 construct showed maximal inhibition superior to that in Eylea, while the VEGFR-anti-IL6-OG1802 construct showed even greater maximal inhibition (VEGFR-anti-IL6-OG1802=79.5%, VEGFR-anti-IL6=67 .0% relative to Eylea=49.6%). These data indicate that the attachment of the OG1802 biopolymer to the VEGFR-antiIL6 construct confers additional inhibitory abilities on VEGF binding to VEGFR. Accordingly, said biopolymer enhances the function of the molecule.

Example 24 IL-6/IL-6R Competitive ELISA Assay

[0659] This analysis was performed as described in Example 3.

[0660] Under these conditions, the VEGFR-anti-IL6-OG1802 construct effectively blocked the IL-6/IL-6R interaction comparable to the VEGFR-anti-IL6 construct, with IC ₅₀ values of 0.26 nM for VEGFR-anti-IL6 and 0.23 nM for VEGFR- antiIL6-OG1802 ( Fig. 35 ). These results indicate that the biopolymer did not alter the inhibitory effect of the dual inhibitor on the binding of IL6 to its receptor. Eilea served as a negative control.

Example 25 Reporter assay of VEGF-stimulated VEGFR in a cellular system

[0661] This assay was performed as described in Example 15.

[0662] Under the conditions tested, the VEGFR-anti-IL6-OG1802 construct inhibited VEGF-stimulated VEGFR reporter cells comparable to the VEGFR-anti-IL6 and Eylea constructs, with mean _IC50 values of 0.35±0.10 nM, 0.51±0. 07 nM and 0.35±0.03 nM, respectively ( Fig. 36 ). There may be a tendency for the VEGR-anti-IL6-OG1802 construct (protein component shown in Figure 27 ) and Eylea to be moderately more potent than the VEGFR-anti-IL6 construct, but this is not a significant finding and is due to assay bias. Based on these data, it can be concluded that the bioconjugate inhibits VEGFR2 signaling in a similar manner to its precursor protein.

Example 26. Lipopolysaccharide-stimulated formation of tubes in ECHF

[0663] 2 subcultures of EHRV cells were cultured in vascular cell basal media supplemented with 1x Bovine Brain Extract endothelial cell growth kit (ATCC; culture media) in 5% CO2 at 37 degrees. Once the cells reached 70% confluency, the cells were separated from the plate using trypsin (0.05%; ATCC) and 0.75 x 10 ⁶ cells were seeded into a T25 flask in culture media.

[0664] The following day, the media were replaced with assay media (2% FBS in vascular cell basal media). After 6 hours, 5 μg/ml Calcein AM was directly added to the media and incubated for 30 minutes at 37 degrees.

[0665] Simultaneously, 200 μl of pre-thawed Matrigel (growth factor-reduced; Corning) was added to each well of an 8-well glass slide and incubated at 37 degrees for 30 minutes.

[0666] During incubation, inhibitors (final concentration 37.5 nM) were mixed with lipopolysaccharide (LPS; Sigma; final concentration 1 μg/ml).

[0667] After 30 minutes of incubation with Calcein AM, cells were washed with 3x PBS and then treated with trypsin. The collected cells were centrifuged for 3 minutes at 1500×g and resuspended in assay medium at a density of 500,000 cells/ml. Then, 150 μl of cells were mixed with 100 μl of the prepared inhibitor/complex solutions and then transferred to Matrigel-coated glass slides and incubated overnight.

[0668] The next day, the tubules were imaged using a 10x objective lens and a GFP filter on an EVOS fluorescence microscope (Life Technologies). For each condition, at least 2 representative images were acquired and then analyzed using the Angiogenesis Analyzer Plugin for ImageJ (NIH), which evaluates and quantifies 20 parameters of tubule network formation.

[0669] Statistical significance was determined for all replicates of independent experiments using an unpaired two-tailed Student's t test.

[0670] Under these conditions, the VEGFR-anti-IL6 construct demonstrated statistically significant inhibition of 15/20 of the measured angiogenesis parameters compared to control, while Eylea significantly inhibited 5/20 and anti-IL6 inhibited 3/20 compared to control-treated cells. Overall, the VEGFR-anti-IL6 construct significantly inhibited all but one parameter that Eylea or anti-IL6 tended to influence or significantly influenced. Moreover, there were several parameters for which the VEGFR-anti-IL6 construct demonstrated significantly better inhibition than Eylea (7/20) and anti-IL6 (10/20), 3 of which the VEGFR-anti-IL6 construct exhibited significantly better inhibition than Eylea and anti-IL6. (Fig.37 and Fig.38 and 11)

[0671] The VEGFR-anti-IL6-OG1802 construct effectively inhibited the formation of ECHV tubes and, interestingly, the VEGFR-anti-IL6-OG1802 construct showed a trend towards increased inhibition of 9/20 parameters compared to the VEGFR-anti-IL6 construct. (Fig. 39). This means that the VEGFR-anti-IL6-OG1802 construct has an additional advantage over its unconjugated counterpart.

Example 27: EHPV Proliferation Assay

[0672] This assay was performed as described in Example 17.

[0673] Under the conditions tested, the VEGFR-anti-IL6-OG1802 construct (protein component shown in FIG. 27 ) inhibited VEGF/IL6-stimulated ECHR proliferation comparable to the VEGFR-anti-IL6 construct, whereas Eylea had no effect ( FIG. 40A ).

[0674] Interestingly, the VEGFR-anti-IL6-OG1802 construct showed increased potency compared to the VEGFR-anti-IL6 construct when cells were seeded into wells at different densities at the start of the experiment. As shown in Fig. 40B , at a density of 2000 cells per well, the VEGFR-anti-IL6 and VEGFR-anti-IL6-OG1802 constructs exhibited virtually identical inhibition of ECHR proliferation. At a density of 4,000 cells per well, the VEGFR-anti-IL6-OG1802 construct showed slightly greater maximum inhibition than the VEGFR-anti-IL6 construct (40% vs. 30%, respectively), and at a density of 6,000 cells per well, the VEGFR-anti-IL6-OG1802 construct inhibited proliferation by 25% relative to the control, whereas the VEGFR-antiIL6 construct had no effect.

[0675] As shown in the examples above, without being limited by any theory, it appears that the biopolymer helps deliver the protein to which it is bound to the surface of the plate and cells. This allows the drug to primarily inhibit the pools of VEGF and IL6 that are most likely to activate cell signaling, and avoid saturation of VEGF and IL6, which are at higher levels in the fluid phase and are less likely to affect cell signaling. As the number of cells increases, this effect will be more pronounced because more drug is needed to affect more cells. Accordingly, in some embodiments, the conjugate provides a superior product because it can promote distribution of the molecule.

[0676] The entire contents of all patent applications, websites, other publications, accession numbers, etc. cited above or below are incorporated herein by reference for all purposes in the same manner as if the contents of each individual item from This list has been specifically included separately. If different sequence variants are associated with an accession number at different times, the variant associated with the accession number as of the effective filing date of this application is meant. Effective filing date means the earlier of the effective filing date or the filing date of the priority application associated with the accession number, as applicable. Likewise, if different versions of a publication, website, etc. published at different times, means the version most recently published on the effective filing date of the application, unless otherwise stated. Any feature, step, element, embodiment or aspect of the present invention may be used in combination with any other unless specifically stated otherwise. Although the present invention has been described in detail by way of illustration and example to ensure a clear and complete understanding thereof, it will be appreciated that certain changes and modifications may be made within the scope of the appended claims.

--->

ANTIBODIES TO IL-6 AND THEIR FUSIONS AND CONJUGATES

<110>KODIAC SCIENCES INC.

JACOBSON, Rachel D.

CORREA, Fernando

LIAN, Hong

PERLROTH, D. Victor

<120>ANTIBODIES TO IL-6 AND THEIR FUSIONS AND CONJUGATES

<130>KDIAK.044WO

<150>62/637575

<151>2018-03-02

<150>62/727950

<151>2018-09-06

<160>269

<170>PatentIn, version 3.5

<210>1

<211>183

<212>PRT/PROTEIN

<213>Homo sapiens

<400>1

Val Pro Pro Gly Glu Asp Ser Lys Asp Val Ala Ala Pro His Arg Gln

1 5 10 15

Pro Leu Thr Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg Tyr Ile Leu

20 25 30

Asp Gly Ile Ser Ala Leu Arg Lys Glu Thr Cys Asn Lys Ser Asn Met

35 40 45

Cys Glu Ser Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro

50 55 60

Lys Met Ala Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe Asn Glu Glu

65 70 75 80

Thr Cys Leu Val Lys Ile Ile Thr Gly Leu Leu Glu Phe Glu Val Tyr

85 90 95

Leu Glu Tyr Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu Gln Ala Arg

100 105 110

Ala Val Gln Met Ser Thr Lys Val Leu Ile Gln Phe Leu Gln Lys Lys

115 120 125

Ala Lys Asn Leu Asp Ala Ile Thr Thr Pro Asp Pro Thr Thr Asn Ala

130 135 140

Ser Leu Leu Thr Lys Leu Gln Ala Gln Asn Gln Trp Leu Gln Asp Met

145 150 155 160

Thr Thr His Leu Ile Leu Arg Ser Phe Lys Glu Phe Leu Gln Ser Ser

165 170 175

Leu Arg Ala Leu Arg Gln Met

180

<210>2

<211>119

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>2

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>3

<211>106

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>3

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Asp Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210>4

<211>205

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>trap

<400>4

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Ile His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

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

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys

195 200 205

<210>5

<211>204

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>trap

<400>5

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr Ile Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu

115 120 125

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

130 135 140

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

145 150 155 160

Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg

165 170 175

Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr

180 185 190

Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys

195 200

<210>6

<211>205

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>trap

<400>6

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Ile Ile Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

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

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys

195 200 205

<210>7

<211>119

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>7

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>8

<211>119

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>8

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>9

<211>119

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>9

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>10

<211>119

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>10

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>11

<211>119

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>11

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>12

<211>119

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>12

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>13

<211>119

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>13

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>14

<211>205

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>trap

<400>14

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

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

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys

195 200 205

<210>15

<211>205

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>trap

<400>15

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Ile His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

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

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys

195 200 205

<210>16

<211>204

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>trap

<400>16

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr Ile Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

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

130 135 140

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

145 150 155 160

Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg

165 170 175

Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr

180 185 190

Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys

195 200

<210>17

<211>205

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>trap

<400>17

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Ile Ile Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

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

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys

195 200 205

<210>18

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>linker

<400>18

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210>19

<211>453

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>19

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Trp 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 Pro 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 Ser Arg Glu Glu Met Thr Lys Thr Lys Asn Gln Val

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Cys Ser Pro Gly Lys

450

<210>20

<211>450

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>20

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

355 360 365

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

435 440 445

Gly Lys

450

<210>21

<211>444

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>21

Glu Val Gln Leu Val 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 Thr Phe Ser Pro Phe Ala

20 25 30

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

35 40 45

Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val Thr

50 55 60

Asp Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr Leu

65 70 75 80

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

85 90 95

Arg Gln Gly Trp Gly Tyr Tyr Ala Leu Asp Ile 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 Thr Val Pro Ser Ser Ser

180 185 190

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

195 200 205

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

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro 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 Arg Ser

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 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 Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Cys Ser Pro Gly Lys

435 440

<210>22

<211>448

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>22

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Glu Ala Ala Gly Ala 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 Pro 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 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

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

435 440 445

<210>23

<211>448

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>23

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 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 Ala 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 Pro 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 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 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 Cys Ser Pro Gly Lys

435 440 445

<210>24

<211>449

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>24

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys

<210>25

<211>448

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>25

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210>26

<211>449

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>26

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys

<210>27

<211>447

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>27

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val Thr

50 55 60

Asp Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr Leu

65 70 75 80

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

85 90 95

Arg Gln Leu Trp Gly Tyr Tyr Ala Leu Asp Val 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 Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210>28

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>28

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Asp Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>29

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>29

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>30

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>30

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Asp Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>31

<211>223

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>31

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Thr 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

210 215 220

<210>32

<211>222

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>32

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

<210>33

<211>221

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>33

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

1 5 10 15

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

20 25 30

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

35 40 45

Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val Thr

50 55 60

Asp Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr Leu

65 70 75 80

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

85 90 95

Arg Gln Gly Trp Gly Tyr Tyr Ala Leu Asp Ile 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

210 215 220

<210>34

<211>222

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>34

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

<210>35

<211>222

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>35

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

<210>36

<211>222

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>36

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

<210>37

<211>222

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>37

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

<210>38

<211>222

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>38

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

<210>39

<211>222

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>39

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

<210>40

<211>225

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>40

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

1 5 10 15

Ala 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 Asp Val Ser His Glu

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Lys

225

<210>41

<211>225

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>41

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Lys

225

<210>42

<211>226

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antobyd

<400>42

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

1 5 10 15

Ala 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 Pro Ala Ile Glu

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Gly Lys

225

<210>43

<211>227

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>43

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

1 5 10 15

Ala 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 Pro 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

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

130 135 140

Leu Thr Cys Leu 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 Tyr 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 Cys Ser

210 215 220

Pro Gly Lys

225

<210>44

<211>227

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>44

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

1 5 10 15

Ala 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 Pro 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

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

130 135 140

Leu Thr Cys Leu 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 Tyr 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 Cys Ser

210 215 220

Pro Gly Lys

225

<210>45

<211>227

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>45

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

1 5 10 15

Ala 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 Pro 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

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

130 135 140

Leu Thr Cys Leu 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 Tyr 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 Cys Ser

210 215 220

Pro Gly Lys

225

<210>46

<211>227

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>46

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

1 5 10 15

Ala 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 Pro 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

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

130 135 140

Leu Thr Cys Leu 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 Tyr 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 Cys Ser

210 215 220

Pro Gly Lys

225

<210>47

<211>227

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>47

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

1 5 10 15

Ala 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 Pro 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

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

130 135 140

Leu Thr Cys Leu 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 Tyr 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 Cys Ser

210 215 220

Pro Gly Lys

225

<210>48

<211>225

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>48

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

1 5 10 15

Ala 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 Val Glu Val His

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Lys

225

<210>49

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>49

Gly Phe Thr Phe Ser Pro Phe Ala Met His

1 5 10

<210>50

<211>19

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>50

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

1 5 10 15

Val Thr Asp

<210>51

<211>12

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>51

Ala Arg Gln Ala Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>52

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>52

Gly Phe Thr Phe Ser Pro Phe Ala Met His

1 5 10

<210>53

<211>19

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>53

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

1 5 10 15

Val Thr Asp

<210>54

<211>12

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>54

Ala Arg Gln Ala Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>55

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>55

Gly Phe Thr Phe Ser Pro Phe Ala Met His

1 5 10

<210>56

<211>19

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>56

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

1 5 10 15

Val Thr Asp

<210>57

<211>12

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>57

Ala Arg Gln Ala Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>58

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>58

Gly Phe Thr Phe Ser Pro Phe Ala Met His

1 5 10

<210>59

<211>19

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>59

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

1 5 10 15

Val Thr Asp

<210>60

<211>12

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>60

Ala Arg Gln Ala Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>61

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>61

Gly Phe Thr Phe Ser Pro Phe Ala Met His

1 5 10

<210>62

<211>19

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>62

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

1 5 10 15

Val Thr Asp

<210>63

<211>12

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>63

Ala Arg Gln Ala Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>64

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>64

Gly Phe Thr Phe Ser Pro Phe Ala Met His

1 5 10

<210>65

<211>19

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>65

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

1 5 10 15

Val Thr Asp

<210>66

<211>12

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>66

Ala Arg Gln Ala Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>67

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>67

Gly Phe Thr Phe Ser Pro Phe Ala Met His

1 5 10

<210>68

<211>19

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>68

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

1 5 10 15

Val Thr Asp

<210>69

<211>12

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>69

Ala Arg Gln Ala Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>70

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>70

Gly Phe Thr Phe Ser Pro Phe Ala Ile Ser

1 5 10

<210>71

<211>19

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>71

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

1 5 10 15

Val Thr Asp

<210>72

<211>12

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>72

Ala Arg Gln Ala Trp Gly Tyr Tyr Ala Leu Asp Val

1 5 10

<210>73

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>73

Gly Phe Thr Phe Ser Pro Phe Ala Trp Ser

1 5 10

<210>74

<211>19

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>74

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

1 5 10 15

Val Thr Asp

<210>75

<211>12

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>75

Ala Arg Gln Ala Trp Gly Tyr Tyr Ala Leu Asp Val

1 5 10

<210>76

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>76

Ser Ala Ser Ile Ser Val Ser Tyr Leu Tyr

1 5 10

<210>77

<211>11

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>77

Leu Leu Ile Tyr Asp Asp Ser Ser Leu Ala Ser

1 5 10

<210>78

<211>9

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>78

Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

15

<210>79

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>79

Ser Ala Ser Ile Ser Val Ser Tyr Leu Tyr

1 5 10

<210>80

<211>11

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>80

Leu Leu Ile Tyr Asp Asp Ser Ser Leu Ala Ser

1 5 10

<210>81

<211>9

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>81

Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

15

<210>82

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>82

Ser Ala Ser Ile Ser Val Ser Tyr Leu Tyr

1 5 10

<210>83

<211>11

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>83

Leu Leu Ile Tyr Asp Asp Ser Ser Leu Ala Ser

1 5 10

<210>84

<211>9

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>84

Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

15

<210>85

<211>431

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>85

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly

50 55 60

Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys

65 70 75 80

Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His

85 90 95

Arg Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly

100 105 110

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

115 120 125

Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser

130 135 140

Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser

145 150 155 160

Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val

165 170 175

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

180 185 190

Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

<210>86

<211>432

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>86

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Ile His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

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

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

<210>87

<211>432

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>87

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Leu Thr Ile

85 90 95

Arg Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly

100 105 110

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

115 120 125

Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser

130 135 140

Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser

145 150 155 160

Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val

165 170 175

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

180 185 190

Met Thr Lys Lys Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

<210>88

<211>430

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>88

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

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

50 55 60

Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala

65 70 75 80

Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Ile Ile Arg Gln

85 90 95

Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile Glu

100 105 110

Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu

115 120 125

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

130 135 140

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

145 150 155 160

Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg

165 170 175

Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Thr Lys

180 185 190

Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr His Thr

195 200 205

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

405 410 415

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

420 425 430

<210>89

<211>119

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>89

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>90

<211>119

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>90

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>91

<211>106

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>91

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Asp Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210>92

<211>106

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>92

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210>93

<211>106

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>93

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Asp Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210>94

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>94

Gly Phe Thr Phe Ser Pro Phe Ala Met Ser

1 5 10

<210>95

<211>17

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>95

Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val Thr

1 5 10 15

Gly

<210>96

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>96

Ser Ala Ser Ile Ser Val Ser Tyr Met Tyr

1 5 10

<210>97

<211>7

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>97

Asp Met Ser Asn Leu Ala Ser

15

<210>98

<211>9

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>98

Met Gln Trp Ser Gly Tyr Pro Tyr Thr

15

<210>99

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>artificial

<400>99

Gly Phe Thr Phe Ser Pro Phe Ala Ile Ser

1 5 10

<210>100

<211>19

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>100

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

1 5 10 15

Val Thr Asp

<210>101

<211>12

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>101

Ala Arg Gln Leu Trp Gly Tyr Tyr Ala Leu Asp Val

1 5 10

<210>102

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>102

Gly Phe Thr Phe Ser Pro Phe Ala Trp Ser

1 5 10

<210>103

<211>19

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>103

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

1 5 10 15

Val Thr Asp

<210>104

<211>12

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>104

Ala Arg Gln Leu Trp Gly Tyr Tyr Ala Leu Asp Val

1 5 10

<210>105

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>105

Ser Ala Ser Ile Ser Val Ser Tyr Leu Tyr

1 5 10

<210>106

<211>11

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>106

Leu Leu Ile Tyr Asp Asp Ser Ser Leu Ala Ser

1 5 10

<210>107

<211>9

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>107

Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

15

<210>108

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>108

Ser Ala Ser Ile Ser Val Ser Tyr Leu Tyr

1 5 10

<210>109

<211>11

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>109

Leu Leu Ile Tyr Asp Ala Ser Ser Leu Ala Ser

1 5 10

<210>110

<211>9

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>110

Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

15

<210>111

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>111

Ser Ala Ser Ile Ser Val Ser Tyr Leu Tyr

1 5 10

<210>112

<211>11

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>112

Leu Leu Ile Tyr Asp Asp Ser Asn Leu Ala Ser

1 5 10

<210>113

<211>9

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>113

Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

15

<210>114

<211>205

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>114

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

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

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys

195 200 205

<210>115

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>115

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210>116

<211>666

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>fuison

<400>116

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

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

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

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

210 215 220

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

225 230 235 240

Gly Phe Thr Phe Ser Pro Phe Ala Ile Ser Trp Val Arg Gln Ala Pro

245 250 255

Gly Lys Gly Leu Glu Trp Val Ala Lys Ile Ser Pro Gly Gly Ser Trp

260 265 270

Thr Tyr Tyr Ser Asp Thr Val Thr Asp Arg Phe Thr Phe Ser Leu Asp

275 280 285

Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

290 295 300

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Leu Trp Gly Tyr Tyr Ala

305 310 315 320

Leu Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

325 330 335

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

340 345 350

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

355 360 365

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

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

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

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

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

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

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

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

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

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

610 615 620

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

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys Gly Lys

660 665

<210>117

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>117

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Asp Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>118

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>118

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

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

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

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

210 215 220

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

225 230 235 240

Gly Phe Thr Phe Ser Pro Phe Ala Ile Ser Trp Val Arg Gln Ala Pro

245 250 255

Gly Lys Gly Leu Glu Trp Val Ala Lys Ile Ser Pro Gly Gly Ser Trp

260 265 270

Thr Tyr Tyr Ser Asp Thr Val Thr Asp Arg Phe Thr Phe Ser Leu Asp

275 280 285

Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

290 295 300

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Leu Trp Gly Tyr Tyr Ala

305 310 315 320

Leu Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

325 330 335

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

340 345 350

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

355 360 365

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

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

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

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

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

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

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

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

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

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

610 615 620

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

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>119

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>119

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>120

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>120

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

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

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

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

210 215 220

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

225 230 235 240

Gly Phe Thr Phe Ser Pro Phe Ala Ile Ser Trp Val Arg Gln Ala Pro

245 250 255

Gly Lys Gly Leu Glu Trp Val Ala Lys Ile Ser Pro Gly Gly Ser Trp

260 265 270

Thr Tyr Tyr Ser Asp Thr Val Thr Asp Arg Phe Thr Phe Ser Leu Asp

275 280 285

Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

290 295 300

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Leu Trp Gly Tyr Tyr Ala

305 310 315 320

Leu Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

325 330 335

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

340 345 350

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

355 360 365

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

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

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

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

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

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

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

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

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

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

610 615 620

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

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>121

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>121

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Asp Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>122

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>122

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

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

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

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

210 215 220

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

225 230 235 240

Gly Phe Thr Phe Ser Pro Phe Ala Trp Ser Trp Val Arg Gln Ala Pro

245 250 255

Gly Lys Gly Leu Glu Trp Val Ala Lys Ile Ser Pro Gly Gly Ser Trp

260 265 270

Thr Tyr Tyr Ser Asp Thr Val Thr Asp Arg Phe Thr Phe Ser Leu Asp

275 280 285

Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

290 295 300

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Leu Trp Gly Tyr Tyr Ala

305 310 315 320

Leu Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

325 330 335

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

340 345 350

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

355 360 365

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

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

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

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

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

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

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

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

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

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

610 615 620

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

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>123

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>123

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Asp Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>124

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>124

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

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

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

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

210 215 220

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

225 230 235 240

Gly Phe Thr Phe Ser Pro Phe Ala Trp Ser Trp Val Arg Gln Ala Pro

245 250 255

Gly Lys Gly Leu Glu Trp Val Ala Lys Ile Ser Pro Gly Gly Ser Trp

260 265 270

Thr Tyr Tyr Ser Asp Thr Val Thr Asp Arg Phe Thr Phe Ser Leu Asp

275 280 285

Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

290 295 300

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Leu Trp Gly Tyr Tyr Ala

305 310 315 320

Leu Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

325 330 335

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

340 345 350

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

355 360 365

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

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

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

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

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

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

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

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

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

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

610 615 620

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

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>125

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>125

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>126

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>126

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

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

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

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

210 215 220

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

225 230 235 240

Gly Phe Thr Phe Ser Pro Phe Ala Trp Ser Trp Val Arg Gln Ala Pro

245 250 255

Gly Lys Gly Leu Glu Trp Val Ala Lys Ile Ser Pro Gly Gly Ser Trp

260 265 270

Thr Tyr Tyr Ser Asp Thr Val Thr Asp Arg Phe Thr Phe Ser Leu Asp

275 280 285

Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

290 295 300

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Leu Trp Gly Tyr Tyr Ala

305 310 315 320

Leu Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

325 330 335

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

340 345 350

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

355 360 365

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

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

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

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

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

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

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

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

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

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

610 615 620

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

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>127

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>127

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Asp Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>128

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>128

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg Pro Phe Val

225 230 235 240

Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg

245 250 255

Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr

260 265 270

Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile

275 280 285

Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys

290 295 300

Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr

305 310 315 320

Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val

325 330 335

Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu

340 345 350

Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe

355 360 365

Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn

370 375 380

Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser

385 390 395 400

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

405 410 415

Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val

420 425 430

Arg Val His Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

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

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

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

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

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

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

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

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

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

610 615 620

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

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>129

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>129

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Asp Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>130

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>130

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg Pro Phe Val

225 230 235 240

Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg

245 250 255

Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr

260 265 270

Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile

275 280 285

Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys

290 295 300

Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr

305 310 315 320

Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val

325 330 335

Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu

340 345 350

Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe

355 360 365

Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn

370 375 380

Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser

385 390 395 400

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

405 410 415

Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val

420 425 430

Arg Val His Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

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

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

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

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

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

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

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

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

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

610 615 620

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

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>131

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>131

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>132

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>132

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg Pro Phe Val

225 230 235 240

Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg

245 250 255

Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr

260 265 270

Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile

275 280 285

Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys

290 295 300

Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr

305 310 315 320

Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val

325 330 335

Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu

340 345 350

Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe

355 360 365

Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn

370 375 380

Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser

385 390 395 400

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

405 410 415

Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val

420 425 430

Arg Val His Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

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

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

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

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

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

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

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

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

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

610 615 620

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

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>133

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>133

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Asp Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>134

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>134

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg Pro Phe Val

225 230 235 240

Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg

245 250 255

Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr

260 265 270

Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile

275 280 285

Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys

290 295 300

Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr

305 310 315 320

Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val

325 330 335

Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu

340 345 350

Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe

355 360 365

Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn

370 375 380

Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser

385 390 395 400

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

405 410 415

Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val

420 425 430

Arg Val His Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

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

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

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

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

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

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

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

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

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

610 615 620

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

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>135

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>135

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Asp Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>136

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>136

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg Pro Phe Val

225 230 235 240

Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg

245 250 255

Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr

260 265 270

Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile

275 280 285

Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys

290 295 300

Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr

305 310 315 320

Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val

325 330 335

Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu

340 345 350

Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe

355 360 365

Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn

370 375 380

Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser

385 390 395 400

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

405 410 415

Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val

420 425 430

Arg Val His Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

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

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

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

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

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

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

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

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

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

610 615 620

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

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>137

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>137

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>138

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>138

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg Pro Phe Val

225 230 235 240

Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg

245 250 255

Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr

260 265 270

Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile

275 280 285

Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys

290 295 300

Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr

305 310 315 320

Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val

325 330 335

Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu

340 345 350

Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe

355 360 365

Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn

370 375 380

Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser

385 390 395 400

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

405 410 415

Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val

420 425 430

Arg Val His Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

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

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

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

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

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

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

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

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

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

610 615 620

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

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>139

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>139

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Asp Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>140

<211>119

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>140

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>141

<211>119

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>141

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>142

<211>106

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>142

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Asp Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210>143

<211>106

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>143

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210>144

<211>106

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>144

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Asp Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210>145

<211>205

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>trap

<400>145

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

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

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys

195 200 205

<210>146

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>146

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Asp Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>147

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>147

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

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

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

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

210 215 220

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

225 230 235 240

Gly Phe Thr Phe Ser Pro Phe Ala Ile Ser Trp Val Arg Gln Ala Pro

245 250 255

Gly Lys Gly Leu Glu Trp Val Ala Lys Ile Ser Pro Gly Gly Ser Trp

260 265 270

Thr Tyr Tyr Ser Asp Thr Val Thr Asp Arg Phe Thr Phe Ser Leu Asp

275 280 285

Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

290 295 300

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Leu Trp Gly Tyr Tyr Ala

305 310 315 320

Leu Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

325 330 335

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

340 345 350

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

355 360 365

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

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

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

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

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

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

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

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

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

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

610 615 620

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

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>148

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>148

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>149

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>149

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

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

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

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

210 215 220

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

225 230 235 240

Gly Phe Thr Phe Ser Pro Phe Ala Trp Ser Trp Val Arg Gln Ala Pro

245 250 255

Gly Lys Gly Leu Glu Trp Val Ala Lys Ile Ser Pro Gly Gly Ser Trp

260 265 270

Thr Tyr Tyr Ser Asp Thr Val Thr Asp Arg Phe Thr Phe Ser Leu Asp

275 280 285

Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

290 295 300

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Leu Trp Gly Tyr Tyr Ala

305 310 315 320

Leu Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

325 330 335

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

340 345 350

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

355 360 365

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

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

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

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

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

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

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

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

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

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

610 615 620

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

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>150

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>150

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Asp Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>151

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>151

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

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

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

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

210 215 220

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

225 230 235 240

Gly Phe Thr Phe Ser Pro Phe Ala Trp Ser Trp Val Arg Gln Ala Pro

245 250 255

Gly Lys Gly Leu Glu Trp Val Ala Lys Ile Ser Pro Gly Gly Ser Trp

260 265 270

Thr Tyr Tyr Ser Asp Thr Val Thr Asp Arg Phe Thr Phe Ser Leu Asp

275 280 285

Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

290 295 300

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Leu Trp Gly Tyr Tyr Ala

305 310 315 320

Leu Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

325 330 335

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

340 345 350

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

355 360 365

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

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

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

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

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

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

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

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

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

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

610 615 620

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

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>152

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>152

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Asp Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>153

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>153

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

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

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

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

210 215 220

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

225 230 235 240

Gly Phe Thr Phe Ser Pro Phe Ala Trp Ser Trp Val Arg Gln Ala Pro

245 250 255

Gly Lys Gly Leu Glu Trp Val Ala Lys Ile Ser Pro Gly Gly Ser Trp

260 265 270

Thr Tyr Tyr Ser Asp Thr Val Thr Asp Arg Phe Thr Phe Ser Leu Asp

275 280 285

Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

290 295 300

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Leu Trp Gly Tyr Tyr Ala

305 310 315 320

Leu Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

325 330 335

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

340 345 350

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

355 360 365

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

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

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

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

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

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

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

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

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

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

610 615 620

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

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>154

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>154

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>155

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>155

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

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

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

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

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

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

210 215 220

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

225 230 235 240

Gly Phe Thr Phe Ser Pro Phe Ala Trp Ser Trp Val Arg Gln Ala Pro

245 250 255

Gly Lys Gly Leu Glu Trp Val Ala Lys Ile Ser Pro Gly Gly Ser Trp

260 265 270

Thr Tyr Tyr Ser Asp Thr Val Thr Asp Arg Phe Thr Phe Ser Leu Asp

275 280 285

Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

290 295 300

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Leu Trp Gly Tyr Tyr Ala

305 310 315 320

Leu Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

325 330 335

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

340 345 350

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

355 360 365

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

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

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

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

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

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

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

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

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

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

610 615 620

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

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>156

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>156

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Asp Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>157

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>157

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg Pro Phe Val

225 230 235 240

Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg

245 250 255

Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr

260 265 270

Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile

275 280 285

Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys

290 295 300

Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr

305 310 315 320

Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val

325 330 335

Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu

340 345 350

Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe

355 360 365

Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn

370 375 380

Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser

385 390 395 400

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

405 410 415

Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val

420 425 430

Arg Val His Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

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

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

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

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

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

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

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

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

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

610 615 620

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

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>158

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>158

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

1 5 10 15

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

20 25 30

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

35 40 45

Asp Asp Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>159

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>159

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Gly

210 215 220

Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg Pro Phe Val

225 230 235 240

Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg

245 250 255

Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr

260 265 270

Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile

275 280 285

Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys

290 295 300

Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr

305 310 315 320

Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val

325 330 335

Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu

340 345 350

Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe

355 360 365

Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn

370 375 380

Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser

385 390 395 400

Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr

405 410 415

Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val

420 425 430

Arg Val His Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

610 615 620

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>160

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>160

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ile Ser Val Ser Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>161

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>161

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val

50 55 60

Thr Asp Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala 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 Arg Gln Leu Trp Gly Tyr Tyr Ala Leu Asp Val Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Gly

210 215 220

Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg Pro Phe Val

225 230 235 240

Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg

245 250 255

Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr

260 265 270

Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile

275 280 285

Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys

290 295 300

Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr

305 310 315 320

Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val

325 330 335

Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu

340 345 350

Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe

355 360 365

Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn

370 375 380

Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser

385 390 395 400

Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr

405 410 415

Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val

420 425 430

Arg Val His Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

610 615 620

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>162

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>162

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ile Ser Val Ser Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Asp Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>163

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>163

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

Ala Trp Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val

50 55 60

Thr Asp Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala 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 Arg Gln Leu Trp Gly Tyr Tyr Ala Leu Asp Val Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Gly

210 215 220

Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg Pro Phe Val

225 230 235 240

Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg

245 250 255

Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr

260 265 270

Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile

275 280 285

Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys

290 295 300

Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr

305 310 315 320

Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val

325 330 335

Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu

340 345 350

Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe

355 360 365

Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn

370 375 380

Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser

385 390 395 400

Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr

405 410 415

Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val

420 425 430

Arg Val His Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

610 615 620

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>164

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>164

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ile Ser Val Ser Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Asp Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>165

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>165

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

Ala Trp Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val

50 55 60

Thr Asp Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala 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 Arg Gln Leu Trp Gly Tyr Tyr Ala Leu Asp Val Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Gly

210 215 220

Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg Pro Phe Val

225 230 235 240

Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg

245 250 255

Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr

260 265 270

Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile

275 280 285

Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys

290 295 300

Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr

305 310 315 320

Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val

325 330 335

Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu

340 345 350

Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe

355 360 365

Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn

370 375 380

Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser

385 390 395 400

Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr

405 410 415

Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val

420 425 430

Arg Val His Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

610 615 620

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>166

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>166

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ile Ser Val Ser Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>167

<211>667

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>167

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

Ala Trp Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val

50 55 60

Thr Asp Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala 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 Arg Gln Leu Trp Gly Tyr Tyr Ala Leu Asp Val Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Gly

210 215 220

Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg Pro Phe Val

225 230 235 240

Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg

245 250 255

Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr

260 265 270

Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile

275 280 285

Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys

290 295 300

Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr

305 310 315 320

Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val

325 330 335

Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu

340 345 350

Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe

355 360 365

Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn

370 375 380

Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser

385 390 395 400

Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr

405 410 415

Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val

420 425 430

Arg Val His Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

610 615 620

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys Pro Gly Lys

660 665

<210>168

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>light chain

<400>168

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ile Ser Val Ser Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Asp Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>169

<211>213

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>169

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ile Ser Val Ser Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Asp Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>170

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>170

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly

210 215 220

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

225 230 235 240

Gly Phe Thr Phe Ser Pro Phe Ala Met His Trp Val Arg Gln Ala Pro

245 250 255

Gly Lys Gly Leu Glu Trp Val Ala Lys Ile Ser Pro Gly Gly Ser Trp

260 265 270

Thr Tyr Tyr Ser Asp Thr Val Thr Asp Arg Phe Thr Phe Ser Leu Asp

275 280 285

Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

290 295 300

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Ala Trp Gly Tyr Tyr Ala

305 310 315 320

Leu Asp Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

325 330 335

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

340 345 350

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

355 360 365

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

370 375 380

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

385 390 395 400

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

405 410 415

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

420 425 430

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

610 615 620

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>171

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>171

Gln Leu Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>172

<211>5

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>172

Pro Phe Ala Met His

15

<210>173

<211>17

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>173

Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val Thr

1 5 10 15

Asp

<210>174

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>174

Gln Ala Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>175

<211>5

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>175

Pro Phe Ala Met His

15

<210>176

<211>17

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>176

Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val Thr

1 5 10 15

Asp

<210>177

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>177

Gln Ser Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>178

<211>5

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>178

Pro Phe Ala Met His

15

<210>179

<211>17

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>179

Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val Thr

1 5 10 15

Asp

<210>180

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>180

Gln Gly Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>181

<211>5

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>181

Pro Phe Ala Met His

15

<210>182

<211>17

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>182

Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val Thr

1 5 10 15

Asp

<210>183

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>183

Gln Thr Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>184

<211>5

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>184

Pro Phe Ala Met His

15

<210>185

<211>17

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>185

Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val Thr

1 5 10 15

Asp

<210>186

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>186

Gln Val Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>187

<211>5

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>187

Pro Phe Ala Met His

15

<210>188

<211>17

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>188

Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val Thr

1 5 10 15

Asp

<210>189

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>189

Gln Gln Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>190

<211>5

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>190

Pro Phe Ala Met His

15

<210>191

<211>17

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>191

Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val Thr

1 5 10 15

Asp

<210>192

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>192

Gln Lys Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>193

<211>5

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>193

Pro Phe Ala Ile Ser

15

<210>194

<211>17

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>194

Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val Thr

1 5 10 15

Asp

<210>195

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>195

Gln Leu Trp Gly Tyr Tyr Ala Leu Asp Val

1 5 10

<210>196

<211>5

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>196

Pro Phe Ala Trp Ser

15

<210>197

<211>17

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>197

Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val Thr

1 5 10 15

Asp

<210>198

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>198

Gln Leu Trp Gly Tyr Tyr Ala Leu Asp Val

1 5 10

<210>199

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>199

Ser Ala Ser Ile Ser Val Ser Tyr Leu Tyr

1 5 10

<210>200

<211>7

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>200

Asp Asp Ser Ser Leu Ala Ser

15

<210>201

<211>9

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>201

Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

15

<210>202

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>202

Ser Ala Ser Ile Ser Val Ser Tyr Leu Tyr

1 5 10

<210>203

<211>7

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>203

Asp Ala Ser Ser Leu Ala Ser

15

<210>204

<211>9

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>204

Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

15

<210>205

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>205

Ser Ala Ser Ile Ser Val Ser Tyr Leu Tyr

1 5 10

<210>206

<211>7

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>206

Asp Asp Ser Asn Leu Ala Ser

15

<210>207

<211>9

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>207

Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

15

<210>208

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>208

Gly Phe Thr Phe Ser Pro Phe Ala Met His

1 5 10

<210>209

<211>19

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>209

Val Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr

1 5 10 15

Val Thr Asp

<210>210

<211>12

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>210

Ala Arg Gln Ala Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>211

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>211

Gly Phe Thr Phe Ser Pro Phe Ala Met His

1 5 10

<210>212

<211>19

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>212

Val Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr

1 5 10 15

Val Thr Asp

<210>213

<211>12

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>213

Ala Arg Gln Ser Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>214

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>214

Gly Phe Thr Phe Ser Pro Phe Ala Met His

1 5 10

<210>215

<211>19

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>215

Val Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr

1 5 10 15

Val Thr Asp

<210>216

<211>12

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>216

Ala Arg Gln Gly Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>217

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>217

Gly Phe Thr Phe Ser Pro Phe Ala Met His

1 5 10

<210>218

<211>19

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>218

Val Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr

1 5 10 15

Val Thr Asp

<210>219

<211>12

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>219

Ala Arg Gln Thr Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>220

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>220

Gly Phe Thr Phe Ser Pro Phe Ala Met His

1 5 10

<210>221

<211>19

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>221

Val Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr

1 5 10 15

Val Thr Asp

<210>222

<211>12

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>222

Ala Arg Gln Val Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>223

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>223

Gly Phe Thr Phe Ser Pro Phe Ala Met His

1 5 10

<210>224

<211>19

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>224

Val Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr

1 5 10 15

Val Thr Asp

<210>225

<211>12

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>225

Ala Arg Gln Gln Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>226

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>226

Gly Phe Thr Phe Ser Pro Phe Ala Met His

1 5 10

<210>227

<211>19

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>227

Val Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr

1 5 10 15

Val Thr Asp

<210>228

<211>12

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>228

Ala Arg Gln Lys Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>229

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>229

Gly Phe Thr Phe Ser Pro Phe Ala Ile Ser

1 5 10

<210>230

<211>19

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>230

Val Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr

1 5 10 15

Val Thr Asp

<210>231

<211>12

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>231

Ala Arg Gln Leu Trp Gly Tyr Tyr Ala Leu Asp Val

1 5 10

<210>232

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>232

Gly Phe Thr Phe Ser Pro Phe Ala Trp Ser

1 5 10

<210>233

<211>20

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>233

Trp Val Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp

1 5 10 15

Thr Val Thr Asp

20

<210>234

<211>12

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>234

Ala Arg Gln Leu Trp Gly Tyr Tyr Ala Leu Asp Val

1 5 10

<210>235

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>235

Ser Ala Ser Ile Ser Val Ser Tyr Leu Tyr

1 5 10

<210>236

<211>11

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>236

Leu Leu Ile Tyr Asp Asp Ser Ser Leu Ala Ser

1 5 10

<210>237

<211>9

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>237

Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

15

<210>238

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>238

Ser Ala Ser Ile Ser Val Ser Tyr Leu Tyr

1 5 10

<210>239

<211>11

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>239

Leu Leu Ile Tyr Asp Ala Ser Ser Leu Ala Ser

1 5 10

<210>240

<211>9

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>240

Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

15

<210>241

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>241

Ser Ala Ser Ile Ser Val Ser Tyr Leu Tyr

1 5 10

<210>242

<211>11

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>242

Leu Leu Ile Tyr Asp Asp Ser Asn Leu Ala Ser

1 5 10

<210>243

<211>9

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>243

Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

15

<210>244

<211>5

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>244

Pro Phe Ala Met Ser

15

<210>245

<211>17

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>245

Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val Thr

1 5 10 15

Gly

<210>246

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>246

Gln Leu Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>247

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>247

Ser Ala Ser Ile Ser Val Ser Tyr Met Tyr

1 5 10

<210>248

<211>7

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>248

Asp Met Ser Asn Leu Ala Ser

15

<210>249

<211>9

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>249

Met Gln Trp Ser Gly Tyr Pro Tyr Thr

15

<210>250

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>250

Gly Phe Thr Phe Ser Pro Phe Ala Met Ser

1 5 10

<210>251

<211>20

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>251

Trp Val Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp

1 5 10 15

Thr Val Thr Gly

20

<210>252

<211>12

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>252

Ala Arg Gln Leu Trp Gly Tyr Tyr Ala Leu Asp Ile

1 5 10

<210>253

<211>10

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>253

Ser Ala Ser Ile Ser Val Ser Tyr Met Tyr

1 5 10

<210>254

<211>11

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>254

Leu Leu Ile Tyr Asp Met Ser Asn Leu Ala Ser

1 5 10

<210>255

<211>9

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>255

Met Gln Trp Ser Gly Tyr Pro Tyr Thr

15

<210>256

<211>119

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>256

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val

50 55 60

Thr Asp Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala 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 Arg Gln Ala Trp Gly Tyr Tyr Ala Leu Asp Ile Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>257

<211>119

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>257

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val

50 55 60

Thr Asp Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala 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 Arg Gln Ser Trp Gly Tyr Tyr Ala Leu Asp Ile Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>258

<211>119

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>258

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val

50 55 60

Thr Asp Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala 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 Arg Gln Gly Trp Gly Tyr Tyr Ala Leu Asp Ile Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>259

<211>119

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>259

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val

50 55 60

Thr Asp Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala 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 Arg Gln Thr Trp Gly Tyr Tyr Ala Leu Asp Ile Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>260

<211>119

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>260

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val

50 55 60

Thr Asp Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala 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 Arg Gln Val Trp Gly Tyr Tyr Ala Leu Asp Ile Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>261

<211>119

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>261

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val

50 55 60

Thr Asp Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala 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 Arg Gln Gln Trp Gly Tyr Tyr Ala Leu Asp Ile Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>262

<211>119

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>antibody

<400>262

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val

50 55 60

Thr Asp Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala 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 Arg Gln Lys Trp Gly Tyr Tyr Ala Leu Asp Ile Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>263

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>263

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly

210 215 220

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

225 230 235 240

Gly Phe Thr Phe Ser Pro Phe Ala Met His Trp Val Arg Gln Ala Pro

245 250 255

Gly Lys Gly Leu Glu Trp Val Ala Lys Ile Ser Pro Gly Gly Ser Trp

260 265 270

Thr Tyr Tyr Ser Asp Thr Val Thr Asp Arg Phe Thr Phe Ser Leu Asp

275 280 285

Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

290 295 300

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Ala Trp Gly Tyr Tyr Ala

305 310 315 320

Leu Asp Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

325 330 335

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

340 345 350

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

355 360 365

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

370 375 380

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

385 390 395 400

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

405 410 415

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

420 425 430

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

610 615 620

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>264

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>264

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly

210 215 220

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

225 230 235 240

Gly Phe Thr Phe Ser Pro Phe Ala Met His Trp Val Arg Gln Ala Pro

245 250 255

Gly Lys Gly Leu Glu Trp Val Ala Lys Ile Ser Pro Gly Gly Ser Trp

260 265 270

Thr Tyr Tyr Ser Asp Thr Val Thr Asp Arg Phe Thr Phe Ser Leu Asp

275 280 285

Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

290 295 300

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Ala Trp Gly Tyr Tyr Ala

305 310 315 320

Leu Asp Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

325 330 335

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

340 345 350

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

355 360 365

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

370 375 380

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

385 390 395 400

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

405 410 415

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

420 425 430

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

610 615 620

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>265

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>trap

<400>265

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly

210 215 220

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

225 230 235 240

Gly Phe Thr Phe Ser Pro Phe Ala Met His Trp Val Arg Gln Ala Pro

245 250 255

Gly Lys Gly Leu Glu Trp Val Ala Lys Ile Ser Pro Gly Gly Ser Trp

260 265 270

Thr Tyr Tyr Ser Asp Thr Val Thr Asp Arg Phe Thr Phe Ser Leu Asp

275 280 285

Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

290 295 300

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Ala Trp Gly Tyr Tyr Ala

305 310 315 320

Leu Asp Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

325 330 335

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

340 345 350

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

355 360 365

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

370 375 380

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

385 390 395 400

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

405 410 415

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

420 425 430

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

610 615 620

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>266

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>266

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val

50 55 60

Thr Asp Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala 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 Arg Gln Ala Trp Gly Tyr Tyr Ala Leu Asp Ile Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Gly

210 215 220

Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg Pro Phe Val

225 230 235 240

Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg

245 250 255

Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr

260 265 270

Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile

275 280 285

Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys

290 295 300

Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr

305 310 315 320

Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val

325 330 335

Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu

340 345 350

Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe

355 360 365

Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn

370 375 380

Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser

385 390 395 400

Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr

405 410 415

Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val

420 425 430

Arg Val His Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

610 615 620

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>267

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>267

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val

50 55 60

Thr Asp Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala 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 Arg Gln Ala Trp Gly Tyr Tyr Ala Leu Asp Ile Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Gly

210 215 220

Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg Pro Phe Val

225 230 235 240

Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg

245 250 255

Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr

260 265 270

Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile

275 280 285

Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys

290 295 300

Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr

305 310 315 320

Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val

325 330 335

Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu

340 345 350

Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe

355 360 365

Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn

370 375 380

Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser

385 390 395 400

Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr

405 410 415

Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val

420 425 430

Arg Val His Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

610 615 620

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>268

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>268

Glu Val Gln Leu Val 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 Pro Phe

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val

50 55 60

Thr Asp Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala 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 Arg Gln Ala Trp Gly Tyr Tyr Ala Leu Asp Ile Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Gly

210 215 220

Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg Pro Phe Val

225 230 235 240

Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg

245 250 255

Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr

260 265 270

Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile

275 280 285

Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys

290 295 300

Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr

305 310 315 320

Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val

325 330 335

Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu

340 345 350

Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe

355 360 365

Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn

370 375 380

Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser

385 390 395 400

Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr

405 410 415

Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val

420 425 430

Arg Val His Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

610 615 620

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<210>269

<211>664

<212>PRT/PROTEIN

<213>Artificial sequence

<220>

<223>merged

<400>269

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly

210 215 220

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

225 230 235 240

Gly Phe Thr Phe Ser Pro Phe Ala Ile Ser Trp Val Arg Gln Ala Pro

245 250 255

Gly Lys Gly Leu Glu Trp Val Ala Lys Ile Ser Pro Gly Gly Ser Trp

260 265 270

Thr Tyr Tyr Ser Asp Thr Val Thr Asp Arg Phe Thr Phe Ser Leu Asp

275 280 285

Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

290 295 300

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Leu Trp Gly Tyr Tyr Ala

305 310 315 320

Leu Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

325 330 335

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

340 345 350

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

355 360 365

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

370 375 380

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

385 390 395 400

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

405 410 415

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

420 425 430

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

435 440 445

Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

565 570 575

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

610 615 620

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Cys Ser Pro Gly Lys

660

<---

Claims (139)

1. A fusion protein for binding VEGF and IL-6, containing: an isolated antagonist antibody that specifically binds to IL-6, wherein the antibody comprises a heavy chain and a light chain; And a VEGF (vascular endothelial growth factor) trap, wherein said VEGF trap contains VEGFR1 domain 2 and VEGFR2 domain 3, wherein said VEGF trap is located either at the N-terminus of said heavy chain containing IL-6 VH, with a linker linking said VEGF trap and said heavy chain; or between the hinge region and the CH1 domain of a heavy chain containing IL-6 VH, with a linker connecting said CH1 domain and said VEGF trap. 2. The fusion protein according to claim 1, characterized in that the VEGF trap is located at the N-terminus of the heavy chain containing IL-6 VH. 3. The fusion protein according to claim 1, characterized in that the VEGF trap is located between the hinge region and after the CH1 domain of the heavy chain containing IL-6 VH. 4. The fusion protein according to claim 1, characterized in that the VEGF trap consists of the sequence SEQ ID NO: 114. 5. Fusion protein according to any one of paragraphs. 1-4, characterized in that the antibody binds human IL-6 with an affinity of from about 10 pM to about 1 nM. 6. Fusion protein according to any one of paragraphs. 1-5, containing a heavy chain constant domain containing a non-native cysteine residue. 7. The fusion protein according to claim 6, characterized in that said non-native cysteine residue is located at a position selected from the group consisting of 347 and 443 in EU numbering. 8. The fusion protein according to claim 7, characterized in that said non-native cysteine residue is located at position 443, in the EU numbering. 9. Fusion protein according to any one of paragraphs. 1-8, characterized in that (i) said heavy chain contains the amino acid sequence of any of SEQ ID NOs: 19-27, or contains a heavy chain variable region (VH) containing the amino acid sequence of any of SEQ ID NOs: 7-13, 89, 90, 256-262 ; and a light chain that contains the amino acid sequence of any of SEQ ID NO: 28-30, or contains a light chain variable region (VL) containing the amino acid sequence of any of SEQ ID NO: 91-93; or (ii) said heavy chain comprises a heavy chain variable region (VH) containing 3 complementarity determining regions (CDRs): VH CDR1, VH CDR2 and VH CDR3, having the amino acid sequences of CDR1, CDR2 and CDR3, respectively, in SEQ ID NO: 256; and the light chain comprises a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3, respectively, in one of SEQ ID NOs: 91-93; or (iii) said antibody contains: CDRH1 containing the amino acid sequence of SEQ ID NO: 172; CDRH2 containing the amino acid sequence of SEQ ID NO: 173; CDRH3 containing the amino acid sequence of SEQ ID NO: 174; CDRL1 containing the amino acid sequence of SEQ ID NO: 199; CDRL2 containing the amino acid sequence of SEQ ID NO: 200; CDRL3 containing the amino acid sequence of SEQ ID NO: 201; And Fc domain containing: at least one of the following mutations (EU numbering): L234A, L235A and G237A; and/or at least one of the following mutations (EU numbering): Q347C or L443C. 10. Conjugate for binding VEGF and IL-6, containing: fusion protein according to any one of claims. 1-9; And a polymer containing phosphorylcholine, wherein said polymer is covalently attached to the heavy chain of the isolated antagonist antibody. 11. The conjugate according to claim 10, characterized in that the polymer contains a zwitterionic monomer, wherein said zwitterionic monomer is selected from the group consisting of HEMA-phosphorylcholine, PEG, biocompatible fatty acids and their derivatives, hydroxyalkyl starch (HAS), hydroxyethyl starch (HES) , polyethylene glycol (PEG), poly(Gly _x -Ser _y ) (HAP), hyaluronic acid (HA), heparosan polymers (HEP), fleximers, dextran, polysialic acids (PSA), Fc domains, transferrin, 25 albumin, elastin-like peptides (ELP), XTEN polymers, PAS polymers, PA polymers, albumin binding peptides, CTP peptides and FcRn binding peptides. 12. Conjugate according to any one of paragraphs. 10-11, characterized in that the polymer contains monomers of 2(methacryloyloxy)ethyl-(2-(trimethylammonium)ethyl)phosphate (MPC). 13. Conjugate according to any one of paragraphs. 10-12, characterized in that the polymer has a peak molecular weight of from 300,000 to 1,750,000 Daltons, measured by size exclusion chromatography with multi-angle light scattering (hereinafter "SEC-MALS"). 14. Conjugate according to any one of paragraphs. 10-13, characterized in that the polymer has a peak molecular weight of from 500,000 to 1,000,000 Daltons, measured by SEC-MALS. 15. The conjugate according to claim 14, characterized in that the polymer has a peak molecular weight of from 600,000 to 800,000 Daltons, measured by SEC-MALS. 16. Conjugate according to any one of paragraphs. 10-15, characterized in that the polymer contains 2 or more branches. 17. Conjugate according to any one of paragraphs. 10-16, characterized in that the polymer contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 branches. 18. The conjugate according to claim 17, characterized in that the polymer contains 9 branches. 19. Conjugate according to any one of paragraphs. 10-18, characterized in that the antibody is IgG. 20. The conjugate according to claim 19, characterized in that the polymer is covalently linked to the sulfhydryl group of a cysteine residue in the IgG heavy chain. 21. Conjugate according to any one of paragraphs. 10-20, characterized in that the polymer is covalently linked to the sulfhydryl group of the cysteine residue at position 347 or 443, in EU numbering. 22. Conjugate according to any one of paragraphs. 10-21, characterized in that the antibody is further conjugated to a polymer to form a bioconjugate, and wherein said bioconjugate has a molecular weight of from about 350,000 to 1,900,000 Daltons. 23. Conjugate according to any one of paragraphs. 10-22, characterized in that the polydispersity index (PDI) is equal to or less than 1.5. 24. Conjugate according to any one of paragraphs. 10-23, characterized in that the polymer contains 9 branches; And the polymer has a molecular weight of from about 600,000 to about 900,000 Da. 25. Conjugate according to any one of paragraphs. 10-24 containing the following structure: Where: each anti-IL-6 antibody heavy chain is designated by the letter H and each anti-IL-6 antibody light chain is designated by the letter L; the polymer is linked to the antibody through the sulfhydryl group C443, in EU numbering; the specified bond is depicted on one of the heavy chains; PC represents where the wavy line shows the point of attachment to the rest of the polymer, where X=a) OR, where R=H, methyl, ethyl, propyl, isopropyl, b) H or c) any halide including Br; And n1, n2, n3, n4, n5, n6, n7, n8 and n9 are the same or different so that the sum of n1, n2, n3, n4, n5, n6, n7, n8 and n9 is equal to 2500 plus or minus 15%. 26. An isolated cell line producing a fusion protein, wherein the cells of said cell line contain an isolated nucleic acid encoding the fusion protein according to any one of claims. 1-9. 27. The isolated cell line according to claim 26, characterized in that said cell line is selected from the group consisting of CHO, k1SV, XCeed, CHOK1SV and GS-KO. 28. An isolated nucleic acid encoding a fusion protein according to any one of claims. 1-9. 29. Recombinant expression vector containing the nucleic acid according to claim 28. 30. A host cell for producing a fusion protein containing the expression vector of claim 29. 31. A method for producing a fusion protein, the method comprising: culturing a cell line that recombinantly produces the fusion protein according to any one of claims. 1-9, under conditions under which the fusion protein is produced; and recovering the fusion protein. 32. A pharmaceutical composition for treating an ocular disorder or for inhibiting VEGF and IL-6 signaling in a patient in need thereof, comprising a fusion protein according to any one of claims. 1-9 and/or conjugate according to any one of paragraphs. 10-25, and a pharmaceutically acceptable carrier. 33. The pharmaceutical composition according to claim 32, characterized in that the composition is a liquid and has an endotoxin level below about 0.2 EU/ml. 34. The pharmaceutical composition of claim 33, characterized in that said composition is a liquid and has an endotoxin level of less than about 2.0, 1, 0.5 or 0.2 EU/ml. 35. A method of treating or preventing a disease in a patient in need thereof, comprising administering to the patient a fusion protein according to any one of claims. 1-9 and/or conjugate according to any one of paragraphs. 10-25, wherein said disease is an ocular disorder selected from wet macular degeneration or neovascular macular degeneration, wet age-related macular degeneration or neovascular age-related macular degeneration, dry age-related macular degeneration, venous, arterial occlusion, or other occlusion of the blood vessels of the eye and/or retina with or without retinal edema, anterior and posterior uveitis, macular edema in uveitis, diabetic retinopathy, non-proliferative diabetic retinopathy, diabetic macular edema, non-proliferative diabetic macular edema, ophthalmic inflammatory disease - scleritis and intraocular tumors. 36. A method of inhibiting VEGF and IL-6 signaling in a patient in need thereof, comprising: identification of a patient with hyperactivity of IL-6 and/or VEGF; And administering to said patient a fusion protein according to any one of claims. 1-9 and/or conjugate according to any one of paragraphs. 10-25. 37. The method of claim 36, characterized in that the patient requires treatment of an ocular disorder by inhibiting VEGF and IL-6 signaling. 38. The method of claim 37, characterized in that said ocular disorder is at least one of: wet macular degeneration or neovascular macular degeneration, wet age-related macular degeneration or neovascular age-related macular degeneration, dry age-related macular degeneration, venous, arterial or other occlusion of the blood vessels of the eye and/or retina with or without retinal edema, anterior and posterior uveitis, uveitis macular edema, diabetic retinopathy, non-proliferative diabetic retinopathy, diabetic macular edema, non-proliferative diabetic macular edema, ophthalmic inflammatory disease - scleritis and intraocular tumors. 39. Method according to any one of paragraphs. 35-38, characterized in that the fusion protein and/or conjugate is administered no more than once a month. 40. Method according to any one of paragraphs. 35-38, characterized in that the fusion protein and/or conjugate is administered no more than once every two months. 41. Method according to any one of paragraphs. 35-38, characterized in that the fusion protein and/or conjugate is administered no more than once every three months. 42. Method according to any one of paragraphs. 35-38, characterized in that the fusion protein and/or conjugate is administered at a frequency of once a year to once every two months. 43. A fusion protein for binding VEGF and IL-6, containing: IL-6 VH; IL-6 VL; Fc region; a VEGF trap, wherein said VEGF trap comprises VEGFR1 domain 2 and VEGFR2 domain 3, wherein said VEGF trap is fused to IL-6 by one of the following methods: with the N-terminus of a heavy chain containing IL-6 VH, a linker linking said VEGF trap and said heavy chain; or between the hinge region and after the CH1 domain of the heavy chain containing IL-6 VH, with a linker connecting said CH1 domain and said VEGF trap. 44. The fusion protein according to any one of paragraphs. 1-9 or a conjugate according to any one of paragraphs. 10-25, containing a mutation at position 94 in the VEGF trap sequence. 45. The fusion protein according to any one of paragraphs. 1-9 or a conjugate according to any one of paragraphs. 10-25, containing a mutation at position 95 in the VEGF trap sequence. 46. Fusion protein according to claims. 1-9 or a conjugate according to any one of paragraphs. 10-25 containing T94I and/or H95I mutations in the VEGF trap sequence. 47. A dual inhibitor of VEGFR and IL-6 signaling, wherein said dual inhibitor comprises a decoy-antibody fusion construct of an anti-IL6 antibody and a VEGF decoy, wherein said anti-IL 6 antibody comprises IL-6 VH and IL-6 VL, wherein said VEGF trap comprises VEGFR1 domain 2 and VEGFR2 domain 3, wherein said dual inhibitor comprises at least one point mutation in the VEGFR sequence to reduce degradation of the VEGFR protein, wherein said VEGF trap is located either at the N-terminus of a heavy chain containing IL-6 VH, with a linker linking said VEGF trap and said heavy chain; or between the hinge region and the CH1 domain of a heavy chain containing IL-6 VH, with a linker connecting said CH1 domain and said VEGF trap. 48. A dual inhibitor according to claim 47, characterized in that the molecular weight of said dual inhibitor is 1.0 MDa. 49. A dual inhibitor according to claim 47, characterized in that said dual inhibitor provides therapy for inflammatory diseases of the retina. 50. The dual inhibitor of claim 47, comprising a heavy chain variable region sequence selected from SEQ ID NOs: 7-13, 89, 90 and/or 256-262. 51. Double inhibitor according to any one of paragraphs. 47-50, characterized in that the VEGF trap contains a sequence selected from at least one of SEQ ID NO: 15, 16 or 17. 52. Double inhibitor according to any one of paragraphs. 47-51, characterized in that the linker sequence is SEQ ID NO: 18. 53. Double inhibitor according to any one of paragraphs. 47-52, characterized in that the heavy chain sequence is selected from at least one of SEQ ID NO: 19-27 or contains a sequence in at least one of SEQ ID NO: 89, 90, 256-262. 54. Double inhibitor according to any one of paragraphs. 47-53, containing a light chain sequence containing at least 3 light chain CDRs from at least one of SEQ ID NO: 76-84. 55. Double inhibitor according to any one of paragraphs. 47-54, characterized in that the heavy chain sequence for the anti-IL-6 molecule contains at least 3 heavy chain CDRs from at least one of SEQ ID NO: 49-75. 56. Double inhibitor according to any one of paragraphs. 47-55 containing the VEGFR-Fc sequence from at least one of SEQ ID NO: 85-88. 57. Double inhibitor according to any one of paragraphs. 47-56, containing one or more sequences in any one or more of SEQ ID NO: 7-13, 145, 15-17, 18-84. 58. The dual inhibitor of claim 47, wherein the IL-6 VH contains a sequence from the IL6 VH sequence in any of SEQ ID NOs: 19-27, 31-39, 89, 90 or 256-262. 59. The dual inhibitor according to claim 47 or 58, characterized in that the IL-6 VL contains a sequence from the sequence of IL6 VL in any of SEQ ID NO: 28-30 or 91-93. 60. Double inhibitor according to any one of paragraphs. 47-59, containing an Fc region containing a sequence from the Fc sequence in any of SEQ ID NO: 40-48. 61. Protein construct for binding VEGF and IL-6, containing: a heavy chain comprising at least 3 heavy chain CDRs selected from the corresponding heavy chain CDRs in any of SEQ ID NOs: 7-13, 19-27, 31-39, 49-75; a light chain containing at least 3 light chain CDRs selected from the corresponding light chain CDRs in any of SEQ ID NOs: 28-30, 76-84; a VEGF trap sequence selected from the corresponding VEGF trap sequence of any one of SEQ ID NO: 14-17, 85-88; And linker sequence SEQ ID NO:18, wherein said VEGF trap is located either at the N-terminus of said heavy chain, with a linker connecting said VEGF trap and said heavy chain; or between the hinge region and the CH1 domain of said heavy chain, with a linker connecting said CH1 domain and said VEGF trap. 62. A fusion protein for binding VEGF and IL-6, containing: IL-6 VH; IL-6 VL; Fc region; And a VEGF trap, wherein said VEGF trap comprises VEGFR1 domain 2 and VEGFR2 domain 3, wherein said VEGF trap is located either at the N-terminus of said heavy chain containing IL-6 VH, with a linker linking said VEGF trap and said heavy chain; or between the hinge region and the CH1 domain of a heavy chain containing IL-6 VH, with a linker connecting said CH1 domain and said VEGF trap, wherein said fusion protein inhibits the proliferation of human umbilical vein endothelial cells (HUEC). 63. The fusion protein of claim 62, characterized in that the change in ECHF proliferation inhibits VEGF/IL6-mediated proliferation. 64. A conjugate for binding VEGF and IL-6 containing a fusion protein conjugated to a polymer containing phosphorylcholine, wherein said fusion protein contains (i) a sequence that is at least 90% identical to SEQ ID NO: 263, wherein said sequence contains a sequence that is at least 90% identical to SEQ ID NO: 114, and a sequence that is at least 90% identical to SEQ ID NO: 256, and (ii) a sequence that is at least 90% identical to SEQ ID NO: : 117, wherein said sequence contains a sequence that is at least 90% identical to SEQ ID NO: 91. 65. The conjugate of claim 64, characterized in that said fusion protein is at least 95% identical to SEQ ID NO: 263 and at least 95% identical to SEQ ID NO: 117. 66. The conjugate according to claim 64 or 65, characterized in that said protein contains at least a) SEQ ID NO: 172, 173, 174, 199, 200 and 201 or b) SEQ ID NO: 172, 173, 174, 199, 200 and/or 201, which contains up to 3 amino acid substitutions, wherein said substitution is a conservative substitution. 67. The fusion protein according to any one of paragraphs. 1-9 or 43-46, characterized in that said fusion protein contains a) an amino acid sequence of SEQ ID NO: 169 and 170 or b) an amino acid sequence that is at least 80% identical to SEQ ID NO: 169 and at least 80% identical to SEQ ID NO: 170. 68. Conjugate according to any one of paragraphs. 10-25, characterized in that said conjugate contains a) an amino acid sequence of SEQ ID NO: 169 and 170 or b) an amino acid sequence that is at least 80% identical to SEQ ID NO: 169 and at least 80% identical to SEQ ID NO: 170. 69. Double inhibitor according to any one of paragraphs. 47-60, characterized in that said dual inhibitor contains an amino acid sequence that is at least 80% identical to SEQ ID NO: 169 and at least 80% identical to SEQ ID NO: 170. 70. The protein construct according to claim 61, characterized in that said construct contains a) an amino acid sequence of SEQ ID NO: 169 and 170 or b) an amino acid sequence that is at least 80% identical to SEQ ID NO: 169 and at least 80% identical to SEQ ID NO: 170. 71. Conjugate for binding VEGF and IL-6, containing: a protein construct comprising two heavy chains and two light chains, each of the heavy chains comprising the amino acid sequence of SEQ ID NO: 170 with or without a C-terminal lysine, and each of the light chains comprising the amino acid sequence of SEQ ID NO: 169; And polymer, wherein the conjugate contains the following structure: Where: each heavy chain of the protein construct is designated by the letter H and each light chain of the protein construct is designated by the letter L; the polymer is linked to the protein construct through the sulfhydryl group C443 (EU numbering), this link is depicted on one of the heavy chains; PC represents , where the wavy line shows the point of attachment to the rest of the polymer; where X=a) OR, where R=H, methyl, ethyl, propyl, isopropyl, b) H, or c) any halide including Br; And wherein n1, n2, n3, n4, n5, n6, n7, n8 and n9 each represent the number n of the following repeating units in formula (17): where the wavy lines show the points of attachment between monomer units in the polymer, and wherein n1, n2, n3, n4, n5, n6, n7, n8 and n9 each represent an integer and are the same or different so that the sum of n1, n2, n3, n4, n5, n6, n7, n8 and n9 is 2500 plus or minus 15%. 72. The conjugate according to claim 70, wherein each heavy chain does not contain a C-terminal lysine. 73. The fusion protein of claim 67, the conjugate of claim 68, the dual inhibitor of claim 69, or the protein construct of claim 70, wherein said amino acid sequence, which is at least 80% identical to SEQ ID NO: 170, does not contain C-terminal lysine.