RU2819228C2 - Connective tissue growth factor antibody and use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention discloses an anti-CTGF antibody, a nucleic acid molecule coding the antibody, a host cell for producing the antibody, a pharmaceutical composition. Invention also relates to a method for determining CTGF and a method of treating or preventing a disease associated with CTGF.

EFFECT: invention can find application in therapy of diseases and conditions associated with CTGF, such as fibrotic diseases, hypertension, diabetes, myocardial infarction, arthritis, cell proliferative disease associated with CTGF, atherosclerosis, glaucoma or cancer.

18 cl, 2 dwg, 27 tbl, 8 ex

Description

This application claims priority to Chinese Patent Application No. 201910480169.4 (title: Anti-connective tissue growth factor antibody and its use, priority date: June 4, 2019).

FIELD OF THE INVENTION

The present disclosure relates to the field of biomedicine and in particular relates to antibodies that bind to connective tissue growth factor (CTGF) and their uses.

BACKGROUND ART

The descriptions herein offer only general information about the present disclosure and do not necessarily constitute prior art.

CTGF expression is induced by members of the transforming growth factor beta (TGFβ) superfamily. This superfamily includes TGFβ-1, -2 and -3, bone morphogenetic protein (BMP)-2 and activin. Many regulators (including dexamethasone, thrombin, VEGF and angiotensin II) and environmental stress (including hyperglycemia and hypertension) also induce CTGF expression (see e.g. , Franklin (1997) Int J Biochem Cell Biol 29:79-89; Wunderlich (2000) Graefes Arch Clin Exp Ophthalmol 238: 910-915; Denton and Abraham (2001) Curr Opin Rheumatol 13: 505-511; ) Blood 97: 3109-3116; Riser et al. (2000) J Am SocNephrol 11: 25-38; and International Publication WO 00/13706).

The stimulatory effect of TGFβ on CTGF expression is rapid and long-lasting, and there is no need for targeted application of TGFβ (Igarashi et al. (1993) Mol BiolCell 4:637-645). TGFβ activates transcription through DNA regulatory elements located in the CTGF promoter, leading to increased expression of CTGF (Grotendorst et al. (1996) Cell Growth Differ 7: 469-480; Grotendorst and Bradham, US Patent No. 6,069,006; Holmes et al. (2001) ) J Biol Chem 276: 10594–10601).

CTGF expression is up-regulated in glomerulonephritis, IgA nephropathy, interfocal and segmental glomerulosclerosis and diabetic nephropathy (see, for example, Riser et al. (2000) J Am Soc Nephrol 11:25-38). An increase in the number of cells expressing CTGF has also been observed in areas of chronic tubulointerstitial lesions, and the level of CTGF correlates with the extent of the lesion (Ito et al, (1998) Kidney Int 53:853-861). In addition, in various types of nephropathy associated with scarring of the renal parenchyma and sclerosis, the level of CTGF expression in the glomerulus or tubulointerstitium also increases. Elevated CTGF levels are also associated with liver fibrosis, myocardial infarction, and pulmonary fibrosis. For example, CTGF is strongly up-regulated in cells from biopsies and bronchoalveolar lavage fluid from patients with spontaneous pulmonary fibrosis (IPF) (Ujike et al, (2000) Biochem Biophys Res Commun 277:448-454; Abou-Shady et al. (2000) ) Liver 20: 296-304; Williams et al. (2000) J Hepatol 32: 754-761). Thus, CTGF represents an effective therapeutic target for the diseases described above.

However, there is currently no effective CTGF-targeting antibody agent in clinical practice, and there is still a need to develop new safe and effective anti-CTGF antibody agents.

SUMMARY OF THE INVENTION

The present disclosure provides an anti-CTGF antibody. Anti-CTGF antibody includes full-length antibodies against human CTGF and antigen-binding fragments thereof.

In some embodiments, an anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, wherein:

i) the heavy chain variable region contains the same sequences HCDR1, HCDR2 and HCDR3 as the sequences HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 6, and the light chain variable region contains the same sequences LCDR1, LCDR2 and LCDR3, as well as the light chain variable region sequences LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 7; or

ii) the heavy chain variable region contains the same HCDR1, HCDR2 and HCDR3 sequences as the heavy chain variable region sequences HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 8,

and the light chain variable region contains the same LCDR1, LCDR2 and LCDR3 sequences as the LCDR1, LCDR2 and LCDR3 sequences of the light chain variable region shown in SEQ ID NO: 9.

In some embodiments, an anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, wherein:

ii-i) the heavy chain variable region contains the same HCDR1, HCDR2 and HCDR3 sequences as the HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region shown in SEQ ID NO: 69, and the light chain variable region contains the same LCDR1, LCDR2 sequences and LCDR3, as well as the light chain variable region sequences LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 70; or

ii-ii) the heavy chain variable region contains the same HCDR1, HCDR2 and HCDR3 sequences as the HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region shown in SEQ ID NO: 85, and the light chain variable region contains the same LCDR1, LCDR2 sequences and LCDR3, as well as the light chain variable region sequences LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 70.

In some embodiments, an anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, wherein:

iii) the heavy chain variable region contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively, and the light chain variable region contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively; or

iv) the heavy chain variable region contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively, and the light chain variable region contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively.

In some embodiments, an anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, wherein:

iv-i) the heavy chain variable region contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 71, SEQ ID NO: 72 and SEQ ID NO: 73, respectively, and the light chain variable region contains LCDR1, LCDR2 and LCDR3, as shown in SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, respectively; or

iv-ii) the heavy chain variable region contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 102, SEQ ID NO: 103 and SEQ ID NO: 104, respectively, and the light chain variable region contains LCDR1, LCDR2 and LCDR3, as shown in SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, respectively.

In some embodiments, the anti-CTGF antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

In some embodiments, an anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, wherein:

(v-1) the amino acid sequence of the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 6, 27, 28 or 29; and the amino acid sequence of the light chain variable region has at least 90% sequence identity to SEQ ID NO: 7, 22, 23, 24, 25 or 26;

(vi-1) the amino acid sequence of the heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 8, 33, 34, 35 or 36; and the amino acid sequence of the light chain variable region has at least 90% sequence identity to SEQ ID NO: 9, 30, 31 or 32; or

(vi-i) the amino acid sequence of the heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 69, 81, 82, 83, 84 or 85, and the amino acid sequence of the light chain variable region has at least 90 % sequence identity to SEQ ID NO: 70, 77, 78, 79 or 80.

In some embodiments, an anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, wherein:

(v) the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or 100% identity to the heavy chain variable region as shown in SEQ ID NO: 6, 27, 28 or 29, and the light chain variable region has at least 90% identity , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the light chain variable region as shown in SEQ ID NO: 7, 22, 23, 24, 25 or 26; or

(vi) the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or 100% identity to the heavy chain variable region as shown in SEQ ID NO: 8, 33, 34, 35 or 36, and the light chain variable region has at least 90% identity -th, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% - identity with the light chain variable region as shown in SEQ ID NO: 9, 30, 31 or 32; or

the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% -, 99% or 100% identity with the heavy chain variable region as shown in SEQ ID NO: 6, and the light chain variable region has at least 90%, 91%, 92% identity 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the light chain variable region as indicated in SEQ ID NO: 7;

the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% -th, 99% or 100% identity with the heavy chain variable region as shown in SEQ ID NO: 27, 28 or 29, and the light chain variable region has at least 90%, 91% identity , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the light chain variable region as shown in SEQ ID NO: 22, 23, 24, 25 or 26;

the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% -, 99% or 100% identity with the heavy chain variable region as shown in SEQ ID NO: 8, and the light chain variable region has at least 90%, 91%, 92% identity 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the light chain variable region as indicated in SEQ ID NO: 9;

the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% -th, 99% or 100% identity with the heavy chain variable region as shown in SEQ ID NO: 33, 34, 35 or 36, and the light chain variable region has at least 90%, 91% identity -th, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with variable light chain region as shown in SEQ ID NO: 30, 31 or 32.

In some embodiments, an anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, wherein:

(vi-ii) the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99%, or 100% identity to the heavy chain variable region as shown in SEQ ID NO: 69, 81, 82, 83, 84, or 85, the light chain variable region having at least at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the light chain variable region as shown in SEQ ID NO: 70, 77, 78, 79 or 80.

In some embodiments, an anti-CTGF antibody, wherein the anti-CTGF antibody is a humanized antibody that comprises a human antibody framework region or framework region variant, and the framework region variant has up to 10 back mutations in each of the light chain framework region and/or framework region human antibody heavy chain;

In some embodiments, an anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, as described below:

(a) the light chain variable region contains LCDR1, LCDR2 and LCDR3, as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively, and contains one or more reverse mutations of the amino acid(s) selected from the group consisting of 4L, 36F, 43S, 45K, 47W, 58V or 71Y, and/or the heavy chain variable region contains HCDR1, HCDR2 and HCDR3, as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively, and contains one or more reverse mutations of an amino acid(s) selected from the group consisting of 28S, 30N, 49A, 75E, 76S, 93V, 94E or 104D; or

(b) the light chain variable region contains LCDR1, LCDR2 and LCDR3, as shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively, and contains one or more back mutations selected from the group consisting of of 36V, 44F, 46G or 49G, and/or the heavy chain variable region contains HCDR1, HCDR2 and HCDR3, as shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively, and contains one or more reverse mutations selected from the group consisting of 44G, 49G, 27F, 48L, 67L, 71K, 78V or 80F.

In some embodiments, an anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, as described below:

(b-i) the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, respectively, and one or more back mutations selected from the group consisting of 45P, 46W, 48Y, 69S or 70Y, and the heavy chain variable region contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 71, SEQ ID NO: 72 and SEQ ID NO: 73, respectively, and one or more reverse mutations selected from the group consisting of 27F, 38K, 48I, 67K, 68A, 70L or 72F; or

(b-ii) the light chain variable region contains LCDR1, LCDR2 and LCDR3, as shown in SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, respectively, and contains one or more back mutations selected from the group consisting of 45P, 46W, 48Y, 69S or 70Y, and the heavy chain variable region contains HCDR1, HCDR2 and HCDR3, as shown in SEQ ID NO: 102, SEQ ID NO: 103 and SEQ ID NO: 104, respectively, and contains one or more reverse mutations selected from the group consisting of 27F, 38K, 48I, 67K, 68A, 70L or 72F.

In some embodiments, an anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, as described below:

(vii) the heavy chain variable region sequence is as shown in SEQ ID NO: 6, and the light chain variable region sequence is as shown in SEQ ID NO: 7;

(viii) the heavy chain variable region sequence is as shown in SEQ ID NO: 27, 28 or 29, and the light chain variable region sequence is as shown in SEQ ID NO: 22, 23, 24, 25 or 26;

(ix) the heavy chain variable region sequence is as shown in SEQ ID NO: 8, and the light chain variable region sequence is as shown in SEQ ID NO: 9; or

(x) the heavy chain variable region sequence is as shown in SEQ ID NO: 33, 34, 35 or 36, and the light chain variable region sequence is as shown in SEQ ID NO: 30, 31 or 32.

In some embodiments, an anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, as described below:

(xi) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 69, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 70; or

(xii) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 81, 82, 83, 84 or 85, and the amino acid sequence of the light chain variable region is as shown in EQ ID NO: 77, 78, 79 or 80.

In some embodiments, the anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, as shown below in Table a, Table b and Table c:

In some embodiments, an anti-CTGF antibody comprises a heavy chain variable region and a light chain variable region, as described below:

(xiii) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 27, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 22;

(xiv) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 34, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 30; or

(xv) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 85, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 77.

In some embodiments, an anti-CTGF antibody, wherein the antibody further comprises a heavy chain constant region and a light chain constant region of the antibody; preferably, the heavy chain constant region is selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4 constant regions and conventional variants thereof, and the light chain constant region is selected from the group consisting of k and A chain constant regions, human antibodies and conventional variants thereof options; more preferably, the antibody contains a heavy chain constant region, as shown in SEQ ID NO: 37 or 38, and a light chain constant region, as shown in SEQ ID NO: 39 or 40. In some embodiments, the anti-CTGF antibody contains:

(c) a heavy chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the heavy chain as shown in SEQ ID NO: 41, 43, 44, 45, 46, 47, or 48, and a light chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% -th, 99% or 100% identity with the light chain as shown in SEQ ID NO: 42, 49, 50, 51, 52 or 53;

(d) a heavy chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the heavy chain as shown in SEQ ID NO: 54, 56, 57, 58, 59, 60, 61, 62, or 63, and light chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or 100% identity with the light chain as shown in SEQ ID NO: 55, 64, 65 or 66;

(e) a heavy chain as shown in SEQ ID NO: 41, 43, 44, 45, 46, 47 or 48, and a light chain as shown in SEQ ID NO: 42, 49, 50, 51, 52 or 53; or

(f) a heavy chain as shown in SEQ ID NO: 54, 56, 57, 58, 59, 60, 61, 62 or 63, and a light chain as shown in SEQ ID NO: 55, 64, 65 or 66.

In some embodiments, an anti-CTGF antibody comprises: (e) a heavy chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95% -th, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 86, 88, 89, 90, 91, 92, 93, 94, 95, 96 or 97, and a light chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 87, 98, 99, 100 or 101; or

(h) a heavy chain as shown in SEQ ID NO: 86, 88, 89, 90, 91, 92, 93, 94, 95, 96 or 97, and a light chain as shown in SEQ ID NO: 87, 98, 99, 100 or 101.

In some embodiments, an anti-CTGF antibody comprises: (j) a heavy chain, as shown in SEQ ID NO: 46, and a light chain, as shown in SEQ ID NO: 49;

(k) a heavy chain as shown in SEQ ID NO: 61 and a light chain as shown in SEQ ID NO: 64; or

(l) a heavy chain as shown in SEQ ID NO: 97, and a light chain as shown in SEQ ID NO: 98.

In other aspects of the present disclosure, an anti-CTGF antibody is provided that competes with an anti-CTGF antibody or antigen-binding fragments thereof, as described above, for binding to human CTGF.

In other aspects of the present disclosure, a nucleic acid molecule encoding an anti-CTGF antibody is provided, as described above.

In other aspects of the present disclosure, a host cell is provided comprising a nucleic acid molecule as described above.

In other aspects, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount or a prophylactically effective amount of an anti-CTGF antibody, as described above, or a nucleic acid molecule, as described above, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

In certain specific embodiments, a therapeutically effective amount or a prophylactically effective amount means that the dosage unit dose of the composition contains 0.1-3000 mg or 1-1000 mg of anti-CTGF antibody as described above.

In other aspects of the present disclosure, a method of immunoassaying or determining CTGF is provided, including the step of using an anti-CTGF antibody as described above.

In other aspects of the present disclosure, a method of immunoassaying or determining CTGF is provided, comprising the step of contacting a subject or a sample thereof with an anti-CTGF antibody as described above.

In other aspects of the present disclosure, a kit is provided containing an anti-CTGF antibody as described above.

In other aspects of the present disclosure, there is provided a method of treating diseases associated with CTGF, comprising administering to a subject a therapeutically effective amount of an anti-CTGF antibody, as described above, or a nucleic acid molecule, as described above, or pharmaceutical compositions, as described above, wherein the disease preferably is fibrotic disease (the fibrotic disease is preferably spontaneous pulmonary fibrosis, diabetic nephropathy, diabetic retinopathy, osteoarthritis, scleroderma, chronic heart failure, liver cirrhosis or renal fibrosis), hypertension, diabetes, myocardial infarction, arthritis, CTGF-related cell proliferative disease, atherosclerosis, glaucoma or cancer (the cancer is preferably acute lymphoblastic leukemia, dermatofibroma, breast cancer, angiolipoma, angioleiomyoma, connective tissue cancer, prostate cancer, ovarian cancer, colon and rectal cancer, prostate cancer, gastrointestinal cancer -intestinal tract or liver cancer).

Other aspects of the present disclosure provide the use of an anti-CTGF antibody as described above, or a nucleic acid molecule as described above, or a pharmaceutical composition as described above, in the preparation of a medicament for the treatment of a CTGF-associated disease, a CTGF-associated disease, includes fibrotic disease (fibrotic disease is preferably spontaneous pulmonary fibrosis, diabetic nephropathy, diabetic retinopathy, osteoarthritis, scleroderma, chronic heart failure, cirrhosis of the liver or renal fibrosis), hypertension, diabetes, myocardial infarction, arthritis, cell proliferative disease associated with CTGF , atherosclerosis, glaucoma or cancer (the cancer is preferably acute lymphoblastic leukemia, dermatofibroma, breast cancer, angiolipoma, angioleiomyoma, connective tissue cancer, prostate cancer, ovarian cancer, colon and rectal cancer, prostate cancer, cancer gastrointestinal tract or liver cancer).

In other aspects, the present disclosure provides an anti-CTGF antibody, as described above, or a nucleic acid molecule encoding an anti-CTGF antibody, as described above, or a pharmaceutical composition, as described above, for use in the treatment of a CTGF-related disease, a disease associated with CTGF, includes fibrotic disease (fibrotic disease is preferably spontaneous pulmonary fibrosis, diabetic nephropathy, diabetic retinopathy, osteoarthritis, scleroderma, chronic heart failure, cirrhosis or renal fibrosis), hypertension, diabetes, myocardial infarction, arthritis, cell proliferative disease, related with CTGF, atherosclerosis, glaucoma or cancer (the cancer is preferably acute lymphoblastic leukemia, dermatofibroma, breast cancer, angiolipoma, angioleiomyoma, connective tissue-derived cancer, prostate cancer, ovarian cancer, colon and rectal cancer, prostate cancer , gastrointestinal cancer or liver cancer).

DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1: Affinity of a humanized mab164-derived antibody for human CTGF as determined by ELISA (enzyme-linked immunosorbent assay).

Fig. 2: Experimental results of a humanized antibody derived from mab147 or mab164 in inhibiting the SU86.86 mouse xenograft tumor.

DETAILED DESCRIPTION OF THE INVENTION

Terminology

To make the present disclosure easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which this disclosure pertains.

The three-letter codes and single-letter codes for amino acids used in the present disclosure are those described in J. Biol. Chem, 243, p3558 (1968).

Connective tissue growth factor (CTGF) is a 36 kDa cysteine-rich heparin-binding secreted glycoprotein that is originally isolated from human umbilical vein endothelial cells (see, for example, Bradham et al. (1991) J Cell BioM 14: 1285- 1294; Grotendorst and Bradham, US Patent No. 5408040). CTGF belongs to the CCN family of proteins (CTGF, Cyr61, Nov) (secreted glycoproteins), and this family includes the serum-induced immediate early gene product Cyr61, the putative Nov oncogene, the ECM-associated protein FISP-12, the src-inducible gene CEF-10, Wnt-induced secreted protein WISP-3 and antiproliferative protein HICP/rCOP (Brigstock (1999) Endocr Rev 20: 189-206; O'Brian et al. (1990) Mol Cell Biol 10: 3569-3577; Joliot et al. ( 1992) Mol Cell Biol 12: 10-21; Ryseck et al. (1990) Cell Growth and Diff 2: 225-233; Proc Natl Acad Sci USA 86: 1178-1182; (1998) Proc Natl Acad Sci USA, 95: 14717-14722; and Zhang et at. (1998) Mol Cell Biol 18: 6131-6141). The CCN protein is characterized by a conserved 38 cysteine residues. 38 Cysteine residues account for more than 10% of the total amino acid content and form a modular structure with N-terminal and C-terminal domains. The modular structure of CTGF includes conserved motifs for insulin-like growth factor binding protein (IGF-BP) and von Willebrand factor (VWC) in the N-terminal domain and thrombospondin (TSP1 - from the English thrombospondin), and a cysteine motif in the C-terminal domain.

As used herein, the term "antibody" refers to an immunoglobulin, and a full-length antibody is a structure of four peptide chains linked together by interchain disulfide bond(s) between two identical heavy chains and two identical light chains. Immunoglobulin heavy chain constant regions exhibit different amino acid compositions and arrangements and therefore present different antigenicity. Accordingly, immunoglobulins can be divided into five types, or called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, and the corresponding heavy chains are μ, δ, γ, α and ε, respectively. According to its amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds, the same type of Ig can be divided into different subtypes; for example, IgG can be divided into IgG1, IgG2, IgG3 and IgG4. Light chains can be subdivided into κ or λ chain, taking into account different constant regions. Each of the five types of IgG can have a kappa chain or a lambda chain.

The approximately 110 amino acid sequences near the N-terminus of the heavy and light chains of an antibody are highly variable, known as variable regions (Fv regions); the remaining amino acid sequences close to the C-terminus are relatively stable, known as constant regions. The variable region includes 3 hypervariable regions (HVR - from the English hypervariable region) and 4 frame regions (FR - from the English framework region) with relatively conservative sequences. These three hypervariable regions, which determine the specificity of the antibody, are also known as complementarity determining regions (CDRs). Each light chain variable region (VL) and each heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. These three light chain CDR regions are LCDR1, LCDR2 and LCDR3; and these three heavy chain CDR regions are HCDR1, HCDR2 and HCDR3.

Antibodies of the present disclosure include murine antibodies, chimeric antibodies and humanized antibodies.

As used herein, the term “mouse antibody” refers to monopoly antibodies against human CTGF prepared in accordance with knowledge and skill in the art. During production, the test subject is injected with CTGF antigen, and a hybridoma expressing an antibody that has the desired sequence or functional characteristics is then isolated. In a preferred embodiment of the present disclosure, the mouse anti-CTGF antibody or antigen binding fragments thereof may further comprise a mouse kappa, lambda light chain constant region, or variants thereof, or further comprise a mouse IgG1, IgG2, IgG3 heavy chain constant region, or variants thereof.

The term "chimeric antibody" is an antibody obtained by fusing the variable region of a mouse antibody with the constant region of a human antibody, and such antibody can mitigate the immune response caused by the mouse antibody. To create a chimeric antibody, a hybridoma secreting a specific mouse monoclonal antibody is first created, and the variable region gene is cloned from the mouse hybridoma. The constant region gene is then cloned from the human antibody if necessary. A mouse variable region gene is fused to a human constant region gene to form a chimeric gene that can subsequently be inserted into an expression vector. Finally, the chimeric antibody molecule will be expressed in a eukaryotic or prokaryotic system. In a preferred embodiment of the present disclosure, the antibody light chain of the chimeric antibody further comprises a kap light chain constant region, human lambda, or variants thereof. The antibody heavy chain of the chimeric anti-CTGF antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, preferably comprising a heavy chain constant region of human IgG1, IgG2 or IgG4 or variant IgG1, IgG2 or IgG4 with mutation(s) amino acids (such as the L234A and/or L235A mutation, and/or the S228P mutation).

The term “humanized antibody,” also known as a CDR-grafted antibody, refers to an antibody created by grafting murine CDR sequences into human antibody variable region frameworks, namely, an antibody produced in different types of human germline antibody framework sequences. The humanized antibody can overcome the heterologous responses caused by a chimeric antibody that carries a large number of murine protein components. Such scaffold sequences can be obtained from a publicly available DNA database covering germline antibody gene sequences or published references. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the human germline sequence database "VBase" (available at www.mrccpe.com.ac.uk/vbase), as well as in Kabat, EA, et al. 1991 Sequences of Proteins of Immunological Interest, 5th Ed. In order to avoid the reduction in activity caused by reduced immunogenicity, framework sequences in the variable region of a human antibody may undergo minimal back mutation(s) or back mutation(s) to maintain or increase activity. Humanized antibodies of the present disclosure also include humanized antibodies subjected to CDR affinity maturation via yeast display.

Due to the residues in contact with the antigens, CDR grafting may result in a decrease in the affinity of the antibody or its antigen-binding fragment for the antigen due to changes in the framework residues with which the antigen is in contact. Such interactions may result from highly somatic mutations. Thus, it may still be necessary to graft such donor scaffold amino acids onto humanized antibody scaffolds. Amino acid residues involved in antigen binding and derived from a non-human antibody or an antigen-binding fragment thereof can be identified by examining the sequence and structure of the variable region of the animal monoclonal antibody. Amino acid residues of the donor CDR scaffold that differ from the germ lines can be considered related. If the most closely related germline cannot be determined, the sequence can be compared to a common sequence shared among subtypes or to an antibody sequence from an animal with a high percentage of similarity. Rare residues in the framework are thought to result from high mutation rates in somatic cells and play an important role in binding.

In one embodiment of the present disclosure, the antibody or antigen binding fragment thereof further comprises a light chain constant region derived from a human or murine k, A chain, or a variant thereof, or further comprises a heavy chain constant region derived from human or murine IgG1, IgG2, IgG3, IgG4, or its variant; it preferably includes a heavy chain constant region derived from human IgG1, IgG2 or IgG4, or a variant of IgG1, IgG2 or IgG4 with amino acid mutation(s) (such as the L234A and/or L235A mutation, and/or the S228P mutation) .

As used herein, "conventional variants" of the human antibody heavy chain constant region and the human antibody light chain constant region refer to variants of the human heavy and light chain constant region disclosed in the prior art that do not change the structure and the function of antibody variable regions. Exemplary variants include heavy chain constant region variants of IgG1, IgG2, IgG3, or IgG4 obtained through site-directed modification and amino acid substitutions on the heavy chain constant region. Specific substitutions are, for example, the YTE mutation, the L234A and/or L235A mutations, the S228P mutation, and/or mutations resulting in a knob-to-valley structure (which results in the antibody heavy chain having a knob-Fc combination and depression-Fc), etc. It turned out that these mutations impart new properties to the antibody without changing the function of the variable region of the antibody.

The terms “human antibody (HiMAb)”, “human-derived antibody”, “full-length human antibody” and “full human antibody” can be used interchangeably and could represent human-derived antibodies or genetically derived antibodies. a modified organism that is "engineered" by any method known in the art to produce specific human antibodies in response to antigen stimulation, and can be produced. In some technologies, elements of the human heavy and light chain loci are introduced into cell lines of organisms derived from embryonic stem cell lines, and the endogenous heavy and light chain loci in these cell lines are targeted and disrupted. The endogenous heavy and light chain target loci included in these cell lines are disrupted. Transgenic organisms can synthesize human antibodies specific for human antigens, and these organisms can be used to produce a hybridoma that secretes human antibodies. A human antibody may also be one in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources. Full length human antibodies can also be constructed using gene or chromosomal transfection techniques and phage display technology, or constructed from in vitro activated B cells, all of which are known in the art.

The terms “full-length antibody,” “whole antibody,” and “full antibody” are used interchangeably herein and refer to an antibody in substantially intact form that is distinct from the antigen-binding fragments defined below. The term specifically refers to antibodies that contain constant regions in the light and heavy chains.

"Antibody" as used herein includes "full-length antibodies" and antigen-binding fragments thereof.

In some embodiments, a full-length antibody of the present disclosure includes full-length antibodies formed by linking a light chain variable region to a light chain constant region, and linking a heavy chain variable region to a heavy chain constant region, as shown in the light and heavy chain combinations listed below in the Tables 1, 2 and 3. Those skilled in the art can select the light chain constant region and the heavy chain constant region from different antibody sources according to actual need, such as the human antibody-derived light chain constant region and the heavy chain constant region. At the same time, different combinations of the light and heavy chain variable regions described in Tables 1, 2 and 3 can form a single-chain variable fragment (scFv), Fab or other forms of antigen-binding fragment , containing scFv or Fab.

The term "antigen-binding fragment" or "functional fragment" refers to one or more antibody fragments that retain the ability to specifically bind an antigen (eg, CTGF). It has been shown that full-length antibody fragments can be used to achieve binding function to a specific antigen. Examples of binding fragments included in the term “antigen binding fragment” of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) fragment F(ab') ₂ , a bivalent fragment containing two Fab fragments connected by disulfide bridge(s) in the hinge region, (iii) fragment Fd, consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VH and VL domains of one arm of the antibody; (v) dsFv, a stable antigen-binding fragment formed by VH and VL through interchain disulfide bond(s); and (vi) a diabody, bispecific antibody and multispecific antibody containing fragments such as scFv, dsFv and Fab. In addition, the two domains, VL and VH domain, of the Fv fragment are encoded by two separate genes, however, they can be linked by a synthetic linker through the use of genetic engineering techniques to form a single protein chain in which a monovalent molecule is formed by combining the VL and VH domain ( called single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. Such single chain antibodies are also intended to be included within the term “antigen binding fragment” of an antibody. Such antibody fragments are prepared using conventional techniques known in the art and are screened for functional fragments using the same method as for the intact antibody. Antigen-binding sites can be obtained through genetic engineering technology or through enzymatic or chemical destruction of intact immunoglobulin. Antibodies can be in the form of different isotypes, for example, IgG antibodies (for example, subtype IgG 1, IgG2, IgG3 or IgG4), lgA1, lgA2, IgD, IgE or IgM.

A Fab is an antibody fragment produced by treating an IgG antibody molecule with papain (which cleaves the amino acid residue at position 224 of the H chain), and the antibody fragment has a molecular weight of approximately 50,000 and has antigen-binding activity in which approximately half of the N-terminal side of the H chain is the L chain is linked together by disulfide bond(s).

"F(ab') ₂ " is an antibody fragment with a molecular weight of approximately 100,000 and having antigen-binding activity, and is obtained by pepsin cleavage of the portion downstream of the two disulfide bonds in the hinge region of IgG. F(ab') ₂ contains two Fabs connected at the hinge region.

Fab' is an antibody fragment having a molecular weight of approximately 50,000 and having antigen-binding activity, which is obtained by cleavage of the disulfide bond in the hinge region of the above-mentioned F(ab') ₂ . The Fab' of the present disclosure can be obtained by treating F(ab') ₂ of the present disclosure, which specifically recognizes CTGF and binds to the amino acid sequences of the extracellular region or its three-dimensional structure, with a reducing agent such as dithiothreitol.

In addition, a Fab' can be produced by inserting DNA encoding a Fab' antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic expression vector for the Fab'.

The term "single chain antibody", "single chain Fv" or "scFv" refers to a molecule containing an antibody heavy chain variable domain (or region; VH) connected to an antibody light chain variable domain (or region; VL) via a linker. Such scFv molecules have the general structure NH ₂ -VL-linker-\/H-COOH or NH ₂ -\/H-linker-VL-COOH. A suitable linker in the prior art consists of the repeated amino acid sequence GGGGS or a variant thereof, for example a variant with 1-4 repeats (Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that may be used in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731; Choi et al. (2001), Eur. J. Immunol. 31:94-106; Hu et al. (1996), Cancer Res. 56:3055-3061; Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

A diabody is an antibody fragment in which the scFv or Fab is dimerized, and it is an antibody fragment that has bivalent antigen-binding activity. In bivalent antigen-binding activity, the two antigens may be the same or different.

Bispecific and multispecific antibodies refer to an antibody that can simultaneously bind to two or more antigens or antigenic determinants, including scFv or Fab fragments that can bind CTGF.

The diabody of the present disclosure can be produced by the following steps: obtaining cDNAs encoding the VH and VL of a monoclonal antibody of the present disclosure that specifically recognizes human CTGF and binds to the extracellular region or its three-dimensional structure, constructing the DNA encoding the scFv such that the length the linker peptide is 8 or fewer amino acid residues, inserting the DNA into a prokaryotic expression vector or eukaryotic expression vector and then introducing the expression vector into a prokaryotic or eukaryotic expression diabody.

dsFv is prepared by replacing one amino acid residue in each of VH and VL with a cysteine residue, and then joining the polypeptides with a disulfide bond between two cysteine residues. The amino acid residues to be replaced by a cysteine residue can be selected based on prediction of the three-dimensional structure of the antibody according to known methods (eg, Protein Engineering, 7, 697 (1994)).

Full-length antibodies or antigen-binding fragments thereof of the present disclosure can be produced by the following steps: preparation of cDNAs encoding an antibody of the present disclosure that specifically recognizes human CTGF and binds to the amino acid sequence of the extracellular region or three-dimensional structure thereof, construction of the DNA encoding the dsFv, insertion of the DNA into a prokaryotic expression vector or eukaryotic expression vector and then introducing the expression vector into a prokaryotic or eukaryotic dsFv expression vector.

The term "amino acid difference" or "amino acid mutation" refers to changes or mutations in an amino acid in a variant protein or polypeptide compared to the parent protein or polypeptide, including 1, 2, 3 or more insertions, deletions or substitutions of amino acids, based on the parent protein or polypeptide.

The term “antibody framework” or “FR region” refers to the portion of a variable domain, either VL or VH, that serves as a framework for the antigen binding loops (CDRs) of that variable domain. Essentially, it is a variable domain without a CDR.

The term “complementarity determining region,” “CDR,” or “hypervariable region” refers to one of the six hypervariable regions present in the variable domain of an antibody that primarily facilitate antigen binding. Typically, three CDRs (HCDR1, HCDR2, HCDR3) are found in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, LCDR3) are found in each light chain variable region. CDR amino acid sequence boundaries can be determined using any of a variety of well-known schemes, including the "Kabat" numbering criteria (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), "Chothia" numbering criteria (see Al-Lazikani et al., (1997) JMB 273:927-948) and ImMunoGenTics (IMGT) numbering criteria (Lefranc MP, Immunologist, 7, 132 - 136 (1999); Lefranc, MP, etc., Dev. Immunol., 27, 55-77 (2003)), etc. For example, in the case of the classical format, following the Kabat criteria, the amino acid residues of the CDR are in the variable the heavy chain (VH) domain is numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). According to Chothia criteria, the amino acid residues of the CDRs in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3). Combining both Kabat and Chothia to define CDRs, CDRs consist of amino acid residues 26–35 (HCDR1), 50–65 (HCDR2), and 95–102 (HCDR3) in human VH and amino acid residues 24–34 (LCDR1), 50 -56 (LCDR2) and 89-97 (LCDR3) in human VL. According to IMGT criteria, CDR amino acid residues in VH are approximately numbered 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and CDR amino acid residues in VL are approximately numbered 27-32 (CDR1), 50-52 (CDR2) and 89-97 (CDR3). According to IMGT criteria, antibody CDR regions can be identified using the IMGT/DomainGapAlign program. Unless otherwise noted, antibody variable regions and CDR sequences included in embodiments of the present disclosure are applicable to the Kabat numbering criteria.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (eg, a specific site on a CTGF molecule). Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-sequential amino acids in a unique tertiary conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol.66, ed. G. E. Morris, Ed. (1996).

The term “specifically binds to,” “selectively binds to,” “selectively binds to,” or “specifically binds to” refers to the binding of an antibody to a given epitope on an antigen. Typically, an antibody binds with an affinity (KD) of less than about 10 ^-8 M, such as less than about 10 ^-9 M, 10 ^-10 M, 10 ^-11 M, 10 ^-12 M, or even less.

The term "KD" refers to the dissociation equilibrium constant for a particular antibody-antigen interaction. Typically, the antibody of the present disclosure binds to CTGF with a dissociation constant (KD) of less than about 10 ^-7 M, for example, less than about 10 ^-8 M or 10 ^-9 M, for example, the affinity of the antibody of the present disclosure for a cell surface antigen determined by measuring the KD value using the FACS method (fluorescence-activated cell sorting).

When the term "competition" is used in the context of antigen-binding proteins (e.g., antigen-binding neutralizing proteins or neutralizing antibodies) that compete for the same epitope, it means that competition occurs between antigen-binding proteins as determined by assays in which the antigen-binding the protein to be tested (eg, an antibody or an immunologically functional fragment thereof) prevents or inhibits (eg, reduces) the specific binding of a reference antigen binding protein (eg, a ligand or a reference antibody) to a common antigen (eg, CTGF antigen or fragments thereof). Many types of competitive binding assays are available to determine whether one antigen binding protein competes with another. These tests are, for example, solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), competitive “sandwich” analysis (see, for example, Stahli et al, 1983, Methods in Enzymology 9: 242-253); solid-phase direct EIA with biotin-avidin (see, for example, Kirkland et al, 1986, J. Immunol. 137: 3614-3619); solid phase direct labeling assay, solid phase direct labeling sandwich assay (see, for example, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct RIA based on G ¹²⁵ labeling (see, for example, Morel et al, 1988, Molec. Immunol. 25: 7-15); solid-phase direct EIA based on biotin-avidin labeling (see, for example, Cheung, et al, 1990, Virology 176: 546-552); and direct labeling-based RIA (Moldenhauer et al, 1990, Scand. J. Immunol. 32: 77-82). Typically, the assay involves the use of purified antigen capable of binding to a solid surface or cell that is loaded with both an unlabeled test antigen binding protein and a labeled reference antigen binding protein. Competitive inhibition is determined by measuring the amount of label bound to a solid surface or cell in the presence of the antigen binding protein being tested. Typically, the antigen binding protein being tested is in excess. Antigen-binding proteins identified by competition analysis (competition with antigen-binding protein) include: antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein; and antigen-binding proteins that bind to an epitope that is sufficiently close to the epitope to which a reference antigen-binding protein binds, where the two epitopes interfere spatially with each other, preventing binding. Additional details regarding methods for determining competitive binding are provided herein in the Examples section. Typically, when a competing antigen binding protein is in excess, it will inhibit (e.g., reduce) by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70 %, 70-75% or 75% or even more specific binding of the reference antigen binding protein to the common antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97% or 97% or even more.

The term "nucleic acid molecule", as used herein, refers to DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, and is preferably double-stranded DNA or single-stranded mRNA or modified mRNA. A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it influences the transcription of that sequence.

The term "amino acid sequence identity" refers to the percentage of amino acid residues that are identical between the first and second sequences when the given amino acid sequences are aligned (introducing gaps as necessary) to achieve the maximum percentage of sequence identity, and any conservative substitution is not considered part of the sequence identity. For the purpose of determining percent amino acid sequence identity, alignment can be achieved in a variety of ways within a given region, for example, using publicly available software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine parameters suitable for measuring alignment, including any algorithm required to achieve optimal alignment over the entire length of the sequences being compared.

The term "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In one embodiment, the vector is a "plasmid" which refers to a loop of circular double-stranded DNA into which additional DNA segments can be ligated. In yet another embodiment, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome. The vectors disclosed herein are capable of self-replication in the host cell into which they are introduced (for example, bacterial vectors having a bacterial replicator and mammalian episomal vectors), or could be integrated into the genome of the host cell when introduced into the cell. host and are thus replicated along with the host genome (e.g. non-episomal mammalian vectors).

Methods for preparing and purifying antibodies and antigen-binding fragments thereof are well known in the art, for example, A Laboratory Manual for Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, chapters 5-8 and 15. For example, mice can be immunized with human CTGF or a fragment thereof, and the resulting antibodies can then be renatured, purified and sequenced for amino acid sequences using conventional methods well known in the art. Antigen binding fragments can also be prepared by conventional methods. Antibodies or antigen binding fragments thereof of the present disclosure are constructed by including one or more human framework regions in the CDR regions derived from a non-human antibody. Human germline FR sequences can be obtained from ImMunoGeneTics (IMGT) via their website http://imgt.cines.fr or from The Immunoglobulin Facts Book, 2001, ISBN 012441351, by alignment against germline antibody variable region gene database sequences human lines using MOE software.

The term "host cell" refers to the cell into which the expression vector has been introduced. Host cells may include bacterial, microbial, plant or animal cells. Bacteria that are easily transformed include members of Enterobacteriaceae, such as strains of Escherichia coil or Salmonella; Baciiiaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary), 293 cells and NS0 cells.

The engineered antibodies or antigen binding fragments of the present disclosure can be prepared and purified by conventional methods. For example, the cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector. Vectors expressing the recombinant immunoglobulin can then be stably transfected into CHO cells. As a more preferred method, well known in the art, mammalian expression systems can result in glycosylation of antibodies, especially at the highly conserved N-terminal regions in the Fc region. Stable clones can be obtained by expressing an antibody that specifically binds to human CTGF. Positive clones can be propagated in serum-free culture medium in bioreactors to produce antibodies. The culture medium into which the antibody has been secreted can be purified using conventional techniques. For example, purification can be performed on a Protein A or G Sepharose FF column that has been modified with buffer. Nonspecific binding components are washed away. The bound antibody is eluted using a pH gradient, and antibody fragments are detected by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and then combined. Antibodies can be filtered and concentrate using conventional techniques. Soluble mixtures and multimers can be effectively removed by conventional techniques such as gel filtration or ion exchange. Then the resulting product must be immediately frozen, for example at -70°C, or lyophilized.

The terms “administration,” “dosing,” and “treatment,” when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to bringing an exogenous pharmaceutical, therapeutic, diagnostic agent or composition into contact with the animal, human , subject, cell, tissue, organ or biological fluid. The terms "administration", "dosing" or "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of the cell includes bringing the reagent into contact with the cell, as well as bringing the reagent into contact with a liquid, where the liquid is in contact with the cell. The term “administration,” “dosing,” or “treatment” also means in vitro or ex vivo treatment of, for example, a cell, reagent, diagnostic, binding compound, or other cell. The term "treatment", when applied to a human subject, veterinary subject or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

The term “treating” means administering a therapeutic agent, such as a composition containing any of the binding compounds of the present disclosure, orally or externally to a patient having one or more symptoms of a disease for which the agent has known therapeutic activity. Typically, the agent is administered in an amount effective to relieve one or more symptoms of a disease in the patient or population being treated by causing regression or inhibiting the progression of such symptom(s) to any clinically measurable extent. The amount of a therapeutic agent that is effective in alleviating any particular symptom of a disease (also called a "therapeutically effective amount") may vary depending on various factors such as the pathological condition, the age and body weight of the patient, and the ability of the drug to produce the desired effect. patient's response. Whether a disease symptom has been alleviated can be assessed by any clinical measurement commonly used by treating physicians or other qualified health care professionals to assess the severity or progression status of that symptom. While one embodiment of the present disclosure (e.g., a treatment method or product of manufacture) may not be effective in alleviating the target disease symptom(s) in every patient, it should alleviate the target disease symptom(s) in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student's t test, the chi-square test, the Mann and Whitney U test, the Kruskal-Wallis test (H test), the Jonckheere-Terpstra test, and the Wilcoxon.

The term "conservative modification" or "conservative substitution or substitution" refers to the replacement of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), thus that these changes can often occur without changing the biological activity of the protein. Those skilled in the art will recognize that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not significantly alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., page 224, (4th edition)). In addition, substitutions with structurally or functionally similar amino acids are less likely to disrupt biological activity. Typical conservative substitutions are outlined below.

The term “effective amount” or “effective dose” refers to the amount of a drug, compound or pharmaceutical composition required to obtain any one or more beneficial or desired results. For prophylactic applications, beneficial or desirable results include eliminating or reducing the risk of, reducing the severity of, or delaying the onset of a disease, including the biological, histological, and behavioral manifestations of a pathological condition, its complications, and intermediate pathological phenotypes throughout the development of the condition. For therapeutic applications, useful or desirable results include clinical results, such as a reduction in the incidence of various conditions associated with the target antigen of the present disclosure, or an improvement in one or more symptoms of a condition, a reduction in the dosage/dose of other agents required to treat the pathological condition, enhancement the effectiveness of another agent and/or delaying the progression of a pathological condition associated with the target antigen of the present disclosure in patients.

The term "exogenous" refers to substances that are formed outside of organisms, cells, or humans, as the case may be. The term "endogenous" refers to substances that are produced in cells, organisms, or the human body, as the case may be.

The term "homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptide sequences. When a position in both of the two sequences being compared is occupied by the same monomeric subunit—a base or an amino acid—for example, when a position in each of two DNA molecules is occupied by an adenine, then the molecules are homologous at that position. The percentage homology of two sequences is a function of the number of matching or homologous positions that the two sequences have in common, divided by the number of positions being compared, and then multiplied by 100. For example, in an optimal alignment of two sequences, if 6 out of 10 positions in the two sequences match or are homologous, then these two sequences have 60% homology; if 95 out of 100 positions in two sequences are the same or homologous, then the two sequences have 95% homology. Typically, when two sequences are aligned, a comparison is made to obtain the maximum percentage of homology. For example, the comparison can be made using the BLAST algorithm, in which the algorithm parameters are selected to provide the maximum match between each sequence over the entire length of each reference sequence. The following references refer to the BLAST algorithm often used for sequence analysis: BLAST algorithm (BLAST ALGORITHMS): Altschul, S. F. et al., (1990) J. Mol. Biol. 215:403-410; Gish, W. et al., (1993) Nature Genet. 3:266-272; Madden, T. L. et al., (1996) Meth. Enzymol. 266:131-141; Altschul, S. Fetal., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J. et al. (1997) Genome Res. 7:649-656. Other conventional BLAST algorithms, such as those available from NCBI BLAST, are also well known to those skilled in the art.

As used herein, the expressions “cell,” “cell line,” and “cell culture” are used interchangeably, and all such designations include progeny. Thus, the terms “transformant” and “transformed cell” include the primary cells under study and the cultures derived from them, without regard to the number of transfers. It should also be understood that all offspring cannot be exactly identical in DNA content due to targeted or random mutations. Also included are progeny with mutations that have the same function or have the same biological activity as shown in the originally transformed cells. Where different designations are intended, this will be clearly understood from the context.

As used herein, the phrase “polymerase chain reaction” or “PCR” refers to a method or technique in which small amounts of a specific nucleic acid fragment, RNA and/or DNA, are amplified as described, for example, in US Patent No. 4683195. There is generally a need to have sequence information available at the ends or outside the region of interest so that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to the complementary strands of the template to be amplified. The 5' terminal nucleotides of these two primers may correspond to the ends of the material being amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA and cDNA transcribed from total cell RNA, bacteriophage or plasmid sequences, etc. Generally see Mullis et al. (1987) Cold Spring Harbor Symp.Ouant.Biol. 51:263; Erlich, ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). PCR as used in the present disclosure is considered one, but not the only, example of a polymerase reaction-based method for amplifying a nucleic acid sample to be analyzed. The method involves using nucleic acid sequences, known as primers, together with a nucleic acid polymerase to amplify or generate a specific nucleic acid fragment.

The term “isolated” refers to a purified state in which the designated molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates or other substances such as cell debris and growth media. In general, the term “isolated” is not intended to mean the complete absence of these substances or the absence of water, buffers or salts unless they are in an amount that significantly interferes with the experimental or therapeutic use of the compound as described herein.

The term "possible" or "perhaps" means that the event or situation that follows may occur, but does not necessarily occur, and the description includes examples in which the event or situation does or does not occur.

The term "pharmaceutical composition" refers to a mixture containing one or more compounds according to the present disclosure or a physiologically/pharmaceutically acceptable salt or prodrug thereof and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration into the body, facilitate absorption of the active substance and thereby produce a biological effect.

The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a composition for the delivery of antibodies or antigen-binding fragments. The carrier may be an antiadhesive, a binder, a coating agent, a disintegrating agent, a filler or diluent, a preservative (such as an antioxidant, antibacterial or antifungal agent), a sweetener, an anti-absorption agent, a humectant, an emulsifier, a buffer, and the like. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyol (such as glycerin, propylene glycol, polyethylene glycol, and the like), dextrose, vegetable oil (such as olive oil), saline, buffer, buffered saline, and isotonic agent (such as e.g. sugars, polyol, sorbitol and sodium chloride).

In addition, the present disclosure includes an agent for treating a disease associated with cells positive for a target antigen (such as CTGF), the agent comprising an anti-CTGF antibody or fragment thereof of the present disclosure as an active ingredient. The active ingredient is administered to a subject in a therapeutically effective amount and is capable of treating a disease associated with CTGF-positive cells in the subject. A therapeutically effective amount means that a single dose of the composition contains 0.1-3000 mg of a full-length antibody or antigen-binding fragment thereof that specifically binds to human CTGF as described above.

The CTGF-related disease of the present disclosure is not limited as long as it is a CTGF-related disease. For example, the therapeutic response induced by a molecule of the present disclosure may play a role by binding to human CTGF and then preventing CTGF from binding to its receptor/ligand or by killing tumor cells overexpressing CTGF. Thus, the molecules of the present disclosure, when in a preparation or composition suitable for therapeutic applications, are very useful for such people suffering from a tumor or cancer, possibly including pulmonary fibrosis, renal fibrosis, tumor development and growth, glaucoma, cell proliferative disease , cataract, choroidal neovascularization, retinal detachment, proliferative vitreoretinopathy, macular degeneration, diabetic retinopathy, corneal scarring, corneal opacification, cyst, reduced vascular calcification, pancreatic ductal adenocarcinoma, pancreatic cancer, melanoma, post-radiation fibrosis (RIF - from the English radiation- induced fibrosis), idiopathic pulmonary fibrosis, a disease based on lung remodeling, selected from the group consisting of asthma, chronic bronchitis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, emphysema, etc., preferably pancreatic cancer, pulmonary fibrosis and renal fibrosis.

In addition, the present disclosure relates to methods for immunoassaying or detecting target antigens (e.g., CTGF), reagents for immunoassaying or detecting target antigens (e.g., CTGF), methods for immunoassaying or detecting cells expressing target antigens (e.g., CTGF), and diagnostic agents for diagnosing diseases associated with a target antigen, for example, CTGF-positive cells, containing an antibody or antibody fragment of the present disclosure (as an active ingredient) that specifically recognizes target antigens (for example, human CTGF) and binds to the amino acid sequences of the extracellular region or to its tertiary structure.

In the present disclosure, the method for detecting or measuring the amount of a target antigen (eg, CTGF) may be any known method. For example, it includes immunoassay or detection methods.

An immunoassay or determination method is a method of detecting or measuring the amount of an antibody or antigen using a labeled antigen or antibody. Examples of immunoassay or detection methods include radiolabeled antibody (RIA) immunoassay, enzyme-linked immunosorbent assay (EIA or ELISA), fluorescence immunoassay (FIA), immunoluminescence assay, Western blotting, physical chemical method, etc.

The above-mentioned diseases associated with CTGF-positive cells can be diagnosed by detecting or measuring CTGF-expressing cells using antibodies or antibody fragments of the present disclosure.

Cells expressing the polypeptide can be detected by known immunodetection methods, preferably by immunoprecipitation, fluorescent cell staining, immunohistochemical staining, and the like. In addition, a method such as a fluorescent antibody staining method using the FMAT8100HTS system (Applied Biosystem) can be used.

In the present disclosure, samples to be detected or measured for a target antigen (e.g., CTGF) are not particularly limited as long as they may contain cells expressing the target antigen (e.g., CTGF), such as tissues cells, blood, plasma, serum, pancreatic secretions, urine, feces, tissue fluid or culture medium.

Depending on the required diagnostic method, a diagnostic agent containing a monoclonal antibody or antibody fragment of the present disclosure may also contain reagents for conducting an antigen-antibody reaction or reagents for detecting this reaction. Reagents for conducting the antigen-antibody reaction include buffers, salts, and the like. Reagents used for detection include those commonly used in immunoassay or immunodetection methods, for example, a labeled secondary antibody that recognizes a monoclonal antibody, antibody fragment, or conjugate thereof, and a substrate corresponding to the label.

Details of one or more embodiments of the present disclosure are set forth in the above description of the invention. Preferred methods and materials are described below, although any method or material similar or identical to the method or material described herein may be used in the practice or analysis of the present disclosure. With reference to the specification and claims, other features, objects and advantages of the present disclosure will become apparent. In the specification and claims, the singular forms are intended to include the plural forms unless the context clearly dictates otherwise. Unless otherwise expressly defined herein, all technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which this disclosure pertains. All patents and publications cited in the specification are incorporated by reference. The following examples are presented to more fully illustrate preferred embodiments of the present disclosure. These examples should not be construed as limiting in any way the scope of the present disclosure, and the scope of the present disclosure is determined by the claims.

EXAMPLES

Example 1: Preparation of CTGF Antigen and Antibody 1: Antigen Construction and Expression

Human CTGF protein (Genbank number: NM_001901.2) was used as a template to construct the amino acid sequence of the antigen, and the protein was used for detection. Optionally, the CTGF protein or fragment thereof was fused with different tags and separately cloned into the pTT5 vector or the pXC vector and transiently expressed in 293 cells or stably expressed in CHO LONZA cells to produce the antigens and proteins used to detect the present disclosure. The following CTGF antigens refer to human CTGF unless specifically stated otherwise.

Human CTGF Full Length Protein (for immunization), SEQ ID NO: 1

CTGF-his (his-tagged human mature CTGF protein fusion protein) can be used to detect SEQ ID NO: 2

(Note: the single underline represents the signal peptide, the italics represent the extracellular region of hCTGF, and the double underline represents the his tag.)

mCTGF-mFc (fusion protein of the coding region of mouse CTGF with a mouse Fc tag that can be used for detection) SEQ ID NO: 3

(Note: single underline represents signal peptide, italics represents mouse CTGF coding region, and double underline represents mFc tag.)

Mouse CTGF-His Tag Protein, SEQ ID NO: 4

(Note: the single underline represents the signal peptide, the italics represent the mouse CTGF coding region, and the double underline represents the his-tag).

Cynomolgus CTGF-Fc (fusion protein of the cynomolgus CTGF coding region with Fc that can be used for detection), SEQ ID NO: 5

(Note: single underline represents signal peptide, italics represents cynomolgus CTGF coding region, and double underline represents Fc tag.)

II. Purification of CTGF-bound recombinant protein and purification of hybridoma antibody against human CTGF and recombinant antibody

1. Isolation and purification of hybridoma supernatant by protein G affinity chromatography

For purification of mouse hybridoma supernatant, protein G affinity chromatography was preferred. The resulting hybridoma after cultivation was centrifuged, and the supernatant was taken out, and, based on the volume of the supernatant, a 10-15% volume of 1 M Tris-HCl (pH 8.0-8.5) was added to adjust the pH of the supernatant to neutral. The protein G column was washed with 3-5 ^x column volume of 6 M guanidine hydrochloride and then washed with 3-5 ^x column volume of pure water; the column was equilibrated with 3-5 ^x column volume 1×PBS (Phosphate buffered saline solution) (pH 7.4); the cell supernatant was loaded at a low flow rate for binding, and the flow rate was controlled such that the retention time was approximately 1 minute or longer; the column was washed with 3-5 ^x column volume of 1 x PBS (pH 7.4) until the UV absorbance dropped to the original level; samples were eluted with 0.1 M acetic acid/sodium acetate buffer (pH 3.0), and elution peaks were combined according to UV detection. The pH of the eluted product was quickly adjusted to pH 5-6 with 1 M Tris-HCl (pH 8.0). The product to be eluted may be subject to solution exchange in accordance with methods well known to those skilled in the art, for example, solution exchange with a desired buffer system through ultrafiltration-concentration using an ultrafiltration tube, or solution exchange with a desired buffer system through the use of molecular exclusion, such as desalting G -25, or removing aggregates from the eluted product to improve sample purity through the use of a molecular size exclusion column such as Superdex 200.

2. Antibody Purification by Protein A Affinity Chromatography

First, the supernatant of the cell culture expressing the antibody was centrifuged at high speed to collect the supernatant. The Protein A affinity chromatography column was washed with 3-5 ^x column volume of 6 M guanidine hydrochloride and then washed with 3-5 ^x column volume of pure water. The column was equilibrated with 3-5 ^x column volume, eg 1 x PBS (pH 7.4). The cell supernatant was loaded at a low flow rate for binding, and the flow rate was controlled such that the retention time was approximately 1 minute or longer; Immediately after completion of binding, the column was washed with 3-5 ^x column volume of 1 x PBS (pH 7.4) until the UV absorbance dropped to the original level. Samples were eluted with 0.1 M acetic acid/sodium acetate buffer (pH 3.0–3.5) and elution peaks were pooled according to UV detection. The pH of the eluted product was quickly adjusted with 1 M Tris-HCl (pH 8.0) to pH 5-6. The eluted product can be subjected to solvent exchange in accordance with methods well known to those skilled in the art, for example, solution exchange with the desired buffer system through ultrafiltration-concentration using an ultrafiltration tube, or solution exchange with the desired buffer system through the use of molecular exclusion, such as desalting G -25, or removing aggregates from the eluted product to improve sample purity through the use of a high resolution molecular size exclusion column such as a Superdex 200.

3. Purification of CTGF-bound recombinant proteins (such as human CTGF-his) via a nickel column

The cell expression supernatant sample was centrifuged at high speed to remove impurities, the buffer was replaced with PBS, and imidazole was added to a final concentration of 5 mM. The nickel column was equilibrated with PBS solution containing 5 mM imidazole and washed with 2-5 ^x column volume. The supernatant sample after replacement was loaded onto a binding column, and nickel columns available from different companies could be selected as media. The column was washed with PBS solution containing 5 mM imidazole until the measured value at A280 dropped to the original level. The chromatography column was washed with PBS plus 10 mM imidazole to remove nonspecifically bound protein impurities, and the effluent was collected. The target protein was eluted with PBS solution containing 300 mM imidazole, and the elution peak was collected.

The collected elution product was concentrated, and then it could be further purified through a Superdex200 size exclusion chromatography (GE) column using PBS as the mobile phase to remove protein impurity aggregates and peaks and to collect the elution peak of the target product. The resulting protein was identified through electrophoresis, peptide mapping and LC-MS (liquid chromatography-mass spectrometry), and the correct proteins were aliquoted for use.

4. Purification of CTGF-related recombinant proteins (such as human CTGF-Fc, cynomolgus CTGF-Fc) by protein A affinity chromatography

First, the culture supernatant was centrifuged at high speed to collect the supernatant. The Protein A affinity chromatography column was washed with 3-5 ^x column volume of 6 M guanidine hydrochloride and then washed with 3-5 ^x column volume of pure water. The column was equilibrated with 3-5 ^x column volume, eg 1 x PBS (pH 7.4). The cell culture supernatant was loaded at a low flow rate for binding, and the flow rate was controlled such that the retention time was approximately 1 minute or longer; Immediately after completion of binding, the column was washed with 3-5 ^x column volume of 1 x PBS (pH 7.4) until the UV absorbance dropped to the original level. Samples were eluted with 0.1 M acetic acid/sodium acetate buffer (pH 3.0–3.5) and elution peaks were pooled according to UV detection.

The collected elution product was concentrated, and it could then be further purified by Superdex200 size exclusion chromatography (GE) using PBS as the mobile phase to remove protein contaminant aggregates and peaks, and to collect the elution peak of the target product. The resulting protein was identified by electrophoresis, peptide mapping, and LC-MS, and the correct proteins were aliquoted for application.

Example 2: Preparation of anti-human monoclonal antibody

CTGF

I. Immunization

Monoclonal antibodies against human CTGF were obtained by immunization of mice. Female albino SJL mice aged 6–8 weeks (Beijing Charles River Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012-0001) were used for the experiment. Feeding environment: SPF level (from the English specific pathogen free - free from specific pathogenic microflora). After acquisition of mice, they were kept in laboratory conditions for 1 week, with a 12/12-hour light/dark cycle, at a temperature of 20-25°C; humidity 40-60%. The mice, which were adapted to the environment, were then immunized according to the following schedules. CTGF (R&D) and mCTGF-mFc were used as the antigen for immunization.

Immunization schedule: Mice were immunized with CTGF protein, 25 micrograms/mouse/time, by intraperitoneal injection. 100 μl/mouse was injected intraperitoneally (i.p.) on day 0, and then boosted once every 14 days. Blood samples were collected on days 21, 35, 49 and 63, and antibody titer in mouse serum was determined by ELISA method. After 7-9 immunizations, mice with high serum antibody titers reaching a plateau were selected for fusion with splenocytes. Three days before fusion with splenocytes, a solution of the CTGF protein antigen obtained in physiological solution was injected intraperitoneally (i.p.), 25 μg/mouse, for repeated immunization.

II. Fusion with splenocytes

Hybridoma cells were generated by fusing spleen lymphocytes with Sp2/0 myeloma cells (ATCC® CRL-8287™) using a PEG (polyethylene glycol)-mediated fusion method. Fusion hybridoma cells are resuspended in complete medium (IMDM medium containing 20% FBS (Fetal Bovine Serum), 1×HAT and 1×OP1) at a density of 0.5×10 ⁶ - 1×10 ⁶ /ml, seeded into a 96-well plate in an amount of 100 µl/well, incubated at 37°C and 5% CO ₂ for 3-4 days, supplemented with 100 µl/well of complete HAT medium and continued to be cultured for 3-4 days before the formation of precisely defined clones. The supernatant was removed, 200 μl/well of complete HT medium (IMDM medium containing 20% FBS, 1×HT and 1×ORI) was added, incubated at 37°C, 5% CO ₂ for 3 days and then subjected to ELISA. identification.

III. Screening for hybridoma cells

According to the growth density of the hybridoma cells, the hybridoma culture supernatant was detected by a binding ELISA method. Cell supernatant in positive wells was detected by ELISA assay for binding to monkey CTGF/mouse CTGF/human CTGF protein (specific methods were the same as those of Example 3). Cells in wells positive for protein binding ELISA were promptly expanded and cryopreserved; and subcloned 2 - 3 times to obtain a single cell clone.

Cells from each subcloning were also analyzed by CTGF binding ELISA. Hybridoma clones were obtained by screening using the above assay. Antibodies were additionally obtained by a serum-free cell culture method. Antibodies were purified according to the purification example for use in the assay examples.

IV. Sequencing of positive hybridoma clones

The methods for cloning sequences from a positive hybridoma were as follows. Hybridoma cells were harvested at logarithmic growth phase. RNA was isolated using Trizol (Invitrogen, cat. no. 15596-018) according to the kit instructions and reverse transcribed using the PrimeScript™ Reverse Transcription Kit (Takara, cat. no. 2680A). The cDNAs resulting from reverse transcription were amplified by PCR using a mouse Ig primer set (Novagen, TB326 Rev. B 0503), and the amplified products were sequenced. After sequencing, mouse anti-CTGF antibodies maM47, maM64 and mab95 were obtained. The amino acid sequences of the variable regions are as follows:

The amino acid sequence of VH mab147 is as shown in SEQ ID NO: 6,

The amino acid sequence of VL mab147 is as shown in EQ ID NO: 7,

The amino acid sequence of VH mab164 is as shown in SEQ ID NO: 8,

The amino acid sequence of VL mab164 is as shown in SEQ ID NO: 9,

The amino acid sequence of VH mab95 is as shown in SEQ ID NO: 69,

The amino acid sequence of VL mab95 is as shown in SEQ ID NO: 70,

In the above-mentioned antibody variable region sequences mab147, maM64 and mab95, italics represent the FR sequence, italics with underlining represent the CDR sequences, and the sequence order is as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

V. Preparation of chimeric human IgG1 antibody

The candidate molecules mab147, mab164 and mab95 were amplified and sequenced to obtain gene sequences encoding the variable regions. Forward and reverse primers were designed based on the sequences obtained by sequencing, the gene to be sequenced was used as a template to construct a VH/VK gene fragment for each antibody by PCR and then inserted into the pHr expression vector (with signal peptide and constant region gene fragment hIgG1/hkappa/hlambda (CH1-Fc/CL)) through homologous recombination to construct an expression plasmid for the full-length recombinant chimeric antibody VH-CH1-Fc-pHr/VL-CL-pHr, which resulted in the production of three chimeric antibodies Ch147, Ch164 and CH95.

VI. Humanization of Mouse Anti-Human CTGF Antibody Germline heavy chain and light chain variable region genes with high homology to mouse antibodies were selected as templates through alignment against the IMGT human germline antibody heavy and light chain variable region gene database (http:// imgt.cines.fr) through the use of MOE (Molecular Operating Environment) software. Mouse CDR antibodies were grafted onto the corresponding human templates to generate variable region sequences in the following order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

1. Humanization mab147

For the mouse antibody mab147, the light chain humanization templates were IGKV3-1T01 and IGKJ2*01, or IGKV1-39*01 and IGKJ2*01, and the heavy chain humanization templates were IGHV3-72*01 and IGHJ1×01. Each of the CDRs of the murine mab147 antibody was grafted onto its human template, and the FR amino acids of the humanized antibody were then reverse mutated. Among them, the back mutation(s) in the light chain variable region includes one or more selected from the group consisting of 4L, 36F, 43S, 45K, 47VV, 58V or 71Y, and back mutation(s) (i) in the heavy chain variable region, comprise(s) one or more selected from the group consisting of 28S, 30N, 49A, 75E, 76S, 93V, 94E, or 104D (back mutation positions in the light and heavy chain variable regions were determined in according to Kabat numbering criteria), resulting in light chain variable regions and heavy chain variable regions with different sequences. Humanized antibodies containing the mab147 CDR were produced, and the sequences of their variable regions are as follows:

The specific sequences of the variable regions of humanized tAM47 antibodies are as follows:

The amino acid sequence of Hu147-VL1 is as shown in SEQ ID NO: 22

The amino acid sequence of Hu147-VL2 is as shown in SEQ ID NO: 23

The amino acid sequence of Hu147-VL3 is as shown in SEQ ID NO: 24

The amino acid sequence of Hu147-VL4 is as shown in SEQ ID NO: 25

The amino acid sequence of Hu147-VL5 is as shown in SEQ ID NO: 26

The amino acid sequence of Hu147-VH1 is as shown in SEQ ID NO: 27

The amino acid sequence of Hu147-VH2 is as shown in SEQ ID NO: 28

The amino acid sequence of Hu147-VH3 is as shown in SEQ ID NO: 29

Note: CDR sequences determined according to Kabat criteria are underlined and the sequence data are arranged in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

2. Humanization of mab164

For the mouse antibody mab164, the light chain humanization templates consisted of IGLV7-43*01 and IGLJ2*01, IGLV8-61×01 and IGLJ2*01, or IGLV1-40*02 and IGLJ2*01, respectively, and the heavy chain humanization template consisted of IGHV2-26*01 and IGHJ6*01 or IGHV4-31×02 and IGHJ6*01, respectively. Each of the CDRs of the murine mab164 antibody was grafted onto its human template, and the FR amino acids of the humanized antibody were then reverse mutated. Among them, the back mutation(s) in the variable region of the light chain includes one or more selected from the group consisting of 36V, 44F, 46G or 49G, and the back mutation(s) in the variable region heavy chain comprise(s) one or more selected from the group consisting of 44G, 49G, 27F, 48L, 67L, 71K, 78V, or 80F (back mutation positions in the light and heavy chain variable regions were determined according to Kabat numbering criteria ), resulting in humanized mab164 heavy and light chains with variable region sequences that look like this:

The specific sequences of the variable regions of humanized tAM64 antibodies are as follows:

The amino acid sequence of Hu164-VL7 is as shown in SEQ ID NO: 30

The amino acid sequence of Hu164-VL8 is as shown in SEQ ID NO: 31

The amino acid sequence of Hu164-VL9 is as shown in SEQ ID NO: 32

The amino acid sequence of Hu164-VH5 is as shown in SEQ ID NO: 33

The amino acid sequence of Hu164-VH6 is as shown in SEQ ID NO: 34

The amino acid sequence of Hu164-VH7 is as shown in SEQ ID NO: 35

The amino acid sequence of Hu164-VH8 is as shown in SEQ ID NO: 36

Note: In the above-mentioned Hi164 antibody sequences, the underline represents the CDR sequence, and the sequence order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where the CDR sequences are defined according to Kabat criteria.

3. Humanization of mab95

For the mouse antibody mab95, the light chain humanization templates were IGKV3-20*02 and IGKV1-40*01, and the heavy chain humanization template was IGHV1-3*01. Each of the CDRs of the murine mab95 antibody was grafted onto its human template, and the FR amino acids of the humanized antibody were then reverse mutated. Among them, the back mutation(s) in the light chain variable region includes one or more selected from the group consisting of 45P, 46W, 48Y, 69S or 70Y, and back mutation(s) in heavy chain variable region comprise(s) one or more selected from the group consisting of 27F, 38K, 48I, 67K, 68A, 70L or 72F (back mutation positions in the light and heavy chain variable regions were determined according to Kabat numbering criteria) , resulting in light chain variable regions and heavy chain variable regions with different sequences. Humanized antibodies containing the mab95 CDR were produced, and the sequences of their variable regions are as follows:

The specific sequences of the variable regions of the humanized mAb95 antibodies are as follows:

The amino acid sequence of Hu95-VL1 is as shown in SEQ ID NO: 77

The amino acid sequence of Hu95-VL2 is as shown in SEQ ID NO: 78

The amino acid sequence of Hu95-VL3 is as shown in SEQ ID NO: 79

The amino acid sequence of Hu95-VL4 is as shown in SEQ ID NO: 80

The amino acid sequence of Hu95-VH1 is as shown in SEQ ID NO: 81

The amino acid sequence of Hu95-VH2 is as shown in SEQ ID NO: 82

The amino acid sequence of Hu95-VH3 is as shown in SEQ ID NO: 83

The amino acid sequence of Hu95-VH4 is as shown in SEQ ID NO: 84

The amino acid sequence of Hu95-VH5 is as shown in SEQ ID NO: 85

Note: CDR sequences determined according to Kabat criteria are underlined and the sequences are arranged in the following order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Among them, the CDR regions of Hu95-VH5 are further modified, where HCDR1 is as shown in SEQ ID NO: 102 (NYAIH), HCDR2 is as shown in SEQ ID NO: 103 (LVYPYTGGTAYNQKFKD), and HCDR3 is such as shown in SEQ ID NO: 104 (WGMIPGTNSYFDV).

4. Construction and expression of a humanized antibody against

CTGF

Primers were designed, the VH/VL gene fragment of each humanized antibody was constructed by PCR and then inserted into the pHr expression vector (with the signal peptide and constant region gene fragment (CH/CL)) through homologous recombination to construct the expression vector for the full-length VH-CH - antibody. pHr/VL-CL-pHr. For a humanized antibody, the constant region may be selected from the group consisting of a human K, X light chain constant region, and may be selected from the group consisting of an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, or a variant thereof. Non-limiting examples include optimizing the constant region of human IgG1, IgG2 or IgG4 to improve antibody function. For example, the half-life of an antibody can be prolonged by point mutations in the constant region, such as mutations at the M252Y/S254T/T256E (YTE) site and mutations such as S228P, F234A and L235A in the constant region.

An exemplary humanized anti-CTGF antibody constant region sequence is as follows:

The amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 37 (the heavy chain of a full-length antibody is named with the suffix: w):

The amino acid sequence of the heavy chain constant region of the engineered YTE antibody is as shown in SEQ ID NO: 38 (the full-length antibody heavy chain is named with the suffix: y):

The amino acid sequence of the kappa light chain constant region of the humanized anti-CTGF antibody is as shown in SEQ ID NO: 39: (the heavy chain of the full-length antibody is named with the suffix: k):

The amino acid sequence of the lambda light chain constant region of the humanized anti-CTGF antibody is as shown in SEQ ID NO: 40: (the heavy chain of the full-length antibody is named with the suffix: l):

The heavy chain variable region of the humanized antibody was linked to the heavy chain constant region as shown in SEQ ID NO: 37, and the light chain variable region of the humanized antibody was linked to the kappa light chain constant region as shown in SEQ ID NO: 39, resulting in to obtain a full-length humanized antibody derived from mab147, including:

The humanized antibody heavy chain variable region was linked to a YTE variant of the heavy chain constant region as shown in SEQ ID NO: 38, and the humanized antibody light chain variable region was linked to the kappa light chain constant region as shown in SEQ ID NO: 39. resulting in a full-length humanized antibody derived from mab147, including:

The heavy chain variable region of the humanized antibody was linked to the heavy chain constant region as shown in SEQ ID NO: 37, and the light chain variable region of the humanized antibody was linked to the lambda light chain constant region as shown in SEQ ID NO: 40, resulting in to obtain a full-length humanized antibody derived from mab164, including:

The humanized antibody heavy chain variable region was linked to a YTE variant of the heavy chain constant region as shown in SEQ ID NO: 38, and the humanized antibody light chain variable region was linked to the lambda light chain constant region as shown in SEQ ID NO: 40. resulting in a full-length humanized antibody derived from mab164, including:

The heavy chain variable region of the humanized antibody was linked to the heavy chain constant region as shown in SEQ ID NO: 37, and the light chain variable region of the humanized antibody was linked to the kappa light chain constant region as shown in SEQ ID NO: 39, resulting in to obtain a full-length humanized antibody derived from mab95, including:

The humanized antibody heavy chain variable region was linked to the heavy chain constant region YTE variant as shown in SEQ ID NO: 38, and the humanized antibody light chain variable region was linked to the kappa light chain constant region as shown in SEQ ID NO: 39. resulting in a full-length humanized antibody derived from mab95, including:

Exemplary amino acid sequences of a full-length anti-CTGF antibody are as follows:

The sequences of the anti-CTGF positive control antibody gpAM (from CN1829740 B, with the antibody name pamrevlumab, also known as FG-3019) are as follows:

Heavy Chain: (SEQ ID NO: 67)

Light chain: (SEQ ID NO: 68)

Example 3 Detection of Anti-CTGF Antibody Binding Activity I. Anti-CTGF Antibody Binding ELISA to Human CTGF Protein

The binding activity of anti-human CTGF antibody to human CTGF protein was analyzed by ELISA and Biacore assays. A 96-well microtiter plate was directly coated with recombinant CTGF protein. The signal intensity upon addition of the antibody was used to determine the binding activity of the antibody to CTGF. The specific experimental methods were as follows:

CTGF protein (R&D, cat. no. 9190-CC) was diluted with PBS buffer, pH 7.4 (Shanghai Yuanpei, cat. no. B320), to a concentration of 0.2 μg/ml, of which 50 μl/well was added to 96- well microtiter plate (Corning, cat. no. CLS3590-100EA), and incubated in an incubator at 37°C for 2 hours or at 4°C overnight (16-18 hours). The liquid was removed, 250 μl/well of blocking solution (5% skim milk (BD Skim Milk, cat. no. 232100) diluted in PBS) was added and incubated in an incubator at 37°C for 3 hours or at 4°C for overnight (16-18 hours) for blocking. After blocking was completed, the blocking solution was removed, the plate was washed 5 times with PBST buffer (PBS containing 0.1% Tween-20, pH 7.4), and 50 μl/well of different concentrations of each of the tested antibodies (antibodies purified from hybridoma or humanized antibodies) diluted with sample dilution solution and incubated in an incubator at 37°C for 1 hour. After incubation, the plate was washed 3 times with PBST, and 50 μl/well of a secondary goat anti-mouse antibody labeled HRP (horseradish peroxidase) (Jackson Immuno Research, cat. No. 109-035-003), or a secondary goat anti-human antibody (Jackson Immuno Research, cat. #109-035-003) diluted in sample dilution solution and incubated at 37°C for 1 hour. The plate was washed 3 times with PBST, 50 µl/well of the chromogenic substrate TMB (tetramethylbenzidine) (KPL, cat. No. 52-00-03) was added, incubated at room temperature for 5-10 min and 50 µl/well 1 M H ₂ S0 ₄ was added to stop the reaction. The absorbance value was read using a microplate reader (Molecular Devices, VERSA max) at a wavelength of 450 nm, and the data was analyzed using GraphPad Prism 5. The binding of anti-CTGF antibody to human CTGF protein was calculated as the EC ₅₀ value (half maximal effective concentration - half-maximal effective concentration). The experimental results are shown in Fig. 1 and Table 12.

The experimental results show that all humanized full length anti-CTGF antibodies of the present disclosure have excellent binding activity against human CTGF protein.

II. ELISA analysis of binding of anti-CTGF antibody to cynomolgus CTGF protein

The binding activity of the anti-monkey CTGF antibody to the monkey CTGF protein was analyzed by ELISA assay. A 96-well microtiter plate was directly coated with anti-mFc protein, and then the Fc-tagged cyno-CTGF protein was bound to the plate, and then the antibody was added. The signal intensity upon addition of the antibody was used to determine the binding activity of the antibody to cyno-CTGF. The specific methods of the experiment were as follows:

Anti-mFc protein (Sigma, cat. no. M4280) was diluted with PBS buffer, pH 7.4 (Shanghai Yuanpei, cat. no. B320) to a concentration of 2 μg/ml, which was added to a 96-well microtiter plate (Corning, cat. no. CLS3590 -100EA) in an amount of 50 μl/well, and incubated in an incubator at 37°C for 2 hours. The liquid was removed, 250 μl/well of blocking solution (5% skim milk (BD Skim Milk, cat. no. 232100) diluted in PBS) was added and incubated in an incubator at 37°C for 3 hours or at 4°C for overnight (16-18 hours) for blocking. After blocking was completed, the blocking solution was removed, the plate was washed 3 times with PBST buffer (PBS containing 0.1% Tween-20, pH 7.4), 50 μp of 1 μg/ml cyno-CTGF-mFc protein was added to each well, and incubated in incubator at 37°C for 1 hour, and then the plate was washed 3 times with PBST buffer (PBS containing 0.1% Tween-20, pH 7.4). 50 μl/well of different concentrations of each of the antibodies tested (hybridoma purified antibodies or humanized antibodies) diluted in sample dilution solution were added and incubated in an incubator at 37°C for 1 hour. After incubation, the plate was washed 3 times with PBST, and 50 μl/well of HRP-labeled goat anti-mouse secondary antibody (Jackson Immuno Research, cat. no. 109-035-003) or goat anti-human secondary antibody (Jackson Immuno Research, cat. No. 109-035-003), diluted with sample dilution solution, and incubated at 37°C for 1 hour. The plate was washed 5 times with PBST, 50 µl/well of TMB chromogenic substrate (KPL, cat. no. 52-00-03) was added, incubated at room temperature for 5-10 min, and 50 µl/well of 1 M H ₂ SO ₄ was added to stop the reaction. The absorbance value was read by a microplate reader (Molecular Devices, VERSA max) at a wavelength of 450 nm, and the data was analyzed by GraphPad Prism 5. The binding of anti-CTGF antibody to cyno-CTGF protein was calculated as an EC ₅₀ value. The experiment results are shown in Table 13.

The experimental data show that all of the humanized anti-CTGF antibodies of the present disclosure have excellent binding activity to the monkey CTGF protein.

III. ELISA assay for binding of anti-CTGF antibody to mouse protein

CTGF

The binding activity of anti-mouse CTGF antibody to mouse CTGF protein was analyzed by ELISA assay. A 96-well microtiter plate was directly coated with mouse CTGF fusion protein. The signal intensity upon addition of the antibody was used to determine the binding activity of the antibody to murine CTGF. The specific methods of the experiment were as follows:

Mouse CTGF-his protein was diluted with PBS buffer, pH 7.4 (Shanghai Yuanpei, cat. No. B320), to a concentration of 0.5 μg/ml, added to a 96-well microtiter plate (Corning, cat. No. CLS3590-100EA) in an amount of 50 μl/well and incubated in an incubator at 37°C for 2 hours. The liquid was removed, 250 μl/well of blocking solution (5% skim milk (BD Skim Milk, cat. no. 232100) diluted in PBS) was added and incubated in an incubator at 37°C for 3 hours or at 4°C for overnight (16-18 hours) for blocking. After blocking was completed, the blocking solution was removed, the plate was washed 3 times with PBST buffer (PBS containing 0.1% Tween-20, pH 7.4), 50 μl/well of different concentrations of each of the analyzed antibodies (antibodies purified from hybridoma, or humanized antibodies) diluted with sample dilution solution and incubated in an incubator at 37°C for 1 hour. After incubation, the plate was washed 3 times with PBST, and 50 μl/well of HRP-labeled goat anti-mouse secondary antibody (Jackson Immuno Research, cat. no. 109-035-003) or goat anti-human secondary antibody (Jackson Immuno Research, cat. No. 109-035-003), diluted with sample dilution solution, and incubated at 37°C for 1 hour. The plate was washed 5 times with PBST, 50 µl/well of TMB chromogenic substrate (KPL, cat. No. 52-00-03) was added, incubated at room temperature for 5-10 min and 50 µl/well of 1 M H ₂ SO ₄ was added to stopping the reaction. The absorbance value was read by a microplate reader (Molecular Devices, VERSA max) at a wavelength of 450 nm, and the data was analyzed by GraphPad Prism 5. The binding of anti-CTGF antibody to mouse CTGF protein was calculated as an EC ₅₀ value. The experiment results are shown in Table 14.

The experimental results show that all humanized full length anti-CTGF antibodies of the present disclosure have excellent binding activity against murine CTGF protein.

Example 4 Anti-CTGF Antibody Function-Blocking Assay

In this assay, the Biacore instrument was used to detect the activity of the humanized anti-CTGF antibody of interest in blocking the function of human TGF-(31.

First, human CTGF antigen was immobilized on the CM5 biosensor chip (Cat. No. BR-1005-30, GE) by binding amino groups with an immobilization level of 1500 RU, and then a high concentration of anti-CTGF antibody (150 μg/ml) was allowed to flow through the chip surface on for 150 s; 50 nM TGF-01 protein was injected over 120 s, and interaction signals were detected in real time using a Biacore T200 instrument to generate a binding and dissociation curve. After completion of dissociation in each experimental cycle, the biosensor chip was washed and regenerated with regeneration buffer.

The obtained experimental data were statistically analyzed using GE Biacore T200 software, trial version 3.0.

Blocking efficiency=[1-(sample response value/blank response value)]* 100%. Specific results are shown in Table 15.

The experimental results show that all humanized anti-CTGF antibodies of the present disclosure have high function-blocking activity.

Determination of Anti-CTGF Antibody Affinity by Biacore In this experiment, the Biacore instrument was used to determine the affinity of humanized anti-CTGF antibodies to be assayed by human CTGF and murine CTGF.

A specified amount of each antibody assay was affinity captured using a Protein A biosensor chip (Cat. No. 29127556, GE), and then a specified concentration of human or murine CTGF was allowed to flow through the surface of the chip. Interaction signals were detected in real time using the Biacore instrument to obtain a binding and dissociation curve. After completion of dissociation in each cycle, the biosensor chip was washed and regenerated with a glycine-HCl regenerating solution, pH 1.5 (Cat. No. BR-1003-54, GE). The running buffer for the assay was 1×HBS-EP buffer solution (Cat. No. BR-1001-88, GE).

The data obtained were fitted to a (1:1) Langmuir model using GE Biacore T200 software evaluation version 3.0, and affinity values were obtained as shown in more detail in Tables 16 and 17.

The experimental results show that all chimeric antibodies CM 47 and CM64 and humanized anti-CTGF antibodies derived from murine antibodies tAM47 and tAM64 can bind to human and murine CTGF with high affinity. Neither substitutions nor reverse mutation(s) found in the FR regions of a human germline antibody interfere with the affinity of the humanized antibody, and some modifications may even enhance the affinity of the antibody for the antigen.

Example 5 Anti-CTGF Antibody Assay for In Vitro Inhibition of TGFβ-Induced C2C12 Cell Migration

C2C12 cells (mouse myoblasts, Chinese Academy of Sciences Cell Bank, #GNM26) were digested with 0.25% trypsin (Gibco, #25200-072), centrifuged, and resuspended in serum-free DMEM (Gibco, #10564-029). The cell-antibody mixture and the TGFβl-antibody mixture were prepared with serum-free DMEM medium, in which the cell density was 2 * 10 ⁵ /ml, the concentration of recombinant human TGFβl (Cell signaling Technology, #8915LC) was 10 ng/ml, and the concentration of the antibody to be analysis was 30 µg/ml.

The lid and filter membrane of the ChemoTx ® Disposable Chemotaxis System (Neuro Probe, #106-8) were opened and the TGFβ antibody mixture was added to the lower chamber, 30 μl/well, 4 pairs of wells for each group. The filter membrane was coated and the cell-antibody mixture was added to the membrane, 50 μl/well, 4 pairs of wells for each group. The lid was closed and placed in an incubator (37°C, 5% CO ₂ ).

After 48 hours of incubation, the liquid on the filter membrane was removed with a clean paper towel, the filtration membrane was opened, 10 µL of pre-cooled 0.25% trypsin was added to each well in the lower chamber, and the filter membrane was covered. After 5 minutes of digestion, samples were centrifuged at 1000 rpm. within 1 minute. The filter membrane was opened, 20 µl of Cell Titer-Glo solution (Promega, #G7573) was added to each well in the lower chamber and incubated at room temperature for 10 minutes. The liquid was transferred to a 384-well white bottom plate (Thermo Scientific, #267462) and the plate was read using a microplate reader (BMG, #PheraStar) by chemiluminescence. Data were analyzed and processed using Graphpad Prism 6.

The experiment results are shown in Table 18.

The results show that the humanized anti-CTGF antibodies of the present disclosure can inhibit the in vitro migratory ability of C2C12 cells induced by TGFβ1 and exhibit a stronger inhibitory effect on the migratory ability of C2C12 cells compared to the migratory ability of C2C12 cells of the positive control antibody tAM.

Example 6 Assay demonstrating in vitro inhibitory effect of anti-CTGF antibody on TGFβ-induced PANC1 cell migration

PANC-1 cells (human pancreatic cancer cells, ATCC, #CRL-1469) were digested with 0.25% trypsin (Gibco, #25200-072), centrifuged and resuspended in DMEM (Gibco, #10564-029), containing 0.1% fetal bovine serum (Gibco, #10099-141). The cell-antibody mixture and the TGFβ-antibody mixture were prepared using DMEM containing 0.1% fetal calf serum, in which the cell density was 4×10 ⁵ /ml, the concentration of recombinant human TGFβ (Cell signaling Technology, #8915LC) was 10 ng /ml, and the concentration of the antibody to be analyzed was 30 μg/ml.

The lid and filter membrane of the ChemoTx® Disposable Chemotaxis System (Neuro Probe, #106-8) were opened and the TGFβ antibody mixture was added to the lower chamber, 30 μl/well, 4 pairs of wells for each group. The filter membrane was coated and the cell-antibody mixture was added to the membrane, 50 μl/well, 4 pairs of wells for each group. The lid was closed and placed in an incubator (37°C, 5% CO ₂ ).

After 48 hours of incubation, the liquid on the filter membrane was removed with a clean paper towel, the filter membrane was opened, 10 µl of pre-cooled 0.25% trypsin was added to each well in the lower chamber, and the filter membrane was covered. After 5 minutes of digestion, samples were centrifuged at 1000 rpm. within 1 minute. Cells that adhered to the bottom of the plate were counted under an inverted microscope (Leica, #DMIL LED). Data were analyzed and processed using Graphpad Prism 6. Inhibition rate = [(TGFβ mean - sample mean)/(TGFβ group mean - untreated group mean)]×100%

The experiment results are shown in Table 19.

The results show that all humanized anti-CTGF antibodies of the present disclosure can inhibit the in vitro migratory ability of PANC1 cells induced by TGFβl.

Example 7 Anti-CTGF Antibody In Vitro Inhibitory Effect on PANC1 Proliferation on Soft Agar

Deionized water was added to agar (BD, #214220) and heated to produce a 1.4% gel solution. 2*DMEM medium (Thermo, #12100046) was mixed with the gel solution in a 1:1 volume ratio, 500 µP was added to each well of a 24-well plate (Costar, #3524) and placed at 4 degrees to cure.

PANC-1 cells (human pancreatic cancer cells, ATCC, #CRL-1469) were digested with 0.25% trypsin (Gibco, #25200-072), centrifuged and adjusted to DMEM (GE, #SH30243.01) containing 4% fetal calf serum (Gibco, #10099-141) to obtain a cell density of 5x10 ⁴ cells/ml. 2 mg/ml antibody was prepared using 2xDMEM medium. Cells, antibody, and 1.4% gel solution were mixed in a 2:1:1 ratio by volume, and 200 μp was added to each well of a 24-well plate coated with the bottom layer of gel. After the top gel had solidified, 200 µl of DMEM containing 2% fetal bovine serum was added to a 24-well plate. After 28 days of incubation in an incubator (37°C, 5% _CO2 ), a high content assay system (Molecular Devices, ImageXpress) was used for imaging development, and the area of the resulting cell clone was analyzed.

Inhibition rate = (1 - sample mean/untreated group mean) x 100%. The experiment results are shown in Table 20.

The results show that humanized antibodies derived from clones tAM47 and tAM64 have a significant inhibitory effect on the proliferation of human pancreatic cancer cells (PANC-1 cells) in vitro.

Example 8: In Vivo Biological Activity of Anti-CTGF Antibodies in Animals

I. Analysis of anti-CTGF antibody for its inhibitory effect on bleomycin-induced mouse lung fibrosis

Experimental protocol: 40 mice were randomly divided into the following 4 groups according to body weight: normal control group (PBS, i.p., once every two days), gpAM group (10 mg/kg, i.p., once once every two days), Hu164-67wl group (10 mg/kg, i.p., once every two days) and Hu164-67yl group (mg/kg, i.p., once every two days). There are 10 animals in each group, and the specific division into groups is shown below in Table 21. On day 1 of the experiment, each of the solvents and antibodies were injected intraperitoneally according to body weight (10 ml/kg, normal control and reference groups were provided with PBS); after 2 hours, aerosolized bleomycin (50 mg/8 mL nebulized for 30 minutes and held for 5 minutes) was used to create the models. Then, the vehicle or antibody was injected intraperitoneally every other day according to the above groups. The experimental period was 21 days, with a total of 11 intraperitoneal administrations. On day 22 of the experiment, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (10 ml/kg) and then fixed on the operating table in a supine position. A 1 cm skin incision was made along the midline of the neck, and the lower end of the incision reached into the chest. Forceps were used to directly separate the subcutaneous connective tissues and muscles, exposing the trachea. The connective tissues on both sides of the trachea and between the trachea and esophagus were separated to isolate the free trachea. A needle was inserted into the vein and secured with sutures and ligation at the site where the cannula entered the trachea. A 1 ml syringe filled with 0.8 ml PBS was connected to the inlet end of a venous needle, PBS was slowly injected into the trachea, left in place for 30 seconds, and then PBS was slowly withdrawn. During the seizure, a milky foaming liquid could be observed. The washing was repeated three times, the washing liquid was transferred into EP tubes, and then 0.5 ml of PBS was withdrawn and the above steps were repeated. The two washing liquids were mixed and centrifuged at 1500 rpm. for 5 min, and the supernatant was stored at -20°C for analysis. The BALF supernatant was centrifuged again at 10,000 rpm. for 10 min, and the supernatant was collected to detect soluble collagen (kit: Biocolor, lot no. AA883).

Excel statistical analysis software was used: the mean was calculated as avg (Average); SD (Standard Deviation) value was calculated as STDEV (standard deviation); the SEM value (standard error of the mean; standard deviation of the sample mean) was calculated as STDEV/SQRT (square root) (number of animals in each group); GraphPad Prism software was used for plotting, two-way ANOVA (two-way analysis of variance) or one-way ANOVA (one-way analysis of variance) was used for statistical analysis of the data.

The experiment results are shown in Table 22.

The results show that Hu164-67wl or Hu164-67yl exhibited potent inhibitory effects on pulmonary fibrosis in mice.

II. Anti-CTGF antibody assay for inhibition of subcutaneously transplanted human pancreatic cancer cell tumor Su86.86

Experimental protocol: 200 μp of SU86.86 cells (ATCC, CRL-1837, 3.0×10 ⁶ cells) were inoculated subcutaneously into the right ribs of nude Nu/Nu mice. 11 days after inoculation, when the tumor volume was approximately 140 mm ³ , mice with too large or small body weight or tumor volume were excluded. The mice were randomly divided into 5 groups according to tumor volume, 10 mice/group, and administration was started on the same day. The antibody was injected intraperitoneally, twice a week, for a total of 18 days. Tumor volume and body weight were measured 1–2 times per week and data were recorded. The division into groups and dosage regimen are shown in Table 23.

Excel statistical analysis software was used: the mean was calculated as avg; SD value was calculated as STDEV; the SEM value was calculated as STDEV/SQRT (number of animals in each group); GraphPad Prism software was used for plotting, two-way ANOVA or one-way ANOVA was used for statistical analysis of data.

Tumor volume (V) was calculated according to the following formula:

Relative tumor proliferation rate T/C (%)=(T _t -T ₀ )/(C _t -C ₀ )×100, where T _t and C _t represent the tumor volumes of the treatment group and the control group at the end of the experiment; T ₀ and C ₀ represent the tumor volume at the beginning of the experiment.

Experiment results:

From D1 to D18, subcutaneous injection was performed twice a week, and the effect of anti-CTGF antibodies on inhibiting SU86.86 tumor growth was determined. The experiment results are shown in Table 24 and FIG. 2. Compared with the blank control group of the same human IgG isotype, the tumor inhibition rates of the tAM-40 mg/kg, Hu164-67yl-40 mg/kg, Hu164-67yl-20 mg/kg and Hu147-33wk (40 mg/kg) groups kg) were 45%, 53%, 47% and 54%, respectively. TAM-40 mg/kg, Hu164-67yl -40 mg/kg, HM64-67yl-20 mg/kg and Hu147-33wk (40 mg/kg) groups can significantly inhibit the growth of Su86.86 tumors (p<0.001), and The tumor inhibitory effect of SU86.86 is dose dependent. In addition, the body weight of mice in each group did not change significantly.

--->

LIST OF SEQUENCES

<110> JIANGSU HENGRUI MEDICINE CO., LTD.;

SHANGHAI HENGRUI PHARMACEUTICAL CO., LTD.

<120> ANTIBODY AGAINST CONNECTIVE TISSUE GROWTH FACTOR AND ITS APPLICATION

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Gln Asn Cys Ser Gly Pro Cys Arg Cys Pro Asp Glu Pro Ala Pro Arg

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

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Val Cys Ala Lys Gln Leu Gly Glu Leu Cys Thr Glu Arg Asp Pro Cys

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Asp Pro His Lys Gly Leu Phe Cys Asp Phe Gly Ser Pro Ala Asn Arg

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Lys Ile Gly Val Cys Thr Ala Lys Asp Gly Ala Pro Cys Ile Phe Gly

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

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Gln Cys Thr Cys Leu Asp Gly Ala Val Gly Cys Met Pro Leu Cys Ser

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Met Asp Val Arg Leu Pro Ser Pro Asp Cys Pro Phe Pro Arg Arg Val

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Lys Leu Pro Gly Lys Cys Cys Glu Glu Trp Val Cys Asp Glu Pro Lys

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Asp Gln Thr Val Val Gly Pro Ala Leu Ala Ala Tyr Arg Leu Glu Asp

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Thr Phe Gly Pro Asp Pro Thr Met Ile Arg Ala Asn Cys Leu Val Gln

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Thr Thr Glu Trp Ser Ala Cys Ser Lys Thr Cys Gly Met Gly Ile Ser

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

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Arg Leu Cys Met Val Arg Pro Cys Glu Ala Asp Leu Glu Glu Asn Ile

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Lys Lys Gly Lys Lys Cys Ile Arg Thr Pro Lys Ile Ser Lys Pro Ile

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Lys Phe Glu Leu Ser Gly Cys Thr Ser Met Lys Thr Tyr Arg Ala Lys

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Phe Cys Gly Val Cys Thr Asp Gly Arg Cys Cys Thr Pro His Arg Thr

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Thr Thr Leu Pro Val Glu Phe Lys Cys Pro Asp Gly Glu Val Met Lys

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Lys Asn Met Met Phe Ile Lys Thr Cys Ala Cys His Tyr Asn Cys Pro

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Val Gln Cys Gln Asn Cys Ser Gly Pro Cys Arg Cys Pro Asp Glu Pro

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

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Cys Cys Arg Val Cys Ala Lys Gln Leu Gly Glu Leu Cys Thr Glu Arg

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Asp Pro Cys Asp Pro His Lys Gly Leu Phe Cys Asp Phe Gly Ser Pro

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Ala Asn Arg Lys Ile Gly Val Cys Thr Ala Lys Asp Gly Ala Pro Cys

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Ile Phe Gly Gly Thr Val Tyr Arg Ser Gly Glu Ser Phe Gln Ser Ser

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Cys Lys Tyr Gln Cys Thr Cys Leu Asp Gly Ala Val Gly Cys Met Pro

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Leu Cys Ser Met Asp Val Arg Leu Pro Ser Pro Asp Cys Pro Phe Pro

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Arg Arg Val Lys Leu Pro Gly Lys Cys Cys Glu Glu Trp Val Cys Asp

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Glu Pro Lys Asp Gln Thr Val Val Gly Pro Ala Leu Ala Ala Tyr Arg

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Leu Glu Asp Thr Phe Gly Pro Asp Pro Thr Met Ile Arg Ala Asn Cys

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Leu Val Gln Thr Thr Glu Trp Ser Ala Cys Ser Lys Thr Cys Gly Met

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Gly Ile Ser Thr Arg Val Thr Asn Asp Asn Ala Ser Cys Arg Leu Glu

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Lys Gln Ser Arg Leu Cys Met Val Arg Pro Cys Glu Ala Asp Leu Glu

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Glu Asn Ile Lys Lys Gly Lys Lys Cys Ile Arg Thr Pro Lys Ile Ser

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Lys Pro Ile Lys Phe Glu Leu Ser Gly Cys Thr Ser Met Lys Thr Tyr

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Arg Ala Lys Phe Cys Gly Val Cys Thr Asp Gly Arg Cys Cys Thr Pro

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His Arg Thr Thr Thr Leu Pro Val Glu Phe Lys Cys Pro Asp Gly Glu

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Val Met Lys Lys Asn Met Met Phe Ile Lys Thr Cys Ala Cys His Tyr

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Asn Cys Pro Gly Asp Asn Asp Ile Phe Glu Ser Leu Tyr Tyr Arg Lys

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Met Tyr Gly Asp Met Ala His His His His His

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Val Gln Cys Gln Asp Cys Ser Ala Gln Cys Gln Cys Ala Ala Glu Ala

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Ala Pro His Cys Pro Ala Gly Val Ser Leu Val Leu Asp Gly Cys Gly

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Cys Cys Arg Val Cys Ala Lys Gln Leu Gly Glu Leu Cys Thr Glu Arg

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Asp Pro Cys Asp Pro His Lys Gly Leu Phe Cys Asp Phe Gly Ser Pro

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Ala Asn Arg Lys Ile Gly Val Cys Thr Ala Lys Asp Gly Ala Pro Cys

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Val Phe Gly Gly Ser Val Tyr Arg Ser Gly Glu Ser Phe Gln Ser Ser

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Cys Lys Tyr Gln Cys Thr Cys Leu Asp Gly Ala Val Gly Cys Val Pro

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Leu Cys Ser Met Asp Val Arg Leu Pro Ser Pro Asp Cys Pro Phe Pro

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Arg Arg Val Lys Leu Pro Gly Lys Cys Cys Glu Glu Trp Val Cys Asp

145 150 155 160

Glu Pro Lys Asp Arg Thr Ala Val Gly Pro Ala Leu Ala Ala Tyr Arg

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Leu Glu Asp Thr Phe Gly Pro Asp Pro Thr Met Met Arg Ala Asn Cys

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Leu Val Gln Thr Thr Glu Trp Ser Ala Cys Ser Lys Thr Cys Gly Met

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Gly Ile Ser Thr Arg Val Thr Asn Asp Asn Thr Phe Cys Arg Leu Glu

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Lys Gln Ser Arg Leu Cys Met Val Arg Pro Cys Glu Ala Asp Leu Glu

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Glu Asn Ile Lys Lys Gly Lys Lys Cys Ile Arg Thr Pro Lys Ile Ala

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

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Arg Ala Lys Phe Cys Gly Val Cys Thr Asp Gly Arg Cys Cys Thr Pro

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His Arg Thr Thr Thr Leu Pro Val Glu Phe Lys Cys Pro Asp Gly Glu

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Ile Met Lys Lys Asn Met Met Phe Ile Lys Thr Cys Ala Cys His Tyr

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Asn Cys Pro Gly Asp Asn Asp Ile Phe Glu Ser Leu Tyr Tyr Arg Lys

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Met Tyr Gly Asp Met Ala Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys

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Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val

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

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Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln

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Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser

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Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys

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Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile

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Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro

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

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Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn

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Val Gln Cys Gln Asp Cys Ser Ala Gln Cys Gln Cys Ala Ala Glu Ala

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Ala Pro His Cys Pro Ala Gly Val Ser Leu Val Leu Asp Gly Cys Gly

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Cys Cys Arg Val Cys Ala Lys Gln Leu Gly Glu Leu Cys Thr Glu Arg

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Asp Pro Cys Asp Pro His Lys Gly Leu Phe Cys Asp Phe Gly Ser Pro

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Ala Asn Arg Lys Ile Gly Val Cys Thr Ala Lys Asp Gly Ala Pro Cys

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Val Phe Gly Gly Ser Val Tyr Arg Ser Gly Glu Ser Phe Gln Ser Ser

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Cys Lys Tyr Gln Cys Thr Cys Leu Asp Gly Ala Val Gly Cys Val Pro

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Leu Cys Ser Met Asp Val Arg Leu Pro Ser Pro Asp Cys Pro Phe Pro

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Arg Arg Val Lys Leu Pro Gly Lys Cys Cys Glu Glu Trp Val Cys Asp

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Glu Pro Lys Asp Arg Thr Ala Val Gly Pro Ala Leu Ala Ala Tyr Arg

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Leu Val Gln Thr Thr Glu Trp Ser Ala Cys Ser Lys Thr Cys Gly Met

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Gly Ile Ser Thr Arg Val Thr Asn Asp Asn Thr Phe Cys Arg Leu Glu

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Lys Gln Ser Arg Leu Cys Met Val Arg Pro Cys Glu Ala Asp Leu Glu

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Glu Asn Ile Lys Lys Gly Lys Lys Cys Ile Arg Thr Pro Lys Ile Ala

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

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Arg Ala Lys Phe Cys Gly Val Cys Thr Asp Gly Arg Cys Cys Thr Pro

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His Arg Thr Thr Thr Leu Pro Val Glu Phe Lys Cys Pro Asp Gly Glu

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Ile Met Lys Lys Asn Met Met Phe Ile Lys Thr Cys Ala Cys His Tyr

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Asn Cys Pro Gly Asp Asn Asp Ile Phe Glu Ser Leu Tyr Tyr Arg Lys

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Met Tyr Gly Asp Met Ala His His His His His

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Val Gln Cys Gln Asn Cys Ser Gly Pro Cys Arg Cys Pro Ala Glu Gln

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

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Cys Cys Arg Val Cys Ala Lys Gln Leu Gly Glu Leu Cys Thr Glu Arg

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Asp Pro Cys Asp Pro His Lys Gly Leu Phe Cys Asp Phe Gly Ser Pro

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Ala Asn Arg Lys Ile Gly Val Cys Thr Ala Lys Asp Gly Ala Pro Cys

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Ile Phe Gly Gly Thr Val Tyr Arg Ser Gly Glu Ser Phe Gln Ser Ser

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Cys Lys Tyr Gln Cys Thr Cys Leu Asp Gly Ala Val Gly Cys Met Pro

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Leu Cys Ser Met Asp Val Arg Leu Pro Ser Pro Asp Cys Pro Phe Pro

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Arg Arg Val Lys Leu Pro Gly Lys Cys Cys Glu Glu Trp Val Cys Asp

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Glu Pro Lys Asp Gln Thr Val Val Gly Pro Ala Leu Ala Ala Tyr Arg

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Leu Glu Asp Thr Phe Gly Pro Asp Pro Thr Met Ile Arg Ala Asn Cys

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Leu Val Gln Thr Thr Glu Trp Ser Ala Cys Ser Lys Thr Cys Gly Met

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Gly Ile Ser Thr Arg Val Thr Asn Asp Asn Ala Ser Cys Arg Leu Glu

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Lys Gln Ser Arg Leu Cys Met Val Arg Pro Cys Glu Ala Asp Leu Glu

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Glu Asn Ile Lys Lys Gly Lys Lys Cys Ile Arg Thr Pro Lys Ile Ser

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

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Arg Ala Lys Phe Cys Gly Val Cys Thr Asp Gly Arg Cys Cys Thr Pro

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His Arg Thr Thr Thr Leu Pro Val Glu Phe Lys Cys Pro Asp Gly Glu

290 295 300

Val Met Lys Lys Asn Met Met Phe Ile Lys Thr Cys Ala Cys His Tyr

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Asn Cys Pro Gly Asp Asn Asp Ile Phe Glu Ser Leu Tyr Tyr Arg Lys

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Met Tyr Gly Asp Met Ala Glu Pro Lys Ser Ser Asp Lys Thr His Thr

340 345 350

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

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

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

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

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

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

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

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

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

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

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

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Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

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

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Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Glu Ser Met

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Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr

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115

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35 40 45

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Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

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Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

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Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

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Gly Val Ile Trp Arg Arg Gly Gly Thr Asp Tyr Asn Ala Ala Phe Met

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Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Arg Ser His Val Phe Phe

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Lys Met Thr Ser Leu Gln Thr Asp Asp Ser Ala Ile Tyr Tyr Cys Ala

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Arg Asp Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val

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Ser Ser

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Thr Val Ile Leu Thr Cys Arg Ser Ser Ile Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly

35 40 45

Leu Ile Gly Gly Thr Ser Asn Arg Ala Pro Gly Val Pro Val Arg Phe

50 55 60

Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Ala Glu Asp Asp Ala Met Tyr Phe Cys Ala Leu Trp Tyr Ser Thr

85 90 95

His Tyr Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu

100 105

<210> 10

<211> 5

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<223> mab147 HCDR1

<400> 10

Thr Tyr Ala Met Asn

15

<210> 11

<211> 19

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<223> mab147 HCDR2

<400> 11

Arg Ile Arg Thr Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser

1 5 10 15

Val Lys Asp

<210> 12

<211> 5

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<223> mab147 HCDR3

<400> 12

Thr Gly Phe Ala Tyr

15

<210> 13

<211> 10

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<223> mab147 LCDR1

<400> 13

Ser Ala Ser Ser Ser Val Ser Tyr Met His

1 5 10

<210> 14

<211> 7

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<223> mab147 LCDR2

<400> 14

Ser Thr Ser Asn Leu Ala Ser

15

<210> 15

<211> 9

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<223> mab147 LCDR3

<400> 15

Gln Gln Arg Ser Ser Tyr Pro Leu Thr

15

<210> 16

<211> 5

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<223> mab164 HCDR1

<400> 16

Thr Phe Gly Val His

15

<210> 17

<211> 16

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<223> mab164 HCDR2

<400> 17

Val Ile Trp Arg Arg Gly Gly Thr Asp Tyr Asn Ala Ala Phe Met Ser

1 5 10 15

<210> 18

<211> 6

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<223> mab164 HCDR3

<400> 18

Asp Gly Gly Phe Asp Tyr

15

<210> 19

<211> 14

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<223> mab164 LCDR1

<400> 19

Arg Ser Ser Ile Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn

1 5 10

<210> 20

<211> 7

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<223> mab164 LCDR2

<400> 20

Gly Thr Ser Asn Arg Ala Pro

15

<210> 21

<211> 9

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<223> mab164 LCDR3

<400> 21

Ala Leu Trp Tyr Ser Thr His Tyr Val

15

<210> 22

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu147-VL1

<400> 22

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 23

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu147-VL2

<400> 23

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 24

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu147-VL3

<400> 24

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 25

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu147-VL4

<400> 25

Asp Ile Gln Met 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 Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

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

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 26

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu147-VL5

<400> 26

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 Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

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

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 27

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu147-VH1

<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 Thr Tyr

20 25 30

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

35 40 45

Gly Arg Ile Arg Thr Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Glu Thr Gly Phe Ala Tyr Trp Asp Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 28

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu147-VH2

<400> 28

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 Asn Thr Tyr

20 25 30

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

35 40 45

Ala Arg Ile Arg Thr Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Glu Thr Gly Phe Ala Tyr Trp Asp Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 29

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu147-VH3

<400> 29

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 Ser Phe Asn Thr Tyr

20 25 30

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

35 40 45

Ala Arg Ile Arg Thr Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Glu Ser Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Glu Thr Gly Phe Ala Tyr Trp Asp Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 30

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu164-VL7

<400> 30

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

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Ile Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Phe Arg Gly

35 40 45

Leu Ile Gly Gly Thr Ser Asn Arg Ala Pro Trp Thr Pro Ala Arg Phe

50 55 60

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

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Thr

85 90 95

His Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 31

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu164-VL8

<400> 31

Glu Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Ile Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Gln Thr Pro Gly Gln Ala Phe Arg Gly

35 40 45

Leu Ile Gly Gly Thr Ser Asn Arg Ala Pro Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Thr

85 90 95

His Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 32

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu164-VL9

<400> 32

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

1 5 10 15

Arg Val Thr Ile Ser Cys Arg Ser Ser Ile Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Gln Leu Pro Gly Thr Ala Phe Lys Gly

35 40 45

Leu Ile Gly Gly Thr Ser Asn Arg Ala Pro Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Thr

85 90 95

His Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 33

<211> 114

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu164-VH5

<400> 33

Glu Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Phe

20 25 30

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

35 40 45

Ala Val Ile Trp Arg Arg Gly Gly Thr Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Asp Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val

100 105 110

Ser Ser

<210> 34

<211> 114

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu164-VH6

<400> 34

Glu Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Phe

20 25 30

Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Arg Arg Gly Gly Thr Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Phe

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Asp Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val

100 105 110

Ser Ser

<210> 35

<211> 114

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu164-VH7

<400> 35

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Ile Ser Thr Phe

20 25 30

Gly Val His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Arg Arg Gly Gly Thr Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val

100 105 110

Ser Ser

<210> 36

<211> 114

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu164-VH8

<400> 36

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Ile Ser Thr Phe

20 25 30

Gly Val His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Arg Arg Gly Gly Thr Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Phe

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val

100 105 110

Ser Ser

<210> 37

<211> 330

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> heavy chain variable domain w

<400> 37

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

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

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

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

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

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

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

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 38

<211> 330

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> heavy chain variable domain y

<400> 38

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

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

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

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

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

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

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

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 39

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> kappa light chain constant region of a humanized antibody

<400> 39

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

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

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 40

<211> 105

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> humanized antibody lambda light chain constant region

<400> 40

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

1 5 10 15

Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Glu Lys Thr Val Ala Pro Thr Glu Cys

100 105

<210> 41

<211> 446

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> heavy chain Ch147

<400> 41

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

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Asn Thr Tyr

20 25 30

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

35 40 45

Ala Arg Ile Arg Thr Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Glu Ser Met

65 70 75 80

Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr

85 90 95

Tyr Cys Val Glu Thr Gly Phe Ala Tyr Trp Asp Gln Gly Thr Leu Val

100 105 110

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

115 120 125

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

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Lys Pro Arg Glu 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 Tyr Lys Cys

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

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

435 440 445

<210> 42

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Ch147 light chain

<400> 42

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

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu 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> 43

<211> 446

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu147-11wk heavy chain

<400> 43

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 Thr Tyr

20 25 30

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

35 40 45

Gly Arg Ile Arg Thr Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Glu Thr Gly Phe Ala Tyr Trp Asp Gln Gly Thr Leu Val

100 105 110

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

115 120 125

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

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Lys Pro Arg Glu 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 Tyr Lys Cys

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

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

435 440 445

<210> 44

<211> 446

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu147-21wk heavy chain

<400> 44

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 Asn Thr Tyr

20 25 30

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

35 40 45

Ala Arg Ile Arg Thr Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Glu Thr Gly Phe Ala Tyr Trp Asp Gln Gly Thr Leu Val

100 105 110

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

115 120 125

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

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Lys Pro Arg Glu 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 Tyr Lys Cys

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

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

435 440 445

<210> 45

<211> 446

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu147-31wk heavy chain

<400> 45

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 Ser Phe Asn Thr Tyr

20 25 30

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

35 40 45

Ala Arg Ile Arg Thr Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Glu Ser Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Glu Thr Gly Phe Ala Tyr Trp Asp Gln Gly Thr Leu Val

100 105 110

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

115 120 125

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

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Lys Pro Arg Glu 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 Tyr Lys Cys

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

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

435 440 445

<210> 46

<211> 446

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu147-11yk heavy chain

<400> 46

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 Thr Tyr

20 25 30

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

35 40 45

Gly Arg Ile Arg Thr Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Glu Thr Gly Phe Ala Tyr Trp Asp Gln Gly Thr Leu Val

100 105 110

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

115 120 125

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

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro

245 250 255

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

260 265 270

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

275 280 285

Lys Pro Arg Glu 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 Tyr Lys Cys

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

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

435 440 445

<210> 47

<211> 446

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu147-21yk heavy chain

<400> 47

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 Asn Thr Tyr

20 25 30

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

35 40 45

Ala Arg Ile Arg Thr Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Glu Thr Gly Phe Ala Tyr Trp Asp Gln Gly Thr Leu Val

100 105 110

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

115 120 125

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

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro

245 250 255

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

260 265 270

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

275 280 285

Lys Pro Arg Glu 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 Tyr Lys Cys

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

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

435 440 445

<210> 48

<211> 446

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu147-31yk heavy chain

<400> 48

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 Ser Phe Asn Thr Tyr

20 25 30

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

35 40 45

Ala Arg Ile Arg Thr Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Glu Ser Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Glu Thr Gly Phe Ala Tyr Trp Asp Gln Gly Thr Leu Val

100 105 110

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

115 120 125

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

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro

245 250 255

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

260 265 270

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

275 280 285

Lys Pro Arg Glu 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 Tyr Lys Cys

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

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

435 440 445

<210> 49

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu147-11wk light chain

<400> 49

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu 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> 50

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu147-12wk light chain

<400> 50

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu 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> 51

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu147-13wk light chain

<400> 51

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu 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> 52

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu147-14wk light chain

<400> 52

Asp Ile Gln Met 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 Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

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

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu 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> 53

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu147-15wk light chain

<400> 53

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 Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

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

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu 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> 54

<211> 444

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Ch164 heavy chain

<400> 54

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Thr Phe

20 25 30

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

35 40 45

Gly Val Ile Trp Arg Arg Gly Gly Thr Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Arg Ser His Val Phe Phe

65 70 75 80

Lys Met Thr Ser Leu Gln Thr Asp Asp Ser Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Asp Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val

100 105 110

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

115 120 125

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

130 135 140

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

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

245 250 255

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

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

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

340 345 350

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

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

385 390 395 400

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

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

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

435 440

<210> 55

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Ch164 light chain

<400> 55

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

1 5 10 15

Thr Val Ile Leu Thr Cys Arg Ser Ser Ile Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly

35 40 45

Leu Ile Gly Gly Thr Ser Asn Arg Ala Pro Gly Val Pro Val Arg Phe

50 55 60

Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Ala Glu Asp Asp Ala Met Tyr Phe Cys Ala Leu Trp Tyr Ser Thr

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Val Ala Pro Thr Glu Cys

210

<210> 56

<211> 444

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu164-57wl heavy chain

<400> 56

Glu Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Phe

20 25 30

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

35 40 45

Ala Val Ile Trp Arg Arg Gly Gly Thr Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Asp Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val

100 105 110

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

115 120 125

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

130 135 140

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

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

245 250 255

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

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

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

340 345 350

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

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

385 390 395 400

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

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

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

435 440

<210> 57

<211> 444

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu164-67wl heavy chain

<400> 57

Glu Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Phe

20 25 30

Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Arg Arg Gly Gly Thr Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Phe

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Asp Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val

100 105 110

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

115 120 125

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

130 135 140

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

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

245 250 255

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

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

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

340 345 350

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

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

385 390 395 400

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

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

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

435 440

<210> 58

<211> 444

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu164-77wl heavy chain

<400> 58

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Ile Ser Thr Phe

20 25 30

Gly Val His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Arg Arg Gly Gly Thr Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val

100 105 110

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

115 120 125

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

130 135 140

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

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

245 250 255

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

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

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

340 345 350

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

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

385 390 395 400

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

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

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

435 440

<210> 59

<211> 444

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu164-87wl heavy chain

<400> 59

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Ile Ser Thr Phe

20 25 30

Gly Val His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Arg Arg Gly Gly Thr Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Phe

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val

100 105 110

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

115 120 125

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

130 135 140

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

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

245 250 255

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

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

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

340 345 350

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

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

385 390 395 400

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

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

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

435 440

<210> 60

<211> 444

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu164-57yl heavy chain

<400> 60

Glu Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Phe

20 25 30

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

35 40 45

Ala Val Ile Trp Arg Arg Gly Gly Thr Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Asp Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val

100 105 110

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

115 120 125

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

130 135 140

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val

245 250 255

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

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

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

340 345 350

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

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

385 390 395 400

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

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

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

435 440

<210> 61

<211> 444

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu164-67yl heavy chain

<400> 61

Glu Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Phe

20 25 30

Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Arg Arg Gly Gly Thr Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Phe

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Asp Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val

100 105 110

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

115 120 125

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

130 135 140

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val

245 250 255

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

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

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

340 345 350

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

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

385 390 395 400

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

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

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

435 440

<210> 62

<211> 444

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu164-77yl heavy chain

<400> 62

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Ile Ser Thr Phe

20 25 30

Gly Val His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Arg Arg Gly Gly Thr Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val

100 105 110

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

115 120 125

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

130 135 140

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val

245 250 255

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

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

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

340 345 350

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

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

385 390 395 400

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

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

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

435 440

<210> 63

<211> 444

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu164-87yl heavy chain

<400> 63

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Ile Ser Thr Phe

20 25 30

Gly Val His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Arg Arg Gly Gly Thr Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Phe

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val

100 105 110

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

115 120 125

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

130 135 140

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val

245 250 255

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

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

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

340 345 350

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

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

385 390 395 400

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

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

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

435 440

<210> 64

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu164-57wl light chain

<400> 64

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

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Ile Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Phe Arg Gly

35 40 45

Leu Ile Gly Gly Thr Ser Asn Arg Ala Pro Trp Thr Pro Ala Arg Phe

50 55 60

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

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Thr

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Val Ala Pro Thr Glu Cys

210

<210> 65

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu164-58wl light chain

<400> 65

Glu Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Ile Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Gln Thr Pro Gly Gln Ala Phe Arg Gly

35 40 45

Leu Ile Gly Gly Thr Ser Asn Arg Ala Pro Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Thr

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Val Ala Pro Thr Glu Cys

210

<210> 66

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu164-59wl light chain

<400> 66

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

1 5 10 15

Arg Val Thr Ile Ser Cys Arg Ser Ser Ile Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Gln Leu Pro Gly Thr Ala Phe Lys Gly

35 40 45

Leu Ile Gly Gly Thr Ser Asn Arg Ala Pro Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Thr

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Val Ala Pro Thr Glu Cys

210

<210> 67

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> heavy chain mAb1

<400> 67

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

1 5 10 15

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

20 25 30

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

35 40 45

Ser Gly Ile Gly Thr Gly Gly Gly Thr Tyr Ser Thr Asp Ser Val Lys

50 55 60

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

65 70 75 80

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

85 90 95

Arg Gly Asp Tyr Tyr Gly Ser Gly Ser Phe Phe Asp Cys Trp Gly Gln

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

435 440 445

Gly Lys

450

<210> 68

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> mAb1 light chain

<400> 68

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 69

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> mab95 VH

<400> 69

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asn Tyr

20 25 30

Asp Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

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

50 55 60

Lys Asp Lys Ala Thr Leu Thr Phe Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Trp Gly Leu Ile Pro Gly Thr Thr Ser Tyr Phe Asp Val Trp

100 105 110

Gly Thr Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 70

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> mab95 VL

<400> 70

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

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Ser Asn Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 71

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> mab95 HCDR1

<400> 71

Asn Tyr Asp Ile His

15

<210> 72

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> mab95 HCDR2

<400> 72

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

1 5 10 15

Asp

<210> 73

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> mab95 HCDR3

<400> 73

Trp Gly Leu Ile Pro Gly Thr Thr Ser Tyr Phe Asp Val

1 5 10

<210> 74

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> mab95 LCDR1

<400> 74

Arg Ala Ser Ser Ser Val Ser Tyr Ile His

1 5 10

<210> 75

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> mab95 LCDR2

<400> 75

Ala Thr Ser Asn Leu Ala Ser

15

<210> 76

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> mab95 LCDR3

<400> 76

Gln Gln Trp Asn Ser Asn Pro Trp Thr

15

<210> 77

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu95-VL1

<400> 77

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

1 5 10 15

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

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Asn Ser Asn Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 78

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu95-VL2

<400> 78

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

1 5 10 15

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

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Asn Ser Asn Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 79

<211> 105

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu95-VL3

<400> 79

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

1 5 10 15

Arg Val Thr Ile Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile His

20 25 30

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

35 40 45

Thr Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys

50 55 60

Ser Gly Thr Ser Tyr Ser Leu Ala Ile Thr Gly Leu Gln Ala Glu Asp

65 70 75 80

Glu Ala Asp Tyr Tyr Cys Gln Gln Trp Asn Ser Asn Pro Trp Thr Phe

85 90 95

Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 80

<211> 105

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu95-VL4

<400> 80

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

1 5 10 15

Arg Val Thr Ile Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile His

20 25 30

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

35 40 45

Thr Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys

50 55 60

Ser Gly Thr Ser Tyr Ser Leu Ala Ile Thr Gly Leu Gln Ala Glu Asp

65 70 75 80

Glu Ala Asp Tyr Tyr Cys Gln Gln Trp Asn Ser Asn Pro Trp Thr Phe

85 90 95

Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 81

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu95-VH1

<400> 81

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asn Tyr

20 25 30

Asp Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

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

50 55 60

Lys Asp Arg Val Thr Leu Thr Phe Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Trp Gly Leu Ile Pro Gly Thr Thr Ser Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 82

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu95-VH2

<400> 82

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asn Tyr

20 25 30

Asp Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

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

50 55 60

Lys Asp Arg Ala Thr Leu Thr Phe Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Trp Gly Leu Ile Pro Gly Thr Thr Ser Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 83

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu95-VH3

<400> 83

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asn Tyr

20 25 30

Asp Ile His Trp Val Lys Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Trp Gly Leu Ile Pro Gly Thr Thr Ser Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 84

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu95-VH4

<400> 84

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asn Tyr

20 25 30

Asp Ile His Trp Val Lys Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

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

50 55 60

Lys Asp Lys Ala Thr Leu Thr Phe Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Trp Gly Leu Ile Pro Gly Thr Thr Ser Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 85

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu95-VH5

<400> 85

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Arg Ala Ser Gly Phe Thr Phe Thr Asn Tyr

20 25 30

Ala Ile His Trp Val Lys Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Trp Gly Met Ile Pro Gly Thr Asn Ser Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 86

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Ch95 heavy chain

<400> 86

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asn Tyr

20 25 30

Asp Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

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

50 55 60

Lys Asp Lys Ala Thr Leu Thr Phe Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Trp Gly Leu Ile Pro Gly Thr Thr Ser Tyr Phe Asp Val Trp

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

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

195 200 205

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

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

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

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

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

275 280 285

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

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

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

435 440 445

Ser Pro Gly Lys

450

<210> 87

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Ch95 light chain

<400> 87

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

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Ser Asn Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Arg Leu 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> 88

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu95-11wk heavy chain

<400> 88

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asn Tyr

20 25 30

Asp Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

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

50 55 60

Lys Asp Arg Val Thr Leu Thr Phe Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Trp Gly Leu Ile Pro Gly Thr Thr Ser Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

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

195 200 205

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

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

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

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

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

275 280 285

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

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

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

435 440 445

Ser Pro Gly Lys

450

<210> 89

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu95-21wk heavy chain

<400> 89

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asn Tyr

20 25 30

Asp Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

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

50 55 60

Lys Asp Arg Ala Thr Leu Thr Phe Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Trp Gly Leu Ile Pro Gly Thr Thr Ser Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

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

195 200 205

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

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

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

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

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

275 280 285

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

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

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

435 440 445

Ser Pro Gly Lys

450

<210> 90

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu95-31wk heavy chain

<400> 90

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asn Tyr

20 25 30

Asp Ile His Trp Val Lys Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Trp Gly Leu Ile Pro Gly Thr Thr Ser Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

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

195 200 205

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

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

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

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

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

275 280 285

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

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

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

435 440 445

Ser Pro Gly Lys

450

<210> 91

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu95-41wk heavy chain

<400> 91

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asn Tyr

20 25 30

Asp Ile His Trp Val Lys Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

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

50 55 60

Lys Asp Lys Ala Thr Leu Thr Phe Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Trp Gly Leu Ile Pro Gly Thr Thr Ser Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

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

195 200 205

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

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

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

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

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

275 280 285

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

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

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

435 440 445

Ser Pro Gly Lys

450

<210> 92

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu95-51wk heavy chain

<400> 92

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Arg Ala Ser Gly Phe Thr Phe Thr Asn Tyr

20 25 30

Ala Ile His Trp Val Lys Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Trp Gly Met Ile Pro Gly Thr Asn Ser Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

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

195 200 205

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

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

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

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

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

275 280 285

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

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

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

435 440 445

Ser Pro Gly Lys

450

<210> 93

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu95-11yk heavy chain

<400> 93

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asn Tyr

20 25 30

Asp Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

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

50 55 60

Lys Asp Arg Val Thr Leu Thr Phe Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Trp Gly Leu Ile Pro Gly Thr Thr Ser Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

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

195 200 205

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

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

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

245 250 255

Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

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

275 280 285

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

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

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

435 440 445

Ser Pro Gly Lys

450

<210> 94

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu95-21yk heavy chain

<400> 94

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asn Tyr

20 25 30

Asp Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

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

50 55 60

Lys Asp Arg Ala Thr Leu Thr Phe Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Trp Gly Leu Ile Pro Gly Thr Thr Ser Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

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

195 200 205

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

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

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

245 250 255

Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

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

275 280 285

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

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

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

435 440 445

Ser Pro Gly Lys

450

<210> 95

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu95-31yk

<400> 95

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asn Tyr

20 25 30

Asp Ile His Trp Val Lys Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Trp Gly Leu Ile Pro Gly Thr Thr Ser Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

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

195 200 205

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

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

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

245 250 255

Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

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

275 280 285

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

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

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

435 440 445

Ser Pro Gly Lys

450

<210> 96

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu95-41yk heavy chain

<400> 96

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asn Tyr

20 25 30

Asp Ile His Trp Val Lys Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

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

50 55 60

Lys Asp Lys Ala Thr Leu Thr Phe Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Trp Gly Leu Ile Pro Gly Thr Thr Ser Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

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

195 200 205

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

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

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

245 250 255

Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

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

275 280 285

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

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

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

435 440 445

Ser Pro Gly Lys

450

<210> 97

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu95-51yk heavy chain

<400> 97

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Arg Ala Ser Gly Phe Thr Phe Thr Asn Tyr

20 25 30

Ala Ile His Trp Val Lys Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Trp Gly Met Ile Pro Gly Thr Asn Ser Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

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

195 200 205

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

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

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

245 250 255

Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

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

275 280 285

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

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

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

435 440 445

Ser Pro Gly Lys

450

<210> 98

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu95-11wk light chain

<400> 98

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

1 5 10 15

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

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Asn Ser Asn Pro Trp Thr

85 90 95

Phe Gly Gly 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> 99

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu95-12wk light chain

<400> 99

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

1 5 10 15

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

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Asn Ser Asn Pro Trp Thr

85 90 95

Phe Gly Gly 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> 100

<211> 212

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu95-13wk light chain

<400> 100

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

1 5 10 15

Arg Val Thr Ile Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile His

20 25 30

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

35 40 45

Thr Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys

50 55 60

Ser Gly Thr Ser Tyr Ser Leu Ala Ile Thr Gly Leu Gln Ala Glu Asp

65 70 75 80

Glu Ala Asp Tyr Tyr Cys Gln Gln Trp Asn Ser Asn Pro Trp Thr Phe

85 90 95

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

100 105 110

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

115 120 125

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

130 135 140

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

145 150 155 160

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

165 170 175

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

180 185 190

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

195 200 205

Arg Gly Glu Cys

210

<210> 101

<211> 212

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<223> Hu95-14wk light chain

<400> 101

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

1 5 10 15

Arg Val Thr Ile Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile His

20 25 30

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

35 40 45

Thr Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys

50 55 60

Ser Gly Thr Ser Tyr Ser Leu Ala Ile Thr Gly Leu Gln Ala Glu Asp

65 70 75 80

Glu Ala Asp Tyr Tyr Cys Gln Gln Trp Asn Ser Asn Pro Trp Thr Phe

85 90 95

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

100 105 110

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

115 120 125

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

130 135 140

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

145 150 155 160

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

165 170 175

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

180 185 190

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

195 200 205

Arg Gly Glu Cys

210

<210> 102

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu95-VH5 HCDR1

<400> 102

Asn Tyr Ala Ile His

15

<210> 103

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu95-VH5 HCDR2

<400> 103

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

1 5 10 15

Asp

<210> 104

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Hu95-VH5 HCDR3

<400> 104

Trp Gly Met Ile Pro Gly Thr Asn Ser Tyr Phe Asp Val

1 5 10

<---

Claims (64)

1. An anti-connective tissue growth factor (CTGF) antibody comprising a heavy chain variable region and a light chain variable region, wherein: i) the heavy chain variable region contains the same sequences HCDR1, HCDR2 and HCDR3 as the sequences HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 6, and the light chain variable region contains the same sequences LCDR1, LCDR2 and LCDR3, as well as the light chain variable region sequences LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 7; ii) the heavy chain variable region contains the same sequences HCDR1, HCDR2 and HCDR3 as the sequences HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 8, and the light chain variable region contains the same sequences LCDR1, LCDR2 and LCDR3, as well as the light chain variable region sequences LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 9; ii-i) the heavy chain variable region contains the same HCDR1, HCDR2 and HCDR3 sequences as the HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region shown in SEQ ID NO: 69, and the light chain variable region contains the same LCDR1 sequences, LCDR2 and LCDR3, as well as the light chain variable region sequences LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 70; or ii-ii) the heavy chain variable region contains the same HCDR1, HCDR2 and HCDR3 sequences as the HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region shown in SEQ ID NO: 85, and the light chain variable region contains the same LCDR1, LCDR2 sequences and LCDR3, as well as the light chain variable region sequences LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 70. 2. Antibody against CTGF according to claim 1, containing a heavy chain variable region and a light chain variable region, where: iii) the heavy chain variable region contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively, and the light chain variable region contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively; iv) the heavy chain variable region contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively, and the light chain variable region contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively; iv-i) the heavy chain variable region contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 71, SEQ ID NO: 72 and SEQ ID NO: 73, respectively, and the light chain variable region contains LCDR1, LCDR2 and LCDR3, as shown in SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, respectively; or iv-ii) the heavy chain variable region contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 102, SEQ ID NO: 103 and SEQ ID NO: 104, respectively, and the light chain variable region contains LCDR1, LCDR2 and LCDR3, as shown in SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, respectively. 3. An anti-CTGF antibody according to claim 1 or 2, wherein said anti-CTGF antibody is a murine antibody, a chimeric antibody, or a humanized antibody. 4. Antibody against CTGF according to any one of paragraphs. 1-3, containing a heavy chain variable region and a light chain variable region, where: (v) the amino acid sequence of the heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 6, 27, 28 or 29, and the amino acid sequence of the light chain variable region has at least 90% sequence identity with SEQ ID NO: 7, 22, 23, 24, 25 or 26; (vi) the amino acid sequence of the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 8, 33, 34, 35 or 36, and the amino acid sequence of the light chain variable region has at least 90% sequence identity sequences with SEQ ID NO: 9, 30, 31 or 32; or (vi-i) the amino acid sequence of the heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 69, 81, 82, 83, 84 or 85, and the amino acid sequence of the light chain variable region has at least 90 % sequence identity to SEQ ID NO: 70, 77, 78, 79 or 80. 5. The anti-CTGF antibody of claim 2, wherein said anti-CTGF antibody is a humanized antibody, wherein said humanized antibody comprises a human antibody framework region or a variant framework region thereof, wherein said framework region variant has up to 10 back mutations on each of the framework regions the light chain region and/or the heavy chain framework region of a human antibody. 6. The anti-CTGF antibody of claim 5, wherein said humanized antibody comprises a light chain variable region and a heavy chain variable region selected from the group consisting of the following (a), (b) or (b-i): (a) a light chain variable region containing: LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively, and one or more back mutations selected from the group consisting of 4L, 36F, 43S, 45K, 47W, 58V and 71Y, and heavy chain variable region containing: HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively, and one or more back mutations selected from the group consisting of 28S, 30N, 49A, 75E, 76S, 93V, 94E and 104D; (b) a light chain variable region comprising: LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively, and one or more back mutations selected from the group consisting of 36V, 44F, 46G and 49G, And heavy chain variable region containing: HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively, and one or more back mutations selected from the group consisting of 44G, 49G, 27F, 48L, 67L, 71K, 78V and 80F; (b-i) a light chain variable region comprising: LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, respectively, and one or more back mutations selected from the group consisting of 45P, 46W, 48Y, 69S and 70Y, and heavy chain variable region containing: HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 71, SEQ ID NO: 72 and SEQ ID NO: 73, respectively, and one or more back mutations selected from the group consisting of 27F, 38K, 48I, 67K, 68A, 70L and 72F; or (b-ii) a light chain variable region comprising: LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, respectively, and one or more back mutations selected from the group consisting of 45P, 46W, 48Y, 69S and 70Y, and heavy chain variable region containing: HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 102, SEQ ID NO: 103 and SEQ ID NO: 104, respectively, and one or more back mutations selected from the group consisting of 27F, 38K, 48I, 67K, 68A, 70L and 72F. 7. Antibody against CTGF according to claim 6, containing a heavy chain variable region and a light chain variable region, in which: (vii) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 7; (viii) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 27, 28 or 29, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 22, 23, 24, 25 or 26; (ix) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 8, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 9; (x) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 33, 34, 35 or 36, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 30, 31 or 32; (xi) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 69, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 70; or (xii) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 81, 82, 83, 84 or 85, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 77, 78, 79 or 80. 8. The anti-CTGF antibody of claim 7, which comprises a heavy chain variable region and a light chain variable region as shown below: (xiii) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 27, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 22; (xiv) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 34, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 30; or (xv) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 85, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 77. 9. Antibody against CTGF according to any one of paragraphs. 1-8, wherein said antibody further comprises a heavy chain constant region and a light chain constant region of the antibody; wherein the heavy chain constant region is selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4 constant regions and variants thereof, and the light chain constant region is selected from the group consisting of human antibody κ and λ chain constant regions and variants thereof. 10. The anti-CTGF antibody of claim 9, wherein the antibody comprises a heavy chain constant region as shown in SEQ ID NO: 37 or 38 and a light chain constant region as shown in SEQ ID NO: 39 or 40. 11. Antibody against CTGF according to any one of paragraphs. 1-10, containing: (c) a heavy chain having at least 85% sequence identity to SEQ ID NO: 41, 43, 44, 45, 46, 47 or 48, and a light chain having at least 85% sequence identity to SEQ ID NO: 42, 49, 50, 51, 52 or 53; (d) a heavy chain having at least 85% sequence identity to SEQ ID NO: 54, 56, 57, 58, 59, 60, 61, 62 or 63, and a light chain having at least 85% sequence identity similar sequence identity to SEQ ID NO: 55, 64, 65 or 66; (e) a heavy chain as shown in SEQ ID NO: 41, 43, 44, 45, 46, 47 or 48, and a light chain as shown in SEQ ID NO: 42, 49, 50, 51, 52 or 53; (f) a heavy chain as shown in SEQ ID NO: 54, 56, 57, 58, 59, 60, 61, 62 or 63, and a light chain as shown in SEQ ID NO: 55, 64, 65 or 66; (g) a heavy chain having at least 85% sequence identity to SEQ ID NO: 86, 88, 89, 90, 91, 92, 93, 94, 95, 96 or 97, and a light chain having at least at least 85% sequence identity to SEQ ID NO: 87, 98, 99, 100 or 101; or (h) a heavy chain as shown in SEQ ID NO: 86, 88, 89, 90, 91, 92, 93, 94, 95, 96 or 97, and a light chain as shown in SEQ ID NO: 87, 98, 99, 100 or 101. 12. Antibody against CTGF according to claim 11, containing: (j) a heavy chain as shown in SEQ ID NO: 46 and a light chain as shown in SEQ ID NO: 49; (k) a heavy chain as shown in SEQ ID NO: 61 and a light chain as shown in SEQ ID NO: 64; or (l) a heavy chain as shown in SEQ ID NO: 97, and a light chain as shown in SEQ ID NO: 98. 13. A nucleic acid molecule encoding an anti-CTGF antibody according to any one of claims. 1-12. 14. A host cell for producing an anti-CTGF antibody, comprising the nucleic acid molecule of claim 13. 15. A pharmaceutical composition for the treatment or prevention of a disease associated with CTGF, containing a therapeutically effective amount of an antibody against CTGF according to any one of paragraphs. 1-12 and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients. 16. A method for determining CTGF, including the step of bringing an antibody against CTGF according to any one of paragraphs. 1-12 into contact with the subject or its sample. 17. A method of treating or preventing a disease associated with CTGF, comprising administering to a subject a therapeutically effective amount or a prophylactically effective amount of an anti-CTGF antibody according to any one of claims. 1-12 or the pharmaceutical composition of claim 15, wherein the CTGF-related disease is fibrotic diseases, hypertension, diabetes, myocardial infarction, arthritis, CTGF-related cell proliferative disease, atherosclerosis, glaucoma or cancer. 18. The method of claim 17, wherein the fibrotic disease is selected from the group consisting of: spontaneous pulmonary fibrosis, diabetic nephropathy, diabetic retinopathy, osteoarthritis, scleroderma, chronic heart failure, liver cirrhosis or renal fibrosis; the cancer is selected from the group consisting of: acute lymphoblastic leukemia, dermatofibroma, breast cancer, angiolipoma, angioleiomyoma, connective tissue cancer, prostate cancer, ovarian cancer, colorectal cancer, pancreatic cancer, gastrointestinal cancer or cancer liver.